Insights into chemistry, extraction and industrial application of lemon grass essential oil -A review of recent advances.

Lemongrass essential oil (LEO), extracted from high-oil lemongrass, gains prominence as a versatile natural product due to growing demand for safe health solutions. LEO comprises beneficial compounds like citral, isoneral, geraniol, and citronellal, offering diverse pharmacological benefits such as antioxidant, antifungal, antibacterial, antiviral, and anticancer effects. LEO finds applications in food preservation, cosmetics, and pharmaceuticals, enhancing profitability across these sectors. The review focuses on the extraction of LEO, emphasizing the need for cost-effective methods. Ultrasound and supercritical fluid extraction are effective in reducing extraction time, increasing yields, and enhancing oil quality. LEO shows promise as a valuable natural resource across industries, with applications in packaging, coating, and film development. LEO's ability to extend the shelf life of food items and impart natural flavors positions it as a valuable asset. Overall, the review emphasizes LEO's therapeutic, antimicrobial, and antioxidant properties, strengthening its potential in the food, pharmaceutical, and cosmetic sectors.

Identification and functional analysis of a deduced geraniol synthase from Camphora officinarum.

The market demand for essential oil containing citral is increasing. Our research group identified a rare chemotype of Camphora officinarum whose leaves are high in citral content by examining over 1000 wild trees across the entire native distribution area of C. officinarum in China. Because C. officinarum is suitable for large-scale cultivation, it is therefore seen as a promising source of natural citral. However, the molecular mechanism of citral biosynthesis in C. officinarum is poorly understood. In this study, transcriptomic analyses of C. officinarum with different citral contents revealed a strong positive correlation between the expression of a putative geraniol synthase gene (CoGES) and citral content. The CoGES cDNA was cloned, and the CoGES protein shared high similarity with other monoterpene synthases. Enzymatic assays of CoGES with geranyl diphosphate (GPP) as substrate yielded geraniol as the single product, which is the precursor of citral. Further transient expression of CoGES in Nicotiana benthamiana resulted in a higher relative content of geranial and the appearance of a new substance, neral. These findings indicate that CoGES is a geraniol synthase-encoding gene, and the encoded protein can catalyze the transformation of GPP into geraniol, which is further converted into geranial and neral through an unknown mechanism in vivo. These findings expand our understanding of citral biosynthesis in Lauraceae plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01463-4.

Growth fitness, virulence, and heat tolerance of Salmonella Typhimurium variants resistant to food preservation methods.

To study potential ramifications of antimicrobial resistance, we carried out adaptive laboratory evolution assays (ALE) to isolate three resistant variants (RVs) of Salmonella enterica Typhimurium, employing three different types of food preservation methods: 1) an emergent technology, plasma-activated water (PAW), leading to variant RV-PAW; a traditional method, heat, leading to variant RV-HT, and a natural antimicrobial compound, carvacrol, leading to variant RV-CAR. The variant resistant to plasma-activated water, RV-PAW, had mutations in rpoA and rpoD; it showed increased tolerance to heat in orange juice but ultimately did not pose a significant threat, as it exhibited a fitness cost at refrigeration temperature (8 °C), whereas its virulence against Caenorhabditis elegans decreased. The variant resistant to heat, RV-HT, had mutations in flhC, dnaJ: it exhibited a fitness cost at high growth temperatures (43 °C) and induced morphofunctional alterations in C. elegans. The variant resistant to carvacrol, RV-CAR, had mutations in sseG, flhA, wbaV, lon; this variant not only exhibited significantly higher thermotolerance in both laboratory media and food models but also effectively increased its growth fitness at refrigeration temperatures while retaining its virulence, evidenced by the highest percentage of Smurf phenotype in C. elegans. To address these challenges, we applied a process combining thermal treatment with citral, with the aim of leveraging the sublethal damage caused in RVs by heat treatments in orange juice. This approach achieves enhanced microbial inactivation without having to escalate the intensity of the thermal treatment. The result was particularly encouraging in the case of RV-CAR, the most challenging strain, for which we improved lethality by up to 3 log10 inactivation cycles.

