New insights into the qualitative phenolic profile of Ficus carica L. fruits and leaves from Tunisia using ultra-high-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry and their antioxidant activity

Abstract

Ficus carica L. fruits have been consumed from the earliest times, and other parts of the tree have been used for traditional medicinal purposes. Nowadays, the beneficial properties of this and other Ficus species are attributed to the presence of key phytochemicals. To increase our knowledge about this topic, the present study has conducted phenolic profiling of the leaves and whole fruits from two Tunisian cultivars, ‘Temri’ and ‘Tounsi’, using reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled to two detection systems: diode-array detection (DAD) and quadrupole time-of-flight (QTOF) mass spectrometry (MS). UV-Vis absorption was a valuable tool for classifying phenolic compounds into families, while MS using electrospray ionization (ESI) and MS/MS allowed the molecular formula to be established and structural information to be obtained. The total phenol content and the antioxidant activity were also assessed. As a result, in the negative ionization mode 91 phenolic compounds were characterized including hydroxybenzoic acids, hydroxycinnamic acids, hydroxycoumarins and flavanoids (flavonols, flavones, flavanones, flavanonols, flavanols and isoflavones). This work was complemented by the detection of other 18 phenolic compounds in the positive ionization mode, including anthocyanins and furanocoumarins. To the best of our knowledge, this is the first time most of these compounds have been tentatively reported in F. carica. These results indicate the complexity of this family of secondary metabolites in F. carica, as well as the potential of this analytical method for characterization purposes. In conclusion, the qualitative phenolic profile, total phenolic content and antioxidant activity differed especially between leaves and fruits.

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1. Introduction

Moraceae is an angiosperm plant family, very rich in edible species and characterized by milky latex in all parenchymatous tissue, unisexual flowers, anatropous ovules, and aggregate drupes or achenes.¹ Ficus is one of the thirty-seven genera of this family, which comprises about 800 species.² Among them, the fig tree or common fig (Ficus carica L.) is the most well known species. This plant is a native of the Middle East and one of the first plants cultivated by humans. Fig fruits are consumed either fresh or dried,^3,4 and today F. carica continues to be an important crop worldwide, especially in the Mediterranean basin,⁵ which includes Tunisia.

In general, figs have the best nutrient score among dried fruit, being an important source of minerals and vitamins,⁴ as well as containing relatively higher amounts of crude fibre than all other common fruits.^6,7 Among its phytochemicals, some phenolic classes have been reported in Spanish, Italian and Turkish commercial figs such as the furanocoumarins psoralen and bergapten (5-methoxypsoralen),⁸ the flavonoid rutin,^8–10 hydroxycinnamic acids like ferulic and chlorogenic acids^8,9,11 and anthocyanins.⁴ The analytical techniques to perform these studies include gas chromatography (GC) coupled to mass spectrometry (MS) and a flame ionization detector (FID),¹² as well as high-performance liquid chromatography (HPLC) coupled to UV, diode array detection (DAD) and mass spectrometry (MS) in a negative or positive ionization mode depending on the target phenolic class.^{4,7,8,10–14}

Regarding the potential health benefits, F. carica exhibits antioxidant,^2,6,7 and remarkably hypolipidemic and hypoglycemic properties¹⁵ that could be of interest for managing metabolic syndrome. In fact, the antidiabetic effects of F. carica leaves extracts have evoked great interest as a natural therapy¹⁵ since diabetes is one of the most common diseases in nearly all countries. It also continues to increase in number and significance as changing lifestyles lead to reduced physical activity and increased obesity.¹⁶ Pèrez and co-workers confirmed that the water extract of fig leaves and its chloroform fraction tend to normalize the antioxidant status of diabetic rats.¹⁷ Although several studies have related the bioactivity of this and other Ficus species to the phenolic constituents,¹⁵ more studies are needed to clarify this issue. Thus, novel analytical methodologies may help in the elucidation of the bioactive molecules.

In the case of Tunisia, more than 70 different fig ecotypes were recently reported with a wide phenotypic diversity and distinguished by taste, colour and flavour of fruits. However, little is known about their bioactivity and minor phytochemical composition. Two examples of cultivars, known as the ‘Temri’ and ‘Tounsi’ cultivars, are commonly cultivated in the centre and south of Tunisia,¹⁸ respectively. Therefore, as potential bioactive markers, the total phenolic content (TPC) and antioxidant capacity of leaves and dried whole fruits from these two Tunisian cultivars of F. carica were firstly evaluated. Secondly, their phenolic profiles were extensively studied by ultra-high-performance liquid chromatography (UHPLC) coupled with two detection systems, DAD and quadrupole time-of-flight (QTOF)-MS using electrospray ionization in complementary negative and positive ionization modes.

2. Results and discussion

Total phenolic content and antioxidant activity of the ‘Tounsi’ and ‘Temri’ fig cultivars

Total phenolic content. In general, the leaves were richer in phenolic compounds than fruits, the TPC value being the highest in the ‘Temri’ cultivar (686.88 mg of gallic acid/100 g of leaves; Fig. 1). However, the dried whole fruits from the ‘Tounsi’ cultivar presented a higher TPC value (200.18 mg of gallic acid/100 g of dried fruits) than ‘Temri’ (124.48 mg of gallic acid/100 g of dried fruits) (Fig. 1). Concerning the fig fruits, Solomon et al.⁷ evaluated the TPC of six common commercial figs, which had values ranging from 48.6 to 281.1 mg of gallic acid/100 g of fresh fruits. These authors showed that cultivars with skins with dark purple colours, such as Mission and Chechick, were richer in phenolic compounds than those with clearer skins, which explain our results since the skin from the ‘Tounsi’ fruits presents a darker purple colour than ‘Temri’ fruits.

Fig. 1 Bar graph of total phenol content (TPC) (mg of gallic acid/100 g sample) of leaves and fruits from F. carica cultivars ‘Tounsi’ and ‘Temri’ and antioxidant activity evaluated by: trolox equivalent antioxidant capacity (TEAC) (mmol eq. trolox/100 g of sample), ferric ion reducing antioxidant power (FRAP) (mmol eq. FeSO₄/100 g sample) and oxygen radical absorbance capacity (ORAC) (mmol eq. trolox/100 g sample) assays. The primary Y axis corresponds to TPC and the secondary Y axis corresponds to antioxidant activity. Data are given as mean ± standard deviation. Caffeic acid was used as the control and expressed as mmol eq. trolox or FeSO₄/mmol of compound.

In vitro antioxidant activity. Three different methods were used to evaluate the antioxidant capacity: trolox equivalent antioxidant capacity (TEAC), which is also known as the ABTS method; ferric ion reducing antioxidant power (FRAP); and oxygen radical absorbance capacity (ORAC). The TEAC and FRAP methods are based on single electron transfer (SET) mechanisms, whereas the ORAC method is based on a hydrogen atom transfer (HAT) reaction. In this regard, it is now recommended that in vitro antioxidants should be determined by at least two methods, preferably with different mechanisms.^19,20 The results are depicted in Fig. 1. Caffeic acid was used as control due to the lack of standardization of these protocols in the literature, with the TEAC, FRAP and ORAC values in agreement with those in studies by Rice-Evans et al.,²¹ Ozgen et al.²² and Ou et al.,²³ respectively.

According to aforementioned results for TPC, the leaves showed higher antioxidant activity values than fruits by the three methods assayed. In the same manner, the highest TEAC, FRAP and ORAC values were measured in the ‘Temri’ cultivar, being 2.58 mmol equivalent of trolox/100 g of sample, 2.93 mmol equivalents of FeSO₄/100 g of sample and 1.56 mmol equivalents of trolox/100 g of sample, respectively. In general, the antioxidant potential of leaves from the Ficus genus is higher than that of the fruits.²⁴

Previous studies on the antioxidant activity were only conducted on fresh fruits, with results ranging from 0.025 to 0.716 mmol equivalent of trolox/100 g for TEAC, and 0.36 to 1.61 mmol equivalent of FeSO₄/100 g for FRAP,^7,25 so it is not appropriate to compare these with our values. Furthermore, the drying process may partially alter the total fruits phenolic content,¹⁰ anthocyanins,²⁶ as well as antioxidant activity.²⁶ In the case of the ORAC, this activity has not been studied before in this fruit. This method is interesting since it is based on the scavenging of peroxyl radicals that are physiologically relevant radicals.¹⁹

Correlation between TPC and antioxidant activity. Overall, the leaves of both cultivars possessed the strongest antioxidant activity and the fruits had the weakest activity. This may be explained by the occurrence of the highest amounts of phenolic compounds in leaves, since our results indicate an excellent correlation between TPC content and TEAC (r = 0.994), FRAP (r = 0.997) and ORAC (r = 0.993) at p < 0.01 (Table 1). On the other hand, the antioxidant activities determined by these three methods also correlated well between each other (r > 0.98; Table 1). These results are in accordance with previous studies that have also shown a strong correlation between the TPC, TEAC⁷ and FRAP²⁵ of fig fruits. However, in other foods little or no relationship has been found and other antioxidant compounds may contribute greatly.²⁰ Thus, our results indicate that phenolic compounds are determinants of antioxidant agents in the F. carica samples.

