Phytochemical and biological studies on rare and endangered plants endemic to China. Part XXXVI. Tsugaforrestiacids A-O: Structurally diverse C-18 carboxylated diterpenoids from the twigs and needles of the 'vulnerable' conifer Tsuga forrestii and their inhibitory effects on ATP-citrate lyase.

An extensive phytochemical investigation on the EtOAc-soluble fraction of the 90% MeOH extract from the twigs and needles of the 'vulnerable' Chinese endemic conifer Tsuga forrestii (Forrest's hemlock) led to the isolation and characterization of 50 structurally diverse diterpenoids, including 15 unreported C-18 carboxylated ones (tsugaforrestiacids A-O, 1-15, resp.). Among them, compounds 1-7 are abieten-18-oic acids, compound 8 is an abieten-18-succinate, and compounds 10-12 are podocarpen-18-oic acids, whereas compounds 13-15 are pimarane-type, isopimarane-type, and totarane-type diterpenoid acids, respectively. Their structures and absolute configurations were determined by a combination of spectroscopic methods, GIAO NMR calculations and DP4+ probability analyses, electronic circular dichroism (ECD) data, and single crystal X-ray diffraction analyses. All the isolates were evaluated for their inhibitory activities against the ATP-citrate lyase (ACL), a key enzyme in cellular metabolism. Tsugaforrestiacids E (5) and H (8) were found to have significant inhibitory effects against ACL, with IC50 values of 5.3 and 6.2 µM, respectively. The interactions of the bioactive molecules with the ACL enzyme were examined by molecular docking studies. The isolated diterpenoids also provide chemotaxonomic evidence to support the delimitation of Tsuga from its closest sister group (Nothotsuga). The above findings highlight the importance of protecting plant species with unique and diverse secondary metabolites, which may be potential sources of new therapeutic agents for the treating ACL-associated diseases.

Undescribed polyketides from endophytes associated with the critically endangered conifer Abies beshanzuensis.

Four undescribed polyketides, beshanzones A (1) and B (2) as well as beshanhexanols A (3) and B (4), along with three known ones (5-7) were isolated from the rice fermentation of two endophytic fungi associated with the critically endangered Chinese endemic conifer Abies beshanzuensis. γ-Butyrolactone derivatives 1, 2, and 5 were isolated from Phomopsis sp. BSZ-AZ-2, an interesting strain that drawn our attention this time. The cyclohexanol derivatives 3, 4, 6, and 7 were obtained during a follow-up investigation on Penicillium commune BSZ-P-4-1. The chemical structures including absolute configurations of compounds 1-4 were determined by spectroscopic methods, Mo2(OAc)4 induced electronic circular dichroism (IECD), GIAO NMR calculations and DP4+ probability analyses. In particular, compound 2 contains a novel 5/5 bicyclic ring system, which might be biogenetically derived from the known compound 5 through hydrolysis followed by an Aldol reaction. All isolates were evaluated for their antimicrobial activities against a small panel of bacterial and fungal pathogens. Compounds 6 and 7 showed moderate inhibitory activities against Candida albicans, with MIC values of 16 and 32 µg/mL, respectively.

Pentacyclic triterpenoids as potential ACL inhibitors from the rare medicinal plant Semiliquidambar cathayensis.

An extensive phytochemical investigation on the rare medicinal plant Semiliquidambar cathayensis (family: Hamamelidaceae) led to the isolation of four new (1-4, named semiliquidacids A-D, respectively) and 25 related known pentacyclic triterpenoids. The new structures with absolute configurations were elucidated by spectroscopic methods, electronic circular dichroism (ECD) calculations, and single-crystal X-ray diffraction analysis. Compound 1 represents the first naturally occurring ursane-type triterpenoid featuring an uncommon C-25 formyl group. Compound 4 and oleanolic acid (13) exhibited remarkable inhibitory effects against the ATP-citrate lyase (ACL, an emerging drug target for hyperlipidemia and related metabolic disorders) with IC50 values of 6.5 and 11.9 µM, respectively. The molecular interaction and binding mode between the bioactive triterpenoids and ACL were elaborated by conducting a molecular docking study. Meanwhile, the chemotaxonomic significance of the isolated triterpenoids has been briefly discussed.

