Inhibitory Effects of <i>Porphyra tenera</i> Extract on Oxidation and Inflammatory Responses

Lee, Chul Won; Ahn, Yong-Tae; Zhao, Rongjie; Kim, Youn Sook; Park, Sang Mi; Jung, Dae Hwa; Kim, Jae Kwang; Kim, Hyung Woo; Kim, Sang Chan; An, Won G.

doi:https://doi.org/10.1155/2021/6650037

Evidence-Based Complementary and Alternative Medicine

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusion Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2021 | Article ID 6650037 | https://doi.org/10.1155/2021/6650037

Inhibitory Effects of Porphyra tenera Extract on Oxidation and Inflammatory Responses

Chul Won Lee,¹Yong-Tae Ahn,¹Rongjie Zhao,²Youn Sook Kim,³Sang Mi Park,⁴Dae Hwa Jung,⁴Jae Kwang Kim,⁵Hyung Woo Kim,⁶Sang Chan Kim,⁴and Won G. An⁶

Academic Editor: Vincenzo De Feo

Received21 Oct 2020

Accepted26 Mar 2021

Published02 Apr 2021

Abstract

Porphyra tenera (laver) has long been a popular and traditional seaweed food in Korea, Japan, and China. Historically, it was known as a marine medicinal herb to treat hemorrhoids and cholera morbus in Donguibogam. We investigated the effects of P. tenera extract (PTE) for its antioxidant and anti-inflammatory activities. These activities were measured using assays for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging and its superoxide dismutase- (SOD-) like activity, and through the inhibitory production of inflammatory mediators (prostaglandin E₂ (PGE₂), NO, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6)) in lipopolysaccharide- (LPS-) stimulated Raw 264.7 cells. The antioxidant assay results showed that PTE displayed DPPH radical scavenging activity (46.44%), NO radical scavenging activity (67.14%), and SOD-like activity (80.29%) at a concentration of 5 mg/mL. In the anti-inflammatory assays, treatment with PTE (1 mg/mL) significantly inhibited expression levels of LPS-induced COX-2 and iNOS, as well as the production of PGE₂, NO, TNF-α, and IL-6. These results show that PTE has antioxidant and anti-inflammatory properties and provide scientific evidence to explain the antioxidative and anti-inflammatory properties of PTE.

1. Introduction

Inflammation is a host response to pathogen attack and is characterized by redness, heat, pain, and swelling. Over the long term, this response can lead to tissue damage and the pathogenesis of diverse disorders, such as atherosclerosis, asthma, and arthritis [1, 2]. Several inflammatory mediators are involved during an inflammatory response. Among them, COX-2, iNOS, and cytokines like IL-6 and TNF-α play significant roles and are considered as significant anti-inflammatory targets [3]. Moreover, when Raw 264.7 cells are activated, they produce reactive oxygen species (ROS), which cause oxidative stress. Oxidative stress is an inflammatory mediator that induces the release of nitric oxide (NO) and inflammatory cytokines [4]. NO is a radical made from L-arginine via NO synthase. NO contributes to the degeneration of inflammatory disorders, suppresses mitochondrial enzymes, and activates cyclooxygenases (COXs) to produce prostaglandins. In particular, the COX-2 enzyme is involved in the production of prostaglandin E₂ (PGE₂) [5, 6]. TNF-α is also a crucial mediator in the inflammatory response that leads to innate immune responses via the release of other inflammatory cytokines [7]. Conversely, IL-6 is produced by macrophages and is an important inflammatory cytokine in the acute phase response [8]. Accordingly, an inhibitor of NO, COX-2, ROS, and inflammatory cytokines is a crucial target for the treatment and prevention of inflammatory diseases.

Porphyra tenera (laver), a type of red algae (phylum: Rhodophyta, class: Bangiophyceae, order: Bangiales), has long been a popular and traditional seaweed food in Korea, Japan, and China [9]. P. tenera is rich in protein, carbohydrates, minerals, and vitamins and low in calories. Unlike other sea algae, P. tenera contains many free sugars, such as the major carbohydrates isofloridoside and floridoside. It also contains dietary fiber, including hemicellulose, which is a cell wall component and an insoluble polysaccharide [10, 11]. Moreover, P. tenera contains diverse inorganic and organic substances, including tocopherols, carotenoids, and polyphenols [12]. Importantly, P. tenera reportedly functions as a marine medicinal herb for the treatment of hemorrhoids and cholera morbus in Donguibogam [13]. It is reported that P. tenera functions as an antioxidant [14], has anti-inflammatory activities [15], and exerts antimutagenic effects [16]. Furthermore, Song et al. [17] reported that P. tenera extract (PTE) activates the immune response in mouse Raw 264.7 cells via NF-κB signaling. However, the molecular mechanisms underlying the antioxidative and anti-inflammatory activities of PTE remain unknown. In the present study, we examined the antioxidant and anti-inflammatory profile of PTE. Our data provide a basis for understanding the mechanisms underlying the inhibitory effects of PTE on oxidation and inflammatory responses.

