Essential Oil Composition and Antioxidant and Antifungal Activities of Two Varieties of Ocimum basilicum L. (Lamiaceae) at Two Phenological Stages

Abstract

Ocimum basilicum is a valuable source of bioactive metabolites with high preventive and therapeutic effectiveness. Here we aimed to investigate the effect of phenological stages (vegetative and flowering stages) on essential oil composition and biological activities of two varieties of O. basilicum (Fino Verde variety and Genovese varieties). To this end, the level of essential oils, flavonoids and phenols, as well as antioxidant and antifungal activities were measured. At the metabolic level, essential oil at vegetative stage of O. bailicum Fino Verde and Genovese variety was constituted by 22 and 26 compounds representing 71.68% and 82.54% of the total oil, respectively. Where germacrene D (10.07%), bicyclogermacrene (6.07%) and β-elemene (4.88%) were the most present components in Fino Verde variety. Moreover, 22.19% are oxygenated monoterpenes represented mainly by the linalool (15.18%) and 1.8 cineole (6.36%) in Genovese variety. The individuals of essential oils were significantly increased to 40 components in Fino Verde variety (98.01% of total essential oil) and decreased to 15 components (95.6% of total essential oil) in Genovese variety at flowering stage. At this stage, the oxygenated monoterpenes (78.4%) were the major fraction represented by linalool (40.1%) and 1.8 cineole (30.96%) in Fino Verde variety, however 64.69% were esters which mainly represented by the methyl cinnamate (64.69%), and 16.83% of oxygenated monoterpenes and Linalool (12.7%) were recorded for Genovese variety. Genovese variety showed the highest levels at both vegetative and flowering stage compared to Fino Verde variety. At flowering stage, the two varieties showed high antioxidant and antifungal activities. Overall, O. basilicum properties offer prospects for their use as a source, particularly at flowering stage to extend new medicines based on natural bioactive molecules.

1. Introduction

Basil (Ocimum basilicum L.) is an important essential oil crop, medicinal plant and culinary herb O. basilicum belongs to the Lamiaceae family [1,2], and grows in tropical and sub-tropical climates and its essential oil is a component of oral health, dental products, and has been used in the fragrance and food industry [3,4]. In Tunisia, this family contains 91 species, or 4% of Tunisian vascular flora [5,6]. The genus Ocimum, the most-known genus of this family, has contains more than 150 species [7]. It is grown in several East Asian countries, Europe, America, and Australia to produce essential oils [8], due to its widely used in traditional medicine and herbal medicine [9]. Out of the 150 species, O. basilum is known as vernacular name “Hbag” and is widely cultivated in the Mediterranean, tropical and subtropical regions [10]. In Tunisia, O. basilicum is grown from the north to the south of the country, in bioclimatic floors ranging from wet to upper arid on silty or sandy soils [11]. Due to high bioactivity, it is frequently exploited in traditional medicine, human food as an additive species [12], as well as for industrial purposes (agri-food, perfumery, cosmetics, pharmaceuticals). This can be attributed to its richness in essential oils, phenols and flavonoids [13]. Thus, this species is used in traditional medicine for its analgesic, anti-inflammatory, antimicrobial, antioxidant, anti-ulcerogenic properties [9,14], hypoglycemic [15]. It is also used as an insecticide [9,16]. In total, six varieties have been identified for O. basilicum which presents significant morphological and biological differences [17]. Out of these 6 varieties the Fino Verde variety and the Genovese variety are the most cultivated ones. They are differing in morphologically by the shape of the leaves, where the leaves are small, thin, smooth, bright green and slightly spicy aroma in the Fino Verde variety, and they are broad green and oval for the Genovese variety which is very popular in southern cuisine. Correlations between chemical composition of essential oils and antimicrobial and antioxidant activities were also studied for other varieties of O. basilicum [18,19]. Several studies have demonstrated that the chemical composition of Ocimum varied among the two varieties. In fact, Mota et al. (2020) [20] revealed that cv. Genovese represents a total of 24 compounds, representing 86.5–95.8% of the essential oils. The major compounds (>10%) of O. basilicum L. cv. Genovese Gigante were linalool (17.0–54.7%), followed by eugenol (7.1–50.8%), trans-α-bergamotene (0.1–15.4%) and methyleugenol (0.2–13.1%). The analyzed essential oils mainly consisted of oxygenated monoterpenes (20.2–64.0%) followed by phenylpropanoids (7.3–59.2%), sesquiterpene hydrocarbons (0.4–32.6%), oxygenated sesquiterpenes (2.4–10.8%) and monoterpene hydrocarbons (0.6–3.8%). While most aromatic molecules in Fino Verde basil are housed in trichomes and are (mono-)terpenes and phenylpropanoids, which are regarded to be the primary constituents of essential oils. Among the latter, linalool and methyl chavicol characterize the Fino Verde aroma of this variety [21]. The antioxidant activity of the essential oils of the leaves of O. basilicum L., genevose growing in arid conditions is higher than that of the flowering tops. Winter is also the season when leaves and flowering tops have the least antioxidant activity [22]. Whereases, the leaves of Fino Verde basil variety exhibited quite good antioxidant activity with relatively high total contents of polyphenols and flavonoids [23]. Moreover, high antifungal activities of essential oil composition for different basil variety has been demonstrated [24]. Whereas chemical composition of essential oil and it pharmacological benefits has been characterized for Genovese variety. Several studies have reported that bioactive compounds levels can vary by genotype and ripening stage [24]. Moreover, harvesting time is another important factor affecting the shelf-life and antioxidant capacity of O. basilicum. In this regards, numerous biochemical and physiological changes occur at different stages of O. basilicum development due to changes in the synthesis, transportation, and degradation of various metabolites such as organic acids and sugars and compounds [9]. Thus the characterization of phytochemical changes in O. basilicum that occur during ripening is essential, since these changes could affect the biological activities (e.g., antioxidant antimicrobial activities), nutritive value (aroma, taste, essential oil), postharvest storage, and ultimately, consumer preference. Furthermore, the influence of the phenological stage in which the O. basilicum is consumed as a potential influencing factor in its bioactive compounds content and antioxidant capacity remains also unknown. The aim of this work was therefore to elucidate the influence of the phenological stage and harvest date on the bioactive compounds content of O. basilicum fruit in order to fill this knowledge gap.

