Enhanced Solubility and Permeability of Salicis cortex Extract by Formulating as a Microemulsion^[*]

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Materials
• Methods
• Preparation of Salicis cortex extract sample solution
• HPLC/diode array detector and HPLC/TOF-MS settings
• Method validation
• Selection of oil and surfactants
• Development of pseudo-ternary phase diagram
• Solubility of Salicis cortex extract into a microemulsion
• Droplet size, homogeneity, and ζ-potential measurements
• Morphological examination
• Chemical and physical stability
• Stability in simulated gastrointestinal fluids
• In vitro parallel artificial membrane permeability assay
• Caco-2 cell culture and cell viability studies
• Caco-2 transport studies
• References

Abstract

A microemulsion system was developed and investigated as a novel oral formulation to increase the solubility and absorption of Salicis cortex extract. This extract possesses many pharmacological activities, in particular, it is beneficial for back pain and osteoarthritic and rheumatic complaints. In this work, after qualitative and quantitative characterization of the extract and the validation of an HPLC/diode array detector analytical method, solubility studies were performed to choose the best components for microemulsion formulation. The optimized microemulsion consisted of 2.5 g of triacetin, as the oil phase, 2.5 g of Tween 20 as the surfactant, 2.5 g of labrasol as the cosurfactant, and 5 g of water. The microemulsion was visually checked, characterized by light scattering techniques and morphological observations. The developed formulation appeared transparent, the droplet size was around 40 nm, and the ζ-potential result was negative. The maximum loading content of Salicis cortex extract resulted in 40 mg/mL. Furthermore, storage stability studies and an in vitro digestion assay were performed. The advantages offered by microemulsion were evaluated in vitro using artificial membranes and cells, i.e., parallel artificial membrane permeability assay and a Caco-2 model. Both studies proved that the microemulsion was successful in enhancing the permeation of extract compounds, so it could be useful to ameliorate the bioefficacy of Salicis cortex.

Introduction

SC, also known as willow bark, consists of the whole or fragmented dried bark of young branches or whole dried pieces of current year twigs of various species of the genus Salix including Salix purpurea L., Salix daphnoides Vill., and Salix fragilis L. (Salicaceae). The main constituents are salicylic derivatives such as salicin and its related compounds, principally, salicortin, 2′-O-acetylsalicortin and tremulacin. Other molecules include flavonoids, in particular, the flavanones eriodictyol-7-O-glucoside, (+)- and (−)-naringenin-5-O-glucoside, and naringenin-7-O-glucoside, catechins, and polyphenols [1], [2]. SC extract resulted in being beneficial for low back pain and in mild osteoarthritic and rheumatic complaints. These activities were proven in vitro, in vivo, and by clinical studies. Commission E of the German Federal Health Agency has approved the use of willow bark preparations for the treatment of “diseases accompanied by fever, rheumatic ailments and headaches”, in a daily dose equivalent to 60 – 120 mg salicin [3], [4], [5], [6], [7]. However, as reported in the literature, the bioavailability of salicin after oral administration of willow bark extracts is low [8]. Flavonoids also contribute to the overall therapeutic effect of the extract, but many studies have shown that poor solubility, fast metabolism, and inadequate bioavailability hinder their clinical use [9].

Novel drug delivery systems could represent a valid tool to overcome these limitations and to improve the efficacy of willow bark extract after oral administration. It has been reported that nanoformulations ameliorate selectivity, increase stability, modulate the release of active principles, and reduce the required dose of extracts [10].

In the present work, an O/W ME was developed to improve the solubility of SC extract, knowing that the solubility is a crucial factor for oral bioavailability. MEs are defined as transparent, thermodynamically stable mixtures of oil and water stabilized by one or more emulsifiers [11]. Despite to the terminology, MEs consist of the smallest droplet sizes found in emulsion systems, specifically, ME droplet sizes range from 10 to 100 nm [12]. Generally, MEs form spontaneously without the input of energy and require high amounts of surfactants or emulsifiers [13]. MEs also possess unique characteristics as drug delivery systems, in particular, they offer the possibility to administer drugs in liquid form and, consequently, increase their absorption rate, avoiding disintegration processes and gastrointestinal enzymatic degradation [14], [15]. In addition, the small droplet sizes increase the surface area to volume ratio, a key factor for drug absorption, and enhance the stability of the system. Furthermore, MEs can incorporate both hydrosoluble and lipophilic molecules frequently present in phytoterapeutics and in herbal extracts due to the presence of a hydrophilic and hydrophobic phase [10], [16], [17], [18], [19], [20], [21]. Finally, the ability of MEs to form spontaneously results in an economical formation process, and they are suitable for almost any route of administration [11].

In previous studies, salicin was incorporated into an ME for topical application [22], and willow bark extract was formulated into nanostructured lipid carriers to increase its antioxidant activity [23]. The present work represents the first attempt to increase the solubility of SC extract and its absorption after oral administration by formulating as an ME.

In this study, an HPLC/DAD analytical method was optimized and validated for qualitative and quantitative characterization of SC extract. The selected components of ME were: triacetin as the oil phase, Tween 20 as the surfactant, and labrasol as the cosurfactant. Empty and extract-loaded MEs were physically and chemically investigated by light scattering techniques, electron microscopy, and chromatographic analyses. Chemical and physical stability were also assessed during storage for 1 month and after incubation in simulated gastrointestinal conditions.

The ability of ME to increase the permeability of SC extract was evaluated in vitro by artificial membranes and Caco-2 cells, after preliminary cytotoxicity studies. The data obtained from a passive diffusion permeability assay and a cell-based test were compared to elucidate the permeation/absorption mechanism of salicylic derivatives and flavanones.

