UPLC-ESI-MS/MS Profiling of Secondary Metabolites from Methanol Extracts of In Vivo and In Vitro Tissues of Daucus capillifolius Gilli (A Comparative Study)

Daucus capillifolius Gilli is a rare annual wild herb grown in Libya. It belongs to the Apiaceae family, which is one of the largest flowering plant families. Plants of this family are outstanding sources of various secondary metabolites with various biological activities. A UPLC-ESI-MS/MS analysis of different extracts of in vivo and in vitro tissues of Daucus capillifolius together with the fruit extract of the cultivated plant in both ionization modes was carried out for the first time in the current study. Our results reveal the tentative identification of eighty-seven compounds in the tested extracts, including thirty-two phenolic acids and their derivatives; thirty-seven flavonoid glycosides and aglycones of apigenin, luteolin, diosmetin, myricetin and quercetin, containing glucose, rhamnose, pentose and/or glucuronic acid molecules; seven anthocyanins; six tannins; three acetylenic compounds; and three nitrogenous compounds. The tentative identification of the above compounds was based on the comparison of their retention times and ESI-MS/MS fragmentation patterns with those previously reported in the literature. For this Apiaceae plant, our results confirm the presence of a wide array of secondary metabolites with reported biological activities. This study is among the first ones to shed light on the phytoconstituents of this rare plant.


Introduction
Recently, great attention has been paid to the chemical and biological investigation of native medicinal plants, which constitute a gold mine of phytoconstituents with exceptional biological activities and represent an essential source of novel bioactive drugs.The extraction and isolation of targeted, safe and potent antimicrobial natural drugs is becoming of vital importance to control the microbial resistance to reported chemically synthesized drugs and food deterioration resulting from fungal or bacterial infections.North Africa is still a very rich source of untapped medicinal plants that are undergoing extensive screening for novel natural drug discovery [1].
The Apiaceae family (previously Umbelliferae), commonly referred to as carrot or parsley family, comprises approximately 3780 species and 434 genera distributed in temperate zones.It includes various herbs and vegetables of variable medicinal and economical importance [2,3].Notably, plants of this family are a rich source of specialized secondary metabolites (furanocoumarins, sesquiterpene lactones and sesquiterpene coumarins) [4] oil of many species of this genus showed a wide range of important pharmacological activities, such as antioxidant, cytotoxicity, insecticidal, antimicrobial and anti-inflammatory activities [19].Daucus capillifolius Gilli from Libya is a rare annual wild herb with an erect and smooth stem which reaches about 50 cm in length [18] Based on the available literature, nothing has been reported on the phytochemical constituents of this plant, except our previous work, which investigated the micropropagation and callus culture of this endangered plant in addition to the GC-MS analysis of its essential-oil constituents [19].The current study aimed to conduct a comparative phytochemical investigation of the methanolic extracts of in vivo (cultivated fruit) and in vitro tissues (calli grown on different media with various hormonal combinations) of this rare plant by using UPLC-ESI-MS/MS analysis in both ionization modes.

Results and Discussion
The methanolic extracts of in vivo (the fruit extract of the cultivated plant) and in vitro tissues of D. Capillifolius Gilli (Figure 1) were analyzed by using UPLC-ESI-MS/MS.The analysis revealed the tentative identification of 87 different phenolic and non-phenolic compounds (Figure 2).Greenish white, friable Bright yellow, friable Yellowish white, compact Greenish white, friable Bright yellow, friable Yellowish white, compact

