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Optimization of Extraction Process and Effect of Time on Chemical Composition and Antimicrobial Activity of Allium sativum (Garlic) and Allium schoenoprasum (Chives) Volatile Fractions

V
Vo Thi Thu Hang1,2
0009-0009-0798-7841
N
Nguyen Thi Van Anh1,*
0000-0003-2242-8094
1Faculty of Engineering and Food Technology, University of Agriculture and Forestry, Hue University, 102 Phung Hung Street, Hue 49000, Vietnam.
2Department of Food Biotechnologies and Agricultural Product Quality, Faculty of Agriculture and Technology, University of South Bohemia in České Budějovice, Studentska 1668, 37005 České Budějovice, Czech Republic.

ABSTRACT

Background: This study optimized the hydrodistillation kinetics of Allium sativum (garlic) and Allium schoenoprasum (chives) bulbs from North and Central Vietnam and evaluated their bioactivity.

Methods: Extraction parameters were investigated by varying pretreatment methods, material-to-water ratios and distillation time. The chemical compositions of the obtained volatile fractions were analyzed via GC-MS, while biological activities were assessed through antioxidant assays and antibacterial testing against Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 12943 using inhibition zone diameters and MIC values.

Result: Ultrasound-assisted pretreatment yielded the best results (p≤0.05) at a 1:3 material-to-water ratio over 3 hours (1.00% for garlic; 1.34% for chives). Chemically, garlic volatile fraction was stable with diallyl disulfide as the major component, whereas chives volatile fraction showed a transient accumulation of allyl propyl sulfide at 3 hours. Biologically, garlic volatile fraction displayed high antioxidant capacity comparable to vitamin C and potent antibacterial activity against Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 12943 (Inhibition zone: 21 mm; MIC: 12.5 µg/mL). In contrast, chives volatile fraction showed significantly lower efficacy (Inhibition zone: 9.0 mm; MIC: 250 µg/mL).

INTRODUCTION

The genus Allium (Amaryllidaceae family), comprising 600-700 species, includes some of the most ancient and widely consumed cultivated plants in the world, most notably garlic (Allium sativum) and chives (Allium schoenoprasum) (Calimpang et al., 2024). Beyond their culinary ubiquity as flavoring agents, these species are renowned for their broad spectrum of biological activities, including antimicrobial, antioxidant, anti-inflammatory and anticancer properties (Danquah et al., 2022; Iwar et al., 2024). These bioactive effects are primarily attributed to their rich content of organosulfur compounds (Dai et al., 2025). Unlike typical essential oils which are predominantly terpene-based, the volatile fractions of Allium species are characterized by a complex matrix of sulfur-containing constituents such as diallyl disulfide (DADS), trisulfides and vinyldithiins. Crucially, these compounds are not inherently present in intact tissues but are formed enzymatically and thermally from non-volatile precursors like alliin upon tissue rupture and extraction processing (Edris and Fadel, 2002).
       
Conventional extraction is often hindered by prolonged processing and thermal degradation of labile sulfur constituents (Stratakos and Koidis, 2016). To mitigate this, green intensification via ultrasound and microwave pretreatments is increasingly adopted. Mechanistically, ultrasound employs acoustic cavitation for cell disruption, whereas microwaves utilize ionic conduction and dipole rotation to induce rapid heating (Mandal et al., 2007; Tiwari, 2015; Patil et al., 2018). However, the comparative efficacy of these interventions on Allium tissues during hydrodistillation requires further investigation.
       
Furthermore, most extraction optimization studies prioritize yield maximization, often overlooking the temporal evolution of chemical constituents. While recent literature has evaluated the chemical profiles of Allium volatile fractions, these studies predominantly rely on end-point analyses after a fixed extraction duration (Dehariya et al., 2021; Tran et al., 2024). Such single-time-point approaches often yield conflicting optimal conditions and fail to capture the dynamic kinetic transformations of the extract. This is particularly critical for Allium volatile fractions, where heat-sensitive precursors like allicin degrade rapidly into various transient polysulfides under continuous thermal stress (Zhu et al., 2025). Consequently, focusing solely on yield plateaus without tracking these time-dependent chemical shifts can lead to over-processing and the loss of potent bioactive intermediates.
       
This study pursues two primary objectives. First, it systematically evaluates the comparative efficacy of mechanical, ultrasound-assisted and microwave-assisted pretreatments on the hydrodistillation of A. sativum and A. schoenoprasum volatile fractions. Second, it investigates how extraction duration influences not only yield but also the kinetic evolution of the chemical profile. By analyzing fractions at 2, 3 and 4-hour intervals via GC-MS, the research aims to elucidate the interplay between thermal processing time, chemical composition and biological activity against Escherichia coli and Staphylococcus aureus. Ultimately, these findings provide a scientific basis for optimizing extraction protocols to prioritize specific bioactive profiles over mere volume maximization.

MATERIALS AND METHODS

Plant materials
 
Fresh garlic (from Son La) and chives (from Quang Tri) bulbs were harvested (April-August, 2024) and identified according to the Vietnamese Pharmacopoeia. The raw materials were cleaned, skinned and air-dried to moisture contents of approximately 58% and 71%, respectively. Samples were stored in sealed polyethylene bags at 4°C and utilized for extraction within two weeks.
 
Bacterial strains
 
Two representative pathogenic bacteria, comprising Gram-negative Escherichia coli ATCC 35218 and Gram-positive Staphylococcus aureus ATCC 12493, were used in this study.
 
Volatile fractions extraction
 
General hydrodistillation protocol
 
Fresh materials (100 g) were finely crushed and thoroughly mixed with varying volumes of distilled water to achieve the specified material-to-water ratios. The mixture was then transferred to a 1000 mL round-bottom flask connected to a Clevenger-type apparatus for hydrodistillation. To prevent boil-over and ensure efficient mass transfer, the total working volume within the flask was strictly maintained at an optimal level. The flask was heated to the boiling point. The mixture of volatile fractions and water vapor was condensed and collected in a separating funnel. Finally, the volatile fractions were dried with anhydrous Na2SO4 to eliminate residual moisture and stored in sealed amber glass bottles at 5°C until analysis.

