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Bio-guided fractionation and characterization of powerful antioxidant compounds from the halophyte Inula crithmoїdes
⁎Corresponding author at: Laboratoire des Plantes Aromatiques et Médicinales (LPAM), Centre de Biotechnologie à la Technopole de Borj Cédria (CBBC), BP 901, 2050 Hammam-lif, Tunisia. phytochimie2009@yahoo.com (Inès Jallali)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Abstract
The investigation of natural and safe antioxidants from natural origin is highly encouraged since it was revealed that synthetic antioxidants have restricted use in foods due to their toxicological effects and suspected carcinogenic potential. Purification of most active phenolics from the halophyte Inula crithmoїdes was the objective fixed for this work. The separation of the flower phenolics was carried using centrifugal partition chromatography (CPC) and yielded 24 fractions. A bio-guided selection of the most active fractions was done based on their antioxidant activities. Fractions 2, 7, 11 and 19 were the most active ones, even more potent than positive controls BHT, BHA and ascorbic acid. The semi-preparative High Performance Liquid Chromatography (HPLC) purification of the antioxidant molecules from these active fractions and their identification by Nuclear Magnetic Resonance spectroscopy (NMR) revealed that the most potent phenolics of I. crithmoїdes are the chlorogenic acid and its two derivatives 3-p-coumaroyl-5-caffeoyl quinic acid and 1,5-di-O-caffeoylquinic acid, in addition to the quercetin and its derivative quercimeritrin. All identified compounds are powerful antioxidants, as they have many biological properties allowing their use in agro-food, pharmaceutical or cosmetic industries.
Keywords
Inula crithmoїdes
CPC
Antioxidant activities
Phenolics
HPLC
MNR
1 Introduction
In recent years, scientific communities have shown an increased interest in plant antioxidants which could be used as natural additives to replace synthetic ones (Jallali et al., 2014). Plant extracts contain various antioxidant compounds belonging to different chemical forms and classes, thus offering a wide range of alternatives to chemical preservatives (Materska, 2008). Phenolics are the most represented dietary antioxidants and have shown various beneficial effects on human health (Moglia et al., 2014). The high antioxidant potential of these secondary metabolites is mainly responsible of diverse biological effects, including anti-inflammatory, antiviral, antiatherogenic, antibacterial, and anticancer effects (Farhoosh et al., 2016). Furthermore, the activity of these compounds as food antioxidants is well known. Their addition in food matrix increases the shelf life, especially of oil and fat containing food products. The investigation of natural and safe antioxidants from natural origin is highly encouraged since it was revealed that synthetic antioxidants, such as BHA and BHT have restricted use in foods due to their toxicological effects and suspected carcinogenic potential (Ksouri et al., 2012).
I. crithmoїdes, belonging to the Asteraceae family, is an underestimated halophyte largely distributed in the Mediterranean countries. In fact, the flowering branches of Inula species are used in traditional medicine for treatment of bronchitis, tuberculosis, anemia, as astringent, for malaria and diseases of urinary system (Abdel-Wahhab et al., 2008). In this study, we tried to separate phenolic compounds with high antioxidant potential from I. crithmoïdes flowers using centrifugal partition chromatography (CPC). A bio-guided selection of the most active fractions obtained from this separation was done based on their antioxidant activities. In a second step, we tried to purify the maximum of antioxidant molecules from these active fractions by semi-preparative HPLC, then to identify them by 1H NMR.
2 Materials and methods
2.1 Chemical and reagents
All organic solvents were HPLC grade and purchased from Scharlau (Sentmenat, Spain). Water was bi-distilled.
2.2 Plant sampling
Flowers of I. crithmoїdes were sampled in October 2011 from El Kalbia salt depression (Kairouan region, N35°48′53; E10°09′07) belonging to the upper arid bioclimatic stage (mean annual rainfall >300 mm). The harvested plants were identified at the Biotechnology Center at the Technopark of Borj-Cédria, and a voucher specimen [PLM78] was deposited at the Herbarium of the Laboratory.
