The first phytochemical investigation of Artemisia nujianensis: Eight new guaianolides and their anti-inflammatory activity.

The first phytochemical investigation of Artemisia nujianensis resulted in the isolation of eight new guaianolides (1-8) and six known analogs. Their structures were determined by extensive analysis of 1D and 2D NMR data, HRESIMS data, DFT NMR calculations, and X-ray diffraction studies. Some compounds were evaluated for their anti-inflammatory activities in LPS-stimulated RAW 264.7 cells. Compounds 5, 7 and 9 showed moderate inhibitory effects on LPS-induced NO production in RAW 264.7 cells, with IC50 values of 12.50 ± 0.21, 9.53 ± 0.14 and 6.85 ± 0.11 µM, respectively.

Secondary Metabolites and Their Cytotoxic Activity of Artemisia nitrosa Weber. and Artemisia marschalliana Spreng.

As a promising source of biologically active substances, the Artemisia species from Kazakhstan have not been investigated efficiently. Considering the rich history, medicinal values, and availability of the Artemisia plants, systematic investigations of two Artemisia species growing in the East Kazakhstan region were conducted. In this study, one new germacrane-type sesquiterpene lactone (11), together with 10 known sesquiterpenes and its dimer, were characterized from A. nitrosa Weber. Additionally, one new chromene derivative (1') with another 12 known compounds, including coumarins, sesquiterpene diketones, phenyl propanoids, polyacetylenics, dihydroxycinnamic acid derivatives, fatty acids, naphthalene derivatives, flavones, and caffeic acid derivatives were isolated from A. marschalliana Spreng. All compounds were isolated and identified for the first time from these two Artemisia species. The structures of new compounds (11, 1') were established by using UV, TOFMS, LC-MS, 1D and 2D NMR spectroscopic analyses. The cytotoxicity of all isolated compounds was evaluated. As a result, all compounds did not show significant inhibition against HL-60 and A-549 cell lines. The sesquiterpenoids isolated from A. nitrosa were tested for their inhibitory activity against the LPS-induced NO release from the RAW624.7 cells, and neither of them exhibited significant activity.

Anti-inflammatory sesquiterpenoid dimers from Artemisia atrovirens.

Eight new sesquiterpenoid dimers, artatrovirenolides A-H (1-8), along with three known analogues (9-11), were isolated from Artemisia atrovirens by using the LC-MS guided isolation. Compound 1 was a compound dimerized from a guaianolide and a 1,10-seco-guaianolide unit while others were from two guaianolide units. Their structures were established by comprehensive analysis of spectroscopic data, and their absolute configurations were determined by the aid of time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculation. Compound 8 showed anti-inflammatory effect in LPS-stimulated BV-2 microglial cells at 1 µM, while compounds 1, 2, 5, and 6 inhibited microglial inflammation at 10 µM.

Ten undescribed cadinane-type sesquiterpenoids from Eupatorium chinense.

Ten undescribed cadinane-type sesquiterpenes (1-10) were isolated from the whole plant of Eupatorium chinense. Their planar structures were mainly elucidated by extensive analysis of spectroscopic data and DFT NMR calculations. The absolute configurations of 1, 2, and 3 were determined by TDDFT ECD calculations while those of compounds 4-7 and 9 were confirmed by single crystal X-ray diffraction experiments. Compounds 2 and 3 are a pair of C-10 epimers, compounds 4 and 5 a pair of C-1 epimers, and compounds 9 and 10 a pair of compounds isomerized at both C-1 and C-10. A possible biosynthetic pathway for these new sesquiterpenes was proposed.

Three new carabrane sesquiterpenoid derivatives from the whole plant of Carpesium abrotanoides L.

Dicarabrols B and C (1 and 2), two new carabrane sesquiterpenoid dimers, along with one new carabrane sesquiterpenoid (3) were isolated from the whole plant of Carpesium abrotanoides L. Their full structures were established by extensive analysis of HR-ESI-MS and NMR spectroscopic data, and time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. Dicarabrol B possesses a novel C30 skeleton featuring a methylene-tethered bridge between two sesquiterpene moieties, while dicarabrol C presents the unique linkage of a cyclopentane ring in the molecule. Dicarabrol C exhibited potent inhibitory effects on HL-60 cells with an IC50 value of 3.7 µmol·L-1.

Guaianolides from Artemisia codonocephala suppress interleukine-1ß secretion in macrophages.

