Polyphenolic Characterization, Antioxidant, Antihyaluronidase and Antimicrobial Activity of Young Leaves and Stem Extracts from Rubus caesius L.

Fruits are the main food part of the European dewberry (Rubus caesius L.), known as a source of polyphenols and antioxidants, while very little attention is paid to leaves and stems, especially young first-year stems. The purpose of this work was to analyze for the first time water and ethanol extracts obtained from young, freshly developed, leaves and stems of the European dewberry to determine their antioxidant and biological activity, whereas most of the papers describe biological properties of leaves collected during summer or autumn. As the phytochemical profile changes during the growing season, the quantitative and qualitative content of flavonoid glycosides and flavonoid aglycones was analyzed using reversed phase liquid chromatography/electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS) with multiple reaction monitoring (MRM). The ability to inhibit hyaluronidase as well as antioxidant activity (2,2 diphenyl-1-picrylhydrazyl: DPPH and ferric antioxidant power: FRAP) were estimated. Extracts were also analyzed against Gram-positive and Gram-negative bacteria. The results of the qualitative phytochemical analysis indicated the presence of flavonoid aglycones and flavonoid glycosides, with the highest amount of tiliroside, hyperoside, isoquercetin, astragalin, rutin and catechin in ethanol extracts. DPPH and FRAP tests proved the high antioxidant activity of the extracts from leaves or stems and the antihyaluronidase assay revealed for the first time that water and ethanol extracts obtained from the stems exhibited the ability to inhibit hyaluronidase activity resulting in an IC50 of 55.24 ± 3.21 and 68.7 ± 1.61 µg/mL, respectively. The antimicrobial activity has never been analyzed for European dewberry and was the highest for Clostridium bifermentans and Clostridium sporogenes-anaerobic sporulation rods as well as Enterococcus faecalis for both water and ethanol extracts.

An In Vitro Anticancer, Antioxidant, and Phytochemical Study on Water Extract of Kalanchoe daigremontiana Raym.-Hamet and H. Perrier.

Kalanchoe species are succulents with anti-inflammatory, antioxidant, and analgesic properties, as well as cytotoxic activity. One of the most popular species cultivated in Europe is Kalanchoe daigremontiana Raym.-Hamet and H. Perrier. In our study, we analyzed the phytochemical composition of K. daigremontiana water extract using UHPLC-QTOF-MS and estimated the cytotoxic activity of the extract on human ovarian cancer SKOV-3 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, flow cytometry, luminometric, and fluorescent microscopy techniques. The expression levels of 92 genes associated with cell death were estimated via real-time PCR. The antioxidant activity was assessed via flow cytometry on human keratinocyte HaCaT cell line. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and FRAP (ferric-reducing antioxidant power) assays were also applied. We identified twenty bufadienolide compounds in the water extract and quantified eleven. Bersaldegenin-1,3,5-orthoacetate and bryophyllin A were present in the highest amounts (757.4 ± 18.7 and 573.5 ± 27.2 ng/mg dry weight, respectively). The extract showed significant antiproliferative and cytotoxic activity, induced depolarization of the mitochondrial membrane, and significantly arrested cell cycle in the S and G2/M phases of SKOV-3 cells. Caspases-3, 7, 8, and 9 were not activated during the treatment, which indicated non-apoptotic cell death triggered by the extract. Additionally, the extract increased the level of oxidative stress in the cancer cell line. In keratinocytes treated with menadione, the extract moderately reduced the level of oxidative stress. This antioxidant activity was confirmed by the DPPH and FRAP assays, where the obtained IC50 values were 1750 ± 140 and 1271.82 ± 53.25 µg/mL, respectively. The real-time PCR analysis revealed that the extract may induce cell death via TNF receptor (tumor necrosis factor receptor) superfamily members 6 and 10.

Cytotoxic activity of standardized extracts, a fraction, and individual secondary metabolites from fenugreek seeds against SKOV-3, HeLa and MOLT-4 cell lines.

CONTEXT: Trigonella foenum-graecum L. (Fabaceae) has many therapeutic properties and anticancer potential. OBJECTIVE: The cytotoxic activities of standardized extracts and a fraction from fenugreek seeds and their compounds (sapogenins, flavone C-glycosides, alkaloid trigonelline) against human cancer SKOV-3, HeLa and MOLT-4 cells were evaluated. MATERIALS AND METHODS: Fenugreek seeds were extracted with 70% methanol (A) or water (B). Furthermore, the seeds were purified with petroleum ether and chloroform and next extracted with methanol to obtain fraction (C). The quantitative analysis of saponins and flavonoids in the extracts was done with HPLC methods. The extracts (5-120 µg/mL) and compounds (1-50 µg/mL) were tested on the cells by MTT assay and RTCA system. The effect of a fraction on ROS production, mitochondrial membrane potential and caspase-3/7 activity in HeLa and SKOV-3 cells was also evaluated by flow cytometry. RESULTS: The strongest cytotoxic activity on cancer cells showed the fraction C (IC50 was 3.91 ± 0.03 for HeLa, 3.97 ± 0.07 for SKOV-3, and 7.75 ± 0.37 for MOLT-4) with the highest content of steroidal saponins (163.18 ± 11.03 µg/mg) and flavone C-glycosides (820.18 ± 0.05 µg/mg). The fraction significantly increased ROS production (up to four times higher than in keratinocytes as control) and caspases activity in the cells. The examined flavonoids did not exhibit the cytotoxic activity in contrast to yamogenin, tigogenin, and diosgenin. CONCLUSIONS: The obtained results complement the data on the cytotoxic activity of Foenugraeci Semen and synergistic effect of flavonoids and saponins complex contained in the plant.