2. Material and Methods

100 seedlings (1 month of growth) of each variety of O. basilicum (Fino Verde and Genovese) collected from a commercial source in Sidi Bouzid were cultivated during a period of 4 months (March–June 2016) the field condition at plot under ambient temperature at National Institute for Research in Rural Engineering, Water and Forest of Gabés South-Eastern Tunisia (33°53′ N, 10°07′ E) with an average rainfall varying between 167 and 176 mm and an average temperature between a maximum of 19.3 °C and a minimum of 18.8 °C. The harvest of plant material was done during two stages of growth, the first vegetative stage (at March) and the second during flowering (at May)

2.1. Essential Oil Isolation

Fresh leaves of 2 varieties of O. basilicum in two phenolic stages were collected. About 100 g of fresh leaves was placed in 1 L of distilled water and subjected to hydrodistillation for 3 h, using a Clevenger-type apparatus [24,25,26,27] (1.5% yield). Anhydrous sodium sulphate was used to eliminate water traces from essential oil. The essential oil is stored at +4 °C until tested and analyzed.

2.2. Gas Chromatography and Gas Chromatography-Mass Spectrometry

Quantitative and qualitative analysis of essential oil was carried out by GC-FID and GC-MS. GC analyses were carried out on a Varian (Les Ulis, France) Star 3400 Cx chromatograph fitted with a fused silica capillary DB-5MS (5% phenylmethyl polysyloxane; 30-m/0.25-mm; film thickness, 0.25 mm) column. Chromatographic conditions were a 60 °C to 260 °C temperature rise with a gradient of 5 °C/min and 15 min isothermal at 260 °C. A second gradient was applied to 340 °C at 40 °C/min. Total analysis time was 57 min. For analysis, essential oil was dissolved in petroleum ether to not saturate the column. One microliter of sample was injected in the split mode ratio of 1:10. Helium (purity 99.999%) was used as the carrier gas at 1 mL/minute. The injector was operated at 200 °C. The mass spectrometer (Varian Saturn GC/MS/MS 4D) was adjusted for an emission current of 10 mA and electron multiplier voltage between 1400 and 1500 V. The trap temperature was 150 °C, and that of the transfer line was 170 °C. Mass scanning was from 40 to 650 atomic mass units. The components were identified based on the comparison of their KI (Kovats indices) and mass spectra with those of standards, Wiley 2001 library data (NIST 02 version 2.62) of the GC-MS system and literature data (Adams). Alkanes (C5–C24) were used as reference points in the calculation of KI. GC and GC-MS analysis results are given in

Table 1. Phenological stage effect on yields of essential oil of two O. basilicum varieties.

