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.

Protective Effect of Nigella sativa and Nigella damascena Fixed Oils Against Aflatoxin Induced Mutagenicity in the Classical and Modified Ames Test.

The antioxidant and mutagenic/antimutagenic activities of the fixed oils from Nigella sativa (NSO) and Nigella damascena (NDO) seeds, obtained by cold press-extraction from the cultivar samples, were comparatively investigated for the first time. The antimutagenicity test was carried out using classical and modified Ames tests. The fatty acid composition of the fixed oils was characterized by gas chromatography-mass spectrometry (GC-MS) while the quantification of thymoquinone in the fixed oils was determined by UPC2 . The main components of the NSO and NDO were found to be linoleic acid, oleic acid, and palmitic acid. The results of the Ames test confirmed the safety of NSO and NDO from the viewpoint of mutagenicity. The results of the three antioxidant test methods were correlated with each other, indicating NDO as having a superior antioxidant activity, when compared to the NSO. Both NSO and NDO exhibited a significant protective effect against the mutagenicity induced by aflatoxin B1 in Salmonella typhimurium TA98 and TA100 strains. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, a significant increase in antimutagenic activity was observed, suggesting that the degradation of aflatoxin B1 epoxides by these oils may be a possible antimutagenic mechanism. It is worthy to note that this is the first study to assess the mutagenicity of NSO and NDO according to the OECD 471 guideline and to investigate antimutagenicity of NDO in comparison to NSO against aflatoxin.

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.

A New Type of Anatolian Propolis: Evaluation of Its Chemical Composition, Activity Profile and Botanical Origin.

This study was undertaken to analyze the phenolic profiles of 19 propolis samples from Turkey by using a high-performance thin-layer chromatographic (HPTLC) method in order to identify their plant origins. Furthermore, their antioxidant and antimicrobial activity profiles were comparatively evaluated. For the appraisal of antioxidant potential, total phenolic (TPC) and total flavonoid contents (TFC) of propolis samples were firstly determined and then their effects on free radicals were evaluated by FRAP, ABTS.+ , CUPRAC, DPPH. and HPTLC-DPPH. methods. Antimicrobial activity of propolis samples against Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 11229) and Candida albicans ATCC 10231 were determined by disc diffusion and broth dilution methods. HPTLC fingerprinting analyses revealed that O-type (botanical origin from Populus nigra L.) was the primarily available propolis type in Turkey. Moreover, 3-O-methylquercetin (3MQ) rich propolis was identified as a new propolis type for the first time. Principal component analysis (PCA) indicated that 3MQ-type propolis differs from the O-type. Antioxidant activity studies showed that O-type of propolis possesses higher antioxidant effect than the other tested propolis types. Quercetin, caffeic acid, caffeic acid phenethyl ester (CAPE) and galangin were determined to contribute significantly to the antioxidant potential of O-type propolis among others. Propolis extracts exerted moderate antimicrobial activity against the tested microorganisms with MIC values between the ranges of 128-512 µg/mL.

Three New Iridoid Glycosides from the Aerial Parts of Asperula involucrata.

Three new iridoid glycosides, named involucratosides A - C (1 - 3), were isolated from the H2 O subextract of crude MeOH extract prepared from the aerial parts of Asperula involucrata along with a known iridoid glycoside (adoxoside), three flavone glycosides (apigenin 7-O-ß-glucopyranoside, luteolin 7-O-ß-glucopyranoside, apigenin 7-O-rutinoside) as well as two phenolic acid derivatives (chlorogenic acid and ferulic acid 4-O-ß-glucopyranoside). Their chemical structures were established by UV, IR, 1D- (1 H, 13 C and JMOD) and 2D- (COSY, HSQC, HMBC and NOESY) NMR experiments and HR-ESI-MS. In addition, the crude extract, subextracts and isolates were evaluated for their xanthine oxidase inhibitory and antioxidant activities in in vitro tests. This is the first report on the chemical composition and bioactivities of A. involucrata.

