As complex matrices comprising hundreds of diverse compounds, essential oils necessitate meticulous analysis to determine their volatile profiles. These profiles are crucial for understanding the therapeutic and aromatic properties of medicinal plants like L. angustifolia, as their chemical composition can be influenced by various factors, including season, the plant’s developmental stage, climate conditions, harvest time, extraction parameters, and extraction techniques6.In this study, we employed two distinct extraction methods, TDH and AH, to investigate the volatile profile of L. angustifolia essential oil. The results in Table 1 reveal the presence of 50 different volatile compounds. While thirty-five of these compounds were detected in both extractions, 15 compounds exhibited method-specific detection. Specifically, eight compounds, including 1-borneol (2.11%), 2-cyclohexen-1-one (0.35%), 2-methyl-3-phenylpropanal (0.45%), 4-carvomenthenol (4.97%), α-bisabolol (0.69%), β-myrcene (0.21%), β-ocimene (0.11%), and hexyl isovalerate (0.24%), were exclusively identified in the MAH extract. Conversely, seven compounds, namely 1-hexanol (0.87%), α-pinene (0.88%), β-pinene (0.16%), cyclooctane (0.07%), levomenol (0.80%), geraniol (0.43%), and hexyl valerate (0.24%), were unique to the TDH extraction.Table 1 Composition of L. angustifolia essential oil.Despite these differences, both TDH and MAH yielded essential oil with α-terpinolene (25.60% and 24.25%, respectively) and (–)-borneol (19.55% and 19.37%, respectively) as the predominant volatile constituents (Fig. 2). This consistency in major compound extraction suggest that both methods are effective in capturing the characteristic aroma and therapeutic potential of L. angustifolia, even though they may differ in their efficiency for extracting minor or trace components. These findings align with previous studies, such as that by Hajhashemi et al.31, which identified camphor (9.5%), borneol (11.5%) and 1,8-cineole (65.4%) as major volatiles in lavender leaves. However, some variations were observed, such as the detection of α-terpineol (0.5%) in their study but not in ours, emphasizing the potential influence of methodological or sample-specific factors on essential oil composition.Fig. 2Molecular structure of (A) α-terpinolene and borneol (B).Essential oil extraction from medicinal plants, such as lavender, inevitably generates substantial by-products, including wastewater. However, previous research has revealed that the organic components of this wastewater possess diverse bioactivities, including anti-inflammatory, antibacterial, antioxidant, antifungal, and enzymatic activities32. This study compared the chemical profile of essential oil and wastewater obtained from L. angustifolia using different extraction methods (TDH and MAH). Table 2 details the phytochemical profile of the wastewater extract, with the MAH method yielding higher total content than the TDH method. According to the results, the total phenolic and total flavonoid content of MAH extraction were significantly higher than that of TDH extraction. The higher phenolic and flavonoid content in MAH extracts compared to TDH can be attributed to enhanced cell disruption, reduced thermal degradation, increased mass transfer, and selective heating mechanisms facilitated by microwave energy, leading to more efficient extraction of these bioactive compounds33,34,35,36. DPPH, CUPRAC, and ABTS assays evaluated the wastewater’s antioxidant activity. Results revealed significant differences in antioxidant activity between the MAH (7.619 ± 0.83 mg TEAC/g, 7.847 ± 0.59 and 7.790 ± 0.80 mg TE/g) and TDH (5.603 ± 0.58 mg TEAC/g, 3.869 ± 0.17 and 4.291 ± 0.43 mg TE/g) methods. Furthermore, the MAH method also resulted in the highest total proanthocyanidins (0.548 ± 0.03 mg CAE/g) and TacC (0.023 ± 0.002 mg C3-G/g). Our results highlight that the choice of extraction method significantly impacts the essential oil yield but also the bioactive composition of the resulting wastewater. Microwave-assisted hydrodistillation (MAH) consistently outperformed traditional hydrodistillation (TDH) in extracting a more comprehensive array of bioactive compounds and producing wastewater with superior antioxidant activity. This suggests that MAH, as an innovative extraction technique, offers a more efficient and comprehensive approach to essential oil isolation, yielding a richer source of valuable compounds in both the oil and its by-product. Beyond its efficacy in extracting bioactive substances, MAH also presents several operational advantages over TDH, including reduced extraction time, minimized solvent usage, and lower energy