Results and Discussion

The first step of this study concerned the qualitative and quantitative characterization of SC commercial extract by an HPLC/DAD analytical method. The wavelengths of 210, 260, 280, and 350 nm were selected for acquiring chromatograms of salicylic derivatives and flavanones. In order to achieve a good resolution, various percentages of mobile phase mixtures at different flows and several RP-C18 columns were tested. Finally, the conditions reported in the Materials and Methods section were selected because the peaks related to the characteristics constituents of the extract were separated. The chromatogram of a sample solution selecting a wavelength of 280 nm with identified compounds [salicin, salicortin, eriodictyol-7-O-glucoside, (+)- and (−)-naringenin-5-O-glucoside, naringenin-7-O-glucoside, isosalipurposide, tremulacin, naringenin] is reported in [Fig. 1].

The identification of SC extract constituents was obtained comparing UV spectrum, retention time, and MS data with those of standards or with the literature data. The structures and UV/MS data of (+)- and (−)-naringenin-5-O-glucoside and naringenin-7-O-glucosidewere are reported in Table S1, Supporting Information [24].

The HPLC/DAD analytical method was validated according to the criteria established by International Conference on Harmonisation guidelines [25], [26], relatively to salicin and naringenin-7-O-glucoside, the most representative compounds of salicylic derivatives and flavanones, respectively.

Firstly, the linearity range of response was determined on five levels of concentration of salicin and naringenin-7-O-glucoside with three injections for each level. Salicin and naringenin-7-O-glucoside showed a linear response from 100 to 650 µg/mL with a coefficient of linear correlation ≥ 0.993.

Regarding the reproducibility, the relative standard deviation for salicin and naringenin-7-O-glucoside solutions was 0.81 and 0.55%, respectively.

The repeatability of the method was evaluated using methanolic solutions of SC extract at three different level of concentration. The RSDs were 1.86, 3.09, and 2.08% for salicylic derivatives and 1.80, 2.42, and 2.19% for flavanones.

Intermediate precision relative to salicin and naringenin-7-O-glucoside was also calculated. For the first sample (2.04 mg/mL), the RSDs of salicin were 1.90, 2.86, and 1.90% and for naringenin-7-O-glucoside, the RSDs were 1.82, 2.14, and 2.03%. Precision inter-day for salicin was 2.25% and for naringenin-7-O-glucoside, it was 1.99%. For the second sample (3.30 mg/mL), the RSDs of salicin were 3.10, 1.95, and 2.03% and for naringenin-7-O-glucoside, they were 2.14, 1.41, and 0.93%. The precision inter-day for salicin was 2.36% and for naringenin-7-O-glucoside, it was 1.49%. For the third sample (4.03 mg/mL), the RSD of salicin were 1.11, 0.73, and 0.72%, and 0.96, 1.42, and 1.44% for naringenin-7-O-glucoside. The precision inter-day for salicin was 0.85% and for naringenin-7-O-glucoside, it was 1.27%.

The accuracy was determined by analyzing the percentage recovery of salicin and naringenin-7-O-glucoside in the solutions of SC extracts in three concentration levels (2.00, 2.67, and 4.00 mg/mL) after spiking 100 µg/mL of salicin and 50 µg/mL of naringenin-7-O-glucoside separately to the extract solutions. The percentage recoveries of salicin were 106.67 ± 8.92, 103.99 ± 1.32, and 106.84 ± 1.45%, and for naringenin-7-O-glucoside, they were 119.70 ± 0.42, 123.56 ± 1.11, and 111.00 ± 0.25%.

Regarding the peak purity (purity factors were 999.996 for salicin and 999.999 for naringenin-7-O-glucoside), no deviations were seen overlaying the UV spectra at the beginning, at the apex, and at the end of the salicin and naringenin-7-O-glucoside peaks.

The limits of detection of salicin and naringenin-7-O-glucoside were 3.25 and 0.25 ng, respectively, (the signal-to-noise ratio was 3), while the limits of quantification (signal-to-noise ratio = 10) of these molecules were 16.25 and 2.51 ng, respectively.

Thus, based on the obtained results, HPLC/DAD conditions could be considered specific and useful for the characterization of SC extract. The percentage w/w of each compound in the SC extract ([Fig. 1]) was as follows: salicin (1) 0.8%; salicin derivative (2) 0.6%; salicin derivative (3) 0.7%; salicin derivative (4) 0.9%; salicin derivative (5) 1.2%; salicortin (6) 4.3%; eriodictyol-7-O-glucoside (7) 3.0%; (+)- and (−)-naringenin-5-O-glucoside (8) 1.1%; naringenin-7-O-glucoside (9) 0.5%; isosalipurposide (10) 0.5%; tremulacin (11) 0.5%; naringenin (12) 0.1%. Total flavanone and salicylic derivative contents were 4.7 and 9.5%, respectively.

The components of the ME were selected on the basis of the solubility study of SC extract in various oils and surfactants ([Table 1]). The selected oils were olive oil, wheat germ oil, sunflower oil, almond oil, hemp oil, borage oil, triacetin, oleic acid, and tocopherol acetate. The surfactants were Tween 20, cremophor EL, labrasol, PEG 400, and ethanol.

Safety of the vehicles and solubility of the extract are the most important factors in choosing an oil and surfactants. Among the oils tested, the solubility of SC extract was highest in triacetin, which was selected as the oil phase. Triacetin, or glyceryl triacetate, is widely present in oral pharmaceutical formulations because it is nontoxic, nonirritating, and biocompatible. Moreover, its water solubility and mixability makes it an ideal oil phase for ME development [27], [28].

