Polyphenolic Extract from Sambucus ebulus L. Leaves Free and Loaded into Lipid Vesicles

The paper deals with the preparation and characterisation of hydroalcoholic polyphenolic extract from Sambucus ebulus (SE) leaves that was further loaded into three-types of lipid vesicles: liposomes, transfersomes, and ethosomes, to improve its bioavailability and achieve an optimum pharmacological effect. For Sambucus ebulus L.-loaded lipid vesicles, the entrapment efficiency, particle size, polydispersity index and stability were determined. All prepared lipid vesicles showed a good entrapment efficiency, in the range of 75–85%, nanometric size, low polydispersity indexes, and good stability over three months at 4 °C. The in vitro polyphenols released from lipid vehicles demonstrated slower kinetics when compared to the free extract dissolution in phosphate buffer solution at pH 7.4. Either free SE extract or SE extract loaded into lipid vesicles demonstrated a cytoprotective effect, even at low concentration, 5 ug/mL, against hydrogen peroxide-induced toxicity on L-929 mouse fibroblasts’ cell lines. However, the cytoprotective effect depended on the time of the cells pre-treatment with SE extract before exposure to a hydrogen peroxide solution of 50 mM concentration, requiring at least 12 h of pre-treatment with polyphenols with radical scavenging capacity.


Establishing the preparation conditions for SE extract-loaded liposomes and transfersomes by using one-factor-at-a-time experiment
Influence of four factors (Phosphatidylcholine from egg yolk (PC)/cholesterol (sodium cholate) ratio, evaporation temperature, stirring rate, SE extract amount) on the entrapment efficiency was assessed for liposomes and transfersomes.

Influence of PC/cholesterol (sodium cholate) ratio on the entrapment efficiency
For PC/cholesterol (sodium cholate) ratio was tested the following values: 10/1, 9/1, 8/2 and 7/3, while the other preparation conditions of liposomes and transfersomes were kept constant (evaporation temperature 35 °C, stirring rate 200 rpm, 10 mg of SE extract). The best results were obtained for 10/1 PC/cholesterol ratio for liposomes and 8/2 PC/sodium cholate ratio for transfersomes ( Figure S1A and S2A). These values were used in further experiments.

Influence of evaporation temperature on the entrapment efficiency
The evaporation temperature was varied in the range of 25-40 °C, and the other preparation conditions of liposomes and transfersomes were kept constant (PC/cholesterol (sodium cholate) ratio 10/1 (8/2), stirring rate 200 rpm, 10 mg of SE extract). One can notice the highest entrapment efficiency for the formulations prepared at 35 °C ( Figure S1B and S2B).

Influence of stirring rate on the entrapment efficiency
The tested stirring rate values were 100 rpm, 150 rpm, 200 rpm and 250 rpm, the other preparation conditions being kept constant (PC/cholesterol (sodium cholate) ratio 10/1 (8/2), evaporation temperature 35 °C, 10 mg of SE extract). A stirring rate of 200 rpm determined the best results for both lipid vesicles ( Figure S1C).

Influence of SE extract amount on the entrapment efficiency
In order to obtain the best entrapment efficiency, different SE extract amounts were tested: 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, the other preparation conditions of liposomes and transfersomes being kept constant (PC/cholesterol (sodium cholate) ratio 10/1 (8/2), 200 rpm stirring rate, 35 °C evaporation temperature). The results were obtained for 20 mg of SE extract for both lipid vesicles ( Figures S1D and S2D).

Establishing the preparation conditions of SE extract-loaded ethosomes by using one-factorat-a-time experiment
Influence of two factors (PC/SE extract ratio and water/ethanol ratio) on the entrapment efficiency was assessed for SE extract-loaded ethosomes.

Influence of water/ethanol ratio on the entrapment efficiency
The tested following water/ethanol ratios were to 9/1, 8/2, 7/3, 6/4 and 5/5, while the other preparation conditions of ethosomes were kept constant. The best entrapment efficiency was obtained for 7/3 water/ethanol ratio ( Figure S3B).

Characterization of SE extract-loaded lipid vesicles
Freeze dried SE extract-loaded liposomes were characterized by scanning electron microscopy (SEM). The SEM image ( Figure S4) showed the uniform size of SE extract loaded liposomes in accordance with DLS analysis. The AFM investigation of SE-loaded lipid vesicles performed on a drop of lipid vesicles suspension in water, deposited on clean Si and dried at room temperature, showed nanosized quasi spherical particles ( Figure S5) with lower dimensions than in the case of SEM analysis performed on freeze-dried samples. The aggregates of liposomes containing SE extract preserve the quasi spherical shape of nanoparticles, ranging in 1-3 m. Also, in the AFM images can be observed the core-shell structure of samples ( Figure S5).
It was also recorded SEM images on SE extract loaded samples prepared from a drop of lipid suspension, which was further dried in vacuum. As it can be observed in figure S6 that the lipid vesicles are not agglomerated having slightly larger diameters than that measured on AFM analysis, probably because of lipid vesicles flattening. The size of SE extract loaded lipid vesicles increase in the following order: liposomes < trasfersomes < ethosomes.