2. Materials and Methods

2.1. Chemicals and Reagents

Two reference standards, chlorogenic acid and palmitic acid, were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). The purity of these reference standards was greater than 98%. Ultra-high-performance liquid chromatography- (UPLC-) grade solution, acetonitrile, methanol, and other reagents were purchased from J. T. Baker Chemical Company (Phillipsburg, NJ, USA). Anti-COX-2 and peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). In addition, anti-β-actin and anti-iNOS antibodies were obtained from Calbiochem (San Diego, USA.). The enzyme-linked immunoassay kit of PGE₂ was obtained from R&D Systems (Minneapolis, USA), and the TNF-α and IL-6 enzyme immunosorbent assay kits were purchased from Pierce Endogen (Rockford, USA). MTT, LPS, sulfanilamide, L-N⁶-(1-Iminoethyl)lysine (L-NIL), NS-398, and all other chemicals were purchased from the Sigma Chemical Co. (St. Louis, MO, USA).

2.2. Preparation of PTE

P. tenera was purchased from Jindoherb Co. (Jindo, Jeollanam-do, Korea). The voucher specimens (Porphyra tenera, PNU10-150) have been deposited into the Herbarium of Ducom in the Korean Medicine, Pusan National University, South Korea. P. tenera (50 g) was extracted with 3 liters of boiling purified water for 3 h long and filtered via a filter paper (Advantec No. 2 Filter Paper; Advantec Toyo Kaisha, Ltd., Tokyo, Japan). The filtrate was then lyophilized in a freeze drier (Ilshin, Seoul, Korea). The yield of lyophilized PTE was 2.32%. The lyophilized PTE powder was dissolved in purified water and filtered via a 0.22 μm filter (Nalgene, USA) prior to use.

2.3. Profiling the Chemical Contents of PTE by UPLC

2.3.1. Chromatography Conditions

We applied an UPLC (Waters Corp., Milford, USA), supplied with a Waters pump ACQUITY™ UltraPerformance LC system (Waters Corp.) and a Waters ACQUITY™ photodiode array (PDA) detector, for the analyses. The Empower Chromatography Data System (Waters Corp.) was used to record the output signal from the detector and a Waters ACQUITY™ BEH C₁₈ column (1.7 μm, 2.1 × 100) was used for separation of the products. The mobile stage was constituted of acetonitrile and water with a gradient system (0.4 mL per min). The volume for injection was 2 μL. The UV wavelength for detection was set up at 280 nm. The temperature for the column was set up at 22–25°C.

2.3.2. Preparation of the Standard Solutions and Samples

Standard stock solutions of the marker components, chlorogenic acid, and palmitic acid, were prepared by dissolving them at a concentration of 1 mg/mL in 10 mL methanol. Working solutions were produced by diluting the stock solution of standard with methanol. The standard stock solutions and working solutions were stored at 4°C. For the sample preparation, the PTE was dissolved in methanol (10 mg/mL). Before UPLC, the sample was filtered via a 0.22 μm filter.

2.4. Antioxidant Assays

2.4.1. DPPH Radical Scavenging Activity Assay

Electron donating ability was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) by the method of Blois [18]. Briefly, 100 μL of DPPH solution (0.4 mM in ethanol) was added to 100 μL of PTE (dissolved in ethanol) at concentrations of 0.1–5 mg/mL. Ethanol was used as the control for the experiments. The mixture was incubated for 15 min at 22–25°C. The optical density was measured at 517 nm by a microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Ascorbic acid was applied as a positive control. The capacity to scavenge the DPPH radical was calculated by the subsequent formula:where absorbance_control and absorbance_treatment are the absorbance of the control and the treatment, respectively.

2.4.2. NO Radical Scavenging Activity Assay

NO radical scavenging activity was measured according to Kato et al. [19]. Briefly, 40 μL of each sample concentration was added to 20 μL of 1 mM NaNO₂ solution, followed by the addition of 140 μL of 0.1 N HCl (pH 1.2). The mixture was allowed to react at 37°C for 1 h. Next, 40 μL of the reaction mixture was added to 200 μL of 2% acetic acid, followed by mixing with 16 μL of Griess reagent. After incubating at 22–25°C for 15 min, the optical density was measured at 520 nm by a microplate reader (Tecan Group Ltd.). Ascorbic acid was applied as a positive control. The NO radical scavenging activity was calculated as (%) = (1 − (A − B)/C) × 100, where A is the optical density of the sample without Griess reagent, B is the absorbance of the sample with Griess reagent, and C is the absorbance of the control.

2.4.3. SOD-like Activity Assay

The SOD-like activity was evaluated by determining the amount of pyrogallol needed to catalyze the conversion to H₂O₂, based on Marklund and Marklund [20]. The reaction mixture contained 20 μL of the sample (10 mg/mL) and 300 μL of 50 mM Tris-HCl buffer (pH 8.5), which were mixed with 10 mM EDTA and 20 μL of 7.2 mM pyrogallol. The mixture was incubated at room temperature for 10 min, and then the reaction was blocked by adding 10 μL 1 N HCl. The optical density was measured at 420 nm by a microplate reader (Tecan Group Ltd.). Ascorbic acid was applied as a positive control. The SOD-like activity was calculated with the following formula:where absorbance_control and absorbance_treatment are the absorbance of the control and the treatment, respectively.

2.5. Cell Culture

Raw 264.7 cells (ATCC, Manassas, USA) were maintained in DMEM (Hyclone; Thermo Fisher Scientific, Waltham, USA) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Aldrich Chemical Co.), 100 μg/mL of streptomycin, and 100 U/mL of penicillin (Gibco-BRL, Grand Island, USA) in a 5% CO₂ incubator at 37°C.

