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Phenolic constituents of Pulicaria undulata (L.) C.A. Mey. sub sp. undulata (Asteraceae): Antioxidant protective effects and chemosystematic significances

One new naturally isoflavone compound, 5,7,2′,3′,4′ penta hydroxyl isoflavone-4′-O-β-glucopyranoside (1) was isolated from the aqueous methanol extract (AME) of Pulicaria undulata subsp. undulata, together with seven known compounds: kaempferol (2), kaempferol 3-O-β-glucoside (3), quercetin (4), quercetin 3-O-β-glucoside (5), quercetin 3-O-β-galactoside (6), quercetin 3,7-di OCH3 (7), and caffeic acid (8). Their structures were established through chemical (acid hydrolysis) and spectral analysis (UV, NMR, and ESIM). The AME and some isolated compounds were evaluated as protective agents. Free radical scavenging using a microscaled 2,2-diphenyl-1-picrylhydrazyl assay was used to assess the direct antioxidant properties that were evaluated by the ability to protect murine Hepa1c1c7 liver cells against damage induced by the organic peroxide tert-butyl hydroperoxide. The neutral red uptake assay (NRU) was used to record the activity. Results of the 2,2-diphenyl-1-picrylhydrazyl assay recorded differential scavenging properties in ascending order: 5,7,2′,3′,4′ penta hydroxyl isoflavone-4′-O-β-glucopyranoside > quercetin > quercetin 3-O-galactoside > caffeic acid > quercetin 3,7-di-OCH3 > kaempferol with 50% inhibitory concentrations of 3.9 μM, 7.5 μM, 11.4 μM, 12.2 μM, 78.1 μM, and 252.3 μM, respectively. The antioxidative potential reveals the potency of AME, quercetin, and quercetin 3,7-di-OCH3. The latter compound showed full protection at 100 μM (33 μg/mL) against the induced toxicant effect where the 50% effective concentration was calculated as 33.6 ± 1.7 μM (11.1 μg/mL). In addition to quercetin, which was extensively shown previously as a cytoprotective agent, AME was less potent; it was capable of protecting 75% at 100 μg/mL with 50% effective concentration of 92.3 ± 4 μg/mL. Moreover, the isolated flavonoids were found to be significantly chemosystematic markers.


Introduction
Many plant genera belonging to the Asteraceae family are used as herbal medicines and as beverage ingredients in Asian countries, especially in China [1]. Among such plants, the genus Pulicaria Gaertn. (Inuleae: Asteraceae), which consists of 100 species widely distributed from Europe to North Africa and Asia [2]. Previous phytochemical studies on Pulicaria species gave rise to the isolation of flavonoids and phenolics [3e10] mono-, di-, and sesquiterpenes [11e17], essential oils and caryophyllene derivatives [18e21]. The plants of this genus are used in traditional medicine as tonic, antispasmodic, and antihypoglycemic drugs, as well as ingredients of perfumes [17,22]. Also, they have antimicrobial [23e25], antioxidant [25,26], and anticancer [28] properties. Pulicaria undulata (L.) C.A. Mey. is one of the most widespread desert plants growing wild in Egypt [29,30]. It is known as "Dethdath", and the flower branches are used for preparing a powerful sneezing powder as an insect repellent and as a herbal tea [29]. Previous studies of P. undulata allowed the isolation of various flavonoids [5,8e10] and sesquiterpenes [13e16]. The present study aimed to evaluate the phenolic constituents of P. undulata (L.) C.A. Mey. in comparison with those previously isolated from P. undulata (L.) Kostel., to find out the chemosystematics relationship between them. In addition, the study aimed to investigate the potential protective activity against oxidative stress.

