Phlorotannins from Undaria Pinnatifida Sporophyll: Extraction, Antioxidant, and Anti-Inflammatory Activities

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Phlorotannins from Undaria pinnatifida Sporophyll: Extraction, Antioxidant, and Anti-Inflammatory Activities

Xiufang Dong Dalian Polytechnic University

Ying Bai Dalian Polytechnic University

Zhe Xu Dalian Polytechnic University

Yixin Shi Dalian Polytechnic University

Yihan Sun Dalian Polytechnic University

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Abstract Undaria pinnatifida sporophyll (U. pinnatifida) is a major byproduct of U. pinnatifida (a brown algae) processing. Its phenolic constituents, phlorotannins, are of special interest due to their intrinsic ability to precipitate proteins. Herein, a high-temperature extraction procedure was used to isolate these biologically active compounds. The heating temperature, heating time, and extraction solvent (ethanol) concentration were examined with response surface analysis to determine the optimal conditions to achieve the maximum extraction rate. The analysis revealed the optimal conditions to be: 170 °C of heating temperature, 5.2 h of heating time, and 52% ethanol concentration for a yield of 10.7 ± 0.2 mg gallic acid equivalent/g dry weight (GAE/g DW) of sample. Compared to epigallocatechin gallate (EGCG), the extracted phlorotannins displayed higher antioxidant activity on H2O2-induced RAW 264.7 cells with improved efficiency. Furthermore, the compounds exhibited strong anti-inflammatory activity. These findings potentially can be utilized to guide development of novel functional foods and food supplements from sea-originated resources such as brown algae.

Keywords Undaria pinnatifida sporophyll, phlorotannin extracts, prepared processing, antioxidant activity, anti- inflammatory activity

Disciplines Amino Acids, Peptides, and Proteins | Bioresource and Agricultural Engineering | Marine Biology

Comments This article is published as Dong, Xiufang, Ying Bai, Zhe Xu, Yixin Shi, Yihan Sun, Srinivas Janaswamy, Chenxu Yu, and Hang Qi. "Phlorotannins from Undaria pinnatifida Sporophyll: Extraction, antioxidant, and anti-inflammatory activities." Marine Drugs 17, no. 8 (2019): 434. DOI: 10.3390/md17080434. Posted with permission.

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Authors Xiufang Dong, Ying Bai, Zhe Xu, Yixin Shi, Yihan Sun, Srinivas Janaswamy, Chenxu Yu, and Hang Qi

This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/abe_eng_pubs/1147 marine drugs

Article Phlorotannins from Undaria pinnatiﬁda Sporophyll: Extraction, Antioxidant, and Anti-Inﬂammatory Activities

Xiufang Dong 1 , Ying Bai 1, Zhe Xu 1, Yixin Shi 1, Yihan Sun 1, Srinivas Janaswamy 2 , Chenxu Yu 3 and Hang Qi 1,*

1 School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian 116034, China 2 Department of Dairy and Food Science, South Dakota State University, Brookings, SD 57007, USA 3 Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA 50011, USA * Correspondence: [email protected]; Tel.: +86-411-8631-8785

Received: 21 June 2019; Accepted: 22 July 2019; Published: 24 July 2019

Abstract: Undaria pinnatifida sporophyll (U. pinnatifida) is a major byproduct of U. pinnatifida (a brown algae) processing. Its phenolic constituents, phlorotannins, are of special interest due to their intrinsic ability to precipitate proteins. Herein, a high-temperature extraction procedure was used to isolate these biologically active compounds. The heating temperature, heating time, and extraction solvent (ethanol) concentration were examined with response surface analysis to determine the optimal conditions to achieve the maximum extraction rate. The analysis revealed the optimal conditions to be: 170 ◦C of heating temperature, 5.2 h of heating time, and 52% ethanol concentration for a yield of 10.7 0.2 mg gallic acid equivalent/g dry weight (GAE/g DW) of sample. Compared to ± epigallocatechin gallate (EGCG), the extracted phlorotannins displayed higher antioxidant activity on H2O2-induced RAW 264.7 cells with improved efficiency. Furthermore, the compounds exhibited strong anti-inflammatory activity. These findings potentially can be utilized to guide development of novel functional foods and food supplements from sea-originated resources such as brown algae.

Keywords: Undaria pinnatiﬁda sporophyll; phlorotannin extracts; prepared processing; antioxidant activity; anti-inﬂammatory activity

1. Introduction Polyphenolics are natural bioactive compounds commonly found in fruits, vegetables, and cereal grains. They are best known for their health beneficial properties and have been attracting considerable attention among food processors and nutritionists [1]. The burgeoning field of substituting synthetic compounds by natural bioactive ingredients in food, pharmaceutical, and nutraceutical applications requires research on natural materials [2,3]. An emerging trend is to extract bioactive compounds such as polyphenolics from inexpensive and abundant sources [4,5], among which seaweeds stand out as promising candidates. Seaweeds have long been part of human diets and medicine [6]. They are among major sources of industrially important metabolites such as polysaccharides and polyphenols [7]. In addition, they also contain a wide range of bioactive secondary metabolites such as phenolic compounds, terpenoids, and nitrogen compounds. Among the seaweeds, algae, composed of a highly heterogeneous group of organisms, are gaining attraction as functional food ingredients [8]. In this family, brown algae (Phaeophyceae) are well-known for possessing phenolic compounds [9] with antioxidant [10,11], anti-inflammatory [12], antidiabetic [13], anti-proliferative [14], and antibacterial [10] activities. Among

Mar. Drugs 2019, 17, 434; doi:10.3390/md17080434 www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, 434 2 of 14 the long list of phenolic compounds, phlorotannins are of special interest because of their intrinsic ability to precipitate proteins. Phlorotannins could be utilized as inhibitors of glucosidase and amylase as well as to delay absorption of dietary carbohydrates [15,16]. They exhibit a high protective effect on fish mince for quality enhancement, especially in terms of controlling lipid oxidation and Ca2+-ATPase [17]. Phlorotannins are generally found as complexes within algae cell wall components [18], which greatly limits its extraction with common protocols used typically for fruits, vegetables, and grains which have less rigid cell wall structures. Hence, utilization of advanced extraction procedures such as centrifugal partition extraction (CPE), supercritical fluid extraction (SFE), pressurized liquid extraction (PLE), and enzyme-assisted extraction (EAE) [18] has become necessary. One such processes is high-temperature extraction. In this method, heat leads to hydrolysis of cross-linked cell wall structures and facilitates the release of heat-resistant bioactive components (e.g., small molecular weight compounds) to increase the overall extraction efficiency. To optimize the operation, different heating temperatures coupled with various solvent concentrations and heating times need to be explored. Undaria pinnatifida (U. Pinnatifida) is a brown alga that normally grows in the seas of China, Korea, and Japan. During 2015, around 193,000 tons were harvested in China [19], while the worldwide production reached 2,359,000 tons (FAO, 2016). The structure of U. pinnatifida can be categorized into blade (lamina), midrib, sporophyll, and root-like formations (haptera). It is also known as wakame, the name of the processed U. pinnatifida [20]. Sporophyll, the reproductive organ of U. pinnatifida, is a processing by-product of the seaweed food industry. It is rich in protein and bioactive compounds; therefore, it is a valuable raw material for further processing to yield functional supplements and products [21]. Sporophyll is rigid, and its tissue structure is compact and dense. To effectively process it, relatively high temperature heating is needed. Herein, in this study, a high-temperature extracting procedure was developed to isolate phlorotannin extracts from U. pinnatifida sporophyll (PEUPS). The heating temperature, heating time, and extraction solvent (i.e., ethanol) concentration were studied by the response surface method (RSM) to determine the optimal conditions to achieve the maximum extraction rate. In addition, PEUPS were evaluated for their antioxidant and anti-inflammatory activities.