Identification of Phenolic Compounds of Methanol Extracts of In Vivo and In Vitro Tissues of D. capillifoliusby UPLC-ESI-MS/MS
Eighty-one compounds were tentatively identified for the first time in the methanol extracts of in vivo and in vitro tissues of Daucus capillifolius fruits.The results are illustrated in Tables 1-3.As shown in Table 1, thirty-two phenolic acids and their derivatives were identified in methanol extracts of in vivo and in vitro tissues of D. capillifolius Gilli as follows.
Compound 1 was suggested to be malic acid due to the presence of the molecular ion peak at m/z of 133 [M + H] + [20].
Compounds 2 and 25 showed the same pseudo-molecular ion peak at m/z of 279 [M-H] − and were suggested to be benzoic acid and coumaric acid derivatives, respectively.This suggestion was based on the presence of MS 2 base peak fragment ions at m/z of 121.0 and 162.6, respectively [21].
Compound 3 exhibited a deprotonated molecular ion peak at m/z of 329.Based on the presence of one main fragment in the MS 2 spectrum at m/z of 131.1 [pentose-H], which resulted from a neutral loss of the syringic acid molecule [M-H−198] − , it was identified as syringic acid pentoside, which has not been reported in Daucus species [22].
Compound 4 had a precursor ion at m/z of 327 [2M-H] − and a protonated molecular ion at m/z of 165.In negative ESI mode, the MS 2 showed a base peak fragment ion at m/z of 163.7 for [M-H] − , so it was identified as coumaric acid [23].Coumaric acid was previously isolated from the genus Daucus [24].
Compounds 5 and 7 had deprotonated molecular ions at m/z of 137 and an MS 2 base fragment ion at m/z of 92.9.Based on the mass fragmentation and the low retention time, as well as previously published reports, these compounds were tentatively identified as hydroxybenzoic acid, which was previously detected in Daucus [25,26].
Compounds 6 and 9 were identified as gallic acid derivatives according to the LC-MS 1 and MS 2 data reported in Table 1.Similarly, compounds 28 and 31 were tentatively identified as benzoic acid and quinic acid derivatives, respectively [21].
Compound 8 had a molecular ion fragment at m/z of 179 [M-H] − and an MS 2 base fragment ion at m/z of 135.2 [M-H-COOH] (Figure 3).Based on mass fragmentation, as well as previously reported data [25], compound 8 was identified as caffeic acid.It was previously isolated from the genus Daucus [24].
Compound 10 showed a deprotonated molecular ion peak at m/z of 279 [M-H] − .It was suggested to be a vanillic acid derivative based on the presence of the MS 2 base peak fragment ion at m/z of 167.4 (Table 1) [22].
Compounds 11, 17 and 29 had deprotonated and protonated molecular ions at m/z of 353 and 355, respectively (Figure 3).They had MS 2 fragment ions at m/z of 191.3 with a relative abundance of 100%.This fragmentation pattern was found to be consistent with previous findings on chlorogenic acid reported by [25].Notably, chlorogenic acid has been detected in Daucus species, as mentioned by [26].
Compound 12 with a molecular ion compound at m/z of 329 [M − -H] − was tentatively identified as a cinnamic acid derivative.The HPLC-ESI-MS spectra of this compound showed an MS 2 base peak fragment ion at m/z of 146.6 for the cinnamic acid moiety after the loss of 182 amu [22].Compounds 13 and 18 were readily detected at m/z of 371 and 367 [M-H] − , respectively.Based on the MS 2 data in Table 1, they were tentatively identified as caffeic acid derivatives, as they produced the MS 2 base peak fragment ions at m/z of 178.9 [M-H−192] − and 135.0 [M-H−232] − (possibly malonyl rhamnoside), respectively.Several caffeic acid derivatives have been previously reported in the genus Daucus [41].
Compounds 14 had ESI-MS with a deprotonated molecular ion at m/z of 271, which fragmented in MS 2 to produce a base peak fragment ion at m/z of 191.0 which was identified as quinic acid derivative [22].
Compound 15 (Rt7.69min), a ferulic acid derivative, was determined with MS 1 [M-H] − at m/z of 273 and an MS 2 base fragment ion at m/z of 192.7.Several ferulic acid derivatives were detected in the genus Daucus [24].
Compound 16 with molecular ion peak at m/z of 185 [M-H] − was tentatively identified as hydroxy gallic acid [27].
Compound 19 showed a molecular ion peak at m/z of 303 [M+H] + and was tentatively identified as ellagic acid.The MS 2 showed typical fragmentation of ellagic acid at m/z Based on the MS 2 data in Table 1, they were tentatively identified as caffeic acid derivatives, as they produced the MS 2 base peak fragment ions at m/z of 178.9 [M-H−192] − and 135.0 [M-H−232] − (possibly malonyl rhamnoside), respectively.Several caffeic acid derivatives have been previously reported in the genus Daucus [41].
Compounds 14 had ESI-MS with a deprotonated molecular ion at m/z of 271, which fragmented in MS 2 to produce a base peak fragment ion at m/z of 191.0 which was identified as quinic acid derivative [22].
Compound 15 (R t 7.69 min), a ferulic acid derivative, was determined with MS 1 [M-H] − at m/z of 273 and an MS 2 base fragment ion at m/z of 192.7.Several ferulic acid derivatives were detected in the genus Daucus [24].
Compound 16 with molecular ion peak at m/z of 185 [M-H] − was tentatively identified as hydroxy gallic acid [27].
Compound 19 showed a molecular ion peak at m/z of 303 [M+H] + and was tentatively identified as ellagic acid.The MS 2 showed typical fragmentation of ellagic acid at m/z of 257.0, 229.0, 201.2 and 164.9 and a base peak fragment at 153.1 [20,28].Ellagic acid has gained a lot of interest due to its anti-inflammatory, antitumor antibacterial and liver protection effects [60].
Compound 20 had a protonated molecular ion fragment at m/z of 193 and gave an MS 2 fragment at m/z of 119.0.This compound was identified as quinic acid [28].Notably, it was previously reported in the genus Daucus [24].
Compounds 21 and 22 were identified as sinapic acid isomers 1 and 2, based on the presence of the molecular ion peak at m/z of 225 [30].
Compounds 23 and 24 were identified as isomers 1 and 2 of methyl gallic acid based on the molecular ion peak at m/z of 185 as reported by [20].Meanwhile, compound 26 (R t of 13.95) was identified as coumaric acid, supported by the presence of a molecular ion peak at m/z of 163 [M-H] − as reported by [21].
Compound 27 was tentatively identified as hydroxy ferulic acid according to [31] and was detected from the ion fragment at m/z of 209 [M-H] − .Further, compound 30 was identified as benzoic acid methyl ester after the detection of the ion fragment at m/z of 137 [M-H] − according to [21].
Finally, compound 32 was identified as coumaric acid glucuronide based on the presence of a molecular ion fragment at m/z of 339 [M-H] − and the fragment ion at 163 comprising coumaric acid after the loss of the glucuronic acid moiety [M-H−176] − [21].

