**E**ff**ects of Food Processing on In Vivo Antioxidant and Hepatoprotective Properties of Green Tea Extracts**

**Xiao-Yu Xu 1, Jie Zheng 1, Jin-Ming Meng 1, Ren-You Gan 2,3,\*, Qian-Qian Mao 1, Ao Shang 1, Bang-Yan Li 1, Xin-Lin Wei <sup>3</sup> and Hua-Bin Li 1,\***


Received: 24 October 2019; Accepted: 19 November 2019; Published: 21 November 2019

**Abstract:** Food processing can affect the nutrition and safety of foods. A previous study showed that tannase and ultrasound treatment could significantly increase the antioxidant activities of green tea extracts according to in vitro evaluation methods. Since the results from in vitro and in vivo experiments may be inconsistent, the in vivo antioxidant activities of the extracts were studied using a mouse model of alcohol-induced acute liver injury in this study. Results showed that all the extracts decreased the levels of aspartate transaminase and alanine aminotransferase in serum, reduced the levels of malondialdehyde and triacylglycerol in the liver, and increased the levels of catalase and glutathione in the liver, which can alleviate hepatic oxidative injury. In addition, the differences between treated and original extracts were not significant in vivo. In some cases, the food processing can have a negative effect on in vivo antioxidant activities. That is, although tannase and ultrasound treatment can significantly increase the antioxidant activities of green tea extracts in vitro, it cannot improve the in vivo antioxidant activities, which indicates that some food processing might not always have positive effects on products for human benefits.

**Keywords:** green tea extract; food processing; tannase; ultrasound; antioxidant activity; liver injury

#### **1. Introduction**

The antioxidant properties of food include the capacities of reducing, scavenging radicals, chelating metal ions, inhibiting oxidative enzymes, and activities as antioxidative enzymes [1–6]. Many methods have been developed for the evaluation of in vitro antioxidant activities of natural products, and some of them showed strong antioxidant activities, such as vegetables, fruits, cereals, algae, and tea [7–16].

Oxidative stress can be caused in the human body due to the overproduction of reactive oxygen species (ROS) over the capability of cells to present an effective antioxidant response [17,18]. The oxidative stress results in cellular dysfunction and is involved in various chronic disease initiation and progression, such as diabetes, cancer, neurodegeneration, aging, cardiovascular diseases, and liver diseases [19–21]. Due to strong in vitro antioxidant activities, some natural products have been regarded as effective agents for the prevention and management of several chronic diseases [22–25]. On the other hand, the formation of ROS in vivo can be stimulated due to alcohol metabolism [26,27]. The animal model with acute alcohol administration has been used to investigate the in vivo antioxidant activities of food [28,29], and it often occurs accompanied by liver injury, which can be used for

hepatoprotection studies [30]. Hence, we used an animal model with acute alcohol-induced liver injury to evaluate the in vivo antioxidant and hepatoprotective activities of green tea extracts with different processing in this study.

Green tea (*Camellia sinensis L.*) has been reported to show multiple bioactivities with health benefits, such as antioxidant, anti-inflammation, hepatoprotection, cardiovascular protection, neuroprotection, and anti-cancer [31–36]. The epidemiological studies showed that green tea consumption can result in a decreased risk of metabolic syndrome, but there is not enough evidence to draw a strong conclusion regarding tea and non-alcoholic fatty liver [37]. Moreover, accumulating in vivo evidence suggested that green tea showed hepatoprotective effects, which can ameliorate the liver injury induced by alcohol, cholesterol, chemicals, or drugs [38–41]. These benefits are mainly due to the richness of bioactive compounds like polyphenols, polysaccharides, and amino acids [42–45]. The major polyphenols in green tea include catechins and phenolic acids [46]. Catechins are mainly composed of (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), and (−)-epigallocatechin gallate (EGCG), and phenolic acids include gallic, coumaric, caffeic acids, etc. [47,48]. Furthermore, many findings have demonstrated that the catechins and phenolic acids are responsible for the antioxidant properties of green tea, which has protective effects against many diseases, such as diabetes, cancer, hypertension, and cardiovascular diseases [49–53]. However, there is potential hepatotoxicity induced by the overdose of EGCG [54,55]. Hence, food processing, such as enzymatic treatment, is used to reduce the content of EGCG in green tea extracts to eliminate its negative effects [56].

Numerous types of enzymes are currently used in food processing to meet the demands of a broad variety of food products [57,58]. Additionally, the use of enzymes in foods produces other substances from enzymatic hydrolysis and improves the quality of food products [59–62]. Due to its capacities in catalyzing hydrolysis of gallic acid esters and hydrolysable tannins, tannase (tannin acyl hydrolase EC 3.1.1.20) is widely used in the production of gallic acid [63,64]. On the other hand, ultrasound has been extensively used in food processing, improving the quality and safety of products [62,65,66]. Also, ultrasound creates cavitation and promotes heat and mass transfer, which accelerates chemical reactions, such as enzymatic reactions [67–69]. However, some compounds in products have been changed during food processing, and they might pose negative effects on the quality and health benefits of foods [70]. Most present studies use in vitro methods to evaluate antioxidant properties of food after they are treated with different processing methods. But fewer studies were found about the in vivo antioxidant activities of processed and original products. Our previous study revealed that tannase and ultrasound treatments markedly increase the antioxidant activities of green tea extracts based on the results of in vitro assays. In this study, we aim to investigate the in vivo effects of green tea extracts processed by tannase and ultrasound against oxidative stress and liver injury induced by alcohol.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

Tannase (200 U/g) was bought from Yuanye Biological Technology Co., Ltd. (Shanghai, China). All the other chemicals or reagents were of analytical grade. The kits of aspartate transaminase (AST), alanine aminotransferase (ALT), triglyceride (TG), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and total protein were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The deionized water was used for all experiments.

#### *2.2. Preparation of Green Tea Extracts*

Green tea was purchased from the local market of Guangzhou, China, and was ground into powder which was filtered through a 100 mesh sieve. The deionized water was used to mix with the powder (50 g/L, *w*/*v*), and then the mixture was heated at 85 ◦C for 30 min in a water bath and centrifugated at 4200× *g* for 30 min. The supernatants were collected as the green tea extracts for further experiments.

According to the previous study, the green tea extracts showed the highest antioxidant activities in vitro under the optimal extraction conditions with 0.1 M citrate-phosphate buffer (pH 4.62), ultrasonic temperature of 44.12 ◦C, ultrasonic time of 12.17 min, tannase concentration of 1 mg/mL, and ultrasonic power of 360 W [71]. The green tea extracts were divided into four groups with different treatments. For the first group, the treatment of ultrasound and tannase (UST) was conducted by mixing the green tea extract with 1 mg/mL tannase in 0.1 M citrate-phosphate buffer (pH 4.62) and using an ultrasonic device (Kejin Ultrasonic Equipment Factory, Guangzhou, China) for 12.17 min at 44.12 ◦C under 360 W. For the second group, the ultrasound treatment (US) was carried out by mixing the extract with 0.1 M citrate-phosphate buffer (pH 4.62) without tannase and treating with ultrasound for 12.17 min at 44.12 ◦C under 360 W. For the next group, the only tannase (TAN) treatment was mixed with 1 mg/mL tannase in 0.1 M citrate-phosphate buffer (pH 4.62), and placed in a water bath at 44.12 ◦C for 12.17 min. The group with the green tea extract (GTE) had a treatment which included the dilution of original extract with the same buffer solution. After the completion of treatment, the mixtures were fully mingled by a vortexing machine. Then they were placed in the water bath at 100 ◦C for 10 min to inactivate tannase and cooled down to room temperature. The mixture was centrifugated at 4200× *g* for 10 min, and the supernatant was collected for further experiments.

The extracts from UST, US, TAN, and GTE groups were later dried via the vacuum rotary evaporator. The dried crude extracts were collected and dissolved in deionized water for the animal study.

#### *2.3. Animal Study*

Male Kunming mice (20–25 g) were obtained from the Experimental Animal Center of Sun Yat-Sen University, Guangzhou, China. All procedures were strictly carried out according to the principles of "laboratory animal care and use" approved by School of Public Health, Sun Yat-Sen University (No. 2019-002; 28 February 2019). The mice were fed in a specific pathogen free (SPF) animal room under a temperature of 22 ± 0.5 ◦C, relative humidity of 40–60%, and 12 h light/dark cycle. After the mice had acclimated for one week, they were randomly divided into different groups (6 mice in each group), including control, model, and treatment groups. The treatment groups were fed intragastrically with the solutions of US (50 mg/kg body weight), TAN (50 mg/kg body weight), GTE (50 mg/kg body weight), and UST (50, 100, 200 mg/kg body weight) for 7 days. The model and control groups received the deionized water. On the seventh day, all treatment and model groups were fed intragastrically with 52% alcohol (*v*/*v*, 10 mL/kg body weight) 30 min after the last administration, while the control group received the deionized water. After 6 h fasting following the last administration of alcohol, all mice were weighed and anaesthetized to sacrifice. Then the blood samples were collected and centrifuged at 3000× *g* for 10 min. The serum was isolated for AST and ALT evaluation which followed the instructions of the commercial kits. The liver was harvested and weighed. In order to control potential oxidation of the sample, the low temperature condition was adopted. That is, the 10% (*w*/*v*) liver homogenate was prepared by mixing the liver and ice-cold 0.9% normal saline solution in a glass tube that was put in the ice box and the liver was grinded with a glass grinder [29,72,73]. The homogenate of liver was centrifuged at 2500× *g* for 10 min to obtain the supernatant which was used for the biochemical assays.

#### *2.4. Biochemical Assays*

The determination of SOD, CAT, GSH, MDA, TG, and total protein followed the instructions of the Nanjing Jiancheng commercial kits produced by Nanjing Jiancheng Bioengineering Institute, Nanjing, China [29,72–76]. (1) Determination of SOD activity: the xanthine and xanthine oxidase reacted to produce superoxide radicals. The radicals oxidated hydroxylamine to induce nitrite that reacted with a color developing agent to produce a purple-red compound. When the sample contained SOD, it reduced the production of nitrite, which was reflected on a decrease in absorbance. The liver

homogenate was diluted by ice-cold saline solution to 0.25% (*w*/*v*), and the 50 μL was mixed with reagents. The mixture was placed in room temperature for 10 min. The absorbance was detected at 550 nm using a spectrophotometer. (2) Determination of CAT activity: CAT catalyzed the H2O2 decomposition, and the remaining H2O2 reacted with ammonium molybdate to produce a light yellow compound. The activity of CAT was calculated based on the change in absorbance. The liver homogenate was diluted by ice-cold saline solution to 0.5% (*w*/*v*), and the 50 μL was mixed with reagents. The absorbance of mixture was detected at 405 nm using the microplate reader. (3) Determination of GSH content: The reaction of GSH and 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) produced a yellow compound. The GSH content was determined by the colorimetry. The 100 μL liver homogenate (10%, *w*/*v*) was mixed with 0.1 mL precipitant, and the mixture was centrifuged at 3500× *g* for 20 min to obtain the supernatant. The 100 μL supernatant was mixed with reagents, and placed in room temperature for 5 min. The absorbance of mixture was determined at 405 nm using the microplate reader. (4) Determination of MDA content: The reaction of MDA with thiobarbituric acid (TBA) led to a red product that had an absorbance peak at 532 nm. The 100 μL liver homogenate (10%, *w*/*v*) was mixed with reagents, and put in a water bath at 95 ◦C for 40 min. After the mixture was cooled down, it was centrifuged at 4000× *g* for 10 min. The absorbance of the supernatant was detected at 532 nm using a spectrophotometer. (5) Determination of TG content: TG was hydrolyzed into glycerol and fatty acids by the lipase. The reaction of glycerol and adenosine triphosphate (ATP) was catalyzed by glycerol kinase (GK) and produced glycerol-3-phosphate, which was further oxidized into H2O2 and dihydroxyacetone phosphate by glycerophosphate oxidase. H2O2 reacted with 4-aminoantipyrine (4-AAP) and p-chlorophenol under the catalysis of peroxidase to produce a red quinone compound, and its color was proportional to the TG content. The 2.5 μL liver homogenate (10%, *w*/*v*) was mixed with reagents and placed in the water bath at 37 ◦C for 10 min. The absorbance of mixture was detected at 510 nm using the microplate reader. (6) Determination of AST activity: AST could act on α-ketoglutaric acid and aspartic acid to produce oxaloacetic acid and glutamic acid. The oxaloacetic acid decarboxylated into pyruvate acid that reacted with 2,4-dinitrophenylhydrazine (DNPH) to produce 2,4-dinitrophenylhydrazone which was a reddish brown compound under alkaline conditions. The 5 μL serum was mixed with the reagents and placed at room temperature for 15 min. The absorbance was detected at 510 nm using a microplate reader. (7) Determination of ALT activity: Under the condition of 37 ◦C and pH 7.4, ALT acted on alanine and α-ketoglutaric acid to produce pyruvate acid and glutamic acid. After 30 min, DNPH in hydrochloric acid solution was added to form acetone phenylhydrazone that was a reddish brown compound under alkaline conditions. The 5 μL serum was mixed with the reagents and placed at room temperature for 15 min. The absorbance was recorded at 510 nm using the microplate reader. (7) Determination of total protein: The protein reduced Cu2<sup>+</sup> to Cu<sup>+</sup> under the alkaline conditions, and Cu<sup>+</sup> reacted with the bicinchoninic acid (BCA) reagent to form a purple complex compound that had an absorbance peak at 562 nm. The absorbance was proportional to the concentration of total protein. The liver homogenate was diluted by ice-cold saline solution to 0.5% (*w*/*v*), and the 10 μL diluted liver homogenate was mixed with the reagents. The mixture was placed at 37 ◦C for 30 min and the microplate reader was used to detect the absorbance at 562 nm.

#### *2.5. Statistical Analysis*

All experiments were conducted independently three times, and the results were presented as mean ± standard deviation (SD). The statistical analysis was performed by using SPSS 19.0 (IBM SPSS Statistics, IBM Corp, Somers, NY, USA). One-way ANOVA plus a post hoc least-significant difference (LSD) test was utilized to analyze the significance of differences for each group, and the statistical significance was defined at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. E*ff*ects of Extracts on Antioxidant Enzymes, GSH, and MDA in the Liver*

The animal experiments were conducted with the model of alcohol-induced liver injury to assess antioxidant activities of the extracts in vivo. The alcohol administration was observed to induce oxidative stress in mice. Compared with the control group, the model group showed an obvious decrease in CAT and SOD activities as well as GSH content, and an increase in MDA content in the mouse liver (*p* < 0.05, Figure 1).

**Figure 1.** Effects of different extracts on catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in the liver. (**A**) CAT, (**B**) SOD, (**C**) GSH, and (**D**) MDA: extracts from different methods (50 mg/kg body weight). One unit of CAT activity is defined as the amount of protein which decomposes 1 μmol H2O2 per second. One unit for SOD activity is defined as the amount of protein necessary to inhibit 50% of the SOD reaction where superoxide radicals oxidize hydrosylamine to produce nitrite. UST, the group treated with ultrasound and tannase; TAN, the group treated with only tannase; US, the group treated with only ultrasound; green tea extract (GTE), the group treated without ultrasound and tannase. The values are presented as means ± SD. Bars with different letters (a–c) are significantly different (*p* < 0.05). \* *p* < 0.05, the model group vs. the control group.

As displayed in Figure 1A, all treatment groups at the same dose (50 mg/kg body weight) significantly increased the CAT activity in comparison with the model group. In addition, there was no significant difference in CAT activity among the UST, US, and GTE groups. However, the TAN group showed a negative effect on CAT activity compared with the GTE group. This is because tannase might induce the degradation of some related bioactive compounds [71]. Seen from Figure 1B, although the activity of SOD in the UST group was significantly higher than that of the GTE group, all treatment groups did not increase SOD activity compared with the model group. However, treatment with 200 mg/kg body weight could increase SOD activity (Figure 2B). Therefore, the dose of 50 mg/kg body weight was too low to increase the SOD activity. From Figure 1C, all treatment groups improved GSH content significantly when they were compared with the model group. In addition, there was no marked difference in the levels of GSH between the US and GTE groups. However, the UST and

TAN groups showed significantly lower levels of GSH than the GTE groups. These results suggest that tannase treatment might pose negative effects on the in vivo antioxidant activities of green tea extract. As shown in Figure 1D, green tea extracts by different methods reversed the alcohol-induced increase in the level of hepatic MDA, but the differences between groups were not significant. For another thing, the effects of UST at different doses (50, 100, and 200 mg/kg body weight) were shown in Figure 2, and they all increased the activities of CAT and SOD as well as GSH content and lowered the level of MDA. There was no distinct dose-dependent response for the levels of CAT, SOD, and MDA. However, a higher dose showed a stronger effect on GSH content.

**Figure 2.** Effects of different doses of UST extracts on CAT, SOD, GSH, and MDA in liver. (**A**) CAT, (**B**) SOD, (**C**) GSH, and (**D**) MDA: UST extract (50, 100, and 200 mg/kg body weight). One unit of CAT activity is defined as the amount of protein which decomposes 1 μmol H2O2 per second. One unit for SOD activity is defined as the amount of protein necessary to inhibit 50% of the SOD reaction where superoxide radicals oxidize hydrosylamine to produce nitrite. UST, the group treated with ultrasound and tannase; TAN, the group treated with only tannase; US, the group treated with only ultrasound; GTE, the group treated without ultrasound and tannase. The values are presented as means ± SD. Bars with different letters (a–c) are significantly different (*p* < 0.05). \* *p* < 0.05, the model group vs. the control group.

Acute alcohol consumption has been reported to induce oxidative stress and stimulate lipid peroxidation, leading to hepatic dysfunction [77]. It produces free radicals and promotes the development of liver diseases. Thus, the activation of antioxidant enzymes for scavenging free radicals is essential for the protection against alcoholic liver disease. SOD and CAT are important antioxidant enzymes in the defence against oxidative damage. SOD acts on removing superoxide, and CAT catalyses the decomposition of hydrogen peroxide [30]. The contents of GSH are also a crucial indicator reflecting the antioxidant and oxidant status in vivo [78]. MDA is an important product of lipid peroxidation, and its content shows the degree of interaction of ROS with polyunsaturated fatty acid [79].

The present study showed that the administration of extracts increased the levels of CAT, SOD, and GSH, and decreased the contents of MDA in the liver as compared to the model group. However, the differences among groups were not significant in the assays of SOD and MDA. On the other hand, the groups with tannase treatment showed low CAT activity and GSH content, indicating that tannase

might degrade some other compounds and affect antioxidant activity negatively in vivo. Overall, the tannase and ultrasound treatment had no significant beneficial effects on the antioxidant enzymes and MDA compared with GTE groups, even posing negative effects on the GSH content.