		Fino Verde Variety		Genovese Variety
		Vegetative Stage	Flower STAGE	Vegetative Stage	Flower Stage
Component	RT
sabinene	4.95	_	0.1	_	_
β-pinene	5.14	_	0.2	_	0.36
Limonene	5.75	_	0.13	_	_
α-pinene	5.84	_	0.09	6.36	_
Eugenol	5.9	_	1.54	_	_
Gamma terpinene	6.19	_	0.1	0.85	_
Myrcene	6.25	_	0.14	0.17	_
Pulegone	9.73	_	0.09	_	_
α-terpinolene	6.64	_	0.12	_	_
Benzene-1-methyle	6.83	_	_	1.15	_
Methyl chavicol	6.9	_	2.06	_	_
Carvone	10.22	1.98	_	_	_
Geraniol	9.04	_	0.27	_	_
α-Terpenol	8.19	_	0.97	_	_
Borneol	7.84	_	0.58	_	_
Fenchyl acetate	8.58	_	3.3	_	_
Cis linalool oxide	6.41	_	0.13	_	_
Estragol	9.5	_	_	1.44	3.62
Endobronyl acetate	10	0.86	_	_	_
Bronyl acetate	10.72	0.86	5.26	0.65	_
Linalool	8.78	7.65	40.1	15.18	12.47
Comphor	11	_	_	_	0.67
1,8cineole	6.59	_	30.96	6.36	3.69
Methyle eugénol	1108	_	0.49	_	_
Β-farnesene	11.37	3.27	0.94	_	_
β-caryophyllene	11.38	_	_	_	0.79
δ-Guaiene	12.01	2.95	1.65	0.73	0.65
Beta patchouléne	12.06	_	0.26	_	_
Isocaryophyllene	12.17	2.53	_	_	_
Cis caryophylene	12.23	_	0.4	_	_
Germacrene B	12.37	2.13	_	_	_
Aromadendrene	12.42	_	0.23	_	_
α-amorphene	12.55	_	0.1	_	_
α-amorphene	12.55	_	01	_	_
α-bergamotene	12.73	_	_	2.74	_
α-Zingiberene	12.73	_	_	2.74	_
α-Guaiene	12.81	3.77	_	0.62	0.59
α-humulene	13.29	2.3	0.71	0.81	0.58
δ-cadinéne	13.38	_	2.63	0.11	2.15
Germacrene D	13.49	10.7	2.23	3.19	1.63
Bicyclogermacrene	13.61	6.07	0.33	1.8	1.18
δ-cadinene	13.52	4.1	_	0.11	_
δ-sélinene	13.24	_	0.3	_	_
α-Gurjunene	13.77	_	1.65	_	_
δ-Gurjunéne	13.93	_	0.1	_	_
Globulol	14.26	3.5	0.58	8.22	_
Methylcinnamate	14.31	3.32	2.36	15.42	64.69
α-cadinol	15.35	_	_	9.83	_
α-cubebene	15.06	2.5	_	1.04	_
Cadina1,4diene	15.06	1.6	_	_	_
Naphtalene	15.09	_	0.38	1.04	_
β-Eudesmol	15.53	_	_	1.11	_
Phenol	19.37	0.58	0.36	_	_
Alcool (phytol):		20	_	0.24	0.44
% Monoterpens hydrocarbons		1.6	1.26	8.42	0.36
%Oxygenated monoterpens		11.35	78.47	22.19	16.83
%Sesquiterpens hydrocarbons		51.34	11.56	14.68	10.1
%Oxygenated sesquiterpenes		3.5	0.58	18.71	_
%Ester		3.32	6.6	15.42	64.69
%Phenol		0.57	3.96	2.59	3.62
Total%		71.68	98.24	82.45	95.6

RT: retention time, _: absent.

. All determinations were performed in duplicate and averaged.

2.3. Determination of Active Biomolecules

2.3.1. Extract Preparation

3 g of leaves powder was crushed and extracted with 30 mL of methanol for 24 h at ambient temperature and darkness. After filtration and removal of methanol under reduced pressure in a rotary evaporator, the dry residue was dissolved in absolute methanol (1 g of extract in 10 mL of methanol) for the conservation and analysis.

2.3.2. Determination of Total Phenolic Compounds

The dosage of total polyphenols was determined with Folin-Ciocalteu reagent using the method of Mau et al. (2001) [28]. The coloration produced, including maximal absorption at 760 nm, is proportional to the quantity of polyphenol present in plant extracts. It has been carried out with a spectrophotometer UV-visible. 1 mL of Diluted samples was mixed with 1 mL of Folin reagent and 4 mL of ultra-pure water. After incubation during 3 min at ambient temperature, 10 mL of sodium carbonate solution, Na₂CO₃ (1 M) was added. This mixture was adjusted to a volume of 20 mL by the ultrapure water. The mixtures were incubated for 90 mn. To ensure the reliability of the results, the dosage of each Phenolic compound was realized in three tests. The phenol standard used is the gallic acid. The levels of total polyphenols were expressed in mg equivalent of gallic acid per g of dry weight of plant.

2.3.3. Determination of Flavonoids

The determination of flavonoids concentration (on the same previous extracts) was carried out by Djeridane et al. (2006) [29] method. The method is based on the formation of a complex aluminum-flavonoid with yellow color in the presence of the soda. This staining absorbs in the visible at 430 nm. 1 mL of each diluted sample was mixed with 1 mL of aluminum chloride solution AlCl₃ (0.3 M), the mixtures were incubated for 15 mn. The concentrations of flavonoids were expressed in mg equivalent of rutin per g of dry weight of plant. Rutin is the used standard flavonoid.

2.4. Antioxidant Activity

2.4.1. DPPH Test

Samples (300 µL of methanolic extract) of the 2 variety of O. basilicum at various dilutions (500, 200, 50 and 10 μg/mL⁻¹) were mixed with 900 µL of DPPH solution (4 × 10⁻⁵ M). After incubation 60 mn in the dark at 37 °C, the absorbance was performed at 517 nm with a spectrophotometer UV-visible. The wavelength of maximum absorbance of DPPH was recorded as A_(sample). A blank experiment was also carried out applying the same procedure to a solution without test material and the absorbance was recorded as A_(blank). The free radical-scavenging activity of each solution was then calculated as percent inhibition according to the following equation: (% inhibition = ((A_(blank) − A_(sample))/A_(blank)) × 100).

Antioxidant activity of standard or samples was expressed as IC₅₀, defined as the concentration of the test material required to cause 50% decrease in initial DPPH concentration. Ascorbic acid was used as a standard. All measurements were performed in triplicate.

