Assessment of the in vitro acaricidal activity of Bravecto® (fluralaner) and a proposed orange oil-based formulation vehicle for the treatment of Sarcoptes scabiei.

BACKGROUND: Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). Fluralaner (Bravecto®) is a novel acaricide that has recently been utilised for treating mange in wombats. The topical 'spot-on' formulation of fluralaner can limit treatment delivery options in situ, but dilution to a volume for 'pour-on' delivery is one practicable solution. This study investigated the in vitro acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei. METHODS: Sarcoptes scabiei were sourced from experimentally infested pigs. In vitro assays were performed to determine the lethal concentration (LC50) and survival time of the mites when exposed to varying concentrations of the test solutions. RESULTS: All compounds were highly effective at killing mites in vitro. The LC50 values of Bravecto, Orange Power, limonene and citral at 1 h were 14.61 mg/ml, 4.50%, 26.53% and 0.76%, respectively. The median survival times of mites exposed to undiluted Bravecto, Orange Power and their combination were 15, 5 and 10 min, respectively. A pilot survival assay of mites collected from a mange-affected wombat showed survival times of < 10 min when exposed to Bravecto and Orange Power and 20 min when exposed to moxidectin. CONCLUSIONS: These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. As well as killing mites via direct exposure, Orange Power could potentially enhance the topical delivery of Bravecto to wombats by increasing drug penetration in hyperkeratotic crusts. Further research evaluating the physiochemical properties and modes of action of Orange Power and its constituents as a formulation vehicle would be of value.

Comprehensive quality assessment of peppermint oils and commercial products: An integrated approach involving conventional and chiral GC/MS coupled with chemometrics.

Peppermint essential oil (EO) has a multitude of applications, such as a fragrance in cosmetics, personal care and industrial products, or as a flavoring ingredient in food and beverages. Despite its popularity and economic significance, peppermint EO is often adulterated to reduce production costs and to increase profits. Although the ISO standard for peppermint EO exists, detecting sophisticated forms of adulteration remains challenging.The current study used conventional and chiral GC/MS analysis of volatiles compounds, and chemometric techniques to evaluate an extensive set of authentic peppermint EO (n = 22) and commercial products (n = 36) purported to contain peppermint EO. Specifically, thirty-six terpenoids were examined in each sample and compared with the ISO standard. Fifty-three percent of the selected commercial products did not meet the ISO specifications and the ratio between menthone/isomenthone was proven to be a good indicator for authentication and adulteration detection. Chiral GC/MS was further employed to measure eight terpenoids: α-pinene, ß-pinene, limonene, menthol, menthone, isomenthone, pulegone, and menthyl acetate. The enantiomeric compositions of 27 commercial products were above or below the norm measured from authentic peppermint EOs. Of the 27 samples, eight met the ISO standard. A sample class prediction (SCP) model based on partial least squares-discriminant analysis (PLS-DA) of conventional GC/MS data was constructed using authentic peppermint EOs and cornmint EOs. The model can distinguish the most common types of peppermint EOs (US, India, and US/India blend) and cornmint EOs sold in the US market. After construction, the SCP model was then used to analyze commercial samples. One sample, which passed both ISO specification and chiral analysis, was identified as outlier by the SCP model. Overall, the applicability of combining both conventional and chiral GC/MS along with chemometric tools has been successfully demonstrated to address the overall quality of peppermint EOs in commerce and may help combat sophisticated adulteration.

Encapsulation of Hemp (Cannabis sativa L.) Essential Oils into Nanoemulsions for Potential Therapeutic Applications: Assessment of Cytotoxicological Profiles.

