Chemical Composition, Antioxidant, and Antimicrobial Activities of Leaves of Ajuga Iva

The main objective of this research was to study the biological characteristics in terms of antioxidant and antimicrobial activities of Ajuga iva and determine the best analytical and extraction methods applicable to this specie and studied compounds. A short screening of its nutritional value in terms of chemical composition is also included. A. iva leaves were analyzed for crude protein (CP), cell wall [neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL)], minerals, fatty acids, essential oils, and phenolic compounds. Mature aerial parts of A. iva were randomly collected during the Spring season from Mograne-Zaghouan, Tunisia. Leaves of A. iva contained 13.4 ± 0.4% CP, 26.3 ± 0.35% NDF, 20.2 ± 0.42% ADF, and 5.13 ± 0.21% ADL. Mineral content (13.0 ± 0.45%) was mainly composed of potassium (4.5% g DM) and magnesium (4.25% DM). Leaves of A. iva had linolenic (26.29 ± 0.760%) and linoleic (37.66 ± 2.35%) acids as the main components of the acid profile. Thymol was found to be the most dominant (23.43%) essential oil, followed by 4-vinylguaiacol (14.27%) and linalool (13.66%). HPLC-PDA-ESI-MS/MS analysis pointed out the presence of phytoecdysteroids. Phenolic acids and flavonoids, such as glycosylated derivatives of naringenin, eriodyctiol, and apigenin, were detected in the methanol extract of A. iva leaves. Our results underline the importance of choosing proper extraction methods and solvents to extract and characterize the described compounds profile of A. iva leaves. Results also show A. iva leaves as a potential source of functional ingredients with beneficial health-promoting properties. Overall, leaves of A. iva have low biological activities (antioxidant and antimicrobial activities) with a chemical composition suitable as a feed for ruminants in rangeland pasture. It also has low-grade antibacterial or medicinal characteristics when fed to ruminants.


Introduction
Determining plants' biological activity and nutritive values is important before they are included in human or animal diets [1,2]. Also, identifying plant species' chemical characteristics help to differentiate them from similar species, which often leads to incorrect identification [3]. Therefore, determining nutrient concentrations and amounts of secondary metabolites in plants helps to create databases useful for nutritionists, which results in hay, collected from mature plants, were air-dried at room temperature (40 ± 2 • C) for one week, ground in Retsch blender mill (Normandie-Labo, Normandy, France), and sieved through a 0.5 mm mesh screen to obtain a uniform particle size. The ground substrates were stored in a dark room at room temperature until used.

Chemical Analysis
Dry matter (DM, method ID 934.01), ash (method ID 942.05), and crude protein (CP, method ID 984.13) contents were determined following the methods of AOAC [20]. Total ash content was determined after calcinating 0.5 g in a muffle furnace Manfredi L9C (Manfredi S.R.L, Torino, Italy) at 500 • C for 24 h. The mineral composition was determined after digestion of the sample (500 mg) with 4 mL concentrated nitric acid and 1 mL hydrogen peroxide, using the microwave digestion system Milestone ETHOS 1 (Sorosole, Italy). After cooling at room temperature, the digested samples were diluted up to 25 mL with deionized water for subsequent determination of minerals. Analysis of the elements (Na, K, Ca, Mg, Cu, Fe, and Zn) was carried out only for leaves of A. iva by inductively coupled plasmaatomic emission spectrometer, ICP-AES (Horiba Jobin-Yvon Ultima 2 CE). Measurements were performed at the following emission line Analysis of cell wall components in terms of neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) was determined with the ANKOM fiber analyzer [21]. Sodium sulfite, but not β-amylase, was added to the solution for the NDF determination.

Total Lipid Content and Fatty Acid Composition
Total lipid extraction was performed according to the AOAC reference method [20], using a Soxhlet apparatus for 8 h with hexane. The final extract was evaporated under reduced pressure in a Heidolph rotary evaporator (Schwabach, Germany), and the total lipids were determined. The defatted raw material was conserved at −20 • C for further analysis.
Prior to analysis by gas chromatography (GC), fatty acids were converted into their corresponding fatty acid methyl esters (FAMEs), using sodium methoxide (3% in methanol) following the method of Cecchi et al. [22]. FAMEs were separated and identified on an Agilent HP 6890 (II) GC equipped with a flame ionization detector (FID) and a polar column TR-FAME (60 m, 0.25 mm i.d., 0.25 µm film thickness). The GC was operated under programmed temperature conditions from 100 • C (5 min) to 240 • C (15 min) at 4 • C/min. The injector and FID detector temperatures were maintained at 240 • C and 206 • C, respectively. Identification of FAMEs was made by comparing their retention time with those of 37 FAMEs Standards (Sigma-Aldrich, Stenheim, Germany). The percent compositions of FAMEs were calculated with reference to the total fatty acids.

