Study of phenanthrenes from their unique mass spectrometric behavior through quantum chemical calculations to liquid chromatographic quantitation.

Phenanthrenes and their derivatives have biological relevance owing to their antimicrobial, antioxidant, and cytotoxic effects on cancer cells. They can be efficiently analyzed through ultrahigh-performance liquid chromatography coupled to tandem high-resolution mass spectrometry (UHPLC-MS/HRMS). Herein, we first studied the unique fragmentation behavior of phenanthrenes based on direct infusion MS/HRMS analysis. As a newly described phenomenon, "organ pipe distribution", we found a structural connection linking their unique fragmentation pattern to serial H radical losses. The bonds responsible for this behavior were identified through quantum chemical calculations using a stepwise approach. Furthermore, the chromatographic aspect of this study was enhanced by developing, validating, and applying a new unscheduled targeted UHPLC-MS/HRMS method for quantifying phenanthrenes in Juncus compressus herb. Targeted compounds were efficiently separated within 4 min upon utilizing the Accucore C30 column, and the unscheduled targeted analytical approach afforded five new isomers. Compounds 1 (effususol), 3 (dehydroeffusol), and 6 (7-hydroxy-1-methyl-2-methoxy-5-vinyl-9,10-dihydrophenanthrene) had their linearity limits determined within 10-5000 nM, and Compounds 2 (effusol), 4 (juncusol), 5 (effususin A), and 7 (compressin A) within 25-5000 nM. The coefficients of variation for precision ranged from 1.4 % to 15.2 %. The obtained matrix effects and accuracy values were also within acceptable ranges. Compounds 2 (effusol) and 3 (dehydroeffusol) were present in both methanolic and dichloromethanolic extracts of Plants 1 and 3 at the highest concentrations. Furthermore, the relationship between phenanthrene fingerprints, obtained through ANOVA statistical analysis of quantitative data, and the geographical location of herbs was also established.

Euphane and Tirucallane Triterpenes with Trypanocidal Activity from Euphorbia desmondii.

The phytochemical investigation of Euphorbia desmondii resulted in the isolation of 15 previously undescribed triterpenoids (desmondiins A, C-P) and 8 already described compounds. The structures of the isolated compounds were determined by extensive spectroscopic analyses. The compounds were identified as tirucallane and euphane triterpenes based on 7-keto-8-ene, 11-keto-8-ene, or 7,11-diketo-8-ene skeletons. Additionally, the selective trypanocidal activities of these compounds against Trypanosoma cruzi were evaluated. Desmondiins A, C, D, F, H, and M exhibited IC50 values in the range of 3-5 µM, and selectivity indices between 5-9, against T. cruzi epimastigotes over the host cell (RAW264.7 macrophages). Furthermore, desmondiin A efficiently inhibited amastigote replication in host cells (IC50 = 2.5 ± 0.3 µM), which was comparable to that of the positive control, benznidazole (3.6 ± 0.4 µM). Overall, the isolated euphane and tirucallane triterpenoids could act as antichagasic lead scaffolds.

Phenanthrenes from Juncus articulatus with Antibacterial and Biofilm Formation Inhibitory Activity.

Continuing our search for bioactive compounds in species from the Juncaceae family, Juncus articulatus was investigated. Ten previously undescribed phenanthrenes─articulins A-J (1-10)─and ten known compounds─juncuenin B, dehydrojuncuenin B, juncatrin B, ensifolins E, F, H, I, K, juncuenin D, and luzulin A (11-20)─along with other compounds, have been isolated and identified. The isolated compounds were evaluated for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus aureus (MRSA). Compounds 12 and 14 exhibited the most potent activity against planktonic and sessile MSSA and MRSA with minimum inhibitory concentration (MIC) values of 15.1 µM (12 for both bacterial strains) and 15.3 µM (14 for both bacterial strains). Compounds 15, 17, and 18 also exhibited activity against both strains, although to a lower extent, with MIC values ranging from 30.0 to 56.8 µM. The inhibition of biofilm formation of these compounds was observed at 15.1-114.3 µM. This study elucidates the phenanthrene composition of J. articulatus and the antibacterial effect of these compounds.

