Phenolic compound profiling and antioxidant potential of different types of Schisandra henryi in vitro cultures.

Schisandra henryi is an endemic species of medicinal potential known from traditional Chinese medicine. As part of this study, a complex biotechnological and phytochemical assessment was conducted on S. henryi with a focus on phenolic compounds and antioxidant profiling. The following in vitro cultures were tested: microshoot agar and callus, microshoot agitated, and suspension, along with the microshoot culture in PlantForm bioreactors. Qualitative profiling was performed by ultra-high-performance liquid chromatography with a photodiode array detector coupled with ion-trap mass spectrophotometry with electrospray ionization and then quantitative analysis by high-performance liquid chromatography with a diode array detector using standards. In the extracts, mainly the compounds from procyanidins were identified as well as phenolic acids (neochlorogenic acid, caffeic acid, protocatechuic acid) and catechin. The highest content of phenolic compounds was found for in vitro agar microshoot culture (max. total content 229.87 mg/100 g DW) and agitated culture (max. total content 22.82 mg/100 g DW). The max. TPC measured using the Folin-Ciocalteu assay was equal to 1240.51 mg GAE/100 g DW (agar microshoot culture). The extracts were evaluated for their antioxidant potential by the DPPH, FRAP, and chelate iron ion assays. The highest potential was indicated for agar microshoot culture (90% of inhibition and 59.31 nM/L TEAC, respectively). The research conducted on the polyphenol profiling and antioxidant potential of S. henryi in vitro culture extracts indicates the high therapeutic potential of this species. KEY POINTS: • Different types of S. henryi in vitro cultures were compared for the first time. • The S. henryi in vitro culture strong antioxidant potential was determined for the first time. • The polyphenol profiling of different types of S. henryi in vitro cultures was shown.

Comparative Assessment of Lignan Profiling and Biological Activities of Schisandra henryi Leaf and In Vitro PlantForm Bioreactor-Grown Culture Extracts.

This research's scope encompassed biotechnological, phytochemical, and biological studies of Schisandra henryi, including investigations into its in vitro microshoot culture grown in PlantForm bioreactors (temporary immersion systems, TISs), as well as extracts from leaves of the parent plant, focusing on anti-inflammatory, antioxidant, anticancer, and antimicrobial activities. The phytochemical analysis included the isolation and quantification of 17 compounds from dibenzocyclooctadiene, aryltetralin lignans, and neolignans using centrifugal partition chromatography (CPC), HPLC-DAD, and UHPLC-MS/MS tandem mass spectrometry with triple quadrupole mass filter methods. Higher contents of compounds were found in microshoots extracts (max. 543.99 mg/100 g DW). The major compound was schisantherin B both in the extracts from microshoots and the leaves (390.16 and 361.24 mg/100 g DW, respectively). The results of the anti-inflammatory activity in terms of the inhibition of COX-1, COX-2, sPLA2, and LOX-15 enzymes indicated that PlantForm microshoot extracts showed strong activity against COX-1 and COX-2 (for 177 mg/mL the inhibition percentage was 76% and 66%, respectively). The antioxidant potential assessed using FRAP, CUPRAC, and DPPH assays showed that extracts from microshoot cultures had 5.6, 3.8, and 3.3 times higher power compared to extracts from the leaves of the parent plant, respectively. The total polyphenol content (TPC) was 4.1 times higher in extracts from the in vitro culture compared to the leaves. The antiproliferative activity against T-cell lymphoblast line Jurkat, breast adenocarcinoma cultures (MCF-7), colon adenocarcinoma (HT-29), and cervical adenocarcinoma (HeLa), showed that both extracts have considerable effects on the tested cell lines. The antimicrobial activity tested against strains of Gram-positive and Gram-negative bacteria and fungi showed the highest activity towards H. pylori (MIC and MBC 0.625 mg/mL).

Plant-based meat substitute analysis using microextraction with deep eutectic solvent followed by LC-MS/MS to determine acrylamide, 5-hydroxymethylfurfural and furaneol.