Isolation and Analysis of Essential Oils
To isolate A. iva essential oils (AIVO), 100 g of ground leaves were submitted to hydrodistillation for 3 h using a Clevenger-type apparatus. The hydrolat was extracted twice with n-pentane (20 mL), dried over anhydrous sodium sulfate, then concentrated up to 1.5 mL at 35 • C using a Vigreux column and subsequently analyzed [23]. Analysis of AIVO was performed by gas chromatography-mass spectrometry (GC-MS) using a gas chromatograph HP 6890 interfaced with an HP 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) with electron impact ionization (70 eV). Separation of the individual compound was achieved by an HP-5MS capillary column (60 m length, 0.25 mm i.d., 0.25 µm film thickness) under the following operating conditions: temperature program: 50 • C (1 min) to 280 • C (8 min) at 5 • C/min. Injection temperature: 250 • C; flow rate of the carrier gas helium: 1.2 mL/min; ion source and quadrupole temperatures: 230 and 150 • C, respectively; ionization voltage: 70 eV; mass range: 50-550 u.
The volatile compounds were identified by comparing their retention indices relative to (C 7 -C 20 ) n-alkanes with the literature and/or with authentic compounds when available. Further identification was made by matching their mass spectra fragmentation patterns with corresponding data (Wiley 275L and NIST05a libraries) and other published mass spectra [24].

Samples Preparation
Five solvents with increasing polarity (petroleum ether, ethyl acetate, ethanol, methanol, and water) were used to prepare different extracts. Dried and ground leaves (1 g) were mixed with 20 mL of solvent in an orbital shaker (150 rpm for 48 h). Each extraction was repeated twice, and the resulting solvent extracts (except for water extract) were filtered through Wattman #1 filter paper (Bärenstein, Germany) and evaporated under reduced pressure in a Heidolph rotary evaporator (Schwabach, Germany). The water extract was frozen and lyophilized in a Christ-Alpha 2-4 freeze drier (Osterode, Germany).

Total Phenolic Content (TPC)
Total phenolics were determined with the Folin-Cieucalteu (FC) assay, according to Lister and Wilson [25]. Briefly, 100 µL of leaf extracts were mixed with 500 µL of freshly diluted 10-fold FC reagent and 1 mL of 20% sodium carbonate solution. After incubation for 1 h in the dark, the absorbance was measured at 760 nm using a Jasco V-630 UV-vis spectrophotometer (Tokyo, Japan). Gallic acid was used as the standard, and results were expressed as micrograms of gallic acid equivalents (µg GAE/g).

Total Flavonoid Content (TFC)
Total flavonoid content was determined by the AlCl 3 colorimetric method [26]. A 500 µL sample aliquot was mixed with 1.5 mL methanol, 0.1 mL of a 10%AlCl 3 solution, 0.1 mL of potassium acetate (1 M), and 2.8 mL of distilled water. After 30 min incubation at room temperature, the absorbance was measured at 415 nm. Quercetin was used as a reference standard, and the total flavonoid content was expressed as micrograms of quercetin equivalents (µg QE/g).

