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.

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.

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).

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.

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.

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.