Experimental design
 
The optimization of the extraction process was carried out through three sequential single-factor experiments:
 
Experiment 1: Effect of pretreatment technologies
 
To evaluate the impact of physical intensification on extraction efficiency, mixtures underwent specific pretreatments prior to hydrodistillation. Microwave-assisted pretreatment involved irradiation at 700 W for 5 min, while ultrasound-assisted pretreatment utilized a Kingsonic bath (Model MC-040S, 300 × 240 × 150 mm) at 40 kHz for 10 min at 35°C. These experimental conditions were subsequently compared against a control group subjected solely to grinding (conventional hydrodistillation).
 
Experiment 2: Effect of material-to-water ratio
 
The influence of the solvent volume was investigated by varying the material-to-water ratio at three levels: 1:2, 1:3 and 1:4 (w/w). During this stage, the pretreatment method was kept constant based on the results from Experiment 1, while the extraction time was fixed at 2 hours.
 
Experiment 3: Effect of extraction time
 
Using the optimized pretreatment and material-to-water ratio, the effect of extraction time was investigated at 2, 3 and 4 hours. Volatile fractions were collected at these intervals to evaluate variations in both yield and chemical composition.
       
The volatile fractions yield (%) was calculated using the following equation:

 
Where,
• Volatile fractions (%): Volatile fractions yield (%).
• m volatile fractions: Mass of obtained volatile fractions (g).
• msample: Mass of the initial raw material (g).
 
GC-MS analysis
 
GC-MS analysis was performed on an Agilent 7890B system coupled with a 5977B detector and a DB-1 column (30 m × 0.25 mm, 0.25 μm). A 1 µL sample was injected at 250°C (split 1:200) with helium carrier gas at 1 mL/min. The temperature program initiated at 45°C, ramped at 5°C/min to 150°C, then 40°C/min to 290°C (held 5 min). MS conditions included 70 eV ionization, source at 230°C, quadrupole at 150°C and a scan range of m/z 30-500. Data were processed using MassHunter. The volatile compounds were tentatively identified by comparing their mass spectra with the NIST 2017 library (match factor >85%). To support this tentative identification, the elution order of the detected compounds on the non-polar DB-1 column was cross-referenced with established literature data. Quantification of the constituents was performed by peak area normalization.

Evaluation of antioxidant activity using the free radical scavenging assay (DPPH)
 
The antioxidant activity was determined using the DPPH radical scavenging assay (Emami et al., 2007). Briefly, 0.5 mL of serially diluted volatile fraction was mixed with 2.5 mL of a 0.004% (w/v) DPPH solution prepared in ethanol. The control contained DPPH solution without volatile fraction, while pure ethanol (99.5%) served as the blank. After incubation in the dark at room temperature for 30 minutes, absorbance was measured at 517 nm against the blank. Ascorbic acid (Vitamin C, 12.5-100 μg/mL) was used as the positive reference standard to validate the assay sensitivity.
       
The percentage of DPPH radical scavenging activity (Inhibition Capacity - IC%) was calculated using the following equation:

 
Where,
• AC: Absorbance of the control reaction (containing all reagents except the volatile fraction).
• AS: Absorbance of the test sample containing the volatile fraction.
       
The antioxidant activity was expressed as the IC50 value, defined as the concentration of the volatile fraction required to scavenge 50% of the DPPH radicals. The IC50 value was determined by plotting the inhibition percentage against the sample concentrations using linear regression analysis. To ensure reliability and method reproducibility, all DPPH assays were performed in independent triplicates (n=3) and the resulting IC50 values were expressed as the mean ± standard deviation (SD).
 
Determination of antibacterial activity and minimum inhibitory concentration (MIC)
 
The antibacterial efficacy of the volatile fractions was assessed against two bacteria: Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 12493.
       
Antibacterial activity was screened via the agar well diffusion method (Balouiri et al., 2016). Briefly, 50 μL of the volatile fraction was dispensed into 6-mm wells on Mueller-Hinton agar seeded with standardized bacterial suspensions (0.5 McFarland; ~1.5×108 CFU/mL). Pure n-hexane served as the solvent control, while Tetracycline (10%) was utilized as the positive reference standard to validate the susceptibility of the tested strains. Inhibition zones were measured after incubation at 37°C for 24 hours. Subsequently, MIC were determined using broth microdilution (Moreira et al., 2007). Serial two-fold dilutions of the volatile fractions in 5% DMSO (range: 3.9-2000 µg/mL) were inoculated to a final density of 5×105 CFU/mL. After incubation at 37°C for 24 hours, the MIC was recorded as the lowest concentration inhibiting visible growth. To ensure reliability and method reproducibility, all antibacterial assays were performed in independent triplicates (n=3). The resulting inhibition zones were expressed as mean ± standard deviation (SD), whereas the MIC values were reported as the consistent values across the replicates.
 
Statistical analysis
 
All experiments were performed in triplicate (n = 3). Data were analyzed using IBM SPSS Statistics 25.0 and expressed as mean ± standard deviation (SD). Prior to one-way ANOVA, data normality and variance homogeneity were verified via the Shapiro-Wilk and Levene’s tests, respectively. Duncan’s multiple range test (DMRT) was selected as the post hoc test for its high sensitivity in detecting differences among multiple groups, with significance set at p≤0.05.

