WO2023174207A1 - Procédé d'obtention d'extraits de spatholobus suberectus dunn (ssd), fractions et compositions de ceux-ci et utilisation contre des maladies virales - Google Patents

Procédé d'obtention d'extraits de spatholobus suberectus dunn (ssd), fractions et compositions de ceux-ci et utilisation contre des maladies virales Download PDF

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WO2023174207A1
WO2023174207A1 PCT/CN2023/081058 CN2023081058W WO2023174207A1 WO 2023174207 A1 WO2023174207 A1 WO 2023174207A1 CN 2023081058 W CN2023081058 W CN 2023081058W WO 2023174207 A1 WO2023174207 A1 WO 2023174207A1
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ssp
ssd
extract
solvent
cov
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Li Liu
Jianping Chen
Zhiwei Chen
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Versitech Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

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  • Disclosed is a method of preparing a natural product of antiviral extract from Spatholobus suberectus Dunn (SSD) , which contains total flavonoid and proanthocyanidins.
  • the disclosure relates to a method for discovering an active proanthocyanidins of Spatholobus suberectus (SSP) as viral entry inhibitor.
  • a method of preventing and treating viral diseases such as coronavirus disease 2019 (COVID-19) .
  • Herbal medicines and purified natural products provide a rich resource for novel antiviral drug development. Identification of the antiviral mechanisms from these natural agents has shed light on where they interact with the viral life cycle, such as viral entry, replication, assembly, and release.
  • Traditional Chinese Medicine TCM
  • Artemisia annua is the representative herb.
  • Qinghaosu an antimalarial drug from Artemisia annual, and subsequent development of several therapies to inhibit the malaria parasite has saved millions of lives.
  • the plant SSD Slightly sweet, warm in nature, belongs to Chinese medicine liver and kidney. "Compendium of Materia Medica” claims that it can nourish the stomach and dry stomach; “Compendium of Compendium of Materia Medica” claims that it can promote blood circulation and warm the waist and knees.
  • Centipede to treat diseases caused by viruses, but it has not been reported in Covid-19. Till now, no publication has indicated SSP as a viral entry inhibitor.
  • the chemical constituents of SSD mainly include flavonoids, terpenes, sterols, lignin, anthraquinones, polyphenols, proanthocyanidins and some trace elements, among which the content of flavonoids is the most.
  • the research on the extraction method of content mostly uses the total flavonoid extraction rate as an indicator and does not consider the effect of the extraction method on the drug efficacy of the extract.
  • a method of obtaining an extract of SSD comprising: (i) cutting SSD into pieces; (ii) immersing and treating SSD pieces in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; and (iv) drying the concentrated extract to obtain the powder of the extract of SSD (SSP) .
  • a method of obtaining an active fraction from SSD comprising: (i) cutting SSD into pieces; (ii) immersing and treating SSD pieces in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; (iv) drying the concentrated extract to obtain the powder of the extract of SSD (SSP) ; (v) dispersing SSP in water to obtain an aqueous solution; (vi) extracting the SSP aqueous solution with a series of solvent with different polarities at equal volume to obtain parts of SSP corresponding to each solvent, the series of solvent comprising n-butanol; and (vii) separating the n-butanol part (SSP-n-BuOH) with eluents to obtain the active fraction.
  • the treating process in (ii) comprises percolation.
  • an extract of SSD and an active fraction from SSD obtained by the above methods as well as a composition comprising the extract or the active fraction.
  • a method of preventing and/or treating a viral disease comprising administering an effective amount of an extract of SSD or an active fraction from SSD obtained by the above methods to a subject in need thereof.
  • the viral disease is caused by SARS-CoV-2 including Omicron variants, Ebola virus (EBOV) , HIV-1 with either CCR5 or CXCR4 tropism, SARS-CoV, H5N1.
  • SARS-CoV-2 including Omicron variants, Ebola virus (EBOV) , HIV-1 with either CCR5 or CXCR4 tropism, SARS-CoV, H5N1.
  • EBOV Ebola virus
  • the effective dose is 0.5 ⁇ g/ml to 5000 ⁇ g/ml.
