CN118900695A - Method for obtaining Solanum sphaerocephalum (SSD) extract, its fractions and compositions and use thereof in combating viral diseases - Google Patents

Abstract

Disclosed is a method for obtaining a preparation of a dense flower bean extract containing total flavonoids and proanthocyanidins. The present disclosure relates to a method for extracting an active fraction from Millettia reticulata. Also provided are SSP, active fractions and compositions thereof. Further provided are methods for treating viral diseases such as COVID-19 disease caused by SARS-CoV-2, and uses of SSP and active fractions in preparing preparations for preventing and/or treating viral diseases.

Description

Method for obtaining extract of Sparassis crispa (SSD), fraction and composition thereof and use for combating viral diseases

Technical Field

A method for preparing an antiviral extract natural product containing total flavonoids and procyanidins from Sparassis crispa (Spatholobus suberectus Dunn, SSD) is disclosed. The present disclosure relates to methods for discovering active procyanidins (SSPs) of spatholobus stem (Spatholobus seberectus) as inhibitors of viral entry. Methods of preventing and treating viral diseases such as 2019 coronavirus disease (COVID-19) are also provided.

Background

Recent outbreaks of epidemic diseases caused by emerging and reappeared viruses have highlighted serious threats to public health from the prevention and treatment of these viruses in recent years with the advent of globalization and rapid urban ization. Examples include the recent outbreak of COVID-19 caused by severe acute respiratory syndrome virus type 2 (SARS-CoV-2). Despite advances in immunization and drug development, many viruses still lack prophylactic vaccines and effective antiviral therapies. Therefore, it is of great importance to find new drugs, especially natural products, that are efficient and cost-effective for the management and control of viral infections. Broad spectrum antiviral agents are particularly beneficial against new variants of the relevant viruses.

The herbal medicine and the purified natural products provide abundant resources for the development of novel antiviral drugs. The identification of the antiviral mechanisms of these natural agents reveals their interactions with viral life cycles such as viral entry, replication, assembly and release. Traditional Chinese medicine (traditional CHINESE MEDICINE, TCM) is particularly important. There are 250,000 to 300,000 plant varieties worldwide, and chinese herbal medicine provides a quick way and important source ^6-10 for drug discovery. Sweet wormwood (ARTEMISIA ANNUA) is a representative herb. The discovery of the antimalarial drug artemisinin (Qinghaosu, artemisinin) from artemisia annua and several subsequently developed therapies to inhibit plasmodium have saved millions of lives.

Plant SSD. Slightly sweet and warm in nature, enter liver and kidney meridians of TCM. The 'materia medica outline' claims that it can nourish and dry the stomach; it is claimed to promote blood circulation and warm the waist and knees by the formula Ben Cao gang mu Shi. There is a great medical need for the treatment of patients suffering from arthralgia, rheumatism, numbness, paralysis, blood deficiency, and chlorosis. In recent years, some reports on common use of centipedes for treating diseases caused by viruses have been made, but have not been reported in Covid-19. To date, there is no publication indicating SSP as a viral entry inhibitor.

The chemical components of SSD mainly comprise flavonoids, terpenes, sterols, lignin, anthraquinones, polyphenols, procyanidins, and some trace elements, wherein the flavonoids are the most abundant. The studies on the extraction method of the contents mainly use the total flavonoid extraction rate as an index, and do not consider the influence of the extraction method on the efficacy of the extract.

Disclosure of Invention

Provided herein are methods of obtaining an SSD extract, the methods comprising: (i) slicing the SSD; (ii) Soaking and treating the SSD tablet in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; and (iv) drying the concentrated extract to obtain a powder (SSP) of the SSD extract.

Provided herein are methods of obtaining an active fraction from an SSD, the active fraction comprising procyanidins, the method comprising: (i) slicing the SSD; (ii) Soaking and treating the SSD tablet in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; (iv) Drying the concentrated extract to obtain a powder (SSP) of SSD extract; (v) dispersing SSP in water to obtain an aqueous solution; (vi) Extracting the aqueous SSP solution with equal volumes of a series of solvents having different polarities to obtain SSP fractions corresponding to each solvent, the series of solvents comprising n-butanol; and (vii) separating the n-butanol fraction (SSP-n-BuOH) with an eluent to obtain an active fraction.

In one embodiment, the treatment in (ii) comprises diafiltration.

Provided herein are SSD extracts and active fractions from SSDs obtained by the above methods, and compositions comprising the extracts or the active fractions.

Provided herein are methods of preventing and/or treating a viral disease comprising administering to a subject in need thereof an effective amount of an SSD extract or an active fraction from an SSD obtained by the above methods.

In one embodiment, the viral disease is caused by SARS-CoV-2 (including Omicron variants), ebola virus (EBOV), HIV-1 with CCR5 or CXCR4 tropism, SARS-CoV, H5N 1.

In one embodiment, the effective dose is from 0.5 μg/ml to 5000 μg/ml.

Provided herein is the use of an SSD extract or an active fraction from an SSD obtained by the above method for the preparation of a formulation for the prevention and/or treatment of viral diseases.

Drawings

FIG. 1. Study flow chart. SSP preparation and quality control was used to determine its broad-spectrum antiviral activity and its underlying mechanisms. The powder SSP is further extracted to obtain the effective active fraction.

Figure 2. Representative picture of ssp quality control. (a) HPLC chromatograms of SSP of different batches after ethyl acetate treatment. (b) TLC chromatograms of SSP and SS positive controls of different batches.

FIG. 3 antiviral activity of SSP against SARS-CoV-2. Serial dilutions of SSP were added to HEK293T-ACE2 cells infected with SARS-CoV-2 (a) and VSV (b), respectively. Luciferase levels were measured 2 or 3 days post infection. To test the cytotoxicity of SSP, cell viability (c) was measured. (d) Pretreatment of SARS-CoV-2 and target cells inhibits viral infection. (e) RBD binds to ACE2 expressing 293T cells but not 293T control cells. (f) SSP inhibits RBD binding to target cells. HEK293T-ACE2 cells pretreated with SSP were incubated with RBD-PD1 for 30min on ice, followed by antibody staining and flow cytometry analysis of ACE2 (upper panel) and RBD (lower panel). Data represent mean ± SEM of triplicate experiments.

