Wild strawberry (Arbutus unedo): Phytochemical screening and antioxidant properties of fruits collected in northern Morocco

2.1 Samples

The fruits of Arbutus unedo L. were collected fully ripened in October 2017 from three different forests: Achakar (AA), Qsar Kbir (AQ), and Chaoun-Qalaa (AC) (northern region of Morocco). Mature berries with seeds were freeze-dried, ground, and stored at –20 °C prior to extraction.

2.2 Chemicals

2,20-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azobis (2-amidinopropane), gallic acid dihydrochloride (AAPH), L-ascorbic acid, trichloroacetic acid (TCA), 1,1,3,3- tetraethoxypropane (TEP), thiobarbituric acid (TBA), and butylated hydroxytoluene (BHT) were purchased from Sigma (St. Lois, MO). Folin-Ciocalteu phenol reagent was obtained from Fluka. Standards (gallic acid, caffeic acid, rutin, catechin, coumaric acid, kaempferol, isorhamnetin, cinnamic acid, apigenin, and vanillic acid) were obtained from Merck Life Science (Merck KGaA, Darmstadt, Germany). LC-MS grade methanol, acetonitrile, acetic acid, acetone, and water were purchased from Merck Life Science (Merck KGaA, Darmstadt, Germany). All other chemicals were of analytical grade and obtained from Sigma (St. Louis, MO).

2.3 Extraction

5 g of lyophilized powder was defatted three times with 50 mL of n-hexane, dried, and were homogenized with 50 mL of two solvents with increased polarity (EtOAc and MeOH:water, 80:20 v/v). Each fraction was extracted by sonication in an ultrasound bath (130 kHz) for 45 min. After centrifugation at 5000 g for 5 min, the supernatant filtered through a paper filter, dried, reconstituted with MeOH:water, 80:20 (v/v) and then filter through 0.45 μm Acrodisc nylon membrane (Merck Life Science, Merck KGaA, Darmstadt, Germany) prior to HPLC-DAD-ESI/MS analysis. The resulting extracts were stored at 4 °C until use.

2.4 Phytochemical screening

Phytochemical screening was made according to the method of Trease and Evans (1989) in order to detect the presence of starch, saponins, flavonoids, tannins, catechic tannins, gallic tannins, anthocyanins, and alkaloids. The tests were based on visual observation of the color change or the formation of a precipitate after the addition of specific reagents.

2.5 Determination of total polyphenols

Total polyphenols content was determined with the Folin-Ciocalteu reagent by spectrophotometry according to the method of Singleton and Rossi. (1965) with some modifications. Gallic acid was used as standard (10, 25, 50, 100, 200 ppm). The total phenolic content was measured at 755 nm and was expressed in mg of gallic acid (GAE) / g dry weight (DW).

2.6 Determination of the total flavonoids

Quantification of flavonoids was made by the method of Zhishen et al., (1999), using AlCl₃ to 10% (w/v), NaOH 4% and NaNO₂ to 5%. The absorbance was determined at 510 nm. A curve of catechin (12.5, 25, 50, 100, 200 ppm) was also carried out. The total flavonoids content was expressed in mg of quercetin (QE)/g dry weight (DW).

2.7 Determination of anthocyanins

Determination of the total anthocyanin content was based on the differential pH (pH = 1 and pH = 4.5) by the method of Giusti and Wrolstad, (2001) with some modifications. Measurement was conducted at 510–700 nm in the UV–Vis spectrophotometer. The absorbance was calculated by the following formula: $$\mathrm{A}=[\left({\mathrm{A}}_{510}-{\mathrm{A}}_{700}\right)\mathrm{t}\mathrm{o}\mathrm{p}\mathrm{H}1.0]-[({\mathrm{A}}_{510}-{\mathrm{A}}_{700})\mathrm{a}\mathrm{t}\mathrm{p}\mathrm{H}4.5]$$