Citral in lemon myrtle, lemongrass, litsea, and melissa essential oils suppress the growth and invasion of breast cancer cells.

OBJECTIVE: Although cancer therapy suppresses recurrence and prolongs life, it may be accompanied by strong side effects; thus, there is a strong demand for the development effective treatments with fewer side effects. Cancer therapy using plant-derived essential oils is attracting attention as one promising method. This study investigated the antitumor effects of essential oil volatiles on breast cancer cells and identifies four essential oils that display antitumor activity. METHODS: Breast cancer cells were cultured in a 96-well plate, then one of twenty essential oils was added dropwise to the central well. The plate was incubated at 37 °C for 48 h and the effect of the volatile components of each essential oil on the surrounding breast cancer cell growth ability was examined using an MTT assay. Gas chromatography was used to investigate the concentration of the transpiration components that may affect cancer cells. RESULTS: Of the 20 essential oils, Lemongrass, Lemon myrtle, Litsea, and Melissa displayed strong anti-tumor effects. These essential oils inhibited the growth of nearby breast cancer cells, even when diluted more than 500-fold. The transpiration component of lemon Myrtle showed the strongest antitumor effect, but was the least cytotoxic to mononuclear cells in normal peripheral blood (PBMC). Each of these essential oils contained a very large amount of citral. The IC50 against breast cancer cells when citral was volatilized from each essential oil was 1.67 µL/mL for geranial and 1.31 µL/mL for neral. Volatilized citral alone showed strong anti-proliferation and infiltration-inhibiting effects. CONCLUSION: The transpiration components of Lemongrass, Lemon myrtle, Litsea, and Melissa are thought to inhibit breast cancer cell proliferation due to their high levels of citral.

Citral and triclosan synergistically silence quorum sensing and potentiate antivirulence response in Pseudomonas aeruginosa.

Among the ESKAPE pathogens, Pseudomonas aeruginosa is an extensively notorious superbug that causes difficult-to-treat infections. Since quorum sensing (QS) directly promotes pseudomonal virulence, targeting QS circuits is a promising approach for disarming phenotypic virulence. Hence, this study scrutinizes the anti-QS, antivirulence, and anti-biofilm potential of citral (CiT; phytochemical) and triclosan (TcN; disinfectant), alone and in combination, against P. aeruginosa PAO1/PA14. The findings confirmed synergism between CiT and TcN and revealed their quorum quenching (QQ) potential. At sub-inhibitory levels, CiT-TcN combination significantly impeded pyocyanin, total bacterial protease, hemolysin, and pyochelin production alongside inhibiting biofilm formation in P. aeruginosa. Moreover, the QQ and antivirulence potential of CiT and TcN was positively correlated by molecular docking studies that predicted strong associations of the drugs with QS receptors of P. aeruginosa. Collectively, the study identifies CiT-TcN as an effective drug combination that harbors QQ, antivirulence, and anti-biofilm prospects against P. aeruginosa.

Antibacterial mechanism of whey protein isolated-citral nanoparticles and stable synergistic antibacterial eugenol encapsulated Pickering emulsion for grapes preservation.

The purpose of this study was to prepare Pickering emulsion with synergistic antibacterial effect using whey protein isolated-citral (WPI-Cit) nanoparticles with eugenol for grape preservation. In this emulsion, eugenol was encapsulated in oil phase. The particle size, ζ-potential, and antibacterial mechanism of the nanoparticles were characterized. The rheological properties, antibacterial effects and preservation effects of WPI-Cit Pickering emulsion were measured. The results showed that the optimal preparation condition was performed at WPI/Cit mass ratio of 1:1, WPI-Cit nanoparticles were found to damage the cell wall and membrane of bacteria and showed more effective inhibition against S. aureus. Pickering emulsion prepared with WPI-Cit nanoparticles exhibited a better antibacterial effect after eugenol was encapsulated in it, which extended the shelf life of grapes when the Pickering emulsion was applied as a coating. It demonstrated that the Pickering emulsion prepared in this study provides a new way to extend the shelf life.

In vitro susceptibility testing of phytochemicals from essential oils against Prototheca species.