FIG. 4 antiviral activity of SSP against SARS-CoV-1, H5N1, and HIV, and EBOV viruses. Serial dilutions of SSP were added to HEK293T-ACE2, MDCK, ghest-CCR 5, and ghest-CXCR 4 infected with SARS-CoV-1 (a), H5N1 (b), HIV _ADA (c), and HIV _HXB2 (d), respectively. Luciferase levels were measured 2 or 3 days post infection. Cell viability was measured (e) to test SSP cytotoxicity. (f) Serial dilutions of SSP were added to 293T cells infected with EBOV. Luciferase levels were measured 2 days after infection. Cell viability was measured to test SSP for cytotoxicity on 293T cells. Data represent mean ± SEM of triplicate experiments.

FIG. 5.SSP mediated inhibition of viral entry mechanism. (a-b) SARS-CoV-1 (a), H5N1 (b), and pretreatment of target cells inhibits viral infection. SARS-CoV-1 or H5N1 pseudoviruses pretreated with serial dilutions of SSP and HEK293T-ACE2 (a) or MDCK (b) cells were recovered and subsequently used to infect target cells or incubated with untreated SARS-CoV-1 pseudoviruses for 48 hours. Luciferase levels were measured 2 or 3 days post infection. (c-d) post-entry assay. GHOST-CD4-CCR5 or CXCR4 cells were incubated with pseudovirus for 2hr, washed and then treated in the presence of 50mg/ml SSP and 1mM AZT as a positive control for 48hr. After viral entry is achieved, SSP does not inhibit HIV-1 _ADA or HIV _HxB2 viral gene replication. (e-f) SSP-virus interaction assay. HIV-1 _ADA and HIV _HxB2 pseudoviruses were pretreated with 50mg/ml SSP and the entry inhibitor enfuvirdine (T-20) as positive controls. SSP pretreatment inhibited both HIV-1 _ADA and HIV _HxB2 pseudovirus infections to a similar extent as T-20. (g-h) SSP-cell binding assay. GHOST cells were pretreated with SSP and CCR5 antagonists maraviroc and CXCR4 antagonist JM2987 as positive controls for 1hr at 37℃before infection. SSP pretreatment of cells did not have antiviral effect, whereas maraviroc and JM2987 pretreatment showed strong inhibition, as expected. Dimethyl sulfoxide (DMSO) was used as a negative control. Data represent mean ± SD of triplicate experiments.

FIG. 6 separation of SSP and UPLC-MS analysis. (a) a UPLC-DAD spectrum of SSP; the UPLC spectrum of (b) fr.b (part I); (c) UPLC spectrum of Fr.G (part II); (d) Extracting Ion Chromatograms (EIC) of procyanidin monomers and dimers in SSP; (e) An Extracted Ion Chromatogram (EIC) of a polymer having an mDP of procyanidins in SSP of between 3 and 7; (f) Extraction Ion Chromatograms (EIC) of polymers with procyanidins in SSP having an mDP between 8 and 10.

FIG. 7 antiviral activity of SSP and its extract on SARS-CoV and VSV. Serial dilutions of SSP, SSP-n-BuOH, fr.b, fr.f, and fr.g were added to HEK293T-ACE2 cells infected with SARS-CoV-2 (a), SARS-CoV (b), and VSV (c), as shown in figures a-d. Luciferase levels were measured 2 or 3 days post infection. To test their cytotoxicity, cell viability (d) was also measured. (e-h) pretreatment of SARS-CoV-2 and target cells inhibits viral infection. SARS-CoV-2 pseudoviruses and HEK293T-ACE2, pretreated with serial dilutions of SSP-n-BuOH (e), fr.b (f), fr.f (g), and fr.g (h), were recovered and then used to infect HEK293T-ACE2, respectively, or incubated with untreated SARS-CoV-2 pseudoviruses for 48hr. As a control HEK293T-ACE2 cells were infected with SARS-CoV-2 pseudovirus while treated with gradient diluted SSP. Data represent mean ± SEM of triplicate experiments.

Figure 8 ssp did not show significant cytotoxicity in various cell lines, or long-term in vivo toxicity in rats. (a-d) to detect SSP cytotoxicity, cell viability in HEK293T-ACE2 (a), MDCK (b), ghest (3) -CD4-CXCR4 (c)/CCR 5 (d) was measured. (e) effect of SSP on SD rat body weight for 4 weeks. (f) Effect of SSP on food intake in SD rats for 4 weeks. (g) effect of SSP on SD rat organ coefficients for 4 weeks.

FIG. 9 SSP shows greater potency and greater broad spectrum of SARS-CoV-2 variant entry than GSE and TP. (A) After 6 days and 13 days of storage of the SSP solution, antiviral activity and cytotoxicity were tested. (B) SSP has a fairly strong inhibitory effect on 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) Grape Seed Extract (GSE) and Tea Polyphenols (TP) antiviral efficacy and cytotoxicity against SARS-CoV-2 and its Omacron variants.

FIG. 10 SSP shows efficacy in protecting mice from nasal challenge with SARS-CoV-2 omicron. (A) flow chart of in vivo experiments. (B) Compared to saline group, omicron ba.2 virus infected cells (green) and inflammatory cells (red) were significantly reduced in mice lung tissue treated with SSP. (C) In lung tissues of mice treated with SSP, the expression levels and viral titers of RNA-dependent RNA polymerase were significantly lower than those treated with saline. * P is less than 0.05.

Detailed Description

The present invention relates to a method of obtaining an SSD extract, the method comprising: (i) slicing the SSD; (ii) Soaking and treating the SSD tablet in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; and (iv) drying the concentrated extract to obtain a powder of an SSD extract comprising procyanidins, known as SSP.