2.4.2. Carotene Bleaching Test

The b-carotene method was carried out according to Wettasinghe and Shahidi (1999) [30]. Two milliliters of b-carotene solution (0.2 mg/mL in chloroform) were pipetted into a round-bottomed flask containing 20 µL linoleic acid and 200 µL Tween 20. The mixture was then evaporated at 40 °C for 10 min to remove the solvent, the addition of distilled water (100 mL) followed immediately. After agitating the mixture, 1.5 mL aliquot of the resulting emulsion was transferred into test tubes containing 150 L of extract and the absorbance was measured at 470 nm against a blank (emulsion without b-carotene). The tubes were placed in a water bath at 50 °C and the oxidation of the emulsion was monitored by measuring absorbance at 470 nm over a 60 min period using spectrophotometry. The same procedure was repeated with the synthetic antioxidant, butylated hydroxytoluene (BHT) as positive control. The antioxidant capacity (AA%) of the solutions tested was calculated as: AA% = (b-carotene content after 2 h assay/initial b-carotene content) _100. An extract concentration providing 50% inhibition (IC50) was obtained plotting inhibition percentage versus extract solution concentrations.

2.5. Antifungal Activity

To estimate the antifungal activity of essential oil and extracts of 2 varieties of O. basilicum against the genus Aspergillus, we adopted the disk scattering method for essential oil and the well method for extracts. In both varieties, antifungal activity of HEs and extracts was tested during the flowering stage. To study the antifungal activity of 2 varieties, a PDA medium (Potato dextrose agar) was prepared, which is a favorable environment for the growth of fungi used for determining the antifungal activity of oils and extracts. It is prepared by dissolving 200 g of potato (cut into small pieces) hot and stirring in 1 tree of water. The resulting filtrate from this mixture is divided into 4 erlens to which 5 g of agar and 5 g of sugar are added. The environment undergoes autoclave sterilization for 15 min. The discs (6 mm in diameter) impregnated with HE (10-L) was deposited aseptically on the PDA medium (20 mL). However, extracts require dilution with DMSO (Dmethoyl sulfoxide) to eliminate the risk of methanol cytotoxicity before testing their activities. The strains tested are isolated from different sources, Aspergillus carbonarius (isolated from oats), Aspergillus teurreus (wheat), Aspergillus flavus (grape), and Aspergillus niger (barley).

To have a final concentration of 106 spore/mL one takes the following volumes: A. carbonarius 1.875 L, A. niger 75-L, A. flavus 167-L and A. Terrors 58 L. The experiment is repeated 2 times for each HE and for each fungal species. Antifungal activity is determined after incubation for 24 h at 37 °C by measure of inhibition zone diameter including disk diameter. The results obtained with a control box, or the disc is soaked by distilled water are compared.

2.6. Statistical Methods

The results were presented as the means ± SEM. To better compare the means of characters between varieties and phenological stages, we conducted analysis to two classification criteria on polyphenol flavonoid and antioxidant activities levels in both varieties. Means of varieties were the mean of the overall observations allowing defining the gap between the different varieties. A comparison of the means by Duncan test was also performed (SAS software v.6.2). A correlation tests (with SAS program) has bine conducted within phenol and flavonoids. To better estimate the divergence between the varieties based on the chemical markers, we conducted analyzes in main components (PCA) (SAS program 2006) [31]. Correlation analyses of all parameters were also carried out using the correlation and regression program in the EXCEL stat.

3. Results and Discussion

3.1. Phenological Stage Affcetd the Yield of Essential oil of Two O. basilicum Varity

The variation of essential oil yield at different phenological stages may be due to genetic factors, developmental stages, plant origin, harvesting, drying and storage methods, extraction and analysis methods [22]. In this present investigation, the content of essential oils (EO) from the two varieties of O. basilum L. (Fino Verde and Genovese) reveals a significant variation in yield over two growth stages. In fact, Fino Verde variety showed the highest yield during the two phenological stages, where it reached 0.7% during the vegetative stage to 0.9% at flowering one. The results are similar to those reported in previous studies [22,32]. In more details, low yield of basil EO in O. basilicum in Tunisia (0.2%) and in locurance [32,33]. On the other hand, O. basilicum grown in India (0.1 to 0.74%), Oman (0.17%), Pakistani basil (ranging from 0.5 to 0.8%) and Egypt (1.7%) [29,30,34]. These differences could be explained by environmental factors in the culture environment (climatic and edaphic conditions) and genetic factors [30,35]. In this context, the climate, the geographical area, the organ used of the plant, the drying period, the extraction method adopted, can have a direct impact on yields and composition in EO [36,37]. Both varieties are grown under the same environmental conditions, indicating that this difference in yield level is attributed to genetic factors [30].

Moreover, the choice of harvest period is paramount in terms of the yield and quality of essential oils [38]. The flowering stage is the richest in EO than the vegetative stage, the high yields obtained at flowering for both varieties of O. basilum studied. Similar results were reported in for Artemisia pallens [39], Thymus vulgaris and Hyptis suaveolens [40] for Origanum majorana [41] and Rosmarinus officinalis L. [42]. The variation in HE yields during plant growth is also verified for Origanum vulgare ssp. hurtim [43]. Mohammadreza [44] reported a variation in the quantity and quality of HE obtained for Artemisia annua during the different stages of growth. Hamrouni et al. [41] suggest that low yield at the vegetative stage is mainly attributed to low biosynthesis of volatile compounds, due to inactivation of enzymes responsible for the synthesis of some compounds. Zaouali et al. [42] also attributed the variation in yields during plant growth to physiological changes in the organs. Indeed, the high yield obtained in flowering is mainly due to flowers and the conversation of apical meristem in floral merits increases the secretion of essential oils. The phenological stages of medicinal and aromatic plants can have a significant impact on the essential oil yield, as evidenced by previous studies synchronizing the preferential essential oil accumulations indicated by high levels in the flowering stage [41,42], which quickly declined thereafter, whereas other studies show that the lowest essential oil yield was found in the vegetative stage. It’s possible that the low amount of volatile compound biosynthesis during the vegetative stage is due to partial inactivation of enzymes required for certain component biosynthesis. While the highest rate in the flowering stage is strongly linked to the high production of biomass and oil content [42].

