Phenolic Profile of Tunisian Opuntia Ficus Indica Thornless form Flowers via Chromatographic and Spectral Analysis by Reversed Phase-High Performance Liquid Chromatography-UV-Photodiode Array and Electrospray Ionization-Mass Spectrometer

Abstract

The qualitative and quantitative analysis of phenolic compounds of the methanol extract of Tunisian thornless form Opuntia ficus indica flowers was described. Reversed phase high-performance liquid chromatography UV photodiode array analysis detected four peaks at 320 nm, attributed to phenolic acids; three of them were quantified to 141 mg.kg⁻¹ of fresh plant material. Electrospray ionization mass spectrometer analysis detected 8 glycosylated flavonols at 350 nm, identified among 11 chromatographic peaks. The amount of flavonoids was 4980 mg.kg⁻¹ of fresh plant material, with isorhamnetin glycosylated derivatives as the main flavonoid components (55.1%), quercetin derivatives as the second (23.7%), and kaempferol derivatives as the third (8.4%).

Keywords:

• Tunisian Opuntia ficus indica
• Flowers of thornless form
• Glycosylated flavonols
• Isorhamnetin
• Phenolic acids
• ESI-MS

INTRODUCTION

Cactus pears are produced by a perennial cactus (i.e., Opuntia ficus indica) associated with the semiarid zones of the world. This cactus is widely distributed in Mexico and in all American hemispheres and grows in many other parts of the world, such as Africa, Australia, and the Mediterranean basin. It is one of the few crops that can be cultivated in areas that offer very little growth possibility for common fruits and vegetables.[1] Cactus pears were largely ignored by the scientific world until the beginning of 1980. These years corresponded to an increase of research works and symposia resulting in a large number of publications. This increasing interest motivated the investigation on the chemical content of the flowers and other organs of the plant. Renewed interest is to be ascribed in part to the multi-functionality of cactus fruits, pads, and flowers. The great number of potentially active nutrients and their multifunctional properties make cactus pears perfect candidates for the production of health promoting food and food supplements. Both cactus fruit and cladode yield high values of important nutrients such as: minerals and vitamins, as well as further antioxidants.[2 ⁻ 5] The main studies on Opuntia fruits were on the chemical analysis of pulp, skin, and seeds;[6] other authors have studied the nutritional significance of both despined cladodes and fruit pulp from Opuntia spp.[7] Several studies have reported the efficiency of fruits and cladodes in the treatment of several diseases. For example, cactus pear fruit and stem extract exhibited anti-tumoral,[8] anti-viral,[9] anti-inflammatory,[10] and antioxidant effects.[11,12] The use of prickly pear fruits is recommended for their beneficial and therapeutic properties.[13] Literature data report that cactus pears and the high fiber content of the cladodes mucilage and pectin are also used in folk medicine, such as emollient, moisturizing, cicatrizing, hypocholesterolemic, hypoglycemic agent, and also as effective in gastric mucosa diseases.[14 ⁻ 19] In Sicilian folk medicine, cactus flower infusion is considered as depurative and particularly used for its diuretic and relaxant action on the renal excretory tract.[20,21] Therefore, it is stipulated that cactus flower infusion may help the expulsion of renal calculus. Galati et al.[22] investigated the diuretic effect of a 15% extract from: flowers, fruits, and peeled cladodes from O. ficus indica. The presence of phenolics has been detected in cactus pulp fruits.[5,23,24] Kuti[25] reported an antioxidative effect due to the major flavonoids encountered in cactus fruits (quercetin, kaempferol, and isorhamnetin). The effects of solvent extracts from natural sources of antioxidants on appropriate cell lines revealed that phenolic compounds were able to delay pro-oxydative effects on proteins, DNA, and lipids while producing stable radicals.[26] Furthermore, O. ficus indica polyphenolic compounds were shown to induce a hyperpolarization of the plasma, and to raise the intracellular pool of calcium in human Jurkat T-cell lines.[27] Flavonol derivatives detected in Opuntia spp. have been recently compiled.[4] When fruits are investigated, it must be taken into account that higher phenolic contents are expected in the peel, rather than the pulp. Compared to fruits and cladodes of O. ficus indica, studies that focus on the phenolic compounds in flowers remained scarce in literature. Some authors explained the anti-diuretic effect of flowers to secondary metabolites including phenolic compounds. Previous studies on the plant flowers reported the isolation of β-sitosterol, fatty acids, some of their esters from a polar extract,[20] and unidentified isorhamnetin glycoside.[28] On the same topic, recent studies on Egyptian cactus flowers enable isolation and identification of some secondary metabolites, such as penduletin, kaempferol, luteolin, quercetin, and rutin.[29] In the methanol extract from O. ficus indica flowers collected in Monasterace (Italy), the isorhamnetin glycosylated was the major component (73%), and it was followed by quercetin derivatives (14%) and quercetin 3-O-rutinoside (8.8%).[30] In this context, the aim of the present study was to provide new findings about the phenolic metabolites of the methanol extract of the flowers of the Tunisian thornless form of O. ficus indica. Identifications and quantifications were achieved thanks to chromatographic, UV-visible, and mass spectrometry data.

