Résumé

Le but de cette étude était l’extraction de composés organiques volatils (COVs) et les composés phénoliques totaux des variétés de figue barbarie «Opuntia ficus indica (L.) Mill» les plus répandues au Maroc à savoir Dellahia, Aissa et Shoul. Les COVs ont été extraits par microextraction sur phase solide (SPME) couplée à la chromatographie en phase gazeuse et à la spectrométrie de masse (GC-MS). Les composés phénoliques dans les extraits ont été étudiés par chromatographie en phase liquide couplée à la spectrométrie de masse (LC-MS). L’étude a permis l’identification de quarante-six composés pour les COVs. Les composés les plus abondants dans les trois variétés étudiées (Dellahia, Aissa et Shoul) sont le n-hexanol et le 2-hexanal avec respectivement un pourcentage de 10,6 %; 10,9 % et 44,0% pour le premier composé et 10,3 %; 59 %; 18,7 % pour le deuxième. L’analyse des composés phénoliques a permis l’identification et la quantification de 15 fractions de flavonoïdes et acides phénoliques. L’acide caféique est l’acide phénolique le plus abondant avec 16,0 et 10,8 mg/100 g de matériel végétal respectivement pour Dellahia et Aissa. Pour les flavonoïdes; isorhamnetin était le principal composé, il représentait respectivement 40,5 % et 43,2 % pour Dellahiaet Aïssa. Nous pouvons conclure que les fruits de figues de barbarie constituent une bonne source naturelle d’antioxydants naturels et par conséquent leurs bienfaits sur la santé sont inestimables.
Mots-clés: Opuntia Ficus Indica, SPME, LC- MS, GC-MS, composés organiques volatils, composés phénoliques

INTRODUCTION 

The Opuntia cactus is a xerophyte of about 200 to 300 species and grows mainly in arid and semiarid zones. Due to their remarkable genetic variability, Opuntia plants show a high ecological adaptivity and can therefore be encountered in places of virtually all climatic conditions (Moßhammer et al., 2006). The Opuntia ficus-indica L. is the most cultivated species of the genera. Several authors confirm that the cactus is native to Mexico, which is the main world producer. The species is also widespread in Mediterranean area, South Africa and central-South America (Inglese et al., 2002).

The fruit is a many-seeded berry with a thick peel, enclosing a delicately flavoured seedy pulp. The flavours of selected cactus pear fruit varieties resemble that of strawberry, watermelon, honeydew melon, fig, banana or citrus (Savio, 1987). Cactus pear fruits are a source of nutrients and vitamins (Sawaya et al., 1983; Teles et al.,1984) and are eaten fresh, dried or preserved in jams, syrups or processed into candy-like products (Hoffman, 1980).

Scientific studies have indicated that several parts of O. ficus-indica have diuretic and antigotous effects (Galati et al., 2002).

The cladodes are utilized to reduce serum cholesterol level and blood pressure, for treatment of ulcers, rheumatic pain, wounds, fatigue, capillary fragility, and liver conditions (Agozzino et al., 2005). In addition, a recent study showed the potential antigenotoxic activities of cactus cladodes against the mycotoxin zearalenone, a potent estrogenic metabolite (Zorgui et al., 2009).

The seeds contained in the pulp produce oil that is composed mainly of fatty acids, sterols and vitamins (Ghazi et al., 2013).

Both cladodes and fruits contain a wide range of secondary metabolites presenting health-promoting activities (Munoz-de-Chavez et al., 1995). Among the minor compounds of O. ficus-indica, the most important are polyphenols (Khatabi et al., 2011) that may act as antioxidants against oxidative damage.

Phenolic compounds are strongly correlated to the antioxidant activity (Cotelle, 2001) and several reports showed a linear correlation between antioxidant activity and polyphenol content (Elhadi Yahia et al., 2011; Khatabi et al., 2011; Kuti, 2004), therefore the knowledge of the polyphenolic composition in the food is of great interest.

The aroma profile in plant is influenced by environment, variety and other factors such as growing geographical location or time of collection (Arena et al., 2001).

In the last years, the analysis of volatile organic compounds (VOCs) is becoming of great interest; it is well known that flavor of food is related to the volatile compounds perception by the nose. In addition, plant VOCs play a key role in the growing of the plants by interactions with other plants or they can be produced as a response against some pathogen or insect pests (Loi et al., 2008).

