Oxidative Stress Modulation by Cameroonian Spice Extracts in HepG2 Cells: Involvement of Nrf2 and Improvement of Glucose Uptake

Oxidative stress plays a relevant role in the progression of chronic conditions, including cardiometabolic diseases. Several Cameroonian plants, including spices, are traditionally used as herbal medicines for the treatment of diseases where oxidative stress contributes to insulin resistance, like type 2 diabetes mellitus. This study evaluated the antioxidant capacity and the effects on oxidative-stress-induced impairment of glucose uptake of 11 Cameroonian spice extracts. H2O2-induced reactive oxygen species (ROS) production by human HepG2 cells was significantly reduced by 8/11 extracts. The most effective extracts, Xylopia parviflora, Echinops giganteus, and Dichrostachys glomerata, showed a concentration-dependent ROS-scavenging activity, which involved Nrf2 translocation into the nucleus. Xylopia parviflora, Tetrapleura tetraptera, Dichrostachys glomerata, Aframomum melegueta, and Aframomum citratum extracts showed the highest antioxidant capacity, according to oxygen radical absorbance capacity (ORAC) (2.52–88 μM Trolox Eq/g of extract), ferric-reducing antioxidant power (FRAP) (40.23–233.84 mg gallic acid Eq/g of extract), and total phenol (8.96–32.96% mg gallic acid Eq/g of extract) assays. In HepG2 cells, glucose uptake was stimulated by 4/11 extracts, similarly to insulin and metformin. H2O2-induced oxidative stress reduced glucose uptake, which was rescued by pretreatment with Xylopia aethiopica, Xylopia parviflora, Scorodophloeus zenkeri, Monodora myristica, and Dichrostachys glomerata extracts. The ROS-scavenging ability of the spice extracts may reside in some secondary metabolites observed by phytochemical profiling (reverse-phase high-performance liquid chromatography coupled to a diode array detector (HPLC-UV-DAD)). Further studies are needed to better clarify their biological activities and potential use to control oxidative stress and promote insulin sensitivity.


Introduction
Oxidative stress is recognized as an important pathophysiological factor leading to molecular and cellular tissue damage [1]. It plays a relevant role in the process of aging and in the pathogenesis of cardiometabolic disorders, including obesity, type 2 diabetes mellitus (T2DM), metabolic syndrome, and insulin resistance [2]. It has been well documented that increased reactive oxygen species (ROS) production and derangement of endogenous antioxidant defense mechanisms play a pathophysiological role in hyperglycemia-induced cellular and tissue damage [3]. Moreover, in T2DM, several studies have shown a strong association between increased intake of exogenous antioxidants, from both pharmacological (i.e., metformin) [4] and nutritional sources, and reduction of oxidative-stress-associated biomarkers [5]. Therefore, the ingestion of dietary antioxidants from foods, herbs, and spices is the most recommended approach to counteract macromolecular damage by oxidative stress, and the search for natural compounds able to contrast oxidative stress is of major interest. In this regard, plant-derived products are widely used in Africa for the treatment of many ailments and traditionally constitute the first line of health support for about 70-80% of the population [6]. The reasons for such massive use of traditional herbal medicines include cultural and economic aspects, such as the high cost and logistical difficulties associated with some pharmaceutical approaches [7]. Although the efficacy of some traditional medicines for the treatment of several ailments has been demonstrated, questions about their safety of use and toxicity have also emerged, highlighting the need for a detailed characterization of the efficacy/safety balance [8].
Interestingly, some spices commonly used in Cameroon for both nutritional and medicinal purposes have been reported to contain high levels of polyphenols with ROS-scavenging properties, and thus potentially to be able to decrease the adverse effects of ROS [9,10]. Based on these observations, this study investigated the antioxidant potential of 11 selected spice extracts from Cameroon and evaluated some associated molecular mechanisms and their ability to modulate glucose uptake in a cell-based model system after exposure to oxidative stress. For this purpose, we selected the human hepatoma HepG2 cell line, which is a widely used experimental model of human liver cells, often utilized in studies on oxidative stress and glucose uptake/insulin resistance [11].