#### *3.2. E*ff*ects of Extracts on AST and ALT in Serum*

The activities of serum aspartate transaminase (AST) and alanine transaminase (ALT) were measured to investigate the effects of extracts on liver injury induced by acute alcohol intake. In Figure 3, the serum AST and ALT activities were increased in the model group compared with the control group (*p* < 0.05). All treatment groups significantly decreased the serum AST activities in comparison with the model group, but there was no significance among the treatment groups. On the other hand, all treatment groups non-significantly decreased the serum ALT activities compared with the model group. As shown in Figure 3C,D, the ingestion of UST extracts significantly ameliorated the alcohol-induced increase in AST activities, but the dose-dependent effect was not significant. For the ALT activity, the highest dose of UST extracts decreased significantly the activity of ALT in comparison with the model group.

**Figure 3.** Effects of different extracts on serum aspartate transaminase (AST) and alanine aminotransferase (ALT) activities. (**A**) AST and (**B**) ALT: extracts from different methods (50 mg/kg body weight). (**C**) AST and (**D**) ALT: UST extracts (50, 100, and 200 mg/kg body weight). UST, the group treated with ultrasound and tannase; TAN, the group treated with only tannase; US, the group treated with only ultrasound; GTE, the group treated without ultrasound and tannase. The values are presented as means ± SD. Bars with different letters (a,b) are significantly different (*p* < 0.05). \* *p* < 0.05, the model group vs. the control group.

AST and ALT are known as effective markers for liver function. In response to liver damage, AST and ALT are released to plasma from hepatocytes, and the levels of serum AST and ALT are enhanced [80]. In this study, the model groups showed higher serum AST and ALT activities than the control group, which indicated the injury in the liver. The extracts with different methods reduced the serum AST and ALT activities, but the differences among groups were not significant. It indicated that the treatment of ultrasound or tannase contributed little to the reduction in serum ALT and AST activities compared with GTE group. In addition, treatment groups decreased non-significantly the

ALT activity in the comparison with the model group. It might be because the doses of extracts were too small to produce a significant effect. In the dose-dependent experiment, a higher dose of UST extract showed a more potent effect on attenuating the abnormal increase in serum ALT activities (Figure 3D). It suggested that using appropriate doses of extracts obtained from the combined treatment of ultrasound and tannase in green tea could diminish the liver dysfunction induced by alcohol in vivo.

#### *3.3. E*ff*ects of Extracts on TG in Liver*

Acute alcohol intake resulted in disturbed lipid metabolism with an increase in hepatic TG. Figure 4 displays the effects of extracts on TG level in liver tissue, and a significant elevation in the level of TG was observed in the model group (*p* < 0.05). The administration of extracts from different methods non-significantly decreased the level of TG in liver tissue compared with the model group. In addition, the results showed that high doses of UST extract could lower the hepatic TG significantly compared with the model group in Figure 4B, but the dose-dependent effect was not obvious.

**Figure 4.** Effects of different extracts on the levels of triglyceride (TG). (**A**) Extracts from different methods (50 mg/kg body weight). (**B**) UST extracts (50, 100, and 200 mg/kg body weight). UST, the group treated with ultrasound and tannase; TAN, the group treated with only tannase; US, the group treated with only ultrasound; GTE, the group treated without ultrasound and tannase. The values are presented as means ± SD. Bars with different letters (a,b) are significantly different (*p* < 0.05). \* *p* < 0.05, the model group vs. the control group.

Excessive drinking can lead to lipid production in the liver, and the accumulation of adipose in liver tissue promotes the progress of relevant diseases [81]. The results revealed that the ultrasound and tannase treatment had little effect in reducing lipid accumulation, and there was no significant difference compared with the GTE group. On the other hand, a high dose of UST extracts could significantly reduce the level of TG in the liver. These results indicated that a certain dose of UST extract could be effective in reducing lipids in the liver.

In our previous study, the treated extracts showed stronger in vitro antioxidant activities than the original extracts, and the contents of several compounds were determined using the HPLC method [71]. HPLC results revealed that tannase and ultrasound treatment increased gallic acid content, while the original extract had a higher EGCG content than the treated extracts. It indicated that the in vitro antioxidant activities of UST and TAN were mainly attributed to the content of gallic acid, while the antioxidant properties of US and GTE mainly depended on the contents of EGCG. On the other hand, this study showed that the differences between treated and original extracts were not significant in vivo, and to our surprise, the food processing even posed a negative effect on in vivo antioxidant activities in some cases. Therefore, the results from in vivo and in vitro studies on antioxidant activities were inconsistent, suggesting that some food processing might not always have positive effects on health benefits. This study also indicated that in the future, the effects of food processing on the quality of products should not be evaluated only using in vitro methods, and in vivo evaluation methods should be adopted.

#### *3.4. Histopathological Observation*

The histopathological analysis on hematoxylin and eosin (H&E)-stained liver tissue slices further confirmed the protective effects of all treatment groups against acute alcohol liver injury (Figure 5). The model group showed obvious pathologic changes such as disordered cell arrangement and lipid droplets accumulation, while the control group had no significant damage (Figure 5A,B). All treatment groups presented less steatosis than the model group (Figure 5C–F), which indicated that the lesion induced by acute alcohol administration was attenuated by green tea extracts from different treatment methods. In addition, there was no obvious difference among different treatment groups.

**Figure 5.** The histopathological observation of hematoxylin and eosin (H&E)-stained liver tissue slices. (**A**) control group; (**B**) model group; (**C**) UST group (50 mg/kg body weight); (**D**) TAN group (50 mg/kg body weight); (**E**) US group (50 mg/kg body weight); (**F**) GTE group (50 mg/kg body weight). UST, the group treated with ultrasound and tannase; TAN, the group treated with only tannase; US, the group treated with only ultrasound; GTE, the group treated without ultrasound and tannase.

#### **4. Conclusions**

This study investigated the effects of food processing (tannase and ultrasound treatments) on in vivo antioxidant and hepatoprotective properties of green tea extracts. Results showed that green tea extracts with ultrasound and tannase treatment could attenuate the oxidative stress induced by acute alcohol administration. It increased the activities of antioxidant enzymes, such as SOD and CAT, and the content of antioxidants such as GSH, and reduced the level of MDA. However, there was no significant difference between the treated and original extracts. To our surprise, the tannase treatment even had negative effects on the in vivo antioxidant activities of extracts, which might be related to its degradation of some compounds. In addition, the dose-effect relationship was not significant in green tea extracts with tannase and ultrasound treatment. Thus, it was indicated that the in vitro and in vivo antioxidant activities could be inconsistent, which might be affected by many other factors, such as metabolism and bioavailability. Moreover, the effects of food processing on properties of products should not be evaluated only using in vitro methods, and more in vivo evaluation methods should be carried out. Also, further studies are needed on the necessity of using different food processing methods to produce functional compounds.

**Author Contributions:** Conceptualization, X.-Y.X., R.-Y.G., and H.-B.L.; data curation, X.-Y.X., and J.-M.M.; formal analysis, X.-Y.X., and J.-M.M.; funding acquisition, X.-Y.X., R.-Y.G., and H.-B.L.; investigation, X.-Y.X., J.-M.M., Q.-Q.M., A.S., and B.-Y.L.; methodology, X.-Y.X., J.Z., Q.-Q.M., A.S., and B.-Y.L.; project administration, H.-B.L.; resources, Q.-Q.M., A.S.; and H.-B.L.; software, X.-Y.X., J.-M.M., and A.S.; supervision, R.-Y.G. and H.-B.L.; validation, X.-Y.X., and B.-Y.L.; visualization, X.-Y.X.; writing—original draft, X.-Y.X., and J.Z.; writing—review and editing, J.Z., R.-Y.G., X-.L.W., and H.-B.L.

**Funding:** This study was supported by the National Key R&D Program of China (No. 2018YFC1604405), the Fundamental Research Funds for the Central Universities (No. 19ykyjs24), the Agri-X Interdisciplinary Fund of Shanghai Jiao Tong University (No. Agri-X2017004), Shanghai Basic and Key Program (No. 18JC1410800), and the Key Project of Guangdong Provincial Science and Technology Program (No. 2014B020205002).

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Narrow-Leafed Lupin (***Lupinus angustifolius* **L.) Seeds Gamma-Conglutin is an Anti-Inflammatory Protein Promoting Insulin Resistance Improvement and Oxidative Stress Amelioration in PANC-1 Pancreatic Cell-Line**

### **Elena Lima-Cabello 1, Juan D. Alché 1, Sonia Morales-Santana 2, Alfonso Clemente <sup>3</sup> and Jose C. Jimenez-Lopez 1,4,\***


Received: 3 November 2019; Accepted: 19 December 2019; Published: 23 December 2019

**Abstract:** (1) Background: Inflammation molecular cues and insulin resistance development are some of the main contributors for the development and advance of the pathogenesis of inflammatory-related diseases; (2) Methods: We isolated and purified γ-conglutin protein from narrow-leafed lupin (NLL or blue lupin) mature seeds using affinity-chromatography to evaluate its anti-inflammatory activities at molecular level using both, a bacterial lipopolysaccharide (LPS)-induced inflammation and an insulin resistance pancreatic cell models; (3) Results: NLL γ-conglutin achieved a plethora of functional effects as the strong reduction of cell oxidative stress induced by inflammation through decreasing proteins carbonylation, nitric oxide synthesis and inducible nitric oxide synthase (iNOS) transcriptional levels, and raising glutathione (GSH) levels and modulation of superoxide dismutase (SOD) and catalase enzymes activities. γ-conglutin induced up-regulated transcriptomic and protein levels of insulin signalling pathway IRS-1, Glut-4, and PI3K, improving glucose uptake, while decreasing pro-inflammatory mediators as iNOs, TNFα, IL-1β, INFγ, IL-6, IL-12, IL-17, and IL-27; (4) Conclusion: These results suggest a promising use of NLL γ-conglutin protein in functional foods, which could also be implemented in alternative diagnosis and therapeutic molecular tools helping to prevent and treat inflammatory-related diseases.

**Keywords:** 7S basic globulins; anti-inflammatory protein; antioxidant protein; cytokines; glutathione; iNOS; nitric oxide; oxidative stress; sweet lupins group

#### **1. Introduction**

The outcomes from epidemiological studies have revealed that an increasing number of health problems are affecting all societies around the globe as diabetes, insulin resistance, obesity, metabolic syndrome and cardiovascular diseases [1], where they have been associated to both scarce physical activity and the ingestion of high sugar–high lipid diets in metropolitan areas [2]. In this regard, there is an increasing demand of plant proteins highly beneficial for human health to be used for foodstuffs

development and production and has prompted an increasing body of research covering diverse nutraceutical aspects in a number of crop plants. There is a strong interest focused in legumes, which are an economical important source of high-quality proteins compared to other plant foods [3].

Interestingly, lupin seeds, and particularly seeds from the species encompassing the "sweet lupin" group have been reported to exert beneficial effects in human health [4]. Thus, the dietary consumption of lupin seed proteins might provide preventive and protective effects (also complementing the current treatments for metabolic diseases) for different human inflammatory-related diseases such as metabolic syndrome, obesity, and high blood pressure (lowering capacity), type 2 diabetes mellitus (T2DM) development and triggered by uncontrolled glycemia throughout increasing insulin resistance, familial hypercholesterolemia and cardiovascular disease [5]. Different factors or stressors promote and stimulate immune-response-mediated inflammation leading to the molecular mechanisms underlying many of these diseases including defective insulin secretion and responses, and finally to the insulin resistance which has the pancreatic tissue as the key target for this disease evolution, progressing with an uncontrolled synthesis of pro-inflammatory mediators. Among them, interleukin 6 (IL-6), interleukin 1 (IL-1), interferon gamma (INFγ), tumor necrosis factor (TNF-α), chemokines (i.e., CCL2, CCL5), reactive oxygen species (ROS) as H2O2, peroxide and superoxide anion, nitric oxide (NO) overproduction, and nitrogen intermediate molecules, as well as adhesion molecules release (i.e., ICAM-1, VCAM-1) facilitating immune system cells attraction and movement through the tissues enhancing the inflammatory response [6]. The most frequently associated stressors are oxidative stress, alterations in gut microbiota that increase lipopolysaccharides (LPS) in blood, lipotoxicity, glucotoxicity and endoplasmic reticulum (ER) stress promoting misfolded proteins that may be deposited in the islets β-cells in form of amyloids [6]; these amyloid deposits enhance inflammatory response mediated by immune cells attracted to the pancreatic tissues [7].

Thus, lowering the synthesis and/or functional role of pro-inflammatory molecules has the advance of potentiate an anti-inflammatory reaction that may also help to the inflammatory-related diseases amelioration.

Searching for naturally-occurring compounds with the potential of anti-inflammatory responses has also increased parallel to the risen number of inflammation-related diseases. Only a few studies have described the anti-inflammatory properties of some seeds-derived bioactive hydrolysates of proteins; however, even more scarce are the studies concerning legume seed compounds with these potential functional activities. Interestingly, enzymatic hydrolysates of field pea seeds showed anti-inflammatory properties at molecular level by inhibiting several inflammation mediators' production, i.e., NO and TNFα [8]. Lunasin, a peptide derived of isolated 2S albumin that was found in soybean, as well in some cereal grains displayed great benefits related to cancer amelioration, cardiovascular disease improvement and lowering cholesterol [9]. In soybean, the anti-inflammatory properties of lunasin have been associated to its ability to suppress the NFκB functional pathway [10]. Seed protein hydrolysates from blue lupin were found to have the potential to inhibit phospholipase A2 and cyclooxygenase-2 enzymes that are involved in the inflammatory pathway [11]. Another further study showed the example of bioactive peptides with high homology with *Arabidopsis thaliana* 2S albumin and *Glycine max* lectin-like protein, which were associated with genes expression modulation of inflammatory molecules [12].

In this work, we have studied the anti-inflammatory properties of narrow-leafed lupin (NLL) γ-conglutin protein from mature seeds using in vitro human PANC-1 pancreatic cell-line in both, an induced inflammation model using bacteria lipopolysaccharide (LPS), and an induced insulin resistance (IR) cell model, with the aim of assessing the capability of NLL γ-conglutin to improve the oxidative stress homeostasis of cells, the inflammatory induced state and the IR improvement at molecular level by decreasing several pro-inflammatory mediators genes expression and proteins levels, as well as up-regulating of insulin signaling pathway gene expression.

#### **2. Material and Methods**

#### *2.1. Isolation and Purification of* γ*-Conglutin from NLL Mature Seeds*

The isolation and purification of γ-conglutin proteins from NLL was accomplished following the Czubi ´nski et al. [13] method. Briefly, NLL seed proteins were extracted using Tris buffer pH 7.5 [20 mmol L<sup>−</sup>1], having 0.5 mol L−<sup>1</sup> NaCl/gr defatted seeds. After sample centrifugation at 20,000<sup>×</sup> *g*, 30 min at 4 ◦C, the supernatant was filtered using a 0.45 μm syringe filter of PVDF. Thus, the sample was ready to be introduced in a desalting column of Sephadex G-25 medium. The desalted crude protein sample was applied to a HiTrap Q HP column (GE Healthcare) previously equilibrated with Tris buffer pH 7.5 [20 mmol L<sup>−</sup>1], where the proteins' separation was possible using a linear gradient [0 to 1 mol L<sup>−</sup>1] of NaCl. Under these conditions, the γ-conglutin proteins were not retained on the media contained in the column. Thus, different fractions that contained γ-conglutin proteins were pooled and introduced on HiTrap SP HP column (GE Healthcare) previously equilibrated with Tris buffer pH 7.5 [20 mmol L<sup>−</sup>1]. γ-conglutin proteins retained in this column were eluted with a linear gradient of NaCl [0 to 0.5 mol L<sup>−</sup>1]. The γ-conglutin proteins were collected and directly used in the further SDS-PAGE analysis and fingerprinting characterization. The remaining protein was kept frozen at −80 ◦C.

#### *2.2. Analysis of Purified* γ*-Conglutin Protein by Peptide Mass Fingerprinting*

The identity proof of the purified γ-conglutin protein was achieved following peptide mass fingerprinting. Briefly, proteins (10 μg) were separated by SDS-PAGE using precast gels of 12% Bis-Tris (Invitrogen) under reduced conditions. Electrophoretic bands corresponding to γ-conglutin protein (bands 1 to 4, Supplementary Figure S1), were cut out from the gel and in-gel trypsin digested. These peptide fragments generated were subjected to desalt and concentration, to be afterward loaded onto the MALDI plate and analyzed. MALDI-MS spectra were generated in a 4700 Proteomics Analyzer (Applied Biosystems, Waltham, MA, USA), and these data were used for proteins ID validation (www.matrixscience.com).

#### *2.3. SDS-PAGE and Immunoblotting*

Analysis of protein extracts were made by mixing the samples sample buffer (6× concentrated) and heated during 5 min up to 95 ◦C. Proteins were separated by SDS-PAGE using gradient TGX gels of 4–20% acrylamide (Bio-Rad). To identify the molecular weight (MW) of separated proteins we used a MW marker for stained gels as Mark12 Unstained Standard (ThermoFisher Scientific), with a MW range between 2.5 to 200 kDa. The resolved protein bands were visualized in a Gel Doc™ EZ Imager (Bio-Rad, Berkeley, CA, USA). For immunoblotting, proteins were transferred to PVDF membranes, which afterward were blocked for 2 h at room temperature (RT) using 5% of non-fat dry milk dissolved in PBST (phosphate-buffered saline, 0.05% Tween-20). Different membranes were incubated with goat anti-TNFα (Abcam, ref. ab8348, Cambridge, UK) at 1:1000 dilution; anti-IL-1β (Abcam, ref. ab9722) at 1:500; anti-iNOS (Invitrogen, ref. PA1-036, Carlsbad, USA) at 1:1000; anti-IRS1 (Sigma-Aldrich, Ref. 06-248, Darmstadt, Germany) at 1:500; anti GLUT4 (Sigma-Aldrich, ref. 07-1401) at 1:1000; and anti-PI3K (Abcam, ref. 86714) at 1:500. All the incubations were made leaving the membranes overnight at 4 ◦C in constant movement. Next day, membranes were washed for 5 times with PBST, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma-Aldrich, ref. A9169) at 1:2500 dilution in 2% non-fat dry milk dissolved in PBST 24 for 2 h at RT. The membranes were then washed 5 times with PBST; signal development was achieved for each antibody by incubation with ECL Plus chemiluminescence following the manufacturer's instructions (Bio-Rad). The reactive bands in the membranes were detected by exposure to C-DiGit Blot Scanner (LI-COR).

#### *2.4. Cell Culture and Treatment*

The PANC-1 pancreatic cells were grown in poly-L-lysine-coated 75 cm<sup>2</sup> flasks (∼2.5 <sup>×</sup> 106 cells/mL) in Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated fetal bovine

serum (10%) and 2 mM glutamine, all at final concentration, in a 5% CO2/95% humidified atmosphere at 37 ◦C.