A novel fluorescence probe for simultaneous detection of mitochondrial viscosity in hepatic ischemia reperfusion injury models.

Acute liver failure caused by hepatic ischemia reperfusion injury (HIRI) poses a severe threat to life, emphasizing the urgent need for precise and timely early diagnosis. Viscosity, a key parameter reflecting active analyte levels at the cellular level, remains underexplored in relation to HIRI. To address this gap, we have developed a groundbreaking near-infrared molecule rotator, PN, exhibiting exceptional characteristics. PN demonstrates remarkable sensitivity, with a 32-fold change in response to viscosity, ranging from PBS to glycerol solution. PN's distinctive features include maximum emission wavelength 790 nm, as well as an impressive Stokes shift 190 nm. Moreover, PN exhibits the ability to sensitively and selectively differentiate nystatin-induced viscosity changes within living cells, and can be used for the detection of viscosity changes in the HIRI mouse model. This capability enhances our understanding of cellular responses, opening avenues for potential applications within disease models. The versatility of PN extends to its potential role in guiding timely monitoring and imaging of viscosity, offering valuable insights into disease progression.

Staphylococcus aureus biofilm: Formulation, regulatory, and emerging natural products-derived therapeutics.

Staphylococcus aureus can readily form biofilm which enhances the drug-resistance, resulting in life-threatening infections involving different organs. Biofilm formation occurs due to a series of developmental events including bacterial adhesion, aggregation, biofilm maturation, and dispersion, which are controlled by multiple regulatory systems. Rapidly increasing research and development outcomes on natural products targeting S. aureus biofilm formation and/or regulation led to an emergent application of active phytochemicals and combinations. This review aimed at providing an in-depth understanding of biofilm formation and regulation mechanisms for S. aureus, outlining the most important antibiofilm strategies and potential targets of natural products, and summarizing the latest progress in combating S. aureus biofilm with plant-derived natural products. These findings provided further evidence for novel antibiofilm drugs research and clinical therapies.

Carbopol 940-based hydrogels loading synergistic combination of quercetin and luteolin from the herb Euphorbia humifusa to promote Staphylococcus aureus infected wound healing.

With the increasing prevalence of Staphylococcus aureus infections, rapid emergence of drug resistance and the slow healing of infected wounds, developing an efficient antibiotic-free multifunctional wound dressing for inhibiting S. aureus and simultaneously facilitating wound healing have become a huge challenge. Due to their excellent biocompatibility and biodegradability, some carbopol hydrogels based on plant extracts or purified compounds have already been applied in wound healing treatment. In China, Euphorbia humifusa Willd. (EuH) has been traditionally used as a medicine and food homologous medicine for the treatment of furuncles and carbuncles mainly caused by S. aureus infection. In an earlier study, EuH-originated flavonoids quercetin (QU) and luteolin (LU) could serve as a potential source for anti-S. aureus drug discovery when used in synergy. However, the in vivo effects of QU and LU on S. aureus-infected wound healing are still unknown. In this study, we found a series of Carbopol 940-based hydrogels loading QU and LU in combination could disinfect S. aureus and also could promote wound healing. In the full-thickness skin defect mouse model infected with S. aureus, the wound contraction ratio, bacterial burden, skin hyperplasia and inflammation score, as well as collagen deposition and blood vessels were then investigated. The results indicate that the optimized QL2 [QU (32 µg mL-1)-LU (8 µg mL-1)] hydrogel with biocompatibility significantly promoted S. aureus-infected wound healing through anti-infection, anti-inflammation, collagen deposition, and angiogenesis, revealing it as a promising alternative for infected wound repair.

Platanosides from Platanus × acerifolia: New molecules, SAR, and target validation of a strong lead for drug-resistant bacterial infections and the associated sepsis.