Preparation of Spice Extracts
Material from the 11 plants (nutritional spices) was extracted according to the reported protocol [12]. The extraction yield from fruits was 6.4-49.2%, from seeds was 7.1-32.7%, and from root samples was 13.1% ( Table 1). Most of the extracts were in solid form (powders or crystals), except for Monodora myristica, which yielded an oily liquid form. The extraction yield was 7.1-32.7% for seeds, 6.4-49.2% for fruits, and 13.1% for the root samples (Table 1).

Effect of Spice Extracts on ROS Production in HepG2 Cells
The protective effect of antioxidants includes their ability to increase the cellular antioxidant defense mechanisms as well as to reduce the level of intracellular ROS generation. To test this latter effect, intracellular ROS generation was evaluated in HepG2 cells using the 5-(and-6)-Chloromethyl-

Effect of Spice Extracts on ROS Production in HepG2 Cells
The protective effect of antioxidants includes their ability to increase the cellular antioxidant defense mechanisms as well as to reduce the level of intracellular ROS generation. To test this latter effect, intracellular ROS generation was evaluated in HepG2 cells using the 5-(and-6)-Chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) assay. A 1 h treatment with 500 µM hydrogen peroxide (H 2 O 2 ) resulted in a two-fold increase of intracellular ROS levels and, as expected, co-treatment with 500 µM Trolox, a potent antioxidant, was able to substantially prevent such an increase ( Table 2). After cell pretreatment for 24 h with spice extracts (all at 10 µg/mL), ROS production induced by H 2 O 2 was reduced to a different extent by all spices except Scorodophloeus zenkeri, Monodora myristica, and Afrostyrax lepidophyllus (Table 2). Furthermore, some of the most effective extracts, like Xylopia parviflora, Echinops giganteus, and Dichrostachys glomerata, were found to exert their ROS-scavenging activity in a concentration-dependent fashion ( Figure 2). Data are expressed as % of control taken as 100; mean ± SD, N = 3. Different letters (a, b, c) refer to significant differences among values at the 5% probability threshold (Waller-Duncan test). All spice extracts were used at 10 μg/mL.

Evaluation of Antioxidant Ability of Spice Extracts
Additional information on the antioxidant properties of Cameroonian spice extracts was obtained through different biochemical assays, including phenol content quantification, oxygen radical absorbance capacity (ORAC), and ferric-reducing antioxidant power (FRAP) assays. Spice extracts, tested at the final concentration of 1 μg/mL, were found to contain different amounts of phenols ( Figure 3A). The values ranged from 0.9% to 34.4% of phenols, expressed as gallic acid equivalents (GAE) per g of extract. The highest phenol content (>18% GAE/g of sample) was found

Nuclear Translocation of Nrf2
Cells counteract the negative effects of ROS through several molecular mechanisms, such as the nuclear factor E2-related factor 2 (Nrf2) [14]. Therefore, the effect of two of the most potent ROS scavenging spices reported above, Dichrostachys glomerata and Xylopia parviflora, on Nrf2 translocation into the nucleus was assessed. According to immunofluorescence analysis, H 2 O 2 treatment increased nuclear Nrf2 abundance. Dichrostachys glomerata treatment resulted in a reduced Nrf2 immunofluorescence in the nucleus compared to controls, but was unable to counteract the effect of H 2 O 2 ( Figure 4). Compared to controls, treatment with Xylopia parviflora elicited an increase of nuclear Nrf2 levels, which was not modified in the presence of H 2 O 2 ( Figure 4). scavenging spices reported above, Dichrostachys glomerata and Xylopia parviflora, on Nrf2 translocation into the nucleus was assessed. According to immunofluorescence analysis, H2O2 treatment increased nuclear Nrf2 abundance. Dichrostachys glomerata treatment resulted in a reduced Nrf2 immunofluorescence in the nucleus compared to controls, but was unable to counteract the effect of H2O2 ( Figure 4). Compared to controls, treatment with Xylopia parviflora elicited an increase of nuclear Nrf2 levels, which was not modified in the presence of H2O2 ( Figure 4).