2.6. MTT Assay for Cell Viability

We followed the methods of Park et al. [21]. Briefly, to determine the cytotoxic concentration of PTE, Raw 264, 7 cells were planted in 96 wells (5 × 10⁴ cells per well). Cells were serum-starved for 16 h, treated with various concentrations of PTE for 1 h, induced by 1 μg/mL of LPS, and then the cells were incubated for 20 h at 37°C in an incubator with 5% CO₂. Following incubation, cells were stained with MTT at the concentration of 0.5 mg/mL for 4 h, and then the media were eliminated and the formazan produced was dissolved by adding 200 μL DMSO. Optical density was measured at 570 nm by an ELISA plate reader (Tecan Group Ltd.). Cell viability was described relative to the untreated control cells, where viability (% control) = 100 × (optical density of treated sample)/(optical density of control).

2.7. PGE₂ and Cytokines (TNF-α and IL-6) Assays

Cells (Raw 264.7 macrophage, 5 × 10⁵ cells/mL) were incubated for 16 h. The cells were then treated with various concentrations of PTE or with a positive control for the production of PGE₂ (NS-398) for 1 h, followed by stimulation with 1 μg/mL of LPS. At 20 h after LPS stimulation the culture supernatants were collected and ELISA was performed according to the manufacturer’s protocol to quantify the amounts of PGE₂, TNF-α, and IL-6 (PGE₂, R&D Systems; TNF-α and IL-6, Pierce Endogen).

2.8. Measurement of NO Production

The Raw 264.7 cells (5 × 10⁵ cells/mL) were incubated for 16 h, after which the cells were pretreated with various concentrations of PTE or with a positive control (L-NIL) for 1 h and induced by LPS (1 μg/mL). Next, the cells were incubated for 20 h at 37°C in a 5% CO₂ incubator, after which the culture supernatants were collected. NO was measured by adding 100 μL of Griess reagent (0.1% N-[1-naphthy]-ethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid; Roche, Switzerland) to the culture supernatant (100 μL) for 15 min at 22–25°C in the dark. Optical density was determined at 540 nm by an ELISA plate reader (Tecan Group Ltd.). A standard curve was generated similar to that of NaNO₂.

2.9. Western Blot Analysis

Control and PTE-treated Raw 264.7 cells were harvested by centrifugation and washed twice with phosphate-buffered saline (PBS). Washed pellets of cells were resuspended in lysis buffer for extraction (0.5 mM dithiothreitol, 5 mM EDTA, 250 mM NaCl, 5 mM NaF, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM sodium orthovanadate, and 50 mM HEPES (pH 7.0)) containing 5 μg/mL each of aprotinin and leupeptin and incubated at 4°C for 20 min. Microcentrifugation was performed to remove the cell debris, followed by rapid freezing of the supernatant. Bio-Rad protein assay reagent was used to measure the protein concentrations by the manufacturer’s instructions. Then, the cellular proteins (30 μg) from cell extracts were separated on 8% sodium dodecyl sulfate–polyacrylamide gels, followed by electroblotting onto nitrocellulose membranes. The membranes were incubated overnight, shaking with 5% skim milk at 4°C and subsequently with primary antibody (2 h). The blots were then washed five times with tween 20/tris-buffered saline (TTBS), incubated for 1 h, shaking with a 1 : 1000 dilution of horseradish peroxidase-conjugated secondary antibody at 22–25°C, and then rewashed three times with TTBS. ECL™ Western Blot Reagents for Detection (Amersham Biosciences, USA) were used to develop the blots.

2.10. Statistical Analysis

All data were recorded as means ± standard deviation (SD). The data were estimated by one-way analysis of variance (ANOVA) tests, Dunnett’s tests, and independent t-tests. SPSS for Windows (release 25.0 K, SPSS Inc., USA) was used for all statistical analyses. Differences were considered significant at < 0.05.

3. Results

3.1. PTE Analysis

UPLC was used to identify two PTE markers: chlorogenic acid and palmitic acid. Their contents were calculated from the standards calibration curve (Figure 1 and Table 1). Validation of the method proved its stability and reliability. UPLC resulted in the successive separation of the two marker components in PTE.

(a)

(b)

3.2. Antioxidant Assay

3.2.1. DPPH Scavenging Activity

Antioxidative activity of PTE was determined using DPPH radicals to ascertain the free radical scavenging activity (0.1–5 mg/mL). The DPPH radical scavenging activity increased significantly as the concentration of PTE increased ( < 0.05, Figure 2(a)). PTE displayed scavenging activities of 18.71% and 46.44% at concentrations of 1 and 5 mg/mL, respectively. Although the DPPH radical scavenging activity of PTE at 5 mg/mL reached 46%, that of the reference compound, ascorbic acid displayed more scavenging activity (∼91%) than PTE at concentrations of 0.1–5 mg/mL.

(a)

(b)

(c)

3.2.2. NO Radical Scavenging Activity

Figure 2(b) presents the NO radical scavenging potential of PTE alongside that of ascorbic acid as the positive control. PTE exhibited scavenging activities of 50.01% and 67.14% at concentrations of 1 and 5 mg/mL ( < 0.05), respectively, whereas ascorbic acid exhibited scavenging activities of 96.09% and 97.06% at concentrations of 1 and 5 mg/mL, respectively.

