A Standardized Cichorium intybus L. Leaf Extract Abrogates P53/caspase-Dependent Apoptosis and Oxidative Stress via Activation of PI3K/Akt/Nrf2/HO-1 Signaling Pathway in Human Embryonic Kidney (HEK293) Cell Line

Introduction

Acute kidney injury (AKI) is characterized by a compromised renal function due to the damage of renal tubules. AKI can range from mild to severe, caused by sepsis, nephrotoxins, pregnancy and renal ischemia-reperfusion.¹ AKI-related mortality has affected the global population with the incidence of occurrence being either community-acquired or hospital-acquired.² In developed countries, the incidence of AKI is common in hospitalized individuals, caused by the medications especially in the intensive care unit.³

Kidney is a dynamic organ that plays a crucial role in the excretory system. Renal function involves more of the oxidation reactions in mitochondria. This makes the organ more susceptible to oxidative stress.^4,5 Progressive oxidative stress leads to the chronic kidney disease (CKD) that can further result in secondary complications such as inflammation and cardiovascular diseases.^6,7 There is plentiful of evidence for the elevating levels of oxidative stress markers with the deteriorating renal function in CKD.^8,9 Dietary supplementation of the antioxidants is an effective strategy in the CKD management. Plant-based therapies have been used to improve the progression of CKD for several years.¹⁰ These complementary and alternative medicines have been used either solely or as complement to the conventional medicines.¹¹

Cichorium intybus L. (Fam. Asteraceae) is a perennial plant native to parts of Asia, Europe and Africa. Chicory is valued not only as a food source but also as a versatile source of medicinal applications. Chicory extracts have significant pharmacological properties that include antidiabetic, analgesic, antimicrobial, anticancer, hepatoprotective and neuroprotective activities.^12–14 Previously, the antioxidant activity of C. intybus leaf extract has been demonstrated, attributing to the presence of pharmacologically active phytochemicals and minerals.¹⁵ Epure et al. reported the cardioprotective and nephroprotective activities of C. intybus extract in rats.¹⁶ Chemically, chicory leaves contain gallic acid, chlorogenic acid, caffeic acid, p-hydroxybenzoic acid, p-coumaric acid and isovanillic acid as major polyphenols.^17,18 In addition, the plant also contains chicoric acid, a derivative of caffeic acid and tartaric acid. In fact, chicoric acid was first identified in C. intybus leaves. However, later it has been charted in several other plant families.¹⁹

In accordance with the available literature, we hypothesized that chicory extract with a standardized content of chicoric acid exert potential nephroprotective activity. To prove the hypothesis, we have used two in vitro cell culture models: cisplatin (CP)-induced toxicity and H₂O₂-induced oxidative stress in HEK293 cells. Herein, we report for the first time that chicory leaf extract has anti-apoptotic and antioxidant activities that can protect the AKI-induced renal cell damage.

Experimental

Chemicals and reagents – Chicoric acid (Purity: 97%), Dulbecco's modified eagle medium (DMEM) (D5648), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) (M2128), acridine orange (318337), 2',7'-dichlorofluorescin diacetate (DCFH-DA) and Radio Immunoprecipitation Assay (RIPA) lysis buffer were procured from Sigma Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from GIBCO^TM, USA. Ethidium bromide (MB071) was obtained from Himedia. Cisplatin (CP, sc-200896), Caspase fluorescent assay substrates (Z-DEVD)²-R110 (sc-477255) and Ac-LEHD-AFC (sc-311277), primary antibodies for P53 (sc-98), PARP-1 (sc-8007), Caspase-9 (sc-73548), caspase 3 (sc-7272), Bcl-2 (sc-7382), Bax (sc-20067), Nrf2 (sc-365949), HO-1 (sc-136960), Akt (sc-56878), Pi3K (sc-1637), phosphorylated Akt (sc-514032) and phosphorylated Pi3K (173665) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Plant extract – The fresh C. intybus (Chicory) leaves were collected during the month of September 2023 from the Northern part of India. The plant material was authenticated at R&D center, Vidya Herbs Pvt Ltd., and the voucher specimen stored in the facility (VH/CH/31/23). The leaves were shade-dried and powdered. A 100 g powdered leaf sample was extracted with 800 mL of 70% (v/v) ethanol in water at 65–70°C for 3 h. After 3 h of extraction, the mixture was cooled and filtered through a Buchner filter under vacuum. The extraction was repeated three more times with 600 mL of 70% (v/v) ethanol each time. The filtrate was pooled and concentrated to dryness using a rotary evaporator under vacuum at 65–70°C. The final extract (20 g) was analyzed for the chicoric acid content.