Evaluation of in vitro antiprotozoal activity of Ajuga laxmannii and its secondary metabolites.

Context Some Ajuga L. (Lamiaceae) species are traditionally used for the treatment of malaria, as well as fever, which is a common symptom of many parasitic diseases. Objective In the continuation of our studies on the identification of antiprotozoal secondary metabolites of Turkish Lamiaceae species, we have investigated the aerial parts of Ajuga laxmannii. Materials and methods The aerial parts of A. laxmannii were extracted with MeOH. The H2O subextract was subjected to polyamide, C18-MPLC and SiO2 CCs to yield eight metabolites. The structures of the isolates were elucidated by NMR spectroscopy and MS analyses. The extract, subextracts as well as the isolates were tested for their in vitro antiprotozoal activities against Plasmodium falciparum, Trypanasoma brucei rhodesiense, T. cruzi and Leishmania donovani at concentrations of 90-0.123 µg/mL. Results Two iridoid glycosides harpagide (1) and 8-O-acetylharpagide (2), three o-coumaric acid derivatives cis-melilotoside (3), trans-melilotoside (4) and dihydromelilotoside (5), two phenylethanoid glycosides verbascoside (6) and galactosylmartynoside (7) and a flavone-C-glycoside, isoorientin (8) were isolated. Many compounds showed moderate to good antiparasitic activity, with isoorientin (8) displaying the most significant antimalarial potential (an IC50 value of 9.7 µg/mL). Discussion and conclusion This is the first report on the antiprotozoal evaluation of A. laxmannii extracts and isolates. Furthermore, isoorientin and dihydromelilotoside are being reported for the first time from the genus Ajuga.

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.

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.

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.

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.

Phytochemical and Bioactivity Studies on Hedera helix L. (Ivy) Flower Pollen and Ivy Bee Pollen.

Bee pollen, known as a 'life-giving dust', is a product of honeybees using flower pollen grains and combining them with their saliva secretions. Thus, flower pollen could be an indicator of the bee pollen botanical source. Identification of bee pollen sources is a highly crucial process for the evaluation of its health benefits, as chemical composition is directly related to its pharmacological activity. In this study, the chemical profiles, contents of phenolic marker compounds and pharmacological activities of Hedera helix L. (ivy) bee pollen samples from Türkiye and Slovenia, as well as ivy flower pollen grains, were compared. High-performance thin-layer chromatography (HPTLC) analyses revealed that pollen samples, regardless of where they were collected, have similar chemical profiles due to the fact that they have the same botanical origins. Marker compounds afzelin, platanoside and quercetin-3-O-ß-glucopyranosyl-(1→2)-ß-galactopyranoside, common to both bee pollen and flower pollen, were isolated from bee pollen, and their structures were elucidated by nuclear magnetic resonance (NMR) and mass spectrometry (MS). These three compounds, as well as chlorogenic acid and 3,5-dicaffeoylquinic acid (found in flower pollen), were quantified using high-performance liquid chromatography (HPLC) analyses. In vitro tests and effect-directed analyses were used to evaluate the xanthine oxidase inhibition and antioxidant activity of the marker compounds and extracts from flower pollen and bee pollen. This is the first report comparing chemical profiles and related bioactivities of the flower pollen and bee pollen of the same botanical origin, as well as the first report of the chemical profile and related bioactivities of ivy flower pollen.

A new depside and a new secoiridoid from the aerial parts of Gentiana olivieri from flora of Turkey.

A new depside, olivieridepside (1), and a new secoiridoid, olivierigenin (2) were isolated from the aerial parts of Gentiana olivieri Griseb. along with four known compounds, gentiopicroside (3), olivierosides A (4) and B (5) and isoorientin (6). The structures of the isolates were determined by extensive 1 D and 2 D NMR spectroscopy and HR-MS analysis. This is the first report on the occurrence of a depside structure in the genus Gentiana. Moreover, a rare type of non-glycosidic secoiridoid (2) lacking an oxygenated group at C-1 is also being reported for the first time from this genus. The chemotaxonomic importance of the isolates was discussed in detail.

