On-Line Screening, Isolation and Identification of Antioxidant Compounds of Helianthemum ruficomum

Many Helianthemum species (Cistaceae) are recognized for their various medicinal virtues. Helianthemum ruficomum is an endemic species to the septentrional Sahara on which no report is available so far. The purpose of this work was to investigate the chemical composition and the radical scavenging capacity of this species and its isolated components. Collected from Mougheul (south-west of Algeria), the aerial parts were macerated with 80% EtOH/H2O, after evaporation, the remaining extract was diluted with H2O and extracted with petroleum ether, chloroform, ethyl acetate and n-butanol. EtOAc and n-BuOH extracts were evaluated for their free radical scavenging capacity by on-line HPLC-ABTS•+ assay. The obtained data which were confirmed by TEAC and ORAC assays, allowed guiding the fractionation of these extracts by CC, TLC and reverse phase HPLC. Among the components, 14 were isolated and identified by spectroscopic analyses: protocatechuic acid (1), trans-tiliroside (2), cis-tiliroside (3), astragalin (4), picein (7), vanillic acid 4-O-β-d-glucopyranoside (8), lavandoside (9), 4-hydroxybenzoic acid 4-O-β-d-glucopyranoside (10), nicotiflorin (11), rutin (12), vicenin-2 (13), narcissin (14) and stigmasterol (5) and β-sitosterol (6) as a mixture (71% and 29%, respectively). Compounds 5, 7, 8, 9, 10 and 14 were new for the genus Helianthemum. The antioxidant power of all the isolated compounds was also evaluated by HPLC-ABTS•+, TEAC and ORAC assays. The results clearly indicated high antioxidant potential of the extracts and tested compounds of this species especially, compounds 1, 4, 8, 9, 10 and 12.


Introduction
Currently, there is an increasing interest in the research and prospection of new sources of natural antioxidants as safe additives in food industry or ingredients of functional foods, neutraceuticals and pharmaceuticals. Free radicals are major contributors in aging and play a key role in degenerative diseases [1][2][3]. Sahara species can develop metabolite responses against drought stress and ROS produced by extensive UV exposition [4][5][6]. According to this finding and the results of our previous studies on Saharan species which showed the presence of high content of bioactive compounds and positive antioxidant and antiproliferative properties [7][8][9][10], we investigate in this study, extracts of an Cistaceae consists of 8 genera and about 180 species [11]. Helianthemum genus (Cistaceae) contains approximately 110 species [12], some of them are important medicinal plants used in several countries for different purposes [13][14][15][16][17]. This genus is reported to possess anti-inflammatory, antimicrobial, antiprotozoal and antioxidant properties [18][19][20][21][22]. Even if this genus was not studied much from the phytochemical viewpoint, some species have been previously examined for bioactive components like flavonoids, phenolic acids, lignans and essential oils [23][24][25][26][27][28].
To the best of our knowledge, the species Helianthemum ruficomum (Viv.) Spreng (synonyms: Cistus ruficomus Viv., Helianthemum arnaizii Sennen, H. desiderii Sennen, H. hirtum subsp. ruficomum (Viv.) Maire, H. eremophilum Pomel, H. hirtum var. deserti Coss., H. desertorum Willk. [29][30][31] has not been previously studied. In this work, ethyl acetate and n-butanol soluble parts of the aqueous-EtOH extract of the aerial parts were investigated using liquid chromatography with post-column reaction allowing a direct on-line detection of radical scavenging power of molecular species. A special focus was done on the evaluation of free radical scavenging capacities (ABTS •+ , TEAC, ORAC assays and on-line HPLC-ABTS •+ ) of extracts and isolated compounds. The structures of the isolated compounds were elucidated using ESI-HRMS, molecular absorption spectroscopy, extensive application of oneand two-dimensional NMR spectroscopy and comparison with literature data.

