Carvacrol as a Stimulant of the Expression of Key Genes of the Ginsenoside Biosynthesis Pathway and Its Effect on the Production of Ginseng Saponins in Panax quinquefolium Hairy Root Cultures.

The accumulation of ginsenosides (triterpenic saponins) was determined in Panax quinquefolium hairy root cultures subjected to an elicitation process using carvacrol at 5, 10, 25, 50, 100, 250, and 500 µM concentrations during 24 and 72 h exposure. This study was the first one in which carvacrol was applied as an elicitor. The content of eight ginsenosides, Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, and Re, was determined using HPLC analysis. Moreover, the quantitative RT-PCR method was applied to assess the relative expression level of farnesyl diphosphate synthase, squalene synthase, and dammarenediol synthase genes in the studied cultures. The addition of carvacrol (100 µM) was an effective approach to increase the production of ginsenosides. The highest content and productivity of all detected saponins were, respectively, 20.01 mg∙g-1 d.w. and 5.74 mg∙L-1∙day-1 after 72 h elicitation. The production profile of individual metabolites in P. quinquefolium cultures changed under the influence of carvacrol. The biosynthesis of most examined protopanaxadiol derivatives was reduced under carvacrol treatment. In contrast, the levels of ginsenosides belonging to the Rg group increased. The strongest effect of carvacrol was noticed for Re metabolites, achieving a 7.72-fold increase in comparison to the control. Saponin Rg2, not detected in untreated samples, was accumulated after carvacrol stimulation, reaching its maximum concentration after 72 h exposure to 10 µM elicitor.

Antioxidant and Anti-inflammatory Effects of α-Lipoic Acid on Lipopolysaccharide-induced Oxidative Stress in Rat Kidney.

α-Lipoic acid (α-LA) is a naturally occurring organosulfur component. Oxidative stress plays an essential role in the pathogenesis of various diseases, such as kidney and cardiovascular diseases, diabetes, neurodegenerative disorders, cancer and aging. Kidneys are especially vulnerable to oxidative stress and damage. The aim of the study was to evaluate the effect of α-LA on lipopolysaccharide (LPS)-induced oxidative stress parameters in rat kidneys. The experimental rats were divided into four groups: I-control (0.9% NaCl i.v.); II-α-LA (60 mg/kg b.w. i.v.); III-LPS (30 mg/kg b.w. i.v.); and IV-LPS + LA (30 mg/kg b.w. i.v. and 60 mg/kg b.w. i.v., respectively). In kidney homogenates the concentration of thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2), sulfhydryl groups (-SH), total protein, superoxide dismutase (SOD), total glutathione (tGSH), reduced glutathione (GSH), glutathione disulphide (GSSG) and the GSH/GSSG ratio were determined. In addition, the levels of tumour necrosis factor (TNF)-α, and interleukin (IL)-6 were measured to assess inflammation and was estimated kidney oedema. Studies have shown that α-LA administered after LPS administration attenuated kidney oedema and significantly decreased TBARS, H2O2, TNF-α, and IL-6 levels in rat kidneys. α-LA also resulted in increase -SH group, total protein, and SOD levels and ameliorated the GSH redox status when compared to the LPS group. The results suggest that α-LA plays an important role against LPS-induced oxidative stress in kidney tissue as well as downregulating the expression of pro-inflammatory cytokines.

Improved efficacy of eugenol and trans-anethole in combination with octenidine dihydrochloride against Candida albicans and Candida parapsilosis.

