Secondary metabolites from the underground parts of Valeriana sisymbriifolia Vahl. and their in vitro cytotoxic activities.

Cytotoxic activity-guided isolation studies on the underground parts of Valeriana sisymbriifolia Vahl. led to the isolation of 12 secondary metabolites including two undescribed iridoids, sisymbriifolivaltrate and sisymbriifolioside, and two unreported sesquiterpene lactones, sisymbriifolins A and B. Chemical structures of the isolates were established by extensive 1D and 2D NMR analyses as well as HR-ESI-MS. The in vitro cytotoxic activities of the extract, sub-fractions and isolates on lung (A549), breast (MCF7), gastric (HGC27) and prostate (PC3) cancer cell lines were evaluated by MTS assay. Sisymbriifolivaltrate, didrovaltrate, valtrate, 7-homovaltrate and 1-α-acevaltrate exhibited promising cytotoxic activity on MCF7 cell line with IC50 values ranging from 2.5 to 12.3 µM, while valtrate demonstrated the best cytotoxicity against A549 cells with the IC50 value of 7.5 µM. Valtrate and 7-homovaltrate were found to exert noteworthy cytotoxicity towards HGC27 cell line (IC50 values: 2.3 and 3.7 µM, respectively), whereas valtrate, 7-homovaltrate and 1-α-acevaltrate (IC50 values: 2.3-9.7 µM) were found to be potent cytotoxic against PC3 cells. Among the tested compounds, particularly valepotriate-type iridoids were found to be the main cytotoxic principles of V. sisymbriifolia.

Cytotoxic and Antimigratory Activity of Retrochalcones from Glycyrrhiza echinata L. on Human Cancer Cells.

Cytotoxic activity-guided fractionation studies on Glycyrrhiza echinata roots led to the isolation of eight compounds (1-8). Chemical structures of the isolates were identified by NMR and MS analysis. Among the tested molecules, retrochalcones namely echinatin (3) (IC50 =23.45-41.83 µM), licochalcone B (4) (IC50 =36.04-39.53 µM) and tetrahydroxylmethoxychalcone (5) (IC50 =7.09-80.81 µM) were the most active ones against PC3, MCF7 and HepG2 cells. Moreover, 5 exhibited selectivity on prostate cancer cells (SI: 5.19). Hoechst staining and Annexin V/PI binding assays as well as cell cycle analysis on the compounds 3 (23 µM) and 5 (5 and 7 µM) demonstrated that these retrochalcones induced apoptosis and significantly suppressed cell cycle in G1 and G2 /M phases. Furthermore, 3 and 5 showed antimigratory effects on PC3 cells by wound healing assay. The results indicated that tested retrochalcones most particularly 5 could be potential anticancer drug candidates that prevent proliferation and migration of cancer cells.

Activity-Guided Isolation of Cytotoxic Non-Glycosidic Ester Iridoids from Valeriana alliariifolia Adams and Unravelling Their Cell Death Mechanisms.

The aim of this study was to isolate the cytotoxic compounds from V. alliariifolia via activity-guided isolation and to determine the mechanism of actions of the most potent ones. The crude EtOH extract as well as CHCl3 and AcOEt subextracts demonstrated remarkable cytotoxic activities against A549, MCF7, HGC27 and PC3 cancer cells. Sequential chromatographic separations on active subextracts yielded 14 secondary metabolites, including 11 iridoids (1-11) most of which belong to non-glycosidic ester iridoids, two phenylpropanoids (12 and 13) and one lignan (14). The chemical structures of purified compounds were elucidated by NMR and MS analysis. Among the isolates, 7-deisovaleroylvaltrate (3) was isolated for the first time as a natural product. According to the cytotoxic assay compounds, 2, 4-6 and 8 were found to be the potent cytotoxic compounds (IC50 <10 µM) against at least one of the tested cancer cell lines. Thus, 2, 4-6 and 8 were investigated for their effects on apoptotic, necrotic and autophagic pathways as well as cell cycle progression. They exerted anticancer activities by inducing different cell death mechanisms depending on the cancer cells. The results demonstrated that 2, 4-6 and 8 could be potential anticancer drug leads that deserve further in vivo and clinical studies on the way to discover novel natural compounds with anticancer properties.