Identification of Flavonoids Flavonoids Aglycones
Ten flavonoid aglycones were identified in methanol extracts of in vivo and in vitro tissues, as well as in the fruit extract of the cultivated plant Daucus capillifolius, as described in the following.
Compound 33 (R t of 13.56 min) was identified as diosmetin from the ESI-MS spectrum, which showed deprotonated and protonated molecular ions at m/z of 299 and 301, respectively.The ESI-MS/MS fragmentation pattern showed fragment ions at m/z of 284.9 (100%) [M + -H-CH 3 ] and 255.6 [M + -CH 3 -CO], in addition to the fragment ions mentioned in Table 3, which are characteristic of fragmentation for diosmetin [32].Flavones such as diosmetin and apigenin were reported in Petroselinum crispum, Apiaceae.It was used in menstrual disorders treatment due its phytoestrogen content [14] Compound 34 produced a mass spectrum [M + -H] at m/z of 281.It was identified as 5,4-dimethoxyflavone through MS 2 fragment ions at m/z of 149.3 (100%) and 132.0, which are characteristic for ring A and ring B, respectively, each with one methoxy group [33].
Compound 35 (R t of 12.07 min) was identified as luteolin based on the [M + -H] at m/z of 285 and the MS 2 fragmentation pattern presented in Table 2, as reported by [34].It is worth noting that luteolin has been previously isolated from Daucus species [61].
Compounds 36 and 37 were identified as apigenin and galangin, respectively, from the fragmentation pattern in both positive and negative modes (Figure 4) of ESI-MS/MS, as shown in Table 2 [25,34,36].
Compound 40 showed an [M + -H] − ion at m/z of 329, with the production of daughter ions at m/z of 314 and m/z of 299, indicating the loss of two methyl groups from the parent 329 ion.Therefore, it was tentatively identified as 5-hydroxy-3 ′ ,4 ′ ,7-trimethoxyflavanone [33,37].
Compound 40 showed an [M + -H] − ion at m/z of 329, with the production of daugh-