INTRODUCTION AND OBJECTIVE: Candidiasis is a fungal infection caused by yeasts from the Ogenus Candida. Considering increasing antifungal resistance rates the activity was analyzed of natural compounds to eradicate Candida spp. The aim of the study was to check the antifungal activity of selected essential oil compounds (EOCs; thymol, menthol, eugenol [E], carvacrol, trans-anethole [TA]) alone, and in combination with octenidine dihydrochloride (OCT) against C. albicans and C. parapsilosis reference, and clinical strains. MATERIAL AND METHODS: Investigated clinical isolates were obtained from skin wounds of patients treated for superficial wounds candidiasis. The following parameters were studied: antifungal susceptibility testing using the VITEK system, antifungal activity of EOCs alone and in combination with OCT using microdilution and checkerboard assays, antifungal efficacy of selected chemicals using time-kill curve assay, and changes in cell permeability in the presence of selected chemicals using crystal violet assay. RESULTS: Clinical isolates of C. albicans and C. parapsilosis were resistant to fluconazole and voriconazole. The highest inhibition activity against Candida isolates was observed for E. The OCT - TA and OCT - E combinations showed synergistic and additive activities against all strains, respectively. These combinations also appeared to affect the rate of yeast cell killing and increasing the permeability of Candida cells. CONCLUSIONS: The study indicates that E and TA potentially used in formulation with OCT might eradicate pathogenic yeasts; however, microbiological and clinical studies are still required.

The Effect of Herbal Supplements on Blood Pressure: Systematic Review and Meta-Analysis.

Herbal supplements rich in phenolic compounds are evidenced to have a protective effect against cardiovascular diseases. Therefore, they are suggested to be included in diets for people with hypertension (HT). HT is a global health problem and is estimated to affect billions of people until the end of 2025. For this reason, every possible and effective solution preventing HT should be considered. The aim was to perform an updated meta-analysis and review of recently published studies to evaluate the effect of selected herbal supplements on blood pressure reduction. We searched the PubMed database with specified selection criteria, analysing the RCT studies from 2011 to 2021. A total of 31 studies were included in the analysis, and the meta-analysis was conducted on the data from 16 of them. The general effect size of all the supplements via placebo was d = 1.45, p < 0.05 for systolic blood pressure (SBP) and d = 0.31, p < 0.05 for diastolic blood pressure (DBP). The meta-analysis and review of the literature demonstrated that herbal supplements, such as resveratrol, cherry juice, beetroot juice, bergamot extracts, barberry, and pycnogenol, can be effective in blood pressure reduction and cardiovascular prevention, but attention should be paid to their appropriate dosage due to the possibility of side effects from the digestive system.

Inhibitory Effect of Eugenol and trans-Anethole Alone and in Combination with Antifungal Medicines on Candida albicans Clinical Isolates.

One of the most common pathogens among yeasts is Candida albicans, which presents a serious health threat. The study aimed to check the antifungal properties of trans-anethole and eugenol with selected antifungal medicines (AMs) against C. albicans clinical isolates. The checkerboard method was used to tests of interactions between these compounds. Achieved results indicated that eugenol showed synergistic and additive activities with miconazole and econazole against investigated clinical isolates, respectively. Moreover, the combination - trans-anethole - miconazole also showed an additive effect against two clinical isolate. We tried to relate the results to changes in C. albicans cell sheaths under the influence of essential oils compounds (EOCs) performing the Fourier transform infrared spectra analysis to confirm the presence of particular chemical moieties in C. albicans cells. Nevertheless, no strong relationships was observed between synergistic and additive actions of used EOC-AMs combinations and chemical moieties in C. albicans cells.

Synergistic effect of fennel essential oil and hydrogen peroxide on bacterial biofilm.

INTRODUCTION: Staphylococcal biofilm formation significantly challenges wound management. The causes of difficult-to-treat wounds are not only methicillin-resistant staphylococci, but also methicillin-sensitive strains with different patterns of resistance. Bacterial biofilm significantly limits the access and activity of antimicrobials used in dermatological infections. AIM: To evaluate the synergistic effect of fennel essential oil (FEO) and H2O2 on biofilm formation by Staphylococcus aureus (MSSA and MRSA) reference strains. MATERIAL AND METHODS: Minimum inhibitory concentration (MIC) values were determined for FEO and H2O2 against S. aureus reference strains by the broth microdilution method. The combined effects of the FEO and H2O2 were calculated and expressed in terms of a fractional inhibitory concentration index (FICI) using the checkerboard method. The FEO composition was analyzed by the GC-MS method. The data were analysed by one-way ANOVA. RESULTS: Decreased MIC values for FEO combined with H2O2 were observed in comparison to FEO itself. The combinations of FEO and H2O2 determined synergistic effects on all S. aureus reference strains. Subinhibitory concentration of FEO alone and in combination with 0.5 MIC of H2O2 significantly decreased the production of biofilm biomass in S. aureus strains and reduced the metabolic activity of attached cells. CONCLUSIONS: Combination of fennel essential oil containing nearly 80% trans-anethole and H2O2 represents a potential for further basic and applied research on wound management.