The integrated use of in silico methods for the hepatotoxicity potential of Piper methysticum.

Herbal products as supplements and therapeutic intervention have been used for centuries. However, their toxicities are not completely evaluated and the mechanisms are not clearly understood. Dried rhizome of the plant kava (Piper methysticum) is used for its anxiolytic, and sedative effects. The drug is also known for its hepatotoxicity potential. Major constituents of the plant were identified as kavalactones, alkaloids and chalcones in previous studies. Kava hepatotoxicity mechanism and the constituent that causes the toxicity have been debated for decades. In this paper, we illustrated the use of computational tools for the hepatotoxicity of kava constituents. The proposed mechanisms and major constituents that are most probably responsible for the toxicity have been scrutinized. According to the experimental and prediction results, the kava constituents play a substantial role in hepatotoxicity by some means or other via glutathione depletion, CYP inhibition, reactive metabolite formation, mitochondrial toxicity and cyclooxygenase activity. Some of the constituents, which have not been tested yet, were predicted to involve mitochondrial membrane potential, caspase-3 stimulation, and AhR activity. Since Nrf2 activation could be favorable for prevention of hepatotoxicity, we also suggest that these compounds should undergo testing given that they were predicted not to be activating Nrf2. Among the major constituents, alkaloids appear to be the least studied and the least toxic group in general. The outcomes of the study could help to appreciate the mechanisms and to prioritize the kava constituents for further testing.

Antiproliferative Activity of Chemically Characterized Propolis from Turkey and Its Mechanisms of Action.

The aim of this study was to evaluate the ethanolic extract of propolis originated from northern Turkey for its antiproliferative, apoptotic and cell cycle arrest promoting effects on MCF7, HGC27, A549 cancer cell lines and a healthy cell line (HUVEC) in terms of DNA content, morphological features, expression of cell cycle checkpoint proteins p21, p53, Cyclin D1 and immune checkpoint protein PD-L1. The extract showed moderate antiproliferative activity against all tested cancer cell lines with IC50 values in the range of 58.6-90.7 µg/mL in MTS assay. Further studies indicated that propolis extract exerted apoptotic effect on cancer cell lines, promoted cell cycle arrest through activation of p21 and resulted in accumulation at G0/G1 phase of cancer cells. Propolis treatment caused increased cell size, according to fluorescent imaging except for MCF7. HPTLC analysis revealed that 3-O-methylquercetin, chrysin, caffeic acid, CAPE, galangin and pinocembrin were the main components of the extract. The amounts of caffeic acid and CAPE in the extract were found to be 5.5 and 11.1 mg/g, respectively, by a validated HPLC method. Our study is the first one, revealing effect of propolis on PD-L1 expression on certain cancer cell lines.

Mechanisms of action of cytotoxic phenolic compounds from Glycyrrhiza iconica roots.