MATERIALS AND METHODS

Plant Material

The thornless form of O. ficus indica is from local pilot cultivar of Bou Argoub region (Nabeul-Cap Bon Region, northeast of Tunisia). The region of Bou Argoub is located at 36° 32′ N latitude, 10° 33′ E longitude, and at an altitude of 62 m. Flowers have been collected during the flowering period in the month of June 2008.

Solvents and Standard Phenolics

Methanol (CH₄O), acetonitrile (CH₃CN), formic acid (CH₂O₂), and acetic acid (CH₃COOH) requested for chromatographic analysis were purchased from Merck (Darmstadt, Germany). Water was purified on a MilliQ system (Millipore S.A., Molsheim, France). The standards used in this work provided by Extrasynthèse S.A. (Lyon, France) and consisted of the following: rutin (quercetin 3-O-rutinoside), myricitrin (myricetin 3-O-rhamnoside), hyperoside (quercetin 3-O-galactoside), kaempferol 3-O-rutinoside, isorhamnetin 3-O-rutinoside, kaempferide (4′-methylkaempferol), rhamnetin (7-methylquercetin), isorhamnetin (3-methylquercetin), quercitrin (quercetin 3-O-rhamnoside), and myricetin.

Direct Solvent Extraction of Polyphenols

Flowers including all parts (petals, sepals, and reproductive organs) were first dried in the dark at room temperature for 48 h, freeze-dried, and reduced into flower powders. Weighted aliquots (30 mg) were placed in eppendrof vials, and 1% methanolic acid acetic (1200 μl) was added. Vials were closed and incubated at room temperature in an ultrasonic water bath for 15 min. The mixture was immediately filtered through a polytetrafluoroethylene (PTFE) membrane (0.45 μm) into insert vials. The samples were ready for analysis by reverse phase-high-performance liquid chromatography (RP-HPLC) coupled to UV-visible detection. The same method was adopted for the preparation of the samples injected on HPLC system equipped with an electrospray ionization mass spectrometer (HPLC-ESI-MS) with only 10 mg of plant material, the weight reduction was recommended to avoid concentrating the solution. We note that the second HPLC system was also equipped with a photodiode array (PDA) detector. RP-HPLC-UV-PDA is used for quantitative investigation and ESI-MS for qualitative analysis.

RP-HPLC-UV-Visible Analysis

The RP-HPLC-UV-visible analyses were performed using a Waters Alliance 2690 HPLC system (Waters, Milford, MA, USA), which included a quaternary gradient pumping system, a thermostated column oven, and a thermostated automatic injection module. The system was coupled to a PDA 996 detector used in the 240–600 nm range (Waters, Milford, MA, USA). The column was a reversed phase column (Merck, Purospher TM C18, 250 × 4 mm, 60 Ǻ, 5 μm) coupled to precolumn containing the same phase (Merck, Purospher TM C18, 4 × 10 mm). The injected volume was 10 μl, and the column was thermostated at 30°C. The elution solvent was a mixture of solvent A consisting of ultrapure water/glacial acetic acid (97.5:2.5, v/v) and solvent B consisting of pure acetonitrile. Helium was used for degassing solvents. The following gradient was applied: initial 3% B, 0–5 min, 9% B linear; 5–15 min, 16% B linear; and 15–45 min, 50% B linear; the gradient was followed by washing and reconditioning of the column. Three particular wavelengths were used for quantification of polyphenols: 280 nm for flavan-3-ols and dihydrochalcones, 320 nm for hydroxycinnamic acids, and 350 nm for flavonols. Acquisition and processing of the data were achieved using Millennium32 chemometric software (version 3.20). Quantitative determination was carried out using calibration curves of standards. Hyperoside (quercetin 3-O-galactoside) and chlorogenic acid were chosen as external standards for quantification of flavanols and hydroxycinnamic acids, respectively. HPLC peaks were identified on chromatograms according to their retention times (RTs) and their UV-V is spectra by comparison with available standard compounds. Quantification of the identified compounds was performed by correlating the measured peak area to the calibration curves obtained with reference compounds. Integration of HPLC peaks was performed at 350 nm for flavonols, using the extinction coefficient of hyperoside, and at 320 nm for phenolic acids, using the extinction coefficient of chlorogenic acid.

