Exploring the Volatile Composition and Antibacterial Activity of Edible Flower Hydrosols with Insights into Their Spontaneous Emissions and Essential Oil Chemistry

In the circular economy framework, hydrosols, by-products of the essential oil industry, are gaining attention for their potential in waste reduction and resource reuse. This study analyzed hydrosols from six edible flowers, investigating their chemical composition (VOC-Hyd) and antibacterial properties alongside volatile organic compounds of fresh flowers (VOC-Fs) and essential oils (EOs). Antirrhinum majus exhibited ketones as major VOC-Fs (62.6%) and VOC-Hyd (41.4%), while apocarotenoids dominated its EOs (68.0%). Begonia cucullata showed alkanes (33.7%) and aldehydes (25.7%) as primary VOC-Fs, while alkanes were prevalent in both extracts (65.6% and 91.7% in VOC-Hyd and in EOs, respectively). Calandula officinalis had monoterpenoids in VOC-Fs and VOC-Hyd (89.3% and 49.7%, respectively), while its EOs were rich in sesquiterpenoids (59.7%). Dahlia hortensis displayed monoterpenoid richness in both VOC-Fs and extracts. Monocots species’ VOC-Fs (Polianthes tuberosa, Tulbaghia cominsii) were esters-rich, replaced by monoterpenoids in VOC-Hyd. P. tuberosa EO maintained ester richness, while T. cominsii EOs contained a significant percentage of sulfur compounds (38.1%). Antibacterial assays indicated comparable minimum inhibitory concentration profiles across VOC-Hyd: B. calcullata and P. tuberosa against Staphylococcus aureus and Salmonella enterica ser. typhimurium, T. cominsii against Escherichia coli and S. enterica, A. majus and C. officinalis against S. aureus, and D. hortensis against S. enterica.


Introduction
The deep bond between humans and flowers has been reported for millennia.Historically, the latter were protagonists of funeral rituals, feasts, offerings, and songs due to their myriad symbolic meanings.Their undeniable aesthetic beauty has made them highly sought-after for decorative functions, paintings, ceramics, and fabrics [1].Gardens, in particular, result from skillful combinations of plant species with a multitude of shapes and colors, whose wonderfulness can be fully observed during blooming.Very recently, the concept of a 'healing garden' has emerged, specifically designed to promote wellbeing, comfort, and pain tolerance both in clinical and non-clinical populations, thereby representing a combination of flower beauty and human health [2].
The flowers' aroma is pivotal, as much as their ornamental value, since it can evoke several emotional responses tied to individual experiences and memories.This is attributed to the varied bouquets of volatile organic compounds (VOCs) emitted by petals, each contributing to the distinctive signature of every flower.Flower extracts as essential oils (EOs) hold particular appeal for industries such as cosmetics, perfume, toiletries, and hygiene [3].
In recent years, the gastronomic and nutritional value of flowers has garnered attention, with several edible flowers (EFs) recognized for their attractiveness, sensory perception [4][5][6][7], nutritional properties [8], and biological activities [9].EFs are very versatile and suitable for various culinary contexts, such as raw flowers without processing, by minimum processing, or in powdered form [10]. Furthermore, their EOs are gaining more and more interest from the food industry: (1) detailed scientific reports highlighting the antimicrobial, antioxidant, and antiparasitic activities of EFs EOs are increasing; (2) they could be used as safer substitutes for chemical preservatives, thus meeting consumer concerns about the use of artificial and harmful compounds in the food sector [11].Therefore, the EOs obtained from EFs are the perfect combination for the preservation of food products and food safety.
Hydrodistillation is widely used to extract EOs from flowers [12].During this process, three main by-products are created: solid residue (plant debris), water residue (non-distilled aqueous phase), and hydrosols (also known as hydrosols or aromatic water).The latter are aqueous aromatic solutions saturated with water-soluble volatile compounds [13].Hydrosols distillate together with the EOs, retaining a good quantity of dissolved compounds that are, however, in lesser amounts than EOs, both in terms of number and concentration.Despite this, oxygenated components (e.g., oxygenated monoterpenes (OM)) are higher in hydrosols, while EOs are richer in highly hydrophobic compounds such as monoterpene and sesquiterpene hydrocarbons [13].Currently, aromatic waters have several applications in the food sector, as well as in the aromatherapy, cosmetics, and perfume industries, representing the most commonly used by-product of EO production [14].
Within this framework, the current study meticulously examined the chemical composition of hydrosols derived from six distinctive flowers tailored for culinary purposes, namely, Antirrhinum majus L., Begonia cucullata Willd, Calendula officinalis L., Dahlia hortensis Guillaumin, Polianthes tuberosa L., and Tulbaghia cominsii Vosa.Our investigation extended beyond the hydrosols, encompassing a thorough analysis of the EOs and spontaneous emissions of these botanical specimens.Furthermore, a comprehensive assessment of the antibacterial properties was conducted, involving six bacterial strains.This study scrutinized the primary phytochemicals inherent in these floral extracts, marking a pivotal stride in revealing the transformative potential encapsulated within these often-neglected by-products.
Full-opened and flawless flowers were collected at the Department of Pharmacy (University of Pisa) and properly analyzed as follows.