The total anthocyanin content was calculated by the molecular weight of pelargonidin-3-glucoside by the following formula: $$[\mathrm{m}\mathrm{g}\mathrm{P}\mathrm{g}-3-\mathrm{g}\mathrm{l}\mathrm{u}/\mathrm{g}\mathrm{M}\mathrm{S}]=\frac{\mathrm{A}\ast \mathrm{M}\ast \mathrm{F}\ast \mathrm{V}\ast 1000}{\epsilon \ast \mathrm{d}\ast \mathrm{Q}}$$

2.8 Determination of tannins

The content of condensed tannins has been determined by the vanillin method described by Julkunen-Tiitto (1985). A volume of 50 μL of each extract was added to 1500 μL of the 4% vanillin (w/v) in MeOH: water, 80:20 (v/v) then mix vigorously. Immediately after that 750 μL of hydrochloric acid concentrated (HCl) was added. The absorbance of the resulting mixture was measured at 550 nm after being allowed to react for 20 min at room temperature. The results were plotted after a (+)-catechin standard made in the same manner.

2.9 Determination of antioxidant activity

DPPH radical scavenging activity of entire fruit extracts was measured according to the method described by Braca et al., (2002) with a few modifications. Briefly, different concentration of extracts (25 µL) were added to 2 mL of 6.25–10 − 5 M DPPH MeOH:water, 80:20 (v/v) solution. After gentle mixing and 30 min of standing at room temperature, the absorbance of the resulting solutions was measured at 517 nm. The antiradical activity is estimated according to this equation: % of antiradical activity = [(Abs control –Abs sample) / Abs control] × 100. IC50 values of the extract i.e., the concentration of extract necessary to decrease the initial concentration of DPPH by 50% was calculated.

2.10 HPLC-DAD-ESI/MS

LC analyses were performed on a Nexera-e liquid chromatograph (Shimadzu, Kyoto, Japan), consisting of a CBM-20A controller, two LC-30AD dual-plunger parallel-flow pumps, a DGU-20A5R degasser, a CTO-20AC column oven, a SIL-30AC autosampler, an SPD-M30A photodiode array detector, and an LCMS-8050 triple quadrupole mass spectrometer, through an ESI source (Shimadzu, Kyoto, Japan), operating in both negative and positive ionization modes.

As chromatographic column, an Ascentis Express RP C18 column (150 × 4.6 mm; 2.7 µm) (Merck Life Science, Merck KGaA, Darmstadt, Germany) was employed. The mobile phase was composed of water/acetic acid (99.85/0.15 v/v, solvent A) and acetonitrile/acetic acid (99.85/0.15 v/v, solvent B), The flow rate was set at 1 mL/min under gradient elution: 0–5 min, 5% B, 5–15 min, 10% B, 15–30 min, 20% B, 30–60 min, 50% B, 60 min, 100% B. DAD detection was applied in the range of λ = 200–400 nm and two different wavelengths were monitored at λ = 280 nm and λ = 350 nm (sampling frequency: 40.0 Hz, time constant: 0.08 s). MS conditions were as follows: scan range and the scan speed were set at m/z 100–800 and 2500 u/sec, respectively, event time: 0.3 sec, nebulizing gas (N₂) flow rate: 1.5 L/min, drying gas (N₂) flow rate: 15 L/min, interface temperature: 350 °C, heat block temperature: 300 °C, DL (desolvation line) temperature: 300 °C, DL voltage: 1 V, interface voltage: −4.5 kV. Calibration curves (R² ≥ 0.997) of eleven polyphenolic standards used for the semi-quantification in sample extracts were obtained using triplicate injections with different concentrations in the range of (1, 5, 10, 50, 100 ppm), and according to the area of peaks acquired at following wavelengths of 270 nm, 277 nm, 278 nm, 280 nm, 321 nm, 330 nm, 336 nm, 365 nm, 370 nm, 355 nm. All protocatechuic acid, gallic acid, ellagic acid, methylellagic acid, ellagitannins, and their derivatives were quantified as gallic acid. Caffeoylquinic acid, caffeic acid and its derivatives, feruloylquinic acid, and syringic acid were quantified as caffeic acid while cinnamic acid derivatives and p-coumaroylquinic acid were quantified as cinnamic acid. Dihydroxyflavone and apigenin derivatives were quantified as apigenin. Epigallocatechin, catechin, and procyanidin were quantified as catechin. Myricetin and kaempferol derivatives were quantified as kaempferol while all quercetin derivatives were quantified as rutin. For the rest of compounds, calibration curves of vanillic acid, coumaric acid and isorhamnetin, were employed. (Mosele et al., (2016).