For the analysis of plant-based meat substitutes and the determination of Maillard reaction products such as acrylamide, 5-hydroxymethylfurfural and furaneol, a novel and effective procedure based on hydrophobic natural deep eutectic solvent and liquid chromatography coupled with tandem mass spectrometry was developed for the first time. The 49 compositions of the deep eutectic solvents were designed and screened to select the most suitable option. The terpenoids eugenol and thymol in a molar ratio of 2:1 were selected as precursors for solvent formation, allowing effective extraction of the target analytes. The developed procedure comprised two main steps: extraction - in which the analytes are isolated from the solid sample due to the salting-out effect and pre-concentrated in the deep eutectic solvent, and back-extraction - in which the analytes are re-extracted into the formic acid solution for subsequent mass spectrometric detection. As the density of the aqueous phases changed during the extraction and back-extraction steps, the phenomenon of inversion of the coalesced organic phase was observed, which simplified the withdrawing of the phases. The linear range was 1-50 ng/mL for acrylamide, 10-1000 ng/mL for 5-hydroxymethylfurfural and 200-1000 ng/mL for furaneol with coefficients of determination above 0.9952. The developed method was fully validated and found recoveries were in the range 83-120%, with CVs not exceeding 4.9%. The method was applied to real sample analysis of pea-based meat substitutes.

Shoot Cultures of Vitis vinifera (Vine Grape) Different Cultivars as a Promising Innovative Cosmetic Raw Material-Phytochemical Profiling, Antioxidant Potential, and Whitening Activity.

The primary purpose of this work was the initiation and optimization of shoot cultures of different Vitis vinifera L. cultivars: cv. Chardonnay, cv. Hibernal, cv. Riesling, cv. Johanniter, cv. Solaris, cv. Cabernet Cortis, and cv. Regent. Cultures were maintained on 30-day growth cycles using two media, Murashige and Skoog (MS) and Schenk and Hildebrandt (SH), with various concentrations of plant growth regulators. Tested media ('W1'-'W4') contained varying concentrations of 6-benzylaminopurine (BA) in addition to indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA). High performance liquid chromatography coupled with mass spectrometry (UPLC-MS) was used for metabolomic profiling. In all tested extracts, 45 compounds were identified (6 amino acids, 4 phenolic acids, 13 flavan-3-ols, 3 flavonols, and 19 stilbenoids). Principal component analysis (PCA) was performed to assess the influence of the genotype and medium on metabolic content. PCA showed that metabolic content was mainly influenced by genotype and to a lesser extent by medium composition. MS media variants induced the amino acid, procyanidin, and flavan-3-ol production. In addition, the antioxidant potential and anti-tyrosinase activity was measured spectrophotometrically. The studies on antioxidant activity clearly reveal very high efficiency in reducing free radicals in the tested extracts. The strongest tyrosinase inhibition capacity was proved for shoots cv. Hibernal cultured in SH medium and supplemented with NAA, with an inhibition of 17.50%. These studies show that in vitro cultures of V. vinifera cvs. can be proposed as an alternative source of plant material that can be potentially used in cosmetic industry.

Optimization of a Fabric Phase Sorptive Extraction protocol for the isolation of six bisphenols from juice pouches to be analysed by high performance liquid chromatography coupled with diode array detector.

Fabric Phase Sorptive Extraction (FPSE) combined with high pressure liquid chromatography using to diode array detection (HPLC-DAD) was applied for the simultaneous determination of bisphenols (BPA, BPB, BPC, BPE, BPF, BPS) in juice pouches. The FPSE procedure was optimized with regards to the critical parameters that affect the performance of the method including the selection of the FPSE membrane type and size, adsorption time, extraction time, solvent volume desorption, magnetic stirring ratio, and salt addition. The FPSE membrane could be reused up to 14 times. The developed FPSE-HPLC-DAD method was validated in terms of linearity, sensitivity, accuracy andprecision. The limits of detection (LODs) were lower than 6.9 ng/mL, while the limits of quantification (LOQs) were lower than 21 ng/mL. The results obtained are satisfactory in terms of precision, accuracy and repeatability, with recoveries above 86% and CV values below 9.5%. The FPSE-HPLC-DAD method was successfully applied in the determination of six bisphenols in juice samples stored in pouches.

