Structures and bioactivities of monomeric and dimeric carvotacetones from Sphaeranthus africanus.

Four previously undescribed carvotacetones including one monomeric (1) and three dimeric (8, 9, 10) derivatives, together with six known compounds were isolated from the n-hexane extract of the aerial parts of Sphaeranthus africanus L. The structures of the previously undescribed compounds were elucidated as 3-angeloyloxy-5-isobutanoyloxy-7-hydroxycarvotacetone (1), 7,7'-oxybis{3-angeloyloxy-5-[(2R*,3R*)-2,3-dihydroxy-2-methylbutanoyloxy]carvotacetone} (8), (2″S*,3″R*)-7-{3-angeloyloxy-5-[(2R*,3R*)-2,3-dihydroxy-2-methylbuta-noyloxy]carvotaceton-7-yloxy}-3-angeloyloxy-5-(2,3-dihydroxy-2-methylbutanoyloxy)carvo-tacetone (9), and 7,7'-oxybis{3-angeloyloxy-5-[(2S*,3R*)-2,3-dihydroxy-2-methylbutanoyl-oxy]carvotacetone} (10). The three dimeric derivatives (8-10) showed potent anti-proliferative activity against human cancer cell lines (CCRF-CEM, MDA-MB-231, U-251, HCT-116) with IC50 values ranging from 0.2 to 2.0 µM. Caspases 3 and 7 were found to be activated by all compounds, indicating apoptosis induction activity. Monomers exhibited a specific inhibition of NO production in BV2 and RAW 264.7 cells with IC50 values ranging from 4.2 to 6.8 µM which were 2-3.5-fold lower than IC50 values causing cytotoxicity. In addition, the carvotacetones reduced NF-κB1 (p105) mRNA expression at concentrations of 10 and 2.5 µM. Altogether, the results indicate that carvotacetones may be interesting lead structures for the development of anti-cancer and anti-inflammatory drugs.

Challenges to Accurate Estimation of Methane Emission from Septic Tanks with Long Emptying Intervals.

Septic tanks in low- and middle-income countries are often not emptied for a long time, potentially resulting in poor pollutant removal efficiency and increased greenhouse gas emissions, including methane (CH4). We examined the impact of long emptying intervals (4.0-23 years) on the biochemical oxygen demand (BOD) removal efficiency of 15 blackwater septic tanks and the CH4 emission rates of 23 blackwater septic tanks in Hanoi. The average BOD removal efficiency was 37% (-2-65%), and the average CH4 emission rate was 10.9 (2.2-26.8) g/(cap·d). The emptying intervals were strongly negatively correlated with BOD removal efficiency (R = -0.676, p = 0.006) and positively correlated with CH4 emission rates (R = 0.614, p = 0.001). CH4 emission rates were positively correlated with sludge depth (R = 0.596, p = 0.002), but against expectation, negatively correlated with BOD removal efficiency (R = -0.219, p = 0.451). These results suggest that shortening the emptying interval improves the BOD removal efficiency and reduces the CH4 emission rate. Moreover, the CH4 emission estimation of the Intergovernmental Panel on Climate Change, which is a positive conversion of BOD removal, might be inaccurate for septic tanks with long emptying intervals. Our findings suggest that emptying intervals, sludge depth, and per-capita emission factors reflecting long emptying intervals are potential parameters for accurately estimating CH4 emissions from septic tanks.

Cytotoxicity of Carvotacetones from Sphaeranthus africanus Against Cancer Cells and Their Potential to Induce Apoptosis.