2.11 Statistical analysis

For each one of the samples, three replicates were analyzed. The results of these assays are expressed as mean values and standard deviation (SD). Differences among treatments were detected by analysis of variance ANOVA (P < 0.05).

3.1 Phytochemical screening

To obtain a general vision of the existing compounds occurring in the extracts, general phytochemical screening of all extracts was carried out. The employed colorimetric method showed the presence of flavonoids, tannins, anthocyanins, anthraquinones, sterols, steroids, deoxysugars, and glycosides, while alkaloids, and saponosides were not detected (Table 1). This finding, except for the presence of alkaloids, was in agreement with a previous work (Dib et al., 2013).

3.2 Polyphenolic content

The determination of the content of phenolic acids, flavonoids, carotenoids, vitamins, and minerals occurring in fruits and vegetables is mandatory for the evaluation of their health-promoting properties and immunity-boosting effects. Table 2 shows the extraction yields, total phenolic, flavonoid, anthocyanins, tannin contents, and antioxidant activity EC50 values of the wild fruits. The higher extract yields for total phenolic, flavonoids, and tannins were attained for the MeOH:water, 80:20 (v/v) extract with respect to EtOAc. On the other hand, the highest concentration of anthocyanins contents was observed for the EtOAc extract. The average of the total contents of phenolic acid, flavonoids, anthocyanins and tannins were respectively 108.41 ± 9.29 mg GAE/g (w/w) (DW), 101.07 ± 5.6 mg QE/g (w/w) (DW), 0.35 ± 0.48 mg Pg-3-glu/g (w/w) (DW) and 0.45 ± 0.48 mg EC/g (w/w) (DW). The results concerning the polyphenolic contents are 5-fold higher than another study which found it between 9.51 and 19.73 mg GAE/g (w/w) (DW) (Ruiz-Rodríguez et al., 2011). Also, this concentration is higher than those obtained in the fruits collected from Portugal (16.7 ± 0.4 mg GAE/g (w/w) (DW); (Mendes et al., 2011). The fruit extract from Chaoun-Qalaa (AC) presented the highest yield (68.33%) and the highest quantity of polyphenols and flavonoids (127.5 ± 7.14 mg GAE/g (w/w) (DW) and 105.9 ± 3.2 mg QE /g (w/w) (DW)). It was higher than the ones found by Barros et al., 2010 (126.83 ± 6.66 mg GAE/g (w/w) (DW) and 34.99 ± 1.55 mg CE/g (w/w) (DW)).

The highest quantity of total anthocyanins and tannins content was obtained from AQ fruit extract by EtOAc (1.42 ± 0.09 mg Pg-3-glu/g (w/w) (DW) and 0.9 ± 0.1 mg EC/g (w/w) (DW) respectively). In another study 0.76 ± 9.85 mg cy-3-glu/g (w/w) (DW) was found as the total amount of anthocyanin (Fortalezas et al., 2010). All extract obtained from EtOAc and MeOH:water, 80:20 (v/v) showed a significant amount of phenolic compounds. Another research realized on aqueous methanol extracts (methanol:water, 80:20 v/v) using sonication exposed an amont of 34.3 ± 1.9 mg GAE/g and 2.1 ± 0.1 mg RE/g respectively for total phenolic acids and total flavonoids (Asma et al., 2019).

