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Abstract Background Natural products can serve as one of the alternatives, exhibiting high potential for the treatment and prevention of COVID-19, caused by SARS-CoV-2. Herein, we report a screening platform to test the antiviral efficacy of a natural product library against SARS-CoV-2 and verify their activity using lung organoids. Methods Since SARS-CoV-2 is classified as a risk group 3 pathogen, the drug screening assay must be performed in a biosafety level 3 (BSL-3) laboratory. To circumvent this limitation, pseudotyped viruses (PVs) have been developed as replacements for the live SARS-CoV-2. We developed PVs containing spikes from Delta and Omicron variants of SARS-CoV-2 and improved the infection in an angiotensin-converting enzyme 2 (ACE2)-dependent manner. Human induced pluripotent stem cells (hiPSCs) derived lung organoids were generated to test the SARS-CoV-2 therapeutic efficacy of natural products. Results Flavonoids from our natural product library had strong antiviral activity against the Delta- or Omicron-spike-containing PVs without affecting cell viability. We aimed to develop strategies to discover the dual function of either inhibiting infection at the beginning of the infection cycle or reducing spike stability following SARS-CoV-2 infection. When lung cells are already infected with the virus, the active flavonoids induced the degradation of the spike protein and exerted anti-inflammatory effects. Further experiments confirmed that the active flavonoids had strong antiviral activity in lung organoid models. Conclusion This screening platform will open new paths by providing a promising standard system for discovering novel drug leads against SARS-CoV-2 and help develop promising candidates for clinical investigation as potential therapeutics for COVID-19. Graphical Abstract

Background: Withania somnifera (Solanaceae), generally known as Indian ginseng, is a medicinal plant that is used in Ayurvedic practice for promoting health and longevity. This study aims to identify the bioactive metabolites from Indian ginseng and elucidate their structures. Methods: Withanolides were purified by chromatographic techniques, including HPLC coupled with LC/MS. Chemical structures of isolated withanolides were clarified by analyzing the spectroscopic data from 1D and 2D NMR, and HR-ESIMS experiment. Absolute configurations of the withanolides were established by the application of NMR chemical shifts and ECD calculations. Anti-adipogenic activities of isolates were evaluated using 3T3-L1 preadipocytes with Oil Red O staining and quantitative real-time PCR (qPCR). Results: Phytochemical examination of the roots of Indian ginseng afforded to the isolation of six withanolides (1–6), including three novel withanolides, withasilolides G–I (1–3). All the six compounds inhibited adipogenesis and suppressed the enlargement of lipid droplets, compared to those of the control. Additionally, the mRNA expression levels of Fabp4 and Adipsin, the adipocyte markers decreased noticeably following treatment with 25 μM of 1–6. The active compounds (1–6) also promoted lipid metabolism by upregulating the expression of the lipolytic genes HSL and ATGL and downregulating the expression of the lipogenic gene SREBP1. Conclusion: The results of our experimental studies suggest that the withasilolides identified herein have anti-adipogenic potential and can be considered for the development of therapeutic strategies against adipogenesis in obesity. Our study also provides a mechanistic rationale for using Indian ginseng as a potential therapeutic agent against obesity and related metabolic diseases.

Kaempferia parviflora Wall. ex Baker (Zingiberaceae), commonly known as Thai ginseng or black ginger, is a tropical medicinal plant in many regions. It has been traditionally used to treat various ailments, including ulcers, dysentery, gout, allergies, abscesses, and osteoarthritis. As part of our ongoing phytochemical study aimed at discovering bioactive natural products, we investigated potential bioactive methoxyflavones from K. parviflora rhizomes. Phytochemical analysis aided by liquid chromatography–mass spectrometry (LC-MS) led to the isolation of six methoxyflavones (1–6) from the n-hexane fraction of the methanolic extract of K. parviflora rhizomes. The isolated compounds were structurally determined to be 3,7-dimethoxy-5-hydroxyflavone (1), 5-hydroxy-7-methoxyflavone (2), 7,4′-dimethylapigenin (3), 3,5,7-trimethoxyflavone (4), 3,7,4′-trimethylkaempferol (5), and 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (6), based on NMR data and LC-MS analysis. All of the isolated compounds were evaluated for their anti-melanogenic activities. In the activity assay, 7,4′-dimethylapigenin (3) and 3,5,7-trimethoxyflavone (4) significantly inhibited tyrosinase activity and melanin content in IBMX-stimulated B16F10 cells. In addition, structure–activity relationship analysis revealed that the methoxy group at C-5 in methoxyflavones is key to their anti-melanogenic activity. This study experimentally demonstrated that K. parviflora rhizomes are rich in methoxyflavones and can be a valuable natural resource for anti-melanogenic compounds.