Original papers
Enhanced radical scavenging activity by interactions between hyaluronan and antioxidants -A study of hyaluronan-containing drinks-
2022 Volume 28 Issue 2 Pages 159-168
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2022 Volume 28 Issue 2 Pages 159-168
Hyaluronan (HA) possesses radical scavenging properties. The low molecular weight HA (LHA)-containing drink (PL) is a dietary supplement that consists of several antioxidants in addition to LHA. The aim of this study is to characterize the hydroxyl radical scavenging activity of LHA through the interactions between the components of PL in in vitro assays. Moreover, the efficacy of LHA toward oxidative stresses (ultraviolet C irradiation, oxidative DNA damage) was evaluated. LHA revealed the following hydroxyl radical-scavenging properties: 1) LHA directly scavenges hydroxyl radicals, 2) LHA activity is enhanced in the presence of other PL components, thereby enabling protection against oxidative damage to DNA, and 3) exposure to UVC-radiation temporarily attenuated the antioxidant activity of PL, which is recovered in an LHA-dependent process. These results suggest that LHA is an excellent material because its antioxidative activity is enhanced in the presence of other antioxidants, which ultimately increases resistance to oxidative stress.
Reactive oxygen species (ROS) are generated in living organisms as a result of normal cellular metabolism (endogenous ROS) and environmental factors such as ultraviolet radiation or air pollutants (exogenous ROS). ROS are highly reactive molecules that have both beneficial and harmful effects on biomolecules. Low levels of ROS production are required to maintain physiological cell functions, including cell proliferation, signal transduction, and gene expression. In contrast, oxidative stress occurs when ROS generation exceeds the intrinsic antioxidant capacity of cells and tissues. Oxidative stress causes oxidative damage to cellular biomolecules including proteins, lipids, and nucleic acids, and consequently triggers the pathogenesis of many diseases, including age-related disorders, cancer, and cardiovascular diseases (Rahman, 2007). In addition, sunlight induces exogenous ROS and is a major cause of exogenous aging (photoaging) of facial skin tissues, and causes appearance changes, such as deep wrinkles, loss of elasticity, and pigmented spots (Uitto, 1997). Thus, in order to prevent oxidative damage to tissues, ROS generation is tightly regulated through several antioxidative defense mechanisms, including enzymes and antioxidants, which originate from both endogenous and exogenous sources. Three key enzymes, superoxide dismutase, glutathione peroxidase, and catalase, are endogenous antioxidants that neutralize ROS and decrease oxidative stress. However, these enzymatic activities decline with aging. Recently, antioxidants have been identified from natural products and food, and these natural antioxidants have been shown to protect cells from oxidative stress by a variety of mechanisms via the conversion of ROS to non-radical species, thereby halting the auto-oxidative chain reaction initiated by ROS. These antioxidants are utilized as medicines and dietary supplements because they are expected to compensate for deficits in endogenous antioxidative activity.
Hyaluronan (HA) is a high molecular weight (105–107 Da) molecule that is composed of repeating polymeric disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine linked by a glucuronide β (1→3) bond. HA is continuously secreted from fibroblasts, keratinocytes, chondrocytes, and other specialized cells throughout the body. Consequently, HA is a major component of the extracellular matrix in tissues, where it regulates cell adhesion and cellular motility. In humans, HA is ubiquitously distributed in the body and is most abundant in the skin, which accounts for 50% of total body HA (Papakonstantinou et al., 2012). HA has excellent properties due to its viscoelasticity and high moisture retention capacity (Jegasothy et al., 2014), which imparts a variety of physiological functions such as promoting wound healing, control of inflammation, and protection of articular cartilage. Due to its physiological properties, HA has been used as a medicinal agent for ophthalmic and arthritic therapies. In the body, HA is degraded by two main mechanisms, enzymatically by hyaluronidases or non-enzymatically via ROS. Excessive degradation of HA by ROS (resulting in depolymerization, decreased concentrations, and reduced molecular weight and viscosity) causes dysfunction of HA-containing tissues, such as in arthritis (Sato et al., 1988). HA possesses antioxidant activity, which imparts a resistance to ROS degradation that enables HA to maintain its biological properties, and also accelerates wound healing and reduces arthritis symptoms (Campo et al., 2003a; Presti and Scott 1994; Sato et al., 1988; Trabucchi et al., 2002). Therefore, HA has received much attention as a functional natural product with utility as an exogenous antioxidant. In addition, HA is a biomaterial with broad use in different dietary supplements and cosmetics due to its unique sensory properties (i.e. tasteless, odorless, and colorless). The origin and molecular weight of HA are chosen depending on its usage. HA derived from rooster combs is frequently used in pharmaceutical materials. Microbial fermentation is used to degrade HA with enzymes and yields low molecular weight fragments (LHA) for the purpose of improving absorption and skin penetration, and is therefore used mainly for cosmetics and dietary supplements. Although HA exhibits the same structure and properties regardless of its origin, it has been reported that there are differences in antioxidant activity associated with its molecular weight (Ke et al., 2011). Moreover, when HA is mixed with other components in a given material, the effect of HA on the functionality of the entire mixture requires clarification.