Identification of Flavonoid Glycosides Identification of O-glycosides
Twenty-two flavone or flavonol-O-glycosides were identified in methanol extracts of in vivo and in vitro tissues of D. capillifolius, as described below.
Compound 44 exhibited a molecular ion compound at m/z of 479 [M-H] − .The fragment ions in MS 2 at m/z of 317.0 [M-H−162] − showed the loss of a hexose moiety.Additionally, other fragment ions at m/z of 161.1 and 159.0 were observed.Based on these findings, this compound was identified as myricetin-3-O-glucoside, as depicted in Figure 5 [39].
Compound 45 presented a molecular ion at m/z of 491 [M-H] − .The MS data show a base fragment signal at m/z of 317.1 [M-H−204] − , indicating the loss of acetyl hexoside.From these results, compound 45 was tentatively identified as myricetin-3-O-acetylglucoside [40].
Compounds 47, 48 and 50 exhibited pseudo-molecular ions at m/z of 625, 463 and 447, respectively.Through analysis of the MS/MS spectrum, these compounds displayed a characteristic product ion at m/z of 301, corresponding to quercetin.This product ion resulted from the loss of diglucoside [M + -H−324] for 47, a glucosyl [M + -H−162] for 48 and a rhamnosyl [M + -H−146] moiety for 50.As a result, these compounds were identified as quercetin diglucoside, quercetin-O-glucoside (Figure 5) and quercetin-O-rhamnoside [41].
Compound 57 was identified as an isomer of compound 54 and identified as an apigenin 7-O-glucoside isomer.Similarly, compound 58 (R t of 10.40 min) showed molecular ions at m/z of 447 and another fragment ion at m/z of 299, which corresponds to [M + -H] after the loss of the rhamnosyl moiety.It was identified as an isomer of compound 50 and identified as a quercetin-O-rhamnoside isomer.
Compounds 59 and 63 were proposed as isomers of diosmetin-O-rutinoside.They exhibited molecular ions at m/z of 609 [M+H] + .The product ion in the MS/MS spectrum was at m/z of 300.9 [M + +H−308], showing loss of the rutinose moiety [46].
Compound 60 (R t of 13.27 min) was tentatively identified as diosmetin-7-O-glucoside which was previously isolated from the genus Daucus [61].This identification was based on the ESI-MS spectrum, which presented a molecular ion peak at m/z of 461 [M + -H] − , and the MS 2 data that show a fragment ion at m/z of 299.3 [diosmetin-H], indicating a loss of the glucose moiety [47].
Compounds 61 and 62 had molecular ion fragments at 567 [M + -H] and 489 [M + -H], respectively.Upon analyzing the fragmentation pattern, MS 2 fragment ions at m/z of 285.0 (100%) [M + -H−282] − were observed for 61, indicating a loss of 282 atomic mass units.For 62, the fragmentation pattern displayed 285 [M + -H−204] − , indicating the loss of an acetyl hexoside.Consequently, compound 61 was identified as a luteolin derivative, while compound 62 was identified as luteolin-7-O-acetyl hexoside [41] Compound 64 exhibited a deprotonated molecular ion compound at m/z of 463 and was tentatively identified as myricetin-3-O-rhamnoside, as it gave the MS 2 base compound fragment ion at m/z of 316.6, corresponding to the neutral loss of the rhamnose moiety (146 amu) [20].
Identification of C-glycosides Four flavone C-glycosides (Table 2) were identified in methanol extracts of in vivo and in vitro tissues of D. capillifolius Gilli, as described below.
Compound 65 (R t of 13.27 min) had the molecular ion peak in the ESI-MS spectrum at m/z of 445 [M + -H] − and an MS 2 fragment ion at m/z of 341.0 [M−104] − , which showed the loss of 104 amu characteristic for the 8-C-rhamnoside of flavone [46,48].Therefore, compound 65 was concluded to be diosmetin-8-C-rhamnoside (Figure 3).It is the first report of compound 65 in the genus Daucus.Diosmetin-di-C-rhamnoside was previously isolated from Daucus carota [35].
Compound 66 (R t of 23.27 min) displayed a molecular ion compound at m/z of 433 [M+H] + and an MS 2 fragment ion at m/z of 313.1 [M+H−120] corresponding to the 0.3 X ion.Another fragment ion signal at m/z of 150.6 and 130.7 (100%) was observed, which, along with the previous fragment, is characteristic for apigenin-8-C-glucoside [48].
Compound 68 was suggested to be diosmetin-8-C-glucoside-O-rhamnoside.This was confirmed by the [M+H] + ion at m/z of 609 and by the MS 2 compound at m/z of 489.2, which indicates a loss of 120 amu ( 0.3 X ion), a characteristic feature of a C-glucoside flavonoid.The fragment at m/z of 462.5 [M + +H−146] and at m/z of 341.9 [aglycone+H+4] for mono C-glycoside flavonoid and the absence of a fragment at [M + -H−18] indicated the presence of 8-C-glycoside rather than 6-C glucoside [48].