Table 1 Correlation between the total phenolic content (TPC) and antioxidant activity of leaves and fruits of F. carica cultivars ‘Temri’ and ‘Tounsi’^a

Correlations
		TPC	TEAC	FRAP	ORAC
a Antioxidant activity: TEAC, trolox equivalent antioxidant capacity; FRAP, ferric ion reducing antioxidant power; ORAC, oxygen radical absorbance capacity.b Correlation is significant at the 0.01 level (2-tailed).
TPC	Pearson correlation	1	0.994^b	0.997^b	0.993^b
	Sig. (2-tailed)		0.000	0.000	0.000
TEAC	Pearson correlation	0.994^b	1	0.991^b	0.985^b
	Sig. (2-tailed)	0.000		0.000	0.000
FRAP	Pearson correlation	0.997^b	0.991^b	1	0.984^b
	Sig. (2-tailed)	0.000	0.000		0.000
ORAC	Pearson correlation	0.993^b	0.985^b	0.984^b	1
	Sig. (2-tailed)	0.000	0.000	0.000

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Qualitative profiling of leaves and fruits

General identification process. In the present work, a qualitative analysis of the phenolic composition was performed using RP-UHPLC-DAD-QTOF-MS and MS/MS, using electrospray ionization in negative and positive ionization modes. Respectively, Tables 2 and 3 show the general results for the following: retention time (RT), molecular monoisotopic mass, experimental m/z, molecular formula, UV data (nm), MS score, error (ppm), main MS/MS fragments and the proposed assignment. Additionally, Tables S1 and S2† provide the species, plant family and previous studies that have reported on each compound.

Table 2 Phenolic compounds characterized using the negative ionization mode in leaves and fruits of F. carica cultivars ‘Tounsi’ and ‘Temri’