Industrial hemp (Cannabis sativa L.), due to its bioactive compounds (terpenes and cannabinoids), has gained increasing interest in different fields, including for medical purposes. The evaluation of the safety profile of hemp essential oil (EO) and its encapsulated form (nanoemulsion, NE) is a relevant aspect for potential therapeutic applications. This study aimed to evaluate the toxicological effect of hemp EOs and NEs from cultivars Carmagnola CS and Uso 31 on three cell lines selected as models for topical and inhalant administration, by evaluating the cytotoxicity and the cytokine expression profiles. Results show that EOs and their NEs have comparable cytotoxicity, if considering the quantity of EO present in the NE. Moreover, cells treated with EOs and NEs showed, in most of the cases, lower levels of proinflammatory cytokines compared to Etoposide used as a positive control, and the basal level of inflammatory cytokines was not altered, suggesting a safety profile of hemp EOs and their NEs to support their use for medical applications.

FEMA GRAS assessment of natural flavor complexes: Lemongrass oil, chamomile oils, citronella oil and related flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, eleventh in the series, evaluates the safety of NFCs characterized by primary alcohol, aldehyde, carboxylic acid, ester and lactone constituents derived from terpenoid biosynthetic pathways and/or lipid metabolism. The scientific-based evaluation procedure published in 2005 and updated in 2018 that relies on a complete constituent characterization of the NFC and organization of the constituents into congeneric groups. The safety of the NFCs is evaluated using the threshold of toxicological concern (TTC) concept in addition to data on estimated intake, metabolism and toxicology of members of the congeneric groups and for the NFC under evaluation. The scope of the safety evaluation does not include added use in dietary supplements or any products other than food. Twenty-three NFCs, derived from the Hibiscus, Melissa, Ricinus, Anthemis, Matricaria, Cymbopogon, Saussurea, Spartium, Pelargonium, Levisticum, Rosa, Santalum, Viola, Cryptocarya and Litsea genera were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

Swertia chirayita: A comprehensive review on traditional uses, phytochemistry, quality assessment and pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Swertia chirayita (Roxb.) H. Karst. is a traditionally used, well-recognized medicinal plant of the family Gentianaceae with significant therapeutic potential. It has been traditionally used to cure various ailments such as fever, vomiting, jaundice, digestive disorders, heart diseases, diabetes, malaria, scorpion bite, and skin diseases. AIM OF REVIEW: The present review emphasized the traditional uses, phytochemistry, pharmacology, toxicology, chemical profiling, and structural identification of isolated compounds by analytical and spectroscopic techniques. This review demonstrates the possibility of advanced ethnopharmacological research. MATERIALS AND METHODS: The literature on S. chirayita was obtained from bibliographic databases like Web of Science, PubMed, Science-Direct, American Chemical Society (ACS), Google Scholar, and SciFinder. The compiled review is covered up until March 2022. RESULTS: Approximately, 123 specialized metabolites including xanthones, seco-iridoids, terpenoids, alkaloids, and flavonoids have been isolated and characterized from S. chirayita. The extract and isolated compounds exhibited a wide spectrum of pharmacological effects such as anti-inflammatory, antioxidant, antitumor, hepatoprotective, antiviral, antimalarial, and antibacterial offering scientific evidence for traditional claims of this medicinal plant. In addition, various analytical methods using HPTLC, UPLC, HPLC, LC-MS, and GC-MS have also been documented to determine the phytochemicals of S. chirayita. CONCLUSION: The current article provides information on traditional usage, phytochemistry, chemical profiling, structure elucidation, pharmacological efficacy, toxicity, and future prospects of S. chirayita. This plant has long been traditionally used in a variety of ways by indigenous people. Numerous phytoconstituents and several pharmacological activities have been reported in S. chirayita. However, there are still some scientific gaps such as identification of bioactive compounds, structure-activity relationship and mechanistic action of isolated bioactive compounds, development of effective analytical methods for comprehensive quality control, and safety profiles that need to be addressed.

Multifaced Assessment of Antioxidant Power, Phytochemical Metabolomics, In-Vitro Biological Potential and In-Silico Studies of Neurada procumbens L.: An Important Medicinal Plant.