2. Results and Discussion

2.1. Single-Factor Experiment

2.1.1. Effect of Ethanol Concentration on Total Phlorotannin Content (TPC) and Antioxidant Activity of the Phlorotannin Extracts from U. pinnatifida Sporophyll (PEUPS) In order to assess the effect of ethanol concentration on the total phlorotannin content (TPC) and antioxidant activity of the PEUPS, four ethanol concentrations (25%, 50%, 75%, and 100%) were tested with heating temperature of 115 ◦C and heating time of 4 h. As shown in Figure1A, the TPC increased from 2.78 0.40 to 4.33 0.57 mg Gallic acid equivalent/g dry weight (GAE/g DW) when ethanol went ± ± from 25% to 50%, but it subsequently decreased to 2.88 0.19 and finally to 0.80 0.18 mg GAE/g ± ± DW at 75% and 100% ethanol, respectively. A similar trend was noticed in the antioxidant activity of the PEUPS; it rose from 0.06 0.01 to 0.11 0.01 mmol Trolox/g DW as ethanol went from 25% to ± ± 50%, but then it dropped to 0.05 0.004 and 0.01 0.01 mmol Trolox/g DW at 75% and 100% ethanol, ± ± respectively. It appeared that the ethanol concentration of 50% was the most appropriate for obtaining the highest amounts of TPC and the highest antioxidant activity. This phenomenon might be due to the solubility differences among phenolic chemicals in solvents with different polarities. To extract the most TPC, a mixed system with a solvent of different polarity performs better than a single solvent. These results agreed very well with the phlorotannin extraction from Eisenia Bicyclis [18], perennial brown algae from the Laminariaceae family that forms an integral part of traditional Japanese cuisine. Mar. Drugs 2019, 17, 434 3 of 14 Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 14

A B C 8 B 0.12 20 E 0.2 20 B C 0.2 Antioxidant Antioxidant activity (mmol Antioxidant activity (mmol

D Antioxidantactibity (mmol D D 16 0.16 Trolox/g DW Trolox/g

6 0.09 DW Trolox/g 15 F 0.15 A e c C Trolox/g DW) 12 b b 0.12 A f b 4 0.06 10 d 0.1 A

b ） 8 0.08 ） B c TPC DW) GAE/g (mg TPC DW) (mg GAE/g a DW) (mg GAE/g TPC A 2 aC0.03 5 0.05 a A b 4 0.04 A a c a a 0 0 0 0 0 0 0 255075100125 0 25 50 75 100 125 150 175 200 225 0246810 Time (h) Ethanol concentration (% ) Temperature (℃)

Total phenolic content Total phenolic content Total phenolic content Antioxidant activity Antioxidant activity Antioxidant activity

FigureFigure 1. TheThe extractionextraction characteristics characteristics of phlorotanninof phlorotannin extracts extracts from U.from pinnatifida U. pinnatifidasporophyll sporophyll (PEUPS) (PEUPS)extracted extracted under di ffundererent extractiondifferent extraction conditions. conditions. The total phenolicThe total content phenolic (TPC) content and antioxidants(TPC) and antioxidantswere determined were bydetermined changing theby ethanolchanging percentage the ethanol in ethanolpercentage concentration in ethanol (A concentration), temperature (A (B),), temperatureand time (C). (B The), and results time were (C). The expressed results as were mg expressed GAE/g DW as and mg mmolGAE/g trolox DW /andg DW, mmol respectively. trolox/g DW, 2.1.2.respectively. Effect of Extraction Temperature on TPC and Antioxidant Activity of the PEUPS

2.1.2. TheEffect TPC of Extraction and antioxidant Temperature activity on of TPC PEUPS and wereAntioxidant monitored Activity at eight of the extraction PEUPS temperatures (25, 50, 75, 100, 115, 125, 150, and 200 C) with 50% ethanol for 4 h. The TPC surged steadily from The TPC and antioxidant activity of◦ PEUPS were monitored at eight extraction temperatures (25, 0.30 0.30 GAE/g DW at 25 C to 12.30 0.66 mg GAE/g DW at 150 C, and it later dropped 50, 75,± 100, 115, 125, 150, and 200◦ °C) with 50%± ethanol for 4 h. The TPC surged◦ steadily from 0.30 ± to 8.62 0.80 mg GAE/g DW at 200 C (Figure1B). Similarly, the antioxidant activity rose from 0.30 GAE/g± DW at 25 °C to 12.30 ± 0.66◦ mg GAE/g DW at 150 °C, and it later dropped to 8.62 ± 0.80 0.02 0.002 mmol Trolox/g DW at 25 C to 0.19 0.01 mmol Trolox/g DW at 150 C and settled at mg GAE/g± DW at 200 °C (Figure 1B). Similarly,◦ the± antioxidant activity rose from 0.02◦ ± 0.002 mmol 0.12 0.01 mmol Trolox/g DW at 200 C. Thus, it appeared that 150 C could be the optimal temperature Trolox/g± DW at 25 °C to 0.19 ± 0.01 ◦mmol Trolox/g DW at 150 °C◦ and settled at 0.12 ± 0.01 mmol for maximum extraction of phlorotannin from U. pinnatiﬁda sporophyll. In the case of Eisenia bicyclis, Trolox/g DW at 200 °C. Thus, it appeared that 150 °C could be the optimal temperature for maximum a gain in yield was noticed with a temperature surge from 20 to 80 C[18], but further increase of extraction of phlorotannin from U. pinnatifida sporophyll. In the case of◦ Eisenia bicyclis, a gain in yield temperature did not enhance the yield, possibly due to heat-induced degradation of some of the was noticed with a temperature surge from 20 to 80 °C [18], but further increase of temperature did phlorotannins. In comparison, previous studies on extraction from the Sargassum muticum [22,23], not enhance the yield, possibly due to heat-induced degradation of some of the phlorotannins. In an invasive brown macroalga widely spread across the European Atlantic coast, suggested 150 C comparison, previous studies on extraction from the Sargassum muticum [22,23], an invasive brown◦ was the optimal temperature. In general, proper heat treatment could enhance the solubility of macroalga widely spread across the European Atlantic coast, suggested 150 °C was the optimal phlorotannins, it can also disrupt the cell wall structure of plant cells and soften plant tissues to ease temperature. In general, proper heat treatment could enhance the solubility of phlorotannins, it can the release of phlorotannins to facilitate their extraction. However, excessive heating seems not to also disrupt the cell wall structure of plant cells and soften plant tissues to ease the release of further increase the extraction yield. phlorotannins to facilitate their extraction. However, excessive heating seems not to further increase the2.1.3. extraction Eﬀect of yield. Extraction Time on TPC and Antioxidant Activity of the PEUPS

