Hesperidin hexosomal loaded nanodispersion: insights of its antimycobacterial, cytotoxic and anti-HCoV effects

Abstract Fruits of Citrus sinensis L. Osbeck var. Valencia contain hesperidin as a major flavanone glycoside. Hesperidin (H) was isolated from the peels of Valencia orange and formulated as hexosomal nanodispersions (F1) adopting the hot emulsification method. The antimycobacterial activity(anti-TB) was evaluated through a microplate Alamar blue (MABA) assay where F1 showed significant activity with MIC = 0.19 µM. To unravel the potential mechanism of the anti-TB, a molecular docking study of H using the Mycobacterial Dihydrofolate reductase (Mtb. DHFR) enzyme was performed. Hesperidin exhibited significant interactions with Mtb. DHFR active site. Sulforhodamine B assay was applied to evaluate cytotoxic activity against the lung cancer cell line (A-549). F1 showed a cytotoxic effect at IC50= 33 µM. It also has potent antiviral activity against Human Coronavirus 229E with IC50= 258.8 μM utilising crystal violet assay. Peels of Valencia orange could be a source of bioactive metabolites to control significant diseases. Graphical Abstract


Introduction
The current approach will focus on three lung disorders which are Tuberculosis (TB), lung cancer and corona virus diseases. Sweet orange fruits (Citrus sinensis (L.) Osbeck var. Valencia) contain many secondary metabolites mainly flavonoids glycosides. Conventionally, sweet orange has been used for treatment of bronchitis, and TB (Favela-Hernandez et al. 2016). Hesperidin is the predominant flavonoid glycoside in Citrus fruits, especially in sweet orange. Hesperidin acts as anti-inflammatory, antioxidant, antimicrobial and anti-carcinogenic (Kunti€ u et al. 2014). Poor solubility and bioavailability of hesperidin may render it, so it is slightly absorbed and requiring a delivery system to reach its therapeutic target (Sulaiman et al. 2020). Hesperidin loaded on gold nanoparticles was a successful delivery system model as an anti-cancer and anti-inflammatory agent (Sulaiman et al. 2020). Hesperidin-loaded PLGA (Poly Lactic co-Glycolic Acid) was previously reported as antimicrobial and cytotoxic agents (Balakrishnan et al. 2021). Hesperidin and its mediated zinc oxide (ZnO) nanoparticles were exhibited antiviral agents against SARS-CoV-2 (Attia et al. 2021). The main target of this study is to offer hesperidin as an effective and low-cost natural metabolite from sweet orange fruits to overcome the risk of the respiratory conditions, besides possible improvement of their medicinal effect by the nano-formulation. Furthermore, docking study will also be accomplished to decipher the potential underlying mechanism of the antimycobacterial effect, the major component of sweet orange peel.

Identification and structure elucidation of hesperidin
Hesperidin was isolated in accordance with the procedures mentioned by Victor et al. (2021) with minor modifications. Results of 1 H-NMR and 13 C-NMR: d (400 and 100 MHz, respectively, DMSO-d6) were justified in Table S1. From spectral data and after comparing with the published data (Lahmer et al. 2015), we have confirmed that the isolated compound (475 mg) white powder is hesperitin-7-rhamnoglucoside (hesperidin)( Figure S1).

Characterisation of hexosomal nanodispersions
Hexosomal nanodispersions were formulated adopting a hot emulsification method (Gabr et al. 2017) with minor modifications. Particle size (PS), polydisperisty index (PDI), and zeta potential (ZP) values of the prepared hexosomal nanoparticles were measured. The prepared hexosomal nanoparticles were found to have an average particle size of 191.7 ± 0.95 nm ( Figure S2). The PDI of the formula was 0.27 indicating small and uniform size distributions and indicating the suitability of the applied preparation method (Gabr et al. 2017). The mean value of ZP was À34.4 ± 0.12 mV ( Figure  S3). The negative ZP values are due to the presence of free oleic acid (OA) in the glyceryl monooleate (GMO), besides ionisation of OA which was already added for the formulation of hexosomes (Abdel-Bar et al. 2020).

Transmission electron microscope
The picture of the transmission electron microscopy (TEM) regarding the nanohexosomal particles is presented in Figure S4. Almost hexagonal non-aggregated nanoparticles were observed in the illustrated photo. The present figure shows particle size smaller than obtained with zeta sizer as the dynamic light scattering technique is a multiangle measuring method which determines the hydrodynamic radius of the particle depending on the intensity of the light scattering.

