Aromatic Profiles of Essential Oil from 5 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 2 Holy basil (Ocimum sanctum) varieties namely, white and red and other basil species, including Tree basil (O. gratissimum), Sweet basil (O. basilicum var. thyrsiflorum) and Lemon basil (O. citriodorum). Oil physicochemical characteristics and volatile chromatograms from Gas Chromatography-Mass Spectrometry (GC-MS) were used to qualitatively and quantitatively describe the chemical compositions. Methyl eugenol, estragole and eugenol were among the major chemicals 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 (β-methyl eugenol, caryophyllene and α-cubebene). The essential oil was also used for descriptive sensorial determination by five trained panelists, using the following developed terms: anisic, citrus, herb, spice, sweet and woody. The panelists were able to differentiate essential oil 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 Sweet 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 future food research and development.


Introduction
The genus of Ocimum (belonging to the Lamiaceae which also recognised as the richest essential oil bearing plant family) is represented by more than 150 species that are grown widely and distributed throughout the 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 (Ocimum sanctum), Sweet 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 for culinary herbs [3,4].Much recently demands are utmost for their essential oils not only for food but also for medicinal industries [1,[5][6][7].There has also been an increasing concern on liable health problems associated with synthetic food flavouring agent.Therefore, food researchers are focusing on the search of natural products that could replace the chemically synthetised food additive [8,9].Essential oil as well as any other plant based natural ingredients have been the beneficiary of legal, regulatory and consumer preference as the result of a shared opinion on food safety [9,10].
Thai food plays an important part of Thai image in global eyes and is the most important reason for travelers to choose Thailand as their holiday destination [11,12].Moreover, the use of genuine ingredients in Thai food is accepted globally as healthy food choice because of its nutritional value and most important factor supporting the authentic Thai flavour [13,14].Thus, it has come as no surprise that food suppliers of ethnic food ingredients in quest for true Thai basils essential oil.However, the varieties of basil species available as raw material for essential oil extraction may leads to the confusion of true basils aroma.The research aim of this study is therefore to describe the aromatic identity of common Thai basils by chemical and sensory analyse as ultimately to set the ideal scent in natural food additive products.

Plant material
To obtain reliable source of raw material supply, the aerial parts of different stages were collected from 5 Ocimum spp.types viz., O. sanctum var.Rama (red), O. sanctum var.Shyama (white), O. citriodorum, O. basilicum var.thyrsiflorum, and O. gratissimum (100 plants/ type) at their flowering stages.Plants were grown at Mae-Hia Agricultural Training and Research Center, Division of Horticulture, Faculty of Agriculture, Chiang Mai University in an open-area (100 x 2,500 cm plot and the plant spacing was 50 cm 2 ) and maintained by watering 2 hr and 3 times per week using drip irrigation, and fertilizing [Urea (46-0-0) and NPK Fertiliser (15-15-15) in the ratio 1:3] once a month until flowering.After collections, plant specimens (leaf and floral specimens) were separated and their morphological appearances were recorded prior to sending them to the Department of Biology, Faculty of Science, Chiang Mai University for confirmation of their scientific and common names by the experts.

Essential oil extraction
At the laboratory, leaf tissues were parted from the stems and inflorescence.Fresh leaves (~300 g) were used for essential oil extraction in a 5 L Clevenger hydro-distillation apparatus containing 2.5 L of distilled-water and the extraction was for 2 hr at 150 °C.After allowing to cool down at room temperature, the essential oil was collected and treated with anhydrous sodium sulfate to remove the remaining water.The yields were averaged and calculated according to fresh weight of plant material [15].

Physical characteristics of essential oil
Colour of the essential oil was determined by physical observation in day light and under ultraviolet radiation using an ultra-violet chamber [16].The essential oil from water distillation was dissolved in methanol (10% v/v) and scanned for the absorbance with the wavelengths ranging from 250 to 500 nm by the UV-visible spectrophotometer (SPECTROstar Nano; BMG LABTECH) [17].

Gas chromatography mass spectrometry (GC-MS)
The GC-MS analyses were accomplished with a Bruker-scion 436 GC-MS equipped with Rxi-5Sil MS columns (restek).Essential oil sample (2 µL at the dilution of 1%, v/v, in dichloromethane with a presence of 0.003 % w/v toluene as an internal standard) was injected in 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 C8 -C20 n-alkane (Fluka® Analytical) in hexane was also used for the calculation of retention indices [19].

