Nepeta cataria L. (catnip) can serve as a chassis for the engineering of secondary metabolic pathways.

OBJECTIVE: Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences. RESULTS: The reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 µmol g-1 s-1, N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 ± 8 µmol g-1 s-1 and 18 ± 4 µmol g-1 s-1, respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides. CONCLUSION: Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes.

The complete chloroplast genome sequence of Nepeta bracteata and comparison with congeneric species.

Nepeta bracteata (N. bracteata) is an important medicinal plant used by Chinese ethnic minorities. However, the lack of knowledge regarding the chloroplast genome of N. bracteata has imposed current limitations on our study. Here, we used Next-generation sequencing to obtain the chloroplast genome of N. bracteata. The findings suggested that the 151,588 bp cp genome of N. bracteata comprises 130 genes, including 35 tRNA genes and 87 protein-coding genes. And its chloroplast genome exhibits a typical quadripartite structure, the largest single copy (LSC; 82,819 bp) and the smallest single copy (SSC; 17,557 bp) separate a pair of inverted repeats IR regions (IRa and IRb; 25,606 bp) from one another. Interestingly, palindromic repeats are more common, as shown by the examination of repetition. In the interim, 18 SSRs were discovered in the interim, the bulk of which were Adenine-Thymine (A-T) mononucleotides. Meanwhile, we compared it with five other species from the Nepeta genus. Five hypervariable areas were found by the study, including ndhH-rps15, accD-psal, ndhG-ndhl, trnH-GUG-psbA, and rpoC1-rpoB. Furthermore, the phylogenetic study revealed that N. bracteata and Nepeta stewartiana (N. stewartiana) were linked to each other most closely. In summary, our findings enrich the resources available for chloroplast genomes in the Nepeta genus. Moreover, these hypervariable regions have the potential to be developed into molecular markers, enabling the rapid identification of species within the Nepeta genus. Comparative analysis of species within the Nepeta genus can help enhance our study of their phylogenetic relationships, potential medicinal properties and bioprospecting.

Superior In Vivo Wound-Healing Activity of Biosynthesized Silver Nanoparticles with Nepeta cataria (Catnip) on Excision Wound Model in Rat.

Silver nanoparticles were biosynthesized with Nepeta cataria plant extract. It was determined that the synthesized Nc-AgNPs gave a strong absorbance peak at 438 nm wavelength in the UV-vis spectrophotometer. SEM and TEM analyses of Nc-AgNPs showed that the synthesized nanoparticles had a spherical morphology. Based on XRD analysis, the average crystallite size of Nc-AgNPs was calculated at 15.74 nm. At the same time, EDS spectrum analysis exhibited dominant emission energy at 3 keV, indicative of Nc-AgNPs. Nc-AgNPs showed an inhibition zone of 12 nm in gram-negative Escherichia coli, 10 nm in gram-positive Enterococcus faecalis, and 11 nm in Staphylococcus aureus. Nc-AgNPs showed high antioxidant properties, with 63% at 5000 µg/mL. The wound-healing properties of Nc-AgNPs were evaluated in vivo in wound models created in a total of 20 Wistar albino male rats, divided into four groups. After 10 days of treatment, the highest wound closure rate was seen in the Nc-AgNP + Vaseline (Group IV) treatment group, at 94%. It was observed that Nc-AgNP + Vaseline nanoformulation significantly increased wound healing, similar to Silverdin®, and Vaseline alone supported healing but did not result in complete closure. Histopathological examination revealed an increase in mature Type 1 collagen in Group IV and positive control (Group II), with better collagen maturation in vehicle control (Group III) compared to negative control (Group I). Immunohistochemical analysis showed complete epithelialization in Group IV and Group II, with distinct cytokeratin expressions, while Group III exhibited mild expressions.

Antimicrobial effects of catnip (Nepeta cataria L.) essential oil against canine skin infection pathogens.