Three undescribed (1-3) and nine known (4-12) platanosides were isolated and characterized from a bioactive extract of the May leaves of Platanus × acerifolia that initially showed inhibition against Staphylococcus aureus. Targeted compound mining was guided by an LC-MS/MS-based molecular ion networking (MoIN) strategy combined with conventional isolation procedures from a unique geographic location. The novel structures were mainly determined by 2D NMR and computational (NMR/ECD calculations) methods. Compound 1 is a rare acylated kaempferol rhamnoside possessing a truxinate unit. 6 (Z,E-platanoside) and 7 (E,E-platanoside) were confirmed to have remarkable inhibitory effects against both methicillin-resistant S. aureus (MIC: ≤ 16 µg/mL) and glycopeptide-resistant Enterococcus faecium (MIC: ≤ 1 µg/mL). These platanosides were subjected to docking analyses against FabI (enoyl-ACP reductase) and PBP1/2 (penicillin binding protein), both of which are pivotal enzymes governing bacterial growth but not found in the human host. The results showed that 6 and 7 displayed superior binding affinities towards FabI and PBP2. Moreover, surface plasmon resonance studies on the interaction of 1/7 and FabI revealed that 7 has a higher affinity (KD = 1.72 µM), which further supports the above in vitro data and is thus expected to be a novel anti-antibacterial drug lead.

Terricoxanthones A-E, unprecedented dihydropyran-containing dimeric xanthones from the endophytic fungus Neurospora terricola HDF-Br-2 associated with the vulnerable conifer Pseudotsuga gaussenii.

An investigation on the secondary metabolites from a rice culture broth of the endophytic fungus Neurospora terricola HDF-Br-2 derived from the vulnerable conifer Pseudotsuga gaussenii led to the isolation and characterization of 34 structurally diverse polyketides (1-34). Seven of them are previously undescribed, including five unprecedented dihydropyran-containing (terricoxanthones A-E, 1-5, resp.) and one rare tetrahydrofuran-containing (terricoxanthone F, 6) dimeric xanthones. The structures were elucidated by spectroscopic methods and single-crystal X-ray diffraction analyses. Terricoxanthones each were obtained as a racemic mixture. Their plausible biosynthetic relationships were briefly proposed. Compounds 6, aspergillusone A (8), and alatinone (27) displayed considerable inhibition against Candida albicans with MIC values of 8-16 µg/mL. 4-Hydroxyvertixanthone (12) and 27 exhibited significant inhibitory activities against Staphylococcus aureus, with MIC values of 4-8 µg/mL. Furthermore, compounds 8 and 27 could disrupt biofilm of S. aureus and C. albicans at 128 µg/mL. The findings not only extend the skeletons of xanthone dimers and contribute to the diversity of metabolites of endophytes associated with the endangered Chinese conifer P. gaussenii, but could further reveal the important role of protecting plant species diversity in support of chemical diversity and potential sources of new therapeutics.

Liriogerphines E-U, further unique sesquiterpene-alkaloid hybrids from the rare Chinese tulip tree.

Seventeen undescribed sesquiterpene-alkaloid hybrids (liriogerphines E-U, 1-17) were isolated and identified during a further phytochemical investigation on the branches and leaves of Chinese tulip tree (Liriodendron chinense), a rare medicinal and ornamental plant endemic to China. These unique heterodimers are conjugates of germacranolide-type sesquiterpenoids with structurally diverse alkaloids [i.e., aporphine- (1-15), proaporphine- (16), and benzyltetrahydroisoquinoline-type (17)] via the formation of a C-N bond. The previously undescribed structures were elucidated by comprehensive spectroscopic data analyses and electronic circular dichroism calculations. Such a class of sesquiterpene-alkaloid hybrids presumably biosynthesized via an aza-Michael addition is quite rare from terrestrial plants. In particular, the sesquiterpene-benzyltetrahydroisoquinoline hybrid skeleton has never been reported until the present study. All the isolates were evaluated for their cytotoxic effects against a small panel of leukemia cell lines (Raji, Jeko-1, Daudi, Jurkat, MV-4-11 and HL-60), and some of them exhibited considerable activities.

Development of a novel near-infrared molecule rotator for early diagnosis and visualization of viscosity changes in acute liver injury models.