Exposure scenario and risk assessment of infants and newborns to bisphenols and their derivatives from diapers.

Newborns and infants are more sensitive to harmful compounds such as bisphenols and their derivatives because of their not fully developed detoxification mechanism. Exposure to these substances can lead to developmental problems and health consequences in adulthood. Since disposable baby diapers are used from the first days of life and remain in contact with the baby skin, it seems important to monitor the levels of endocrine disrupting chemicals (EDCs) in such products. Ultrasound assisted solvent microextraction of porous membrane-packed solid sample (UASE-PMSS) was used in sample preparation. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used at determination step. Bisphenol A was quantified with the highest frequency at 81 % of samples tested, ranging from 5.0 to 520 ng/g. BADGE·2HCl was also quantified in high concentrations (from 6.8 to 530 ng/g), but was found in only 15 % of the tested samples. The daily exposure dose (DED) of bisphenols was calculated. In addition health risk assessment was conducted using previous (4 µg/kg BW) and actual (0.2 ng/kg BW) values of tolerable daily intake (TDI) of bisphenol A recommended by European Food Safety Authority (EFSA).

Determination of Glycerol, Propylene Glycol, and Nicotine as the Main Components in Refill Liquids for Electronic Cigarettes.

Refill liquids for electronic cigarettes are an important area of research due to the health safety and quality control of such products. A method was developed for the determination of glycerol, propylene glycol, and nicotine in refill liquids using liquid chromatography, coupled with tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode with electrospray ionisation (ESI). Sample preparation was based on a simple dilute-and-shoot approach, with recoveries ranging from 96 to 112% with coefficients of variation < 6.4%. Linearity, limits of detection and quantification (LOD, LOQ), repeatability, and accuracy were determined for the proposed method. The proposed sample preparation and the developed chromatographic method using hydrophilic interaction liquid chromatography (HILIC) were successfully applied for the determination of glycerol, propylene glycol, and nicotine in refill liquid samples. For the first time, the developed method using HILIC-MS/MS has been applied for the determination of the main components of refill liquids in a single analysis. The proposed procedure is rapid and straightforward and is suitable for quick determination of glycerol, propylene glycol, and nicotine. The nicotine concentrations corresponded to the labelling of samples (it varied from

Phytochemical Profiling, Antioxidant and Tyrosinase Regulatory Activities of Extracts from Herb, Leaf and In Vitro Culture of Achillea millefolium (Yarrow).

Achillea millefolium L. is one of the most known medicinal plants with a broad spectrum of applications in the treatment of inflammation, pain, microbial infections and gastrointestinal disorders. In recent years, the extracts from A. millefolium have also been applied in cosmetics with cleansing, moisturizing, shooting, conditioning and skin-lightening properties. The growing demand for naturally derived active substances, worsening environmental pollution and excessive use of natural resources are causing increased interest in the development of alternative methods for the production of plant-based ingredients. In vitro plant cultures are an eco-friendly tool for continuous production of desired plant metabolites, with increasing applicability in cosmetics and dietary supplements. The purpose of the study was to compare phytochemical composition and antioxidant and tyrosinase inhibitory properties of aqueous and hydroethanolic extracts from A. millefolium obtained from field conditions (AmL and AmH extracts) and in vitro cultures (AmIV extracts). In vitro microshoot cultures of A. millefolium were obtained directly from seeds and harvested following 3 weeks of culture. Extracts prepared in water, 50% ethanol and 96% ethanol were compared for the total polyphenolic content, phytochemical content using the ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-hr-qTOF/MS), antioxidant activity by DPPH scavenging assay and the influence on the activity of mushroom and murine tyrosinases. The phytochemical content of AmIV extracts was significantly different from AmL and AmH extracts. Most of the polyphenolic compounds identified in AmL and AmH extracts were present in AmIV extracts only in trace amounts and the major constituents presented in AmIV extracts were fatty acids. The total content of polyphenols in AmIV exceeded 0.25 mg GAE/g of dried extract, whereas AmL and AmH extracts contained from 0.46 ± 0.01 to 2.63 ± 0.11 mg GAE/g of dried extract, depending on the solvent used. The low content of polyphenols was most likely responsible for the low antioxidant activity of AmIV extracts (IC50 values in DPPH scavenging assay >400 µg/mL) and the lack of tyrosinase inhibitory properties. AmIV extracts increased the activity of mushroom tyrosinase and tyrosinase present in B16F10 murine melanoma cells, whereas AmL and AmH extracts showed significant inhibitory potential. The presented data indicated that microshoot cultures of A. millefolium require further experimental research before they can be implemented as a valuable raw material for the cosmetics industry.