ESI-MS Analysis

The ESI-MS system consisted of a LCQ DECA ion trap mass spectrometer (Thermofinnigan, San Jose, CA, USA) equipped with an ESI source and run by Xcalibur (version 1.2) software. ESI-MS analysis were performed in the negative mode using the following parameters for the ESI source: ion spray voltage, 3,69 kV; capillary voltage, –70.78 V; capillary temperature, 240.4°C; sheath nitrogen gas flow rate, 66.65 arbitrary units; auxiliary gas flow rate, 3.81 arbitrary units; scan range of m/z 50–2000. Samples corresponding to collected HPLC peaks were directly introduced into the ESI source by a built-in the syringe pump at the flow rate of 3 μl.mn⁻¹. For the generation of MSⁿ data, the precursor ions were fragmented by helium gas collision in the ion trap by optimizing the collision energy in order to obtain the intensity of the precursor ion close to 10% of the relative scale spectrum.

RESULTS AND DISCUSSION

The purified methanol extract of O. ficus indica of thornless form flowers was analyzed by means of RP-HPLC coupled with UV-visible diode array detection and ESI-MS. To understanding the phenolic composition of grape vine leaves, chromatographic assays were applied by the HPLC-RP-PDA method to create a data base of the principal polyphenolic compounds.[31] Two classes of phenolic compounds (i.e., phenolic acids and flavonols) were detected according to UV chromatograms at 320 nm (Fig. 1a) and 350 nm (Fig. 1b). A total of four peaks numbered from a₁ to a₄ (Fig. 1c) and eleven peaks numbered from f₁ to f₁₁ (Fig. 1d) were considered for identification and quantification. Peaks numbered a₁ to a₄ showed typical UV spectra with maximum absorbance at the 320–330-nm region in accordance with hydroxycinnamic acid structures (Table 1), whereas peaks numbered f₁ to f₁₁ showed absorbance in the 255–263- and 350–355-nm regions, in accordance to flavonol structures (Table 2). De Leo et al.[30] analyzed the methanolic extracts of O. ficus indica grown in Italy, the resulting chromatogram, showed nine peaks, with only seven identified as flavonol glycosylated derivatives. The results of this work provided further information on phenolic acids (four peaks: a₁–a₂) and revealed more peaks in flavonol glycoside region chromatogram (eleven peaks: f₁–f₁₁). The RP-HPLC-UV-PDA results displayed the presence of the secondary metabolites in O. ficus indica flowers belonging to the flavonol glycoside class indicated by absorbance at 255–263 nm and 350–355 nm were in accordance to Wollenweber's work.[32] The chromatograms at 350 nm revealed eight peaks (f₃, f₄, f₅, f₆, f₈, f₉, f₁₀, and f₁₁) corresponding to flavanols available as standard compounds. Peaks were identified by comparison of their HPLC RTs, UV-visible spectrum with authentic standards compounds, and confirmation of their identity was given by their

Table 1 RT, spectral UV, and amount compounds (a₁–a₄) data from flowers' methanol extract of Tunisian O. ficus indica thornless form

Peaks	Compound	RT (min)	λmax (nm)	Amount (mg kg^−1 MF) M ± SE ^a
a1	Unidentified	9.6	320	ND ^b
a2	Unidentified	11.0	320	32.8 ± 0.5
a3	Unidentified	11.5	320	69.7 ± 0.9
a4	Unidentified	12.9	320	38.2 ± 0.9
^aM: mean (n = 3); SE: standard error.
^bND = not detected.

