Preparation and standardization of O. stamineus ethanolic extract
OS dried leaves were obtained from specialized supplier of herbal products in Malaysia (Herbagus Trading, Kepala Batas, Pulau Pinang). OS ethanolic extract was prepared by the maceration method as the following; 100 g of OS powder was added to 1 L of 96% ethanol, mixed continuously on a magnetic stirrer for 48 h, filtered, concentrated at 60°C by rotavapor and further freeze-dried to 10.6 g of solid material. Total glycosaponins content of the extract was analyzed as previously described , and total phenolics content was estimated as previously described by Aisha et al., . Concentration of 4 marker compounds including rosmarinic acid (RA), sinensetin (SIN), eupatorin (EUP), and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone (TMF) was determined by reverse phase HPLC as previously described ; analysis was carried out using Agilent 1100 HPLC system, using Nucleosil C18 column (250 mm × 4.6 mm, 5 μm), column temperature was 25°C, injection volume was 20 μl, the mobile phase was isocratic which consisted of methanol (55%): tetrahydrofuran (5%): 0.1% H3PO4 (40%). Flow rate was 0.7 ml/min, separation time 25 min, and wavelength was 330 nm.
UV–vis spectrophotometry was carried out in the wavelength range 500–200 nm using Perkin Elmer UV spectrophotometer.
Preparation of soybean phospholipids
The unpurified soybean phospholipids were prepared from food grade soybean lecithin as previously described with minor modifications . In brief; crude lecithin (500 g) was refluxed in 96% ethanol (2.5 L) for 30 min and cooled to RT. Subsequently, the supernatant was collected by decantation and concentrated at 60°C using rotavapor. The residue was then washed 5× with acetone (1 L) to give 50 g of semi solid material; this phospholipid was called PH-Et. In another experiment, 500 g of the food grade lecithin was refluxed for 30 min in acetone (2.5 L), cooled down to RT, and the precipitate was saved and further washed with acetone (5×) to give 220 g and named PH-Ac. In addition, a fraction containing higher concentration of phosphatidylcholine was prepared as mentioned previously ; this phospholipid fraction was named PH-Fr.
The crude lecithin was characterized by measuring concentration of the acetone insoluble phosphatides and by RP-HPLC [20, 32]. The soybean phospholipid fractions were analyzed by RP-HPLC as previously described with some modifications ; Agilent 1100 HPLC system was used, and separation was achieved on Agilent Eclipse C18 column (250 mm × 4.6 mm, 5 μm) at 35°C, the mobile phase was isocratic which consisted of isopropyl alcohol, methanol and water (70:22:8 v/v) at 0.5 ml/min. The samples were prepared in methanol at 1 mg/ml and filtered through 0.45-μm syringe filter, injection volume was 10 μl, and detection was done at 205 nm.
Preparation of OS liposomes
Liposomes of OS extract (OS-L) were prepared by the film method as the following; soybean phospholipids was dissolved in chloroform and OS extract was dissolved in ethanol or methanol, the solutions were mixed, and the solvent was evaporated under vacuum using rotary evaporator at 45°C for 30 min, followed by drying in oven at 60°C for 1 h.
Determination of aqueous solubility
Solubility was evaluated by UV–vis spectrophotometry; OS extract, and OS liposomes were dissolved at theoretical concentration of 4 mg/ml in deionized water, vortexed for 2 min, and sonicated for 10 min. Subsequently, the solutions were centrifuged at 8000 rpm and 25°C for 10 min. Concentration of OS extract in the supernatant was determined at 286 nm. A calibration curve was prepared at the same time in order to calculate concentration of soluble OS extract (y = 0.0135x – 0.0423, R2 = 0.9999). All experiments were carried out in triplicates. Concentration of 4 marker and 4 unknown compounds was also determined by HPLC as described previously.
Effect of pH on liposomes stability
Effect of pH on liposome’s stability was studied as previously described with some modifications . OS liposomes were dissolved in water at 4 mg/ml and further diluted at 1:4 ratio in phosphate buffered saline (PBS) at pH 1.6, 5.5, 7.4 and water, and incubated for overnight (16 h) at 37°C. Subsequently, the solutions were centrifuged at 8000 rpm and 25°C for 10 min, and concentration of OS extract in the supernatant was determined by UV spectrophotometry at 286 nm. The precipitate obtained at pH 1.6 was resuspended in PBS (pH 1.6, 5.5 and 7.4), vortexed for 5 min, centrifuged, and the supernatant was analyzed by UV spectrophotometry. The results are presented as percentage of soluble fraction relative to liposomes diluted in water.
Determination of entrapment efficiency
Entrapment efficiency was determined by precipitation of OS liposomes at pH 1.6. It is noteworthy that this pH caused precipitation of OS liposomes, but not the free extract. The OS liposomes was dissolved in water at 4 mg/ml, sonicated for 10 min, and centrifuged at 8000 rpm for 10 min. The supernatant (1 ml) was added to the same volume of PBS at pH 1.6, mixed thoroughly by vortex, centrifuged at 8000 rpm for 10 min, the supernatant containing the free extract was decanted, the precipitate was washed with PBS (pH 1.6) and resuspended in 1 ml PBS (pH 7.4). After sonication for 10 min, the extract content was determined by UV spectrophotometry at 286 nm. Concentration of RA, TMF, SIN and EUP was measured by HPLC as described previously [14
]. The entrapment efficiency was calculated as the following:
Partition between n-octanol and water
OS extract and OS liposomes were prepared in deionized water at 1 mg/ml and mixed with the same volume of n-octanol. The mixtures were then mixed by several manual inversions (30 times), allowed to settle for 4 h, and centrifuged at 5000 rpm for 5 min to allow separation of the 2 layers. Concentration of OS extract in both layers was determined by UV–vis spectrophotometry at 286 nm. The results are presented as the ratio of extract concentration in aqueous phase relative to that in n-octanol.
