Simultaneous quantification of phenolic acids and flavonoids in Chamaerops humilis L. using LC–ESI-MS/MS

Flavonoids and phenolic acids, a class of polyphenol compounds, are widely distributed in plants kingdom. Flavonoids include over 6000 identified family members. They play an important role in protection of plants from microbial and insect attack. Many studies reported that flavonoids exhibit various effects as antioxidant (Siahpoosh et al., 2016; Balci & Özdemir, 2016), anti-cancer (Androutsopoulos et al., 2010; Ma et al., 2015), anti-allergic (Park et al., 2006), anti-thrombotic and vasodilatory (Rahimi et al., 2010), anticholinesterase (Ertas et al., 2016) actions. Finally, because of their UV-absorbing properties, flavonoids protect plants from the UV radiation of the sun and scavenge UV-generated reactive oxygen species (Shirley, 1996).


Introduction
Flavonoids and phenolic acids, a class of polyphenol compounds, are widely distributed in plants kingdom. Flavonoids include over 6000 identified family members. They play an important role in protection of plants from microbial and insect attack. Many studies reported that flavonoids exhibit various effects as antioxidant (Siahpoosh et al., 2016;Balci & Özdemir, 2016), anti-cancer (Androutsopoulos et al., 2010;Ma et al., 2015), anti-allergic (Park et al., 2006), anti-thrombotic and vasodilatory (Rahimi et al., 2010), anticholinesterase (Ertas et al., 2016) actions. Finally, because of their UV-absorbing properties, flavonoids protect plants from the UV radiation of the sun and scavenge UV-generated reactive oxygen species (Shirley, 1996).
Chamaerops humilis L., var. argentea, belonging to the Arecaceae family, is a palm widely distributed in the Mediterranean basin especially in Algeria which has been located mainly in Tlemcen and Dranian coast. On folk medicine, this plant has been widely applied by decoction in Algerian populations for a variety of illnesses as stomachache, toning (Hasnaoui et al., 2011), diabetes (Bnouham et al., 2002). Moreover, previous studies reported Chamaerops to contain phenolic compounds, such as tannins, flavonoids, saponins, quinons, coumarines (Benahmed-Bouhafsoun et al., 2013), sterols and terpenoids (Hasnaoui et al., 2013).
The flavonoids were previously reported as constituents of the Arecaceae family plants, but literature lacks detailed information on the phytochemical composition of C. humilis. This is the first study for the identification and quantification of phenolic acids and flavonoids of C. humilis. Therefore, the objective of the present study was to characterize the chemical composition of water and 80% methanol extracts of C. humilis leaflets, rachis and fruits by using liquid chromatography coupled with mass spectrometry (LC-ESO-MS/MS) as a potent analytical technique.

Plant material
Chamaerops humilis L. Var. argentea was collected by Dr. A. Bouhafsoun from western Algeria (Dran city) in June of 2014.

Extraction under continuous reflux
Three grams of dried samples were soaked separately in 50 ml of 80% aqueous methanol and ultrapure water at 60 °C for 30 min. The extracts were filtered through nylon filter. The extraction was repeated twice. The collected filtrates were dried under vacuum using a rotary evaporator at 30 °C until dry extracts were obtained. Dry filtrates were diluted to 1000 mg/L and filtrated with 0.2 µm microfiber filter prior to LC-MS/MS analysis.

MS instrumentation
MS detection was performed using Shimadzu LCMS 8040 model triple quadrupole mass spectrometer equipped with an ESO source operating in both positive and negative ionization modes. LC-MS/MS data were collected and processed by LabSolutions software (Shimadzu, Kyoto, Japan). The multiple reaction monitoring (MRM) mode was used to quantify the analytes: the assay of investigated compounds was performed following two or three transitions per compound, the first one for quantitative purposes and the second and/or the third one for confirmation. The optimum ESO conditions were determined as interface temperature; 350 °C, DL temperature; 250 °C, heat block temperature; 400 °C, nebulizing gas flow (nitrogen); 3 L/min and drying gas flow (nitrogen); 15 L/min.

Method validation parameters
On this study, twenty-four phenolic compounds (flavonoids, flavonoid glycosides, phenolic acids, phenolic aldehyde, coumarin) and three non-phenolic organic acids that are widespread in edible plant materials were qualified and quantified in two edible plants. Rectilinear regression equations and the linearity ranges of the studied standard compounds were given in Table 1. Correlation coefficients were found to be higher than 0.99. The limit of detection (LDD) and limit of quantitation (LDQ) of the reported analytical method were shown in Table 1. For the studied compounds, LDD ranged from 0.05 to 25.8 μg/L and LDQ ranged from 0.17 to 85.9 μg/L ( Table 1) (Ertas et al., 2015). Moreover, the recoveries of the phenolic compounds ranged from 96.9% to 106.2%.

Statistical analysis
All experiments were conducted in triplicate and the data was presented as the mean value ± standard deviation (SD).

Results and discussion
On this study, twenty seven compounds (authentic markers) were studied for their dominant fragmentation pathways ( Figure 1).