New Members of the Centrapalus Coumarin and Pauciflorin Series from Centrapalus pauciflorus.

Monoterpene and 5-methylcoumarin- or 5-methylchromone-coupled meroterpenoids occurring mainly in the Asteraceae species proved to have high potency against protozoans, worms, and various tumor cells, which make them interesting targets for searching for new bioactive compounds. The African plant Centrapalus pauciflorus was applied in traditional medicine for healing chest pain and stomach aches. Three new meroterpenoids named centrapalus coumarin N (2), pauciflorins P (3), and Q (4), and the already known cyclohoehnelia coumarin (1), were isolated from the chloroform extract of C. pauciflorus, together with centrapalus coumarin O (5), which was obtained for the first time from a natural source. The structures were established from HRESIMS, 1D (1H NMR, 13C NMR JMOD) and 2D NMR (HSQC, HMBC, 1H-1H COSY, NOESY) spectroscopies, and the absolute stereochemistry of 5 was determined by single-crystal X-ray diffraction. Compounds 1, 2, and 5 are hybrid molecules of 5-methylcoumarin-monoterpene origin. Centrapalus coumarin N is the first example of meroterpenoids, where a monoterpene is fused with a coumarin and an acetophenone unit. Pauciflorins P and Q are dimeric meroterpenoid isomers. Centrapalus coumarins N and O were tested for antiproliferative activity against human adherent breast (MCF-7, MDA-MB-231), cervical (HeLa, SiHa), and ovarian (A2780) cancer cell lines, and were additionally included to obtain data concerning cancer selectivity. Both compounds exhibited moderate (IC50 > 10 µM) but selective activity against A2780 cells.

Diverse diterpenoids and a triterpenoid from Euphorbia spinidens Bornm. ex Prokh.

Four previously unreported diterpenoids including three ent-atisanes (1-3) and one ent-abietane (4), along with one known linear triterpenoid (5) and five known diterpenoids including four myrsinanes (6-9), and one abietane (10) have been isolated from the roots of Euphorbia spinidens Bornm. ex Prokh. The structures were determined on the basis of extensive spectroscopic analyses including HR-ESI-MS, 1D and 2D NMR and comparison of the data with those reported in the literature. Antimicrobial potential of isolated compounds were also evaluated. Guionianol B (10) showed good antibacterial activity against Staphylococcus aureus and Bacillus subtilis with MIC value of 6.25 µg/mL.

Rapid Detection of Adulteration in Boswellia Extracts with Citric Acid by UPLC-HRMS and 1H NMR.

Boswellia serrata ole-gum-resin extracts (BSEs) are commonly used as food supplements, especially in osteoarthritis management. The quality standard is established by determining 11-keto-ß-boswellic acid (KBA) and acetyl-11-keto-boswellic acid (AKBA) content using high-performance liquid chromatography (HPLC) or assessing the total boswellic acid (TBA) content by titrimetry. The limited geographical distribution of Boswellia species and increasing industrial demand could increase the risk of adulteration in Boswellia-containing products. In this study, 14 BSEs from commercial sources, used in food supplements, were analyzed in comparison with a USP Reference Standard extract. The KBA and AKBA content was determined by HPLC, whereas the TBA content was determined by titration. Targeted UHPLC-high-resolution mass spectrometry (HRMS) was applied to identify the carboxylic acid content in the samples. The 1H NMR spectra of extracts were also analyzed. Only two products met the criteria for KBA and AKBA content. Although, the TBA content complied with the expected amount, 10 extracts contained citric acid levels of 6-11% even though citric acid is not a cha-racteristic component of BSEs. Our results suggest undeclared addition of citric acid to comply with declared contents of TBA when using titration methods. Incorporation of citric acid to industrial samples - in order to alter the outcomes of the titration analysis - was demonstrated for the first time.

Indonesian Euphorbiaceae: Ethnobotanical Survey, In Vitro Antibacterial, Antitumour Screening and Phytochemical Analysis of Euphorbia atoto.