3.2.3. SOD-like Activity

PTE at concentrations of 0.1–5 mg/mL exhibited a slight dose-dependent effect in terms of SOD-like activity (Figure 2(c)). PTE exhibited SOD-like activities of 79.84% and 80.29% at 1 and 5 mg/mL, respectively. The reference compound ascorbic acid exhibited SOD-like activities of 52.53% and 88.85% at 1 and 5 mg/mL, respectively ( < 0.05).

3.3. Inhibitory Effects of PTE on LPS-Induced Production of NO and PGE₂

Different concentrations of PTE (0.25–1 mg/mL) were used to evaluate the inhibitory effects of PTE on LPS-stimulated production of NO and PGE₂ in Raw 264.7 cells. Compared to the control, treatment with LPS resulted in significantly increased NO production. However, treatment with L-NIL (10 μM), a positive control, significantly reduced the production ( < 0.001) of LPS-induced NO. In addition, treatment with PTE resulted in significantly reduced production of LPS-induced NO at concentrations of 0.5 and 1 mg/mL ( < 0.05 for 0.5 mg/mL, < 0.01 for 1 mg/mL; Figure 3(a)). Furthermore, we observed the same inhibitory effects of PTE on LPS-induced PGE₂ production (Figure 3(b)). Compared to the control, treatment with LPS resulted in significantly increased PGE₂ production. However, treatment with NS-398 (10 μM), a positive control, significantly reduced the production ( < 0.001) of LPS-induced PGE₂. Moreover, treatment with PTE resulted in significantly reduced LPS-induced PGE₂ production at a concentration of 1 mg/mL ( < 0.01 for 1 mg/mL; Figure 3(b)). Thus, PTE exhibited an inhibitory effect on the induction of NO and PGE₂ in Raw 264.7 cells.

(a)

(b)

Figure 3

Inhibitory effects of PTE on LPS-induced production of NO (a) and PGE₂ (b) in Raw 264.7 cells. Cells (5 × 10⁵ cells/mL) were treated with 0.25, 0.5, or 1 mg/mL PTE for 1 h followed by continuous incubation with 1 μg/mL LPS for the next 20 h L-N⁶-(1-iminoethyl)lysine (L-NIL) and NS-398 (10 μM each) were used as positive controls, respectively. Concentrations of NO and PGE₂ in the culture medium were monitored as described in methods. Data show the means ± SD from three experiments. < 0.01 compared with the control; < 0.001, < 0.01, and < 0.05 compared with LPS alone.

3.4. Cell Viability

We used the MTT assay to test for possible cytotoxic effects of PTE in Raw 264.7 cells. There were no changes in cell viability after exposure to 0.25, 0.5, and 1 mg/mL PTE (Figure 4), indicating that PTE displayed no cell toxicity.

3.5. Inhibitory Effects of PTE on LPS-Stimulated Expression of COX-2 and iNOS

Western blot analysis was carried out to determine whether the effects of PTE against PGE₂ and NO production were related to modulation of COX-2 and iNOS. There were marked increases in the levels of COX-2 and iNOS proteins in response to LPS (Figure 5). PTE (1 mg/mL) showed significant suppression of LPS-stimulated COX-2 protein levels ( < 0.01 for 1 mg/mL). In addition, treatment with PTE (0.5 and 1 mg/mL) resulted in significant inhibition of LPS-stimulated iNOS protein levels ( < 0.05 for 0.5 mg/mL, < 0.01 for 1 mg/mL). These data confirmed the inhibitory effects of PTE on the production of PGE₂ and NO in LPS-induced Raw 264.7 macrophage cells.

Figure 5

Inhibitory effects of PTE on LPS-stimulated expression of COX-2 and iNOS. Raw 264.7 macrophage cells (5 × 10⁵ cells/mL) were treated with PTE (0.25–1 mg/mL) for 1 h followed by continuous incubation with 1 μg/mL LPS for the next 20 h. The cells of control were incubated with the vehicle only. Western blot analysis was carried out to determine the COX-2 and iNOS protein levels. β-Actin was used as a control. The blots are representative results of three blots. COX-2 and iNOS versus β-actin were determined through densitometry. Data show the means ± SD from three experiments. < 0.01 between the control and LPS-treated cells; < 0.05 and < 0.01 between the LPS-treated cells with, or without PTE.

3.6. Inhibitory Effects of PTE on LPS-Stimulated TNF-α and IL-6 Production

We performed enzyme immunoassays to evaluate the effects of PTE on the LPS-inducible production of TNF-α and IL-6. Compared to the control, LPS treatment showed a significant increase of TNF-α and IL-6 in the culture supernatants of Raw 264.7 macrophage cells ( < 0.01). However, treatment with 1 mg/mL PTE showed significant inhibition in LPS-induced TNF-α and IL-6 production (Figures 6(a) and 6(b)). These findings indicate that PTE might inhibit the expression of the specific genes involved in the inflammation response, such as TNF-α and IL-6.

(a)

(b)

Figure 6

Inhibitory effects of PTE on LPS-stimulated TNF-α (a) and IL-6 (b) production in Raw 264.7 macrophage cells. Cells (5 × 10⁵ cells/mL) were treated with PTE (0.25–1 mg/mL) for 1 h followed by continuous incubation with 1 μg/mL LPS for the next 20 h TNF-α and IL-6 concentrations in the culture supernatants were recorded as described in Section 2. Data show the means ± SD from three experiments. < 0.01 compared to the control; < 0.05 compared with LPS alone.

