Cytotoxicity, genotoxicity, and gene expression changes induced by methanolic extract of Moringa stenopetala leaf with LC-qTOF-MS metabolic profile.

Moringa stenopetala (Baker f.) Cuf.and other Moringa species have traditionally been used to treat various diseases. The purpose of this study was to determine the cytotoxic and genotoxic effects of the methanolic extract of M. stenopetala leaf and its fractions on selected tumor cells. Cytotoxicity was determined by MTT assay. The comet assay was used toassess DNA damage, and gel electrophoresis was used to determine DNA fragmentation. Gene expression was analyzed by qPCR using two specific genes for each cancer cell line. Fractionation of the methanolic extract (E-1) on Diaion HP-20 yielded five fractions (Fr-2 to Fr-6); only Fr-4 and Fr-6 were cytotoxic to breast cancer cells (MCF-7; IC50 = 58.3 ± 0.93 and 35.8 ± 2.44 µg/mL, respectively), human hepatocellular carcinoma cells (HepG2; IC50 = 57.8 ± 1.57 and 39.3 ± 1.90 µg/mL, respectively), and Fr-4 was cytotoxic to human colon cancer cells (HCT-116; IC50 = 94.2 ± 4.9 µg/mL). In addition, exposure of the cancer cells to Fr-4 and Fr-6 resulted in a high level of DNA damage. Moreover, relative expression of MTAP and CDKN2A in MCF-7 were increased, whereas expression of p21 and p53 in HCT-116, and APC and TERT in HepG2 were decreased, similar to that of doxorubicin. LC-qTOF-MS was used to identify metabolites in E-1, the majority of which were enriched in Fr-4. Two terpenes (loliolide and dihydroactinidiolide), the majority of the flavonoids, and niazirin were about two fold enriched in Fr-4, whereas the majority of the lipids were 4-10 fold enriched. However, Fr-6 hardly showed compounds other than the two terpenes that were enriched 1.5 and 7 fold. The findings suggest that Fr-4 and Fr-6 are promising sources of compounds possessing cytotoxic and genotoxic properties.

An Unsuccessful Attempt to Confirm the Occurrence of 4'-O-ß- d-Glucosyl-9-O-(6″-deoxysaccharosyl)olivil in Valerian Root.

The lignan 4'-O-ß- d-glucosyl-9-O-(6″-deoxysaccharosyl)olivil had previously been discovered in a methanolic extract of valerian root (Valeriana officinalis agg.) and characterized as a potent partial agonist at the A1 adenosine receptors. Today, countless scientific sources, webpages, and press articles mention this compound and discuss it as an active constituent for the sedative effect of this herbal drug. As no second report confirmed the occurrence of this lignan in valerian root during the 20 years since its first description in 1998, we intended to re-prove its presence by means of LCMS using other genuine or added lignans as a quantitative benchmark. Whilst those lignans were clearly detectable in methanolic valerian extracts of all six investigated batches of valerian root, no positive proof of 4'-O-ß- d-glucosyl-9-O-(6″-deoxysaccharosyl)olivil was achieved. Our result suggests that this compound does not occur regularly in valerian root in the amounts expected from the single report on the occurrence of this compound.

Lignans and sesquiterpene lactones from Hypochaeris radicata subsp. neapolitana (Asteraceae, Cichorieae).