Indonesia is among the countries with the most significant biodiversity globally. Jamu, the traditional medicine of Indonesia, predominantly uses herbal materials and is an integral component of the Indonesian healthcare system. The present study reviewed the ethnobotanical data of seven Indonesian Euphorbiaceae species, namely Euphorbia atoto, E. hypericifolia, Homalanthus giganteus, Macaranga tanarius, Mallotus mollissimus, M. rufidulus, and Shirakiopsis indica, based on the RISTOJA database and other literature sources. An antimicrobial screening of the plant extracts was performed in 15 microorganisms using the disk diffusion and broth microdilution methods, and the antiproliferative effects were examined in drug-sensitive Colo 205 and resistant Colo 320 cells by the MTT assay. The antimicrobial testing showed a high potency of M. tanarius, H. giganteus, M. rufidulus, S. indica, and E. atoto extracts (MIC = 12.5-500 µg/mL) against different bacteria. In the antitumour screening, remarkable activities (IC50 0.23-2.60 µg/mL) were demonstrated for the extracts of H. giganteus, M. rufidulus, S. indica, and E. atoto against Colo 205 cells. The n-hexane extract of E. atoto, with an IC50 value of 0.24 ± 0.06 µg/mL (Colo 205), was subjected to multistep chromatographic separation, and 24-methylene-cycloartan-3ß-ol, jolkinolide E, tetra-tert-butyl-diphenyl ether, α-tocopherol, and ß-sitosterol were isolated.

Anticancer Meroterpenoids from Centrapalus pauciflorus leaves: Chromone- and 2,4-Chromadione-Monoterpene Derivatives.

Eight previously undescribed chromones, named pauciflorins F-M and two 5-methyl-2,4-chromadione derivatives named as pauciflorins N and O, were isolated from the methanol extract of the leaves of Centrapalus pauciflorus (Willd.) H.Rob. together with the known (+)-spiro-ethuliacoumarin. The structures were determined via extensive spectroscopic analyses, including HRESIMS, 1D NMR (1H, 13C JMOD), and 2D NMR (HSQC, HMBC, 1H-1H COSY, and NOESY) experiments. Through an MTT assay, seven isolated compounds were tested for their antiproliferative properties against human adherent breast (MCF-7, MDA-MB-231), cervical (HeLa, SiHa), and ovarian (A2780) cancer cell lines. Pauciflorin F was effective against MCF-7 breast cancer cells, its activity (IC50 5.78 µM) was comparable to that of the reference agent cisplatin (IC50 5.78 µM).

Isolation of the Lanostane Triterpenes Pholiols L-S from Pholiota populnea and Evaluation of Their Antiproliferative and Cytotoxic Activities.

Pholiols L-S (1−8), eight undescribed triterpenes were isolated from the sporocarps of the mushroom Pholiota populnea. Various chromatographic techniques, such as open column chromatography, flash chromatography, gel filtration, preparative thin layer chromatography, and HPLC, were applied to purify the compounds. The structure elucidation was carried out by spectroscopic analysis, including 1D (1H NMR and 13C JMOD) and 2D NMR (1H-1H COSY, HSQC, HMBC and NOESY) and HRESIMS experiments. The isolated compounds had lanostane (1−7) or trinorlanostane (8) skeletons; all of them were substituted with 3-hydroxy-3-methylglutaroyl group or its 6-methyl ester. Five compounds (1, 2, 4, 6, and 8) were investigated for their antiproliferative and cytotoxic activity in vitro by MTT assay on breast cancer (MCF-7), human colon adenocarcinoma (sensitive Colo 205, and resistant Colo 320), non-small cell lung cancer (A549), and human embryonic lung fibroblast (MRC-5) cell lines. Pholiols M (2) and O (4) showed antiproliferative activity against the MCF-7 cell line with IC50 of 2.48 and 9.95 µM, respectively. These compounds displayed tumor cell selectivity on MCF-7 cells with SI values of >40 (2) and 4.3 (4), but they did not show a cytotoxic effect, proving their action exclusively on tumor cell proliferation. Pholiols L (1) and Q (8) were found to have selective cytotoxicity on drug resistant cells in comparison to their effects on Colo320 and Colo205 cells [relative resistance values 0.84 (1) and 0.62 (8)].