Physical activity of future health care professionals: adherence to current recommendations.

BACKGROUND: When assessing physical activity (PA), particular attention should be paid to medical university students who are taught to be health care professionals (HCPs) responsible for maintaining health in humans. However, different studies have shown that HCPs exhibit the same unhealthy behaviors as the general population. This study analyzed PA among medical university students of different faculties and their adherence to current PA recommendations. MATERIAL AND METHODS: Data from 216 medical university students of physiotherapy, dietetics and pharmacy, including males (N = 44) and females (N = 172), the mean age of 22.3±1.8 years, were collected. The International Physical Activity Questionnaire in its long form (IPAQ-LF) was used to assess and classify PA behaviors. The results were analyzed in accordance with World Health Organization recommendations regarding PA. RESULTS: Over 60% of all the students were classified as active during all-day activity. However, while analyzing PA in different domains, the same shares of all the students were still insufficiently active during leisure time, and so they did not meet the recommendation of >75 min/week of vigorous PA, >150 min/week of moderate PA or an equivalent combination. All the students self-reported PA mainly in the work and transport domains. Generally, physiotherapy students were the most active and performed PA with higher intensity. CONCLUSIONS: This study revealed a low level of leisure time PA among the students, and no habit of regular PA. Some changes in medical education should be suggested to include physical education as a long-term subject in medical school curricula. Future research is needed to investigate the exercise barriers that students perceive, which can guide future interventions aimed at improving their PA, and thereby impact on the quality of health care which they will provide. Med Pr. 2020;71(5):539-49.

Panax quinquefolium L. Ginsenosides from Hairy Root Cultures and Their Clones Exert Cytotoxic, Genotoxic and Pro-Apoptotic Activity towards Human Colon Adenocarcinoma Cell Line Caco-2.

American ginseng, Panax quinquefolium (L.), is traditionally used in folk medicine. It exhibits a range of anti-inflammatory, hepatoprotective, anti-diabetic, anti-obesity, anti-hyperlipidemic and anti-carcinogenic effects. Its main components are ginsenosides, also known as panaxosides or triterpene saponins. In order to obtain high yields of ginsenosides, different methods of controlled production are involved, i.e., with hairy root cultures. However, they are still employed under in vitro conditions. Our studies revealed that hairy root cultures subjected to an elicitation process can be considered as a potent source of ginsenosides. The present study examines the biological activity of ginseng hairy root cultures against the Caco-2 human adenocarcinoma cell line. Among our six different clones of P. quinquefolium hairy roots, extracts B and Be (treated with elicitor) were the strongest inhibitors of the cellular metabolic activity. While all extracts induced DNA damage, B and Be also generated reactive oxygen species (ROS) in a concentration-dependent manner, which was correlated with the depletion of the mitochondrial membrane potential and induction of apoptosis. These findings indicate that further research concerning P. quinquefolium hairy root cultures should focus on the activity of rare ginsenosides and other biologically active compound profiles (i.e., phenolic compounds).

The time spent sitting does not always mean a low level of physical activity.

