Comparative Analysis of Volatile Components and Sensory Profiles of Four Basil Varieties Based on HS-SPME and SD Coupled with GC-MS

Highlights

What are the main findings?

• Forty-seven and sixty-six volatiles were found in fresh basils and EOs, respectively.
• Drying and extraction process of basils reduced ethers and altered aroma quality.

What is the implication of the main finding?

• PCA and OPLS-DA revealed differences between fresh basil and EO volatile profiles.
• Sensory analysis also showed processing affecting aromas intensity and quality.

Abstract

This study utilized gas chromatography-mass spectrometry (GC-MS) to analyze the volatile components and sensory profiles of four basil varieties, both in their fresh state and as essential oils (EOs) extracted via steam distillation (SD). By employing headspace solid-phase microextraction (HS-SPME) and SD/GC-MS, a comprehensive comparison was conducted to elucidate the changes in volatile profiles before and after drying and extraction processes. In total, 47 volatile components were identified in fresh basil samples. Methyl chavicol was predominant in Thai basil (66.53%), lemon basil (90.18%), and sweet basil (89.19%), whereas linalool (58.56%) was the major component in purple basil. For EOs, 66 volatile components were detected, with methyl chavicol remaining significant in Thai basil (65.27%) and lemon basil (81.03%), though its proportion decreased in sweet basil (29.34%). Purple basil EOs showed a higher proportion of alcohols (54.54%) and terpenoids (31.31%), with the notable presence of linalool (20.08%) and τ-juniper alcohol (18.18%). Multivariate analyses, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), revealed significant variations in volatile profiles among basil varieties. Sensory evaluation, supported by radar fingerprinting, demonstrated that the drying and extraction processes notably impacted the aroma profiles. Distinctive aroma profiles revealed that Thai basil was noted for its aniseed aroma, lemon basil for its lemon scent, sweet basil for its pungency, and purple basil for its floral notes. These findings highlight the diverse applications of basil varieties and their EOs, providing valuable insights into flavoring, fragrance, and therapeutic products based on their volatile compositions and sensory attributes.

1. Introduction

Basil (Ocimum basilicum), a member of the Lamiaceae family, is renowned for its aromatic properties and essential oils (EOs). Widely cultivated in tropical regions of Asia and Africa, basil serves both medicinal and culinary purposes [1]. Research has demonstrated that basil’s volatile compounds offer a range of benefits, including aromatic, soothing, antioxidant, anti-allergic, and antibacterial properties [2]. Consequently, basil EOs are extensively utilized in the perfume, cosmetic, food, medicinal, and healthcare industries [3]. The diverse aromatic volatile components presented in the stems, leaves, and inflorescences of various basil varieties contribute significantly to its sensory profile, enhancing its popularity and broad applicability [4].

For instance, the tender stems and leaves of Thai basil (Ocimum basilicum var. thyrsiflora) are noted for their subtle aroma and high content of methylpiperol and eugenol, which are primarily used as additives in confectionery and baked goods [5]. Lemon basil (Ocimum basilicum var. citriodorum) is recognized for its spicy scent and is traditionally employed to alleviate symptoms such as congestion, fever, headache, and chest discomfort associated with the common cold [6]. Sweet basil (Ocimum basilicum var. sweet) is predominantly utilized in culinary applications and for its dried leaves and seeds, which are used to improve vision and enhance high-quality tea blends [7]. Purple basil (Ocimum basilicum var. Purple Ruffles) is valued for its distinctive leaf color and pesticidal properties, making it a popular choice for ornamental garden planting [8]. In South and Southeast Asia, these basil varieties are often used interchangeably as both culinary ingredients and herbal remedies for respiratory and gastrointestinal disorders [9]. Given the unique aromatic profiles of different basil varieties, a systematic comparison of their aroma characteristics could provide valuable insights to aid in their diverse applications.

Additionally, significant variations in aromatic compositions and sensory characteristics are observed between fresh and dried basil. Basil EOs are typically extracted from dried plant material for preservation and use. Various extraction methods have been developed to efficiently capture these aromatic components [5]. Steam distillation (SD) is a traditional technique that utilizes volatile compounds from plants using steam, which are then condensed and collected [7]. Due to its efficiency and cost-effectiveness, SD is widely used to extract less polar and more volatile components [8]. For example, Ma et al. employed SD combined with gas chromatography–mass spectrometry (GC-MS) to study the effects of pretreatment on the chemical composition and aroma profile of EOs from dried Thai basil. Their study revealed that methyl chavicol and methyl eugenol were the predominant components, with fennel- and lemon-like notes being the primary aromas associated with Thai basil EOs obtained via SD extraction [10].

Headspace solid-phase microextraction (HS-SPME) is an advanced technique based on adsorption–desorption principles [11]. Its advantages, including solvent-free sample processing, high sensitivity, reliable qualitative and quantitative results, and enrichment capabilities, have made HS-SPME coupled with GC-MS a widely adopted method for detecting volatile and semi-volatile components in gaseous, liquid, or solid samples [12,13]. For example, Xie et al. employed HS-SPME/GC-MS to identify key volatile organic compounds in various parts of fresh and dried Perilla frutescens, resulting in the identification of 115 volatile organic compounds [14]. Tarchoune et al. used HS-SPME/GC-MS to analyze the volatile compounds emitted by living leaves of Ocimum basilicum L. cv. Genovese, comparing them with EOs obtained via hydrodistillation. Their study found that the EO was rich in linalool (45.9%), 1,8-cineole (16.7%), eugenol (10.3%), aromadendrene (4.9%), and epi-α-cadinol (4.9%), while the primary volatile in fresh basil leaves was linalool (29.8%), followed by 1,8-cineole (19.2%), trans-bergamotene (10.0%), and eugenol (7.0%) [15]. Additionally, a study identified 30 volatile compounds from the postharvest waste of Ocimum basilicum L. using HS-SPME/GC-MS, with β-linalool, methyl eugenol, methyl cinnamate, and methyl chavicol being the predominant compounds [16]. While there are existing studies that utilize LC-HRMS for the characterization of plant natural products [17], for volatile substances, especially samples obtained through headspace solid-phase microextraction (HS-SPME), GC-MS analysis is more appropriate. Moreover, the process of combining these two techniques is relatively more established.