RESULTS AND DISCUSSION

Optimization of extraction conditions
 
Effect of pretreatments
 
Fig 1 illustrates that physical intensification significantly enhanced extraction efficiency relative to conventional methods (p≤0.05). For garlic, yields rose from a baseline of 0.29% (grinding) to 0.40% with microwave treatment, peaking at 0.67% under ultrasound. Chives exhibited a similar trend, improving from 0.34% to 0.55% and 0.59% for microwave and ultrasound-assisted pretreatments, respectively. Consequently, ultrasound was established as the optimal protocol for subsequent trials.

Fig 1: Effect of pretreatment methods on the yield of garlic and chives volatile fractions.


 
Effect of material-to-water ratio
 
Following the ultrasound-assisted pretreatment, the influence of the material-to-water ratio on the volatile fraction yield was investigated. Fig 2 reveals a non-linear trend for both plant species. Garlic yield peaked at 0.83% (1:3) before declining to 0.74% (1:4). Similarly, chives yield rose from 0.59% (1:2) to 1.07% (1:3), then dropped significantly to 0.69% (1:4). These results suggest that a material-to-water ratio of 1:3 provides the ideal equilibrium for extraction efficiency.

Fig 2: Effect of material-to-water ratio on the yield of garlic and chives volatile fractions.


 
Effect of extraction time
 
Using the optimized pretreatment (ultrasound) and material-to-water ratio (1:3), the kinetics of volatile fraction recovery were evaluated over a period of 4 hours. The time-dependent yield profiles are presented in Fig 3. For garlic, the yield increased progressively from 0.83% at 2 hours to a maximum of 1.00% at 3 hours. However, extending the distillation further to 4 hours resulted in a notable decline in oil recovery to 0.79%. A similar trend was observed for chives, where the yield peaked at 1.34% after 3 hours (up from 1.07% at 2 hours) before slightly decreasing to 1.28% at the 4-hour mark. These results indicate that 3 hours is the optimal duration for the hydrodistillation of these Allium species.

Fig 3: Effect of time extraction on the yield of garlic and chives volatile fractions.


 
Effect of time on chemical compositions
 
Chemometric analysis confirmed that volatile profiles were driven primarily by species differentiation rather than extraction duration. As shown in the PCA (Fig 5), species formed distinct clusters along PC1 (58.3%), with garlic occupying the positive quadrant and chives the negative quadrant. This separation was corroborated by heatmap data (Fig 6), which contrasted the allyl-sulfide dominance in garlic against the propyl- and methyl-rich signatures of chives. Kinetically, sulfur distribution followed a non-linear trajectory (Fig 4), with bioactive diallyl sulfides peaking at 3 hours across both species; specifically, garlic reached a maximum concentration of >25% before declining. Extending the process to 4 hours triggered a qualitative shift, marked by the conversion of acyclic sulfides to vinyldithiins in garlic and the near depletion of diallyl sulfides in chives (sample 4HC).

Fig 4: Effect of extraction time on the major sulfur-containing volatile groups of garlic (A) and chives (B)**.



Fig 5: PCA score plot of volatile profiles in garlic (G) and chives (C) across different extraction times (2, 3 and 4 hours)**.



Fig 6: Heatmap visualization of volatile compounds in garlic and chives**.


 
Antioxidant and antimicrobial activity of optimized volatile fractions
 
The biological assays revealed a significant contrast in the antimicrobial efficacy of the two Allium volatile profiles. To validate the assay reliability, standard controls were evaluated: the positive control (10% Tetracycline) exhibited strong inhibition against E. coli (25.5±0.35 mm) and S. aureus (29.5±0.25 mm), while the negative control (n-hexane) showed no inhibition (NI). Furthermore, the solvent control (5% DMSO) exhibited no bacteriostatic effect (MIC > 2000 µg/mL), confirming that the observed antimicrobial activities were exclusively attributed to the volatile profiles (Table 1).

Table 1: Antioxidant and antimicrobial activity of garlic and chives optimized volatile fractions.


       
The biological assays revealed a significant contrast in the antimicrobial efficacy of the two Allium volatile fractions. Garlic volatile fraction demonstrated potent, broad-spectrum antibacterial activity against both E. coli and S. aureus, with inhibition zones of 21.0±0.28 mm and 21.0±0.32 mm, respectively. This high potency was quantitatively confirmed by the MIC values, which were remarkably low at 12.5 µg/mL for both bacterial strains. In comparison, the chives extracts exhibited significantly weaker inhibitory effects, with zone diameters limited to 9.0±0.34 mm (E. coli) and 9.0±0.51 mm (S. aureus). Correspondingly, the MIC values for chives reached 250 µg/mL, representing a 20-fold difference in the concentration required to achieve bacteriostasis compared to garlic.
       
Regarding antioxidant potential, however, both species showed strong activity. The DPPH assay revealed that garlic volatile fraction possessed high antioxidant potential with an IC50 value of 26.57 µg/mL, closely approaching the efficacy of the positive control, vitamin C (23.95 µg/mL). In contrast, chives volatile fraction exhibited moderate activity, with an IC50 of 35.28 µg/mL compared to its respective vitamin C standard (24.95 µg/mL).
 
Effects of extraction parameters on yield
 
Based on these findings, the ultrasound-assisted method was the most effective technique among those tested, resulting in a significantly higher yield compared to conventional grinding. This enhancement can be primarily attributed to the acoustic cavitation phenomenon induced by ultrasonic waves (Sindumathi et al., 2025). The collapse of cavitation bubbles in the vicinity of the plant matrix generates micro-jets and shock waves, which effectively disrupt the rigid cell walls of the bulbs, thereby facilitating the release of intracellular volatile fractions glands into the solvent (Panda and Manickam, 2019). Although microwaves also improved yields significantly through rapid internal heating and electroporation-like mechanisms (Hu et al., 2021; Thanh et al., 2025), it was generally less effective than sonication in this study. Kimbaris et al. (2006) demonstrated that ultrasound-assisted extraction outperforms traditional thermal procedures by significantly mitigating the damage to highly reactive sulfur molecules and maintaining the stability of acyclic compounds of Allium sativum (Kimbaris et al., 2006). Besides, while Putra et al., (2019) reported higher yields using combined microwave-ultrasound extraction, our results suggest that sonication alone provided adequate extraction efficiency for the Allium bulbs used in this study. The additional thermal input from microwaves did not yield proportional benefits, possibly due to the specific structural characteristics of the plant matrix and the risk of degrading thermolabile organosulfur compounds.
       