The pancreatic cells were maintained by serial passage in culture flasks and used in the experimental studies when the exponential phase was reached. Cells were grown to confluence and the monolayer culture was washed two times with phosphate-buffered solution (PBS, Sigma). The cells were then treated with trypsin-EDTA (Lonza) at 0.25% for 10 min. After 5 min centrifugation at 1000× *g* and two times PBS washing, PANC-1 cells were collected. Afterward, cells counting and viability assessment were achieved by using a Countess II FL Automated Cell Counter (Thermo Fisher) at both, the initial and final step of each experiment. Viability of cells was higher than 95%. Cell cultures were stablished at 80% of confluence and treated with LPS (1 μg/mL) for 24 h. PANC-1 cells were challenged with purified γ-conglutin protein for 24 h alone or in combination adding LPS. Aliquots of γ-conglutin protein stored at −20 ◦C in PBS were thawed just before use and dissolved in culture media to target concentrations and to be added to the cultures. After treatment, cells were harvested for further analyses.

#### *2.5. MTT Assay for Cell Viability*

Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) following the manufacturer's instructions (Roche). Briefly, 96-well microtitre plates were inoculated at a density of 1 <sup>×</sup> 10<sup>3</sup> PANC-1 cells per well in 300 <sup>μ</sup>L of growth media. Plates were incubated overnight under 5% CO2 in humidified air to allow the cells to adhere to the wells. After incubation, cells were treated for 24 h with either LPS or γ-conglutin protein, and washed three times with PBS in order to prevent any interfering issue because of the phenolic compounds when making the MTT assay. A volume of 200 μL of free red-phenol DMEM containing 1 mg mL−<sup>1</sup> of MTT was added to the cells, and these were incubated for 3 h. Metabolically active viable cells are able to convert MTT into formazan crystals (purple color), and the former compound was solubilized with 200 μL of DMSO to absorb at 570 nm (test) and 690 nm using a iMark microplate reader (Bio-Rad, USA).

#### *2.6. Insulin Resistance PANC-1 Cell Model and Glucose Uptake*

Culture PANC-1 control cells were seeded in DMEM supplemented with 10% (v/v) FBS, using 96-well microtiter plates under standard conditions (5% CO2 and 37 ◦C in humidified air), and a density of 2 <sup>×</sup> 10<sup>4</sup> cells per mL in 200 mL. Optimal dose of insulin and treatment time as requisite to establish insulin-resistant IR\_PANC-1 (IR-C) cells. Cells display reduced glucose uptake, and this is one of the main feature of the insulin resistance impaired glucose uptake since decreasing cells responses to glucose uptake to increasing levels of insulin. Thus, the cell culture was separated into two groups having six independent replicates per each group: (1) Cultured cells in 200 μL complete medium (control cells, group C); (2) Treated cells with insulin (10−<sup>5</sup> to 10−<sup>9</sup> nmol L<sup>−</sup>1) when the cells became adherent (group IR-C). These PANC-1 cells were then cultured for 24, 48, and 72 h and the concentration of glucose in the media was measured using the glucose oxidase method (Abcam, UK). The concentration required to stablish IR-C PANC-1 cells was 10−<sup>7</sup> nmol L−<sup>1</sup> and cultured for 24 h. At this IR stage, it was evaluated whether cells were sensitive to insulin and to evaluate whether γ-conglutin protein can improve the insulin-dependent glucose uptake capacity of IR-C PANC-1 cells. Thus, these cells were separated in three groups, each one with six replicates: The control group (C), IR-C and the IR-C + γ-conglutin groups. After 24 h, 2 μL of culture supernatant was collected from each sample and glucose concentration was determined as described above. Cultures of IR-C cells were stablished to 80% confluence and challenged with γ-conglutin protein for 24 h. After the treatments, the cells were harvested for further analyses.

#### *2.7. Quantitative Real-Time PCR*

GLUT-4, IL-1β, iNOS, IRS-1, PI3K and TNFα mRNA expression were assayed by mean of Real-time quantitative PCR for each experimental group. Total RNA was isolated from group C using the RNeasy Tissue RNA isolation kit (Qiagen, Hilden, Germany). First strand cDNA was synthesized using a

High-Capacity cDNA Archive Kit (Applied Biosystems, Waltham, MA, USA). cDNA was prepared, diluted and subjected to real-time polymerase chain reaction (PCR), and amplified using TaqMan technology (LightCycler 480 quantitative PCR System, Roche, Basel, Switzerland) for gene expression assays. Primers and probes were used from the commercially available TaqMan Gene Expression Assays [IRS-1: Assay ID Hs00178563\_m1, GLUT-4: Hs00168966\_m1, PI3K: Hs00898511\_m1, TNFα: Hs01555410\_m1, IL-1β: Hs01075529\_m1, iNOS: Hs00174128\_m1, respectively]. Gene expression levels relative changes were assessed using the 2−ΔΔCt method. The cycle number where the transcripts were detectable (CT) was normalized to the cycle number of β-actin detection as housekeeping gene (Assay ID: Hs99999903\_m1, Applied Biosystems), and referred to as ΔCT, where the relative mRNA levels are presented as unit values of 2<sup>∧</sup> [CT (<sup>β</sup>-actin)–CT (gene of interest)], and displaying CT as the threshold cycle value. This parameter was defined as the fractional cycle number at which the target fluorescent signal passes a fixed threshold above baseline. PCR efficiency was assessed by TaqMan analysis on a standard curve for targets and endogenous control amplifications, which were highly similar.

#### *2.8. ELISA Assays for INF*γ *and Cytokines Quantification*

The cell cultured were prepared by cell counting and plated in six-well plates including 10<sup>6</sup> cells per well, and a duplicated well per group. After 24 h incubation, the media from treated culture was eliminated and cells were washed with PBS at 4 ◦C. To achieve proteins extraction, temperature of the plates was kept closely to 4 ◦C by placing these on ice, thus avoiding the denaturation of cytokines. One hundred microliters of buffer (150 mM sodium chloride, 1% NP-40, 50mM Tris pH 8) was added to each well and supplemented with 1 μL of protease inhibitor (Sigma) for 15 s. Scraped cells from the bottom of the wells were transferred to microcentrifuge tubes. These tubes were centrifuged at 12,500× *g* for 15 min at 4 ◦C. After this step, every supernatant was collected and diluted to a 1:4 ratio used for the ELISA quantification test of INFγ, IL-6, IL12p70, IL-17, and IL-27 (Diaclone). Data were statistically analyzed using the *t*-test.

#### *2.9. Antioxidant Enzymatic Activity Assays*

The cell cultures were prepared and after 24 h of incubation of the treated culture, growing media was removed, and cells washed with PBS at 4 ◦C. Cells from C, IR-C and IR-C cultures challenged with γ-conglutin protein were collected and used for the enzymatic activity assessment of SOD and catalase, as well as the GSH measurement (Canvax, Córdoba, Spain), following manufacturer's instructions. Data were analyzed by the statistical t-test.

#### *2.10. Determination of Intracellular ROS and Nitric Oxide (NO)*

C and IR-C cell cultures, challenged or not with γ-conglutin protein, were used for proteins extraction and following company instructions either for control or treatment samples (EMD Millipore, USA). A total proteins quantity of 25 μg was loaded onto polyacrylamide gels at 12% for proteins separation by SDS-PAGE. Achieved this step, proteins were transferred to PVDF membranes to be used for protein oxidation detection by using The OxyBlot™ Kit (EMD Millipore, Burlington, MA, USA) according to the manufacturer's instructions, This kit was used for the detection of carbonyl groups present into proteins because the proteins reaction with ROS. Measurements were developed at 485 nm and 530 nm excitation and emission wavelengths, respectively.

The total amount of NO, including nitrite/nitrate content, was measured using a commercial assay kit [ab65328, Abcam, Cambridge, UK] from C and IR-C culture cells before and after γ-conglutin protein challenges. Briefly, samples including every experimental group were deproteinized according to the manufacturer's instructions. An equal amount of sample (30 μL) and standards were loaded into 96-well microtiter plates. Nitrate reductase, enzyme cofactor and assay buffer were added following a 1 h of incubation at RT with Enhancer, Griess Reagent R1 and Griess Reagent R2. Just after incubation, samples were used to measure absorbance at 540 nm with an i-Mark microplate reader (Bio-Rad, USA). The value of the blank control (medium without cells) was subtracted to the samples' values. Total nitrite/nitrate concentrations were calculated by using a standard curve.

#### *2.11. Statistical Analysis*

Data obtained from each experimental were expressed as means ± standard deviation (SD). Experimental assessment was developed at least three times. The one-way variance analysis was implemented using SPSS statistical software (SPSS Inc., Chicago, IL, USA). Statistical significance of differences (*p* < 0.05) in the analyzed data was evaluated with the use of SPSS software by analysis of variance and Dunnett analysis afterward.

#### **3. Results and Discussion**

#### *3.1. Isolation and Purification of the NLL Anti-Inflammatory* γ*-Conglutin Protein*

The γ-conglutin protein extraction, isolation and purification were accomplished following the methodology from Czubinski et al. [13] using mature NLL seeds as starting material. A representative SDS-PAGE is shown in supplementary Figure S1. The sample from γ-conglutin purification went to the electrophoretically separation under reduced conditions; several different electrophoretic bands were found for this protein. The most abundant forms were the separated α and β subunits, followed by the unreduced γ-conglutin (α + β subunits) and the uncleaved γ-conglutin precursor [14]. The γ-conglutin monomer is integrated by two subunits (α + β) linked by a single disulphide bridge, which is highly resistant to be broken under reducing conditions due to the structure of the monomeric protein [15].

The expected MW of the γ-conglutin monomer from these sequences is ∼45 kDa. After reduction of the disulphide bridge, two electrophoretic bands of 30 kDa (α-subunit) and 17 kDa (β-subunit) were detected, in addition to a ∼56.0 kDa band corresponding to the uncleaved γ-conglutin precursor (Supplementary Figure S1, Supplementary Table S1). The purity of this isolated protein assayed by SDS-PAGE under reducing conditions (Supplementary Figure S1) reached a 95%.

In order to identify the different bands showed in the SDS-PAGE gel corresponding to the isolated and purified γ-conglutin (Supplementary Figure S1), we performed an in-gel tryptic digestion of the cut bands, and these were subjected to separation of the peptides and MS-based analysis. The peptide mass data generated was searched against the MS protein sequence database enabled the unambiguous identification by mass peptide fingerprinting as γ-conglutin (NLL 7S-basic globulin) (Supplementary Table S1).

#### *3.2. Cell Viability Assessment of the PANC-1 Cells Treated with* γ*-Conglutin Protein*

In this study, we assessed the viability of PANC-1 cells under treatment of the γ-conglutin protein and the potential cytotoxicity of this protein. In order to evaluate whether inflammation inductor LPS and γ-conglutin produce cell cytotoxicity effects, the viability MTT assay was achieved on PANC-1 cells under separate treatments with LPS adding γ-conglutin, at increasing concentrations to complete the conditions of DMEM culture medium + FBS + antibiotic for 24 h. The LPS plus γ-conglutin had no significant (*p* > 0.05) effects on cell viability (Supplementary Table S2), when compared with the control (untreated) group. The cell cultures used as positive control lacked LPS and γ-conglutin protein. In order to complete the usefulness of the γ-conglutin protein study, trypan blue staining was also used for assessing PANC-1-pancreatic cells viability after treatment with LPS (1 μg/μL) and increasing concentrations (from 10 to 50 μg) of γ-conglutin for 24 h, finding significant differences (*p* < 0.05) in cell viability after 24 h of incubation only at 50 μg compared to the control (Supplementary Table S2).

Furthermore, a parallel study was made to assess the cell viability and cytotoxicity of increasing concentrations of insulin in order to know whether an insulin resistance model could be performed in PANC-1 pancreatic cells and to know the actual insulin concentration that should be used to stablish the model. An MTT assay was developed on PANC-1 cell finding that an important change in the percentage of viability was induced for insulin concentrations higher than 10−<sup>7</sup> nmol L−<sup>1</sup> (Supplementary Table S3). Afterward, IR-C cells were assayed for viability using MTT kit when performed the addition of γ-conglutin protein for 24 h. No significant (*p* > 0.05) effect on cell viability (when treated with 25 μg of γ-conglutin protein) (Supplementary Table S4) was found after comparison with unchallenged IR-C group. When insulin was added alone (in the absence of γ-conglutin), these samples were used as a positive control. We also performed the cell viability assessment using trypan blue exclusion in IR-C pancreatic cells treated with increasing concentrations of this protein for a period of 24 h. No cell viability differences were found after 24 h of incubation in the presence of γ-conglutin.

These results suggest that γ-conglutin do not affect to the PANC-1 pancreatic cell integrity in both, the induced (LPS treatment) inflammation and the IR-C cell models.

#### *3.3. E*ff*ect of* γ*-Conglutin Protein on the Inflammatory Process*

Inflammatory-related illnesses as metabolic syndrome, T2DM, obesity and cardiovascular diseases are well known to be developed and chronically associated to a continuously sustained inflammatory state. Among different mechanisms hidden in the inflammatory-based diseases, different molecules namely stressors affect functional pancreatic tissues physiology, particularly β-islets, promoting the course of pathology, which also of course mainly depend of particular genetic backgrounds and environmental factors [16].

Nowadays, there is an increasing number of diabetes associated to obesity named "Diabesity epidemic", which is frequently coincidental with a pancreatic islet cells failure unable to generate enough amount of insulin and/or a developed decreasing sensitivity to insulin by tissues able to metabolize glucose. During the establishment of T2DM, sustained high levels of glucose may lead to organ damage, which is mediated by pancreatic β-cells tissue damage, and the enhancement of immune system inflammatory response because the synthesis and release of pro-inflammatory mediators as cytokines and chemokines (cells chemotactic factors). These processes create feed-forward progressive steps that further increases immune system cell content, promoting a chronic inflammatory state [17]. Thus, increasing levels of multiple factors as IL-1β, TNFα, and iNOS are important contributors for the development of inflammation since IL-1β-mediates β-cell dysfunction during the development of T2DM, while are able to activate the expression of iNOS with the result of an exacerbate synthesis of NO, promoting the up-regulation of pro-inflammatory genes [18]. In this regard, we evaluated the ability of γ-conglutin protein to modulate the mRNA levels of genes of pro-inflammatory mediators as potential anti-inflammatory targets (TNFα, IL-1β, and iNOS mRNA) in PANC-1 cells (Figure 1). Induced inflammatory state by LPS was significantly inhibited (*p* < 0.05) by γ-conglutin proteins at mRNA expression level in PANC-1 [−694, −2733, and −4208–fold, respectively, *versus* LPS treated culture cells] (Figure 1A). No statistically significant differences were observed in IL-1β cytokine, TNFα, and iNOS mRNA levels (*p* > 0.05) when challenges were performed with γ-conglutin + LPS as compared to the control group (Figure 1A). These results highlight the potential implications of γ-conglutin to decrease the pro-inflammatory capacity in PANC-1 cells by decreasing cytokines and iNOS genes expression levels, thus supporting the inflammatory process amelioration at molecular level. In this study, this lowering in the cellular pro-inflammatory capacity could be the result of the antioxidant capacity of γ-conglutin since changes in GSH levels, SOD and catalase activities was shown, helping to keep redox homeostasis in T2DM and other inflammatory-dependent diseases also affected by the oxidative stress [19]. On this line, the above results on PANC-1 pancreatic cells are in agreement with previous studies that shown a similar reduction in the expression levels of iNOS and IL-1β mRNA in T2DM blood culture [20].

**Figure 1.** Narrow-leafed lupin (NLL) γ-conglutin decreases the mRNA expression and protein levels of TNFα, IL-1β, and iNOS on lipopolysaccharide (LPS)-induced inflammation pancreatic cells. PANC-1 cells were incubated for 24 h with LPS alone or γ-conglutin + LPS. (**A**) The bar graph shows mRNA levels determined by real-time RT-qPCR of TNFα, iNOS, and IL1β. (**B**) The bar graph shows protein levels determined by immunoblotting of TNFα, iNOS, and IL1β. Average value from triplicate experiments of each biomarker were relativized to the average value of their housekeeping actin protein in control samples. Then, average values from challenge experiments (calculated in the same way than controls) are relativized to these from their respective control values previously calculated. Data represent mean ± SD from three independent experiments. C: Untreated control culture cells; LPS: LPS-treated culture cells; LPS + γ: LPS + γ-conglutin challenge. *p* < 0.05 represents statistically significant differences associated with each figure. *p*\* < 0.05 LPS *versus* C; *p*\*\* < 0.05 LPS + γ-conglutin *versus* LPS. Challenges were made with LPS and/or γ-conglutin protein at 1 μg/mL and 25 μg, respectively.

It is well established that systemic production of IL-1β at local tissues plays a fundamental role in the progression of pancreatic dysfunction as β-cell apoptosis in T2DM. The advance of this disease is facilitated by a continued production of inflammatory molecular mediators that would have an initial development stage and further progression promoted by TNFα-/IL-1β-mediated iNOS synthesis and NO production [21]. We have also demonstrated that NLL γ-conglutin can reverse this state by decreasing the levels of TNFα, IL-1β and iNOS functional protein levels in PANC-1 [−158, −144, and −164–fold, respectively, *versus* LPS treated culture cells] (Figure 1B, Supplementary Table S5), while no statistically significant differences (*p* > 0.05) were observed in TNFα, IL-1β and iNOS protein levels when challenges were accomplished with γ-conglutin (LPS + γ) when compared to the control group (Figure 1B).

#### *3.4.* γ*-Conglutin Protein Inhibits the Production of Di*ff*erent Cytokines and Pro-Inflammatory Mediators*

Physiological circulating levels of cytokines have important implications in the functional regulation of pancreatic β-cells, although these produce different cytokines itself in response to physio-pathological states, playing also important roles in its own β-cells function [18]. When insulin resistance is stablished, increasing production of dangerous pro-inflammatory circulating mediators is also stablished. During the T2DM state progression, this non-physiological condition is characterized by an imbalance pro-inflammatory cytokines and mediators profile, led by the β-cell dysfunction and T2DM sustainable situation, which on the other hand, is based on the crosstalk among cytokines in pancreatic β-cells and immune tissues [22]. Thus, restoring the balance back to the increased levels of protective plasma circulating and β-cells cytokines could prevent and promote the treatment of this β-cell dysfunctional statement, and for extension the T2DM progression.