Phytochemicals isolated from essential oils are effective alternatives for inhibiting microbial pathogens. Bovine protothecal mastitis is the cause of a reduction in milk production and the secretion of thin, watery milk with white flakes. In the present study, we performed in vitro susceptibility testing of the phytochemicals carvacrol, citral, and thymol in Prototheca strains isolated from cases of protothecosis in small animals and cow feces. The susceptibility of the algae to carvacrol, citral, and thymol was assessed using the modified CLSI M27-A3 broth microdilution method. The ranges of the minimum inhibitory concentrations (MIC%) of the phytochemicals in all isolates were 0.03% to 0.125% for carvacrol, 0.03% to 0.25% for citral, and 0.06% to 0.25% for thymol. Based on these results, carvacrol, citral, and thymol appear effective against Prototheca species at the tested concentrations, and may thus be useful for environmental disinfection in barns.

Neuroinflammation and acetylcholinesterase inhibition potentials of acyclic monoterpenoids isolated from Cymbopogon distans (Nees ex Steud.) Will. Watson.

Cymbopogon distans (Nees ex Steud.) Will. Watson (Poaceae) is a promising aromatic plant distributed in the Himalayas. In this study, five acyclic monoterpenoids, namely geranyl acetate (RS1), neral (RS2), geranial (RS3), citral (RS4) and geraniol (RS5) were isolated from the essential oil of C. distans. The isolated compounds were tested for in-vitro neuroinflammation inhibitory potential in LPS-stimulated BV2 microglial cells. RS1-RS4 exhibited significant neuroinflammation inhibition without any cytotoxic effect at the dose of 10 µM. RS4, the most active anti-neuroinflammatory compound (TNF-α 31.48 ± 1.00%; IL-6 24.02 ± 0.63%; IL-1ß 42.15 ± 1.76%) was also able to inhibit acetylcholinesterase (AChE) in a dose-dependent manner. The results showed that RS4 (an isomeric mixture of neral and geranial) has the potential to inhibit neuroinflammation and AChE, which are the biomarkers of neurodegenerative disorders.

Citral relaxes umbilical vessels of normotensive and preeclamptic parturients.

INTRODUCTION: Citral is a low-toxicity monoterpene that has a vasodilator effect on various smooth muscles, and The present study aimed to evaluate its vasorelaxant effect on umbilical vessels of normotensive parturients (NTP) and with preeclampsia parturients (PEP). METHOD: Segments of human umbilical artery (HUA) and vein (HUV) of NTP or PEP were mounted in a bath to record the force of contraction, under tension of 3.0 gf and contracted with the contracting agents: K+ (60 mM), 5 -HT (10 µM) and Ba2+ (1-30 mM). Next, the effect of citral (1-3000 µM) on these contractions and on basal tone was evaluated. RESULTS: In HUA and HUV, citral (1-1000 µM), in NTP condition, inhibited contractions evoked by K+ (IC50 of 413.5 and 271.3, respectively) and by 5-HT (IC50 of 164.8 and 574.3). In the PEP condition, in HUA and HUV, citral also inhibited the contractions evoked by K+ (IC50 of 363.3 and 218.3, respectively) and 5-HT (IC50 of 432.1 and 520.4). At a concentration of 1000 µM, citral completely or almost completely (>90 %) inhibited all contractions. At a concentration of 100-1000 µM, citral, in general, was already able to reduce the contraction induced by 1-3 mM Ba2+ in both AUH and VUH, under NTP and PEP conditions. DISCUSSION: Citral has been shown to be an effective HUA and HUV vasodilator in NTP and PEP. As its toxicity is low, it suggests that this substance can be considered a potential therapeutic agent.

[Study on mechanism of inhibiting proliferation of head and neck cancer cells by citral, active ingredient of lemon essential oil].