Thai basil (O. basilicum var. thyrsiflora), lemon basil (Ocimum basilicum var. citriodorum), sweet basil (Ocimum basilicum var. sweet), and purple basil (Ocimum basilicum var. purple ruffles) are four widely cultivated basil varieties with significant regional presence. The commercial demand for aromatic plants is rapidly increasing across sectors such as perfumery, pharmaceuticals, natural cosmetics, urban landscaping, and household gardening [10]. The distinctive and unique flavor of basil results from the complex interplay of numerous compounds present in specific ratios and proportions. Variations in basil types and processing methods can significantly impact the composition of volatile compounds and, consequently, the flavor profile [14,18]. Despite growing interest, comprehensive studies comparing the volatile fractions of these four basil varieties remain limited. Only one recent study has investigated the chemical composition and potential health benefits of six samples from five different basil cultivars [14]. Therefore, a comparative analysis of the volatile components and sensory profiles between fresh leaves and EOs of these four basil varieties is a valuable and underexplored area of research.

In this study, HS-SPME and SD coupled with GC-MS were employed to analyze the primary volatile profiles of fresh basil and EOs extracted from the dried material of four typical basil varieties. The collected data were processed and analyzed using both supervised and unsupervised multivariate statistical methods. Principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and variable importance for the projection (VIP) were utilized to identify distinct volatile compound fingerprints that characterize each basil variety. Subsequently, the impact of these differential components on the characteristic aroma of each basil type was further explored through sensory evaluation and represented by radar fingerprints. This comprehensive study on the differentiation of volatile components and aroma-related indices among various basil varieties, both fresh and in EOs, provides valuable insights into the processing and utilization of different basil types.

2. Materials and Methods

2.1. Materials and Reagents

Eighteen samples of fresh Thai basil (Ocimum basilicum var. thyrsiflora), sweet basil (Ocimum basilicum var. sweet), and purple basil (Ocimum basilicum var. Purple Ruffles) were collected from Zhongheng Ecological Agriculture Co., Ltd., Lianyungang, Jiangsu Province, China, between April 2023 and May 2023. Additionally, six samples of lemon basil (Ocimum basilicum var. citriodorum) were sourced from Hefei Zhishun Agricultural Development Co., Ltd., Hefei, Anhui Province, China. The four samples were identified by A.P. Hao Jiang (the College of horticulture, Anhui Agricultural University) as Thai basil, sweet basil, purple basil, and lemon basil. To ensure uniformity, all basil varieties were harvested around their first flowering and transported to the laboratory immediately. Prior to EO extraction, the fresh basil samples were shade-dried at room temperature until they reached a constant weight. All samples were stored at 4 °C and processed within one week of collection for HS-SPME/GC-MS and SD/GC-MS analyses.

2.2. Volatile Extraction Using the HS-SPME Method and Analyses

To ensure reproducibility in sample collection, fresh basil samples were lyophilized using a vacuum freeze dryer (FD-1E-110, Biocool, Beijing, China). The lyophilized material was then ground into a fine powder and stored at −20 °C. For analysis, 100 ± 1.0 mg aliquots of the powdered basil were placed into screw-capped headspace vials with adhesive gaskets (LBSV410C, Membrane Solutions, Seattle, WA, USA) and equilibrated at 65 °C for 5 min. Subsequently, an SPME needle (57316-U, Supelco, Bellefonte, PA, USA) coated with a polydimethylsiloxane/divinylbenzene/carbon (PDMS/DVB/CAR) fiber (30 μm thickness, Supelco, Bellefonte, PA, USA) was activated for 30 min. The fiber was then inserted into the headspace of the vials and allowed to interact with the volatile components at 65 °C for 45 min, with the vial stirred at 100 rpm throughout. After sampling, the SPME fiber was retracted into the needle and inserted into the injection port of the GC-MS system, where it was desorbed at 250 °C for 5 min.

2.3. EO Extraction Using the SD Method and Analyses

EOs from four basil varieties were extracted using a hydrodistillation apparatus (JY 009, Lishui Yidinfang Distillation Equipment Co., Ltd., Lishui, China) following the SD method optimized in our previous study [10]. Fifty grams of shade-dried basil were placed in the evaporating flask of a glass round-bottom apparatus, and 1.0 L of deionized water was added. After approximately 20 min for the water to reach boiling, the distillation process continued for a total of 80 min. The yellowish EO was condensed in the cooling column and collected in the EO separator tube. The collected EO was then dehydrated using anhydrous Na₂SO₄. The resulting EOs were stored in dark conditions at 4 °C until further use. Each extraction was performed in triplicate to ensure reproducibility [10].

The yield of EO from each basil variety was calculated using the formula:

EO Yield (Y, %) = Weight of EO (W_EO, g)/Mass of dried basil (M_db, g) × 100

(1)

2.4. GC-MS Analysis

GC-MS analysis of the extracted volatile compounds was conducted using a Trace1300-ISQ GC-MS system (Chromeleon 7, Thermo Fisher, Waltham, MA, USA) equipped with a DB-WAX capillary column (30 m × 0.25 mm internal diameter, 0.25 mm film thickness, Supelco, USA). Helium was used as the carrier gas at a flow rate of 1 mL/min. The injector temperature was set to 250 °C, with samples introduced in splitless mode at a split ratio of 20:1. The initial column temperature was maintained at 60 °C for 1 min, then increased to 150 °C at a rate of 4 °C/min over 5 min, and further raised to 240 °C at a rate of 4 °C/min over 3 min. The GC was operated with an ionization energy of 70 eV. The transfer line and ionization source temperatures were set to 280 °C and 230 °C, respectively [10].

Volatile components were identified by comparing their retention times with known standards, matching against commercial spectral libraries (Wiley and NIST), and consulting MS literature data. Retention indices were determined using a C8-C20 n-alkane series (Fluka Analytical, Munich, Germany) under the same GC conditions and compared to values in the literature to further confirm component identities [18]. The relative proportions of volatile components were calculated from the total ion flow chromatograms using the peak area normalization method.

2.5. Sensory Evaluation

According to the requirements of International Standard ISO 8589:2007 [19], the sensory quality of the four basil varieties, both fresh and as EOs, was evaluated using a modified version of a previously reported method [20]. A panel of 10 trained assessors conducted the evaluations in the Sensory Evaluation Laboratory at Hefei University of Technology, Hefei, China, in individual testing booths under controlled white lighting at room temperature.