Essential Oil (EO) and Hydrosol Extraction
The extraction was conducted using 20 g of fresh flowers through a 2 h hydrodistillation process employing a micro-Clevenger-type apparatus.During hydrodistillation, 1 mL of HPLC-grade n-hexane was added to the separator funnel.This precaution was taken due to concerns about the possibility of obtaining an EO yield as low as one drop.The extraction process resulted in obtaining both EO dissolved in hexane and hydrosol, which were then separated based on density difference.The dissolved EO was directly analyzed by GC-MS, while the hydrosol was stored in sealed vials at 4 • C in the dark.

Headspace Solid-Phase Microextraction (HS-SPME) for VOC Analyses
The HS-SPME procedure, as outlined by Najar et al. [18], involved both spontaneous emissions of fresh EFs (VOC-Fs) and hydrosols (VOC-Hyd).Briefly, 1 g of fresh flower or 1 mL of hydrosol was placed into a 50 mL headspace glass vial, sealed with aluminum foil, and stored at room temperature.HS-SPME extractions utilized a DVB/CAR/PDMS SPME fiber exposed to the headspace for 30 min.Post-extraction, the fiber was introduced into the GC-MS injection system at 220 • C for 3 min for thermal desorption of analytes.All analyses were performed in triplicate (n = 3).The SPME fiber underwent thermal conditioning before use and daily 10 min conditioning before the initial extraction to ensure the absence of carryover effects.

Phytochemical Analysis: GC-MS Analysis
Chromatographic analyses were conducted on VOC-Fs, EOs, and VOC-Hyd extracted from edible flowers using an Agilent 7890B gas chromatograph system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent HP-5MS capillary column (30 m × 0.25 mm; Santa Clara, CA, USA).Helium served as carrier gas, with a flow rate of 1 mL/min and a column head pressure of 13 psi.The injector temperature was set at 220 • C, with a split ratio of 1:25.The oven temperature programming ranged from 60 to 240 • C at a rate of 3 • C/min.Full scan MS detection was conducted using an Agilent 5977B single quadrupole inert mass selective detector (Agilent Technologies Inc., Santa Clara, CA, USA) with an electron impact (EI) ion energy of 70 eV, and the mass acquisition range was set to 30-300 m/z.
Identification of the constituents was based on a comparison of the retention times (Rt) with those of the authentic samples, comparing their calculated Kovats Index (KI) (determined using the van Den Dool and Kratz equation) to the series of a C8-C22 n-hydrocarbons.Computer matching was also used against commercial [19] and laboratory-developed mass spectra library built up from pure substances and components of commercial essential oils of known composition and MS literature data [20][21][22][23][24]. Analysis was conducted in triplicate for each sample to ensure reliability.