For the relevant assessment of O. ficus-indica quality, main compounds responsible for the aroma must be known and an appropriate analytical method is needed. Analysis of food aroma could be approached by different techniques (solvent extraction, distillation, solid-phase microextraction (SPME), headspace techniques, adsorption traps) each of them having its inherent problems and possible bias (Belitz et al., 2004; Hofmann et al., 2005; Kaiser, 2006; Stephan et al., 2000). However, SPME coupled with gas chromatography-mass spectrometry (GC-MS) and comparison of spectra and retention indices of reference compounds, stays the best technique for the analysis of the volatile compounds (Molyneux, 2007; Zhou et al., 1999).

To our knowledge, this study is the first conducted to analyze volatile organic compounds in Moroccan prickly pear.

Therefore, the objectives of this study are the identification of the chemical composition of volatile fraction and quantification of polyphenols of three cultivars of O. ficus-indica from Morocco, namely Dellahia, a white cultivar, Aissa, a red cultivar and Shoul, a yellow cultivar.

MATERIAL AND METHODS

Plant material

Ripe fruits of three important cultivars of Morocco; Aissa from Sidi Ifni (South of Morocco), Dellahia from El-Hoceima (North of Morocco) and Shoul (in the central region) were collected during the month of September.

Harvesting of fruits from wild plant was done by done by simple random sampling; the spines were removed manually using brushes. They were immediately transported and stored in a freezer.

Extraction and identification of volatile organic compounds

SPME is based on the adsorption of volatile compounds present in the head space of food on a thin fiber covering the removable internal needle of a microsyringe. The adsorbed vapors are then desorbed by direct introduction of the microsyringe into the injector of a gas chromatograph (Arena et al., 2001).

SPME analysis: A 100 μm PDMS/DVB/CAR coated fiber was conditioned prior to use according to manufacturer instructions. The conditioned fiber was injected through the septum, and suspended in the headspace of a 40 mL glass vial containing 5 g of homogenized whole fruit (pulp, seeds and skin), to which was added 5mL of saturated aqueous NaCl solution. The fiber was exposed for extracting volatiles for 60 min at 40 °C under stirring. The fiber was then retracted and removed from the vial, and placed immediately into the injector of the GC. Thermal desorption was performed into the injector at a temperature of 250 °C for 5 min in splitless injection mode.

An aliquot of 2 μL was injected into the GC–MS equipment and analyzed under the same conditions mentioned before to determine the retention time of each n-alkane necessary for the Kovats indexes calculations (Elhadi Yahia and Mondragon-Jacobo, 2011).

The GC-MS analysis was carried out using a Hewlett Packard 5890 GC, equipped with a Hewlett Packard 5971 MS system (Palo Alto, USA) operating in the EI mode at 70 eV, using the column HP5.

Identification of the individual components was performed by comparison with the co-injected pure compounds or by matching the MS fragmentation patterns and retention indices with the built in libraries or literature data and commercial mass spectral libraries (NIST/EPA/NIH 1999).

The formula used to obtain Kovats indexes was:

I = 100n + 100z (log t RA− log t Rn) / (log t RN− log t Rn)

Where I is the Kovats index, A is the unknown compound, n is the number of carbon atoms in the smaller n-alkane, N is the number of carbon atoms in the larger n-alkane, z is the difference in the carbon atoms in the smaller and larger n-alkanes, and tR is the retention time.

Extraction and determination of phenolic compounds by LC-MS

The phenolic fraction of each O. ficus-indica (pulp) sample was extracted with a solution composed of ethanol:water 70:30 (v/v); using a ratio of sample to solvent 1:25 w/v at room temperature overnight. The resulting extract was then filtered through paper filter and the solvent was evaporated using a rotary evaporator under vacuum at 40°C.

LC-MS analysis conditions: Full scan ESI-MS and collision induced dissociation (CID) ESI-MS/MS analyses of samples were performed on an ABSciex API2000 spectrometer. The analytical parameters were optimized (declustering potential -22 eV, focusing potential -316 eV, and entrance potential -6 eV) by infusing a standard solution of chlorogenic acid (1 mg/ ml in methanol 50%) into the source at a flow rate of 10 mL/min. Data were acquired in the negative ion MS and MS/MS modes.