Background and Aim: Catnip essential oils have antimicrobial effects against bacteria, yeast, and fungi; however, there is limited information regarding their antimicrobial activity against pathogens that cause canine skin infections. This study aimed to identify the phytochemical constituents of catnip essential oil and assay its antimicrobial activity against Staphylococcus pseudintermedius, Malassezia pachydermatis, Microsporum canis, Microsporum gypseum, Microsporum gallinae, and Trichophyton mentagrophytes. Materials and Methods: Catnip essential oil was extracted by hydrodistillation, and its chemical constituents were analyzed by gas chromatography-mass spectrometry (GC-MS). In vitro antimicrobial activity was investigated using broth microdilution and time-kill tests. To evaluate the effect of catnip essential oil on microbial morphology and cell membrane integrity, scanning electron microscopy (SEM) and leakage studies were conducted. Results: GC-MS analysis revealed that the principal components of catnip essential oil were cis- and trans-nepetalactone (57.09% of peak area), trans-, cis-nepetalactone (39.69% of peak area), trans-caryophyllene (1.88% of peak area), and caryophyllene oxide (1.34% of peak area). The minimum inhibitory concentration, minimum bactericidal concentration, and minimum fungicidal concentration values determined by broth microdilution ranged from 0.0625 mg/mL to 4.0 mg/mL. Time-kill testing showed that the germicidal effects of catnip essential oil were time and concentration-dependent, respectively. Environmental SEM and cell leakage analysis indicated that catnip essential oil disrupted the integrity of cell membranes in the tested microorganisms. Conclusion: Catnip essential oil has potential as an alternative antimicrobial against a wide range of canine skin infection pathogens, including S. pseudintermedius, M. pachydermatis, Mi. canis, Mi. gypseum, Mi. gallinae, and T. mentagrophytes.

Comparative Effect of Antioxidant and Antibacterial Potential of Zinc Oxide Nanoparticles from Aqueous Extract of Nepeta nepetella through Different Precursor Concentrations.

Antibiotic resistance is a global health crisis caused by the overuse and misuse of antibiotics. Accordingly, bacteria have developed mechanisms to resist antibiotics. This crisis endangers public health systems and medical procedures, underscoring the urgent need for novel antimicrobial agents. This study focuses on the green synthesis of ZnO nanoparticles (NPs) using aqueous extracts from Nepeta nepetella subps. amethystine leaves and stems, employing different zinc sulfate concentrations (0.5, 1, and 2 M). NP characterization included transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray diffraction (XRD), along with Fourier transform infrared spectroscopy (FTIR) analysis. This study aimed to assess the efficacy of ZnO NPs, prepared at varying concentrations of zinc sulfate, for their capacity to inhibit both Gram-positive and Gram-negative bacteria, as well as their antioxidant potential using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. SEM and TEM results showed predominantly spherical NPs. The smallest size (18.5 ± 1.3 nm for leaves and 18.1 ± 1.3 nm for stems) occurred with the 0.5 M precursor concentration. These NPs also exhibited remarkable antibacterial activity against both Gram-positive and Gram-negative bacteria at 10 µg/mL, as well as the highest antioxidant activity, with an IC50 (the concentration of NPs that scavenge 50% of the initial DPPH radicals) of 62 ± 0.8 (µg/mL) for the leaves and 35 ± 0.6 (µg/mL) for the stems. NPs and precursor concentrations were modeled to assess their impact on bacteria using a 2D polynomial equation. Response surface plots identified optimal concentration conditions for antibacterial effectiveness against each species, promising in combating antibiotic resistance.

Patterns of Genetic Variation of Nepeta nuda L. from the Central Balkans: Understanding Drivers of Chemical Diversity.