Quantification of chicoric acid by High performance liquid chromatography (HPLC) – The chicoric acid content in the chicory leaf extract was quantified using the Shimadzu LC2030 C Prominence-i (Japan) system, equipped with a quaternary low-pressure gradient pump. A separation was carried out in a Kinetex XB C-18 column (100 Å, 150 mm × 4.6 mm, 5 µm pore size). Reverse phase elution was performed with 50 mM sodium acetate (solvent A) and acetonitrile (solvent B) with a flow rate of 0.5 mL/min and an injection volume of 10 µL. The column oven temperature was maintained at 28°C, monitored with UV detection at 335 nm. The gradient elution consisted of 10 to 90% of solvent B at 0.01 to 1 min, followed by 90% of solvent B from 1 to 4 min, and return to the initial condition from 4 to 8 min. A 10% of solvent B continued up to 10 min. The limit of detection (LOD) and limit of quantification (LOQ) were 0.9 ppm and 2.7 ppm, respectively. The detailed quantification method of chicoric acid is provided in Supplementary file.

Cell culture – Human embryonic kidney (HEK293) cell line was procured from Cell repository, National Center for Cell Sciences, Pune, Maharashtra, India. The cells were cultivated in DMEM supplemented with 10% FBS and 100 µg/mL streptomycin and 100 units/mL penicillin, in a humidified CO₂ incubator at 37°C.

Cell viability test – Cytotoxicity assessment in HEK293 cells was performed using MTT assay. Briefly, the cells were seeded at a density of 1.5 × 10⁴ cells/well in a 96-well plate and incubated overnight at 37°C. Later, the cells were exposed to different concentrations of chicory extract (0.025–0.5 mg/mL) for 24 h. Subsequently, the media was removed and 100 µL of MTT solution (5 mg/mL) added. After 4 h of incubation, the formazan crystals were solubilized, and absorbance measured at 570 nm in a microplate reader (Tecan Infinite 200 Pro).

CP-induced HEK293 nephrotoxicity model in HEK293 cells – HEK293 cells were seeded onto 6-well plate at a density of 4 × 10⁵ cells/well. The cells were pre-incubated with 12.5 and 25 µg/mL of chicory extract for 4 h at 37°C in CO₂ incubator. Later, 20 µM CP was added and further incubated for 24 h. The cells were observed for morphological changes under the microscope and the images captured.

H₂O₂-induced oxidative stress model in HEK293 cells – HEK293 cells were seeded onto 6-well plate at a density of 2.5 × 10⁵ cells/well. The cells were pre-incubated with 12.5 and 25 µg/mL of chicory extract for 1 h at 37°C in CO₂ incubator. Later, 500 µM H₂O₂ was added and further incubated for 24 h. The cellular morphology was observed under the microscope.

Acridine orange (AO) and Ethidium bromide (EB) dual staining – The AO/EB staining was performed as described by Ribble et al.²⁰ After the experimental treatments as described above, the culture media was removed, and the cells detached using trypsin-EDTA solution. The cell pellets were resuspended in PBS (25 µL) and 2 µL of AO/EB dye mix (100 µg/mL each). The stained cells were observed under a fluorescence microscope (Lawrence and Mayo India Pvt Ltd.) for the apoptotic changes.

Western blotting – The cell lysates (20 µg protein) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinyldenefluoride (PVDF) membrane (Pall Life Sciences). The membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and later probed with primary antibodies overnight at 4°C. Then, the blots were incubated with HRP-conjugated secondary antibody for an hour at room temperature and developed using enhanced chemiluminescence (ECL), visualized and photographed in ImageQuant^TM LAS 500 (GE Healthcare, Chicago, USA).

Caspase activity assay – The caspase activation in the cells was studied by fluorescence-based assays described elsewhere, with slight modifications.^21,22 Briefly, the cell lysate protein samples (10-50 µg) were incubated with (Z-DEVD)₂-R110 and Ac-LEHD-AFC respectively for caspase 3/7 and caspase-9 activities for 2 h at 37°C. Later, the reaction mixtures were measured for fluorescence at excitation and emission wavelengths of 485 and 535 nm for caspase 3/7 activity, and 400 and 505 nm for caspase 9 activity.

Cellular ROS assay – The formation of intracellular reactive oxygen species (ROS) was evaluated using DCFH-DA as per the method described elsewhere with slight modifications.²³ Briefly, HEK293 cells were seeded on to black 96-well plate at 1.5 × 10⁴ cells/well. After overnight culture, the cells were treated with the non-toxic concentrations of chicory extract for 4 h. Later, the cells were washed with phosphate buffered saline (PBS) and further incubated for 45 min with 20 µM DCFH-DA in the CO₂ incubator. The supernatant was removed, and the cells were incubated with 500 µM of H₂O₂ solution in DMEM for 1 h. The fluorescence was measured with excitation and emission wavelengths of 485 nm and 530 nm respectively using a microplate reader (Tecan Infinite 200 Pro).