In general, these differences could be due to environmental characteristics, the period of harvesting, cultivar variability, or fruit maturity (Ancos et al., 2000). For testing the antiradical activity of a large variety of the existing compounds in all extracts, the scavenging activity on DPPH was used as screening method. The results presented in Fig. 1 show that all A. unedo samples do have antiradical ability (lowest EC50 values). This efficiency was 10-fold higher in samples extracted by MeOH:water, 80:20 (v/v) than other extracted by EtOAc. AC fruits presented the highest phenolic content which is compatible to its lower EC50 (11.42 ± 0.13 and 1.37 ± 0.2 µg/mL (w/v) for MeOH:water, 80:20 (v/v) and EtOAc extracts). The value of EC50 indicates that the investigated sample extracts show significantly higher antioxidant capacity. The results are superior than the ones obtained in other studies (EC50 447.92 ± 0.81, Barros et al., 2010 and 790 ± 0.016 µg/mL (w/v), Mendes et al., 2011).

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Fig. 1

Scavenging activity on DPPH radicals of EtOAc (a) and MeOH:water, 80:20 (v/v) (b) extracts of A. unedo fruits from three different regions.

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3.3 Phytochemical profile by HPLC-DAD-ESI/MS

Fruits of strawberry tree were partitioned with two different solvents in increasing order of polarity with the aim to identify the maximum number of compounds with different solubility. Concerning the qualitative composition of A. unedo fruits, all extracts were analyzed by - HPLC-DAD-ESI/MS (Tables 3 and 4) and a large variety of compounds were tentatively identified based on their retention times, MS data and comparison with available references and literature survey. The main compounds present in the strawberry tree were represented by organic acids (malic acid and citric acid), phenolic acids (quinic acid, protocatechuic acid, gallic acid, caffeic acid, ferulic acid, cinnamic acid, ellagic acid, syringic acid, and hydroxycoumarin), flavones (dihydroxyflavone), flavanols (catechins, epicatechins, procyanidin dimer with respective gallate and prodelphinidin), flavonols (hexoside of isorhamnetin, myricetin, quercetin, kaempferol and apigenin) and other compounds (ascorbic acid, benzyl alcohol pentose, and hydroxyphenylvaleric). Most of these compounds were already described in A.unedo by Pawlowska et al., (2006), Fortalezas et al., (2010), Mendes et al., (2011), Guimarães et al., (2013) and Mosele et al., (2016). The number of determined compounds is the highest one compared to previously published data reporting many new compounds for the first time.

AQ fruits presented the highest phytochemical profile (34 compounds) followed by AC (28 compounds) and the last was AA (24 compounds) without considering the unknown compounds. EtOAc extract was the only one that presented two organic acids, namely citric acid (peak 2) and malic acid (peak 5) identified according to their UV spectra (λ_max = 254 nm) and pseudo molecular ion (m/z at 191 and 133, respectively).