Acute liver injury leading to acute liver failure can be a life-threatening condition. Therefore, timely and accurate early diagnosis of the onset of acute liver injury in vivo is critical. Viscosity is one of the key parameters that can accurately reflect the levels of relevant active analytes at the cellular level. Herein, a novel near-infrared molecule rotator, DJM, was designed and synthesized. This probe exhibited a highly sensitive (461-fold from PBS solution to 95% glycerol solution) and selective response to viscosity with a maximum emission wavelength of 760 nm and a Stokes shift of 240 nm. Furthermore, DJM has exhibited a remarkable capacity to discern viscosity changes induced by nystatin in viable cells with sensitivity and selectivity and further applied in the zebrafish and mouse model of acute liver injury. Additionally, DJM may potentially offer direction for the timely observation and visualization of viscosity in more relevant disease models in the future.

Specific volatiles of tea plants determine the host preference behavior of Empoasca onukii.

Empoasca onukii is a major pest that attacks tea plants. To seek effective and sustainable methods to control the pest, it is necessary to assess its host preference among different species of tea and understand the critical factors behind this behavior. In this study, the behavioral preference of E. onukii for volatile organic compounds (VOCs) of three potted tea species was evaluated. The VOCs released by the three tea species were analyzed using gas chromatography-mass spectrometry, and the major components were used to test the pest's preference. Transcriptome analysis was used to infer the key genes that affect the biosyntheses of the VOCs. The results showed that the tendency of E. onukii toward the VOCs of the three tea species was the strongest in green tea, followed by white tea, and the weakest in red tea. This behavioral preference was significantly and positively correlated with the relative levels of hexanol, linalool, and geraniol in tea volatiles. Relative hexanol was significantly and positively correlated with the expression of genes TEA009423 (LOX2.1), TEA009596 (LOX1.5), TEA008699 (HPL), TEA018669 (CYPADH), and TEA015686 (ADHIII). Relative linalool was significantly and positively correlated with the expression of genes TEA001435 (CAD) and Camellia_sinensis_newGene_22126 (TPS). Relative geraniol was significantly and positively correlated with the expression of genes TEA001435 (CAD), TEA002658 (CYP76B6), TEA025455 (CYP76T24), and Camellia_sinensis_newGene_22126 (TPS). The above findings suggested that three volatiles (hexanol, linalool, and geraniol) determined the behavioral preference of E. onukii toward tea plants, and their biosynthesis was mainly affected by nine genes (TEA009423, TEA009596, TEA008699, TEA018669, TEA015686, TEA001435, TEA002658, TEA025455, and Camellia_sinensis_newGene_22126).

Structurally Diverse Triterpene-26-oic Acids as Potential Dual ACL and ACC1 Inhibitors from the Vulnerable Conifer Keteleeria fortunei.

A preliminary phytochemical investigation on the 90% MeOH extract from the twigs and needles of the vulnerable conifer Keteleeria fortunei led to the isolation and characterization of 17 structurally diverse triterpen-26-oic acids, including nine previously undescribed ones (fortunefuroic acids A-I, 1-9) featuring a rare furoic acid moiety in the lateral chain. Among them, 1-5 are uncommon 9ßH-lanostane-type triterpenoic acids. Friedo-rearranged triterpenoids 6 and 7 feature a unique 17,14-friedo-lanostane skeleton, whereas 9 possesses a rare 17,13-friedo-cycloartane-type framework. Their structures and absolute configurations were elucidated by extensive spectroscopic (e.g., detailed 2D NMR) and computational (NMR/ECD) calculations and the modified Mosher's method. In addition, the absolute structure of compound 1 was ascertained by single-crystal X-ray diffraction analyses. Fortunefuroic acids B (2), G (7), and I (9), along with isomangiferolic acid (12) and 3α,27-dihydroxycycloart-24E-en-26-oic acid (14), exhibited dual inhibitory effects against the adenosine triphosphate (ATP)-citrate lyase (ACL, IC50s: 5.7-11.4 µM) and acetyl-CoA carboxylase 1 (ACC1, IC50s: 7.5-10.5 µM), both of which are key enzymes for glycolipid metabolism. The interactions of the bioactive triterpenoids with both enzymes were examined by molecular docking studies. The above findings reveal the important role of protecting plant species diversity in support of chemical diversity and potential sources of new therapeutics for ACL-/ACC1-associated diseases.