Regarding the selection of surfactants, it was demonstrated that the hydrophilic-lipophilic balance value appropriate to form a stable O/W ME must be greater than 10 [29]. For this reason, Tween 20 and labrasol were chosen as the surfactant and cosurfactant, respectively, as well as considering their high solubilizing capacity toward the SC extract. Tween 20 (also known as polysorbate 20) is a nonionic surfactant used to increase oral bioavailability of drugs due to its capacity to improve solubility and inhibit ABC transporter-mediated cellular efflux [30], [31]. Labrasol consists of a small fraction of mono-, di-, and triglycerides and mainly PEG-8 (MW 400) mono- and diesters of caprylic and capric acid. In this research, labrasol was used together with Tween 20 to achieve transient negative interfacial tension and fluid interfacial film. It is also reported that labrasol can increase membrane permeability and inhibit secretory systems in the intestinal epithelium [32], thus, it could be useful to increase oral absorption of SC extract.

To obtain the existing region of ME, water was added dropwise to the oil/surfactant-cosurfactant (S_m) mixture. Ternary compositions were visually analyzed to determine if a transparent ME, gel, cloudy, or milky emulsion was formed. [Fig. 2] depicts the phase diagram constructed using different combinations of oil, surfactants, and water.

The final composition of developed ME was 2.5 g of triacetin (20% w/w) chosen as the oil phase, 2.5 g of Tween 20 (20% w/w) as the surfactant, 2.5 of labrasol (20% w/w) as the cosurfactant, and 5 mL of deionized water (40% w/w). The ME became light brown after the addition of the extract, but it resulted in being transparent, proof of the stability of the formulation.

The solubility of SC extract into a developed ME was evaluated by adding increasing amounts (from 5 mg/mL until 50 mg/mL) to the system. Resulting samples were visually checked and analyzed by HPLC/DAD after the elimination of any undissolved extract. No phase separation or turbidity was detected, and the SC extract resulted in being completely solubilized into an ME at the concentration of 40 mg/mL, while in water, the amount of dissolved extract was below 7 mg/mL.

Furthermore, as shown in [Table 2], the ME increased considerably the solubility of salicylic derivatives and flavanones in respect to water (about 3.6 times for the salicylic derivatives and about 2 times for the flavanones). This is probably due to a synergistic effect between triacetin, water, and S_m, and to chemical interactions between ME ingredients and extract compounds.

DLS analysis revealed that the empty ME was composed of dispersed droplets with a mean diameter of around 30 – 40 nm and a PdI below 0.2, and confirmed that in the presence of 40 mg/mL of SC extract, size and homogeneity of the system did not change significantly. ζ-Potential values ranged from − 11 to − 14 mV, indicating that the developed ME were negatively charged, signifying the stability of the formulations ([Table 3]). Indeed, it is known that an increase of electrostatic repulsive forces between ME droplets prevents coalescence phenomena.

TEM observations proved the presence of spherical and non-aggregated droplets with a size less 50 nm, both for empty and SC-MEs ([Figs. 3] and [4]).

According to the data reported in the literature about physical and morhpological properties of MEs [33], these results suggest that the optimized formulation represents a suitable vehicle for oral delivery of SC extract.

Stability of empty and SC-MEs was evaluated at 4 and 25 °C for 4 weeks. During this period, the systems were stable, as no phase separation or creaming was observed. Moreover, physical parameters, i.e., size, homogeneity, and ζ-potential values, were unchanged. The concentration of salicylic derivatives and flavanones, calculated by HPLC analysis, was not changed at 4 and 25 °C. The obtained results suggested that the developed formulations were stable during the storage period and they were able to prevent the degradation of loaded molecules.

It is known that gastrointestinal enzymes will digest coarse emulsions, so one of the aims of this work was the development of an innovative formulation able to protect SC extract from an unfavorable environment after oral administration. For this purpose, physical and chemical properties of the ME were assessed after exposure to SGF and SIF.

After 2 h of incubation in gastric conditions followed by 2 h of incubation in a simulated intestinal environment, the size remained around 40 nm with a PdI lower than 0.25 ([Table 4]). Coalescence or separation phenomena were not observed after visual inspection. This stability in acidic pH and in intestinal conditions may be due to the presence of nonionic surfactants, Tween 20, and labrasol, which reduce the flocculation and coalescence phenomena.

The ME is able to prevent the enzymatic degradation of the entrapped extract before intestinal absorption. In fact, the concentration of the main constituents of SC did not decrease during the time of the assay ([Table 4]).

Furthermore, the optimized ME demonstrated excellent physical stability in gastrointestinal conditions, thus it represents a promising strategy to deliver a high quantity of extract and to enhance its intestinal permeability when orally administered.

PAMPA was performed to estimate passive transcellular permeability. PAMPA is a non-cell-based permeability model, but it is considered robust, reproducible, or fast [34], [35], [36]. It was demonstrated that PAMPA can be used to predict the passive permeability through the gastrointestinal tract of constituents in a complex plant extract [37]. Based on the results obtained in our previous studies [19], [20], [21], [38], this in vitro assay was useful not only in the preformulation studies of single molecules but also for herbal extracts loaded into innovative drug delivery systems, i.e., MEs and solid lipid nanoparticles. Considering that most compounds reach the blood stream by passive mechanism, PAMPA has been proposed as a helpful complement to the Caco-2 assay to predict the oral absorption of SC extract.

The maximum P_e values were found after 4 h of incubation. Furthermore, the amount of SC extract diffused into the acceptor compartment and the P_e of salicylic derivatives and flavanones has improved considerably in the case of ME formulation with respect to water solution. This may be due to the increased lipophilicity of the extract. The P_e values and the permeated amounts of salicylic derivatives and flavanones are reported in [Table 5].