RT^a (min)	[M − H]^−	Formula	Score	Error^a (ppm)	UV^a (nm)	Main fragments via MS/MS	Proposed compound	Presence
								‘Tounsi’		‘Temri’
								L^a	F^a	L^a	F^a
a RT, retention time; L, leaves; F, fruits. The UV data agreed with Gómez-Romero et al.;^28 Lin et al.;^36 Tsimogiannis et al.^37b Identification confirmed by comparison with standards.c Compounds described here for first time in family Moraceae with several saccharide combinations and conjugation positions reported in different plant families (see KNApSAck, Reaxys or SciFinder databases).d Apigenin C-hexoside pentoside could be schaftoside (apigenin 6-C-glucoside 8-C-arabinoside) or isochaftoside (apigenin 6-C-arabinoside 8-C-glucoside). The latter were previously described in F. carica leaves (Takahashi et al.^32).e The identification was based on the elution pattern under similar analytical conditions (Tahir et al.^39).f Compounds described here for first time in family Moraceae and common in the family Fabaceae (see KNApSAck, Reaxys or SciFinder databases).g 6-, 8- and 3′-prenylgenistein were previously reported in other Ficus species.
Hydroxybenzoic acids and derivatives
10.61	359.0976	C15H20O10	94.7	1.6	280	197.0455; 179.0346; 153.0549; 135.0452; 85.0292	Syringic acid hexoside I	+	−	+	−
10.76	315.0725	C13H16O9	98.9	−1.2	—	153.0190; 152.0109; 108.0212; 109.0293	Dihydroxybenzoic acid hexoside I	+	+	+	+
10.76	313.0569	C13H14O9	84.0	−1.2	280	197.0462; 167.0354; 153.0559; 135.0454; 133.0145; 123.0455; 115.0039	Syringic acid malate I	+	−	+	−
10.94	359.0993	C15H20O10	89.3	−2.9	280	197.0458; 179.0352; 153.0352; 135.0454; 123.0453; 85.0297	Syringic acid hexoside II	+	−	+	−
11.07	329.0886	C14H18O9	80.3	−2.4	255; 291	167.0345; 152.0111; 123.0450; 108.0213	Vanillic acid glucoside	+	+	−	+
11.07	313.0573	C13H14O9	82.7	−2.7	280	179.0350; 135.0450; 133.0142; 115.0037	Syringic acid malate II	+	−	+	−
11.09	475.1473	C20H28O13	86.7	−3.8	—	329.0880; 167.0347; 109.0293	Vanillic acid hexoside deoxyhexoside	+	−	+	−
11.19	433.1002	C17H22O13	95.3	−3.2	280	301.0564; 169.0138; 168.0061; 151.0035; 125.0242	Gallic acid di-pentoside I	+	−	+	−
11.23	315.0726	C13H16O9	98.9	−1.5	—	153.0188; 109.0294	Dihydroxybenzoic acid hexoside II	+	+	+	+
11.50	433.0996	C17H22O13	98.2	−1.6	280	301.0568; 169.0130; 168.0064; 151.0041; 125.0245	Gallic acid di-pentoside II	+	+	+	+
11.57	447.1143	C18H24O13	99.3	0.4	305	315.0714; 271.0816; 152.0112; 109.0291; 108.0217	Dihydroxybenzoic acid hexoside pentoside I	+	+	+	+
12.32	447.1143	C18H24O13	97.4	0.2	260; 297	152.0114; 109.0291	Dihydroxybenzoic acid hexoside pentoside II	+	−	+	−
12.54	153.0198	C7H6O4	92.5	−4.1	260; 290	109.0296; 108.0220	Dihydroxybenzoic acid	+	+	+	+
12.56	315.0723	C13H16O9	99.7	−0.5	—	153.0194; 152.0194; 109.0291; 108.0219	Dihydroxybenzoic acid hexoside II	+	−	+	−
12.62	285.0613	C12H14O8	97.3	1.2	260; 300	152.0115; 153.0191; 108.0217; 109.0295	Dihydroxybenzoic acid pentoside I	+	+	+	+
12.68	447.1151	C18H24O13	94.3	−1.6	—	153.0192; 109.0295	Dihydroxybenzoic acid hexoside pentoside III	+	+	+	+
13.17	285.0621	C12H14O8	85.2	−1.4	230; 300	153.0191; 152.0113; 109.0294; 108.0219	Dihydroxybenzoic acid pentoside II	+	+	+	+
13.25	417.1043	C17H22O12	98.2	−1.0	310	285.0613; 241.0718; 153.0165; 152.0119; 108.0217; 109.0287	Dihydroxybenzoic acid di-pentoside	+	+	+	+
14.66	137.025	C7H6O3	96.1	−5.0	—	109.0294; 108.0221; 93.0349; 92.0273	Hydroxybenzoic acid I	+	−	+	−
15.17	137.0245	C7H6O3	95.3	−1.7	—	93.0344	Hydroxybenzoic acid II	+	+	+	+
15.90	167.0349	C8H8O4	96.9	0.9	261; 292	152.0110; 123.0431; 124.0163; 108.0214	Vanillic acid^b	+	+	+	+
Hydroxycinnamic acids and derivatives
11.19	515.1408	C22H28O14	88.4	−0.9	264; 327	353.0881; 191.0560; 179.0346	Caffeoylquinic acid hexoside I	+	−	+	−
11.75	515.1408	C22H28O14	98.2	−0.7	262; 324	341.0872; 323.0771; 191.0559; 179.0348; 173.0451; 135.0451	Caffeoylquinic acid hexoside II	+	+	+	+
12.31	353.0884	C16H18O9	98.3	−1.3	264; 328	191.0560; 179.0349; 135.0448	Caffeoylquinic acid I	+	+	+	+
12.37	343.1043	C15H20O9	95.9	−2.7	282	181.0508; 163.0400; 137.0609; 135.0443	Dihydrocaffeic acid hexose	+	+	+	+
12.64	515.1408	C22H28O14	98.5	−1.0	280; 320	341.0773; 323.0773; 191.0560; 179.0347; 135.0446	Caffeoylquinic acid hexoside III	+	+	+	+
12.89	355.1038	C16H20O9	99.1	−1.0	322	193.0502; 178.0267; 149.0606; 134.0369	Ferulic acid hexoside I	−	+	−	+
13.79	337.0926	C16H18O8	81.3	0.8	300; 320	191.0557; 173.0454; 163.0399	Coumaroylquinic acid I	+	−	+	−
13.90	353.0883	C16H18O9	98.2	−1.3	298; 325	191.0566; 179.0347	Caffeoylquinic acid II^b (chlorogenic acid)	+	+	+	+
14.11	325.0929	C15H18O8	92.4	−0.5	326	163.0397; 119.0499	Comaroyl hexoside	−	+	−	+
14.21	353.0882	C16H18O9	83.7	−1.1	325	—	Caffeoylquinic acid III	+	+	+	+
14.72	355.1036	C16H20O9	99.3	−0.4	323	193.0502; 178.0267; 149.0602; 134.0370	Ferulic acid hexoside II	−	+	−	+
15.28	353.0885	C16H18O9	83.2	−1.6	272; 328	191.0565	Caffeoylquinic acid IV	+	+	+	+
15.61	337.0929	C16H18O8	98.4	0.5	272; 328	191.0568	Coumaroylquinic acid II	+	−	+	−
15.84	179.036	C9H8O4	94.5	−5.5	295; 324	135.056; 134.0377; 89.0399	Caffeic acid^b	−	+	−	+
16.03	295.0467	C13H12O8	95.9	−2.5	298; 330	179.0350; 133.0143; 115.0038	Caffeoylmalic acid	+	−	+	−
16.83	337.0929	C16H18O8	99.8	0.0	272; 326	191.0558	Coumaroylquinic acid III	+	−	+	−
17.30	385.1144	C17H22O10	82.0	−0.9	268; 326	267.0724; 249.0617; 223.0458; 205.0353; 147.0294; 113.0241; 91.0551; 85.0294	Sinapic acid hexoside	+	+	+	+
18.01	279.0513	C13H12O7	99.0	−1.0	291; 324	163.0398; 133.0139; 119.0499; 115.0033	Coumaroylmalic acid I	+	−	+	−
18.10	339.0729	C15H16O9	98.1	−1.7	286; 330	309.0621; 223.0616; 208.0372; 193.0507; 164.0480; 149.02543; 133.0142; 115.0039	Sinapic acid malate	+	−	+	−
18.25	279.051	C13H12O7	99.2	0.0	298; 320	163.0401; 133.0139; 119.0500; 115.0033	Coumaroylmalic acid II	+	−	+	−
18.40	309.0625	C14H14O8	96.0	−2.8	286; 325	193.0510; 178.0267; 149.0607; 133.0146; 115.0039	Ferulic acid malate I	+	−	+	−
18.67	309.0623	C14H14O8	98.0	−2.0	288; 320	193.0556; 134.0371	Ferulic acid malate II	+	−	+	−
19.09	193.0511	C10H10O4	98.3	−2.0	293; 325	134.0373	trans-Ferulic acid^b	−	+	−	+
19.62	193.0503	C10H10O4	84.7	0.3	282; 325	134.0373	Ferulic acid isomer	−	+	−	+
Flavonoids–flavonols
13.09	771.2002	C33H40O21	97.5	−1.7	356	609.1459; 462.0801; 463.0871; 301.0352; 300.0258	Quercetin O-deoxyhexoside di-hexoside^c	+	+	+	+
13.39	625.141	C27H30O17	87.5	−1.0	346	463.0893; 462.0814; 301.0360	Quercetin O-di-hexoside^c	+	+	+	+
15.59	755.2052	C33H40O20	94.1	−1.6	356	301.0359; 300.0279	Quercetin di-deoxyhexoside hexoside^c	+	+	+	+
17.18	609.1486	C27H30O16	93.8	−1.9	354	463.0890; 300.0278; 273.0398; 257.0448; 229.0502; 178.9983; 121.0297; 151.0036; 107.0142	Quercetin-3-O-rutinoside^b (rutin)	+	+	+	+
17.94	463.0888	C21H20O12	99.8	−0.3	354	301.0349; 300.0278; 151.0037	Quercetin-3-O-glucoside^b (isoquercetin)	+	+	+	+
18.68	549.0882	C24H22O15	99.2	0.6	354	505.0986; 463.0874; 301.0351; 300.0276	Quercetin 3-O-(6′′-malonyl)glucoside	+	+	+	+
23.05	301.0373	C15H10O7	83.2	−0.8	371	273.0399; 178.9983; 151.0034; 121.0296; 107.0139	Quercetin^b	−	+	−	+
Flavonoids–flavones
14.76	579.1367	C26H28O15	87.3	−3.3	344	561.1251; 519.1156; 489.1044; 459.0938; 429.0834; 399.0727; 369.0623; 285.0499; 133.0289	Luteolin C-hexoside C-pentoside I	+	+	+	+
14.89	579.136	C26H28O15	96.3	−0.7	354	561.1254; 519.1153; 489.1049; 459.0939; 429.0834; 399.0723; 369.0624; 285.0400; 133.0297	Luteolin C-hexoside C-pentoside II	+	+	+	+
15.10	563.1415	C26H28O14	98.3	−1.5	336	545.1321; 503.1212; 473.1097; 443.0988; 383.0786; 353.0669; 325.0733; 297.0766; 117.0347	Apigenin C-hexoside C-pentoside I^d	+	+	+	+
15.60	563.1435	C26H28O14	88.3	−4.7	335	545.1312; 503.1203; 473.1104; 443.0999; 383.07858; 353.0680; 325.0726; 297.0778; 117.0343	Apigenin C-hexoside C-pentoside II^d	+	+	+	+
16.00	447.0937	C21H20O11	98.7	−1.0	350	429.0821; 387.2027; 357.0615; 327.0512; 285.0404; 133.0138	Luteolin 6-C-glucoside (isoorientin)^e	+	+	+	+
16.21	563.142	C26H28O14	84.5	−3.3	330	545.1302; 503.1195; 473.1092; 443.0989; 383.0777; 353.0670; 297.0766; 117.0357	Apigenin 6-C-hexose-8-C-pentose III^d	+	+	+	+
16.58	447.0938	C21H20O11	98.7	−1.3	350	357.0608; 327.0507; 285.0398; 133.0291	Luteolin 8-C-glucoside (orientin)^e	+	+	+	+
16.80	577.1579	C27H30O14	98.2	−2.0	330	457.1140; 413.0880; 293.0455	Apigenin C-hexoside C-deoxyhexoside	+	+	+	+
17.42	431.0989	C21H20O10	99.4	−1.2	326	341.0663; 311.0553; 283.0603; 269.0444; 268.0372; 117.0342	Apigenin 8-C-glucoside (vitexin)	+	+	+	+
17.82	447.0932	C21H20O11	89.9	−1.0	352	285.0406; 284.0327; 197.0806; 175.0282; 133.0294	Luteolin 7-O-glucoside^b (cynaroside)	+	+	+	+
24.29	269.0459	C15H10O5	98.8	0.0	336	241.0495; 227.0352; 225.0553; 201.0551; 183.0445; 181.650; 159.0457; 151.0033; 149.0240; 117.0344; 107.0137	Apigenin^b	+	+	+	+
22.46	285.0407	C15H10O6	95.7	−1.8	349	267.0298; 257.0453; 243.0297; 241.504; 217.0506; 213.0549; 199.0396; 197.0604; 175.0395; 151.0031; 133.0295	Luteolin^b	+	+	+	+
Flavonoids–flavanones
16.09	611.1624	C27H32O16	94.2	−1.5	280	449.1094; 287.0563; 151.0036; 135.0445	Eriodictyol di-hexoside^c	−	−	−	+
17.95	449.1099	C21H22O11	96.2	−2.3	280	287.0565; 151.0039; 135.0451; 107.0142	Eriodictyol hexoside I^c	−	−	−	+
19.87	449.1086	C21H22O11	96.0	0.9	286	287.058; 151.0033; 135.0450; 107.0138	Eriodictyol hexoside II^c	−	−	−	+
22.91	287.0569	C15H12O6	97.7	−2.5	282	151.0039; 135.0449; 125.0241; 107.0139; 83.0137	Eriodictyol	−	+	−	+
24.46	271.0617	C15H12O5	98.8	−2.0	289	177.0183; 151.0034; 119.0499; 107.0137	Naringenin	+	+	+	+
Flavonoids–flavanols
14.52	289.0717	C15H14O6	81.5	0.6	278	245.0821; 205.0497; 203.0707; 161.0606; 125.0245	(+)-Catechin^b	+	+	+	+
Flavonoid–flavanonols
19.50	303.0510	C15H12O7	98.7	−0.1	283	285.0399; 151.0034; 125.0240	Dihydroquercetin (taxifolin)	−	+	−	+
Flavonoid–isoflavones
22.68	547.1093	C25H24O14	88.6	−0.7	—	503.1204; 299.0558; 284.0320; 165.0191; 149.9954; 133.0294; 121.0292	Hydroxygenistein methyl ether malonylhexoside	+	−	+	−
24.46	269.0459	C15H10O5	85.5	−0.9	260; 330	241.0492; 225.0556; 201.0552; 151.0031; 133.0292; 119.0504; 117.0349; 107.0139	Genistein^b	−	−	+	−
25.82	299.0555	C16H12O6	99.6	2.2	260; 335	298.0475; 285.0357; 284.0310; 256.0370; 240.0419; 239.0343; 165.0190; 149.9955; 133.0289; 121.0291	7-Methoxy 2′-hydroxy genistein (cajanin)	+	+	+	+
26.49	353.1037	C20H18O6	97.9	−1.9	266	325.1074; 298.0472; 283.0604; 219.0655; 175.0397; 133.0290; 133.0658	Prenylhydroxygenistein I^f	+	−	+	−
27.19	353.1034	C20H18O6	84.2	−2.2	264	325.1074; 285.1127; 284.0322; 219.0657; 175.0398; 151.0761; 133.0657; 133.0295	Prenylhydroxygenistein II^f	+	+	+	+
27.62	353.1037	C20H18O6	97.5	−2.0	264; 344	325.1078; 285.1127; 284.0320; 219.0660; 175.0762; 151.0761; 151.0032; 133.0657; 133.0293	Prenylhydroxygenistein III^f	+	+	+	+
27.69	337.1087	C20H18O5	94.8	−2.7	—	293.0462; 282.0534; 269.1190; 254.0516; 133.0658; 117.0346	Prenylgenistein I^g	+	+	+	+
27.82	283.0614	C16H12O5	99.7	−0.5	—	268.0374; 239.0348; 151.0040; 132.0214; 107.0133	Genistein 4′-methyl ether (biochanin A)	+	+	+	+
28.54	337.1082	C20H18O5	98.2	0.2	265; 339	293.0449; 282.0526; 269.0436; 268.0368; 254.0564; 238.0622; 225.0469; 133.0287	Prenylgenistein II^g	+	+	+	+
29.10	337.1084	C20H18O5	99.0	−0.3	266; 340	293.0452; 282.0528; 269.0446; 268.0370; 253.0500; 254.0574; 238.0624; 133.0923	Prenylgenistein III^g	+	+	+	+
Hydroxycoumarins
13.09	339.0728	C15H16O9	94.8	−2.4	279; 330	177.0191; 133.0293	Esculetin hexoside I	+	+	+	+
13.71	339.075	C15H16O9	83.4	−0.1	279; 335	177.0197	Esculetin hexoside II	+	−	+	−
15.39	177.0187	C9H6O4	97.0	−3.9	—	149.0241; 133.0293; 105.0346	Dihydroxycoumarin I	+	+	+	+
18.32	205.0146	C10H6O5	98.6	−1.8	286	161.0243; 133.0295; 117.0348; 105.0347; 89.0396; 77.0398	6-Carboxyl-umbelliferone	+	−	+	−
19.34	161.0244	C9H6O3	97.4	−1.7	283; 324	133.0291; 117.0342; 105.034	7-Hydroxycoumarin^b (umbelliferone)	+	+	+	+
20.86	177.0194	C9H6O4	93.5	0.7	285	149.0247; 133.02937; 105.0346	Dihydroxycoumarin II	+	+	+	+
22.60	205.0517	C11H10O4	92.2	−5.2	244; 252sh; 289; 338	187.0400; 161.0607; 146.0372; 133.0657; 118.0419; 105.0709	Phellodenol A/hydrated form of 4′,5′-dihydropsoralen	+	−	+	−
22.94	235.0616	C12H12O5	97.8	−1.7	255; 282	217.0499; 201.0189; 191.0712; 176.0477; 161.0241; 148.0523; 133.0293; 117.0345	Murrayacarpin B/di-hydrated form of bergapten	+	−	+	−
27.95	229.0872	C14H14O3	99.5	−0.4	—	213.0553; 185.0603; 146.0368; 130.0420; 118.0426	Prenyl-7-hydroxycoumarin	+	+	+	−
Others
17.88	365.0964	C17H18O9	97.8	−1.7	244; 288; 334	203.0347; 159.0453; 131.0497; 130.0421; 103.0552	(2Z)-3-[6-(β-D-Glucopyranosyloxy)-1-benzofuranyl]-2-propenoic acid (psoralic acid glucoside)	+	−	+	−