Fingerprinting of Spice Extracts
The spice extract compositional profiles were evaluated via reverse-phase high-performance liquid chromatography coupled to a diode array detector (HPLC-UV-DAD) ( Figure 5). The chromatogram of Xylopia aethiopica fruit hydroalcoholic extract was dominated by three peaks generated by substances with UV absorption maxima in the range of 223 to 230 nm. Xylopia parviflora fruits showed the presence of two major hydrophilic substances generating peaks at low RT values (1.5 min and 2.3 min) with UV absorption profiles typical of chlorogenic ester derivatives (λmax = 325 nm), followed by a peak series with a Gaussian-like distribution shape and λmax = 270 nm typical of gallotannin derivatives. The compositional profile of Scorodophloeus zenkeri was dominated by a major peak at RT = 10.4 min and UV absorption at λmax = 273 nm, and a shoulder at λ = 320 nm with minor peaks at higher RT with similar spectroscopic profiles. Monodora myristica seeds were characterized by a major peak at RT = 12.2 min and λmax1 = 264 nm and λmax2 = 300 nm, typical of coumarin derivatives. The composition of Tetrapleura tetraptera fruits was characterized by a major peak at RT = 8.8 min with a spectroscopic profile like that of caffeic acid, but with shorter λmax (312 nm), typical of amide caffeic derivatives. Echinops giganteus roots showed a major peak at RT = 11.4 min (λmax = 268 nm and secondary maxima in the range 288-348 nm) and at least two additional minor peaks at lower RT (9.8 min and 7.8 min) with UV absorptions (λmax = 246 nm, 321 nm, 335 nm), suggestive of substituted quinoline alkaloids. Gingerol, shogaol, and paradol (λmax = 280 nm) were clearly detectable in the chromatographic profile of Aframomum melegueta fruits, while in that of Dichrostachys glomerata, several poorly resolved peaks were observed (λmax = 220 nm, 276-280 nm). Aframomum citratum showed the presence of several components with different UV absorption profiles, among

Fingerprinting of Spice Extracts
The spice extract compositional profiles were evaluated via reverse-phase high-performance liquid chromatography coupled to a diode array detector (HPLC-UV-DAD) ( Figure 5). The chromatogram of Xylopia aethiopica fruit hydroalcoholic extract was dominated by three peaks generated by substances with UV absorption maxima in the range of 223 to 230 nm. Xylopia parviflora fruits showed the presence of two major hydrophilic substances generating peaks at low RT values (1.5 min and 2.3 min) with UV absorption profiles typical of chlorogenic ester derivatives (λ max = 325 nm), followed by a peak series with a Gaussian-like distribution shape and λ max = 270 nm typical of gallotannin derivatives. The compositional profile of Scorodophloeus zenkeri was dominated by a major peak at RT = 10.4 min and UV absorption at λ max = 273 nm, and a shoulder at λ = 320 nm with minor peaks at higher RT with similar spectroscopic profiles. Monodora myristica seeds were characterized by a major peak at RT = 12.2 min and λ max1 = 264 nm and λ max2 = 300 nm, typical of coumarin derivatives. The composition of Tetrapleura tetraptera fruits was characterized by a major peak at RT = 8.8 min with a spectroscopic profile like that of caffeic acid, but with shorter λ max (312 nm), typical of amide caffeic derivatives. Echinops giganteus roots showed a major peak at RT = 11.4 min (λ max = 268 nm and secondary maxima in the range 288-348 nm) and at least two additional minor peaks at lower RT (9.8 min and 7.8 min) with UV absorptions (λ max = 246 nm, 321 nm, 335 nm), suggestive of substituted quinoline alkaloids. Gingerol, shogaol, and paradol (λ max = 280 nm) were clearly detectable in the chromatographic profile of Aframomum melegueta fruits, while in that of Dichrostachys glomerata, several poorly resolved peaks were observed (λ max = 220 nm, 276-280 nm). Aframomum citratum showed the presence of several components with different UV absorption profiles, among which that eluting at around RT = 7.1 min, with λ max1 = 210 nm and λ max2 = 280 nm was dominant. By contrast, the HPLC-UV-DAD profile of the Zanthoxylum leprieurii did not show any detectable peak (data not shown).
Metabolites 2020, 10, x 8 of 19 which that eluting at around RT = 7.1 min, with λmax1 = 210 nm and λmax2 = 280 nm was dominant. By contrast, the HPLC-UV-DAD profile of the Zanthoxylum leprieurii did not show any detectable peak (data not shown).