2.
Materials and methods

General
Nuclear magnetic resonance (NMR) experiments were recorded on a Jeol EX-500 spectroscopy (JOEL Inc., Tokyo, Japan):

2.4.
Cell-free antioxidant assay (anti-2,2-diphenyl-1picrylhydrazyl) AME and the isolated flavonoids from P. undulata were prepared in dimethyl sulfoxide (DMSO). The method used in the present study was based on a modified procedure [32] based on previously published methods [33]. The AME/compound stock solutions (20 mL/well) were pipetted in triplicate onto flat-bottomed, 96-well plates (Nunc TM, Thermo Fisher Scientific, NY, USA). The assay was started with the addition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent (0.004% weight/ volume in methanol, 180 mL/well). Appropriate blanks were prepared using the sample vehicle only (i.e., 20 mL DMSO) in addition to the same amount of DPPH reagent to eliminate any inherent vehicle activity. Negative control (replacing DPPH with methanol) was also run simultaneously to eliminate the optical density (OD) values resulting from colored samples. The plate was immediately shaken for 30 seconds and incubated in the dark for 30 minutes at room temperature. The remaining DPPH was measured in a Fluostar Optima microplate reader (BMG LABTECH GmbH, Ortenberg, Germany) at 540 nm. The percentage of antioxidant activity was calculated using the following equation: percentage of antioxidant activity ¼ (OD (blank) À OD (sample) /OD (blank) ) £ 100. To determine the 50% inhibitory concentration (IC 50 ; concentration of sample producing 50% scavenging of the DPPH radical present in the blank), the mean percentage scavenging to control were plotted against concentrations, and the resulting plot was fitted to a nonlinear regression curve using GraphPad Prism version 5.0 software (GraphPad Software Inc., San Diego, USA).

Cell culture
Monolayer cultures of murine hepatoma cell line Hepa1c1c7 (generously provided by Professor M.S. Denison, University of California, CA, USA) were grown in 75-cm 2 flasks at 37 C and 5% CO 2 in a humidified CO 2 incubator (Certomat-20S, Sartorius group, NY, USA). At 75e85% confluence, the monolayer was routinely subcultured using trypsineversene (EDTA) solution and maintained in a-minimal essential medium supplemented with 10% fetal bovine serum, 2mM L-glutamine and triple antibiotic mixture containing 100 U/mL penicillin, 100 mg/mL streptomycin sulfate, and 250 ng/mL amphotericin B. All culture reagents were purchased from Lonza, Verviers, Belgium.

Bioassay procedure
We followed the published antitert-butyl hydroperoxide (anti-TBHP) bioassay procedure [32] with some modifications. Hepa1c1c7 (2 Â 10 5 cells/well) were seeded onto 24-well culture plates (Greiner Bio-one, Frickenhausen, Germany) and left to adhere for 24 hours to form semiconfluent monolayers. Monolayers were treated with DMSO (vehicle control, 0.5% volume/volume), AME, or test compounds. In the screening experiment, a single final concentration of 100 mg/mL of the extract or 100mM of the compounds was separately examined; the insufficient amount of compound 1 allowed only the examination of 11.2 mM. Quercetin dihydrate (4 mg/mL) was run in parallel as a positive control for cytoprotection. Treated plates were incubated for 24 hours prior to exposure to 125 mM TBHP as an inducer dose for oxidative cytotoxicity [34]. The neutral red uptake assay (NRU) was performed as the endpoint for cell viability measurement to monitor the cytoprotection produced by tested samples [35]. The sample that was able to protect > 50% of Hepa1c1c7 cells was further subjected to a concentration-dependent response curve to determine its 50% effective concentration (EC 50 ; the concentration that protected 50% of the oxidative induced culture). The obtained data were analyzed using GraphPad Prism. The EC 50 values were determined from the average of two independent experiments using nonlinear regression concentrationeresponse curve fit.