RESULTS AND DISCUSSION

Identification of volatile organic compounds

The analysis of VOCs of Moroccan cactus fruits using SPME- GC-MS technique resulted in the identification of forty-six compounds (Table 1). The identified molecules represented 84.8% of the whole volatiles for Dellahia, 92.2 % for Aissa and 92.1 % for Shoul varieties. These compounds were tentatively identified by comparison of retention index and MS fragmentation pattern both with pure standard coinjection and, when pure standard was not available, by comparison with built in libraries and literature data.

According to the literature, sixteen volatile compounds were found in Italian fruits of O. ficus-indica (Arena et al., 2001) and 61 in Mexican fruits of O. ficus-indica (Robert and Takahashi, 1978), representing successively 100% and 95% of whole volatiles in fruits.

The comparison between the chemical compositions of the VOCs using SPME- GC-MS of the three cultivars showed that Shoul cactus differs from Dellahia and Aissa. Indeed, the aroma profile of Shoul cultivar differed both qualitatively and quantitatively with respect to Dellahia and Aissa cactus.

Many terpenes derivatives have been found in the analyzed samples like camphene. Results showed that it represented 5.3% and 0.6% of the aroma profile of Dellahia and Aissa, respectively, while it was not detected in Shoul.

The most abundant compound in Aissa cultivars was (2E)-nonenal with a percentage of 16.7, while it was absent in Shoul variety and it exists in limited quantity in Dellahia with a 4.8%.

The n-hexanol represented also an important percentage in different cultivars analyzed with respectively 10.3 %, 18.7 % and 5.9 % for Dellahia, Shoul and Aissa. According to studies conducted by Arena et al., (2001), this compound exists in larger amount in Italian fruit (26.4%) (Table 2).

One of the aldehyde responsible for the aroma profile of citrus was Decanal a, this compound exists in trace amounts in fruits of Opuntia ficus indica with 0.2%, 0.6% and 0.3% for successively Dellahia, Aissa and Shoul, whereas, it was not detected in Italian fruit (Arena et al., 2001).

For nonanal, it was more present in Aissa (2.8%) followed by Dellahia (1.4%) and Shoul (1.1%), while it was not detected in Italian fruit (Arena et al., 2001).

E-2 hexen-1-ol was the major compound in Italian fruit (56.8%) (Arena et al., 2001), however, this compound was detected in its stereoisomers form (2 hexen-1-ol Z) in Moroccan fruit in small amounts (4.3%).

According to the literature, our results were in agreement with those reported by Robert and Takahashi (1978). These researchers found that the alcohols were the major class of compound represented with trans-2-Hexen-1-ol and n-hexanol even though numerous esters and carbonyl compounds are also present at low concentrations. However, Ammar et al., (2012) analyzed two species of prickly pear flowers Opuntia ficus indica (L.) Mill. and Opuntia stricta (Haw.) and concluded that carboxylic acid was the main compound (28-97%), followed by terpenes (0.2-57%), esters (0.2-27%) and alcohols (<1.8%).

Another study was conducted by Bergaoui et al., (2007) on cladodes, flowers and fruits of Opuntia lindheimeri var. linguiformis L. Benson from Tunisia. The results showed that the saturated fatty acid were the most important compounds in the three organs represented by tetradecanoic acid (3.2-13.6%) and hexadecanoic acid (8.5-17.3%). The authors have identified also butyl tetradecanoate (8.1-21.5%) and (E)-3-butyldiene phthalide (6.9-15.8%).

Determination of phenolic compounds

In order to investigate the occurrence of phenolic compounds, the qualitative analysis of Opuntia ficus-indica was performed by LC-MS allowing to identify 15 compounds belonging to two families as shown in Figure 1 and 2: flavonoids (kaempferol, isorhamnetin, quercetin, rutin) and phenolic acids (quinic acid, caffeic acid, acetylcaffeic acid, caffeic acid derivative, chlorogenic acid, caffeoylquinic acid, dicaffeoylquinic acid, rosmarinic acid, p-coumaric acid, ferulic acid and syringic acid).

The amount of the identified compounds was determined in different cactus pear cultivars using a rapid and sensitive LC-MS/MS (MRM) method.

According to figure 1, the overall polyphenolic content in Dellahia was higher than that of Aissa.

In terms of phenolic compounds identified, the most abundant was caffeic acid with respectively 16.0 and 10.8 mg/100g of plant material for Dellahia and Aissa.

