Evaluation of the Toxicity Potential of the Metabolites of Di-Isononyl Phthalate and of Their Interactions with Members of Family 1 of Sulfotransferases-A Computational Study.

Di-isononyl phthalates are chemicals that are widely used as plasticizers. Humans are extensively exposed to these compounds by dietary intake, through inhalation and skin absorption. Sulfotransferases (SULTs) are enzymes responsible for the detoxification and elimination of numerous endogenous and exogenous molecules from the body. Consequently, SULTs are involved in regulating the biological activity of various hormones and neurotransmitters. The present study considers a computational approach to predict the toxicological potential of the metabolites of di-isononyl phthalate. Furthermore, molecular docking was considered to evaluate the inhibitory potential of these metabolites against the members of family 1 of SULTs. The metabolites of di-isononyl phthalate reveal a potency to cause liver damage and to inhibit receptors activated by peroxisome proliferators. These metabolites are also usually able to inhibit the activity of the members of family 1 of SULTs, except for SULT1A3 and SULT1B1. The outcomes of this study are important for an enhanced understanding of the risk of human exposure to di-isononyl phthalates.

Copper Accumulation Efficiency in Different Recombinant Microorganism Strains Available for Bioremediation of Heavy Metal-Polluted Waters.

The aim of this research was to investigate the bioremediation conditions of copper in synthetic water. In the present study, copper ions accumulation efficiency was determined using various genetically modified strains of Saccharomyces cerevisiae (EBY100, INVSc1, BJ5465, and GRF18), Pichia pastoris (X-33, KM71H), Escherichia coli (XL10 Gold, DH5α, and six types of BL21 (DE3)), and Escherichia coli BL21 (DE3) OverExpress expressing two different peroxidases. Viability tests of yeast and bacterial strains showed that bacteria are viable at copper concentrations up to 2.5 mM and yeasts up to 10 mM. Optical emission spectrometry with inductively coupled plasma analysis showed that the tolerance of bacterial strains on media containing 1 mM copper was lower than the tolerance of yeast strains at the same copper concentration. The E. coli BL21 RIL strain had the best copper accumulation efficiency (4.79 mg/L of culture normalized at an optical density of 1.00), which was 1250 times more efficient than the control strain. The yeast strain S. cerevisiae BJ5465 was the most efficient in copper accumulation out of a total of six yeast strains used, accumulating over 400 times more than the negative control strain. In addition, E. coli cells that internally expressed recombinant peroxidase from Thermobifida fusca were able to accumulate 400-fold more copper than cells that produced periplasmic recombinant peroxidases.

Assessment of the Effects of Triticonazole on Soil and Human Health.

Triticonazole is a fungicide used to control diseases in numerous plants. The commercial product is a racemate containing (R)- and (S)-triticonazole and its residues have been found in vegetables, fruits, and drinking water. This study considered the effects of triticonazole on soil microorganisms and enzymes and human health by taking into account the enantiomeric structure when applicable. An experimental method was applied for assessing the effects of triticonazole on soil microorganisms and enzymes, and the effects of the stereoisomers on soil enzymes and human health were assessed using a computational approach. There were decreases in dehydrogenase and phosphatase activities and an increase in urease activity when barley and wheat seeds treated with various doses of triticonazole were sown in chernozem soil. At least 21 days were necessary for the enzymes to recover the activities. This was consistent with the diminution of the total number of soil microorganisms in the 14 days after sowing. Both stereoisomers were able to bind to human plasma proteins and were potentially inhibitors of human cytochromes, revealing cardiotoxicity and low endocrine disruption potential. As distinct effects, (R)-TTZ caused skin sensitization, carcinogenicity, and respiratory toxicity. There were no significant differences in the interaction energies of the stereoisomers and soil enzymes, but (S)-TTZ exposed higher interaction energies with plasma proteins and human cytochromes.

Assessment of the Effects of Chitosan, Chitooligosaccharides and Their Derivatives on Lemna minor.