Fourier transform infrared spectroscopy
FTIR analysis of OS extract, soybean phospholipids and OS liposomes was carried out using Spectrum 400 spectrometer (Perkin Elmer, USA). The IR spectra were recorded in the range of 4000 – 400 cm−1 (n = 3).
Measurement of particle size and zeta potential
Particle size, polydispersity index (PDI) and zeta potential (ζ) were determined by Photon Correlation Spectroscopy (PCS) using a Zetasizer nano zs (Malvern Instruments Ltd, UK). The samples were dissolved in ultra pure water (18 MΩ) at 1 mg/ml and filtered through 0.45-μm syringe filters to remove any insoluble matter. Measurements were carried out in triplicates.
Determination of critical micellar concentration
Critical micellar concentration (CMC) of OS liposomes was estimated using the fluorescent probe pyrene . Pyrene was dissolved in dimethylsulfoxide (DMSO) at 10 mM and diluted in PBS (pH 6.8) to 10 μM. Concentration of liposomes was maintained in the range 1–280 μg/ml in PBS. Pyrene was added at a final concentration of 0.1 μM and incubated for 30 min in the dark at RT. Fluorescence intensity was measured at excitation of 336 nm and emission of 375 and 384 nm by LS 45 fluorescence spectrometer (Perkin Elmer, USA). The intensity ratio (I384/I375) was calculated and plotted versus log concentration. The resulting curve was used to calculate the CMC (n = 3).
Transmission electron microscopy
Studies in transmission electron microscopy (TEM) were carried out in order to confirm presence of the liposomal structures. One drop of OS liposomes (1 mg/ml in water) was deposited on a 400 mesh copper grid coated with 5 nm layer of carbon, air-dried at RT for 3 min, and stained with 2% uranyl acetate for 1 min. The samples were dried and studied using CM12 TEM (Philips, Netherlands).
In vitro release and DPPH scavenging effect
In vitro release study was performed by dialysis method using dialysis bags with molecular weight cut off value 8200 Dalton. The experiment was carried out using magnetic stirrer in PBS at pH 6.8, and 37°C with continuous stirring at 100 rpm. Briefly, 10 ml of OS liposomes or non-formulated OS extract (in water) containing 10 mg of extract was filled in dialysis bag. The bags were hermetically sealed and kept in the receiver compartment containing 200 ml of same medium. Samples (3 ml) were collected at 0, 0.25, 0.5, 1.0, 2.0, 3.0, 6.0, 12, and 24 h, and immediately replaced with 3 ml fresh medium. Concentration of extract was determined spectrophotometrically at 286 nm. The results are presented as average percentage of cumulative release ± SD (n = 3). DPPH scavenging effect of released extracts was then investigated as described previously ; DPPH (9 mg/100 ml) was added to the same volume of released extract, incubated at RT for 30 min, and absorbance was measured at 516 nm. Median inhibitory concentration (IC50) was then calculated from the dose response curves (n = 3).
Absorption through the everted rat intestine
Absorption through everted rat intestinal sacs was carried out as described previously with some modifications . Overnight fasted male Sprague–Dawley rats were anesthetized by diethyl ether and euthanized by cervical dislocation. The first two thirds of the small intestine were collected immediately after euthanasia, everted gently using a glass rod, washed thoroughly, and were kept in Tyrodes solution (NaCl, 8 g; KCl, 0.2 g; NaHCO3, 1 g; CaCl2, 0.2 g; MgCl2.6H2O, 0.1 g; NaH2PO4, 0.05 g; and glucose, 1 g dissolved in 1 L deionized water) at 37°C with aeration. The tissues were cut into 6-cm length, sealed at one end using surgical thread and filled with 1 ml aerated Tyrodes solution. The intestinal sacs were kept for 1 h in a medium containing OS liposomes or OS non-formulated extract (120 μg/ml) in Tyrodes solution at 37°C with continuous agitation at 100 rpm using magnetic stirrer and with continuous aeration with air. Subsequently, sacs were removed, washed thoroughly from exterior with water, the content was collected and centrifuged at 10000 rpm for 5 min, and the supernatant was analyzed by HPLC . Peak area of the marker compounds was recorded, and the fold change in absorption was calculated by dividing peak area of compounds in OS liposomes by that of non-formulated extract (n = 3–4).
Male Sprague–Dawley rats were obtained from USM animal breeding facility, and were allowed to acclimatize for one week before the experiment. Experiment was performed according to the guidelines of USM Animal Ethics Committee (Ref. No.: USM/Animal Ethics Approval/2012/ (78) (399)).
Results are presented as average ± SD of triplicate experiments unless otherwise mentioned. Differences between groups were considered significant at P < 0.05 using Student’s t-test or One way ANOVA.