  • an extract of SSD or an active fraction from SSD obtained by the above methods in the manufacture of preparations for prevention and/or treatment of a viral disease.
  • Figure 1 Flow chart of the study. Preparation and quality-controlled of SSP which is used to determine the broad-spectrum antiviral activity and its underlying mechanism. The powder SSP was further extracted to obtain the effective active parts.
  • FIG. 1 Representative pictures of SSP's quality control.
  • FIG. 3 Antiviral activity of SSP against SARS-CoV-2. Serially diluted SSP was added to HEK293T-ACE2 cells infected with SARS-CoV-2 (a) and VSV (b) respectively. The luciferase level was measured 2 or 3 days post-infection. To test SSP cytotoxicity, cells viability (c) was measured. (d) Pre-treatment of SARS-CoV-2 and target cells inhibited viral infection. (e) Binding of RBD to ACE2 expressing 293T cells, but not 293T control cells. (f) SSP inhibited RBD binding to target cells.
  • SSP pre-treated HEK293T-ACE2 cells were incubated with RBD-PD1 for 30 mins on ice, followed by antibody staining of ACE2 (upper panel) and RBD (lower panel) and flow cytometry analysis.
  • the data represent the mean ⁇ SEM of triplicate experiments.
  • FIG. 4 Antiviral activity of SSP against SARS-CoV-1, H5N1, and HIV, and EBOV viruses.
  • Serially diluted SSP was added to HEK293T-ACE2, MDCK, GHOST-CCR5, and GHOST-CXCR4 infected with SARS-CoV-1 (a) H5N1 (b) HIV ADA (c) , and HIV HXB2 (d) respectively.
  • the luciferase level was measured 2 or 3 days post-infection. Cells viability (e) was measured to test SSP cytotoxicity.
  • Serially diluted SSP was added to 293T cells infected with EBOV. The luciferase level was measured 2 days post infection. Cells viability was measured to test SSP cytotoxicity against the 293T cells. The data represent the mean ⁇ SEM of triplicate experiments.
  • FIG. 1 Mechanisms of SSP mediated virus entry inhibition.
  • (a-b) Pre-treatment of SARS-CoV-1 (a) , H5N1 (b) and target cells inhibited viral infection.
  • (c-d) Post-entry assay.
  • GHOST-CD4-CCR5 or CXCR4 cells were co-incubated with pseudovirus for 2 hrs, washed, and then treated with the presence of 50 mg/ml SSP and 1 mM AZT as a positive control for 48 hr.
  • SSP does not inhibit either HIV-1 ADA or HIV HxB2 virus gene replication after the viral entry is achieved.
  • (e-f) SSP-virus interaction assay. HIV-1 ADA and HIV HxB2 pseudovirus pre-treated with 50 mg/ml SSP and entry inhibitor enfuvirtide (T-20) as a positive control.
  • SSP pre-treatment inhibited both HIV ADA and HIV HxB2 pseudovirus infection to a similar degree as T-20.
  • FIG. 7 Antiviral activity of SSP and their extracted parts against SARS-CoVs, and VSV.
  • serially diluted SSP, SSP-n-BuOH, Fr. B, F, and G were added to HEK293T-ACE2 cells infected with SARS-CoV-2 (a) , SARS-CoV (b) , and VSV (c) .
  • the luciferase level was measured 2 or 3 days post-infection. To test their cytotoxicity, cells viability (d) was also measured.
  • e-h Pre-treatment of SARS-CoV-2 and target cells inhibited viral infection.
  • HEK293T-ACE2 cells were infected with SARS-CoV-2 pseudovirus and treated with gradient diluted SSP simultaneously. The data represent the mean ⁇ SEM of triplicate experiments.
  • FIG. 8 SSP did not show significant cytotoxicity in multiple cell lines, or long-term in vivo toxicity in rats.
  • (a ⁇ d) To test SSP cytotoxicity, cells viability was measured in HEK293T-ACE2 (a) , MDCK (b) , GHOST (3) -CD4-CXCR4 (c) /CCR5 (d) .