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Table 3 Other phenolic compounds characterized using the positive ionization mode in leaves and fruits of F. carica cultivars ‘Tounsi’ and ‘Temri’

RT^a (min)	[M + H]^+	Formula	Score	Error (ppm)	UV^a (nm)	Main fragments via MS/MS	Proposed compound	Presence
								‘Tounsi’		‘Temri’
								L^a	F^a	L^a	F^a
a RT, retention time; L, leaves; F, fruits. The UV data agreed with Dueñas et al.;^4 Teixeira et al.;^8 Frérot et al.;^55 Tang et al.^65b Hydroxypsoralen hexoside could be 5-hydroxypsoralen hexoside (bergaptol hexoside) or 8-hydroxypsoralen hexoside (xanthoxol hexoside).c Marmesin was previously described in F. carica and its enantiomeric form nodakenetin in Ficus tsiangii.d Hydroxypsoralen could be 5-hydroxypsoralen (bergaptol) or 8-hydroxypsoralen (xanthoxol) according to Yang et al.^52e Compounds described here for first time in F. carica but described in the family Moraceae and other families (see KNApSAck, Reaxys or SciFinder databases).f Non detected in the negative ionization mode.g Compounds described here for first time in the family Moraceae and common in the family Fabaceae (see KNApSAck, Reaxys or SciFinder databases).
Anthocyanins
11.51	611.1603	C27H31O16	93.9	−0.3	520	449.1078; 287.0565	Cyanidin 3,5-diglucoside	−	+	−	+
13.13	595.1667	C27H31O15	98.5	0.5	282; 520	449.1073; 287.0547	Cyanidin 3-rutinoside	−	+	−	+
Furanocoumarins
15.97	365.0872	C17H16O9	98.6	−0.9	250; 264; 308	203.0336; 175.0438; 147.0438; 131.0387; 119.0487; 101.0387; 91.0540	Hydroxypsoralen hexoside I^b	+	−	+	−
16.65	365.0871	C17H16O9	96.9	−1.0	252; 264; 310	203.0336; 175.0389; 147.0440; 131.0395; 119.0485; 91.0539	Hydroxypsoralen hexoside II^b	+	−	+	−
17.58	247.0969	C14H14O4	94.3	−2.4	—	229.0845; 213.0548; 189.0574; 175.0393; 147.0438; 119.0489; 103.0545	Marmesin isomer I^c	+	+	+	+
17.64	409.1496	C20H24O9	96.2	−1.0	—	247.0962; 229.0862; 213.0545; 185.0602; 175.0389; 147.0348; 119.0487; 91.0543	Marmesinin	+	−	+	−
17.77	235.0606	C12H10O5	93.2	−3.3	256; 303	217.0505; 202.0259; 174.0547; 131.0489; 115.0537	Methoxypsoralen derivative (hydrate)	+	−	+	−
21.8	189.0549	C11H8O3	86.8	−1.5	250; 290	161.0605; 147.0441; 133.0644; 119.0489; 105.0700	4′,5′-dihydropsoralen	+	−	+	−
22.05	247.0971	C14H14O4	95.0	−2.6	255	229.0858; 213.0545; 189.0537; 175.0392; 147.0442; 119.0492; 103.0544	Marmesin isomer II^c	+	+	+	+
22.30	305.1030	C16H16O6	95.7	−3.0	257; 266; 310	203.0344; 175.0391; 159.0441; 147.0438; 131.0489; 119.0490	Oxypeucedanin hydrate	+	−	+	−
22.48	203.0343	C11H6O4	85.8	−2.0	254; 269; 306	147.0442; 131.0494; 129.0332; 119.0496; 101.0376; 91.0541	Hydroxypsoralen^d^,^e	+	−	+	−
24.46	187.0317	C11H6O3	80.0	−1.7	254; 296; 328	159.0440; 131.0492; 115.0542; 103.0543	Psoralen	+	+	+	+
26.01	217.0502	C12H8O4	97.6	−2.4	258; 266; 310	202.0259; 174.0311; 159.0447; 146.0359; 131.0490; 118.0410; 115.0486	Methoxypsoralen	+	+	+	+
26.26	287.0918	C16H14O5	99.4	−1.2	—	203.0338; 175.1124; 159.0429; 147.0430; 131.0477; 119.0487; 103.0550	Oxypeucedanin	+	−	+	−
28.25	271.0980	C16H14O4	91.8	−5.3	—	229.0503; 215.0349; 203.0349; 201.0554; 187.0397; 173.0603; 159.0448; 131.0495; 117.0702	Isopentenoxypsoralen^e	+	−	+	−
31.16	285.1131	C17H16O4	95.5	−3.4	268; 309	243.0638; 229.0478; 217.0473; 201.0530; 186.0293; 115.0521	Prenyl methoxypsoralen	+	+	+	+
Isoflavones^f
30.85	299.0906	C17H14O5	96.5	2.9	262; 329	284.0660; 267.0633; 256.0711; 243.0998; 166.0242; 137.0576	Hydroxy-dimethoxyisoflavone^g	+	+	+	+
Others
17.21	205.0502	C11H8O4	96.1	−3.5	255; 290; 335	187.0401; 133.0648; 131.0491; 115.0537; 107.0491; 105.0700; 103.0541	Psoralic acid/dihydro-hydroxypsoralen	+	−	+	−