2.3 Preparation of flower extract
I. crithmoїdes flowers were air-dried in shadow at room temperature, reduced to a fine powder then subjected to a differential extraction. A solid/liquid extraction of 1 kg of flower’s powder by 60% aqueous acetone (v/v) (2 × 2l) was carried out. Mixture was kept in frequent agitation at ambient temperature for two hours then filtered through a Whatman N°4 filter paper. After filtration, the aqueous acetone extracts were combined and concentrated at 35 °C under reduced pressure. The residual aqueous phase (1l) was extracted by petroleum ether (3 × 1l) for two hours to eliminate pigments and waxes then subjected to a liquid/liquid extraction by ethyl acetate (4 × 1l). Mixture was transferred in a funnel and left to stand until two phases were observed. Aqueous phase was discarded and the organic phase was collected and evaporated under vacuum ay 35 °C to dryness then dissolved in water to be freeze-dried. The ethyl acetate phase yielded 10.55 g (1.06% of the primary used plant material). Dry residue was stored in the darkness at 4 °C until analysis.
2.4 Fractionation of I. Crithmoїdes flower extract by CPC
Separation procedure was carried out using an FCPC200® apparatus (Kromaton Technologies (Angers, France) that is equipped with a rotor made of 20 circular partition disks enclosing 1320 partition cells (0.130 mL per cell); with a total column capacity of 204 mL. The dead volume is about 75 mL. Rotation speed is adjustable in a range of 0–2000 rpm. The resulting centrifugal force field in the partition cell is nearly 1200 ms−2 at 1100 rpm and 4200 ms−2 at 2000 rpm. The CPC is equipped with a Gilson 321-H1 2-way binary high-pressure gradient pump and a high pressure injection valve (3725(i)038 Rheodyne) equipped with a 10 mL sample loop. The effluent was monitored with an ICS UV-Lambda 1010 detector equipped with a preparative flow cell. Fractions were collected by a Gilson FC 204 fraction collector. The experiments were conducted at room temperature. The quaternary biphasic solvent systems were prepared at room temperature by n-heptane(n-Hept), ethyl acetate (EtOAc), methanol (MeOH), and water in the convenient proportions for systems J (2:5:2:5; v/v/v/v) and M (5:6:5:6; v/v/v/v) of Arizona-Margraff systems (Foucault and Chevolot, 1998). The resulting two phases were separated just before use. As a first step, aqueous phase (methanol/water) was used as a stationary phase to fill up the rotor in the ascending mode without rotating. After that, 1 g of dry matter was dissolved in 10 mL of the organic/aqueous phase mixture (1:1) then injected in the apparatus. The pumping of the organic mobile phase into the column was then allowed. The flow-rate was 10 mL min−1, and rotation speed was fixed at 300 rpm. Once the column was filled with the mobile phase, the rotation speed was increased from 300 to 1200 rpm and the flow-rate was fixed at 9 mL min−1. The pressure was maintained at 23 bars along the experience. Fractions of 9 mL were collected every minute. The content of the outgoing organic phase was monitored by online UV absorbance measurement at λ = 280 nm.
2.5 Characterization of I. Crithmoïdes flower polyphenols
2.5.1 Thin layer chromatography (TLC) analysis
One of the chromatographic tools used in the analysis of I. crithmoïdes flower fractions is TLC. Ten µL of each fraction were deposed on pre-coated silica gel 60 F254 plates (Merck). Plates were then partially immersed in a migration solution consisted of CHCl3–methanol–acetic acid (80/20/3) and left for 15 min to be developed. Revelation of the separated bands was achieved by anisaldehyde sulphuric reagent (5 mL p-anisaldehyde, 90 mL ethanol and 5 mL sulphuric acid) permitting their detection at 254 and 366 nm.
2.5.2 HPLC analysis
HPLC analysis was performed on Agilent apparatus equipped with an autosampler model 1100, a Prostar Pump model 1100, diode array detector model 1100, A C18 column (Prontosil, 250 mm × 4.0 mm, 5 µm, Bischoff) was used for analysis. The mobile phase was composed of two solvents: A, 0.025% TFA in H2O and B, acetonitrile (MeCN). The sample was dissolved in MeOH/H2O (1:1) and filtered through a 0.45 µm Millipore filter. The elution program at 1 mL/min was as follows: 10% B (0–5 min), 10–100% B (5–55 min), 100–10% B (55–60 min), 10% B (60–65 min), 10 µL of each sample was directly injected and chromatograms were monitored at 280 nm.
