A traditional Chinese medicine formula NRICM101 to target COVID-19 through multiple pathways: A bedside-to-bench study

2.1. Clinical setting, participants and data analysis

Tri-Service General Hospital and Taichung Veterans General Hospital were among the 52 response and isolation hospitals designated by Taiwan Centers for Disease Control for treatment of severe COVID-19 cases. Thirty-five patients were admitted to the two medical centers between January and April 2020. They received symptomatic treatment and/or hydroxychloroquine until 13 of them shifted to NRICM101 on a voluntary basis and with consent from the care teams. NRICM101 was administered three times daily, each time 100 mL 30 min after a meal until discharge. In Taiwan, the discharge criteria during the research period was 3 consecutive negative results for SARS-CoV-2 of respiratory tract samples, with each sample collected at least 24 h apart (3 N). Adverse events, if any, would be recorded on a standard form.

The bedside study retrospectively examined demographic and clinical data of all the 35 patients. We used descriptive statistics to summarize our sample data by NRICM101 and non-NRICM101 groups. After excluding 2 cases (one received TCM intermittently and the other received intravenous immunoglobulin therapy), there were 12 in the NRICM101 group and 21 in the non-NRICM101 group. Distribution of age, gender, disease severity, comorbidity, length of hospital stay before 3 N of the 2 groups is presented.

2.2. Preparation and composition of the formula

The decoction of NRICM101 was prepared by the TCM pharmacies in the two medical centers, after slightly modifying the formula for mild cases included in the TCM Treatment Guideline for COVID-19 developed by NRICM (Supplementary 1). It consisted of 10 herbs: Scutellaria Root (Scutellaria baicalensis, HA, 18.75 g), Heartleaf Houttuynia (Houttuynia cordata, HC, 18.75 g), Mulberry Leaf (Morus alba, NB, 11.25 g), Saposhnikovia Root (Saposhnikovia divaricata, NC, 7.50 g), Mongolian Snakegourd Fruit (Trichosanthes kirilowii, ND, 18.75 g), Indigowoad Root (Isatis indigotica, NE, 18.75 g), baked Liquorice Root (Glycyrrhiza glabra, NG, 7.50 g), Magnolia Bark (Magnolia officinalis, NK, 11.25 g), Peppermint Herb (Mentha haplocalyx, NL, 11.25 g), and Fineleaf Nepeta (Nepeta tenuifolia, NR, 11.25 g). For a patient’s daily dose, a full set of herbs and 1 L of water were placed in a boiler, boiled and simmered for the decoction to reduce to 300 mL.

2.3. HPLC analysis of NRICM101

Herbal materials (10 items) were obtained from local licensed herbal stores and the TCM pharmacy of Tri-Service General Hospital. Each of them (1.0 g) was extracted with water (30 mL) at 100 ℃ for 1 h and centrifuged at 1000 rpm for 10 min. The supernatant of all samples was further filtered through a 0.22 μm of PTFE filter. All the samples were stored at -80 ℃ before HPLC analysis. Twelve batches of NRICM101 decoction were collected at different times (2020/04/03, 2020/04/18, 2020/04/19, 2020/04/20) from the TCM pharmacies in the two medical centers. The decoction was dried with a freeze-dryer, yielding the crude extract of 15.9 ± 0.9 g per 300 mL. HPLC was performed on a Shimadzu Nexera-i LC-2040C 3D Liquid Chromatograph (Shimadzu, Kyoto, Japan) equipped with an ODS COSMOSIL 5C₁₈-AR-II column (4.6 mm × 250 mm) at 40 ℃. In HPLC fingerprint analysis, the mobile phase consisted of D.D water with 0.3 % phosphoric acid and acetonitrile (ACN) using a gradient condition as follows: 0.01–7.00 min, 3–10 % ACN; 7−15 min, 10–20 % ACN; 15−35 min, 20 % ACN; 35−50 min, 20–35 % ACN; 50−60 min, 35–100 % ACN. The mobile phase flow rate and the injection volume were 1.0 mL/min and 10 μL, respectively.