  • (e) Effects of SSP administration for 4 weeks on the bodyweight of SD rats.
  • (f) Effects of SSP administration for 4 weeks on food intake of SD rats.
  • FIG. 9 SSP shows higher potency and broader spectrum against SARS-CoV-2 variants entry in comparison with GSE and TP.
  • A After keeping SSP solution for 6 and 13 days, the anti-viral activity and cyto-toxicity were tested.
  • B SSP had a fairly strong inhibitory effect against major SARS-CoV-2 variants [including Alpha (B. 1.1.7) , Beta (B. 1.351) , Gamma (P1) , Delta (B. 1.617.2) , and Omicron (BA. 1, BA. 2, BA2.1.2.1 and BA. 4/5) ] .
  • C ⁇ D The anti-viral efficacy of grape seed extract (GSE) and tea polyphenol (TP) against SARS-CoV-2 and Omicron variants, as well as their cyto-toxicity.
  • FIG. 10 SSP shows efficacy in protection against nasal challenge of SARS_CoV-2 omicron in mice.
  • A Flowchart of the in vivo experiments.
  • B Omicron BA. 2 virus-infected cells (green) and inflammatory cells (red) were significantly reduced in the lung tissues of mice treated with SSP compared with the saline group.
  • C In the lung tissue of mice treated with SSP, the expression level of RNA-dependent RNA polymerase and the virus titer were significantly lower than those of mice treated with saline. *P ⁇ 0.05.
  • the present invention is related to a method of obtaining an extract of SSD, said method comprising: (i) cutting SSD into pieces; (ii) immersing and treating SSD pieces in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; and (iv) drying the concentrated extract to obtain the powder of the extract of SSD, the extract of SSD including proanthocyanidins, named SSP.
  • SSD Spatholobus suberectus Dunn.
  • SSP refers to the extract of SSD including proanthocyanidins.
  • SSP can be obtained by the above method comprising steps (i) to (iv) .
  • suitable treating process can comprise percolation, ultrasonic, heating and refluxing, decoction, and/or solvent extraction.
  • the treating process in (ii) is percolation.
  • the solvent used can comprise water, and/or an organic solvent.
  • the solvent in (ii) comprises water and ethanol, such as 60%ethanol/water (v/v) .
  • step (ii) comprises treating SSD pieces via a percolation process with a 60%ethanol (in water, v/v) at room temperature.
  • the percolation process can be performed for 12 hours.
  • the concentrating process can comprise reduced pressure rotary evaporation, centrifugation, and/or nitrogen blowing.
  • the concentrating process in (iii) is reduced pressure rotary evaporation.
  • the concentrating process can be performed at a temperature under 50°C.
  • the drying process can comprise freeze drying, spray drying, microwave drying, and/or infrared heating drying.
  • the drying process in (iv) is freeze drying.
  • the invention also relates to the SSP obtained by the above method.
  • SSP exhibited significant inhibitory ability against SARS-CoV-2 IC 50 values of 3.574 ⁇ g/mL and 3.648 ⁇ g/mL.
  • SSP uniquely inhibited the entry of SARS-CoV-2, HIV-1, Ebola virus (EBOV) , SARS-CoV-1 and influeunza H5N1 infections but failed to block vesicular stomatitis virus (VSV) .
  • VSV vesicular stomatitis virus
  • SSP had no effects on post-entry events of replication. It blocked SARS-CoV-2, HIV-1, H5N1, EBOV and SARS-CoV-1 entry by acting on viral envelope directly.
  • SSP is a novel entry inhibitor, which has a potential for preventing and treating COVID-19, also can fight EBOV, HIV-1, H5N1 and SARS-CoV infection, and stockpiling for possible use against future pandemic caused by related virus.
  • the invention is related to a method of obtaining an active fraction from SSD, the active fractions including proanthocyanidins, said method comprising: (i) cutting SSD into pieces; (ii) immersing and treating SSD pieces in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; (iv) drying the concentrated extract to obtain the powder of the extract of SSD (SSP) ; (v) dispersing SSP in water to obtain an aqueous solution; (vi) extracting the SSP aqueous solution with a series of solvent with different polarities at equal volume to obtain parts of SSP corresponding to each solvent, the series of solvent comprising n-butanol; and (vii) separating the n-butanol part (SSP-n-BuOH) with eluents to obtain the active fraction.
