Phytochemical investigation of Phoenix canariensis Hort . ex Chabaud leaves and pollen grains

Article history: Received on: 08/09/2016 Revised on: 09/10/2016 Accepted on: 22/11/2016 Available online: 28/12/2016 Phoenix canariensis is a commonly grown, yet understudied, palm plant. The phytochemical screening of leaves and pollens revealed the presence of flavonoids, saponins, tannins, sterols and/or triterpenes. Quantitative estimation of constituents, revealed that the total polyphenolics were higher in the leaves (69.9) than in pollens (29.98) expressed in mg gallic acid equivalent/g d.wt, the total flavonoids calculated as rutin equivalent were (23.86 mg/g) in leaves and (17.20 mg/g) in pollens, the total tannins content were 55.18 and 3.31 mg tannic acid equivalent/g fresh wt, while the total steroids content were 2.6 and 12.4 mg β-sitosterol equivalent/g d.wt, in leaves and pollens, respectively. Eighteen phenolic compounds and ten flavonoids were identified by HPLC. GLC analysis of lipids, revealed the identification of phytosterols (4.93 and 28.90%), saturated (35.35 and 40.56%) and unsaturated (62.42 and 59.01%) fatty acids in leaves and pollens, respectively. Proximate analysis revealed a total moisture content of (6.4 and 7.7 %), crude fiber (32.22 and 39.50%), total ash (12.1 and 8.1%) and acid insoluble ash (4.7 and 7.6 %) for leaves and pollens, respectively. Moreover, spathe headspace volatile analysis combined with GC-MS revealed the presence of fifty-two compounds constituting 72.84% of the total oil composition where αcopaene predominates (18.72%).


INTRODUCTION
Palms are among the best known and extensively cultivated plant families and they are of great economic importance.They all belong to family Arecaceae (Johnson, 1998).Palms have been cultivated in the Middle East and North Africa for at least 5000 years (Zohary and Hopf, 2000).The genus Phoenix (Family Arecaceae) comprises about 19 species, distributed in tropical and subtropical areas southern Asia and Africa (Adawy et al., 2002, Mohtasheem et al., 2005) Phoenix canariensis, the Canary date palm, is native to the Canary Islands.It is widely planted as ornamental in the US and the Mediterranean area (Krueger, 2001).It can be identified by its single, upright, thick trunk topped with a crown of leaves in 2.5 to 4.5 meters long.The stalks of inconspicuous flowers are replaced with clusters of one inch diameter, orange yellow, datelike, ornamental fruits, which ripen in summer (Gilman and Watson, 2006).The inflorescence of most palm trees in its early stage of growth is enclosed in hard covering envelop known as spathe, which splits open as the flowers reach maturation (Zaid and Arias-Jimenez, 2002).
In Egypt, pollens were believed to enhance fertility and to be an aphrodisiac.Several constituents have been identified and isolated from the genus including lipids, phenolic acids, flavonoids, procyanidins, saponins, triterpenes, sterols and carbohydrates (Elgindi et al., 2015).
To the best of our knowledge, no published data was found concerning the pollen grains and spathe of the plant while only few reports were found concerning the isolation of flavonoids, phenolic acid and steroidal saponin from the leaves (Garcia et al., 1981;Asami et al., 1991) and isolation of an alkaloid from the seeds as well as seed oil chemical composition (Hammami et al., 2010;Nehdi et al., 2011).This study includes the determination of pharmacopoeial constants, phytochemical screening, spectroscopic analysis of different classes, HPLC analysis of polyphenolics and GLC analysis of lipids for leaves and pollen grains of Phoenix canariensis in addition to its spathe volatile analysis.

Plant material
Phoenix canariensis hort.ex Chabaud leaves and pollens were collected over the years 2013-2015 from El-Orman Botanical Garden, Egypt.The plant was identified by Dr. Mohamed el Gebaly (Taxonomist) and Mrs. Therese Labib, consultant of plant taxonomy at the Ministry of Agriculture.Voucher specimens numbered (5.10.2015), were deposited at the Herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.Spathe used for volatiles analysis was collected in summer.

Proximate analysis
Moisture, crude fiber, total ash and acid insoluble ash were determined (AOAC, 2000).Each value was an average of 3 determinations.

Spectrophotometric determination of total polyphenolic content
The total polyphenolic content was performed using Folin-Ciocalteu reagent.A calibration curve (R 2 =0.9987) was prepared using gallic acid standard (Sigma Co., St. Louis, MO, USA) at concentrations of 50 μg to 0.9 mg/ml.Total content of phenolic compounds was calculated as gallic acid equivalents (GAE) (Singleton et al.,1999).For each concentration, three replicates were carried out.