2.1.3. InEffect order of toExtraction investigate Time the on eﬀ TPCect ofand extraction Antioxidant time Activity on the TPCof the and PEUPS antioxidant activity of the PEUPS, heating times of 1, 2, 4, 6, and 8 h in the condition of 150 ◦C and 50% ethanol were used. FigureIn1 orderC shows to investigate an increasing the trend effect for of both extraction TPC and time the on antioxidant the TPC and activity antioxidant from 1 to activity 4 h. The of TPC the PEUPS,increased heating from 2.77times of0.51 1, to2, 11.644, 6, and0.99 8 h mgin the GAE condition/g DW, and of 150 antioxidant °C and 50% activity ethanol enhanced were used. from ± ± Figure0.06 1C0.01 shows to 0.18 an increasing0.01 mmol trend Trolox for/g both DW. TPC Subsequent and the antioxidant increase of activity heating from time 1 to to 8 4 h h. resulted The TPC in increased± from 2.77± ± 0.51 to 11.64 ± 0.99 mg GAE/g DW, and antioxidant activity enhanced from 0.06 decreased TPC to 9.65 0.52 mg GAE/g DW and antioxidant activity to 0.13 0.01 mmol Trolox/g DW, ± 0.01 to 0.18 ± 0.01 mmol± Trolox/g DW. Subsequent increase of heating± time to 8 h resulted in respectively. Thus, it appeared that 4 h of heating time was optimal. This observation agrees well with decreased TPC to 9.65 ± 0.52 mg GAE/g DW and antioxidant activity to 0.13 ± 0.01 mmol Trolox/g phlorotannin extraction from the seaweed Sargassum muticum [22]. Excessive heating seems to have DW, respectively. Thus, it appeared that 4 h of heating time was optimal. This observation agrees a negative impact on the extraction of PEUPS, most likely due to heat-induced degradation of some well with phlorotannin extraction from the seaweed Sargassum muticum [22]. Excessive heating seems of phlorotannins. to have a negative impact on the extraction of PEUPS, most likely due to heat-induced degradation of2.2. some Response of phlorotannins. Surface Method (RSM) Model

2.2. ResponseTo optimize Surface the Method controlling (RSM) parametersModel of the PEUPS extraction, the independent variables (extraction time, ethanol concentration, temperature) were analyzed by RSM (Table1). The PEUPS To optimize the controlling parameters of the PEUPS extraction, the independent variables (extraction time, ethanol concentration, temperature) were analyzed by RSM (Table 1). The PEUPS

Mar. Drugs 2019, 17, 434 4 of 14 were extracted with the 17 selected combinations of the controlling parameters, as shown in Table2. Table3 presents the predicted TPCs by the RSM model. ANOVA was used to examine the statistical signiﬁcance of the model. The p-value Prob > F for the regression model of TPC was 0.0399, indicating that the model was a reasonably good ﬁt to the experimental data. The experimental values for the TPC matched well with the predicted values, indicating a satisfactory model (Table4).

Table 1. Codes and levels of factors for response surface method (RSM) experiments.

Level Factor 1 0 1 − Temperature (◦C) 100 150 200 Ethanol concentration (%) 25 50 75 Time (h) 1 4 7

Table 2. Experimental design and results for optimization of extraction parameters by RSM.

No. Temperature Ethanol Concentration Time TPC (mg GAE/g DW) 1 0 1 1 1.7 − 2 1 1 0 1.6 − − 3 0 1 1 8.9 − 4 0 0 0 10.3 5 1 1 0 6.7 6 1 0 1 2.6 − 7 1 0 1 6.9 8 0 0 0 9.5 9 0 1 1 10.3 10 1 1 0 6.6 − 11 0 0 0 10.3 12 0 1 1 1.9 − − 13 1 0 1 0.9 − − 14 1 0 1 10.4 − 15 1 1 0 1.2 − 16 0 0 0 10.2 17 0 0 0 10.1

Table 3. Analysis of variance for the ﬁtted regression equation.

Sum of Mean p-Value Signiﬁcance Source df F Value Squares Square Prob > F Level Model 205.06 9 22.78 4.03 0.0399 * A-Temperature 73.80 1 73.80 13.04 0.0086 ** B-Ethanol concentration 0.16 1 0.16 0.028 0.8715 C-Time 24.36 1 24.36 4.31 0.0767 AB 0.068 1 0.068 0.012 0.9159 AC 6.81 1 6.81 1.20 0.3089 BC 0.67 1 0.67 0.12 0.7403 AA 45.65 1 45.65 8.07 0.0250 * BB 32.95 1 32.95 5.82 0.0466 * CC 11.07 1 11.07 1.96 0.2046 Residual 39.61 7 5.66 Lack of Fit 39.20 3 13.07 127.14 0.062 Pure Error 0.41 4 0.10 Cor Total 244.67 16 * p < 0.05; ** p < 0.01. Mar. Drugs 2019, 17, 434 5 of 14

Table 4. Predicted and experimental values of PEUPS.

