Potential natural product 3,4-seco-schitriterpenoids from Kadsura japonica L. as anti-neuroinflammatory agents.

In the present study, the undescribed schitriterpenoids, kadsujanonols A-I (1-9), and eleven reported compounds (10-20) were isolated from K. japonica L. vines. Their structures of 3,4-seco-schitriterpenoids were elucidated mainly by spectroscopic analyses including 1H-, 13C-, and 2D-NMR, IR, HRESIMS spectra. The spatial configurations were determined by the single-crystal X-ray diffraction analysis of kadsujapnonol A (1), 15, 17, and 18, CD data and computational analysis. Furthermore, all isolates were evaluated for the anti-neuroinflammatory activity on LPS-stimulated NO production in BV2 microglial cells and compounds 2, 4, 5, 7, 9, 11, 13-16, and 18 exposed better or comparable suppression abilities than PDTC. Among them, kadlongilactone B (14) showed the best significant inhibiting ability (IC50 = 0.87 µg/mL) and the effect is through the attenuation of the inflammatory transcription factor p65NF-κB. Preliminary structure-activity relationship revealed that δ-lactone at the side chain and 7-member lactone at C-3/C-4, and 3,4:9,10 ring opening are important.

Anti-inflammatory effect of euphane- and tirucallane-type triterpenes isolated from the traditional herb Euphorbia neriifolia L.

The Euphorbiaceae plant Euphorbia neriifolia L. is distributed widely in India, Thailand, Southeastern China, and Taiwan and used as a carminative and expectorant to treat several inflammation-related diseases, such as gonorrhoea, asthma, and cancer. In the course of our search for potential anti-inflammatory agents from the titled plant, 11 triterpenes from the stem of E. neriifolia were isolated and reported in our previous endeavor. Given its rich abundance in triterpenoids, the ethanolic extract in this follow-up exploration has led to the isolation of additional eight triterpenes, including six new euphanes-neritriterpenols H and J-N (1 and 3-7)-one new tirucallane, neritriterpenol I (2), and a known compound, 11-oxo-kansenonol (8). Their chemical structures were elucidated on the basis of spectroscopic data, including 1D- and 2D NMR, and HRESIMS spectra. The absolute stereochemistry of neritriterpenols was determined by single-crystal X-ray diffraction analysis, ICD spectra, and DP4+ NMR data calculations. Compounds 1-8 were also evaluated for their anti-inflammatory activity by using lipopolysaccharide (LPS)-stimulated IL-6 and TNF-α on RAW 264.7 macrophage cells. Intriguingly, the euphane-type triterpenes (1 and 3-8) showed an inhibitory effect on LPS-induced IL-6 but not on TNF-α, while tirucallane-type triterpene 2 showed strong inhibition on both IL-6 and TNF-α.

NRICM101 ameliorates SARS-CoV-2-S1-induced pulmonary injury in K18-hACE2 mice model.

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a challenge for public health globally since transmission of different variants of the virus does not seem to be effectively affected by the current treatments and vaccines. During COVID-19 the outbreak in Taiwan, the patients with mild symptoms were improved after the treatment with NRICM101, a traditional Chinese medicine formula developed by our institute. Here, we investigated the effect and mechanism of action of NRICM101 on improval of COVID-19-induced pulmonary injury using S1 subunit of the SARS-CoV-2 spike protein-induced diffuse alveolar damage (DAD) of hACE2 transgenic mice. The S1 protein induced significant pulmonary injury with the hallmarks of DAD (strong exudation, interstitial and intra-alveolar edema, hyaline membranes, abnormal pneumocyte apoptosis, strong leukocyte infiltration, and cytokine production). NRICM101 effectively reduced all of these hallmarks. We then used next-generation sequencing assays to identify 193 genes that were differentially expressed in the S1+NRICM101 group. Of these, three (Ddit4, Ikbke, Tnfaip3) were significantly represented in the top 30 enriched downregulated gene ontology (GO) terms in the S1+NRICM101 group versus the S1+saline group. These terms included the innate immune response, pattern recognition receptor (PRR), and Toll-like receptor signaling pathways. We found that NRICM101 disrupted the interaction of the spike protein of various SARS-CoV-2 variants with the human ACE2 receptor. It also suppressed the expression of cytokines IL-1ß, IL-6, TNF-α, MIP-1ß, IP-10, and MIP-1α in alveolar macrophages activated by lipopolysaccharide. We conclude that NRICM101 effectively protects against SARS-CoV-2-S1-induced pulmonary injury via modulation of the innate immune response, pattern recognition receptor, and Toll-like receptor signaling pathways to ameliorate DAD.

