Optimization of extracted essential oil from clove buds (Syzygium aromaticum) using microwave-assisted hydro distillation

Introduction

Zanzibar clove buds (Syzygium aromaticum L., family Myrtaceae) represent the finest quality clove source globally, contributing approximately 80% of world production and generating significant economic value for the island nation (Cortés-Rojas et al., 2014). The essential oil of Zanzibar clove is highly valued in pharmaceutical, cosmetic, food, and flavor industries due to its superior chemical composition, particularly high eugenol content (82-89%) and documented antimicrobial, antioxidant, and analgesic properties (Kamatou et al., 2012; Radünz et al., 2019) [1,2,3] . Traditional extraction methods such as steam distillation and hydro distillation require prolonged extraction times (2-4 hours) and consume significant thermal energy, resulting in relatively low oil yields (12-14%) and potential loss of heat-sensitive bioactive compounds (Einfalt, 2020). Microwave-assisted hydro distillation (MAH-D) represents an innovative technology combining selective microwave heating with water distillation, offering potential advantages in extraction speed, efficiency, and product quality (Rutkowska et al., 2017). The selective absorption of microwave energy by polar molecules within plant tissues generates rapid internal heating, facilitating cell disruption and enhanced mass transfer kinetics (Gomez et al., 2020) [4,5,6] . Systematic optimization of extraction parameters is essential to identify conditions that maximize oil yield while maintaining superior product quality. This study employs a structured three-level factorial experimental design to rigorously investigate the effects of microwave power, extraction duration, and sample ratio on essential oil yield and physicochemical properties of Zanzibar clove buds. Understanding the relationships between extraction parameters and oil quality indicators will facilitate implementation of this technology at industrial scale.

Materials and Methods

Raw Material and Moisture Content

Dried Zanzibar clove buds (Syzygium aromaticum L.) were procured from Kerala. The cloves were ground using the grinder machine available in the Department of Processing and Food Engineering. The ground samples were then packed in zip-lock pouches and stored for further analysis. Moisture content of raw clove buds was determined by standard oven drying method according to. Approximately 5.0 g of ground clove sample was placed in a pre-weighed aluminium dish and dried in a hot air oven at 105°C±2°C until constant weight was achieved. The dish was cooled in a desiccator and weighed. Moisture content was calculated as follows (Najafian, 2014):

Where,  W₁ = initial weight of fresh sample (g),  W₂ = final weight of dried sample (g)  Moisture content of the raw material was determined to be 8.2±0.3% (w/w). All extraction experiments were conducted using this standardized moisture content clove material.

Physical and Frictional Properties of clove buds

The frictional and particle characterization properties of whole clove buds were evaluated to determine their packing behaviour, density characteristics, porosity, and flow properties. All determinations were performed in triplicate and expressed as mean±standard deviation.

Bulk Density

Bulk density was determined by gently filling a graduated measuring cylinder with a known weight of whole clove buds without applying external compaction. The bulk volume occupied by the sample was recorded, and the bulk density was calculated using the following equation:

Where,  𝜌 𝑏 = Bulk density (g/cm³)  𝑀= Mass of sample (g)  𝑉 𝑏 = Bulk volume (cm³)

True Density

True density was determined using the liquid displacement method, where the actual volume occupied by the whole clove buds excluding void spaces was measured. The true density was calculated using the equation:

Angle of Internal Friction:

The internal friction angle of whole clove buds was determined using a shear testing method by measuring the resistance between particles under applied stress conditions. The internal friction coefficient was expressed as:

Where,  𝜇 𝑖 = Coefficient of internal friction  𝜙 𝑖 = Internal friction angle (°)

Angle of External Friction

External friction was evaluated by measuring the resistance between whole clove buds and the contact surface material. The coefficient of external friction was calculated using the equation:

Where,  𝜇 𝑒 = Coefficient of external friction  𝜙 𝑒 = External friction angle (°)  The obtained parameters were used to assess the handling, storage, flowability, and processing characteristics of whole clove buds.

Physicochemical properties of extracted essential oil 2.3.1 Oil Yield:

The essential oil from clove buds was extracted by hydro distillation using a Clevenger-type apparatus. A known quantity of dried clove buds was subjected to distillation for a fixed duration, and the extracted oil was separated and measured volumetrically. The percentage oil yield was calculated based on the dry weight of the plant material using the following equation (Singh et al., 2007) [17] :

Where,  Volume of extracted oil = volume of oil obtained after extraction (mL)  Weight of dried clove buds = weight of sample used for extraction (g)

Specific Gravity

Specific gravity of the extracted clove bud essential oil was determined at 20±0.1 °C using a digital densitometer. The measurement was based on the ratio of oil density to the density of distilled water at the same temperature (Burt, 2004) [12] .

SG = ρ oil ρ water

Where,  SG= specific gravity  ρ oil = density of oil at 20 °C (g/cm³)  ρ water = density of water at 20 °C (0.998 g/cm³)

Refractive Index:

The refractive index of clove bud essential oil was measured at 20 °C using a hand-held refractometer. Prior to analysis, the refractometer was calibrated with distilled water ( n D 20 = 1.3330 ). A few drops of oil sample were placed on the prism surface and the refractive index was recorded directly from the instrument scale (Jirovetz et al., 2006) [13] .

Saponification Value:

The saponification value of clove bud essential oil was determined by refluxing a known quantity of oil sample with excess alcoholic potassium hydroxide solution followed by titration of the remaining alkali with standardized hydrochloric acid using phenolphthalein as an indicator (Adejumo et al., 2013) [14] .