  • SSP n-butanol part
  • the term “active fraction” refers to the fraction extracted from SSD which is active as entry inhibitor and/or has anti-virus activity.
  • one or more active fractions can be obtained by the above method.
  • the active fraction (s) can include concentrated proanthocyanidins than SSP.
  • the active fraction (s) exhibits entry inhibitory, anti-virus, immunoregulation and/or anti-inflammatory effects as SSP.
  • the active fraction (s) can have different mean degrees of polymerization (mDP) .
  • the active fraction (s) can have higher mDP than SSP.
  • one active fraction can have a mDP of 7 ⁇ 10.
  • Each of the steps (i) - (iv) can have any of the features as described above in the method of obtaining an extract of SSD.
  • the series of solvent can further comprise water, ethanol, petroleum ether (PE) , and/or ethyl acetate.
  • the series solvent is petroleum ether (PE) , ethyl acetate and n-butanol.
  • the parts of SSP corresponding to each solvent comprise petroleum ether part (SSP-PE) , ethyl acetate part (SSP-EA) , ethyl acetate insoluble part (SSP-EAin) , and n-butanol part (SSP-n-BuOH) .
  • suitable separating process can comprise macroporous resin columns separation, fractional precipitation, crystallization, normal phase chromatography, reverse phase chromatography, and/or ion exchange resin columns separation.
  • the separating process is macroporous resin columns separation.
  • suitable eluents can comprise water, and/or an organic solvent.
  • the eluents in (vii) are gradient ethanol-water from 10%to 95%.
  • the method of obtaining an active fraction from SSD can further comprise: (viii) concentrating the active fraction; and (ix) drying the concentrated active fraction.
  • the concentrating process in step (viii) can have any of the features of the corresponding process in the above step (iii) .
  • the drying process in step (ix) can have any of the features of the corresponding process in the above step (iv) .
  • the invention also relates to the active fraction obtained by the above method.
  • the above active fraction from SSD related nature products from SSD may be useful in this disclosure.
  • the prescript or formula similar to SSP can also have anti-viral function against COVID-19.
  • the invention further relates to a composition comprising the SSP or the active fraction obtained by the above methods.
  • the composition can further comprise auxiliary materials.
  • the auxiliary materials can include, but are not limited to fillers, such as starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc. ; disintegration agents, such as, starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked polymethyl cellulose sodium, etc.
  • composition can further comprise herbs other than SSD.
  • SSP is an entry inhibitor for SARS-CoV-2 via direct binding to viral envelope and downregulating viral receptor ACE2 on target cell surface.
  • the invention relates to a method of preventing and/or treating a viral disease comprising administering an effective amount of the SSP or the active fraction obtained by the above methods to a subject in need thereof.
  • the effect dose of SSP can be ranging from 0.5 ⁇ g/mL to 5000 ⁇ g/mL.
  • the invention also relates to use of SSP or the active fraction obtained by the above methods in the manufacture of preparations for prevention and/or treatment of a viral disease.
  • the preparations can be in the form of injections, tablets, granules, emulsions, gels, sustained-release preparations, nasal wash/spray, oral liquids, throat spray, like-tea pills/candy, nano preparations, “throat lozenges” , “throat sprays” , “nasal washes” , and/or “nasal sprays” .
  • the viral disease include that caused by coronaviruses, e.g., SARS-CoV-2 and SARS-CoV-1 and their variants, Ebola virus (EBOV) , HIV-1, H5N1 and other enveloped viruses except for VSV.
  • coronaviruses e.g., SARS-CoV-2 and SARS-CoV-1 and their variants, Ebola virus (EBOV) , HIV-1, H5N1 and other enveloped viruses except for VSV.