HPLC-ESI-MS/MS Analysis
The chromatographic separation and mass spectrometric analyses of phenolics contained in the methanolic extracts were carried out on an Agilent 1100 series HPLC system equipped with a photodiode array detector (PDA) and a triple quadrupole mass spectrometer type Micromass Autospec Ultima Pt interfaced with an ESI ion source. Separation was achieved using a Superspher ® 100 (12.5 cm × 2 mm i.d, 4 µm, Agilent Technologies, Rising Sun, MD, USA) at 40 • C. The samples (20 µL) were eluted through the column with a gradient mobile phase consisting of A (0.1% acetic acid) and B (acetonitrile) with a flow rate of 0.25 mL/min. The following multi-step linear solvent gradient was employed: 0-5 min, 2% B, 5-60 min, 40% B, 60-85 min, 100% B, 85-85.1 min, 2% B, 85.1-100 min, 2% B. PDA detection was performed in the 250-550 nm wavelength range and the mass spectra were recorded in positive ion mode, under the following operating conditions: capillary voltage, 3.2 kV; cone voltage, 40 V; probe temperature, 350 • C; ion source temperature, 120 • C. The spectra were acquired in the m/z range of 150-750 amu. Tentative identification of phenolic compounds was based on co-chromatography with authentic standards when available. PDA spectra and mass spectra were used to confirm the identity of compounds previously reported in the literature [27][28][29].
2.6. Antioxidant Activity 2.6.1. DPPH Radical Scavenging Activity The DPPH assay followed a reported method [30] with some modifications. Briefly, 1 mL of different extracts was added to 2 mL of a 0.1 mM methanol DPPH solution. The mixture was shaken vigorously and left to stand at room temperature in the dark for 1 h. Thereafter, the absorbance was measured at 515 nm. The scavenging activity was measured as the decrease in absorbance of the samples versus the DPPH standard solution. Results were expressed as radical scavenging activity percentage (%) of the DPPH using the following formula: where A 0 and A s are the absorbance of the control and the sample, respectively. The effective concentration having a 50% radical inhibition (IC 50 ) expressed as mg extract/mL was determined from the graph of the free radical scavenging activity (%) against the extract concentration.

ABTS Scavenging Activity
The ABTS assay was based on the procedure described by Re et al. [31]. The solution consisting of 7 mM of ABTS and 2.4 mM potassium persulfate (1:1 v/v) was reacted in the dark for 12 h at room temperature. Then it was diluted with methanol to obtain an absorbance of 0.7 at 734 nm. The diluted ABTS solution (2850 µL) was mixed with 150 µL of sample extracts or trolox standard. The mixture was left to stand at room temperature in the dark for 15 min, and then the absorbance was measured at 734 nm. Results were expressed as µmol trolox equivalent (µmol eq. trolox/g), based on the trolox calibration curve.

Ferric Reducing Antioxidant Power (FRAP)
The FRAP assay was carried out as described by Benzie and Strain [32] with slight modifications. Stock solutions of 300 mM acetate buffer, 10 mM TPTZ (2,4,6-tripyridyls-triazine) in 40 mM HCl, and 20 mM FeCl 3 . 6H 2 O were prepared. The working FRAP reagent was prepared by mixing the stock solutions in a 10:1:1 ratio. The solution was maintained at 37 • C and pH 3.6. Then, 150 µL of the sample was mixed with 2850 µL of the working solution and left standing at room temperature for 30 min in the dark. The absorbance of the mixture was then measured spectrophotometrically at 593 nm. The change in absorbance was calculated and related to the standard curve generated with trolox. Results were expressed as micromole of trolox equivalent per gram of extract (µmol TE/g).

Microbial Strains and Growth Conditions
The different extracts from A. iva leaves were individually tested against a panel of 6 microorganisms, including the gram + Staphylococcus aureus ATCC8739 and Streptococcus agalactiae isolated in the Institute Pasteur (Tunis, Tunisia), the gram − Enterococcus faecium ATCC19434, Salmonella typhimurium ATCC14028, and Escherichia coli ATCC8739, as well as the yeast Candida albicans ATCC10231. Microbial strains were cultured overnight at 37 • C in Muller-Hinton agar (MHA). For the antimicrobial screening, the disk diffusion method was employed [33].

Disk Diffusion Method
In this assay, a suspension of the test micro-organisms (10 8 CFU/mL) was spread on the MHA media plate. The filter paper disk (6 mm in diameter) was soaked with 15 µL of the extracts dissolved in dimethyl sulfoxide (DMSO) and placed on the inoculated plates. After being kept at 4 • C for 2 h, they were incubated at 37 • C for 24 h for bacteria and at 30 • C for 48 h for the yeast. The definite zone of inhibition of any dimension surrounding the paper disk was measured accurately.