Spectrophotometric determination of flavonoid content
Spectrophotometric determination of total flavonoid content was based on measuring the intensity of the yellow colour developed when flavonoids were complexed with aluminum chloride reagent at 420 nm using UV spectrophotometer expressed as rutin equivalent.A standard calibration curve (R 2 = 0.9984) was prepared using different aliquots of standard rutin (Sigma Co., St. Louis, MO, USA) equivalent to 20-180 µg/ml (Geissman, 1962).Three replicates were carried out, for each concentration.

Spectrophotometric determination of total tannin content
Tannin content was estimated by Folin-Denis reagent using tannic acid as standard (Sigma Co., St. Louis, MO, USA) (Earp et al., 1981).A standard calibration curve (R 2 = 0.9998) was prepared using tannic acid concentrations ranging from 0 -100μg/ml.The values were expressed in micrograms (μg) of tannic acid equivalents (TAE) per gram fresh weight.Three replicates were carried out, for each concentration.

Spectrophotometric determination of total steroidal content
The determination of total steroidal content of leaves and pollen grains was based on measuring the intensity of the green colour developed when steroidal compound complexes with Libermann-Burchard , s reagent using β-sitosterol as a standard.A standard calibration curve (R 2 =0.9981) was prepared using different aliquots of β-sitosterol solution (E-Merk, Darmstadt, Germany) equivalent to 0.1 to 0.5 mg/ml.The concentration of the total steroids was calculated as β-sitosterol equivalent, Daksha et al., 2010.The results were average of three determinations.
The volume injected was 10µl.Phenolic compounds were assayed by external standard calibration at 280nm and expressed in mg/g dry matter of equivalent (+)-catechin for flavan-3-ols, equivalent coumarin for apolar aromatic compounds and equivalent quercetin-3-rutinoside for flavonols.
Identification of the major phenolic components were performed by comparing their retention times to those obtained for the standards (prepared as 50-600 ug/ml solutions in methanol).The concentration of individual phenolic component was calculated on the basis of peak area measurements of both standards and samples by adopting the external standard method.Results were the average of triplicate experiments.All standards used were obtained from Sigma.

HPLC analysis of flavonoids
The 70% ethanolic extracts of leaves and pollen grains were subjected to HPLC, adopting the conditions described in (Mattila et al., 2000).Flavonoid analysis was done on Inertsil (GL Sciences, Inc., Japan) using ODS-3 (4.0×150 mm, 3 um) column with a C-18 guard column.Temperature of the column oven was set at 35 °C.Gradient elution was employed for flavonoids with a mobile phase consisting of 50 mM H 3 PO 4 , pH 2.5 (solution A) and acetonitrile (solution B) as follows: Isocratic elution 95% A/5% B, 0-5 min; linear gradient from 95% A/5% B to 50% A/ 50% B, 5-55 min; isocratic elution 50% A/50% B, 55-65 min; linear gradient from 50% A/50% B to 95% A/5% B, 65-67 min; post-time 6 min before next injection.The flow rate of the mobile phase was 0.7 mL/min, and the injection volumes were 10 uL of each the standards and sample extracts.Identification of major flavonoids was done by comparing their retention times to those obtained for the standards.Peak areas were used for quantification of both standards and samples using the external standard method.Standard flavonoids calibration curves were made by diluting stock standards in methanol to yield 2-20 ug/mL.Results were the average of triplicate experiments.Standards of flavonoid aglycones were obtained from different manufacturers and must be in HPLC purity grade.

Investigation of lipid content
100 g of each of the air-dried powdered leaves and pollen grains were, separately, extracted with petroleum ether till exhaustion.The extract was evaporated under vacuum to give 0.84g and 0.38g residue, respectively.The unsaponifiable and saponifiable fractions were obtained from the petroleum ether fraction and the liberated fatty acids were methylated according to Finar, 1973 andVogel, 1967.GLC conditions for the analysis of unsaponifiable matter (USM) was carried out on a capillary column (30 m × 0.32 mm I.D. × 0.25 um film) and packed with HP-5 (5% phenyl methyl siloxane), the injected volume was 2 ul.The analysis was carried out at a programmed temperature.Initial temperature was 80 C for 5 min.then increased to 280 C by the rate of 8 C/min., injector and detector (FID) temperatures were 240 C and 300 C, respectively.
Flow rate of nitrogen was 20 ml/min.Fatty acid methyl ester was analysed on the same column used for USM with the same carrier gas but at flow rate of 30ml/min.The initial temperature was 120 C increased to 240 C by the rate of 4 C/min.the injector and detector (FID) temperatures were 250 C and 280 C, respectively.Identification of compounds was carried out by comparing the retention times of their peaks with those of the available authentic compounds (Sigma Chemical Co.St. Louis, MO, USA) similarly analysed.Quantification was based on peak area measurement using a computing integrator.