This work was undertaken to explore the phytochemical composition, antioxidant, and enzyme-inhibiting properties of Neurada procumbens L. extracts/fractions of varying polarity (methanol extract and its fractions including n-hexane, chloroform, n-butanol, and aqueous fractions). A preliminary phytochemical study of all extracts/fractions, HPLC-PDA polyphenolic quantification, and GC-MS analysis of the n-hexane fraction were used to identify the phytochemical makeup. Antioxidant (DPPH), enzyme inhibition (against xanthine oxidase, carbonic anhydrase, and urease enzymes), and antibacterial activities against seven bacterial strains were performed for biological investigation. The GC-MS analysis revealed the tentative identification of 22 distinct phytochemicals in the n-hexane fraction, the majority of which belonged to the phenol, flavonoid, sesquiterpenoid, terpene, fatty acid, sterol, and triterpenoid classes of secondary metabolites. HPLC-PDA analysis quantified syringic acid, 3-OH benzoic acid, t-ferullic acid, naringin, and epicatechin in a significant amount. All of the studied extracts/fractions displayed significant antioxidant capability, with methanol extract exhibiting the highest radical-scavenging activity, as measured by an inhibitory percentage of 81.4 ± 0.7 and an IC50 value of 1.3 ± 0.3. For enzyme inhibition experiments, the n-hexane fraction was shown to be highly potent against xanthine oxidase and urease enzymes, with respective IC50 values of 2.3 ± 0.5 and 1.1 ± 0.4 mg/mL. Similarly, the methanol extract demonstrated the strongest activity against the carbonic anhydrase enzyme, with an IC50 value of 2.2 ± 0.4 mg/mL. Moreover, all the studied extracts/fractions presented moderate antibacterial potential against seven bacterial strains. Molecular docking of the five molecules ß-amyrin, campesterol, ergosta-4,6,22-trien-3ß-ol, stigmasterol, and caryophyllene revealed the interaction of these ligands with the investigated enzyme (xanthine oxidase). The results of the present study suggested that the N. procumbens plant may be evaluated as a possible source of bioactive compounds with multifunctional therapeutic applications.

Quantitative assessment of enzymatic processes applied to flavour and fragrance standard compounds using gas chromatography with flame ionisation detection.

The performance of different enzymes towards the bioprocessing of aroma-related compounds was investigated and a strategy based on GC-FID analysis was developed to facilitate assessment of the stages of characterisation, screening and optimisation, including chiral ratio determination. Characterisation included activity assays (UV-Vis and GC-FID), protein quantification (NanoDrop spectrophotometry) and molar mass estimation (SDS-PAGE electrophoresis). Screening experiments assessed different enzymes, substrates, solvents, acyl donors or mediators. Aroma-related substrates comprised terpene and phenolic compounds. The enzymes tested included the lipases CALA (Sigma-Aldrich), NZ-435, LZ-TLIM, NC-ADL, LZ-CALBL and the laccases NZ-51003 and DL-IIS (all from Novozymes). Among those, NZ-435 and NZ-51003 had the highest activities in the characterisation stage and, along with CALA, achieved conversions above 70% for citronellol (lipases) or 50% for eugenol (laccases) at the screening stage. The lipases had preference for the primary alcohol and laccases for phenolic compounds, among the tested substrates. The transesterification reaction between the lipase CALA and the standards mixture (citronellol, menthol, linalool) was used to demonstrate the optimisation stage, where the best levels of temperature, enzyme and acyl donor concentrations were investigated. Optimum conditions were found to be 37-40 °C, 3-4 mg/mL of enzyme and 58-60% (v/v) vinyl acetate. Additional confirmation experiments using the same terpene standards mixture and citronella oil sample, gave a conversion of > 95% for citronellol after 1 h (for both, standards mixture and sample), and 20% or 74% for menthol after 1 h or 24 h, respectively. None of the tested enzymes demonstrated significant enantioselectivity under the tested conditions. The GC-FID approach demonstrated here was suitable to determine the reaction profiles and chiral ratio variations for biocatalysed reactions with aroma compounds in low complexity samples. Advanced separations will be applied to more complex samples in the future.