Bersaldegenin-1,3,5-orthoacetate induces caspase-independent cell death, DNA damage and cell cycle arrest in human cervical cancer HeLa cells.

CONTEXT: Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. OBJECTIVE: The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate - a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. MATERIALS AND METHODS: The cytotoxic activity of the compound (at 0.1-20.0 µg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. RESULTS: The compound had strong effect on the cells (IC50 = 0.55 µg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. CONCLUSIONS: Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides.

Identification of Flavonoids and Bufadienolides and Cytotoxic Effects of Kalanchoe daigremontiana Extracts on Human Cancer Cell Lines.

Kalanchoe species are well-known medicinal plants used in traditional medicine as anti-inflammatory and analgesic remedies. Recently, it has been reported that Kalanchoe plants have cytotoxic properties; however, data on traditional use of these plants in tumor treatment are extremely limited. Kalanchoe daigremontiana is one of the most popular species cultivated in Europe, and it is used, among other things, as a remedy in treating skin injuries and wounds. Studies on the biological activity of this species are scarce, and there is a lack of data on the cytotoxic activity of K. daigremontiana extracts on epithelial cancer cells in the literature. In our present study, we analyzed the phytochemical composition of K. daigremontiana ethanol extract and fractions-water and dichloromethane-by the HPLC-DAD-ESI-MS method and estimated cytotoxic activity of the extracts on human adenocarcinoma (HeLa), ovarian (SKOV-3), breast (MCF-7), and melanoma (A375) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, real-time cell analyzer (RTCA), and flow cytometry. We identified 6 bufadienolide compounds and 19 flavonoids, mostly kaempferol, quercetin, isorhamnetin, and myricetin glycosides, of which only 3 flavonoids have been identified in K. daigremontiana to date. Other flavonoids that were characterized in our study have not yet been found in this plant. The ethanol extract and water fraction of K. daigremontiana did not show significant cytotoxic activity on the tested cell lines. In contrast, the dichloromethane fraction showed the strongest activity against all cell lines with IC50 values of ≤ 10 µg/mL. The results indicated that this activity is mainly due to the presence of bersaldegenin-1,3,5-orthoacetate.

Securinine from Phyllanthus glaucus Induces Cell Cycle Arrest and Apoptosis in Human Cervical Cancer HeLa Cells.

BACKGROUND: The Securinega-type alkaloids occur in plants belonging to Euphorbiaceae family. One of the most widely distributed alkaloid of this group is securinine, which was identified next to allosecurinine in Phyllanthus glaucus (leafflower). Recently, some Securinega-type alkaloids have paid attention to its antiproliferative potency towards different cancer cells. However, the cytotoxic properties of allosecurinine have not yet been evaluated. METHODS: The cytotoxicity of the extract, alkaloid fraction obtained from P. glaucus, isolated securinine and allosecurinine against HeLa cells was evaluated by real-time xCELLigence system and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and 7-amino-actinomycin (7-AAD) staining and confirmed with fluorescent Hoechst 33342 dye. The assessment of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, the level of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), caspase-3/7 activity and cell cycle analysis were measured by flow cytometry. The enzymatic activity of caspase-9 was assessed by a luminometric assay. The expression of apoptosis associated genes was analyzed by real-time PCR. RESULTS: The experimental data revealed that securinine and the alkaloid fraction were significantly potent on HeLa cells growth inhibition with IC50 values of 7.02 ± 0.52 µg/ml (32.3 µM) and 25.46 ± 1.79 µg/ml, respectively. The activity of allosecurinine and Phyllanthus extract were much lower. Furthermore, our study showed that the most active securinine induced apoptosis in a dose-dependent manner in the tested cells, increased the percentage of ROS positive cells and depolarized cells as well as stimulated the activity of ERK1/2, caspase-9 and -3/7. Securinine also induced cell cycle arrest in S phase. Real-time PCR analysis showed high expression of TNFRSF genes in the cells stimulated with securinine. CONCLUSIONS: Securinine induces apoptosis and activates cell cycle checkpoints in HeLa cells which is associated with oxidative stress. The results indicate that the mitochondrial pathway is involved in the programmed cell death.

Isolation and identification of cytotoxic compounds from the rhizomes of Paris quadrifolia L.