2.5.3 Purification of phenolic compounds by semi-preparative HPLC
Phenolic compounds contained in the active fractions were purified by preparative HPLC. The apparatus is equipped by a Prostar 218 2-way binary high-pressure gradient pump, Prostar 345 UV detector and a column (Microsorb 100-5 250 mm × 21.4 mm, 5 µm particle size, Varian). Solvent A was used as mobile phase and it consisted of 0.025% TFA in water while solvent B was composed of pure MeCN. The gradient elution program was: 10% B (0–5 min), 10–100% B (5–55 min), 100% B (55–60 min), 100–10% B (60–65 min). Flow rate was fixed at 3 mL/min. Detection was performed at 280 nm.
2.5.4 Spectroscopic analysis
All isolated compounds were analyzed by 1H and 13C NMR and spectra were recorded on Bruker Avance 3 (1H at 600 MHz and 13C at 150 MHz) in acetone-d6. ESI-MS was obtained on a Micromass Q-TOF micro spectrometer (Manchester, UK). Esquire 3000+ Ion Trap mass spectrometer (Bruker Daltonics, Germany) was used to study the mass of pure molecules. Sample solutions were infused directly into ESI source with a syringe pump (74900 Series, Cole-Parmer Instrument) at a constant flow-rate of 180 µL/h. Mass spectra were recorded with m/z = 150–2000 in positive ionization mode. Normal scan resolution (13,000 m/z s−1) was selected. The source parameters were −4000 V; nebulizer gas (N2), 10 psi; drying gas (N2), 7 l/min; dry temperature, 300 °C.
2.6 Antioxidant activities of I. Crithmoїdes flower fractions
2.6.1 Evaluation of total antioxidant capacity
Corresponding to Koleva et al. (2002), the total antioxidant capacity of an extract is reflected by the number of reduced molybdate ions by the action of the extract antioxidant. The importance of this activity is positively correlated to the formation of green phosphate/Mo (V) complex at acid pH. First, a reagent solution consisted of 0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate is prepared. One mL of this solution is combined with 0.1 mL of each fraction at the concentration of 100 µg mL−1. The mixture was then incubated in a thermal block at 95 °C for 90 min. Samples have to be cooled to room temperature before measuring the absorbance at 695 nm against a blank. The antioxidant capacity was expressed as mg GAE g−1 DW. The calibration curve range was 0–500 µg mL−1. All samples were analyzed in triplicate.
2.6.2 DPPH assay
In this study, we chose to adopt the method described by Hatano et al. (1988) to evaluate the DPPH (2,2-Diphenyl-1-picrylhydrazyl) quenching ability of I. crithmoїdes flower fractions. This method principal consists on DPPH radical reduction by accepting hydrogen from antioxidant, reflected by the change of the DPPH solution color from the deep violet to almost a colorless one. This color reduction is correlated to the radical scavenging activity of the antioxidant. To carry out this test, 1 mL of the samples properly diluted at different concentrations was added to 250 µL of DPPH solution at 0.2 mM. After 30 min of incubation at room temperature and in the dark, the absorbance was read against a positive control (methanol in place of the sample) at 517 nm. Inhibition of DPPH radical in percent (I%) was calculated as follows:
2.6.3 FRAP assay
Determination of ferric reducing antioxidant power (FRAP) is a simple direct test for measuring antioxidant capacity. The iron (III) reductive capacity of the fractions was assessed as described by Oyaizu (1986). Briefly, 1 mL of extract at different concentrations was mixed with 2.5 mL phosphate buffer (0.2 mol l−1, pH 6.6) and 2.5 mL of potassium ferricyanide (III) (K3Fe (CN)6) solution (1%). After 20 min at 50 °C, 2.5 mL (10%) Trichloroacetic acid (TCA) was added and the mixture was centrifuged for 10 min at 650g in a centrifugation apparatus type SIGMA. Finally, a 2.5 mL aliquot was mixed with 2.5 mL ultra-pure water and 0.5 mL (0.1 g 100−1 mL) of iron(III) chloride anhydrous (FeCl3) and the absorbance was recorded at 700 nm and AscA was used as a positive control. A higher absorbance indicates a higher reducing power. EC50 value (µg mL−1) was obtained from linear regression analysis.