Discussion
Exploitation of novel nutraceuticals for the prevention and treatment of chronic cardiometabolic diseases like T2DM and the metabolic syndrome is currently very relevant and is the focus of intense research [15][16][17][18][19][20]. An important source of these products is represented by extracts derived from plants traditionally used in some African countries for nutritional and medicinal purposes [21], and currently under characterization in terms of biochemical and nutraceutical properties [6,[8][9][10]. Moreover, since these plant extracts may exhibit a relevant antioxidant activity [6,9,10], they could represent a specific countermeasure to control excessive ROS generation, which is linked to insulin resistance in different pathological conditions (obesity, T2DM, and metabolic syndrome) [22,23].
Based on these observations, the present study aimed to evaluate the ability of selected traditional Cameroonian spices to counteract oxidative-stress-induced reduction of glucose uptake, using human HepG2 hepatoma cells as the experimental model. In addition, we implemented a comprehensive characterization of the antioxidant properties of these spice extracts. Among the 11 spice extracts included in the study, 8 were found to reduce H 2 O 2 -induced ROS production in HepG2 cells by 34-65%, and the most effective were Xylopia aethiopica, Aframomum citratum, Echinops giganteus, Dichrostachys glomerata, and Xylopia parviflora. The potential involvement of Nrf2, a master regulator of the transcription of genes involved in antioxidation, antioxidant biosynthesis, and metabolic shift, was then highlighted [24]. HepG2 cells showed a relatively high basal level of nuclear Nrf2, in agreement with previous studies [25], which was further increased by H 2 O 2 exposure, but was not affected when the latter was preceded by treatment with either Dichrostachys glomerata or Xylopia parviflora. Interestingly, exposure to Dichrostachys glomerata alone resulted in a reduced Nrf2 accumulation, whereas Xylopia parviflora extract increased Nrf2 translocation, suggesting the involvement of different pathways of antioxidant homeostasis. The antioxidant capability of these spice extracts assessed by ORAC and FRAP analyses was in agreement with total phenol content, indicating the highest antioxidant power for the same extracts (in decreasing order: Xylopia parviflora, Dichrostachys glomerata, Aframomum melegueta, Aframomum citratum, Echinops giganteus, and Tetrapleura tetraptera). These results suggest that the antioxidant effects of phenolic compounds, and particularly polyphenols, present in some extracts could be the consequence of metal ion chelation and free radical scavenging [10,26]. In agreement with these findings, some studies have observed that extracts with higher phenolic compound levels also showed a higher antioxidant activity [27]. A similar trend was observed by Abdou et al. [28], Etoundi et al. [29], and Ene-Obong et al. [30], who studied the antioxidant effect of different spices. Similar experimental procedures and findings were also reported by Somanah et al. [31] when investigating the antioxidant effects of Carica papaya (Sol Var) on H 2 O 2 -induced oxidative stress in HepG2 cells. The antioxidant properties of phenolic compounds are directly related to their structure, which is most often composed of one (or more) aromatic rings carrying one or more hydroxyl groups, making them potentially capable of scavenging free radicals by forming resonance-stabilized phenoxyl radicals [32,33].
The phytochemical profiling of extracts deriving from the different species described indicated a wide compositional variability, with specimens (fruits or seeds) from some species showing the presence of a few or single dominating components (e.g., Monodora myristica seeds and Tetrapleura tetraptera fruits) and others containing a complex mix of structurally related and unrelated substances (e.g., fruits from Xylopia parviflora and Aframomum citratum). A few previous studies have reported inconsistent information regarding some of the plant parts from the species investigated in this study, and the details have been discussed elsewhere [12]. However, it is worth noting that the hydroalcoholic extract of Xylopia parviflora, which exhibited the simultaneous presence of potent antioxidant compounds such as caffeic acid derivatives and gallotannins, was the only spice extract found to be mostly active in all the assays employed in this study. These findings further support that its positive effects on the tested cell system are mediated by an antioxidant action against ROS. The results obtained suggest that the extracts act at the intracellular level and prevent the formation of free radicals, or that they can indirectly modulate the activity and expression of antioxidant and detoxifying enzymes [34,35]. In this study, we used HPLC-UV-DAD analysis to obtain fast and detailed information on the qualitative and quantitative differences existing among phytochemical extracts. However, a limitation of this technological approach is that it is not fully able to obtain immediate identification of all the observed species (especially when authentic standards are not available), which deserves further investigation.