For the fresh basil samples, leaves of similar size were cut from freshly harvested stems and placed into 10 mL odor-free covered cups. Assessors were instructed to tear the basil leaves, inhale the aroma, and score the sensory attributes based on their experience [21]. For the EO samples, the EOs were prepared at a 2.0% concentration in ethanol and dispensed into identical cups to a height of 1–2 cm. Smelling paper strips were dipped into the samples within the odor-free cups and mounted on a test rack for evaluation by the trained panelists [5]. Each sample was evaluated in triplicate on three separate days.

To maintain anonymity, basil varieties and their corresponding EOs were coded for each panelist. Eight representative aroma descriptors of aniseed, floral, pungent, woody, lemon-like, light, sweet, and mint-like were used for the evaluation [21]. Panelists rated the intensity of each aroma on a scale from 0 (no odor) to 10 (maximum intensity) [22]. The average scores for each aroma descriptor were calculated and presented in radar charts to visually represent the sensory profiles.

2.6. Data Processing and Statistical Analyses

Data from HS-SPME/GC-MS and SD/GC-MS analyses were processed using SIMCA 14.1 software (Umetrics, Malmö, Sweden). PCA was initially employed to reduce data dimensionality, identify significant factors (principal components) influencing volatile aroma components, and examine the interrelationships among variables. Subsequently, OPLS-DA was utilized to further differentiate between volatile samples, providing additional dimensionality reduction and developing predictive models to visualize the distribution patterns and trends across different basil groups. Moreover, a key metabolite dataset with VIP values was derived from the supervised OPLS-DA model. VIP values were used to identify the most distinguishing components between groups, offering insights into the differences among basil varieties. These statistical methods were selected for their accuracy and widespread application in similar research [13,23].

Results were presented as means ± SD from six or three replicates. One-way analysis of variance (ANOVA), conducted with SPSS software version 20.0 (SPSS Inc., Chicago, IL, USA), was employed to assess differences between samples. Statistical significance was indicated by different letters in tables and figures, with differences considered significant at p < 0.05. Additionally, bar charts illustrating the yields of basil EOs, types, and relative proportions of volatile components were created using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA).

3. Results and Discussion

3.1. Volatile Components of Four Fresh Basil Varieties Analyzed by HS-SPME/GC-MS

Table 1. Categories and relative area percentages * (%) of volatile compounds identified by HS-SPME/GC-MS in the four typical basil varieties.

Categories	ID	Compounds	Linear Retention Index	CAS Number	Relative Mass fraction (%)
					Thai Basil	Lemon Basil	Sweet Basil	Purple Basil
Ketone	1	Fechone	1385	4695-62-9	ND	ND	ND	0.12 ± 0.01 a
	2	Camphor	1515	76-22-2	1.08 ± 0.07 a	ND	0.11 ± 0.01 c	0.19 ± 0.02 b
Subtotal					1.08	-	0.11	0.31
Ester	3	Fenchyl acetate	1559	13851-11-1	1.72 ± 0.03 a	ND	ND	0.38 ± 0.01 b
	4	Bornyl acetate	1591	76-49-3	0.23 ± 0.02 a	ND	ND	0.26 ± 0.04 a
	5	Methyl cinnamate	2069	103-26-4	0.59 ± 0.02 a	ND	ND	ND
Subtotal					2.54	-	-	0.64
Ether	6	2-Methyl-5-(prop-1-en-2-yl)cyclohexanol	1377	619-01-2	ND	ND	ND	0.08 ± 0.02 a
	7	Methyl chavicol	1671	140-67-0	66.53 ± 1.65 b	90.18 ± 0.33 a	89.19 ± 0.15 a	0.74 ± 0.02 c
	8	Isoestragole	1988	104-46-1	ND	ND	0.11 ± 0.01 b	0.55 ± 0.03 a
	9	Methyl eugenol	2010	93-15-2	0.76 ± 0.03 d	1.36 ± 0.05 c	1.85 ± 0.04 b	2.14 ± 0.05 a
	10	Eugenol	2138	97-53-0	Tr	ND	Tr	1.45 ± 0.04 a
Subtotal					67.29	91.54	91.15	5.74
	11	Sesquisabinen	1668	58319-04-3	0.14 ± 0.04 a	ND	ND	ND
	12	Germacrene D	1721	23986-74-5	0.98 ± 0.02 d	1.05 ± 0.04 c	1.74 ± 0.06 b	2.75 ± 0.05 a
Terpene	13	β-Myrcene	1153	123-35-3	ND	Tr	ND	0.27 ± 0.05 a
	14	γ- Terpinene	1228	99-85-4	ND	ND	ND	0.09 ± 0.01 a
	15	(Z)-13,7-Dimethyl-3,6-octatriene	1244	3338-55-4	ND	ND	ND	0.08 ± 0.02 a
	16	Terpinolene	1267	586-62-9	Tr	Tr	ND	0.16 ± 0.03 a
	17	α-Copaene	1488	3856-25-5	0.08 ± 0.01 c	0.23 ± 0.01 b	0.24 ± 0.01 b	0.27 ± 0.03 a
	18	β-Cubebene	1544	13744-15-5	0.15 ± 0.03 a	0.15 ± 0.03 a	0.14 ± 0.01 a	0.16 ± 0.01 a
	19	α- Bergamotene	1593	17699-05-7	0.10 ± 0.04 b	Tr	0.07 ± 0.03 b	0.24 ± 0.04 a
	20	β-Caryophyllene	1601	87-44-5	0.30 ± 0.03 c	2.62 ± 0.04 b	2.80 ± 0.06 a	0.20 ± 0.03 d
	21	(-)-β-Elemene	1603	515-13-9	0.33 ± 0.07 b	Tr	ND	0.75 ± 0.07 a
	22	α-Cedrene	1604	469-61-4	ND	ND	ND	0.08 ± 0.02 a
	23	α-Caryophyllene	1607	6753-98-6	1.00 ± 0.11 a	0.48 ± 0.02 c	0.56 ± 0.06 c	0.68 ± 0.03 b
	24	trans-a-Bergamotene	1608	13474-59-4	7.23 ± 0.66 b	0.78 ± 0.03 c	0.82 ± 0.03 c	19.77 ± 1.01 a
	25	α-Guaiene	1614	3691-12-1	0.32 ± 0.03 b	ND	ND	1.12 ± 0.13 a
	26	β-Longipinene	1622	41432-70-6	ND	ND	ND	0.46 ± 0.08 a
	27	cis-β-Farnesene	1666	28973-97-9	1.12 ± 0.09 a	0.20 ± 0.03 b	0.24 ± 0.02 b	ND
	28	trans -β-Famesene	1695	18794-84-8	0.33 ± 0.04 a	0.09 ± 0.01 b	0.10 ± 0.02 b	0.09 ± 0.01 b
	29	β-Selinene	1717	17066-67-0	0.66 ± 0.04 a	0.08 ± 0.02 b	ND	ND
	30	α-Bulnesene	1727	3691-11-0	0.32 ± 0.04 b	ND	ND	1.15 ± 0.22 a
	31	(+)-Bicyclogermacrene	1749	24703-35-3	ND	ND	Tr	1.61 ± 0.25 a
	32	β-Bisabolene	1751	495-61-4	ND	0.94 ± 0.07 a	0.12 ± 0.01 b	ND
	33	Naphthalene,1,2,3,5,6,8α-hexahydro-4,7-dimethyl-1-(1-methylethyl)	1765	16729-01-4	ND	0.27 ± 0.03 a	ND	ND
	34	β-Sesquiphellandrene	1779	20307-83-9	0.09 ± 0.01 b	ND	0.05 ± 0.03 b	0.30 ± 0.05 a
	35	α-Bisabolene	1785	25532-79-0	ND	0.98 ± 0.21 a	ND	ND
	36	cis-Calamenene	1838	22339-23-7	1.54 ± 0.05 a	ND	ND	ND
Subtotal					14.69	7.87	6.88	30.23
Alcohol (includes a phenolic compound)	37	2-Methoxy-3-(2-propenyl) phenol	2065	1941-12-4	ND	ND	ND	0.78 ± 0.02 a
	38	2,7-dimethyl-2,6-Octadien-1-ol	1175	22410-74-8	ND	ND	ND	0.15 ± 0.01 a
	39	Eucalyptol	1210	470-82-6	1.09 ± 0.02 a	0.08 ± 0.01 b	0.08 ± 0.01 b	0.43 ± 0.07 c
	40	Bicyclo[3.1.0]hexan-2-ol,2-methyl-5-(1-methylethyl)-	1374	546-79-2	0.19 ± 0.03 b	ND	ND	0.36 ± 0.02 a
	41	trans-β-Terpineol	1438	7299-41-4	0.36 ± 0.04 a	Tr	ND	ND
	42	Linalool	1545	78-70-6	9.19 ± 1.11 b	ND	ND	58.56 ± 1.66 a
	43	δ-Terpineol	1586	7299-42-5	0.07 ± 0.01 b	0.08 ± 0.01 b	Tr	0.24 ± 0.05 a
	44	Terpinene-1-ol-4	1606	20126-76-5	0.75 ± 0.01 a	ND	ND	ND
	45	(+)-α-Terpineol	1704	7785-53-7	ND	ND	ND	1.19 ± 0.20 a
	46	Borneol	1712	507-70-0	0.06 ± 0.01 a	ND	Tr	ND
	47	τ-Juniper alcohol	1736	5937-11-1	ND	0.09 ± 0.03 b	ND	1.20 ± 0.19 a
Subtotal					11.71	0.25	0.08	62.13
Total					97.31	99.66	98.22	99.05