3.2. Essential Oil Composition of the Tow O. basilicum Variety Was Affected by Developmental Stage

The phenological variations on the chemical composition of essential oils from O. basilicum varieties have been renovated in a striking way, whereby it is conceivable that these changes are accompanied by the modifications of the secondary metabolism. The hypothesis put forward corroborates with some study in which it was reported that the effect of different phenological stages on the essential oil and its composition may be due to its influence on the enzymatic activity and metabolism of the essential oil production [45]. In the present study, the result of the GC/MS analysis of the essential oil composition of two varieties of O. basilicum reveals a wide variation at both growth stages (Table 1). The HeD of the Fino Verde variety consisted of 22 compounds, representing 71.68% of the total oil during the vegetative stage, where hydrocarbon sesquiterpenes were the main fraction (51.34%), with high levels of germacren D (10.07%), bicyclogermacrene (6.07%) and elements (4.88%). Moreover, oxygenated monoterpenes (11.35%), hydrocarbon monoterpene (5.22%), oxygenated sesquiterpenes (3.50%), phenolic compounds (0.53%) esters (3.32%) were presented in lower amount (Table 1). During the flowering stage, 40 compounds representing 98.01% total EO were identified. Oxygenated monoterpens (78.4%) were the majority chemical class represented by linalool (40.1%), the 1.8 cineole (30.96%) and bronylactate (5.26%). On the other hand, sesquiterpenes hydrocarbons (11.56%), esters (6.6%) hydrocarbon monoterpene (1.26%), oxygenated sesquiterpenes (0.58%), phenolic compounds (3.96%), alcohols (0.27%) and acids (0.20%) were presented in low proportions.

Regarding Genovese variety, analysis of the chemical composition at the vegetative stage revealed the presence of 26 compounds of 82.54% of the total EO. The highest fraction was the oxygenated monoterpenes (22.19%) represented mainly by linalool (15.18%) and 1.8 cineole (6.36%), sesquiterpenes (18.71%), esters (15.42%), hydrocarbon sesquiterpenes (14.68%), hydrocarbon monoterpenes (8.42%), phenolic compounds (2.59%) and alcohol (0.44%). About 15 compounds representing 95.6% total EO were recorded during flowering stage, the major fraction of which esters (64.69%) were represented mainly by methyl cinnamate (64.69%). The oxygenated mono terpenes (16.83%) were mainly represented by linalool (12.7%). In contrast, hydrocarbon sesquiterpenes (10.1%), phenolic compounds (3.62%), and hydrocarbon monoterpenes (0.36%) are relatively negligible. Methyl cinnamate (esters) is present in the Genovese variety during both stages of growth, as a major compound of EO, is detected in low level for Fino Verde variety EO. Vernin metzger [45] reported that meyhl cinnamate is a major component of O. basilicum EO grown in India, Guatemala and Pakistan.

Our results are relatively different from the chemical composition of O. basilicum grown in Tunisia, where methylchavicol and linalool as a major component [32]. Linalool is the major compound for sweet basil from Albania [46], Omani [34,47] and Turkey [48], Korea [49]. Parkaystha and Nath (2006) [50] reported that the major constituents of O. basilicum L. from India are comphor, limonene and β-selinene [51], the majority compounds of O. basilicum L.Eo from Turkey are methyl eugenol. Other work in the world has shown the existence of different chemotypes of EO of O. basilicum. Indeed, Saliou et al. (2012) [52] found that the major constituents of EO of O. basilicum of the Republic of Senegale are estragol. This difference in the EO composition of O. basilicum L. is explained by the variation in environmental conditions (climatic, seasonal, geographical) and genetic factors [53,54].

Analysis of essential oils of the Tunisian variety revealed the presence of chemical compounds that have not been identified as habitual constituents of O. basilicum in Mediterranian region such as the patchouléne beta, the pulegone, and the Bicyclogermacrene.

Vieira et al., 1990 [55], showed that O. basilicum flowers are richer in EO compounds than leaves. Our results are similar to other works for Mentha piperita [56], Salvia officinalis [57] and Coriandrum sativum [58], which have confirmed that the physiological stage of growth affects EO composition and yield. Essential oil composition depends on the stage of development, genetic factors and ecological conditions of growth. However, a correlation between morphological characteristics and chemotypes has been detected for O. basilicum varieties [14]. Indeed, Sénatore et al., 2000 [59] attributed the variation in terpene composition to the combination of genetic factors that are related to the reproduction modality of the plant. In fact, the effect of genotype-medium interaction may play an important role in this variation.

3.3. How Developmental Stages Affect Polyphenol and Flavonoid Levels

Compounds that contribute to the total antioxidant activity in O. basilicum are numerous and phenolic compounds, mainly flavonoids which are a group of phenolics. These two compounds are thus important in assessing their quality.