BACKGROUND: Glycyrrhiza (licorice) species are rich in bioactive secondary metabolites and their roots are used traditionally for the treatment of several diseases. In recent years, secondary metabolites of licorice are gaining popularity, especially due to their significant cytotoxic and antitumor effects. However, Glycyrrhiza iconica, an endemic species to Turkey, was not investigated in terms of its anticancer secondary metabolites previously. PURPOSE: This study aimed to isolate the cytotoxic compounds from G. iconica through bioactivity-guided fractionation and to elucidate mechanisms of action of the most potent compounds. METHODS: Total MeOH extract and CHCl3, EtOAc, n-buOH and rH2O subextracts were prepared from G. iconica roots. Sequential chromatographic techniques were conducted for the isolation studies. The chemical structures of the isolates were established based on NMR and HR-MS analysis. Sulforhodamine B assay was used to evaluate the cytotoxic activity of extracts, main fractions as well as isolates against hepatocellular (Huh7), breast (MCF7) and colorectal (HCT116) cancer cell lines. The mechanisms underlying the cytotoxicity of the most active compounds in Huh7 cells were elucidated by using Hoechst staining, Fluorescence-activated cell sorting and Western blot assays. RESULTS: A new dihydrochalcone, iconichalcone (1) along with 15 known phenolic compounds were isolated from the active CHCl3, EtOAc and n-buOH subextracts. Compounds 2-5, 7-16 were found to be responsible for the in vitro cytotoxic activity of G. iconica against all tested cancer cell lines with IC50 values ranging from 2.4 to 33 µM. Amongst these compounds, licoricidin (10), dehydroglyasperin C (12), iconisoflaven (13) and 1-methoxyficifolinol (15) were found to be the most active compounds according to SRB and real time bioactivity assays and submitted to further mechanistic investigations in Huh7 cells. Compounds 10, 12, 13 and 15 caused accumulation of cells in different phases of cell cycle. Moreover, 10, 12, 13 and 15 induced apoptosis through caspase activation. Besides, 12 showed activation of p53 expression and thus G2/M arrest as well as a condensed nuclei, established very promising results. CONCLUSION: The results demonstrated that the aforementioned compounds, particularly 12 could be potential lead molecules for anticancer drug development that deserve further in vivo and clinical investigations.

Phenolic compounds from the aerial parts of Clematis viticella L. and their in vitro anti-inflammatory activities.

Phytochemical investigations on the EtOH extract of Clematis viticella led to the isolation of six flavonoid glycosides, isoorientin (1), isoorientin 3'-O-methyl ether (2), quercetin 7-O-α-L-rhamnopyranoside (3), quercetin 3,7-di-O-α-L-rhamnopyranoside (4), manghaslin (5) and chrysoeriol 7-O-ß-D-glucopyranoside (6), one phenylethanol derivative, hydroxytyrosol (7), along with three phenolic acids, caffeic acid (8), (E)-p-coumaric acid (9) and p-hydroxybenzoic acid (10). The structures of the isolates were elucidated on the basis of NMR and HR-MS data. All compounds were isolated from C. viticella for the first time. Compounds 7 and 8 showed significant anti-inflammatory activity at 100 µM by reducing the release of NO in LPS-stimulated macrophages comparable to positive control indomethacin. Compounds 3 and 7 exhibited anti-inflammatory activity through lowering the levels of TNF-α while 1, 3 and 5 decreased the levels of neopterin better than the positive controls.

New iridoids from the roots of Valeriana dioscoridis Sm.

Four new iridoids (1-4), together with three known iridoids (5-7), one known flavonoid glycoside, three phenolic acids and one phytosterol were isolated from the roots of Valeriana dioscoridis. Their structures were elucidated by means of NMR spectroscopy and mass spectrometry. This is the first report on the phytochemical composition of the non-volatile constituents of V. dioscoridis and the occurrence of a bis-iridoid glycoside in the genus Valeriana. The antiproliferative effects of the iridoids (1-7) were evaluated against three human cancer cell lines of gynacological origin (HeLa, A2780 and T47D) at 10, 30 and 60 µM concentrations, using the MTT assay and they elicited modest antiproliferative activity when compared to the reference agent, cisplatin.

