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[This corrects the article DOI: 10.3389/fpls.2024.1514614.].

Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characterized by specialized metabolite profiles predominated by iridoids and phenolics, are differentiated according to the stereochemistry of major iridoid aglycone nepetalactone (NL). For the first time, the present study provides a comparative analysis of antimicrobial and immunomodulating activities of the two Nepeta species and their major iridoids isolated from natural sources—cis,trans-NL, trans,cis-NL, and 1,5,9-epideoxyloganic acid (1,5,9-eDLA), as well as of phenolic acid rosmarinic acid (RA). Methanol extracts and pure iridoids displayed excellent antimicrobial activity against eight strains of bacteria and seven strains of fungi. They were especially potent against food-borne pathogens such as L. monocytogenes, E. coli, S. aureus, Penicillium sp., and Aspergillus sp. Targeted iridoids were efficient agents in preventing biofilm formation of resistant P. aeruginosa strain, and they displayed additive antimicrobial interaction. Iridoids are, to a great extent, responsible for the prominent antimicrobial activities of the two Nepeta species, although are probably minor contributors to the moderate immunomodulatory effects. The analyzed iridoids and RA, individually or in mixtures, have the potential to be used in the pharmaceutical industry as potent antimicrobials, and in the food industry to increase the shelf life and safety of food products.

Nepeta spp. generate volatile nepetalactone iridoids that have cat-attractant and insect-repellent activities. They differ from typical mint family (Lamiaceae) iridoids, which are non-volatile glucosides, and also vary from other species in the Nepetoideae sub-family, which do not generate iridoids. The chemistry and evolution of Nepeta make it suitable for further investigation. However, the lack of transgenic technology hampers the molecular and genetic investigations in Nepeta . Virus-induced gene silencing (VIGS) is a powerful tool to detect gene functions in vivo . Here, we constructed a modified VIGS method in Nepeta cataria , using cotyledon infiltration, with the gene silencing effect spreading to the first two pairs of true leaves. The VIGS efficiency reached as high as 84.4%, and the procedure takes only 3 weeks. We employed this method to validate the role of geraniol 8-hydroxylase in nepetalactone biosynthesis with ChlH as a visual marker in N. cataria . The method is also applicable to Nepeta mussinii . Thus, we developed an easy and effective VIGS approach, which will be advantageous for endogenous gene studies in two Nepeta species and holds the potential for application in other plants.

Main conclusionUsing virus-induced gene silencing, we demonstrated that the enzymes GES, ISY, and MLPL are responsible for nepetalactone biosynthesis in Nepeta cataria.Nepetalactone is the main iridoid that is found in the Nepeta genus and is well-known for its psychoactive effect on house cats. Moreover, there is a burgeoning interest into the effect of nepetalactone on insects. Although the enzymes for nepetalactone biosynthesis have been biochemically assayed in vitro, validation of the role that these enzymes have in planta has not been demonstrated. Virus-induced gene silencing (VIGS) is a silencing method that relies on transient transformation and is an approach that has been particularly successful when applied to a variety of non-model plants. Here, we use a recently designed visual-marker dependent VIGS system to demonstrate that the nepetalactone biosynthetic enzymes GES, ISY, and MLPL impact nepetalactone biosynthesis in Nepeta cataria.