2.4. Identification of the components from NRICM101

NRICM101 was isolated by Diaion column (HP-20, EtOH/H₂O), C₁₈ flash column, and further purified by preparative HPLC, yielding 17 compounds. The chemical structures of all isolated compounds were determined by NMR (1D and 2D experiments) and HRMS, and compared with the reported data. For preparation of standard solutions for HPLC fingerprint of the decoction, each compound was accurately weighed and dissolved in MeOH/D.D. H₂O, and the concentration was 1 mg/mL. All the tested solutions were filtered through a 0.22 μm filter before use. The injection volume of the isolates was 5 μL.

2.5. Quantitation of major compound

The standard solution of the major compound, baicalin (Sigma-Aldrich, 95 %) was prepared in the DMSO, at the concentration of 10 mg/mL. The stock solution with 50 % ACN in D.D. H₂O was diluted to obtain the developing solutions, including seven concentrations in the range of 0.015–1.0 mg/mL. The worked solutions were filtered through a 0.22 μm filter before HPLC injection. Linear regression analysis was applied to achieve linearity by the integrated peak and the concentration at seven different worked concentrations. The analysis condition of HPLC was the same with those of the NRICM101 decoction.

2.6. ACE2-spike protein inhibition enzyme-linked immunosorbent assay (ELISA)

The ACE2-spike protein inhibition ELISA was carried out to determine the ability of NRICM101 to block the interaction between ACE2 and SARS-CoV-2 spike receptor-binding domain (RBD) protein (Sino Biological). In brief, microplate wells were coated with spike RBD protein (0.2 μg/well). After blocking with 1% bovine serum albumin (BSA), plates were washed with phosphate-buffered saline with Tween detergent (PBST). Then, 5-, 10-, 20-, 40-, 80-, 160-, 320-, 640-, 1280-, 2560-, 5120-fold diluted samples were added to the wells to react with spike RBD protein. After washing with PBST, ACE2 protein (0.2 μg/mL, Sino Biological) was added to each well for incubation. Next, a rabbit anti-His tag antibody conjugated with horseradish peroxidase (HRP) was used (Immunology consultants laboratory, Inc.) for detection. Finally, each well was given the 3,3′,5,5′ tetramethylbenzidine (TMB, SeraCare) for color development. After stopping the reaction with 1 N HCl, signal intensity was then measured at OD 450 nm (SPECTROstar Nano, BMG LABTECH).

2.7. Surface plasmon resonance (SPR)

Binding reactivity to spike RBD protein of SARS-CoV-2 was performed by SPR (OpenSPR, Nicoyalife). The recombinant spike RBD protein was diluted to 25 μg/mL with PBS and captured by a NTA chip (Nicoyalife). The analytes were tested in different dilutions in PBS and maintained the flow rate at 20 μL/min to analyze. The NTA sensor chip was regenerated with 10 mM glycine−HCl, pH2.2 (Nicoyalife). The results were analyzed using TraceDrawer software (Nicoyalife).

2.8. Inhibition assay of 3CL protease

The assay, modified from previous research [15], was performed with a volume of 20 μL in a 384-well white microplate (NUNC, Roskilde, Denmark). Briefly, a total of 50 ng of 3CL protease (final concentration: 75 nM) was incubated with the sample in the reaction buffer (25 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.3) on ice for 30 min. Subsequently, the reaction was initiated by adding 0.25 μg of the protease fluorogenic substrate peptide Dabcyl-KTSAVLQSGFRKME-Edans (final concentration of 6 μM), and monitored at 538 nm with excitation at 355 nm at 37℃ using the CLARIOstar microplate reader (BMG LABTECH, Ortenberg, Germany) for 1 h. The inhibition was calculated by the following formula [16]: Inhibition (%) = {1 - [(S – S₀)/(C– C₀) x 100 [C: fluorescence of control (enzyme, buffer, and substrate) after 1 h incubation; C₀: fluorescence of control at time zero; S: fluorescence of samples (enzyme, tested sample solution, buffer, and substrate) after 1 h incubation; S₀: fluorescence of samples at time zero]. The recombinant SARS-CoV-2 3CL protease was obtained from Pharmtekx (Taipei, Taiwan). The protease fluorogenic substrate peptide was purchased from Kelowna International Scientific Inc. (New Taipei City, Taiwan). Chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