As used herein, the term "SSD" refers to spathiphyllum (Spatholobus suberectus Dunn). As used herein, the term "SSP" refers to an SSD extract comprising procyanidins. Herein, SSP can be obtained by the above-described method comprising steps (i) to (iv).

In step (ii), suitable treatment processes may include diafiltration, ultrasonication, heating and refluxing, decoction, and/or solvent extraction. Suitably, the treatment in (ii) is diafiltration.

In step (ii), the solvent used may comprise water and/or an organic solvent. In one embodiment, the solvent in (ii) comprises water and ethanol, such as 60% ethanol/water (v/v).

For example, step (ii) includes treating the SSD tablet with 60% ethanol (v/v in water) at room temperature by a diafiltration process. The diafiltration process may be for 12 hours.

In step (iii), the concentration process may include reduced pressure rotary evaporation, centrifugation, and/or nitrogen purging. In one embodiment, the concentration process in (iii) is reduced pressure rotary evaporation. The concentration process may be carried out at a temperature below 50 ℃.

In step (iv), the drying process may comprise freeze drying, spray drying, microwave drying, and/or infrared heating drying. In one embodiment, the drying process in (iv) is freeze drying.

The invention also relates to an SSP obtained by the above method. SSP showed remarkable inhibitory capacity against SARS-CoV-2, IC ₅₀ values of 3.574 μg/mL and 3.648 μg/mL. Innovative, SSP uniquely inhibits the entry of SARS-CoV-2, HIV-1, ebola virus (EBOV), SARS-CoV-1, and influenza H5N1 infection, but does not block Vesicular Stomatitis Virus (VSV). Furthermore, we demonstrate that SSP has no effect on replicated post-entry events. It blocks SARS-CoV-2, HIV-1, H5N1, EBOV, and SARS-CoV-1 entry by acting directly on the viral envelope. Our findings indicate that SSP is a novel entry inhibitor, has the potential to prevent and treat COVID-19, can also combat EBOV, HIV-1, H5N1, and SARS-CoV infections, and is reserved for use against future pandemics caused by related viruses.

Our findings provide scientific evidence for the potential use of SSP for the prevention and treatment of COVID-19 (SARS-CoV-2) infection, and for reserves that can be used to combat future pandemics caused by related viruses, and for the search for constituent small molecule compounds as entry inhibitors.

Furthermore, the present invention relates to a method for obtaining an active fraction from an SSD, the active fraction comprising procyanidins, the method comprising: (i) slicing the SSD; (ii) Soaking and treating the SSD tablet in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; (iv) Drying the concentrated extract to obtain a powder (SSP) of SSD extract; (v) dispersing SSP in water to obtain an aqueous solution; (vi) Extracting the aqueous SSP solution with equal volumes of a series of solvents having different polarities to obtain SSP fractions corresponding to each solvent, the series of solvents comprising n-butanol; and (vii) separating the n-butanol fraction (SSP-n-BuOH) with an eluent to obtain an active fraction.

As used herein, the term "active fraction" refers to a fraction extracted from an SSD that has activity as an entry inhibitor and/or has antiviral activity. Herein, one or more active fractions may be obtained by the above-described methods. The active fraction may comprise concentrated procyanidins compared to SSP. Thus, the active fraction exhibits the same entry inhibiting, antiviral, immunomodulatory, and/or anti-inflammatory effects as SSP.

The active fractions may have different average degrees of polymerization (mDP). The active fraction may have a higher mDP than SSP, e.g. the mDP of one active fraction may be 7-10.

Each of steps (i) - (iv) may have any of the features described above in the method of obtaining an SSD extract.

In step (vi), the series of solvents may further comprise water, ethanol, petroleum Ether (PE), and/or ethyl acetate. In one embodiment, the series of solvents is Petroleum Ether (PE), ethyl acetate, and n-butanol. In this case, the SSP portion corresponding to each solvent includes a petroleum ether portion (SSP-PE), an ethyl acetate portion (SSP-EA), an ethyl acetate insoluble portion (SSP-EAin), and an n-butanol portion (SSP-n-BuOH).

In step (vii), suitable separation processes may include macroporous resin column separation, fractional precipitation, crystallization, normal phase chromatography, reverse phase chromatography, and/or ion exchange resin column separation. In one embodiment, the separation process is macroporous resin column separation.

In step (vii), suitable eluents may include water and/or organic solvents. In one embodiment, the eluent in (vii) is a gradient of from 10% to 95% ethanol-water.

The method of obtaining an active fraction from an SSD may further comprise: (viii) concentrating the active fraction; and (ix) drying the concentrated active fraction. The concentration process in step (viii) may have any of the features described above for the corresponding process in step (iii). The drying process in step (ix) may have any of the features of the corresponding process in step (iv) described above.

The invention also relates to an active fraction obtained by the above method.

In addition to the SSP extracts described above, the active fractions from SSDs described above, related natural products from SSDs, may also be used in the present disclosure. Also, a formulation or recipe similar to SSP may have antiviral effects on COVID-19.

The invention further relates to a composition comprising SSP or active fraction obtained by the above method. The composition may further comprise auxiliary materials. Auxiliary materials may include, but are not limited to: fillers such as starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, and the like; disintegrants such as starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, crosslinked sodium polymethyl cellulose, and the like; additives such as magnesium stearate, sodium lauryl sulfate, talc, and/or silica suspending agents, binders, and the like; suspending agents such as polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; binders such as starch slurry, polyvinylpyrrolidone, and the like. The composition may further comprise herbs other than SSD.

SSP is disclosed herein as an entry inhibitor of SARS-CoV-2 via a viral receptor ACE2 that binds directly to the viral envelope and down-regulates on the surface of target cells. The present invention relates to a method for preventing and/or treating viral diseases, which method comprises administering to a subject in need thereof an effective amount of SSP or an active fraction obtained by the above method. Preferably, an effective dosage of SSP can range from 0.5 μg/mL to 5000 μg/mL.