Identification of Anthocyanins
Six anthocyanin compounds were identified in methanol extract of in vivo and in vitro tissues of D. capillifolius Gilli.They were identified as glycosides or acylated glycosides of cyanidin, pelargonidin and malvidin, as shown in Table 3 and described below.
Compound 69 exhibited a molecular ion peak [M+H] + at m/z of 610, which on MS 2 produced a fragment ion at m/z of 271.3 corresponding to pelargonidin aglycone [M + +H −338] with the loss of glucose and glucuronide moieties [50].From the previous results, compound 69 was identified as pelargonidin-3-O-glucuronosyl-O-glucoside.The position of the glucouronide moiety could not be identified.
Compounds 70 and 71 had a [M] + at m/z of 449 (Figure 3), which on MS 2 produced an ion at m/z of 287 (cyanidin, [M + −162]), with the loss of a glucose moiety.From the previous results, compounds 70 and 71 were identified as isomers of cyanidin-3-glucoside [51].
Compound 74 was also suggested to be a cyanidin derivative.This was confirmed by the [M + ] ion at m/z of 721 and by the MS 2 compound ion at m/z of 287 (100%) [49,52].
Compound 75 (R t of 28.31 min) was suggested to be malvidin-3-O-glucoside malonyl glucoside, which was confirmed by the [M + ] ion at m/z of 741 and by the MS 2 compound at m/z of 331.4 [M + −410] indicated the loss of glucoside malonylglucoside [54].Anthocyanins of different cultivars of black carrot are relatively stable under low-acid conditions and could be used as natural food-coloring agents [24] 2.1.4.Identification of Tannins Six flavanes compounds were identified in methanol extracts of in vivo and in vitro tissues of Daucus capillifolius as shown below.
Compounds 76, 77 and 78 exhibited characteristic features in their mass spectra.Compound 76 (gallocatechin) displayed [M + -H] − at m/z of 305 and MS 2 fragments at m/z of 261.0, 119.0 and 97.0 (100%).Similarly, compound 77 (epigallocatechin) exhibited [M + -H] − at m/z of 305 and [M+H] + at m/z of 307 and shared the same MS 2 fragment ions as compound 76.On the other hand, compound 78, an epigallocatechin derivative, showed [M + -H] − at m/z of 721 and a specific MS 2 fragment ion at m/z of 304.7 [55].
Compound 79 (R t of 11.25 min) was suggested to be catechin-3-O-hexosidepentoside, which was confirmed by the [M+H] + ion at m/z of 585 and by the MS 2 compound at m/z of 294 [M + −291] − which indicates a loss of 291 amu [catechin+H] (Table 3) [55].The fragment at m/z of 294 [162+132] is a characteristic feature of hexose and pentose moieties.
Compounds 80 and 81 displayed distinct molecular ion signals.Compound 80 exhibited an [M-H] − signal at m/z of 625, while compound 81 showed an [M+H] + signal at m/z of 291.The fragmentation pattern for compound 80 revealed MS 2 fragments at m/z of 288.5 (100%), indicating a loss of 336 atomic mass units.On the other hand, compound 81 displayed MS 2 fragments at m/z of 174.9, 147.3, 137.3, 121.0 and 106.9, which are typical fragmentation patterns associated with catechin.As a result, compound 80 was identified as catechin-O-acetyl glucoside pentoside, while compound 81 was identified as catechin.These compounds are not common in daucus species but have been isolated and identified in green tea [50].
In the extract obtained from the calli grown in media A and C, falcarindiol-8-O-methyl ether (84) was present at a higher concentration.Conversely, it was found at a lower concentration in medium B and cultivated fruits.On the other hand, compound 82 showed a higher concentration in the extract obtained from cultivated fruits compared with the calli grown on the three types of media, where it was present in very minimal amounts.Compound 83 was found in minor quantities in all the extracts.