Descriptive analysis
Prior to sensory testing, 5 trained panelists (2 males, 3 females and age ranged from 25 -37 years old) who had completed 3 days of orientation and a 12 h descriptive training course.Thereafter, they had developed attribute terms describing the odour of 5 essential oil on cotton ball (200 µL).Panelists tasted all samples and discussed the attribute definitions, attribute references, reference intensities, and evaluation procedures.Most references were consistent with those of the previous studies as described in Table 1, although some additional terms (anisic, 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 panelists.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 sec was sufficient to the individual odour assessments.

Statistical analysis
Differences of the descriptive analysis data was determined using Analysis Of Variance (ANOVA) using SPSS 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.

Plant identification and physiochemical characteristics of the essential oil
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 colour, seed structure and 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 their distinct morphological characteristics.The O. sanctum of white and red varieties (viz., Rama and Shyama) possesed different leaf colours.O. citriodorum and O. basilicum var.thyrsiflorum illustrated unique seed characteristic which was mucilaginous after soaking in water.O. gratissimum gave 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 physicochemical characteristics were shown in Table 2. White Holy basil (O.sanctum var.Rama) and Sweet basil (O.basilicum var.thyrsiflorum) illustrated maximum yield ~ 0.4%, Lemon basil (O.citriodorum) and red Holy basil (O.sanctum var.Shyama ) yielded the essential oil of ~ 0.33%.However, Tree basil (O.gratissimum) gave the least essential oil content (< ~ 0.2%).The colour (orange, yellow and colourless) of essential oil under day light were different from species to species (O.gratissimum, O. citriodorum, O. sanctum and O. basilicum var.thyrsiflorum) but not from variety to variety (O.sanctum var.Rama and Shyama).Variation of essential oil colours not only depends upon taxonomical characteristics but also rely on age of the plants as well as time of harvesting and different extraction techniques [25][26][27].Siddique et al., [27] suggested that the chemical compositions of essential oil contribute largely on its colour.Moreover, the alteration 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 oil 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 oil reflected high UV light intensity, followed by moderate reflection intensity (Tree and Sweet 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 [32][33][34].Dighe et al., [35] advised that UV spectrum of eugenol gave the maximum absorption at 220 -230 nm and a smaller peak at 278 nm which are in accordant to our results.However, we found the tiny peaks at 269 -275 instead of 278 nm (Figure 1).
Methyl eugenol (3) is the principal composition in O. sanctrm var Shyama [44], O. uttricifolium [45] and O. campechianum Mill.[46].These results are in line with our work, however, the quantitative compositions of such chemicals may vary.Joshi and Sl [47] and Murarikova et al., [48] described that variation of the qualitative and quantitative of chemical profiles of the basil essential oil may be due to the variety of plant and their growing conditions such as season, climate or soil conditions.
The PCA between the volatile compositions and the essential oil types revealed three major clustering groups.The first group included Sweet basil and Lemon basil with the evident aromatic compounds (viz., estragole, geranial and neral) which represent anise, lime-like and fresh aroma [49].
The second cluster was of the white Holy basil and red Holy basil group with methyl eugenol, βcaryophyllene and α-cubebene (herb and spice) are distinctive compounds [50].White and red Holy basils are also variety related species (O.sanctum).The last group is the Tree basil that do not correlate with any groups described previously.The PCA was able to split aromatic profile of white Holy basil relating with the higher intensity of anisic attribute.However, there was no correlation of woody and sweet attributes, with 5 essential oil types.attributes than the essential oil extracted from fresh leaves.In our study citrus attribute is a principle characteristic of essential oil 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 [52] who studied the relationship between growth stages and volatile compositions of Lemon basil (O.citriodorum Vis.).They found that Citral, Linalool and Estragole major compounds of the essential oil and they contributed to the citrusy aroma.

Conclusions
From this research, we conclude that the major compositions of essential oil from the five types of basils used as food ingredient in Thailand are estragole, eugenol and methyl eugenol.From this chemical analysis we can then distinguish these Ocimum spp.plants from the odour characteristics into two groups: citrus and spice-like.Sensory analysis also confirm that citrus is the main feature in these Ocimum spp.essential oil.It is also possible to sensorially separate the aromatic scent of the two Holy basil (var.red and white) essential oils by using anisic attribute.

Overall sensory scores obtained by the different 5
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 Sweet 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 Sweet 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.

Table 1 .
Attributes and references used in evaluating in 5 essential oil odour

Table 3 .
Chemical composition of the essential oils from 5 Ocimum spp.plants

Table 4 .
The mean intensity values of the six attributes for the five Ocimum spp.essential oil in descriptive sensory evaluation*