2.6.4 β-Carotene bleaching test (BCBT
The capacity of different fractions to inhibit β-carotene bleaching was evaluated using Koleva et al. (2002) method, with slight modifications. β-carotene (2 mg) was dissolved in 20 mL chloroform. After that, 4 mL of this solution were combined with 40 mg of linoleic acid and 400 mg of Tween 40 and well mixed together before evaporating the chloroform under vacuum at 40 °C. The residual phase was diluted in 100 mL of oxygenated ultra-pure water, and then the emulsion was vigorously shaken. Aliquots of 150 µL of the β-carotene/linoleic acid emulsion were distributed in the microtitre plates and then 10 µL of tested fractions at different concentrations were added. Analyses were performed in triplicates. The absorbance of all samples was measured immediately (t = 0 min) using an EAR 400 microtitre reader (Labsystems Multiskan MS) at 470 nm. The microtitre plates were incubated at 50 °C for 120 min. After that, the absorbance was measured once again (t = 120 min). The antioxidant activity (AA) of the fractions was evaluated in term of β-carotene bleaching using the following formula:
2.7 Statistical analysis
Means were statistically compared using the STATISTICA program. A one-way analysis of variance (ANOVA) and Duncan’s multiple range tests were carried out to perceive any significant differences between parameters at P < 0.05.
3 Results and discussion
3.1 Pre-purification of I. Crithmoïdes flower extract
HPLC chromatogram of I. crithmoïdes flower extract (Fig. 1) contains many compounds, probably belonging to phenolics, by reference to their UV spectrum. A good separation of these compounds by CPC implies the use of adequate solvent system. Therefore, we tested the 23 Arizona-Margraff solvent systems (Hept/EtOAc/MeOH/water) [7] to select the best one permitting a quasi-equal partition of the compounds between the two phases of the solvent system. Results of this experience indicated that J system is the more convenient for this separation.
HPLC chromatograms of flower extract of I. crithmoïdes and its most active fractions and sub-fractions, monitored at 280 nm.
Preparative CPC separation of I. crithmoïdes flower extract yielded 131 fractions. TLC analysis was used to compare these fractions and those enclosing the same compounds were gathered to reduce the fraction number to 24 ones: 18 fractions from the ascending mode and 6 fractions from the descending one (Table 1). Obtained fractions were evaporated to dryness then analyzed by HPLC. Means followed by the same letter are not significantly different at P ≤ 0.05.
Fraction N°
Fraction yield (mg)
TAA (mg GAE g−1) at 100 µg mL−1
DPPH test (CI50 µg mL−1)
FRAP assay (CE50 µg mL−1)
BCBT (CI50 µg mL−1)
Ascending mode
F1 (8)
28.1 ± 0.0gh
0.48 ± 0.03 h
7.3 ± 0.24ef
153 ± 8.2d
260 ± 3.3r
F2 (9)
40.1 ± 0.6d
0.74 ± 0.03 cdef
2.25 ± 0.14l
43 ± 4o
285 ± 4.3p
F3 (10–12)
62.6 ± 0.5c
0.56 ± 0.15 gh
3.1 ± 0.09kl
82 ± 7.2ij
266 ± 6.7q
F4 (13–17)
74.2 ± 0.6b
0.55 ± 0.07 gh
6.4 ± 1.03fgh
65 ± 3kl
80 ± 5.1t
F5 (18)
4.6 ± 0.1q
0.47 ± 0.02 h
5 ± 0.33hij
107 ± 6.8g
45 ± 3.7v
F6 (19–22)
17.6 ± 0.3k
0.53 ± 0.05 gh
5.4 ± 0.82ghi
174 ± 2.7c
48 ± 2.7u
F7 (23–25)
7.6 ± 0.2o
0.73 ± 0.06 cdef
3.8 ± 0.1jkl
62 ± 5.7klm
34 ± 2.1w
F8 (26–32)
26.1 ± 0.2h
0.49 ± 0.11 h
6.9 ± 0.42efg
102 ± 4.5g
385 ± 7.4o
F9 (33–34)
5.5 ± 0.3p
0.80 ± 0.14 cd
7.4 ± 1.55ef
99 ± 11.3gh
90 ± 5.7s
F10 (35–39)