Antiprotozoal activity of Melampyrum arvense and its metabolites.

An activity guided isolation of the H(2)O subextract of the crude extract of Melampyrum arvense L. afforded iridoid glucosides: aucubin (1), melampyroside (2), mussaenoside (3), mussaenosidic acid (4), 8-epi-loganin (5); flavonoids: apigenin (6), luteolin (7), luteolin 7-O-ß-glucopyranoside (8); a lignan glycoside dehydrodiconiferyl alcohol 9-O-ß-glucopyranoside (9); and benzoic acid (10). ß-Sitosterol (11) and a fatty acid mixture (12) were identified as the active principles of the CHCl(3) subextract. The structures of the isolates were elucidated by spectroscopic methods, while the composition of 12 was identified by GC-MS after methylation. Luteolin (7) appeared as the most active compound against Trypanosoma brucei rhodesiense and Leishmania donovani (IC(50) values 3.8 and 3.0 µg/mL). Luteolin 7-O-ß-glucopyranoside (8) displayed the best antiplasmodial activity against Plasmodium falciparum (IC(50) value 2.9 µg/mL). This is the first detailed phytochemical study on Turkish M. arvense and the first report of the antiprotozoal effect of Melampyrum species and its constituents.

Anti-inflammatory secondary metabolites from Scrophularia kotschyana.

The species belonging to Scrophularia genus grow mainly in Irano-Turanian and Mediterranean regions and have been used as folk remedy for inflammatory-related diseases since ancient times. The present study was aimed to evaluate the anti-inflammatory activity of the extracts of Scrophularia kotschyana as well as the isolated compounds. The aerial parts and the roots of the plant were separately extracted with methanol. Anti-inflammatory activities of both extracts were evaluated with formalin test in mice. As the methanolic extract of the aerial parts significantly (p < .05) inhibited inflammation, it was then submitted to successive solvent extractions with n-hexane, dichloromethane, ethyl acetate and n-butanol to yield subextracts. Anti-inflammatory activities of the subextracts were evaluated within the same test system. Among the subextracts tested, the n-butanol subextract produced a significant (p < .05) anti-inflammatory activity at all doses (5, 10, and 30 mg/kg, ip.). Sequential chromatographic separation of the n-butanol subextract yielded 8-O-acetyl-4'-O-(E)-p-coumaroylharpagide, 8-O-acetyl-4'-O-(Z)-p-coumaroylharpagide, ß-sitosterol 3-O-ß-glucopyranoside, apigenin 7-O-ß-glucopyranoside, apigenin 7-O-rutinoside, luteolin 7-O-ß-glucopyranoside and luteolin 7-O-rutinoside. The anti-inflammatory activities of the isolates were evaluated at 5 mg/kg dose. Luteolin 7-O-ß-glucopyranoside and apigenin 7-O-rutinoside caused a significant (p < .05) inhibition of oedema formation.

Secondary metabolites from the aerial parts of Sideritis germanicopolitana and their in vitro enzyme inhibitory activities.

Three iridoid glycosides, 5-allosyloxy-aucubine (1), melittoside (2), ajugol (3), five phenylethanoid glycosides, verbascoside (4), martynoside (5), leucoseptoside A (6), lamalboside (7), decaffeoylverbascoside (8), four flavonoids, xanthomicrol (9), isoscutellarein 7-O-[6'''-O-acetyl-ß-allopyranosyl-(1→2)]-ß-glucopyranoside (10), 4'-O-methylisoscutellarein 7-O-[6'''-O-acetyl-ß-allopyranosyl-(1→2)]-ß-glucopyranoside (11), 3'-hydroxy-4'-O-methylisoscutellarein 7-O-[6'''-O-acetyl-ß-allopyranosyl-(1→2)]-ß-glucopyranoside (12), and two lignan glycosides dehydrodiconiferylalcohol 4-O-ß-D-glucopyranose (13) and pinoresinol 4'-O-ß-glucopyranoside (14) were isolated from the aerial parts of Sideritis germanicopolitana. Their structures were determined on the basis of detailed NMR and HRESIMS analyses. To our knowledge, all compounds are being reported for the first time from S. germanicopolitana, while the isolated lignans (13 and 14) are new for the genus Sideritis. In vitro evaluation of AChE, BChE and LOX inhibitory effects of all the tested compounds (1-14) resulted in low to moderate activities.