Chemical Composition, Fatty Acid (FA), and Essential Oil Constituents (VOAI) of A. iva Leaves
Data on the chemical composition of leaves of A. iva and alfalfa hay is shown in Table 1. As compared with alfalfa hay, A. iva leaves contain low total mineral (13.0 ± 0.45%), CP (13.4 ± 0.4%), NDF (20.2 ± 0.42%) and ADF (20.2 ± 0.42%). However low lipid contents (2.18 ± 0.58%) were detected in the leaves of A. iva. The ICP-AES analysis ( Table 2) allowed the detection of 7 major elements from which K was the most dominant (3201.0 ± 143.88 mg/kg DM) and Cu the lowest (87.9 ± 3.34 mg/kg DM). Analytical GC analysis showed that unsaturated fatty acids (UFA) contributed more than 89% of the total fatty acid profile (Table 3). Linolenic (37.66%), linoleic (26.29%), and oleic (15.91%) were the most abundant UFA. Palmitic (16.82%) and stearic (2.18%) acids were the principal saturated fatty acids (SFA).  The chemical composition of AIVO was determined by GC-MS analysis. The essential oil compositions, along with their retention index and percentage composition, are listed in Table 4. Within the 21 identified volatile compounds, thymol was found to be the most dominant (23.43%), followed by 4-vinylguaiacol (14.27%) and linalool (13.66%) ( Table 4).

Extract Yield, Total Phenolic, Flavonoid Contents of Different Solvent Extracts
A total of five solvents (petroleum ether, ethyl acetate, ethanol, methanol, and water) were used to extract polyphenolic compounds from A. iva. As shown in Table 5, depending on extraction solvent, the extract yield ranged from 0.82 ± 0.27% (petroleum ether) to 12.85% ± 1.40% (water). The contents of phenolic compounds were variable, with maximum levels found in methanol (177 GAE/g extract) followed by aqueous (144 GAE/g extract) and ethanol (143 GAE/g extract) extracts. On the other hand, total flavonoids assumed maximum amounts in ethyl acetate extract (52.61 QE/g extract). An intermediate value was revealed in methanol extracts (40.23 QE/g extract).

HPLC-PDA-MS/MS Analysis of the Methanol Extract
The total ion chromatogram of the methanol extract of A. iva is shown in Figure 1, and the peak characteristics as well as tentative identification are summarized in Table 6. Twenty compounds were tentatively identified based on their UV and mass spectral data as well as by comparison of their retention time (RT) with those of available commercial standards. As expected, the main components showed a maximum absorbance of around 247 nm which represents the UV spectral characteristic of phytoecdysteroids (peaks 1, 7, 9, 11, 12, 16, and 20), typical components of the genus Ajuga.

Antioxidant Activity
The screening of the antioxidant capacity of the methanolic extracts was evaluated by three methods, namely the DPPH, ABTS radical scavenging capacity, and the ferric reducing antioxidant power (FRAP) ( Table 7). A significant variation was observed between extracts, regarding their ability to scavenge DPPH•. The IC 50 values of the DPPH radical scavenger ranged from 411.83 to 3328.42 µg/mL for the five extracts tested, with the highest scavenging capacity for water and methanol (IC50 411.83-729.98 µg/mL). The same trend was found in the ABTS test (1161.52-148.51 µg/mL). Moreover, the most important reducing power was interestingly observed also in aqueous and methanol extract (0.12-0.13 TE/g extract). However, they were by far less effective than the synthetic antioxidant trolox.  50 , the scavenging of the free anionic 2,2-diphenyl-1-picrylhydrazyl radical expressed in µg/mL. 2 FRAP, ferric reducing antioxidant power value expressed as mM TE/g extract.

Antimicrobial Activity
The in vitro antimicrobial activity of the A. iva extracts was qualitatively evaluated by the presence or absence of an inhibition zone (IZ). As can be seen in Table 8, the most susceptible strain was found to be S. agalactiae (Gram + ) which was particularly sensitive to the most of extracts with methanol, ethanol, and water is the most active. The Gram + E. faecium, the Gram-S. typhimurium, and E. coli were also found to be sensitive to most of the extracts with the highest inhibition observed with ethanol extract. The latter extract showed weak activity against the yeast C. albicans. All tested microorganisms were more susceptible to Ampicillin (20-44 mm) than to the extracts.