The material-to-water ratio had a significant effect on the volatile fractions yield of both garlic and chives. At the lower ratio (1:2), the solvent volume appeared insufficient to fully immerse the plant material, leading to a higher viscosity of the slurry which hinders the convective mass transfer and the diffusion of volatile compounds from the internal matrix to the surface (Mandal et al., 2007; Milojević et al., 2008). Conversely, an excessive volume of water (1:4) not only requires higher energy input to reach the boiling point but may also promote the hydrolysis of susceptible thiosulfinates or facilitate the solubilization of polar sulfur-containing derivatives into the hydrosol phase, thereby reducing the volume of the separated volatile fraction (Olascuaga-Castillo et al., 2024). Therefore, the 1:3 ratio was established as the optimal parameter for the subsequent study.
 
Temporal evolution of volatile profiles
 
The initial increase in yield up to 3 hours corresponds to the time required for the steam to penetrate the plant matrix and diffuse the volatile fractions from the internal glands to the surface (the diffusion-controlled phase). The subsequent decrease observed at 4 hours, particularly evident in garlic, suggests that the process is not merely reaching equilibrium but may be suffering from thermal degradation or volatilization losses (Zhou et al., 2025). Prolonged exposure to high temperatures can lead to the decomposition of thermolabile sulfur compounds or their conversion into water-soluble derivatives that are lost in the hydrosol, thereby reducing the final volume of the hydrophobic volatile fraction (Mungwari et al., 2025). Consequently, a 3-hour extraction time was deemed sufficient to maximize yield while minimizing potential thermal damage.
       
The distinct volatile profiles observed were primarily attributed to species-specific biosynthetic precursors. The dominance of allyl derivatives in garlic confirmed the enzymatic breakdown of alliin (Yamaguchi and Kumagai, 2019), whereas the methyl- and propyl-rich composition of chives aligned with the high levels of isoallin and methiin in the tissue matrix (Yamazaki et al., 2011). Regarding kinetics, extraction efficiency peaked at 3 hours, a phase that was governed by diffusion mechanisms and cellular rupture  (Milojević et al., 2008). Extending the process beyond this window shifted the kinetics toward degradation; notably, the rise in vinyldithiins at 4 hours suggested that prolonged heating triggered the rearrangement of unstable acyclic thiosulfinates into heterocyclic artifacts. This chemical transformation aligns with the observed decrease in total physical yield at 4 hours. Consequently, the 3-hour duration represented the optimal condition for maximizing bioactive recovery while minimizing thermal artifacts.

Linking chemical composition to biological activity
 
The stronger antimicrobial activity antimicrobial footprint of garlic volatile fraction (MIC: 12.5 µg/mL) is directly linked to the dominance of diallyl sulfides and thiosulfinates identified in its volatile profile. Mechanism-wise, these allyl-derivatives, particularly allicin, possess a highly reactive thiosulfinate group capable of penetrating bacterial membranes and irreversibly reacting with the thiol groups of essential enzymes (Borlinghaus et al., 2021). A strong correlation was observed between the concentration of major allyl sulfides (specifically DADS) and the antibacterial efficacy. Furthermore, the highest concentration of DADS, observed at the 3-hour mark, coincided with the strongest antimicrobial activity. Garlic volatile fraction, which possessed the higher content of DADS, compared with chives volatile fraction, exhibited significantly stronger inhibition zones and lower MIC values compared to chives volatile fraction (Fig 4). This suggests that DADS is a primary bioactive compound contributing to bactericidal activity, consistent with previous studies (Jin et al., 2021) and supports the selection of the 3-hour extraction time to limit the thermal degradation of this active component.
       
Interestingly, the radical scavenging data (DPPH assay) did not mirror the sharp contrast seen in antimicrobial testing, both volatile fractions exhibited nearly identical antioxidant profiles. This decoupling suggests that while antimicrobial activity is highly specific to the allyl-structure (found in garlic), antioxidant capacity is likely a collective property of organosulfur compounds regardless of their specific substituents. Both the diallyl polysulfides in garlic and the dipropyl polysulfides in chives appear to act as effective hydrogen donors or electron scavengers, successfully interrupting free radical chains (Osipova et al., 2021).

CONCLUSION

This study demonstrates that hydrodistillation with ultrasound-assisted pretreatment is a  is a highly effective extraction method, achieving optimal recovery at a 1:3 material-to-water ratio over 3 hours. A clear distinction in extraction dynamics was observed: while the composition of the garlic volatile fraction remained relatively stable over the tested extraction periods, the chives fraction exhibited significant time sensitivity. This variation is likely driven by the transient nature of mixed allyl-propyl sulfides, highlighting the need for precise temporal control during extraction. Exhibiting strong antioxidant capacity and a low MIC (12.5 µg/mL) against the tested strains, the garlic volatile fraction shows promising potential for use in natural preservative formulations. However, this study has certain limitations, including the use of a single-factor experimental design and evaluation against a limited panel of microorganisms. Future research should incorporate multivariate approaches to investigate parameter interactions, expand the microbial testing spectrum and conduct comprehensive long-term storage stability analyses to fully validate the industrial viability of these volatile fractions.