In this regard, we evaluated by ELISA method the potential anti-inflammatory effects of γ-conglutin protein through its capacity to modulate the amount of important pro-inflammatory mediator as INF-γ and cytokines (IL-6, IL-12p70, IL-17A, and IL-27) in both, an induced inflammation model (Figure 2, Supplementary Tables S5 and S6), and in an IR-C cell model (Supplementary Figure S2, Supplementary Tables S5 and S6) using PANC-1-pancreatic cells. Levels of INFγ and the above cytokines were assessed under basal conditions, after cell treatment with LPS, by challenging the cell culture with γ-conglutin protein after LPS and by adding LPS + γ-conglutin together, or alternatively with γ-conglutin after IR-C model is stablished (as explained in material and methods, Section 2.6). The protein levels of INF-γ and cytokines (IL-6, IL-12p70, IL-17A, and IL-27) significantly (*p* < 0.05) augmented (several-fold) after LPS challenges [LPS: +11335, +2979, +12127, +5632 and +5676-fold *versus* C, respectively] (Figure 2, Supplementary Table S6); and IR-C model [+8994, +1881, +11592, +5553, +5231-fold *versus* C, respectively] (Supplementary Figure S2, Supplementary Table S6) whereas the LPS + γ -conglutin protein challenges showed a significant reduction (several-folds) in protein levels [LPS: −256, −1849, −11786, −5339 and −6100-fold *versus* LPS treated cells, respectively], and IR-C model [−8644, −1839, −11409, −5659 and −5339-fold *versus* IR-C cells, respectively] (Supplementary Figure S2, Supplementary Table S5). These results are in agreement with these obtained from RT-qPCR, where reduced mRNA and proteins levels of the pro-inflammatory mediators TNFα, IL-1β, and iNOS were found in PANC-1-cells culture (Figure 1A, Figure 1B, Supplementary Tables S5 and S6). No statistically significant differences (*p* > 0.05) were observed in INF-γ and cytokines (IL-6, IL-12p70, IL-17A, and IL-27) protein levels when challenges were accomplished with γ-conglutin (LPS + γ) after comparison to the control group (Figure 2, Supplementary Figure S2).

Currently, scarce studies have showed results concerning the anti-inflammatory effects of plant peptides, usually promoted by the modulation of the balance regulation of pro-inflammatory interleukins, INFγ, TNFα and NO. In the case of studied soybean peptides, these inhibited mRNA iNOS expression levels and TNFα and NO production, while also reduced the pro-inflammatory enzymatic activity of COX-2 in LPS-induced macrophages [8]. Moreover, lunasin was shown to reduce the ROS production in macrophages induced by LPS while inhibiting the release of IL-6 and TNFα [11,12]. In this regard, we demonstrated that NLL γ-conglutin protein lowered the pro-inflammatory mediators' levels assayed. This anti-inflammatory capacity would be capable to manage the diseases developmental states promoting feed-forward process for the establishment of these chronic inflammatory-derived diseases as T2DM. Thus, lupin γ-conglutins may be capable to promote the improvement from the detrimental effects of several inflammatory molecular developments as follows:

(i) Lipotoxicity as a sustained high lipid diet induces the production of IL-1β, IL-6, which β-cells continued exposure induces exacerbate synthesis and release of ROS, while secretion of insulin is also inhibited. This combination promotes the apoptosis of the pancreatic β-cells [23]. Based in our research, challenging pancreatic β-cells with γ-conglutin decreased the mRNA expression of IL-1β, and protein levels of IL-1β and IL-6 in LPS-induced inflammation [LPS + γ: −2749; −146 and −1100-fold *versus* LPS treated cells, respectively] (Figure 1; Figure 2, Supplementary Table S5); and IR-C model [IR-C + γ: −177; −97 and −1849-fold *versus* IR-C cells, respectively] (Figure 3, Supplementary Figure S2, Supplementary Table S5).

**Figure 2.** Effect of NLL γ-conglutin on the protein levels of pro-inflammatory cytokines. PANC-1 cells were incubated for 24 h with LPS alone, or γ-conglutin + LPS. The bar graph shows protein levels determined by ELISA of INFγ, IL-6, IL-12, IL-17, and IL-27. Data represent mean ± SD from three independent experiments. C: Untreated control culture cells; LPS: LPS-treated culture cells; LPS + γ: LPS + γ-conglutin challenge. *p* < 0.05 represents statistically significant differences associated with each figure. *p*\* < 0.05 LPS *versus* C; *p*\*\* < 0.05 LPS + γ-conglutin *versus* LPS. Challenges were made with LPS and/or γ-conglutin at 1 μg/mL and 25 μg, respectively.

(ii) Apoptosis of islets β-cells prompted by IL-1β and INFγ is stimulated by endoplasmic reticulum stress [24]. In this regard, β-cell apoptosis is also activated by the join action of INFγ and TNFα, together with the activation of Ca2<sup>+</sup> channels. This situation induces the NO synthesis and consequently the endoplasmic reticulum stress pathway activation [25], leading to caspases activation and mitochondrial dysfunction [26]. In this concern, γ -conglutin may be able to prevent these mechanisms by suppressing the TNFα, IL-1β and INFγ mRNA and protein levels (Figures 1–3, Supplementary Figure S2, Supplementary Tables S5 and S6).

(iii) The synergistic action of IL-1β + INFγ, or even IL-1β + INFγ + TNFα cytokines in pancreatic tissues increases NO production as consequence of direct increasing of iNOS, resulting in islet β-cell destruction [27]. We have shown that mRNA expression levels of TNFα and IFNγ (apoptosis mediated molecules) were lowered after treatment with γ-conglutin (Figures 1–3, Supplementary Figure S2, Supplementary Tables S5 and S6), which may have a positive effect on the survival of islet β-cells [28].

(iv) IL-12 mRNA expression levels are increased by the effect of INFγ, while IL-12 promote signaling positive feed-back effect for raising levels of INFγ [29]. The γ-conglutin protein reduction effect of IL-12 mRNA levels (Figure 2, Supplementary Figure S2, Supplementary Tables S5 and S6) may decrease INFγ levels and its negative inflammatory effects.

**Figure 3.** NLL γ-conglutin decreases the mRNA expression and protein levels of TNFα, IL-1β and iNOS on an insulin-resistance IR-C cell model. Control PANC-1 cells, and IR-C pancreatic cells were cultured for 24 h alone, or the former culture with γ-conglutin. (**A**) The bar graph shows mRNA levels determined by real-time RT-qPCR of TNFα, iNOS and IL1β. (B) The bar graph shows protein levels determined by immunoblotting of TNFα, iNOS and IL1β. Average value from triplicate experiments of each biomarker were relativized to the average value of their housekeeping actin protein in control samples. Then, average values from challenge experiments (calculated in the same way than controls) are relativized to these from their respective control values previously calculated. Data represent mean ± SD from three independent experiments. Control: Untreated control PANC-1 culture cells; IR-C: insulin resistant culture cells; IR-C + γ: IR-C + γ-conglutin challenge. *p* < 0.05 represents statistically significant differences associated with each figure. *p*\* < 0.05 IR-C *versus* control PANC-1 cells; *p*\*\* < 0.05 IR-C + γ-conglutin *versus* IR-C. Challenges were made with 25 μg of γ-conglutin.

(v) Important inflammatory cytokine, IL-17A, involved in the T2DM progressing, is able to induce ROS production, which also greatly affects to insulin resistance. A join action from IL-17 and INFγ acts as diabetes chronic state development [30]. Overall, IL-17A has pleiotropic functional effects comprising synthesis of IL-6 and TNFα, and chemokines (chemotaxis effect) on a diversity of cells [31]. Thus, the lowering of the IL-17 protein level (Figure 2, Supplementary Figure S2, Supplementary Tables S5 and S6) might reduce pro-inflammatory effects of IL-6 and TNFα (Figure 2, Supplementary Figure S2, Supplementary Tables S5 and S6), avoiding islet β-cell apoptosis and the recruitment of immune cells to local tissues, enhancing feed-forward mechanism of inflammation progression in islets [32] as preventive action for inflammation based T2DM progression.

#### *3.5.* γ*-Conglutin Reverses the Insulin Resistance through Inflammation Amelioration while Improving Insulin Signalling Pathway in Pancreatic IR-C Cells*

Insulin resistance is another consequence of a sustained inflammation, which has been observed in several pathophysiological processes, including metabolic disorders as hyperinsulinemia, hyperglycemia, and hypertriglyceridemia, being IR also an important cause of pre-diabetes establishment and T2DM development and obesity [33], affecting to different insulin target organs. Thus, amelioration of IR by NLL γ-conglutin may constitute a major approach to prevent and treat these metabolic disorders.

In this study, it was established an in vitro insulin-resistant (IR-C) cell model using PANC-1 cells to evaluate the insulin effects on glucose uptake and metabolism in IR-C cell. To evaluate glucose uptake, control cells were incubated with a range of insulin concentrations (between 10−<sup>5</sup> to 10−<sup>9</sup> nmol L−1) for 24 h (Figure 4). Following an insulin concentration of 10−<sup>7</sup> nmol L−1, we found the most statistically significant reduction in the extracellular glucose depletion (*p* < 0.05) in comparison to control cells (without insulin treatment) (Figure 4A). The addition of 10−<sup>7</sup> nmol L−<sup>1</sup> of insulin promoted a time-dependent lowering (*p* < 0.05) of glucose consumption between 24–48 h when compared to control cells (Figure 4B). These results clearly showed the maintenance of the insulin resistance by IR-C cells for a period of 48 h after insulin treatment. Following 48 h, cells acquired a normal condition as control cells (C). These results are consistent with the increasing glucose uptake shown in Figure 3B after 72 h, while no statistically significant differences (*p* > 0.05) in glucose consumption was observed when compared to control cells without insulin treatment. Furthermore, the molecular mechanisms leading to glucose homeostasis and/or IR are still uncertain. However, NLL γ-conglutin might be able to contribute in this process of glucose homeostasis, as we have demonstrated in the current study that glucose uptake by IR-C cells is clearly induced by treatment with γ-conglutin protein, reaching higher glucose uptake levels after IR-C cells challenged with 25 μg of γ-conglutin protein, which glucose uptake increased more than 60% in comparison to IR-C cells (*p* < 0.05), which were assayed without γ-conglutin protein challenge (Figure 4C).

**Figure 4.** Insulin-resistant IR\_PANC-1 cell model and glucose consumption promoted by γ-conglutin. (**A**) Increasing concentrations of insulin from 10−<sup>9</sup> to 10−<sup>5</sup> nmol/L showed that cell culture did uptake the lower level of glucose at 10−<sup>7</sup> nmol/L in comparison to C cell culture, taking this concentration as the level of insulin where cells acquired the resistance state. (**B**) C cells were cultured for 24, 48 and 72 h, testing the glucose uptake of cultures including 10−<sup>7</sup> nmol/L (white bars), in comparison to control C cells (black bars). In these assays were showed that insulin resistance state is preserved for 48 h. *p*\* < 0.05 IR-C *versus* C. (**C**) Glucose consumption by IR-C cells promoted by γ-conglutin at 0, 10, 25 and 50 μg was assayed after 24 h of culture. Values are shown as the mean ± SD from three independent experiments. *p* < 0.05 represents statistically significant differences associated with each figure. *p*\* < 0.05 treated cells (μg) *versus* control.

The treatment of pancreatic IR-C cells with γ-conglutin was also accomplished to determine whether this protein had effects on insulin resistance improvement throughout recovering the control-like associated mRNA expression levels of IRS-1, GLUT-4, and PI3K, key upstream and glucose transport mediators in the insulin signaling pathway [20], which would also be the reflect of a potential improvement in the glucose uptake and the inflammatory state on IR-C cells. The analysis of IRS-1, GLUT-4 and PI3K showed their up-regulation in their mRNA expression after γ-conglutin treatment in IR-C cells (Figure 5) [IRS-1: +70; GLUT-4: +97%; and PI3K: +90-fold, respectively], which differences were statistically significant compared to IR-C untreated cells (*p* < 0.05) (Figure 5A), as well as the mRNA expression level reduction of IRS-1, GLUT-4 and PI3K in IR-C cells [IRS-1: −93; GLUT-4: −84%; and PI3K: −89-fold, respectively] compared to control cells PANC-1 (Figure 5A).

**Figure 5.** NLL γ-conglutin increases mRNA expression and protein levels of the insulin signaling pathway mediators IRS-1, PI3K and GLUT-4. PANC-1 cells or IR-C cell culture were incubated for 24 h alone, or the former culture with γ-conglutin. (**A**) The bar graph shows mRNA levels determined by real-time RT-qPCR of IRS-1, PI3K and GLUT-4. (**B**) The bar graph shows protein levels determined by immunoblotting of IRS-1, PI3K and GLUT-4. Average value from triplicate experiments of each biomarker were relativized to the average value of their housekeeping actin protein in control samples. Then, average values from challenge experiments (calculated in the same way than controls) are relativized to these from their respective control values previously calculated. Data represent mean ± SD from three independent experiments. Control: Untreated control PANC-1 culture cells; IR-C: insulin resistant culture cells; IR-C + γ: IR-C + γ-conglutin challenge. *p* < 0.05 represents statistically significant differences associated with each figure. *p*\* < 0.05 IR-C *versus* control PANC-1 cells; *p*\*\* < 0.05 IR-C + γ-conglutin *versus* IR-C. Challenges were made with 25 μg of γ-conglutin.

We have also demonstrated that NLL γ-conglutin can reverse this state by up-regulating the IRS-1, GLUT-4 and PI3K functional protein levels in IR-C cells [IRS-1: +266; GLUT-4: +185; and PI3K: +144-fold, respectively] (Figure 5B), after decreased proteins levels showed when PANC-1 control cells acquired the IR-C statement compared to the control group [IRS-1: −302; GLUT-4: −310; and PI3K: −166-fold, respectively] (Figure 5B). These results confirm that γ-conglutin protein would be capable to reduce significantly the blood glucose level by promoting glucose uptake by insulin sensitive tissues while ameliorating hyperglycemia via increasing GLUT-4 glucose transporter protein level and plasma membrane recruitment [34], and insulin signaling pathway upstream mediators IRS-1 and PI3K [20].

Furthermore, at the same time we also evaluated the capability of γ-conglutin protein to regulate the mRNA and protein levels of pro-inflammatory molecules as potential mechanism helping to reverse the IR-C cell statement. TNFα, IL-1β and iNOS were analyzed in IR-C culture (Figure 3). These pro-inflammatory mediators were significantly lowered in γ-conglutin protein treated IR-C cells, at the mRNA expression levels [TNFα: −158; IL-1β: −144; and iNOS: −164-fold, respectively, *versus* IR-C untreated cells] (Figure 3A), and at the protein levels [TNFα: −189; IL-1β: −146; and iNOS: −97-fold, respectively, *versus* IR-C untreated cells] (Figure 3B, Supplementary Table S6). No statistically significant differences (*p* > 0.05) were found for TNFα, IL-1β and iNOS levels in IR-C cells treated with γ-conglutin in comparison to the PANC-1 control group (Figure 3). These results highlight the potential implications of γ-conglutin to improve insulin resistance through inflammation amelioration at molecular level in PANC-1 pancreatic cells by decreasing cytokines and iNOS levels [20].

In this study, we have demonstrated for the first time that NLL γ-conglutin protein is able to help improving the insulin resistance state in PANC-1 cell line targeting two major molecular signaling cross-roads, restoring functional levels of insulin activation pathway mediators while decreasing several pro-inflammatory mediators' levels that worthwhile reinforces the first effect on PANC-1 cells. These outcomes are vital knowledge to be considered for successful anti-inflammatory insulin sensitizing new alternative therapies from natural plant sources.

#### *3.6. Oxidative Stress Modulation by* γ*-Conglutin Protein as Anti-Inflammatory and Insulin Resistance Improvement Mechanism*

Oxidative stress, understood as the cellular statement of excess reactive oxygen species (ROS) production, is a main factor in the T2DM development [35], through promoting IR development. Afterward, high amounts of blood glucose sustained long time causes damage on the enzymes superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathione molecule as the most important elements of the cell antioxidant defense system [36]. Thus, an excessive ROS production contributes to oxidative stress, a pro-inflammatory state, and mitochondrial dysfunction that in turn exacerbates IR [37]. It would be necessary a comprehensive knowledge about the relationship between oxidative stress and T2DM risk factors (inflammation and IR) in order to improve diabetes prevention and its associated complications. In this regard, signaling molecules as nitric oxide (NO) play a critical role of the inflammation pathogenesis acting as a pro-inflammatory molecule, together with cytokines and chemokines (e.g., TNFα, IL-6, IL-12), under oxidative stress situations because of the excessive NO and ROS production, i.e., IR [38], promoting islet β-cell apoptosis [39] and the progression of diseases concomitant with inflammation [40].

In the present study, we evaluated the oxidative homeostasis in inflammatory LPS-induced PANC-1 cells, as well as in IR-C cell model, after treatment with γ-conglutin protein. In both cases, we assessed the ROS production by measuring the levels of protein carbonylation, the covalent modifications of proteins induced by ROS, i.e., H2O2 or other derived molecules from the oxidative stress process by using an OxyBlot protein oxidation detection and immunoassay [41], and comparing them with control cells, LPS treated cells and IR-C cells, respectively, without any challenge with γ-conglutin. Very low levels of protein oxidation, generated through normal metabolic activity, were observed in untreated (control) cells with LPS (Supplementary Figure S3A), as well as in control PANC-1 cells before IR-C statement induction (Figure 6A). However, ROS production was significantly increased (*p* < 0.05) after LPS cells treatment (+677-fold, Supplementary Figure S3A), and in IR-C cells (+445-fold, Figure 6A), as significant (*p* < 0.05) increased levels of proteins carbonylation was detected. Treatments of these type of cells with γ-conglutin protein restored oxidative balance in both situations (LPS-induced cells: −423-fold, and IR-C cells: −445-fold, respectively; Supplementary Figure S3A, Figure 6A), in comparison to their respective inflammatory induced stages. These results suggest that γ-conglutin protein efficiently avoid at certain levels the ROS production (oxidative stress) in PANC-1 cells after inflammatory statement incensement, and that γ-conglutin exhibited strong anti-oxidant effect since this protein ameliorated the oxidative stress induced by LPS and in IR-C

cell model. Interestingly, the present and future related studies would benefit from the comparative further analyses using other types of cell cultures, like primary islets and/or pancreatic β-cells and/or adipocyte cells to determine actions related to insulin secretion and islet inflammation.

**Figure 6.** Effect of γ-conglutin on proteins oxidative modifications, antioxidant enzymatic activities and production of glutathione (GSH) and NO. (**A**) Changes in protein carbonyl formation were measured in IR-C cells after 24 h of incubation with γ-conglutin. Protein carbonyls were measured using an OxyBlot kit. Representative blots show basal carbonylation levels in C control PANC-1 cells, IR-C cells, and IR-C culture cells challenged with γ-conglutin. Graph y-axis represents arbitrary densitometry units. *p*\* < 0.05 IR-C cells *versus* C cells. (**B**) IR-C pancreatic cells were incubated for 24 h with γ-conglutin protein. GSH and NO production, as well as SOD and catalase activities were measured. Data represent mean ± SD from three independent experiments. *p* < 0.05 represents statistically significant differences associated with each figure. *p*\* < 0.05 IR-C *versus* control PANC-1 cells; *p*\*\* < 0.05 IR-C + γ-conglutin *versus* IR-C. Challenges were made with 25 μg of γ-conglutin.