32.5 ± 0.3f
1.69 ± 0.2 a
3.7 ± 0.36jkl
51 ± 0.65no
910 ± 8b
F11 (40–45)
81.5 ± 0.5a
1.63 ± 0.01 a
2.7 ± 0.09 l
46 ± 4.1o
805 ± 9.8f
F12 (46–47)
30.9 ± 0.0g
1.15 ± 0.12b
4.0 ± 0.1ijkl
52 ± 4.1mno
725 ± 3h
F13 (48–50)
38.3 ± 0.4e
0.6 ± 0.06 fgh
3 ± 0.08l
72 ± 2jk
713 ± 4.4i
F14 (51–58)
29.6 ± 0.4g
0.73 ± 0.03 cdef
4.1 ± 0.35ijkl
80 ± 2.9ij
670 ± 5.3j
F15 (59–70)
19.4 ± 0.0j
0.62 ± 0.05 efgh
12.4 ± 1.48cd
91 ± 5.8hi
469 ± 6n
F16 (71–83)
9.5 ± 0.4mn
0.25 ± 0.1 i
16.7 ± 1.6b
236 ± 13.5a
870 ± 5.3c
F17 (84–98)
8.9 ± 0.3n
0.53 ± 0.06 gh
13.4 ± 2.23c
134 ± 2e
847 ± 5.5e
F18 (99–125)
11.5 ± 0.5l
0.7 ± 0.19 cdefg
8.4 ± 1.03e
77 ± 1.3j
590 ± 6.3m
Descending mode
F19 (126)
8.4 ± 0.3n
0.76 ± 0.08 cdef
4.7 ± 0.56ijk
60 ± 1.7lmn
714 ± 5.7i
F20 (127)
8.5 ± 0.3n
0.68 ± 0.11 cdefg
5.3 ± 1.11hij
81 ± 1.3ij
885 ± 6.6d
F21 (128)
10.1 ± 0.2m
0.84 ± 0.1 c
11.7 ± 0.46d
81 ± 0.7ij
650 ± 3.3k
F22 (129)
9.8 ± 0.2m
0.66 ± 0.02 defg
12.9 ± 1.59cd
121 ± 1.3f
610 ± 3.9l
F23 (130)
20.6 ± 0.4j
0.78 ± 0.1 cde
8.1 ± 0.84e
175 ± 1.7c
957 ± 6.3a
F24 (131)
24.4 ± 0.2i
0.47 ± 0.12 h
22.6 ± 1.49a
219 ± 1.1b
740 ± 5.9f
Positive control
BHT
–
11.5 ± 0.0d
–
–
AscA
–
–
37.33 ± 0.0p
–
BHA
–
–
–
48 ± 0.0u
3.2 Antioxidant activities of I. Crithmoïdes flower fractions
Results of the antioxidant activities of the 24 obtained fractions are listed in Table 1. Total antioxidant activity was performed on 100 µg mL−1for each fraction and obtained results disclose the significant variability of this activity among the 24 fractions, with a pre-eminence of fractions 10 and 11 whose activities are in a range of 1.69 and 1.63 mg GAE g−1DW, respectively. Fraction 12 comes in the second array with a total antioxidant activity equal to 1.15 mg GAE g−1DW. As for the antiradical activity, results displayed that all tested fractions exhibited high scavenging activity of the DPPH radical, exceeding in the major cases that of the positive control BHT (Table 1). The lowest IC50 (2.25 and 2.7 µg mL−1) were depicted in fractions 2 and 11, followed by fraction 7 (3.8 µg mL−1) statistically a little bit less active. Both these three fractions belong to the ascending mode, while only the fraction 19 expressed important antiradical activity (4.7 µg mL−1) among those of the descending one. However, fractions 3, 10, 12, 13, and 14 expressed also a very interesting antiradical activity (Table 1), probably due to the presence of some remaining proportions of the active compounds eluted in the fractions 2 and 11. The analysis of the FRAP results showed that almost the same fractions expressing high total antioxidant and antiradical activities have the best reducing power potential. In fact, fraction 2 and 11 exhibited the lowest EC50 values (43 et 46 µg mL−1, respectively) followed by fractions 10 and 12 (51 and 52 µg mL−1, respectively), while fractions 7 and 19 come in a third range, which does not underplay their strong ability to reduce ferric iron corresponding to EC50 values of 60 and 62 µg mL−1, respectively (Table 1). On the other hand, fraction ability to inhibit β-carotene bleaching was significantly variable and seems to be different from the other antioxidant tests. In fact, fraction 7 was distinguished by its high bleaching inhibition potential (34 µg mL−1), even more important than BHA (48 µg mL−1), together with fraction 5 (45 µg mL−1) while fraction 6 comes behind with an IC50 = 48 µg mL−1.