Chitosan, chitooligosaccharides and their derivatives' production and use in many fields may result in their release to the environment, possibly affecting aquatic organisms. Both an experimental and a computational approach were considered for evaluating the effects of these compounds on Lemna minor. Based on the determined EC50 values against L. minor, only D-glucosamine hydrochloride (EC50 = 11.55 mg/L) was considered as "slightly toxic" for aquatic environments, while all the other investigated compounds, having EC50 > 100 mg/L, were considered as "practically non-toxic". The results obtained in the experimental approach were in good agreement with the predictions obtained using the admetSAR2.0 computational tool, revealing that the investigated compounds were not considered toxic for crustacean, fish and Tetrahymena pyriformis aquatic microorganisms. The ADMETLab2.0 computational tool predicted the values of IGC50 for Tetrahymena pyriformis and the LC50 for fathead minnow and Daphnia magna, with the lowest values of these parameters being revealed by totally acetylated chitooligosaccharides in correlation with their lowest solubility. The effects of the chitooligosaccharides and chitosan on L. minor decreased with increased molecular weight, increased with the degree of deacetylation and were reliant on acetylation patterns. Furthermore, the solubility mainly influenced the effects on the aqueous environment, with a higher solubility conducted to lower toxicity.

A Cheminformatics Study Regarding the Human Health Risks Assessment of the Stereoisomers of Difenoconazole.

Difenoconazole is a chemical entity containing two chiral centers and having four stereoisomers: (2R,4R)-, (2R,4S)-, (2S,4R)- and (2S,4S)-difenoconazole, the marketed product containing a mixture of these isomers. Residues of difenoconazole have been identified in many agricultural products and drinking water. A computational approach has been used to evaluate the toxicological effects of the difenoconazole stereoisomers on humans. It integrates predictions of absorption, distribution, metabolism, excretion and toxicity (ADMET) profiles, prediction of metabolism sites, and assessment of the interactions of the difenoconazole stereoisomers with human cytochromes, nuclear receptors and plasma proteins by molecular docking. Several toxicological effects have been identified for all the difenoconazole stereoisomers: high plasma protein binding, inhibition of cytochromes, possible hepatotoxicity, neurotoxicity, mutagenicity, skin sensitization potential, moderate potential to produce endocrine disrupting effects. There were small differences in the predicted probabilities of producing various biological effects between the distinct stereoisomers of difenoconazole. Furthermore, there were significant differences between the interacting energies of the difenoconazole stereoisomers with plasma proteins and human cytochromes, the spectra of the hydrogen bonds and aromatic donor-acceptor interactions being quite distinct. Some distinguishing results have been obtained for the (2S,4S)-difenoconazole: it registered the highest value for clearance, exposed reasonable probabilities to produce cardiotoxicity and carcinogenicity and negatively affected numerous nuclear receptors.

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A.

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl ß-d-N,N',N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2-8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

Computational Assessment of Chito-Oligosaccharides Interactions with Plasma Proteins.

It is widely recognized that chitin and chitosan are potential sources of bioactive materials and that their oligosaccharides reveal various biological activities (including antimicrobial) that are correlated with their structures and physicochemical properties. This study uses the molecular docking approach to assess the interactions of small chito-oligosaccharides (MW< 1500 Da) with plasma proteins in order to obtain information regarding their fate of distribution in the human organism. There are favorable interactions of small chito-oligomers with plasma proteins, the interactions with human serum albumin being stronger than those with α-1-acid glycoprotein. The interaction energies increase with increasing the molecular weight, decrease with increasing deacetylation degrees and are reliant on the deacetylation pattern. This study could inform the application of chito-oligosaccharides with varying molecular weights, degrees, and patterns of deacetylation in human health.

In silico Assessment of Pharmacological Profile of Low Molecular Weight Oligo-Hydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are a large class of polyesters that are biosynthesized by microorganisms at large molecular weights (Mw > 80 kDa) and have a great potential for medical applications because of their recognized biocompatibility. Among PHAs, poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxyvalerate), poly(4-hydroxyvalerate), and their copolymers are proposed to be used in biomedicine, but only poly(4-hydroxybutyrate) has been certified for medical application. Along with the hydrolysis of these polymers, low molecular weight oligomers are released typically. In this study, we have used a computational approach to assess the absorption, distribution, metabolism, and excretion (ADME)-Tox profiles of low molecular weight oligomers (≤32 units) consisting of 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxyvalerate, 3-hydroxybutyrate-co-3-hydroxyvalerate, and the hypothetical PHA consisting of 4-hydroxybutyrate-co-4-hydroxyvalerate. According to our simulations, these oligomers do not show cardiotoxicity, hepatotoxicity, carcinogenicity or mutagenicity, and are neither substrates nor inhibitors of the cytochromes involved in the xenobiotic's metabolism. They also do not affect the human organic cation transporter 2 (OCT2). However, they are considered to be inhibitors of the organic anion transporters OATP1B1, and OATP1B3. In addition, they may produce eye irritation, and corrosion, skin irritation and have a low antagonistic effect on the androgen receptor.

Understanding the Factors Influencing Chitosan-Based Nanoparticles-Protein Corona Interaction and Drug Delivery Applications.