TPC of PEUPS (mg GAE/g DW) Reaction Condition Observed Predicted

A = 169.2 ◦C, B = 52%, C = 5.2 h - 11.0 A = 170 ◦C, B = 52%, C = 5.2 h 10.7 -

The mean experimental TPC varied from 1.2 to 10.3 mg GAE/g DW. Using the RSM model mentioned above, three-dimensional (3D) response surface plots were obtained to spot the region of optimal TPC extraction (Figure2). Temperature appeared to have the greatest influence on TPC of the PEUPS. On the other hand, a subtle, significant, negative quadratic effect of extraction time (p < 0.1) for the TPC extraction was noticed, indicating that there was a maximum yield point in the TPC at an extraction time of 5.2 h (Figure2B,C). TPC yield started to decrease at higher extraction time. Further increase in the extraction time may lead to degradation of the phlorotannins through heat-induced chemical degradation or reaction with other cell components [24]. The ethanol concentration was another important parameter in the extraction procedure. As shown in Figure2A,C, the TPC increased with ethanol concentration up to 52%, followed by gradual reduction with a minimum at 100%. A remarkable drop in the TPC at 100% ethanol concentration revealed the low phlorotannin solubility in pure ethanol. This observation indicated that the extraction of phlorotannins largely depended on the polarity of the solvent. Thus, it appeared that one single solvent might not be effective to isolate the total bioactive compounds. The extraction temperature also appeared to be significant. As shown in Figure2A,B, the linear and quadratic interactions with time were significant on the TPC. With a rise in the temperature, the TPC adopted a linear effect for temperatures less than 170 ◦C, indicating the role of temperature on the overall yield of TPC. Overall, the optimal conditions obtained were extraction temperature of 169.2 ◦C, time of 5.2 h, and ethanol concentration of 52%, with a predicted 11.0 mg GAE/g DW TPC. These values correlated well with the experimental values of TPC 10.7 0.2 mg GAE/g DW at a temperature of 170 C with an extraction time of 5.2 h using 52% ± ◦ ethanol. These conditions were used for further extraction of the PEUPS. Based on this optimal condition, the extracted PEUPS were analyzed using an Ultra-high performance liquid chromatography-triple four-stage bar-time-of-flight/mass spectrometry (UPLC-Triple-TOF/MS) system (AcquityTM ultra, Waters, USA). There were nine small molecular weight polyphenolic compounds identified including 3,5-dihydroxyphenol, 3,4-dihydroxybenzaldehyde, 5-acetoxy-2-hydroxybenzaldehyde, hydroquinone, hydroquinone monoacetate, 4-hydroxy-benzaldehyde, C8H8O3, 1-(3-methoxy-4-hydroxyphenyl) ethenone, and 4-methoxy-phenol (as shown in supplementary Figure S1). These small molecular weight phenolic compounds were thermal stable. Although high-temperature extraction tends to become less effective due to possible heat-induced chemical degradation when the heating becomes excessive, as shown by our data, it appeared to be an efficient way of harvesting small molecular weight phenolic compounds, which, as shown below, are also quite valuable as antioxidant and anti-inflammatory agents. Mar. Drugs 2019, 17, 434 6 of 14 Mar. Drugs 2018, 16, x FOR PEER REVIEW 6 of 14

Figure 2. Standardized Box–Behnken design for the three response variables studied in the experimental Figure 2. Standardized Box–Behnken design for the three response variables studied in the design and their corresponding response surfaces. (A)Effects of ethanol concentration (25%, 50%, experimental design and their corresponding response surfaces. (A) Effects of ethanol concentration and 75%) and temperature (100, 150, and 200 C) on TPC. (B)Effects of time (1, 4, and 7 h) and (25%, 50%, and 75%) and temperature (100, 150,◦ and 200 °C) on TPC. (B) Effects of time (1, 4, and 7 h) temperature (100, 150, and 200 C) on TPC. (C)Effects of time (1, 4, and 7 h) and ethanol concentration and temperature (100, 150,◦ and 200 °C) on TPC. (C) Effects of time (1, 4, and 7 h) and ethanol (25%, 50%, and 75%) on TPC. concentration (25%, 50%, and 75%) on TPC. 2.3. Antioxidant Activity of the PEUPS Evaluated with RAW 264.7 Macrophages 2.3. Antioxidant Activity of the PEUPS Evaluated with RAW 264.7 Macrophages Before the activity analysis, the PEUPS at six selected concentrations of 2.5, 5, 10, 20, 40, and 80 µBeforeg/mL werethe activity cultured analysis, with cells the for PEUPS 24 h to at demonstrate six selected thatconcentrations PEUPS do notof 2.5, exert 5, any10, 20, toxic 40, e ffandect. 80 Weμ foundg/mL were the RAW cultured 264.7 with macrophages cells for 24 viabilityh to demonstra was notte significantlythat PEUPS do (p not> 0.05) exert influenced any toxic effect. except We forfound the 80 theµg /RAWmL concentration. 264.7 macrophages However, viability the cell was viability not significantly was much (p higher > 0.05) thaninfluenced 80% (as except shown for in the supplementary80 μg/mL concentration. Figure S2). To However, investigate the the cell antioxidant viability was and anti-inflammatorymuch higher than properties80% (as shown of the in PEUPS,supplementary we used RAW Figure 264.7 S2). macrophage To investigate cells the as a antioxidant model system and and anti-inflammatory epigallocatechin gallateproperties (EGCG) of the as aPEUPS, positive we control. used RAW We 264.7 designed macrophage and conducted cells as a our model experiments system and (3.8 epigalloca and 3.9)techin for two gallate scenarios: (EGCG) (1)as “post-stimuli a positive control. (procedure We designed 1)”, where and the conducted PEUPS/ EGCGour experiments were administered (3.8 and 3.9) to for the two cells scenarios: after the (1) “post-stimuli (procedure 1)”, where the PEUPS/EGCG were administered to the cells after the stimuli stimuli (H2O2 or lipopolysaccharide (LPS)) was introduced, was used to mimic a therapeutic treatment scenario(H2O2 inor whichlipopolysaccharide the agents were (LPS)) used was to repairintroduced, damages was already used to caused mimic by a thetherapeutic stimuli; andtreatment (2) “pre-stimuliscenario in (procedure which the 2)”,agents where were the used PEUPS to repair/EGCG damages were administered already caused to the by cells the stimuli; before the and stimuli (2) “pre- stimuli (procedure 2)”, where the PEUPS/EGCG were administered to the cells before the stimuli (H2O2 or LPS) was introduced, was used to mimic a preventive treatment scenario in which the agents were(H used2O2 or to LPS) protect was the introduced, cells from was damages used to to mimic be caused a preventive by the stimuli. treatment Both scenario scenarios in are which meaningful the agents in termswere ofused evaluating to protect the bioactivitiesthe cells from of thedamages PEUPS. to be caused by the stimuli. Both scenarios are meaningful in terms of evaluating the bioactivities of the PEUPS. As shown Figure 3A, under the post-stimuli scenario, the viability of RAW 264.7 cells (blank) decreased markedly (p < 0.05) upon exposure to 1 mmol/L of H2O2. After treatment with different

Mar. Drugs 2019, 17, 434 7 of 14

Mar. Drugs 2018, 16, x FOR PEER REVIEW 7 of 14 As shown Figure3A, under the post-stimuli scenario, the viability of RAW 264.7 cells (blank) decreasedconcentrations markedly of the ( pPEUPS< 0.05) (2.5–80 upon exposure μg/mL), toor 1EGCG mmol /Lat ofa Hconcentration2O2. After treatment of 80 μg/mL with di(positivefferent concentrationscontrol), the viability of the PEUPS of the RAW (2.5–80 264.7µg/mL), cells orim EGCGproved. at Approximately, a concentration of the 80 cellµg/ mLviability (positive at 40 control), μg/mL thePEUPS viability treatment of the was RAW already 264.7 cellsat the improved. same level Approximately, of the positive thecontrol. cell viabilityFor the pre-stimuli at 40 µg/mL scenario, PEUPS treatmentas shown wasFigure already 3B, the at theviability same levelin the of RAW the positive 264.7 cells control. also Fordecreased the pre-stimuli markedly scenario, (p < 0.05), as shownand it Figurewas much3B, the lower viability than inthat the in RAW Figure 264.7 3A. cells Different also decreased concentrations markedly of PEUPS ( p < 0.05), (2.5–80 and μg/mL) it was muchcould lowerprotect than the cells that into Figuredifferent3A. degrees. Di fferent EGCG concentrations at a concentration of PEUPS of 80 (2.5–80 μg/mLµ galso/mL) had could approximately protect the cellsthe same to di cellfferent viability degrees. as the EGCG PEUPS at a at concentration 80 μg/mL. However, of 80 µg/ overall,mL also the had preventive approximately mode the worked same less cell viabilitywell than as the the therapeutic PEUPS at 80modeµg/mL. (Figure However, 3A), suggestin overall, theg these preventive agents modewere better worked at lessrepair well than than at theprevention. therapeutic Reactive mode (Figureoxygen3 A),species suggesting (ROS) theseare key agents mediators were better for atcell repair death. than Elimination at prevention. of Reactivestimulation-induced oxygen species ROS (ROS) through are the key use mediators of antioxidan for cellts such death. as Eliminationpolyphenol could of stimulation-induced protect cells from ROSthose through stimuli [25]. the use Hence, of antioxidants it is reasoned such that as the polyphenol antioxidant could activities protect of cellsthe PEUPS from those play stimulikey roles [25 in]. Hence,reducing it iscell reasoned deaths. that Our the results antioxidant were activities consistent of the with PEUPS previous play key studies, roles inwhich reducing reported cell deaths. that Ourphlorotannins results were can consistent be developed with previous into a potential studies, whichbio-molecular reported candidate that phlorotannins to inhibit can ROS be developedformation into[26]. a potential bio-molecular candidate to inhibit ROS formation [26].

