Antioxidant activity of phenolic compounds from extracts of Eucalyptus globulus and Melaleuca styphelioides and their protective role on D-glucose-induced hyperglycemic stress and oxalate stress in NRK-49Fcells

Abstract Phytochemicals serve as potential therapeutic agents for the prevention and treatment of diseases. In this study, we elucidate the renoprotective activity of compounds isolated from Eucalyptus globulus and Melaleuca styphelioides extracts in glucose- and oxalate-challenged NRK-49F cell model. The antioxidant potential of isolated compounds was evaluated based on their effect on antioxidant enzyme activities and lipid peroxidation levels. The results demonstrated that exposure of NRK-49F cells to glucose and oxalate stress augmented cell damage and attenuated antioxidant enzyme activities. The phytochemicals 2,2,8-trimethyl-6-formyl-chrom-3-ene-7-O-β-D-glucopyranoside, Cornusiin B and tellimagrandin I treatment restored antioxidant enzyme activity, significantly lowered lipid peroxidation levels and effectively protected cells from glucose and oxalate stress equivalent to the known antioxidant, N-acetyl cysteine. Pterocarinin A significantly reversed cellular damage owing to glucose stress. In conclusion, the compounds isolated from E. globulus and M. styphelioides showed potential cytoprotective and anti-oxidative property against glucose- and oxalate-induced oxidative stress in NRK-49F cells.


Introduction
Oxidative stress and impaired antioxidant system have been identified as critical processes in the pathophysiology of diverse diseases. Increased free radical levels in the system causes deterioration of renal function and subsequently leads to tubulointerstitial fibrosis. In diabetic condition, hyperglycemia-mediated oxidative stress plays a pivotal role in the progression of disease (Giacco and Brownlee 2010). In the pathophysiology of calcium oxalate stone disease, oxalate augments the generation of free radicals ensuing increased levels of lipid peroxidation in the kidneys, leading to renal tubular injury and tubulointerstitial fibrosis (Khan 2014). Current therapeutic options for the prevention of kidney damage resulting from glucose and oxalate toxicity are at preliminary levels. Since most diseases can be effectively managed by the quality of diet, several phytotherapeutic agents have been proposed as useful alternatives for the prevention of various ailments.
Herbal treatment in the management of diseases has gained more attention among researchers owing to its biocompatibility and efficiency. This can be attributed to various phytochemicals such as, triterpenes, flavonoids and alkaloids. The biological properties of herbal extracts attract researchers to depend on plants for developing potential therapeutic drugs. Various Eucalyptus plants are rich sources of bioactive natural compounds, including ellagitannins, galloyl esters, flavonoids, chromones and terpenoids (Ito et al. 2000;Al-Sayed et al. 2010, 2014bTian et al. 2010). Eucalyptus globulus Labill. (Myrtaceae) is the most cultivated species of this genus (Brooker 2002). Even though Eucalyptus extracts are known to possess potent antioxidant effect and antibacterial properties (Salem et al. 2015;Belfeki et al. 2016), the antioxidant activity of the isolated compounds of E. globulus has not been extensively investigated.
Melaleuca styphelioides Sm. (Myrtaceae) is a medium-sized ornamental tree rich in bioactive compounds, including ellagitannins, galloyl esters and flavonoids Al-Sayed et al. 2014a). Previously, we demonstrated the antioxidant and hepatoprotective effect of total extract M. styphelioides against liver toxicity in CCl 4-induced mice (Al-Sayed et al. 2014a). The active principles responsible for the antioxidant and hepatoprotective activity of the extract were identified in HepG2 cells challenged with CCl 4 in vitro (Al-Sayed and Esmat 2016).
To the best of our knowledge, only a few studies have been reported on the antioxidant activity of isolated compounds from E. globulus and M. styphelioides. Hence, the present study is aimed to determine the possible mechanistic action of compounds isolated from E. globulus (1-4) and M. styphelioides (5-8) leaves extract.

Cytotoxic effect of glucose and oxalate on NRK-49F cells
The results showed that cellular viability under glucose and oxalate stress was significantly (p < 0.05) decreased with increasing concentration of glucose and potassium oxalate ( Figure S4a-d). A 50% decrease in cellular viability was observed at 30 mM glucose after 48 h and 1500 μM of oxalate after 18 h exposure when compared to the negative control (cell survival 100%). Emerging evidences suggest that excessive reactive oxygen species (ROS) generation under pathophysiologic conditions plays a critical role in the initiation and progression of fibrotic diseases. Increased accumulation of reactive oxygen radicals alters enzyme activities and exerts deleterious effect on mitochondrial membranes. Overproduction of ROS leads to the reduction in endogenous antioxidant function that eventually causes development of oxidative stress which has been proposed as the root cause mechanism underlying the development of insulin resistance, β-cell dysfunction, impaired glucose tolerance and type 2 diabetes mellitus (T2DM) (Wright et al. 2006;Stefano et al. 2016). Similarly, persistent oxalate exposure leads to increased production of ROS which in turn damages kidney tissue resulting in calcium oxalate stone formation (Khan 2013