Oxidative stress, as mentioned above, is correlated with insulin resistance on an individual basis [22,36], as also observed in an animal model with genetic deficiency of the H 2 O 2 -scavenging enzymes, which triggers adipose tissue oxidative stress and subsequent insulin resistance [37]. Moreover, several studies have demonstrated the contribution of increased ROS production to the development of diabetic vascular complications such as atherosclerosis [38]. Therefore, counteracting this pathophysiological event on a long-term basis may be beneficial for subjects at risk of T2DM as well as for patients with insulin resistance, T2DM, and metabolic syndrome, together with drug therapy if appropriate. Interestingly, the antioxidant properties of some Cameroonian plants, including 7 out of 11 spices considered here, have been evaluated in experimental models [12,[27][28][29][30], although no information in this respect was available for the remaining four plants. Based on these considerations, the effect of spice extracts on oxidative-stress-induced reduction of glucose uptake, as an experimental paradigm of insulin resistance, was assessed in HepG2 cells. Four spice extracts (Aframomum melegueta, Scorodophloeus zenkeri, Monodora myristica, and Tetrapleura tetraptera) were found to increase glucose uptake, similarly to metformin at the concentration of 1.3 µg/mL. Moreover, pretreatment of HepG2 cells with extracts and subsequent H 2 O 2 challenge unveiled the ability of Xylopia aethiopica, Xylopia parviflora, Scorodophloeus zenkeri, Monodora myristica, and Dichrostachys glomerata to fully rescue H 2 O 2 -induced reduction of glucose uptake, suggesting a potentially beneficial effect of these spices on insulin resistance through direct scavenging of ROS. The other spice extracts, although not fully effective, displayed some partial effectiveness in restoring normal glucose uptake.
The novelty of our findings is thus two-fold: (1) stimulation of glucose uptake is an important cellular event promoted by all spices tested, and (2) four spices (Xylopia parviflora, Scorodophloeus zenkeri, Echinops giganteus, and Zanthoxylum leprieurii) were shown to possess relevant antioxidant activity, in addition to that reported previously and herein for the remaining seven plants. These processes ultimately appear to be significant in terms of counteracting the impairment of glucose uptake produced by oxidative stress, thereby restoring insulin sensitivity. In support of our results, the hypoglycemic activity of four of the spices studied here, Dichrostachys glomerata, Tetrapleura tetraptera, Xylopia aethiopica, and Aframomum melegueta, was reported by other studies conducted in animal models of diabetes and in clinical trials [39][40][41][42][43].
Similarities and discrepancies among the findings of the present study and those from previous investigations on the same spice products may arise from different experimental protocols, especially different extraction procedures. The qualitative and quantitative analysis of biological molecules as well as the standardization of herbal medicines is a fundamental process to evaluate and possibly improve their safety, stability, and efficacy.
In conclusion, in the present study, we found that some Cameroonian spice extracts were able to rescue the ROS-induced intracellular impairment of glucose uptake. The observed potent antioxidant capability of most spice extracts may also be beneficial for several pathological conditions involving increased oxidative stress (chronic inflammation, atherosclerosis, cancer), in addition to improving insulin sensitivity, and may be coupled with our recent work showing their positive modulation of enzymes relevant to carbohydrate/lipid digestion and cardiometabolic diseases [12]. Moreover, the analytical data allowed the identification of some bioactive compounds that could justify the observed biological activities and that should be further characterized using more detailed analytical technologies. These results raise important questions for further research on spice extracts, aiming to validate their efficacy, toxicity, and safety with preclinical models and well-designed clinical trials, to demonstrate the health value of this nutraceutical approach, which may be placed beyond diet and before drug treatment, thus being complementary to pharmacological interventions.