A. B. 120 120

100 100

* ** ** 80 80 ** * * * * * * ** 60 # * 60 ** ** ** ** # *** 40 40 Cell viability (%) viability Cell Cell viability (%) viability Cell

20 20

0 0 PEUPS (μg/mL) ﹣﹣2.5 5 10 20 40 80 ﹣ PEUPS (μg/mL) ﹣﹣2.5 5 10 20 40 80 ﹣ EGCG(80μg/mL) ------+ EGCG (80μg/mL) ------+ H2O2 (1mmol/L) - + + + + + + + + H2O2 (1mmol/L) - + + + + + + + +

Figure 3. EEffectsffects of PEUPS onon cellcell viabilityviability inin HH22O2-induced-induced RAW RAW 264.7 cells. ( A))E Effectffect of PEUPS on cell viability inin HH22O2-induced-induced RAW RAW 264.7 264.7 cells; the cells were treated using procedure 1.1. ( B))E Effectffect of PEUPS on cell viability in HH22O2-induced-induced RAW RAW 264.7 264.7 cells; cells; the the cells cells were were treated using procedure 2. Cells (4 × 105 cells/mL)cells/mL) were were indicated indicated (2.5, (2.5, 5, 5, 10, 10, 20, 40, and 80 μµg/mL)g/mL) of PEUPS and 1 mmolmmol/L/L of × H22O2.. The The cell cell viability viability assay assay was was performed performed using using me methylthyl thiazolyl thiazolyl tetrazolium tetrazolium (MTT) (MTT) assay. assay. Values are meansmeans ± SE (n = 3). # p < 0.010.01 compared compared with control group; * p << 0.05, ** p < 0.01 compared with ± H2O2 groupgroup alone. alone. 2.4. Anti-Inflammatory Activity of PEUPS in the RAW 264.7 Macrophages 2.4. Anti-Inflammatory Activity of PEUPS in the RAW 264.7 Macrophages The chronic presence of nitric oxide (NO) is associated with various carcinomas and inflammatory The chronic presence of nitric oxide (NO) is associated with various carcinomas and conditions [27]. The anti-inflammatory characteristics of phlorofucofuroeckol A isolated from Ecklonia inflammatory conditions [27]. The anti-inflammatory characteristics of phlorofucofuroeckol A cava has been studied in LPS-stimulated RAW 264.7 cells [28]. Since NO produced by inducible isolated from Ecklonia cava has been studied in LPS-stimulated RAW 264.7 cells [28]. Since NO nitricoxide synthase (iNOS) is one of the inflammatory mediators, the effects of various PEUPS produced by inducible nitricoxide synthase (iNOS) is one of the inflammatory mediators, the effects concentrations on NO production in LPS-activated RAW 264.7 cells were evaluated in vitro. As shown of various PEUPS concentrations on NO production in LPS-activated RAW 264.7 cells were evaluated in Figure4, lipopolysaccharide (LPS) stimulation resulted in a significant NO hike in the cell culture. in vitro. As shown in Figure 4, lipopolysaccharide (LPS) stimulation resulted in a significant NO hike However, treating affected cells with PEUPS (post-stimuli scenario) decreased the production of NO in the cell culture. However, treating affected cells with PEUPS (post-stimuli scenario) decreased the at all tested concentrations (Figure4). In addition, as confirmed by the immunomodulatory activity production of NO at all tested concentrations (Figure 4). In addition, as confirmed by the assessment, the PEUPS did not show any cytotoxic effect on the RAW 264.7 cells. The NO production immunomodulatory activity assessment, the PEUPS did not show any cytotoxic effect on the RAW of PEUPS at the concentration of 80 µg/mL (procedure 1) was 0.57 0.02 µmol/mL. Cells treated 264.7 cells. The NO production of PEUPS at the concentration of 80 μ±g/mL (procedure 1) was 0.57 ± with EGCG at a concentration of 80 µg/mL had approximately the same NO production as those 0.02 μmol/mL. Cells treated with EGCG at a concentration of 80 μg/mL had approximately the same treated with the PEUPS at 10 µg/mL. The NO production decreased significantly, and this trend agreed NO production as those treated with the PEUPS at 10 μg/mL. The NO production decreased well with the findings reported from Kim et al. [27] on the effect of phlorofucofuroeckol A, dieckol, significantly, and this trend agreed well with the findings reported from Kim et al. [27] on the effect of phlorofucofuroeckol A, dieckol, and dioxinodehydroeckol on NO production in the LPS-induced RAW 264.7 cells: phlorofucofuroeckol A was found to significantly reduce LPS-induced NO

Mar. Drugs 2019, 17, 434 8 of 14

Mar. Drugs 2018, 16, x FOR PEER REVIEW 8 of 14 and dioxinodehydroeckol on NO production in the LPS-induced RAW 264.7 cells: phlorofucofuroeckol Aproduction was found in to a significantly dose-dependent reduce manner LPS-induced at the NOconcentration production of in 5–20 a dose-dependent μM [29]. As for manner preventive at the concentrationeffects shown of in 5–20 FigureµM[ 4B,29 the]. As trend for preventive of NO produc effectstion shown under in scenario Figure4B, “post-stimuli” the trend of NO was production similar to underFigure scenario 4A: the “post-stimuli”higher the exposure was similar to the toPEUPS, Figure the4A: less the higherNO production the exposure in the to cells; the PEUPS, hence, thea larger less NOanti-inflammatory production in the effect cells; hence,can be a largerachieved. anti-inflammatory The NO production effect can bewith achieved. PEUPS The treatment NO production at the withconcentration PEUPS treatment of 80 μg/mL at the was concentration 3.21 ± 0.11 of μ 80mol/mL,µg/mL wasand 3.21it appeared0.11 µ molthat/ mL,the andPEUPS it appeared was a better that ± theanti-inflammatory PEUPS was a better agent anti-inflammatory than EGCG at the agent same than concentration. EGCG at the The same PEUPS concentration. can be considered The PEUPS as a canpotential be considered agent for as suppressing a potential agentthe NO for production suppressing without the NO any production cytotoxic without effect, both any as cytotoxic a therapeutic effect, bothagent as and a therapeutic as a preventive agent agent, and as though a preventive the preventive agent, though effect theis less preventive significant. effect is less significant.