ACKNOWLEDGEMENT

The present study was supported by the subject of the Code B2024-DHH-08, Ministry of Education and Training, Vietnam.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Optimization of Extraction Process and Effect of Time on Chemical Composition and Antimicrobial Activity of Allium sativum (Garlic) and Allium schoenoprasum (Chives) Volatile Fractions

    V
    Vo Thi Thu Hang1,2
    0009-0009-0798-7841
    N
    Nguyen Thi Van Anh1,*
    0000-0003-2242-8094
    1Faculty of Engineering and Food Technology, University of Agriculture and Forestry, Hue University, 102 Phung Hung Street, Hue 49000, Vietnam.
    2Department of Food Biotechnologies and Agricultural Product Quality, Faculty of Agriculture and Technology, University of South Bohemia in České Budějovice, Studentska 1668, 37005 České Budějovice, Czech Republic.

    ABSTRACT

    Background: This study optimized the hydrodistillation kinetics of Allium sativum (garlic) and Allium schoenoprasum (chives) bulbs from North and Central Vietnam and evaluated their bioactivity.

    Methods: Extraction parameters were investigated by varying pretreatment methods, material-to-water ratios and distillation time. The chemical compositions of the obtained volatile fractions were analyzed via GC-MS, while biological activities were assessed through antioxidant assays and antibacterial testing against Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 12943 using inhibition zone diameters and MIC values.

    Result: Ultrasound-assisted pretreatment yielded the best results (p≤0.05) at a 1:3 material-to-water ratio over 3 hours (1.00% for garlic; 1.34% for chives). Chemically, garlic volatile fraction was stable with diallyl disulfide as the major component, whereas chives volatile fraction showed a transient accumulation of allyl propyl sulfide at 3 hours. Biologically, garlic volatile fraction displayed high antioxidant capacity comparable to vitamin C and potent antibacterial activity against Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 12943 (Inhibition zone: 21 mm; MIC: 12.5 µg/mL). In contrast, chives volatile fraction showed significantly lower efficacy (Inhibition zone: 9.0 mm; MIC: 250 µg/mL).

    INTRODUCTION

    The genus Allium (Amaryllidaceae family), comprising 600-700 species, includes some of the most ancient and widely consumed cultivated plants in the world, most notably garlic (Allium sativum) and chives (Allium schoenoprasum) (Calimpang et al., 2024). Beyond their culinary ubiquity as flavoring agents, these species are renowned for their broad spectrum of biological activities, including antimicrobial, antioxidant, anti-inflammatory and anticancer properties (Danquah et al., 2022; Iwar et al., 2024). These bioactive effects are primarily attributed to their rich content of organosulfur compounds (Dai et al., 2025). Unlike typical essential oils which are predominantly terpene-based, the volatile fractions of Allium species are characterized by a complex matrix of sulfur-containing constituents such as diallyl disulfide (DADS), trisulfides and vinyldithiins. Crucially, these compounds are not inherently present in intact tissues but are formed enzymatically and thermally from non-volatile precursors like alliin upon tissue rupture and extraction processing (Edris and Fadel, 2002).
           
    Conventional extraction is often hindered by prolonged processing and thermal degradation of labile sulfur constituents (Stratakos and Koidis, 2016). To mitigate this, green intensification via ultrasound and microwave pretreatments is increasingly adopted. Mechanistically, ultrasound employs acoustic cavitation for cell disruption, whereas microwaves utilize ionic conduction and dipole rotation to induce rapid heating (Mandal et al., 2007; Tiwari, 2015; Patil et al., 2018). However, the comparative efficacy of these interventions on Allium tissues during hydrodistillation requires further investigation.
           
    Furthermore, most extraction optimization studies prioritize yield maximization, often overlooking the temporal evolution of chemical constituents. While recent literature has evaluated the chemical profiles of Allium volatile fractions, these studies predominantly rely on end-point analyses after a fixed extraction duration (Dehariya et al., 2021; Tran et al., 2024). Such single-time-point approaches often yield conflicting optimal conditions and fail to capture the dynamic kinetic transformations of the extract. This is particularly critical for Allium volatile fractions, where heat-sensitive precursors like allicin degrade rapidly into various transient polysulfides under continuous thermal stress (Zhu et al., 2025). Consequently, focusing solely on yield plateaus without tracking these time-dependent chemical shifts can lead to over-processing and the loss of potent bioactive intermediates.
           
    This study pursues two primary objectives. First, it systematically evaluates the comparative efficacy of mechanical, ultrasound-assisted and microwave-assisted pretreatments on the hydrodistillation of A. sativum and A. schoenoprasum volatile fractions. Second, it investigates how extraction duration influences not only yield but also the kinetic evolution of the chemical profile. By analyzing fractions at 2, 3 and 4-hour intervals via GC-MS, the research aims to elucidate the interplay between thermal processing time, chemical composition and biological activity against Escherichia coli and Staphylococcus aureus. Ultimately, these findings provide a scientific basis for optimizing extraction protocols to prioritize specific bioactive profiles over mere volume maximization.

    MATERIALS AND METHODS

    Plant materials
     
    Fresh garlic (from Son La) and chives (from Quang Tri) bulbs were harvested (April-August, 2024) and identified according to the Vietnamese Pharmacopoeia. The raw materials were cleaned, skinned and air-dried to moisture contents of approximately 58% and 71%, respectively. Samples were stored in sealed polyethylene bags at 4°C and utilized for extraction within two weeks.
     
    Bacterial strains
     
    Two representative pathogenic bacteria, comprising Gram-negative Escherichia coli ATCC 35218 and Gram-positive Staphylococcus aureus ATCC 12493, were used in this study.
     
    Volatile fractions extraction
     
    General hydrodistillation protocol
     
    Fresh materials (100 g) were finely crushed and thoroughly mixed with varying volumes of distilled water to achieve the specified material-to-water ratios. The mixture was then transferred to a 1000 mL round-bottom flask connected to a Clevenger-type apparatus for hydrodistillation. To prevent boil-over and ensure efficient mass transfer, the total working volume within the flask was strictly maintained at an optimal level. The flask was heated to the boiling point. The mixture of volatile fractions and water vapor was condensed and collected in a separating funnel. Finally, the volatile fractions were dried with anhydrous Na2SO4 to eliminate residual moisture and stored in sealed amber glass bottles at 5°C until analysis.