Sambulin A and B, non-glycosidic iridoids from Sambucus ebulus, exert significant in vitro anti-inflammatory activity in LPS-induced RAW 264.7 macrophages via inhibition of MAPKs's phosphorylation.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Sambucus ebulus L. (Adoxaceae) are widely used in Turkish folk medicine particularly against inflammatory disorders. The fresh leaves after wilted over fire or the poultices prepared are directly applied externally to heal burns, edema, eczema, urticarial and abscess. Two iridoids were recently isolated (sambulin A, sambulin B) from the leaves of S. ebulus. AIM OF THE STUDY: This study aims to investigate the in vitro anti-inflammatory activities of these iridoids on LPS-induced RAW 264.7 macrophages. MATERIALS AND METHODS: Raw 264.7 macrophages were treated with 12.5, 25 and 50µg/ml Sambulin A and 6.25, 12.5 and 25µg/ml Sambulin B and induced with 1µg/ml lipopolysaccaharides (LPS). Effect of the compounds on nitric oxide (NO) production and cytokines (TNFα, IL-6) were determined by Griess and ELISA assays respectively. iNOS and the phosphorylation levels of MAPKs (ERK, JNK) were examined by Western Blot. RESULTS: Sambulin A and sambulin B inhibited 52.82% and 72.88% of NO production at 50 and 25µg/ml concentrations respectively. The levels of iNOS were significantly decreased by both molecules, sambulin B at 25µg/ml almost completely decreased iNOS levels (97.53%). Both molecules significantly inhibited TNFα productions. However, only sambulin B inhibited IL-6 production. Consequently, it was shown that sambulin B exerted its effect through the inhibition of ERK and JNK phosphorylations. CONCLUSION: The prominent bioactivities exerted by two iridoids will contribute to explanation of the usage of S. ebulus in traditional medicine against rheumatoid diseases.

Secondary Metabolites from the Leaves of Digitalis viridiflora.

A new phenylethanoid glycoside, named digiviridifloroside (1), was isolated from the leaves of Digitalis viridiflora Lindley along with a known phenylethanoid glycoside, calceolarioside A (2), two flavonoid glycosides, scutellarein 7-Ο-ß-D-glucopyranoside (3) and hispidulin 7-0-ß-D-glucopyranoside (4), two cleroindicins, cleroindicins B (5) and F (6), a nucleoside, adenosine (7), as well as a mixture of ß-glucopyranosyl-(1-6)-4-O-caffeoyl-α/ß glucopyranose and 3,4-dihydroxyphenylethanol. The structure of the new compound was established as 3,4-dihydroxy-ß-phenylethoxy-6-O-(E)-feruloyl-ß- glucopyranosyl-(l->6)-4-0-(E)-caffeoyl-ß-glucopyranoside (1) based on extensive ID- and 2D-NMR spectroscopy, as well. as HR-ESI-MS. Digiviridifloroside represents a rare type of phenylethanoid glycoside which bears two aromatic acyl units in its structure. In addition to phytochemical studies, the isolates were evaluated for their in vitro antimicrobial activities against three pathogenic bacteria and three yeast strains using a microdilution method. Among the tested compounds, 5 exhibited moderate antibacterial activity against Bacillus cereus NRRLB 3711 with a MIC value of 25 µg/mL, whereas compounds 5 and 6 showed relatively high anticandidal activity against Candida strains with MIC values down to 12.5 µg/mL, in comparison to the standard antimicrobial compounds.

Bioactivites of two common polyphenolic compounds: Verbascoside and catechin.

CONTEXT: Natural products can present remarkable biological and pharmacological activities. In traditional medicine, plants have been used historically in treating cancer, infections, and other inflammatory conditions. OBJECTIVE: Verbascoside and catechin are widespread polyphenolic plant compounds that could play a role in the anti-inflammatory and health-promoting effects of plants and plant extracts. MATERIALS AND METHODS: This study compares the potential cytotoxic effects of polyphenols verbascoside and catechin (6.25-200 µM) on human peripheral blood mononuclear cells (PBMC) for 48 h and myelomonocytic THP-1 and THP-1 Blue cells for 24 h. The effects of the compounds on immune activation markers such as indoleamine 2,3-dioxygenase (IDO) activity as well as on neopterin formation and nuclear factor-κB (NF-κB) activation were investigated. Cytotoxicity of the compounds was tested using Cell-Titer Blue assay. RESULTS: Verbascoside exhibited significant suppressive effects in mitogen-stimulated PBMC on tryptophan breakdown (>50 µM; IC50 value: 58.6 µM) and the production of neopterin (>6.25 µM; IC50 value: 217 µM). These effects correlated with a decline in cell viability, while THP-1 Blue cells were less sensitive. NF-κB activity was slightly enhanced at lower concentrations (<50 µM verbascoside) in stimulated cells and at the highest concentration used in unstimulated cells. Catechin had no relevant effects on cell viability and on the tested inflammation markers, except NF-κB activation in THP-1 Blue cells. DISCUSSION AND CONCLUSION: The results obtained show that verbascoside and catechin represent effective compounds which interfere with immunobiochemical pathways that are highly relevant for immunosurveillance and competing virus infections.