Four undescribed lignans and two undescribed sesquiterpenic acids, together with three known compounds (hypochoeroside C, hypochoeroside D, and 5-O-caffeoylshikimic acid) were isolated from the roots of Hypochaeris radicata subsp. neapolitana (Asteraceae, Cichorieae). The lignans were identified as 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranosyl-2'-O-methacrylate, (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-7,8,7',8'-tetrahydronaphtho [8,8'-c]furan-1(3H)-one, and (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-8'-(hydroxymethyl)-7,8,7',8'-tetrahydronaphthalen-8-carboxylic acid. The two sesquiterpenic acids were identified as the ring open precursors of hypochoerosides C and D. Structures were elucidated using NMR and HRMS. Absolute configurations of (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-7,8,7',8'-tetrahydronaphtho [8,8'-c]furan-1(3H)-one and (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-8'-(hydroxymethyl)-7,8,7',8'-tetrahydronaphthalen-8-carboxylic acid were determined using electronic circular dichroism (ECD) spectroscopy. 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside was evaluated for its anti-proliferative activity against myeloma cell lines MM1S, U266, and NCI-H929 and showed cytotoxicity at 100 mM against MM1S strain. No neurotoxicity was observed for major compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, hypochoeroside C, and hypochoeroside D in a fluorescence assay measuring neurite outgrowth in dorsal root ganglion (DRG) neurons. Additionally, compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, hypochoeroside C, hypochoeroside D, and hypochoerosidic acid D were quantified in unstressed and drought-stressed plants using HPLC-DAD. Drought-stressed plants were found to contain lower concentrations of the lignan 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside and sesquiterpene lactone hypochoeroside C.

Influence of Cranberry Extract on Tamm-Horsfall Protein in Human Urine and its Antiadhesive Activity Against Uropathogenic Escherichia coli.

LC-MS characterized cranberry extract from the fruits of Vaccinium macrocarpon inhibited under in vitro conditions the bacterial adhesion of Escherichia coli strain 2980 uropathogenic E. coli (UPEC strains UTI89, NU14) to T24 bladder cells and adhesion of UPEC strain CFT073 to A498 kidney cells in a concentration-dependent manner. Within a biomedical study, urine samples from 16 volunteers (8 male, 8 female) consuming cranberry extract for 7 d (900 mg/d) were analyzed for potential antiadhesive activity against UPEC by ex vivo experiments. Results indicated inhibition of adhesion of UPEC strain UTI89 to human T24 bladder cells. Subgroup analysis proved significant inhibition of bacterial adhesion in case of urine samples obtained from male volunteers while female urine did not influence the bacterial attachment. Differences between antiadhesive capacity of urine samples from male/female volunteers were significant. Protein analysis of the urine samples indicated increased amounts of Tamm-Horsfall protein (THP, syn. uromodulin) in the active samples. Inhibition of bacterial adhesion by the urine samples was correlated to the respective amount of THP. As it is known that THP, a highly mannosylated glycoprotein, strongly interacts with FimH of UPEC, this will lead to a decreased interaction with uroplakin, a FimH-binding transmembrane protein of urothelial lining cells. From these data it can be concluded that the antiadhesive effect of cranberry after oral intake is not only related to the direct inhibition of bacterial adhesins by extract compounds but is additionally due to an induction of antiadhesive THP in the kidney.

Traditionally used medicinal plants against uncomplicated urinary tract infections: Hexadecyl coumaric acid ester from the rhizomes of Agropyron repens (L.) P. Beauv. with antiadhesive activity against uropathogenic E. coli.

The rhizomes from Agropyron repens are traditionally used for the treatment of uncomplicated urinary tract infections. Extracts prepared with solvents of different polarity did not show any cytotoxic effects against different strains of uropathogenic E. coli (UPEC) and human T24 bladder cells under in vitro conditions. Significant antiadhesive activity against the bacterial attachment to human T24 bladder cells was found for an acetone extract (AAE) at concentrations >250µg/mL. More hydrophilic extracts did not influence the bacterial attachment to the eukaryotic host cells. Bioassay guided fractionation of AAE led to the identification of (E)-hexadecyl-3-(4-hydroxyphenyl)-acrylate (hexadecyl-coumaric acid ester) 1 as the compound responsible for inhibiting the UPEC adhesion to T24 bladder cells. 1 reduced the bacterial invasion into the bladder cells as shown by a specific invasion assay. Additionally, 1 was obtained by chemical synthesis, and also the synthetic structural analogs 2 and 3 were tested for their potential antiadhesive activity, indicating that a shorter alkyl chain at the ester function as well as the lack of hydroxylation of the phenyl moiety will abolish the antiadhesive activity.