Discussion
It is pertinent to mention that the nutritional value of A. iva cultivated from different Tunisian areas was earlier studied [4]. According to these authors, cultivar collected from Mograne had the lowest cell wall contents and the highest digestibility. Therefore, this cultivar collected from the Mograne area was the subject of the determination of biological activities reported in this current research. On the other hand, it is worth remembering that our main objective is to present complementary data to the nutritional value of the most appropriate cultivar, as animal fodder, on its medicinal characteristics in terms of antioxidant and antimicrobial activity, when different solvents are studied. Crude protein content (13.4%) is comparable to that of alfalfa hay and is considered enough to meet the minimum CP requirement (8% of DM) for optimal microbial function and the maintenance of adequate ruminal fibrolytic activity [6,34]. In this same context, Sampaio et al. [34] reported that increasing the dietary CP concentration close to 10% DM was found to optimize the use of low-quality forage and considered supplying enough nitrogen to maintain the microbial activity in the rumen. The low ADL content (5.1%) of A. iva is an indicator of a low lignification of its cell wall and thus a high digestibility is expected.
On the other hand, high values in mineral content (>10%) seem to be characteristic of the Lamiaceae family. This result was consistent with those reported for the genus Ocimum, Perilla, Hyptis, Scuttelaria, Mentha, and Nepeta [35][36][37]. As mentioned in Table 2, the ICP-AES analysis allowed detection of 7 major elements with the following trend: K > Mg > Ca > Na > Fe > Zn > Cu.
To the best of our knowledge, this is the first report on the mineral composition of A. iva. Nevertheless, the only chemical investigation of A. bracteosa revealed that Ca, Mg, K, and Na were the main macro-elements, while Fe, Cr, Mn, Cu, and Zn were the major microelements [38]. Collectively, these results indicate that A. iva leaves could be considered an excellent source of essential minerals and encourage its use in diet and/or dietary supplement. The abundance of K, and to a less extent Mg, Ca and Fe was expected if we bear in mind that these elements have structural and functional roles in plants [39].
From a medicinal point of view, the presence of some essential minerals which have pivotal physiological and biochemical roles in the human body (e.g., enzymatic reactions, energy production, and transmission of nerve impulses, among others) could justify the traditional medicinal uses of A. iva. Support for this assumption was given by the low value of the Na/K ratio, suggesting the potential of A. iva leaves for managing hypertension. Additionally, it has been reported that incorporation of Mg-rich extract or Zn-rich extracts of Atriplex balimus in the diet was associated with better insulin release, high uptake of glucose by fat cells, and reduced arterial pressure, with the effects being modulated by the presence of K and Ca [40]. The lipid-lowering properties and anti-obesity effects of Zn and Ca have also been reported [41].
As mentioned before, the average total lipid content was 2.18% (w/w) ( Table 1). This value was lower than those reported for A. reptans L. (13.14%), A. chia (Poir) Schreb (12.03%), and A. genevensis L. (10.20%) [42]. However, it was 3.2-fold higher than the total lipid content in the aerial part of A. turkestanica (Rgl.) Briq [43]. These low levels would reflect the low essential oil contents of A. iva.
The fatty acid profile (C18:3 > C18:2 > C16:0 > C18:1) differed from those reported in A. reptans, A. chia, and A. genevensis where the following profile was obtained C18:2 > C18:3 > C18:1 > C16:0 [42]. Studies reported by Khidoyatova et al. [43] revealed that linolenic, oleic, linoleic, and palmitic acids were found to be the main components of neutral lipids, glycolipids, and phospholipids of A. turkestanica aerial parts. However, the profile C16:0 > C18:2 > C18:1 was found in A. genevensis and A. chia [44]. Such discrepancies are likely due to genetic and environmental factors [45]. Moreover, given the importance of polyunsaturated fatty acids (i.e., linolenic and linoleic acids) as health-promoting nutrients and their potential in alleviating cardiovascular, inflammatory, heart, and neurodegenerative diseases, as well as atherosclerosis, autoimmune disorder, and diabetes [46], the high amounts of the essential linolenic and linoleic acids, makes the lipids of A. iva leaves important for nutritional applications and support their use as a food supplement.
The chemical composition of AIVO revealed the presence of 21 compounds covering more than 91% of the GC profile (Table 3). Similarly, results reported by Chouitah et al. [47] on A. iva collected from Algeria identified 22 compounds with a cover rate of 99.92%. However, studies reported by Bouyahya et al. [48] pointed out the presence of 28 volatile compounds in essential oils of A. iva harvested at different phonological stages (vegetative, flowering, and post-flowering stages) from Morocco. It appears, therefore, that some factors, such as the origin and plant growing environment [19] and phenological stage [48] regulate the secretion of essential oils.
On the other hand, it is pertinent to notice that the identified essential oils belonged to five different groups classified according to the importance of their presence as follows: phenol (39.53%) > alkane (28.65%) > oxygenated monoterpen (16.86%) > alcohol (4.78%) > phenylpropen (2.1%). The phenol group was mostly represented by thymol (23.43%) and the alkane group was represented by heptacosane (4.14%) and hexacosane (3.96%). Linalool was the main component (13.66%) of the oxygenated monoterpene group, while octen-3-ol was the chief component (4.02%) of the alcohol group. Phenylpropenes were represented by eugenol (2.1%). Studies reported by Bouyahya et al. [48] pointed out that, at the vegetative stage of A. iva, three major essential oils were identified, namely carvacrol (32.72%), octadecane (17.28%) and methyl chavicol (8.18%); while thymol (1.25%) and linalool (1.17%) were less abundant, as compared by those reported in our present study. Moreover, these same authors revealed that the most dominant group was oxygenated monoterpen (62.24%), which was far higher than its presence in our study. Chemical composition of two varieties of A. chamaepitys subsp. chia var. chia and var. ciliate studied by Baser et al. [49] revealed that β-pinene (20.8%) and germacrene D (12.6%) were the main components in the former variety, while germacrene D (14.6%) and β-pinene (14%) were dominants in the latter variety. In this same context studies reported by Azizan et al. [50] on A. chamaepitys growing in Iran reported that β-pinene and α-pinene are by far the dominant compounds (34.3% and 16.1%, respectively). These same authors pointed out that other compounds present in rather appreciable amounts were the monoterpene hydrocarbons y-terpinene (7.7%), limonene (6.1%), and mycrene (1.4%).
