3.1. Phytochemical Screening
The extraction used in the first place is of the solid–liquid type, in this method the solvent must cross the barrier of the solid–liquid interface and dissolve the active ingredient (organic compounds) that the solvent contains inside. Secondly, a liquid–liquid extraction was carried out which makes it possible to diffuse the phenolic compounds into a liquid by applying solvents of increasing polarity allowing a selection and separation of the secondary metabolites contained in the crude extracts.
According to
Figure 1, the crude hydroethanolic extract represents a high yield of 17.9 ± 1.177%. In the work of Kalantari et al. [
9], hydro-alcoholic extract of
Capparis spinosa L. leaves had a higher percentage (15.75%) yield of extraction compared to the ethyl acetate and chloroform extracts, which is the same in relation to our results.
The extraction yields by fractionation of the raw extract of leaves of
Capparis spinosa L. fluctuate significantly between 2.17 ± 0.149% and 12.03 ± 1.621% in the following increasing order: ethyl acetate (CSAC), hexane (CSHex), diethyl ether (CSDE) n-butanol (CSBu) and aqueous (CSAQ). These results indicate a relatively interesting for the extraction carried out by butanol and the fraction of the residual aqueous solution. This could presume the richness of the leaves of
Capparis spinosa L. in compounds of high polarity including phenolic compounds, given their richness in hydroxyl groups [
24]. Therefore, the difference between the extraction yields obtained with the five fractions depends on the chemical properties of the molecules to be extracted, the physicochemical characteristics of the solvents used, and in particular their polarity which influences the solubility of the chemical constituents of a sample [
5].
The crude extract and the five fractions were quantitatively characterized by spectrophotometer, in order to determine their contents in total polyphenols (TP), flavonoids (TF), and condensed tannins (TC), then underwent phytochemical characterization by FT-IR and Py-GC-MS.
The results of the colorimetric analysis of the phenolic compounds are summarized in
Table 2. The results revealed that the crude hydro-ethanolic extract is rich in a significant number of bioactive compounds. When compared to other extracts, the hydro-ethanolic crude fraction presented significantly higher values of condensed tannins (17.93 ± 0.15 mg CE/g) (
p < 0.5). This can be explained by the differential solubility of the bimolecular extracted from the plant material and the selectivity of the solvents used. The liquid-liquid extraction method can dilute or on the contrary increase the contents of phenolic compounds in the crude extract [
25,
26].
Khojasteh Rad et al. [
27] reported that the content of condensed tannins in the hydroalcoholic extract of
Capparis spinosa L. leaves harvested in July 2016, Iran is 2.54 mg CE/g DM. This quantity of condensed tannins is less important compared to our results.
However, we found that the values determined in the fractions depended significantly on the solvents used. The ethyl acetate fraction (CSAC) showed the significantly highest levels of polyphenols and total flavonoids 259.31 ± 6.177 mg GAE/g and 107.291 ± 8.65 mg QE/g, respectively (
p < 0.05). This suggests that the hydroxyl-aromatic concentrates in the fraction (CSAC). While the lowest amount of these compounds was found in the aqueous fraction (CSAQ) with a content of 54.253 ± 0.406 mg GAE/g for polyphenols and 13.546 ± 1.547 mg QE/g for flavonoids which means that this fraction is the least hydroxyl-aromatic. Our results are in agreement with research by Hyun et al. [
28], who found that the ethyl acetate fraction, n-butanol fraction, and crude extract were more efficient in recovering phenolic compounds. Furthermore, previous studies showed that the flavonoids of
Capparis leaves mainly concentrate in the ethyl acetate extract (1.6 ± 0.016 mg RE/g MS), followed by the hexane extract (0.61 ± 0.009 mg ER/g MS), and a low amount was detected in the aqueous extract (0.3 ± 0.018 mg RE/g MS) [
29]. These results are similar to the conclusions drawn by our study. The difference in the amount of total phenolic compounds may be due to differences in environmental conditions, plant growth, and experimental conditions [
30].
The results of the quantitative analysis of the condensed tannins indicate that these molecules are important constituents of the phenolic compounds of the different fractions, their quantity varies between 8.036 ± 0.253 mg EC/g and 16.786 ± 0.101 mg EC/g.