Antiviral activity of hydroalcoholic extract from Eupatorium perfoliatum L. against the attachment of influenza A virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Eupatorium perfoliatum have been traditionally used by American natives as a treatment for fever and infections. Also modern phytotherapy in Europe documents the use of hydroalcoholic extracts of this herbal material for the treatment of infections of the upper respiratory tract. AIM OF THE STUDY: The aim of the present study was to characterize the anti-influenza A virus (IAV) potential of extracts derived from the aerial parts of E. perfoliatum and to identify their antiviral mode of action and potential active fraction's compounds of the extract. MATERIALS AND METHODS: The inhibitory effects of extracts obtained by different organic solvents with different polarities on the cytopathic effect induced by IAV replication was determined in a Madin-Darby Canine Kidney Epithelial (MDCK II) cell-based assay measuring cell viability by MTT stain (MTTIAV assay). Plaque reduction assays were used for investigation of antiviral activity. The mode of action was investigated by different incubation and treatment cycles as well as hemagglutination inhibition assays. Influence of the test extract on tumor necrosis factor (TNF-α) and epidermal growth factor (EGF)-induced cell signaling was analyzed in human lung epithelial (A549) cells. Analytical characterization of extract and fractions obtained from the extract was performed by UHPLC-MS. RESULTS: Hydroalcoholic extracts from the aerial parts of E. perfoliatum were shown to inhibit growth of a clinical isolate of IAV(H1N1)pdm09 I1 and the influenza virus A/Puerto Rico/8/34 (PR8; H1N1) with a half-maximal inhibitory concentration (IC50) of 7µg/mL and 14µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 52 and 26, respectively. Extracts prepared with dichloromethane and methanol were inactive. At concentrations >1-10µg/mL of the hydroalcoholic extract plaque formation of IAV(H1N1)pdm09 was abrogated. The extract was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. The extract blocked attachment of IAV and interfered with virus-induced hemagglutination. TNF-α-induced signal transduction in A549 cells was not affected, while the EGF-induced signaling to phosphorylated ERK was slightly upregulated by the extract. Bioassay-guided fractionation and subsequent LC-MS analysis indicated that the antiviral activity might be due to polyphenolic compounds with higher molecular weights, which strongly interact with stationary phases of different chromatographic systems. These still unknown compounds with probably high molecular weight could not be isolated in the present study. A variety of different flavonoid glycosides and caffeoyl quinic acids obtained from E. perfoliatum did definitely not contribute to the antiviral effect of the extract and its respective fractions. CONCLUSION: Hydroalcoholic extracts from the aerial parts of E. perfoliatum and its main active polyphenolic constituents protect cells from IAV infection by inhibiting viral attachment to the host cells. The extract appears to be a promising expansion of the currently available anti-influenza agents.