Peak 1 (m/z 153) showed a λ_max at 295 nm in the extract of AQ-EtOAc and AA- MeOH: water, 80:20 (v/v), respectively and was identified as protocatechuic acid. Peak 15 (λ_max at 357 and 364) presented m/z of 315 and gave a fragment at m/z 153; it was identified as protocatechuic glucoside. It was presented in all the extracts of AA, in AQ- MeOH: water 80:20 (v/v) extract and AA-EtOAc extract. Peaks 3a,3b,3c and 3d showed the same [M − H]⁻ fragment ions at m/z 191(quinic acid) and different [M − H]⁻ parent ion at m/z 383, 287,371 and 533 respectively, with maximum UV spectra at 325 nm typical of quinic acid derivative. Except for AQ- MeOH: water, 80:20 (v/v), all extracts presented peak 6 that showed the same parent ion at m/z 267, MS fragment ion at m/z 147 and similar λ_max at 277 nm, characteristics of cinnamic acid derivatives. Similarly, peak 51 (AC-EtOAc) had the parent ion at m/z 343, MS fragment ions at m/z 267, 147, and UV spectra identical to peak 6 identified as cinnamic acid derivative. All peaks 7,9,11,21,24,27,30(a,b,c),52,54,63 yielded a fragment ion at m/z 169 typical of gallic acid. Gallic acid and its derivatives (such as glucosides, gallic acid gallate, galloylquinic acid, galloyl shikimic acid, trimethyl gallic acid glucuronide, galloyl quercetin and gallotannins) were the dominating peaks in all extracts. The isomers identification was carried out by their comparison to the following references: Mosele et al., 2016; Tavares et al., 2010; Mendes et al., 2011. According to MS fragment at m/z 179 and UV spectra, all peaks 13a, 13b, 13c;13d (λ_max at 321,350,380 and 356 nm respectively) were related to caffeic acid and showed different parent ions at m/z 357, 359, 665 and 601 respectively. Caffeic acid isomers were identified thanks to their comparison to the analytical standards. Ellagic acid, is a dimeric derivative of gallic acid, and is generated by the hydrolysis of the ellagitannins. Its derivatives (ellagic acid glucoside, methylellagic acid rhamnoside, methylellagic acid rhamnopyranoside, strictinin ellagitannin, and ellagic acid arabinoside/xyloside) were distributed differently in the fruits on the regions studied. The content of gallic acid and its derivatives could have scientific value in various fields of medicine and biotechnology. They have also been implicated as anticarcinogenic, antimicrobial, antimutagenic, antiangiogenic and anti-inflammatory agents besides their use in treating critical diseases like depression, cancer, microbial infections, lipid-related diseases, etc (Choubey et al., 2015). Peak 53 showed UV spectra at 275–350 nm and molecular ion at m/z 161, characteristic of 4-hydroxycoumarin. AA- MeOH: water 80:20 (v/v) fruits showed peaks 60 and 65 (m/z 337 and 329; λ_max at 292 nm and 277 nm and MS fragments m/z 191 and 167 respectively) were identified as p-coumaroylquinic acid vanillic acid hexoside, respectively by comparison of their mass fragment and UV spectra profiles with previously published data Stanoeva et al., (2017), Mena et al., (2012).

Only AQ-EtOAc extract showed a peak 4 (m/z 253; λ_max at 287 nm) that was identified as dihydroxyflavone. Flavan-3-ols presented in this sample were catechin and its derivatives (epigallocatechin, procyanidin dimer type B, (-)-epicatechin gallate, catechin glucose, prodelphinidin dimer, procyanidin gallate). These compounds were abundant only in the fruits of AQ regions.

Also, 24 flavonols were found in this species and were distributed differently depending on the extraction solvent (14 and 15 compounds in EtOAc and MeOH: water, 80:20 (v/v) respectively) and the regions of harvest (22, 19 and 22 compounds in AA, AC and AQ respectively). Their UV spectra are in fact characteristic of the flavonols structure, with two absorption bands. The first band, determined by the benzene moiety, in 250–270 nm range, the second one in the 340–350 nm range, with intensities and relative positions reflecting hydroxylation pattern and degree of substitution (Dugo et al. 2009). The presence of MS fragments at m/z 301, 285, 315, 317, and 269 indicate the presence of aglycones as quercetin, kaempferol, isorhamnetin, myricetin, and apigenin. Accordingly, to these MS fragments ions, these compounds were identified as results from glycosylation of the flavonols at various positions. All studied samples were rich in quercetin derivatives. Peaks 33, 35, and 41 respectively for quercetin pentoside, quercetin glucoside, quercetin rhamnoside ([M−H]^- at m/z 433, 463 and 447, respectively) were found in all the studied fruits. In ethyl acetate extract (AC and AQ), two compounds were tentatively identified as quercetin arabinose/xyloside (peak 36) and quercetin hexoside (peak 38) detected a similar [M − H]⁻ ion at m/z 433 also similar UV spectra (252–347 nm and 254–351 nm, respectively). Also, all methanolic samples presented quercetin glucuronide (m/z 477; 235–355 nm). Other quercetin derivatives were assigned according to pseudo molecular ions, MS fragments, UV spectra, and by comparison with literature data. Peak 31 ([M − H]⁻ at m/z 609) detected in all samples was identified as quercetin galloyl hexoside derivative. Peak 37 assigned to quercetin rhamnoside-glucoside ([M−H]^- at m/z 609) was present in AQ-EtcOH and (AA/AC)- MeOH:water, 80:20 (v/v). Only the samples of AA-EtcOH presented peak 47 ([M − H]⁻ at m/z 551) which was identified as quercetin hexose protocatechuic acid. Peak 48 in ethyl acetate samples of AA and AC was tentatively identified as dihydroquercetin rhamnoside. Other detected peaks 67, 68, and 69 were identified as quercetin galloyl glucoside, quercetin galloyl hexuronide ([M − H]⁻ at m/z 629), quercetin galloyl pentoside ([M−H]^- at m/z 585) respectively.