The invention also relates to the use of SSP or active fractions obtained by the above-described method for producing a preparation for the prophylaxis and/or treatment of viral diseases. The preparation can be in the form of injection, tablet, granule, emulsion, gel, sustained release preparation, nasal lotion/nasal spray, oral liquid, throat spray, tea-like pill/candy (like-tea pills/candy), nanometer preparation, "throat containing tablet", "throat spray", "nasal wash", and/or "nasal spray".

Viral diseases include diseases caused by coronaviruses such as SARS-CoV-2 and SARS-CoV-1 and variants thereof, etc., ebola virus (EBOV), HIV-1, H5N1, and enveloped viruses other than VSV.

The following examples further illustrate the findings of the present disclosure.

Examples

The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

Materials and methods

SSP extract of SSD

Dried SS stems were purchased from conmei pharmaceutical company limited (guangxi, china) and plants were identified by inspectors at the university of hong Kong, chinese medical college. Briefly, SS was sectioned and extracted with 10 volumes (v/w) of 60% ethanol using a diafiltration unit for 12 hours at room temperature. The extract was concentrated under reduced pressure below 50 ℃ and freeze-dried by vacuum freeze-dryer to give diafiltration powder, designated SSP (figure 1). SSP can be dissolved in Dimethylsulfoxide (DMSO) to a concentration of 40mg/mL for future use.

2. Active fraction extraction

About 200g of SSP extract was weighed and uniformly dispersed in a suitable amount of water to obtain an aqueous solution, followed by extraction with equal volumes of Petroleum Ether (PE), ethyl Acetate (EA), and n-butanol (n-BuOH) to obtain the following fractions: 2g of petroleum ether fraction (SSP-PE), 21g of ethyl acetate fraction (SSP-EA), 8.2g of EA insoluble fraction (SSP-EAin), 62g of n-butanol fraction (SSP-n-BuOH), and 113g of aqueous layer residue (SSP-W). The n-butanol fraction (SSP-n-BuOH) was then passed through a macroporous resin column for ethanol-water gradient elution to give fractions A-J (FIG. 1).

3. Quality control

The quality control method of the SSP extract comprises the following steps: 1) Determining the content of procyanidine by vanillin-hydrochloric acid method; 2) Determining the content of a reference compound by UPLC; 3) TLC identification.

1) The PAC (procyanidin) content in SSP was determined by a slightly modified vanillin-hydrochloric acid method (y. Cheng et al 2011). 2) UHPLC analysis of SSP was performed by Ultimate3000,3000 system (Thermo FISHER SCIENTIFIC inc.) with Diode Array Detector (DAD). Chromatography was performed using an ACE Excel 2C ₁₈ column (100 mm x 2.1mm id, scotland, uk) with mobile phases comprising acetonitrile (solvent a) and Milli-Q water containing 0.1% formic acid (solvent B), eluting with the following gradient: 0min 95% B,3min 92% B,20min 85% B,26min70% B,30min 40% B,35min 5% B and hold at 5% B for more than 5 minutes. The solvent flow rate was set at 0.4mL/min and the analysis wavelength was 280nm (FIG. 2 a). The method has been validated. Flavonoids and procyanidins are present in the UPLC spectrum of SSP extracts. 3) SSP was identified by Thin Layer Chromatography (TLC) according to the chinese pharmacopoeia (2015 edition). Based on the color spots on the chromatogram, compounds of SSP were identified according to the corresponding positions of the control (fig. 2 b).

4. Cell culture

The highly sensitive and transferable cell lines 293T, TZM-bl, and MDCK (available from ATCC (American type culture Collection) and passaged less than 10 times) were cultured in Du 'S modified eagle' S medium (DMEM, gibco, grand Ai Lan, N.Y.) containing 10% inactivated fetal bovine serum (FBS, gibco, grand Ai Lan (GRAND ISLAND), 1% penicillin and streptomycin mixtures (P/S, sigma-Aldrich, st.Louis, misu, U.S.A.). HEK293FT-ACE2 was incubated in DMEM containing 10% FBS, 1% P/S and 1. Mu.g/mL puromycin (Sigma-Aldrich, st. Louis, mitsui, USA). GHOST (3) -CD4-CCR5/CXCR4 cell line was cultured in DMEM containing 10% FBS, 1% P/S and 100. Mu.g/mL hygromycin B, 500. Mu.g/mL G418, and 1. Mu.g/mL puromycin (Sigma-Aldrich, st. Louis, mitsui, U.S.A.).

5. Pseudovirus generation

We inserted the optimized full-length S gene of SARS-CoV-2 (QHR 63250,63250) into the pVAX-1 vector, i.e., pVax-1-S-COVID. The construct was confirmed by sequence analysis. The single cycle luciferases SARS-CoV-2, SARS-CoV-1, HIV _ADA、HIV_HXB2, H5N1, and vesicular stomatitis Indiana virus (VSV) pseudoviruses were constructed as described previously (L.Liu et al, 2007; yi, ba, zhang, ho, & Chen, 2005). Briefly, pseudoviruses were generated by co-transfecting 293T cells with HIV-1NL4-3 ΔEnv Vpr luciferase (pNL 4-3.Luc. R-E-) or HIV-1NL4-3 ΔEnv EGFP reporter vector (NIH AIDS reagent program, catalog nos. 3418 and 11100) and envelope proteins from different viral strains (polyethylenimine [ PEI ]; polysciences Inc., wolington, pa.). 48h after transfection, cell-free supernatant was collected and frozen at-80 ℃. To determine viral titers, approximately 10,000 HEK293T-ACE2 cells per well were seeded in 96-well plates in media containing 10% FBS. Cells were infected with serial dilutions of SARS-CoV-2 pseudovirus in a final volume of 200. Mu.L. After 48 hours, the infected cells were lysed using a commercial kit (Promega, E1500, madison, wisconsin, usa) to measure luciferase activity. The previously described "TCID" macros were used to calculate the 50% tissue culture infection dose (TCID 50) (Richards & Clapham, 2006).