Therefore, removal of free radicals is strongly dependent of enzymatic activities as superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione (GSH) levels, representing crucial indicators of the cellular anti-oxidant capacity, and the oxidative stress cell state [35]. In the current study, we assessed the modulation of these antioxidant factors by γ-conglutin in the inflammatory LPS-induced PANC-1 cells, as well as in IR-C cell model, by measuring SOD and catalase activities, GSH levels and NO production, before and after the treatment with γ-conglutin (Supplementary Figure S3B, Figure 6B). We found a statistically significant (*p* < 0.05) decreased levels of GSH (LPS-induced inflammation cells: −660-fold; IR-C cells: −949-fold, respectively) (Supplementary Figure S3B, Figure 6B). Furthermore, the levels of SOD and catalase activity were strongly reduced after the same treatments with γ-conglutin protein in LPS-induced inflammatory statement (SOD: −677-fold; catalase: −142-fold, respectively) (Supplementary Figure S3B) and IR-C cells (SOD: −183-fold; catalase: −33-fold, respectively) (Figure 6B). These data showed that high GSH and low SOD levels and catalase activities might be regulated by γ-conglutin protein through direct or indirect marked effects in avoiding lipids and protein oxidative modifications, which is also supported by the concomitant large reduction of oxidative carbonylation (Supplementary Figure S3B, Figure 6B), and an overall oxidative stress balance improvement, translated also to an inflammation molecular cellular statement amelioration by γ-conglutin protein as an anti-oxidant protein.

Furthermore, we analyzed the NO production again in both induced inflammation cell models treated with γ-conglutin protein for 24 h. Statistically significant decreased levels of NO were found (*p* < 0.05) in the LPS-induced cells (−351-fold, Supplementary Figure S3B) and IR-C cells (−91-fold, Figure 6B), in comparison to inflammation induced cells without γ-conglutin protein treatment, showing again how γ-conglutin is able to ameliorate the inflammatory state of cells promoting lowering NO [42] and iNOS expression levels, showing potential uses in the improvement of T2DM and other inflammatory-based diseases.

These novel results clearly indicated that oxidative stress is a major point targeted by NLL γ-conglutin protein effects causing an improved stress balancing through reduced ROS-related pro-inflammatory mediators and increased anti-oxidative molecules. Indeed, such data can be helpful for the development of future antioxidant and new anti-inflammatory therapeutics avoiding the oxidative stress activation of inflammatory mediators involved in several chronic diseases, with the advantage of being a natural product from lupin seeds that can be implemented as a functional food.

#### **4. Conclusions**

In this study, treatment with NLL γ-conglutin protein to inflammation LPS-induced and IR-C in the PANC-1 pancreatic cell-line promoted: (i) Lowering expression of mRNA and proteins levels of key pro-inflammatory mediators as TNFα, IL-1β, and iNOS; ii) the up-regulation mRNA expression and increasing protein levels of IRS-1, and p85-PI3K, and GLUT-4 transporter, which are crucial biomarkers of the insulin signaling pathway activation. This up-regulation makes possible the recovery of the physiological condition of the cells as control cell-like situation from an induced inflammatory statement; (iii) glucose uptake in IR-C cells; (iv) a significant decrease (*p* < 0.05) in proteins levels of pro-inflammatory mediators INFγ, IL-6, IL-12, IL-17 and IL-27; (v) significant dropping oxidative stress in inflammation LPS-induced and IR-C pancreatic cells, as indicated by a reduced levels of protein carbonylation, improved glutathione (GSH) levels and lower SOD and catalase antioxidant enzymatic activities; (vi) reduction of NO production and down-regulation of iNOS in both, LPS-induced inflammation and IR-C pancreatic cells. This study is the first describing the anti-inflammatory effects at molecular level of the legume protein family 7S basic globulins or γ-conglutin, constituting strong evidences that NLL γ-conglutins play a crucial role in the development of novel functional foods and therapeutic options for the prevention and treatment of inflammatory-related diseases.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/1/12/s1, Figure S1: Isolation and purification of NLL seed γ-conglutin protein; FigureS2: Effect of NLL γ-conglutin on the protein levels of pro-inflammatory cytokines; Figure S3: Effect of γ-conglutin on proteins oxidative

modifications, antioxidant enzymatic activities and production of GSH and NO; Table S1: γ-conglutin peptides mass fingerprinting characterization; Table S2: Cell viability (%) and dose effects of NLL γ-conglutin protein; Table S3: Cell viability (%) on insulin resistance IR-C cell model; Table S4: Cell viability (%) and dose effects of purified NLL γ-conglutin protein on insulin resistance cell (IR-C) model; Table S5: Fold-change in protein levels of pro-inflammatory cytokines and iNOS; Table S6: Fold-change in protein levels of pro-inflammatory cytokines and iNOS.

**Author Contributions:** Conceptualization: J.C.J.-L. and E.L.-C.; Methodology: J.C.J.-L., E.L.-C., A.C.; Data Analysis: J.C.J.-L., E.L.-C., A.C.; Resources: J.C.J.-L., A.C., J.D.A.; Writing—Original Draft Preparation: J.C.J.-L.; Writing—Review and Editing: J.C.J.-L., A.C., J.D.A., S.M.-S.; Funding Acquisition: J.C.J.-L., A.C., J.D.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by the European Research Program MARIE CURIE (FP7-PEOPLE-2011-IOF) grant ref. number PIOF-GA-2011-301550; The Spanish Ministry of Economy, Industry and Competitiveness for the grant ref. number RYC-2014-16536 (Ramon y Cajal Research Program); the grant ref. number BFU2016-77243-P; and the grant ref. number AGL2017-83772-R (AEI/FEDER, UE) funded by the Spanish Ministry of Science, Innovation and Universities" and "The APC was funded by the grant ref. number BFU2016-77243-P".

**Acknowledgments:** This study has been funded by the European Research Program MARIE CURIE (FP7-PEOPLE-2011-IOF) for through the grant ref. number PIOF-GA-2011-301550 to J.C.J.-L. and J.D.A.; The Spanish Ministry of Economy, Industry and Competitiveness for the grant ref. number RYC-2014-16536 (Ramon y Cajal Research Program) to J.C.J.-L.; the grant ref. number BFU2016-77243-P to J.D.A. and J.C.J.-L.; and the grant ref. number AGL2017-83772-R (AEI/FEDER, UE) funded by the Spanish Ministry of Science, Innovation and Universities to A.C.

**Conflicts of Interest:** The authors have declared that no competing interests exist.

#### **References**


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### *Article* **In Vitro Antioxidant, Antiinflammation, and Anticancer Activities and Anthraquinone Content from** *Rumex crispus* **Root Extract and Fractions**

### **Taekil Eom 1, Ekyune Kim 2,\* and Ju-Sung Kim 1,\***


Received: 10 July 2020; Accepted: 7 August 2020; Published: 10 August 2020

**Abstract:** *Rumex crispus* is a perennial plant that grows in humid environments across Korea. Its roots are used in traditional Korean medicine to treat several diseases, including diseases of the spleen and skin and several inflammatory pathologies. In this study, different solvent fractions (*n*-hexane, dichloromethane, ethyl acetate, *n*-butanol, and aqueous fractions) from an ethanol extract of *R. crispus* roots were evaluated for the presence and composition of anthraquinone compounds and antioxidants by checking for such things as free radical scavenging activity, and electron and proton atom donating ability. In addition, anti-inflammatory activity was measured by NO scavenging activity and inflammatory cytokine production; furthermore, anti-cancer activity was measured by apoptosis-inducing ability. Polyphenolic and flavonoid compounds were shown to be abundant in the dichloromethane and ethyl acetate fractions, which also exhibited strong antioxidant activity, including free radical scavenging and positive results in FRAP, TEAC, and ORAC assays. HPLC analysis revealed that the dichloromethane fractions had higher anthraquinone contents than the other fractions; the major anthraquinone compounds included chrysophanol, emodin, and physcione. In addition, results of the anti-inflammatory assays showed that the ethyl acetate fraction showed appreciable reductions in the levels of nitric oxide and inflammatory cytokines (TNF-α, IL-1β, and IL-6) in Raw 264.7 cells. Furthermore, the anthraquinone-rich dichloromethane fraction displayed the highest anticancer activity when evaluated in a human hepatoma cancer cell line (HepG2), in which it induced increased apoptosis mediated by p53 and caspase activation.

**Keywords:** anthraquinone; free radical scavenging; inflammatory cytokines; apoptosis; *Rumex crispus*

#### **1. Introduction**

Reactive oxygen species (ROS), also known as oxygen-centered free radicals, are produced during normal metabolic processes and play an essential role in maintaining cellular homeostasis. ROS levels can increase as a result of exposure to chemical substances or other environmental stress resulting in oxidative stress [1]. When the intrinsic antioxidant system within an organism is damaged, it is not possible to remove these free radicals and the resulting oxidative stress can lead to various chronic diseases. A state of chronic oxidative stress can cause oxidative damage to various cellular components, including cell membranes, DNA, and proteins. It can also result in the activation of systemic chronic inflammatory responses via a number of different intracellular signaling pathways, ultimately exacerbating a variety of pathological conditions, including cardiovascular diseases, cancer, dementia, diabetes, autoimmune disorders, and aging [2]. One of the underlying signaling mechanisms triggered by excessive ROS generation is the activation of nuclear transcription factor κB (NF-κB), which acts as a transcriptional regulator of the innate immune system and can stimulate the release of a variety of pro-inflammatory cytokines from various tissues [3,4].

Inflammation is one of the self-defense responses used by organisms to defend against a wide range of external stimuli; however, excessive or prolonged inflammation can lead to the development of serious pathologies. The inflammatory response is characterized by the activation of macrophages and subsequent increases in the secretions of nitric oxide (NO), pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and cell adhesion molecules [5]. ROS-mediated chronic inflammatory responses can be inhibited by antioxidants. Various antioxidant compounds have been described in the literature with varying degrees of efficacy. The versatility of these compounds makes them ideal candidates for novel therapies. Various natural products have been shown to exhibit antioxidant properties and hence this is a growing field of interest. Plants have been identified as an especially rich source of antioxidant compounds, with most containing phenolic groups, which are known to play a crucial role in the removal of ROS. These phenolic compounds are generally secondary metabolites involved in stress responses and are known to perform various physiological functions, including antioxidant, anti-inflammatory, and anticancer functions [6,7]. The *Rumex* genus belongs to the Polygonaceae family and includes *R. crispus*, *R. acetosella, R. acetosa, R. aquatica, R. longifolius, R. gmelini, R. conglomeratus*, and *R. maritimus*. *R. crispus* is a perennial plant endemic to Korea which is found growing in humid environments. Its roots have been used as traditional medicinal materials in the treatment of several pathological conditions, including bladder infections, gallbladder disease, skin disease, and lymph node disorders. They have also been used as an adjuvant therapy in oriental medicine strategies used to treat cancer [8,9]. Several bioactive components of *R. crispus* have been identified and include saponins, tannins, flavonoids, essential oils, and anthraquinone derivatives such as chrysophanol and emodin [10–12]. This study was designed to evaluate the potential of new bioactive substances from *R. crispus* identified by analyzing their antioxidant, anti-inflammatory, and anticancer activities using root extracts and various extract fractions.

#### **2. Materials and Methods**

#### *2.1. Materials*

2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2 -azobis(2-methylpropionamidine) dihydrochloride (AAPH), 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2 ,7 dichlorofluorescin (DCFH), trolox, Folin-Ciocalteu reagent, 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ), gallic acid, quercetin, trichloroacetic acid (TCA), aluminum chloride hexahydrate (AlCl3·6H2O), phenazine methosulphate (PMS), β-nicotinamide adenine dinucleotide reduced disodium salt (NADH), nitro blue tetrazolium tablet (NBT), thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The human hepatoma cancer cell line HepG2 and mouse macrophage cell line Raw 264.7 were obtained from the American Type Culture Collection (Manassas, VA, USA). All western blot antibodies were obtained from Santa Cruz Biotechnology (Logan, UT, USA). All other chemicals used in this study were at least 99% pure.

#### *2.2. Preparation of Extracts and Fractions*

Dried *R. crispus* root powder was extracted three times using ten times its weight of ethanol and subjected to reflux for 12 h. After drying by evaporation in a vacuum rotary evaporator, the extract was suspended in water and fractionated with *n*-hexane (HF), dichloromethane (DCMF), ethyl acetate (EAF), *n*-butanol, (BF), and water (AF) three times, respectively. A total of five fractions were obtained after the solvents were removed.

#### *2.3. Determination of Total Phenol and Flavonoid Contents*

The total phenolic content was quantified using Folin–Ciocalteu reagent and a Gallic acid standard [13]. Briefly, 100 μL of each sample solution (1 mg/mL) was mixed in a test tube containing 3.5 mL distilled water and 500 μL 50% Folin–Ciocalteu reagent. The mixture was then allowed to react for 2 h, after which 500 μL of 20% Na2CO3 was added. The mixture was then placed in a dark room for 1 h, and the absorbance at 720 nm was recorded using a SpectraMax M2e microplate reader (Molecular Device, Sunnyvale, CA, USA). The total phenolic contents are expressed as gallic acid equivalents (mM GAE/g).

To analyze total flavonoid content of each sample, 500 μL of each sample (1 mg/mL) was mixed with 100 μL of 10% (*w*/*v*) aluminum chloride and 100 μL of 1.0 M potassium acetate. Then, 1.5 mL of ethanol and 2.8 mL of distilled water were added and mixed in. The mixture was then placed in a dark room for 1 h, and the absorbance at 415 nm was recorded using a SpectraMax M2e microplate reader [14]. The total flavonoid content is expressed as quercetin equivalents (mM QE/g).

#### *2.4. HPLC Analysis of Anthraquinone Derivative*

Anthraquinone derivatives in the *R. crispus* extracts and solvent fractions were analyzed using high performance liquid chromatography coupled with a PDA detection system (Shimadzu Prominence, Japan). The analysis was performed on a Triat C-18 column (250 mm × 4.6 mm, 5 μm) from YMC Co., Ltd. The column temperature was set to 40 ◦C and the detection wavelength was set to 450 nm. The mobile phase consisted of water containing 0.1% TFA (Trifluoro aceticacid) and 0.1% TFA containing methanol (B) with the gradient program set as follows: isocratic 20% B at 0–5 min, linear gradient 20–80% B at 5–15 min, linear gradient 80–90% B at 15–30 min, linear gradient 90–100% B at 30–35 min, isocratic 100% B at 35–40 min with flow rate of 1.0 mL/min.

#### *2.5. DPPH Radical Scavenging Activity*

The DPPH radical scavenging effect was evaluated using the published method with slight modifications [15]. Briefly, 160 <sup>μ</sup>L of 1.5 <sup>×</sup> 10−<sup>4</sup> M DPPH solution was mixed with 40 <sup>μ</sup>L of a sample solution, incubated at room temperature for 30 min, and then absorbance at 540 nm was evaluated using a SpectraMax M2<sup>e</sup> microplate reader. The scavenging activity of the DPPH radicals was calculated as follows: ((Abs blank−Abs sample)/Abs blank) × 100. The radical scavenging activity was expressed as a concentration that inhibited the radicals by 50%.

#### *2.6. Hydroxyl Radical Scavenging Activity*

The hydroxyl radical scavenging activity was determined using the method described by Label and Bondy [16]. Briefly, the sample was mixed with 1 mM H2O2 and 0.2 mM FeSO4, and incubated at 37 ◦C for 5 min. Esterase-treated 2 μM DCHF-DA was then added and the change in fluorescence was monitored on a SpectraMax M2<sup>e</sup> microplate reader, with excitation and emission wavelengths of 460 nm and 530 nm, respectively, for 30 min. The scavenging activity of the hydroxyl radicals was calculated as follows: ((FLU blank−FLU sample)/FLU blank) × 100. The radical scavenging activity was expressed as a concentration that inhibited the radicals by 50%.

#### *2.7. Superoxide Radical Scavenging Activity*

The superoxide radical scavenging effect was evaluated using the method reported by Liu et al. [17] with minor modifications. Briefly, the reagent mixture containing a 50 μL aliquot of a sample solution, 50 μL of 150 μM NBT, 50 μL of 468 μM NADH, and 50 μL of 60 μM phenazine methosulfate was incubated at room temperature for 5 min. The absorbance was measured at 560 nm and compared to the blank, and the superoxide anion radical scavenging activity was then calculated using the following equation: Scavenging effect, % = ((Abs sample − Abs blank)/Abs blank) × 100. The radical scavenging activity was expressed as a concentration that inhibited the radicals by 50%.

#### *2.8. TEAC Assay*

The TEAC method is based on the reaction of ABTS•<sup>+</sup> ions and was carried out according to the method described by Zulueta et al. [18] with minor modifications. An ABTS•<sup>+</sup> working solution was prepared daily by diluting the ABTS•<sup>+</sup> stock solution with distilled water to get an absorbance of 0.07 ± 0.02 at 734 nm. Briefly, 50 μL aliquots of the sample solutions were each mixed with 1.0 mL ABTS•<sup>+</sup> working solution. Each mixture was incubated at 25 ◦C in the dark for 5 min and absorbance was measured using a SpectraMax M2<sup>e</sup> microplate reader at 734 nm. The sample extract activity was expressed as mM trolox/g dry sample and all determinations were carried out in triplicate.

#### *2.9. ORAC Assay*

ORAC measures the antioxidant inhibition of peroxyl-radical-induced oxidations and reflects radical chain-breaking antioxidant activity by H-atom transfer. This assay is based on the scavenging of peroxyl radicals generated by AAPH, which prevents the degradation of the fluorescein probe, and consequently, prevents the loss of fluorescence. For this study, we used the method described by Zulueta et al. [18]. A 75 mM phosphate buffer (pH 7.4) was used for all sample dilutions and reagent preparations. Aliquots of the sample extractions (50 μL) and the 150 μL 75 nM fluorescein solutions were placed in 96-black well microplates. The mixture was preincubated for 10 min at 37 ◦C. The reaction was initiated by adding 25 μL of 120 mM AAPH solution and the changes in the fluorescence were monitored using a SpectraMax M2<sup>e</sup> microplate reader, with excitation and emission wavelengths of 460 nm and 530 nm, respectively, for 60 min. The sample extract activity was expressed as mM of trolox/g dry sample and all determinations were carried out in triplicate.

#### *2.10. FRAP Assay*

The FRAP value was determined using the method described by Benzie et al. [19] with slight modifications. Briefly, 50 μL aliquots of the sample extracts each were mixed with 1.5 mL FRAP working reagent prepared fresh daily. The FRAP working reagent consisted of 10 volumes of 300 mM acetate buffer (pH 3.6) mixed with 10 volumes of 20 mM FeCl3. In addition, one volume of 10 mM TPTZ in 40 mM HCl, was also added to each sample and the final mixture was incubated at 37 ◦C in the dark for 30 min. Absorbance was measured after 30 min at 593 nm. The activities of each extract are expressed as mM of FeSO4/g dry sample and all determinations were carried out in triplicate.

#### *2.11. Cell Culture and Cell Viability Assays*

HepG2 and Raw 264.7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heated-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 ◦C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed every other day. Cell viability was measured using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases. Cells were cultured in 96-well plates (2.0 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well) with serum free media and treated with different concentrations of sample for 24 h. The *R. crispus* extracts and its solvent fractions were dissolved in 10% DMSO. The final concentration of DMSO in the culture medium never exceeded 0.1%. For the assay, 100 μL of MTT solution was added to each well and incubated for 4 h. Finally, 200 μL of DMSO was added to dissolve the formazan crystals and the absorbance was measured using a SpectraMax M2<sup>e</sup> microplate reader at 540 nm.