    Experimental design
     
    The optimization of the extraction process was carried out through three sequential single-factor experiments:
     
    Experiment 1: Effect of pretreatment technologies
     
    To evaluate the impact of physical intensification on extraction efficiency, mixtures underwent specific pretreatments prior to hydrodistillation. Microwave-assisted pretreatment involved irradiation at 700 W for 5 min, while ultrasound-assisted pretreatment utilized a Kingsonic bath (Model MC-040S, 300 × 240 × 150 mm) at 40 kHz for 10 min at 35°C. These experimental conditions were subsequently compared against a control group subjected solely to grinding (conventional hydrodistillation).
     
    Experiment 2: Effect of material-to-water ratio
     
    The influence of the solvent volume was investigated by varying the material-to-water ratio at three levels: 1:2, 1:3 and 1:4 (w/w). During this stage, the pretreatment method was kept constant based on the results from Experiment 1, while the extraction time was fixed at 2 hours.
     
    Experiment 3: Effect of extraction time
     
    Using the optimized pretreatment and material-to-water ratio, the effect of extraction time was investigated at 2, 3 and 4 hours. Volatile fractions were collected at these intervals to evaluate variations in both yield and chemical composition.
           
    The volatile fractions yield (%) was calculated using the following equation:

     
    Where,
    • Volatile fractions (%): Volatile fractions yield (%).
    • m volatile fractions: Mass of obtained volatile fractions (g).
    • msample: Mass of the initial raw material (g).
     
    GC-MS analysis
     
    GC-MS analysis was performed on an Agilent 7890B system coupled with a 5977B detector and a DB-1 column (30 m × 0.25 mm, 0.25 μm). A 1 µL sample was injected at 250°C (split 1:200) with helium carrier gas at 1 mL/min. The temperature program initiated at 45°C, ramped at 5°C/min to 150°C, then 40°C/min to 290°C (held 5 min). MS conditions included 70 eV ionization, source at 230°C, quadrupole at 150°C and a scan range of m/z 30-500. Data were processed using MassHunter. The volatile compounds were tentatively identified by comparing their mass spectra with the NIST 2017 library (match factor >85%). To support this tentative identification, the elution order of the detected compounds on the non-polar DB-1 column was cross-referenced with established literature data. Quantification of the constituents was performed by peak area normalization.

    Evaluation of antioxidant activity using the free radical scavenging assay (DPPH)
     
    The antioxidant activity was determined using the DPPH radical scavenging assay (Emami et al., 2007). Briefly, 0.5 mL of serially diluted volatile fraction was mixed with 2.5 mL of a 0.004% (w/v) DPPH solution prepared in ethanol. The control contained DPPH solution without volatile fraction, while pure ethanol (99.5%) served as the blank. After incubation in the dark at room temperature for 30 minutes, absorbance was measured at 517 nm against the blank. Ascorbic acid (Vitamin C, 12.5-100 μg/mL) was used as the positive reference standard to validate the assay sensitivity.
           
    The percentage of DPPH radical scavenging activity (Inhibition Capacity - IC%) was calculated using the following equation:

     
    Where,
    • AC: Absorbance of the control reaction (containing all reagents except the volatile fraction).
    • AS: Absorbance of the test sample containing the volatile fraction.
           
    The antioxidant activity was expressed as the IC50 value, defined as the concentration of the volatile fraction required to scavenge 50% of the DPPH radicals. The IC50 value was determined by plotting the inhibition percentage against the sample concentrations using linear regression analysis. To ensure reliability and method reproducibility, all DPPH assays were performed in independent triplicates (n=3) and the resulting IC50 values were expressed as the mean ± standard deviation (SD).
     
    Determination of antibacterial activity and minimum inhibitory concentration (MIC)
     
    The antibacterial efficacy of the volatile fractions was assessed against two bacteria: Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 12493.
           
    Antibacterial activity was screened via the agar well diffusion method (Balouiri et al., 2016). Briefly, 50 μL of the volatile fraction was dispensed into 6-mm wells on Mueller-Hinton agar seeded with standardized bacterial suspensions (0.5 McFarland; ~1.5×108 CFU/mL). Pure n-hexane served as the solvent control, while Tetracycline (10%) was utilized as the positive reference standard to validate the susceptibility of the tested strains. Inhibition zones were measured after incubation at 37°C for 24 hours. Subsequently, MIC were determined using broth microdilution (Moreira et al., 2007). Serial two-fold dilutions of the volatile fractions in 5% DMSO (range: 3.9-2000 µg/mL) were inoculated to a final density of 5×105 CFU/mL. After incubation at 37°C for 24 hours, the MIC was recorded as the lowest concentration inhibiting visible growth. To ensure reliability and method reproducibility, all antibacterial assays were performed in independent triplicates (n=3). The resulting inhibition zones were expressed as mean ± standard deviation (SD), whereas the MIC values were reported as the consistent values across the replicates.
     
    Statistical analysis
     
    All experiments were performed in triplicate (n = 3). Data were analyzed using IBM SPSS Statistics 25.0 and expressed as mean ± standard deviation (SD). Prior to one-way ANOVA, data normality and variance homogeneity were verified via the Shapiro-Wilk and Levene’s tests, respectively. Duncan’s multiple range test (DMRT) was selected as the post hoc test for its high sensitivity in detecting differences among multiple groups, with significance set at p≤0.05.