consumption. These findings underscore the potential of MAH as a sustainable and economically viable alternative to conventional methods for essential oil production.Table 2. Phytochemical profile of L. angustifolia wastewater.Several studies have investigated the composition and bioactivity of lavender by-products, specifically wastewater generated during essential oil extraction. Our research builds upon these findings, providing a more comprehensive analysis of the chemical profile and antioxidant activity of L. angustifolia wastewater. Previous studies have reported lower total phenolic and flavonoid content in lavender wastewater compared to our findings. For example, Turrini et al.37 found levels of 0.75 mg GAE/g and 0.002 mg QE/g, respectively, using traditional distillation. This discrepancy may be attributed to differences in extraction methods, plant varieties, or environmental conditions. Ciocarlan et al.38 focused on the antimicrobial activity of L. angustifolia by-products, demonstrating their effectiveness against various pathogens, including Bacillus sp., B. subtilis, Xanthomonas campestris, Pseudomonas fluorescens, Candida utilis, Erwinia carotovora, E. amylovora, Penicillium chrysogenum, Aspergillus niger, Alternaria alternata, and Pseudomonas aeroginosa38. Our study builds upon previous research by Turrini et al.37 and Ciocarlan et al.38, providing a more in-depth analysis of lavender wastewater. Specifically, we assessed the total phenolic and flavonoid content and identified and quantified individual phenolic and flavonoid compounds. Furthermore, our study investigated the antioxidant activity of the wastewater, offering a more comprehensive understanding of its bioactive potential.Additionally, Méndez-Tovar et al.39 assessed the total phenolic content, antioxidant activity, and phenolic profiles of solid residues, a distinct by-product generated during essential oil extraction, from 12 different populations of L. latifolia collected across two seasons. Their findings revealed a total phenolic content of 3.54 mg GAE/g in these residues, which aligns with the range observed in our study for lavender wastewater. However, the individual phenolic levels, specifically apigenin, luteolin, and rosmarinic acid, were lower in their study (55.9, 102.6, and 3040 mg/kg, respectively) compared to our results. This discrepancy can be attributed to variations in both plant species (L. latifolia vs. L. angustifolia) and the type of by-product analyzed (solid residues vs. wastewater). Dobros et al.40 demonstrated that the total phenolic content of extracts from various L. angustifolia cultivars obtained through different extraction methods (ultrasonically assisted extraction, maceration, and decoction), ranged from 14.89 to 32.82 mg GAE/g. Similarly, they found a range of 15.55 to 30.79 mg GAE/g for extracts from two L. x intermedia cultivars using different methods. These findings highlight the substantial impact of extraction methodology on the recovery of bioactive substances from lavender, suggesting that the choice of extraction technique can significantly influence the phenolic content of the final product. A separate study analyzing inflorescence powder samples of L. x intermedia collected from 30 different populations found a range of 0.31–1.03 mg GAE/g for total phenolic content and 0.28–0.71 mg QE/g for total flavonoid content41. In comparison to our findings, the study by41 reported lower total phenolic and flavonoid content in L. x intermedia inflorescence powder extract than what we observed in the L. angustifolia wastewater by-product. In contrast, a study by42 focusing on the total phenolic content and antioxidant activity (using the DPPH method) of L. angustifolia essential oil itself reported values of 1.22 ± 0.04 mg GAE/g and 6.522 ± 0.069 mg/mL, respectively. Our study, however, found higher total phenolic content (1.7776 mg GAE/g for TDH and 3.2803 mg GAE/g for MAH) and antioxidant activity (7.619 mg TE/g) in the wastewater by-product. This highlights a key finding: the by-product generated during essential oil extraction from L. angustifolia may contain a higher concentration of phenolic compounds and exhibit stronger antioxidant activity than the essential oil. This result underscores the potential value of this by-product as a source of bioactive compounds. This observation aligns with numerous other studies demonstrating the diverse biological activities of wastewater generated from essential oil isolation of various medicinal plants. These activities include not only antioxidant properties43,44,45 but also antiacetylcholinesterase44, antifungal effects45, and antibacterial46. For