Salicylic acid (SA) is involved in plant defense responses to environmental stressors by modulating gene expression and specialized metabolites production, enhancing plant adaptive resilience through systemic signaling pathways. This study investigates the impact of exogenous application of SA on the metabolism of iridoids and phenolic compounds—characteristic specialized metabolites of the Nepeta species, associated with diverse biological activities. Nepetalactone (NL) is a characteristic monoterpene iridoid, while rosmarinic acid (RA) represents the most abundant phenolic compound within the genus. We explored the effects of varying SA concentrations (2 µM, 5 µM, 10 µM, and 20 µM) on iridoid and phenolic metabolism in in vitro-grown Nepeta nuda, following 7 days and 28 days of elicitation. A significant increase in trans,trans-NL content was observed after 7-day exposure to 2 µM SA, while prolonged exposure led to a decrease in its levels, particularly at higher SA concentrations. Gene expression analysis revealed that 7 days of exposure to lower concentrations of SA upregulated genes coding for NAD-dependent nepetalactol-related short-chain dehydrogenase/reductases (NEPSs), key regulatory enzymes catalyzing the final steps of NL biosynthesis. In contrast, prolonged exposure to 20 µM SA downregulated genes coding for geraniol 8-hydroxylase (NnG8H) and 8-hydroxygeraniol oxidoreductase (Nn8HGO), which resulted in reduced iridoid content. Conversely, SA treatment notably increased RA content after prolonged exposure to 20 µM SA, which is a result of the enhanced expression of all analyzed RA biosynthesis-related genes. These findings demonstrate that both concentration and duration of SA treatment are critical determinants of elicitation outcomes in N. nuda. Strategic manipulation of these parameters can redirect metabolic flux toward either iridoid or phenolic compounds production, and enhance biotechnological production of specialized metabolites in N. nuda.

Cyclodextrin inclusion complexes have been successfully used to encapsulate essential oils, improving their physicochemical properties and pharmacological effects. Besides being well-known for its effects on cats and other felines, catnip (Nepeta cataria) essential oil demonstrates repellency against blood-feeding pests such as mosquitoes. This study evaluates the tick repellency of catnip oil alone and encapsulated in β-cyclodextrin, prepared using the co-precipitation method at a 1:1 molar ratio. The physicochemical properties of this inclusion complex were characterized using GC-FID for encapsulation efficiency and yield and SPME/GC-MS for volatile emission. Qualitative assessment of complex formation was done by UV-Vis, FT-IR, 1H NMR, and SEM analyses. Catnip oil at 5% (v/v) demonstrated significant tick repellency over time, being comparable to DEET as used in commercial products. The prepared [catnip: β-CD] inclusion complex exerted significant tick repellency at lower concentration of the essential oil (equivalent of 1% v/v). The inclusion complex showed that the release of the active ingredient was consistent after 6 h, which could improve the effective repellent duration. These results demonstrated the effective tick repellent activity of catnip essential oil and the successful synthesis of the inclusion complex, suggesting that β-CDs are promising carriers to improve catnip oil properties and to expand its use in repellent formulations for tick management.

Nepeta persica is a medicinal plant with significant pharmacological potential, primarily attributed to its high nepetalactone content. Understanding the environmental drivers of nepetalactone biosynthesis is essential for optimizing both cultivation and conservation strategies. In this study, we combined machine learning algorithms (random forest, support vector machines, gradient boosting machines) with a hybrid ensemble model (RF-SVM-GBM), alongside statistical approaches (generalized linear models [GLM] and partial least squares [PLS]) and geospatial analyses (GIS, remote sensing, habitat suitability modeling) to assess the influence of climatic, topographic, and edaphic factors on nepetalactone concentration in N. persica across Fars province, Iran. The results identified elevation, south-facing slopes, and mean annual temperature as the most critical determinants of nepetalactone accumulation. The hybrid ensemble model demonstrated the highest predictive accuracy, reducing RMSE by 21.1% (RMSE = 0.015) compared to individual models. Habitat suitability maps revealed Marvdasht and Shiraz counties as the most favorable regions for cultivating N. persica with high nepetalactone concentrations, followed by smaller high-suitability zones in Northeast Firozabad and Northern Kazerun. In contrast, areas such as Abadeh, Eqlid, and Khorrambid exhibited lower suitability. These findings provide actionable insights for precision agriculture, resource-efficient cultivation, and climate-adaptive conservation of medicinal plants. By integrating ecological modeling with machine learning, this research offers a scalable, data-driven framework to support the sustainable production of high-value secondary metabolites in environmentally challenging regions.