Assessment of spent hemp biomass as a potential ingredient in ruminant diet: nutritional quality and effect on performance, meat and carcass quality, and hematological parameters in finishing lambs.

Spent hemp biomass (SHB), a byproduct of cannabinoid extraction from the production of industrial hemp has not been approved by FDA-CVM since its effects on animal health, performance, and product quality are unknown. Our objective was to investigate the effects of feeding two levels of SHB and a 4-wk withdrawal period on performance, carcass characteristic, meat quality, and hematological parameters in finishing lambs. A total of 35 weaned, Polypay male lambs kept in single pens were randomly assigned to five feeding treatments (n = 7) and fed diets containing either no SHB (CON) or SHB at 10% (LH1) or 20% (HH1) for 4 wk with 4 wk of clearing period from SHB, or SHB at 10% (LH2) or 20% (HH2) for 8 wk. Chemical analysis revealed SHB to have a nutritive quality similar to alfalfa with no mycotoxin, terpenes, or organic residuals as a result of the extraction process. Feed intake of lambs was negatively affected by 20% SHB in period 1 but not in period 2 where feed intake was the greatest in HH1 and LH2. In contrast, none of the performance data, including liveweight gains, were different across the groups and periods. In period 1, blood glucose, cholesterol, calcium, paraoxonase, and tocopherol were decreased by the level of SHB fed, while bilirubin and alkaline phosphatase (ALP) were increased. In period 2, the concentration in blood of urea, magnesium, bilirubin, ALP, and ferric reducing ability of the plasma (FRAP) were higher in LH2 and HH2 as compared with CON, while ß-hydroxybutyrate was lower in HH2. Blood parameters related to liver health, kidney function, immune status, and inflammation were unaffected by feeding SHB. Most carcass and meat quality parameters did not differ across feeding groups either. Except carcass purge loss and meat cook loss were larger in lambs that were fed 20% SHB. Although lower feed intake of lambs that were fed 20% SHB initially in period 1 suggested SHB was not palatable to the lambs, increased feed intake at a lower level of inclusion at 10% in period 2 may point to a positive long-term effect of feeding SHB.

The use of hemp by-products in livestock diets holds promise for reducing feed costs and achieving greater resource-use efficiency through integration of livestock production and rapidly growing hemp farming. Spent hemp biomass (SHB), the byproduct of the extraction process of cannabidiol from hemp can potentially be included in the ruminant diets due to its desirable nutritional properties. However, the potential accumulation of tetrahydrocannabinol­a psychotropic compound in animal tissues and its effect on animal health, production, and product quality are still unknown. Therefore, we conducted an indoor feeding study to investigate the effects of varying levels of SHB and a withdrawal period on feed intake, performance, health, and meat quality of lambs at Oregon State University. Our findings indicated that SHB can be included in lamb diets without causing any major detrimental effects on performance, meat quality, or health of the lambs.

The fabrication and assessment of mosquito repellent cream for outdoor protection.