Note: * Percentages are mean values from six replicates of HS-SPME/GC-MS analysis; ND stands for not detected and Tr denotes trace amounts. ^a–d Different letters in the same row indicate significant statistical differences (Duncan’s multiple range test, p < 0.05), while identical lowercase letters indicate non-significant differences (p > 0.05) [24]. The same applies below.

presents the detailed chemical composition of the headspace volatiles from the four fresh basil varieties. A total of 47 volatile components were identified, accounting for 97.31% to 99.66% of the total ion chromatogram. The analysis highlighted significant chemical variability among the basil varieties, with each sample displaying a complex mixture of ethers, terpenoids, alcohols, esters, and ketones, consistent with previous research [18].

The headspace volatile compositions of the four fresh basil varieties are summarized as follows: Thai basil was predominantly characterized by methyl chavicol (7, 66.53%), linalool (42, 9.19%), and trans-α-bergamotene (24, 7.23%). Lemon basil exhibited a high proportion of methyl chavicol (7, 90.18%), with β-caryophyllene (20, 2.62%) and methyl eugenol (9, 1.36%) also present. Sweet basil, which had the fewest number of identified volatile components (16), featured methyl chavicol (7, 89.19%), β-caryophyllene (20, 2.80%), and methyl eugenol (9, 1.85%) as its major constituents. Purple basil, with the highest diversity of volatile components (36), was distinguished by linalool (42, 58.56%), trans-α-bergamotene (24, 19.77%), and methyl eugenol (9, 2.14%). The dominance of methyl chavicol in Thai basil, lemon basil, and sweet basil suggests that they may belong to the methyl chavicol chemotype, while purple basil, with its high linalool content, likely represents the linalool chemotype [5,25].

The results presented in Table 1 indicated significant differences (p < 0.05) in the relative contents of methyl eugenol (9) and β-caryophyllene (20) among the fresh basil varieties. Specifically, the proportion of methyl chavicol (7) in fresh lemon basil was significantly different from that in Thai basil and purple basil, although it was not significantly different from sweet basil. Linalool (42) was nearly absent in lemon basil and sweet basil, but a notable difference was observed between Thai basil and purple basil in terms of linalool (42) content. These variations in volatile compound concentrations are critical for sensory profiling and application recommendations for different basil varieties [26].

For instance, methyl chavicol (7) and methyl eugenol (9), both featuring the allylbenzene structure, are valuable intermediates in drug synthesis. Consequently, Thai basil, lemon basil, and sweet basil, with their high levels of methyl chavicol and/or methyl eugenol, are promising candidates for developing anti-inflammatory drugs. Additionally, allylbenzene-containing compounds contribute distinctive aromas and flavors to basil, making them key ingredients in perfumes, flavors, and seasonings [22]. Moreover, β-caryophyllene (20), a natural sesquiterpenoid, is essential as an intermediate in organic chemistry and drug synthesis. Therefore, lemon basil and sweet basil hold potential for exploring and developing novel pharmacological substances [27]. Linalool (42), a chain terpene alcohol, is used as a raw material for synthesizing various fragrances, industrial flavors, and drugs. Therefore, Thai basil and purple basil may be valuable in developing perfumes, insecticides, antimicrobial agents, and other high-value products [28].