Identification of Nitrogenous Compounds
Compound 85 (R t of 1.35 min) exhibited protonated molecular ions [M + +H] + with an m/z value of 132 with a fragment ion at m/z of 76, which are characteristic of 3-methyl indole [58].
Compounds 86 and 87 (R t of 1.77 and 2.23 min) were tentatively suggested to be 4-(aminoethyl) benzoic acid isomers based on the presence of a protonated molecular ion fragment at m/z of 166 [M+H] + along with another fragment at m/z of 119.9 [M + +H-COOH] and a base peak fragment at m/z of 103 [119.9-NH 2 ] + , in addition to other characteristic fragments at m/z of 93.0, 91.0 and 76.9 [59].Notably, this is the first report about the presence of nitrogenous compounds in this genus.

Plant Materials
The fruits of the cultivated Daucus capillifolius Gilli plant were collected in the fruiting stage in 2016 from the Farm of the Pharmacognosy Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt.The plant was kindly identified by the late Prof Dr. Hussein Abdel Basset, Professor of Taxonomy, Faculty of Science, Zagazig University.A voucher specimen (D.C 2016/12) was deposited at the herbarium in the Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University, Egypt.D. capillifolius fruits were air-dried and ground into coarse particles for use.Additionally, 50 g of 100-day-old calli grown on three different media, medium A [M&S + NAA (1 mg/L) + BAP (0.1 mg/L)], medium B [M&S + TDZ (0.5 mg/L) + 2,4 D (1 mg/L) + BAP (0.1 mg/L)] and medium C [M&S + 2, 4D (2 mg/L) + K (1 mg/L)], was prepared from the seedling explants of D. capillifolius fruits for the analysis.

UPLC-ESI-MS/MS Analysis and Separation Method of D. capillifolius Extracts
UPLC-ESI-MS/MS (ultra-performance liquid chromatography-electrospray tandem mass spectrometry) in both ionization modes was carried out as described by [62] on an aXEVO-TQD triple-quadruple instrument (Waters Corporation, Milford, MA, USA) mass spectrometer (ACQUITY UPLC BEH C18 (1.7 µm, 2.1-50 mm) column; column flow rate of 0.2 mL/min).The solvent system consisted of (A) water and (B) methanol, both containing 0.1% formic acid (Ain Shams University, Cairo, Egypt).The gradient was programmed as follows: 0 min, 10% B; 5 min, 30% B; 15 min, 70% B; 22 min, 90% B; 25 min, 90% B; 26 min, 100% B; 29 min, 100% B; 32 min, 10% B. Finally, the initial conditions were held for 3 min as a re-equilibration step.The flow rate was 0.2 mL/min, and the sample at a concentration of 100 g/ml was prepared in HPLC-grade methanol, degassed, and filtered by using a 0.2 µm membrane disc filter before being subjected to LC-ESI-MS analysis.The injection volume was 10 µL.The parameters for analysis in negative ion mode were as follows: source temperature of 150 • C, cone voltage of 30 eV, capillary voltage of 3 kV, desolvation temperature of 440 • C, cone gas flow of 50 L/h and desolvation gas flow of 900 L/h.Mass spectra were detected in the ESI negative and/or positive ion modes between 50 m/z and 900 m/z.The peaks and spectra were processed by using MassLynx 4.1 software and tentatively identified by comparing their retention time (R t ) and mass spectrum with the reported data.A fragmentation collision energy of 40 eV was used.

Conclusions
Daucus capillifolius Gilli, grown in Libya, is an endangered plant.Its micropropagation and callus culture were successfully established in our previous work with GC-MS analysis of its essential oil.In the current study, we investigated its phytoconstituents for the first time by using UPLC-ESI-MS/MS analysis.Our results revealed that D. capillifolius fruit extract is a rich source of phenolic compounds, including simple phenolic acids, anthocyanidins, tannins, flavonoids, flavonoids -O-and -C-glycoside, and acetylenic compounds.Moreover, the extracts from the in vitro calli grown on media A, B and C with different hormonal combinations showed the accumulation of less phenolic acids, acid derivatives tannins, compared with the cultivated fruit extract.All the tested extracts exhibited the formation of acetylenic compounds, but only the extracts of the in vitro calli.showed the accumulation of nitrogenous compounds.Notably, only luteolin was detected in the extract of the in vitro calli grown on medium C, while calli grown on medium B did not show any flavonoidal aglycons.In summary, this variation in the accumulation of secondary metabolites based on the investigated hormonal combination requires further future studies to achieve the required amounts of secondary metabolites compared with the wild and cultivated D. capillifolius plant.