Where,  SV= saponification value (mg KOH/g)  V 0 = volume of HCl used for blank (mL) [15] .

Iodine Value

The iodine value of clove bud essential oil was determined using the Wijs method. The oil sample was treated with Wijs solution under dark conditions followed by titration with standardized sodium thiosulfate solution using starch as an indicator (Chaieb et al., 2007) [16] .

Where,  IV= iodine value (g/100 g)  V 0 = volume of sodium thiosulfate used for blank (mL)  V 1 = volume of sodium thiosulfate used for sample (mL)  N= normality of sodium thiosulfate solution  W= weight of oil sample (g)

Experimental Design

A three-level factorial experimental design was employed with three independent variables: microwave power, extraction time, and sample ratio. Each variable was set at three levels, resulting in 27 unique treatment combinations. Each treatment was replicated 5 times (n=135 total observations) to provide statistical power for estimation of main effects and two-factor interactions. The experimental design is presented in Table 1. per treatment *Three-level design with three independent variables results in 27 treatment combinations. Each treatment was replicated 5 times for a total of 135 observations 2.5 Statistical Analysis: All experimental data were analysed using Microsoft Excel and expressed as mean±standard deviation (SD) from triplicate determinations ( 𝑛 = 5 ). Statistical significance between the obtained values was evaluated using p-values, and differences were considered statistically significant at 𝑝 < 0.05. The calculated mean and standard deviation values were used to assess the variability and reproducibility of the experimental results.

Results and Discussion

Physical and Frictional Properties of clove buds

Effect of Extraction Time on Oil Yield

Table 3 presents the effect of extraction time on mean oil yield, averaged across all power and ratio combinations.

Extending extraction from 10 to 20 minutes resulted in a 20.0% increase in oil yield (10.93% to 13.12%). [7] .

Effect of Sample Ratio on Oil Yield

Table 4 presents the effect of sample ratio (clove mass per unit water volume) on mean oil yield. Increasing the sample ratio from 0.5:1 to 0.7:1 g:ml resulted in a 12.1% increase in oil yield (11.27% to 12.62%). Sample ratio (mass of plant material per unit volume of solvent) affects extraction through its influence on the concentration gradient driving mass transfer. At 0.5:1 ratio (50 g clove per 100 mL water), abundant solvent relative to sample mass provides high solvent capacity but lower local substrate concentration. At 0.7:1 ratio (70 g clove per 100 mL water), increased substrate concentration enhances the driving force for oil diffusion into the extraction medium. However, further increases in sample ratio beyond 0. would likely result in diminished returns due to saturation of solvent capacity and reduced circulation within the extraction vessel. The modest effect range for sample ratio (1.35% increase from 0.5:1 to 0.7:1) indicates that this parameter has secondary importance compared to power and time (Aspé & Fernández, 2011) [8] .

Interaction Effects: Power × Time

Table 5 presents the two-way interaction between microwave power and extraction time, showing mean oil yield values for all nine combinations of these two factors (averaged across the three sample ratio levels and 5 replicates). [9] .

Interaction Effects: Power × Sample Ratio

Table 6 presents the two-way interaction between microwave power and sample ratio, with mean oil yield values for all nine combinations of these factors. The Power × Ratio interaction demonstrates that microwave power shows a consistent positive effect on oil yield across all sample ratio levels, with power increases from 250 W to 450 W providing 37.4%, 38.9%, and 30.1% improvements at 0.5:1, 0.6:1, and 0.7:1 ratios, respectively. At lower power (250 W), sample ratio has a modest effect (11.3% improvement from 0.5:1 to 0.7:1). At 350 W, the ratio effect is similar (8.1% improvement). At 450 W, an interesting plateau occurs where 0.6:1 and 0.7:1 ratio shows nearly equivalent yields (13.37%), suggesting that at high microwave power, increasing sample mass beyond 60 g per 100 mL water provides minimal additional benefit. This interaction indicates that the optimization should favour 450 W power combined with either 0.6:1 or 0.7:1 sample ratio for practical considerations (Kutner & Nachtsheim, 2005) [10] .

Physicochemical properties of extracted clove essential oil

Table 7 presents the physicochemical quality parameters of essential oil extracted at optimal conditions (450 W, 20 minutes, 0.7:1 sample ratio). All parameters conform to international pharmacopeial standards. [11] .

Conclusion

This comprehensive three-level factorial experimental design study successfully optimized microwave-assisted hydrodistillation for essential oil extraction from Zanzibar clove buds. Systematic evaluation of microwave power, extraction time, and sample ratio with appropriate replication (n=135 total observations) revealed that microwave power is the dominant factor influencing oil yield, followed by extraction time and sample ratio. Optimal extraction conditions of 450 W microwave power, 20 minutes extraction time, and 0.7:1 sample ratio (70 g clove per 100 mL water) yielded 14.8±0.6% essential oil with excellent physicochemical properties meeting international pharmacopeial standards. Significant interactions between power and time, and between power and ratio, indicate that optimal parameter selection requires consideration of multiple variables simultaneously. The extracted oil demonstrated specific gravity of 1.048±0.008 g/cm³, refractive index of 1.531±0.003, saponification value of 187±4 mg KOH/g, iodine value of 96±3 g/100g, and acid value of 1.2±0.2 mg KOH/g. Particle characterization of residual material showed bulk density of 0.654±0.028 g/cm³, porosity of 42.0±2.1%, and angle of repose of 38.4±1.2°. These results provide a quantitative foundation for implementing microwave-assisted hydrodistillation at industrial scale for Zanzibar clove essential oil production.

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