3.2. Extraction of EOs from Four Basil Varieties Using the SD Method

The yields of EOs extracted from the four basil varieties using the SD method are presented in Figure 1. Lemon basil yielded the highest amount of EO at 0.62%, while purple basil had the lowest yield at 0.07%. These findings are consistent with previously reported data for various basil varieties extracted using the SD method [29]. It has been known that the yield and composition of EOs are influenced by multiple factors, including plant genetics, the specific plant organ used, developmental stage, and environmental conditions [30,31]. In this study, the observed differences in EO yield and composition among the basil varieties are mainly attributed to genetic variations. Thus, lemon basil is recommended for EO-based product production due to its higher economic efficiency.

3.3. Volatile Components of EOs from Four Basil Varieties Analyzed by GC-MS

The EOs from the four basil varieties were analyzed using GC-MS, and the volatile constituents are detailed in

Table 2. Categories and relative area percentages * (%) of volatile compounds identified by SD/GC-MS in the four typical basil varieties.

Categories	ID	Compounds	Linear Retention Index	CAS Number	Relative Mass Fraction (%)
					Thai Basil	Lemon Basil	Sweet Basil	Purple Basil
Ketone	1	Fenchone	1384	7787-20-4	0.36 ± 0.02 a	0.08 ± 0.01 b	0.09 ± 0.02 b	0.25 ± 0.04 a
	2	Camphor	1513	76-22-2	0.15 ± 0.04 b	ND	0.39 ± 0.08 a	ND
	48	Menthone	1482	10458-14-7	ND	ND	ND	0.14 ± 0.05 a
	49	D-(+)-Carvone	1717	2244-16-8	Tr	ND	0.19 ± 0.04a	ND
Subtotal					0.51	0.08	0.67	0.39
Ester	50	β-Terpinyl acetate	1366	10198-23-9	ND	ND	ND	0.14 ± 0.04 a
	3	Fenchyl acetate	1558	13851-11-1	0.14 ± 0.04 a	ND	ND	ND
	4	Bornyl acetate	1592	76-49-3	0.54 ± 0.07 b	0.17 ± 0.05 c	1.79 ± 0.44 a	ND
	5	Methyl cinnamate	2070	103-26-4	ND	ND	0.1 ± 0.03 b	3.22 ± 0.40 a
	51	Glyceryl linolenate	2165	18465-99-1	ND	ND	ND	0.40 ± 0.06 a
Subtotal					0.68	0.17	1.89	3.76
Ether	7	Methyl chavicol	1671	140-67-0	65.27 ± 0.94 b	81.03 ± 1.7 a	29.34 ± 0.91 c	3.71 ± 0.43 d
	9	Methyl eugenol	2010	93-15-2	12.35 ± 0.80 a	7.22 ± 0.82 c	8.94 ± 0.48 b	0.49 ± 0.05 d
	10	Eugenol	2138	97-53-0	Tr	Tr	13.26 ± 0.82 a	0.83 ± 0.03 b
Subtotal					77.62	88.25	51.54	5.03
Terpene	12	Germacrene D	1721	23986-74-5	ND	Tr	ND	3.64 ± 0.43 a
	52	α -Pinene	1015	80-56-8	0.22 ± 0.04 b	ND	ND	0.29 ± 0.03 a
	53	β-Phellandrene	1142	555-10-2	0.22 ± 0.04 a	ND	ND	ND
	13	β-Myrcene	1153	123-35-3	0.61 ± 0.03 a	ND	0.09 ± 0.02 c	0.21 ± 0.04 b
	54	Ocimene	1227	13877-91-3	0.94 ± 0.05 a	0.30 ± 0.04 b	0.30 ± 0.03 b	ND
	55	4-Thujanol	1355	546-79-2	ND	ND	ND	0.18 ± 0.06 a
	56	(-)-α-Cubebene	1455	17699-14-8	ND	Tr	ND	0.09 ± 0.02 a
	17	α-Copaene	1490	3856-25-5	ND	0.19 ± 0.02 a	ND	ND
	18	β-Cubebene	1544	13744-15-5	0.95 ± 0.05 a	0.28 ± 0.05 b	ND	ND
	57	Cedrene	1568	11028-42-5	ND	0.17 ± 0.03 a	ND	ND
	58	β-Copaene	1592	18252-44-3	ND	1.58 ± 0.02 a	Tr	ND
	19	α-Bergamotene	1593	17699-05-7	ND	Tr	ND	0.27 ± 0.04 a
	23	α-Caryophyllene	1594	6753-98-6	ND	0.42 ± 0.10 a	0.40 ± 0.08 a	ND
	59	5-Germacratriene	1598	37839-63-7	ND	ND	1.52 ± 0.11 a	ND
	21	(-)-β-Elemene	1600	515-13-9	0.90 ± 0.07 b	Tr	0.8 ± 0.08 b	4.34 ± 0.47 a
	60	β-Guaiene;	1602	88-84-6	0.38 ± 0.06 b	ND	0.21 ± 0.05 c	1.12 ± 0.13 a
	61	Aromadendrene	1608	489-39-4	3.92 ± 0.10 b	0.71 ± 0.06 d	2.77 ± 0.43 c	13.19 ± 1.32 a
	20	β-Caryophyllene	1609	87-44-5	0.86 ± 0.08 b	1.73 ± 0.11 a	0.13 ± 0.03 d	0.48 ± 0.06 c
	26	β-Longipinene	1622	41432-70-6	0.32 ± 0.04 b	0.11 ± 0.02 c	0.17 ± 0.05 c	1.31 ± 0.09 a
	28	trans-β-Farnesene	1693	18794-84-8	ND	0.19 ± 0.03 a	0.12 ± 0.05 b	ND
	62	γ-Muurolene	1698	10208-80-7	1.40 ± 0.13 a	ND	ND	ND
	29	β-Selinene	1716	17066-67-0	ND	ND	ND	0.16 ± 0.07 a
	63	β-Bulnesene	1730	3772-93-8	0.74 ± 0.05 a	ND	0.63 ± 0.03 b	ND
	64	α-Selinene	1734	473-13-2	ND	Tr	Tr	0.40 ± 0.03 a
	65	δ-Guaiene	1745	3691-11-0	ND	ND	ND	2.58 ± 0.10 a
	31	(+)Bicyclogermacrene	1747	24703-35-3	0.75 ± 0.08 c	ND	0.83 ± 0.03 b	2.17 ± 0.03 a
	66	(+)-δ-Cadinene	1768	483-76-1	0.23 ± 0.05 c	0.11 ± 0.02 c	1.46 ± 0.04 b	4.52 ± 0.47 a
	67	cis-α-Bisabolene	1785	17627-44-0	ND	1.79 ± 0.03 a	ND	ND
	36	cis-Calamenene	1838	22339-23-7	ND	ND	0.40 ± 0.08 a	ND
	68	Zingiberene	2039	495-60-3	ND	0.22 ± 0.04 a	ND	ND
Subtotal					12.44	7.80	9.83	34.95
Alcohol	69	Pogostol	1822	21698-41-9	ND	ND	ND	0.33 ± 0.04 a
	39	Eucalyptol	1212	470-82-6	3.26 ± 0.09 b	1.20 ± 0.09 d	4.25 ± 0.05 a	2.30 ± 0.52 c
	70	α-Acorenol	1534	28400-11-5	ND	0.09 ± 0.02 a	ND	ND
	42	Linalool	1559	78-70-6	3.33 ± 0.02 c	0.52 ± 0.07 d	26.95 ± 0.37 a	20.08 ± 1.14 b
	71	Spathulenol	1580	6750-60-3	ND	ND	0.09 ± 0.02 a	0.76 ± 0.08 a
	72	4-Thujanol	1610	546-79-2	ND	ND	0.60 ± 0.01 a	ND
	73	α-Terpineol	1701	98-55-5	ND	Tr	ND	0.58 ± 0.01 a
	74	(-)-α-Terpineol	1705	10482-56-1	0.21 ± 0.04 c	0.12 ± 0.16 c	0.96 ± 0.03 b	1.12 ± 0.13 a
	46	Borneol	1712	507-70-0	0.10 ± 0.02 a	ND	ND	ND
	47	τ-Juniper alcohol	1735	5937-11-1	1.17 ± 0.03 b	0.26 ± 0.03 c	0.37 ± 0.03 c	18.18 ± 0.80 a
	75	13-Heptadecyn-1-ol	1742	56554-77-9	ND	0.14 ± 0.03 a	ND	ND
	76	Terpinen-4-ol	1755	562-74-3	ND	ND	0.11 ± 0.02 a	0.09 ± 0.02 a
	77	α-Bisabolol	1786	515-69-5	Tr	ND	ND	1.58 ± 0.07 a
	78	Nerol	1809	106-25-2	ND	Tr	ND	0.82 ± 0.03 a
	79	Costol	1936	515-20-8	ND	ND	ND	0.46 ± 0.06 a
	80	(-)-Epiglobulol	2012	88728-58-9	ND	ND	ND	0.23 ± 0.04 a
	81	(-)-Cubenol	2059	21284-22-0	0.20 ± 0.03 a	ND	0.73 ± 0.04 a	ND
	82	Piperonyl alcohol	2065	495-76-1	ND	Tr	ND	0.21 ± 0.06 a
	83	Cubebol	2070	23445-02-5	ND	ND	Tr	0.33 ± 0.02 a
	84	9-Decen-1-ol	2086	13019-22-2	ND	ND	ND	2.63 ± 0.05 a
	85	13-Heptadecyn-1-ol	2116	56554-77-9	ND	ND	ND	0.13 ± 0.03 a
	86	α-Cadinol	2184	481-34-5	Tr	Tr	0.32 ± 0.04 b	4.59 ± 0.06 a
	87	Ergosterol	2218	57-87-4	ND	ND	ND	0.23 ± 0.04 a
	88	Androstenediol	2232	521-17-5	ND	ND	ND	0.22 ± 0.08 a
Subtotal					8.27	2.33	34.38	54.87
Total					99.52	98.43	97.41	99.00