A recovery above 80% was achieved for the SC-ME and for the aqueous solution ([Table 5]), as is required for an acceptable permeation prediction [39], [40], [41]. Moreover, this value means that there was neither relevant SC extract membrane retention nor binding to the surface of the filter. The results confirm that developed formulation represents a successful attempt to ameliorate passive permeation of SC.

Permeation studies were also performed using a cell-based model in order to complete in vitro characterization of the SC-ME. Indeed, an understanding of oral absorption mechanisms is crucial for the elaboration of novel oral formulation. For this purpose, Caco-2 cells are considered the most predictive in vitro model to estimate not only passive intestinal diffusion, as previously described for PAMPA, but also active transport processes, paracellular permeability, and active efflux [39], [40], [41]. However, for the differentiation and preparation of a fully functional monolayer, cells require around 3 weeks.

Firstly, the potential cytotoxicity of the SC-ME was investigated to select the concentration for permeation studies. As reported in [Fig. 5], the optimized formulation had a very low toxicity, proven by the cell viability value above 80%. The SC-ME was diluted 50-fold for permeation studies considering that after 24 h of incubation at this concentration, the percentage of cell death was negligible compared to the positive control. This is an encouraging result considering that the ME undergoes a dilution from 1 : 200 to 1 : 1000 with biological fluids after oral administration.

Furthermore, LY was used to prove the absence of monolayer cleavage before performing the transport experiments. The LY passage resulted in less than 2%, confirming the integrity of the layer.

P_app values are given in [Table 6]. It can be observed that the permeation of salicylic derivatives and flavanones was improved when SC extract is formulated into the ME with respect to the saturated aqueous solution. P_app values were found above 1 × 10⁻⁵, signifying the high permeability properties of the ME. This is probably due to the small droplets of ME characterized by a big surface area, which leads to an increased solubility and absorption. Moreover, nonionic surfactants, such as labrasol and Tween 20, also contributed to increase extract solubility, avoid degradation phenomena, extend the contact time with the absorption site, increase endocytic and transcellular pathways, and inhibit P-glycoprotein efflux activity [30], [31], [32]. As in the case of PAMPA, also in this case, the recovery resulted above 80%, as required for acceptable in vitro permeation prediction ([Table 6]).

The obtained results clearly demonstrated that both in vitro models, i.e., PAMPA and Caco-2 cell line, could be synergistically applied for the correct estimation of drug permeability and intestinal absorption, and future studies will be aimed to evaluate pharmacokinetic parameters to confirm the potential of the developed ME.

Materials and Methods

Materials

SC (willow bark) dry extract was supplied by Bionorica SE (sample number: PM 14 – 168, BNO 1455, Batch number: 0000083735, DEV: 8 – 17 : 1). The extract was standardized according to the Ph. Eur. criteria (minimum 5.0 percent of total salicylic derivatives, expressed as salicin). Storage: tightly closed, dry, protected from light, 15 – 25 °C. Voucher specimen: 54 – 2015.

Salicin (purity ≥ 99%, HPLC), naringenin (purity ≥ 99%, HPLC), naringenin-7-O-glucoside (purity ≥ 99%, HPLC), and eriodictyol-7-O-glucoside (purity ≥ 99%, HPLC) were from Extrasynthese; tremulacin (purity ≥ 82%, HPLC) was from Sigma-Aldrich. Olive oil was from Olivia, wheat germ oil was purchased from Aboca, sunflower oil was from Coop, and Tween 20, Tween 40, cremophor EL, DL- α -Tocopherol acetate (Vitamin E), triacetin, and PEG 400 were purchased from Sigma-Aldrich. Hempseed oil, almond oil, borage oil, and propylene glycol were from Galeno srl, oleic acid was purchased from Carlo Erba Spa, and labrafil andlabrasol ECH were a gift from Gattefossé.

Ethanol analytical reagent, acetonitrile and methanol HPLC grade, formic acid (98%), cholesterol, lecithin, dichloromethane, DMSO, 1,7-octadiene (98%), PBS bioperformance certified, lipase from porcine pancreas, pepsin from porcine gastric mucose, bile salts, HCl, and NaOH were purchased from Sigma-Aldrich. All the substances for electron microscopy were purchased from Electron Microscopy Sciences.

Methods

Preparation of Salicis cortex extract sample solution

Twenty millilitres of methanol were added to 500 mg of SC extract. The resulting mixture was sonicated for 30 min in an ultrasonic bath, filtered, and dried under vacuum. The obtained residue, exactly weighted, was solubilized in methanol for HPLC/DAD/TOF-MS studies.

HPLC/diode array detector and HPLC/TOF-MS settings

Qualitative and quantitative analyses were performed using an HP 1100 liquid chromatograph with a DAD controlled with an HP 9000 workstation (Agilent Technologies). A 150 mm × 4.6 mm i. d., 5 µm Zorbax Eclipse XDB, RP18 column was used. The eluents were (A) HCOOH/water pH 3.2 and (B) CH₃CN. The multistep linear solvent gradient applied was: 0 – 5 min 15% B; 5 – 10 min 15 – 20% B; 10 – 15 min 20 – 30% B; 15 – 20 min 30 – 40% B; 20 – 25 min 40 – 15% B; equilibration time 10 min; oven temperature 30 °C; flow rate 0.6 mL/min. The chromatograms were registered at different wavelengths: 210, 260, 280, and 350 nm.