In vitro antioxidant assays – The free radical scavenging action of chicory extract was determined using in vitro assays such as DPPH, ABTS, nitric oxide and hydroxyl radical scavenging activities.^24-27 Oxygen radical absorbance capacity (ORAC) assay was performed in accordance with Lucas-Abellán et al. with modifications.²⁸ The ORAC value was expressed as µM Trolox Equivalence/g.

Statistical analysis – The data were analyzed using GraphPad Prism 10.2.0 (GraphPad Software Inc.) and presented as mean ± standard deviation. The data were statistically analyzed by one way ANOVA followed by Tukey's test. p < 0.05 were considered statistically significant.

Results and Discussion

CKD is characterized by the progressive loss of renal function while AKI refers to the sudden reduction in the kidney function.²⁹ Use of plant-based compounds as nephroprotective agents is well-documented.^30,31 In the present study, we have used the in vitro cell culture models to demonstrate the nephroprotective effect of a standardized chicory extract.

Firstly, the standardized chicory leaf extract was quantified for the presence of 3.64% of chicoric acid. The details of chicoric acid quantification are provided in Supplementary file. The retention time of chicoric acid in the reference standard and chicory extract was found to be 2.667 min and 2.625 min respectively (Fig. 1).

Fig. 1.

HPLC chromatograms of chicoric acid. (A) Reference standard; (B) Chicory extract.

In the present study, we have used the CP-induced HEK293 cell culture model to demonstrate the protective effect of chicory extract. CP is a well-known anticancer drug used for treating several types of cancer. The drug, however, is associated with unwanted side effects including AKI and other kidney diseases.³² CP-induced toxicity in HEK293 cells is a well-known model to study the nephroprotective effects of drugs/natural agents against AKI.³³

The cell viability was assessed in the presence of different concentrations of chicory extract, to establish the cytotoxicity in HEK293 cells (Fig. 2). There was no significant change in the cell viability upto 50 µg/mL of the plant extract. However, the viability % was considerably reduced at 100–500 µg/mL of chicory extract as compared to the untreated control cells (F_(7,16) = 14.86, p < 0.01). Based on these results, we used 12.5 and 25 µg/mL as the non-cytotoxic concentrations of chicory extract for further experiments.

Fig. 2.

Cytotoxicity assessment of chicory extract in HEK293 cells. The cells were exposed to different concentrations of chicory extract for 24 h followed by the cell viability assessment using MTT assay. The values are mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001 and ****p < 0.0001 Vs. control.

As shown in Fig. 3A, a 24 h treatment of HEK293 cells with CP (20 µM) caused noticeable changes in cellular morphology such as rounded cell shape and cell shrinkage. Interestingly, these CP-induced changes were markedly reduced in the cells pretreated with chicory extract. We further examined the apoptotic changes in the cells using AO/EB fluorescent staining. The normal cells remained AO-stained with green fluorescence whereas the CP-induced cells showed obvious signs of early and late apoptosis along with necrotic cells. The CP-induced apoptosis was markedly reduced in chicory extract-treated cells (Fig. 3B). We further confirmed the anti-apoptotic effect of chicory extract by examining the CP-induced caspase activation using fluorescence assays. As expected, CP treatment induced 3.02-fold (F_(3,8) = 186.5, p < 0.0001) increase in the caspase 3/7 activity relative of control (Fig. 3C). However, the caspase activity was significantly reduced in the cells treated with chicory extract at 12.5 and 25 μg/mL (p < 0.0001). Similarly, the caspase 9 activity of CP-treated cells was markedly increased (F_(3,8) = 181.9, p < 0.0001) as compared to the control cells. Pretreatment of the cells with chicory extract at 12.5 (p < 0.05) and 25 μg/mL (p < 0.0001) significantly inhibited the caspase 9 activity in CP-induced cells. These results clearly suggest the possible protective role of chicory extract against CP-induced apoptosis in HEK293 cells.

Fig. 3.

Effect of chicory extract on the cellular morphology in cisplatin-induced HEK293 cells. (A) Representative photomicrographs of cells exposed to 20 μM of cisplatin (CP) in presence or absence of chicory extract (12.5 and 25 μg/mL). (B) Fluorescent images of acridine orange/ethidium bromide stained HEK293 cells following a 24 h exposure to CP (20 μM). Yellow arrows: healthy cells; blue arrows: cells showing late apoptosis. Cells were viewed and images captured using a fluorescent microscope (Lawrence & Mayo). Scale bar: 50 μm. The cell lysates were measured for (C) caspase 3/7 activity and (D) caspase 9 activity using fluorescent substrates. The values are mean ± SD of three independent experiments. ####p < 0.0001 Vs. control. *p <0.05 and ****p < 0.0001 Vs. CP group.