4. Discussion

P. tenera has long been a popular and traditional seaweed food in Korea, Japan, and China [9]. It is also reported to have antioxidative [14] and anti-inflammatory effects [15]. However, there is little scientific evidence to demonstrate the effects of PTE. Consequently, we examined the molecular mechanisms underlying the antioxidative and anti-inflammatory effects of PTE.

ROS consist of hydroxyl radicals, peroxynitrite, singlet oxygen, peroxyl radicals, and superoxide, which cause oxidative stress, leading to cellular damage [22]. Identifying the free radical-quenching abilities and antioxidant activities of antioxidant compounds sourced from plants is essential [23]. In the present study, we used DPPH [18] and NO [19] radical scavenging, as well as SOD-like activity [20], assays to estimate the antioxidant activity of PTE. DPPH is commonly used in antioxidant assays [24]; in this study, the DPPH radical scavenging activity was significantly increased as the concentration of PTE increased. PTE had a DPPH radical scavenging activity of 46.44% at a concentration of 5 mg/mL; however, the reference compound, ascorbic acid, showed higher scavenging activity (91.53%) compared to PTE at a concentration of 5 mg/mL. NO is unstable in the aerobic state; it reacts with O₂ to make stable nitrite and nitrate products via the N₃O₄, N₂O₄, and NO₂ intermediates [25]. Our results indicated that 5 mg/mL PTE exhibited a NO radical scavenging activity of 67.14%, whereas 5 mg/mL ascorbic acid showed a scavenging activity of 97.06%. SOD is a significant antioxidant enzyme catalyzing the conversion of the superoxide radical into H₂O₂ and O₂ [26]. We measured the SOD-like activity of PTE at different concentrations (0.1–5 mg/mL) and observed slight dose-dependent responses. At 5 mg/mL, PTE showed a SOD-like activity of 80.29%, whereas 5 mg/mL ascorbic acid exhibited a SOD-like activity of 88.85% ( < 0.05). Collectively, these results suggest that the effects of PTE on the DPPH, NO radical scavenging, and SOD-like activities might have important implications for strategies used to manage pathological stress.

In wounds, inflammation is associated with microbiological toxins or chemicals [1]. LPS stimulates the production of the NO, PGE₂, and cytokines by initiating the NF-κB transcription factor in Raw 264.7 cells [27–29]. The inflammatory cytokines TNF-α and IL-6 are involved in various immunological interactions and reactions with diverse target cells [6, 7, 30]. In this study, we evaluated the inhibitory effects of PTE on LPS-stimulated TNF-α and IL-6 by ELISA, which revealed that PTE (1 mg/mL) significantly inhibited LPS-induced levels of cytokines (TNF-α and IL-6). Thus, the inhibitory effects of PTE on inflammatory cytokines could form the basis for treatments of pathological inflammation. Moreover, iNOS causes damage to cells via NO production in macrophages stimulated by LPS [31]. In this study, PTE significantly suppressed LPS-induced iNOS protein expression and NO production.

Furthermore, treatment with L-NIL (10 μM), a positive control, significantly reduced the production of LPS-induced NO. In particular, the NO production value for treatment with PTE (1 mg/mL) was somewhat higher than that of the L-NIL. This finding indicates that PTE, similar to the L-NIL, can inhibit NO production. In addition, these results suggest that the preventive effects of PTE on the inflammatory response are due, in part, to its suppression of iNOS expression and NO production. Furthermore, PGE₂, made via COX-2 from arachidonic acid, plays crucial regulatory roles in the inflammatory responses and in brain injuries [32]. PGE₂ is released from blood vessel walls in response to inflammation or infection to induce fever [33]. Therefore, inhibition of PGE₂ is useful to help identify beneficial plant extracts that have anti-inflammatory properties [34, 35]. Our data showed that PTE resulted in significant inhibition of PGE₂ and the COX-2 protein induced by LPS. Moreover, treatment with NS-398 (10 μM), a positive control, significantly reduced the production of LPS-induced PGE₂. In particular, the PGE₂ production value for treatment with PTE (1 mg/mL) was higher than that of the NS-398. This result shows that PTE can weakly inhibit PGE₂ production compared to the NS-398. Therefore, these findings suggest that PTE might have preventive and therapeutic effects in the treatment of pathogenic pain, heat, and inflammation.

PTE is an aqueous extract of the red algae P. tenera. Zhang et al. [36] reported that the sulfated galactan fraction isolated from the red seaweed Porphyra haitanensis had significant in vivo antioxidant activity in aging mice. In addition, Senevirathne et al. [37] showed that enzymatic extracts from P. tenera effectively inhibited LPS-stimulated production of NO in Raw 264.7 macrophage cells. Furthermore, the red algae Porphyra yezoensis reportedly showed high antitumor activity against Ehrlich carcinoma [38]. According to Jung et al. [39], high-performance liquid chromatography showed that PTE contained Porphyra-334. However, our chromatographic results revealed that the main markers of PTE were chlorogenic acid and palmitic acid (Table 1 and Figure 1). Chlorogenic acid, an abundant polyphenol compound, possesses multiple biological activities, including anti-inflammation, immunomodulation, antivirus, and cardiovascular protection [40–42]. In addition, chlorogenic acid reportedly has a protective effect on myocardial infarction via its antioxidant activity and reducing the inflammatory response [43]. Meanwhile, palmitic acid is a saturated fatty acid discovered in microorganisms, plants, and animals. It is naturally produced by a wide range of plants and organisms, but typically at low levels [44]. From the findings from this study, we suggest that the antioxidative and anti-inflammatory activities of PTE are likely due to chlorogenic acid.