Chitosan is a polymer that is extensively used to prepare nanoparticles (NPs) with tailored properties for applications in many fields of human activities. Among them, targeted drug delivery, especially when cancer therapy is the main interest, is a major application of chitosan-based NPs. Due to its positive charges, chitosan is used to produce the core of the NPs or to cover NPs made from other types of polymers, both strategies aiming to protect the carried drug until NPs reach the target sites and to facilitate the uptake and drug delivery into these cells. A major challenge in the design of these chitosan-based NPs is the formation of a protein corona (PC) upon contact with biological fluids. The composition of the PC can, to some extent, be modulated depending on the size, shape, electrical charge and hydrophobic / hydrophilic characteristics of the NPs. According to the composition of the biological fluids that have to be crossed during the journey of the drug-loaded NPs towards the target cells, the surface of these particles can be changed by covering their core with various types of polymers or with functionalized polymers carrying some special molecules, that will preferentially adsorb some proteins in their PC. The PC's composition may change by continuous processes of adsorption and desorption, depending on the affinity of these proteins for the chemical structure of the surface of NPs. Beside these, in designing the targeted drug delivery NPs one can take into account their toxicity, initiation of an immune response, participation (enhancement or inhibition) in certain metabolic pathways or chemical processes like reactive oxygen species, type of endocytosis of target cells, and many others. There are cases in which these processes seem to require antagonistic properties of nanoparticles. Products that show good behavior in cell cultures may lead to poor in vivo results, when the composition of the formed PC is totally different. This paper reviews the physico-chemical properties, cellular uptake and drug delivery applications of chitosan-based nanoparticles, specifying the factors that contribute to the success of the targeted drug delivery. Furthermore, we highlight the role of the protein corona formed around the NP in its intercellular fate.

Deeper inside the specificity of lysozyme when degrading chitosan. A structural bioinformatics study.

Lysozymes that were used in numerous in vitro experiments of chitosan degradation were regularly from hen egg-white. Human lysozyme has been proved to be more active than hen egg-white lysozyme when exerting its bacteriostatic effect and there is possible that the two enzymes have some different structural properties. Consequently, within this study, we compared the structural and physicochemical properties of the human and egg-white lysozymes and used the molecular docking approach to obtain information concerning the specificity of the interactions between chitooligosaccharides with these enzymes. There is 60.47% of identity between the sequences of the human and the hen egg-white lysozymes, but the amino acids that are involved in the interactions of considered lysozymes with N-acetylchitohexaose are well conserved. Superimposition of the structures of investigated lysozymes reveals their structural similarity, the RMSD value being 1.198 Å for 118 equivalent carbon alpha atom pairs from a total of 129. There are some local physicochemical properties like the distribution of the electrostatic potential and the hydrophobicity of the catalytic cavities of the enzyme that are quite different. Substrate specificities of human and hen egg-white lysozyme with respect to chito-oligosaccharides are not clearly distinguishable but they are dependent on the molecular weight, deacetylation degree and pattern of deacetylation.

Evaluation of Ecotoxicology Assessment Methods of Nanomaterials and Their Effects.

This paper describes the ecotoxicological effects of nanomaterials (NMs) as well as their testing methods. Standard ecotoxicity testing methods are applicable to nanomaterials as well but require some adaptation. We have taken into account methods that meet several conditions. They must be properly researched by a minimum of ten scientific articles where adaptation of the method to the NMs is also presented; use organisms suitable for simple and rapid ecotoxicity testing (SSRET); have a test period shorter than 30 days; require no special equipment; have low costs and have the possibility of optimization for high-throughput screening. From the standard assays described in guidelines developed by organizations such as Organization for Economic Cooperation and Development and United States Environmental Protection Agency, which meet the required conditions, we selected as methods adaptable for NMs, some methods based on algae, duckweed, amphipods, daphnids, chironomids, terrestrial plants, nematodes and earthworms. By analyzing the effects of NMs on a wide range of organisms, it has been observed that these effects can be of several categories, such as behavioral, morphological, cellular, molecular or genetic effects. By comparing the EC50 values of some NMs it has been observed that such values are available mainly for aquatic ecotoxicity, with the most sensitive test being the algae assay. The most toxic NMs overall were the silver NMs.

Solubility and ADMET profiles of short oligomers of lactic acid.