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On the one hand, the UV-Vis was a valuable tool for classifying phenolic compounds into families and subfamilies according to the presence of one or two characteristic absorption bands in the UV: band I and band II that come from the B-ring cinnamoyl structure and the A-ring benzoyl or benzene structure, respectively. In this regard, the wavelength of maximum absorption for the characterized phenolic compounds is depicted in Tables 2 and 3, as commented above. Besides, as an example, Fig. S1† shows the UV spectra of several phenolic types from F. carica, where band I ranged from 325–371 nm, approximately, and band II was around 260–298 nm. In the case of flavonoids, at the same time that the heterocyclic C-ring structure serves for their sub-classification, the most intensive band also depends on this ring. For example quercetin (flavonol) showed a prominent band I with a maximum at 371 nm, whereas naringenin (flavanone) presented a maximum at 289 nm that comes from the band II (Fig. S1†). Genistein (isoflavone) was characterized by a maximum around 260 nm with higher intensity than the second maximum at 330 nm. This UV absorption behaviour enabled to differentiate isoflavones from flavones, preliminarily. In addition, anthocyanins presents a maximum absorption at visible wavelengths, around 520 nm, that is a characteristic feature of this flavonoid subclass.

On the other hand, the QTOF mass analyzer delivers accurate mass measurements and isotopic fidelity (see Experimental section) that allow the molecular formula of the target compound to be obtained. Therefore, in order to procure confident formula assignments for target molecular ions, the lower mass error value and the higher MS score the better (see values in Tables 2 and 3). Afterwards, databases as well as literature were consulted for the retrieval of chemical structure information taking the MS and UV data into account. Finally, using MS/MS analyses, the structure of the parent compound may be tentatively confirmed through studying the fragmentation pattern: fragment ions and neutral losses, which are also accurately measured. As an example, this general identification process is summarized in Fig. S2.† Moreover, the RT served as criterion of polarity and elution order. In this way, a total of 13 phenolic compounds were confirmed with standards by comparison of the RT, UV spectra and MS/MS data in order to validate our characterization process (see Table 2).

Briefly, with a concise data mining, 91 phenolic compounds were characterized in the negative ionization mode (method 1) including hydroxybenzoic acids, hydroxycinnamic acids, flavonoids that were represented by flavonols, flavones, flavanones, flavanonols, flavanols and isoflavones subclasses, and hydroxycoumarins (Table 2). Several of these compounds were also detected using the analytical method 2 (data not shown). These data were complemented with 18 phenolic compounds tentatively characterized using the positive ionization mode, belonging to anthocyanins, furanocoumarins and a isoflavone (Table 3), which were either poorly ionized in the negative ionization mode or undetectable. Furthermore, the major part of the characterized phenolic compounds were tentatively reported in F. carica for the first time in this work (Fig. S3†), and other unreported phenolic structures were proposed as well according to their UV, MS and MS/MS information. Several previous studies on F. carica applied GC and HPLC coupled to several detectors, including mass analyzers such as quadrupole and ion trap.^{4,7,8,10–14} However, there were few compounds identified in these works, which are in the range from 4 to 15. One of the reason is that the authors focused on a particular phenolic subclass, e.g. anthocyanins,^4,7 or target phenolic compounds.^8,11 Therefore, at this point, our findings remark the potential of RP-UHPLC-DAD-QTOF-MS in order to perform successful and extensive characterization works of plant extracts and as starting point for structure elucidation of new molecules. In this regard, the MS analysis via electrospray ionization in the negative and positive ionization modes was complementary and enabled the detection and characterization of a large number of compounds. However, the analyst must be cautious in offering interpretations until all the information is evaluated. It is probably the most critical and long time-consuming part since, although there are efforts to generate spectral libraries using standards, unfortunately LC-ESI-MS methods often lack the consistency, standardization or reproducibility that characterizes GC-MS or nuclear magnetic resonance spectroscopy.²⁷

The chromatographic profiles are depicted in Fig. 2 that show the base peak chromatograms (BPC) of leaves and fruits of both cultivars that represent the ions detected in negative ionization mode using method 1, and the UV chromatograms at 254 and 520 nm, at which furanocoumarins and anthocyanins, respectively, show absorption,^4,8 using method 2 (see Experimental section). These chromatographic profiles, BPC and UV at 254 nm, show that the leaves presented richer qualitative and quantitative profiles, explaining our previous results for TPC and antioxidant activity. However, not surprisingly, anthocyanins were only detected in fruits.

Fig. 2 Chromatographic profiles of the leaves and fruits from F. carica cultivars ‘Tounsi’ and ‘Temri’ obtained by RP-UHPLC-DAD-QTOF-MS: (a and b) base peak chromatogram (BPC) in negative ionization mode using analytical method 1 and (c and d) UV chromatograms at 254 and (e and f) at 520 nm using analytical method 2. In each figure, the intensity was scaled to the largest area.

Regarding non-phenolic compounds, several organic acids, amino acids and other compounds were also characterized, and these are additionally described in the ESI, see Table S1† and non phenolic compounds information. Furthermore, Table S3† also contains information about certain unknown compounds that their MS/MS spectra is related to the MS/MS of hydroxybenzoic derivatives. Double bond equivalents (DBE) are reported in this table since this value is related to the total number of combined rings and double bonds in the molecule, and so it is useful as indicator of aromaticity or unsaturation. For example, a benzene ring has 4 DBE, that is one ring and three double bonds.

Phenolic acids: hydroxybenzoic, hydroxycinnamic acids and others. Overall, 45 phenolic acids were found in F. carica (Tables 2 and S1†), belonging to hydroxybenzoic and hydroxycinnamic acids. The main qualitative differences were found between leaves and fruits. The first phenolic class with a more polar feature eluted over a period of 10.61 to 15.90 min, whereas the second class compounds eluted between 11.19 and 19.62 min. In general, phenolic acids and their derivatives ionized better in the negative ionization mode and most of them presented a loss of 18.0106 u (H₂O) and 43.9898 u (CO₂) in MS/MS, which is consistent with previous findings in Gómez Romero et al.²⁸ and Abu-Reidah et al.²⁹ Interestingly, the leaves were richer in phenolic derivatives formed by conjugation with sugars and organic acids, including malic and quinic acid. However, the free forms of hydroxybenzoic, caffeic and ferulic acids, except vanillic acid, were only present in fruits.