Prenylated polyphenolic compounds from Glycyrrhiza iconica and their antimicrobial and antioxidant activities.

A new prenylated isoflavan, iconisoflavan (1), and a new prenylated isoflav-3-ene, iconisoflaven (2) were isolated from the roots of Glycyrrhiza iconica together with four known ones namely (3S)-licoricidin (3), licorisoflavan A (4), topazolin (5) and glycycoumarin (6). The structures were elucidated on the basis of extensive spectroscopic analysis including 1D and 2D NMR as well as HR-MS. Furthermore, the absolute configurations of compounds 1, 3 and 4 were established by electronic circular dichroism (ECD). All the isolated compounds (1-6) were evaluated for their in vitro antimicrobial activities against five pathogenic bacteria and one yeast (Candida albicans) using an in vitro microdilution method. Compounds 1 and 3-5 displayed significant activity against Salmonella typhimurium ATCC 13311 with MIC values ranging from 2 to 8 µg/mL. Additionally, all compounds were screened for their in vitro free radical scavenging activities using an in vitro microdilution DPPH assay spectrofotometrically. The tested compounds exhibited IC50 values in the range of 0.18-0.56 mg/mL, suggesting an activity comparable with that of ascorbic acid (IC50: 0.07 mg/mL). To the best of our knowledge, the present study constitutes the first phytochemical and bioactivity investigation on G. iconica.

Steroidal glycosides with antiproliferative activities from Digitalis trojana.

The phytochemical investigation of Digitalis trojana led to the isolation of two cardiac glycosides (1, 2), one pregnane glycoside (3), three furostanol type saponins (4-6), along with three cleroindicins (7-9), four phenylethanoid glycosides (10-13), two flavonoids (14, 15) and two phenolic acid derivatives (16, 17). The structure elucidation of the isolates was carried out by NMR experiments as well as ESI-MS. The cytotoxic activity of compounds 1-13 against a small panel of cancer cell lines, namely MCF-7, T98G, HT-29, PC-3, A375 and SH-SY5Y, was investigated. Compounds 1-6 showed antiproliferative activity against human breast MCF-7 and colon HT-29 cancer cell lines with IC50 values ranging from 8.3 to 50 µM. In order to understand the mechanism involved in the cell death, the active compounds were tested as pro-apoptotic agents using propidium iodide staining by flow cytometry method. No significant increase was observed in the apoptosis of the MCF-7 and HT-29 cancer cells. Moreover, the effects of the active compounds on cell proliferation were assessed on the same cancer cell lines by cell cycle analysis of DNA content using flow cytometry. No significative changes were observed in the cell cycle of MCF-7, while significant changes in G2 /M cell cycle phase of HT-29 cells were observed after treatment with digitalin (1), cariensoside (3) and 22-O-methylparvispinoside B (6) at 10 µM.

Effects of globularifolin on cell survival, nuclear factor-κB activity, neopterin production, tryptophan breakdown and free radicals in vitro.