Figure 1 UV spectra of phenolic acids (a) and flavonols (b), RP-HPLC of phenolic acids at 320 nm (c) and flavonols at 350 nm (d) of Tunisian thornless form of O. ficus indica flowers methanol extract. (Color figure available online.)

mass spectrometry data (MS and MS² fragments) obtained by infusion of the collected fractions in the ESI-MS system (Table 2). In order to complete the structural characterization of some flavonols compounds, ESI-MS analysis was performed on series of collected chromatographic fractions corresponding to well-resolved peaks in HPLC-UV analysis. Each collected fraction was infused in the ESI source used in the negative mode. Then, the MS:MS spectrum of each molecular ions was registered. The pseudomolecular ions [M-H]⁻ corresponding to the deprotonated molecules were clearly detected by mass spectrometry used in the negative mode. Thus, [M-H]⁻ were observed at m/z 769, 755, 609, 463, 463, 593, 623, 623, 477, 477, 417 for peaks f₁ to f₁₁, respectively. Accordingly to the available standards, f₃ was identified as quercetin 3-O-rutinoside, f₄ as quercetin 3-O-galactoside, f₅ as quercetin 3-O-glucoside, f₆ as kaempferol, f₈ as isorhamnetin 3-O-rutinoside, f₉ as isorhamnetin 3-O-galactoside, f₁₀ as isorhamnetin 3-O-glucoside, and f₁₁ as kaermpferol 3-O-arabinoside. The predominant flavonoids in the flowers of Tunisian O. ficus indica were isorhamnetine 3-O-rutinoside (f₈: major peak) and a small concentrations of flavonoids, quercetin (f₃, f₄, and f₅), and kaempferol (f₆ and f₁₁) core aglycone. The flavonol quercetin is one of the most commonly consumed flavonoids and has been well studied for its health benefits potential. Quercetin possesses anti-proliferate, anti-carcinogenic, and antioxidant activities.[33] The chemical structural of identified glycosylated flavonols was reported in Fig. 2. The other flavonols peaks f₁, f₂, and f₇, their RT and UV-visible did not correspond to those of available standards. These selected peaks were therefore characterized by MS in the negative mode. It showed the presence of the pseudomolecular ion [M-H]⁻ corresponding to the deprotonated molecule of the flavonoid glycosides. The fragmentation pattern of the constituents, which remained not identified, suggested a structural correlation with isorhamnetin derivatives. The f₁ peak showed a pseudomolecular ion [M-H]⁻ at m/z 769 (Table 2). The corresponding MS² spectrum exhibited a main product ion at m/z 605 and 314 (Fig. 3a). The former at m/z 605 corresponded to the loss of 146 in accordance with the mass of a deoxyhexose moiety ([M-H-146]⁻) followed by one water molecule ([MH-146-18]⁻). The latter at m/z 314 ion was indicative of isorhamnetin moiety. The MS spectrum of peak f₂ produced a pseudomolecular ion [M-H]⁻ at m/z 755. The MS² spectrum showed produced ions at m/z 605 and 315 (Fig. 3b), similar to MS² spectrum of the compound of peak f₁. This allowed the supposition that peak f₂ was also an isorhamnetin diglycoside containing one pentose molecule. The mass spectrum of peak f₇ exhibited a pseudomolecular ion [M-H]⁻ at m/z 623 as peak f₈. The MS² analysis spectrum showed the characteristic ions m/z 315 and 314 (Fig. 3c), which was identified as isorhamnetin. Peak f₇ and peak f₈ present the same spectrum of mass MS and MS² and the same UV-visible spectrum, whereas RTs are close to the level of the chromatographic elution (0.31 min). It is very probable there are two isomers of isorhamnetin 3-O-rutinoside. Two isomers isorhamnetin 3-Orutinoside and isorhamnetin 3-O-robinobioside were identified in extracts and fractions of major flavonoids contained in Nitraria retusa leaves in the work of Hadj Salem in 2009.[34] However, Halim et al.[35] showed that the Nitraria retusa contains another flavonoid not available, isorhamnetin 3-O-robinobioside, which is an isomer of the isorhamnetin 3-O-rutinoside. These two compounds will hardly be separable by the chromatographic method. Arcoleo et al.[36] first reported the presence of isorhamnetin in flowers of O. ficus indica. Other reports have indicated that plants in the Cactaceae family produce flavonol 3-O-glycosides (quercetin, kaempferol, and isorhamnetin), dihydroflavonols, flavonones, and flavanonols.[37 ⁻ 40] Nearly all reports on flavonoids found in Opuntia cacti have dealt with extraction from the floral tissue.[41,42] Yang et al.[43] showed the pharmacological interest of isorhamnetin 3-O-robinobiose, obtained from O. ficus indica flowers extraction, as a testosterone 5 α-reductase inhibitor. The total amount of phenolic acids and flavonoids of Tunisian thornless form of O. ficus indica flowers were, respectively, 141 mg and 4980 mg.kg⁻¹ of fresh plant material. Three of phenolic acids peaks were quantified to 140.7 mg.kg⁻¹ of fresh plant material, it was apparent that the name compound a₃ is the main phenolic acid (49.6%) in the thornless form flower of Tunisian O. ficus indica (Table 1). The methanol extract of flavonols resulted, showed a high percentage of isorhamnetin glycosylated derivatives, isorhamnetin 3-O-rutinoside was the major component (34.7%), and it was followed by isorhamnetin 3-O-galactoside (12.5%), quercetin 3-O-rutinoside (10.5%), isorhamnetin 3-O-glucoside (7.9%), quercetin 3-O-galactoside (7.2%), kaempferol 3-O-arabinoside (7.1%), quercetin 3-Oglucoside (6%), and kaempferol 3-O-rutinoside (1.3%).