The available literature suggest that AKI is associated with the activation of apoptotic signaling in the cells.³⁴ CP intoxication induces apoptosis by increasing the expression of P53 and proapoptotic Bax while downregulating Bcl-2 protein.³⁵ Hence, in the present study, we have examined the levels of these key apoptotic proteins in the cells following a pretreatment with the non-cytotoxic concentrations of chicory extract. In line with the previous findings, we observed that CP induced the apoptotic changes in the cells. Interestingly, chicory extract could significantly reduce the P53 expression and Bax/Bcl-2 ratio in the CP-exposed cells indicating the strong anti-apoptotic effect. As shown in Fig. 4, CP induced a 1.15-fold increase in the relative expression of P53 (F_(3,8) = 11.52, p < 0.01) in the cells, compared to control. Interestingly, the P53 expression was downregulated to a significant extent by chicory extract at 12.5 μg/mL compared to CP-alone treated cells (1.19-fold, p < 0.01). Further analysis of the downstream pro-and anti-apoptotic proteins showed a significant increase in the Bax/Bcl-2 ratio in the CP-induced cells compared to normal cells (F_(3,8) = 39.54, p < 0.0001). Pretreatment with chicory extract significantly reduced the Bax/Bcl-2 ratio (p < 0.01), compared to CP-alone treated cells.

Fig. 4.

Effect of chicory extract on caspase-dependent apoptotic pathway in cisplatin-induced HEK293 cells. The cells were pretreated with chicory extract (12.5 and 25 μg/mL) and exposed to CP (20 μM) for 24 h. The cell lysates (20 μg) were analyzed by western blotting. (A) Representative blots showing the expression of apoptosis-related proteins. (B) Quantification of proteins by densitometry analysis (ImageJ software). The values are mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01 and ####p < 0.0001 Vs. control group. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 Vs. CP group.

Further, the CP-induced caspase activation was markedly reduced in the chicory extract-treated cells. The exposure of CP significantly activated the caspase-9 (F_(3,8) = 8.69, p < 0.05) and caspase 3 proteins (F_(3,8) = 38.56, p < 0.0001) compared to the control cells. Subsequent to caspase activation, it was further observed that CP treatment induced PARP-1 cleavage (F_(3,8) = 122.5, p < 0.0001) compared to the control cells. Pretreatment with chicory extract significantly reduced the caspase-9 (p < 0.05) and caspase 3 cleavage (p < 0.01) compared to CP-alone treated cells. Further, the chicory extract significantly reduced the CP-induced PARP-1 cleavage (p < 0.0001). These data provided the first line of evidence that chicory extract was effective in protecting the cells against CP-induced toxicity via suppression of P53-dependent intrinsic apoptotic pathway.

Further, to investigate the antioxidant effect of the extract, we used H₂O₂ as a stable source of free radicals inducing oxidative stress in HEK293 cells.³⁶ In addition, H₂O₂ triggers apoptosis by reducing the mitochondrial membrane potential and increased cytochrome c release and caspase activation.^37,38 The cells treated with H₂O₂ showed obvious signs of stress resulting in the altered cellular morphology compared to the control. Pretreatment with chicory extract protected the cells from H₂O₂-induced stress (Fig. 5A). Further, the H₂O₂-induced cellular apoptotic changes were considerably reduced in the cells treated with respective concentrations of chicory extract (Fig. 5B).

Fig. 5.

Effect of chicory extract on the cellular morphology in H2O2-induced HEK293 cells. (A) Representative photomicrographs of cells exposed to 500 μM of H2O2 in presence or absence of chicory extract (12.5 and 25 μg/mL). (B) Fluorescent images of acridine orange/ethidium bromide stained HEK293 cells following a 24 h exposure to 500 μM of H2O2. Yellow arrows: healthy cells; blue arrows: cells showing late apoptosis. Cells were viewed and images captured using a fluorescent microscope (Lawrence & Mayo). Scale bar: 50 μm.

Fig. 6 shows the relative expression of apoptotic proteins in H₂O₂-induced HEK293 cells. The H₂O₂-induced cells showed a marked increase in the Bax/Bcl2 ratio compared to the control cells (F_(3,8) = 50.18, p < 0.001). Further, there was a significant increase in the relative expression of cleaved caspase 3 (F_(3,8) = 33.79, p < 0.001) and caspase 9 (F_(3,8) = 62.44, p < 0.01) in the H₂O₂-induced cells compared to the control. The expression of these marker proteins was noticeably reduced in the chicory extract-treated cells (p < 0.001).