5. Conclusion

We showed that PTE has antioxidative and anti-inflammatory properties using DPPH, NO radical scavenging, and SOD-like activity assays and that it acts through the inhibitory actions of inflammatory mediators (NO, PGE₂, TNF-α, and IL-6) in LPS-induced Raw 264.7 macrophage cells. Our results provide scientific evidence that explains the antioxidant and anti-inflammatory properties of PTE.

Data Availability

The data justifying the conclusions of this study are all statistically analyzed and presented in the Results section and are also available from the corresponding authors.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

CWL and SCK contributed to the design and supervision of this study. YTA, YSK, and SMP participated in the in vitro studies. Experimental and laboratory analysis was carried out by RZ, DHJ, and JKK. RZ performed the statistical analysis. The manuscript was written by CWL and WGA. The editing of the manuscript was done by WGA, SCK, HWK, and CWL. All authors approved and read the final version of this manuscript. Chul Won Lee and Yong-Tae Ahn contributed equally to this work.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (no. 2018R1A6A3A11051245) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (no. 2018R1A5A2025272).

References

T. J. Guzik, R. Korbut, and T. Adamek-Guzik, “Nitric oxide and superoxide in inflammation and immune regulation,” Journal of Physiology and Pharmacology: An Official Journal of the Polish Physiological Society, vol. 54, no. 4, pp. 469–487, 2003.
View at: Google Scholar
F. Runjic, D. Martinovic-Kaliterna, I. Salamunic, I. Kristic, M. Ljubkovic, and J. Marinovic, “Association of anticardiolipin antibodies, complement and leptin with the severity of coronary artery disease expressed as syntax score,” Journal of Physiology and Pharmacology, vol. 71, 2020.
View at: Publisher Site | Google Scholar
T. Lawrence, D. A. Willoughby, and D. W. Gilroy, “Anti-inflammatory lipid mediators and insights into the resolution of inflammation,” Nature Reviews Immunology, vol. 2, no. 10, pp. 787–795, 2002.
View at: Publisher Site | Google Scholar
C. S. Nworu and P. A. Akah, “Anti-inflammatory medicinal plants and the molecular mechanisms underlying their activities,” African Journal of Traditional, Complementary and Alternative Medicines, vol. 12, no. 6, pp. 52–61, 2015.
View at: Publisher Site | Google Scholar
S. Cuzzocrea, D. P. Riley, A. P. Caputi, and D. Salvemini, “Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury,” Pharmacological Reviews, vol. 53, no. 1, pp. 135–159, 2001.
View at: Google Scholar
J. Y. Park, M. H. Pillinger, and S. B. Abramson, “Prostaglandin E2 synthesis and secretion: the role of PGE2 synthases,” Clinical Immunology, vol. 119, no. 3, pp. 229–240, 2006.
View at: Publisher Site | Google Scholar
B. Beutler and A. Cerami, “The biology of cachectin/TNF—a primary mediator of the host response,” Annual Review of Immunology, vol. 7, no. 1, pp. 625–655, 1989.
View at: Publisher Site | Google Scholar
A. Yoshimura, “Signal transduction of inflammatory cytokines and tumor development,” Cancer Science, vol. 97, no. 6, pp. 439–447, 2006.
View at: Publisher Site | Google Scholar
E.-J. Lee, G.-R. Kim, H.-J. Lee, and J.-H. Kwon, “Monitoring microbiological contamination, pre-decontamination, and irradiation status of commercial dried laver (Porphyra sp.) products,” Korean Journal of Food Science and Technology, vol. 49, no. 1, pp. 20–27, 2017.
View at: Publisher Site | Google Scholar
K.-W. Kim, J.-H. Hwang, M.-J. Oh, M.-Y. Kim, M.-R. Choi, and W.-M. Park, “Studies on the major nutritional components of commercial dried lavers (Porphyra yezoensis) cultivated in Korea,” Korean Journal of Food Preservation, vol. 21, no. 5, pp. 702–709, 2014.
View at: Publisher Site | Google Scholar
J. Cao, J. Wang, S. Wang, and X. Xu, “Porphyra species: a mini-review of its pharmacological and nutritional properties,” Journal of Medicinal Food, vol. 19, no. 2, pp. 111–119, 2016.
View at: Publisher Site | Google Scholar
S. Chanda, R. Dave, M. Kaneria, and K. Nagani, “Seaweeds: a novel, untapped source of drugs from sea to combat infectious diseases,” in Current Research, Technology and Education Topics in Applied Microbiology and Microbial, pp. 473–480, Formatex Research Center, Badajoz, Spain, 2010.
View at: Google Scholar
J. Heo, Donguibogam, Bubin-Munwhasa, Seoul, South Korea, 1999.
N. Lee and K. Oh, “Screening of radical scavenging effects from marine algae,” Cheju Journal of Life Science, vol. 3, pp. 95–101, 2000.
View at: Google Scholar
J. Y. Lee, J. W. Choi, M. K. Lee, Y. M. Kim, I. H. Kim, and T. J. Nam, “Anti-inflammatory effects of Pyropia yezoensis extract in LPS-stimulated raw 264.7 cells,” Korean Journal of Fisheries and Aquatic Sciences, vol. 47, no. 6, pp. 757–764, 2014.
View at: Publisher Site | Google Scholar
M. L. Cornish and D. J. Garbary, “Antioxidants from macroalgae: potential applications in human health and nutrition,” Algae, vol. 25, no. 4, pp. 155–171, 2010.
View at: Publisher Site | Google Scholar
J.-H. Song, H.-B. Kang, S.-H. Park et al., “Extracts of porphyra tenera (nori seaweed) activate the immune response in mouse RAW264.7 macrophages via NF-κB signaling,” Journal of Medicinal Food, vol. 20, no. 12, pp. 1152–1159, 2017.
View at: Publisher Site | Google Scholar
M. S. Blois, “Antioxidant determinations by the use of a stable free radical,” Nature, vol. 181, no. 4617, pp. 1199-1200, 1958.
View at: Publisher Site | Google Scholar
H. Kato, I. E. Lee, N. Van Chuyen, S. B. Kim, and F. Hayase, “Inhibition of nitrosamine formation by nondialyzable melanoidins,” Agricultural and Biological Chemistry, vol. 51, no. 5, pp. 1333–1338, 1987.
View at: Publisher Site | Google Scholar
S. Marklund and G. Marklund, “Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase,” European Journal of Biochemistry, vol. 47, no. 3, pp. 469–474, 1974.
View at: Publisher Site | Google Scholar