To explore the active substances exerting anti-tumour effect in lemon essential oil and the molecular mechanism inhibiting the proliferation of head and neck cancer cells SCC15 and CAL33, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay(MTT) was utilized to identify the active component inhibiting the proliferation of head and neck cancer cells, namely citral. The IC_(50) of citral inhibiting the proliferation of head and neck cancer cells and normal cells were also determined. In addition, a 5-ethynyl-2'-deoxyuridine(EdU) staining assay was used to detect the effect of citral on the proliferation rate of head and neck cancer cells, and a colony formation assay was used to detect the effect of citral on tumor sphere formation of head and neck cancer cells in vitro. The cell cycle arrest and apoptosis induction of head and neck cancer cells by citral were evaluated by flow cytometry, and Western blot was used to detect the effect of citral on the expression levels of cell cycle-and apoptosis-related proteins in head and neck cancer cells. The findings indicated that citral could effectively inhibit the proliferation and growth of head and neck cancer cells, with anti-tumor activity, and its half inhibitory concentrations for CAL33 and SCC15 were 54.78 and 25.23 µg·mL~(-1), respectively. Furthermore, citral arrested cell cycle at G_2/M phase by down-regulating cell cycle-related proteins such as S-phase kinase associated protein 2(SKP2), C-MYC, cyclin dependent kinase 1(CDK1), and cyclin B. Moreover, citral increased the cysteinyl aspartate-specific proteinase-3(caspase-3), cysteinyl aspartate-specific proteinase-9(caspase-9), and cleaved poly ADP-ribose polymerase(PARP). It up-regulated the level of autophagy-related proteins including microtubule associated protein 1 light chain 3B(LC3B), sequestosome 1(P62/SQSTM1), autophagy effector protein Beclin1(Beclin1), and lysosome-associate membrane protein 1(LAMP1), suggesting that citral could effectively trigger cell apoptosis and cell autophagy in head and neck cancer cells. Furthermore, the dual-tagged plasmid system mCherry-GFP-LC3 was used, and it was found that citral impeded the fusion of autophagosomes and lysosomes, leading to autophagic flux blockage. Collectively, our findings reveal that the main active anti-proliferation component of lemon essential oil is citral, and this component has a significant inhibitory effect on head and neck cancer cells. Its underlying molecular mechanism is that citral induces apoptosis and autophagy by cell cycle arrest and ultimately inhibits cell proliferation.

Effect of polyethoxylated flavonoids (PMFs)-loaded citral and chitosan composite coatings on citrus preservation: From the perspective of fruit resistance.

Previous studies have shown that polymethoxylated flavonoids-loaded citral emulsion (PCT) can inhibit the growth and reproduction of Penicillium in citrus; however, PCT is difficult to apply to fruit preservation due to its high fluidity and volatility. Therefore, in this study, we combined PCT with chitosan (CS) to investigate the effect of a composite coating on citrus preservation. The results showed that compared to the control group, the CS-PCT group could effectively reduce the decay rate and maintain moisture availability, color difference, and hardness. Moreover, the contents of nonenzymatic antioxidants and volatile substances with antimicrobial activity were better preserved. In addition, the activities of related antioxidant enzymes were greater in the treatment group, and the expression of the corresponding enzyme-encoding genes was upregulated. Consequently, CS-PCT treatment could effectively maintain fruit quality and improve the resistance of citrus fruits during storage; moreover, it can be considered a nontoxic and efficient citrus preservative.

Identification of key genes controlling monoterpene biosynthesis of Citral-type Cinnamomum bodinieri Levl. Based on transcriptome and metabolite profiling.

The citral-type is the most common chemotype in Cinnamomum bodinieri Levl (C. bodinieri), which has been widely used in the daily necessities, cosmetics, biomedicine, and aromatic areas due to their high citral content. Despite of this economic prospect, the possible gene-regulatory roles of citral biosynthesis in the same geographic environment remains unknown. In this study, the essential oils (EOs) of three citral type (B1, B2, B3) and one non-citral type (B0) varieties of C. bodinieri were identified by GC-MS after hydrodistillation extraction in July. 43 components more than 0.10% were identified in the EOs, mainly composed of monoterpenes (75.8-91.84%), and high content citral (80.63-86.33%) were identified in citral-type. Combined transcriptome and metabolite profiling analysis, plant-pathogen interaction(ko04626), MAPK signaling pathway-plant(ko04016), starch and sucrose metabolism(ko00500), plant hormone signal transduction(ko04075), terpenoid backbone biosynthesis (ko00900) and monoterpenoid biosynthesis (ko00902) pathways were enriched significantly. The gene expression of differential genes were linked to the monoterpene content, and the geraniol synthase (CbGES), alcohol dehydrogenase (CbADH), geraniol 8-hydroxylase-like (CbCYP76B6-like) and 8-hydroxygeraniol dehydrogenase (Cb10HGO) were upregulated in the citral-type, indicating that they were associated with high content of geraniol and citral. The activities of CbGES and CbADH in citral type were higher than in non-citral type, which was corroborated by enzyme-linked immunosorbent assay (ELISA). This study on the accumulation mechanism of citral provides a theoretical basis for the development of essential oil of C. bodinieri.