  • SSP Dried SS stems were purchased from KangMei Pharmaceutical Company Ltd (Guang Xizhou, China) and the plants were authenticated by inspectors in the School of Chinese Medicine, the University of Hong Kong, Hong Kong. Briefly, SS was cut into pieces and extracted using a percolating device with 10 times volume (v/w) of 60%ethanol with 12 hours at room temperature. The extract was then concentrated by reduced pressure under 50°C and freeze-dried by vacuum freeze dryer to obtain the percolation powder, named SSP ( Figure 1) . SSP can be dissolved in dimethyl sulfoxide (DMSO) to reach a concentration of 40 mg/mL for future use.
  • DMSO dimethyl sulfoxide
  • the quality control methods for SSP extract contains: 1) the contents of proanthocyanidins by vanillin-hydrochloric acid method; 2) content determination of references compounds by UPLC; 3) TLC identification.
  • GHOST (3) -CD4-CCR5/CXCR4 cell lines were cultivated in DMEM with 10%FBS, 1%P/S, and 100 ⁇ g/mL hygromycin B, 500 ⁇ g/mL G418, and 1 ⁇ g/mL puromycin (Sigma-Aldrich, St.Louis, MO, USA) .
  • pseudoviruses were generated by co-transfection of 293T cells with HIV-1 NL4-3 ⁇ Env Vpr Luciferase (pNL4-3. Luc. R-E-) or HIV-1 NL4-3 ⁇ Env EGFP Reporter Vector (NIH AIDS Reagent Program, Cat #3418 and 11100) and envelope protein from different strains of viruses (polyethyleneimine [PEI] ; Polysciences Inc., Warrington, PA) . Cell-free supernatant was collected 48h post-transfection and frozen at -80 °C. To determine the viral titer, around 10,000 HEK293T-ACE2 cells per well in 96-well plates were seeded in 10%FBS-containing media.
  • TCID50 50%tissue culture infective dose
  • the inhibitory activities of the SSP against viruses are evaluated as previously described (L. Liu et al., 2007; X. Lu et al., 2012) . Briefly, serially diluted SSP was tested against 100TCID50 viral infection. HEK 293FT-ACE2 were used for SARS-CoV-1/2; MDCK was used for H5N1; GHOST (3) -CD4-CCR5/CXCR4 were used for HIV ADA and HIV HXB2 infection. On day three, the viral infection was determined by measuring the reporter luciferase activity in target cells post-infection using commercially available kits (Promega, Wisconsin, USA) . Antiviral data are reported as the concentration of drugs required to inhibit viral replication by 50% (EC 50 ) .
  • the different SARA-CoV-2 variants including Alpha (B. 1.1.7) , Beta (B. 1.351) , Gamma (P1) , Delta (B.1.617.2) , and Omicron (BA. 1, BA. 2, BA2.1.2.1 and BA. 4/5) were also used to test the inhibition rate of SSP.
  • the anti-viral activity of grape seed extract (GSE) and tea polyphenol (TP, CAS: 84650-60-2) (Shanghai Yuanye Company) were also tested by the same method.
  • Acute toxicity experiment observe and study the toxicity of SD rats and NIH mice given single or multiple times by intragastric administration of SSP within 24 hours to determine the acute toxic dose and the maximum tolerated dose and possible toxicity for target organs for its clinical safety.
  • Method Several SD rats and NIH mice were selected, half male and female, and randomly divided into vehicle control group and SSP group. The rats were fasted for 12h-16h before administration and weighed.
  • the SSP group was given SSP powder 0.25g /mL was given to rats by 20mL /kg, and the vehicle control group was given an equal volume of purified water, orally or twice a day (each dosing interval 8h) , continuous observation for 4 hours after each administration , D1 ⁇ D14 were observed once a day in the morning and afternoon; D1, D4, D7, D14 weighed the rats and their situations were also recorded. After animals sacrifice and visual observation, any changes in volume, color, and texture of any tissues and organs were recorded and histopathological checked.
  • Nasal spray irritation test-Rabbits This experiment was completed in the CFDA-certified GLP-compliant Drug Safety Evaluation Research Center (Shandong Xinbo Drug Research Co., Ltd. ) , and the code of this experiment was KY22233.