Pholiols E-K, lanostane-type triterpenes from Pholiota populnea with anti-inflammatory properties.

Investigation of the chloroform and ethyl acetate extracts of the edible mushroom Pholiota populnea led to the isolation of eight triterpenes, the undescribed natural products pholiols E-K and the known (+)-clavaric acid. HRESIMS and 1D and 2D NMR spectroscopy were employed to determine the structures of the undescribed compounds. The NOESY spectra were used to assign the relative configurations of triterpenes. The isolated compounds were screened for their anti-inflammatory activity on cyclooxygenase (COX-1 and COX-2), and lipoxygenase (5-LOX and 15-LOX) inhibitory assays. Dose-response investigations revealed that lanostane derivatives exhibited moderate 5-LOX and COX-2 inhibitory activities, with pholiol F (IC50 194.5 µM against 5-LOX and 439.8 µM against COX-2) the most active among the isolated compounds. Our findings indicated that P. populnea is an abundant source of new bioactive lanostane-type triterpenes.

Secondary metabolites from Gentiana cruciata L. and their anti-inflammatory and analgesic activities.

A previously unreported secoiridoid glycoside, cruciatoside (1) was isolated from the aerial parts of Gentiana cruciata L. along with ten known compounds eustomoside (2), eustomorusside (3), gentiopicroside (4), 6'-O-ß-D-glucopyranosyl gentiopicroside (5), loganic acid (6), isoorientin (7), isovitexin (8), isovitexin 2''-(E)-ferulate (9), mangiferin (10), and 2-methyl-inositol (11). The chemical structures of the isolates were elucidated based on extensive 1 D and 2 D NMR experiments as well as HRMS analysis. All isolates were evaluated for their in vitro anti-inflammatory and analgesic activities. Compounds 9, 4, and 7 (200 µM) showed moderate anti-inflammatory activity by inhibiting nitrite production from LPS-induced RAW 264.7 macrophage cells, with the inhibition rates of 39.5%, 25.8% and 22.9% respectively without exhibiting substantial cytotoxicity. Besides, 1, 2, 4, and 7 exerted the highest decrease in IL-6 levels. Moreover, compound 4 showed in vitro analgesic activity by decreasing the PGE2 level comparable to the reference drugs.

Concentration-dependent effects of effusol and juncusol from Juncus compressus on seedling development of Arabidopsis thaliana.

Juncus species are valuable sources of phenanthrene compounds that have been used in traditional Chinese medicine for thousands of years. Effusol and juncusol are the most investigated compounds reported to have antimicrobial and anticancer effects; however, to date, their effects on higher plants have not been investigated. In this study, we examined the effects of effusol and juncusol on the growth and other biochemical parameters of the dicot model plant Arabidopsis thaliana in a concentration-dependent manner with a focus on polyamine metabolism. Phenanthrene induced toxic effects on plant growth and development, while effusol and juncusol induced higher biomass and maintained antioxidant defence mechanisms associated with reduced polyamine degradation. Taken together, our results suggest that these compounds could be good candidates for new biopesticide or biostimulant plant growth regulators in the future.

Unique Phenanthrenes from Juncus ensifolius and Their Antiproliferative and Synergistic Effects with the Conventional Anticancer Agent Doxorubicin against Human Cancer Cell Lines.