In a study performed by Albano et al., (2015) on Italian prickly pear fruit, they found that the total phenol content was 89.2 mg GAE (Gallic Acid Equivalents)/100 g of fresh weight in purple fruits and 69.8 mg GAE /100 g of fresh weight in orange fruit. These values were similar to those found in prickly pear cultivars from Saudi Arabia, California, and Tunisia (Abdel-Hameed et al., 2014; Stintzing et al.,2005; Yeddes et al., 2013) and lower than that reported by El Mostapha et al., (2014). Fernandez-Lopez et al., (2010) reported a much higher phenolic content but it referred to a whole (skin and pulp) red-skinned fruit.

The total phenols of some prickly pear analyzed are similar to clarified apple or white grape juices (Mullen et al., 2007). Other juices extracted from cranberries, pomegranate, tropical fruits, purple grape, and apple contained more than three times the total phenolics determined in the set of prickly pear juices analyzed (Butera et al., 2002).

In terms of flavonoid composition, isorhamnetin derivatives was the major component, it accounted for 40.5 % (0.8 mg/100g of plant material), quercetin derivatives for 33.1%, kaempferol derivatives for 17.3 % and rutin derivatives accounted for 8.9% of total flavonoids in Dellahia.

For Aissa cultivar, isorhamnetin derivatives accounted for 43.2 % (0.4 mg/100 g of plant material), quercetin derivatives for 21.6%, rutin derivatives for 18.5% and kaempferol derivatives accounted for 16.4 % of total flavonoids. These values were lower than those reported by El Mostapha et al., (2014) (4.94; 9.00 and 0.78 mg/100 g of plant material in of isorhamnetin, quercetin and kaempferol, successively).

Kuti (2004) analyzed different types of prickly pears and concluded that purple skinned fruits contained the highest amounts of flavonoids.

Almost half of flavonoid compounds is composed of isorhamnetin for both studied varieties with 40.5 % and 43.2%, for Dellahia and Aissa, successively. These values are higher than those found by Kuti (2004) in American prickly pear fruit: the isorhamnetin was about 34.6 % for green skinned and 2.0 %, for purple skinned, while it was not detectable in red and yellow skinned. However, the total flavonoids were composed of about 96.8% quercetin (90.5 ± 11.5 mg/g fresh weight) in purple-skinned, 93.1% quercetin (51.0 ± 4.6 mg/g fresh weight) in red-skinned, and 100% quercetin (9.8 ± 3.0 mg/g fresh weight) in yellow-skinned cactus pear fruits, respectively.

It appears that Opuntia ficus indica contains flavonoids common to other fruits and vegetables (Peterson and Dwyer, 1998). However, the flavonoid types and contents, as in other fruits and vegetables, vary with the cultivars (Bilyk and Sapers, 1986).

One of the more interesting findings in this study regarding the phytochemical content of Moroccan prickly pear is that they had relatively high content of phenolic acids (e.g. caffeic acid) and flavonols (e.g. isorhamnetin).

The isorhamnetin and caffeic acid which showed the highest contents in this study have a lot of health benefits; the first compound may reduce the risk of cancer, improve heart health and ease diabetes complications b, and the second is able to protect cells against damage caused by free radicals (Chung et al., 2006).

Ndhlala et al., (2007) analyzed the total phenolics and flavonoids of ethanol extracts of prickly pears from Zimbabwe belonging to Opuntia megacantha and found that the pulp contained approximately 180 µg/g GAE (gallic acid equivalents) and 10 µg/g cathecin. However, Chang et al., (2008) reported that they found 915 µg/g GAE and 292 µg quercetin/g in the Opuntia dillenii fruits extracts from Taiwan.

CONCLUSION

The chemical composition of Moroccan prickly pear was determined for the first time by SPME coupled with GC-MS. Major compounds responsible of flavor in the variety studied of Opuntia Ficus Indica are 2-Hexanal and n-hexanol.

For phenolic compounds, this investigation showed the potential value of cactus pear fruits as a good source of natural antioxidants and that consumption of cactus pear fruit may contribute substantial amounts of antioxidants to the diet. Based on the available data in this study and the phytochemical contents of cactus pear fruits, there is a high likelihood that cactus pear fruits may provide the types of nutritional and health benefit associated with consumption of fruits and vegetables in general.

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