Fig. 6.

Effect of chicory extract on the expression of apoptotic proteins in H2O2-induced HEK293 cells. The cells were pretreated with chicory extract (12.5 and 25 μg/mL) and exposed to H2O2 (500 μM) for 24 h. The cell lysates (20 μg) were analyzed by western blotting. Quantification of proteins by densitometry analysis (ImageJ software). The values are mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01 and ####p < 0.0001 Vs. control group. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 Vs. CP group.

It is well-known that activation of Pi3K/Akt signaling is a crucial survival mechanism to abrogate the oxidative stress and cell injury.³⁹ Accumulating evidence suggest that natural products can play a protective role against pathological manifestations via activation of Pi3K/Akt pathway.⁴⁰ In agreement with these observations, the cells exposed to the respective concentrations of chicory extract showed a certain increase in the Pi3K/Akt phosphorylation. As shown in Fig. 7, there was a significant downregulation of Akt signaling in the H₂O₂-induced cells compared to the control (F_(3,8) = 269.1, p < 0.0001). Pretreatment with chicory extract at 12.5 µg/mL (p < 0.0001) and 25 µg/mL (p < 0.001) significantly activated the Akt signaling in the cells. Similar trend was observed in the Pi3K expression. However, the data were not significant. These data clearly indicate the anti-apoptotic activity of chicory extract in H₂O₂-induced cells via activation of Akt/Pi3K signaling.

Fig. 7.

Effect of chicory extract on Akt/Pi3K signaling pathway in H2O2-induced HEK293 cells. The cells were pretreated with chicory extract (12.5 and 25 μg/mL) and exposed to H2O2 (500 μM) for 24 h. The cell lysates (20 μg) were analyzed by western blotting. Quantification of proteins by densitometry analysis (ImageJ software). The values are mean ± SD of three independent experiments. ####p < 0.0001 Vs. control group. ****p < 0.0001 Vs. H2O2 group.

Further, Pi3K/Akt signaling activated the downstream Nrf-2/HO-1 antioxidant signaling pathway in the chicory extract-treated cells (Fig. 8A). treatment of cells with 500 µM H₂O₂ considerably reduced the expression of Nrf2 (F_(3,8) = 65.20, p < 0.001) and HO-1 proteins (F_(3,8) = 17.86, p < 0.01), compared to control cells. The antioxidant protein expression was however restored in the cells pretreated with chicory extract. The relative expression of Nrf2 was significantly increased in the chicory extract-treated cells as compared to the H₂O₂ treated cells (p < 0.0001). Similarly, the extract treatment at 12.5 µg (p < 0.001) and 25 µg/mL (p < 0.01) markedly upregulated the expression of HO-1 protein in the cells. Our findings are in line with the previous reports suggesting an involvement of Nrf-2 activation via Pi3K/Akt signaling as the cell survival mechanism against oxidative stress.⁴¹

Fig. 8.

Antioxidant effect of chicory leaf extract in H2O2-induced HEK293 cells. (A) Effect of pretreatment with chicory extract (12.5 and 25 μg/mL) on Nrf2-HO-1 antioxidant pathway in the cells. The cell lysates (20 μg) were analyzed by western blotting and the protein expression was quantified by densitometry analysis (ImageJ software). (B) Quantification of cellular ROS by DCFH-DA assay. The values are mean ± SD of three independent experiments. ##p < 0.01, ###p < 0.001 and ####p < 0.0001 Vs. control group. ****p < 0.0001 Vs. H2O2 group. **p < 0.01, ***p < 0.001 and ****p < 0.0001 Vs. H2O2 group.

The effect of pretreatment of cells with chicory extract on the intracellular ROS generation in H₂O₂-induced HEK293 cells was determined using the DCFH-DA fluorescent probe (Fig. 8B). As expected, the fluorescent intensity of the H₂O₂-induced cells was increased significantly compared to the control cells (F_(3,8) = 832.9, p < 0.0001), indicating an increment in the intracellular ROS production. However, chicory extract-treated cells showed a concentration-dependent reduction in the ROS level compared to the H₂O₂-alone treated cells (p < 0.0001).

Further examination of the antioxidant signaling showed that treatment of cells with 500 µM H₂O₂ considerably reduced the expression of Nrf2 (F_(3,8) = 65.20, p < 0.001) and HO-1 proteins (F_(3,8) = 17.86, p < 0.01), compared to control cells. The antioxidant protein expression was however restored in the cells pretreated with chicory extract. The relative expression of Nrf2 was significantly increased in the chicory extract-treated cells as compared to the H₂O₂ treated cells (p < 0.0001). Similarly, the extract treatment at 12.5 µg (p < 0.001) and 25 µg/mL (p < 0.01) markedly upregulated the expression of HO-1 protein in the cells.