Phenanthrenes are the main special metabolites of Juncaceae species from phytochemical, pharmacological, and chemotaxonomical points of view. The present study focused on the isolation, structure determination, and pharmacological investigation of phenanthrenes from Juncus ensifolius. Nineteen compounds, including 17 phenanthrenes, were identified from the methanol extract of the plant. Thirteen compounds, namely, ensifolins A−M (1−13), were obtained for the first time from natural sources. Four phenanthrenes [2-hydroxy-1,7-dimethyl-5-vinyl-9,10-dihydrophenanthrene (14), juncuenin B (15), juncatrin B (16), and sylvaticin A (17)], 4-hydroxybenzaldehyde (18) and luteolin (19) were isolated for the first time from J. ensifolius. Ensifolins A (1) and B (2) are structurally unique phenanthrenes, considering that they are flavonoid- (1) or benzaldehyde-adducts (2). The antiproliferative activity of all isolated compounds against HeLa, COLO 205, and COLO 320 cancer cells and a non-tumor (MRC-5) cell line was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assay. The luteolin-substituted phenanthrene ensifolin A (1) proved to be the most active against all three cancer cell lines (IC50 values 3.9−12.7 µM) and showed good selectivity (SI = 4.95) in the case of COLO 205. The best selectivity was recorded for ensifolins D (4, SI > 5.15, HeLa), H (8, SI > 8.13, HeLa), and 17 (SI > 9.43, HeLa). The synergistic activity of the compounds with doxorubicin was also tested on HeLa cells, and ensifolins E (5) and H (8) exhibited very strong synergism (CI < 0.1). In conclusion, these phenanthrenes are worthy of further investigation.

Juncaceae Species as Promising Sources of Phenanthrenes: Antiproliferative Compounds from Juncus maritimus Lam.

Juncaceae family represents an abundant source of phenanthrenes. In continuation of our work aiming at the isolation of biologically active compounds from Juncaceae species, Juncus maritimus Lam. was subjected to phytochemical and pharmacological investigations. The isolation process was carried out by using combined extraction and chromatographic methods. The structures of the obtained chemical compounds were elucidated by spectroscopic analysis, including HRESIMS, 1D (1H, 13C-JMOD), and 2D (1H-1H-COSY, HSQC, HMBC, NOESY) NMR spectra. Four new [maritins A-D (1-4)] and seven known phenanthrenes (5-11) were isolated from the plant, of which two (4 and 11) are phenanthrene dimers composed of effusol monomers. Maritin C (3) has an unusual 4,5-ethanophenanthrene skeleton most likely produced by biosynthetic incorporation of a vinyl group into a cyclohexadiene ring. Compounds 1-11 were tested for their antiproliferative activity on seven human tumor cell lines (HeLa, HTM-26, T-47D, A2780, A2780cis, MCF-7, KCR) and one normal cell line (MRC-5) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The dimeric phenanthrenes showed strong antiproliferative activity against T-47D cells with IC50 values of 9.1 and 6.2 µM, respectively.

Gerardiins A-L and Structurally Related Phenanthrenes from the Halophyte Plant Juncus gerardii and Their Cytotoxicity against Triple-Negative Breast Cancer Cells.

Species in the Juncaceae accumulate different types of secondary metabolites, among them phenanthrenes and 9,10-dihydrophenanthrenes in substantial amounts. These compounds have chemotaxonomic significance and also possess interesting pharmacological activities. The present study has focused on the isolation, structure determination, and pharmacological investigation of phenanthrenes from Juncus gerardii. Twenty-six compounds, including 23 phenanthrenes, have been isolated from a methanol extract of this plant. Twelve compounds, the phenanthrenes gerardiins A-L (1-12), were obtained as new natural products. Eleven phenanthrenes [effusol (13), dehydroeffusol (14), effususin A (15), compressin A, 7-hydroxy-2-methoxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene, juncusol, 2-hydroxy-7-hydroxymethyl-1-methyl-5-vinyl-9,10-dihydrophenanthrene, 2,7-dihydroxy-5-formyl-1-methyl-9,10-dihydrophenanthrene, effususol A, 2,7-dihydroxy-5-hydroxymethyl-1-methyl-9,10-dihydrophenanthrene, and jinflexin C], 1-O-p-coumaroyl-3-O-feruloyl-glycerol, and the flavones apigenin and luteolin were isolated for the first time from this plant. The cytotoxicity of the 23 isolated phenanthrenes in both mouse (4T1) and human (MDA-MB-231) triple-negative breast cancer cells and in a nontumor (D3, human cerebral microvascular endothelial) cell line was tested using an MTT viability assay. The results obtained showed that the dimeric compounds gerardiins I (9), J (10), K (11), and L (12), derived biogenetically from effusol and dehydroeffusol, were cytotoxic to both tumor and nontumor cell lines, while the monomeric compounds exerted no or very low cytotoxicity. Impedance measurements were consistent with the results of the MTT assays performed.