Phytochemical screening and radioprotective potential of Jasminum grandiflorum methanol extract against gamma irradiation-induced oxidative damage and diverse inflammatory mediators in lungs of male Swiss Albino Rats

Abstract Rising awareness about the ecological burden of agricultural by-products and their potential industrial and medicinal applications spurred the research of waste valorization. Egypt is a chief producer of jasmine oils. However, jasmine-dense foliage is an agricultural waste that merits exploitation. Herein, Jasminum grandiflorum leaves were profiled using UPLC-MS, revealing a promising phytochemical composition rich in secoiridoids. A good safety profile was confirmed in vitro on cancerous and normal cell lines and in vivo (LD50>4000mg/kg). The radioprotective effect of 3500 mg/kg jasmine leaves extract against irradiation-induced lung injury was demonstrated. Biochemical analysis indicated that jasmine extract decreased malondialdehyde level and enhanced the endogenous antioxidants GSH-Px, SOD and CAT levels. Jasmine alleviated the inflammatory response by downregulating the expression of COX-2 and iNOS and reducing the inflammatory mediators, TNF-α, IL-6, NO, and PGE2. Jasmine leaves offer interesting application prospects that add economic and industrial value to agricultural waste. Graphical abstract


Introduction
Although radiotherapy plays central role in cancer treatment, it can damage irradiated normal tissues especially the lungs which can significantly affect the success of radiotherapy and cancer patient's prognosis (Owis et al. 2022).Cellular exposure to ionizing radiation (IR) leads to radiolysis of water inducing the overproduction of reactive oxygen species (ROS) leading to inflammation and damage of biological macromolecules e.g.DNA, proteins and lipids (Wei et al. 2019).
Literature reviews indicated that a single dose ranged between 11-13 Gy γ-rays is enough to imply radiation-induced pulmonary toxicity such as pneumonitis (Almeida et al. 2013).Natural radioprotective agents can be useful adjunct to radiotherapy to differentially prevent radiation-induced tissue damage on normal rather than cancerous cells (Akbari et al. 2018;Mun et al. 2018;Mulinacci et al. 2019;Abdallah et al. 2022;Begum et al. 2022).Genus Jasminum of the olive family Oleaceae comprises 200 species (Arun et al. 2016).Jasminum grandiflorum was extensively used in traditional medicine.Largely the polar extracts from the leaves of the plant have been analyzed for antioxidant, antimicrobial, anti-inflammatory, and other activities (Al-Snafi 2018; Balkrishna et al. 2021).Agricultural waste recycling has gained considerable interest as affordable source of phytochemicals with myriads of industrial and pharmaceutical applications (Ibrahim and Moussa 2021).To capitalize jasmine green biomass, in this work, the prophylactic effect of J. grandiflorum leaves methanol extract (MeJLE) was evaluated against irradiation-induced lung oxidative stress and its deleterious effect on diverse inflammatory mediators.Acute toxicity was investigated in vitro and in vivo.Metabolite profiling was performed via UPLC-ESI-MS.

Metabolite profiling of Jasminum grandiflorum methanol extract
UPLC-ESI-MS analysis revealed a compositional profile dominated by secoiridoids and their glycosides.Some examples are discussed herein and in Supplementary material (Table S1 and Figures S1-S4).Peak 1 (R t 1.18 min) presented a base peak [M + Na] + at m/z 339 and a less abundant ion at m/z 323 (Supplementary material Table S1).In the negative ionization mode, [M-H] -was detected at m/z 315, and the ions at m/z 153 and 179 correspond to the deprotonated hydroxytyrosol [M-H-162] -and the deprotonated hexose ion [M-H-136] -(Figure S1).This coincides with the phenylethanoid hydroxytyrosol-O-hexoside (C 14 H 20 O 8 ) (El-Shiekh et al. 2020).Peak 3 (R t 2.94 min), assigned as jaslanceoside A/C (C 27 H 32 O 15 ), showed a sodium adduct at m/z 619 [M + Na] + in the positive ionization mode.This parent ion gave rise to a further ion at m/z 457 (base peak) formed via the loss of a hexose [M + Na-162] + (Figure S2).A series of ions at m/z 439 [M + Na-162-18], 413 [M + Na-162-44] + and 263 [M + Na-162-194] + suggest the dehydration, decarboxylation and loss of ferulic acid moiety of the aglycone.This was corroborated by the negative ionization mode (Table S1) (Shen and Lin 1996).
The compound eluting at R t 5.83 min was assigned as hydroxy oleuropein based on its base peak at m/z 579 [M + Na] + .The sodium adduct gave rise to a daughter ion at m/z 563 by loss of oxygen to yield oleuropein [M + Na-16] + .Another daughter ion was detected at m/z 443 ascribed to the loss of hydroxytyrosol group [M + Na-136] + .In the negative mode, a base peak at m/z 377 was attributed to the loss of hexose and oxygen [M-H-162-16] -(El-Shiekh et al. 2020).A major peak at R t 8.12 min was identified as oleuropein based on the molecular ion detected in the negative mode at m/z 539 [M-H] -, the deprotonated aglycone at m/z 377 [M-H-162] -(oleuropeinenal-(E)-enol), as well as the deprotonated glycosylated elenolic acid at m/z 403 [M-H-136] -due to loss of hydroxytyrosol and the deprotonated hexose ion at m/z 179, as illustrated by the suggested fragmentation pattern (Figure S3).The sodium adduct was detected at m/z 563 [M + Na] + in the positive ionization mode (Ventura et al. 2019).