In the present study we examined the in vitro antioxidant activity of chicory extract. As shown in Fig. 9, chicory extract showed a concentration-dependent moderate antioxidant activity with the IC₅₀ values of 708.7 µg/mL, 47.87 µg/mL, 228.8 µg/mL and 781.1 µg/mL respectively for DPPH, ABTS, nitric oxide and hydroxyl radical scavenging. The reference compound ascorbic acid showed profound inhibition of DPPH (IC₅₀ = 18.13 µg/mL) and ABTS radicals (53.18 µg/mL). The reference standard curcumin inhibited the NO radical formation with the IC₅₀ value of 72.02 µg/mL while mannitol showed a hydroxyl radical scavenging activity at an IC₅₀ of 967.3 µg/mL.

Fig. 9.

Determination of in vitro free radical scavenging activity of chicory leaf extract. The experiment was performed in triplicates and the IC50 was calculated using non-linear regression analysis.

Fig. 10.

Determination of oxygen radical absorbance capacity (ORAC) of chicory leaf extract. The fluorescence decay curves of fluorescein in the presence of different concentrations of (A) Ascorbic acid and (B) chicory leaf extract.

Further, the antioxidant potential of chicory extract was confirmed by the peroxyl radical scavenging activity. The ORAC value of the extract was found to be 2100 ± 90.15 µM TE/g while it was 2379 ± 112.1 µM TE/g for the reference compound ascorbic acid.

In the present study, the observed effects of chicory leaf extract could be attributed to the presence of bioactive constituents such as phenolic acids, flavonoids and sterols. The extract has a standardized content of chicoric acid, an effective natural antioxidant with reported pharmacological activities.^42,43 Experimental data from previous studies have highlighted the nephroprotective properties of chicoric acid. Abd El-Twab et al. reported that chicoric acid ameliorated the methotrexate-induced AKI in rats through the activation of Nrf-2/ARE/HO-1 signaling and down-regulation of NLRP3 inflammasome signaling.⁴⁴ In a recent study, chicoric acid has been reported to attenuate the renal tubular injury in high fat diet-induced CKD mice.⁴⁵ Based on the previous literature it can be speculated that chicoric acid with other bioactive components in the chicory extract could synergistically contribute to the observed cytoprotective effects in HEK293 cells.

In conclusion, the present study demonstrated that chicory leaf extract with a standardized content of chicoric acid could significantly subside the apoptosis in HEK293 cells. Also, the plant extract effectively abrogates the oxidative stress in the cells via activation of Pi3K/AKT/Nrf-2/HO-1 signaling. These findings strongly suggest the possible nephroprotective role of chicory extract. However, to recommend its candidature as a functional ingredient it is important to evaluate the efficacy using the preclinical models of nephrotoxicity and further validate the claims in humans.

Acknowledgments

The authors thank Biomedicinal Research and analytical team, R&D Center for Excellence, Vidya Herbs Pvt Ltd., Bengaluru, Karnataka, India, for the technical support.