6. Cytotoxicity test

Cells were incubated in 5% CO ₂ for 48 hours at 37 ℃ with or without serial dilutions of SSP. And then useThe viability of the cells was measured using a luminescent cell viability assay kit (Promega, wisconsin, usa). Cytotoxicity was generated based on the sample concentration and luminescence values. The results were analyzed according to the following formula:

7. Antiviral assay

The inhibitory activity of SSP against viruses was evaluated as described previously (L.Liu et al, 2007; X.Lu et al, 2012). Briefly, serial dilutions of SSP were tested for 100TCID50 virus infection. HEK 293FT-ACE2 for SARS-CoV-1/2; MDCK for H5N1; GHOST (3) -CD4-CCR5/CXCR4 is used for HIV _ADA and HIV _HXB2 infections. The third day, viral infection was determined by measuring the reported luciferase activity of target cells after infection using a commercially available kit (Promega, wisconsin, usa). Antiviral data is reported as the drug concentration required to inhibit viral replication by 50% (EC ₅₀). Similarly, 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 of SSP. The antiviral activity of Grape Seed Extract (GSE) and tea polyphenol (TP, CAS: 84650-60-2) (Shanghai field Co.) was also tested by the same method.

8. Security assessment

And (3) model: acute toxicity-mice and rats; long-term toxicity-rats; nasal spray irritation test-rabbits

Acute toxicity experiment: toxicity in SD rats and NIH mice was observed and studied for clinical safety of SSP by intragastric administration of SSP one or more times over a 24 hour period to determine acute toxic and maximum tolerated doses and possible toxicity to target organs. The method comprises the following steps: several SD rats and NIH mice, half male and half female, were selected and randomized into vehicle control and SSP groups. Rats were fasted for 12-16 h and weighed prior to administration. The SSP group was given 0.25g/mL of SSP powder, rats at 20mL/kg, and the vehicle control group was given an equal volume of purified water, orally or twice daily (8 h dosing interval) with continuous 4 hours after each administration, once daily in the morning and afternoon for D1-D14; d1, D4, D7, D14 rats were weighed and recorded. After the animals were sacrificed and visual observations were made, any changes in volume, color and texture of any tissues and organs were recorded and histopathological examination was performed.

Long-term toxicity: 24 male and female Sprague-Dawley rats (150-180 g) were used to determine the long term adverse effects of SSP. All experiments were approved by the living animal committee for use in guidelines and teaching and research of the laboratory animal care institution at the agricultural university of south China, guangzhou, china. Animals were randomly divided into four groups of three males and three females. SSP was dissolved in water and administered by gavage, once daily, at doses of 2, 4, 6g/kg or water. Their body weight, general behavior, signs of poisoning, food intake, and mortality were recorded at every two to four day intervals. After 25 days, animals were sacrificed by over anesthesia. Necropsy, organ index calculations (such as brain, heart, lung, liver, kidney, etc.), organ pathology, blood collection, and analysis were performed.

Nasal spray irritation test-rabbits: the experiment was completed in CFDA certified GLP standard drug safety evaluation research center (shandong new boy drug research limited) and the experimental code was KY22233.

9. Stability of

The stability of the SSP powder was determined by measuring the procyanidin content by the vanillin-hydrochloric acid method mentioned in 3-1), the test samples being an aqueous solution of SSP prepared immediately and an aqueous solution of SSP after standing at room temperature for more than 1 month.

In addition, antiviral activity was found after 6 days and 13 days of storage of the aqueous SSP solution.

10. In vivo antiviral assay

The efficacy of SSP on in vivo intranasal SARS-CoV-2omicron ba.2 challenge was tested in K18-hACE2 mice. Viral units of 2 x 10a 4moi are administered into the nasal cavity of mice. As shown in fig. 10a, one dose of SSP intervention (40 mg/ml 20 μl,800 μg) was administered 30 min before virus infection, control group was normal saline group (n=5 per group). 3 days after virus infection, lung tissue was collected from mice.

Results

SSP inhibits SARS-CoV-2 entry

To determine the antiviral activity of SSP, SSP was weighed as described and dissolved in 40 mg/mL. As described above, SARS-CoV-2 pseudovirus was produced (L.L.Liu et al, 2019; X.). To test the antiviral activity of SSP against SARS-CoV-2, HE293T-ACE2 cells were subsequently infected with 100TCID50 SARS-CoV-2 pseudovirus in the presence of serial dilutions of SSP. A virus of VSV-G pseudotyped with a second unrelated viral envelope glycoprotein was used as a control to reduce false positives. As shown in FIG. 3, SSP showed 3.5 μg/mL EC ₅₀ (FIG. 3 a) upon inhibition of SARS-CoV-2 pseudovirus infection, and no significant cytotoxicity (FIG. 3 c). No inhibition was observed when SSP was tested against VSV pseudoviruses (fig. 3 b). Because SARS-CoV-2 and VSV pseudoviruses share a common genetic HIV backbone that expresses Proteases (PR), reverse Transcriptase (RT), and Integrase (IN) and differ only IN the glycoproteins on the viral surface. The results indicate that SSP antagonizes SARS-CoV-2 pseudovirus entry, rather than antagonizing post-entry events (e.g., reverse transcription). Furthermore, the lack of antiviral activity against VSV also precludes the possibility that SSP can inactivate SARS-CoV-2 virus by acting on viral lipids only as a disinfectant.