  • the stability of SSP powder is determined by vanillin-hydrochloric acid method as mentioned in 3-1) to detect the content of proanthocyanidins, and the detection samples are the SSP aqueous solution prepared immediately, and the SSP aqueous solution after keeping at room temperature for over 1 month.
  • Efficacy of SSP against live intranasal SARS-CoV-2 Omicron BA. 2 challenge were tested in K18-hACE2 mice. The units of 2*10 ⁇ 4 moi of viruses were administered to the nasal cavity of the mice. As shown in Figure 10a, one dose of SSP intervention (40 mg/ml *20 ⁇ L, 800 ⁇ g) was given at the time of 30 minutes before virus infection, and the control group was saline group (n 5 per group) . After 3 days of virus infection, the lung tissues of the mice were collected.
  • SSP SARS-CoV-2 pseudoviruses were generated (L. L. Liu et al., 2019; X. ; ) .
  • HE293T-ACE2 cells were then infected with 100 TCID50 SARS-CoV-2 pseudoviruses in the presence of serially diluted SSP.
  • Virus pseudotyped with a second, unrelated viral envelope glycoprotein of VSV-G was included as a control to reduce the false positives.
  • SSP displayed an EC 50 of 3.5 ⁇ g/mL in the inhibition of SARS-CoV-2 pseudovirus infection (Figure 3a) and was devoid of overt cytotoxicity (Figure 3c) .
  • SARS-CoV-2 and VSV pseudoviruses share the common genetic HIV backbone expressing protease (PR) , reverse transcriptase (RT) , and integrase (IN) and differ only in their glycoproteins on the surface of the viruses.
  • PR protease
  • RT reverse transcriptase
  • I integrase
  • SSP antagonizes SARS-CoV-2 pseudovirus entry rather than post-entry events (e.g., reverse transcription) .
  • the lack of antiviral activity against VSV also ruled out the possibility that SSP simply inactivates the SARS-CoV-2 virus by acting on viral lipids as a disinfectant.
  • SSP acts by blocking the attachment of SARS-CoV spike envelope protein to entry receptor ACE2
  • SARS-CoV-2 enter cells in the host through a virus surface-anchored S-protein.
  • the S protein mediates viral entry through the RBD in the S1 subunit that specifically recognizes ACE2 as its receptor and then fuses the viral into host membranes through the S2 subunit.
  • Entry inhibitors usually target protein-protein interactions within the viral envelope proteins or between viral envelope proteins and host cells or inhibit protein-lipid interactions.
  • the viruses and HEK-293T-ACE2 cells were then recovered and subsequently subjected to infect HEK293T-ACE2 or incubated with untreated SARS-CoV-2 pseudovirus (Rapista et al., 2011) .
  • HEK293T-ACE2 cells were infected with SARS-CoV-2 pseudovirus and then treated with gradient diluted SSP immediately or 2-hours post-infection (hpi) .
  • SSP treatment at 2-hours post-infection showed limited anti-SARS-CoV-2 activity compared with the virus and SSP being added simultaneously. This result confirmed the antiviral entry activity of SSP against SARS- CoV-2.
  • pre-treatment of pseudovirus with SSP resulted in increased inhibition activities with the EC 50 value of 2.3 ⁇ g/mL, suggesting that SSP targets SARS-CoV-2 viral envelop S protein to inhibit SARS-CoV-2 entry.
  • pre-treatment of target cells with SSP halted SARS-CoV-2 infection with the EC 50 value of 2.1 ⁇ g/mL, suggesting that SSP also targets cellular components to inhibit SARS-CoV-2 entry.
  • HEK-293T-ACE2 cells were treated with serially diluted SSP for 2 hours at 37°C, washed, and were then incubated with supernatant collected from RBD-PD1 expressing plasmid transfected HEK293T cells or a goat polyclonal antibody against ACE2 for 30 min on ice. Cells were then washed and stained with a fluorescent-labeled secondary antibody against goat-IgG or PD-1, respectively.