References

• Linkermann, A.; Chen, G.; Dong, G.; Kunzendorf, U.; Krautwald, S.; Dong, Z. J. Am. Soc. Nephrol. 2014, 25, 2689–2701. [https://doi.org/10.1681/ASN.2014030262]
• Lewington, A. J. P.; Cerdá, J.; Mehta, R. L. Kidney Int. 2013, 84, 457–467. [https://doi.org/10.1038/ki.2013.153]
• Hoste, E. A.; Bagshaw, S. M.; Bellomo, R.; Cely, C. M.; Colman, R.; Cruz, D. N.; Edipidis, K.; Forni, L. G.; Gomersall, C. D.; Govil, D.; Honoré, P. M.; Joannes-Boyau, O.; Joannidis, M.; Korhonen, A.-M.; Lavrentieva, A.; Mehta, R. L.; Palevsky, P.; Roessler, E.; Ronco, C.; Uchino, S.; Vazquez, J. A.; Vidal Andrade, E.; Webb, S.; Kellum, J. A. Intensive Care Med. 2015, 41, 1411–1423. [https://doi.org/10.1007/s00134-015-3934-7]
• Che, R.; Yuan, Y.; Huang, S.; Zhang, A. Am. J. Physiol. Renal. Physiol. 2014, 306, F367–F378. [https://doi.org/10.1152/ajprenal.00571.2013]
• Sureshbabu, A.; Ryter, S. W.; Choi, M. E. Redox Biol. 2015, 4, 208–214. [https://doi.org/10.1016/j.redox.2015.01.001]
• Gouroju, S.; Rao, P. V. L. N.; Bitla, A. R.; Vinapamula, K. S.; Manohar, S. M.; Vishnubhotla, S. Indian J. Nephrol. 2017, 27, 359–364. [https://doi.org/10.4103/ijn.IJN_71_17]
• Popolo, A.; Autore, G.; Pinto, A.; Marzocco, S. Free Radic. Res. 2013, 47, 346–356. [https://doi.org/10.3109/10715762.2013.779373]
• Tbahriti, H. F.; Kaddous, A.; Bouchenak, M.; Mekki, K. Biochem. Res. Int. 2013, 358985. [https://doi.org/10.1155/2013/358985]
• Modaresi, A.; Nafar, M.; Sahraei, Z. Iran J. Kidney Dis. 2015, 9, 165–179.
• Zhong, Y.; Deng, Y.; Chen, Y.; Chuang, P. Y.; Cijiang He, J. Kidney Int. 2013, 84, 1108–1118. [https://doi.org/10.1038/ki.2013.276]
• Gobe, G. C.; Wojcikowski, K. Clin. Nephrol. 2020, 93, 49–54. [https://doi.org/10.5414/CNP92S108]
• Janda, K.; Gutowska, I.; Geszke-Moritz, M.; Jakubczyk, K. Molecules 2021, 26, 1814. [https://doi.org/10.3390/molecules26061814]
• Al-Malki, A.; Abo-Golayel, M. K. Life Sci. J. 2013, 10, 140–157.
• Hasannejad, F.; Ansar, M. M.; Rostampour, M.; Mahdavi Fikijivar, E.; Khakpour Taleghani, B. J. Physiol. Sci. 2019, 69, 465–476. [https://doi.org/10.1007/s12576-019-00659-8]
• Abbas, Z. K.; Saggu, S.; Sakeran, M. I.; Zidan, N.; Rehman, H.; Ansari, A. A. Saudi J. Biol. Sci. 2015, 22, 322–326. [https://doi.org/10.1016/j.sjbs.2014.11.015]
• Epure, A.; Pârvu, A. E. E.; Vlase, L.; Benedec, D.; Hanganu, D.; Gheldiu, A.-M.; Toma, V. A.; Oniga, I. 2020, 10, 64. [https://doi.org/10.3390/plants10010064]
• Nwafor, I. C.; Shale, K.; Achilonu, M. C. ScientificWorldJournal 2017, 2017, 7343928. [https://doi.org/10.1155/2017/7343928]
• Perović, J.; Tumbas Šaponjac, V.; Kojić, J.; Krulj, J.; Moreno, D. A.; García-Viguera, C.; Bodroža-Solarov, M.; Ilić, N. Food Chem. 2021, 336, 127676. [https://doi.org/10.1016/j.foodchem.2020.127676]
• Lee, J.; Scagel, C. F. Front Chem. 2013, 1, 40. [https://doi.org/10.3389/fchem.2013.00040]
• Ribble, D.; Goldstein, N. B.; Norris, D. A.; Shellman, Y. G. BMC Biotechnol. 2005, 5, 12. [https://doi.org/10.1186/1472-6750-5-12]
• Carrasco, R. A.; Stamm, N. B.; Patel, B. K. R. Biotechniques 2003, 34, 1064–1067. [https://doi.org/10.2144/03345dd02]
• Chang, C.-Y.; Chiang, C.-Y.; Chiang, Y.-W.; Lee, M.-W.; Lee, C.-Y.; Chen, H.-Y.; Lin, H.-W.; Kuan, Y.-H. Polymers 2020, 12, 1398. [https://doi.org/10.3390/polym12061398]