BACKGROUND: Paris quadrifolia L. is a medicinal plant which contains steroidal saponins. The present study reports isolation and structural identification of six pennogenyl saponins obtained from P. quadrifolia rhizomes. The four spirostan saponins were obtained from P. quadrifolia for the first time. The cytotoxic effects of the sub-fractions and six compounds isolated from the plant extract were evaluated on tumour cells. MATERIALS AND METHODS: Ethanol extract from the rhizomes of P. quadrifolia were partinioned using column chromatography. The saponins were isolated from the obtained sub-fractions by isocratic RP HPLC and their structures were determined by means of 1D and 2D NMR spectroscopy and MALDI TOF MS. The cytotoxic effects of the sub-fractions and the isolated compounds were tested against human promyelocytic leukaemia cells (HL-60), human cervical adenocarcinoma cells (HeLa) and human breast cancer cells (MCF-7) using the [(3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Six pennogenyl saponins were isolated from P. quadrifolia rhizomes: pennogenin 3-O-ß-D-glucopyranoside (1), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranoside (2), pennogenin 3-O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranoside (3), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranoside (4), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside (5), pennogenin 3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranoside (6). Pennogenyl saponins 5 and 6 exhibited cytotoxic activity against HL-60, HeLa and MCF-7 tumour cells with IC50 values of 1.0 ± 0.04 µg/ml, 1.8 ± 0.072 µg/ml and 2.4 ± 0.096 µg/ml respectively, and 2.0 ± 0.08 µg/ml, 2.5 ± 0.125 µg/ml and 3.2 ± 0.128 µg/ml respectively. CONCLUSION: Compounds 1-4 were isolated from this species for the first time.

The phytochemical and genetic survey of common and dwarf juniper (Juniperus communis and Juniperus nana) identifies chemical races and close taxonomic identity of the species.

Juniperus communis L. (= J. communis var. communis) and Juniperus nana Willd. (= J. communis var. SAXATILIS) are subspecies of juniper. J. communis grows widely in both hemispheres, primarily in lower elevations while J. nana is mainly observed in high mountains. Although they can be distinguished by morphological features, it is not known whether they are genetically and phytochemically distinct entities. We aimed to check whether it is possible to distinguish these two plants (i) by pharmaceutically important chemical traits and (ii) on the basis of intraspecifically highly polymorphic fragment of chloroplast DNA. We used GC with achiral as well as with enantioselective stationary phase columns to identify the main monoterpenes of the essential oil. Sequence analysis of the TRNL (UAA)- TRNF (GAA) intergenic spacer of the chloroplast genome was used as a genetic marker of taxonomic identity between these two subspecies. The chromatographic analysis showed the existence of three chemical races - the alpha-pinene type, the sabinene type and one with intermediate contents of these terpenes among both J. communis and J. nana. Surprisingly, sequence analysis of TRNL (UAA)- TRNF (GAA) revealed 100 % similarity between the common and the dwarf juniper. Thus, the monoterpene pattern is related to geographical origin, and not to the species identity. We suggest that the three chemical races identified in the present study should be considered as separate sources of pharmaceutical raw material. Our results demonstrate that the contents of alpha-pinene and sabinene may be applied as a quick diagnostic test for preliminary evaluation of plant material.

Molecular genetic survey of European mistletoe (Viscum album) subspecies with allele-specific and dCAPS type markers specific for chloroplast and nuclear DNA sequences.

The qualitative and quantitative content of mistletoe metabolites, and bioactivity of extracts is related to the subspecies of Viscum album L. These were indicated to be genetically distinct and host specific. We aimed to check (i) whether the specificity is strict and (ii) how frequently hybridization occurs among the subspecies. We designed two sets of allele-specific and dCAPS molecular genetic markers that would facilitate identification of Viscum album L. subspecies and their hybrid derivatives on the basis of chloroplast trnH(GUG)- trnK(UUU) and nuclear rDNA ITS1&2 sequences. Out of 118 plants surveyed, 103 displayed characteristics that confirmed strict host specificity of the subspecies, in addition, the results were compliant between nuclear and chloroplast markers showing no indication of hybridization among subspecies. From 15 samples that showed deviations from this model 13 came from the Mediterranean Sea basin, and only two originated from Central and Western Europe. Abbreviations. dCAPS:derived Cleaved Amplified Polymorphic Sequence ITS1&2:Internal Transcribed Spacers 1&2 MAMA:Mismatch Amplification Mutation Assay

Antibacterial and antifungal activity of juniper berry oil and its selected components.

Juniper berry oil is stated to possess a wide spectrum of pharmacological activities and its monographs are included in some National Pharmacopoeias. The antibacterial and antifungal activity of the oil was reported by some authors. In our study we estimated the antibacterial and antifungal activity of three different juniper berry oils and their main components. All the micro-organisms used in this experiment were isolated from patients of Regional Hospital of Gdansk and some of them showed resistance against commonly used antibiotics. Only one of the oils (labelled A) revealed good antimicrobial properties. None of the single oil components was a stronger antibacterial and antifungal inhibitor than the oil A itself. Our data suggest that the antimicrobial activity of juniper oil A is the result of either the specific composition of the oil A (highest concentration of (-)-alpha-pinene, p-cymene and beta-pinene) or activity of a single non-identified compound. The presence of an adulterant in the oil was excluded.