Chemical Composition and Biological Activities of the Aqueous Fraction of Parkinsonea aculeata L. Growing in Saudi Arabia

2.4.1 Antioxidant activity (Yen and Duh, 1994)

According to Yen and Duh (1994), the antioxidant activity of the aqueous fraction of the ethanolic extract of aerial parts of P. aculeata was determined at different concentration, 10, 20, 40, 80, 160 and 320 μg/ml methanol. The assay was performed by the DPPH (0.1 mM) solution 2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging assay in triplicates and average values were considered. The percentage of DPPH radical scavenging was calculated according to the formula: % DPPH radical scavenging = (AC-AS)/AC × 100, Where AC is the absorbance of the control and AS is the absorbance of the sample in DPPH solution. SC₅₀ (μg/ml) of both of fraction and the standard (ascorbic acid) were calculated and the results were shown in Figs. 5 and 6.

2.4.2 Antitumor activity (Al-Enazi et al., 2018; Kameyama et al., 2005)

The cell lines HepG-2 (Hepatocellular carcinoma) and MCF-7 (Breast carcinoma) were used for determination of antitumor activity of P. aculeata by the MTT assay and according to the method and statistics reported by Al-Enazi et al. (2018), Kameyama et al. (2005).

3.1.1 Identification of flavone, flavonol-C-glycosides

Sixteen C-flavonoidal glycoside (1–3, 5, 7, 8, 10, and 12, 14–16, 18–20 and 32–33) of apigenin, luteolin, kaempferol, chrysoeriol and diosmetin were identified in the aqueous fraction of P. aculeata L. To our knowledge apigenin, kaempferol, luteolin, chrysoeriol and diosmetin aglycones were reported in P. aculeata L. (Divya et al., 2011; Nabil et al., 1991).

• Characterization of Luteolin/kaempferol -C- glycosides:

Peak 1 showed pseudo molecular ion peak at m/z 447 [M−H]. MS² showed fragment ions at m/z 357.4 [M−H-90], 326.9 (100%) [M−H-120] and 297.0 [M−H-150] corresponding to fragments ^O,4X_O, ^O,3X_O and ^O,2X_O respectively which revealed the presence of hexose nature of C-glycoside flavone. Moreover, from the observed base peak fragment at m/z 326.9 (aglycone + 41) and short retention time at 0.81 it could be concluded that compound 1 was kaempferol -8-C- β-D-glucopyranoside (Brito et al., 2014; Geng et al., 2016). Similarly compounds 8, 32 and 33 were assigned as luteolin-8-C-β-D-glucopyranoside (orientin), while compound 12 was identified as luteolin-6-C-β-D-glucopyranoside (isoorientin). To the best of our knowledge orientin and isoorientin were reported in P. aculeata L. (Divya et al., 2011; Sharma and Vig, 2014). Compound 14 (R_t 8.54 min), The ESI-MS¹ at m/z 593 [M−H] and MS² ion at m/z 473.4 [M−H-120] indicated the presence of mono- C- glucoside. Another fragment at m/z 326.7 [M−H -120–146 (rha)] showed loss of ^O,3X_O fragment and one rhamnose moity. From these results and based on the previous reports (Brito et al., 2014; Geng et al., 2016). Compound 14 was suggested to be luteolin-8-C-β-D-glucopyranoside-7-O-rhamnoside.

• Characterization of apigenin -C-glycosides:

Compounds 7, 18 (R_t 5.72 and 9.22 mins) exhibited the same [M−H] ion at m/z 431, and the MS² spectra showed typical fragment ions of mono-C-glycosides as they exhibited fragment ions at m/z 310.7 and 311.0 (100%) corresponding to [M−H-120] respectively. In addition the MS² spectra showed no loss of water molecule which is characteristic for C-6- glycoside, so these compounds were identified as apigenin-8- C-β-D-glucopyranoside (vitexin) (Llorent-Martínez et al., 2015), to our knowledge vitexin, was previously isolated from P. aculeata L. (Divya et al., 2011; Sharma and Vig, 2014). Compound 19 at R_t 9.37 min and molecular ion peak at 577 [M−H], its MS² data exhibited a fragment ion at m/z 341.0 [(M−H)–90–146]⁻ corresponding to the loss of rhamnose and 90 amu (^O,3X_O). Also the fragment ion at m/z 322.9 which displayed the loss of [90 + 146 + 18] which is characteristic for 6-C-glucoside -O- rhamnoside (Sakalem et al., 2012). From the previous studies and through comparison with the present study, compound 19 was identified as apigenin-6-C-hexoside 2″-rhamnoside (isovitexin-2′-rhamnoside) (Abu-Reidah et al., 2013). Similarly compound 15 was identified as vitexin-2″-rhamnoside from [M−H] at m/z 577 and MS² fragment ions at m/z 356.7 [M−H -120], 311.0 [M−H -120–146] (Ag + 41), 292.9 (100%) (Ferreres et al., 2007; Karar MGE, 2015; Slimestad, 2003). These compounds were previously isolated from P. aculeata (Divya et al., 2011). Based on previous literature (Benayad et al., 2014; Brito et al., 2014; Cao et al., 2014; Qiao et al., 2011), another apigenin derivatives at R_t of 0.92 and 9.13 mints were tentatively identified as apigenin 8‐C‐(‐6′′‐O‐feruloyl‐)glucoside and apigenin-7-O-acetylglucoside-6-C-hexoside-8-C-pentoside for compounds 5 and 16 respectively. This is the first report about the presence of these compounds in P. aculeata L. Compound 10 with retention time of 7.58 min was tentatively identified as apigenin 6,8-di-C-glucosides (vicenin 2) (Brito et al., 2014).

• Characterization of chrysoeriol and diosmetin -C-glycosides:

Peak at R_t of 0.87 min showed two components with the molecular ion peaks at m/z 461 and 593 [M−H] respectively. The MS² spectrum of the first component produced fragment ion at m/z 340.9 [M−H -120]. This fragment is equal to (aglycone + 41) which is characteristic for mono-C- glycoside of aglycone with m/z at 300 amu (diosmetin). These results suggest that, this component (compound 2) was diosmetin -8-C-β-D-glucopyranoside (Brito et al., 2014; Gattuso et al., 2007), while the second component (compound 3) MS² displayed fragment ion at m/z 473.0 [M−H-120], indicating the presence of 8-C- glucoside. The characteristic fragment ion at m/z 412.9 [M−H-120-60] gave the loss of 60 amu for C-pentose, this fragment represents aglycone plus 113 and indicates the presence of di-C-glycoside with diosmetin aglycone (Benayad et al., 2014; Geng et al., 2016). According to the previous studies, compound 3 was suggested to be diosmetin -8-C- hexoside -6-C pentoside (Brito et al., 2014; Cao et al., 2014) and compound 20 was identified as chrysoeriol 8-C-glucoside (scoparin) (Brito et al., 2014).

3.1.2 Identification of flavone, flavonol-O-glycosides

The LC-MS analyses of aqueous fraction obtained from the ethanolic extract of the aerial parts of P. aculeata L. revealed the presence of twenty-two flavonoid-O- glycosides.

• Characterization of luteolin/kaempferol -O-glycosides:

Compounds 17 and 24 (R_t 9.13 and 10.44 min) with the same [M−H]- at m/z 739 were tentatively identified as kaempferol -3-O- (3″, 6″-dicoumaroyl glucoside) and kaempferol-3-O-rutinoside-7-O-rhamnoside respectively. Based on the ESI-mass data in Table 1 and the one step neutral loss of (146 + 146 + 162 amu) for two coumaroyl and one glucose (compound 17) and two losses of 308 amu and 146 amu (compound 24) leaving fragment ion at m/z 285.0 kaempferol aglycone (Lin and Harnly, 2010). Compound 13 (R_t 8.48 min) with [M−H]- at m/z 461 was tentatively identified as kampferol 3-O-glucuronide from ESI- mass data in (Table 1) and the neutral losses of 176 amu for glucouronyl moiety (Lin and Harnly, 2010). By the same manner compounds 25, 30 and 26 (R_t 10.50, 11.55 and 10.58 mins) were identified as luteolin -7-O-rutinoside (25 and 30) (Llorent-Martínez et al., 2015; Omezzine and Haouala, 2017) and kaempferol-3-O-β-D-glucopyranoside (26) (Llorent-Martínez et al., 2015; Omezzine and Haouala, 2017). Kaempferol-3-O-rhamnoside (compound 31) was tentatively identified from The MS profile of peak at retention time of 11.55 min with [M−H] at m/z 431 and MS² base peak fragment ion at m/z 285.0 which gave the loss of 146 Da (rhamnose moiety) (Downey and Rochfort, 2008; Kajdžanoska et al., 2010; Plazonić et al., 2009). In addition compound 40 was identified as luteolin 7, 4′ dimethyl ether-O- β-D-glucopyranoside. It is worth mentioning that luteolin 7, 4′ dimethyl ether-6-C-glucopyranoside was previously isolated from P. aculeata L which confirmed the presence of luteolin 7, 4′ dimethyl ether as aglycone in compound 40 (Nabil et al., 1991).