A. B. 6 6 # # 5 ** 5 ** ** ** ** ** 4 4 ** ** ** 3 3 ** ** ** 2 ** 2

1 1 ** Concentration of NO (μmol/mL)

Concentration 0 of NO (μmol/mL) 0 PEUPS (μg/mL) ﹣﹣2.5 5 10 20 40 80 ﹣ PEUPS (μg/mL) ﹣﹣2.5 5 10 20 40 80 ﹣ EGCG(80μg/mL) ------+ EGCG(80μg/mL) ------+ LPS (2μg/mL) - + + + + + + + + LPS (2μg/mL) - + + + + + + + + . Figure 4. Effects of PEUPS on NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. (FigureA)Effect 4. Effects of PEUPS of PEUPS on cell on viability; NO production the cells werein lipopol treatedysaccharide using procedure (LPS)-stimulated 1. (B)Eff ectRAW of PEUPS264.7 cells. on NO(A) production;Effect of PEUPS the cellson cell were viability; treated the using cells procedure were treated 2. LPSusing was procedure the control 1. (B and) Effect epigallocatechin of PEUPS on gallateNO production; (EGCG) was the the cells positive were treated control. using Cells proced (4 105urecells 2. /LPSmL) was were the indicated control concentrationsand epigallocatechin (2.5, 5, × 5 10,gallate 20, 40, (EGCG) and 80 wasµg/ mL)the positive of PEUPS control. and 2 Cellsµg/mL (4 of× 10 LPS, cells/mL) respectively. were Theindicated nitrite concentrations concentration (2.5, in the 5, medium10, 20, 40, was and determined 80 μg/mL) of using PEUPS Griess and reagent. 2 μg/mL Values of LPS, are respectively. means SE Th (ne nitrite= 3). # concentrationp < 0.01 compared in the ± withmedium control was group; determined * p < 0.05, using ** p Griess< 0.01 reagent. were compared Values are with means the control ± SE ( group.n = 3). # p < 0.01 compared with control group; * p < 0.05, ** p < 0.01 were compared with the control group. In order to further characterize the inhibitory effect of the PEUPS on the NO production, a western blot analysisIn order was to further carried outcharacterize to evaluate the the inhibitory expression effect ofkey of the proteins PEUPS/enzymes on the (iNOSNO production, and COX-2) a inwestern the cells. blot iNOSanalysis is awas catalytic carried enzyme out to evaluate to produce the expression NO, and cyclooxygenase-2 of key proteins/enzymes (COX-2) (iNOS is one and of theCOX-2) inducible in the enzymescells. iNOS in is excessive a catalytic inflammatory enzyme to produce responses NO, thatand cyclooxygenase-2 can regulate the production(COX-2) is one of prostaglandins.of the inducible iNOS enzymes and COX-2in excessive gene expressions inflammatory are responses primarily regulatedthat can regulate at the transcriptional the production level, of andprostaglandins. their inductions iNOS are and largely COX-2 dependent gene expressions on activities are ofprimarily transcription regulated factors at the that transcriptional interact with respectivelevel, and cognatetheir inductions cis-acting are elements largely withindependent the iNOSon activities or COX-2 of transcription promoters [27 factors,30,31]. that iNOS interact and COX-2with respective protein expressions cognate cis-acting were markedly elements increased within whenthe iNOS the RAWor COX-2 264.7 promoters cells were treated[27,30,31]. with iNOS LPS comparedand COX-2 to protein the control expressions without LPSwere (Figure markedly5). As in showncreased in when Figure 5theA, theRAW iNOS 264.7 protein cells expressionswere treated werewith significantlyLPS compared inhibited to the control by the PEUPSwithout in LPS a dose-dependent (Figure 5). As shown manner. in Figure Moreover, 5A, thethe iNOSiNOS proteinprotein expressionexpressions was were almost significantly completely inhibited suppressed by the at PEUPS a concentration in a dose-dependent of 40 µg/mL ofmanner. PEUPS. Moreover, As shown the in FigureiNOS 5proteinB, pretreatment expression with was the almost PEUPS completely (10, 20, and suppressed 40 µg/mL) at predominantlya concentrationinhibited of 40 μg/mL iNOS of PEUPS. protein expression,As shown in and Figure iNOS 5B, protein pretreatment expressions with were the PEUPS almost completely(10, 20, and suppressed40 μg/mL) predominantly at both 20 and 40inhibitedµg/mL ofiNOS PEUPS. protein All expression, these results and clearly iNOS pointed protein out expressions that the PEUPS were almost reduce completely the production suppressed of NO mainlyat both by20 and down-regulating 40 μg/mL of PEUPS. iNOS proteinAll these expression. results clearly It seemedpointed notout that to inhibit the PEUPS COX-2 reduce protein the expressionproduction (Figureof NO 5mainlyA,B) much, by down-regulating di fferent from EGCG, iNOS whichprotein down-regulates expression. It seemed both iNOS not andto inhibit COX-2. COX-2 Nonetheless, protein PEUPSexpression is clearly (Figure a good 5A,B) anti-inflammatory much, different from agent EG thatCG, could which reduce down-regulates stimulated NO both production iNOS and inCOX-2. RAW 264.7Nonetheless, macrophages. PEUPS is clearly a good anti-inflammatory agent that could reduce stimulated NO production in RAW 264.7 macrophages.