Identification of Chromatographic Peaks and Antioxidant Activity of Plant Extracts and Pure Compounds
On-line HPLC-ABTS •+ profiles of ethyl acetate and n-butanol extracts of H. ruficomum are reported in Figures 2 and 3, respectively. These profiles showed a wealth of the two extracts in phenolic compounds detected by their absorbancy at 280 nm. The identification of chromatographic peaks was carried out after separation and purification by chromatographic techniques and re-injection of the pure isolated compounds (HPLC-ABTS •+ ) under the same conditions as the extracts. Regarding antioxidant activity, compounds protocatechuic acid (1), astragalin (4), vanillic acid 4-O-β-D-glucopyranoside (8), lavandoside (9), 4-hydroxybenzoic acid 4-O-β-D-glucopyranoside (10) and rutin (12) showed relatively high radical scavenging capacity (Tables 1 and 2 Table 2). The antioxidant activity of the n-BuOH extract is largely due to the presence of the three phenolic acids 8, 9, 10 and the flavonoid glycoside rutin (12) which represent 88.15% of the total activity ( Table 2). The phenolic compound 1 and the flavonoid glucoside 4 represent only 10.34% of the total antioxidant activity of EtOAC extract (Table 1) as the compounds A, B, C, D and E which also showed high antioxidant activity ( Figure 2) could not be isolated in pure state and then were not identified. These five components represented 62.21% of the ethyl acetate extract antioxidant activity (Table 1). trans-Tiliroside (2), cis-tiliroside (3), nicotiflorin (11) and narcissin (14) which showed a relatively high molecular absorbance on the upper chromatogram (Figure 3), exhibited little or no radical scavenging capacities. These results were confirmed off-line by ABTS, ORAC and TEAC assays. The measure of the free radical scavenger capacity of the two studied extracts of Helianthemum ruficomum by off-line TEAC test, confirmed these results and showed that EtOAc and n-BuOH extracts exhibited comparable activities (TEAC 432 and 431 µMol TE /mg, respectively, Table 3). This may be in relation with the strongest amount of phenolic and flavonoid compounds in these extracts. The off-line ABTS assay of the isolated compounds showed also that protocatechuic acid  (Table 3) and TEAC assay values: 469, 106, 408 and 556 µMol TE /mg ( Table 3). The higher activity of rutin (12) in comparison to the other flavonoid glycosides present in these extracts may be due to the presence of the ortho di-OH system on the ring B of this molecule [44]. In addition, the higher activity of protocatechuic acid (1) in comparison with vanillic acid 4-O-β-D-glucopyranoside (8) and 4-hydroxybenzoic acid 4-O-β-D-glucopyranoside (10) may be due the lack of glycosylation, which has been found to diminish the radical scavenging activity [45]. This was also observed for ferulic acid glucoside (9), which showed relatively low antioxidant activity in comparison with ferulic acid [46,47].
The presence in this species, of phenolic acids and flavonoids was in good agreement with major studies reported on Helianthemum species [14,24,26,27]. The strong accumulation by this species, of trans-tiliroside which has been demonstrated to exert multiple biological effects [48,49] and the high antioxidant potential of its extracts and tested compounds, emphasized the possible relevance of this plant for Algerian traditional medicine. Moreover, it is important to note that no report has been published so far on eventual ethnomedical uses of this species. This may be due to a low distribution of this species. trans-tiliroside which has been demonstrated to exert multiple biological effects [48,49] and the high antioxidant potential of its extracts and tested compounds, emphasized the possible relevance of this plant for Algerian traditional medicine. Moreover, it is important to note that no report has been published so far on eventual ethnomedical uses of this species. This may be due to a low distribution of this species.   trans-tiliroside which has been demonstrated to exert multiple biological effects [48,49] and the high antioxidant potential of its extracts and tested compounds, emphasized the possible relevance of this plant for Algerian traditional medicine. Moreover, it is important to note that no report has been published so far on eventual ethnomedical uses of this species. This may be due to a low distribution of this species.

On-Line HPLC-ABTS •+ Assay
The ABTS •+ assay was based on the procedure described by Re (1999) [50] and Siddhuraju (2006) [51]. ABTS (7 mM) was dissolved in 20 mL of Milli-Q water, to which potassium persulfate (2.5 mM) was added, generating the radical cation ABTS •+ overnight. The solution was left overnight at 4 • C protected from light exposure. This solution was used within 4 days, dilutes in the Phosphate Buffer Saline (PBS) solution (pH 7.4) in order to reach an absorbance of 1.2 at 412 nm. Phosphate Buffer Saline (PBS) was prepared by dissolving in Milli-Q water, 80 g NaCl, 14.4 g Na 2 HPO 4 , 2.4 g KH 2 PO 4 and 2 g KCl; the volume was completed with Milli-Q water to 1L. The pH was adjusted to 7.4 using NaOH 0.1 mol/L. The radical cation ABTS •+ solution (10 mL) was diluted in PBS (50 mL) and completed to 500 mL with Milli-Q water before use. The diode array detector (DAD) was connected to a mixing tee followed by a reaction coil (Peek, 20 m × 0.25 mm) loaded in a temperature controlled oven. Post-column reaction is operated by delivering (0.5 mL/min) the ABTS •+ reagent with an Ultimate 3000 variable wavelength detector through the mixing tee. After the reaction coil, the flow pass through a second molecular absorption photometric detector set at 412 nm to detect the reduced form of ABTS •+ radical and thus a reduced absorbancy. The result is presented as a double chromatogram, the upper part representing the phenolic compounds, detected by their absorbancy at 280 nm, while the lower part representing the free radical scavenging activity of these phenolic compounds. A negative peak indicates that a compound having radical scavenging activity elutes out of the chromatographic column and react with the ABTS •+ radical cation. The area of the chromatographic negative peak gives an indication on the radical-scavenging activity of the considered compound. The column used in the separation of EtOAc and n-BuOH extracts, was a Kromasil C 18 with a 5 µm particle size, 4.60 mm × 250 mm (column temperature: 25 • C). The mobile phase delivered at 1 mL/min, was composed of 0.1% formic acid in H 2 O milli-Q (solvent A) and acetonitrile containing 1% formic acid (solvent B). Gradient was as follow: 0 min, 10% B; 10 min, 20% B; 20 min, 20% B; 50 min, 50% B; 55 min, 50% B; 56 min, 80% B; 66 min, 80% B; 67 min, 10% B, maintained during 13 min. Each phenolic compound was injected into the LC-ABTS •+ and quantified by reference to its appropriate authentic standard by absorption at 280 nm, whereas the antioxidant potential was calculated as the concentration of trolox required to produce an equivalent negative peak area by absorption at 412 nm and expressed as trolox equivalent antioxidant capacity (TEAC) or µMol TE /mg.