• Characterization of chrysoeriol and diosmetin -O-glycosides:

Compound 4 with a [M−H]⁻ ion at m/z 751 was tentatively identified as diosmetin-7-O-acetyl hexoside -malonyl hexoside (Table 1). The base peak fragment ion at m/z 298.7 [M−H-204-248] showed the loss of (acetylglucose and malonyl glucoside) (Brito et al., 2014; Kajdžanoska et al., 2010). Diosmetin -7-arabinosyl (1‴- 6″)-β-D-glucopyranoside was proposed for compound 11, R_t 8.18 min, (m/z 593 [M−H]). In the MS/MS spectra, the loss of pentosyl-hexoside (294 Da) gave base peak fragment ions at m/z 299.1 (diosmetin) (Al-Shammari et al., 2015). Compound 27 with R_t 10.74 min, molecular ions at m/z 607 and fragments at m/z 299.0 and 283.8, suggesting that it might be chrysoeriol 7-O-neohesperidoside (Brito et al., 2014; Plazonić et al., 2009). Compound 29 shows a molecular ion at m/z 461 [M−H]⁻ (Table 1). The MS data showed fragment signal at m/z: 299.1 [(M−H-162) which indicate glucose moiety, this result shows that compound 29 could be chrysoeriol-7-O- glucopyranoside (Plazonić et al., 2009). Compound 36 with R_t 16.10 min, molecular ion peak at m/z 739 [M−H] and MS/MS fragments at m/z 461.0 [M−H-132-146], showed the loss of rhamnose and pentose moieties, while the base peak fragment ion at m/z 308. 3 [M−H-431] show the presence of rutinose as sugar moiety and leaving chrysoeriol-pentoside, the MS² showed another fragment at m/z 299.2 corresponding to chrysoeriol aglycone. From these results compound 36 was tentatively identified as Chrysieriol 4′-O-pentoside-7′-O-rutinoside (Brito et al., 2014; Plazonić et al., 2009).

• Characterization of methyl apigenin -O-glycosides

Peaks at 1.43 and 7.13 min (compounds 6 and 9) (Table 1), gave the [M−H]^– ion at m/z 577. The main fragmentation step of this compound in MS/MS spectrum yielded one type of ion with 100% abundant [M−H – aglycone (284)]⁻ ions at m/z 293.3 which showed the loss of 284 amu which corresponding to methyl apigenin as an aglycone (Marczak et al., 2010). The fragment at m/z 293.3 [162 + 132-H] displayed the presence of hexose and pentose moieties interlinkage with each other as reported by Al-Shammari et al. (2015), so compounds 6 and 9 were tentatively characterized as Methyl apigenin-7-O-arabinosyl (1‴-6″)-β-D-glucopyranoside. Compound 28 with molecular ion peak at m/z 607 was characterised as acacetin-7-O-di glucoside based on the MS² base peak fragment ion at m/z 283.0 which showed the loss of two hexose moieties (Lin and Harnly, 2010) To the best of our knowledge, this is the first characterization of these compounds in P. aculeata. L.