Synthesis, characterization, and application of 2D/2D TiO2-GO-ZnFe2O4 obtained by the fluorine-free lyophilization method for solar light-driven photocatalytic degradation of ibuprofen.

In this study, we report the potential of 2D/2D TiO2-GO-ZnFe2O4 photocatalyst obtained using the fluorine-free lyophilization technique for the degradation of ibuprofen belonging to the group of active pharmaceutical ingredients (API). The improved ibuprofen degradation under simulated solar light was achieved in the presence of a composite of 2D TiO2 combined with GO and embedded ZnFe2O4, which additionally provides superparamagnetic properties and enables photocatalyst separation after the photodegradation process. After only 20 min of the photodegradation process in the presence of 2D/2D TiO2-GO-ZnFe2O4 composite, more than 90% of ibuprofen was degraded under simulated solar light, leading to non-toxic and more susceptible to biodegradation intermediates. At the same time, photolysis of ibuprofen led to the formation of more toxic intermediates. Furthermore, based on the photocatalytic degradation analysis, the degradation by-products and possible photodegradation pathways of ibuprofen were investigated. The photodegradation tests and electronic spin resonance analyses indicated the significant involvement of superoxide radicals and singlet oxygen in the ibuprofen photodegradation process.

Stimulation of Lignan Production in Schisandra rubriflora In Vitro Cultures by Elicitation.

The study investigated the effect of elicitation with: chitosan (CH) (200 mg/L), yeast extract (YeE) (3000 mg/L), ethephon (ETH) (25 µM/L), and methyl jasmonate (MeJA) (50 µM/L), on lignan accumulation in agitated and bioreactor (Plantform temporary immersion systems) microshoot cultures of female (F) and male (M) Schisandra rubriflora Rehd. et Wils. (Schisandraceae) lines. The elicitors were supplemented on the 10th day of culture. Biomasses were collected at 24 h and 48 h, and 4, 6, and 8 days after the addition of each elicitor. The 24 compounds from the dibenzocyclooctadiene, aryltetralin, dibenzylbutane, and tetrahydrofuran lignans and neolignans were determined qualitatively and quantitatively in biomass extracts using the UHPLC-MS/MS method. The highest total contents [mg/100 g DW] of lignans were: for CH-95.00 (F, day 6) and 323.30 (M, 48 h); for YeE 104.30 (F, day 8) and 353.17 (M, day 4); for ETH 124.50 (F, 48 h) and 334.90 (M, day 4); and for MeJA 89.70 (F, 48 h) and 368.50 (M, 24 h). In the biomass extracts of M cultures grown in bioreactors, the highest total lignan content was obtained after MeJA elicitation (153.20 mg/100 g DW). The maximum total lignan contents in the biomass extracts from agitated and bioreactor cultures were 3.29 and 1.13 times higher, respectively, than in the extracts from the non-elicited cultures. The poor understanding of the chemical composition and the lack of studies in the field of plant biotechnology of S. rubriflora emphasize the innovativeness of the research.

Phenylpropanoid Glycoside and Phenolic Acid Profiles and Biological Activities of Biomass Extracts from Different Types of Verbena officinalis Microshoot Cultures and Soil-Grown Plant.