Flavonoid glycosides from Olax mannii: Structure elucidation and effect on the nuclear factor kappa B pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Olax mannii Oliv. (Olacaceae) is among the many medicinal plants used in Nigeria for the ethnomedicinal management of both cancer and inflammation. Such plants represent potential sources of innovative therapeutic agents for the treatment of cancer and other malignant disorders. While the majority of medicinal plants exert their anticancer effects by direct cytotoxicity on tumor cells, it is important that other mechanisms through which these plants can exhibit anticancer effects are investigated. Preliminary studies indicated that Olax mannii leaves are rich sources of novel flavonoid glycosides. The detailed chemistry as well the mechanisms through which these flavonoid constituents may exert their cancer chemo-preventive and therapeutic effects are, however, not yet investigated. AIM OF THE STUDY: The aim of this study is to carry out a detailed chemical investigation of Olax mannii leaves and the effects of the isolated constituents on the nuclear factor kappa B (NF-κB) pathway. MATERIALS AND METHODS: A methanol leaf extract was subjected to various chromatographic separations to achieve isolation of flavonoid glycosides and the structures of the isolated compounds were elucidated by a combination of 1D and 2D NMR and high resolution mass spectrometry. Biological activities were assessed by measurement of cellular viability and proliferation using quantitative IncuCyte videomicroscopy, trypan blue staining and by quantification of the number of metabolically active K562 cells based on quantitation of ATP. The effect of the compounds on the inhibition of the NF-κB pathway as well as toxicity towards peripheral blood mononuclear cells to evaluate differential toxicity was also assayed. RESULTS: Chemical investigation of the methanol leaf extract of the plant material led to the isolation of three new flavonoid triglycosides, kaempferol 3-O-[α-D-apiofuranosyl-(1 → 2)-α-L-arabinofuranoside]-7-O-α-L-rhamnopyranoside (1), kaempferol 3-O-[ß-D-glucopyranosyl-(1 → 2)-α-L-arabinofuranoside]-7-O-α-L-rhamnopyranoside (2), kaempferol 3-O-[ß-D-arabinopyranosyl-(1→4)-α-L-rhamnopyranoside]-7-O-α-L-rhamnopyranoside (3), in addition to fourteen known flavonoid glycosides (4-17). Of all the tested compounds, only compound 9 (kaempferol 3-O-α-L-rhamnopyranoside) exhibited promising and specific antiproliferative activity on human K562 chronic myelogenous leukemia cells and dose-dependently inhibited NF-κB transactivation. CONCLUSION: The presence of this flavonoid glycoside and derivatives may account for the reported efficacy of Olax mannii leaf extract in the ethnomedicinal management of cancer and inflammation.

Isolation and Quantification of Oligomeric and Polymeric Procyanidins in the Aerial Parts of St. John's Wort (Hypericum perforatum).

Proanthocyanidins (condensed tannins) constitute a class of oligomeric and polymeric polyphenols with flavan-3-ols as monomeric building blocks. Despite the high impact of proanthocyanidins, these polyphenols are mostly quantified by colorimetric methods or by chromatographic determination of the flavan-3-ols as cleavage products or low molecular oligomers as lead compounds. For St. John's wort (Hyperici herba) from Hypericum perforatum, a protocol for preparative isolation of oligomeric and polymeric proanthocyanidins from an acetone-water extract by chromatography on Sephadex®LH20 in combination with preparative high-performance liquid chromatography on the diol stationary phase was developed, yielding procyanidin reference clusters with a defined degree of polymerization from 3 to 10. Identity and purity of these clusters was proven by high-performance liquid chromatography (RP18 and diol phase) and mass spectrometry. For identification and quantification of proanthocyanidin clusters from St. John's wort, an ICH-Q2 (International harmonized guideline for analytical validation) validated high-performance liquid chromatography method with fluorimetric detection was developed using an acetone-water extract of the herbal material, purified by solid-phase extraction for the removal of naphthodianthrones. The method enabled the quantification of procyanidin clusters with a degree of polymerization from 2 to 10. Analysis of nine batches of Hyperici herba from different sources indicated a high variability of proanthocyanidin content in the range from 8 to 37 mg/g. In all of the batches investigated, the trimer cluster DP3 was the dominant proanthocyanidin (about 40 %), followed by DP 4 (about 15 %) and DP5 (about 12 %). Monitoring of procyanidin distribution during seasonal growth of fresh plants of H. perforatum indicated the highest proanthocyanidin content in young plants (about 50 mg/g) and a time-dependent decrease during the growing season to about 16 mg/g. The highest proanthocyanidin content was found in young leaves and flowers, while the fruits were proanthocyanidin-free; older parts of the stem and the herb had a lower proanthocyanidin content. From these data, it can be concluded that proanthocyanidins serve as part of the plant defense system in the reproductive organs.

Covalent modification of human serum albumin by the natural sesquiterpene lactone parthenolide.

The reactivity of parthenolide (PRT), a natural sesquiterpene lactone from Tanacetum parthenium (Asteraceae), with human serum albumin (HSA) was studied by UHPLC/+ESI-QqTOF MS analysis after tryptic digestion of albumin samples after incubation with this compound. It was found that the single free cysteine residue, C34, of HSA (0.6 mM) reacted readily with PRT when incubated at approximately 13-fold excess of PRT (8 mM). Time-course studies with PRT and its 11ß,13-dihydro derivative at equimolar ratios of the reactants revealed that PRT under the chosen conditions reacts preferably with C34 and does so exclusively via its α-methylene-γ-lactone moiety, while the epoxide structure is not involved in the reaction.