It is worth mentioning that all hydroxybenzoic acids except dihydroxybenzoic acid were reported for the first time in F. carica. The new compounds were derivatives of hydroxybenzoic, dihydroxybenzoic and trihydroxybenzoic acid (e.g. gallic acid), being O-methylated (e.g. vanillic and syringic acids) or conjugated with hexose, pentose and malic acid. These moieties were assigned according to their respective neutral losses established on the basis of the fragmentation pattern in MS/MS, as previously reported.^28–31 As an example, Fig. 3a shows the MS/MS spectra of the isomer of syringic acid malate (isomer I) (m/z 313.0569): 197.0462 ([C₉H₁₀O₅ − H]⁻), 133.0145 ([C₄H₆O₅ − H]⁻) and 115.0039 ([C₄H₄O₄ − H]⁻), which correspond to free syringic acid ion after the loss of malic acid, malic acid ion and its fragment ion generated by the loss of H₂O, respectively. In addition, the presence of fragments at m/z 167.0354 and 153.0559 indicated the loss of CH₂O and CO₂ from the methoxy group and the carboxylic acid moiety of the phenolic acid, respectively. This compound was detected only in the leaves of both cultivars.

Fig. 3 Examples of MS/MS spectra of phenolic compounds highlighting the main fragments from F. carica: (a) syringic acid malate (isomer I), (b) quercetin 3-O-(6′′-malonyl) glucoside, and (c) methoxypsoralen.

A total of 24 hydroxycinnamic acids were derivatives of coumaric, caffeic, ferulic and sinapic acids. Overall, hydroxycinnamics also presented a higher signal in leaves than in fruits (Fig. S4a and d†). The presence of caffeic acid and trans-ferulic acid in fruits and chlorogenic acid in fruits and leaves was confirmed with standards and presented the same RT, molecular formula, UV maximum and fragmentation pattern. These compounds have been previously reported in this species.^8,9,32–34 Moreover, other three isomers of chlorogenic acid were also found. Recently, Oliveira et al.⁹ described the isomers 3-O- and 5-O-caffeoylquinic acids (chlorogenic acid) in Portuguese white fig samples.

Interestingly, as for the aforementioned phenolic acid class, several conjugated forms were reported for the first time in F. carica and as well as in the Moraceae family (Tables 2 and S1†). For example, three isomers of caffeoylquinic acid hexoside were characterized based on their molecular formula and UV and MS/MS spectra, which was in agreement with previous studies on other plant families.^30,35,36 In a similar manner, the fragmentation pattern of caffeic, coumaric and ferulic acids conjugated with hexose or organic acids were in accordance with previous studies.^{28–30,32,35} Overall, these conjugations could be established on the basis of the MS/MS spectra, because the moieties of the latter and/or the free phenolic acid were observed (Tables 2 and S1†). For example, fragment ions with m/z values of 191.0561 ([C₇H₁₂O₆ − H]⁻) (quinic acid) and 179.0350 ([C₉H₈O₄ − H]⁻) (caffeic acid) were released from caffeoylquinic acid isomers.

Finally, a phenylpropanoid acid related to furanocoumarin psoralen was assigned as psoralic acid glucoside, according to the recent findings in F. carica leaves (Takahashi et al.³²), which also suggested that this compound could be a precursor of psoralen. Their fragmentation pattern agreed with the Takahashi's study, as we also observed the loss of glucose (m/z 203.0347) and the loss of CO₂ (m/z 159.0453) as the main fragments in MS/MS. This compound was detected in leaves (Fig. S4b and e†). Furthermore, a compound related to this was detected in negative and positive ion modes (e.g. see compound with m/z value at 205.0502 and RT 17.21 min in Tables 3 and S2†), which could be the aglycone or a dihydro form of hydroxypsoralen. The MS data, the UV spectra and published literature were not enough to elucidate the structure of this compound.

Flavonoids. As commented above, UV-Vis spectra can be used as an indicative tool for the primary characterization of flavonoids, whereas MS and MS/MS information can provide additional and significant information.³⁷ In this way, the flavonols, flavones, flavanones, flavanonols and isoflavones were characterized in the negative ionization mode (Tables 2 and S1†) and two anthocyanins in positive ionization mode (Tables 3 and S2†).

The flavonols quercetin, quercetin-3-O-glucoside and quercetin-3-O-rutinoside were confirmed by standards and previously reported in fresh and dried figs.^{8–10,32–34,38} A malonyl derivative of quercetin was found at RT 18.68 min, the fragmentation pattern (Fig. 3b) being in agreement with Takahashi et al.³² and so characterized as quercetin 3-O-(6′′-malonyl)glucoside. In the case of quercetin di-deoxyhexoside hexoside, it is tentatively described here in F. carica for the first time, and has been previously reported in other plant families (e.g. Table S1†). In general, new quercetin derivatives in F. carica probably contain at least a sugar at the position 3 of the C-ring that produces a shift of λ_max from band I, which comes from the B-ring cinnamoyl structure, of quercetin to a lower wavelength (<20 nm).³⁶

Flavones were among the most qualitatively abundant fig flavonoids and presented slight distribution differences between leaves and fruits. In the case of luteolin-7-O-glucoside, luteolin and apigenin, their identification was resolved by means of comparison of the RT, UV absorption and MS/MS spectra with commercial standards. When standards were unavailable, MS/MS helps in the assignment, together with the previous literature. For example, consecutive neutral losses of 18.0106 u (H₂O), 60.0211 u (C₂H₄O₂), 90.0317 u (C₃H₆O₃), 120.0423 u (C₄H₈O₄), 180.0634 u (C₆H₁₂O₆) and/or 210.0740 u (C₇H₁₄O₇) are considered to be characteristic of the fragmentation pattern of C-glycosylated compounds. The MS/MS spectra for these compounds are in good agreement with previous studies.³⁹ In contrast to C-glycosides, the MS/MS spectra of the O-glycosidic forms of apigenin and luteolin showed more abundant fragment ions corresponding to the aglycones after the neutral loss of 162.0526 u (hexose) and 308.1122 u (hexose–deoxyhexose), respectively (Tables 2 and S1†).

The third group of flavonoids identified was flavanones (Tables 2 and S1†). Among them, eryodictiol and naringenin have been previously reported in other Ficus species.^40,41 It should be mentioned that the glycosylated flavanones were reported here for the first time in the Moraceae family. For example, eriodictyol di-hexoside (m/z 611.1624, RT 16.09 min) was characterized according to its fragmentation pattern, which agrees with the findings of Iswaldi et al.⁴² for eriodictyol 5,3′-di-O-glucoside in Aspalathus linearis (Fabaceae). Moreover, the UV-Vis spectra of these compounds showed a main maximum close to 280 nm related to a strong UV band II absorption from the A ring benzoyl structure.^36,37 Two isomers of eriodictyol hexoside, with m/z at 449.1099 and 449.1086 and RT 17.95 and 19.87, respectively, were reported in the Cucurbitaceae family.⁴³ It was not possible to distinguish between both isomers, since no commercial standards were available for these compounds. Interestingly, the last three compounds were found only in fruits of cultivar ‘Temri’, being putative characteristic biomarkers of its consumption.

