Chemical variability of Centaurium erythraea essential oils at three developmental stages and investigation of their in vitro antioxidant, antidiabetic, dermatoprotective and antibacterial activities

Industrial Crops & Products 132 (2019) 111–117

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Chemical variability of Centaurium erythraea essential oils at three developmental stages and investigation of their in vitro antioxidant, antidiabetic, dermatoprotective and antibacterial activities

T

Abdelhakim Bouyahyaa, , Omar Belmehdib, Meryem El Jemlic, Ilias Marmouzic, Ilhame Bouraisa, Jamal Abrinib, My El Abbes Faouzic, Nadia Dakkaa, Youssef Bakria ⁎

a

Laboratory of Human Pathologies Biology, Department of Biology, Faculty of Sciences, and Genomic Center of Human Pathologies, Faculty of Medicine and Pharmacy, Mohammed V University of Rabat, Morocco Biology and Health Laboratory, Department of Biology, Faculty of Science, Abdelmalek Essaadi University, Tetouan, Morocco c Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy, Mohammed V University of Rabat, Morocco b

ARTICLE INFO

ABSTRACT

Keywords: Centauruim erythraea Essential oils Antidiabetic Dermatoprotective Antioxidant Antibacterial

This work is aimed the study of volatile compounds of Centauruim erythraea Raphin at three developmental stages (vegetative, flowering and post-flowering) and the investigation of their in vitro antioxidant, antidiabetic, dermatoprotective and antibacterial properties. The chemical composition of Centauruim erythraea essential oils (CEEO) was determined using GC/MS analysis. 39 volatile compounds were identified, belonging mainly to oxygenated monoterpenes, 51.63%, 44.10% and 53.69% at the vegetative, flowering and post-flowering stage, respectively. Menthol, carvacrol and tricosane were the main compounds of CEEO at the three developmental stages. The antioxidant effects of CEEO were determined by DPPH, FRAP and ABTS assays. CEEO at the flowering stage showed the best antioxidant effects by an IC50 = 47.18 ± 3.62 μg/mL, IC50 = 53.25 ± 2.19 μg/mL, IC50 = 65.34 ± 3.71 μg/mL determined by DPPH, FRAP and ABTS assays, respectively. The in vitro antidiabetic effect was evaluated by α-amylase and α-glucosidase enzymes inhibitory. CEEO at vegetative stage demonstrated a remarkable α-amylase (IC50 = 31.91 ± 0.336 μg/mL) and α-glucosidase (IC50 = 56.77 ± 1.02 μg/ mL) inhibitory activities. Moreover, CEEO at the flowering and post-flowering stage strongly inhibited the tyrosinase enzyme with IC50 of 41.863 ± 0.031 μg/mL IC50 = 49.183 ± 0.298 μg/mL, respectively. The antibacterial effects were evaluated by determining the diameters of inhibition, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). CEEO showed important antibacterial inhibition at three seasonal stages and particularly against Staphylococcus aureus and Listeria monocytogenes. Moreover, CEEO at post-flowering stage presented bactericidal effects especially against S. aureus (MIC = MBC = 0.125% (v/v)), L. monocytogenes (MIC = MBC = 0.125% (v/v)), and Proteus mirabilis (MIC = MBC = 0.125% (v/v)). The results obtained in this study show that the biological properties of CEEO are mainly depending to phenological stages, during which volatile bioactive synthesis occurs.

1. Introduction

synthesized by so-called aromatic plants as secondary metabolites to execute certain functions such as defense against microorganisms and cellular stress (Bakkali et al., 2008). In recent years, a particular interest has been given to essential oils and their derivatives. Indeed, several works have shown today that essential oils have numerous biological properties such as antioxidant, antibacterial and antidiabetic activity (Burt, 2004). The synthesis of essential oils is influenced by several parameters such as the plant in question, the part used, the geographical situation and the phenological stage (Aboukhaled et al., 2017; Bouyahya et al., 2017a).