3.
Results and discussion

Identification of isolated compounds
One new naturally isoflavone compound; 5,7,2 0 ,3 0 ,4 0 pentahydroxyl isoflavone-4 0 -O-b-glucopyranoside (1) was isolated and identified, together with seven known compounds: (7), and caffeic acid (8) (Figure 1). Their purity was determined using high-performance liquid chromatography by calculating the percentage of peak area in relation to the total area of peaks and ranged from 98% to 99%. Their chemical structures were established through chemical and spectral analyses. Compounds 1e3 and 6e8 were isolated for the first time from this species. The chemical structures of the known compounds were determined by complete acid hydrolysis, UV, one-dimensional NMR and mass spectrometry techniques and confirmed by comparing their spectral data with those from the literature [36,37]. Compound 1 was isolated as colorless crystals. Negative ESIMS analysis showed a molecular ion peak at 463.2 m/z (C 21 H 20 O 13 ). Complete acid hydrolysis (2N HCl, 1 hour, 100 C) revealed the presence of an isoflavones aglycone (UV, 1 H NMR) and glucose as the sugar moiety [Co-PC]. The UV, lmax/nm, methanol (262,294 sh, 332 sh), 1 H NMR (d 8.59, s, for H-2) and 13 C NMR (156.9 for C-2 and 125.2 for C-3) were consistent with an isoflavone skeleton. In addition, the 1 H NMR spectra showed two meta-couplings (J ¼ 1.5 Hz) of Ring A at d 6.09 and d 6.27 for H-6 and H-8, respectively. The aromatic protons of the B ring appeared as two doublets with ortho-coupling (J ¼ 8.5 Hz) at d 6.77 and d 7.18 assigned to H-6 0 and H-5 0 , respectively. The anomeric proton signal resonated at d5.09 (J ¼ 7.5 Hz) indicated the b configuration of the glucopyranose unit [36]. The heteronuclear multiple-bond correlation spectroscopy spectrum confirmed the isoflavone structure [38]; H-2 (d 8.59) showed cross-correlations with C1 0 (115.9), C4 (177.6), and C9 (159). H-6 0 showed three-bond heteronuclear multiplebond correlation spectroscopy correlations; with C-3 (156.9), C-2 0 (145.2), and C4 0 (148.9). Further correlation between H-1 00 (d 5.09) and C-4 0 (148.9) confirmed the occupation of OH at position 4 0 .

Direct antioxidant assay (DPPH assay)
The cell-free direct antioxidant assay using DPPH revealed the potency of the tested samples to scavenge free radicals in the order of 7,2 0 ,3 0 ,4 0 penta hydroxyl isoflavone-4 0 -O-b-glucopyranoside > quercetin > quercetin 3-O-galactoside > caffeic acid > quercetin 3,7-di-OCH 3 > kaempferol. Their IC 50 values were calculated as: 3.9mM, 7.5mM, 11.4mM, 12.2mM, 78.1mM, and 252.3mM, respectively, while the IC 50 of AME was calculated to be 27.5 mg/mL (Table 1). Comparing the structure of the tested compounds (Figure 1), the anti-DPPH activity showed a chemical structural dependence where, in case of compounds 1 (5,7,2 0 ,3 0 ,4 0 pentahydroxyl isoflavone-4 0 -O-b-glucopyranoside) and 4 (quercetin), both shared the presence of 5 and 7 hydroxyl groups in Ring A, along with the ortho-dihydroxyl basic structure of Ring B, which seemed essential for the displayed activity. Furthermore, either glycosylation or methylation of the 3-hydroxy group (quercetin 3-O-b-galactoside; 6 and quercetin 3,7-di OCH 3 ; 7) decreased the radicalscavenging activities of flavonoids. These interpretations are supported by previous reports that concluded that the glycosylation and/or methylation of any hydroxyl group in flavonol dihydroxyl basic structure might condense the activity of flavonoids against free radicals [39]. Moreover, the tested phenolic acid (caffeic acid; 8) showed a significant activity with IC 50 of 12.2 mM. This activity was also due to the orthodihydroxyl pattern, which is generally considered as important for the radical-scavenging activity of phenolic acids [40].

