Preparation and in vivo evaluation of nano sized cubosomal dispersion loaded with Ruta graveolens extracts as a novel approach to reduce asthma-mediated lung inflammation

Abstract

Asthma is a chronic disease affecting people of all ages. Asthma medications are associated with adverse effects restricting their long-term usage, demanding newer alternative therapies. This study aimed to investigate the anti-asthmatic properties of Ruta graveolens extract and its prepared nano-cubosomal dispersion (Ruta-ND). Firstly, the R. graveolens methanolic extract exhibited higher anti-inflammatory activity on Lipopolysaccharide (LPS)-activated BEAS-2B cells. To ensure best bioavailability and hence best cellular uptake, R. graveolens extract was loaded in nano-cubosomal dispersion (ND). Then, the anti-asthmatic effects of Ruta extract and ND were simultaneously evaluated in rats' model with ovalbumin-induced allergic asthma. R. graveolens extract and Ruta-ND subsided asthma score and improved lung function by restoring FEV1/FVC ratio to the expected values in control rats. Also, it showed strong antioxidant and anti-inflammatory activities manifested by lowering levels of malondialdehyde (MDA), IL-4, IL-7, TGF-β, and Ig-E, and increasing levels of superoxide dismutase (SOD) and INF-γ in bronchoalveolar lavage fluid. Our research findings also indicate autophagy induction and apoptosis inhibition by Ruta extract and Ruta-ND. Finally, the HPLC MS/MS phytochemical profiling of R. graveolens extract evident production of various alkaloids, flavonoids, coumarins, and other phenolics with reported pharmacological properties corresponding to/emphasize our study findings. In conclusion, R. graveolens exhibited promise in managing Ova-induced allergic asthma and could be developed as an alternative anti-allergic asthma drug.

Keywords: Apoptosis; Autophagy; Beclin-1; Behavior scoring; Ovalbumin Allergic airway; Respiratory function.

Fig. 1

IC₅₀ of butanol fraction (But), ethyl acetate fraction (EtOAc), methanol fraction (MeOH), and TE of R. Graveolens on BEAS-2B cells.

Fig. 2

Real-time PCR analysis data depicting the relative normalized expression of IL-17, IL-4, TGF-β, and IFN-γ after BEAS-2B treatment with IC₅₀ and ½ IC₅₀ of But., EtOAc, MeOH, and TE of R. graveolens. P-values on the graph reflect the statistical significance of various treatments compared to positive control (stimulated with LPS). The relative expression was calculated based on the 2 − ΔΔCt method. * Significance from positive control at P < 0.0001, # Significance from IC₅₀ of TE treatment at P < 0.0001.

Fig. 3

HPLC-PDA-ESI-MS/MS chromatogram (positive ionization mode) of total MeOH extract of R. graveolens.

Fig. 4

(A). Size distribution, (B). Zeta potential (ZP) of Ruta-ND, and (C). Transmission electron micrograph of the nano- cubosomal system.

Fig. 5

A quantitative analysis of lung function tests shows (A) vital capacity, (B) forced expiratory volume 1 (FEV1), and (C) FEV1/FVC ratio. Data represented as mean ± SD.

Fig. 6

A quantitative analysis of (A) MDA and (B) SOD in the bronchioalveolar lavage (BALF). Data represented as mean ± SD.

Fig. 7

A quantitative analysis of (A) IL-4, (B) IL-17, (C) TGFβ, (D) INF-γ, and (E) IgE in the BALF. Data represented as mean ± SD.

Fig. 8

Autophagy parameters: a quantitative analysis of (A) Beclin-1 and (B) P-62 in the lung tissue. Data represented as mean ± SD.

Fig. 9

Photomicrograph of lungs (Immune staining) showing limited mTOR expression in (a) control group, increased expression in (b) Ova group, moderate expression in both Ruta 100 mg (c) and Ruta 200 mg (d), marked reduction in mTOR expression in Ruta-ND 100 mg (e) and Ruta-ND 200 mg groups (f).

Fig. 10

Chart represents mTOR expression in lung tissue (area %) in different groups, data are presented as mean ± SEM. Significant difference was considered at P < 0.05.

Fig. 11

Photomicrographs displayed the expression of Caspase 3 Antibody on lung sections among inspected groups as follows: (A) Lung Section from control group demonstrated scarce Caspase 3 nuclear reaction (arrow) along lung tissue. (B) Lung Section from Ova group highlighted the strongest Caspase 3 nuclear and cytoplasmic expression along collapsed alveoli as well as encircling bronchiole area (arrow). (C) Lung Section from Ruta 100 mg group underscored high Caspase 3 nuclear and cytoplasmic reaction (arrow) along alveolar wall and encircling bronchiole. (D) Lung Section from Ruta 200 mg group marked moderate Caspase 3 nuclear and cytoplasmic expression along collapsed alveoli and surrounded bronchiole (arrow). (E) Lung Section from Ruta-ND 100 mg group exposed moderate Caspase 3 nuclear expression (arrow) with a value beneath Ruta total MeOH extract 200 mg group. (F) Lung Section from Ruta-ND 200 mg group existed with a few Caspase 3 nuclear expression (arrow) along alveolar wall. (Caspase 3 Antibody, Magnification Power = x200 & Scale Bar = 100 μm).

Fig. 12

Photomicrographs presented the histopathological alterations along lung tissue sections of examined groups as follows: (A) Lung Section from control group displayed the standard assembly of alveoli (arrowhead) and bronchiole (thick arrow) inside lung tissue. (B) Lung Section from Ova group highlighted severe lung degeneration with collapsed alveoli and serious hemorrhage with infiltration of inflammatory cells in all tissue (circle). Bronchioles epithelium marked with desquamation (wave arrow), excessive hyperplasia in addition to its detachment from basement membrane (thick arrow). Subepithelial edema (thin arrow), aggregation of inflammatory cells (star), obvious increase in fibers amount (curve arrow), and congestion inside blood vessels (rectangle) were also detected. (C) Lung Section from Ruta 100 mg group emphasized deteriorating changes less than Ova group which demonstrated by little recovery of the collapsed alveoli, obvious decrease in serious hemorrhage as well as the number of inflammatory cells along alveolar wall (circle), partial decline in epithelial hyperplasia (thick arrow) and few amount of submucosal inflammatory cells (star). However clear expansion in edema (thin arrow) and fibers amount encircling bronchiole (curve arrow) were noticed in combination with congestion in blood vessels (rectangle). (D) Lung Section from Ruta 200 mg group disclosed moderate improvement that marked in thin irregular alveolar wall (arrowhead), few hemorrhage (circle), obvious normal bronchial epithelium (thick arrow), low number of aggregated inflammatory cells (star), in addition to edema leading to dispersion between fibers surrounding bronchiole (thin arrow). (E) Lung Section from Ruta-ND 100 mg group revealed high enhancement along alveolar wall architecture (arrowhead), excluding some hemorrhage dispersed throughout lung tissue (circle). Bronchial epithelium revealed desquamation (wave arrow) and some hyperplasia (thick arrow). Inflammatory cells (star) and fibers (thin arrow) are presented in a small amount. Observe the normal existence of blood vessels (rectangle). (F) Lung Section from Ruta-ND 200 mg group marked great development with normal existence of most alveoli (arrowhead). Hemorrhage (circle), inflammatory cells (star), and accumulated fibers (curve arrow) were recorded in tiny mounts. Bronchial epithelium recorded in its normal assembly (thick arrow). (Hematoxylin & Eosin staining, Magnification Power = x200 & Scale Bar = 100 μm).