Yields of different extracts obtained from leaves of Tunisian Ajuga iva varied significantly. The highest extract yield was obtained by methanol extraction. The petroleum ether efficiency as a solvent was lower than the efficiencies of all other solvents. This finding is in accordance with some studies which demonstrate that maximum extract yield, was obtained with methanol and water [9]. The extraction yield appears to be influenced by the polarity degree of the solvent, and by the degree of the polarity of the various extract components such as phenolic constituents [54]. It has been also reported that the efficiency of the extraction depends on the extraction time and temperature [55].
Furthermore, the contents of phenolic compounds of Ajuga iva varied according to the solvent used for the extraction process. Results revealed that methanol extract had the highest contents of total phenols. Our results were higher than those obtained in the same species from Morocco [5,56] and Algeria [55,57,58]. These variations may be due to many factors, including geographical, genetic, and ecological factors [59]. The lower polarity solvents, particularly: Petroleum Ether, and ethyl acetate showed much lower ability in extracting the phenolic compounds as compared to the polar solvents. In contrast, ethyl acetate extracts had more condensed flavonoids than the remaining extracts. These results were in line with those reported by Anokwuru et al. [60] and Senhaji et al. [5] where ethyl acetate was found to be the best solvent for the extraction of flavonoids. So, solvent polarity will play a key role in increasing phenolic solubility [61]. Besides, the solubility of phenolic compounds is governed by the type of solvent used, the degree of polymerization of phenolics, and their interaction [55].
Prompted by these results showing high total phenolic content in methanol extracts, we decided to analyze the phenolic composition to get a deeper insight into specific components of the polyphenolic mixture.
Many reports have been published on the chemical composition of A. iva, mentioning the evidence of numerous bioactive compounds, such as neo-clerodanediterpenoids, phytoecdysteroids, tannins, and iridoids [66]. Our results revealed that 20-hydroxyecdysone was the main component identified in the extracts. Moreover, high levels of campestanol trans-ferulate were found. This is consistent with the general abundance of Ajuga plants in phytoecdysteroids 20-hydroxyecdysone [67,68].
Among the revealed flavonoids, apigenin-dihexoside is the predominant one. In line with that, Boudjelal et al. [66] revealed that two flavonoids appeared to predominate, apigenin. and naringenin, in the aqueous infusion of A. iva. However, Khattelia et al. [69] reported the presence of caffeic acid, p-coumaric acid, rutin, and luteolin in the extract of the aerial part of A. iva.
Regarding the antioxidant activity, it can be inferred that total phenolic content may be indicative of the antioxidant capacity of A. iva leaves. In fact, polar extracts namely water and methanol were found as the most effective antioxidant in the DPPH, ABTS, and FRAP assays. Less promising results were revealed by Sönmez et al. [70] for methanol extracts of A. postii and A. relicta species. Compared to results reported on A. iva from Morocco, the Tunisian species exhibited less activity [5]. Differences in the antioxidant activity could be explained by the presence of some phenolic compounds that may enhance the antioxidant capacity of extracts [71]. In fact, Mamadaliva et al. [72] revealed that the low antioxidant activity of A. turkestanica extracts was attributed to the presence of phytoecdysteroids, recognized for their weak antioxidant activity [72]. However, major and minor components as well as the synergetic or antagonistic interactions between constituents could give rise to the exhibited biological activities. The presence of phytoecdysteroids makes A. iva suitable for other applications including hypoglycaemic [73], antidiabetic [18], hypolipidemic [74], and wound healing [75], among others.
The antimicrobial activity estimated by the diameter of inhibition varied according to extracts and microbial strains. The inhibition zones ranged between 8 and 15 mm. The antimicrobial activity of all extracts depends largely upon the concentration of extracts, the bacterial strains, and the type of solvent. The ethanol extract was slightly more effective than the remaining extracts. In line with our results, Toiu et al. [76] revealed that the bacterial strains were more sensitive to the ethanol extract of A. laxmannii. The most active antimicrobial compounds are rather extracted by ethanol. In fact, ethanol extracts contain higher amounts of iridoids which had a wide range of biological and pharmacological activities [71]. However, our results are contradictory to those reported by Makni et al. [9] who reported strong antimicrobial activity against E. coli, S. aureus, and Fusarium Sp. of the methanol extract of A. iva. These discrepancies might be linked to differences in the sample preparation and bioassays procedures.
Overall, this study's results provide a baseline for assessing the biological activities of A. iva (i.e., the antioxidant and antimicrobial activities) of A. iva and the nutritional value in terms of chemical composition. Moreover, such results help to catalog other fodder species such as alfalfa. Hence, the introduction of A. iva becomes a valuable addition to the available information as this plant has high nutraceutical value. Therefore, based on previous studies [4] and data from the present study, further research should consider feeding A. iva leaves to different ruminants and determine their effects on nutrient degradability and animal's productive performance. Then, the next step could be to test A. iva leaves in on-farm conditions.