The content phenolic compounds in the plants methanolic extracts depends essentially on the variety, stage of growth, growing and harvesting season, climatic and environmental conditions, geographical location and the different diseases that can affect the plants [60]. During both stages of growth, the highest content of phenolic compounds were found in the methanolic extract of the Genovese variety. The highest level of flavonoids was also observed in the Genovese variety varying from 16.11 mgER/gDW at the vegetative stage to 16.46 mgER/g DW at flowering. Moreover, the concentration of total phenolic compounds was around 42.81 mgEAG/gDW during the vegetative stage and 53.14 mgEAG/gDWat flowering stage (Figure 1). On the other hand, the Fino Verde variety showed lower level of phenolic compounds during the two vegetative and flowering stages (23.28 mgEAG/g DW and 16.81 mgEAG/g DW respectively) (Figure 1).

This is the first report in which the effect of the phenological stage on total phenol content has been analyzed, and the results show that this content was increased at the flowering stage. According to the results, it is appeared that the season in which O. basilicum vegetative develop influences changes in phenolic compounds content. Nevertheless, the developmental conditions, as well as the cultivar analyzed, are decisive factors influencing antioxidants like phenolic compounds. Similarly, flavonoids levels were relatively low for the Fino Verde variety, varying from 8.57 mgER/gDW at the vegetative stage to 10.67 mgER/g DW at the flowering stage (Figure 1). The highest flavonoid contents are observed during flowering for both varieties (Figure 1).

ANOVA analysis of variance with 2-way criteria (variety effects and stage of growth) shows significant variations between the 2 varieties (p < 0.05). And the growth stage effect is significant for both varieties (p < 0.05) (

Table 2. Correlation test between all parameters measured for the two varieties of O. basilicum., (*) (p < 0.05), ns: non significative (p ˃ 0.05).

Variety		Flavonoids	DPPH	β-Carotene
Basilicum v. Fino Verde	polyphenols	−0.25618 ns	0.781 ns	−0.577 ns
	flavonoids		0.791 *	−0.84 *
Basilicum v. Genovese	polyphenols	0.689 *	0.572 ns	−0.858 *
	falvenoids		−0.747 ns	0.435

). These results agree with those found by Kwee et al. (2011) [41] for the different varieties of O. basilicum grown in America. In this context, Kwee et al. (2011) [61] and Javanmardi et al. (2003) [62] reported the same level of polyphenols in basil that grown in America (17.58 mg EAG/g DM) and in Iran (ranging from 22.9 to 65.5 mg EAG/g DM). On the other hand, different levels of phenolic compounds in O. basilicum grown in other countries are reported. For example, O. basilicum grown in Poland and Romania are relatively higher compared to our study. These differences in polyphenol content are probably related to the variation in the bioclimatic and edaphic conditions of the growing region.

Regarding the growth stages, the highest content of polyphenols was recorded in Fino Verde variety, at the vegetative stage (23.28 mgEAG/gDW) (Figure 1). These results are similar to those found by Zaouali et al. (2013) [42] for Rosmarinus officinalis. On the other hand, the highest polyphenol content in the tissue of Genovese variety was reported at the flowering stage (53.14 mgEAG/gMS). These results agree with those found by Hamrouni et al. (2009) [41] and Chauhan et al. (2013) [63] for Origanum majorana (Asteraceae). Study of Males et al. (2003) [64] stated that Crithmum maritimum (Apiaceae) has the highest leaves of polyphenols at the flowering stage. Similarly, Ayan et al. (2007) [65] also suggested that the phenolic content reached its maximum at the flowering stage for Hypericum hyssipophylum and Hypericum scabrum. This variation in phenolic concentration during the 2 stages can be explained by physiological and climate factors [38]. In this regard, Zaouali et al. (2013) [42] showed a differential accumulation of phenol in the leaves relative to specific tissues and cells (mesophile, epidermis, cuticle, chloroplast) and to physiological changes during growth. The accumulation of phenolic compounds during the vegetative stage allows the plant to prepare for the budding phase and maintains better protection during this stage, for instance phenols are involved in the mechanisms of defense and attraction of pollinators [33]. The accumulative abilities of bioactive compounds such as flavonoids in plants appear to produce marginality due to phenological stage synchronization, implicating specific organs in relation to others. Indeed, in accordance with the intrinsic capacities of the organ migration, each phenological stage accumulates varying amounts of bioactive chemicals [30]. This theory of bioactive molecules has been well-documented, with plant age being a factor in determining the composition of bioactive compounds in both qualitative and quantitative dimensions.

Correlation between total polyphenols and flavonoids contents for the Fino Verde variety is not significant for the two stages of growth (r = −0.256, n = 6, p ˂ 0.624) (Table 2). Indeed, the contents of polyphenols vary inversely with the contents of flavonoids. During both stages of growth, the flavonoid content does not depend on the polyphenol concentration. However, for the Genovese variety, a positive and significant correlation was shown during the two stages (r = 0.689, n = 6, p < 0.012) (Table 2). The variation in the content of flavonoids depends on the content of phenolic compounds. These differences between the two varieties of O. basilicum in terms of their content of polyphenols and flavonoids are explained by genetic factors [66].

3.4. Antioxidants Activities

3.4.1. DPPH Test

It is well known that O. basilicum has a strong antioxidant activity [67]. In our study, both varieties of O. basilicum were also characterized by strong antioxidant activities at the two stages of growth (

Table 3. DPPH activity and β-carotene content of two varieties in different stage. Values are represented by mean ± standard deviation of at least three independent replicates. Within the same stage, different letters on the bars indicate significant differences at p < 0.05.