Although isoflavonoids are widely distributed in the Moraceae family,^44,45 there is no mention in the literature about this class in F. carica. Our methodology allows ten isoflavones to be tentatively characterized (Tables 2 and S1†), including several prenylated forms. Genistein and methylated derivatives of genistein and prenylgenistein have been previously described in other Ficus species.^45–47 Only genistein (RT 24.46 min, m/z 269.0459) could be confirmed with standards and was found in the leaves of the cultivar ‘Temri’. The UV data clearly show a main maximum close to 260 nm, which is in accordance with the findings of Shen et al.⁴⁸ Overall, among other fragments found in the MS/MS spectra of the genistein derivatives, aglycone at m/z value of 269.1190 (even electron) or 268.0374 (odd electron) were detected, and also the characteristic fragment ions at m/z 151.0031 (^1,3A⁻), 133.0658 (^0,3B⁻) (even electron) or 132.0214 (odd electron) and 117.0346 (^1,3B⁻) released from the breakage of genistein backbone. In the case of the malonylhexoside derivative of hydroxygenistein methyl ether, the loss of CO₂ from the malonyl group and the subsequent loss of 204.0634 u (C₈H₁₂O₆, acetyl hexosyl rest) were also observed in MS/MS. A dimethyl ether isoflavone was only detected in positive ionization mode (Tables 3 and S2†), exhibiting the UV maximums related to the isoflavone core, and the MS/MS spectrum was in agreement with 7-hydroxy-6,4′-dimethoxyisoflavone (afromosin).⁴⁹ Since there was no information about this compound in the Moraceae literature and the MS/MS data were not enough to distinguish the exact position of the free hydroxyl or methoxy groups, the compound was denominated as hydroxy-dimethoxyisoflavone.

Interestingly, the prenylated isoflavones presented a remarkably higher signal in leaves than in fruits (Fig. S4c and f†). Not surprisingly, they eluted at higher RT due to the presence of this lipophilic prenyl side chain, with an RT from 26.49 to 29.10 min. The UV data and main MS/MS fragments agreed with prenylated forms of genistein (6-, 3′-, and 8-prenylgenistein) present in the Lupinus species.⁴⁸ In this regard, 6- and 8-prenylgenistein was reported in stem barks and fruits of other Ficus species (e.g. Ficus tikoua).^47,50 Furthermore, related prenylated forms linked to hydroxygenistein were also tentatively characterized, and the UV data agreed with the findings of Shen et al.⁴⁸ In general, these prenylated compounds were characterized by the neutral loss of C₄H₇ (55.0548 u) and C₅H₉ (69.0704 u) from the prenyl moiety, amongst others. In this regard, prenylated flavonoids possess unique bioactivities relative to their unmodified parent compounds, particularly potent antifungal activity.^47,48

Finally, using the analytical method 1, the flavanol (+)-catechin, which was confirmed with the standard, and the flavanonol dihydroquercetin were also detected, in accordance with previous studies on the Ficus species.^{10,13,33,40,41}

Using analytical method 2, two anthocyanins could be detected in dried fig fruits at 520 nm (see Fig. 2e and f). According to the MS and MS/MS data obtained in the positive ionization mode and the published studies on fig fruits,⁴ they were assigned to cyanidin 3,5-diglucoside (m/z 611.1613, C₂₇H₃₁O₁₆) and cyanidin 3-rutinoside (m/z 595.1667, C₂₇H₃₁O₁₅) (Tables 3 and S2†).

Hydroxycoumarins. The presence of 7-hydroxycoumarin (umbelliferone) was confirmed with the standard. This compound was previously reported in F. carica.⁴⁵ and it is suggested that it is the precursor of furanocoumarins.⁵¹ The rest of the hydroxycoumarins were putatively characterized on the basis of the MS/MS spectra, UV data and literature.^40,45 All of these compounds were found in the leaves and some of them in fruits, too. Their fragmentation pattern was characterized by the loss of CO (27.9949 u), CO₂ (43.9898 u) and, subsequently, C₂H₄ (28.0313 u) from the aglycone, in agreement with our previous findings for lettuce (Lactuca sativa) leaves.²⁹ A prenylated form of 7-hydroxycoumarin was also tentatively characterized at RT 27.95 min and m/z 229.0872 (C₁₄H₁₄O₃), which showed the characteristic loss of C₄H₇ (55.0548 u) from the prenyl moiety in MS/MS at m/z 174.0319, as observed above. Several fragments were also in agreement with the findings of Yang et al.⁵²

Furanocoumarins. There are two type of furanocoumarins in nature, linear and angular ones.⁵³ F. carica contains mainly the first class, with psoralen and 8-methoxypsoralen (bergapten) being the major representatives.^12,24,32 In this regard, a total of 14 furanocoumarins were tentatively characterized in Tunisian figs in positive ionization mode, including the aforementioned compounds (Tables 3 and S2†). The major part of the characterized furanocoumarins are described here for the first time in F. carica and several of them in the Moraceae family (e.g. isopentenoxypsoralen, at RT 28.25 min and m/z 271.098). Alternatively, others have been previously reported in other Ficus species, such as marmesin isomers, 4′,5′-dihydropsoralen and oxypeucedanin hydrate (Table S2†). In agreement with Yang et al.'s study on Radix glehniae,⁵² we detected a characteristic series of fragment ions for furanocoumarins that were mainly generated by consecutive losses of CO (e.g. Fig. 3c). As stated above, in a similar way the loss of C₃H₆ (42.0470 u), C₄H₈ (56.0626 u) and C₅H₈ (68.0626 u) from the prenyl moiety was also observed in the MS/MS spectra of prenylated furanocoumarins, isopentenoxypsoralen and prenyl methoxypsoralen.^52,54 In addition, the UV data of furanocoumarins also agreed with that of Frérot et al.^55,56

Linear furocoumarins have received great attention since these compounds, used medicinally and in a controlled way, may represent a novel class of natural drugs that are potentially useful for the photodynamic treatment of several skin diseases.¹² In this regard, F. carica leaves could be of interest, thanks to their qualitatively rich profiles (Tables 3 and S2;† Fig. 2).

3. Conclusions

Despite the popularity of the consumption of dried fig fruits, there is little information about its antioxidant activity. Moreover, in the case of its qualitative phenolic composition, previous studies have only focused on target phenolic compounds. In our study, a total of 109 phenolic compounds were characterized in F. carica samples. Most of them were reported for the first time in F. carica species. In addition, fig leaves presented a richer phenolic qualitative profile with also a higher total phenol content in comparison to fruits. In this regard, phenolic acids conjugated with sugars and organic acids as well as furanocoumarins were mainly present in leaves, but not in fruits. Concurrently, F. carica leaves exhibited stronger antioxidant capacity by both electron or hydrogen transfer mechanisms. Therefore, our results are of interest to further studies on the phytochemical composition of F. carica and the Moraceae family; additionally, the antioxidant values may be used as references for future researches to make comparisons with other fig cultivars. Overall, these results contribute to explaining the past and current usage of F. carica in folk medicine, as leaves extracts can be regarded as a promising source of antioxidant phenolic compounds for further uses in pharmacology and cosmetology.

4. Experimental

Chemical and reagents

Ethanol, acetonitrile, formic acid and glacial acetic acid were purchased from Fisher Chemicals (ThermoFisher, Waltham, MA, USA). Solvents used for extraction and analysis were of analytical and HPLC-MS grades, respectively. Ultrapure water was obtained by a Milli-Q system (Millipore, Bedford, MA, USA).

The reagents used to measure the TPC and the antioxidant capacity were Folin & Ciocalteu's, sodium carbonate (Na₂CO₃), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) diammonium salt, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), fluorescein, potassium persulphate (K₂S₂O₈) and ferric sulphate (FeSO₄). They were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dehydrated sodium phosphate, trihydrated sodium acetate, sodium acetate, ferric chloride (FeCl₃·6H₂O) and hydrochloric acid (HCl) were obtained from Panreac (Barcelona, Spain). Phenolic standards available in our laboratory were bought from Sigma-Aldrich: chlorogenic acid, caffeic acid, vanillic acid, trans-ferulic acid, rutin, quercetin-3-O-glycoside, quercetin, luteolin-7-O-glucoside, apigenin, luteolin, (+)-catechin, genistein, 7-hydroxycoumarin and gallic acid. The degree of purity of the standards was around 95% (w/w).

Fig samples

Leaves and fruits from the F. carica cultivars ‘Tounsi’ and ‘Temri’ were collected in Sfax region (southeast Tunisia) in August 2013. The samples (about 0.5 kg) was randomly harvested and immediately transferred to the laboratory where they were dried at room temperature in the dark, and then they were finely ground prior to extraction.

Sample preparation

Dried fig leaves and fruits (3 g) were put in amber glass bottle homogenized in 100 mL of 70 : 30 (v/v) ethanol–water placed on a stirring hot plate for 24 hours at 37 °C and 150 rpm. Each mixture was centrifuged at 8000 rpm for 15 min and the supernatant was collected. Afterwards, the solvent was evaporated to dryness using a rotary evaporator under vacuum at 40 °C, and the residue was redissolved in 3 mL of 70 : 30 (v/v) ethanol–water. Finally, the supernatants were filtered with a syringe filter (regenerated cellulose, 0.45 μm pore size) and stored at −20 °C until analysis. The extraction was repeated in duplicate.