2.9. SARS-CoV-2 infection and immunofluorescent assay (IFA)

Vero E6 cells and SARS-CoV-2 (TCDC#4 from Taiwan CDC) were treated with samples at various dilutions for 1 h at 37 °C. The treated viruses were then added to the treated cells for infection (MOI = 0.01) at 37 °C for 2 days. The cells were fixed with 10 % formalin and permeabilized with 0.5 % Triton X-100 in PBS. The cells were stained with a human anti-SARS-CoV-2 N protein monoclonal antibody (provided by Dr. An-Suei Yang in Genomics Research Center, Academia Sinica, Taipei, Taiwan) and goat anti-human IgG-Alexa Fluor 488 (A11013, Invitrogen). Cell nucleus was stained with DAPI (D1306, Invitrogen). The signals were observed and photographed under an immunofluorescent microscope. To quantify viral infection, images were acquired and analyzed using an ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices). For cell viability test, Vero E6 cells were treated at different dilutions for 1 day at 37 °C. The cell viability was determined by Cell Counting Kit-8 (CCK-8). 50 % inhibition concentration (IC₅₀) and 50 % cytotoxic concentration (CC₅₀) were calculated by Prism software.

2.10. SARS-CoV-2 plaque reduction neutralization test (PRNT)

Vero E6 cells and SARS-CoV-2 (TCDC#4) were treated with samples at various dilutions for 1 h at 37 °C. The treated viruses were added to the treated cells for viral adsorption at 37 °C for 1 h. The virus inoculants were removed and the cells were overlaid with medium containing 1% methylcellulose for 4-day incubation at 37 °C. The cells were fixed with 10 % formalin and stained with crystal violet.

2.11. Inhibition of cytokine inhibition assay

5 × 10⁵ MH-S murine alveolar macrophages (ATCC CRL-2019) were cultured in 12-well culture plates for 24 h. The cells were treated with or without the samples in different dilutions in the presence of LPS (1 μg/mL). The production of IL-6 and TNF-α in the cell cultures was determined by using commercial ELISA kits (R&D Systems).

3.1. Clinical data

Table 1 summarizes the demographic and clinical characteristics of 33 patients (54.5 % female). Participants had a median age of 40 years, with 10 below 30 and 8 above 60. The majority (87.9 %) were mild cases. Disease severity was categorized as either mild, severe, or critical following the interim guidance proposed by the United States Centers for Disease Control and Prevention upon hospitalization.

Descriptive statistics show that the NRICM101 group (n = 12) had a median age of 57 years, 8 (66 %) with comorbidities, and 4 (33.3 %) severe or critical cases. On the other hand, those without NRICM101 (n = 21) had a median age of 33 years, 3 (14.3 %) with comorbidities and no severe or critical cases.

The non-NRICM101 group receiving symptomatic care achieved 3 N at a median of 22 days after the onset of hospitalization. NRICM101 was administered to the rest of patients who had not shown signs of improvement after a median of 21.5 days in the hospital. 3 N was observed 9 days (median) after intervention.

3.2. Laboratory findings of NRICM101

Using varying levels of NRICM101 dilution as analytes in the surface plasmon resonance (SPR) analysis, the binding affinity of NRICM101 for RBD protein was found to be dose-dependent (Fig. 3 A). In the ACE2-spike protein inhibition enzyme-linked immunosorbent assay (ELISA), NRICM101 interrupted affinity of the SARS-CoV-2 spike to the human ACE2 receptor with an IC₅₀ at 128-fold dilution (0.41 mg/mL) (Fig. 3B). In the SARS-CoV-2 3CL protease activity assay, our assessment suggested that NRICM101 displayed a prominent inhibitory effect on 3CL protease with an IC₅₀ at 248-fold dilution (0.22 mg/mL) (Fig. 3C).