according to their order of elution. A total of 67 volatile components were identified in the EOs, comprising 97.41% to 99.52% of the total ion chromatograms. In comparison, only 47 volatile components were identified in the fresh basil samples using HS-SPME/GC-MS. Despite this difference, principal compounds such as methyl chavicol (7) and linalool (42) were consistently present across both EOs and fresh basil samples.

As observed with fresh basil, the relative proportions of volatile components in the basil EOs varied significantly among the four basil varieties. Specifically, methyl chavicol (7) showed notable differences in proportion across the EOs. Lemon basil EO had the highest proportion of methyl chavicol (7) at 81.03%, followed by Thai basil EO at 65.27% and sweet basil EO at 29.43%, while purple basil EO had the lowest proportion at 3.71%. The high levels of methyl chavicol (7) in Thai basil and lemon basil EOs indicate their classification as methyl chavicol chemotypes. Additionally, both Thai basil and lemon basil EOs were distinguished by significant proportions of methyl eugenol (9), with concentrations of 12.35% and 7.22%, respectively.

In contrast to the exceptionally high proportion of methyl chavicol (7, 89.19%) found in fresh sweet basil, the EO of sweet basil exhibited a significantly lower proportion of this compound (7, 29.34%). Instead, its major volatile constituents were methyl chavicol (29.34%), linalool (42, 26.95%), methyl eugenol (9, 8.94%), and aromadendrene (61, 2.77%). Purple basil EO was characterized by notable proportions of linalool (42, 20.08%), τ-juniper alcohol (47, 18.18%), aromadendrene (61, 13.19%), and (+)-δ-cadinene (66, 4.52%). Additionally, purple basil EO displayed the highest diversity, with 44 distinct volatile components and remarkably high proportions of alcohols (54.87%) and terpenoids (34.95%).

These findings diverged somewhat from those reported in previous studies (Tangpao et al., 2018). For instance, Tangpao et al. identified methyl chavicol (47.66%), geraniol (19.01%), and neral (15.91%) as the predominant components in Thai basil EO, while lemon basil EO was primarily composed of methyl chavicol (68.35%), citral (6.65%), and neral (4.40%). The observed variations in the qualitative and quantitative profiles of basil EOs are likely due to differences in growing conditions, including seasonal, climatic, and soil factors.