  • SSP shows broad-spectrum antiviral activities against SARS-CoV, H5N1, EBOV and HIV-1
  • SSP broad-spectrum antiviral activities
  • a panel of pseudoviruses was generated in our previous studies, including SARS-CoV (L. Liu et al., 2019) , H5N1 Turkey (Xiao et al., 2013) , CCR5-tropic HIV-1 ADA , and CXCR4-tropic HIV-1 HXB2 (Liang et al., 2013) , were utilized to test the inhibition rate of SSP.
  • SSP inhibited their infection with EC 50 around 3.64, 5.13, 3.61, and 8.15 ⁇ g/mL, respectively, and was devoid of overt cytotoxicity (Figure 4e) .
  • SSP blocks SARS-CoV-1 and H5N1 entry by binding to viral envelope and its target cells.
  • SSP-virus binding and SSP-cell binding assays were performed.
  • pseudovirus was initially pre-treated with 50 ⁇ g/mL of SSP. The viruses were then recovered by ultracentrifugation and subjected to infect target cells. We found that SSP inhibited SARS-CoV-1 pseudovirus infection at a similar degree as when the virus and SSP were added into cells simultaneously ( Figure 5) .
  • the SSP-cell binding assays were performed by pre-treatment of the target cells with SSP for 1 hour at 37°C. After washing by PBS, cells were incubated with SARS-CoV-1 at 37 °C for 48 hrs (Rapista et al., 2011) .
  • pre-treatment of SSP on the target cells inhibited virus infection ( Figure 5) .
  • the same approach was also used to test the inhibitory effect of SSP against H5N1 with a single high dose of 50 ⁇ g/mL. Similar to SARS-CoV-1, SSP treatment with virus particles or their target cells efficiently blocked H5N1 virus entry (Figure 5b) .
  • SSP blocks HIV-1 entry by binding to the viral envelope.
  • AZT but not SSP, significantly inhibited HIV-1 infection when the treatment was initiated at 2 hours post-infection.
  • pre-treatment of the virus with 50 ⁇ g/mL of SSP significantly inhibited HIV-1 infection at a similar degree as T-20, which blocks HIV-1 fusion (Figure 5c ⁇ 5d) .
  • the SSP-cell binding assays were performed by treating the target GHOST cells with SSP or with control compounds the CCR5 antagonist Marvaroc (MVC) or the CXCR4 antagonist JM2987 for 2 hours at 37°C. After washing by PBS, cells were subjected to HIV-1 ADA or HIV-1 HXB2 infection and cultivated at 37 °C for 48 hrs (Rapista et al., 2011) .
  • MVC and JM2987 showed potent viral inhibition against the respective ADA and HXB2 pseudoviruses at 1 ⁇ M as expected ( Figures 5e ⁇ 5f) .
  • This evidence demonstrated that SSP inhibited HIV-1 infection by actions on the viral envelope glycoprotein gp160, which mediates the viral entry into the host target cells.
  • Fr. G inhibited viral entry with lower EC 50 value than adding the virus and fragments simultaneously, suggesting that those fragments target both SARS-CoV-2 viral envelop S protein and target cells to inhibit SARS-CoV-2 entry. More importantly, Fr. G exhibited more outstanding bioactivity when compared to Fr. B, suggesting that Part II of SSP contains more effective antiviral activities than Part I. These results revealed that Fr. G contains an intermediate polymer of proanthocyanidins, which may have better antiviral effects.
  • the maximum tolerated dose for one-time administration is 67 g crude drug /kg in mice (Table 1) .
  • the maximum tolerated dose for one-time administration is 107 g crude drug /kg in rats (Table 2) .
  • the proanthocyanidins contained in the immediately prepared SSP aqueous solution were 794.794 ⁇ 13.619 mg/g, while the content in the SSP aqueous solution after being placed for 1 month was 778.964 ⁇ 63.589mg/g, which was within the quality standard range of 5%, so the SSP aqueous solution is stable in terms of proanthocyanidin content.