Preparation and Storage of Bacterial Cultures
Three Gram-positive bacteria, Staphylococcus aureus ATCC 6538, Listeria monocytogenes ATCC 7644, and Enterococcus faecalis V583E, and three Gram-negative bacteria, Escherichia coli ATCC 15325, Pseudomonas aeruginosa ATCC 27853, and Salmonella enterica serovar Typhimurium ATCC 14028, were selected as test organisms.The bacterial cultures were prepared in Tryptone Soy Agar (TSA, pH 7.3 ± 0.2, Oxoid, UK) and stored at −20 • C in BHI broth (pH 7.4 ± 0.2, Merck, Germany) with 20% glycerol as cryoconservant.To revitalize bacterial cultures, frozen stocks were subcultured in BHI broth and incubated at 37 • C for 24 h.

In Vitro Antibacterial Testing of Hydrosols
MIC values for hydrosols against diverse bacterial strains were determined using a modified twofold serial microdilution method in a 96-well microplate.Hydrosols prepared in BHI with DMSO followed a 1:3:4 v/v ratio.Incubation at 37 ± 2 • C for 24 h was performed after bacterial suspension addition.MIC values, evaluated in triplicate, were mode-determined for each isolate.MBC values, indicating the lowest hydrosols concentration preventing bacterial growth, were assessed on TSA plates after 24 h incubation.Results, expressed as v/v, were mode-reported for both MIC and MBC.The entire process was executed in triplicate to ensure reliability.

Statistical Analysis
Volatile compositions of hydrosols underwent comprehensive multivariate statistical analyses utilizing JMP software (SAS Institute Inc. JMP ® .Version 16, SAS Institute Inc., Cary, NC, USA, 2021).Principal Component Analysis (PCA) was performed on a covariance data matrix of 58 × 6 (58 compounds × 6 samples = 348 data).The resulting PCA was plotted by selecting the two principal components with the highest variance explained, obtained by the linear regressions operated on mean-centered, unscaled data.As an unsupervised method, this analysis aimed at reducing the dimensionality of the multivariate data of the matrix whilst preserving most of the variance [25].Additionally, a two-way Hierarchical Cluster Analysis (HCA) was carried out using Ward's method, and the squared Euclidean distances were used as a measure of similarity.Spontaneous emission of its flowers was characterized by ketones (62.6%), mainly acetophenone (59.9%, ( 15)) (Table 1), reported for its sweet, pungent odor and taste [26].This class of constituents was followed by esters (10.8%) and OM (9.2%), of which methyl benzoate (5.4%, (17)) and linalool (8.3%, (18)) were, respectively, the most abundant compounds.In coherence with our results, Suchet et al. [27] reported the presence of acetophenone in the floral scent of wild snapdragon, with a contribution of 69.0% of the absolute emissions.