Mar. Drugs 2019, 17, 434 9 of 14 Mar. Drugs 2018, 16, x FOR PEER REVIEW 9 of 14

FigureFigure 5.5. EffectEffect of of PEUPS PEUPS on on LPS-induced LPS-induced iNOS iNOS and and COX-2 COX-2 protein protein expression expression in RAW in RAW 264.7 264.7macrophages. macrophages. (A) The (A cells) The were cells treated were treated using procedure using procedure 1. (B) The 1. cells (B) The were cells treated were using treated procedure using procedure2. Then, cell 2. lysates Then, cellwere lysates electrophoresed, were electrophoresed, and the expression and the levels expression of iNOS levels and ofCOX-2 iNOS were and detected COX-2 werewith detected specific withantibodies. specific antibodies. 3. Materials and Methods 3. Materials and Methods 3.1. Samples and Chemicals 3.1. Samples and Chemicals U. pinnatifida sporophyll was obtained from Dalian Aquatic Culture Group Company of LiaoningU. pinnatifida province (China) sporophyll in October was 2014.obtained Gallic from acid, Dalian phloroglucinoldihydrate, Aquatic Culture Group lipopolysaccharide Company of Liaoning province (China) in October 2014. Gallic acid, phloroglucinoldihydrate, lipopolysaccharide (LPS), and 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS, P99%) were purchased from Sigma-Aldrich(LPS), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic (St. Louis, MO, USA). The Folin–Ciocalteu phenolacid (ABTS, reagent P99%) was were provided purchased by Merck from (Shanghai,Sigma-Aldrich China). (St. Dulbecco’s Louis, MO, modified USA). Eagle’sThe Folin–Cio mediumcalteu (DMEM) phenol and reagent penicillin-streptomycin was provided by solution Merck were(Shanghai, obtained China). from Dulbecco’s HyClone (Logan, modified UT, Eagle’s USA). Fetalmedium bovine (DMEM) serum and (FBS) penicillin-streptomycin was obtained from Sangonsolution Biotech were obtained Co., Ltd. from (Shanghai, HyClone China). (Logan, Dimethyl UT, USA). sulfoxide Fetal (DMSO) bovine wasserum obtained (FBS) was from obtained Yeasen Biotechfrom Sangon Co., Ltd. Biotech (Shanghai, Co., Ltd. China). (Shanghai, NO was China) obtained. Dimethyl from sulfoxide Jiancheng (DMSO) Institute was of Biotechnology obtained from (Nanjing,Yeasen Biotech China). Co., Anti-mouse Ltd. (Shanghai, iNOS, anti-mouse China). NO COX-2, was anti-obtainedβ-actin from rabbit Jiancheng polyclonal Institute antibody, of anti-rabbitBiotechnology IgG, and(Nanjing, horseradish China). peroxidase Anti-mouse (HRP)-linked iNOS, anti-mouse antibody COX-2, were purchased anti-β-actin from rabbit Cell polyclonal Signaling Technology,antibody, anti-rabbit BCG, Boston, IgG, MA,and horseradish USA. Other peroxida reagentsse were (HRP)-linked obtained fromantibody Sangon were Biotech purchased Co., from Ltd. (Shanghai,Cell Signaling China). Technology, BCG, Boston, MA, USA. Other reagents were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). 3.2. Sample Preparation 3.2. Sample Preparation U. pinnatifida sporophyll was dried in an oven at 80 ◦C for 48 h. Then it was put into a dusting machineU. pinnatifida and sifted throughsporophyll a 40-mesh was dried sieve. in an The oven powder at 80 was °C rehydratedfor 48 h. Then for 4it hwas at aput mass:water into a dusting ratio ofmachine 1:10, centrifuged and sifted at through 10,000 ag ,40-mesh 4 C for 10sieve. min, The and powder the pallet was was rehydrated collected andfor 4 stored h at a at mass:water4 C for × ◦ − ◦ furtherratio of use. 1:10, centrifuged at 10,000× g, 4 °C for 10 min, and the pallet was collected and stored at −4 °C for further use. 3.3. Extraction of PEUPS 3.3. Extraction of PEUPS Extractions were carried out using an accelerated solvent extractor that was equipped with a solventExtractions controller were unit. carried Ultrapure out us watering an and accelerated ethanol were solvent used extractor as solvents. that Initially, was equipped solvents with were a sonicatedsolvent controller for 10 min. unit. For Ultrapure each experiment, water and 1 g ethanol of the rehydrated were used powder as solvents. of U. Initially, pinnatifida solventssporophyll were wassonicated loaded for into 10 amin. 50 mLFor stainlesseach experiment, steel extraction 1 g of the cell rehydrated with 10 mL powder solvent. of U. The pinnatifida experiments sporophyll were carriedwas loaded out by into varying a 50 mL the staticstainless extraction steel extraction time (1, 2,cell 4, with 6, and 10 8 mL h), extractionsolvent. The temperature experiments (25, were 50, 75,carried 100, 115,out by 125, varying 150, and the 200 static◦C), extraction and percentage time (1, of 2, ethanol4, 6, and in 8 theh), extraction extraction temperature solvent mixture (25, (25%,50, 75, 50%,100, 75%,115, 125, and 150, 100%). and After200 °C), treatment and percentage at different of et conditions,hanol in the ethanolextraction was solvent removed mixture using (25%, a rotary 50%, evaporator,75%, and and100%). phlorotannin After treatment was reconstituted at different with conditions, water. The ethanol PEUPS was were removed then kept using at 4 ◦ Ca beforerotary useevaporator, within 24 and h. phlorotannin was reconstituted with water. The PEUPS were then kept at 4 °C before use within 24 h.

3.4. Experimental Design The extraction was optimized using a three-level factorial design (including two center points). The effects of temperature (100, 150, and 200 °C), percentage of ethanol (25%, 50%, and 75%), and

Mar. Drugs 2019, 17, 434 10 of 14

3.4. Experimental Design The extraction was optimized using a three-level factorial design (including two center points). The effects of temperature (100, 150, and 200 ◦C), percentage of ethanol (25%, 50%, and 75%), and heating time (1, 4, and 7 h) on TPC (mg GAE/g DW) were investigated. A total of 17 experiments were conducted in a randomized order. Design and data analysis were carried out using response surface method with Design-Expert 8.0.5. The effects of the independent variables on these response variables in the extraction process were assessed using the pure error, considering a level of confidence of 95% for all the variables. The quadratic model proposed for each response variable (Y) was:

Y = β0 + β1 A + β2 B + β3 C + β12 A B + β13 A C + β23 B C + × × × × × × × × × (1) β11 A2 + β22 B2 + β33 C2, × × × where A is the solvent composition (percentage of ethanol in the mixture); B is the temperature; C is the heating time; β0 is the intercept; β1, β2, and β3 are the linear coefficients; β11, β22, and β33 are the quadratic coefficients; and β12, β13, and β23 are the linear-by-linear interaction coefficients. The significant criteria for each polynomial model were based on the correlation coefficients (R2), RSM, and the lack-of-fit tests from the analysis of variance table. The effect of each factor and its statistical significance were analyzed from the standardized Pareto chart. The response surfaces of the respective mathematical models were also obtained, and the significances were accepted at p < 0.05 (level of confidence of 95%). A multiple-response optimization was carried out by the combination of experimental factors and maximizing the desirability function.

3.5. TPC Measurement (Folin–Ciocalteu Method) TPC was determined spectrophotometrically by using the Folin–Ciocalteu method, with some modifications. Briefly, 50 µL of extract solution and 600 µL of ultrapure water were mixed, and 50 µL of undiluted Folin–Ciocalteu reagent was added. After 1 min, 150 µL of 20% (w/v) Na2CO3 was added, and the volume was made to 1 mL with water. The samples were incubated for 2 h at 25 ◦C in the dark. Later, 200 µL of reaction mixture was transferred to a 96-well microplate. The absorbance was measured at 760 nm by Microplate Reader (Tecan Infinite, Switzerland, M200). Standard curves with serial gallic acid solutions were generated for calibration. The phenolic content was expressed as milligrams of gallic acid equivalent per gram of dried weight (mg GAE/g DW) of sample. All analyses were done in triplicate and average values reported.