Mosquito-borne infections like dengue, malaria, chikungunya, etc. are a nuisance and can cause profound discomfort to people. Due to the objectional side effects and toxicity associated with synthetic pyrethroids, N,N-diethyl-3-methylbenzamide (DEET), N,N-diethyl phenylacetamide (DEPA), and N,N-di ethyl benzamide (DEBA) based mosquito repellent products, we developed an essential oil (EO) based mosquito repellent cream (EO-MRC) using clove, citronella and lemongrass oil. Subsequently, a formulation characterization, bio-efficacy, and safety study of EO-MRC were carried out. Expression of Anti-OBP2A and TRPV1 proteins on mosquito head parts were studied by western blotting. In-silico screening was also conducted for the specific proteins. An FT-IR study confirmed the chemical compatibility of the EOs and excipients used in EO-MRC. The thermal behaviour of the best EOs and their mixture was characterized by thermogravimetric analysis (TGA). GC-MS examination revealed various chemical components present in EOs. Efficacy of EO-MRC was correlated with 12% N,N-diethyl benzamide (DEBA) based marketed cream (DBMC). Complete protection time (CPT) of EO-MRC was determined as 228 min. Cytotoxicity study on L-132 cell line confirmed the non-toxic nature of EO-MRC upon inhalation. Acute dermal irritation study, acute dermal dose toxicity study, and acute eye irritation study revealed the non-toxic nature of EO-MRC. Non-target toxicity study on Danio rerio confirmed EO-MRC as safer for aquatic non-target animals. A decrease in the concentration of acetylcholinesterase (AChE) was observed in transfluthrin (TNSF) exposed Wistar rats. While EO-MRC did not alter the AChE concentrations in the exposed animals. Results from western blotting confirmed that Anti-OBP2A and TRPV1 proteins were inhibited in TNSF exposed mosquitoes. Mosquitoes exposed to EO-MRC showed a similar expression pattern for Anti-OBP2A and TRPV1 as the control group. In silico study revealed eight identified compounds of the EOs play significant roles in the overall repellency property of the developed product. The study emphasizes the mosquito repellent activity of EO-MRC, which could be an effective, eco-friendly, and safer alternative to the existing synthetic repellents for personal protection against mosquitoes during field conditions.

Assessment of the genotoxic potential of mintlactone.

Mintlactone (chemical name 3,6-dimethyl-5,6,7,7a-tetrahydro-1-benzofuran-2(4H)-one, CAS Number 13341-72-5) is a fragrance and flavor ingredient with reported uses in many different cosmetics, personal care, and household products. In order to evaluate the genotoxic potential of mintlactone, in vitro and in vivo genotoxicity tests were conducted. Results from bacterial mutagenicity tests varied across different batches of differing purity with positive results observed in TA98 only. An in vivo comet assay was also considered to be positive in livers of female mice but negative in male mice. In contrast, in vitro and in vivo micronucleus tests, as well as 3D skin comet/micronucleus tests, were negative, indicating no chromosomal or DNA damage. The underlying causes for these contradictory results are not clear. It appears that the purity and/or stability of the test material may be an issue. In the absence of dependable scientific information on the purity and/or storage stability of mintlactone, its safety for use as a fragrance ingredient cannot be substantiated.

Assessment of Chemopreventive Potential of the Plant Extracts against Liver Cancer Using HepG2 Cell Line.

The edible parts of the plants Camellia sinensis, Vitis vinifera and Withania somnifera were extensively used in ancient practices such as Ayurveda, owing to their potent biomedical significance. They are very rich in secondary metabolites such as polyphenols, which are very good antioxidants and exhibit anti-carcinogenic properties. This study aims to evaluate the anti-cancerous properties of these plant crude extracts on human liver cancer HepG2 cells. The leaves of Camellia sinensis, Withania somnifera and the seeds of Vitis vinifera were collected and methanolic extracts were prepared. Then, these extracts were subjected to DPPH, α- amylase assays to determine the antioxidant properties. A MTT assay was performed to investigate the viability of the extracts of HepG2 cells, and the mode of cell death was detected by Ao/EtBr staining and flow cytometry with PI Annexin- V FITC dual staining. Then, the protein expression of BAX and BCl2 was studied using fluorescent dye to determine the regulation of the BAX and BCl2 genes. We observed that all the three extracts showed the presence of bioactive compounds such as polyphenols or phytochemicals. The W. somnifera bioactive compounds were found to have the highest anti-proliferative activity on human liver cancer cells.

Inherent Safety Assessment of Industrial-Scale Production of Chitosan Microbeads Modified with TiO2 Nanoparticles.

