Rumex vesicarius L. was collected from Al-Fayoum Governorate in Egypt, during March, 2010. Rumex dentatus L. was collected from Al-Sharqia Governorate in Egypt, during April, 2010. The two plant species were identified by Professor Kamal Zayed, Botany Department, Cairo University. A voucher specimen was kept in the herbarium of the National Research Center of Egypt. Aerial parts of the plant species were air dried and ground into fine powder.

The air dried powder of each plant species (10 g) were, separately, extracted at room temperature for 48 h in percolators (shown in Figure 1) using 150 mL chloroform/methanol (2:1, by volume) as described by McCloud, 2010 [10]. The process was repeated three times and the combined extracts were evaporated at reduced pressure using a rotary evaporator at a temperature below 50 °C. Residual brown extracts were kept under argon at −20 °C until further analysis. Extracted yield was 0.8578 and 0.8313 g for R. vesicarius L. and R. dentatus L., respectively.

Three 35 g replicate of each sample were subjected to hydrodistillation in a Clevenger-type apparatus according to the European Pharmacopoeia [11] until there was no significant increase in the volume of oil collected. The oil phase was separated, dried over anhydrous sodium sulfate, and kept in a dark glass bottle at 4 °C until the analysis.

GC/MS analysis of the essential oil was carried out using an HP5890 Series II Gas Chromatograph, HP 5972 Mass Selective Detector and Agilent 6890 Series Autosampler (Agilent Technologies, Santa Clara, CA, USA). A Supelco MDN-5S, 30 m × 0.25 mm capillary column with a 0.5 µm film thickness was used with helium as the carrier gas at a flow rate of 1.0 mL/min. The GC oven temperature was programmed at an initial temperature of 40 °C for 5 min, then heated up to 140 °C at 5 °C/min and held at 140 °C for 5 min, then heated to 280 °C at 9 °C/min and held for five additional minutes. Injector and detector temperatures were set at 250 °C. Mass spectrometry was run in the electron impact mode (EI) at 70 eV. The identification of the chemical constituents of the oil was determined by their GC retention times (tR), retention indices (KI) and interpretation of their mass spectra and confirmed by mass spectral library search using the National Institute of Standards and Technology (NIST) database with those of authentic samples or published mass spectral data [12]. The retention indices were calculated for all of the volatile constituents using a homologous series of C₈–C₂₀ n-alkanes. The quantitative data were expressed as relative percentage of the oil constituents calculated from the GC peak areas without using correction factors, and each oil was analyzed three times.

50 mg of each sample extract was dissolved in 2 mL of 1.5% H₂SO₄ in methanol, vortexed, and left for 3 h in a heating box at 90 °C. The hydrolysis reaction was stopped by the addition of 2 mL water to each tube. The fatty acids were then extracted by shaking the aqueous layer with 5 mL of hexane, then passing the hexane layer over anhydrous sodium sulfate to remove water traces. The concentrated hexane layers were analyzed by GC/MS. GC/MS was carried out using the instrument described above. The GC oven temperature was programmed at an initial temperature of 130 °C for 1 min, then heated up to 300 °C at 5 °C/min and held at 300 °C for 5 min. Injector and detector temperatures were set at 250 °C. Mass spectrometry was run in the electron impact (EI) at 70 eV. The identification of the chemical constituents were determined by their GC retention times, interpretation of their mass spectra and confirmed by mass spectral library search using the NIST database.