The search for new bioactive molecules require the investigation of a large number of substances from different regions such as synthetic products, natural molecules and those derived from biotechnology. In this context, medicinal plants are an inexhaustible source for dissecting new bioactive molecules. Indeed, many drugs come from the products of medicinal plants (Lahlou, 2013). Among medicinal plants derived natural products, the volatile compounds, especially essential oils are of interest. These molecules are

⁎

Corresponding author. E-mail address: [email protected] (A. Bouyahya).

https://doi.org/10.1016/j.indcrop.2019.01.042 Received 8 September 2018; Received in revised form 19 January 2019; Accepted 22 January 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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Centaurium erythraea (C. erythraea) is a plant that belongs to the family of Gentillaceae. This species is widely used in traditional Moroccan medicine to treat certain diseases such as diabetes, digestive illnesses, hepatitis, asthma, allergy, and rheumatism (Jouad et al., 2001; El-Hilaly et al., 2003; Fakchich and Elachouri, 2014; Bouyahya et al., 2017b). It is also in the traditional pharmacopeia of other countries for the treatment of diabetes and diseases related to oxidative stress (El Hamsas El Youbi et al., 2016). Several studies have shown that the organic extracts of this plant are endowed with valuable pharmacological activities, in particular their antidiabetic, antioxidant, antihyperglycemic activity, diuretic effects and antimicrobial effects (Haloui et al., 2000; Valentão et al., 2001; Jerković et al., 2012; Benhamza et al., 2013; Bouyahya et al., 2017c). However, essential oils of C. erythraea are poorly studied concerning their biological effects. The studies carried out tested the essential oils only in flowering stage, whereas the chemical composition varies between the phenological stages of the same plant. In this context, we studied the variation of the chemical composition of the essential oils of C. erythraea according to the stages of development (vegetative, flowering, post-flowering), thus the investigation of their antioxidant, antidiabetic, dermatoprotective and antibacterial properties.

different concentrations of CEEO at three phenological stages with αamylase enzyme and starch solution (Hashim et al., 2013). A mixture of 250 μL of samples and 250 μL of 0.02 M sodium phosphate buffer (pH = 6.9) containing the enzyme α-amylase (240 U/mL) were incubated at 37 °C for 20 min. Then, 250 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH = 6.9) were added to the reacting mixture. Therefore, the reaction mixture was incubated at 37 °C for 15 min. Thereafter, 1 mL of dinitrosalicylic acid (DNS) was added and the reaction mixture was then incubated in a boiling water bath for 10 min. Then, the reaction mixture was diluted by adding 2 mL of distilled water, and absorbance was measured at 540 nm in the spectrophotometer. Acarbose was used as positive control. 2.5.2. α-Glucosidase inhibitory assay The α-glucosidase inhibitory activity of CEEO at three phenological stages was determined using the substrate pNPG according to the method described by Kee et al. (2013), with some modification. Briefly, a mixture of 200 μL of the samples and 100 μL of 0.1 M sodium phosphate buffer (pH = 6.7) containing the enzyme α-glucosidase solution (0.1 U/mL) was incubated at 37 °C for 10 min. After preincubation, 200 μL of 1 mMpNPG solution in 0.1 M sodium phosphate buffer (pH = 6.7) was added. The reaction mixtures were incubated at 37 °C for 30 min. After incubation, 1 mL of 0.1 M of Na2CO3 was added and the absorbance was recorded at 405 nm using the spectrophotometer. The α-glucosidase inhibitory activity was expressed as percentage inhibition, and the IC50 values were determined. Acarbose was used as positive control.

2. Materials and methods 2.1. Chemicals and reagents p-Nitrophenyl-α-D–D-glucopyranoside (p-NPG), 3,4-dihydroxy phénylalanine (L-DOPA), Acarbose, 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 3,4dihydroxyphénylalanine (L-DOPA), and ascorbic acid were purchased from Sigma-Aldrich (France). α-glucosidase from Saccharomyces cerevisiae, αamylase from Bacillus licheniformis. All other reagents were obtained from commercial sources.