Different types of microshoot cultures (agar, stationary liquid, agitated, and bioreactors) of Verbena officinalis were optimized for biomass growth and the production of phenylpropanoid glycosides and phenolic acids. Using ultra-high performance liquid chromatography with high-resolution time-of-flight mass spectrometry, the presence of verbascoside, isoverbascoside, leucoseptoside A/isomers, and cistanoside D/isomer was confirmed in the methanolic extracts obtained from all types of in vitro cultures. The compound's content was determined by ultra-high-performance liquid chromatography. The main metabolites in biomass extracts were verbascoside and isoverbascoside (maximum 4881.61 and 451.80 mg/100 g dry weight (DW)). In the soil-grown plant extract, verbascoside was also dominated (1728.97 mg/100 g DW). The content of phenolic acids in the analyzed extracts was below 24 mg/100 g DW. The highest radical scavenging activity was found in the biomass extract from agitated cultures, the most effective reducing power in agar culture extract, and the highest chelating activity in extract from bioreactor cultures. The extracts showed significantly stronger bacteriostatic and bactericidal activity against Gram-positive bacteria (minimum inhibitory concentration (MIC) of 0.3-2.2 mg/mL and minimum bactericidal concentration (MBC) of 0.6-9 mg/mL) than against Gram-negative bacteria (MIC 0.6-9 mg/mL, MBC of 0.6-18 mg/mL). The biomass extract from liquid stationary culture showed the strongest antibacterial activity, while the extract from soil-grown herb had the lowest.

Illicium verum (Star Anise) and Trans-Anethole as Valuable Raw Materials for Medicinal and Cosmetic Applications.

Illicium verum Hook f. (star anise) is considered an important species in Traditional Chinese Medicine and is also used in contemporary medicine in East Asian countries. It occurs in natural habitats in southeastern parts of China and Vietnam, and is cultivated in various regions in China. The raw materials-Anisi stellati fructus and Anisi stellati aetheroleum obtained from this species exhibit expectorant and spasmolytic activities. The European Pharmacopoeia (4th edition) indicates that these raw materials have been used in allopathy since 2002. The biological activities of the above-mentioned raw materials are determined by the presence of valuable secondary metabolites such as monoterpenoids, sesquiterpenoids, phenylpropanoids, and flavonoids. Recent pharmacological studies on fruit extracts and the essential oil of this species have confirmed their antibacterial, antifungal, anti-inflammatory, and antioxidant activities and thus their medicinal and cosmetic value. The aim of this review was to examine the progress of phytochemical and pharmacological studies that focused on possible cosmetic applications. In addition to fruit extracts and essential oil, the current consensus on the safety of trans-anethole, which is the main compound of essential oil used in cosmetology, is underlined here.

In Vitro Cultures of Some Medicinal Plant Species (Cistus × incanus, Verbena officinalis, Scutellaria lateriflora, and Scutellaria baicalensis) as a Rich Potential Source of Antioxidants-Evaluation by CUPRAC and QUENCHER-CUPRAC Assays.

Comparative estimations of the antioxidant activity of methanolic extracts from biomasses of different types of in vitro cultures of Cistus × incanus, Verbena officinalis, Scutellaria lateriflora, and S. baicalensis and also from plant raw materials were performed. The antioxidant measurements were based on the modern assays-cupric ion reducing antioxidant capacity (CUPRAC) and quick, easy, new, cheap, and reproducible CUPRAC (QUENCHER-CUPRAC). The total extractable antioxidants (CUPRAC assay) ranged from 10.4 to 49.7 mmol (100 g)-1 of dry weight (DW) expressed as Trolox equivalent antioxidant capacity (TEAC), and the global antioxidant response (QUENCHER-CUPRAC assay) ranged from 16.0 to 79.1 mmol (100 g)-1 DW for in vitro cultures, whereas for plant raw materials the total extractable antioxidants ranged from 20.9 to 69.5 mmol (100 g)-1 DW, and the global antioxidant response ranged from 67.2 to 97.8 mmol (100 g)-1 DW. Finally, the in vitro cultures could be regarded as an antioxidant-rich alternative resource for the pharmaceutical, health food and cosmetics industries.