• Baret, P.; Septembre-Malaterre, A.; Rigoulet, M.; Lefebvre D’Hellencourt, C.; Priault, M.; Gonthier, M. P.; Devin, A. Int. J. Biochem. Cell Biol. 2013, 45, 167–174. [https://doi.org/10.1016/j.biocel.2012.10.007]
• Blois, M. S. Nature 1958, 181, 1199–1200. [https://doi.org/10.1038/1811199a0]
• Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Free Radic. Biol. Med. 1999, 26, 1231–1237. [https://doi.org/10.1016/S0891-5849(98)00315-3]
• Marcocci, L.; Maguire, J. J.; Droy-Lefaix, M. T.; Packer, L. Biochem. Biophys. Res. Commun. 1994, 201, 748–755. [https://doi.org/10.1006/bbrc.1994.1764]
• Kunchandy, E.; Rao, M. N. A. Int. J. Pharmaceut. 1990, 58, 237–240. [https://doi.org/10.1016/0378-5173(90)90201-E]
• Lucas-Abellán, C.; Mercader-Ros, M. T.; Zafrilla, M. P.; Fortea, M. I.; Gabaldón, J. A.; Núñez-Delicado, E. J. Agric. Food Chem. 2008, 56, 2254–2259. [https://doi.org/10.1021/jf0731088]
• Tienda-Vázquez, M. A.; Morreeuw, Z. P.; Sosa-Hernández, J. E.; Cardador-Martínez, A.; Sabath, E.; Melchor-Martínez, E. M.; Iqbal, H. M. N.; Parra-Saldívar, R. Plants 2022, 11, 818. [https://doi.org/10.3390/plants11060818]
• Sujana, D.; Saptarini, N. M.; Sumiwi, S. A.; Levita, J. J. Appl. Pharm. Sci. 2021, 11, 113–127.
• Basist, P.; Parveen, B.; Zahiruddin, S.; Gautam, G.; Parveen, R.; Khan, M. A.; Krishnan, A.; Shahid, M.; Ahmad, S. J. Ethnopharmacol. 2022, 283, 114743. [https://doi.org/10.1016/j.jep.2021.114743]
• Landau, S. I.; Guo, X.; Velazquez, H.; Torres, R.; Olson, E.; Garcia-Milian, R.; Moeckel, G.W.; Desir, G.V.; Safirstein, R. Kidney Int. 2019, 95, 797–814. [https://doi.org/10.1016/j.kint.2018.11.042]
• Zhou, Y.-D.; Hou, J.-G.; Yang, G.; Jiang, S.; Chen, C.; Wang, Z.; Liu, Y.-Y.; Ren, S.; Li, W. Biomed. Pharmacother. 2019, 109, 2309–2317. [https://doi.org/10.1016/j.biopha.2018.11.108]
• Mi, X.-J.; Hou, J.-G.; Wang, Z.; Han, Y.; Ren, S.; Hu, J.-N.; Chen, C.; Li, W. Sci. Rep. 2018, 8, 15922. [https://doi.org/10.1038/s41598-018-34156-6]
• Ju, S. M.; Kang, J. G.; Bae, J. S.; Pae, H. O.; Lyu, Y. S.; Jeon, B. H. Evid. Based Complement Alternat. Med. 2015, 2015, 186436. [https://doi.org/10.1155/2015/186436]
• Liu, J.; Chen, Z.; He, J.; Zhang, Y.; Zhang, T.; Jiang, Y. Food Funct. 2014, 5, 3179–3188. [https://doi.org/10.1039/C4FO00665H]
• Viola, H. M.; Arthur, P. G.; Hool, L. C. Circ. Res. 2007, 100, 1036–1044. [https://doi.org/10.1161/01.RES.0000263010.19273.48]
• Singh, M.; Sharma, H.; Singh, N. Mitochondrion 2007, 7, 367–373. [https://doi.org/10.1016/j.mito.2007.07.003]
• Sun, Z.; Sun, L.; Tu, L. J. Alzheimers Dis. 2020, 76, 1513–1526. [https://doi.org/10.3233/JAD-191032]
• Long, H.-Z.; Cheng, Y.; Zhou, Z.-W.; Luo, H.-Y.; Wen, D.-D.; Gao, L.-C. Front. Pharmacol. 2021, 12, 648636.
• Xu, W.; Zheng, H.; Fu, Y.; Gu, Y.; Zou, H.; Yuan, Y.; Gu, J; Liu, Z.; Bian, J. Toxins 2022, 14, 733. [https://doi.org/10.3390/toxins14110733]
• Peng, Y.; Sun, Q.; Park, Y. J. Med. Food 2019, 22, 645–652. [https://doi.org/10.1089/jmf.2018.0211]
• Liu, Q.; Hu, Y.; Cao, Y.; Song, G.; Liu, Z.; Liu, X. J. Agric. Food Chem. 2017, 65, 338–347. [https://doi.org/10.1021/acs.jafc.6b04873]
• Abd El-Twab S. M.; Hussein O. E.; Hozayen, W. G.; Bin-Jumah, M.; Mahmoud, A. M. Inflamm. Res. 2019, 68, 511–523. [https://doi.org/10.1007/s00011-019-01241-z]
• Ding, X.-Q.; Jian, T.-Y.; Gai, Y.-N.; Niu, G.-T.; Liu, Y.; Meng, X.-H.; Li, J.; Lyu, H.; Ren, B.-R.; Chen, J. J. Agric. Food Chem. 2022, 70, 2923–2935. [https://doi.org/10.1021/acs.jafc.1c07795]