When comparing the 24 obtained fractions on the basis of their antioxidant efficiency, we find that fractions 2, 7, 11, and 19 are mainly the most important ones, independently from the antioxidant test kind. Besides, the first three ones belong to the ascending mode while only fraction 19 belongs to the descending one. In accordance with our results, the study of Trabelsi et al. (2013), for example, showed that the separation of the ethyl acetate phase of Limoniastrum guyonianum gave 10 fractions of which the biological activities were positively correlated with high amounts of proanthocyanidins.
The analysis of these results showed that CPC permitted an efficient separation of I. crithmoïdes phenolic compounds as it yielded fractions with high antioxidant potential. In fact, saturation of the stationary phase allows CPC to be efficient in preparative separation. In addition, the CPC is described as “soft” technique because it does not cause loss of sample by irreversible adsorption, such as in the case with other chromatographic techniques using a solid support (usually silica gel) as stationary phase. The absence of the adsorbent eliminates the risk of sample denaturation by interaction with silica (Intes et al., 2001; Zga et al., 2009). This criterion justifies the wide use of CPC as an excellent chromatographic technique for the separation of fragile biomolecules such as low-molecular-mass heparin (Intes et al., 2001), protoberberine quaternary alkaloids (Bourdat-Deschamps et al., 2004), stilbenoids (Zga et al., 2009), and phenolic compounds (Bourdat-Deschamps et al., 2004). According to these findings, we proposed to go deep in this research until we identify the molecules responsible of the high antioxidant potential of these 4 fractions.
3.3 Analysis of active fraction phenolic composition by analytical and preparative HPLC and 1H NMR spectroscopy
HPLC chromatograms of I. crithmoïdes flower extract and its more potent fractions are represented in Fig. 1.
The analysis of fraction 2 chromatogram showed that compounds of this fraction cannot be separated with this solvent system. Fortunately, the yield of this fraction was high enough to allow a second CPC separation of its molecules using M Arizona solvent system (Foucault and Chevolot, 1998). This CPC fractionation yielded 16 sub-fractions divided in 10 ones belonging to the ascending mode and 6 sub-fractions from the descending mode (Table 2). Means followed by the same letter are not significantly different at P ≤ 0.05.