S. M. Park, J. K. Kim, E. O. Kim et al., “Hepatoprotective effect of Pericarpium zanthoxyli extract is mediated via antagonism of oxidative stress,” Evidence-Based Complementary and Alternative Medicine, vol. 2020, Article ID 6761842, 15 pages, 2020.
View at: Publisher Site | Google Scholar
H. Bayr, “Reactive oxygen species,” Critical Care Medicine, vol. 33, pp. S498–S501, 2005.
View at: Publisher Site | Google Scholar
Y. Y. Lee, E. Saba, M. Kim, M. H. Rhee, and H.-K. Kim, “Antioxidant and anti-inflammatory properties of raw and processed fruits and vegetables,” Biomedical Science Letters, vol. 24, no. 3, pp. 196–205, 2018.
View at: Publisher Site | Google Scholar
O. P. Sharma and T. K. Bhat, “DPPH antioxidant assay revisited,” Food Chemistry, vol. 113, no. 4, pp. 1202–1205, 2009.
View at: Publisher Site | Google Scholar
A. Patel, A. Patel, A. Patel, and N. Patel, “Determination of polyphenols and free radical scavenging activity of Tephrosia purpurea linn leaves (Leguminosae),” Pharmacognosy Research, vol. 2, no. 3, pp. 152–158, 2010.
View at: Publisher Site | Google Scholar
C. Y. Kim, Q. M. Yu, H. J. Kong, J. Y. Lee, K. M. Yang, and J. S. Seo, “Antioxidant and anti-inflammatory activities of Agrimonia pilosa Ledeb. extract,” Evidence-Based Complementary and Alternative Medicine, vol. 2020, Article ID 8571207, 10 pages, 2020.
View at: Publisher Site | Google Scholar
A. K. Lee, S. H. Sung, Y. C. Kim, and S. G. Kim, “Inhibition of lipopolysaccharide-inducible nitric oxide synthase, TNF-α and COX-2 expression by sauchinone effects on I-κ Bα phosphorylation, C/EBP and AP-1 activation,” British Journal of Pharmacology, vol. 139, no. 1, pp. 11–20, 2003.
View at: Publisher Site | Google Scholar
M. Higuchi, N. Higashi, H. Taki, and T. Osawa, “Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages,” Journal of Immunology (Baltimore, Md.: 1950), vol. 144, no. 4, pp. 1425–1431, 1990.
View at: Google Scholar
M. G. Rittig, A. Kaufmann, A. Robins et al., “Smooth and rough lipopolysaccharide phenotypes ofBrucellainduce different intracellular trafficking and cytokine/chemokine release in human monocytes,” Journal of Leukocyte Biology, vol. 74, no. 6, pp. 1045–1055, 2003.
View at: Publisher Site | Google Scholar
A. V. Delgado, A. T. McManus, and J. P. Chambers, “Production of tumor necrosis factor-alpha, interleukin 1-beta, interleukin 2, and interleukin 6 by rat leukocyte subpopulations after exposure to substance P,” Neuropeptides, vol. 37, no. 6, pp. 355–361, 2003.
View at: Publisher Site | Google Scholar
L. Tomé, L. Yu, I. de Castro et al., “Beneficial and harmful effects of L-arginine on renal ischaemia,” Nephrology Dialysis Transplantation, vol. 14, no. 5, pp. 1139–1145, 1999.
View at: Publisher Site | Google Scholar
J. Zhang and S. Rivest, “Anti-inflammatory effects of prostaglandin E2 in the central nervous system in response to brain injury and circulating lipopolysaccharide,” Journal of Neurochemistry, vol. 76, no. 3, pp. 855–864, 2001.
View at: Publisher Site | Google Scholar
K. Nakatani, M. Atsumi, T. Arakawa et al., “Inhibitions of histamine release and prostaglandin E2 synthesis by mangosteen, a Thai medicinal plant,” Biological and Pharmaceutical Bulletin, vol. 25, no. 9, pp. 1137–1141, 2002.
View at: Publisher Site | Google Scholar
C. H. Jung, J.-H. Kim, S. Park, D.-H. Kweon, S.-H. Kim, and S.-G. Ko, “Inhibitory effect of Agrimonia pilosa Ledeb. On inflammation by suppression of iNOS and ROS production,” Immunological Investigations, vol. 39, no. 2, pp. 159–170, 2010.
View at: Publisher Site | Google Scholar
M. M. Mohy El-Din, A. M. Senbel, A. A. Bistawroos et al., “A novel COX-2 inhibitor pyrazole derivative proven effective as an anti-inflammatory and analgesic drug,” Basic & Clinical Pharmacology & Toxicology, vol. 108, no. 4, pp. 263–273, 2011.
View at: Publisher Site | Google Scholar
Q. Zhang, N. Li, X. Liu, Z. Zhao, Z. Li, and Z. Xu, “The structure of a sulfated galactan from Porphyra haitanensis and its in vivo antioxidant activity,” Carbohydrate Research, vol. 339, no. 1, pp. 105–111, 2004.
View at: Publisher Site | Google Scholar
M. Senevirathne, C.-B. Ahn, and J.-Y. Je, “Enzymatic extracts from edible red algae, Porphyra tenera, and their antioxidant, anti-acetylcholinesterase, and anti-inflammatory activities,” Food Science and Biotechnology, vol. 19, no. 6, pp. 1551–1557, 2010.
View at: Publisher Site | Google Scholar
H. Noda, H. Amano, K. Arashima, and K. Nisizawa, “Antitumor activity of marine algae,” Hydrobiologia, vol. 204-205, no. 1, pp. 577–584, 1990.
View at: Publisher Site | Google Scholar
S. J. Jung, H. Y. Jang, E. S. Jung et al., “Effects of Porphyra tenera supplementation on the immune system: a randomized, double-blind, and placebo-controlled clinical trial,” Nutrients, vol. 12, p. 1642, 2020.
View at: Google Scholar
M. Naveed, V. Hejazi, M. Abbas et al., “Chlorogenic acid (CGA): a pharmacological review and call for further research,” Biomedicine & Pharmacotherapy, vol. 97, pp. 67–74, 2018.
View at: Publisher Site | Google Scholar
R. Gao, H. Yang, S. Jing et al., “Protective effect of chlorogenic acid on lipopolysaccharide-induced inflammatory response in dairy mammary epithelial cells,” Microbial Pathogenesis, vol. 124, pp. 178–182, 2018.
View at: Publisher Site | Google Scholar
M. D. dos Santos, M. C. Almeida, N. P. Lopes, and G. E. P. de Souza, “Evaluation of the anti-inflammatory, analgesic and antipyretic activities of the natural polyphenol chlorogenic acid,” Biological and Pharmaceutical Bulletin, vol. 29, no. 11, pp. 2236–2240, 2006.
View at: Publisher Site | Google Scholar
D. Wang, L. Tian, H. Lv et al., “Chlorogenic acid prevents acute myocardial infarction in rats by reducing inflammatory damage and oxidative stress,” Biomedicine & Pharmacotherapy, vol. 132, Article ID 110773, 2020.
View at: Publisher Site | Google Scholar
F. D. Gunstone, J. L. Harwood, and A. J. Dijkstra, The Lipid Handbook, CRC Press, Boca Raton, FL, USA, 3rd edition, 2007.