Nepeta nuda L., a notable medicinal species in the tradition of the Balkan region, is a rich source of bioactive iridoids and phenolics previously described as high-resolution taxonomical classifiers for the genus Nepeta. However, their potential in investigating intra-species differentiation is here described for the first time. The aim was to recognize the sources of natural chemical diversity and their association with the genetic variability both within and among N. nuda populations in the Central Balkans. Chemical diversity was assessed from methanol extracts and essential oils through untargeted and targeted metabolomics using state-of-the-art analytical tools, covering a broad spectrum of compounds that represent the N. nuda metabolome. We found that chemodiversity primarily resides within populations of N. nuda, and similar results were obtained at the DNA level using microsatellite markers. The low genetic and chemical differentiation of the studied N. nuda populations implies that their metabolomic profiles may be less influenced by geographic distance and variable environmental conditions within the Central Balkans, as they are under the pivotal control of their genetic backgrounds. Screening the distribution of the major bioactive compounds belonging to phenolics (phenolic acids and flavonoids) and iridoids (both aglycones and glycosylated forms), within and among N. nuda populations, is able to guarantee mass spectrometry-based tools for the selection of elite representative genotypes with practical importance. The knowledge acquired will allow us to delve deeper into the molecular background of N. nuda chemical diversity, which is the course of our further work.

Functional iridoid synthases from iridoid producing and non-producing Nepeta species (subfam. Nepetoidae, fam. Lamiaceae).

Iridoids, a class of atypical monoterpenes, exhibit exceptional diversity within the Nepeta genus (subfam. Nepetoidae, fam. Lamiaceae).The majority of these plants produce iridoids of the unique stereochemistry, with nepetalactones (NLs) predominating; however, a few Nepeta species lack these compounds. By comparatively analyzing metabolomics, transcriptomics, gene co-expression, and phylogenetic data of the iridoid-producing N. rtanjensis Diklic & Milojevic and iridoid-lacking N. nervosa Royle & Bentham, we presumed that one of the factors responsible for the absence of these compounds in N. nervosa is iridoid synthase (ISY). Two orthologues of ISY were mined from leaves transcriptome of N. rtanjensis (NrPRISE1 and NrPRISE2), while in N. nervosa only one (NnPRISE) was identified, and it was phylogenetically closer to the representatives of the Family 1 isoforms, designated as P5ßRs. Organ-specific and MeJA-elicited profiling of iridoid content and co-expression analysis of IBG candidates, highlighted NrPRISE2 and NnPRISE as promising candidates for ISY orthologues, and their function was confirmed using in vitro assays with recombinant proteins, after heterologous expression of recombinant proteins in E. coli and their His-tag affinity purification. NrPRISE2 demonstrated ISY activity both in vitro and likely in planta, which was supported by the 3D modeling and molecular docking analysis, thus reclassification of NrPRISE2 to NrISY is accordingly recommended. NnPRISE also displays in vitro ISY-like activity, while its role under in vivo conditions was not here unambiguously confirmed. Most probably under in vivo conditions the NnPRISE lacks substrates to act upon, as a result of the loss of function of some of the upstream enzymes of the iridoid pathway. Our ongoing work is conducted towards re-establishing the biosynthesis of iridoids in N. nervosa.

Yield and Composition of the Essential Oil of Clinopodium nepeta subsp. spruneri as Affected by Harvest Season and Cultivation Method, i.e., Outdoor, Greenhouse and In Vitro Culture.

Clinopodium nepeta subsp. spruneri is an aromatic herb with a mint-oregano flavor, used in Mediterranean regions in traditional medicine. The aerial parts of the plant are rich in essential oil that has antioxidant, antimicrobial and anti-inflammatory properties as well as insecticidal activity. The aim of our work was to determine the yield and composition of the essential oil of the plant, in relation to the harvest season and cultivation method, i.e., outdoor, greenhouse and in vitro culture, using gas chromatography-mass spectrometry (GC-MS) as an analytical tool. Essential oil yield fluctuated similarly in outdoor and greenhouse plants during the year (0.9-2.6%), with higher percentages (2.1-2.6%) in the hottest periods June-October (flowering stage) and April (vegetative stage), and was similar to the yield in in vitro plants (1.7%). More compounds were identified in the oil of outdoor and greenhouse plants (35) compared to that of in vitro plants (21), while the main compounds were the same, i.e., pulegone (13.0-32.0%, highest in February-April, 15.0% in vitro), piperitenone oxide (3.8-31.8%, lowest in February, 34.2% in vitro), piperitone epoxide (4.6-16.4%, highest in February, 15.5% in vitro), D-limonene (2.1-8.8%, lowest in February, 10.0% in vitro), isomenthone (2.3-23.0%, highest in February, 4.6% in vitro), germacrene D (1.9-6.5% highest in December-April, 2.9% in vitro) and dicyclogermacrene (2.1-5.3%, highest in December-April, 5.2% in vitro). Therefore, greenhouse and in vitro cultures were equally efficient in yielding essential oil and its constituents as outdoor cultivation, while in outdoor and greenhouse cultivations, the harvest season, mainly due to the prevailing ambient temperatures, affected the essential oil yield and its percentage composition.