HPLC-MS analyses were conducted by the same liquid chromatograph apparatus with a DAD coupled to a TOF mass spectrometer equipped with an electrospray interface (all from Agilent Technologies). Analysis parameters were set using the negative ion mode with spectra acquired over a mass range of 100 – 1000 m/z. The conditions of the electrospray source were as follows: drying gas (N₂) temperature, 350 °C; drying gas flow rate, 6 L/min; nebulizer 20 psi; capillary voltage, 4000 V; fragmentation, 150 V; skimmer, 60 V. The other parameters were the same as for the HPLC/DAD analyses.

Method validation

HPLC/DAD method validation was performed according to international regulatory guidelines [25], [26]. Linearity, accuracy, precision, reproducibility, repeatability, limit of quantification, and limit of detection were estimated using salicin and naringenin-7-O-glucoside reference standards as representative compounds of salicylic derivatives and flavanones, respectively. Linearity was investigated after three injections of each level of five different concentrations. The regression parameters were calculated by linear regression analysis.

Accuracy was determined by evaluating the percentage recovery of salicin and naringenin-7-O-glucoside from three preparations of SC extract at different concentrations (2.00, 2.67, and 4.00 mg/mL). Each sample was injected three times. Then, 100 µg/mL of salicin and 50 µg/mL of naringenin-7-O-glucoside were added separately to the three batches of the extract. The reproducibility was calculated after 10 injections of salicin (0.65 mg/mL) and naringenin-7-O-glucoside (0.50 mg/mL) methanolic solutions. The repeatability was evaluated estimating the RSD of salicylic derivatives and flavanone content after three injections of each SC extract solution at three different concentrations (2.04, 3.30, and 4.03 mg/mL).

The intermediate precision was determined by injecting the samples previously used for the repeatability evaluation six times on three different days. The peak purity of salicin and naringenin-7-O-glucoside was investigated overlaying the UV spectra at the beginning, at the apex, and at the end of the peaks.

The limit of detection and limit of quantification of salicin and naringenin-7-O-glucoside were estimated by the means of the signal-to-noise ratio. A signal-to-noise ratio of 3 : 1 is generally considered acceptable for estimating the detection limit. The sample that produces a signal-to-noise ratio of approximately 10 : 1 corresponds to the concentration at which the analyte can be reliably quantified.

Selection of oil and surfactants

Solubility studies of SC extract were performed in various oils (olive oil, wheat germ oil, sunflower oil, almond oil, hemp oil, borage oil, triacetin, oleic acid, and tocopherol acetate) and surfactants (tween 20, cremophor EL, labrasol, peg 400, and ethanol) to select the best components for ME formulation. Increased amounts of SC extract were added to an exact volume of each selected vehicle until saturation and the resulted samples were stirred for 24 h at room temperature. Then, the mixtures were centrifuged at 13 148 × g for 10 min, and the concentration of salicylic derivatives and flavanones was determined using the chromatographic method reported in the previous section, after appropriate dilution with a CH₃OH/CH₂Cl₂ (3 : 2 volume ratio) mixture.

Development of pseudo-ternary phase diagram

Based on solubility studies, a pseudo-ternary phase diagram was constructed by a water titration technique using triacetin as oil the phase, labrasol as the surfactant, and Tween 20 as the cosurfactant. Labrasol and Tween 20 were mixed at different ratios under vigorous stirring to obtain the S_m. Then, various combinations (from 1 : 9 to 9 : 1 volume ratio) of triacetin and S_m were tested, and water was added dropwise to each blend, under stirring at room temperature. During the addition of water, the changes in the samples feature were monitored to determinate if the emulsion, gel, or ME was present. The points from transparent to cloudy and cloudy to transparent were considered as the emulsion and ME, respectively.

Solubility of Salicis cortex extract into a microemulsion

After the elaboration of the pseudo-ternary phase diagram, the maximum loading content of SC extract into the formulation was evaluated adding increasing amount of extract powder to the ME under stirring. The samples were shaken for 24 h at room temperature and sheltered from light. Afterwards, the undissolved powder was removed by centrifugation at 13 148 × g for 10 min and salicylic derivatives and flavanone content were quantified by chromatographic analyses after appropriate dilution with a CH₃OH/CH₂Cl₂ (3 : 2 v/v) mixture.

Droplet size, homogeneity, and ζ-potential measurements

The Z_average, particle size distribution expressed as PdI, and ζ-potential were analyzed by a light scattering apparatus (Ζsizer Nano series ZS90; Malvern Instruments). All measurements were performed in triplicate after proper dilution with distilled water.

Morphological examination

Morphological features of the formulations were examined by TEM JEOL 1010. PTA 1% w/v was used as a negative stain for the empty and SC-ME.

Chemical and physical stability

The empty and SC-ME were stored in sealed glass containers at 4 and 25 °C for 30 days to estimate the shelf life of the optimized formulations. Chemical and physical properties were checked periodically to monitor transparency, phase separation, and color variation as well as the changes in particle size, ζ-potential, and extract concentration by visual observations, light scattering, and HPLC analyses.

Stability in simulated gastrointestinal fluids

Stability of the developed ME in simulated gastrointestinal fluids was evaluated by adding an equal volume of SGF. The composition of SGF was 0.32% w/v pepsin, 2 g of NaCl, and 7 mL HCl dissolved in 1 L water (pH adjusted to 2.0 using 1 M HCl). The resulting mixture was kept at 37 °C under continuous shaking for 2 h, after which droplet size, PdI, and SC extract content were measured [42].

After digestion in SGF, the sample was incubated in SIF containing digestive enzymes (lipase 0.4 mg/mL, bile salts 0.7 mg/mL, pancreatin 0.5 mg/mL, and CaCl₂ 750 mM at pH 7.0) for 2 h at 37 °C. At the end of the experiment, physical and chemical stability were investigated by DLS and HPLC analyses [42].