In AQ samples, two molecules resulted from glycosylation of isorhamnetin: peak 23 (isorhamnetin glucoside; m/z 477) and peak 56 (isorhamnetin rutinoside; m/z 461). These two compounds showed an identical pattern fragment at m/z 315, but two different range of UV spectra at 278–385 nm and 277–350 nm respectively. Myricetin and its derivatives present in fruits of AA and AQ. Peaks 26, 57, and 66 were tentatively identified as myricetin, dihydromyricetin rhamnoside, and myricetin rhamnoside. Other detected flavonols corresponded to apigenin and kaempferol derivatives. Apigenin pentoside (peak 45 in AA and AC), apigenin glucuronide (peak 62 in AA), and apigenin glucoside (peak 63 in AA) were identified according to their mass spectra and UV range. Peak 39 identified as kaempferol galloyl glucoside in accordance with [M − H]⁻ at m/z 599, MS fragment at m/z 447 and by comparison with results reported by Mendes et al., (2011). Peak 42 detected in all ethyl acetate extract was tentatively identified as kaempferol xyloside ([M−H]^- at m/z 417).

The investigated samples showed other compounds that were tentatively identified according to their mass spectra, MS fragments, and λ_max. These compounds were detected as ascorbic acid and its derivatives, benzyl alcohol hexose pentose, and hydroxyphenylvaleric acid (Mosele et al., 2016).

The content of phenolic acids, flavones, flavan-3-ols and flavonols in the A. unedo fruit is presented in Table 5. Regarding the profile composition, the most abundant group was represented by hydroxybenzoic acids with 76.02 ± 1.3 mg/100 g (w/w) (DW) founded in AA fruit followed by AQ fruit 71.78 ± 1.5 mg/100 g (w/w) (DW) and in the last AC fruit 49.41 ± 1.01 mg/100 g (w/w) (DW) caffeic acid and its derivatives were abundant in AA (3.57 mg/100 g (w/w) (DW)) and AC (1.18 mg/100 g) (w/w) (DW). The highest content of flavan-3-ols was in AQ (67.19 mg/100 g (w/w) (DW)), instead. These results do not match other studies which showed that proanthocyanidins present the highest content (De Pascual al., 2000; Pallauf et al., 2008). However, in agreement with Pallauf et al., (2008) quercetin derivatives are abundant flavonols in all samples. On the other hand, flavonol derivates are 10-fold higher than one found in the fruits of Portugal (10.86 ± 0.24 mg/100 g (w/w) (DW); Guimarães et al., 2013). Semi-quantification by HPLC-DAD analysis gave the higher content of antioxidant compounds for the AQ fruits (162.74 ± 27.41 mg/100 g (w/w) (DW)) followed by AA fruits (120.43 ± 27.41 mg/100 g (w/w) (DW)).