In some cases, the analysis by gas chromatography is unsuitable due to the non-volatility of the heaviest compounds. In this case, the GC-MS can be coupled upstream to the pyrolysis (Py-GC-MS). In this technique, the sample is pyrolyzed, which causes the degradation of the macromolecular structures creating volatile fragments. Thus, this method makes it possible to quantify and identify the thermal degradation products of the samples [
31,
32,
33]. The four organics fractions were analyzed by Py-GC-MS. To facilitate the comparison of the organic fractions, we grouped all the pyrolysis fragments identified and classified according to several categories: phenols, proteins, polysaccharides, oxygenated compounds (fatty acids, fatty acid ester, alcohols, and furan), terpenes, halogenated compounds, aliphatic hydrocarbons (alkanes and alkynes) steroids, as well as the compounds that could not be identified. All these results are represented in
Table 3 and
Table 4. The pyrolysis components are partially similar, the most relevant difference, between the distributions of compounds, comes from the fact that the ethyl acetate fraction (CSAC) is richest in phenols with a relative content of 36.50% and contain more polysaccharides (2.66%) and furanics (coumaran, 5-methylfurfural, furfural) (10.19%). Hexane fraction (CSHex) has lower amounts of phenolic compounds (8.43%) and more ester fatty acids (11.65%) and steroids (2.43%) as well as slightly fewer furanics (1.37%) and significant protein (38.95%) and halogenated compounds (5.74%). This suggests that hexane is a good solvent for separating the organic fraction of hydroxyl-lean residuals (for example, terpenes, waxes, fatty acids, oils) from the raw mixture. Fractions CSBu and CSDE were the most protein condensed compared to the samples analyzed with proportions 62.68%, 60.09%, respectively, and contain other organic constituents, in smaller proportions. Our results are similar to previous works which have demonstrated the richness of
Capparis spinosa L. in biologically active substances such as phenols, fatty acids, terpenes, sterols, polysaccharides, flavonoids [
34], alkaloids [
35], and proteins [
12], [
36].
The analysis of the composition showed the presence of numerous secondary metabolites with or without pharmacological and/or therapeutic activity, belonging to different chemical classes could represent a fingerprint for the evaluation of the quality of the different organic fractions of the hydro-alcoholic extract of the leaves.
The chemical structures of the different samples were analyzed through FTIR and their corresponding spectrums are shown in
Figure 2a. For a better understanding, the fingerprint region of the spectrums is represented in
Figure 2b. The analysis of different samples shows several similarities. However, the IR spectrum of the crude extract shows a band at 3679 cm
−1 corresponding to the –OH group of free alcohols, the peak at 1740 cm
−1 indicates the presence of the carbonyl group, which are not visible in fractions [
37]. Comparing the five spectra we noticed that they have the same organic functions but with different transmissions. Indeed, all the spectra have shown a wide absorption band at 3430 cm
−1 which is associated with the presence of –OH stretching vibrations which is attributed to the content of bioactive compounds (alcoholic and phenolic compounds [
38]), and intense bands around 2991 cm
−1 and 2903 cm
−1 could be assigned to C–H of alkanes and cycloalkanes. The main differences between the fractions were found first in the CSDE fraction, exhibiting less intensity on the –OH bands at 3430 cm
−1, on vibrator elongation band C=C at 1636 cm
−1, and more intensity on the band 1023 cm
−1 characteristic of the C–O vibrator which confirms the richness of the CSDE fractions with the hydrocarbons. In the second, the CSAQ fraction presented the widest band located at 3320 cm
−1, most intense band at around 1635 cm
−1 corresponds to the vibrator C=C and show disappearance of the bands included in the region of 1000–650 cm
−1. The spectra of CSHex, CSDE, CSAC, and CSBu showed a signal at 1311 cm
−1, 1023 cm
−1, 951 cm
−1, and 895 cm
−1 was respectively characteristic of the –OH, C–O, C–H, and deformation of the C–H groups indicating the predominance of phenolic such as tannins and flavonoids as well as oxygenated substances [
39,
40]. These results are in agreement with the Py-GC-MS results.
3.2. Antioxidant Activity
The antioxidant capacity of the CSECE extract and the five fractions of this extract could inhibit the free radical DPPH, in comparison with positive antioxidant control, quercetin. The results are illustrated in
Figure 3. The antioxidant activity exerted on the free radical DPPH by the extracts and fractions is dose dependent.