3.6. Trolox Equivalents Antioxidant Capacity Assay (TEAC) Trolox equivalents antioxidant capacity (TEAC) assay was employed to measure the antioxidant capacity. ABTS+ radical was produced by reacting 7 mM ABTS and 2.45 mM potassium persulfate in the dark at room temperature for 16–24 h before use. The aqueous ABTS+ solution was diluted with 200 mM phosphate buffer (pH 7.4) to an absorbance of 0.7 0.02 at 734 nm. Ten microliters of ± sample (5 different concentrations ranging from 0.25 to 2 mg/mL) and 1 mL of ABTS+ solution were mixed in an Eppendorf vial, and 200 µL of the mixture was transferred into a 96-well microplate. The absorbance was measured at 734 nm every 5 min for 45 min in a M200 Microplate Reader (Tecan Infinite, Switzerland). Trolox was used as reference, and results were expressed as TEAC values (mmol of trolox/g the dried weight of sample). The values were obtained from five different concentrations of each tested in the assay giving a linear response between 20% and 80% of the blank absorbance. All analyses were done in triplicate and average values reported.

3.7. Cell Culture The macrophage-like cell line, RAW 264.7, has previously been used to characterize the immunomodulatory action of various components at the molecular level [18,22]. RAW 264.7 cell line was obtained from Shanghai Institute of Cell Biology (Shanghai, China) and maintained in DMEM Mar. Drugs 2019, 17, 434 11 of 14 supplemented with heat-inactivated 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 ◦C. When cells reached sub-confluence, they were harvested with trypsin-EDTA and diluted to a suspension in a fresh medium. The cells were seeded in 96-well plates with 4 105 cells/mL and allowed to adhere for 24 h at 37 C in a humidified × ◦ atmosphere containing 5% CO2.

3.8. Antioxidant Activity Analysis Cells were treated following two protocols: Procedure 1 (post-stimuli type), the number of cells from the pretreated wells was quantified, then the growth medium was replaced with 100 µL medium containing 1 mmol/LH2O2 for 1 h. Then, the medium containing H2O2 was sucked out, and the cells were washed with HBSS (Hank’s balanced salt solution) twice, and the growth mediums containing different concentration of PEUPS (2.5, 5, 10, 20, 40, and 80 µg/mL) were added to the cells and cultured for 3 h. Procedure 2 (pre-stimuli), the number of cells from the pretreated wells was quantified, and the growth medium was replaced with 100 µL medium containing different concentrations of PEUPS (2.5, 5, 10, 20, 40, and 80 µg/mL) for 23 h. Then, the medium containing PEUPS was sucked out, the cells were washed with HBSS twice, and the growth medium containing 1 mmol/LH2O2 was added to the cells and cultured for 1 h. After treatment under each procedure, the cells were washed with HBSS twice and measured by a methyl thiazolyl tetrazolium (MTT) assay to evaluate their viability. Absorption values were recorded at 540 nm using M200 Microplate Reader (Tecan Infinite, Switzerland). The viability of RAW 264.7 cells in each well was calculated as the percentage of live cells. All analyses were done in triplicate and average values reported.

3.9. Determination of Nitric Oxide Production Cells were treated the same as in Section 3.8 with pre-stimuli and post-stimuli procedures with 1 mmol/LH2O2 replaced by 2 µg/mL LPS. NO production was assessed indirectly by the accumulated nitrite in the culture supernatant, which is the stable end product of NO reacted with oxygen. Supernatant from each well was evaluated for NO with a commercial kit. All measurements were conducted in triplicate. The optical densities of the samples were detected at 540 nm. The NO production of RAW 264.7 cells in each well was calculated as the percentage of NO production with respect to nontreated LPS-stimulated RAW 264.7 cells. All analyses were done in triplicate and average values reported.

3.10. Western Blot Analysis After cells were treated following Section 3.9, the RAW 264.7 cells were washed twice with ice-cold PBS and lysed with a buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% Tween-20, 0.1% SDS, 10 g/mL leupeptin, 50 mM NaF, and 1mM phenylmethylsulfonyl fluoride) on ice for 1 h. After centrifugation at 14,000 g for 20 min, the protein content of supernatant was measured, × and aliquots (20 µg) of protein solution were subject to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline Tween-20 (TBS-T) buffer for 1 h and incubated for 2 h with primary antibody (iNOS = 1:1000 dilution, monoclonal mouse anti-iNOS antibody, No. 2982S; COX-2 = 1:1000 dilution, anti-COX-2 polyclonal antibody, No. 4842S; β-actin = 1:1000 dilution, anti-β-actin polyclonal antibody, No. 4967S; Cell Signaling Technology, BCG, Mass, USA) in TBS-T buffer containing 5% BSA. The blots were treated with horseradish peroxidase-conjugated secondary antibody (1:2000 dilution goat anti-rabbit-IgG-HRP; Cell Signaling Technology, BCG, Mass, USA) in TBS-T buffer containing 5% BSA for 1 h, and an immune complex was detected using the ECL detection kit. Mar. Drugs 2019, 17, 434 12 of 14

3.11. Statistical Analysis IBM SPSS Statistics software v.19 was employed for data elaboration and statistical analysis using a level of significance set at 95%. One-way analysis of variance (ANOVA) together with response surface experiments were employed to group extracts, based on statistically significant differences; different letters suggest significant differences (p < 0.05).

4. Conclusions In the current study, a group of single-factor experiments coupled with RSM modeling were utilized to optimize the extraction conditions for phlorotannins from U. pinnatifida sporophyll. The optimal conditions obtained are: heating temperature of 170 ◦C, heating time of 5.2 h, and an ethanol concentration of 52% with a yield of 10.7 0.2 mg GAE/g DW. The PEUPS exhibited strong antioxidant ± and anti-inflammatory activities in vitro. These findings suggest that there is a good potential for the utilization of PEUPS in the design and development of novel functional foods and food supplements.

Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/8/434/s1, Figure S1: Effects of PEUPS on cell viability in RAW 264.7 cells, Figure S2. Nine polyphenolic compounds in extracted PEUPS based on the optimal condition. Author Contributions: H.Q. conceived, designed, and supervised the study. X.D. performed the experiments and drafted the manuscript. Y.B., Y.S. (Yixin Shi), and Y.S. (Yihan Sun) participated in the experiments. Z.X. performed experiments and data analysis. S.J. and C.Y. participated the experimental design and reviewed the manuscript. All authors read and approved the final manuscript. Funding: This research was funded by Young Top Talents in the Xingliao Talents Program of Liaoning Province, grant number XLYC 1807166 and Dalian Outstanding Young Science and Technology Talents Project. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

PEUPS Phlorotannins extracts from U. pinnatiﬁda sporophyll RSM Response surface method EGCG Epigallocatechin gallate ROS Reactive oxygen species iNOS Inducible nitricoxide synthase COX-2 Cyclooxygenase-2 TPC Total phlorotannin content GAE Gallic acid equivalent TEAC Trolox equivalents antioxidant capacity assay HBSS Hank’s Balanced Salt Solution NO Nitric oxide TBST Tris-buﬀered saline Tween-20 GAE/g DW Gallic acid equivalent/g Dry weight

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