Sub-fraction N°
Sub-fraction yield (mg)
IP% at 10 µg mL−1
IC50 (µg mL−1)
Ascending mode
F1 (1–9)
0 ± 0.0m
0 ± 0.0m
–
F2 (10–13)
4.1 ± 0.3g
0 ± 0.0m
–
F3 (14–16)
9 ± 0.4e
31.9 ± 0.6j
–
F4 (17–21)
22.1 ± 0.5b
38.3 ± 0.7i
–
F5 (22–27)
2.9 ± 0.2h
32.6 ± 1.0j
–
F6 (28–37)
37.6 ± 0.7a
83.9 ± 2.7c
2.4 ± 0.4d
F7 (38–44)
15.5 ± 0.6c
85.5 ± 1.4c
1.9 ± 0.2e
F8 (45–48)
2 ± 0.3j
17.3 ± 2.1l
–
F9 (49–70)
1.4 ± 0.1k
29.2 ± 2.0k
–
F10 (71–77)
1.8 ± 0.3j
49.3 ± 1.9h
–
Descending mode
F11 (78–79)
2.1 ± 0.4ij
61.3 ± 2.8f
–
F12 (80–81)
6 ± 0.2f
74.2 ± 0.3e
6.7 ± 0.41a
F13 (82)
1.9 ± 0.3j
81.4 ± 0.5d
5.4 ± 0.17b
F14 (83–84)
11.3 ± 0.4d
90.2 ± 1.0b
4.25 ± 0.17c
F15 (85)
0.3 ± 0.0l
53.9 ± 0.8g
–
F16 (86–87)
2.4 ± 0.2i
50.6 ± 0.8h
–
Fraction 2
–
93.6 ± 0.4a
2.25 ± 0.1de
Once again, we made a bio-guided selection of the most active sub-fractions by the DPPH test using, in a first step, a unique sub-fraction concentration fixed at 10 µg.mL−1. Results of this quick test revealed that only sub-fractions 6, 7, 12, 13, and 14 exhibited high inhibitions percentages (up to 70%). The antiradical activity of the remaining sub-fractions was lower suggesting that compounds contained in are not important contributors to the antioxidant efficiency of fraction 2. In a second step, we compared the IC50 values of the selected 5 sub-fractions and results displayed that only sub-fraction 7 exhibited higher antiradical activity (IC50 = 1.9 µg mL−1) than fraction 2 (IC50 = 2.25 µg mL−1) (Table 2). This fact is symptomatic of the presence of highly active compounds in sub-fraction 7, probably representing the major contributor to the antioxidant activity of fraction 2. HPLC analysis of the sub-fraction 7 revealed the presence of two major peaks (1) and (2) (Fig. 1). Their purification was carried out by preparative HPLC and afforded two pure compounds. After that, their structures were analyzed by 1 and 2D NMR spectroscopy. The structure of compound (1) was unfortunately undetermined because the yield of this compound was insufficient to carry out the experiments. However, compound (2) was identified as quercetin (Fig. 2A). As for the fraction 7 (obtained from the separation of I. crithmoïdes flower extract), HPLC chromatogram showed the presence of 2 major compounds (Fig. 1). Their preparative HPLC purification yielded compounds (3) and (4). The same as compound (1), compound (3) couldn’t be identified, while the structure elucidation of compound (4) disclosed that it was about a chlorogenic acid derived: the 3-p-coumaroyl-5-caffeoyl quinic acid (Fig. 2B). Concerning fraction 11, its chromatographic profile (Fig. 1) contains one main peak (5) corresponding to the major compound of I. crithmoïdes flowers (Fig. 1). Preparative HPLC purification of this molecule yielded a pure product (98% purity) which was identified as 1,5-di-O-caffeoylquinic acid (Fig. 2C) described, to the best of our knowledge, for the first time in the flowers of this species. Finally, HPLC chromatogram of fraction 19 (Fig. 1) contains two major peaks (6) and (7) which were isolated by preparative HPLC and identified as chlorogenic acid and quercimeritrin, respectively (Fig. 2D and E). To summarize, the major antioxidant compounds of I. crithmoïdes flowers belong to the phenolic acids and flavonoids and are clorogenic acid and its two derivatives 3-p-coumaroyl-5-caffeoyl quinic acid and 1, 5-di-O-caffeoylquinic acid from one hand, and quercetin and its glycoside derivative quercimeritrin from the other hand. All identified compounds are described in the literature to be powerful antioxidant and multifunction bioactive compounds.
Structures of identified phenolic compounds from I. crithmoïdes flowers. (A) quercetin, (B) quercimeritrin, (C) chlorogenic acid, (D) 1,5-di-O-caffeoyl quinic acid, and (E) 3-O-p-coumaroyl-5-O-caffeoyl quinic acid.
3.3.1 Clorogenic acid and its derivatives
The chlorogenic acids express a variety of biological activities including antioxidant, antibacterial (Guzman, 2014), anti-inflammatory (Yuan et al., 2012), anti-HIV, anti-HBV, have the capacity to scavenge radicals and inhibit mutagenesis and carcinogenesis (Jaiswal et al., 2011). The dicaffeoyl quinic acids have anti- hepatotoxic activity, effectively suppress melanogenesis (Moglia et al., 2014); have antispasmodic and anti-inflammatory activities in vitro and exhibit selective inhibition of HIV replication (Yuan et al., 2012). Besides, caffeoyl derivatives showed immune-stimulating properties which appear to promote the phagocytic activity, in addition to the protective effect against collagen degradation induced by free radicals (Bazylko and Stolarczyk, 2012).