2.6. Dermatoprotective activity 2.6.1. Tyrosinase inhibitory assay The tyrosinase inhibitory activity was performed to evaluate the dermatoprotective effect following the method described by Batubara et al. (2010). Briefly, the sample (25 μL) was added to the tyrosinase solution (100 μL, 333 unit/mL in phosphate buffer 50 mM, pH 6.5) and kept at 37 °C for 10 min. Then, 300 μL of substrates (L-DOPA, 5 mM) were added. After 30 min of incubation at 37 °C, the absorbance was determined at 510 nm using a spectrophotometer. The percent inhibition of tyrosinase activity was calculated at the concentrations of 40, 60, 120 and 160 μg/mL, and the 50% inhibitory concentrations were calculated (IC50). Quercetin was used as a positive control.

2.2. Plant collection and essential oils extraction Centauruim erythraea was collected from the North-West of Morocco (Zoumi area 2016) at three developmental stages (vegetative stage, flowering stage and post-flowering stage). The sample were collected in May (2016) and dried under dark. CEEO were extracted using hydrodistillation method, and then were stored at 4 °C until experimental use.

2.7. Antibacterial activity

2.3. Chemical composition analysis

2.7.1. Bacteria strains The antibacterial effect of CEEO was evaluated against Escherichia coli K12, Staphylococcus aureus CECT 994, Listeria monocytogenes serovar 4b CECT 4032, Proteus mirabilis CECT, Pseudomonas aeruginosa IH and Bacillus subtilis 6633 DSM.

The chemical composition of CEEO was analyzed using GC–MS analysis as described by our previous study (Bouyahya et al., 2017a). The identification of volatile compounds was carried based on literature and retention index (RI). The confirmation of each compound was made by comparison of its mass spectra with those of NIST02 library data.

2.7.2. Determination of inhibition diameters To determine the inhibition diameters of CEEO against the tested bacteria, we have used agar-well diffusion assays as described by Bouhdid et al. (2008). The results; are expressed as inhibition zones; were measured in millimeters around the wells.

2.4. Antioxidant activity assays The antioxidant activity of CEEO at three phenological stages was estimated by three complementarily methods DPPH, FRAP and ABTS assays. All experiments were carried out as described in our previous works (Bouyahya et al., 2018, 2019). The results are expressed as CEEO concentration providing 50% inhibition (IC50) was calculated by plotting the inhibition degrees against the sample concentrations. Trolox and ascorbic acid were used as positive controls. The test was carried out in triplicate and IC50 values were reported as means ± SD.

2.7.3. Determination of Minimal inhibitory concentration (MIC) and Minimal bactericidal concentration The MIC and MBC of CEEO were determined by the broth microdilution assay as described by Bouhdid et al. (2009). Briefly, 50 μL of CEEO at different concentrations were deposed in each well. Then, 50 μL of bacteria at 106 CFU/mL were added to each well. After a period of incubation of 18 h at 37 °C, 10 μL of resazurin were added to each well for accessing bacterial growth. Bacteria growth was revealed by detecting the reduction of blue dye resazurin to pink resorufin after 2 h of incubation at 37 °C. The MIC was determined as the lowest

2.5. In vitro antidiabetic activity 2.5.1. α-amylase inhibitory assay The α-amylase inhibitory potentials were investigated by reacting 112

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Table 1 Chemical composition of C. erythraea essential oils at three developmental stages. N°

RI

1 964 2 1122 3 1135 4 1148 5 1157 6 1166 7 1171 8 1178 9 1184 10 1193 11 1204 12 1237 13 1244 14 1155 15 1279 16 1286 17 1308 18 1321 19 1340 20 1352 21 1374 22 1398 21 1417 22 1431 23 1448 24 1455 25 1462 26 1576 27 1593 28 1617 29 1629 30 1642 31 1658 32 1665 33 1677 34 1691 35 1714 36 1740 37 1753 38 1771 39 1787 Total No identified Monoterpene hydrocarbons Sysquiterpene hydrocarbons Oxygenated monoterpenes Oxygenated Sysquiterpenes Ketones Acids Aldehyde Alcanes