Three carvotacetones (1 - 3: ) isolated from Sphaeranthus africanus were screened in 60 cancer cell lines at the National Cancer Institute (NCI) within the Developmental Therapeutics Program (DTP). At the concentration of 10-5 M, compound 1: (3,5-diangeloyloxy-7-hydroxycarvotacetone) turned out to be the most active compound against ACHN and UO-31 renal cancer cell lines with growth percent values of - 100% (all cells dead). Compound 2: (3-angeloyloxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone) showed strong effects in SK-MEL-5 melanoma and ACHN renal cancer cells with inhibition values of 93% and 97%, respectively. Compound 3: (3-angeloyloxy-5-[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxy-carvotacetone) exhibited a quite strong effect on renal cancer cells with a growth inhibitory effect of 96% against ACHN and UO-31 cells. When treated with five different concentrations of 1: (1 × 10-8, 1 × 10-7, 1 × 10-6, 1 × 10-5, and 1 × 10-4 M), HOP-92 cells were found to be most sensitive with GI50, TGI, and LC50 values of 0.17, 0.40, and 0.96 µM, respectively. When using the ApoTox-Glo triplex assay to evaluate the apoptosis inducing effects of seven carvotacetones isolated from S. africanus in CCRF-CEM cells, compounds 1:  - 6: increased caspase-3/7 activity with 1, 2: , and 4: (3-angeloyloxy-5,7-dihydroxycarvotacetone) exhibiting the highest activitiy, indicating induction of caspase-dependent apoptosis.

Assessment of In vitro Antioxidant, Antidiabetic and Cytotoxic Activities of Sphaeranthus africanus Extracts

Sphaeranthus africanus is commonly used as a traditional remedy for sore throats and pain treatment in Vietnam. The aerial parts have been studied for its anti-inflammatory and anti-proliferative properties. However, the antioxidant and antidiabetic potential of the plant has not been explored. In this work, hydrophilic extracts of the plant's aerial parts were prepared in order to investigate its antioxidant and anti-diabetic properties. Also, the cytotoxicity of the root was evaluated and compared to that of the aerial parts. All of the extracts inhibited lipid peroxidation with IC 50 values ranging from 2.05 to 3.56 µg/mL, indicating substantial antioxidant activity. At an IC 50 value of 4.80 μg/mL, the 50% ethanol extract exhibited the most potent inhibition of α-glucosidase. The cytotoxic activity of root extracts is 2 to 5-fold less than that of the aerial parts. Nevertheless, dichloromethane and ethyl acetate extracts of the root demonstrated a selective effect on leukemia cells, with no harm towards the normal HEK-293 cell line. This work provides a scientific support for the antioxidant and antidiabetic activity of the plant. Hence, it may find a promising material for the development of novel antioxidant and antidiabetic agents. More research can be conducted on the phytochemistry and anticancer activities of the plant’s root.

Greenhouse Gas Emissions from Blackwater Septic Systems.

Septic systems are potentially a significant source of greenhouse gases (GHGs). The present study investigated GHGs from the blackwater septic systems that are widely used especially in low- and middle-income countries. Ten blackwater septic tanks in Hanoi, Vietnam, were investigated using the floating chamber method. The average methane and carbon dioxide emission rates measured at the first compartment (65% of total capacity) of the septic tanks were 11.92 and 20.24 g/cap/day, respectively, whereas nitrous oxide emission was negligible. Methane emission rate was significantly correlated with septage oxidation-reduction potential (ORP) (R = -0.67, p = 0.034), chemical oxygen demand mass (R = 0.78, p = 0.007), and biochemical oxygen demand mass (R = 0.78, p = 0.008), whereas it was not significantly correlated with water temperature (R = 0.26, p = 0.47) and dissolved oxygen (R = -0.59, p = 0.075) within the limited range: 30.6-31.7 °C and 0.03-0.34 mg-O2/L. The methane emission rates from septic tanks accumulating septage for >5 years were significantly higher than those at 0-5 years (p = 0.016). These results suggest that lower ORP and higher biodegradable carbon mass, in association with longer septage storage periods are key conditions for methane emissions. To the best of our knowledge, this is the first study to characterize GHG emissions from septic systems.

Antimicrobial and Efflux Pump Inhibitory Activity of Carvotacetones from Sphaeranthus africanus Against Mycobacteria.