Ssp works by blocking the binding of SARS-CoV spike envelope protein to the access receptor ACE2

It is well known that SARS-CoV-2 enters host cells via the viral surface anchored S protein. The S protein mediates viral entry via RBD in the S1 subunit, which S1 subunit specifically recognizes ACE2 as its receptor, and then fuses the virus into the host membrane via the S2 subunit. Entry inhibitors typically target protein-protein interactions or inhibit protein-lipid interactions within the viral envelope protein or between the viral envelope protein and the host cell. To determine whether SSP works by targeting SARS-CoV-2S protein or host cells, we pre-treated pseudovirus and HEK-293T-ACE2 cells with serial dilutions of SSP at 37 ℃ for 2 hours. The virus and HEK-293T-ACE2 cells are then recovered and subsequently infected with HEK293T-ACE2 or incubated with untreated SARS-CoV-2 pseudovirus (Rapista et al, 2011). As a control, HEK293T-ACE2 cells were infected with SARS-CoV-2 pseudovirus and then treated with gradient diluted SSP immediately after infection or 2 hours after infection (hours post-infection. As shown in FIG. 3d, SSP treatment 2 hours after infection showed limited anti-SARS-CoV-2 activity compared to the simultaneous addition of virus and SSP. This result demonstrates the antiviral entry activity of SSP against SARS-CoV-2. Furthermore, pretreatment of pseudoviruses with SSP resulted in increased inhibitory activity, with an EC ₅₀ value of 2.3 μg/mL, indicating that SSP targets the SARS-CoV-2 viral envelope S protein to inhibit SARS-CoV-2 entry. Notably, pretreatment of target cells with SSP terminated SARS-CoV-2 infection with an EC ₅₀ value of 2.1 μg/mL, indicating that SSP also targets cellular components to inhibit SARS-CoV-2 entry.

To determine whether SSP targets ACE2 receptors to block virus entry, RBD binding assays were performed. HEK-293T-ACE2 cells were treated with serial dilutions of SSP at 37 ℃ for 2 hours, washed and then incubated on ice for 30min with supernatant collected from HEK293T cells transfected with RBD-PD1 expressing plasmids or goat polyclonal antibodies against ACE 2. The cells were then washed and stained with a fluorescent-labeled secondary antibody directed against goat IgG or PD-1, respectively. As shown in fig. 3e, untreated HEK-293T-ACE2 cells stained positively for both ACE2 and RBD-PD1, but negatively for antibodies against goat IgG and PD-1, indicating that HEK-293T-ACE2 cells have high ACE2 expression levels and can bind to RBD of SARS-CoV-2S protein. The SSP treated cells showed a significant decrease in geometric mean signal to ACE2 staining, but no significant decrease in the percentage of ACE2 ⁺ cells, indicating the possibility that SSP partially blocked ACE 2-specific antibody binding rather than down-regulating ACE2 expression. In contrast, SSP treatment reduced the geometric mean signal and positive cell percentage of RBD staining when compared to untreated cells, indicating that SSP treatment partially blocked RBD binding to ACE2 protein (fig. 3 f).

SSP exhibits broad-spectrum antiviral activity against SARS-CoV, H5N1, EBOV and HIV-1

To determine whether SSPs have broad-spectrum antiviral activity, a panel of pseudoviruses 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 generated in our earlier studies to test the inhibition of SSPs. As shown in FIGS. 4a-d, SSPs inhibited their infection, with EC ₅₀ at about 3.64, 5.13, 3.61, and 8.15 μg/mL, respectively, and no significant cytotoxicity (FIG. 4 e). This result suggests that SSP has broad-spectrum antiviral activity against SARS-CoV, H5N1 and HIV-1. Furthermore, the recent recurrence of ebola virus disease in western africa prompted us to investigate whether SSP also inhibited EBOV infection. EBOV pseudoviruses are produced in 293T cells, as previously described. We tested pseudovirus EBOV and VSV as a control. 293T cells were infected with EBOV and VSV, respectively, in the presence of serial dilutions of SSP. We found that SSP inhibited EBOV, EC ₅₀ value of about 4 μg/ml, and no cytotoxicity was observed (FIG. 4 f). Consistently, SSP did not inhibit VSV infection, indicating that SSP blocked EBOV entry into target cells.

SSP blocks SARS-CoV-1 and H5N1 entry by binding to the viral envelope and its target cells. To determine whether SSP inhibits viral entry by binding to the SARS-COV-1 envelope or its receptor on the target cell, SSP-viral binding and SSP-cell binding assays were performed. In the SSP-virus binding assay, pseudoviruses are first pretreated with 50. Mu.g/mL SSP. The virus is then recovered by ultracentrifugation and used to infect target cells. We found that SSP inhibited SARS-CoV-1 pseudovirus infection to a similar extent as when the virus and SSP were added to cells simultaneously (FIG. 5). Meanwhile, SSP-cell binding assays were performed by pre-treating target cells with SSP at 37 ℃ for 1 hour. After washing with PBS, the cells were incubated with SARS-CoV-1 at 37℃for 48hr (Rapista et al, 2011). We found that pretreatment of SSP on target cells inhibited viral infection (FIG. 5). The inhibitory effect of SSP against H5N1 with a single high dose of 50 μg/mL was also tested using the same method. Like SARS-CoV-1, SSP treatment of the viral particles or their target cells effectively blocks H5N1 virus entry (FIG. 5 b).

SSP blocks HIV-1 entry by binding to the viral envelope. We tested HIV-1 using a similar but slightly modified protocol that includes four anti-HIV drugs: AZT (potent NRTI), HIV-1 fusion blocker T20, the CCR5 antagonist Marvaroc (MVC), and the CXCR4 antagonist JM2987 as positive controls. As shown in figure 5, AZT, but not SSP significantly inhibited HIV-1 infection when treatment was started 2 hours after infection. In contrast, pretreatment of the virus with 50. Mu.g/mL SSP significantly inhibited HIV-1 infection to a similar extent as T-20 blocking HIV-1 fusion (FIGS. 5 c-5 d). Subsequently, SSP cell binding assays were performed by treating target ghest cells with SSP or with control compound CCR5 antagonist Marvaroc (MVC) or CXCR4 antagonist JM2987 for 2 hours at 37 ℃. After washing with PBS, the cells were infected with HIV-1 _ADA or HIV-1 _HXB2 and incubated at 37℃for 48hr (Rapista et al, 2011). We found that pretreatment of target cells with SSP showed little antiviral effect, whereas MVC and JM2987 showed potent viral inhibition as expected against ADA and HXB2 pseudoviruses, respectively, at 1. Mu.M (FIGS. 5 e-5 f). This evidence suggests that SSP inhibits HIV-1 infection by acting on the viral envelope glycoprotein gp160, which mediates viral entry into host target cells.