Effect of compounds against D-glucose stress
In an effort to reduce renal injury, researchers have used plant polyphenols due to their antihyperglycemic property and minimal side effects. Among various phenolic compounds isolated, quercetin an extensively studied poly phenolic compound has gained much attention owing to its anti-inflammatory, antioxidative and neuroprotective activities. Previous reports have clearly demonstrated the protective role of quercetin in hyperglycemic stress conditions (Rizvi and Mishra 2009;Aguirre et al. 2011). In our study, treatment of NRK-49F cells with known antioxidant N-acetyl cysteine (NAC), a positive control under glucose stress, restored cell viability close to that of normal cells ( Figure S5). A significant (p < 0.05) level of cytoprotection against glucose stress was observed at 100 μM concentration of compounds 1 and 7, whereas a decreased protective effect was noticed at the concentrations of 75 μM or lesser. The cytoprotective effect of compounds 2, 3, 4, 5, 6 and 8 significantly (p < 0.05) increased at 75 μM concentration against glucose stress ( Figure S6). Interestingly, no significant difference was observed in cells supplemented with compounds 1, 3 and 6 compared to NAC treatment. Catalase and superoxide dismutase activities were significantly (p < 0.05) reduced in NRK-49F cells exposed to glucose when compared to non-exposed cells. However, no significant alteration in catalase activity was observed in NRK-49F cells treated with compounds 1, 2, 3, 5, 6 and 8. Superoxide dismutase activity was significantly increased (p < 0.05) in NRK-49F cells treated with compounds 1, 2, 3, 5, 6 and 8 when compared to cells exposed to glucose stress (Table S1). Similarly, NAC treatment markedly (p < 0.05) reversed antioxidant enzyme activities compared to cells exposed to glucose stress. The compounds 3 and 6 of class ellagitanins exhibited similar antioxidant property as compared to NAC, indicating these compounds have effective antioxidant potential. In agreement with Poljsak (2011), the toxic effect of ROS-mediated cellular injuries is attenuated by enhancing oxidative defence capacity through supplementation of antioxidants. Plant metabolites are known to facilitate the stimulation of various antioxidant defence mechanisms in the human system against oxidative stress-induced tissue damage (Rahal et al. 2014). The MDA level was elevated in cells (p < 0.05) pre-incubated with glucose when compared to control. However, treatment with 1, 2, 3, 5, 6 and 8 reversed lipid peroxidation levels drastically when compared to other compounds (Table S1). Therefore, treatment with phenolic compounds conferred renoprotective effect by restoring elevated lipid peroxidation levels and improving the antioxidant defence system in renal fibroblast cells. High glucose-induced oxidative stress and chronic inflammation in diabetic tissues result in the accumulation of advanced glycation end products (AGEs), lipid peroxidation products and protein carbonyls that cause adverse effects in the cardiovascular system, retina, kidney, peripheral limbs and other parts of the body (Pandey and Rizvi 2014). Previous studies have demonstrated that supplementation of agents which could decrease oxidative stress is able to rescue the cells from glucose (Aguirre et al. 2011;Kumagai et al. 2015).

Effect of compounds against oxalate stress
Oxalate, an important component of stone formation, is acquired from both dietary sources and endogenous synthesis. The excess of oxalate aggravates ROS generation and subsequently induces injuries in renal tissue (Chaiyarit and Thongboonkerd 2012). Phytotherapeutic agents were proposed as an alternative or complementary therapy for the management of urolithiasis, in part due to anti-oxidative effects. The combination of quercetin and lactic acid bacteria serves as a vital functional food ingredient in the prevention of CaOx crystal formation (Gomathi et al. 2015). Supplementation of Tamarix gallica L extract, gallotannins and NAC significantly inhibited ROS generation and subsequently prevented stone formation (Bensatal and Ouahrani 2008;Lee et al. 2012;Sharma et al. 2016). In the present study, NRK-49F cells' viability was significantly (p < 0.05) improved with the treatment of compound 1 at 100 μM in an oxalate-exposed microenvironment, whereas the protective effect was decreased at concentrations less than 75 μM. Treatment with 75 μM of 2, 3, 6, 7 and 8 reduced the detrimental effect of oxalate in NRK-49F cells. Co-treatment with compounds 4 and 5 at 50 μM showed higher cytoprotective effect ( Figure S7). Catalase and superoxide dismutase activities were significantly (p < 0.05) restored in NRK-49F cells treated with compounds 1, 2, 3, 5, 6 and 8 compared to oxalate-exposed cells (Table S2). In concordance with previous reports (Shimoda et al. 2008;Al-Sayed and Esmat 2016), the compound tellimagrandin I exhibits highest antioxidant property thereby significantly protecting cells against oxalateinduced cellular damage. NAC treatment significantly reversed cell viability (p < 0.05) ( Figure S5) and antioxidant enzyme activities compared to cells exposed to oxalate toxicity (Table S2). The supplementation of compound 7, an ellagitanin, exhibited similar cytoprotective and antioxidant properties compared to NAC. Moreover, treatment with tellimagrandin I ameliorated the oxalate-induced lipid peroxidation levels and thus restored cell viability (Table S2). Even though various strategies have been employed to tackle the oxalate-mediated oxidative stress, supplementation of natural products can promote health and prevent the occurrence of stone disease. Phytochemicals isolated from E. globulus and M. styphelioides protected the cells against high glucose and oxalate stress which stimulates the reninangiotensin system (RAS). ROS generation increases in response to RAS activation that further promotes renal cell injury and apoptosis. The supplementation of bioactive compounds probably functions as a renin-angiotensin inhibitory and anti-oxidative inductive molecule that enables cellular longevity. Our results revealed that compounds isolated from extracts of E. globulus and M. styphelioides exhibited potential therapeutic properties for ameliorating oxidative stress and renal cell injury in hyperglycemic and hyperoxaluric stress conditions.

Conclusion
The results of the present study provide evidence that extracts of E. globulus and M. styphelioides highly abundant with ellagitannins exhibited maximum antioxidant property and can serve as a potential therapeutic option in oxidative stress-mediated kidney damage. The study also demonstrates the nephroprotective role of phenolic compounds against diabetes mellitus and kidney stone disease. Thus, further detailed studies are needed to confirm the role of compounds as novel therapeutic agents against free radical-induced tissue damage in various physiological conditions.

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

Funding
This work was supported by the UGC-meritorious fellowship and DBT-IPLS program, New Delhi, India.