Enhanced Production of ß-Caryophyllene by Farnesyl Diphosphate Precursor-Treated Callus and Hairy Root Cultures of Artemisia vulgaris L.

Artemisia vulgaris L. produces a wide range of valuable secondary metabolites. The aim of the present study is to determine the effects of various concentrations of farnesyl diphosphate (FDP) on ß-caryophyllene content in both callus and hairy root (HR) cultures regeneration from leaf explants of A. vulgaris L. Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4D; 4-13 µM), α-naphthaleneacetic acid (NAA; 5-16 µM), and FDP (1 and 3 µM) was used for callus induction and HR regeneration from leaf explants of A. vulgaris L. In this study, precursor-treated (2,4D 13.5 µM + FDP 3 µM) callus displayed the highest biomass fresh weight (FW)/dry weight (DW): 46/25 g, followed by NAA 10.7 µM + FDP 3 µM with FW/DW: 50/28 g. Two different Agrobacterium rhizogenes strains (A4 and R1000) were evaluated for HR induction. The biomass of HRs induced using half-strength MS + B5 vitamins with 3 µM FDP was FW/DW: 40/20 g and FW/DW: 41/19 g, respectively. To determine ß-caryophyllene accumulation, we have isolated the essential oil from FDP-treated calli and HRs and quantified ß-caryophyllene using gas chromatography-mass spectrometry (GC-MS). The highest production of ß-caryophyllene was noticed in HR cultures induced using A4 and R1000 strains on half-strength MS medium containing 3 µM FDP, which produced 2.92 and 2.80 mg/ml ß-caryophyllene, respectively. The optimized protocol can be used commercially by scaling up the production of a ß-caryophyllene compound in a short span of time.

Evaluation of the Leaf Essential Oil from Artemisia vulgaris and Its Larvicidal and Repellent Activity against Dengue Fever Vector Aedes aegypti-An Experimental and Molecular Docking Investigation.

Aedes aegypti is a mosquito vector that spreads dengue fever and yellow fever worldwide in tropical and subtropical countries. Essential oil isolated from Artemisia vulgaris is found to have larvicidal and repellent action against this vector. The dried leaves were subjected to hydrodistillation using a clevenger-type apparatus for 4 h. The isolated essential oil was analyzed by using gas chromatography-mass spectrometry, and the major insecticidal compounds were identified as α-humulene (0.72%), ß-caryophyllene (0.81%), and caryophyllene oxide (15.87%). Larvicidal activity results revealed that the essential oil exposure for 24 h period against the third stage larvae was LC50 = 6.87, LC90 = 59.197 ppm and for the fourth stage larvae LC50 = 4.269, LC90 = 50.363 ppm. Highest mortality rates were observed at 24 h exposure period of third and fourth stages, and the exposed A. aegypti larvae were subjected to histo chemical studies, and the studies revealed that larvae cells got totally damaged (midgut and cortex). The repellent activity results revealed that at 50% concentration of the essential oil showed the highest repellent activity at 60 min protection time against the A. aegypti female mosquitoes. To gain further insights into the insecticidal activity, density functional theory and molecular docking calculations were performed with the active components of this essential oil as the ligand and NS3 protease domain (PDB ID: 2FOM) as a receptor. Molecular docking calculation results show that (E)-ß-caryophyllene strongly binds with NS3 protease domain than (Z)-ß-caryophyllene, α-humulene, and ß-caryophyllene oxide and is the major active component for the insecticidal action. It primarily interacts with the receptor through hydrophobic and ionic forces and using water bridges between the amino acid residues in the binding pocket and (E)-ß-caryophyllene.

Development of nanoemulsion from Vitex negundo L. essential oil and their efficacy of antioxidant, antimicrobial and larvicidal activities (Aedes aegypti L.).

It is believed that nanoemulsions were emerged as a promising candidate to improve the qualities of natural essential oil towards antimicrobial and insecticidal applications. In the present study, we have focused on the encapsulation of Vitex negundo L. leaf essential oil using Polysorbate80 for its different biological activities including antioxidant, bactericidal and larvicidal activity against dengue fever vector Aedes aegypti L. Initially, the nanoemulsion was prepared by low energy method and droplet size of the formulated nanoemulsion was characterized by using Dynamic Light Scattering analysis. The freshly prepared V. negundo essential nanoemulsion was observed with the mean droplet size of below 200 nm indicating its excellent stability. Further, the larvicidal activity of essential oil and nanoemulsion with various concentrations (25, 50, 100, 200 and 400 ppm). The larvicidal activities were tested 2nd and 3rd instar larval mortality rate that was observed against the 12 and 24 h exposure period. After a 12 h exposure period, the larvicidal activities of 2nd instar larva were observed as essential oil (73.33 ± 1.88), nanoemulsion (81.00 ± 0.88) and the larvicidal activities of 3rd instar larva were displayed essential oil (70.33 ± 2.60) and nanoemulsion (79.00 ± 3.70). Likewise, after a 24 h exposure period, the larvicidal activities of 2nd instar larva were observed as essential oil (90.30 ± 2.15), nanoemulsion (94.33 ± 1.20) and the larvicidal activities of 3rd instar larva were essential oil (80.66 ± 0.66) and nanoemulsion (93.00 ± 1.25) respectively. We finally concluded that the developed plant-based emulsion essential oil systems were thermodynamically stable. Owing to its improved bioavailability and biocompatibility, formulated nanoemulsion can be used in various biomedical applications including drug delivery as well as disease transmitting mosquito vector control. Graphical abstract ᅟ.