Assessment of antimycobacterial activity
Hesperidin (H) exhibited a strong inhibition of Mycobacterium tuberculosis (MTB) with MIC (minimum inhibitory concentration to kill 100% of MTB) 1.95 mg/mL (3.19 mM) (when compared to Isoniazid MIC ¼ 0.24 mg/mL (0.39 mM). While hesperidin loaded nanodispersions (F1) showed MIC ¼ 0.12 mg/mL (0.19 mM) prevailed a potent inhibitory effect on MTB nearly double the potency as isoniazid. MIC of Blank formula (F2) was also determined. Results are displayed at Table S2 and Figure S5 revealed that H and F1 have bactericidal effect against MTB. Formulated hesperidin exhibited more potent activity against MTB than hesperidin alone. Hexosomal nanodispersions are efficient carriers for different drug molecules through different route of administration (Madheswaran et al. 2019). Hesperidin methyl chalcone exhibited an inhibition of granulomas formation during the treatment of spinal tuberculosis (Zhao et al. 2020).

Molecular docking study of antimycobacterial activity
A molecular docking design has been constructed based on analysis of the crystallographic structures of Mtb. DHFR, complexed with NADPH and methotrexate inhibitor. Analysis of the interactions of co-crystallised methotrexate as well as its redocked form revealed that the following amino acid residues are involved: ASP 27, and ILE 94, as H-bond donners; whereas ARG 32 and ARG 60, as H-bond acceptors ( Figure S6). Moreover, ionic interactions are also observable with these amino acid residues and p-H interaction between the phenyl ring of methotrexate and PHE 31 and VAL 54. Subsequent, docking procedures have been accomplished for the investigated hesperidin. The study determined that binding poses ( Figure S7) with higher docking scores (-29.9763 Kcal/mol), indicating that the studied compound could potentially bind to the active site of Mycobacterial Dihydrofolate reductase (Mtb. DHFR), attaining mostly similar orientations. Significant binding interactions involve the following amino acid residues: ILE 94, GLY 18 as H-bond donners; H 2 O and ARG 45 as H-bond acceptors, in addition to PHE 31 and LEU 50 which bind through p-H interaction. The results of the docking study revealed that hesperidin could bind to the active site of Mtb. DHFR, so it could be considered as one of its mode of actions against M. tuberculosis. Our findings may need further in vitro enzyme inhibition assay for hesperidin against Mtb. DHFR.

Evaluation of cytotoxic activity against A-549 cell line
Our results demonstrated that (H) is cytotoxic to the lung cancer cells A-549 only at high concentration IC 50 ¼ 195.40 mg/mL (324 mM). Regarding hesperidin loaded nanohexosomal particles (F1), they showed remarkable reduction in cell viability, where IC 50 ¼ 20.95 mg/mL (33.27 mM) which is statistically different from hesperidin alone IC 50 ¼ 195.40 mg/mL (324 mM). All results of IC 50 were compared to standard Doxorubicin (IC 50 ¼ 0.012 mg/mL). Quick screening at two concentrations (10 and 100 mg/mL) of blank formula (F2) was performed to eliminate any interference (Table S3). The % viability of cancer cells ( Figures S8 and S9) was significantly reduced (p < 0.05) in dose dependent manner, p < 0.05 (one-way ANOVA followed by Tukey's multiple comparison test). Higher activity of nanoformula may be due to their amphiphilic nature which facilitate penetration though cells, besides accumulation of drug inside the cancerous cell (Abdel-Bar et al. 2020).

In vitro cytotoxicity and antiviral against human coronavirus 229E on VERO E6 cells
The results presented in Table S4 and Figure S10 showed that the CC 50 values of H and F1 are 997.91 and 157.92 lg/mL (1635.9 and 258.8 lM), respectively comparing to standard antiviral Ribavirin V R (680.86 lg/mL). Analysis of cytotoxicity was executed to ensure that the cell death was due to direct inhibition of Human coronavirus 229E (HCOV 229E). Hesperidin showed moderate antiviral activity against HCOV 229E with a promising selectivity index (CC 50 /IC 50 ¼ 997.90/40.25 ¼ 24.7) compared to standard antiviral Ribavirin V R (CC 50 /IC 50 ¼ 680.86/18.81 ¼ 36.1). Hexosome nanoformula exhibited more observable reduction in virus titre with higher selectivity index (CC 50 /IC 50 ¼ 157.91/5.93 ¼ 26.6) than hesperidin alone.

Conclusion
Based on the current consequences, hesperidin considered a promising antiviral agent especially if formulated as nanohexosome. We have utilised cheap agriculture waste of citrus fruits (peel) to isolate the bioactive metabolite (hesperidin). To the extent of our knowledge, it is the first time for hesperidin to be loaded on hexosomal nanodispersion and be tested against Mycobacterium tuberculosis and HCoV 229E.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.