Total phenol content (TPC)

The TPC of the extracts was determined in triplicate by the colorimetric assay using the Folin–Ciocalteu reagent⁵⁷ modified according to Romero-de Soto et al.⁵⁸ with 96-well polystyrene microplates (ThermoFisher) and a Synergy Mx Monochromator-Based Multi-Mode Microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The absorbance of the solution at a wavelength of 760 nm was measured after incubation for 2 hours in the dark and compared with a calibration curve of serially diluted gallic acid, which was elaborated in the same manner. The results were expressed as the equivalents of gallic acid.

Antioxidant capacity assays

TEAC assay. This antioxidant method measures the reduction of the radical cation of ABTS by antioxidants, and is based on Miller et al.'s approach⁵⁹ The method was modified as described Laporta et al.⁶⁰ Briefly, the ABTS radical cation (ABTS⁺˙) was produced by reacting the ABTS stock solution with 2.45 mM of potassium persulfate and keeping the mixture in darkness at room temperature for 12 to 24 h before use. For the antioxidant assay with vegetable extracts, the ABTS⁺˙ solution was diluted with water until an absorbance value of 0.70 (±0.02) at 734 nm was reached. Afterwards, 300 μL of the ABTS⁺˙ solution and 30 μL of the extract were mixed for 45 s and measured immediately after 5 min (absorbance did not change significantly up to 10 min). The readings were performed at 734 nm and 25 °C. The result of each sample was then compared with a standard curve made from the corresponding readings of trolox (0.625–30 μM in the microplate wells). Caffeic acid was used as a positive control. The results are expressed in mmol of trolox equivalents/100 g of sample.

FRAP assay. The FRAP assay was conducted following the method described by Benzie and Strain.⁶¹ The stock solutions included 300 mM acetate buffer (1.23 g C₂H₃NaO₂ + 0.8 mL C₂H₄O₂ + 49.2 mL of water, pH = 3.6 adjusted with HCl), 10 mM of TPTZ solution in 40 mM HCl and 20 mM FeCl₃ in water. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL of TPTZ and 2.5 mL of FeCl₃ solutions. Briefly, 40 μL of the extracts was mixed with 250 μL of freshly prepared FRAP reagent on a 96-well plate. Samples were incubated for 10 min at 37 °C; then, absorbance was recorded at 593 nm for 4 min on the microplate reader. The final absorbance of each sample was compared with those from the standard curve made from FeSO₄·7H₂O (12.5–200 μM, final concentration in wells). Caffeic acid was used as a positive control. The results are expressed in mmol of FeSO₄ equivalents/100 g of sample.

ORAC assay. The method used was based on that of Ou et al.²³ modified by Laporta et al.⁶⁰ The reaction was carried out in 75 mM phosphate buffer (pH = 7.4), and the final reaction mixture was 200 μL fluorescein and AAPH, which was used at 40 nM and 19 mM, respectively. A freshly prepared AAPH solution was used for each experiment. The temperature of the incubator was set at 37 °C and the fluorescence was recorded 15 min after the addition of AAPH. The microplate was immediately placed in the reader and the fluorescence recorded every minute for 180 min. The microplate was automatically shaken prior to each reading. All the fluorescent measurements are expressed relative to the initial reading (AUC for each well). A blank (phosphate buffer instead of the antioxidant solution), several dilutions of trolox (0.625–15 μM, final concentration in wells) and samples (at least four valid dilution points) were measured. All the reaction mixtures were prepared in triplicate, and at least two independent assays were performed for each sample. The net area under curve (AUC) corresponding to the trolox or samples was calculated by subtracting the AUC corresponding to the blank. Caffeic acid was used as a positive control. ORAC values were expressed as trolox equivalents by using the standard curve calculated for each assay. The final results were in mmol of trolox equivalents/100 g of samples.

Characterization of phenolic compounds by UHPLC-DAD-QTOF-MS

Analyses were made with an Agilent 1200 series rapid resolution (Palo Alto, CA, USA) equipped with a binary pump, an autosampler and a DAD. The system was coupled to a 6540 Agilent Ultra-High-Definition (UHD) Accurate-Mass Q-TOF LC/MS, equipped with an Agilent Dual Jet Stream electrospray ionization (Dual AJS ESI) interface.

To characterize phenolic compounds, the mobile phases consisted of water plus 0.5% acetic acid (phase A) and acetonitrile (phase B), according to the approach of Abu-Reidah et al.⁶² (method 1). The following multistep linear gradient was applied: 0 min, 0% B; 10 min, 20% B; 15 min, 30% B; 20 min, 50% B; 25 min, 75% B; 30 min, 100% B; 31 min, 100% B; 34 min, 0% B; 40 min, 0% B. The flow rate was set at 0.50 mL min⁻¹ throughout the gradient. To characterize anthocyanins and furanocoumarins, the mobile phases were water plus 5% formic acid (phase A) and acetonitrile (phase B), according to the approach of Gómez-Caravaca et al.⁶³ (method 2). Separation was carried out with a Zorbax Eclipse Plus C18 column (150 × 4.6 mm, 1.8 μm of particle size) at room temperature. The UV spectra were recorded from 190 to 600 nm. The injection volume was 5 μL. Samples were diluted by 1/4 with an ethanol–water mix of 70 : 30 (v/v).

The operating conditions in negative ionization mode were as follows: gas temperature, 325 °C; drying gas, nitrogen at 10 L min⁻¹; nebulizer pressure, 20 psig; sheath gas temperature, 400 °C; sheath gas flow, nitrogen at 12 L min⁻¹; capillary voltage, 4000 V; skimmer, 45 V; octopole radiofrequency voltage, 750 V; focusing voltage, 500 V, with the corresponding polarity automatically set. Spectra were acquired over a mass range from m/z 100 to 1700 and for MS/MS experiments from m/z 70 to 1700. In the case of anthocyanins and furanocoumarins, MS analyses were performed in positive ionization mode based on several studies,^8,64 with the parameters set as commented above, but with the corresponding polarity. Reference mass correction of each sample was performed with a continuous infusion of Agilent TOF mixture containing trifluoroacetic acid (TFA) ammonium salt (m/z 112.9856 corresponding to TFA) and hexakis (1H,1H,3H-tetrafluoropropoxy) phosphazine (m/z 1033.9881 corresponding to the TFA adduct) for negative ionization mode, while using purine (m/z 121.0508) and hexakis (1H,1H,3H-tetrafluoropropoxy) phosphazine (m/z 922.0098) for positive ionization mode. The detection window was set to 100 ppm.

Data analysis was performed on a Mass Hunter Qualitative Analysis B.06.00 (Agilent technologies). For characterization, the isotope model selected was common organic molecules with a peak spacing tolerance of m/z 0.0025 and 7 ppm. Then, the characterization of the compounds was done taking into account the generation of candidate molecular formula with a mass error limit of 5 ppm and also considering RT, experimental and theoretical masses, and MS/MS spectra. The MS score related to the contribution to mass accuracy, isotope abundance and isotope spacing for the generated molecular formula was set at ≥80. Confirmation was made through a comparison with standards, whenever these were available in-house. Consequently, the literature on Moraceae and the following chemical structure databases were consulted: PubChem (http://pubchem.ncbi.nlm.nih.gov), ChemSpider (http://www.chemspider.com), SciFinder Scholar (https://scifinder.cas.org), Reaxys (http://www.reaxys.com), Phenol-Explorer (http://www.phenol-explorer.eu) and KNApSAcK Core System (http://kanaya.naist.jp/knapsackjsp/top.html).

Statistical analysis

Microsoft Excel 2007 (Redmond, WA, USA) was employed for statistical analysis. The correlation between TPC and antioxidant activity was performed using SPSS Statistics 22 (Armonk, NY, USA).

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

The authors are grateful to the Andalusian Regional Government Council of Innovation and Science for the Excellence Project P11-CTS-7625, and M. del M. Contreras to the Spanish Ministry of Economy and Competitiveness (MINECO) for the Juan de la Cierva contract. The authors would like also to thank the Ministry of Higher Education, Scientific Research and Information and Communication Technologies, Tunisia, for its support of this research work.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra16746e

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