Novel Aporphine- and Proaporphine-Clerodane Hybrids Identified from the Barks of Taiwanese Polyalthia longifolia (Sonn.) Thwaites var. pendula with Strong Anti-DENV2 Activity.

Hybrid natural products produced via mixed biosynthetic pathways are unique and often surprise one with unexpected medicinal properties in addition to their fascinating structural complexity/diversity. In view of chemical structures, hybridization is a way of diversifying natural products usually through dimerization of two similar or dissimilar subcomponents through a C-C or N-C covalent linkage. Here, we report four structurally attractive diterpene-alkaloid conjugates polyalongarins A-D (1-4), clerodane-containing aporphine and proaporphine alkaloids, the first of its kind from the barks of Taiwanese Polyalthia longifolia (Sonn.) Thwaites var. pendula. In addition to conventional spectroscopic analysis, single crystal X-ray crystallography was employed to determine the chemical structures and stereo-configurations of 1. Compounds 1-4 were subsequently subjected to in vitro antiviral examination against DENV2 by evaluating the expression level of the NS2B protein in DENV2-infected Huh-7 cells. These compounds display encouraging anti-DENV2 activity with superb EC50 (2.8-6.4 µM) and CC50 values (50.4-200 µM). The inhibitory mechanism of 1-4 on NS2B was further explored drawing on in-silico molecular docking analysis. Based on calculated binding affinities and predicted interactions between the functional groups of 1-4 and the allosteric-site residues of the DENV2 NS2B-NS3 protease, our analysis concludes that the clerodane-aporphine/proaporphine-type hybrids are novel and effective DENV NS2B-NS3 protease inhibitors.

Anti-inflammatory sesquiterpene and triterpene acids from Mesona procumbens Hemsley.

Mesona procumbens Hemsley is a plant conventionally processed to provide popular food materials and herbal medicines in Asia. In this study, six triterpene acids, including five new ones (mesonaic acids D-H, 1-5), and one proximadiol-type sesquiterpene (7) were isolated from the methanolic extract of the air-dried M. procumbens. Chemical structures of 1‒7 were established by spectroscopic methods, especially 2D NMR techniques (1H-1H COSY, HSQC, HMBC, and NOESY) and HRESIMS. Concerning their biological activities, compounds 1, 2, 6, and 7 were examined manifesting high inhibition toward the pro-inflammatory NO production with EC50 values ranging from 12.88 to 21.21 µM, outrunning the positive control quercetin (24.12 µM). The mesoeudesmol B (7) identified from M. procumbens is the very first example, which exhibited high anti-inflammatory activity diminishing the level of the lipopolysaccharide-induced NO in RAW264.7 macrophage cells, thereby suppressing the secretion of pro-inflammatory cytokines TNF-α and IL-6 and the level of two critical downstream inflammatory mediators iNOS and COX-2.

Neritriterpenols A-G, euphane and tirucallane triterpenes from Euphorbia neriifolia L. and their bioactivity.