Mosquitoes (Diperta: Culidae) act as vectors for several diseases, including malaria, dengue fever and yellow fever. Mosquito repellents represent one of the primary measures used to reduce the risk of these diseases in humans by reducing mosquito landing and biting events. Nepetalactone, a natural insect repellent primarily found in the essential oil of catnip (Lamiaceae; Nepeta cataria ), has established mosquito repellence properties, but has not been widely exploited as a mosquito repellent in malaria-endemic regions such as Sub-Saharan Africa. Here, we evaluated the potential of a locally produced lotion containing catnip essential oil (comprising > 92% nepetalactone) for use as a mosquito repellent in Eastern Uganda. Using the human landing catch method in field trials, we analysed the effectiveness of a lotion containing 2% or 6% catnip oil at repelling mosquitoes compared to a lotion lacking catnip essential oil as a negative control and to a commercial repellent containing 15% DEET as a positive control. We found that lotions containing both concentrations of catnip essential oil were highly effective at preventing mosquito landing, with 6% catnip oil performing as well as 15% DEET. Our findings suggest that nepetalactone could be used as a natural, locally sourced and effective alternative to synthetic commercial mosquito repellents, thereby representing a viable import substitution option for protection against mosquito-borne diseases.

Catnip (Nepeta cataria L.) plants produce a wide array of specialized metabolites with multiple applications for human health. The productivity of such metabolites, including nepetalactones, and natural insect repellents is influenced by the conditions under which the plants are cultivated. In this study, we assessed how field-grown catnip plants, transplanted after being propagated via either single-node stem cuttings or seeds, varied regarding their phytochemical composition throughout a growing season in two distinct environmental conditions (Pittstown and Upper Deerfield) in the state of New Jersey, United States. Iridoid terpenes were quantified in plant tissues via ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-QqQ-MS), and phenolic compounds (phenolic acids and flavonoids) were analyzed via UHPLC with diode-array detection (UHPLC-DAD). The highest contents of total nepetalactones in Pittstown were found at 6 weeks after transplanting (WAT) for both seedlings and cuttings (1305.4 and 1223.3 mg/100 g, respectively), while in Upper Deerfield, the highest contents for both propagules were at 11 WAT (1247.7 and 997.1 mg/100 g, respectively) for seed-propagated and stem cuttings). The highest concentration of nepetalactones was associated with floral-bud to partial-flowering stages. Because plants in Pittstown accumulated considerably more biomass than plants grown in Upper Deerfield, the difference in nepetalactone production per plant was striking, with peak productivity reaching only 598.9 mg per plant in Upper Deerfield and 1833.1 mg per plant in Pittstown. Phenolic acids accumulated in higher contents towards the end of the season in both locations, after a period of low precipitation, and flavone glycosides had similar accumulation patterns to nepetalactones. In both locations, rooted stem cuttings reached their maximum nepetalactone productivity, on average, four weeks later than seed-propagated plants, suggesting that seedlings have, overall, better agronomic performance.

The common bed bug, Cimex lectularius L., resurged as an important urban pest in the last 20 yr. Yet, there are no commercial repellent products labeled for bed bugs available in the United States. We evaluated the repellency of two catnip oils from newly developed cultivars, CR3 and CR9, and compared each to 10 and 25% N,N-diethyl-meta-toluamide (DEET). CR3 contains 63.4% E,Z-nepatalactone and 27.5% Z,E-nepatalactone, and CR9 contains 94.9% Z,E-nepatalactone. Arena studies showed that CR3 and CR9 oils were more effective than DEET within a 24-h period. At 10% concentration, both CR3 and CR9 oils exhibited a repellency of over 94% during the first 8 h. At 25% concentration, the repellency of CR3 and CR9 oils increased to 100%, and repellency of DEET was 92% during the first 24 h. Repellency of 25% CR3 and CR9 oils became lower than 25% DEET after being aged for 3 d. After 28-d aging, repellency of 25% CR3, CR9, and DEET reduced to 25, 64, and 92%, respectively. Soiled socks were placed above repellent treated bands to determine if the repellent can protect soiled socks from being infested. The 20% CR3 oil prevented 100% of bed bugs from infesting soiled socks showing that it was more effective than DEET. These results indicate that catnip oils from CR3 and CR9 cultivars are more repellent than DEET over a 24-h period following application, but their longevity is shorter than DEET after 72 h.