    RESULTS AND DISCUSSION

    Optimization of extraction conditions
     
    Effect of pretreatments
     
    Fig 1 illustrates that physical intensification significantly enhanced extraction efficiency relative to conventional methods (p≤0.05). For garlic, yields rose from a baseline of 0.29% (grinding) to 0.40% with microwave treatment, peaking at 0.67% under ultrasound. Chives exhibited a similar trend, improving from 0.34% to 0.55% and 0.59% for microwave and ultrasound-assisted pretreatments, respectively. Consequently, ultrasound was established as the optimal protocol for subsequent trials.

    Fig 1: Effect of pretreatment methods on the yield of garlic and chives volatile fractions.


     
    Effect of material-to-water ratio
     
    Following the ultrasound-assisted pretreatment, the influence of the material-to-water ratio on the volatile fraction yield was investigated. Fig 2 reveals a non-linear trend for both plant species. Garlic yield peaked at 0.83% (1:3) before declining to 0.74% (1:4). Similarly, chives yield rose from 0.59% (1:2) to 1.07% (1:3), then dropped significantly to 0.69% (1:4). These results suggest that a material-to-water ratio of 1:3 provides the ideal equilibrium for extraction efficiency.

    Fig 2: Effect of material-to-water ratio on the yield of garlic and chives volatile fractions.


     
    Effect of extraction time
     
    Using the optimized pretreatment (ultrasound) and material-to-water ratio (1:3), the kinetics of volatile fraction recovery were evaluated over a period of 4 hours. The time-dependent yield profiles are presented in Fig 3. For garlic, the yield increased progressively from 0.83% at 2 hours to a maximum of 1.00% at 3 hours. However, extending the distillation further to 4 hours resulted in a notable decline in oil recovery to 0.79%. A similar trend was observed for chives, where the yield peaked at 1.34% after 3 hours (up from 1.07% at 2 hours) before slightly decreasing to 1.28% at the 4-hour mark. These results indicate that 3 hours is the optimal duration for the hydrodistillation of these Allium species.

    Fig 3: Effect of time extraction on the yield of garlic and chives volatile fractions.


     
    Effect of time on chemical compositions
     
    Chemometric analysis confirmed that volatile profiles were driven primarily by species differentiation rather than extraction duration. As shown in the PCA (Fig 5), species formed distinct clusters along PC1 (58.3%), with garlic occupying the positive quadrant and chives the negative quadrant. This separation was corroborated by heatmap data (Fig 6), which contrasted the allyl-sulfide dominance in garlic against the propyl- and methyl-rich signatures of chives. Kinetically, sulfur distribution followed a non-linear trajectory (Fig 4), with bioactive diallyl sulfides peaking at 3 hours across both species; specifically, garlic reached a maximum concentration of >25% before declining. Extending the process to 4 hours triggered a qualitative shift, marked by the conversion of acyclic sulfides to vinyldithiins in garlic and the near depletion of diallyl sulfides in chives (sample 4HC).

    Fig 4: Effect of extraction time on the major sulfur-containing volatile groups of garlic (A) and chives (B)**.



    Fig 5: PCA score plot of volatile profiles in garlic (G) and chives (C) across different extraction times (2, 3 and 4 hours)**.



    Fig 6: Heatmap visualization of volatile compounds in garlic and chives**.


     
    Antioxidant and antimicrobial activity of optimized volatile fractions
     
    The biological assays revealed a significant contrast in the antimicrobial efficacy of the two Allium volatile profiles. To validate the assay reliability, standard controls were evaluated: the positive control (10% Tetracycline) exhibited strong inhibition against E. coli (25.5±0.35 mm) and S. aureus (29.5±0.25 mm), while the negative control (n-hexane) showed no inhibition (NI). Furthermore, the solvent control (5% DMSO) exhibited no bacteriostatic effect (MIC > 2000 µg/mL), confirming that the observed antimicrobial activities were exclusively attributed to the volatile profiles (Table 1).

    Table 1: Antioxidant and antimicrobial activity of garlic and chives optimized volatile fractions.


           
    The biological assays revealed a significant contrast in the antimicrobial efficacy of the two Allium volatile fractions. Garlic volatile fraction demonstrated potent, broad-spectrum antibacterial activity against both E. coli and S. aureus, with inhibition zones of 21.0±0.28 mm and 21.0±0.32 mm, respectively. This high potency was quantitatively confirmed by the MIC values, which were remarkably low at 12.5 µg/mL for both bacterial strains. In comparison, the chives extracts exhibited significantly weaker inhibitory effects, with zone diameters limited to 9.0±0.34 mm (E. coli) and 9.0±0.51 mm (S. aureus). Correspondingly, the MIC values for chives reached 250 µg/mL, representing a 20-fold difference in the concentration required to achieve bacteriostasis compared to garlic.
           
    Regarding antioxidant potential, however, both species showed strong activity. The DPPH assay revealed that garlic volatile fraction possessed high antioxidant potential with an IC50 value of 26.57 µg/mL, closely approaching the efficacy of the positive control, vitamin C (23.95 µg/mL). In contrast, chives volatile fraction exhibited moderate activity, with an IC50 of 35.28 µg/mL compared to its respective vitamin C standard (24.95 µg/mL).
     
    Effects of extraction parameters on yield
     
    Based on these findings, the ultrasound-assisted method was the most effective technique among those tested, resulting in a significantly higher yield compared to conventional grinding. This enhancement can be primarily attributed to the acoustic cavitation phenomenon induced by ultrasonic waves (Sindumathi et al., 2025). The collapse of cavitation bubbles in the vicinity of the plant matrix generates micro-jets and shock waves, which effectively disrupt the rigid cell walls of the bulbs, thereby facilitating the release of intracellular volatile fractions glands into the solvent (Panda and Manickam, 2019). Although microwaves also improved yields significantly through rapid internal heating and electroporation-like mechanisms (Hu et al., 2021; Thanh et al., 2025), it was generally less effective than sonication in this study. Kimbaris et al. (2006) demonstrated that ultrasound-assisted extraction outperforms traditional thermal procedures by significantly mitigating the damage to highly reactive sulfur molecules and maintaining the stability of acyclic compounds of Allium sativum (Kimbaris et al., 2006). Besides, while Putra et al., (2019) reported higher yields using combined microwave-ultrasound extraction, our results suggest that sonication alone provided adequate extraction efficiency for the Allium bulbs used in this study. The additional thermal input from microwaves did not yield proportional benefits, possibly due to the specific structural characteristics of the plant matrix and the risk of degrading thermolabile organosulfur compounds.
           