BACKGROUND: The problem of spending most of the day in a sitting position concerns all people, regardless of their age. Unfortunately, this trend is more and more often observed among young people. The aim of the study was to assess self-reported physical activity and time spent sitting among students of different fields of health related faculty. METHODS: The study group included 216 students (22.3 ± 1.8 years of age) of the Medical University of Lodz: physiotherapy students (n = 101), pharmacy students (n = 73), and dietetics students (n = 42). The time spent sitting and physical activity level were assessed based on the International Physical Activity Questionnaire-long version. RESULTS: The time spent sitting among health related faculty students was on average more than 46 h a week (2781.8 ± 1238.5 MET-minutes/week). Regarding all the students the pharmacy students spent most time sitting (3086.0 ± 1032.1 MET-minutes/week), while the dietetics students spent the least (2215.7 ± 1230.1 MET-minutes/week). Taking into account the physical activity level almost 65% of all the students were in a high category (mainly physiotherapy students). Only 1.4% of all the surveyed students were classified as the low physical activity category. Statistical analysis showed no significant differences (P = 0.6880) between the time spent sitting and level of physical activity among all students. CONCLUSIONS: Students of medical universities spend too much hours on sitting, mostly 5-8 h a day. Despite this, they undertake various activities due to which their level of physical activity is moderate or even high. Therefore, it cannot be unequivocally stated that there is a relationship between the time spent sitting and physical activity level.

Computer-Aided Saturation Mutagenesis of Arabidopsis thaliana Ent-Copalyl Diphosphate Synthase.

Ent-copalyl diphosphate synthase controls the biosynthesis of gibberellin plant hormones, which in turn coordinate the expression of numerous enzymes. Some gibberellin-dependent genes encode enzymes coordinating the biosynthesis of tanshinones: diterpene derivatives with broad medical applications. New biotechnological approaches, such as metabolic engineering using naturally occurring or mutated enzymes, have been proposed to meet the growing demand for tanshinones which is currently met by the Chinese medicinal plant Salvia miltiorrhiza Bunge. These mutants may be prepared by directed evolution, saturation mutagenesis or rational enzyme design. In the presented paper, 15,257 non-synonymous variants of Arabidopsis thaliana ent-copalyl diphosphate synthase were obtained using the SNAP2 tool. The obtained forms were screened to isolate variants with potentially improved biological functions. A group of 455 mutants with potentially improved stability was isolated and subjected to further screening on the basis of ligand-substrate affinity, and both secondary structure and active site structure stability. Finally, a group of six single mutants was obtained, which were used to construct double mutants with potentially improved stability and ligand affinity. The potential influence of single mutations on protein stability and ligand affinity was evaluated by double mutant cycle analysis. Finally, the procedure was validated by in silico assessment of the experimentally verified enzyme mutants with reduced enzymatic activity.

American Ginseng (Panax quinquefolium L.) as a Source of Bioactive Phytochemicals with Pro-Health Properties.

Panax quinquefolium L. (American Ginseng, AG) is an herb characteristic for regions of North America and Asia. Due to its beneficial properties it has been extensively investigated for decades. Nowadays, it is one of the most commonly applied medical herbs worldwide. Active compounds of AG are ginsenosides, saponins of the glycosides group that are abundant in roots, leaves, stem, and fruits of the plant. Ginsenosides are suggested to be primarily responsible for health-beneficial effects of AG. AG acts on the nervous system; it was reported to improve the cognitive function in a mouse model of Alzheimer's disease, display anxiolytic activity, and neuroprotective effects against neuronal damage resulting from ischemic stroke in animals, demonstrate anxiolytic activity, and induce neuroprotective effects against neuronal damage in ischemic stroke in animals. Administration of AG leads to inhibition of hypertrophy in heart failure by regulation of reactive oxygen species (ROS) in mice as well as depletion of cardiac contractile function in rats. It also has an anti-diabetic and anti-obesity potential as it increases insulin sensitivity and inhibits formation of adipose tissue. AG displays anti-cancer effect by induction of apoptosis of cancer cells and reducing local inflammation. It exerts antimicrobial effects against several pathogenic strains of bacteria. Therefore, AG presents a high potential to induce beneficial health effects in humans and should be further explored to formulate precise nutritional recommendations, as well as to assess its value in prevention and therapy of some disorders, including cancer.

Abscisic Acid Regulates the 3-Hydroxy-3-methylglutaryl CoA Reductase Gene Promoter and Ginsenoside Production in Panax quinquefolium Hairy Root Cultures.