Essential oil of the leaves of psychotria asiatica L.: chemical composition, antioxidant, anti-inflammatory, and cytotoxic properties.

This study is focused on investigating the chemical composition and bioactive properties of the essential oil extracted from Psychotria asiatica L., a plant species known for its medicinal properties. Utilising gas chromatography-mass spectrometry (GC-MS) analysis, the essential oil from P. asiatica was found to contain 53 distinct constituents. Major compounds identified include (E)-citral (20.6%), 10-epi-γ-eudesmol (15.9%), (Z)-citral (10.5%), geraniol (7.4%), α-cadinol (6.7%), 7-epi-α-eudesmol (4.4%), linalool (3.7%), and α-muurolol (3.4%). The essential oil did not exhibit antioxidant activity, as indicated by an IC50 value of > 100 µg/mL, whereas the positive control L-Ascorbic acid had an IC50 of 7.37 ± 0.27 µg/mL in the DPPH model. Assessment of its anti-inflammatory potential revealed an inhibitory effect on NO production, with an IC50 value of 29.08 ± 1.54 µg/mL in Lipopolysaccharide-induced RAW264.7 macrophage cells. Furthermore, the essential oil demonstrated significant cytotoxicity against the SK-LU-1 cancer cell line, with an IC50 value of 39.75 ± 1.79 µg/mL according to the sulforhodamine B (SRB) assay.

Synthesis, antifungal activity and action mechanism of novel citral amide derivatives against Rhizoctonia solani.

BACKGROUND: Rice sheath blight caused by Rhizoctonia solani is a severe threat to the yield and quality of rice. Due to the unscientific abuse of common fungicides causing resistance and environmental issues, the development of new fungicides is necessary. In this study, we used citral as the lead compound, designed and synthesized a series of novel citral amide derivatives, and evaluated their antifungal activity and mode of action against R. solani. RESULT: Bioassay results indicated that the antifungal activities of most citral amide derivatives against R. solani were significantly improved compared to citral, with EC50 values ranging from 9.50-27.12 mg L-1. Among them, compound d21 containing the N-(pyridin-4-yl)carboxamide group exhibited in vitro and in vivo fungicidal activities, with curative effects at 500 mg L-1 as effectively as the commercial fungicide validamycin·bacillus. Furthermore, d21 prolonged the lag phase of the growth curve of R. solani, reduced the amount of growth, and inhibited sclerotium germination and formation. Mechanistically, d21 deformed the mycelia, increased cell membrane permeability, and inhibited the activities of antioxidant and tricarboxylic acid cycle (TCA)-related enzymes. Metabolome analysis showed the abundance of some energy-related metabolites within R. solani increased, and simultaneously the antifungal substances secreted by itself reduced. Transcriptome analysis showed that most genes encoding ATP-binding cassette (ABC) transporters and peroxisomes upregulated after the treatment of d21 and cell membrane destruction. CONCLUSION: This study indicates that novel citral amide derivatives possess antifungal activity against R. solani and are expected to develop an alternative option for chemical control of rice sheath blight. © 2024 Society of Chemical Industry.

Influence of Curcuma Longa extract in citral addition on functional properties of thin films with triple-layer structure based on furcellaran and gelatin.