Stepwise changes in flavonoids in spores/pollen contributed to terrestrial adaptation of plants.

Protecting haploid pollen and spores against UV-B light and high temperature, 2 major stresses inherent to the terrestrial environment, is critical for plant reproduction and dispersal. Here, we show flavonoids play an indispensable role in this process. First, we identified the flavanone naringenin, which serves to defend against UV-B damage, in the sporopollenin wall of all vascular plants tested. Second, we found that flavonols are present in the spore/pollen protoplasm of all euphyllophyte plants tested and that these flavonols scavenge reactive oxygen species to protect against environmental stresses, particularly heat. Genetic and biochemical analyses showed that these flavonoids are sequentially synthesized in both the tapetum and microspores during pollen ontogeny in Arabidopsis (Arabidopsis thaliana). We show that stepwise increases in the complexity of flavonoids in spores/pollen during plant evolution mirror their progressive adaptation to terrestrial environments. The close relationship between flavonoid complexity and phylogeny and its strong association with pollen survival phenotypes suggest that flavonoids played a central role in the progression of plants from aquatic environments into progressively dry land habitats.

Forrestiacids E-K: Further [4 + 2]-Type Triterpene-Diterpene Hybrids as Potential ACL Inhibitors from the Vulnerable Conifer Pseudotsuga forrestii.

Seven [4 + 2]-type triterpene-diterpene hybrids derived from a rearranged or a normal lanostane unit (dienophile) and an abietane moiety (diene), forrestiacids E-K (1-7, respectively), were further isolated and characterized from Pseudotsuga forrestii (a vulnerable conifer endemic to China). The intriguing molecules were revealed with the guidance of an LC-MS/MS-based molecular ion networking strategy combined with conventional phytochemical procedures. Their chemical structures with absolute configurations were established by spectroscopic data, chemical transformation, electronic circular dichroism calculations, and single-crystal X-ray diffraction analysis. They all contain a rare bicyclo[2.2.2]octene motif. Both forrestiacids J (6) and K (7) represent the first examples of this unique class of [4 + 2]-type hybrids that arose from a normal lanostane-type dienophile. Some isolates remarkably inhibited ATP-citrate lyase (ACL), with IC50 values ranging from 1.8 to 11 µM. Docking studies corroborated the findings by highlighting the interactions between the bioactive compounds and the ACL enzyme (binding affinities: -9.9 to -10.7 kcal/mol). The above findings reveal the important role of protecting plant species diversity in support of chemical diversity and potential sources of new therapeutics.

Unprecedented spirodioxynaphthalenes from the endophytic fungus Phyllosticta ligustricola HDF-L-2 derived from the endangered conifer Pseudotsuga gaussenii.

Four undescribed palmarumycin-type spirodioxynaphthalenes (phyligustricins A-D) and a known biogenetic precursor (palmarumycin BG1) were isolated from a solid fermentation of Phyllosticta ligustricola HDF-L-2, an endophyte associated with the endangered Chinese conifer Pseudotsuga gaussenii. The structures were elucidated by spectroscopic methods, single-crystal X-ray diffraction analyses, and electronic circular dichroism calculations. Both phyligustricins A and B have an unprecedented spirodioxynaphthalene-derived skeleton containing an extra 4H-furo [3,2-c]pyran-4-one moiety, while phyligustricins C and D are p-hydroxy-phenethyl substituted spirodioxynaphthalenes. The plausible biosynthetic relationships of the isolates were briefly proposed. Phyligustricins C and D and palmarumycin BG1 showed considerable antibacterial activity against Staphylococcus aureus, each with an MIC value of 16 µg/mL. Palmarumycin BG1 displayed significant inhibitory effects against ACL and ACC1, with IC50 values of 1.60 and 8.00 µM, respectively.

Squamabietenols A-F, undescribed abietane-O-abietane dimeric diterpenoids from the ornamental conifer Juniperus squamata and their ATP-citrate lyase inhibitory activities.