Panax quinquefolium hairy root cultures synthesize triterpenoid saponins named ginsenosides, that have multidirectional pharmacological activity. The first rate-limiting enzyme in the process of their biosynthesis is 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). In this study, a 741 bp fragment of the P. quinquefolium HMGR gene (PqHMGR), consisting of a proximal promoter, 5'UTR (5' untranslated region) and 5'CDS (coding DNA sequence) was isolated. In silico analysis of an isolated fragment indicated a lack of tandem repeats, miRNA binding sites, and CpG/CpNpG elements. However, the proximal promoter contained potential cis-elements involved in the response to light, salicylic, and abscisic acid (ABA) that was represented by the motif ABRE (TACGTG). The functional significance of ABA on P. quinquefolium HMGR gene expression was evaluated, carrying out quantitative RT-PCR experiments at different ABA concentrations (0.1, 0.25, 0.5, and 1 mg·L-1). Additionally, the effect of abscisic acid and its time exposure on biomass and ginsenoside level in Panax quinquefolium hairy root was examined. The saponin content was determined using HPLC. The 28 day elicitation period with 1 mg·L-1 ABA was the most efficient for Rg2 and Re (17.38 and 1.83 times increase, respectively) accumulation; however, the protopanaxadiol derivative content decreased in these conditions.

The Increase of Triterpene Saponin Production Induced by Trans-Anethole in Hairy Root Cultures of Panax quinquefolium.

In vitro cultivation is an effective way to increase pharmaceutical production. To increase ginsenoside production in hairy root cultures of American ginseng, the present study uses trans-anethole as an elicitor. The content of nine triterpene saponins was determined: Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, Re and Rf. Trans-anethole was found to stimulate saponin synthesis regardless of exposure time (24 and 72 h). Twenty-four hour exposure to 1 µmol trans-anethole in the culture medium resulted in the highest increase of total saponin content (twice that of untreated roots), and optimum accumulation of Rb-group saponins, with ginsenoside Rc dominating (8.45 mg g-1 d.w.). In contrast, the highest mean content of protopanaxatriol derivatives was obtained for 10 µmol trans-anethole. The Re metabolite predominated, reaching a concentration of 5.72 mg g-1 d.w.: a 3.9-fold increase over untreated roots. Elicitation with use of trans-anethole can therefore be an effective method of increasing ginsenoside production in shake flasks.

Preliminary Study on the Antibacterial Activity of Essential Oils Alone and in Combination with Gentamicin Against Extended-Spectrum ß-Lactamase-Producing and New Delhi Metallo-ß-Lactamase-1-Producing Klebsiella pneumoniae Isolates.

AIM: The aim of the study was to investigate possible synergistic effects between several selected, commercially available essential oils and gentamicin against extended-spectrum ß-lactamase (ESBL)-producing and New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae isolates. MATERIALS AND METHODS: ESBLs production was confirmed by double-disk synergy test. Isolates positive for blaNDM-1 gene were found among the tested strains. K. pneumoniae ATCC® BAA-1705™ strain was used as a control. The checkerboard method was applied to assess the synergistic and additive action of nine essential oils: caraway, fennel, peppermint, geranium, basil, clove, thyme, clary sage, and lavender, respectively, in combination with gentamicin. RESULTS: Our results indicated that peppermint oil combined with gentamicin showed synergistic activity against both control, ESBL-producing and NDM-1-producing isolates. Caraway essential oil demonstrated synergy with gentamicin toward ESBL-producing and additionally gentamicin-resistant strains. The additive effect was observed for gentamicin combined with thyme, fennel, basil, and clary sage. CONCLUSIONS: Because of their synergistic activity with gentamicin, peppermint, and caraway oils in particular, can be considered as an alternative or an addition for the control of infections with limited therapeutic options due to multidrug resistance.

Yeast Extract Stimulates Ginsenoside Production in Hairy Root Cultures of American Ginseng Cultivated in Shake Flasks and Nutrient Sprinkle Bioreactors.