Sesquiterpene lactones supply a variety of scaffolds for the development of anti-inflammatory drugs. In this study, eight undescribed guaianolides, i.e., lavandolides A‒H, were isolated from the whole plants of Artemisia codonocephala, together with five known analogues. Their planar structures and relative configurations were elucidated by spectroscopic measurements, and their absolute configurations were determined by electronic circulardichroism spectra and single crystal X-ray diffraction experiments. The nitric oxide inhibitory effect of all the isolates was assessed on lipopolysaccharide stimulated THP-1 macrophages. Lavandolide D showed a potent inhibitory effect on NO production, with IC50 values of 3.31 ± 0.74 µM. Furthermore, lavandolide D inhibited NOD-, LRR- and pyrin domain-containing protein 3 inflammasome-mediated interleukin-1ß production through activating autophagy.

3, 4-seco-Isopimarane and 3, 4-seco-pimarane diterpenoids from Callicarpa nudiflora.

A phytochemical investigation was carried out on the extract of a medicinal plant Callicarpa nudiflora, resulting in the characterization of five new 3, 4-seco-isopimarane (1-5) and one new 3, 4-seco-pimarane diterpenoid (6), together with four known compounds. The structures of the new compounds were fully elucidated by extensive analysis of MS, 1D and 2D NMR spectroscopic data, and time-dependent density functional theory (TDDFT) calculation of electronic circular dichroism (ECD) spectra, and DFT calculations for NMR chemical shifts and optical rotations.

Noreudesmane sesquiterpenoids from Artemisia hedinii and their anti-inflammatory activities.

Ten undescribed noreudesmane-type sesquiterpenoids, including eight 12,13-dinoreudesmanes and a pair of 11,12,13-trinoreudesmane epimers were isolated from the whole plant of Artemisia hedinii. Their structures were elucidated by extensive analysis of spectroscopic data, including MS, 1D and 2D NMR, and their absolute configurations were confirmed by X-ray diffraction experiments and DFT calculations. Compounds 1-5, 7-10 were evaluated for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated murine macrophages RAW264.7 cells, and all of them could significantly inhibit the LPS induced CCL2 mRNA expression in a dose-dependent manner.

Anti-inflammatory Eudesmane Sesquiterpenoids from Artemisia hedinii.

Fourteen new eudesmane sesquiterpenoids (1, 3-5, 7-16) and seven known analogues were isolated from the whole plant of Artemisia hedinii. Their structures were elucidated by spectroscopic data analysis and comparison with published NMR data, and their absolute configurations were confirmed by X-ray diffraction experiments and TDDFT ECD calculation. Compounds 1-15 were identified as eudesmane acids, which represent a kind of lactone ring-opening eudesmane-type sesquiterpenes with an acetoxyl or a hydroxy group attached to C-9. Compounds 1 and 2, 5 and 6, and 7 and 8 are three pairs of epimers isomerized at C-3, C-5, and C-11, respectively. Compounds 1-9, 11-13, 15-19, and 21 could influence the proinflammatory phenotype of the M1 macrophage. Among them, compounds 5, 8, 9, 12, 16, and 19 consistently exhibited anti-inflammatory effects, as evidenced by downregulating classic pro-inflammatory cytokines TNF-α, IL-12, IL-6, and IFN-γ in LPS-induced primary bone marrow derived M1 macrophages.

Dimeric 9,10-dihydrophenanthrene derivatives from Bletilla striata and their atropisomeric nature.

Four pairs of undescribed racemic bi(9,10-dihydro) phenanthrene and phenanthrene/bibenzyl atropisomers, bletistriatins A-D (1-4), along with 22 known compounds were isolated from the rhizomes of Bletilla striata. These dimeric derivatives were constructed through direct C-C connection or an oxygen bridge. The structures of new compounds were fully established by extensive analysis of MS, and 1D and 2D NMR spectroscopic data. Owing to sterically hindered rotation around the biaryl axis, these dimeric 9,10-dihydrophenanthrene derivatives can exist as a pair of enantiomers, but were isolated as racemates. Their racemates were separated to yield enantiomerically pure compounds by HPLC on an optically active stationary phase, and were stereochemically characterized on-line by circular dichroism (CD) spectroscopy (LC-CD coupling). Some isolates were evaluated for cytotoxicity against human cancer cell lines HL-60 and A549. Compounds 13, 17, and 20 showed cytotoxicity against HL-60 and A-549 cell lines with IC50 values ranging from 2.56 to 8.67 µM.

Cytotoxic guaianolides and seco-guaianolides from Artemisia atrovirens.