#### *2.12. NO Production*

Raw 264.7 cells were cultured in 96-well plates using media without phenol red and pre-treated for 1 h with each of the test substrates. Cellular NO production was induced by adding 1 μg/mL LPS and incubating the mixture for 24 h. After incubation, 50 μL of conditioned media containing nitrite (primary stable oxidation product of NO) was mixed with the same volume of Griess reagent and incubated for 15 min. Absorbance of the mixture was measured using a SpectraMax M2e microplate reader at 550 nm.

#### *2.13. Cytokine Analysis*

Production of IL-1β, IL-6, and TNF-α in Raw 264.7 cells was evaluated using Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA) as per the manufacturer's instructions. Cells were treated with different concentrations of test materials for 1 h and production of IL-1β, IL-6, and TNF-α was stimulated by adding 1 μg/mL LPS and incubating for a further 24 h. The supernatant was collected and the concentrations of IL-1β, IL-6, and TNF-α were quantified using the relevant kit protocol.

#### *2.14. Annexin V-FITC*/*PI Analysis*

To determine the magnitude of the apoptosis induced by DCMF, an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Pharmingen, San Diego, CA, USA) was used. Briefly, the cells were harvested, washed with PBS and binding buffer, and then stained with FITC-conjugated Annexin V and propidium iodide (PI) for 30 min in the dark. The mixture was then analyzed using an LSR Fortessa flow cytometer (Becton Dickinson, San Jose, CA, USA) according to the manufacturer's protocol.

#### *2.15. Western Blot*

HepG2 cells were cultured in DMEM at a density of 1 <sup>×</sup> 104 cells in 10 cm2 cell culture dishes and incubated for 24 h. The cells were treated with different concentrations of DCMF for 24 h. The cells were lysed using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) and supernatants were treated with a protease inhibitor cocktail and centrifuged at 2300× *g* for 10 min to remove the insoluble fraction. The protein concentrations of the supernatants were determined using a BCA protein assay kit (Thermo Science, Rockford, IL, USA).

The same amounts of cell lysates were analyzed on 10% SDS-PAGE and the proteins were blotted onto immuno-blot nitro-cellulose membranes and blocked with 5% BSA in TBS containing 0.1% Tween 20 (TBS-T) for 1 h. Then the primary monoclonal antibodies were added to the TBS-T (1:1000 dilutions) and incubated overnight. Antibody binding was detected using a horseradish peroxidase secondary antibody and enhanced using a chemi-luminescence ECL assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions and imaged on a FUJIFILM LAS-4000 mini system (Tokyo, Japan). The basal levels of the proteins were normalized against β-actin or β-tubulin.

#### *2.16. Statistical Analysis*

Each experiment was performed at least three times and results are presented as means ± SDs (standard deviations). Statistical comparisons of the mean values were performed using one-way ANOVA followed by Duncan's multiple range test using Minitab 17 software (Minitab Inc., IL, USA, State College, PA, USA). Differences were considered significant at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Analysis of Polyphenol, Flavonoid, and Anthraquinone Contents*

Polyphenols are aromatic compounds containing more than two phenolic hydroxyl groups. They are classified into phenolic acids (e.g., caffeic acid and chlorogenic acid) and flavonoids (e.g., kaempferol and catechin) [20]. The total polyphenol and flavonoid contents for each of the extracts are described in Table 1. Analysis of the total polyphenol and flavonoid content in the *R. crispus* extracts and solvent fractions revealed that polyphenol content was highest in the ethyl acetate fraction (EAF), followed by the dichloromethane fraction (DCMF), ethanol extract (EE), aqueous fraction (AF), butanol fraction (BF), and finally, the hexane fraction (HF). The highest flavonoid content was detected in the DCMF, followed by HF, EAF, EE, AF, and BF. The antioxidant activity of polyphenolic

compounds is attributed to their activities as electron donors and free radical scavengers. Therefore, the antioxidant effects of various plant extracts have been shown to be strongly linked with the relative phenolic content [21,22].


**Table 1.** Total phenolic and flavonoid contents of *Rumex crispus* L. root extracts and fractions 1.

<sup>1</sup> Values are each expressed as a mean ± SD (*n* = 3). <sup>2</sup> EE: ethanol extracts. HF: *n*-hexane fractions. DCMF: dichloromethane fraction. EAF: ethyl acetate fraction. BF: *<sup>n</sup>*-buthanol fractions. AF: aqueous fraction. a–e Means with different superscripts in the same column are significantly different at *<sup>p</sup>* <sup>&</sup>lt; 0.05.

An HPLC-DAD method was applied to identify the five anthraquinones, including aloeemodin, chrysophanol, emodin, physcion, and rhein, in *R. crispus* extracts and solvent fractions. Figure S1 shows the typical chromatograms of the standard solution containing the five anthraquinones and the *R. crispus* extracts and solvent fractions. The retention times of the aloeemodin, rhein, emodin, chrysophanol, and physcion were 22.2, 23.7, 27.3, 29.9, and 33.2 min, respectively (Figure S1). The concentrations of the major anthraquinones from *R. crispus* extracts and solvent fraction are summarized in Table 2. The major anthraquinones found in the samples analyzed in this study were chrysophanol, emodin, and physcion. The anthraquinone content was highest in the DCMF, followed by HF, EE, EAF, and BF. One gram of DCMF contained 66.96 mg chrysophanol, 160.43 mg emodin, and 34.90 mg physcion. One gram of HF contained 48.64 mg chrysophanol, 14.64 mg emodin, and 15.43 mg physcion. However, none of the anthraquinone compounds were detected in the AF. Anthraquinone derivatives are naturally occurring quinone compounds including naphthoquinones and benzoquinones, and are present in large quantities in plants such as Polygonaceae (*Rheum*, *Rumex*), Fabaceae (*Cassia*), Liliaceae (*Aloe*), Rhamnaceae (*Rhamnus*), and Rubiaceae (*Asperula*, *Coelospermum*, *Coprosma*, *Galium*, *Morinda*, and *Rubia*) [23]. Lim et al. [24] analyzed the anthraquinone contents of various *Rumex* species and found that emodin was highest in *R. crispus*. In addition, Smolarz et al. [25] investigated the anthraquinone contents of various *Rumex* species and found that the highest anthraquinone concentrations were found in the root extracts, with these extracts having substantially higher concentrations than those of the fruit extracts (70-fold) and the leaf extracts (10-fold). Most of these compounds are nonpolar with a 9,10-anthracenedione basic structure—a tricyclic aromatic organic compound with a formula of C14H8O2, which is extracted by polar solvents like ethanol/water mixtures, ethanol, methanol, and acetone [26]. It has also been reported that these compounds are well dispersed in nonpolar solvents, such as hexane and dichloromethane [27].

**Table 2.** Anthraquinone derivative contents of *Rumex crispus* L. root extracts and fractions 1.


<sup>1</sup> Values are each expressed as a mean ± SD (*n* = 3). <sup>2</sup> EE: ethanol extracts. HF: *n*-hexane fractions. DCMF: dichloromethane fraction. EAF: ethyl acetate fraction. BF: *<sup>n</sup>*-buthanol fractions. AF: aqueous fraction. a–e Means with different superscripts in the same column are significantly different at *<sup>p</sup>* <sup>&</sup>lt; 0.05.

#### *3.2. Radical Scavenging Activities of R. crispus Extracts and Fractions*

The DPPH radical scavenging assay is used to assess the electron-donating ability of antioxidants to quench free radicals. In *R. crispus* root extracts and fractions, we observed that DPPH radical scavenging was the highest in the EAF, followed by the EE, BF, DCMF, and HF (Table 3). While the antioxidant activities of *R. crispus* leaf and fruit extracts have been extensively studied, there is limited information on these activities in its root extracts. Yildirim et al. [28] have reported that DPPH radical scavenging activity is higher in *R. crispus* fruit extracts with high polyphenol content than in leaf extracts. Consistent with the findings of this study, the radical scavenging ability of *R. japonica* extracts and fractions has also been found to be highest in extracts with high polyphenol content and low in extracts with low polyphenol content [29].

We measured the scavenging capacities of these extracts for hydroxyl radicals using the Fenton reaction assay, which is based on fluorescence emission after hydroxyl radicals generated by H2O2 and Fe2<sup>+</sup> via the Fenton reaction with DCFH. The scavenging ability increased in all the extracts and fractions in a concentration-dependent manner. This is similar to the results for Trolox, a well-known antioxidant. Anusuya et al. [30] have reported that the hydroxyl radical scavenging abilities of *Rubus nepalensis* extracts are closely related to their polyphenol contents. Moreover, phenolic hydroxyl groups are known to rapidly quench hydroxyl radicals by donating hydrogen atoms or electrons, as evidenced by measuring the hydroxyl radical scavenging abilities of various phenolic acids [31]. This study also demonstrated that the EAF and DCMF which both had high polyphenol and flavonoid contents also had the highest hydroxyl scavenging abilities (Table 3).

Superoxide radicals have very low reactivity; however, within the body, they are rapidly transformed into H2O2, and then via the Fenton reaction, to highly reactive hydroxyl radicals, which interact with biomolecules, causing tissue damage. As with hydroxyl radical scavenging, the superoxide radical scavenging ability of *R. crispus* root extracts was strongest in the EAF and DCMF, followed by EE, BF, AF, and HF (Table 3). In a study of the antioxidant properties of *Rumex hastatus* extracts and fractions, superoxide radical scavenging ability has been linked to flavonoid content rather than phenolic content [32]. The present study showed that the EAF and DCMF fractions, with high flavonoid content, exhibited the highest superoxide radical scavenging ability.


**Table 3.** Free radical scavenging activity of *Rumex crispus* L. root extracts and fractions 1.

<sup>1</sup> Values are expressed as a mean ± SD (*<sup>n</sup>* <sup>=</sup> 3). <sup>2</sup> EE: ethanol extracts. HF: *<sup>n</sup>*-hexane fractions. DCMF: dichloromethane fraction. EAF: ethyl acetate fraction. BF: *n*-buthanol fractions. AF: aqueous fraction. <sup>3</sup> Effective concentration of substance that causes 50% inhibition. a–e Means with different superscripts in the same column are significantly different at *p* < 0.05.

#### *3.3. Antioxidant Capacities of R. crispus Extracts and Fractions*

The FRAP assay determines antioxidant activity by measuring electro transport. In this assay, the antioxidant activity was assessed by the reduction of Fe3<sup>+</sup>-TPTZ to Fe2+-TPTZ. The FRAP value of the extracts and fractions was the highest in the EAF, followed by the EE, AF, DCMF, BF, and HF (Table 4). The TEAC assay measures scavenging of ABTS<sup>+</sup> radicals by sulfur oxides through donation of hydrogen atoms or electrons, which are expressed as trolox equivalents. The highest electron-donating capacity was recorded for the EAF (5.65 mM TE/g), followed by the DCMF, EE, AF, BF, and HF

(Table 4). The ORAC assay is an experimental method recommended by the United States Department of Agriculture for the quantification of food antioxidant content based on electron transport capacity. Decomposition of AAPH produces peroxyl radicals, which react with fluorescein, leading to decreased fluorescence. Peroxyl radical scavenging measured as fluorescence reduction is then converted to trolox equivalents (TE). The ORAC value was highest in the EAF (4817 mM TE/g) and decreased in order from DCMF, to EE, then HF, then AF, and finally BF (Table 4).

The antioxidant capacities of *R. crispus* were highest in the EAF and DCMF, which is consistent with their total phenol and flavonoid contents. Similarly, the antioxidant activity of *R. hastatus* extracts has been reported to be highest in the EAF which has high total phenol and flavonoid concentrations [32]. Sahidi and Ambigaipalan [33] have reported that food antioxidant capacity is closely related to the total phenolic and flavonoid contents and have attributed it to the electron or H-atom-donating ability of phenolic hydroxyl groups. In this study, the antioxidant capacities were highest in the EAF and DCMF, which is consistent with their higher concentrations of phenolic and flavonoid compounds.


**Table 4.** FRAP, TEAC, and ORAC values of *Rumex crispus* L. root extracts and fractions 1.

<sup>1</sup> Values are expressed as a mean ± SD (*<sup>n</sup>* <sup>=</sup> 3). <sup>2</sup> EE: ethanol extracts. HF: *<sup>n</sup>*-hexane fractions. DCMF: dichloromethane fraction. EAF: ethyl acetate fraction. BF: *n*-buthanol fractions. AF: aqueous fraction. a–e Means with different superscripts in the same column are significantly different at *p* < 0.05.

#### *3.4. Anti-Inflammation Activities of R. crispus Extracts and Fractions*

To assess the anti-inflammatory activities of *R. crispus* extracts and fractions, we measured their cytotoxicities in mouse leukemic monocyte macrophage cells (Raw 264.7). Raw 264.7 cells were treated with 25–400 μg/mL of *R. crispus* extracts and fractions, and cell survival was measured after 24 h using the WST-1 assay. Cell viabilities of *R. crispus* extracts and fractions are summarized in Figure 1A. The EE, BF, and AF did not show cytotoxicity within the indicated concentration range. The EE and HF were not cytotoxic at a concentration of 25–200 μg/mL, but at 400 μg/mL cell survival decreased to 75.24% and 60.94%, respectively. The EAF showed the highest cytotoxicity, decreasing cell survival to 60.20%, 2.62%, and 17.33% at 100, 200, and 400 μg/mL, respectively (Figure 1A).

Excessive NO production from L-arginine due to an overexpression of inducible NO synthase (iNOS) during acute or chronic inflammation is known to accelerate inflammatory responses [34]. We assessed the inhibition of NO production induced by LPS at non-cytotoxic concentrations (25 and 50 μg/mL) for each of the extract fractions. Consistent with the results of the antioxidant assays the EAF and DCMF showed the highest inhibitions of NO production, while other fractions did not exhibit any inhibitory effects (Figure 1B). Previous studies on the anti-inflammatory activities of *R. crispus* leaf extracts and fractions have also reported high levels of inflammatory activity in the DCMF and EAF, which agrees with the findings of this study [35]. Based on the results of the NO inhibition assay, we used the EAF for further cell-based experiments.

Cytokines play a pivotal role in inflammatory responses by directly affecting the proliferation and activity of immune cells [36]. Among the pro-inflammatory cytokines, TNF-α is an essential mediator in the development of systemic inflammatory responses, and its synthesis is increased by NO generated by LPS-stimulated macrophages. TNF-α increases the expressions of chemokines and cell adhesion molecules, thereby accelerating pro-inflammatory responses [37]. IL-1β and IL-6 are multifunctional cytokines secreted by macrophages activated by various pro-inflammatory stimuli; these cytokines are also implicated in the induction of autoimmune diseases, and they act by accelerating

inflammatory responses through autocrine signaling [38]. Figure 2 shows the inhibition of IL-1β, IL-6, and TNF-α secretion following EAF treatment using the enzyme-linked immunosorbent assay (ELISA). The secretion of pro-inflammatory cytokines by Raw 264.7 cells was sharply increased following LPS stimulation and decreased after treatment with EAF in a concentration-dependent manner. IL-1β, IL-6, and TNF-α were suppressed by 28%, 65%, and 68%, respectively, when treated with 50 μg/mL EAF.

**Figure 1.** Cell viabilities of *R crispus* extracts and solvent fractions. (**A**) NO production (**B**) in Raw 264.7 cells. RAW 264.7 cells were treated with various concentrations (25, 50, 100, 200, 400 μg/mL) of *R. crispus* extracts and fractions for 24 h. Cell viability was measured by MTT assay. RAW 264.7 cells were pre-incubated with 12.5, 25, and 50 μg/mL of extracts and fractions for 1 h and then treated with 1 μg/mL of LPS for 24 h. The NO production was measured by the Griess reagent system. Data are represented as means ± SEMs. The different superscripts are significantly different at *p* < 0.05. \* Statistical significance of the difference between LPS and LPS + sample treatment groups: \* *p* < 0.05.

**Figure 2.** Inhibition of LPS induced IL-1b, IL-6 and TNF-a in the EAF. RAW 264.7 cells were preincubated with 12.5 or 25 μg/mL of EAF for 1 h and then treated with 1 μg/mL of LPS for 24 h. The IL-1β, IL-6, and TNF-α production was measured by ELISA, as described in Materials and Methods. Data are represented as means ± SEMs. \* Statistical significance of the difference between LPS and LPS + sample treatment groups: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

In a study of the anti-inflammatory activity in *R. crispus* leaf extracts and fractions, Im et al. [35] found that the EAF displayed higher concentration-dependent activity than the extracts in inhibiting the expression of COX-2 and iNOS involved in the production of PGE2 and NO. In this study, the anti-inflammatory effects were verified by the reduced expression of all the pro-inflammatory cytokines apart from TNF-α [39].

#### *3.5. Anticancer Activities of R. crispus Extracts and Fractions*

We measured the cytotoxic activities of the fractions against the HepG2 human hepatoma cancer cell line. Among the extracts and fractions, anthraquinone-rich fractions HF and DCMF appeared to be the most potent inhibitors of HepG2 cell proliferation, but the other fractions showed no cytotoxicity. The DCMF inhibited cell growth in a dose-dependent manner. Treatment with HF for 24 h inhibited

cell viability with rates of approximately 97%, 90%, 70%, 55%, and 35% at concentrations of 25, 50, 100, 200, and 400 μg/mL, respectively. Treatment with DCMF for 24 h resulted in inhibition of cell viability with rates of approximately 95%, 82%, 61%, 53%, and 27% at concentrations of 25, 50, 100, 200, and 400 μg/mL, respectively (Figure 3A).

**Figure 3.** Cell viability of *R crispus* extracts and solvent fractions. (**A**) apoptosis induced (**B**) in HepG2 cells. HepG2 cells were treated with various concentrations (25, 50, 100, 200, 400 μg/mL) of *R. crispus* extracts and fractions for 24 h. Cell viability was measured by MTT assay. Flow cytometry analysis of apoptosis after exposure to various concentrations (25, 50, 100, 200, 400 μg/mL) of DCMF for 24 h, using annexin V-FITC/PI. The lower right indicates the percentage of early apoptotic cells; the upper right indicates the percentage of necrotic and late apoptotic cells.

A previous study has shown that anticancer activities of *Rumex* species are closely related to their anthraquinone contents [8,40]. In this study, anticancer activity was highest in the HF and DCMF, which is consistent with their anthraquinone content. Based on these results we used the DCMF for further cell-based experiments. As a decrease in cell proliferation may result from the induction of apoptosis, we investigated whether treatment with the DCMF induced apoptosis in HepG2 cells. HepG2 cells were treated with DCMF at various concentrations; then Annexin V-FITC and PI fluorescence was determined by flow cytometry (Figure 3B). After treatment with 50, 100, 150, and 200 μg/mL DCMF for 48 h, the percentages of apoptotic cells were 13.3%, 15.9%, 25.6%, and 71.3%, respectively. These results suggest that the DCMF inhibited the proliferation of HepG2 cells by inducing apoptosis in a concentration-dependent manner.