Oxygen Radical Absorbance Capacity (ORAC)
The ORAC assay, developed and validated by Ou et al. (2001) [52], was performed as described by Davalos (2004) [53] with minor modification Volden (2008) [54]. The assay measures the oxidative degradation of fluorescein by peroxyl radicals initiated by 2 ,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) at 37 • C. Free radical scavenging molecules protect fluorescein from the oxidative degradation and until exhaustion, slow reduction of the fluorescence signal by inducing latency. The area under the curve of the kinetics of fluorescence is directly proportional to the amount and effectiveness of the free radical scavengers present in a sample. The results are therefore expressed as trolox equivalent (µMol TE /mg) of dry extract. The products are dissolved in a mixture of water/methanol (70/30) at 1 mg/mL (1000 ppm), and then have to be diluted with water (between 25 and 500 ppm) before being placed in triplicate in 96 wells micro-plate up to 10 µL/well. A trolox standard range between 25 and 500 µMol/L was also filed in triplicate. An aqueous solution of 150 µL fluorescein (8.5 × 10 −6 mol/L) was added per well. An automatic dispenser then permits the initiation of the reaction by the addition of AAPH (30 µL, 153 µMol/L) to each well from the initiation of the generation of radicals by the addition of AAPH, the intensity of fluorescence emitted is measured every 5 min for 2 h with a wavelength of excitation between 400 and 600 nm.

Conclusions
The present study allowed the isolation, structural elucidation and antioxidant evaluation of phenolics and flavonoids from Helianthemum ruficomum, an endemic Saharan species on which no report is available so far. In this work, 14 compounds were isolated and identified from the ethyl acetate and n-butanol soluble parts of the aqueous EtOH extract, five phenolics: protocatechuic acid (1), picein (7), vanillic acid 4-O-β-D-glucopyranoside (8), lavandoside (9), 4-hydroxybenzoic acid 4-O-β-D-glucopyranoside (10); seven flavonoid glycosides: trans-tiliroside (2), slightly contamined by its stereoisomer cis-tiliroside, cis-tiliroside (3) contamined by trans-tiliroside, astragalin (4), nicotiflorin (11), rutin (12), vicenin-2 (13), narcissin (14); and a mixture (71-29%) of stigmasterol (5) and β-sitosterol (6) respectively. All the compounds were identified by spectral analysis, mainly ESI-HRMS, UV and NMR experiments ( 1 H, 13 C, DEPT, DOSY, COSY, NOESY, HSQC and HMBC) and comparison of their spectroscopic data with those reported in the literature. Compounds 5, 7, 8, 9, 10 and 14 were new for the genus Helianthemum. The investigated extracts and isolated compounds were evaluated for their free radical scavenging capacity by on-line HPLC-ABTS •+ screening. The antioxidant properties were confirmed by ORAC and TEAC assays. The results clearly indicated high antioxidant potential of the extracts and tested compounds of this species and agreed with literature data that free radical scavenging activity depends on the molecular structure, the number and position of the hydroxyl groups of tested compounds. Moreover, given the large amounts isolated and purified in this work, of trans-tiliroside and rutin which besides its numerous recognized biological activities, is used as oral complement; it becomes important to note that this Helianthemum species might be developed industrially for its rich content of these bioactive components. For this reason, this plant could be a good candidate for culture as a crop.