All the extracts can reduce the stable radical DPPH to 2,2-diphenyl-1-picrylhydrazine. The results indicate that the CSECE extract has a significant and active antioxidant capacity or even less than the positive control. Among the fractions, it appears that they have a significant antioxidant capacity (anti-DPPH) and that the activity is not found in the same way in all the fractions at the same concentration tested. At the concentration of 2 mg/mL, the maximum scavenging activity of the free radical DPPH was observed for all the samples. Our result clearly indicated that the ethyl acetate fraction presented an inhibition percentage (84.02%) quite higher than the other fractions and close to the reference antioxidant ascorbic acid (87.32%) at the same concentration. These findings are in agreement with the results reported in the literature [
29] indicating better DPPH free radical scavenging activity in the ethyl acetate solvent. The high antioxidant capacity could be attributed to the presence of high amounts of phenolic compounds, which exceed 36% in this fraction. While the activity of CSHex, CSDE, and CSAQ was 71.47%, 75.23%, and 52.97% respectively. With the exception, the CSBu fraction was the less effective at neutralizing the DPPH radical with 46.37% inhibition at 2 mg/mL, this might be attributed to the low amount of phenolic compounds (5.82%). The antioxidant activity is expressed significantly in a dose-dependent manner, each time the concentration of extract is increased, the percentage of inhibition is significantly increased. This phenomenon is interpreted by the transfer of single electrons which are localized in the external orbital of the DPPH, and after having reached a given concentration, the antioxidant will react completely with the radical, and when we increase the concentration, the activity antioxidant will remain constant since this is accompanied by the saturation of the electronic layers of the radical [
41]. The inhibition rates of the DPPH radical recorded in the presence of the hydroalcoholic extract and the various fractions are lower than those of ascorbic acid. This could be explained by the presence of methoxy groups which increase the accessibility of the center of the DPPH radical to ascorbic acid [
42,
43,
44]. This result could also be interpreted as the total content of phenolic compounds in the raw extract that does not incorporate all the antioxidants [
45]. Moreover, the fact that the synergistic effect of polyphenols makes the antioxidant activity of extracts weaker than that of isolated natural phytochemicals [
46]. The results of our study illustrate that the antioxidant capacity of the extracts of the leaves of
Capparis spinosa L. was strongly affected by the extraction solvent.
The antioxidant activity of
Capparis Spinosa L. extracts was assessed using the FRAP method. It is based on the capacity of the extracts to reduce ferric iron Fe
3+ to ferrous iron Fe
2+. The results of the FRAP tests (
Table 5) were in agreement with those of the DPPH tests, the ethyl acetate fraction having stronger antioxidant activity, with a reducing power of 4.275 ± 0.011 mmol Fe
2+/g of sample, followed by the diethyl ether fraction 1.38 ± 0.024 mmol Fe
2+/g of sample and the hydroethanolic extract 1.354 ± 0.043 mmol Fe
2+/g. while, the CSHex, CSBu, and CSAQ fractions showed a comparatively lower activity with reducing values of 1.11 ± 0.060, 0.898 ± 0.047, and 0.744 ± 0.045 mmol of Fe
2+/g of sample, respectively. According to the literature, these results are in agreement with the data found by Mohebali et al. [
47], which revealed that the extract of leaves of
Capparis spinosa L. has a significant reducing activity using the FRAP test (3606.75 ± 0.01 μmol/g). The reducing power of
Capparis spinose L. is probably due to the presence of a hydroxyl group in phenolic compounds which can serve as an electron donor. Additionally, these results suggest that the FRAP values reported in our study may be related to the content of thiols and sulfur compounds [
48]. Thus, antioxidants can be considered as reductants, and inactivate oxidants by reductants can be demonstrated as redox reactions [
49]. Some previous studies have also shown that the reducing power of a compound can serve as a significant indicator of its potential antioxidant activity [
50].
3.3. Antifungal Activity
The methods used to study the antifungal effect of CSECE and its fractions against food contaminants were designed to assess the effectiveness of the compounds in inhibiting fungal growth and secondly, to study the sensitivity of microorganisms with extracts and to measure their antifungal power. The results of exposure of
Aspergillus niger to various doses of leaf samples of
Capparis spinosa L. are presented in
Figure 4. The results of
Figure 4 indicate that the samples studied did not inhibit fungal growth in a dose-dependent manner. The crude extract and the fractions (CSHex, CSBu) tested at a dose of 20 μL were effective in controlling the fungal growth (growth intensity = 1). At a dose of 10 µL, the CSAQ fractions were more effective than CSAC and CSDE (growth intensity = 2, 3, respectively).
Table 6 shows the percentage inhibition of fungal growth, and the viability measured after 7 days of incubation at 25 °C using the Cellometer
® software. It was clear from the obtained results that the inhibition of fungal growth depended on the type of solvent used. The crude hydro-ethanolic extract inhibited 46.56% of fungal growth. CSHex, CSBu, CSAQ revealed considerable inhibition with 57.9%, 52.18%, and 58.01%, respectively. Regarding the CSAC fraction, did not show inhibition (FGI, 12.71%) not considered fungistatic, in contrast of CSDE fraction showed a higher inhibition effect (FGI, 58.78%) against the growth of
Aspergillus niger. The positive behavior of CSHex, CSBu, and CSDE against
A. niger was probably due to its high percentage of protein which was 38.95%, 60.09%, and 62.68% respectively. These results were consistent with previous research that concluded that the proteins extracted from
Capparis spinosa L. act as antifungal agents that cause growth retardation in the fungal strain [
36,
51]. The antifungal efficacy of extracts is particularly due to the composition of these in various bioactive compounds belonging to different chemical classes, which can be used to reduce the process of biodegradation [
52,
53].