Results and Discussion
EO evidenced a lesser number of constituents (13) in comparison with spontaneous emission (18) and VOC-Hyd (21).Sixty-eight percent of the identified portion was represented by apocarotenoids uniquely constituted by hexahydrofarnesylacetone (40).A significant decrease in acetophenone was noticed after hydrodistillation, whose percentage passed from 59.3% in VOC-Fs to 5.7% in EO.
Ketone turned out to be the main class in VOC-Hyd (41.4%), and acetophenone (40.2%) was also confirmed as the main compound by excellence here.Aldehydes constitute more than a quarter of the whole VOC-Hyd composition (26.7%), with nonanal being the main constituent (23.6%, (19)).
Regarding the major compounds found in the EO, we highlighted the presence of p-cymene (18.9%, (11)) and limonene (19.3%, (12)).The EO also showed the largest number of compounds (34) compared to both the molecules that were spontaneously perceived (12) and the VOC-Hyd (13).The latter was dominated by MH (95.5%), and p-cymene (71.3%) was again confirmed to be the molecule par excellence of these flowers, regardless of the type of extract.Besides p-cymene, limonene was also found to have a high percentage of VOC-Hyd (19.2%).
To the best of the authors' knowledge, no previous paper has been published about the volatilome of this plant.The available literature primarily investigates other species within the same genus, with a predominant focus on EOs.A unique paper investigated D. pinnata Cav.specifically for its VOC-Fs composition using static headspace volatiles extraction and revealed the extract's richness in myrcene (28.5%), γ-muurolene (27.8%), and (E)-β-ocimene (17.5%) [32].The method used cannot be directly compared with our extraction technique or our approach to spontaneous emission evaluation.Nevertheless, it is noteworthy that the primary compounds belonged to MH and SH, as observed herein in both VOC-Fs and EO.Flower EO of D. pinnata was also investigated by Wang et al. [33], who reported an EO rich in 4-terpineol (25.7%), methallyl cyanide (14.0%), and D-limonene (10.5%).Only limonene was found in our EO, while the other two compounds were omitted.Within the same genus, the capitulum (flower head) EO of Dahlia imperialis Roezel ex Ortgies was rich in β-pinene (27.7%), α-phellandrene (26.2%), and α-pinene (12.4%).A recent study on flower EO of the same species confirmed its dominance of β-pinene (27.7%), α-phellandrene (26.2%), and α-pinene (12.4%) as major chemicals [34].Although all these compounds were also present in our studied flowers, they were found in lesser amounts.In a study conducted by Manah et al. [35] on the flowers EO of Dahlia E'veline', it was evidenced that more than 80% of the identified fraction was composed of anethole.However, this compound was omitted in the studied species.P. tuberosa spontaneous emissions predominantly consisted of esters (76.5%), mirroring its EO composition (90.1%).Methyl benzoate (57.3%, ( 14)) was the main ester in spontaneous emissions, constituting, together with methyl salicylate (13.0%, ( 18)), over 70% of the identified portion (Table 5).The presence of lactones (14.4%) was observed, reported uniquely by two compounds: jasminelactone (13.8%, ( 27)) and δ-decalactone (0.6%, ( 26)) (Table 5).It is important to highlight that this study partially differs from previous research, which reported the presence of methyl benzoate and methyl salicylate in six out of seven studied cultivars of P. tuberosa [37,38].Methyl salicylate was present in the studied species.Kutty and Mitra [38] reported the presence of lactones.Even though the identified lactones were different from the ones reported in this work, their presence is similar to what was reported herein.On the contrary, the main esters found in the EO were methyl icosanoate (24.4%, (32)) and methyl heneicosanoate (58.4%, (33)).Additionally, chromene compounds (precocene II ( 28)) were detected, contributing only 4.0% to the overall composition.The EO composition in half-opened and fully-opened flowers was rich in methyl benzoate (37.9 and 28.6%, respectively) [39], a compound also present in our flowers but in a lesser amount (7.3%, (13)).