#### *3.6. Modulation of Apoptotic Regulation*

Bcl-2 proteins play a complex regulatory role in apoptosis [41]. Treatment with DCMF resulted in decreased Bcl-2 expression, while Bax protein expression was increased in a dose dependent manner. DCMF showed increased p53 tumor suppressor protein expression in HepG2 cells (Figure 4A). These results indicate that part of the DCMF-mediated inhibition of HepG2 cells is related to apoptosis through its effects of p53 and Bcl-2 protein expression. In order to determine whether this inhibition is related to the induction of apoptosis, HepG2 cells were exposed to DCMF and their caspase activity was evaluated. DCMF treatment resulted in increased levels of cleaved caspase-3, -8, and -9. This result was confirmed by the progressive proteolytic cleavage of the poly (ADP-ribose) polymerase (PARP), a downstream target of activated caspase, in HepG2 cells treated with DCMF (Figure 4B). These data agree with the other experiments that suggest that DCMF treatment induces apoptosis in HepG2 cells.

DCMF-induced apoptosis in HepG2 cell was confirmed by the characteristic pattern of Annexin V/PI staining, activation of caspases (-3, -8, and -9), and cleavage of PARP. Activated caspases regulate the execution-phases of cell apoptosis by degrading specific structural, regulatory, and DNA repair proteins within the cell [42]. Activated caspase-9 then initiates the proteolytic activity of other downstream caspases, such as caspase-3. The activation of caspase-3 results in the cleavage of key cellular proteins, such as PARP [43]. Tumor suppressor protein p53 also plays key roles in cell fate, cell growth, and death, via the regulation of the cell cycle proteins [44] and apoptosis induced proteins (Apaf1, Bad, Bax, and Fas) [45]. Treatment with DCMF decreased anti-apoptotic Bcl-2 protein and increased pro-apoptotic Bax protein expression. In addition, DCMF treatment increased p53 tumor suppressor protein levels. These results demonstrate that DCMF-induced apoptosis in HepG2 human hepatoma cancer cells is affected via the induction of p53, activation of Bax, inhibition of Bcl-2, processing of caspases, and cleavage of PARP.

**Figure 4.** Effects of DCMF on the Bcl-2 family and p53 (**A**) and caspase family (**B**) protein expression in HepG2 cells. HepG2 were treated with the indicated concentrations of DCMF for 24 h. The equal amounts of cellular proteins were probed with the indicated antibodies, and the proteins were visualized using an ECL detection system. Actin was used as an internal control.

#### **4. Conclusions**

Several studies have demonstrated that natural polyphenol-containing products reduce ROS which are risk factors for age-related diseases. This study examined whether *R. crispus* extracts and fractions could exert any inhibitory effects on oxidative stress-related reactions and inflammation in vitro. The ethanol extracts of *R. crispus* were separated into hexane, dichloromethane, ethyl acetate, butanol, and aqueous fractions based on polarity. Antioxidant activity was evaluated using various assays and was shown to be highest in the DCMF and EAF, corresponding to their high polyphenol and flavonoid contents. In addition, the anti-inflammatory tests revealed that high antioxidant activity correlated with inhibitory effects on NO production, and that the EAF also reduced the secretion of pro-inflammatory cytokines in a concentration-dependent manner. In addition, DCMF was shown to inhibit HepG2 human hepatoma cancer cell growth and induce cellular apoptosis. DCMF-induced apoptosis is facilitated by p53 tumor suppressor protein-mediated Bcl-2 family protein regulation and caspase family protein activation. The results of this study suggest that *R. crispus* could be used as a natural alternative to synthetic antioxidants and anti-inflammatory agents.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/8/726/s1, Figure S1: Chromatogram of *Rumex crispus* root extract and fractions at 420 nm. a: Aloe-emodin, b: Rhein, c: Emodin, d: Chrysophanol. e: Physcion (EE).

**Author Contributions:** Conceptualization, T.E. and J.-S.K.; formal analysis, J.-S.K.; investigation, E.K.; methodology, T.E. and E.K.; project administration, J.-S.K. and E.K.; writing—original draft, T.E. All authors have read and agreed to this version of the manuscript.

**Funding:** This research was supported as a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number 2013R1A1A2065215).

**Conflicts of Interest:** The authors have no conflicts of interest to declare.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Phytochemical Characterization of** *Dillenia indica* **L. Bark by Paper Spray Ionization-Mass Spectrometry and Evaluation of Its Antioxidant Potential Against t-BHP-Induced Oxidative Stress in RAW 264.7 Cells**

**Md Badrul Alam 1,2,**†**, Arif Ahmed 3,**†**, Syful Islam 3, Hee-Jeong Choi 1, Md Abdul Motin 4, Sunghwan Kim 3,5,\* and Sang-Han Lee 1,2,6,\***


Received: 6 October 2020; Accepted: 5 November 2020; Published: 9 November 2020

**Abstract:** The antioxidant effects of the ethyl acetate fraction of *Dillenia indica* bark (DIBEt) and the underlying mechanisms were investigated in *tert*-butyl hydroperoxide (t-BHP)-stimulated oxidative stress in RAW 264.7 cells. Paper spray ionization-mass spectroscopy with positive-ion mode tentatively revealed 27 secondary metabolites in *D. indica* bark extract; predominant among them were alkaloids, phenolic acids, and flavonoids. A new triterpenoid (nutriacholic acid) was confirmed in DIBEt for the first time. DIBEt had strong free radical-scavenging capabilities and was also able to reduce t-BHP-induced cellular reactive oxygen species (ROS) generation in RAW 264.7 cells. DIBEt was found to prevent oxidative stress by boosting the levels of heme oxygenase-1 (HO-1) through the up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) via the regulation of extracellular signal-regulated kinase (ERK) phosphorylation in RAW 264.7 cells. These results support the potential of DIBEt for defense against oxidative stress-stimulated diseases.

**Keywords:** antioxidant; *Dillenia indica*; heme oxygenase 1 (HO-1); nuclear factor erythroid 2-related factor 2 (Nrf2); RAW 264.7 cells

#### **1. Introduction**

Among the various signaling molecules, reactive oxygen species (ROS) and reactive nitrogen species (RNS) play critical roles in maintaining cellular homeostasis. Redox imbalance precisely participates in the pathogenesis and pathophysiology of numerous chronic diseases [1]. However, macrophage cells serve as the first line of defense in infected cells, and activated macrophages are a major source of ROS and RNS triggers epigenetic modifications, leading to the pathogenesis of chronic diseases [2]. Thus, activated macrophage models can identify the active components for functional diet development through a multiple-target strategy [2]. Natural medicinal products have been exploited

in medical practice for centuries. Phytochemicals with inherent antioxidant potential orchestrate innumerable cellular defensive signaling cascades directly or indirectly and might have remedial applications for oxidative stress-induced disorders [3]. Thus, it is essential to understand and validate the bioactivities of natural compounds and their molecular mechanisms to form concrete scientific evidence for their clinical use and effectiveness and to meet regulatory challenges.

Nuclear factor erythroid 2-related factor 2 (Nrf2) activation triggers the induction of various detoxifying and antioxidant enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1) [4]. In the resting state, cytosolic Kelch-like ECH-associated protein 1 (Keap1) causes the degradation of Nrf2 through the ubiquitin-proteasome system. During stress conditions or xenobiotic challenge, the reactive cysteine residue of Keap1 is modified, causing the conformational change of Keap1 structure that prevents Nrf2 degradation, which is then free to translocate to the nucleus and bind to antioxidant-related elements (AREs) in the promoter regions of antioxidant and cytoprotective genes [4]. Furthermore, the activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/Akt (PI3K/AKT), and protein kinase C (PKC) also boosts Nrf2 nuclear translocation [5].

Instrumental analytical techniques, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) coupled with mass spectrometry (MS), have been applied to qualitatively and quantitatively explore the secondary metabolites of medicinal plants or foods. Although these techniques are very accurate and precise, they are very time-consuming and require laborious sample preparation and high costs. In contrast, paper spray ionization-MS (PSI-MS) requires minimal sample preparation time, boosts the ionization of compounds under mild experimental conditions, and furnishes ultrafast examinations of complex matrices at low cost [6]. Thus, PSI-MS has been widely accepted in resveratrol evaluation in red wine [7], chemical composition and fraud verification of whiskey and beer [8], medicines, pesticide analysis in fruits and vegetables, and food additives and their byproducts [9].

*Dillenia indica* (family Dilleniaecae) is commonly known as elephant apple. The pulp of the fruit is applied on the scalp to cure dandruff and hair loss, and the sepal has been used to treat stomach disorders since ancient times [10]. Evidence suggested that *D. indica* possesses various medicinal properties, such as anticancer [11], antimicrobial, antioxidant [12], analgesic, anti-inflammatory [13], and antidiabetic and its associated complications, such as hyperlipidemia [14], diabetic nephropathy, and neuropathy. However, little information is available on the chemical composition of *D. indica* bark (DIB). Therefore, this study aimed to provide further information on the chemical composition of the ethyl acetate fraction of DIB (DIBEt) through the determination of total phenolic and total flavonoid contents and in-vitro antioxidant capacity. The bioactive components of DIBEt were screened using PSI-MS. Furthermore, the focus was on the regulatory role of DIBEt on the expression of antioxidant enzymes in RAW 264.7 cells and the underlying mechanisms.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Extraction*

DIB was collected from Jahangirnagar University, Bangladesh, in August 2018 and taxonomically identified by the National Herbarium of Bangladesh (voucher specimen no. 49403) and retained in the laboratory for future reference. Dried and coarsely powdered barks (100 g) were extracted by shaking with ethanol at 60 ◦C for 12 h (three times) and dried in a rotary vacuum evaporator. The ethanolic extract (DIBE; 18.21 g) was suspended in 200 mL deionized water and partitioned sequentially with n-hexane, chloroform, and ethyl acetate using separating funnels in a stepwise manner. After vacuum filtration, the solutions were concentrated in a rotary vacuum evaporator. The n-hexane fraction (DIBH; 1.70 g), chloroform extract (DIBC; 2.44 g), ethyl acetate fraction (DIBEt; 8.55 g), and aqueous fraction (DIBW; 5.18 g) were dissolved in deionized water at 30 mg/mL concentration.

DIBEt was dissolved in deionized water and then diluted with HPLC-grade ethanol at 10 mg/mL concentration for PSI-tandem MS (MS/MS). The sample solution was vortexed for 1 min and sonicated for 5 min in a Powersonic 410 sonication bath (Hwashin Technology Co., Gyeonggi, Korea) for a homogeneous mixture.

#### *2.2. PSI-MS*

A 2 μL stock solution (10 mg/mL) was loaded using a disposable glass Pasteur pipette (Volac; Poulten & Graf Ltd., Barking, UK) onto the center of a chromatographic paper tip (Whatman 1 Chr., Kent, UK). The positive-ion mode of the Q-Exactive orbitrap MS (Thermo Fisher Scientific, Inc., Rockford, IL, USA) was used to collect the data over the range of *m*/*z* 50–600. To make a sharp tip, the chromatographic paper was cut into dimensions of 6 mm base and 14 mm height. A syringe pump (Fusion 100T; Chemyx, Stafford, TX, USA) was used to load the ethanol solvent onto the sample-loaded paper at a flow rate of 15 μL/min. A spray voltage of 4.5 kV was directly applied to the paper tip for the ionization of the sample. The other parameters for the PSI experiment were as follows: capillary temperature 300, S-lens RF level 50, mass resolution 140,000 (full-width at half-maximum), and maximum injection time 150 ms. The automatic gain control was set to 1 <sup>×</sup> 106.

To perform the MS/MS experiments, three different stepped normalized collision energies (10, 30, and 50) were used with the same instrument. The instrument was operated in the positive-ion mode. The other operative parameters for the MS/MS experiments were as follows: sheath and auxiliary gas flow rate 10 and 0 (arbitrary units), respectively; spray voltage 3.60 kV; capillary temperature 300; and S-lens RF level 50.

#### *2.3. Data Processing*

Mass spectral data obtained from the orbitrap MS were processed using Xcalibur 3.1 with Foundation 3.1 (Thermo Fisher Scientific). Compounds were tentatively identified by matching their exact (calculated) masses of protonated (M + H) adducts with measured *m*/*z* values and PSI-MS/MS fragmentation patterns from the in-house MS/MS database, and online databases such as the Human Metabolome Database [15] and METLIN [16], and the literature. The compound structures were drawn using ChemDraw Professional 15.0 (PerkinElmer, Waltham, MA, USA).

#### *2.4. Radical-Scavenging Activity Assays*

DPPH, ABTS, superoxide, and hydroxyl radical-scavenging assays were conducted to evaluate the free radical-scavenging potential of DIB extract following the procedures outlined by Alam et al. [3]. Ascorbic acid and quercetin were used as positive controls for DPPH and ABTS and superoxide and hydroxyl radical-scavenging assays, respectively. The following equation was adapted to calculate the percent inhibition:

$$\text{Radical-scavenging activity} \left( \% \text{ inhibition} \right) = \left( \frac{\text{Abs}\_{\text{control}} - \text{Abs}\_{\text{sample}}}{\text{Abs}\_{\text{control}}} \right) \times 100$$

where Abscontrol is the absorbance of the control sample and Abssample is the absorbance of the experimental sample. All samples were analyzed in triplicate.

To determine the reducing power potential, ferric reducing antioxidant power (FRAP) and cupric reducing antioxidant capacity (CUPRAC) assays were performed according to the method described by Alam et al. [17]. The reducing power potential was expressed as the ascorbic acid-equivalent antioxidant value (μM) calculated from the standard curve of ascorbic acid. The oxygen radical absorbance capacity (ORAC) assay [18] was performed using Trolox, a water-soluble analog of vitamin E, as a positive control. The antioxidant potentiality was calculated as the Trolox-equivalent antioxidant value (μM).

#### *2.5. Cell Culture and Cell Viability Assay*

RAW 264.7 cells (American Type Culture Collection, Rockville, MD, USA) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and streptomycin-penicillin (100 μg/mL each; Hyclone, Logan, UT, USA) at 37 ◦C and 5% CO2. The cells were seeded in 96-well plates (5×105 cells/mL) for 24 h and subsequently treated with DIBEt (1–100 <sup>μ</sup>g/mL) for the next 24 h. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously [19].

#### *2.6. Measurement of Intracellular ROS*

The generation of *tert*-butyl hydroperoxide (t-BHP)-induced ROS as a cellular oxidative stress biomarker was determined by the 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) method [3].

#### *2.7. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)*

Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA). RT & Go Mastermix (MP Biomedicals, Seoul, Korea) was used to prepare cDNA by implementing the manufacturer's protocols. As described in Supplementary Table S1, various primers were used to perform RT-PCR using a PCR Thermal Cycler Dice TP600 (Takara Bio, Inc., Otsu, Japan) [17].

#### *2.8. Western Blot Analysis*

Cells were lysed and harvested using radioimmunoprecipitation assay buffer. Nuclear and cytosolic proteins were extracted by applying the nuclear and cytoplasmic extraction kit (Sigma-Aldrich, St. Louis, MO, USA). The bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA) was used to confirm the protein content. An adequate amount of protein (30 μg) was subjected to Western blot analysis, as described in a previous report using various antibodies (Supplementary Table S2) [20].

#### *2.9. Statistical Analysis*

Statistical analysis was performed using SigmaPlot version 12.5 (Systat Software, Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation (SD; *n* = 3). One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was performed to determine the significance of the differentiation and fusion indices. *p* < 0.05 was considered significant.

#### **3. Results and Discussion**

#### *3.1. Identification of Secondary Metabolites of DIBEt*

The identification and characterization of the related compounds from DIBEt were performed in two steps. In the first step, PSI-MS was used to identify the major *m*/*z* peaks with a full-scan MS, and then characterized using PSI-MS/MS to obtain the MS/MS fragment of the obtained *m*/*z* from the first step. Figure 1 corresponds to the total ion chromatogram of DIBEt in PSI-MS in positive-ion mode, revealing 27 secondary metabolites presented with their molecular formula, monoisotopic mass of experimental ions, and calculated ions in positive modes (Table 1). All compounds identified in DIBEt were classified into nitrogen compounds, phenolic acids, flavonoids, amino acids, triterpenoids, and others.

**Figure 1.** PSI-MS parent ion peak of the identified compounds of DIBEt.

Three phenolic acids (3,4-dihydroxy-5-methoxybenzoic acid, 2-caffeoylisocitric acid, and 2-*O*caffeoylhydroxycitric acid) and seven flavonoids (naringenin, kaempferol, 5,7-dimethoxyapigenin, 6,7,3 -trihydroxy-2 ,4 -dimethoxyisoflavan (bryaflavan), formononetin 7-glucoronide, amoradinin, and mallotus B (isoallorottlerin), along with the Na and K adducts of glucose) were identified. Twelve nitrogen compounds [γ-aminobutyric acid (GABA), *N*-isopropylhydrazinecarboxamide, hydroxymethylserine, triethanolamine, 5-acetyl-2,4-dimethylthiazole, dialanine, 4-methylthiazole-5-propionic acid, L-α-aminosuberic acid, 1,3-bis(carbamoylcarbamoyl)urea (carbonyldibiuret), linamarin, 2-(glucosyloxy)isobutyraldoxime, and *N*-acetyl-3,5,11,18-tetrahydroxyoctadecyl-2-amine] were also confirmed. One fatty acid (11-dodecenoic acid) and one triterpenoid (nutriacholic acid) were confirmed for the first time in this genus (Figure 2). Peaks 1–3, 5–8, 10, 14–16, and 23 were characterized as nitrogen compounds, such as GABA, *N*-isopropylhydrazinecarboxamide, hydroxymethylserine, triethanolamine, 5-acetyl-2,4-dimethylthiazole, dialanine, 4-methylthiazole-5-propionic acid, L-α-aminosuberic acid, 1,3-bis(carbamoylcarbamoyl)urea, linamarin, 2-(glucosyloxy)isobutyraldoxime, and *N*-acetyl-3,5,11,18-tetrahydroxyoctadecyl-2-amine, with the parent ion peak at *m*/*z* 104.1075, 118.0866, 136.0619, 150.1131, 156.0428, 161.0966, 172.0434, 190.1081, 233.0633, 248.1138, 266.1233, and 376.2597, respectively [21]. The detailed fragmentation patterns are given in Supplementary Figure S1. Seven flavonoids [naringenin (17), kaempferol (18), 5,7-dimethoxyapigenin (19), 6,7,3 -trihydroxy-2 ,4 -dimethoxyisoflavan (20), formononetin 7-glucoronide (25), amoradinin (26), and mallotus B (27)] were also confirmed. Naringenin and kaempferol yielded a major fragment ion at *m*/*z* 153.01 and 119.05 and/or 121.02 due to 1,3A and 0,2B fragmentation, respectively. Polyphenolics also produced at *m*/*z* (M + H-44 u) and (M + H-18 u) by losing CO2 and water molecules, respectively, in positive-ion mode due to the abundance of carboxyl or hydroxyl groups. The detailed fragmentation patterns are given in Supplementary Figure S2. Furthermore, peaks 9, 21, and 22 were confirmed as 3,5-dihydroxy-4-methoxybenzoic acid, 2-caffeoylisocitric acid, and 2-*O*-caffeoylhydroxycitric acid with the parent ion peak at *m*/*z* 185.0445, 355.0697, and 371.0754, respectively (fragmentation patterns in Supplementary Figure S3). Peak 24 was suggested as nutriacholic acid [*m*/*z* 391.2841 (M + H)] and yielded a major fragmentation ion at *m*/*z* 207.14 due to the cleavage of C8–C14 and C9–C11 followed by water loss at *m*/*z* 189.13 (Supplementary Figure S4) [22].