Production of Verbascoside, Isoverbascoside and Phenolic Acids in Callus, Suspension, and Bioreactor Cultures of Verbena officinalis and Biological Properties of Biomass Extracts.

Callus, suspension and bioreactor cultures of Verbena officinalis were established, and optimized for biomass growth and production of phenylpropanoid glycosides, phenolic acids, flavonoids and iridoids. All types of cultures were maintained on/in the Murashige and Skoog (MS) media with 1 mg/L BAP and 1 mg/L NAA. The inoculum sizes were optimized in callus and suspension cultures. Moreover, the growth of the culture in two different types of bioreactors-a balloon bioreactor (BB) and a stirred-tank bioreactor (STB) was tested. In methanolic extracts from biomass of all types of in vitro cultures the presence of the same metabolites-verbascoside, isoverbascoside, and six phenolic acids: protocatechuic, chlorogenic, vanillic, caffeic, ferulic and rosmarinic acids was confirmed and quantified by the HPLC-DAD method. In the extracts from lyophilized culture media, no metabolites were found. The main metabolites in biomass extracts were verbascoside and isoverbascoside. Their maximum amounts in g/100 g DW (dry weight) in the tested types of cultures were as follow: 7.25 and 0.61 (callus), 7.06 and 0.48 (suspension), 7.69 and 0.31 (BB), 9.18 and 0.34 (STB). The amounts of phenolic acids were many times lower, max. total content reached of 26.90, 50.72, 19.88, and 36.78 mg/100 g DW, respectively. The highest content of verbascoside and also a high content of isoverbascoside obtained in STB (stirred-tank bioreactor) were 5.3 and 7.8 times higher than in extracts from overground parts of the parent plant. In the extracts from parent plant two iridoids-verbenalin and hastatoside, were also abundant. All investigated biomass extracts and the extracts from parent plant showed the antiproliferative, antioxidant and antibacterial activities. The strongest activities were documented for the cultures maintained in STB. We propose extracts from in vitro cultured biomass of vervain, especially from STB, as a rich source of bioactive metabolites with antiproliferative, antioxidant and antibacterial properties.

Verbena officinalis (Common Vervain) - A Review on the Investigations of This Medicinally Important Plant Species.

Verbena officinalis (common vervain) is a medicinal plant species widely distributed in the world and commonly used in folk medicine of different countries, including traditional Chinese medicine. Monographs on "Verbenae herba" have been included in the European Pharmacopoeia since 2008, and in the Chinese Pharmacopoeia since 1995. This work presents botanical characteristics of this species. It reviews the current knowledge of its chemical composition, which is a rich source mostly of iridoids, phenylpropanoid glycosides, phenolic acids, flavonoids, terpenoids, and essential oil. A large part of this article summarizes traditional medicinal uses and professional pharmacological in vitro and in vivo studies that prove new important applications, e.g., antioxidant, antimicrobial, anti-inflammatory, neuroprotective anticancer, analgesic, or anticonvulsant of verbena herb extracts and individual metabolites. Moreover, emphasis is put on the use of V. officinalis in the food and cosmetics industries, especially due to its antioxidant, antibacterial, and anti-inflammatory properties, and the presence of essential oil with an attractive fragrance composition. This paper also presents the state of biotechnological studies of this species.

Polyphenols of Frangula alnus and Peganum harmala Leaves and Associated Biological Activities.

Frangula alnus and Peganum harmala populations growing in Saudi Arabia might be rich sources of natural compounds with important biological activities. A high performance liquid chromatography diode array revealed several polyphenols in the leaf extracts for the first time, including p-coumaric acid, rosmarinic acid, chlorogenic acid, ferulic acid, quercitrin, rutoside, quercetin and trifolin in F. alnus; and hydrocaffeic acid, protocatechuic acid, rosmarinic acid, caffeic acid and cynaroside in P. harmala. F. alnus and P. harmala showed strong antioxidant effects attributed to the polyphenolic composition of leaves and reduction of reactive oxygen species (ROS) accumulation. F. alnus and P. harmala leaf extracts showed cytotoxic effects against Jurkat, MCF-7, HeLa, and HT-29 cancer cells using MTT and flow cytometry assays. These activities were attributed to the polyphenolic composition of leaves including quercitrin, trifolin and cymaroside, as well as the activation of caspase family enzymes 2, 6, 8 and 9 in treated cancer cells compared to control. The current findings of this study include a novel comprehensive investigation on the polyphenol composition and anticancer effects of leaf extracts of F. alnus and P. harmala from natural populations in Saudi Arabia.