In vitro parallel artificial membrane permeability assay

The experiments were performed using the optimized conditions reported in our recent works with slight modifications [20], [21]. A lecithin and cholesterol mixture, 10 and 8 g/L, respectively, previously dissolved in 1,7-octadiene was applied to each PVDF (Polyvinylidene difluoride) filter as an artificial membrane. A DMSO/PBS (0.05 mL/mL, pH 7.4) combination was chosen as the acceptor buffer. Immediately after the deposition of the artificial membrane (5 µL), 250 µL of extract containing samples (saturated aqueous solution and SC-ME) were added to the donor compartments. After 4 h of incubation, flavanone and salicylic derivative content into the donor and receptor compartments was evaluated by HPLC. The compoundsʼ permeability and recovery were calculated according the equation reported in the literature [21], [43], [44]:

where

P_e = effective permeability (cm/s). A = effective filter area (cm²), V_D and V_A = volumes in the acceptor and the donor compartments (mL), t = incubation time (s), [Compound]_A and [Compound]_D = concentrations of the compound in the acceptor and donor compartments at time t, [Compound]_D0 = compound concentration in the donor compartment at time 0.

Caco-2 cell culture and cell viability studies

The human colon carcinoma Caco-2 cell line was kindly provided by Professor Emanuela Masini (University of Florence). Caco-2 cells were seeded in DMEM (or DME) supplemented with 20% heat-inactivated fetal calf serum, 1% L-glutamine, and 1% penicillin/streptomycin, grown at 37 °C in a humidified atmosphere containing 5% CO₂ and passaged at a confluence of 80% [20], [21].

To assess cell viability after sample exposure, the MTS assay was performed. Cells were transferred to flat bottomed 96-well tissue culture plates at a seeding density of 5 × 10³ cells/well and allowed to grow for 24 h. When the cells were approximately 80% confluent, they were incubated with different concentrations of the SC-ME for 24 h. Then, the cells were exposed to MTS solution and allowed to incubate for 24 h at 37 °C. The product of the reaction was measured at 490 nm using a spectrophotometer. Cell death was expressed as a percentage compared to the values obtained from the positive controls, i.e., cells incubated only in DMEM [20], [21].

Caco-2 transport studies

For permeability assays, cells were seeded at a density of 50 × 10³ cells/well in cell culture inserts with PET membranes (0.4 µm pore size, 1.12 cm² growth surface area; BRAND). DMEM was added into the apical (A) and basolateral (B) compartments and changed daily. After seeding, Caco-2 cells were let to differentiate for 3 weeks.

LY (λ _ex = 485 nm, λ _em = 530 nm) at a concentration of 100 mM was used as an integrity control marker [45]. After dilution in transport medium (HBSS with Ca²⁺, Mg²⁺, 25 mM HEPES, pH 7.4), LY was put into A and after 1 h of incubation, the amount permeated into the B chamber was calculated using a fluorescence plate reader to estimate the percentage of cleavage. The maximum permeability of LY to prove the integrity of the layers was established to be not more than 3% with respect to the initial amount added into the AP compartment.

Before the transport study, DMEM was replaced with preheated (37 °C) transport medium. After the cell monolayer was equilibrated for 30 min at 37 °C, Caco-2 cells were exposed to 0.5 mL SC-ME diluted in buffer medium and SC aqueous solution, while the B chamber contained only HBSS. At the end of the assay, the concentration of salicylic derivatives and flavanones was calculated both in the A and B chambers by HPLC and expressed as salicin and naringenin 7-O-glucoside, respectively, as reported in the “Method validation” section. Furthermore, the integrity of the layer was reevaluated with the LY permeability assay as described above.

P_app coefficients (cm/s) and recovery for active constituents of SC extract were calculated according to the equation reported in the literature [39], [40], [41].

where ΔQ/Δt indicates linear appearance rate of mass in B, C₀ is the initial concentration in A, and S is the surface area (i.e., 1.12 cm²). Recovery was calculated as reported in the “In vitro parallel artificial membrane permeability assay” section.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

Financial support was granted by the Global Research Initiative 2013/2014 of Bionorica SE, Neumarkt, Germany. TEM observations were thanks to Dr. Maria Cristina Salvatici, Centro di Microscopie Elettroniche “Laura Bonzi”, ICCOM, National Research Council (CNR), Florence, Italy.

^* Dedicated to Professor Dr. Robert Verpoorte in recognition of his outstanding contribution to natural products research.

• References

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  CrossrefPubMedSearch in Google Scholar