Headspace GC/MS analysis of spathe volatiles
Headspace volatiles analysis using solid phase microextraction (SPME) was adopted from (Farag and Wessjohann, 2012) with slight modification.Frozen spathe sample (3 g) was cut into small pieces and transferred to SPME screw cap vials (20 ml).Vials were then capped and SPME fiber left to sample then the head space heated above the sample at 50 °C for 30 min, the fiber withdrawn into the needle and transferred to the injector port of the GC/MS instrument.A system blank containing no plant material was run as a control.

GC/MS parameters
SPME fibers were desorbed at 210 C for 1 min in the injection port of a shimadzu model GC-17A gas chromatograph interfaced with a shimadzu model QP-5050 mass spectrometer.Volatiles were analysed on a SLB-5 MS column (30 m, 0.25 mm I.D., 0.25µm film) (Supelco, Oakville, ON, Canada).Injections were made in the splitless mode for 30s. the gas chromatograph was operated under the following conditions: injector 220 C, column oven 38 C for 3 min, then programmed at a rate of 12 C/min to 180 C, kept at 180 C for 5 min and finally ramped at a rate of 40 C / min.the transfer line and ion source temperatures were adjusted at 230 and 180 C, respectively.The HP quadrupole mass spectrometer was operated in the electron ionization mode at 70 eV.The scan range was set at m/z 40-500.Volatile components were identified using the procedure described in (Farag and Wessjohann, 2012) and peaks were first deconvoluted using AMDIS software (www.amdis.net)and identified by its retention indices (RI) relative to n-alkanes (C6-C20), mass spectrum matching to NIST, WILEY library database and with authentic standards when available.

Phytochemical screening
In the present investigation, preliminary phytochemical screening of leaves and pollens of the plant revealed the presence of flavonoids, saponins, tannins, carbohydrates and/or glycosides, sterols and / or triterpenes.

Proximate analysis
Pharmacopoeial constants for both organs were determined viz.total moisture content (6.4% and 7.7 % in leaves and pollens, respectively), crude fiber (32.22% and 39.50%, respectively), total ash (12.1% and 8.1% in leaves and pollens, respectively) and acid insoluble ash (4.7 and 7.6% in leaves and pollens, respectively).These constants can serve as standards for the purity of leaves and pollens as well as for the differentiation from other Phoenix species.

Spectrophotometric determination of different classes of the plant
The total polyphenolic content were highest in the leaves (69.9 mg/g) than the pollens (29.98 mg/g) expressed in GAE per gram dry weight; the concentration of total flavonoids calculated as rutin was higher in leaves (23.86 mg/g) than in pollens (17.20 mg/g).Also the total tannins content, were 55.18 mg and 3.31 mg of TAE per gram fresh weight in leaves and pollens, respectively.While the total steroids content, were 2.6 and 12.4 mg/g per gram dry weight expressed as β-sitosterol equivalents in leaves and pollens, respectively.The results showed that the leaves were rich in polyphenolics, flavonoids and tannins as compared to pollens while pollens were rich in steroids (Table 1).The total polyphenolic content in 21 Egyptian date fruit varieties of Phoenix dactylifera was previously recorded to vary from 2.33 to 18.97mg/g dry weight (Farag et al., 2014).While the phenolic profile of the Algerian type date palm fruit range from 2.49-8.36mg/100 g fresh weight GAE (Mansouri et al., 2005).The total phenolic content of seeds of seven different varieties ranges between 1.98 to 4.65 mg/g GAE (Al-Juhaimi et al., 2012)