Table 2 RT, spectral UV, characteristic ions (MS and MS²), and amount compounds (f₁–f₁₁) data from flowers' methanol extract of Tunisian O. ficus indica thornless form

Peaks	Compound	RT (min)	[M-H]^− (m/z)	MS^2 fragments (m/z)	λmax (nm)	Amount (mg kg^−1 MF) M ± SE ^a
f1	Unidentified	20.5	769	605, 314	255–352	92.1 ± 0.8
f2	Unidentified	20.9	755	605, 314	255–352	166.9 ± 1.6
f3	Quercetin 3-O-rutinoside	21.3	609	300, 301	255–353	522 ± 1.8
f4	Quercetin 3-O-galactoside	21.8	463	301,300	263–352	358.6 ± 0.8
f5	Quercetin 3-O-glucoside	22.2	463	300, 301	263–352	301.1 ± 1.6
f6	Kaempferol 3-O-rutinoside	23.5	593	285	263–352	64.2 ± 2
f7	Unidentified	23.6	623	315, 314	253–355	377.9 ± 1.5
f8	Isorhamnetin 3-O-rutinoside	23.9	623	315	253–355	1727.2 ± 16.9
f9	Isorhamnetin 3-O-galactoside	24.4	477	314, 315	260–354	624.4 ± 1.4
f10	Isorhamnetin 3-O-glucoside	24.8	477	314, 315	260–354	392.1 ± 3.7
f11	Kaermpferol 3-O-arabinoside	25.2	417	284, 285	263–350	353.8 ± 1.8
^aM: mean (n = 3); SE: standard error.

Figure 2 Chemical structures of flavonol glycosides found in flowers' methanol extract of Tunisian O. ficus indica thornless form.

Figure 3 ESI-MS² product ion spectra of peaks f₁ (a), f₂ (b), and f₇ (c).

CONCLUSION

In this study, results indicate the presence of interesting phenolic compounds in the composition of Tunisian thornless form of O. ficus indica flowers. These components were characterized via chromatographic and spectral analysis by RP-HPLC-UV-PDA and ESI-MS. The main flowers polyphenols were flavonols—more precisely, isorhamnetin derivatives, quercetin derivatives, and kaempferol derivatives, as well as the presence of compounds from the phenolic acids family. These finding make flowers a promising source of biologically active polyphenolic mixtures. These investigations have to be completed by identification of these unknown peaks and compounds for which structures were not elucidated. Otherwise, polyphenols concentrations in Tunisian thornless form of O. ficus indica flowers could contribute to antioxidant status and would be interesting for further exploitation.