• Characterization of myricetin –O-glycosides:

Compounds 22 (R_t 9.68 min) described as myricetin-3-O-β-D- glucuronyl-glucoside (Marczak et al., 2016). Depending on the data obtained by MS¹ molecular ion peak at m/z 655 and MS² fragment ion at m/z 317.1 [M−H-162-176] indicating the loss of glucose and glucoronyl moieties. Compound 23 (R_t 9.78 min) was suggested to be myricetin-3-O-glucuronide, As its ESI-MS spectrum displayed MS¹ at m/z 493 [M−H] in addition, the MS² data showed base peak fragment at m/z 317 [M−H -176] corresponding to the loss of glucoronyl moiety (Abu-Reidah et al., 2015). To the best of our knowledge, this is the first characterization of compounds 22 and 23 in P. aculeata L.

• Characterization of pinocembrin –O-glycosides:

Compound 21 R_t 9.44 min exhibited a molecular ion peak [M−H] at 593, we observed characteristic losses of 338 Da for feruloyl glucoside (Marczak et al., 2016). The aglycone fragment at 255.2 [aglycone – H] is characteristic for dihydroxyflavone (pinocembrin). From the previous results compound 21 was tentatively identified as pinocembrin -7-O- feruloyl glucoside (Simirgiotis et al., 2015).

• Characterization of tricin –O-glycosides:

Tricin-7-O-dipentoside (compound 34 and 35) is proposed to peak (R_t 14.04 and 14.43 min, m/z 593, [M−H]). In the MS/MS spectra, the loss of dipentose (264 Da) moieties gave a fragment ion at m/z 329.0 which corresponds to tricin in structure (Goławska et al., 2012).

• Characterization of naringenin -O-glycoside:

Compound 39 R_t 26.58 min exhibited a molecular ion peak [M−H] at 475, we observed characteristic losses of 272 Da for an aglycone (naringenin) leaving a base peak fragment at m/z 203.3 which is a characteristic fragment for acetyl hexoside [acetyl glucoside -H]. From this result compound 39 could be naringenin-7-O- acetyl glucopyranoside (Simirgiotis et al., 2015). This is the first report in P. aculeata L.

• Characterization of polymethoxylated compounds:

Compound 37 R_t 17.60 min showed the precursor ion at m/z 377 [M−H]. The compound had been already identified as tetrahydroxy trimethoxy dihydroflavone (may be dihydroquercetin derivative) from the MS² base peak fragment at m/z 303.0 [100%, M−H -74 (2OCH₃ + CH₃)] (Zhang et al., 2011) Similarly compound 38 with R_t 23.54 min with the same molecular ion peak was tentatively identified as an isomer of trimethoxy tetrahydroxy dihydroflavone from the MS² base peak fragment at 270.7 [100%, M−H -106 (M−3OCH₃ –OH)] (may be naringenin derivative) (Zhang et al., 2011).

3.2.1 Antioxidant activity

The DPPH scavenging potential of the aqueous P. aculeata L. aqueous fraction showed significant antioxidant activity with IC₅₀ = 48.3 ± 1.5 μg/mL compared to ascorbic acid 14.2 ± 0.5 μg/mL. These results support the previous literature about the antioxidant potential of P. aculeata L. and confirmed its ethnomedical use in the treatment of jaundice (Mruthunjaya and Hukkeri, 2008).

3.2.2 Antitumor activity

The antitumor activity of P. aculeata L. aqueous fraction was investigated against HepG-2 (Hepatocellular carcinoma) and MCF-7 (Breast carcinoma) cell lines using Cisplatin as a positive standard. NCI screening showed that, the crude extract and the pure compounds are cytotoxic with IC₅₀ less than 20 µg/ml and less than 4 µg/ml respectively (Boik, 2001). Unfortunately the aqueous fraction of P. aculeata L. exhibited a very weak cytotoxic activity against HepG-2 and MCF-7 cell lines with IC₅₀ of 222 ± 1.8 and 304 ± 9.2 μg/ml respectively, when compared to cisplatin 3.67 ± 8.1 and 5.71 ± 3.8 μg/mL (Figs. 7, 8 and Table 2).

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Fig. 5

DPPH scavenging capacity of P. aculeata L. aqueous fraction (PK a) and ascorbic acid.

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Fig. 6

IC₅₀ of P. aculeata L. aqueous fraction (PK a) and ascorbic acid.

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Fig. 7

Cytotoxic activity of P. aculeata L. aqueous fraction (PK a) against Hep-G2 cell line.

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Fig. 8

Cytotoxic activity of P. aculeata L. aqueous fraction (PK a) against MCF-7 cell line.

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