Antioxidant and Biological Activities of Acacia saligna and Lawsonia inermis Natural Populations.

Acacia saligna and Lawsonia inermis natural populations growing in Northern Saudi Arabia might be a valuable source of polyphenols with potent biological activities. Using high-performance liquid chromatography-diode array detection (HPLC-DAD), several polyphenols were detected tentatively in considerable amounts in the methanolic leaf extracts of A. saligna and L. inermis. A. saligna mainly contained rutoside, hyperoside, quercetin 3-glucuronide, gallic acid and p-coumaric acid, whereas those of L. inermis contained apigenin 5-glucoside, apigetrin and gallic acid. Strong antioxidant activities were found in the leaf extracts of both species due to the presence of hyperoside, quercetin 3-glucuronide, gallic acid, isoquercetin, p-coumaric acid, quercitrin and rutoside. A. saligna and L. inermis leaf extracts as well as hyperoside, apigenin 5-glucoside, and quercetin 3-glucuronide significantly reduced reactive oxygen species accumulation in all investigated cancer cells compared to the control. Methanolic leaf extracts and identified polyphenols showed antiproliferative and cytotoxic activities against cancer cells, which may be attributed to necrotic cell accumulation during apoptotic periods. Antibacterial activities were also found in both species leaf extracts and were twice as high in A. saligna than L. inermis due to the high composition of rutoside and other polyphenols. Finally, strong antifungal activities were detected, which were associated with specific phenols such as rutoside, hyperoside, apigenin 5-glucoside and p-coumaric acid. This is the first study exploring the polyphenolic composition of A. saligna and L. inermis natural populations in northern Saudi Arabia and aiming at the detection of their biological activities.

Determination of bromhexine and its metabolites in equine serum samples by liquid chromatography - Tandem mass spectrometry: Applicability to the elimination study after single oral dose.

Bromhexine (BH), expectorant used in the treatment of respiratory disorders associated with viscid or excessive mucus, is not permitted for use in the competing horse by many authorities in horseracing and Olympic disciplines. Metabolic studies are of the great importance in anti-doping field because they allow for updating the selection of the most appropriate markers for prohibited substances, such as metabolites present at higher concentration levels and/or lasted for a longer period of time in biological samples than a parent drug. This study describes LC-MS/MS-based method for simultaneous determination of BH and its metabolites, including 4-(2-amino-3,5-dibromobenzylamino)cyclohexanol (4-HDMB), 3-(2-amino-3,5-dibromobenzylamino)cyclohexanol (3-HDMB), in equine serum samples. The 2-(2-amino-3,5-dibromobenzylamino)cyclohexanol (2-HDMB) was monitored as well. The assay was validated in terms of linearity (R2 greater than 0.9951), intra- and inter-assay accuracy (91.6 - 109.1%) and precision (CV < 9.6%) as well as recovery (94.8 - 105.65%). The LODs were 0.0052, 0.0053, 0.0056 and 0.0043 ng/mL for BH, 2-HDMB, 3-HDMB and 4-HDMB, respectively. The developed method was applied to determine the time curses of BH and its metabolites concentrations in equine serum collected for 95.25 h following a single oral administration of BH to two healthy mares (in dose of 0.8 mg/kg). The parent drug was found at higher concentration levels than 3-HDMB (major metabolite) and 4-HDMB (minor metabolite), however, both BH metabolites lasted for a longer period of time in equine serum than the parent drug. Thus, both metabolites of BH can be considered as BH abuse markers.