Compounds

β-Thujone Linalool Camphor Menthone Isomenthone Borneol Menthol Terpinen-4-ol p-cymen-8-ol α-terpineol Decanal Pulegone Carvone Piperitone Bornyl acetate trans-anethole Menthyl acetate Thymol Carvacrol α-copaene β-Damascenone β-Bourbonene β-caryophyllene Germacrene D β-bisabolene γ-cadinene δ-cadinene Caryophyllene oxide Hexadecane Tetradecanal α-cadinol Pentadecanal Tetradecanoic acid Octadecane Neophytadiene Hexahydrofarnesyl acetone Hexadecanoic acid Heneicosane Linoleic acid Tricosane Tetracosane

C. erythraea essential oils

Identification

Vegetative stage

Flowering stage

Post-flowering stage

0.83 2.13 1.34 2.52 2.86 1.57 12.74 0.62 1.43 1.26 2.03 2.61 0.53 3.52 1.46 3.52 0.73 1.19 11.60 0.54 1.85 0.78 2.04 1.76 0.63 1.17 0.78 1.45 1.02 0.96 0.74 0.61 1.03 0.82 1.92 2.47 3.64 2.50 2.38 10.12 2.03 98.73 1.27 0.83 7.7 51.63 4.72 4.32 5.07 2.99 16.49

1.73 1.94 2.13 1.82 3.09 1.68 20.82 1.12 0.86 0.96 3.31 0.73 1.17 2.58 0.75 1.42 1.20 0.83 8.73 0.74 1.69 nd 0.74 2.17 1.59 0.92 nd 0.58 2.21 1.63 1.05 1.30 0.79 1.21 0.80 1.03 2.02 2.32 1.83 15.27 1.36 94.09 5.91 1.73 8.15 44.10 3.73 2.72 4.64 4.94 16.49

0.62 1.37 1.94 0.95 2.70 1.76 9.46 1.13 0.58 0.81 1.26 1.94 nd 1.74 0.59 2.07 nd 1.04 25.61 1.22 1.53 0.60 1.87 1.04 0.98 0.64 1.31 0.87 0.66 1.01 0.65 nd 0.64 0.56 2.52 1.83 1.47 3.11 1.63 17.70 1.52 98.29 1.71 0.62 8.28 53.69 4.04 3.36 3.74 2.27 16.49

MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS,

IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR IR

RI: Retention index. Bold values indicates the main representative compounds.

concentration of CEEO that has induced changes in resazurin color. The MBC was then determined by transferred 10 μL from negative subcultures to a non-selective medium and MBC values were deduced after a period of incubation of 24 h at 37 °C (Bouyahya et al., 2017d).

3. Results 3.1. Chemical variability The chemical composition of CEEO at three phenological stages expressed as the percentage of each compound is summarized in (Table 1). Using the GCeMS analysis, 39 compounds were identified at the vegetative stage representing 98.73%, 37 compound at the flowering stage with a total of 94.09%, and 36 elements at the post-flowering stage with a total of 98.29%. These biochemical elements were classified in eight groups: monoterpenes hydrocarbons, sesquiterpenes hydrocarbons, oxygenated monoterpenes, oxygenated sesquiterpenes, ketones, acids, aldehyde and alkanes. The most dominated compounds of CEEO at the three phonological stages were oxygenated

2.8. Statistical analysis Data were analyzed using SPSS 21. The experiments were carried out in triplicates and the results were expressed as the average of the three measurements ± SD. The comparison of means between groups was performed with one-way analysis of variance (ANOVA) followed by Tukey test. Differences were considered significant when p < 0.05

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Table 2 IC50 values (μg/mL) of the antioxidant activities of CEEO at three developmental stages. Values represent means ± SD (standard deviations) for triplicate experiments. Assays