instance, a study examining wastewater generated during essential oil isolation from various medicinal plants (including Dittrichia viscosa, Salvia officinalis, Origanum vulgare, Thymus camphoratus, Foeniculum vulgare, Thymbra capitata, Thymus carnosus, and Thymus mastichina) reported total phenolic content values ranging from 5.3 to 45.2 mg/mL46. This wide range demonstrates plant species’ significant impact on wastewater’s phenolic composition. In our study, this species-specific variation can be attributed to the lower total phenolic content observed in L. angustifolia wastewater (1.7776 mg GAE/g for TDH and 3.2803 mg GAE/g for MAH).This study aimed to evaluate the impact of different essential oil extraction methods (TDH and MAH) on the recovery of phenolic compounds from wastewater. Previous research suggests that the chosen distillation method can significantly affect phenolic content. Therefore, analyzing the phenolic profiles of wastewater samples obtained using these methods is crucial. In addition to standard spectrophotometric analyses, we employed LC–MS/MS to identify 32 individual phenolics in the wastewater samples. The results were further evaluated using PCA. LC–MS/MS analysis revealed that several individual phenolics, including gallocatechin + epigallocatechin, (−)-epicatechin, peonidin chloride, epicatechin gallate + gallocatechin gallate, and myricetin, were not detected in either sample. However, minor amounts of specific phenolics were found to be method-dependent. For instance, caffeic acid phenethyl ester, quercitrin, hesperidin, catechin + epicatechin, chlorogenic acid, and pinobanksin were only detected in the TDH wastewater, while quercetin, galangin, and trans-caffeic acid were exclusive to the MAH method. Notably, despite these differences in individual phenolic composition, the total phenolic content values for both methods were quite similar (489.322 µg/kg for TDH; 537.803 µg/kg for MAH).This study compared the profile of polyphenols in wastewater extracts obtained using the TDH and MAH methods. The data are presented in Table 3. Both methods identified protocatechuic acid as the major phenolic compound, with concentrations of 141.852 ± 4.868 μg/kg and 181.397 ± 28.652 μg/kg in TDH and MAH extracts, respectively. While the overall distribution of phenolic compounds was similar between the two methods, p-coumaric acid was found in significantly higher concentrations in the TDH extract. Conversely, apigenin and luteolin were present at significantly higher concentrations in the MAH extract.Table 3 Phenolic profile of L. angustifolia wastewater (µg/Kg).Phenolic compounds, as secondary metabolites in plants, possess various biological activities, making their qualitative and quantitative determination in different plant products crucial for understanding their potential health benefits. Our study revealed that L. angustifolia wastewater contains a notable diversity of individual phenolic compounds. Previous studies have reported that the L. angustifolia by-product is particularly rich in syringic acid (26.082 µg/g) and rutin (56.081 µg/g) compared to other targeted phenolics37. In our study, rutin was identified as a major phenolic compound in lavender wastewater, consistent with previous reports highlighting its abundance in L. angustifolia by-products37. While limited research specifically focuses on the phenolic content of lavender wastewater, studies on other types of lavender extracts have identified a diverse range of phenolic compounds. These include gallic acid, vitexin, luteolin 7-O-glucoside, umbelliferone, chlorogenic acid, and isoquercitroside, which have been found in extracts obtained using various extraction methods47.Additionally, flower extracts obtained through maceration have been shown to contain high amounts of gallic acid, ellagic acid, and chlorogenic acid48. Moreover, the bioactive potential of wastewater from other plant species has also been investigated. For instance, studies on wastewater from Rosa species revealed a phenolic content of 0.37 mg/mL and flavonoid content of 7.2 to 7.8 mg/mL49. This wastewater also contained various phenolic compounds, including brevifolincarboxylic acid, gallic acid, hyperoside, ellagic acid, catechin, glucogallin, epicatechin, chlorogenic acid, avicularin, miquelianin, and kaempferol, along with their derivatives. Notably, the total flavonoid content in Rosa wastewater was lower than that observed in our lavender wastewater, whereas the total phenolic content was higher. These discrepancies can be attributed to variations in plant species and the specific extraction methods employed, highlighting the complex interplay between