In vitro toxicity
MeJLE had no cytotoxicity on Vero (normal) cells up to the high concentration (100 µg/ ml) (p > 0.05).It has been reported that secoiridoid glycosides isolated from the twigs of Ligustrum obtusifolium were non-toxic to the C6 rat glioma cell line (Suh et al. 2018).A considerable reduction in the viability of MCF-7 and HepG-2 cell lines was observed in a time and concentration dependent manner (Figure S5 a & b) (Abdel-Razik et al. 2016).A significant reduction in MCF-7 cell viability was observed after 48h treatment (p < 0.006 and p < 0.001 at concentrations 50 and 100 µg/ml).The highest reduction in cell viability was observed in HepG-2 cell lines (p < 0.025, 0.001, 0.0001, 0.0001 at concentrations of 12.5-100 µg/ml) after 48h treatment (Figure S5b).

Acute toxicity
Administration of different doses of MeJLE (250-3000 mg/kg) did not induce mortality (Table S3) or changes in external appearance with normal appetite and excretion suggesting normal processing of metabolic activity and physiological function (Carreiro et al. 2016).Gross analyses of lungs, kidneys and liver of dosed rats revealed no abnormal physical changes in size, shape, color, and texture up to 3000 mg/kg compared with control group.At 4000 mg/kg, 33.3% mortality in days 7 and 13 was observed with LD 50 >4000 mg/kg BW.

Selection of MeJLE optimum radioprotective dose
Based on the 30 days experiment of survival (Figure S6), the positive control irradiated group (IR) displayed signs and symptoms of radiation toxicity (slow gait, reduced food and water intake, loss in body weight, irritability, diarrhea, lethargy, and ruffling of hair) within 1-12 days post-exposure (average survival time 5.9 ± 4 days).Administration of MeJLE before IR exposure (MeJLE + IR) delayed symptoms of irradiation sickness and onset of mortality in dose-dependent manner compared with positive control.The longest delay was observed with 3500 mg/kg MeJLE.At the end of experiment, there was 90% survival (average survival time 29.4 ± 1 days) in rats which received 3500 mg/kg MeJLE compared with 80% (27.9 ± 3), 60% (25.8 ± 5), 40% (20.1 ± 8), 20% (19.2 ± 7) and 10% (13.6 ± 8.6) survival in rats receiving 3000, 2500, 2000, 1500 and 1000 mg/kg MeJLE, respectively.The dose 3500 mg/kg was selected as the optimum dose to be used in the rest of our study.

Prophylactic effect against γ rays-induced lung oxidative stress and inflammatory mediators
The radioprotective effect of MeJLE at a dose of 3500 mg/kg once daily, five times/ week for 14 days was estimated via evaluation of oxidation and inflammatory mediators in the lung tissues of rats.
In this part of our study, 28 animals were used, and divided into four groups.TableS4 indicates the rate of survived and dead animals per each group during the experimental time (14 days).
Whole-body irradiation (IR) resulted in significant increase in the levels of the lipid peroxidation product MDA and NO (p < 0.001) (Figure S7).Treatment with MeJLE prior to IR exposure markedly normalized MDA (p = 0.002), while NO value approached the negative control (p = 0.356).Radiation therapy caused profound reduction in GSH-Px (p < 0.001) and SOD (p = 0.004) in irradiated rats lung tissue compared to negative control rats (Figure S8,a&b).Combination of MeJLE and radiotherapy markedly ameliorated lung tissue levels of GSH-Px (p = 0.003) and SOD (p = 0.004) compared to negative control group.Radiotherapy significantly upregulated iNOS and COX-2 expression (p < 0.001) (Figure S9) and increased the inflammatory cytokines (p < 0.001) (Figure S10 a).Pretreatment with MeJLE mitigated the radiotherapy-induced acute inflammation as suggested by the significant attenuation of iNOS and COX-2 expression (p < 0.001) (FigureS9) and decreased TNF-α, IL-6 (and PGE2 levels(p < 0.001) compared to irradiated (IR) group (Figure S10 a & b).
Oleuropein, one of the main secoiridoids identified in MeJLE, displayed a potent antioxidant activity may be ascribed to its ortho-dihydroxyphenyl substitution that can sequester ROS via hydrogen donation and stabilize oxygen free radicals through hydrogen bonding (Romani et al. 2017).Moreover, it has been reported that oleuropein reduced COX-2, iNOS, IL-6 and NO levels in mice (Giner et al. 2013).

Conclusion
Jasmine leaves displayed the presence of several secoiridoids as illustrated by the UPLC-MS analysis and proved non-toxic with no or very low in vitro cytotoxicity and good safety profile in vivo.Jasmine extract effectively prevented radiation-induced oxidative stress and inflammation in γ-irradiated rat lungs as evidenced by the significant decrease in MDA, NO, TNF-α, IL-6 and PGE2, significant increase in SOD, GSH-Px and CAT and downregulation of iNOS and COX-2 expression.This is the first investigation on the plausible radioprotective exploitation of Jasminum waste and we expect that it will change the industrial perspective of this crop in the future.