    The material-to-water ratio had a significant effect on the volatile fractions yield of both garlic and chives. At the lower ratio (1:2), the solvent volume appeared insufficient to fully immerse the plant material, leading to a higher viscosity of the slurry which hinders the convective mass transfer and the diffusion of volatile compounds from the internal matrix to the surface (Mandal et al., 2007; Milojević et al., 2008). Conversely, an excessive volume of water (1:4) not only requires higher energy input to reach the boiling point but may also promote the hydrolysis of susceptible thiosulfinates or facilitate the solubilization of polar sulfur-containing derivatives into the hydrosol phase, thereby reducing the volume of the separated volatile fraction (Olascuaga-Castillo et al., 2024). Therefore, the 1:3 ratio was established as the optimal parameter for the subsequent study.
     
    Temporal evolution of volatile profiles
     
    The initial increase in yield up to 3 hours corresponds to the time required for the steam to penetrate the plant matrix and diffuse the volatile fractions from the internal glands to the surface (the diffusion-controlled phase). The subsequent decrease observed at 4 hours, particularly evident in garlic, suggests that the process is not merely reaching equilibrium but may be suffering from thermal degradation or volatilization losses (Zhou et al., 2025). Prolonged exposure to high temperatures can lead to the decomposition of thermolabile sulfur compounds or their conversion into water-soluble derivatives that are lost in the hydrosol, thereby reducing the final volume of the hydrophobic volatile fraction (Mungwari et al., 2025). Consequently, a 3-hour extraction time was deemed sufficient to maximize yield while minimizing potential thermal damage.
           
    The distinct volatile profiles observed were primarily attributed to species-specific biosynthetic precursors. The dominance of allyl derivatives in garlic confirmed the enzymatic breakdown of alliin (Yamaguchi and Kumagai, 2019), whereas the methyl- and propyl-rich composition of chives aligned with the high levels of isoallin and methiin in the tissue matrix (Yamazaki et al., 2011). Regarding kinetics, extraction efficiency peaked at 3 hours, a phase that was governed by diffusion mechanisms and cellular rupture  (Milojević et al., 2008). Extending the process beyond this window shifted the kinetics toward degradation; notably, the rise in vinyldithiins at 4 hours suggested that prolonged heating triggered the rearrangement of unstable acyclic thiosulfinates into heterocyclic artifacts. This chemical transformation aligns with the observed decrease in total physical yield at 4 hours. Consequently, the 3-hour duration represented the optimal condition for maximizing bioactive recovery while minimizing thermal artifacts.

    Linking chemical composition to biological activity
     
    The stronger antimicrobial activity antimicrobial footprint of garlic volatile fraction (MIC: 12.5 µg/mL) is directly linked to the dominance of diallyl sulfides and thiosulfinates identified in its volatile profile. Mechanism-wise, these allyl-derivatives, particularly allicin, possess a highly reactive thiosulfinate group capable of penetrating bacterial membranes and irreversibly reacting with the thiol groups of essential enzymes (Borlinghaus et al., 2021). A strong correlation was observed between the concentration of major allyl sulfides (specifically DADS) and the antibacterial efficacy. Furthermore, the highest concentration of DADS, observed at the 3-hour mark, coincided with the strongest antimicrobial activity. Garlic volatile fraction, which possessed the higher content of DADS, compared with chives volatile fraction, exhibited significantly stronger inhibition zones and lower MIC values compared to chives volatile fraction (Fig 4). This suggests that DADS is a primary bioactive compound contributing to bactericidal activity, consistent with previous studies (Jin et al., 2021) and supports the selection of the 3-hour extraction time to limit the thermal degradation of this active component.
           
    Interestingly, the radical scavenging data (DPPH assay) did not mirror the sharp contrast seen in antimicrobial testing, both volatile fractions exhibited nearly identical antioxidant profiles. This decoupling suggests that while antimicrobial activity is highly specific to the allyl-structure (found in garlic), antioxidant capacity is likely a collective property of organosulfur compounds regardless of their specific substituents. Both the diallyl polysulfides in garlic and the dipropyl polysulfides in chives appear to act as effective hydrogen donors or electron scavengers, successfully interrupting free radical chains (Osipova et al., 2021).

    CONCLUSION

    This study demonstrates that hydrodistillation with ultrasound-assisted pretreatment is a  is a highly effective extraction method, achieving optimal recovery at a 1:3 material-to-water ratio over 3 hours. A clear distinction in extraction dynamics was observed: while the composition of the garlic volatile fraction remained relatively stable over the tested extraction periods, the chives fraction exhibited significant time sensitivity. This variation is likely driven by the transient nature of mixed allyl-propyl sulfides, highlighting the need for precise temporal control during extraction. Exhibiting strong antioxidant capacity and a low MIC (12.5 µg/mL) against the tested strains, the garlic volatile fraction shows promising potential for use in natural preservative formulations. However, this study has certain limitations, including the use of a single-factor experimental design and evaluation against a limited panel of microorganisms. Future research should incorporate multivariate approaches to investigate parameter interactions, expand the microbial testing spectrum and conduct comprehensive long-term storage stability analyses to fully validate the industrial viability of these volatile fractions.

    ACKNOWLEDGEMENT

    The present study was supported by the subject of the Code B2024-DHH-08, Ministry of Education and Training, Vietnam.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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