Schisandra rubriflora Plant Material and In Vitro Microshoot Cultures as Rich Sources of Natural Phenolic Antioxidants.

Schisandra rubriflora is a dioecious, underestimated medicinal plant species known from traditional Chinese medicine. The present study was aimed at characterising the polyphenolic profile composition and the related antioxidant capacity of S. rubriflora fruit, stem and leaf and in vitro microshoot culture extracts. Separate analyses of material from female and male specimens were carried out. This study was specifically aimed at detailed characterisation of the contribution of phenolic compounds to overall antioxidant activity using ultra-high-performance liquid chromatography with a photodiode array detector coupled to electrospray ionization ion trap mass spectrometry (UHPLC-DAD-ESI-MS3) and a high-performance liquid chromatography-diode array detector (HPLC-DAD). Using UHPLC-DAD-ESI-MS3, twenty-seven phenolic compounds from among phenolic acids and flavonoids were identified. Concentrations of three phenolic acids (neochlorogenic, chlorogenic and cryptochlorogenic acids) and eight flavonoids (hyperoside, rutoside, isoquercitrin, guaijaverin, trifolin, quercetin, kaempferol, and isorhamnetin) were determined using HPLC-DAD using reference standards. The highest total phenolic content was confirmed for the stem and leaf extracts collected in spring. The contents of phenolic compounds of in vitro biomasses were comparable to that in the fruit extracts. The methanolic extracts from the studied plant materials were evaluated for their antioxidant properties using various in vitro assays, namely free radicals scavenging estimation using 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), ferric-reducing antioxidant power (FRAP) and cupric-reducing antioxidant capacity (CUPRAC) as well as QUick, Easy, New, CHEap, and Reproducible CUPRAC (QUENCHER-CUPRAC) assays. A close relationship between the content of polyphenolic compounds in S. rubriflora and their antioxidant potential has been documented.

The influence of light quality on the production of bioactive metabolites - verbascoside, isoverbascoside and phenolic acids and the content of photosynthetic pigments in biomass of Verbena officinalis L. cultured in vitro.

In vitro callus cultures of Verbena officinalis L. were maintained on solid Murashige and Skoog medium, enriched with 1 mg dm-3 BA and 1 mg dm-3 IBA under LED lights (red, blue, red/blue 70%/30%), in darkness and under control fluorescent lamps. The measurements of 2 phenylpropanoid glycosides (verbascoside and isoverbascoside) and 23 phenolic acids were performed in methanolic extracts from the biomass collected after 2-, 3- and 4-week growth cycles using the HPLC-DAD method. The presence of verbascoside, isoverbascoside and additionaly 7 phenolic acids (protocatechuic, chlorogenic, vanillic, caffeic, ferulic, o-coumaric and m-coumaric acids) was confirmed in all extracts. Blue and red/blue lights stimulated the accumulation of verbascoside (max. of 6716 and 6023 mg 100 g-1 DW after a 4-week growth cycle) and isoverbascoside (max. 333 and 379 mg 100 g-1 DW also after 4 weeks). The maximum amounts of verbascoside and isoverbascoside were respectively 1.8- and 7.0-fold higher than under the control conditions. Phenolic acids were accumulated in different amounts, and the maximum total amounts ranged from 36 to 65 mg 100 g-1 DW. LED lights also stimulated their accumulation in comparison with darkness and control. The main phenolic acids included: m-coumaric acid (max. 39 mg 100 g-1 DW), ferulic acid (max. 12 mg 100 g-1 DW), and protocatechuic acid (max. 13 mg 100 g-1 DW). Additionally, the quantities of photosynthetic pigments (chlorophyll a, b and carotenoids) were estimated in acetonic extracts using spectrophotometry. Red/blue light stimulated the biosynthesis of pigments (max. total content 287 µg g-1 FW after 4-week growth cycles). This is the first study describing the effect of LED lights on the production of phenylpropanoid glycosides and phenolic acids in V. officinalis callus cultures. Very high amounts of verbascoside and isoverbascoside are interesting from a practical point of view.