In this study, the inherent safety analysis of large-scale production of chitosan microbeads modified with TiO2 nanoparticles was developed using the Inherent Safety Index (ISI) methodology. This topology was structured based on two main stages: (i) Green-based synthesis of TiO2 nanoparticles based on lemongrass oil extraction and titanium isopropoxide (TTIP) hydrolysis, and (ii) Chitosan gelation and modification with nanoparticles. Stage (i) is divided into two subprocesses for accomplishing TiO2 synthesis, lemongrass oil extraction and TiO2 production. The plant was designed to produce 2033 t/year of chitosan microbeads, taking crude chitosan, lemongrass, and TTIP as the primary raw materials. The process was evaluated through the ISI methodology to identify improvement opportunity areas based on a diagnosis of process risks. This work used industrial-scale process inventory data of the analyzed production process from mass and energy balances and the process operating conditions. The ISI method comprises the Chemical Inherent Safety Index (CSI) and Process Inherent Safety Index (PSI) to assess a whole chemical process from a holistic perspective, and for this process, it reflected a global score of 28. Specifically, CSI and PSI delivered scores of 16 and 12, respectively. The analysis showed that the most significant risks are related to TTIP handling and its physical-chemical properties due to its toxicity and flammability. Insights about this process's safety performance were obtained, indicating higher risks than those from recommended standards.

Development and Optimization of Terpene-Enriched Vesicles (Terpesomes) for Effective Ocular Delivery of Fenticonazole Nitrate: In vitro Characterization and in vivo Assessment.

OBJECTIVE: The aim of the current study was to load fenticonazole nitrate, a slightly water-soluble antifungal agent, into terpene-enriched phospholipid vesicles (terpesomes) as a potential delivery system for the management of ocular fungal infection. METHODS: Thin film hydration method was used to prepare terpesomes according to a 32 full factorial design to inspect the effect of several variables on vesicles' features. The investigated factors were terpenes type (X1) and terpenes amount (X2) while the dependent responses were encapsulation efficiency percent (Y1), particle size (Y2) and polydispersity index (Y3). Design Expert® program was used to chose the best achieved formula. The selected terpesomes were further optimized via incorporation of a positive charge inducer (stearylamine) to enhance adhesion to the negatively charged mucus covering the eye surface. The in vivo performance of the optimized fenticonazole nitrate-loaded terpesomes relative to drug suspension was evaluated by measuring the antifungal activity (against Candida albicans) retained in the tear's fluid at different time intervals after ocular application in albino rabbits. RESULTS: The optimized terpesomes showed spherical vesicles with entrapment efficiency of 79.02±2.35%, particle size of 287.25±9.55 nm, polydispersity index of 0.46±0.01 and zeta potential of 36.15±1.06 mV. The in vivo study demonstrated significantly higher ocular retention of the optimized fenticonazole nitrate-loaded terpesomes relative to the drug suspension. Moreover, the histopathological studies proved the safety and biocompatibility of the prepared terpesomes. CONCLUSION: The obtained results verified the potential of the terpesomes for safe and effective ocular delivery of fenticonazole nitrate.

A bio-inspired cell-free system for cannabinoid production from inexpensive inputs.

Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l-1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.

Biochemical characterization and inhibition of the alternative oxidase enzyme from the fungal phytopathogen Moniliophthora perniciosa.

Moniliophthora perniciosa is a fungal pathogen and causal agent of the witches' broom disease of cocoa, a threat to the chocolate industry and to the economic and social security in cocoa-planting countries. The membrane-bound enzyme alternative oxidase (MpAOX) is crucial for pathogen survival; however a lack of information on the biochemical properties of MpAOX hinders the development of novel fungicides. In this study, we purified and characterised recombinant MpAOX in dose-response assays with activators and inhibitors, followed by a kinetic characterization both in an aqueous environment and in physiologically-relevant proteoliposomes. We present structure-activity relationships of AOX inhibitors such as colletochlorin B and analogues which, aided by an MpAOX structural model, indicates key residues for protein-inhibitor interaction. We also discuss the importance of the correct hydrophobic environment for MpAOX enzymatic activity. We envisage that such results will guide the future development of AOX-targeting antifungal agents against M. perniciosa, an important outcome for the chocolate industry.