Copyright

Copyright © 2021 Chul Won Lee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Evidence-Based Complementary and Alternative Medicine

Inhibitory Effects of Porphyra tenera Extract on Oxidation and Inflammatory Responses

Abstract

1. Introduction

2. Materials and Methods

2.1. Chemicals and Reagents

2.2. Preparation of PTE

2.3. Profiling the Chemical Contents of PTE by UPLC

2.3.1. Chromatography Conditions

2.3.2. Preparation of the Standard Solutions and Samples

2.4. Antioxidant Assays

2.4.1. DPPH Radical Scavenging Activity Assay

2.4.2. NO Radical Scavenging Activity Assay

2.4.3. SOD-like Activity Assay

2.5. Cell Culture

2.6. MTT Assay for Cell Viability

2.7. PGE2 and Cytokines (TNF-α and IL-6) Assays

2.8. Measurement of NO Production

2.9. Western Blot Analysis

2.10. Statistical Analysis

3. Results

3.1. PTE Analysis

3.2. Antioxidant Assay

3.2.1. DPPH Scavenging Activity

3.2.2. NO Radical Scavenging Activity

3.2.3. SOD-like Activity

3.3. Inhibitory Effects of PTE on LPS-Induced Production of NO and PGE2

3.4. Cell Viability

3.5. Inhibitory Effects of PTE on LPS-Stimulated Expression of COX-2 and iNOS

3.6. Inhibitory Effects of PTE on LPS-Stimulated TNF-α and IL-6 Production

4. Discussion

5. Conclusion

Data Availability

Conflicts of Interest

Authors’ Contributions

Acknowledgments

References

Copyright

2.7. PGE₂ and Cytokines (TNF-α and IL-6) Assays

3.3. Inhibitory Effects of PTE on LPS-Induced Production of NO and PGE₂