Carvotacetones (1-7) isolated from Sphaeranthus africanus were screened for their antimycobacterial and efflux pump (EP) inhibitory potential against the mycobacterial model strains Mycobacterium smegmatis mc2 155, Mycobacterium aurum ATCC 23366, and Mycobacterium bovis BCG ATCC 35734. The minimum inhibitory concentrations (MICs) of the carvotacetones were detected through high-throughput spot culture growth inhibition (HT-SPOTi) and microbroth dilution assays. In order to assess the potential of the compounds 1 and 6 to accumulate ethidium bromide (EtBr) in M. smegmatis and M. aurum, a microtiter plate-based fluorometric assay was used to determine efflux activity. Compounds 1 and 6 were analyzed for their modulating effects on the MIC of EtBr and the antibiotic rifampicin (RIF) against M. smegmatis. Carvotacetones 1 and 6 had potent antibacterial effects on M. aurum and M. bovis BCG (MIC ≤ 31.25 mg/L) and could successfully enhance EtBr activity against M. smegmatis. Compound 1 appeared as the most efficient agent for impairing the efflux mechanism in M. smegmatis. Both compounds 1 and 6 were highly effective against M. aurum and M. bovis BCG. In particular, compound 1 was identified as a valuable candidate for inhibiting mycobacterial efflux mechanisms and as a promising adjuvant in the therapy of tuberculosis or other non-tubercular mycobacterial infections.

Melodamide A from Melodorum fruticosum - Quantification using HPLC and one-step-isolation by centrifugal partition chromatography.

Melodamide A, a phenolic amide from the leaves of Melodorum fruticosum Lour., has previously shown pronounced anti-inflammatory activity. In order to rapidly isolate larger quantities for biological testing, a fast, one-step isolation method by centrifugal partition chromatography was developed within this study. Fractionation of the dichloromethane extract was performed with a two-phase solvent system consisting of n-hexane, ethyl acetate, methanol, and water (3:7:5:5, v/v), leading to the isolation of melodamide A with a purity of >90% and a yield of 6.7 w% within 32 min. The developed method can also be used in dual mode for the enrichment of further constituents like flavonoids or chalcones. In order to support the centrifugal partition chromatography method development, additionally, a high-performance liquid chromatography method was established and validated to determine quantities of melodamide A in plant material and crude extracts. Analysis of M. fruticosum leaves and a dichloromethane extract obtained from this plant material showed a total melodamide A content of 0.19 ± 0.008 and 8.9 ± 0.249 w%, respectively.

Anti-inflammatory and antiproliferative compounds from Sphaeranthus africanus.

BACKGROUND: Sphaeranthus africanus has been used in traditional Vietnamese medicine to treat sore throat, and to relieve pain and swelling. However, the anti-inflammatory activity of this plant had not yet been investigated. Previously, we isolated five carvotacetones (1-5) from this plant that displayed cytotoxicity against several cancer cell lines. PURPOSE: The objective of this study was to isolate further constituents from S. africanus and to investigate the anti-inflammatory activity of all constituents. Furthermore, the anti-proliferative activity of the newly isolated compounds was evaluated. STUDY DESIGN AND METHODS: Compounds were isolated from the upper parts of S. africanus by chromatographic methods. Structures were determined using spectroscopic techniques, like NMR and MS. All nine compounds isolated from S. africanus were evaluated for inhibitory activity against COX-1 and COX-2 isoenzymes in-vitro, COX-2 mRNA expression and influence on NO production. The anti-proliferative activities of newly isolated compounds (6-9) were evaluated by XTT viability assay with four cancer cell lines, namely CCRF-CEM, MDA-MB-231, HCT-116, and U-251 cells. RESULTS: Two diastereomeric carvotacetones (3-angeloyloxy-5-[2″S,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (6) and 3-angeloyloxy-5-[2″R,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (7), asperglaucide (8) and chrysoplenol D (9) were isolated from S. africanus. COX-1 and COX-2 assays of compounds 1-9 revealed that compounds 1 and 2 possess potent and selective COX-2 inhibitory activity with IC50 values of 3.6 and 0.5 µM, respectively. COX-2 gene expression assay showed that some carvotacetones exhibited inhibitory effects on COX-2 gene expression in THP-1 macrophages. Compound 4 is the most active compound inhibiting the synthesis of COX-2 by 55% at 2.06 µM. In the iNOS assay, all seven carvotacetones inhibited NO production in BV2 and RAW cell lines with IC50 values ranging from 0.2 to 2.9 µM. Compound 4 showed potent inhibitory activity with IC50 values of 0.2 µM in both BV2 and RAW cell lines. Molecular docking studies revealed the binding orientations of 1 and 2 in the active sites of COX-2. XTT assay of the newly isolated compounds revealed that the two isomeric carvotacetones (6-7) exhibited considerable anti-proliferative activity against four cancer cell lines (CCRF-CEM, MDA-MB-231, HCT-116, U-251) with IC50 values ranging from 1.23 to 8 µM. CONCLUSION: For the first-time, the diastereomeric carvotacetones (6-7) were isolated as separate compounds, and their anti-proliferative activity was determined. Selective COX-2 inhibitory, COX-2 mRNA expression and NO production inhibitory activities by some of the major constituents of S. africanus supports the traditional medical application of this plant for the treatment of inflammation-related disorders.