Most of the compounds in MS exhibited abundant [M -H] in negative ion mode and [M + H]+ in the positive ion mode
were subjected to MS/MS analysis, retention time (RT) and mass spectral characteristics of all marker compounds were given in Table 1.
The variables considered during reflux extraction process including 80% methanol and ultrapure water were tested for the extraction of polyphenols from rachis, leaflets and fruits of Chamaerops, in order to achieve high extraction efficiency of phenolic acids and flavonoids. After LC-MS/MS analysis, the results indicated that phenolic acids were among the most abundant polyphenols detected in all plant parts including quinic, malic, chlorogenic, protocatechuic, p-hydroxybenzoic, p-coumaric, tr-aconitic, gallic and tannic acids, with quinic acid being equally predominant in both methanol and water leaflets extracts.
As shown in Table 2, a high content of quinic acid identified in leaflets was almost equal to 2.5 times of that found in rachis (37690 ± 1809 against 13198 ± 634 µg g -1 extract) respectively. On this context, it could be said that C. humilis is a good source of quinic acid. Many studies in the literature showed that quinic acid has a potent broad spectrum antioxidant (Pero et al., 2009), hepatoprotective (Xiang et al., 2001) and can be used to combat prostate cancer (Onbathamizh & Padmini, 2013).
Furthermore, malic acid was the second most prevalent compound after quinic acid in leaflets and fruits. However, in rachis, it was the major organic acid with higher concentration in methanol extract (RM and RW contained 21747 ± 1153 against 17614 ± 933 µg g -1 of malic acid, respectively).
Previous work has already demonstrated in other Arecaceae that malic acid was identified as the major compound in Phoenix canariensis (Al-Farsi et al., 2005), this organic acid was thought to play an important role in cardioprotective properties (Khazanov et al., 2008).
Methanol showed the highest extraction capacity for chlorogenic acid which took the third place after quinic and malic acid in all extracts. Ots presence was dominant in leaflets and rachis. Previous studies have shown that chlorogenic acid blocked chemically induced carcinogens in the large intestine (Mori et al., 1986).
On addition, protocatechuic acid was quantified in all Chamaerops extracts. However, in water solvents approximately equal amounts of protocatechuic acid was obtained (with 33 ± 2 µg g -1 in LW as high concentration). This value was equal to vanillin found also in LW (33 ± 2 µg g -1 ), and identified slightly less in LM (22 ± 1 µg g -1 ). The other reflux extracts contained lower concentrations of that molecule. This aldehyde, contributes to the original natural flavour of vanilla and is a very popular flavouring agent used in large range of foods and as fragrance ingredients (Mitra et al., 2002).
Different types of flavonoids such as flavones, flavonols, and flavanones were found in Chamaerops extracts. Flavonoids identified included various flavone-C-glycosides of apigenin and luteolin.
Dur results showed that heat reflux was a good attractive procedure for the extraction of luteolin in both methanol and water extracts of leaflets. However, it was not identified in rachis and fruits parts.
Flavone C-glycosides and tricin were previously identified in C. humilis leaves (Williams et al., 1973;Hirai et al., 1986). These results showed that the rutin and kaempferol were identified in all Chamaerops extracts, a higher content was obtained in methanol extracts of leaflets (33 ± 2 µg g -1 ), water was extracted three times less rutin LW (10.9 ± 0.54 µg g -1 ). On a previous study rutin was identified from the C. humilis leaves (Hirai et al., 1986) and Phoenix dactylifera L. fruits (Hamad et al., 2015).
According to the literature, kaempferol glycosides are widely distributed in the Arecaceae family (Williams et al., 1973).
LC-ESO-MS-MS detected traces of fisetin, rhamnetin and myricitin in only some Chamaerops extracts. The presence of quercetin traces has been present only in methanol extracts. Nonetheless, flavonoid C-glycosides were already identified from plants of the Arecaceae family. Flavone C-glycosides (84%), tricin (51%), luteolin (30%) and quercetin glycosides (24%)  were found out in the leaves of the 125 species of the Palmae (Williams et al., 1973).
Regarding the flavanone group, naringenin was weakly identified in all Chamaerops extracts. Ot is important to note that hesperitin was not identified, while its glycoside hesperidin (which is conjugates with rhamnosyl-α-1,6-glucose) was found. However, its concentration has decreased about 6 times less in water extracts (35 ± 2 and 6.2 ± 0.3 µg g -1 in LM and LW respectively), this reflects the solubility behaviour of hesperidin (Grandi et al., 1994).
Finally, methanol was more effective extraction solvent, which resulted in the coextraction of lots of compounds ( Figure 2).

Conclusion
On the present study, phenolic composition of leaflets, rachis and fruits parts of C. humilis var. argentea were identified by using heated reflux extraction and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis techniques. The LC-MS/MS results revealed that quinic, malic and chlorogenic acids and rutin and hesperidin were the major phenolic compounds in leaflets and fruits extracts. Besides, the methanol extract was detected to be the most efficient solvent to identify phenolic compounds in C. humilis. This approach showed that Chamaerops was a great promising source of different bioactive components, particularly phenolic acids and flavonoids. On vivo studies are required to determine its benefits as potential food ingredients and being agents for protection against various diseases. Therefore, the growing use of Chamaerops in foods was encouraged.