SSP and antiviral Activity of its active ingredient

To illustrate SS-containing materials, the feasible UPLC method was used to isolate SSP, where SSP was split into three parts, namely part I, II, and part III (fig. 6 a). UPLC-ESI-MS/MSn experiments were performed in the same manner (FIGS. 6 d-6 f) and found: (1) The enrichment of monomers and dimers is mainly carried out for 3-9 min (part I); (2) 8-10 polymers are mainly enriched (part II) in 23-33 min; (3) the 3-7 mer is enriched in both parts. However, polymers having a Degree of Polymerization (DP) higher than 10 cannot be detected due to the limited range of molecular weights. Based on this result, extraction was performed with solvents of different polarities, and the n-butanol fraction (SSP-n-BuOH) was selected for further separation by using a macroporous resin column. By UPLC detection, fr.b and fr.g are separated into two parts as mentioned before, part I and part II respectively (fig. 6 b-6 c). In this study we extracted and enriched the procyanidins in SSP to a concentration of about 60%. Thiolysis experiments have also shown that the average degree of polymerization (mDP) of SSP, SSP-n-BuOH, fr.b and fr.g are 3.49, 4.57, 2.95 and 7.52, respectively, which means that macroporous resin columns can be efficiently separated and fractions with different mdps can be obtained. Using the methods as described previously, we found that SSP, SSP-n-BuOH and Fr.G exhibited lower EC ₅₀ than Fr.B in inhibiting SARS-CoV and SARS-CoV-2 pseudoviral infection (FIGS. 7 a-7 b). No significant inhibition of VSV pseudovirus (fig. 7 c) or cytotoxicity (fig. 7 d) was observed. Furthermore, pretreatment of pseudoviruses or cells with SSP-n-BuOH, fr.f, or fr.g inhibited viral entry and had lower EC ₅₀ values than the simultaneous addition of viruses and fragments, indicating that these fragments target the SARS-CoV-2 viral envelope S protein and target cells to inhibit SARS-CoV-2 entry. More importantly, fr.g showed more prominent bioactivity when compared to fr.b, indicating that part II of SSP had more potent antiviral activity than part I. These results indicate that fr.g contains an intermediate polymer of procyanidins, which may have a better antiviral effect.

15. Safety evaluation result

To assess the safety of SSP, its cytotoxicity in a variety of cell lines was determined by cytotoxicity assays (IC ₅₀ = 181.2-339.8 μg/mL, as shown in figures 8 a-8 d).

To test for acute toxicity in vivo, NIH mice and SD rats were used to obtain the maximum tolerated dose. The maximum tolerated dose in mice for one administration was 67g crude drug/kg (Table 1). The maximum tolerated dose for one administration in rats was 107g crude drug/kg (Table 2).

To further study long-term toxicity in vivo, 24 female and male SD rats were divided into four groups: water (blank), 2g/kg, 4g/kg, 6g/kg SSP. The drug or water was administered by oral gavage once a day for 25 days. During the monitoring period, no mortality was observed in any group of rats (fig. 8). Body weight and food intake are shown in figures 8e-8 f. SSP administered at this dose showed no signs of toxicity to rats, except for weight loss in the high dose group. No significant difference in organ index (ratio of organ weight to body weight) was observed compared to control mice (fig. 8 g). No abnormalities were observed in the level of blood biochemical parameters, indicating that SSP was not significantly toxic to the experimental rats (table 3).

Mouse results:

TABLE 1 acute toxicity of SSP in mice (NIH)

Rat results:

TABLE 2 acute toxicity of SSP in rats (SD)

TABLE 3 blood Biochemical index test projects in Male and female rats

16. Stability results

According to the same method, the procyanidin contained in the immediately prepared aqueous SSP solution is 794.794 ± 13.619mg/g, and the content of the procyanidin in the aqueous SSP solution after 1 month of standing is 778.964 ± 63.589mg/g, which are all within the quality standard range of 5%, so that the aqueous SSP solution is stable in terms of procyanidin content.

In addition, it was found that there was no significant decrease in antiviral activity of aqueous SSP solutions after 6 and 13 days of standing and no change in cytotoxicity (fig. 9 a).

17. In vitro antiviral assay against SARS-CoV-2 Virus variants

As shown in FIGS. 9a-9b, SSP also has a relatively potent inhibitory activity against SARS-CoV-2 virus variants, while being less cytotoxic. GSE (fig. 9 c) and TP (fig. 9 d), which also contained PAC, exhibited much weaker inhibition compared to SSP, and not all variants were inhibited. SSP showed higher potency and broader spectrum of SARS-CoV-2 variant entry, unlike GSE and TP. In summary, SSP is the only drug with higher potency and broader spectrum against viruses, worth further exploration.

18. In vivo antiviral assay

As shown in FIG. 10b, the Omicron BA.2 virus-infected cells (green) and inflammatory cells (red) were significantly reduced in the lung tissue of mice treated with SSP compared to the saline group. Furthermore, the expression level and viral titer of RNA-dependent RNA polymerase in lung tissue of mice treated with SSP was significantly lower than those treated with saline (fig. 10 c). In general, SSP has been shown to be effective in protecting mice from nasal challenge with SARS-CoV-2omicron variant virus.