In this study, we obtained triple-layer films based on furcellaran and gelatin, in which the middle layer was enriched with extract of Curcuma longa in citral. This newly developed material underwent a comprehensive characterisation process to identify significant improvements in its functional properties. Both SEM, XRD and FTIR analyzes indicated the formation of interactions not only between the components but also between the film layers. Notably, the incorporation of the natural extract led to a significant reduction in solubility, decreasing it from 74.79 % to 57.25 %, while enhancing thermal stability expressed as a melting point elevating it from 147.10 °C in the control film to 158.80 °C in the film with the highest concentration of the active ingredient. Simultaneously, the addition of this active ingredient resulted in decreased water contact angle (WCA) values, rendering the film more hydrophilic. The produced films exhibit great promise as packaging materials, particularly within the food industry, and the conducted research is marked by its forward-looking and developmental approach.

Geraniol and citral: recent developments in their anticancer credentials opening new vistas in complementary cancer therapy.

About 10 million people are diagnosed with cancer each year. Globally, it is the second leading cause of death after heart disease, and by 2035, the death toll could reach 14.6 million. Several drugs and treatments are available to treat cancer, but survival rates remain low. Many studies in recent years have shown that plant-derived monoterpenes, particularly geraniol and citral, are effective against various cancers, including breast, liver, melanoma, endometrial, colon, prostate, and skin cancers. This trend has opened new possibilities for the development of new therapeutics or adjuvants in the field of cancer therapy. These monoterpenes can improve the efficacy of chemotherapy by modulating many signaling molecules and pathways within tumors. Analysis of reports on the anticancer effects published in the past 5 years provided an overview of the most important results of these and related properties. Also, the molecular mechanisms by which they exert their anticancer effects in cell and animal studies have been explained. Therefore, this review aims to highlight the scope of geraniol and citral as complementary or alternative treatment options in cancer therapy.

Enhancing acid stability of citral through internal structure modulation in nanostructured lipid carriers with solid lipids and phospholipids.

In this current study, the internal structure of nanostructured lipid carriers was modulated by phospholipids (lecithin PC, hydrogenated soybean phospholipid HPC) and solid lipids to achieve stable encapsulation of citral. The presence of high melting point HPC could construct α-crystalline type with more lattice defects and effectively inhibit ß-ization. The HPC group could maintain the particle size at 155.9-186.9 nm, the polydispersity index (PDI) at 0.182-0.321, the Zeta potential at -57.58 mV to -49.35 mV and the retention rate of citral at 91.33-98.49 % in the acidic environments of 2 mM and 20 mM hydrochloric acid solutions. The recrystallization index (RI) of NLC increased with the number of solid lipid ester bonds (from 3.57 % to 16.58 % in the PC group and from 0.82 % to 12.47 % in the HPC group). The results illustrated that the number of solid lipid ester bonds and the melting point of phospholipids affected crystallinity of the lipid matrix and thus the stability of encapsulated citral. Hydrogenated phospholipid with high melting points was more beneficial in stabilizing citral. The present study improved the acidic stability of citral and provided a new thought for the application of citral in acidic beverages.

Influence of Lemongrass Essential Oil (Cymbopogon flexuosus) Supplementation on Diabetes in Rat Model.

(1) Background: Species of the genus Cymbopogon and its essential oil are known for their antioxidant and hypoglycemic effects. This study aimed to investigate the impact of the essential oil of Cymbopogon flexuosus (EOCF), and its major component, citral, on glycemic, lipid, antioxidant parameters, and oxidative stress in a type 1 diabetes (DM1) rat model. (2) Methods: Initially, EOCF was analyzed by Gas chromatography-mass spectrometry (GC-MS) and the antioxidant activity of EOCF and citral was evaluated. Next, male Wistar rats (3 months old, 200-250 g) induced with DM1 using Streptozotocin (STZ) were divided into four groups: negative control supplemented with an 80% Tween solution, two groups of animals supplemented with EOCF (32 mg/kg and 64 mg/kg) and with citral (32 mg/kg), and treated for 14 days. Measurements of blood glucose levels and body weight were taken; after euthanasia, biochemical markers, including lipid profile, uric acid, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were evaluated. (3) Results: The predominant compounds in EOCF were α-citral (53.21%) and neral (19.42%), constituting 72.63% citral. EOCF showed good antioxidant activity, significantly greater than citral. EOCF supplementation demonstrated a mitigating effect on glycemic, lipid, and hepatic abnormalities induced by DM1. (4) Conclusions: EOCF emerges as a promising therapeutic option for the management of DM1.