#### *Antioxidants* **2020** , *9*, 1099

#### *3.2. Radical-Scavenging Activities of DIB Extracts*

Various molecular mechanisms and/or the synergism between them may cause the attribution of antioxidant activity of the secondary metabolites present in plants. Thus, the evaluation of the antioxidant activity of plant extracts should be performed via several methods. DPPH, ABTS, superoxide and hydroxyl radical-scavenging assays, and FRAP, CUPRAC, and ORAC assays were performed to assess the antioxidant potential of various organic and aqueous DIB extracts. All organic and aqueous DIB extracts significantly scavenged DPPH and ABTS radicals in a dose-dependent manner (Figure 3A; Supplementary Figure S5). DIBEt showed 7.34- and 3.81-fold higher DPPH and ABTS radical-scavenging activities, respectively, than ascorbic acid used as a positive control, with IC50 of 1.87 ± 0.13 and 7.58 ± 0.10 μg/mL for DIBEt and 13.79 ± 0.87 and 28.90 ± 0.16 μg/mL for ascorbic acid, respectively. Other extracts showed DPPH and ABTS radical-scavenging activities in the following order: DIBE (IC50, 5.12 ± 0.68 μg/mL) > DIBW (IC50, 5.82 ± 0.18 μg/mL) > DIBC (IC50, 50.13 ± 1.08 μg/mL) > DIBH (IC50, 63.79 ± 2.19 μg/mL) and DIBE (IC50, 9.95 ± 0.05 μg/mL) > DIBW (IC50, 17.54 ± 1.15 μg/mL) > DIBC (IC50, 91.00 ± 0.86 μg/mL) > DIBH (IC50, >100 μg/mL), respectively. Previous studies revealed that the methanolic extracts of *D. indica* fruits showed strong DPPH radical-scavenging activity followed by petroleum ether, ethyl acetate, and water extract with IC50 of 31.25, 65.77, 97.25, and 106.95 μg/mL, respectively [12,23]. Compared to previous studies, this study revealed that DIB is more powerful to scavenge DPPH radicals. The superoxide and hydroxyl radical-scavenging abilities of DIB extracts were evaluated by the PMS-NADH superoxide-generating system and Fenton reaction in a dose-dependent manner, respectively (Figure 3B; Supplementary Figure S6). DIBEt had 5.70- and 7.10-fold higher superoxide and hydroxyl radical-scavenging potential than quercetin, with IC50 of 2.47 ± 0.05 and 1.58 ± 0.06 μg/mL for DIBEt and 14.12 ± 0.77 and 11.21 ± 1.06 μg/mL for quercetin, respectively. Other extracts had superoxide and hydroxyl radical-scavenging activities in the following order: DIBE (IC50, 14.78 ± 1.15 μg/mL) > DIBW (IC50, 14.48 ± 0.17 μg/mL) > DIBC(IC50, >100 μg/mL) > DIBH (IC50, >100 μg/mL) and DIBE (IC50, 7.85 ± 0.02 μg/mL) > DIBW (IC50, 9.54 ± 0.09 μg/mL) >DIBC (IC50, 22.41 ± 2.17 μg/mL) > DIBH (IC50, 23.85 ± 0.47 μg/mL), respectively. Das et al. [23] reported that the methanolic extract of *D. indica* fruits had powerful superoxide and hydroxyl radical-scavenging activities with IC50 of 51.49 and 51.82 μg/mL, respectively. In contrast, this study showed that various organic and aqueous DIB extracts are more powerful to scavenge superoxide and hydroxyl radicals.

**Figure 3.** *Cont.*

**Figure 3. Radical-scavenging e**ff**ects of DIBEt.** DPPH and ABTS (**A**) and superoxide and hydroxyl (**B**) radical-scavenging activities of DIBEt. Ascorbic acid (ASC) and quercetin (QRC) were considered as standard antioxidant molecules. The reducing power of DIBEt was examined by CUPRAC, FRAP, and ORAC assays (**C**). The ascorbic acid-equivalent antioxidant capacity was calculated for CUPRAC and FRAP assays, and (**D**) the ORAC activity was expressed as the Trolox-equivalent antioxidant capacity. Mean ± SD (*n* = 3). \*\* *p* < 0.01.

CUPRAC, FRAP, and ORAC assays were performed to determine whether DIBEt is capable to donate electrons and establish that DIBEt has a strong reducing power potential in a dose-dependent manner (Figure 3C; Supplementary Figure S7). At 10 μg/mL, DIBEt showed 34.52 ± 0.37 and 81.37 ± 0.57 μM ascorbic acid-equivalent reducing power for CUPRAC and FRAP assays, respectively. Other extracts showed ascorbic acid-equivalent reducing power activities in the following order: DIBE (30.64 ± 0.58 μM) > DIBW (24.51 ± 1.13 μM) > DIBC (11.85 ± 2.03 μM) > DIBH (4.22 ± 0.28 μM) and DIBE (21.08 ± 1.25 μM) > DIBW (19.72 ± 0.80 μM) > DIBC (6.80 ± 0.89 μM) > DIBH (3.30 ± 1.57 μM) for CUPRAC and FRAP assays, respectively. DIBEt showed 9.43 ± 1.97 μM Trolox-equivalent antioxidant capacity at 10 μg/mL in the ORAC assay. Other extracts showed Trolox-equivalent antioxidant capacity in the following order: DIBE (7.87 ± 0.04 μM) > DIBW (5.55 ± 0.27 μM) > DIBC (3.10 ± 0.20 μM) > DIBH (2.74 ± 0.13 μM). Based on these interpretations, DIBEt has a very strong potential to donate/transfer hydrogen/electrons to oxidants to neutralize them.

Studies revealed that phenolic compounds, such as phenol and flavonoids, have strong redox properties capable of quenching singlet and triplet oxygen, adsorbing and neutralizing free radicals, and/or decomposing peroxides, resulting in superior antioxidant potential [17,24,25]. Thus, total phenolic and flavonoid contents were found in the plant chosen for the study (Supplementary data S8). In addition, Pearson coefficient (ρ) and linear regression analyses were performed to correlate the polyphenol, flavonoid, and antioxidant activities of DIB extracts. The results showed substantial correlation for DPPH, ABTS, and hydroxyl radical-scavenging activities (ρ = −0.939, −0.918, and −0.853, respectively) and moderate correlation for superoxide radical-scavenging activity (ρ = −0.622). A negative ρ-value (−1) stands for a perfect positive correlation, as a correlation between polyphenol and free radical-scavenging abilities was found using IC50. The data were also supported by previous studies describing that the antioxidant activity is highly influenced by the presence of total phenol content, and has a linear correlation between phenolic content and antioxidant activity, but the total flavonoid content provided a mixed function [3,26].

The radical-scavenging and reducing power activities of the identified constituents of DIBEt were also tested. The identified compounds showed a strong radical-scavenging activity (IC50) in the order of naringenin > kaempferol > 3,4-dihydroxy-5-methoxybenzoic acid > ethyl maltol > dialanine > GABA linamarin. Naringenin also showed the highest reducing power activity, with an ascorbic acid-equivalent of 33.32 ± 0.31 and 54.34 ± 0.29 μM for CUPRAC and FRAP assays, respectively, followed by kaempferol > 3,4-dihydroxy-5-methoxybenzoic acid > ethyl maltol > dialanine > GABA - linamarin (Table 2).


**Table 2.** Antioxidant activities of commercially available identified compounds from DIBEt.

<sup>a</sup> Radical-scavenging activities as IC50 (μg/mL). <sup>b</sup> Ascorbic acid-equivalent reducing power (μM). GABA, ethyl maltol, dialanine, 3,4-dihydroxy-5-methoxybenzoic acid, naringenin, and kaempferol were purchased from Sigma-Aldrich (catalog nos. A2129, W348708, A9502, CDS003720, N5893, and 60010, respectively). Linamarin was purchased from Cayman Chemical (Ann Arbor, MI, USA). All standards of purity were >98%.

Studies revealed that 3,4-dihydroxy-5-methoxybenzoic acid and 2-*O*-caffeoylhydroxycitric acid have strong free radical-scavenging activities, with IC50 of 10.69 and 6.05 μg/mL and 10.23 and 4.32 μg/mL for DPPH and ABTS assays, respectively [27,28]. Flavonoids, such as naringenin, kaempferol, and 6,7,3 -trihydroxy-2 ,4 -dimethoxyisoflavan, also have very strong free radical-scavenging activities as reported by previous studies [29,30]. Triethanolamine, dialanine, and linamarin have also been studied for their antioxidant properties [31,32]. GABA- and mallotus B-containing plant extracts also showed excellent antioxidant properties and protect RIN-m5F pancreatic cells from hydrogen peroxide-induced oxidative stress [33,34].

#### *3.3. DIBEt Attenuates t-BHP-Induced Cellular Oxidative Stress*

Among all extracts, DIBEt has the highest antioxidant potential triggered to evaluate the potential of DIBEt to scavenge oxidative stress-induced cellular ROS generation with cellular toxicity. t-BHP, a short-chain analog of lipid peroxide, is widely accepted as a model substance to induce oxidative stress in cells and tissues and evaluate the molecular mechanisms of cellular alterations caused by oxidative stress [35]. More than 95% of cell viability was noted at DIBEt concentrations up to 50 μg/mL (Figure 4A; Supplementary Figure S9). In addition, Figure 4B demonstrates that DIBEt has an immense potential to attenuate oxidative stress-induced cellular ROS generation in a concentration-dependent manner with that of gallic acid (30 μg/mL) without showing cellular toxicity. Excessive ROS formation triggers oxidative stress, leading to cell death. Detailed research has revealed that antioxidants mitigated the deleterious effects of ROS and retard many effects that cause cellular death [3,36,37].

#### *3.4. E*ff*ects of DIBEt on Antioxidant Enzyme Expression in RAW 264.7 Cells*

The first-line antioxidant defense system, including superoxide dismutase, catalase, glutathione peroxidase (GPx), and glutathione, critically maintains cellular redox homeostasis [37]. Various stimuli can induce HO-1 expression, conferring cell protection from oxidative stress by maintaining antioxidant/oxidant homeostasis [38]. In Figure 4C,D, the mRNA expression and protein levels of the first-line antioxidant enzymes SOD1, catalase, and GPx-1 and the phase II enzyme HO-1 were extremely extenuated in t-BHP-treated cells, respectively, but DIBEt treatment dose-dependently reversed this trend. Naringenin also enhanced the mRNA expression and protein levels of first-line antioxidant enzymes in the t-BHP model. These data suggest that increased mRNA and protein levels of antioxidant enzymes by DIBEt plays a critical role in cellular homeostasis and protects against oxidative stress-induced cell death.

Studies revealed that medicinal plants or foods possess abundant polyphenolic compounds that boost SOD1, CAT, and GPx activities, decreasing oxidative stress [39,40]. Evidence showed that polyphenolic acids, such as naringenin, kaempferol, apigenin, and formononetin aglycone, and their glycosidic form and caffeoylisocitric acid have a potential to boost first-line antioxidant proteins, leading to cell protection against oxidative stress [41–45]. Therefore, the presence of phenolic acids

and flavonoids in DIBEt may play a major role in boosting the expression of first-line antioxidant enzymes/proteins as the principal mechanism accounting for protecting DIBEt against oxidative stress.

**Figure 4.** Protective effects of DIBEt against t-BHP-induced cell toxicity and intracellular ROS generation through the up-regulation of antioxidant enzymes via Nrf2 activation. Cells were treated with DIBEt and gallic acid (GA) at the indicated concentrations for 12 h and then exposed to 100 μM t-BHP for 6 h. Cell viability percentage (**A**) and intracellular ROS (**B**) were determined by MTT assay and the DCFH-DA method, respectively. Mean ± SD (*n* = 3). # *p* < 0.001, compared to no treatment; \*\* *p* < 0.05, compared to t-BHP treatment. SOD1, catalase, GPx, and phase II antioxidant enzyme (such as HO-1) mRNA (**C**) and protein expression (**D**) were analyzed by RT-PCR and Western blot, respectively. Nrf2 protein expression (**E**) was measured by Western blot. Cells were treated with an Nrf2 inhibitor (brusatol) with and without DIBEt and naringenin (NAR). Nrf2 and HO-1 protein levels were analyzed by Western blot (**F**). Mean ± SD (*n* = 3). # *p* < 0.001, compared to no treatment; \*\* *p* < 0.05, compared to sample treatment. (+): presence; (-): absence.

#### *3.5. E*ff*ects of DIBEt on Phase II Enzymes Mediated by Nrf2 Nuclear Translocation in RAW 264.7 Cells*

An important transcription factor, Nrf2, binds to the ARE in the promoter regions of cytoprotective genes and acts as a master regulator of antioxidative responses [46]. Thus, immunoblotting was performed to evaluate the role of DIBEt on Nrf2 regulation. In Figure 4E, the nuclear Nrf2 content was markedly increased in association with decreased cyto-Nrf2 levels after DIBEt treatment. Furthermore, brusatol, a pharmacological inhibitor of Nrf2, was used to confirm the role of DIBEt on the activation of phase II detoxifying enzymes through Nrf2 regulation. Nrf2 protein levels were greatly diminished by brusatol treatment, which was not reestablished despite the application of DIBEt and naringenin (Figure 4F). Furthermore, DIBEt and naringenin treatment was also unable to restore the basal HO-1 protein levels in brusatol-treated cells (Figure 4F). This observation conferred that DIBEt might disrupt the proteasomal degradation of Nrf2 in the cytoplasm by Keap1 and facilitate Nrf2 nuclear translocation, resulting in the up-regulation of HO-1 expression. Studies revealed that extracts from various medicinal plants or foods, such as *Nymphaea nouchali* flowers, *Lannea coromandelica* bark, and *Ginkgo biloba* bark, can activate Nrf2-mediated phase II enzyme expression in RAW 264.7, Hepa-1c1c7, and Hep G2 cells [3,17,47]. Furthermore, naringenin, kaempferol, apigenin, and formononetin aglycone and their glycosidic form can modulate the Nrf2/ARE/HO-1 signaling cascade, leading to the attenuation of oxidative stress-mediated melanocytes and kidney and neuronal death [43–45].

#### *3.6. DIBEt Regulates Nrf2 Translocation Via Activation of MAPK to Lessen Oxidative Stress*

MAPKs act as a downstream effector in antioxidant responses. The activation of MAPKs can manifest the activation of Nrf2. In-vitro and in-vivo studies have revealed that extracellular signal-regulated kinase (ERK), JNK, and p38 MAPK positively regulate ARE-containing reporter or detoxifying genes via Nrf2-dependent mechanisms [48,49]. In Figure 5A, ERK1/2 was phosphorylated from 15 to 180 min and peaked at 30 min after DIBEt exposure, whereas p38 and JNK phosphorylation was absent in DIBEt-treated cells. Furthermore, to confirm the role of ERK1/2 phosphorylation in Nrf2 translocation to the nucleus and induction of HO-1 expression, U0126, a specific inhibitor of ERK1/2, was used in DIBEt-treated cells. As expected, in Figure 5B, Nrf2 nuclear translocation and subsequently HO-1 expression were successfully enhanced in DIBEt-treated cells, which were strongly mitigated in U0126-treated cells, suggesting that ERK activation plays a critical role in DIBEt-induced Nrf2 translocation into the nucleus and subsequent boost of HO-1 expression in RAW 264.7 cells. Moreover, according to previous reports, dietary antioxidants can cause MAPK activation accountable for cell protection against oxidative stress [50].

**Figure 5. DIBEt facilitates Nrf2 translocation by activating ERK1**/**2.** RAW 264.7 cells were pretreated with DIBEt (10 μg/mL) at the indicated times. Immunoblotting was performed to evaluate kinase activity (**A**). Cells were treated with DIBEt in the presence and absence of the specific inhibitor U0126. The protein levels of Nrf2 and HO-1 were analyzed by Western blot (**B**). Mean ± SD (*n* = 3). # *p* < 0.001; \*\* *p* < 0.05, compared to no treatment. Statistical analysis was performed using one-way ANOVA.

#### **4. Conclusions**

Oxidative stress is a major causative condition for the development and progression of numerous acute and chronic clinical disorders. Thus, antioxidants may cause health benefits as prophylactic agents. In this study, DIBEt contained various polyphenolic compounds as confirmed by PSI-MS/MS and showed superior antioxidant activity in cell-free and cellular levels. DIBEt treatment successfully lessened oxidative stress and cell death, most likely by (i) reducing ROS generation and (ii) boosting the expression of endogenous antioxidant enzymes and/or Nrf2-mediated HO-1 expression. DIBEt pretreatment may cause the activation of ERK1/2 signaling pathways involved

in the cytoprotective effects of DIBEt. The findings provide new insights into the cytoprotective effects and mechanisms of DIB against oxidative stress, which may be used as treatment for oxidative stress-induced disorders.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/11/1099/s1, Figure S1: Mass fragmentation of nitrogen compounds in DIB extracts. Figure S2: Mass fragmentation of flavonoids in DIB extracts. Figure S3: Mass fragmentation of phenolic acids in DIB extracts. Figure S4: Mass fragmentation of ethyl maltol, 11-dodecenoic acid, and the triterpenoid nutriacholic acid in DIB extracts. Figure S5: DPPH and ABTS radical-scavenging activities of various organic and aqueous DIB extracts. Figure S6: Superoxide and hydroxyl radical-scavenging activities of various organic and aqueous DIB extracts. Figure S7: CUPRAC and FRAP activities of various organic and aqueous DIB extracts. Figure S8: Total phenol (A) and flavonoid (B) content of various organic and aqueous DIB extracts. Figure S9: Cell viability of DIBEt. Table S1: List of primer sequences used in this study. Table S2: List of antibodies used in this study.

**Author Contributions:** M.B.A., A.A., S.I. and H.-J.C. performed the research. M.B.A., A.A., M.A.M., S.K. and S.-H.L. designed the research study and analyzed the data. M.B.A., A.A., S.K. and S.-H.L. wrote the paper. M.B.A. and S.-H.L. revised the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by a National Research Foundation of Korea grant funded by the Ministry of Science and ICT (2020R1A2C2011495).

**Acknowledgments:** M.B.A. and H.-J.C. were supported by the BK21Plus Creative Innovative Group for Leading Future Functional Food Industry, Kyungpook National University.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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