Methoxyflavones from Black Ginger (Kaempferia parviflora Wall. ex Baker) and their Inhibitory Effect on Melanogenesis in B16F10 Mouse Melanoma Cells.

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.

Development of a screening platform to discover natural products active against SARS-CoV-2 infection using lung organoid models.

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.

Identification of anti-adipogenic withanolides from the roots of Indian ginseng (Withania somnifera).

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.

Withasomniferol D, a New Anti-Adipogenic Withanolide from the Roots of Ashwagandha (Withania somnifera).

Withania somnifera (Solanaceae), well-known as 'Indian ginseng' or 'Ashwagandha', is a medicinal plant that is used in Ayurvedic practice to promote good health and longevity. As part of an ongoing investigation for bioactive natural products with novel structures, we performed a phytochemical examination of the roots of W. somnifera employed with liquid chromatography-mass spectrometry (LC/MS)-based analysis. The chemical analysis of the methanol extract of W. somnifera roots using repeated column chromatography and high-performance liquid chromatography under the guidance of an LC/MS-based analysis resulted in a new withanolide, withasomniferol D (1). The structure of the newly isolated compound was elucidated by spectroscopic methods, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and high-resolution (HR) electrospray ionization (ESI) mass spectroscopy, and its absolute configuration was established by electronic circular dichroism (ECD) calculations. The anti-adipogenic activities of withasomniferol D (1) were evaluated using 3T3-L1 preadipocytes with Oil Red O staining and quantitative real-time polymerase chain reaction (qPCR). We found that withasomniferol D (1) inhibited adipogenesis and suppressed the enlargement of lipid droplets compared to the control. Additionally, the mRNA expression levels of adipocyte markers Fabp4 and Adipsin decreased noticeably following treatment with 25 µM of withasomniferol D (1). Taken together, these findings provide experimental evidence that withasomniferol D (1), isolated from W. somnifera, exhibits anti-adipogenic activity, supporting the potential application of this compound in the treatment of obesity and related metabolic diseases.

Ergopyrone, a Styrylpyrone-Fused Steroid with a Hexacyclic 6/5/6/6/6/5 Skeleton from a Mushroom Gymnopilus orientispectabilis.

A styrylpyrone-fused ergosterol derivative, ergopyrone (1), was isolated and structurally characterized from a mushroom, Gymnopilus orientispectabilis, along with five biosynthetically related metabolites (2-6). Compound 1 features an unprecedented hexacyclic 6/5/6/6/6/5 skeleton that would be formed from ergosterol and styrylpyrone precursors via [3 + 2] cycloaddition. The chemical structure of 1 was elucidated by conventional spectroscopic and spectrometric data analysis coupled with computational methods including DP4+ probability and ECD simulation and an NOE/ROE-based interproton distance measurement technique via peak amplitude normalization for the improved cross-relaxation (PANIC) method. Plausible biosynthetic pathways of 1-6 are proposed, and compound 6 significantly regulated lipid metabolism in adipocytes through the upregulation of the mRNA expression of Adipsin, Fabp4, SREBP1, and ATGL.

Detection of proximal caries using quantitative light-induced fluorescence-digital and laser fluorescence: a comparative study.

PURPOSE: The purpose of this study was to evaluate the in vitro validity of quantitative light-induced fluorescence-digital (QLF-D) and laser fluorescence (DIAGNOdent) for assessing proximal caries in extracted premolars, using digital radiography as reference method. MATERIALS AND METHODS: A total of 102 extracted premolars with similar lengths and shapes were used. A single operator conducted all the examinations using three different detection methods (bitewing radiography, QLF-D, and DIAGNOdent). The bitewing x-ray scale, QLF-D fluorescence loss (ΔF), and DIAGNOdent peak readings were compared and statistically analyzed. RESULTS: Each method showed an excellent reliability. The correlation coefficient between bitewing radiography and QLF-D, DIAGNOdent were -0.644 and 0.448, respectively, while the value between QLF-D and DIAGNOdent was -0.382. The kappa statistics for bitewing radiography and QLF-D had a higher diagnosis consensus than those for bitewing radiography and DIAGNOdent. The QLF-D was moderately to highly accurate (AUC = 0.753 - 0.908), while DIAGNOdent was moderately to less accurate (AUC = 0.622 - 0.784). All detection methods showed statistically significant correlation and high correlation between the bitewing radiography and QLF-D. CONCLUSION: QLF-D was found to be a valid and reliable alternative diagnostic method to digital bitewing radiography for in vitro detection of proximal caries.

Comparative analysis of serum proteomes to discover biomarkers for ossification of the posterior longitudinal ligament.

STUDY DESIGN: Serum proteomes from normal subjects and the patients with ossification of the posterior longitudinal ligament (OPLL) were analyzed by using proteomics. OBJECTIVES: To identify novel serologic biomarkers for diagnosing OPLL. SUMMARY OF BACKGROUND DATA: OPLL can compress the spinal cord, and special planning is required for surgeries that are done from the front of the cervical spine. However, the definitive serologic biomarkers for OPLL are still unclear. METHODS: The 2-dimensional electrophoresis patterns of sera from OPLL patients and normal subjects were compared. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization quadruple time-of-flight mass spectrometry. RESULTS: Nine spots that were differentially expressed in the sera of OPLL patients were found and were identified. PRO2675, human serum albumin in a complex with myristic acid and tri-iodobenzoic acid, an unknown protein, chain B of the crystal structure of deoxy-human hemoglobin beta6, pro-apolipoprotein, ALB protein, retinol binding protein and chain A of human serum albumin mutant R218h complexed with thyroxine (3,3',5,5', tetraiodo-L-thyronine), were up-regulated in the sera of OPLL patients, whereas alpha1-microglobulin/bikunin precursor was down-regulated. CONCLUSIONS: These proteins could be used as diagnostic biomarkers of OPLL.

The comparative analysis of serum proteomes for the discovery of biomarkers for acute myeloid leukemia.

OBJECTIVE: Acute myeloid leukemia (AML) develops as the consequence of a series of genetic changes in a hematopoietic precursor cell. However, the definitive diagnostic protein biomarkers for AML are still unclear. In our study to identify the biomarkers for an initial diagnosis, detection of relapse, and monitoring the minimal residual disease in AML by a less invasive method, serum proteins reflecting alterations in their proteomes were analyzed. MATERIALS AND METHODS: We compared the two-dimensional electrophoresis patterns of human sera of 12 patients with AML with those of 12 normal subjects. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization quadupole time-of-flight mass spectrometries. RESULTS: Eight proteins that expressed differentially in the AML group were found. The expression levels of alpha-2-HS-glycoprotein, complement-associated protein SP-40, 40, RBP4 gene product, lipoprotein C-III, and an unknown protein were downregulated in serum of AML patients, whereas the other three proteins, including immunoglobulin heavy-chain variant, proteosome 26S ATPase subunit 1, and haptoglobin-1 were upregulated. CONCLUSION: These results suggest that these proteins can be used as less invasive diagnostic and monitoring biomarkers of AML if further studies are done.