The volatile aroma compounds in basil primarily originate from the glandular structures of the leaves, which accounts for their presence in both fresh basil and its EO. Hence, the emission of these compounds into the atmosphere is influenced by their biosynthesis and release rates within the plant tissues. Drying treatments, however, can accelerate the release of certain highly volatile components due to increased volatility. Additionally, changes observed in individual compounds may result from their sensitivity to irreversible oxidative processes and thermal degradation during water evaporation [32]. For instance, aromadendrene (61), which was present in higher proportions in fresh Thai basil (7.23%) and purple basil (19.77%), was not detected in their corresponding EOs. Similarly, β-caryophyllene (20) decreased from 2.62% and 2.80% in fresh lemon basil and sweet basil, respectively, to 1.73% and 0.13% in their EOs. Moreover, the SD process, which thoroughly disrupts plant tissues, facilitates the comprehensive release of volatile components into the distilled water, resulting in a more complex composition of the EOs.

The present study also observed that certain compounds present in EOs were either undetectable or detected only in trace amounts in fresh basil. For instance, β-elemene (21) increased from 0.75% in fresh purple basil to 4.34% in purple basil EO. Similarly, methyl eugenol (9) was detected at a significantly higher proportion of 12.35% in Thai basil EO, whereas it was present only in trace amounts (0.76%) in fresh Thai basil. This increase in phenolic content in the EOs is likely attributed to the inactivation of polyphenol oxidase during the drying process, as reported previously [33].

Additionally, the ANOVA of the volatile components in the EOs from the four basil varieties showed significant differences in the proportions of methyl chavicol (7), methyl eugenol (9), β-caryophyllene (20), aromadendrene (61), eucalyptol (39), and linalool (42) among the EO groups (p < 0.05). Significant variations were also noted in the proportions of eugenol (10) between sweet basil EO and purple basil EO (p < 0.05). These distinct proportions of volatile components highlight the varied potential applications of these EOs. For instance, the high proportion of eugenol (10) at 13.26% in sweet basil EO suggests its potential utility in developing acaricidal products, owing to eugenol’s well-documented acaricidal properties [32]. Additionally, τ-juniper alcohol (47), with its trans-conjugated ring structure known to be effective against fungal growth, and (+)-δ-cadinene (66), known for its anti-inflammatory, antibacterial, and antioxidant activities, were present in notable proportions (18.18% and 4.52%, respectively) in purple basil EO. This suggests that purple basil EO could be effectively utilized in the development of antifungal and antioxidant products [34].

3.4. Comparative Analysis of Volatile Fractions Across Different Basil Varieties

Integration of the data from Section 3.1 and Section 3.3 and Figure 2 indicates notable differences in volatile fractions among the basil varieties. Thai basil exhibited the presence of three additional ester components and one additional alcohol component in its fresh state compared to its EO. Conversely, the EO of Thai basil contained an additional ketone that was not found in the fresh basil. Moreover, the EO demonstrated a higher relative abundance of ether components, while the levels of other components were generally lower than those observed in fresh basil [35]. Furthermore, following drying, lemon basil showed a reduction in ester and ether components in its EO, accompanied by an increase in ketones, terpenes, and alcohols compared to the fresh basil. Sweet basil exhibited the most pronounced changes, with a significant decrease in ether components and an increase in alcohols in its EO after drying. In contrast, the EO of purple basil had a lower alcohol content but higher levels of ketones, esters, ethers, and terpenes compared to the fresh basil.

After undergoing drying and EO extraction processes, basil experienced a general reduction in volatile substances. Similar observations were reported by Li et al. for Tricholoma matsutake Singer [36]. This reduction can be attributed to the Maillard reaction, which is influenced by temperature and water activity [37]. Zhang et al. also demonstrated that the Maillard reaction between amino acids and sugars in chestnuts promotes the formation of certain volatile flavor substances, such as heterocyclic compounds and aldehydes [38]. While differences in volatile fractions between fresh basil and its EOs may be partly due to assay techniques and the volatility of individual compounds, these findings suggest a trend indicating that certain biochemical reactions occur during the extraction process, particularly with the dry steam distillation method.

3.5. PCA-OPLS-DA to Determine Differential Components of Different Basil Varieties

PCA is a statistical technique used for unsupervised pattern recognition in multidimensional datasets [39]. As depicted in Figure 3A, a PCA model (R²x = 0.977, Q² = 0.947) was constructed for various fresh basil varieties. This model extracted two principal components, which together accounted for 90.9% of the cumulative variance, representing the primary dataset. In Figure 3B, another PCA model (R²x = 0.976, Q² = 0.957) was developed for different basil EOs. This model incorporated three principal components, explaining 97.1% of the cumulative variance and characterizing the main dataset. The contributions of PC1, PC2, and PC3 were 0.537, 0.231, and 0.203, respectively, as illustrated in Figures S1 and S2.

In the analysis of fresh basil samples (Figure 3A), the 24 samples were distributed across the first, second, and third quadrants. Significant differences were observed between lemon basil and sweet basil, which were located in the third quadrant, and Thai basil and purple basil, situated in the second and first quadrants, respectively. In contrast, the 24 EO samples (Figure 3B) were distributed across all four quadrants. Notable differences were evident between the EOs of Thai basil and lemon basil, positioned in the first quadrant, and those of sweet basil and purple basil.

To assess the contribution of each variable to sample classification, VIP values were calculated using the supervised OPLS-DA model [13]. According to established criteria, volatile compounds with VIP values exceeding 1 are considered significant for classification [40]. This study analyzed volatile components detected by both HS-SPME/GC-MS and SD/GC-MS separately using the OPLS-DA model. As illustrated in Figure 4, the OPLS-DA model effectively distinguished between the four basil cultivars. The HS-SPME/GC-MS analysis produced fit indices of 0.996 for R²x (independent variable), 0.997 for R²y (dependent variable), and 0.996 for Q² (model prediction). Similarly, the SD/GC-MS results yielded values of 0.993 for R²x, 0.998 for R²y, and 0.997 for Q². All R² and Q² values exceeded 0.5, indicating a satisfactory model fit. Furthermore, after 200 permutation tests, the intersection of the Q² regression line with the vertical axis was below 0, confirming that the model was neither overfitted nor underfitted, thereby validating its robustness.

In this study, the volatile components of four fresh basil varieties and four basil EOs were first analyzed separately using HS-SPME/GC-MS and SD/GC-MS methods. Based on the VIP values (VIP > 1) from the OPLS-DA models, a total of 10 and 14 differential volatile compounds were identified as characteristic components for the fresh basil and basil EOs, respectively (Figure 5). The results demonstrated that the SD/GC-MS method is optimized for extracting polar, highly volatile components, while the HS-SPME/GC-MS method is more effective for capturing volatile or semi-volatile compounds. Additionally, the HS-SPME/GC-MS method exhibited reduced sensitivity to low-concentration volatile substances.