Gastroprotection as an example: antiadhesion against Helicobacter pylori, anti-inflammatory and antioxidant activities of aqueous extracts from the aerial parts of Lippia integrifolia Hieron.

ETHNOPHARMACOLOGICAL RELEVANCE: The aerial parts of Lippia integrifolia (Gris.) Hieronymus (Verbenaceae), known as incayuyo, are used by the peasant population of Argentina for treatment of diseases related to a gastrointestinal system, mainly for inflammation of the stomach and have also been included into the Argentina Food Code. This study aimed to investigate the phytochemical profile of hydrophilic extracts from the herbal material by LC-MS and to evaluate potential pharmacological mechanisms rationalizing the traditional use of incayuyo aqueous extracts potential anti-inflammatory treatment of gastrointestinal disorders. MATERIALS AND METHODS: Phytochemical profiling: LC-MS of an aqueous decoction. Antiadhesive effects against Helicobacter pylori: in vitro FACS assay using FITC-labeled bacteria and AGS human stomach cells. Influence of extracts on stomach cells and RAW 264.7 macrophages: MTT viability assay and BrdU proliferation ELISA. Influence of extracts on IL-6 and IL-8 secretion from stomach cells was quantitated by ELISA after infection of the cells with Helicobacter pylori. Influence of test extracts on macrophages: phagocytosis of FITC-labeled Zymosan particles and NO production. Antioxidative capacity: DPPH assay and O2-induced caroten oxidation. RESULTS: LC-MS profiling indicated the presence of compounds 1-20 with flavonoid hexosides, phenylethanoides (acteoside, isoacteoside) and sesquiterpenes [(epi)lippidulcine, peroxylippidulcine] in the decoction extract and subfraction PhF. The extract exhibits strong in vitro antioxidative capacity and inhibited adhesion of Helicobacter pylori to stomach cells up to 40%, while an EtOH-soluble fraction showed inhibition rates of up to 60%. Decoction increased the cellular viability of AGS cells significantly at >10 µg/mL, while proliferation rate was not influenced. Helicobacter pylori induced IL-8 secretion was significantly reduced by coincubation of AGS cells with the extracts. Aqueous extracts stimulated phagocytosis rate of macrophages and inhibited the LPS-induced NO-secretion. CONCLUSIONS: The traditional use of aqueous extracts from Lippia integrifolia for gastric inflammation seems to be rationalized: besides anti-inflammatory effects on stomach cells antiadhesive properties of the extracts against the main bacterial inductor of gastritis Helicobacter pylori were identified. Additional effects for stimulation of innate immunity and potential radical scavenging effects may additionally contribute to the activity of the extracts.

Arabinogalactan protein cluster from Jatropha curcas seed embryo contains fasciclin, xylogen and LysM proteins.

An non-GPI-anchored AGP cluster (Y2) was isolated from the seeds of Jatropha curcas L. (Euphorbiaceae) composed of 4.8% polypeptides (mainly Ala, Ser, Gly, Hyp, Glu) and a carbohydrate moiety composed of Gal, Ara, GlcA, Rha, Man and GlcN. Besides the typical structural features of arabinogalactan proteins, typical N-glycan linker of the complex type (GlcNAc4Man3Gal2Fuc1Xyl1) were identified. O-glycosylation occurred mainly via Hyp and to a lesser extent via Thr and Ser. N-glycans from the complex type, carrying at the innermost GlcNAc at position O-3 one α-Fuc-residue, were also present. MS analysis of the tryptic digest assigned peptides of three major protein groups: fasciclin-like arabinogalactan proteins, xylogen-like proteins and LysM domain-containing proteins. They could not be separated further and it is indicated that various homologous protein forms co-exist. Histological investigation of J. curcas seeds revealed the presence of AGPs in the vessels of cotyledons and in the procambium ring of the embryo.