Antibacterial Activity of Hydrosols
Due to the limited availability of plant material and consequently very low yields of EOs, the hydrosols, a by-product obtained during the removal of volatile oil through steam distillation [43], underwent antibacterial activity testing.The tests were conducted on six strains, three of which were Gram-positive and three were Gram-negatives.
Among the Gram-positive strains, Staphylococcus aureus exhibited significant susceptibility, displaying a MIC mode value of 1:2 (Table 7) for all tested hydrosols, except for A. majus and D. hortensis.Nonanal and decanal, two aldehydes from green leave volatiles family, were observed in all active hydrosols, except for T. cominsii, where nonanal was completely omitted; these phytochemicals have been granted a 'generally recognized as safe' status [44].The antibacterial efficacy of decanal was assessed against S. aureus strains, including both methicillin-resistant and susceptible strains.However, its effect was less pronounced when compared to the EO of Ducrosia anethifolia Boiss, where decanal was the primary compound [45].Nonanal, a saturated aldehyde, has been documented to induce notable changes in membrane permeability, functioning as an effective antibacterial agent [46].Limonene, present in all studied species, exhibited significant inhibition of S. aureus growth [47] and demonstrated activity against isolated methicillin-resistant strains [48].Additionally, p-cymene, a precursor of carvacrol, present in C. officinalis hydrosols, and γ-terpinene, identified in all active hydrosols (AH), displayed antibacterial and anti-biofilm activities [49].Furthermore, eucalyptol, found in substantial amounts in the hydrosols of P. tuberosa and C. officinalis, has reported effects on membrane integrity and implications in oxidative stress in methicillin-resistant S. aureus [50].
As regards Gram-negative bacteria, Salmonella enterica emerged as the most sensitive strain, showing susceptibility to the hydrosols of B. cucullata, D. hortensis, P. tuberosa, and T. cominsii.Additionally, the hydrosol of T. cominsii exhibited a MIC value of 1:2 against E. coli.Fenchone, the primary compound detected in T. cominsii, was the focal point of a study evaluating its antibacterial and anti-biofilm properties through in vitro and in silico approaches.In silico predictions revealed interactions with E. coli proteins, which were validated by determining MIC and MBC values [51].The same work evidenced that fenchone reduced biofilm formation in E. coli.Additionally, the MIC value of this phytochemical against E. coli was 2 [52].On the other hand, the same hydrosol was rich in viridiflorol, known for its anticancer, antioxidant, anti-inflammatory, and antibacterial activities [52][53][54].
Methoxyphenyl-oxime, found in a high percentage in P. tuberosa but also present in all other hydrosols except for D. hortensis, albeit in lesser abundance, is an alkaloid reported to be isolated from Conocarpus lancifolius Engl.[55].The authors of this paper demonstrated its antibacterial activity against Gram-negative strains.Further research [56] explored its antiviral effects and confirmed its potential as a potent drug-like compound against capripox viruses, utilizing methanolic extract of Leucas aspera.

