Aromatic Profiles of Essential Oils from Five Commonly Used Thai Basils

The research objectives of this study are to analyse the volatile compositions of different basil types available in Thai markets and to descriptively determine their aromatic qualities. Essential oils were hydro-distillated from fresh leaves of two Holy basil (Ocimum sanctum) varieties namely, white and red and other basil species, including Tree basil (O. gratissimum), Thai basil (O. basilicum var. thyrsiflorum), and Lemon basil (O. citriodorum). Oil physiochemical characteristics and volatile chromatograms from Gas Chromatography–Mass Spectrometry (GC-MS) were used to qualitatively and quantitatively describe the chemical compositions. Estragole, eugenol, and methyl eugenol were among the major volatiles found in the essential oils of these basil types. Classification by Principal Component Analysis (PCA) advised that these Ocimum spp. samples are grouped based on either the distinctive anise, citrus aroma (estragole, geranial and neral), or spice-like aroma (methyl eugenol, β-caryophyllene, and α-cubebene). The essential oils were also used for descriptive sensorial determination by five semi-trained panellists, using the following developed terms: anise, citrus, herb, spice, sweet, and woody. The panellists were able to differentiate essential oils of white Holy basil from red Holy basil based on the intensity of the anisic attribute, while the anise and citrus scents were detected as dominant in the Lemon basil, Tree basil, and Thai basil essential oils. The overall benefit from this research was the elucidation of aromatic qualities from Thai common Ocimum species in order to assess their potential as the raw materials for new food products.


Introduction
The genus Ocimum (belonging to the Lamiaceae which is recognised as the richest essential oil-bearing plant family) is represented by more than 150 species that are grown widely and distributed throughout tropical and temperate regions [1]. They are collectively known as the "basils" which are in commercial demand for their nutritional, aromatic, ornamental, culinary, religious, and medicinal importance [2]. Among those, Holy basil (O. sanctum), Sweet or Thai basil (O. basilicum), Lemon basil (O. citriodorum), and Tree basil (O. gratissimum) are frequently cultivated in several countries of South and South-East Asia including Thailand as culinary herbs [3,4]. There has been an increasing concern on liable health problems associated with synthetic food flavouring agents. Therefore, food researchers are focusing on the search for natural products that could replace chemically synthetised food additives [5,6]. 0.003% w/v toluene as an internal standard) were injected in a split mode (1:20). The oven temperature was set at 60 • C for 3 min, and increased by 2.5 • C/min until 240 • C where it was held at this temperature for 10 min. The carrier gas was helium with a flow rate of 1.1 mL /min. The interface with MS was at 200 • C and mass spectra were taken at 70 eV in electron impact ionisation mode, with a scanning speed of 0.5 scans/s from m/z 20-350 [18]. The standard solution of C 8 -C 20 n-alkane (Fluka ® Analytical, Munich, Germany) in hexane was also used for the calculation of retention indices (RI) [19]. The identification of the volatile compositions was by comparison with mass spectra in NIST 05.L and NIST 98.L libraries with >70% similarity. The compounds were confirmed by their RI as well as those from the literature [11]. The amount in µg/mL of essential oil was calculated as relative to that of internal standard.

Descriptive Analysis
Prior to sensory testing, five semi-trained panellists with extensive experience in Thai food (two males, three females, and age ranged from 25 to 37 years old) were chosen based on their abilities to discriminate differences and ranking/rating the intensity scents. They also completed a 3-day orientation and a 12 h descriptive training course. Thereafter, they then developed attribute terms describing the odour of five essential oils on cotton balls (200 µL). Panellists tasted all samples and discussed the attribute definitions, attribute references, reference intensities, and evaluation procedures. References were consistent with those of the previous studies as described in Table 1, although some additional terms (anise, herb, and woody) were added.
At the day of testing, the essential oil (200 µL oil pipetted on clean and deodourised cotton ball) and reference attributes were presented in front of the same group of the panellists. They then evaluated the intensity of the given attributes in triplicates on the 15-point interval scale (0 = none, 15 = extra strong) [20]. Clean air was obtained between each assessment. A gap of 20 s was sufficient to the individual odour assessments.