One of the most effective strategies to enhance metabolite biosynthesis and accumulation in biotechnological systems is the use of elicitation processes. This study assesses the influence of different concentrations of yeast extract (YE) on ginsenoside biosynthesis in Panax quinquefolium (American ginseng) hairy roots cultivated in shake flasks and in a nutrient sprinkle bioreactor after 3 and 7 days of elicitation. The saponin content was determined using HPLC. The maximum yield (20 mg g-1 d.w.) of the sum of six examined ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) in hairy roots cultivated in shake flasks was achieved after application of YE at 50 mg L-1 concentration and 3 day exposure time. The ginsenoside level was 1.57 times higher than that attained in control medium. The same conditions of elicitation (3 day time of exposure and 50 mg L-1 of YE) also favourably influenced the biosynthesis of studied saponins in bioreactor cultures. The total ginsenoside content was 32.25 mg g-1 d.w. and was higher than that achieved in control medium and in shake flasks cultures. Obtained results indicated that yeast extract can be used to increase ginsenoside production in hairy root cultures of P. quinquefolium.

The ability of selected plant essential oils to enhance the action of recommended antibiotics against pathogenic wound bacteria.

The aim of this work was to characterize the ability of essential oils to support antibiotics against pathogenic bacteria in wounds. Gram-positive and Gram-negative bacteria obtained from wound infections were identified according to standard microbiological methods. Essential oils were analysed by GC-FID-MS. The susceptibility of bacteria to antibiotics, essential oils and their combination was assessed using the disc-diffusion method. The Minimal Inhibitory Concentration and Minimum Bactericidal Concentration of the essential oils were established by the micro-dilution broth method. Although cinnamon, clove, thyme and lavender essential oils were found to have the greatest antibacterial activity when used alone, the greatest additive and synergistic effects against pathogenic wound bacteria in combination with recommended antibiotics were demonstrated by basil, clary sage and rosemary oils.

The production of ginsenosides in hairy root cultures of American Ginseng, Panax quinquefolium L. and their antimicrobial activity.

Panax quinquefolium, American ginseng, is valued for its triterpene saponins, known as ginsenosides. These constituents possess a number of pharmacological properties and hairy root cultures can synthesize similar saponins to those of field-cultivated roots. The antibacterial activity of extracts from three hairy root clones of P. quinquefolium L. was tested against a range of standard bacterial and yeast strains. The agar diffusion method was used to evaluate inhibition of microbial growth at various extract concentrations. Commercial antibiotics were used as positive reference standards to determine the sensitivity of the strains. Susceptibility testing to antibiotics was also tested using the disk diffusion method. The minimal inhibitory concentration values of the extracts, obtained by agar diffusion, ranged from 0.8 to 1.4 mg/ml. The results showed that extracts from hairy root cultures inhibited the growth of bacteria and yeast strains and suggest that they may be useful in the treatment of infections caused by pathogenic microorganisms.

Field cultivation and in vitro cultures, root-forming callus cultures and adventitious root cultures, of Panax quinquefolium as a source of ginsenosides.

The content of six ginsenosides (Rb1, Rb2, Rc, Rd, Rg1, and Re) was studied in the roots of field-grown plants, as well as in root-forming callus cultures and adventitious root cultures of Panax quinquefolium using high-performance liquid chromatography (HPLC). The highest level of saponins was isolated from root hairs (128 mg/g dry weight). The examined in vitro culture synthesized all identified saponins, although in smaller amounts than those obtained from field cultivation. Metabolites Rb1 and Re dominated in the ginseng biomass from both field crops and in vitro culture.

Ginsenoside contents in Panax quinquefolium organs from field cultivation.

Quantitative composition of saponins (ginsenosides) in Panax quinquefolium originating from field cultivation in Poland and harvested at the beginning of the growing season, during the blossoming period and at the end of growth was determined. A colourimetric method gave lower values compared to results of HPLC analysis, but the tendency of change in the content of saponins was similar in both instances. Ginsenoside amounts in P. quinquefolium organs changed depending on the specific time during the vegetation season the samples were taken. This study found that the highest content of these metabolites--66 mg/g d. w.-- occurred in the roots of the plant at the time of bloom. Two among the six metabolites examined in our study were dominant independently of the vegetation season. These were Rb1 and Re, with values of 25.4-33.8 mg/g d. w. and 16.4-19.7 mg/g d. w., respectively.