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  • Botany (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La divulgation concerne un procédé d'obtention (préparation) d'un extrait de Spatholobus suberectus Dunn, qui contient un flavonoïde total et des proanthocyanidines. La divulgation concerne un procédé d'extraction d'une fraction active à partir de Spatholobus suberectus. La divulgation concerne également le SSP, la fraction active et une composition de ceux-ci. La divulgation concerne en outre un procédé de traitement de maladies virales telles que la maladie COVID-19 provoquée par le SARS-CoV-2, et l'utilisation du SSP et de la fraction active dans la fabrication de préparations pour la prévention et/ou le traitement d'une maladie virale.
PCT/CN2023/081058 2022-03-15 2023-03-13 Procédé d'obtention d'extraits de spatholobus suberectus dunn (ssd), fractions et compositions de ceux-ci et utilisation contre des maladies virales WO2023174207A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005314316A (ja) * 2004-04-30 2005-11-10 Kikkoman Corp 抗ｓａｒｓコロナウイルス剤
CN101474247A (zh) * 2008-12-26 2009-07-08 中国人民解放军军事医学科学院卫生学环境医学研究所 鸡血藤提取物在制备抗流感病毒及抗肠道病毒药物中的应用
CN113925890A (zh) * 2020-06-29 2022-01-14 香港大学 鸡血藤提取物的提取方法、鸡血藤提取物及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005314316A (ja) * 2004-04-30 2005-11-10 Kikkoman Corp 抗ｓａｒｓコロナウイルス剤
CN101474247A (zh) * 2008-12-26 2009-07-08 中国人民解放军军事医学科学院卫生学环境医学研究所 鸡血藤提取物在制备抗流感病毒及抗肠道病毒药物中的应用
CN113925890A (zh) * 2020-06-29 2022-01-14 香港大学 鸡血藤提取物的提取方法、鸡血藤提取物及其用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DONG PAN, LUO ZEXIN, WANG DONGMEI: "Extraction and antioxidant activity of Proanthocyanidins from Spatholobus suberectus Dunn", ACTA SCIENTIARUM NATURALIUM UNIVERSITATIS SUNYATSENI, vol. 56, no. 1, 1 January 2017 (2017-01-01), pages 8 - 13, XP093091474 *
FU, YING; CHENG, YUE; CHEN, JIAN-PING; WANG, DONG-MEI: "Advances in studies on chemical constituents in Spatholobi Caulis and their pharmacological activities", CHINESE TRADITIONAL AND HERBAL DRUGS, TAINJIN ZHONGCAOYAO ZAZAHISHE, CN, vol. 42, no. 6, 12 June 2011 (2011-06-12), CN , pages 1229 - 1234, XP009549028, ISSN: 0253-2670 *
LIU QINGQING, KWAN KA‐YI, CAO TIANYU, YAN BINGPENG, GANESAN KUMAR, JIA LEI, ZHANG FENG, LIM CHUNYU, WU YAOBIN, FENG YIBIN, CHEN ZH: "Broad-spectrum antiviral activity of Spatholobus suberectus Dunn against SARS-CoV-2, SARS-CoV-1, H5N1, and other enveloped viruses", PHYSIOTHERAPY RESEARCH, JOHN WILEY & SONS LTD. CHICHESTER., GB, vol. 36, no. 8, 1 August 2022 (2022-08-01), GB , pages 3232 - 3247, XP093091470, ISSN: 0951-418X, DOI: 10.1002/ptr.7452 *
ZENG, FANLI; CHENG, YUE; CHEN, JIANPING; FU, YING; WANG, QIAOLI; YANG, DEPO; CHEN, ZHENPING; XIANG, YANGFEI; WANG, YIFEI; WANG DON: "Study on in vitro Antiviral Activities of Different Extracts from Caulis Spatholobi", ZHONGYAO XINYAO YU LINGCHUANG YAOLI - TRADITIONAL CHINESE DRUG RESEARCH & CLINICAL PHARMACOLOGY, GUANGZHOU ZHONGYIYAO DAXUE, CHINA, vol. 22, no. 1, 31 January 2011 (2011-01-31), China , pages 16 - 20, XP009549017, ISSN: 1003-9783, DOI: 10.19378/j.issn.1003-9783.2011.01.005 *

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