Thermo finnigan

Plumbago indica and P. auriculata aerial parts were obtained from El-Mazhar botanical garden, Giza, Egypt after obtaining a permission to acquire the plant materials. The plants were authenticated by Mrs. Therease Labib, Consultant on Plant Identification at El-Orman Botanical Garden. Voucher specimens (Numbers: 14062020 and 15062020) were kept at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. The air-dried materials (500 g, each) were subjected to exhaustive cold maceration using ethanol (70%) with frequent agitation. The extracts were separately evaporated under vacuum at 40 °C till complete dryness and furnished extraction yields of 10.8% and 9.8% for P. indica and P. auriculata, respectively.
A Thermofinnigan (Thermo Electron Corporation, USA) coupled with an LCQ-Duo ion trap mass spectrometer with an ESI source (ThermoQuest) system was utilized. The MS operated in the negative mode. The ions were detected in a full scan mode and mass range of 50–2000^{10 (link)}.

The LC system was ThermoFinnigan (Thermo Electron Corporation, Waltham, MA, USA) coupled with an LCQ-Duo ion trap mass spectrometer with an ESI source (ThermoQuest). The separation was achieved using a C18 reversed-phase column (Zorbax Eclipse XDB-C18, rapid resolution, 4.6 × 150 mm, 3.5 µm, Agilent, Santa Clara, CA, USA). A gradient of water and acetonitrile (ACN) (0.1% formic acid each) was applied from 5% to 30% ACN over 60 min with a flow rate of 1 mL/min with a 1:1 split before the ESI source. The samples were injected automatically using an autosampler surveyor ThermoQuest. The instrument was controlled by Xcalibur software (Xcalibur™ 2.0.7, Thermo Scientific, Waltham, MA, USA). The MS operated in the negative mode for better detection of the phenolic compounds with a capillary voltage of—10 V, a source temperature of 200 °C, and high-purity nitrogen as a sheath and auxiliary gas at a flow rate of 80 and 40 (arbitrary units), respectively. Collision energy of 35% was used in MS/MS fragmentation. The ions were detected in a full scan mode over a mass range of 50–2000 m/z.

High performance liquid chromatography–mass spectrometry (HPLC-PDA-MS/MS) was used to identify the chemical composition of the extracts. The LC system was Thermofinnigan (Thermo Electron Corporation, Waltham, MA, USA) coupled with an LCQDECA XP Plus ion trap mass spectrometer with an ESI source. The separation was achieved using a C18 reversed-phase column (Zorbax Eclipse XDB-C18, Rapid resolution, 4.6 × 150 mm, 3.5 µm, Agilent, Santa Clara, CA, USA). A gradient of water and acetonitrile (0.1% formic acid each) from 5% to 70% ACN in 45 min was applied and then kept for 5 min at the last conditions. The flow rate was 1 mL/min and a splitter was used to deliver only 50% of the sample into the analyzer. The samples were injected automatically using autosampler surveyor ThermoQuest. The instrument was controlled by Xcalibur software to collect the UV chromatogram using PDA mode and the MS data. The MS operated in the positive mode with a capillary voltage of −10 V, a source temperature of 275 °C, and high purity nitrogen as a sheath and auxiliary gas at a flow rate of 80 and 40 (arbitrary units), respectively. The ions were detected in a full scan mass range of 50–2000 m/z [43 (link)].

A Thermofinnigan (Thermo Electron Corporation, USA) coupled with an LCQ-Duo ion trap mass spectrometer with an ESI source (ThermoQuest) system was utilized. A C18 reversed-phase column (Zorbax Eclipse XDB-C18, rapid resolution, 4.6 × 150 mm, 3.5 µm, Agilent, USA) was used to separate the analytes. A gradient of water and acetonitrile (ACN) (0.1% formic acid each) was applied from 5% to 30% ACN over 60 min with flow rate of 1 mL/min with a 1:1 split before the ESI source. The samples were injected automatically using autosampler surveyor ThermoQuest. The instrument was controlled by Xcalibur software (Xcalibur^TM 2.0.7, Thermo Scientific). The MS operated in the negative mode as before^{8 (link)}. The ions were detected in a full scan mode and mass range of 50–2000.

HPLC-ESI-MS/MS was employed to investigate the chemical constituents of the extract. The LC system was Thermo Finnigan (Thermo electron Corporation, USA), coupled with an LCQ Duo ion trap mass spectrometer with an ESI source (ThermoQuest). A Silica gel C18 reversed-phase column (Zorbax Eclipse XDB-C18, Rapid resolution, 4.6 × 150 mm, 3.5 µm, Agilent, USA) was used for the separation process. Water with a gradient increase from 5% to 50% of acetonitrile (ACN) (with 1% formic acid each in the positive mode) was applied in 60 min, with a flow rate 1 mL/min, and then increased to 90% ACN in the next 30 min. The samples were injected automatically using auto sampler surveyor ThermoQuest. The instrument was controlled by Xcalibur software. The MS operating conditions were applied in the negative ion mode, as previously described by us [25 (link)]. The ions were detected in a full scan mode and mass range of 50–2000 m/z.

HPLC-DAD-ESI-MS/MS was employed to investigate the chemical constituents of the extract. The LC system was Thermo Finnigan (Thermo electron Corporation, OK, USA), coupled with an LCQ Duo ion trap mass spectrometer with an ESI source (ThermoQuest). A Silica gel C18 reversed-phase column (Zorbax Eclipse XDB-C18, Rapid resolution, 4.6 × 150 mm, 3.5 µm (Agilent, CA, USA) was used for the separation process. Water with a gradient increase from 5% to 50% of acetonitrile (ACN) (with 1% formic acid each in the positive mode) was applied in 60 min, with a flow rate 1 mL/min, and then increased to 90% ACN in the next 30 min. The samples were injected automatically using auto sampler surveyor ThermoQuest. The instrument was controlled by Xcalibur software (Thermo Fisher Scientific Inc., OK, USA). The MS operating conditions were applied in the negative ion mode, as previously described by us [29 (link)]. The ions were detected in a full scan mode and mass range of 50–2000 m/z.

The chemical constituents of the tested extract were tentatively identified using a Thermo Finnigan (Thermo electron Corporation, OK, USA), coupled with an LCQ Duo ion trap mass spectrometer with an ESI source in negative ionization mode (ThermoQuest Corporation, Austin, TX, USA)^{54 (link)}. The ethyl acetate extract (20.0 g) was consequently fractionated via using column chromatography (5 × 60 cm) packed with polyamide 6S as a stationary phase and eluted via a gradient mix elution system (Water: MeOH). A total of five major sub-fractions (F1–F5) were obtained. The obtained sub-fractions (F1–F4) were separately subjected to further extra purification using a multiple Sephadex LH-20 sub-columns (2 × 30 cm) eluted with (Water: MeOH with MeOH gradient) to obtain gallic acid, methyl gallate, p-coumaric acid, quercetin, taxifolin, naringenin, and quercetin 3-O-glucoside. While F5 (5.2 g) eluted by 85% MeOH from the main polyamide column was subjected to multiple Sephadex LH-20 sub-columns for extra purification eluted with methanol:water in gradient mix elution system to obtain ESC.