The potential effects of globularifolin, an acylated iridoid glucoside, on cell survival, inflammation markers and free radicals scavenging were investigated. Viability assay on human myelomomonocytic cell line THP-1 and human peripheral blood mononuclear cells (PBMC) using the Cell-Titer Blue assay proved that globularifolin had no toxic effect at the tested concentrations. Conversely, it is proportional to the dose globularifolin increased growth of THP-1 cells (p <0.01). On human PBMC, globularifolin at 6.25 and 12.5 µM concentrations showed a stimulatory effect, while at 12.5-200 µM it suppressed response of PBMC to stimulation with phytohemagglutinin (PHA). Globularifolin (50-200 µM) enhanced neopterin formation dose-dependently, whereas tryptophan breakdown was not influenced. At 50-200 µM in unstimulated PBMC in THP-1 cells, globularifolin induced a significant expression of nuclear factor-κB (NF-κB) as was quantified by Quanti-Blue assay. By contrast, in lipopolysaccharide (LPS)-stimulated cells, the higher concentrations of globularifolin suppressed NF-κB expression dose-dependently and a significant decrease was observed at 200 µM concentration. A positive correlation was found between increased neopterin and NF-κB activity (p <0.01). Similarly, a positive correlation was observed between neopterin levels in mitogen-induced cells and NF-κB activity in LPS-stimulated cells after treatment with globularifolin (p=0.001). The free radical scavenging capacity of globularifolin evaluated by Oxygen Radical Absorbance Capacity (ORAC) assay showed relative ORAC values of 0.36±0.05 µmol Trolox equivalent/µmol. All together, results show that natural antioxidant globularifolin might represent a potential immunomodulatory as well as proliferative agent, which deserves further in vitro and in vivo studies.

Appraisal of in vitro and in vivo antioxidant activity potential of cornelian cherry leaves.

The antioxidant activity of the 80% methanolic extract of Cornus mas L. leaves (CMM) was evaluated by various methods both in vitro and in vivo. In vitro screening tests indicated that CMM had high antioxidant activity in terms of free radical scavenging and metal reducing activity. In vivo antioxidant activity studies in normal healthy rats demonstrated that the total antioxidant capacity of liver homogenates were increased, although no changes were observed in the activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase or in the level of lipid peroxidation. Studies on CCl4-treated rats also showed that CMM restored the activities of antioxidant enzymes, lowered the level of lipid peroxidation and elevated the total antioxidant capacities of both the total blood and liver homogenates of the animals. Further activity-guided fractionation studies led to the isolation of gallic acid, a well-known antioxidant, as one of the active components.

Iridoid, phenylethanoid and flavonoid glycosides from Sideritis trojana.

From the MeOH extract of Sideritis trojana, a new iridoid glycoside, 10-O-(E)-feruloylmelittoside (1) was obtained in addition to four known iridoid glycosides [melittoside (2), 10-O-(E)-p-coumaroylmelittoside (3), stachysosides E (4) and G (5)]. Moreover, five phenylethanoid glycosides [verbascoside (6), isoacteoside (7), lamalboside (8), leonoside A (9), isolavandulifolioside (10), three flavone glycosides (isoscutellarein 7-O-[6'''-O-acetyl-ß-allopyranosyl-(1→2)]-ß-glucopyranoside (11), 4'-O-methyisoscutellarein 7-O-[6'''-O-acetyl-ß-allopyranosyl-(1→2)]-ß-glucopyranoside (12), 3'-hydroxy-4'-O-methyisoscutellarein 7-O-[6'''-O-acetyl-ß-allopyranosyl-(1→2)]-ß-glucopyranoside (13) and a benzylalcohol derivative (di-O-methylcrenatin) were obtained and identified. The structures were elucidated on the basis of NMR and HRMS data. All compounds were tested for their antioxidant activity by in vitro TEAC assay and some of them exhibited moderate activity (0.97-1.44 mM) when compared with the reference compound (quercetin 1.86 mM). Glycosides 6-13, the most active compounds in the TEAC assay, were also tested by flow cytometry to evaluate their ability to affect the levels of reactive oxygen species (ROS) in human prostate cancer cells (PC3).