GLC analysis of lipoidal content
The percentage of the USM represents 29.98% and 13.04% of the total lipids of leaves and pollens, respectively.The GLC analysis of the unsaponifiable fraction resulted in the identification of 83.47% and 84.68% of the USM of the leaves and pollens, respectively.The percentage of total hydrocarbons identified was 58.68% and 47.26% in the leaves and pollens, respectively.N-docosane (C22) was the major hydrocarbon (37.54%) of the leaves while n-tricosane (C23) was the major hydrocarbon in the pollens (13.43%).On contrary phytosterols percent was much higher in the pollens (28.90%) than in the leaves (4.93%).Concerning the leaves, α-amyrin was the major triterpene (19.61%), while β-sitosterol and campesterol were the main phytosterols (2.82% and 1.30%, respectively).In the pollens βamyrin was the main triterpene (5.30%) while the main phytosterols were campesterol and β-sitosterol (10.45% and 9.96%, respectively) (Table 2).The percentage of fatty acids represents 70.02% and 86.96% of the total lipids of leaves and pollens, respectively.--: not detected The GLC analysis of fatty acids resulted in the identification of 17 and 19 compounds in leaves and pollens, respectively.Palmitic acid constituted the highest percentage of the saturated fatty acids in the pollens (27.71%) and in the leaves (25.47%).Concerning the unsaturated fatty acids the α-linolenic acid was the major constituent of the leaves (29.12%), while oleic acid was the major unsaturated fatty acid in the pollens (20.52%) (Table 3).Several reports were found concerning the fatty acid profile of seeds and fruits genus Phoenix while little is known about the leaves and pollens.Oleic acid was the major fatty acid in most reports of Phoenix dactylifera seeds and fruits (Al-Shahib and Marshall, 2003, Nehdi et al., 2010, Ogungbenle 2011, Akbari et al., 2012, Ben Salah et al., 2012), while linoleic acid was the major in fruits grown in Tunisia (Saafi et al., 2008).In Phoenix theophrasti fruits palmitic acid predominate (Liolios et al., 2009) while in the leaves of Phoenix dactylifera hexadecanoic acid ethyl ester was reported the major (Azmat et al., 2010).

HPLC analysis of polyphenolics
The HPLC analysis of the 70% ethanolic extracts of leaves and pollens separately, revealed quantitative variation in phenolic and flavonoids composition (Tables 4 and 5).Eighteen different phenolic compounds were identified, where ethyl vanillic acid was the major phenolic acid identified in both organs, its antioxidant (Tai et al., 2011) andantiinflammatory (Jung et al., 2010) activities were previously reported.
Concerning the flavonoids, ten flavonoids were identified in both organs, where isorhamnetin predominates at a concentration of 17785.44 ppm and 4349.10 ppm in leaves and pollens, respectively.Isorhamnetin was reported to exhibit antiinflammatory (Seo et al., 2014), anti-obesity (Lee et al., 2009), anti-oxidant (Pengfei et al., 2009), and antitumor (Teng et al., 2006) activities.The metabolomics fingerprints of 21 Egyptian date fruit varieties of Phoenix dactylifera were previously studied by Farag et al., 2014.Luteolin and apigenin glycosides predominates the flavones identified while quercetin conjugates were the principle flavonols.Caffeoyl shikimic acid was the main hydroxylcinnamic conjugate.

Headspace GC/MS analysis of spathe volatiles.
Headspace volatile analysis combined with GC/MS was utilized for detection of spathe volatiles (Table 6).The percentage of total hydrocarbons, monoterpenes and sesquiterpene hydrocarbons as well as the percentage of total oxygenated compounds and oxygenated monoterpenes were calculated (Table 7).A total of 52 compounds were detected.Sesquiterpene hydrocarbons constituted the most dominant class with 41.51% where α-copaene predominates (18.72%).Concerning oxygenated compounds, total oxygenated compounds were 23.73%, where 2ethylhexanol (4.77%) was the major oxygenated compound, while oxygenated monoterpenes were found to be 2.03%, In conclusion, the results provide the first study on volatiles of spathe from the plant while several reports were found concerning the spathe of the well-known species Phoenix dactylifera (Hamedi et al., 2013, Jahromi et al., 2014).

CONCLUSION
This study is the first report that shed light on leaves and pollens of Phoenix canariensis Hort.ex Chabau cultivated in Egypt, in addition to its spathe volatile analysis.The leaves are rich in polyphenolics, flavonoids and tannins as compared to pollens while pollens are rich in steroids.The main components identified by GLC and HPLC might suggest their incorporation in antioxidant and anti-inflammatory preparations.Further studies are required to evaluate the plant biological activities and to isolate the bioactive compounds.
Financial support and sponsorship: Nil.

Table 1 :
Quantification of different chemical classes of plant constituents.

Table 2 :
GLC analysis of the identified USM of the leaves and pollen grains.Relative retention time to n-Docosane C-22 with Rt= 20.662 min.

Table 3 :
GLC analysis of the identified FAME of the leaves and pollen grains.

Table 4 :
HPLC analysis of phenolics in Phoenix canariensis hort ex.Chabaud, leaves and pollen grains.

Table 5 :
HPLC analysis of flavonoids in Phoenix canariensis hort ex.Chabaud, leaves and pollen grains.

Table 7 :
The calculated percentage of different classes of the spathe oil compounds.