All HPTLC experiments were performed using the CAMAG automatic system programmed through WIN CATS software. Half gram of each sample extract was dissolved in 10 mL chloroform and 2 g anhydrous sodium sulfate was added, vortexed, and filtered. 100 µL of each chloroform extract was diluted to 1 mL and subjected to HPTLC under the following conditions:

Solutions of samples (equivalent to 50 µg of oil) were applied on HPTLC Nano-silica gel 60 F254 (10 × 10 cm) glass plates (Sorbent Technologies Inc., Norcross, GA, USA). Each sample was applied as a 10 mm band onto the plate using a CAMAG Automatic TLC Sampler 4 System and developed in a CAMAG automatic developing chamber (ADC2) with developing solvent of petroleum ether/diethyl ether/formic acid (90:10:2 by volume) for separation of nonpolar compounds. In a separate experiment, another developing solvent was used (toluene/ethyl acetate 7:3, by volume) for separation of relatively polar compounds. After drying the plates, they were viewed under ultraviolet light at wavelengths of 254 nm and 366 nm, and documented by photography. Vanillin/sulfuric reagent solution was used for the derivatization of the plates, which was prepared by adding 0.7 g of vanillin to 196 mL of methanol and 7 mL of sulfuric acid. The derivatization reagent was added to the tank designed for the CAMAG Immersion Device III. The plates were then attached to the device and lowered into the solution for 3 s, removed, and dried. The plates were then heated for 5 min in the oven at 110 °C until optimal colorization was observed. After cooling the plates, pictures were taken under 366 nm and white light to record the results. The documentation of the TLC plates was carried using the CAMAG Reprostar 3 System equipped with a DXA252 camera with a 16 mm lens. Plates were also derivatized in CuSO₄ reagent (prepared by dissolving 20 g of copper sulfate pentahydrate in 200 mL methanol at less than 20 °C, then, under cooling with ice, 8 mL of 98% sulfuric acid and 8 mL of 85% orthophosphoric acid were added). The plates were dipped in the copper sulfate reagent for 5 s and allowed to dry for 1 min then heated for 30 min at 140 °C. Reverse phase HPTLC was carried out using 10 × 10 cm C₁₈ silica HPTLC plates w/uv 100% silanization, glass backed 200 µ and developed with dichloromethane/acetic acid/acetone (20:40:50, by volume). Plates were also derivatized in 1% vanillin reagent, (1 g of vanillin, 100 mL of ethanol mixed with 5 mL of concentrate sulfuric acid, and 95 mL of ethanol), and the plate was dried in the oven at 100 °C for 5 min, after which spots were visualized under white light and 366 nm. Densitometry scanning was done using TLC scanner III (CAMAG, Wilmington, NC, USA) in the absorbance mode at 296 nm.

Solutions of the essential oils in methanol were applied to silica gel plates in 1 cm bands at 20 µg/band. Plates were developed with toluene/ethyl acetate (95:5, by volume). Plates were dried and observed at a UVwavelength of 254 nm to detect spots that have the ability to absorb UV light, which appear as a dark spot with green background. UV light of 366 nm was used to detect spots with fluorescent ability. Plates were also derivatized in vanillin reagent and the plates were dried in the oven at 100 °C for 5 min, after which spots were visualized under white light and 366 nm wavelengths.

TLC plates were developed as described above but then dipped in a 0.2% methanolic DPPH for one second and kept in the dark for 30 min, then observed under white light using CAMAG Camera. Active spots were seen as yellow bands on a violet background.

HPLC of the crude Rumex extracts were performed on Agilent 1100 HPLC using 250 mm × 4.6 mm C18 column with acetonitrile/water (40:60, by volume) as the mobile phase at a flow rate of 1 mL/min and detected at 288 nm. High Performance Liquid Chromatography and Mass Spectrometry (HPLC/MS) was used to determine the chemical composition of each the crude extracts. HPLC was performed on Agilent 1100 HPLC/MSD SL and VL using Phenomenex Kinetic 2.6 µC₁₈100A100 × 4.16 mm column with atmospheric pressure chemical ionization (APCI) and electrospray (ES), respectively. Each system is composed of the following units: a solvent delivery module, an automatic sampler injector, (100-well capacity), a controller module, a column oven, and a model series 1100 MSD mass spectrometer. Methanol/water/formic acid (90:10:1, by volume) was used as mobile phase A and 0.1% formic acid for mobile phase B at a gradient flow rate of 0.5 mL/min. Solvent A = 55% 0.1 formic acid (CH₂O₂), solvent B = 45% MeOH/H₂O/CH₂O₂ (90:10:1, by volume). Starting at time 0, 45% B, at 15 min, increased to 65% B, at 15.5 min decreased to 45% B and held at 45% B for 5 min, and the run ended after 20.5 min. Diodearray detection at 288 nm, mass spectroscopy was performed using single ion monitoring in the negative modeat ions 327.10 and 505.10, using quasi molecular ions [M − H]⁻¹ with a dwell time of 294 ms. Nitrogen was used both as drying gas and nebulizing gas at flow rates of 12 mL/min) and 35 (psig) respectively. The temperature of the drying gas was set to 350 °C. Data collection was handled using Chemstation V.B.04.02.