these factors in determining the bioactive composition of wastewater.In this study, principal component analysis (PCA) was employed to analyze the phenolic profile results of L. angustifolia. PCA is a statistical technique that simplifies complex datasets by reducing the number of variables while retaining most of the original information. It achieves this by transforming the original variables into a new set of uncorrelated variables called principal components (PCs), which are linear combinations of the original variables. The eigenvalues and variance percentages of the PCA analysis are presented in Table 4 and Fig. 3. Eigenvalues indicate the amount of variance explained by each principal component, with higher values representing greater importance. Based on Kaiser’s rule, eigenvalues greater than 1.0 are considered significant and retained as principal components50. In our analysis, five principal components (PC1-PC5) met this criterion, with eigenvalues of 15.352, 7.329, 5.979, 2.925, and 1.415, respectively. Previous studies using PCA on lavender samples have reported varying numbers of principal components explaining the majority of the variance. For example, Gök and ErdoÄŸdu51 identified two principal components accounting for 98.29% of the total variance, while Komes et al.52 found that PC1 and PC2 explained 84.29% of the variance in their study of medicinal aromatic plants. These differences highlight the influence of sample composition and experimental conditions on the PCA results.Table 4 Eigenvalues and percentage of variance for phenolics of PCA analysis.Fig. 3PCA score plot based on the individual phenolic results of MAH and TDH methods.The Kaiser–Meyer–Olkin (KMO) test was employed to assess the suitability of the data for principal component analysis (PCA). The KMO statistic measures sampling adequacy, indicating the proportion of variance among variables that might be common variance. According to Kaiser50, KMO values range from 0 (unacceptable) to 1 (excellent), with values above 0.7 considered suitable for factor analysis. Based on the KMO test results and the evaluation of the phenolic profile of L. angustifolia, five principal components were identified in this study. According to the PCA results obtained in our study, PC1 consists of gallic acid, neochlorogenic acid, p-coumaric acid, trans-ferulic acid, catechin + epicatechin, hesperidin, rutin, kaempherol, cyanidin chloride, luteolin, pinobanksin, apigenin, pinocembrin, chrysin, trans-caffeic acid and artepillin C, while PC2 consists of protocatechuic acid, chlorogenic acid, peonidin chloride, quercitrin, naringenin and caffeic acid phenethyl ester are the main components. In addition, PC3 was composed of epicatechin gallate + gallocatechin gallate, gallocatechin + epigallocatechin, (−)-epicatechin, hyperoside, myricetin, t-cinnamic acid, PC4 was composed of caffeine, quercetin, galangin, and for PC5 catechol alone constituted a major component.In recent years, various tools have been developed to assess the greenness of analytical procedures28,29,30. These approaches are often general and need to pay more attention to the analytical steps responsible for a non-green analytical procedure. The evaluation criteria are based on the ten principles of green sample preparation: selection of solvents, materials, and reagents; waste generation; energy consumption; sample size; and yield. The AGREEprep pictograms in Table 5 represent different levels of environmental sustainability. The central score of the pictograms is 0.57 for the microwave-assisted method and 0.55 for traditional hydrodistillation. The BAGI tool, on the other hand, presents a definitive pictogram and score using 10 BAGI standards, indicating the utility and effectiveness of an analytical procedure. The color of an approach, as shown in Table 5, indicates its proximity to meeting the requirements. A technique is considered applicable if it scores 60 or higher. Table 5 shows the proposed pictograms for BAGI indices. The microwave method scored 70, while traditional hydrodistillation scored 65. Green analytical chemistry properties are used to assess analytical methods in ComplexGAPI. The ComplexGAPI graph has been expanded to include pre-analytical procedures by adding hexagon to the GAPI. A color scale is used in this evaluation, with red indicating a high harmful impact on the ecosystem, while yellow and green reflect moderate and low effects, respectively. As shown in Table 5, the microwave method has three red zones, 11 yellow zones, and 11 green zones; the traditional method has three red zones, 10 yellow zones, and 12 green zones.Table 5 Green assessment tool pictograms.