MTS Cell Viability Assay
Cell viability assay was measured using the Cell Titer 96 aqueous non-radioactive cell proliferation assay (Promega, Madison, WI, USA) according to the method described by Kim and Jang [44] with slight modifications. In brief, HepG2 cells were seeded in a sterile flat-bottomed 96 well plate and incubated at 37 • C for 24 h in a humidified incubator containing 5% CO 2 . Different concentrations of extracts (1, 10, 50, and 100 µg/mL) were prepared directly in fresh serum-free MEM media and 100 µL of each treatment was added to each well and incubated for 24 and 48 h. Next, 20 µL of the MTS reagent in combination with the electron coupling agent phenazine methosulfate was added into each well and allowed to react for 1 h at 37 • C. After 2 min of shaking at minimal intensity, the absorbance at 490 nm was measured using the EnSpire PerkinElmer Multimode Plate Reader. Controls (cells with media containing DMSO (≤0.1%)) and blanks (wells containing media without cells) were assessed under the same conditions. The cell viability values were determined using the equations below.

Morphological Analysis
Cells were cultured as reported above in sterile flat-bottomed 6 cm 2 dishes and incubated at 37 • C for 24 h in a humidified incubator containing 5% CO 2 . Two different concentrations of extracts (1 and 100 µg/mL) were prepared directly in fresh serum-free media and 3 mL of each treatment was added to each dish and incubated for 24 h and 48 h. After the treatment, cells were visualized on a ZEISS microscope (ZEISS, VA, USA) using 10× and 32× magnifications.

ROS Modulation Analysis in Cells
A fluorometric assay was used to determine the intracellular ROS level in HepG2 cells, using the oxidant-sensitive fluorescence probe CM-H2DCFDA according to the method described by Somanah et al. [31]. Cells were seeded in 96 well black plates, and, after reaching 90% confluence, incubated with spice extracts at a different concentrations (0-20 µg/mL) for 24 h. Cells were then exposed to 20 µM CM-H2DCFDA and subsequently incubated with 500 µM hydrogen peroxide for 1 h to induce ROS production. The resulting fluorescence intensities were measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using the EnSpire PerkinElmer Multimode Plate Reader [31].

Determination of the Total Phenolic Content of the Extracts
The determination of the phenols was carried according to the method previously described by Anim et al. [45], with minor modifications. An aliquot of 50 µL of extract stock solution (1.0 mg/mL) previously diluted in 750 µL of distilled water was introduced into a test tube. Next, 50 µL of 2N Folin-Ciocalteu solution was added, followed by incubation at room temperature for 4 min. Freshly prepared Na 2 CO 3 (20%) solution (150 µL) was added and the tubes were further incubated for 30 min at 40 • C. Absorbance was measured at 755 nm and for each sample, three tests and three independent analyses were performed in the same conditions. The concentration calibration range from 0 to 30 µg/mL was performed using a 1.0 mM gallic acid stock solution following sample procedure. The results were expressed in µg of gallic acid equivalent/g of extract and in percentage of phenolic compounds expressed as gallic acid equivalent. The final concentration of the extracts was 50 µg/mL.

Oxygen Radical Absorbance Capacity Assay
The oxygen radical absorbance capacity (ORAC) test was performed according to the method previously described in Reference [46] with minor modifications. An aliquot of 20 µL of 1 µg/mL stock solution of each extract was introduced into a polypropylene fluorescence 96 well microplate. Subsequently, 120 µL of fluorescein solution (70 nM final concentration) previously prepared with a phosphate buffer (pH 7.4) was added to each well. The final concentration of each extract was 0.1 µg/mL. The microplate was incubated for 15 min at (37 • C and 60 µL of an AAPH reagent solution (40 mM) previously prepared with phosphate buffer (pH 7.4) was rapidly added. After 10 s of shaking at maximum intensity, the microplate was immediately placed in a microplate reader and the fluorescence was recorded every 2 min for 60 min at 485 nm (length excitation wave) and at 528 nm (emission wavelength). Each sample was analyzed three times in the same conditions and three distinct readings were performed. The calibration curve (concentrations ranging from 0-50 µM using 0.004 M Trolox stock solution) was then prepared. The calibration curve was prepared using 0.004 M Trolox stock solution following sample procedure. The ORAC values were calculated using the area under the curve (AUC) and net AUC of the standards and samples, determined using the equations below.
where R1 is the fluorescence reading at the initiation of the reaction and Rn is the last measurement.