Wound-healing plants from TCM: in vitro investigations on selected TCM plants and their influence on human dermal fibroblasts and keratinocytes.

Wound-healing plants from Traditional Chinese Medicine and described for wound healing in the Pharmacopoeia of People's Republic of China (2005 ed.) were investigated by in vitro bioassay on human skin cells. Therefore water and EtOH-water extracts (6:4, v/v) from 12 plants were tested on human primary dermal fibroblasts (pNHDF) and human HaCaT keratinocyte cell line by quantification of cell viability (MTT assay) and cellular proliferation (BrdU incorporation ELISA). No functional activity was found for extracts from Achyranthis bidentatae rhizoma, Cimicifugae rhizoma, Corydalis rhizoma, Gardeniae fructus, Houttuyniae herba, Lonicerae japonicae caulis, Paeoniae rubrae radix and Rehmanniae radix. Extracts from Notoginseng radix et rhizoma, Angelicae sinensis radix and Lonicerae japonicae flos showed moderate activity, while extracts from Moutan cortex (the root bark of Paeonia suffruticosa Andr., Ranunculaceae) increased cell viability of HaCaT keratinocytes and pNHDF in a dose-dependent manner significantly. Bioassay-guided fractionation yielded paeonol 1, the flavan-3-ols catechin 2 and epicatechin-3-O-gallate 3, the dimeric proanthocyanidin epicatechin-(4ß→8)-catechin 4, a mixture of trigalloyl-glucoses 5 and 1,2,3,4,6-penta-O-galloyl-ß-d-glucose (PGG) 6. The proanthocyanidin-containing fractions as well as PGG-containing fractions contributed substantially to the stimulating effects. Especially PGG-containing fractions enhanced cell viability and cellular proliferation of HaCaT keratinocytes at concentration of 100nM. From these data we conclude that indication claims for TCM herbal materials must be carefully investigated in order to establish evidence-driven use of such plants. In case of Moutan cortex skin cell stimulating effects have clearly been proven. These effects can be related to the polyphenol fractions of condensed and hydrolysable tannins.

Regulation of dioxin receptor function by different beta-carboline alkaloids.

The dioxin receptor, also known as arylhydrocarbon receptor (AhR), is a ligand-activated transcription factor that mediates the toxicity of dioxins and related environmental contaminants. In addition, there is a growing list of natural compounds, mainly plant polyphenols that can modulate AhR function and downstream signaling with quite unknown consequences for cellular function. We investigate the potential of four different beta-carboline alkaloids to stimulate AhR signaling in human hepatoma cells and keratinocytes. Three test substances, namely rutaecarpine, annomontine and xestomanzamine A, increase AhR-driven reporter gene activity as well as expression of two AhR target genes in a dose-dependent and time-dependent manner. Additionally, the three test alkaloids stimulate cytochrome P450 (CYP) 1 enzyme activity without showing any antagonistic effects regarding benzo(a)pyrene-stimulated CYP1 activation. The AhR-activating property of the beta-carbolines is completely abrogated in AhR-deficient cells providing evidence that rutaecarpine, annomontine and xestomanzamine A are natural stimulators of the human AhR. The toxicological relevance of beta-carboline-mediated AhR activation is discussed.

Chromones from the endophytic fungus Pestalotiopsis sp. isolated from the chinese mangrove plant Rhizophora mucronata.

Six new chromones, named pestalotiopsones A-F (1-6), and the known derivative 7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone (7) were obtained from the mycelia and culture filtrate of the mangrove endophytic fungus Pestalotiopsis sp., which was isolated from leaves of the Chinese Mangrove plant Rhizophora mucronata. Their structures were elucidated on the basis of spectroscopic data. Pestalotiopsones A-F are chromones having both an alkyl side chain substituted at C-2 and a free or substituted carboxyl group at C-5. Compound 6 exhibited moderate cytotoxicity against the murine cancer cell line L5178Y, whereas the other investigated compounds proved to be inactive.