NRICM101 also showed the ability to reduce SARS-CoV-2 viral growth. In the immunofluorescence assay (IFA), viral infection was quantified by SARS-CoV-2 N protein expression, and cytotoxicity of the extract was determined by a CCK-8 assay. NRICM101 showed excellent anti-SARS-CoV-2 activity with an IC₅₀ at 187-fold dilution (0.28 mg/mL) and CC₅₀ at 30-fold dilution (1.77 mg/mL). A plaque reduction neutralization test (PRNT) further demonstrated its ability to reduce viral growth. Similar to the IFA data, NRICM101 blocked plaque formation of SARS-CoV-2 (Fig. 3D,E).

In the analysis of cytokines in cell culture, NRICM101 demonstrated repressive effects on the secretion of IL-6 and TNF-α as measured by lipopolysaccharide stimulated murine alveolar macrophages with an IC₅₀ at 128- (0.42 mg/mL) and 45-fold (1.18 mg/mL) dilutions, respectively (Fig. 3F,G).

3.3. Single herb analyses

Individual components of NRICM101 were analyzed. First, HC and NL demonstrated the ability to block binding between ACE2 and spike protein in a 40-fold dilution (1.33 mg/mL) in the ACE2-spike protein inhibition ELISA assay. HC, in particular, showed a significant >70 % inhibition rate in a 40-fold dilution (Fig. 4 A).

Herbs in NRICM101 presented excellent to moderate inhibitory activity against 3CL protease. Among these herbs, HA exhibited the most potent inhibitory activity at the rates of 101.2 %, 97.1 %, 92.0 %, and 83.7 % in the 5-, 10-, 20-, and 40-fold dilutions, respectively, suggesting that HA was the major contributor for NRICM101 activity (Fig. 4B). NR, NB, NK, and NL also showed strong inhibitory activity against 3CL protease with 65.0 %, 54.3 %, 61.2 %, and 68.2 % in the 20-fold (2.65 mg/mL) dilutions, respectively.

HC and HA were selected for further antiviral assays. Minor antiviral activity was noted for HA in the IFA (IC₅₀ at a 71-fold dilution and CC₅₀ at a 17-fold dilution), but not for HC (Fig. 4C). In PRNT, HA blocked plaque formation of SARS-CoV-2, while HC did not (Fig. 4D).

In the cytokine inhibition assays, each of the 10 herbs were individually analyzed. The extracts of HC and HA with 20-fold dilutions were able to inhibit TNF-α production at a rate greater than 50 %. HA continued to inhibit production of TNF-α and IL-6 in a 40-fold dilution, suggesting that HA could be the major contributor to NRICM101 activity (Fig. 4E,F).

3.4. Identification of the isolated compounds and HPLC profile

Finally, HPLC fingerprint analyses of NRICM101 and its 10 constituent herbs were conducted for quality control and the isolation and identification of lead compounds (Fig 5 A,B). Seventeen compounds, including three caffeoylquinic acids (CQA), 3-CQA, 4-CQA, and 5-CQA; four quercetin glycosides, quercetin 3-galactoside, quercetin 3-glucoside, quercetin 3-rhamnoside, and rutin; two chrysin C-glucosides, chrysin 6-C-glucoside-8-C-arabinoside and chrysin 6-C-arabinoside-8-C- glucoside; baicalien-type flavone glucuronide, baicalin, scutellarin and oroxyloside; two norwogonin glucuronides, wogonoside and norwogonin 7-O-glucuronide; a flavanone glycoside, liquiritin; and the others, epigoitrin and acetoside, were successfully isolated from the TCM decoction. The isolated compounds were further assigned on HPLC fingerprint profile of TCM decoction. Quality assessments across 12 different batches of NRICM101 demonstrated high consistency when comparing similarities of the HPLC fingerprints and lead compounds. On the other hand, the major compound baicalin (14) also showed consistency when comparing similarities of the HPLC fingerprints and lead compounds (Fig. 5C). In the 12 batches of NRICM101 decoction, baicalin (14) was quantified by the calibration curves (R² = 0.9998) and the content of baicalin was 2.3865 mg/mL.