The differential volatile compounds identified by both methods included methyl chavicol, linalool, germacrene D, and methyl eugenol. Specific differential volatiles identified in fresh basil varieties using HS-SPME/GC-MS and the OPLS-DA model included trans-α-bergamotene, terpinolene, β-caryophyllene, fenchyl acetate, and squalene. Conversely, SD/GC-MS identified specific differential volatiles in different basil EOs, such as eugenol, τ-cadinol, cis-α-bisabolene, eucalyptol, γ-muurolene, β-copaene, δ-cadinene, β-elemene, and methyl cinnamate. These findings are consistent with previous reports by Mahmoud et al., where linalool, methyleugenol, methylcinnamate, and methyl chavicol were identified as predominant in basil wastes treated by different drying methods [16].

The PCA-OPLS-DA analysis conducted here further corroborates the aforementioned conclusions. The complementary use of both analytical methods facilitated a more comprehensive identification of differential volatiles in various basil cultivars and their EOs, thereby providing a robust theoretical foundation for the practical application of these basil varieties.

3.6. Sensory Evaluation of Aroma in Different Basil Varieties

As shown in Figure 6, the aroma characteristics of various fresh basil cultivars and their EOs were assessed through sensory evaluation, and radar fingerprints were generated. Eight distinct aroma components of aniseed, floral, pungent, woody, lemony, light, sweet, and minty were identified across the basil varieties. Fresh Thai basil was noted for its prominent spicy and aniseed notes, while fresh lemon basil and purple basil were distinguished by their intense lemon and floral scents, respectively. Furthermore, fresh sweet basil had an even more intense aniseed aroma compared to Thai basil. The EO of Thai basil exhibited a more pronounced aniseed aroma and pungent aroma than its fresh counterpart, with an additional cleaner scent. Lemon basil EO had a more intense lemon aroma compared to fresh lemon basil. Additionally, the EOs of sweet basil and purple basil were characterized by strong floral and woody aromas, respectively. Other aroma attributes showed minimal differences among the four basil varieties.

The volatile components are crucial for the distinctive aroma and flavor of basil, which vary among different varieties, imparting unique aroma profiles to each [21,26]. As detailed in Table 1 and Table 2, methyl chavicol plays a pivotal role in producing pungent and aniseed notes. Thai basil, with a high concentration of methyl chavicol (65.27%), exhibits a pronounced aniseed and pungent aroma. The absence of linalool in Thai basil may explain the lack of a sweet undertone [5]. Additionally, methyl eugenol, present at 12.35% in Thai basil EO, contributed a pronounced refreshing fragrance.

Ali et al. attributed the dominant lemon aroma of lemon basil to volatile compounds such as limonene and citral [26]. However, these compounds were not detected in this study, potentially due to limitations in detection methods and the volatility of the compounds [18]. Fresh sweet basil exhibited the most pronounced pungent and aniseed aroma, attributed to its high methyl chavicol content (89.19%) and the presence of β-caryophyllene (2.8%), which further intensified the pungency. In addition, it has been known that linalool, an acyclic monoterpene alcohol, imparts a floral aroma, while eugenol provides a clove-like floral scent [5,29]. Consequently, the EO of sweet basil, characterized by its high content of linalool (26.95%) and eugenol (13.26%), exhibited the most intense floral aroma among the basil varieties.

Fresh purple basil displayed a more pronounced floral aroma compared to the other three varieties, largely due to its high linalool content (58.56%). However, its volatile oil exhibits a strong woody aroma, likely due to the presence of woody aromatic terpenes such as cis-α-bergamotene, β-elemene, and pinene [41,42].

Several factors contribute to the significant variations in composition and aroma among different fresh basil cultivars and their EOs. The SD process introduces various physical and chemical changes that affect the final product’s composition and quality. Firstly, high temperatures during vaporization and extraction can lead to the thermal degradation of sensitive compounds, such as monoterpenes and sesquiterpenes, resulting in a loss of therapeutic properties and alterations in the oil’s aroma. Additionally, heat-induced isomerization can convert compounds into different isomers with altered odors and properties, impacting the oil’s quality. Moreover, oxidation, driven by heat and oxygen exposure, can generate new compounds that may introduce off-notes and diminish efficacy. Furthermore, the extraction process may solubilize non-volatile substances, such as plant waxes and resins, into the EO, affecting its purity and viscosity [30]. Thus, precise control of extraction parameters is crucial for preserving the EO’s desired aroma and characteristics.

The integration of sensory evaluation with chemical composition analysis in the present study provided a comprehensive understanding of the aroma profiles and volatile compositions across various basil varieties and their EOs. This combined approach enables more informed control over product quality and facilitates the development of tailored applications. Additionally, sensory evaluation provides a robust framework for assessing and categorizing different basil varieties and their EOs, offering valuable insights for selecting basil products optimized for culinary and spice applications.

4. Conclusions

The present study provided a comprehensive and detailed analysis of the volatile components in four basil varieties, Thai basil, lemon basil, sweet basil, and purple basil, both in the fresh state and as EOs, using HS-SPME/GC-MS and SD/GC-MS methods. The findings revealed that Thai basil was characterized by high levels of methyl chavicol, while lemon basil also showed a predominance of this compound along with notable variations in other components such as β-caryophyllene. Sweet basil was distinguished by its high concentration of methyl chavicol, and purple basil was notable for its high linalool content. Significant differences were observed in the volatile profiles of the EOs compared to their fresh counterparts. The drying and SD processes led to significant alterations in the volatile profiles due to thermal degradation, oxidation, and the extraction process, with fresh basil generally containing higher levels of certain compounds compared to their EOs. Sensory evaluation revealed distinct aroma profiles for each basil variety. Thai basil had pronounced aniseed and spicy notes, lemon basil was noted for its strong lemon aroma, sweet basil was pungent with aniseed notes, and purple basil exhibited a floral aroma. These sensory attributes closely align with the predominant volatile compounds identified. Overall, the findings underscore the potential applications of these basil varieties in pharmaceuticals, food flavoring, and fragrance. They also highlight the importance of considering both fresh and processed forms of basil in product development to optimize their desirable properties.