Six undescribed naturally occurring abietane-O-abietane dimers (squamabietenols A-F) together with one 3,4-seco-totarane-type, a pimarane-type, and 17 related known mono-/dimeric diterpenoids were isolated and characterized from the needles and twigs of the ornamental conifer Juniperus squamata. The undescribed structures and their absolute configurations were established by extensive spectroscopic methods, GIAO NMR calculations with DP4+ probability analyses, and ECD calculations. Squamabietenols A and B showed significant inhibitory effects against ATP-citrate lyase (ACL, a novel drug target for hyperlipidemia and other metabolic disorders), with IC50 values of 8.82 and 4.49 µM, respectively. A molecular docking study corroborated the findings by highlighting the interactions between the bioactive compounds and the ACL enzyme (binding affinities: -7.1 to -9.0 kcal/mol). The unique abietane-O-abietane dimeric diterpenoids are quite rare in the vegetable kingdom, and they are of chemotaxonomic significance for the Cupressaceae family.

Antibacterial and antibiofilm efficacy of the preferred fractions and compounds from Euphorbia humifusa (herba euphorbiae humifusae) against Staphylococcus aureus.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia humifusa Willd., known as Di-Jin-Cao in Chinese, has long been utilized as a traditional herb for the treatment of furuncles and carbuncles mainly caused by Staphylococcus aureus infection. Despite extensive chemical and pharmacological studies reported previously for E. humifusa, the antibacterial and antibiofilm activities against S. aureus as well as the related mechanism of action (MoA) remain largely obscure. AIM OF THE STUDY: To investigate the antibacterial and antibiofilm activities of the preferred fractions and compounds from E. humifusa against S. aureus and assess the associated MoA. MATERIALS AND METHODS: The bioactive fractions and compounds were obtained from the 75% ethanol extract of E. humifusa (75%-EEEH) with the assistance of the related antibacterial and antibiofilm screening. Their antibacterial activities were determined using the broth microdilution method, whilst the inhibition of biofilm formation and the disruption of preformed biofilm were assessed by crystal violet staining and confocal laser scanning microscopy (CLSM). To achieve more effective therapies, the combinatory effects of different components were also studied. The biofilm metabolic activities of isolated compounds were evaluated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. The scanning electron microscopy (SEM) and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to explore the antibiofilm mechanism. RESULTS: Fractions DJC06 and DJC07 collected from the ethyl acetate extract of the 75%-EEEH exhibited antibacterial activity (MIC = 256 µg/mL) against S. aureus and further separation of these two fractions led to the isolation and characterization of 22 compounds. Among the isolates, luteolin (LU), quercetin (QU), and kaempferol (KA) are the verified components associated with the antibacterial and antibiofilm activities by displaying individual or combinational MIC values of 8-128 µg/mL and 70.9-99.7% inhibition for biofilm formation. Importantly, QU and KA can work in synergy with LU to significantly enhance the efficacy via destroying cell integrity, increasing membrane permeability, and down-regulating the biofilm-related gene expression. CONCLUSIONS: The preferred fractions and compounds from E. humifusa exerted desired antibacterial and antibiofilm efficacy against S. aureus via a MoA involving cell morphology disruption and altered genes expression. The findings herein not only support its traditional use in the treatment of furuncles and carbuncles, but reveal E. humifusa is a potential source for producing promising antibiofilm alternatives against S. aureus and highlight the isolated components (LU, QU, KA) that can potentiate the efficacy when used in synergy.

Anti-neuroinflammatory sesquiterpenoids from Chloranthus henryi.

Hupelactones A (1) and B (2), two new eudesmanolide-type enantiomers of the corresponding compounds, along with four mono- (3-6) and nine dimeric- (7-15) known sesquiterpenoids were isolated from the whole plant of Chloranthus henryi var. hupehensis (syn. C. henryi). The new structures including the absolute configurations were determined by comparison with previously reported enantiomers, extensive spectroscopic methods in combination with electronic circular dichroism (ECD) calculations. All the isolates were evaluated for their inhibitory activities against the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine BV-2 microglial cells. Among them, the dimeric lindenane sesquiterpenoids shizukaols F (8) and G (11) exhibited the most potent activities, with IC50 values of 2.65 and 4.60 µM, respectively.