Statistical Analysis
Differences of the descriptive analysis data was determined using Analysis of Variance (ANOVA) by SPSS (IBM, Armonk, NY, USA) with a significance level of 0.05. Principal Component Analysis (PCA) was used to summarise graphical differences of volatile components of the essential oil and the descriptive data among the Ocimum spp. using XLSTAT ver. 2018.5 (New York, NY, USA).

Plant Identification and Physiochemical Characteristics of the Essential Oils
Basils are widely distributed in tropical areas and are likely to have originated in South Asia (India). These herbaceous plants are of annual type, usually propagated through seeds [4]. The genus of Ocimum comprises of more than 65 species and is the biggest genera in Lamiaceae family worldwide [24]. Different basil species can be identified by morphological characterisations such as leaf shape and its colour, flower structures and its colour, seed structures and its characteristics (Table 2). However, due to extensive cultivation, inter and intra-specific cross hybridisation has occurred leading to polyploidy and different numbers of species, subspecies, and varieties that are not significantly different in their appearances [24]. In our study, five Ocimum spp. types had distinct morphological characteristics. The O. sanctum of white and red varieties (viz., Rama and Shyama) possessed different leaf colours. O. citriodorum and O. basilicum var. thyrsiflorum illustrated unique seed characteristic which was mucilaginous after soaking in water. O. gratissimum possessed a large leaf size about 45 cm 2 while O. citriodorum conferred leaf size around 3.5 cm 2 .
To obtain the essential oil, fresh basil leaves were extracted by hydro-distillation and the physiochemical characteristics were shown in Table 2 [25][26][27]. Siddique et al. [27] suggested that the chemical compositions of essential oil depend largely on its colour. Moreover, the alteration of the essential oil colour as a result of their compositions is suggested to be due to thermal degradation, oxidation, isomerisation, dehydrogenation and polymerisation [17,[27][28][29].
Essential oils with different chemical compositions can absorb UV light at different wavelength therefore, illustrating variation of light reflection intensity [30,31]. In our experiment, white and red Holy basils essential oils reflected high UV light intensity, followed by moderate reflection intensity (Tree and Thai basils) and low reflection intensity (Lemon basil) ( Table 2). These were confirmed by absorbance spectrum patterns under various UV-Visible wavelengths (220-500 nm) ( Figure 1). The active chemical components of essential oil from Ocimum spp. plants are estragole (methyl chavicol), eugenol, and methyl eugenol [11,32,33]. Dighe et al. [34] advised that UV spectrum of eugenol gave the maximum absorption at 220-230 nm and a smaller peak at 278 nm which agree with our results. However, we found the tiny peaks at 269-275 instead of 278 nm ( Figure 1).  [25][26][27]. Siddique et al. [27] suggested that the chemical compositions of essential oil depend largely on its colour. Moreover, the alteration of the essential oil colour as a result of their compositions is suggested to be due to thermal degradation, oxidation, isomerisation, dehydrogenation and polymerisation [17,[27][28][29].
Essential oils with different chemical compositions can absorb UV light at different wavelength therefore, illustrating variation of light reflection intensity [30,31]. In our experiment, white and red Holy basils essential oils reflected high UV light intensity, followed by moderate reflection intensity (Tree and Thai basils) and low reflection intensity (Lemon basil) ( Table 2). These were confirmed by absorbance spectrum patterns under various UV-Visible wavelengths (220-500 nm) ( Figure 1). The active chemical components of essential oil from Ocimum spp. plants are estragole (methyl chavicol), eugenol, and methyl eugenol [11,32,33]. Dighe et al. [34] advised that UV spectrum of eugenol gave the maximum absorption at 220-230 nm and a smaller peak at 278 nm which agree with our results. However, we found the tiny peaks at 269-275 instead of 278 nm ( Figure 1).
The PCA between the volatile compositions and the essential oil types revealed three major clustering groups (Figure 2). The first group included Thai basil and Lemon basil with the evident aromatic compounds (viz., estragole, geranial, and neral) which represent anise, lime-like, and fresh aroma [51]. The second cluster was of the white Holy basil and red Holy basil group with methyl eugenol, β-caryophyllene, and α-cubebene (herb and spice) as distinctive compounds [52]. White and red Holy basils are also variety related species (O. sanctum). The last group is the Tree basil that did not correlate with any groups described previously.