Phytochemical Composition and Pharmacological Efficacy Evaluation of Calamintha nepeta, Calamintha sylvatica, Lavandula austroapennina and Mentha piperita Essential Oils for the Control of Honeybee (Apis mellifera) Varroosis.

Varroa destructor is currently considered the parasite that causes the greatest damage and economic losses to honeybee farms. Its presence is often associated with that of viral and bacterial pathogens, which ultimately leads to colony collapse. Careful control of the parasitic load is therefore necessary to avoid the onset of these events. Although chemical treatments are often in easily and quickly administered formulations, in recent years, there have been increasingly frequent reports of the onset of drug resistance phenomena, which must lead to reconsidering their use. Furthermore, chemical compounds can easily accumulate in the food matrices of the hive, with possible risks for the final consumer. In such a condition, it is imperative to find alternative treatment solutions. Essential oils (EOs) prove to be promising candidates due to their good efficacy and good environmental biodegradability. In this study, the acaricidal efficacy of the EOs of Calamintha sylvatica Bromf., Calamintha nepeta Savi, Lavandula austroapennina N.G. Passal. Tundis & Upson and Mentha piperita L., extracted from botanical species belonging to the Lamiaceae family, was evaluated. The test chosen for the evaluation was residual toxicity by contact. The examined EOs were diluted in Acetone to a concentration of 2, 1 and 0.5 mg/mL. At the highest concentration, the EOs demonstrated an acaricidal activity equal to 52% for C. nepeta, 60% for C. sylvatica, 80% for L. austroapennina and 68% for M. piperita. Of the EOs tested, therefore, Lavender proves to be a good candidate for subsequent evaluations in semi-field and field studies.

Additive effect of the probiotics Lactobacillus exopolysaccharides and the Satureja calamintha extracts on enteropathogenic Escherichia coli adhesion

Abstract This study assessed the inhibitory potential of the probiotics Lactobacillus (LB) exopolysaccharides (EPS) with or without extracts of Satureja calamintha on enteropathogenic Escherichia coli (EPEc) responsible for gastroenteritis. Methanolic and hydromethanolic extracts were prepared by cold maceration and subjected to phytochemical screening. The compounds of the extracts were determined with the colorimetric assays and identified using high-performance liquid chromatography coupled with diode array detector (HPLC-DAD). Antioxidant activities of the extracts were also evaluated by using 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging. Antibacterial effect on EPEc was evaluated by using both agar disc diffusion and microdilution methods. The in vitro test of auto-aggregation was investigated. Microbiological analysis showed that 63% of the isolated LB were producing EPS, with the amount ranging from 8.21 to 43.13 mg/L. Chemical analysis of the extracts revealed the presence of polyphenols and flavonoids, more abundant in the hydromethanolic extract, which presented the highest content with 2.11 mg EGA/g of polyphenol and 1.64 mg EC/g of flavonoids and 1.71 mg EGA/g of polyphenol and 1.15 mg EC/g of flavonoids in the methanolic extract. Hydromethanolic extracts and EPS exhibited a more important activity than did the methanolic extract against EPEc. The combined action of EPS and extracts reduced the aggregation ability of EPEc and decreased the rate of their adhesion.