Correspondence

• References

• 1 Meier B, Lehmann D, Sticher O, Bettschart A. Identifkation und Bestimmung von je acht Phenolglykosiden in Salix purpurea und Salix daphnoides mit moderner HPLC. Pharm Acta Helv 1985; 60: 269-275
  PubMedSearch in Google Scholar
• 2 ESCOP. Monograph: Salicis cortex (willow bark). Fascicule 4. Exeter, UK: European Scientific Cooperative on Phytotherapy; 1997
  Search in Google Scholar
• 3 Blumenthal M, Busse WR. American Botanical Council, Integrative Medicine Communications, Germany Bundesgesundheitsamt. Commission E. The complete German Commission E monographs: therapeutic Guide to herbal Medicines. Boston, Ma.: American Botanical Council; 1998
  Search in Google Scholar
• 4 Chrubasik S, Eisenberg E, Balan E, Weinberger T, Luzzati R, Conradt C. Treatment of low back pain exacerbations with willow bark extract: a randomized double-blind study. Am J Med 2000; 109: 9-14
  CrossrefPubMedSearch in Google Scholar
• 5 Chrubasik S, Künzel O, Black A, Conradt C, Kerschbaumer F. Potential economic impact of using a proprietary willow bark extract in outpatient treatment of low back pain: an open non-randomized study. Phytomedicine 2001; 8: 241-251
  CrossrefPubMedSearch in Google Scholar
• 6 Mills SY, Jacoby RK, Chacksfield M, Willoughby M. Effect of a proprietary herbal medicine on the relief of chronic arthritic pain: a double-blind study. Rheumatology 1996; 35: 874-878
  CrossrefPubMedSearch in Google Scholar
• 7 Long L, Soeken K, Ernst E. Herbal medicines for the treatment of osteoarthritis: a systematic review. Rheumatology 2001; 40: 779-793
  CrossrefPubMedSearch in Google Scholar
• 8 Pentz R, Busse HG, König R, Siegers CP. Bioverfügbarkeit von Salicylsäure und Coffein aus einem phytoanalgetischen Kombinationspräparat. Z Phytother 1989; 10: 92-96
  PubMedSearch in Google Scholar
• 9 Thilakarathna SH, Rupasinghe HP. Flavonoid bioavailability and attempts for bioavailability enhancement. Nutrients 2013; 5: 3367-3387
  CrossrefPubMedSearch in Google Scholar
• 10 Bilia AR, Piazzini V, Guccione C, Risaliti L, Asprea M, Capecchi G, Bergonzi MC. Improving on nature: The role of nanomedicine in the development of clinical natural drugs. Planta Med 2017; 83: 366-381
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Callender SP, Mathews JA, Kobernyk K, Wettig SD. Microemulsion utility in pharmaceuticals: Implications for multi-drug delivery. Int J Pharm 2017; 526: 425-442
  CrossrefPubMedSearch in Google Scholar
• 12 McClements DJ. Nanoemulsions versus microemulsions: terminology, differences, and similarities. Soft Matter 2012; 8: 1719-1729
  CrossrefPubMedSearch in Google Scholar
• 13 Rosen MJ, Kunjappu JT. Surfactants and interfacial Phenomena. 4th ed. New York: John Wiley & Sons; 2012
  Search in Google Scholar
• 14 Shen Q, Li X, Li W, Zhao X. Enhanced intestinal absorption of daidzein by borneol/menthol eutectic mixture and microemulsion. AAPS PharmSciTech 2011; 12: 1044-1049
  CrossrefPubMedSearch in Google Scholar
• 15 Kee JL, Hayes ER, McCuistion LE. Pharmacology: a patient-centered nursing Process Approach. 8th ed. St. Louis, Missouri: Elsevier Health Sciences; 2014
  Search in Google Scholar
• 16 Liu W, Zhai Y, Heng X, Che FY, Chen W, Sun D, Zhai G. Oral bioavailability of curcumin: problems and advancements. J Drug Target 2016; 24: 694-702
  CrossrefPubMedSearch in Google Scholar
• 17 Gupta S, Kesarla R, Omri A. Formulation strategies to improve the bioavailability of poorly absorbed drugs with special emphasis on self-emulsifying systems. ISRN Pharm 2013; 2013: 1-13
  PubMedSearch in Google Scholar
• 18 Bilia AR, Isacchi B, Righeschi C, Guccione C, Bergonzi MC. Flavonoids loaded in nanocarriers: an opportunity to increase oral bioavailability and bioefficacy. Food Nutr Sci 2014; 5: 1212-1227
  CrossrefPubMedSearch in Google Scholar
• 19 Bergonzi MC, Hamdouch R, Mazzacuva F, Isacchi B, Bilia AR. Optimization, characterization and in vitro evaluation of curcumin microemulsions. LWT-Food Sci Technol 2014; 59: 148-155
  PubMedSearch in Google Scholar
• 20 Piazzini V, Monteforte E, Luceri C, Bigagli E, Bilia AR, Bergonzi MC. Nanoemulsion for improving solubility and permeability of Vitex agnus-castus extract: formulation and in vitro evaluation using PAMPA and Caco-2 approaches. Drug Deliv 2017; 24: 380-390
  CrossrefPubMedSearch in Google Scholar
• 21 Piazzini V, Rosseti C, Bigagli E, Luceri C, Bilia AR, Bergonzi MC. Prediction of permeation and cellular transport of Silybum marianum extract formulated in a nanoemulsion by using PAMPA and Caco-2 cell models. Planta Med 2017; 56: 222-248
  Thieme ConnectPubMedSearch in Google Scholar
• 22 Schonrock U, Steckel F, Kux U, Inoue K. Use of salicin as an anti-irritative active compound in cosmetic and topical dermatological preparations. U.S. Patent 5, 876, 737; 1999. Available at: https://patents.google.com/patent/US5876737A/e Accessed April 20, 2018
  PubMed
• 23 Mitrea E, Lacatusu I, Badea N, Ott C, Oprea O, Meghea A. New approach to prepare willow bark extract – lipid based nanosystems with enhanced antioxidant activity. J Nanosci Nanotechnol 2015; 15: 4080-4089
  CrossrefPubMedSearch in Google Scholar