Polylactic acid (PLA) is a polymer with an increased potential to be used in different medical applications, including tissue engineering and drug-carries. The use of PLA in medical applications implies the evaluation of the human organism's response to the polymer inserting and to its degradation products. Consequently, within this study, we have investigated the solubility and ADMET profiles of the short oligomers (having the molecular weight lower than 3000 Da) resulting in degradation products of PLA. There is a linear decrease of the molar solubility of investigated oligomers with molecular weight. The results that are obtained also reveal that short oligomers of PLA have promising pharmacological profiles and limited toxicological effects on humans. These oligomers are predicted as potential inhibitors of the organic anion transporting peptides OATP1B1 and OATP1B3, they present minor probability to affect the androgen and glucocorticoid receptors, have a weak potential of hepatotoxicity, and may produce eye injuries. These outcomes may be used to guide or to supplement in vitro and/or in vivo toxicity tests such as to enhance the biodegradation properties of the biopolymer.

Chitosan as a Wound Dressing Starting Material: Antimicrobial Properties and Mode of Action.

Fighting bacterial resistance is one of the concerns in modern days, as antibiotics remain the main resource of bacterial control. Data shows that for every antibiotic developed, there is a microorganism that becomes resistant to it. Natural polymers, as the source of antibacterial agents, offer a new way to fight bacterial infection. The advantage over conventional synthetic antibiotics is that natural antimicrobial agents are biocompatible, non-toxic, and inexpensive. Chitosan is one of the natural polymers that represent a very promising source for the development of antimicrobial agents. In addition, chitosan is biodegradable, non-toxic, and most importantly, promotes wound healing, features that makes it suitable as a starting material for wound dressings. This paper reviews the antimicrobial properties of chitosan and describes the mechanisms of action toward microbial cells as well as the interactions with mammalian cells in terms of wound healing process. Finally, the applications of chitosan as a wound-dressing material are discussed along with the current status of chitosan-based wound dressings existing on the market.

Computational Assessment of the Pharmacological Profiles of Degradation Products of Chitosan.

Chitosan is a natural polymer revealing an increased potential to be used in different biomedical applications, including drug delivery systems, and tissue engineering. It implies the evaluation of the organism response to the biomaterial implantation. Low-molecular degradation products, the chito-oligomers, are resulting mainly from the influence of enzymes, which are found in the organism fluids. Within this study, we have performed the computational assessment of pharmacological profiles and toxicological effects on human health of small chito-oligomers with distinct molecular weights, deacetylation degrees, and acetylation patterns. Our approach is based on the fact that regulatory agencies and researchers in the drug development field rely on the use of modeling to predict biological effects and to guide decision making. To be considered as valid for regulatory purposes, every model that is used for predictions should be associated with a defined toxicological endpoint and has appropriate robustness and predictivity. Within this context, we have used FAF-Drugs4, SwissADME, and PreADMET tools to predict the oral bioavailability of chito-oligomers and SwissADME, PreADMET, and admetSAR2.0 tools to predict their pharmacokinetic profiles. The organs and genomic toxicities have been assessed using admetSAR2.0 and PreADMET tools but specific computational facilities have been also used for predicting different toxicological endpoints: Pred-Skin for skin sensitization, CarcinoPred-EL for carcinogenicity, Pred-hERG for cardiotoxicity, ENDOCRINE DISRUPTOME for endocrine disruption potential and Toxtree for carcinogenicity and mutagenicity. Our computational assessment showed that investigated chito-oligomers reflect promising pharmacological profiles and limited toxicological effects on humans, regardless of molecular weight, deacetylation degree, and acetylation pattern. According to our results, there is a possible inhibition of the organic anion transporting peptides OATP1B1 and/or OATP1B3, a weak potential of cardiotoxicity, a minor probability of affecting the androgen receptor, and phospholipidosis. Consequently, these results may be used to guide or to complement the existing in vitro and in vivo toxicity tests, to optimize biomaterials properties and to contribute to the selection of prototypes for nanocarriers.

Polyelectrolyte nanocomplexes based on chitosan derivatives for wound healing application.

Wound healing, when compromised, may be guided by biological cues such as Arg-Gly-Asp (RGD), a peptide known to induce cell adhesion and migration, eventually combined with adapted nanocarriers. Three different formulations were prepared and investigated in vitro for topical application. All formulations were based on carboxylated and trimethylated chitosan (CMTMC) displaying RGD. The polyelectrolyte nanocomplexes were prepared by mixing two oppositely charged polymers of CMTMC and chondroitin sulfate at different polymer ratios and subsequently characterized by dynamic light scattering and scanning electron microscopy. Hydrogels and foams with a high concentration of RGD-functionalized chitosan (3%) and hyaluronic acid (1.5%) that formed gel-embedded nanocomplexes were developed. In vitro assays showed absence of toxicity, ability to promote proliferation over 7 days and promotion of migration of human dermal fibroblasts treated with any of our formulations. These formulations were shown to be suitable for easy topical application and have the potential to accelerate wound healing.