Revealing the mechanism of citral induced entry of Vibrio vulnificus into viable but not culturable (VBNC) state based on transcriptomics.

Citral has attracted much attention as a safe and effective plant-derived bacteriostatic agent. However, the ability of citral to induce the formation of VBNC state in Vibrio vulnificus has not been evaluated. In the present study, V. vulnificus was shown to be induced to form the VBNC state at 4.5 h and 3 h of citral treatment at 4MIC and 6MIC. Moreover, the citral-induced VBNC state of V. vulnificus maintained some respiratory chain activity and was able to recover well in both APW media, APW media supplemented with 5 % (v/v) Tween 80 and 2 mg/mL sodium pyruvate. Field emission and transmission electron microscopy showed that the external structure of the citral-induced VBNC V. vulnificus cells was shortened to short rods, with folded cell membrane, rough cell surface, and dense cytoplasm and loose nuclear material in the internal cell structure. In addition, the possible molecular mechanisms of citral-induced formation and recovery of V. vulnificus in the VBNC state were explored by transcriptomics. Transcriptome analyses revealed that 1118 genes were significantly altered upon entry into the VBNC state, and 1052 genes were changed after resuscitation. Most of the physiological activities related to energy production were inhibited in the citral-induced VBNC state of V. vulnificus; however, the bacteria retained its pathogenicity. The citral-induced resuscitation of V. vulnificus in the VBNC state selectively restored the activity of some genes related to bacterial growth and reproduction. Meanwhile, the expression levels of other genes may have been influenced by citral-induced resuscitation after the formation of the VBNC state. In conclusion, this study evaluated and analyzed the ability and possible mechanism of citral on the formation of VBNC state and the recovery of VBNC state of V. vulnificus, and made a comprehensive assessment for the safety of citral application in food production.

Untargeted metabolomics uncovers prime pathways linked to antibacterial action of citral against bacterial vaginosis-causing Gardnerella vaginalis: An in vitro and in vivo study.

Global increase in recurrence of bacterial vaginosis (BV) and worrisome rise in antimicrobial resistance pose an urgent call for new/novel antibacterial agents. In light of the circumstance, the present study demonstrates the in vitro and in vivo antibacterial activity of a phytochemical citral, with a particular emphasis to elucidate its mechanistic action against Gardnerella vaginalis -a potential cause of BV. Out of 21 phytochemicals screened initially against G. vaginalis, citral was envisaged to be a phenomenal antibacterial agent showing MIC and MBC at 128 µg/mL. Citral's rapid killing ability was revealed by a time-killing kinetics assay supported by CFU, signifying that it completely killed the given inoculum of planktonic G. vaginalis cells within 60 min. Further, citral was found to exhibit 1 min contact-killing efficacy together with mature-biofilm disintegrating ability at increasing MICs. To further understand the molecular action of citral, in vitro investigations such as ROS estimation, PI staining and intracellular protein release assay were performed, which demonstrated that citral deteriorated the membrane integrity of G. vaginalis. Galleria mellonella, a simple invertebrate model used to evaluate citral's non-toxic and antibacterial activity in vivo, demonstrates that citral completely restored the larvae from G. vaginalis infection. The metabolite level investigation using LC-MS revealed that citral had negative impact on biotin metabolism (via., biotin), spermidine metabolism (via., 5'-methylthioadenosine and spermidine) and nucleotide metabolism (via., guanine, adenine and uridine). Since that biotin is associated with seven different metabolic pathways, it is conceivable that citral could target biotin biosynthesis or its metabolism and as a result, disrupt other metabolic pathways, such as lipid and fatty acid synthesis, which is essential for the creation of cell membranes. Thus, the current study is the first of its kind to delineate the promising in vitro and in vivo antibacterial efficacy of citral and decipher its plausible antibacterial action mechanism through metabolomic approach, which concomitantly emphasizes citral as a viable natural therapeutic alternative to manage and control BV.