• 24 Kammerer B, Kahlich R, Biegert C, Gleiter CH, Heide L. HPLC-MS/MS analysis of willow bark extracts contained in pharmaceutical preparations. Phytochem Anal 2005; 16: 470-478
  CrossrefPubMedSearch in Google Scholar
• 25 Guideline ICH Harmonized Tripartite. Validation of analytical procedures: methodology. Text and Methodology Q2 (R1) (2005). Available at: http://www.ich.org/cache/compo/363-272-1.html Accessed 2010
  PubMed
• 26 European Medicines Agency. European Medicines Agency (EMA): Note for Guidance on Validation of Analytical Procedures: Text and Methodology (CPMP/ ICH/381/95). EMA Guidelines 2012; 44: 1-23 Available at http://www.ema.europa.eu Accessed April 20, 2018
  PubMedSearch in Google Scholar
• 27 Sharma G, Wilson K, Van der Walle CF, Sattar N, Petrie JR, Kumar MR. Microemulsions for oral delivery of insulin: design, development and evaluation in streptozotocin induced diabetic rats. Eur J Pharm Biopharm 2010; 76: 159-169
  CrossrefPubMedSearch in Google Scholar
• 28 Fiume MZ. Final report on the safety assessment of triacetin. Int J Toxicol 2003; 22: 1-10
  CrossrefPubMedSearch in Google Scholar
• 29 Shinde UA, Modani SH, Singh KH. Design and development of repaglinide microemulsion gel for transdermal delivery. AAPS PharmSciTech 2018; 19: 315-325
  CrossrefPubMedSearch in Google Scholar
• 30 Nielsen CU, Abdulhussein AA, Colak D, Holm R. Polysorbate 20 increases oral absorption of digoxin in wild-type Sprague Dawley rats, but not in mdr1a (−/−) Sprague Dawley rats. Int J Pharm 2016; 513: 78-87
  CrossrefPubMedSearch in Google Scholar
• 31 Woodcock DM, Linsenmeyer ME, Chojnowski G, Kriegler AB, Nink V, Webster LK, Sawyer WH. Reversal of multidrug resistance by surfactants. Br J Cancer 1992; 66: 62-68
  CrossrefPubMedSearch in Google Scholar
• 32 Sha X, Yan G, Wu Y, Li J, Fang X. Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells. Eur J Pharm Sci 2005; 24: 477-486
  CrossrefPubMedSearch in Google Scholar
• 33 Zhang P, Liu Y, Feng N, Xu J. Preparation and evaluation of self-microemulsifying drug delivery system of oridonin. Int J Pharm 2008; 355: 269-276
  CrossrefPubMedSearch in Google Scholar
• 34 Kansy M, Senner F, Gubernator K. Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes. J Med Chem 1998; 41: 1007-1010
  CrossrefPubMedSearch in Google Scholar
• 35 Alvarez-Figueroa MJ, Pessoa-Mahana CD, Palavecino-González ME, Mella-Raipán J, Espinosa-Bustos C, Lagos-Muñoz ME. Evaluation of the membrane permeability (PAMPA and skin) of benzimidazoles with potential cannabinoid activity and their relation with the Biopharmaceutics Classification System (BCS). AAPS PharmSciTech 2011; 12: 573-578
  CrossrefPubMedSearch in Google Scholar
• 36 Nekkanti V, Wang Z, Betageri GV. Pharmacokinetic evaluation of improved oral bioavailability of valsartan: proliposomes versus self-nanoemulsifying drug delivery system. AAPS PharmSciTech 2016; 17: 851-862
  CrossrefPubMedSearch in Google Scholar
• 37 Petit C, Bujard A, Skalicka-Wozniak K, Cretton S, Houriet J, Christen P, Carrupt PA, Wolfender JL. Prediction of the passive intestinal absorption of medicinal plant extract constituents with the parallel artificial membrane permeability assay (PAMPA). Planta Med 2016; 82: 424-431
  Thieme ConnectPubMedSearch in Google Scholar
• 38 Graverini G, Piazzini V, Landucci E, Pantano D, Nardiello P, Casamenti F, Pellegrini-Giampietro DE, Bilia AR, Bergonzi MC. Solid lipid nanoparticles for delivery of andrographolide across the blood-brain barrier: in vitro and in vivo evaluation. Colloid Surf B Biointerfaces 2018; 161: 302-313
  CrossrefPubMedSearch in Google Scholar
• 39 Hubatsch I, Ragnarsson EG, Artursson P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat Protoc 2007; 2: 2111-2119
  CrossrefPubMedSearch in Google Scholar
• 40 Palmgrén JJ, Mönkkönen J, Korjamo T, Hassinen A, Auriola S. Drug adsorption to plastic containers and retention of drugs in cultured cells under in vitro conditions. Eur J Pharm Biopharm 2006; 64: 369-378
  CrossrefPubMedSearch in Google Scholar
• 41 Heikkinen AT, Mönkkönen J, Korjamo T. Kinetics of cellular retention during Caco-2 permeation experiments: role of lysosomal sequestration and impact on permeability estimates. J Pharmacol Exp Ther 2009; 328: 882-892
  CrossrefPubMedSearch in Google Scholar
• 42 Aditya NP, Shim M, Lee I, Lee Y, Im MH, Ko S. Curcumin and genistein coloaded nanostructured lipid carriers: in vitro digestion and antiprostate cancer activity. J Agric Food Chem 2013; 61: 1878-1883
  CrossrefPubMedSearch in Google Scholar
• 43 Wohnsland F, Faller B. High-throughput permeability pH profile and high-throughput alkane/water log P with artificial membranes. J Med Chem 2001; 44: 923-930
  CrossrefPubMedSearch in Google Scholar
• 44 Sugano K, Hamada H, Machida M, Ushio H. High throughput prediction of oral absorption: improvement of the composition of the lipid solution used in parallel artificial membrane permeation assay. J Biomol Screen 2001; 6: 189-196
  CrossrefPubMedSearch in Google Scholar
• 45 Iacomino G, Fierro O, DʼAuria S, Picariello G, Ferranti P, Liguori C, Addeo F, Mamone G. Structural analysis and Caco-2 cell permeability of the celiac-toxic A-gliadin peptide 31–55. J Agric Food Chem 2013; 61: 1088-1096
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material