Conclusions
This study was carried out to highlight the dual characteristics of leaves of Tunisian A. iva, in terms of nutritional value and biochemical characteristics (antioxidant and antimicrobial activities) and the main essential oils using different extracts. Based on its reasonable CP content (>13% DM) and the low lignification of the cell wall, leaves of A. iva collected from the Mograne area can be considered as a feed for ruminants as an alternative to common forages such as alfalfa (~13% CP). The contents of phenolic compounds varied between extractors, with maximum levels found in methanol followed by aqueous and ethanol extracts, demonstrating the most suitable extractors to use when doing analytical analysis. The analytical data showed that phytoecdysteroid, 20-hydroxyecdysone, was the most abundant compound in A. iva methanol extracts. The presence of these components recognized for their low antioxidant and antimicrobial activities could justify the weak biological activities obtained herein. Despite that, leaves of A. iva might represent an excellent source of functional food ingredients that could be used in food and pharmaceutical applications as a medicinal plant. Further studies need to evaluate how the low-grade antimicrobial effects and the higher polar extracts' effectiveness (especially aqueous) of A. iva leaves will affect rumen microflora activity, and how different rumen microorganisms and pathways (i.e., methanogenesis, protein synthesis) are affected by diverse rates of inclusion in the diet. Hence researchers should consider feeding A. iva leaves to different ruminant species and determine their effects on nutrient degradability and animal productive performance.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.