		Fino Verde Variety	Genovese Variety
Vegetative	IC50 DPPH (µg/mL)	110 ± 10 a	6.13 ± 4.8 b
stage	IC50 β-carotene (µg/mL)	460 ± 20 a	400 ± 12 a
Flower stage	IC50 DPPH (µg/mL)	153.33 ± 30.5 b	133.33 ± 23.09 b
	IC50 β-carotene (µg/mL)	566.66 ± 23.09 a	560 ± 40 a

). These results are comparable to those found for Malaysian O. basilicum [67]. The two varieties were differentially responded at the vegetative stage, where the highest and lowest anti-free radical activity were recorded for Genovese variety (6.13 ± 4.8 μg/mL) and Fino Verde variety (110 ± 10 µg/mL) (Table 3), respectively. Moreover, the Genovese variety was characterized by the highest antioxidant activity also at flowering stages (IC50: 133.33 ± 23.09 µg/mL). In agreement, the study of Tarchoune et al. (2012) [11] stated high antioxidant activity in Genovese variety at flowering stage. The analysis of variance with two-way classification criteria (variety effects and growth stage) shows significant differences between the two varieties (p < 0.05) and a stage effect. A significant growth at (p ˂ 0.001) (Table 3). Furthermore, in Fino Verde and Genovese varieties, there was no significant correlations (r = −0.146, p < 0.78 and r = 0.572, p < 0.23, respectively) between total polyphenols and IC50 values was detected (Table 3). Likewise, the correlations were positive and not significant between the IC50 values and the flavonoid contents (r = 0.791, p < 0.060, r = −0.024, p < 0.963, respectively) (Table 3). Indicating that the increases in antioxidant activity was not dependent on phenolic and flavonoid compounds. Our results are similar to those found by Tarchoune et al., 2012 [11] who suggested that the lack of correlation between the phenolic compounds of basil in Tunisia and the total antioxidant activity. Tarchoune et al., 2012 [11] also indicated levels of single antioxidant does not necessarily reflect their total antioxidant activity and the change in antioxidant capacity depend on the synergistic and redox interactions between several antioxidant molecules such as chlorophylls which contributed by around 40% of basil’s antioxidant activity. Similarly, Janvanmardi et al. (2003) [62] found that the antioxidant activity for O. basilicum grown in Iran was not limited to phenolic compounds, and increased antioxidant capacity was explained by the accumulation of other secondary metabolites, such as carotenoids, and vitamins, which contribute 29% of antioxidant capacity. These correlations are also based on structure and the interaction of these antioxidants [68]. Koleva et al. (2002) [69] claimed that the antioxidant activity mainly uses lipophilic compounds (apolar pigments, neutral lipids, fatty acids, phospholipids, etc.). Our antioxidant activity results along the phenological stages show for the first time that the contribution of both fractions is inversely proportional as O. basilicum develops. Indeed, the increase in antioxidant activity along the phenological stage could be related to increases in phenolic compounds. On the other hand, the drastic decrease in antioxidant activity levels seem to be mediated by the loss of some lipophilic compounds along the phenological stages studied [68,69]. Furthermore, enhanced O. basilicum at the fully flowering stage of both O. basilicum varieties reflects the nutritional and functional importance of consuming the leaves at this stage.

3.4.2. β-Carotene Bleaching Test

O. basilicum has good antioxidant activity against β-carotene discoloration [62]. Here, Genovese variety was characterized by the lowest IC50 values during the vegetative stage (400 µg/mL) and flowering (560 ± 40 µg/mL) (

Table 4. ANOVA to two-way analysis of variance (variety and phenological stage) performed to different performed parameters measured for the two O. basilicum varieties. (**) (p < 0.001), (*) (p < 0.05), ns: non significative (p ˃ 0.05).

	Variation	CM	SC	ddl	F	Probability (p)
HE	Variety	0.607	0.6283	1	7.25	0.024 *
	Stage	0.02	0.7541	1	0.25	0.63 ns
	Interaction	0.45	1.3823	1	3.75	0.065 ns
Phenol	Variety	2287.48	111.916	1	16.99	0.002 *
	Stage	7.52	37.2462	1	0.06	0.018 *
	Interaction	0.65	149.162	1	8.52	0.008 *
Flavonoïds	Variety	99.93	111.916	1	24.15	0.008 *
	Stage	11.98	37.2462	1	2.89	0.123 ns
	Interaction	0.75	149.1626	1	13.52	0.001 *
IC50 DPPH	Variety	11,433.01	33,142.026	1	12.24	0.006 *
	Stage	21,709.01	8406.053	1	23.24	0.0009 **
	Interaction	0.79	49,466.66	1	17.74	0.0008 **
IC50β-Carotene	Variety	1200	2400	1	0.23	0.643 ns
	Stage	1200	47,066.66	1	0.23	0.643 ns
	Interaction	0.048	49,466.66	1	0.23	0.799 ns