Secondary metabolites from Verbascum bugulifolium Lam. and their bioactivities.

Five iridoid glycosides catalpol (1), specioside (2), ajugol (3), ajugoside (4), 8-O-acetylharpagide (5), two phenylethanoid glycosides, verbascoside (6) and glucopyranosyl-(1→Gi-6)-martynoside (7), four flavonoids, luteolin (8), luteolin 7-O-ß-glucopyranoside (9), luteolin 7-O-rutinoside (10), apigenin 7-O-rutinoside (11), quercetin 3-O-rutinoside (12) and ß-sitosterol (13) were isolated from the aerial parts of Verbascum bugulifolium Lam. for the first time. Their structures were elucidated by NMR and MS experiments. The extracts, and the isolates were evaluated for their in vitro antioxidant (DPPH•, ABTS• and CUPRAC), anti-inflammatory (LOX inhibition) and antimicrobial activities. Compounds 6 and 8 showed the highest antioxidant activity in all tests, where luteolin (8) showed the relatively best anti-inflammatory activity compared to other samples with 54.1 ± 5.0% inhibition at 1 µg/mL. All the tested compounds showed weak antimicrobial activity against tested microorganisms. This is the first phytochemical and bioactivity study on V. bugulifolium.[Figure: see text].

Comparison of antioxidant and anti-inflammatory activity profiles of various chemically characterized Turkish propolis sub-types: Which propolis type is a promising source for pharmaceutical product development?

Propolis shows a great variation in its chemical content depending on the vegetation around the beehive. Determination of its botanical origin and the chemical characterization are the most important issues for the standardization and the quality evaluation for propolis samples that are intended to be used in the pharmaceutical industry. This study has focused on the identification of the botanical origin of 47 propolis samples collected from different locations in the Black Sea Region of Turkey. Firstly, palynological and chromatographic analyses were carried out. Then, the major distinguishing components were identified by high-performance thin-layer chromatography (HPTLC), or by nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry (MS) after isolation of the components. Based on the results, the samples were categorized into three main groups as black poplar-type, Euroasian aspen-type, and non-phenolic-type. Key markers of black poplar-type were assigned as phenolic acids and flavonoids, whereas lasiocarpin B and C (phenolic glycerides) were determined as markers for Euroasian aspen-type propolis. The total phenolics and flavonoid contents (TPC and TFC) and antioxidant capacities of the samples were comparatively assessed by free radical-scavenging activity (DPPH) and metal-reducing activity (CUPRAC and FRAP) methods. Additionally, HPTLC-direct bioautography was applied to determine the contribution of components to antioxidant activity. Hierarchical clustering analysis revealed similarities in TFC, TPC values, and antioxidant activity related to the sample origins' geographic proximity. The anti-inflammatory activities of the black poplar sub-type and Euroasian aspen-type propolis samples were comparatively investigated on RAW 264.7 macrophage cells. The black poplar-type propolis extract dominated by caffeic acid, caffeic acid phenethyl ester, apigenin, quercetin, kaempferol, pinocembrin, and galangin exhibited the highest anti-inflammatory and antioxidant activities. Therefore, chemically characterized black poplar-type propolis may be suggested as a good candidate to develop pharmaceutical products.

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.

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.