3.3.2 Quercetin and its derivatives
Numerous in vitro studies have shown that quercetin is an excellent antioxidant. It is even the most powerful scavenger of reactive oxygen species of all the flavonoids. Quercetin offers a variety of therapeutic potentials used primarily in the prevention and treatment of various diseases such as allergies, asthma, hay fever (Rogerio et al., 2007), urticaria, diabetic complications, eye disorders, osteoporosis, ulcers and viral infections (Chang et al., 2012). Quercetin also has antibacterial activity against almost all known bacteria strains causing respiratory, gastrointestinal, skin and urinary tract problems (Materska, 2008; Rogerio et al., 2007). In addition, the antioxidant intake of quercetin protects against coronary heart disease caused by oxidized LDL, as it is also used in the treatment of gout (Lakhanpal and Rai, 2007). Furthermore, this falvonoid interferes in neurodegenerative diseases (Martínez De Morentin et al., 2010), and it has anticancer and anti-inflammatory properties (Materska, 2008). The quercimeritrin is a flavonoid rare in nature. It has been proven that it shows good inhibitory capacity of the DPPH radical (Bazylko and Stolarczyk, 2012) and a high antioxidant activity (Legault et al., 2011). In addition, this flavonoid exhibits a very good vaso-relaxing (Penso et al., 2014) and healing wounds properties (Anuradha et al., 2008). It also has an anti-inflammatory activity via the inhibition of interleukin 5 (IL-5) (Legault et al., 2011). The quercimeritrin enjoys anti-diabetic properties resulting in the inhibition of aldose reductase in rats (Li et al., 2003).
4 Conclusion
The separation of the phenolics from I. crithmoïdes flowers by CPC, guided at every step by the results of their antioxidant capacities, led to the isolation of three chlorogenic acids and two flavonoids. The biological properties, essentially antioxidant, of these molecules are attracting more and more interest of many health-related industries which tend to shift preferences to natural sources of natural antioxidants that can be employed as food additives (Jallali et al., 2014). The isolation of phenolic compounds from natural matrices and the estimation of their effects on human health are the fashion wave that invades the world of research in food, pharmaceutical, and medicinal applications. In this context, I. crithmoïdes is a promising halophyte, potentially rich in bioactive compounds that can replace the carcinogenic synthetic additives in agro-food, pharmacologic and cosmetic industries. The molecules identified in I. crithmoïdes flowers and their related biological activities have been described in previous works. However, the originality of this work resides in the choice of the vegetal matrix, which is part of a national project aiming the valorization of the very important biological potential of the underestimated vegetation of the marginal zones, mainly as source of bioactive molecules. The primary goal set for this work was not to fall on new bio-products, but rather to highlight unconventional sources of molecules of various interests that are easily accessible through spontaneous plants widely represented in these unexploited territories. In addition, the technique described for the isolation of these pure products, from the pre-purification by the set of solvents till the CPC, HPLC and NMR analyses guided at each step by the results of the antioxidant activities, is a refined technique that allows to rapidly discriminate the maximum of these molecules in relation to the biological activity sought, while respecting the chemical nature of these natural products, their links with other molecules as well as their interactions.
5 Contributors
Inès Jallali: Conducted experiments and wrote the manuscript.
Pierre Waffo Téguo: Has ensured the development and result treatment of chromatographic experiences (CPC, HPLC, and MNR).
Abderrazek Smaoui: The botanist who authenticated specimens of the plant in their native ecosystems and established their taxonomic identification.
Jean-Michel Mérillon, Chedly Abdelly and Riadh Ksouri: Scientific and administrative directors of the laboratories where was conducted the research work.
All authors read and approved the manuscript.
Conflicts of interest
None.
Acknowledgments
A special thank goes to Dr. Ons Talbi for her help in the treatment of the statistical analysis and, above all, for her kindness and advices.
Role of the funding source
This work was supported by the Tunisian Ministry of Higher Education and Scientific Research (LR10CBBC02).
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