Inhibition of Pro-Inflammatory Functions of Human Neutrophils by Constituents of Melodorum fruticosum Leaves.

In an initial screening, the dichloromethane extract from the leaves of Melodorum fruticosum showed distinct inhibitory effects on the release of interleukin-8 (IL-8) in human neutrophils. Therefore, the aim of the present study was the phytochemical and pharmacological investigation of this extract, to better understand which compounds might be responsible for the anti-inflammatory effect. Phytochemical analysis led to the isolation of 12 known compounds and two new natural products, 5-hydroxy-6-(2-hydroxybenzyl)-4',7-dimethoxyflavanone (13) and 2',4'-dihydroxy-3'-(2-hydroxybenzyl)-4,6'-dimethoxychalcone (14). The influence of the isolated compounds on the production and release of the pro-inflammatory factors IL-8, tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS), and adhesion molecules (CD62L and CD11b) in human neutrophils was evaluated. Three constituents, melodamide A, 2',4'-dihydroxy-4,6'-dimethoxychalcone, and 2',6'-dihydroxy-4'-methoxychalcone, showed significant inhibition of IL-8 release (IC50 =6.6, 8.6, and 11.6 µm, respectively) and TNF-α production (IC50 =4.5, 13.3, and 6.2 µm, respectively).

Antiproliferative Carvotacetones from Sphaeranthus africanus.

Five carvotacetone derivatives, including two known ones, 3,5-diangeloyloxy-7-hydroxycarvotacetone (1) and 3-angeloyloxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (2), along with three new compounds, 3-angeloyloxy-5-[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone (3), 5-angeloyloxy-7-hydroxy-3-tigloyloxycarvotacetone (4), and 3-angeloyloxy-5,7-dihydroxycarvotacetone (5), were isolated from the aerial parts of Sphaeranthus africanus collected in Vietnam. Bioassay-guided fractionation was monitored by the antiproliferative activity on CCRF-CEM human cancer cells. The structures of compounds 1-5 were determined on the basis of NMR spectroscopic and mass spectrometric data. Activities of compounds 1-5 were evaluated in vitro against the human cancer cell lines CCRF-CEM, MDA-MB-231, U-251, and HCT-116. All compounds exhibited significant antiproliferative activity against all four cancer cell lines. CCRF-CEM was most sensitive to the compounds, with IC50 values ranging from 0.6 to 1.5 µM. Compounds 3 and 4 possessed the highest activity, with IC50 values in the four cell lines ranging from 0.6 to 2.9 µM and 1.3 to 2.5 µM, respectively. These compounds also showed inhibitory activity toward the HEK-293 human embryonic kidney cells with IC50 values ranging from 2.5 to 5.5 µM. This is the first time that antiproliferative activity of S. africanus has been reported, and 1-5 are the most cytotoxic carvotacetone derivatives reported so far.