GC/MS analysis of the essential oil of the two Rumex species revealed five major monoterpenes, however, their concentrations in the two species were greatly different (Table 1). Total ion chromatograms of the essential oils of the two species and the chemical structures of the identified monoterpenes are shown in (Figure 2).

GC/MS analysis of the fatty acid methyl esters of the two species also showed significant difference between the two species although they were similar to some extent in their chemical composition but their percentages were different as seen in (Figure 3). Both species contain high level of palmitic acid, which is known to be found in all plants. Oleic and linoleic acids were also found in the two species. Rumex vesicarius was uniquely rich in the omega-3-fatty acids and Rumex dentatus showed the presence of C₂₄ and C₂₆ fatty acids where they were minor in the R. vesicarius. Table 2 presents the chemical composition of the fatty acids and related compounds in the two species.

The HPTLC analysis of the chloroform/methanol extracts of Rumex species showed eightresolved bands when plates were developed in petroleum ether/diethyl ether/formic acid (90:10:2, by volume) and at least 10 resolved bands when using toluene/ethyl acetate (70:30, by volume) as developing solvent (Figure 4). Both species showed similarity in their compositions but each species has unique finger printing pattern that can be used to distinguish each sample by using copper sulfate and anisaldehyde as the derivatizing reagent. HPTLC finger printing is a valuable quality assessment tool for the evaluation of botanical materials, it allows for the analysis of a broad number of compounds both efficiently and cost effectively. The HPTLC method is simple, rapid, accurate, reproducible, selective and economic and can be used for quality control analysis and for quantitative determination of the plant metabolites. Reversed phase HPTLC of the two species revealed more differences among the two different species, as shown in Figure 4, using phosphomolybdic acid or anisaldehyde as derivatizing agent. The difference is clearly seen when plates were examined under UV light of 366 nm both for normal phase and reverse phase HPTLC, as shown in Figure 4. Bioautography experiment using DPPH radical quenching assay showed that Rumex vesicarius extract is stronger antioxidant than the Rumex dentatus extract, however, in both of them antioxidants were mostly found in the polar fractions (low R_f values near the origin in the normal phase and high R_f values near the solvent front in the reverse phase). This relative high antioxidant activity for the Rumex vesicarius is also consistent with the GC/MS analysis that showed higher level of omega 3-fatty acids and higher flavonoid content than those found in Rumex dentatus.

HPLC and LC/MS of the two species showed unique properties for both species with the diodarray detector (ultra violet and visible absorptions) as well as in the positive and negative total ion spectra. R. dentatus was characterized by the presence of high level of anthaquinones, chromones and naphthoquinones having distinguished UV absorbance at 450 nm as shown in Figure 5. On the other hand R. vesicarius is rich in flavonoides with a strong absorbance at 288 nm. Electrospray mass spectrometry of the two isomers both in the positive and negative modes as well as their retention times, relative percentage and base peak ions in their mass spectra are shown in (Table 3 and Table 4). ESI-MS negative mode was found to be more sensitive than positive mode for both species. Total ion chromatograms of the two species are shown in Figure 6. Several anthraqinones and chromones are characteristic markers for R. dentatus (Figure 7), however, flavonoids are characteristic markers for R. vesicarius (Figure 8) [13].