Indirect antioxidant assay (anti-TBHP cytoprotection assay)
In this assay, we used the murine hepatoma Hepa1c1c7 cell line because it is well known to be inducible for antioxidant phase II genes such as quinone reductase and glutathione-Stransferase that detoxify free radicals [41,42]. The cells have functional promoter of the antioxidant response element that encodes many cytoprotective genes [43]. The results of the anti-TBHP cytoprotection screen are displayed in Figure 2. Exposure to 125 mM TBHP caused a dramatic loss (92%) of Hepa1c1c7 cell viability relative to the non-TBHP treated DMSO control. Quercetin dihydrate as a positive standard produced full cytoprotection at 4 mg/mL. The pretreatment of Hepa1c1c7 cells with 100 mg/mL AME produced 74% viability (i.e., protecting 66% of their intact cells), while quercetin (4) Figure 1 e Chemical structure of compounds 1e8   showed 80% protection (88% viability), which was previously demonstrated as a liver cytoprotective [44,45]. The toxicity of TBHP was completely inhibited by 100 mM quercetin 3,7-di OCH 3 (7) with EC 50 ¼ 33.6 ± 1.7 mM (Figure 3). The remaining tested compounds were not able to protect Hepa1c1c7 cells against the cytotoxicity of organic peroxide. A nonpolar solvent extract from Egyptian P. undulata was recently shown to induce the activity of the Nrf2-dependent cytoprotective enzyme quinone reductase in Hepa1c1c7 cells [46]. Nevertheless, the 70% aqueous methanolic extract of the same plant was devoid of the inducer activity up to a concentration of 50 mg/mL. In the same study, however, the nonpolar extract of Pulicaria incisa strongly upregulated both the Nrf2dependent cytoprotective enzyme quinone reductase activity and protein expression in addition to upregulation of other cytoprotective proteins including glutathione S transferase and hemoxygenase-1. These findings about the Pulicaria genus could support our results for the protective effect of P. undulata constituents against TBHP injury.
T€ ackholm [49] deals with the two species morphologically as P. undulata (L.) Kostel. and F. crispa (Forssk.) Cass (¼ P. undulata (L.) C. A. Mey.). El-Kamali and Mahjoub [50] studied the antibacterial activity of both taxa and showed a different activity. Moreover, Liu et al [27], in their review on the phytochemical and biological activities of Pulicaria species, were transacting with the species without demonstrating the authority of each species. Furthermore, through studying the genetic variation between P. undulata and P. crispa (Forssk.) Benth and Hook (¼ P. undulata (L.) C. A. Mey.), the authors did not clarify the authority of P. undulata [51]. Also, Metwally et al [21], Abdel-Mogib et al [9], and Liu et al [27] characterized the chemical constituents of P. undulata without mentioning the authority.
From the flavonoids point of view, few studies have dealt with the isolation and identification of flavonoids from P. undulata (L.) C.A. Mey. and its synonyms (Table 2) [6,8e10].
Two flavonol aglycones (kaempferol and quercetin) and their methyl ether derivatives either at position 3, 7, or 6 were common. Glucopyranose is the main sugar moiety; the glucosylation of OH group at position 3 is common, while the glucosylation of that at position 7 or 6 is rare and is represented as quercetin 7-O-glucopyranoside and 6hydroxykaempferol 3-methyl ether 6-O-glucopyranoside [6,8]. One di-O-glucopyranoside flavonol (pulicaroside) was also identified [6]. Only one study has dealt with the isolation and identification of five flavonoids from P. undulata (L.) Kostel. [10]. Moreover, the two species are completely different; the methylation of flavonols in P. undulata (L.) C.A. Mey. takes place at position 3 or 3,6, or 3,7, while in P. undulata (L.) Kostel., it occurs at position 7 or 3,7. In order to avoid this confusion over nomenclature, the authority of the scientific plant names must be used in the upcoming studies.