Evaluation of macro and microminerals in crude drugs and infusions of five herbs widely used as sedatives

It has been determined the concentration of fourteen micro and macrominerals (Al, Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, P, Se, and Zn) in both crude drugs and infusions of Melissa officinalis L., Lamiaceae, Nepeta cataria L., Lamiaceae, Passiflora caerulea L., Passifloraceae, Tilia x moltkei Späth ex C.K. Schneid., Tiliaceae, and Valeriana officinalis L., Caprifoliaceae. These herbs are widely consumed by its sedative properties, either alone or in herb mixtures. All measurements were performed using an inductively coupled plasma optical emission spectrometer (ICP-OES). The products were obtained from regional markets, mainly in San Luis province (Argentina). The estimated daily intake was compared with current recommendations. All products and its infusions were included within the upper tolerable limits for minerals, in trace elements such as toxic elements present at low levels.

Anti-nociceptive and anti-inflammatory actions of Nepeta cataria L.var. citriodora (Becker) Balb. essential oil in mice / Efeitos antinociceptivos e anti-inflamatórios do óleo essencial de Nepeta cataria L. var. citriodora (Becker) Balb. em camundongos

Objective - Nepeta cataria (catnip) is a plant used to treat human diseases and is also found in pet toys. This study was performed to analyze the anti-nociceptive and anti-inflammatory effects of N. cataria essential oil (NCEO) in female mice. Methods - Phytochemical analyses of NCEO were performed. In addition, female mice treated with the oil were observed in an open for its general activity and to investigate the dose and time responses. The anti-nociceptive effects were evaluated by tail immersion and acetic acid writhing reflex tests.The anti-inflammatory oil properties were investigated by the carrageenan-induced edema test. Results - The results showed that 0.0005 and 0.001 mL/kg i.p. doses of NCEO increased the general activity of female mice, and the 0.0005 mL/kg dose reduced their immobility. Moreover, NCEO (0.0005 mL/kg) has anti-nociceptive properties, as the treated animals exhibited an increased latency of tail withdrawal and reduced acetic acid-induced abdominal constrictions. Furthermore, NCEO (0.0005 mL/kg) presented peripheric anti-inflammatory properties by reducing the induced edema after carrageenan injection. Conclusions - These effects may be due to the nepetalactone trans-trans and trans-cis nepetalactone isomers, which were detected as the predominant active components in the phytochemical analysis. It was suggested that the main effect of NCEO occurs on the central nervous system mechanism of pain.

Objetivo - A Nepeta cataria (catnip) é uma planta utilizada para tratar doenças humanas e também é encontrada em brinquedos de animais de estimação. O objetivo deste estudo foi analisar os efeitos antinociceptivos e anti-inflamatório do óleo essencial de N. cataria (NCEO) em camundongos fêmeas. Métodos - A análise fitoquímica do NCEO foi realizada. Além disso, os animais que foram tratados como óleo e foram observados em um campo aberto para mensurar a sua atividade em geral e investigar a dose e tempo de respostas. Os efeitos antinociceptivos foram avaliados pelos testes de imersão da cauda e reflexo de contorções abdominais causadas pelo ácido acético. As propriedades anti-inflamatórias do óleo foram investigadas pelo teste de edema induzido por carragenina. Resultados - Os resultados mostraram que 0,0005 e 0,001mL/ kg doses ip. de NCEO aumentou a atividade geral de camundongos fêmeas, e dose de 0,0005 mL/kg reduziu sua imobilidade. Além disso, NCEO (0,0005 mL/kg), tem propriedades antinociceptiva, como os animais tratados apresentaram uma maior latência de retirada de cauda e reduziu as contorções abdominais induzidas pelo ácido acético. Além disso, NCEO (0,0005mL/kg) apresentou efeito periférico e propriedades anti-inflamatórias, reduzindo o edema induzido após a injeção de carragenina. Conclusões - Estes efeitos podem ser devido aos isômeros nepetalactone trans-trans e nepetalactone trans-cis, que foram detectados como os componentes ativos predominante na análise fitoquímica. Foi sugerido que o principal efeito da NCEO ocorre no mecanismo do sistema nervoso central da dor.