). In contrast, Fino Verde variety was characterized by the relatively highest IC50s during the two stages, vegetative (460 ± 20 µg/mL) and flowering (566.66 ± 23.09 µg/mL) revealing a slightly lower antioxidant activity (Table 4). This also indicating that, the protective role was more powerful in the Genovese variety, which effectively protects the β–carotene against oxidation. Javanmardi et al. (2003) [62] evaluated an important antioxidant capacity of the Iranin O. basilicum according to the β-carotene method (71% inhibition). Seeram et al. (2006) [70] also showed that the antioxidant activity of phenolic compounds was mainly due to their redox properties (ROS trapping) and may have metal chelation potential (capture of oxidizing metal ions). Two way analysis of variance (variety effects and growth stage) performed on the β-carotene bleaching test revealed a non-significant variation between the two stages and the two varieties (p ˃ 0.05) (Table 4). On the other hand, a significant correlation test between IC50 values of b-carotene bleaching test and polyphenol contents in the Fino Verdevariety (r = −0.858, p < 0.028) (Table 3). The percentage of inhibition of antioxidant activity by the β-carotene/linoleic acid system was proportional to the concentration of phenolic compounds. Indicating that phenolic compounds strongly contributed in β-carotene bleaching. These flavonoids have good activity, are considered first-class antioxidant agents, and can be used for therapeutic applications, knowing that antioxidants contribute very effectively to the prevention of diseases such as cancer and cardiovascular disease [71]. On the other hand, for the Genovese variety the correlation is negative and significant between the IC50 values and polyphenol contents (r = −0.858, p < 0.0286) (Table 3). The anti-radical power was proportional to the concentration of phenolic compounds. Concerning flavonoids, the correlation is negative and not significant (r = −0.68, p < 0.129). Antioxidant activity revealed by both varieties (Genovese and Fino Verde) was similar to previous work in species of Lamiaceae family [72,73,74]. Antioxidant activity depends on the substrate used and that the linoleic acid used in the β-carotene method can influence the degree of dissociation of acidic antioxidants [69].

3.4.3. Antifungal Activity

O. basilicum is rich in essential oils (EOs) and phenolic compounds. These compounds are well known for their antifungal activity [75]. In our study, the essential oil of O. basilicum showed strong inhibitory activity against all tested Aspergillus species (

Table 5. Antifungal activity of essential oil of two O. basilicum varieties in flower stage; values are represented by mean ± standard deviation of at least three independent replicates. Within the same variety, different letters on the bars indicate significant differences at p < 0.05.

	Inhibition Zone Diameters (mm)
	Aspergillus flavus	Aspergillus niger	Aspergillus teurreus	Aspergillus carbonarius
Fino Verde variety	15 ± 0.7 a	20 ± 0.5 a	10 ± 0.2 b	30 ± 0.2 a
Genovese variety	10 ± 0.3 b	20 ± 0.2 a	15 ± 0.3 a	20 ± 0.1 b

and Figure 2). However, the two varieties showed differential sensitivity to tested microorganisms. Fino Verde variety had the greatest activity against A. carbonarius (inhibition zone of 30 ± 0.2 mm) followed by A. niger (20 ± 0.5 mm). Moderate activity against A. flavus (inhibition zone of 15 ± 0.2 mm) and relatively low activity against A. teurreus (5 ± 0.7 mm) were reported. Moreover, A. carbonarius and A. niger are the most sensitive to EOs of the Genovese variety (20 ± 0.1 and 20 ± 0.2 mm respectively), however these EOs have a low activity aginast A. teurreus (15 ± 0.3 mm) and A. flavus (10 ± 0.3 mm). The EO of the Fino Verde variety gave the best result for reduction of spore’s germination of pathogenic fungi tested (A. carbonaris, A.niger, A.flavus). This important antifungal activity can be explained by the fact that the bioactive compounds of these plants may differ in quantity and quality among different varieties [76]. The sensitivity of microorganisms to EOs depends on EOs composition and the microorganism types [77]. Janvanmardi et al. (2003) [62] showed that the EO of O. basilicum from Oman inhibits the growth of A. niger (the inhibition diameter of 35 mm). Also, Jeff-Agboola et al. (2012) [76] proved that EOs of Nigerian O. basilicum possesses an important antifungal property against A. flavus, it caused the cytoplasm retraction, restriction of mycelium and prevented spores’ germination. The antifungal activity of O. basilicum EOs against the 4 microorganisms was mainly attributed to their major compounds [32,78,79]. In this regard, Eos acted on the cell membrane of strains and lead to their destruction. Phenolic terpenes work by binding to amino and hydroxylamine groups of fungal membrane proteins impaired permeability and leakage of intracellular constituents [80,81]. Basilicum EO therefore inhibits the production of mycotoxins by Aspergillus [82]. Moreover, they caused morphological changes in Aspergillus sporulation and pigmentation, and induced hyphal destruction [83]. Minority compounds also play an important role in the activity of EOs and appear to act synergistically with the main compounds [84].

4. Conclusions

Large variations of essential oil yield and composition were observed between the both growth stages (vegetative and flowering stages) of O. bacilium. Higher yield was noticed at flowering stage. Both varieties are rich in essential oils and phenolic acids which is related to the strong measured antioxidant and antifungal properties of both varieties. Besides, depending on the phenologic stage, a considerable difference in phenolic content was discovered between the two kinds. In fact, during flowering, both cultivars contained high level of flavonoids. The Genovese variety showed the highest overall polyphenol content. Futhermore, the antifungal activity of EOs of both varieties was revealed against the 4 selected microorganisms. However, this antifungal activity depend on varieties and could be attributed to thier major compounds. Our findings support the potential of using the EOs of O. bacilium as natural antimicrobial in food and pharmaceutical industries. These basil chemotypes may be agronimacaly valorised and may therefore be desirable for plant breeding programs in which growers seek to optimize phenolic levels.