Ferric-Reducing Antioxidant Power Assay
The ferric-reducing antioxidant power (FRAP) assay, adapted from Reference [47], was modified to evaluate the reducing power of spice extracts. At low pH, ferric tripyridyltriazine complex is reduced to its ferrous form, the resulting intense blue color being linearly related to the amount of reductant present. The FRAP reagent consisting of 2,2,6 tripyridyl-5-triazine (TPTZ, 10 mM) in 40 mM HCl and 20 mM ferric chloride in 200 mL of sodium acetate buffer (pH 3.6, 0.25 M) was freshly prepared and warmed at 37 • C prior to analysis. Two milliliters of FRAP reagent and 75 µL of the extracts were left to react for 20 min at room temperature (final concentration of the extracts: 37 µg/mL). The absorbance was read at 593 nm. The concentration calibration range from 0 to 250 µg/mL was prepared using a 1.0 mM gallic acid stock solution following sample procedure. The results were expressed as µg of gallic acid equivalent/g of extract.

Nrf2 Immunofluorescence Assay
Nrf2 localization was assessed by immunofluorescence (IF) analysis. Cells were grown on 12 mm polylysine-coated glass slides and then treated with 10 µg/mL spice extract and/or 500 µM H 2 O 2 (without FBS). After 4 h at 37 • C in a humidified incubator containing 5% CO 2 , cells were fixed in 4% formaldehyde for 5 min at RT and the subcellular localization of the Nrf2 was examined by IF staining. The formaldehyde-fixed cells were preincubated for 20 min with 10% normal serum in PBS and then incubated with a diluted solution of the primary antibody (1:100; Invitrogen, cat. PA527882) for 1 h at RT. Cells were then washed in PBS and incubated for 1 h with the Alexa Fluor 488 F(ab')2 fragment of anti-rabbit antibody (1:400; Invitrogen, cat. A32731). Nuclei were counterstained with 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Fluoroshield Sigma-Aldrich, St. Louis, MO, USA). For negative controls, the primary antibody was omitted. Preparations were then examined with a fluorescent microscope (magnification 32×; Carl Zeiss, Thornwood, NY, USA) and images were recorded.

HPLC-UV-DAD Analysis
Separation of the compounds present in extracts was carried out using a Varian LC-940 high-performance liquid chromatography (HPLC) instrument (Varian, Turin, Italy) coupled to a diode array detector (DAD). The extracts were solubilized in a hydroalcoholic solvent system (20 mg/mL; 70% w/vol) and then sonicated for sixty minutes (60 min). After stirring, the mixture was centrifuged at 4000 rpm for 10 min and filtered through paper (Whatman™ paper No 1) to separate insoluble material. The obtained solutions were separated on a Kinetex™ biphenyl 100 Å (100 × 4.6 mm; 2.6 µm) column and the elution gradient was performed with 0.05% aqueous formic acid as mobile phase A and 0.05% formic acid in acetonitrile as phase mobile B, at a flow rate of 1.6 mL/min. The elution gradient used for the separation was 5% B 0-3 min; from 5% B to 60% B 3-15 min; from 60% B to 80% 15-20 min. The sample tray temperature and the injection volume were, respectively, set at 4 • C and 5 µL. The acquisition wavelength was adjusted depending on the specific sample.

Glucose Uptake Assay
Glucose uptake activity was analyzed by measuring the uptake of 2-NBDG. Cells were cultured in 96 well black plates for 48 h. Cells were then incubated with spice extracts (1-20 µg/mL) or 500 µM metformin for 24 h. Finally, 500 µM H 2 O 2 was then added for the last incubation hour. Subsequently, to determine 2-NBDG uptake, cells were incubated with 80 µM 2-NBDG and 0.1 mM insulin (dissolved in glucose-free medium) for 1 h, and then were washed twice with ice-cold PBS to stop further uptake. 2-NBDG fluorescence intensities were measured using a microplate reader (EnSpire, PerkinElmer) at excitation and emission wavelengths of 465 and 540 nm, respectively. Glucose uptake increase rate was calculated as follows.

Statistical Analysis
Results from at least three independent experiments carried out in triplicate or quadruplicate were expressed as mean (±SD) values or as a mean percentage (%) compared to a control. Mean differences were determined by one-way ANOVA using SPSS (Version 25). The Waller-Duncan test was used to test differences of means. Pearson's linear test was used to test for correlation and values of less than 0.05 were considered statistically significant. All graphs were generated using GraphPad Prism (Version 7).