19. Nasal spray irritation test

The experiment was completed in CFDA certified GLP standard drug safety evaluation research center (shandong singbo drug research limited) and the experimental code was KY22233. The animals were in good overall status during the trial period and no mortality occurred; no significant abnormalities were observed after each application. Necropsy was performed 24 hours after the last administration and 14 days after recovery to visualize the local mucosal tissues (oral, nasal, laryngeal, tracheal and bronchial). No significant abnormalities were observed in the negative control group and the test product group. Histopathological examination also showed that no drug-related histopathological changes were found at all animal sites in the negative control group and the test product group 24 days after the end of the last administration and recovery period.

Reference to the literature

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The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the relevant art (including the documents cited herein and the contents incorporated by reference), readily modify and/or adapt for various applications such specific embodiments without undue experimentation without departing from the general concept of the present disclosure. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by one of ordinary skill in the relevant art in light of the teachings and guidance presented herein.

While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example, and not limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the disclosure. Thus, the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Claims (35)

1. A method for obtaining a Solanum sibiricum (SSD) extract, the method comprising: (i) slicing the SSD; (ii) soaking and treating the SSD slices in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; and (iv) drying the concentrated extract to obtain a powder of the SSD extract, wherein the SSD extract contains proanthocyanidins (SSP). 2. A method for obtaining an active fraction from Solanum sphaerocephalum (SSD), the active fraction comprising proanthocyanidins, the method comprising: (i) slicing SSD; (ii) soaking and treating SSD slices in a solvent at room temperature to obtain an extract; (iii) concentrating the extract; (iv) drying the concentrated extract to obtain a powder of the SSD extract (SSP); (v) dispersing SSP in water to obtain an aqueous solution; (vi) extracting the SSP aqueous solution with equal volumes of a series of solvents having different polarities to obtain an SSP portion corresponding to each solvent, the series of solvents comprising n-butanol; and (vii) separating the n-butanol portion (SSP-n-BuOH) with an eluent to obtain the active fraction. 3. The method of claim 1 or 2, wherein the treatment process in (ii) comprises diafiltration, ultrasonic treatment, heating and refluxing, decoction, and/or solvent extraction. 4. The method of claim 3, wherein the treatment process in (ii) comprises diafiltration. 5. The method of claim 1 or 2, wherein the solvent in (ii) comprises water and/or an organic solvent. 6. The method of claim 5, wherein the solvent in (ii) comprises water and ethanol. 7. The method of claim 6, wherein the solvent in (ii) is 60% ethanol/water (v/v). 8. The method of claim 1 or 2, wherein the concentration process in (iii) comprises reduced pressure rotary evaporation, centrifugation, and/or nitrogen purging. 9. The method of claim 8, wherein the concentration process in (iii) comprises rotary evaporation under reduced pressure. 10. The method of claim 1 or 2, wherein the concentration process is carried out at a temperature below 50°C. 11. The method of claim 1 or 2, wherein the drying process in (iv) comprises freeze drying, spray drying, microwave drying, and/or infrared heating drying. 12. The method of claim 11, wherein the drying process in (iv) comprises freeze drying. 13. The method of claim 1 or 2, wherein the series of solvents in (vi) further comprises water, ethanol, petroleum ether (PE), and/or ethyl acetate. 14. The method of claim 1 or 2, wherein the separation process in (vii) comprises macroporous resin column separation, fractional precipitation, crystallization, normal phase chromatography, reverse phase chromatography, and/or ion exchange resin column separation. 15. The method of claim 14, wherein the separation process in (vii) comprises separation using a macroporous resin column. 16. The method of claim 1 or 2, wherein the eluent in (vii) comprises water and/or an organic solvent. 17. The method of claim 16, wherein the eluent in (vii) is a gradient ethanol-water from 10% to 95%. 18. The method of claim 2, further comprising: (viii) concentrating the active fraction. 19. The method of claim 18, further comprising: (ix) drying the concentrated active fraction. 20. A SSD extract obtained by the method of any one of claims 1 and 3-17. 21. An active fraction from SSD obtained by the method of any one of claims 2 to 19. 22. A composition comprising an SSD extract obtained by the method of any one of claims 1 and 3-17, or an active fraction from SSD obtained by the method of any one of claims 2-19. 23. A method for preventing and/or treating a viral disease, comprising administering to a subject in need thereof an effective amount of an SSD extract obtained by the method of any one of claims 1 and 3-17. 24. A method for preventing and/or treating a viral disease, comprising administering to a subject in need thereof an effective amount of an active fraction from SSD obtained by the method of any one of claims 2 to 19. 25. The method of claim 23 or 24, wherein the effective dose ranges from 0.5 μg/mL to 5000 μg/mL. 26. Use of the SSD extract obtained by the method of any one of claims 1 and 3-17 in the preparation of a preparation for preventing and/or treating viral diseases. 27. Use of an active fraction from SSD obtained by the method according to any one of claims 2 to 19 in the preparation of a preparation for preventing and/or treating viral diseases. 28. The method/use of any one of claims 23-26, wherein the viral disease is caused by coronaviruses, including SARS-CoV-2 and SARS-CoV-1 and variants thereof, Ebola virus (EBOV), HIV-1, H5N1, and other enveloped viruses except VSV. 29. The use of claim 26 or 27, wherein the preparation is in the form of an injection, tablet, granule, emulsion, gel, sustained-release preparation, nasal wash/nasal spray, oral liquid, throat spray, tea-like pill/candy, nano preparation, "throat lozenge", "throat spray", "nasal wash" and/or "nasal spray". 30. The use according to claim 26 or 27, wherein the preparation further comprises a filler, a disintegrant, an additive, a suspending agent, and/or a binder. 31. The use of claim 30, wherein the filler comprises starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, and/or sucrose. 32. The use of claim 30, wherein the disintegrant comprises starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, and/or cross-linked polymethylcellulose sodium. 33. The use of claim 30, wherein the additive comprises magnesium stearate, sodium lauryl sulfate, talc, and/or silicon dioxide. 34. The use of claim 30, wherein the suspending agent comprises polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, and/or hydroxypropyl methylcellulose. 35. The use of claim 30, wherein the binder comprises starch slurry and/or polyvinyl pyrrolidone.