A phytochemical investigation of a medicinal plant Artemisia atrovirens was carried out, resulting in the characterization of a novel bis-nor seco-guaianolide, seco-atrovirenolide A (1), a new 1,10-seco-guaianolide derivative, seco-atrovirenoic acid A (2), and a new artifact 10-methanoyloxy-seco-atrovirenoic acid A (3), together with eight known guaianolide and seco-guaianolide derivatives (4-11). The structures of new compounds were fully established by extensive analysis of MS, 1D and 2D NMR spectroscopic data. The absolute configurations of the isolated compounds were confirmed by TDDFT ECD calculation, Mosher's method, and X-ray crystal diffraction experiment. All the compounds were tested in vitro for their cytotoxicity against HL-60 and A549 cell lines. Some of them showed moderate inhibitory activity against HL-60 cell lines with IC50 values ranging from 5.99 to 11.74 µM.

Sesquiterpene lactone dimers from Artemisia lavandulifolia inhibit interleukin-1ß production in macrophages through activating autophagy.

Twelve new sesquiterpene lactone dimers, lavandiolides A-L (1-12), were isolated from the whole plants of Artemisia lavandulifolia. Among them, compounds 1-6 are 1,3-linked Diels-Alder adducts between two guaianolide monomers, and 7-12 are 2,4-linked sesquiterpene lactone dimers. Their structures were elucidated by comprehensive analysis of HRESIMS, 1D and 2D NMR spectra. Their absolute configurations were determined by ECD spectra and single-crystal X-ray diffraction analyses with Cu Kα radiation. The nitric oxide (NO) inhibitory effect of all the isolates was assessed on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Compounds 1, 3, 7 and 9 showed potent inhibitory effects on NO production, with IC50 values of 0.61 ± 0.15, 1.64 ± 0.04, 1.89 ± 0.16, and 1.40 ± 0.23 µM, respectively. Furthermore, compound 1 inhibited NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome-mediated interleukin-1ß (IL-1ß) production through activating autophagy.

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients.

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

Phosphocreatine attenuates Gynura segetum-induced hepatocyte apoptosis via a SIRT3-SOD2-mitochondrial reactive oxygen species pathway.

Purpose: To investigate the mitochondria-related mechanism of Gynura segetum (GS)-induced apoptosis and the protective effect of phosphocreatine (PCr), a mitochondrial respiration regulator. Methods: First, the mechanism was explored in human hepatocyte cell line. The mitochondrial oxidative stress was determined by fluorescence assay. The level of sirtuin 3 (SIRT3), acetylated superoxide dismutase 2 (Ac-SOD2), SOD2, and apoptosis were detected by Western blotting. Mito-TEMPO and cell lines of viral vector-mediated overexpression of SIRT3 and SIRT3H248Y were used to further verify the mechanism of GS-induced apoptosis. GS-induced liver injury mice models were built by GS through intragastric administration and interfered by PCr through intraperitoneal injection. A total of 30 C57BL/6J mice were assigned to 5 groups and treated with either saline, PCr (100 mg/kg), GS (30 g/kg), or PCr (50 or 100 mg/kg)+GS (30 g/kg). Liver hematoxylin and eosin (HE) staining, immunohistochemical analysis, and blood biochemical evaluation were performed. Results: GS induced hepatocyte apoptosis and elevated levels of mitochondrial ROS in L-02 cells. The expression of SIRT3 was decreased. Downregulation of SIRT3 was associated with increased levels of Ac-SOD2, which is the inactivated enzymatic form of SOD2. Conversely, when overexpressing SIRT3 in GS-treated cells, SOD2 activity was restored, and mitochondrial ROS levels and hepatocyte apoptosis declined. Upon administration of PCr to GS-treated cells, they exhibited a significant upregulation of SIRT3 and were protected against apoptosis. In animal experiments, serum ALT level and mitochondrial ROS of the mice treated with GS and 50 mg/kg PCr were significantly attenuated compared with only GS treated. The changes in SIRT3 expression were also consistent with the in vitro results. In addition, immunohistochemical analysis of the mouse liver showed that Ac-SOD2 was decreased in the PCr and GS co-treated group compared with GS treated group. Conclusion: GS caused liver injury by dysregulating mitochondrial ROS generation via a SIRT3-SOD2 pathway. PCr is a potential agent to treat GS-induced liver injury by mitochondrial protection.

Pretreatment with broad-spectrum antibiotics alters the pharmacokinetics of major constituents of Shaoyao-Gancao decoction in rats after oral administration.