Figure 1 .Figure 1 .
Figure 1.Morphological characters of callus produced from leaf explants of D. capillifolius Gilli on different culture media (media A, B &C) after 40 days of cultivation.

Figure 1 .Figure 2 .
Figure 1.Morphological characters of callus produced from leaf explants of D. capillifolius Gilli on different culture media (media A, B &C) after 40 days of cultivation.

2. 1 . 1 .
Identification of Phenolic Acids and Acid Derivatives in Methanol Extracts of In Vivo and In Vitro Tissues of D. capillifolius Gilli

Figure 3 .
Figure 3. Fragmentation pattern mass spectra of some identified phenolic compounds from methanolic extracts of cultivated fruits and calli grown on different media of Daucus capillifolius Gilliin in positive (+) and negative (−) ionization modes.

Figure 3 .
Figure 3. Fragmentation pattern mass spectra of some identified phenolic compounds from methanolic extracts of cultivated fruits and calli grown on different media of Daucus capillifolius Gilliin in positive (+) and negative (−) ionization modes.Compounds 13 and 18 were readily detected at m/z of 371 and 367 [M-H] − , respectively.Based on the MS 2 data in Table1, they were tentatively identified as caffeic acid derivatives, as they produced the MS 2 base peak fragment ions at m/z of 178.9 [M-H−192] − and 135.0 [M-H−232] − (possibly malonyl rhamnoside), respectively.Several caffeic acid derivatives have been previously reported in the genus Daucus[41].Compounds 14 had ESI-MS with a deprotonated molecular ion at m/z of 271, which fragmented in MS 2 to produce a base peak fragment ion at m/z of 191.0 which was identified as quinic acid derivative[22].Compound 15 (R t 7.69 min), a ferulic acid derivative, was determined with MS 1 [M-H] − at m/z of 273 and an MS 2 base fragment ion at m/z of 192.7.Several ferulic acid derivatives were detected in the genus Daucus[24].Compound 16 with molecular ion peak at m/z of 185 [M-H] − was tentatively identified as hydroxy gallic acid[27].Compound 19 showed a molecular ion peak at m/z of 303 [M+H] + and was tentatively identified as ellagic acid.The MS 2 showed typical fragmentation of ellagic acid at m/z of 257.0, 229.0, 201.2 and 164.9 and a base peak fragment at 153.1[20,28].Ellagic acid

Figure 4 .
Figure 4. Fragmentation pattern mass spectra of some identified acetylenic compounds from methanolic extracts of cultivated fruits and calli grown on different media of Daucus capillifolius Gilliin in positive (+) and negative (−) ionization modes.

Figure 4 .
Figure 4. Fragmentation pattern mass spectra of some identified acetylenic compounds from methanolic extracts of cultivated fruits and calli grown on different media of Daucus capillifolius Gilliin in positive (+) and negative (−) ionization modes.Compound 42 (R t of 22.92 min) was identified as a dihydroxy flavone from its ESI-MS spectrum with [M + +H] at m/z of 255.MS 2 fragmentation showed the presence of one hydroxyl group in both of rings A and B, where it showed a base compound fragment ion at m/z of 119.0 [M + +H−136] and a fragment at m/z of 135.0 [33].

Figure 5 .
Figure 5.Chemical structures of the tentatively identified compounds from methanolic extracts of cultivated fruits and calli grown on different media of D. capillifolius Gilli.

Figure 5 .
Figure 5.Chemical structures of the tentatively identified compounds from methanolic extracts of cultivated fruits and calli grown on different media of D. capillifolius Gilli.

Table 1 .
Phenolic compounds tentatively identified in in vivo and in vitro tissues of D. capillifolius Gilli by using UPLC-ESI-MS/MS analysis in positive (+) and negative (−) ionization modes.

Table 2 .
Tentatively identified flavonoid compounds from the in vivo and in vitro tissues of D. capillifolius Gilli by using UPLC-ESI-MS/MS analysis in positive (+)
I: methanol extract of cultivated fruit; II: methanol extract of callus grown on medium A; III: methanol extract of callus grown on medium B; IV: methanol extract of callus grown on medium C. (+) present, (−) absent.