DPPH FRAP ABTS

C. erythraea Essential oils

Controls

Vegetative stage

Flowering stage

Post-flowering stage

Ascorbic acid

Trolox

61.53 ± 2.08 73.42 ± 5.83 134.81 ± 4.73

47.18 ± 3.62 53.25 ± 2.19 65.34 ± 3.71

69.25 ± 4.31 82.06 ± 6.57 101.62 ± 6.74

22.61 ± 1.08 31.63 ± 1.42 44.37 ± 2.32

34.12 ± 2.13 55.25 ± 4.19 54.74 ± 3.85

monoterpenes (51.63% 44.10% and 53.69% at the vegetative, flowering and post-flowering stages, respectively), flowed by alkanes then sesquiterpenes hydrocarbons. In the other hand, the other families existed in low concentrations. The major compounds were carvacrol (11.60%, 8.73% and 25.61% at the vegetative, flowering and postflowering stages, respectively), menthol (12.74%, 20.82% and 9.46% at the vegetative, flowering and post-flowering stages, respectively) and tricosane (10.12%, 15.27% and 17.70% at the vegetative, flowering and post-flowering stages, respectively). The percentage of carvacrol was very high at the post-flowering stage (25.61%) compared to the vegetative and flowering stages (11.60% and 8.73%, respectively). In the other hand, the concentration of menthol was important at the flowering stage (20.82%).

Furthermore, CEEO at vegetative stage showed significantly the highest inhibitory effect of α-amylase and α-glycosidase compared with CEEO at flowering and post-flowering stages with IC50 of 31.91 ± 0.336 and 56.77 ± 1.02 μg/mL, respectively. Moreover, CEEO at this stage was 12 time more active than acabrose. In addition, CEEO at flowering and post-flowering stages showed interesting anti-α-glucosidase activity by inhibition values of IC50 = 87.18 ± 0.422 and IC50 = 71.83 ± 0.72 μg/mL, respectively. 3.4. Dermatoprotective effect The in vitro inhibition of tyrosinase test was used to evaluate the dermatoprotective effect of CEEO at three phenological stages. The results of tyrosinase inhibition by CEEO are expressed as IC50 (Table 3). Remarkably, it was noticed that the CEEO at the flowering stage exhibited significantly the highest inhibitory effect of tyrosinase with IC50 value of 41.83 ± 0.031 μg/mL. Indeed, as listed, the three EOs of C. erythraea showed tyrosinase inhibition significantly higher than quercetin used as standard. In fact, CEEO at flowering stage was six time more active than quercetin. Moreover, CEEO at vegetative and postflowering stages showed important inhibition of tyrosinase with IC50 values of 103.592 μg/mL and 49.183 ± 0.298 μg/mL, respectively.

3.2. Antioxidant activity The antioxidant effects of C. erythraea essential oils at three phenological stages were evaluated by FRAP, DPPH and ABTS methods. The results are expressed in IC50 and listed in (Table 2). As shown in the table, the three essential oils of C. erythraea showed significant antioxidant activity with significant variability between EOs and the used methods. Compared to the positive controls (ascorbic acid and trolox) the CEEO at the three phonological stages possess important antioxidant potential, and by comparison the three types of EOs, it has been noticed that the essential oil at the flowering stage was the most active as antioxidant with antioxidant capacity values of IC50 = 47.18 ± 3.62 μg/mL, IC50 = 53.25 ± 2.19 μg/mL, and IC50 = 65.34 ± 3.71 μg/mL revealed by DPPH, FRAP and ABTS assays, respectively. In addition, by comparing the three methods used, the DPPH scavenging activity assay was the most sensitive showed reduced IC50.

3.5. Antibacterial activity The antibacterial activity of CEEO was tested against six bacterial strains. The results expressed as halo of inhibition are summarized in (Table 4). The results showed that except E. coli, CEEO at the three phonological stages were active against all tested strains. The largest inhibitory zone was obtained against S. aureus by CEEO extracted at the vegetative stage (34 ± 0.5). While the smallest zone was obtained against P. aeruginosa by CEEO obtained at the flowering stage (11 ± 0.5). Importantly, it was noticed that CEEO at vegetative and post-flowering stages showed almost the same antibacterial effect. To

3.3. Antidiabetic activity To evaluate the in vitro antidiabetic effect of CEEO at three phenological stages, α-amylase and α-glycosidase enzymes inhibition assays were used. The results are expressed as the concentration that inhibited the half enzymes activity (Table 3). CEEO at three phenological stages exhibited important inhibitions of α-amylase and α-glycosidase compared with acarbose (α-amylase and α-glycosidase inhibitor standard).