The influence of broad-spectrum antibiotics on the pharmacokinetics and biotransformation of major constituents of Shaoyao-Gancao decoction (SGD) in rats was investigated. The pharmacokinetic behaviors of paeoniflorin (PF), albiflorin (AF), liquiritin (LT), isoliquiritin (ILT), liquiritin apioside (LA), isoliquiritin apioside (ILA), and glycyrrhizic acid (GL), seven major constituents of SGD, as well as glycyrrhetinic acid (GA), a major metabolite of GL, were analyzed. A 1-week pretreatment with broad-spectrum antibiotics (ampicillin, metronidazole, neomycin, 1 g L-1; and vancomycin, 0.5 g L-1) via drinking water reduced plasma exposure of the major constituents. The AUC0-24 h of PF and LT was significantly decreased by 28.7% and 33.8% (P < 0.05 and P < 0.005), respectively. Although the differences were not statistically significant, the AUC0-24 h of AF, ILT, LA, ILA, and GL was decreased by 31.4%, 50.9%, 16.9%, 44.1%, and 37.0%, respectively, compared with the control group. In addition, the plasma GA exposure in the antibiotic-pretreated group was significantly lower (P < 0.005) than the control group. The in vitro stability of the major constituents of SGD in the rat intestinal contents with or without broad-spectrum antibiotics was also investigated. The major constituents were comparatively stable in the rat duodenum contents, and the biotransformation of GL mainly occurred in the rat colon contents. In summary, broad-spectrum antibiotics suppressed the absorption of the major constituents of SGD and significantly inhibited the biotransformation of GL to GA by suppressing the colon microbiota. The results indicated a potential clinical drug-drug interaction (DDI) when SGD was administered with broad-spectrum antibiotics.

Ainsliatriolides A and B, Two Guaianolide Trimers from Ainsliaea fragrans and Their Cytotoxic Activities.

Ainsliatriolides A (1) and B (2), two guaianolide sesquiterpenoid trimers possessing an unprecedented skeleton, were isolated from Ainsliaea fragrans. Their structures were elucidated through extensive analysis of spectroscopic data and confirmed by single-crystal X-ray diffraction experiment. Ainsliatriolides A and B are first examples of compound trimerized from guaianolide sesquiterpenoids through two different C-C linkages (type A, 4-2'/15-14'; type B, 15'-15″). Ainsliatriolide A displayed potent cytotoxicity with an averaged IC50 value of 1.17 µM against four cancer cells.

7α,8α-Epoxynagilactones and their glucosides from the twigs of Podocarpus nagi: Isolation, structures, and cytotoxic activities.

A phytochemical investigation of twigs of Podocarpus nagi resulted in the identification of eight new type B nagilactones (1-8), all bearing a 7α,8α-epoxy-9(11)-enolide substructure, along with two known analogs (9-10). Their structures were determined on the basis of spectroscopic analysis, including HRESIMS, IR and NMR experiments, and X-ray crystallographic analysis. In vitro cytotoxic assay exhibited that compounds 1, 2, 9 and 10 could induce antiproliferation against three different types of human cancer cells while compounds 3 and 5 were inactive. Notably, the IC50 value of compound 1 is 0.208µM for A431 human epidermoid carcinoma cells, reaching the same level as the positive control combretastatin A-4 (0.104µM). Furthermore, compound 1 performed a strong inhibition of cancer cells by triggering apoptosis and arresting the cell cycle at G1 phase. These results unfold potential anticancer therapeutic applications of type B nagilactones.

ent-Labdane diterpenes from the stems of Mallotus japonicus.

Eight new ent-labdane diterpenoids, mallonicusins A-H (1-8), were isolated from the stems of Mallotus japonicus. Their structures, including the absolute configurations, were determined by extensive analyses of spectroscopic data and the ECD spectra of the Pr(FOD)3 complex of substrates in CCl4. The absolute configuration of compound 1 was confirmed by single-crystal X-ray crystallography using Cu Kα radiation.

Limonoids from the fruits of Melia toosendan.

Fifteen limonoids, meliatoosenins E-S (1-15), and 10 known compounds were isolated from the fruits of Melia toosendan. Their structures were elucidated on the basis of extensive spectroscopic methods including DEPT, HSQC, HMBC, (1)H-(1)H COSY, and ROESY experiments. All the compounds were evaluated for antiproliferative activity using A-549 and HL-60 cell lines.

Isoflavonoid glycosides from the flowers of Pueraria lobata.

Pueraria lobata flower is a medicinal herb for treating intoxication, hepatic, and gastrointestinal tract lesions induced by alcohol. This study aims to investigate the isoflavonoid glycosides in P. lobata flowers. Two new isoflavone compounds were isolated from the extract of P. lobata flowers. Their structures were determined to be 5,6,7,4'-tetrahydroxyisoflavone-6,7-di-O-ß-D-glucopyranoside and 5,6,7-trihydroxy-4'-methoxyisoflavone-6,7-di-O-ß-D-glucopyranoside on the basis of spectroscopic means including HR-ESI-MS, UV, IR, ¹H, and ¹³C NMR.