IC50s: 3.39 μM for A/FM-1/1/47, 2.16 μM for A/Puerto Rico/8/34 H274Y, 5.32 μM for A/Aichi/2/68

Dendrobine, a major component of Dendrobium nobile, increasingly draws attention for its wide applications in health care. Dendrobine possessed antiviral activity against influenza A viruses, including A/FM-1/1/47 (H1N1), A/Puerto Rico/8/34 H274Y (H1N1), and A/Aichi/2/68 (H3N2) with IC50 values of 3.39 ± 0.32, 2.16 ± 0.91, 5.32 ± 1.68 μg/mL, respectively. Mechanism studies revealed that dendrobine inhibited early steps in the viral replication cycle. Notably, dendrobine could bind to the highly conserved region of viral nucleoprotein (NP), subsequently restraining nuclear export of viral NP and its oligomerization. In conclusion, dendrobine shows potential to be developed as a promising agent to treat influenza virus infection. More importantly, the results provide invaluable information for the full application of the Traditional Chinese Medicine named "Shi Hu" [1].

Neuraminidase Inhibition Assay[1]
The influence of dendrobine on the release of viral particles was evaluated by NA inhibition assay. Briefly, as described, 15 μL of influenza virus A/Aichi/2/68 (H3N2) solution was mixed with 5 μL of 2-fold diluted dendrobine or zanamivir (a positive control) in a 96-well black plate at 37 °C for 30 min. Then, 30 μL of 20 μM MU-NANA (2-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid sodium, Sigma-Aldrich) substrate solution dissolved in diluted buffer (32.5 mM MES and 4 mM CaCl2, pH 6.5) was added to each well. The plate was further incubated at 37 °C for 1 h in the dark, followed by adding 50 μL of 14 mM NaOH to end the enzyme reaction. Fluorescence intensity of the product 4-methylumbelliferone was recorded at the excitation wavelength of 340 nm and emission wavelength of 440 nm using a microplate reader.

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Mini-replicon Assay[1]
The effect of dendrobine on the activity of the viral ribonucleoprotein complex (vRNP) was determined by using a mini-genome assay as previously described. Briefly, 293T cells grown in 24-well plates were transfected with 50 ng of NP and viral polymerase plasmids (pHW2K-NP, pHW2K-PA, pHW2K-PB1, pHW2K-PB2) and pPolI-Fluc (a firefly luciferase reporter plasmid) together with 10 ng of hRluc-TK (Renilla luciferase plasmid) using Lipofectamine 2000. At 5 h after transfection, the supernatant was displaced by fresh DMEM with 10% FBS containing various concentrations of dendrobine, zanamivir, or 0.1% DMSO. At 24 h post-transfection, cells were lysed by cell lysates (Promega) for 20 min, and luciferase activity was measured as mentioned above.[1]

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Surface Plasmon Resonance (SPR) Analysis[1]
The binding affinity of the influenza NP and dendrobine was detected by the PlexArray HT system according to our previous paper with minor modifications. After dendrobine was immobilized on a chip surface using photo-cross-linking, recombinant influenza NP at 2-fold serial dilutions was injected at a flow rate of 2 μL/s with a contact time of 300 s and a dissociation time of 300 s. The running buffer is 10 mM phosphate buffer with 137 mM NaCl, 2.7 mM KCl, and 0.05% Tween-20. The chip platform was regenerated with glycine–HCl (pH 2.0) and washed with the running buffer. The affinity or KD value was calculated with PlexeraDE software by curve fitting using the Langmuir equation.

Cytotoxicity Assay[1]
The MTT assay was used to evaluate the cytotoxicity of dendrobine. Briefly, approximately 90% confluent cells in 96-well plates were exposed to dendrobine at 2-fold serial dilutions. After 48 h of incubation, 100 μL of MTT solution, which was diluted by the medium to 0.5 mg/mL, was added and retained at 37 °C for 4 h. Subsequently, the supernatant was removed, and 150 μL of DMSO was added to dissolve the formazan product. The absorbance for each well was measured at 570 nm using the Tecan Genios Pro microplate reader (Bedford, MA, USA). The half-maximal cytotoxic concentration (CC50) was calculated using CalcuSyn software.[1]

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Antiviral Assay and Microscopy[1]
To determine the antiviral activity of dendrobine, confluent MDCK cells were infected with the virus at a multiplicity of infection (MOI) of 0.01 at 37 °C for 1 h. Subsequently, dendrobine of non-cytotoxic concentrations was added to the cells after washing away the unabsorbed virus with PBS, and the cells were cultured for another 48 h. At the end of the culture, the MTT-based assay as previously described was assessed for the antiviral activity of dendrobine. The cytopathic effect (CPE) in virus-infected cells was observed through microscopy.[1]

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Plaque Assay[1]
Confluent monolayers of MDCK cells were inoculated with A/Aichi/2/68 (H3N2) strain at an MOI of 0.01 for 1 h at 37 °C. After removing the unbound virus, the cells were cultivated in 1.5 mL of serum-free MEM (2×) containing 1 μg/mL TPCK-trypsin (Sigma-Aldrich), 2% agar (Sigma-Aldrich), and different concentrations of dendrobine for 72 h as previously described. The effect of dendrobine on viral plaque formation was determined by the number of plaques.

[1]. Anti-influenza A Virus Activity of Dendrobine and Its Mechanism of Action. J Agric Food Chem. 2017 May 10;65(18):3665-3674.

Dendrobine is a member of indoles.
Dendroban-12-one has been reported in Dendrobium linawianum, Dendrobium chrysanthum, and Dendrobium nobile with data available.

C16H25NO2

263.3752

263.188

C, 72.97; H, 9.57; N, 5.32; O, 12.15

2115-91-5

442523

White to yellow solid powder

1.1±0.1 g/cm3

374.6±25.0 °C at 760 mmHg

135-136℃

132.8±14.0 °C

0.0±0.8 mmHg at 25°C

1.526

Pyrrole Alkaloids from Dendrobium nobile

1.88

0

3

1

19

435

7

O1C([C@@]2([H])[C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])[C@]1([H])[C@]1([H])[C@@]3(C([H])([H])[H])[C@@]([H])(C([H])([H])N1C([H])([H])[H])C([H])([H])C([H])([H])[C@]32[H])=O

RYAHJFGVOCZDEI-UFFNCVEVSA-N

InChI=1S/C16H25NO2/c1-8(2)11-12-10-6-5-9-7-17(4)14(16(9,10)3)13(11)19-15(12)18/h8-14H,5-7H2,1-4H3/t9-,10+,11+,12-,13-,14-,16+/m1/s1

(2aS,2a1R,4aS,5R,8R,8aS,9S)-9-isopropyl-2a1,4-dimethyldecahydro-7H-6-oxa-4-aza-5,8-methanocyclopenta[cd]azulen-7-one

Dendrobine; (–)-Dendrobine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~33.33 mg/mL (~126.55 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.49 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.49 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.49 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)