Assessment of the properties of chitin deacetylases showing different enzymatic action patterns.

Chitin deacetylases are a group of enzymes catalysing the conversion of chitin to chitosan. Obtaining chitosan with established deacetylation degree and pattern is important for biomedical and biotechnological applications. Understandings of the structural properties of chitin deacetylases and the specificity of their interactions with chitin may conduct to the control of the pattern of deacetylation of chitosan. Our study is focused on the characterization and comparison of the structural and physicochemical properties of chitin deacetylases from fungi and marine bacteria. Despite the low sequences identity for the investigated chitin deacetylases, there are amino acids belonging to their active sites that are strongly conserved. Moreover, they reveal an increased structural similarity of their catalytic domains, reflecting the common biological function of these enzymes. The studied enzymes present dissimilar local physicochemical properties of their catalytic cavities that could be responsible of their distinct deacetylation patterns. Molecular docking studies reflect that deacetylation efficiency is also distinct for the chitin and partially deacetylated chitin oligomers and that N-acetylglucosamine units and some partially deacetylated chitin oligomers could have inhibitory effect against chitin deacetylases belonging to fungi and marine bacteria.

Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H.

Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0-5.0 and within the temperature range 50-60 °C. With colloidal chitin as the substrate, the Km (2.307 mM) and Vmax (0.024 mM min-1) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli. We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants.

A Meta-Analysis on Randomised Controlled Clinical Trials Evaluating the Effect of the Dietary Supplement Chitosan on Weight Loss, Lipid Parameters and Blood Pressure.

Background and objectives: Erratic results have been published concerning the influence of the dietary supplement chitosan used as a complementary remedy to decrease the body weight of overweight and obese people. The published articles mention as secondary possible benefits of usage of chitosan the improvement of blood pressure and serum lipids status. We performed a meta-analysis evaluating body weight, body mass index, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides, systolic and diastolic blood pressure among overweight and obese patients. Materials and Methods: Searching MEDLINE, Cochrane up to December 2017 on clinical trials that have assessed the influence of chitosan used as a dietary supplement on overweight and obese patients. An additional study was identified in the References section of another meta-analysis. A total of 14 randomised control trials (RCT) were used to assess the effect on body weight, serum lipids and blood pressure. Results: The usage of chitosan as a dietary supplement up to 52 weeks seems to slightly reduce the body weight (-1.01 kg, 95% CI: -1.67 to -0.34). Considering the other parameters studied, the most significant improvement was observed in systolic and diastolic blood pressure: -2.68 mm Hg (95% CI: -4.19 to -1.18) and -2.14 mm Hg (95% CI: -4.14 to -0.14) in favour of chitosan versus a placebo. Conclusions: Based on the meta-analysis realized with 14 RCT we concluded that the usage of chitosan as a dietary supplement can lead to a slight short- and medium-term effect on weight loss and to the improvement of serum lipid profile and cardiovascular factors.

Computational Assessment of Pharmacokinetics and Biological Effects of Some Anabolic and Androgen Steroids.

PURPOSE: The aim of this study is to use computational approaches to predict the ADME-Tox profiles, pharmacokinetics, molecular targets, biological activity spectra and side/toxic effects of 31 anabolic and androgen steroids in humans. METHODS: The following computational tools are used: (i) FAFDrugs4, SwissADME and admetSARfor obtaining the ADME-Tox profiles and for predicting pharmacokinetics;(ii) SwissTargetPrediction and PASS online for predicting the molecular targets and biological activities; (iii) PASS online, Toxtree, admetSAR and Endocrine Disruptomefor envisaging the specific toxicities; (iv) SwissDock to assess the interactions of investigated steroids with cytochromes involved in drugs metabolism. RESULTS: Investigated steroids usually reveal a high gastrointestinal absorption and a good oral bioavailability, may inhibit someof the human cytochromes involved in the metabolism of xenobiotics (CYP2C9 being the most affected) and reflect a good capacity for skin penetration. There are predicted numerous side effects of investigated steroids in humans: genotoxic carcinogenicity, hepatotoxicity, cardiovascular, hematotoxic and genitourinary effects, dermal irritations, endocrine disruption and reproductive dysfunction. CONCLUSIONS: These results are important to be known as an occupational exposure to anabolic and androgenic steroids at workplaces may occur and because there also is a deliberate human exposure to steroids for their performance enhancement and anti-aging properties.