Sensory Profile of the Essential Oil
Overall sensory scores obtained by the different five Ocimum spp. essential oils are illustrated in Table 4. The intensity (0-15) represent the maximum possible aroma quantity of each identified attributes judged by the panelists. It was observed that the sweet attribute (scores~2.4-3.4) was not significantly different among all types of the essential oils. The essential oils of Tree basil and Thai basil gave the highest intensity of herb odour (score~9.0-11.0). Essential oils of white and red Holy basils provided the maximum woody scent (score~2.2-3.4), while the Tree basil and white Holy basil dominated anisic attribute (score~5.9-7.4). Tree basil and Thai basil oils illustrated the highest spice aroma intensity (score~6.4-7.3). The citrus scent was the highest in the oil of Lemon basil with the score of 12.8.   [11].     The PCA was able to split aromatic profile of white Holy basil relating with the higher intensity of anisic attribute (Figure 3). However, there was no correlation of woody and sweet attributes across the five essential oil types.  A study on the attribute of essential oil from Ocimum spp. (O. basilicum L.) by Calín-Sánchez et al. [53] evaluated the same attributes that are herbaceous (herb), spice, woody, and sweet. These attributes were also identified in our study. This group of researchers also found that the Sweet basil (O. basilicum) essential oil extracted from the dried leaves, gave stronger sweet and woody attributes than the essential oil extracted from fresh leaves. In our study, the citrus attribute is a principle characteristic of essential oils from most Thai basil types analyses especially the Lemon basil as confirmed by both chemical and sensory evaluations. This is consistent with the previous research by Al-Kateb and Mottram [54] who studied the relationship between growth stages and volatile compositions of Lemon basil (O. citriodorum Vis.). They found that citral, linalool, and estragole are major compounds of the essential oil and they contributed to the citrusy aroma. Active variables Active observations A study on the attribute of essential oil from Ocimum spp. (O. basilicum L.) by Calín-Sánchez et al. [53] evaluated the same attributes that are herbaceous (herb), spice, woody, and sweet. These attributes were also identified in our study. This group of researchers also found that the Sweet basil (O. basilicum) essential oil extracted from the dried leaves, gave stronger sweet and woody attributes than the essential oil extracted from fresh leaves. In our study, the citrus attribute is a principle characteristic of essential oils from most Thai basil types analyses especially the Lemon basil as confirmed by both chemical and sensory evaluations. This is consistent with the previous research by Al-Kateb and Mottram [54] who studied the relationship between growth stages and volatile compositions of Lemon basil (O. citriodorum Vis.). They found that citral, linalool, and estragole are major compounds of the essential oil and they contributed to the citrusy aroma.

Conclusions
From this research, we conclude that the major components of essential oils from the five types of basils used as food ingredients in Thailand are estragole, eugenol, and methyl eugenol. From this chemical analysis we can then distinguish Ocimum spp. plants from the odour characteristics into two groups: citrus and spice-like. Sensory analysis also confirmed that citrus is the main feature in these Ocimum spp. essential oils. It is also possible to sensorially separate the aromatic scent of the two Holy basil (var. red and white) essential oils by using anisic attributes. Future research could be extended to the use of the natural products from these plant species in food as additives active against food microbials and in agriculture (i.e., methyl eugenol) as bio-control agent.