The Multivariate Analysis of Hydrosol and Key Compound Insights
In addition to assessing their antibacterial activity, hydrosols are being explored as valuable by-products, requiring a comprehensive evaluation of their composition.This necessitates the use of advanced analytical tools such as multivariate analysis, including Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA), which provide profound insights into the characteristics of hydrosols.HCA (Figure 1a) analysis revealed two main clusters, with the first one being homogenous, comprising uniquely D. hortensis hydrosol.The second group encompassed the remaining samples, which can be further divided into two subclusters: Subcluster 1, including A. majus and B. cucullata, and the second subcluster comprised the others.According to the PCA plot (Figure 1b), the first axis (PC 1), accounting for over 45% of the variability, clearly distinguished D. hortensis hydrosol from the others.
Meanwhile, PC 2 (accounting for 34.5% of the variability) differentiated A. majus and B. cucullata from the rest.These findings were corroborated by the cluster analysis, where the Ward method clustered D. hortensis separately, while A. majus and B. cucullate formed another cluster, and the remaining species grouped, including T. cominsii, P. tuberosa, and C. officinalis.
Examining the biplot of the PCA analysis (Figure 1c), we observed that D. hortensis stood out due to its high percentage in p-cymene, a common compound found in all aromatic waters.p-cymene, an alkyl-substituted aromatic compound, is known for its diverse beneficial activities, such as antioxidant, anti-inflammatory, antiparasitic, antidiabetic, antiviral, antitumor, antibacterial, and antifungal activities [57].p-Cymene has also been recognized to act as an analgesic, antinociceptive, immunomodulatory, vasorelaxant, neuroprotective agent, and anticancer [57].Moreover, it found applications in the food industry as a flavor/fragrance agent [58] and served as an intermediate constituent in the chemical syntheses of fragrances [59].Additionally, sabinene and (E)-β-ocimene were exclusively present in Dahlia aromatic water.These compounds represented 1% of the identified fraction.(E)-β-ocimene was recommended for fragrance use at levels up to 3% and is known as a pheromone involved in social regulation in the honeybee colony [60].Sabinene was utilized as a perfume additive and possessed anti-fungal activity [61].Also noticed is the presence of β-pinene reported for broad biological activity, including fungicidal, antimicrobial, and antiviral agents, in addition to its use in the flavor and fragrance industry [62].
Nonanal, a common compound found in both A. majus and B. cucullata, contributed to their clustering in the bottom left quadrant of the PCA analysis.It is renowned for its antifungal properties [63] and is widely used in perfumery products for its green-floral fragrance [64].Acetophenone, a preliminary compound of A. majus, serves as both an attractant and repellent for blood-sucking insects such as mosquitoes, flies, and ticks, serving as a crucial component in the manipulation of skin microbiota by vector-borne parasites and as an ingredient in trap crops for economically important crop pests [26].Furthermore, it serves as a flavoring agent and intermediate compound in perfumes and cosmetics [65].
On the other hand, 1-octene-3-ol and bornyl acetate were characteristic compounds of B. cucullata.They exhibited strong antibacterial activity, especially against Gram-positive strains [66,67], which could be one of the reasons why the hydrosols of this species were effective on S. aureus.Bornyl acetate is also documented for its insecticidal activity [68] and anti-inflammatory effect in human chondrocytes [69].On the contrary, A. majus was characterized by 1-hexanol, a volatile alcohol known to have an effect against food-related Gram-negative bacteria [70].In the upper left quadrant of PCA analysis, the remaining hydrosols were clustered together due to their high content in eucalyptol, which is known for its pleasant spicy aroma and taste.Eucalyptol found applications as a flavoring agent, fragrance ingredient, and cosmetics additive [71].Additionally, it is reported to alleviate pain and inflammation associated with monosodium urate [72].Notably, methoxy-phenyl-oxime constituted at least 5.0% of the identified fraction of these three hydrosols and was found in all the others, imparting a scent reminiscent of fresh shrimp and crabs [73].This compound also exhibited antibacterial activity [55].Oximes naturally occur in plants and animals and are known for their anti-inflammatory, antioxidant, and anticancer activities [74].Thymol, a characteristic constituent of T. cominsii, is versatile and used in therapeutic applications [75] and food [76].Decanal, another prominent compound in this hydrosol, is utilized in perfume [77].In contrast, methyl benzoate and 6-methyl-5-hepten-2-one were present in P. tuberosa.Methyl benzoate is suggested as an efficient green pesticide [78].6-Methyl-5-hepten-2-one is considered an important chemical intermediate and flavor compound crucial for fruit flavor in tomato, papaya, and guava [79].C. officinalis evidenced the highest content of linalool, a compound mainly used for its anti-inflammatory, anticancer, antimicrobial, and antioxidant properties, alongside its use in cosmetics, food additives, and perfume [77,80].Furthermore, this hydrosol is characterized by its high content of camphor and 4-terpineol, both of which are reported for their antibacterial activity [81,82].
Limonene, a shared compound in all hydrosols, although it may not be considered a discriminant compound in PCA due to its presence in significant percentages across all samples, is recognized for its therapeutic effect, including anti-inflammatory, antioxidant, and antiviral effects, besides its use as flavor and fragrance additive owing to its pleasant lemon-like odor [83].
Overall, the studied hydrosols offer various chemical compositions with promising applications across multiple industries, including skincare, fragrance, and therapeutics.

Figure 1 .
Figure 1.Hierarchical Cluster Analysis (HCA) (a), Principal Component Analysis (PCA) performed on the hydrosols of the studied species.(b): Score plot; (c): loading plot.Nonanal, a common compound found in both A. majus and B. cucullata, contributed to their clustering in the bo om left quadrant of the PCA analysis.It is renowned for its
Values and mode (in bold) of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of each hydrosol against the six ATCC bacterial strains used in the test.