Euphorbia neriifolia L. is widely distributed in India, Thailand, and China and has been used to treat diseases such as rotten sores and asthma as well as for its antidiabetic and anticancer effects. In this study, seven undescribed triterpenes, including six euphanes, neritriterpenols A-B and D-G, and a tirucallane, neritriterpenol C, together with four known triterpenes, were isolated from ethanolic extracts of E. neriifolia stems. Their structures with absolute configurations were determined through detailed spectroscopic data, including 1D and 2D NMR data analyses, single-crystal X-ray diffraction analysis, ECD spectra, and DP4+ NMR data calculations as well as Mo2(OAc)4-induced ECD analysis. Furthermore, preliminarily evaluation of the anti-inflammatory and anti-proliferative effects of the isolated triterpenes leads to the structure-activity relationship (SAR) studies implying that the unsaturated functional group at the end of the C17 side chain on euphane-type triterpenes may be correlated with the increase of anti-inflammatory and anti-proliferative activities.

New Diterpenoids from Mesona procumbens with Antiproliferative Activities Modulate Cell Cycle Arrest and Apoptosis in Human Leukemia Cancer Cells.

Mesona procumbens is a popular material used in foods and herbal medicines in Asia for clearing heat and resolving toxins. However, phytochemical research on this plant is very rare. In this study, eleven new diterpenoids, mesonols A-K (1-11), comprising seven ent-kauranes, three ent-atisanes, and one sarcopetalane, were isolated from its methanolic extract. Structural elucidation of compounds 1-11 was performed by spectroscopic methods, especially 2D NMR, HRESIMS, and X-ray crystallographic analysis. All isolates were assessed for their antiproliferative activity, and compounds 1-4 showed potential antiproliferative activities against A549, Hep-3B, PC-3, HT29, and U937 cancer cells, with IC50 values ranging from 1.97 to 19.86 µM. The most active compounds, 1 and 2, were selected for further investigation of their effects on cell cycle progression, apoptosis, and ROS generation in U937 human leukemia cancer cells. Interestingly, it was found that compounds 1 and 2 induced antiproliferative effects in U937 cells through different mechanisms. Compound 1 caused cell cycle arrest at the G2/M phase and subsequent cell death in a dose- and time-dependent manner. However, 2-mediated antiproliferation of U937 cells triggered ROS-mediated mitochondrial-dependent apoptosis. These results provide insight into the molecular mechanism involved in the antiproliferative activities of compounds 1 and 2 in U937 cells. Altogether, the study showed that new diterpenoid compounds 1 and 2 from M. procumbens are potent and promising anticancer agents.

Blockage of glutamine-dependent anaplerosis affects mTORC1/2 activity and ultimately leads to cellular senescence-like response.

The purpose of study was to explore the role of glutamine-dependent anaplerosis in cell fate determination (proliferation and senescence) and the potential associated mechanism by employing a pharmacological inhibitor of glutamine-dependent anaplerosis, amino-oxyacetate (AOA). Using the WI38 normal human embryonic fibroblast cell line, we found that exposure to AOA induced mTORC1 inactivation-mTORC2 activation (within day 1), cell cycle arrest (day 2-6) and cellular senescence (day 4-6). These AOA effects were blocked by concomitantly providing anaplerotic factors [α-ketoglutarate (αKG), pyruvate or oxaloacetate], and not affected by ROS scavenger N-acetyl-cysteine (NAC). Moreover, AOA-induced cellular senescence in WI38 cells is associated with elevated protein levels of p53, p21CIP1 and p16INK4A and decreased Rb protein level, which was blocked by αKG supplementation. In p16INK4A-deficient U2OS human osteosarcoma cells and p16INK4A-knockdown WI38 cells, AOA exposure also induced similar effects on cell proliferation, and protein level of P-Rb-S807/811 and Rb. Interestingly, no AOA induction of cellular senescence was observed in U2OS cells, yet was still seen in p16INK4A-knockdown WI38 cells accompanied by the presence of p16 antibody-reactive p12. In summary, we disclose that glutamine-dependent anaplerosis is essential to cell growth and closely associated with mTORC1 activation and mTORC2 inactivation, and impedes cellular senescence particularly associated with p16INK4A.