Table 4 Antibacterial activity of C. erythraea essential oils at three developmental stages using agar-well diffusion assay. Values represent means ± SD (standard deviations) for triplicate experiments. Microorganismsa

Table 3 In vitro antidiabetic and dermatoprotective activities of C. erythraea essential oils at three phenological stages. C. erythraea essential oils

α-amylase

α-glucosidase

Tyrosinase

Vegetative stage Flowering stage Post-Flowering stage Acarbose Quercetin

31.91 ± 0.336a 168.62 ± 0.636b 94.99 ± 1.263c

56.77 ± 1.02a 87.18 ± 0.422b 71.83 ± 0.72b

103.592 ± 0.0a 41.863 ± 0.031b 49.183 ± 0.298c

396.42 ± 3.54d –

199.53 ± 2.45d –

– 246.90 ± 1.90d

S. aureus 994 P. aeruginosa L. monocytogenes B. subtilis 6633 P. mirabilis E. coli K12

Inhibition zone diameter (mm ± SD)b Vegetative stage

Flowering stage

Post-flowering stage

34 13 31 24 21 14

28 11 26 23 19 na

33 15 30 27 24 25

± ± ± ± ± ±

0.5 1.33 0.66 0.5 1.5 1.5

± ± ± ± ±

1.5 0.5 2.5 1.5 0.33

± ± ± ± ± ±

1.5 1.5 2.33 0.66 2.0 2.66

SD: standard deviation and values are means ± standard deviation of three separate experiments. na: not active. a Final bacterial density was around 106 CFU/mL. b Diameter of inhibition zone including well diameter of 6 mm, by the agarwell diffusion method at a concentration of 50 μL of oil/well.

In each column, different letters indicate significant differences (p < 0.05; n = 3). 114

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Table 5 The MIC and MBC values of C. erythraea essential oils against bacterial strains using microdilution assay. Microorganisms

C. erythraea essential oils Vegetative stage

S. aureus 994 P. aeruginosa L. monocytogenes B. subtilis 6633 P. mirabilis E. coli K12

Flowering stage

Post-flowering stage

Chloramphenicol (μg/mL)

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

0.25 1 0.25 0.5 0.25 1

0.5 <2 0.5 1 0.5 <2

0.25 2 0.25 1 0.5 1

1 <2 1 2 1 1

0.125 1 0.25 0.25 0.25 1

0.125 1 0.25 0.5 0.25 2

8 64 16 64 64 8

32 64 32 128 64 32

MIC: Minimum inhibitory concentration (as% (v/v)). MBC: Minimum bactericidal concentration (as% (v/v)). Final bacterial density was around 106 CFU/mL.

stage is rich in antioxidant molecules. These data are important allowing specifying the time of harvest if the antioxidant molecules are targeted in these oils. On the other hand, CEEO has also tested against enzymes involved in hyperglycemia and diabetes mellitus. Indeed, the α-amylase catalyses the cleavage of α-(1–4) glycosidic to dextrin, maltose or maltotriose. Whereas, the α-glucosidase catalyses the hydrolysis of 1–4 linked α-glucose and generates the glucose molecules. Therefore, the inhibition of these two enzymes can contribute to decrease the intestinal level of glucose. In our study, CEEO inhibited interestingly the α-amylase and α-glycosidase with some difference between EOs and the tested enzymes. Effectively, at vegetative stage, CEEO revealed the most inhibition than the flowering and post-flowering stages. The obtained difference is certainly due to the difference between chemical components. Previous studies have shown that organic extracts of C. erythraea possess in vitro and in vivo antidiabetic effects (Stefkov et al., 2014; Hamza et al., 2015). However, CEEO have not yet been tested for their in vitro antidiabetic activity. In our study, CEEO revealed a remarkable inhibition of α-amylase and α-glycosidase with slight modifications depending on EO and inhibited enzyme. Several EOs reported in other studies have shown important in vitro activities in particularly on enzymes inhibitory such as α-amylase and α-glycosidase (Lekshmi et al., 2012; Ceylan et al., 2016; Adefegha et al., 2017). Moreover, these effects were better than those proved by acarbose used as standard drug. This medicament is used mainly as an important inhibitor of αglucosidase and α-amylase. Whereas, numerous studies have shown that this molecules has some side effects such as diarrhea in the gastrointestinal tract (Kast, 2002; Nakhaee and Sanjari, 2013). On the other hand, enzymatic browning in human skin involves mainly the melanin formation. The synthesis of melanin is carried out by several steps, which the first two steps involves tyrosinase (Karioti et al., 2007). It is a monophenol monooxygenase (E:C:1.14. 18.1) known as polyphenol oxidase. Therefore, the inhibition of tyrosinase activity is an important strategy for skin protection. Indeed, the tyrosinase inhibitory method is the most used in vitro method today for revealing the dermatoprotective activity of medicinal plant products. At the best of our knowledge, no study has evaluated the tyrosinase inhibitory of CEEO. From our results, CEEO showed important tyrosinase inhibitory effects, in particularly with CEEO at flowering stage. The difference between three EOs is due to the variability in volatile functional components present in each oil. Moreover, we have revealed some positive correlation between antioxidant and anti-tyrosinase effects. Indeed, CEEO at flowering stage, which showed remarkable antioxidant activity had the strong tyrosinase inhibitory effect. These results were supported by other previous, which reported the positive correlation anti-tyrosinase and antioxidant properties of medicinal plant products and their derivates (Masuda et al., 2005). Infectious diseases constitute another problem of public health. This situation is in increasing way especially with the development of

estimate the bacteriostatic and the bactericidal effects of CEEO, the MIC and MBC values were determined (Table 5). The results showed that the CEEO at the post-flowering stage was the most active against the strains tested. The lowest MIC and MBC were obtained against S. aureus by the same oil. Moreover, at the post-flowering stage MIC was equal to MBC against S. aureus indicating thus a bactericidal action at MIC values. In general, Gram-positive bacteria were more sensitive to CEEO than Gram-negative ones. 4. Discussion Currently, several studies focus on the natural products such as medicinal plants in order to identify new bioactive molecules with specific pharmacological properties (Lahlou, 2013). In this context, several approaches could be used to identify, isolate and characterize novel bio-molecules. In this regard, medicinal plants are considered as a major and important source for identification and discovery of novel drugs. These plants synthesize and release secondary metabolites such as EOs to fight against different biotic and abiotic agents. However, numerous factors could affect the concentration and the diversity of these compounds (Lahlou, 2013; Bouyahya et al., 2017a; Aboukhalid et al., 2016). In our study, we have revealed that C. erythraea contains EOs and the composition of these oils depends dramatically on the phenological stages. The chemical composition of CEEO has been reported by some previous studies (Valentão et al., 2001; Jerković et al., 2012). However, this was the first study that has compared the volatile compounds of CEEO at three developmental stages. The results of this study showed effectively that CEEO varied between these three developmental stages. This fluctuation in the chemical components could be explained by the fact that the synthesis of secondary metabolites including EOs is influenced by growth stages of plants. Whereas, other previous studies have revealed that EOs are synthesized and secreted differentially depending on developmental stage of plant (Porres-Martinez et al., 2015; Bouyahya et al., 2017a; Raudane et al., 2017; Salem et al., 2018). The synthesis of EOs by aromatic plants is controlled by several factors including seasonal fluctuations. Indeed, developmental stages offer differential expression in gene regulation including those implicated in volatile compounds (Sangwan et al., 2001; Prins et al., 2010). Furthermore, CEEO at three stages were subjected to in vitro biological investigations by the evaluation of their antioxidant, antibacterial, antidiabetic and dermatoprotective activities. Concerning the antioxidant activity, CEEO showed interested potential to scavenge free radical such DPPH and ABTS. Other published studies have also reported the antioxidant activity of CEEO (Valentão et al., 2001). The difference between the obtained effects is certainly attributed to the variability between the three oils and the methods used to test the antioxidant activity. In fact, the most important antioxidant activity was obtained at the flowering stage, which explains that the EOs at this 115

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resistance towards antibiotics. In our study, the antibacterial activity of CEEO showed important inhibition of the tested bacteria. Focusing on the inhibitory zones and except of E. coli, no significant differences were noticed between the three phonological stages against the tested strains, but the sensitivity of the bacteria to CEEO was different from strain to another. In fact, the determination of the inhibitory zone is just a qualitative assay. In this regard, The MIC and MBC were determined, which give indication about the bacteriostatic and the bactericidal effect of these oils. Focusing now on MIC and MBC, the post-flowering stage proved the highest bactericidal effect against the bacterial strains tested compared with vegetative and flowering stages. Moreover, in the case of S. aureus the MIC was equal to MBC at the post-flowering stage indicating a bactericidal effect at MIC of this oil. The CEEO at the postflowering stage was the richest in carvacrol and it was the major compound of this oil (25.61%) which can explains its strong antibacterial activity (Chung et al., 2018; Carvalho et al., 2018; Gaio et al., 2017). However, the effect of the minor elements is not negligible, because they can act synergistically on the antibacterial action. On the other hand, Gram-negative bacteria were most resistance towards CEEO than Gram-positive ones. These results are in concordance with several works that demonstrated the resistance of Gram-negative bacteria to EOs (Blair et al., 2014; Bouhdid et al., 2008). The action of EOs against bacteria can be due to different mechanisms inducting membrane permeability, enzymes inhibitory and morphological perturbation of bacteria (Bouhdid et al., 2009; Vasconcelos et al., 2018). Moreover, EOs and their derivates can also induce antibacterial action by targeting quorum sensing regulation (Bouyahya et al., 2017e).

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5. Conclusion It can be concluded from this work that the chemical profile of CEEO vary between the three phenological stages. The main compounds identified were carvacrol, menthol and tricosane, with a percentage varying from a stage to another. CEEO showed an important antioxidant activity, which was variable depending on the developmental stage. The EO at the flowering stage was the most active as antioxidant. These EOs exhibited also a strong antidiabetic activity by inhibiting α-amylase and α-glycosidase. Moreover, with a strong inhibition of tyrosinase, CEEO can be used as dermatoprotective. CEEO exhibited an interesting antibacterial activity, with high sensitivity of Gram positives bacteria. The EO at the post-flowering stage proved the highest bactericidal effect against the strains tested. These data may serve as guideline to target specifically the bio-molecules wanted. References Aboukhalid, K., Lamiri, A., Agacka-Mołdoch, M., Doroszewska, T., Douaik, A., Bakha, M., Casanova, J., Tomi, F., Machon, N., Al Faiz, C., 2016. Chemical polymorphism of Origanum compactum grown in all natural habitats in Morocco. Chem. Biodiv. 13, 1126–1139. Adefegha, S.A., Olasehinde, T.A., Oboh, G., 2017. Essential oil composition, antioxidant, antidiabetic and antihypertensive properties of two afromomum species. J. Oleo Sci. 66, 51–63. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils-a review. Food Chem. Toxicol. 46, 446–475. Batubara, I., Darusman, L.K., Mitsumaga, T., Rahminiwati, M., Djauhari, E., 2010. Potency of indonesian medicinal plants as tyrosinase inhibitor and antioxidant agent. J Biol Sciences. 10, 138–144. Benhamza, L.M., Djerrou, Z., Pacha, Y.H., 2013. Evaluation of anti-hyperglycemic activity and side effects of Centaurium erythraea (L.) Pers. In rats. Afr. J. Biotechnol. 12, 6980–6985. Blair, J.M., Richmond, G.E., Piddock, L.J., 2014. Multidrug efflux pumps in Gram-negative bacteria and their role in antibiotic resistance. Future Microbiol. 9, 1165–1177. Bouhdid, S., Skali, S.N., Idaomar, M., Zhiri, J., Baudoux, A., Abrini, J., 2008. Antibacterial and antioxidant activities of Origanum compactum essential oil. Afr. J. Biotech. 7, 1563–1570. Bouhdid, S., Abrini, J., Zhiri, A., Espuny, M.J., Manresa, A., 2009. Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil. J. Appl. Microbiol. 106, 1558–1568. Bouyahya, A., Dakka, N., Talbaoui, A., Et-Touys, A., El-Boury, H., Abrini, J., Bakri, Y.,

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