The LHA (M.W. = 2×105−5×105 Da) -containing drink (Melsmon® Platinum Liquid: PL™) is categorized as a dietary supplement and that is composed of multiple ingredients, including equine placental extract (EPE). The principal components of EPE are 18 kinds of free amino acids, which are present at a concentration of 836 mg/100 g per serving of PL (10 mL). Placental extracts (PE) such as EPE have been reported to possess antioxidant activity (Shinde et al., 2006). The main substances contributing to the antioxidant activity of PE are amino acids, peptides, and nucleic acids (Togashi et al., 2000; Togashi et al., 2002; Watanabe et al., 2002).
The main purpose of this study was to characterize the antioxidant properties of HA toward hydroxyl radicals, which are highly reactive cytotoxic species that attack the majority of biomolecules, and the interactions between the components of PL. The hydroxyl radical scavenging activity of LHA-containing PL was compared to that of LHA-free PL [PL (-)] to evaluate the contribution of LHA to the hydroxyl radical scavenging activity of PL. Additionally, LHA was combined with individual antioxidants present in PL, and their cumulative effects on hydroxyl radical scavenging were evaluated. The responsiveness of LHA to oxidative stress in the presence of other PL components was further examined using photochemically generated hydroxyl radicals in a solution under UVC (254 nm) irradiation (Hosoya et al., 2017). PL and PL (-) were exposed to UVC light, and the effect of UVC-induced ROS on antioxidant activity was evaluated over time. Moreover, the protective effect of LHA on oxidative stress-induced DNA degradation was evaluated using λ phage DNA (λ DNA) and hydroxyl radicals. The oxidation of ribose or deoxyribose by hydroxyl radicals leads to the dissociation of sugar-phosphate bonds and the eventual degradation of DNA. λ DNA is a double-stranded, linear molecule of 48 502 base pairs (48.5 kbp) that is cleaved into smaller DNA fragments upon oxidative damage. Structural changes to DNA are readily visualized as mobility transitions in agarose gel electrophoresis. The results of the present study indicate that LHA exhibits hydroxyl radical scavenging activity that is synergistically enhanced in the presence of other antioxidants (such as EPE). Thus, LHA provides beneficial antioxidant activity against toxic stimuli, such as eliminating UVC-induced ROS, thereby preventing oxidative DNA damage, making PL an excellent functional material.
Materials Hyaluronan from microbial fermentation (Kewpie Corporation, Tokyo, Japan), PL (Melsmon Pharmaceutical Co., Ltd., Tokyo, Japan), hydrogen peroxide (30%), iron (III) chloride hexahydrate, hyaluronic acid sodium salt (from rooster comb), edaravone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), isoluminol (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), microperoxidase (MP-11) sodium salt, ethidium bromide solution (Sigma-Aldrich, St. Louis, MO, USA), ascorbic acid (AsA; IWAKI SEIYAKU Co., Ltd., Tokyo, Japan), equine placental extract (Sankyo Biochemicals Co., Ltd., Tokyo, Japan), λ DNA, λ-Hind III digest, 6× loading buffer (Takara Bio Inc., Shiga, Japan), 2Na (EDTA/2Na) (DOJINDO LABORATORIES, Kumamoto, Japan), agarose KANTO LM, and L-type amino acid reagents employed for the preparation of amino acid mixture solutions containing 18 amino acids (KANTO CHEMICAL Co., Inc., Tokyo, Japan) were used for subsequent experiments.
Preparation of sample solutions in this study Just prior to use, PL was adjusted to pH 7.0 and then serially diluted (1/100–1/10) with distilled water. Preparation of control samples: AsA was dissolved in phosphate-buffered saline (PBS) at a concentration of 0.1 mol/L and adjusted to pH 7.0, then subsequently diluted with PBS to working concentrations. LHA and EPE were dissolved in distilled water and PBS, respectively. EPE and LHA were weighed and dissolved in PBS to prepare an EPE + LHA mixture.
Preparation of amino acid mixtures (18 a.a.) The eighteen amino acids contained in EPE were individually weighed, dissolved in distilled water, and adjusted to pH 7.0.
Preparation of edaravone solution for DNA degradation assays Edaravone was dissolved in a 1% acetic acid solution and adjusted to pH 7.0 to obtain a 1.5 mg/mL solution.
Reagent preparation An isoluminol/microperoxidase (mPOD) reagent solution was prepared as a 1:1 (v/v) mixture of 0.25 mmol/L isoluminol solution and 1 µmol/L mPOD in 0.8 mol/L carbonate buffer, pH 9.5. The isoluminol/mPOD mixture was stored at 4 °C until use.
Evaluation of hydroxyl radical scavenging activity by chemiluminescence The hydroxyl radical scavenging activity of samples was evaluated according to the method reported by Arakawa et al. (2015). The samples were diluted with distilled water for use in the assay. Briefly, 50 µL of the sample solution was mixed in a glass tube with 80 µL of PBS, 15 µL of 1×10−5 mol/L hydrogen peroxide, and 200 µL of the isoluminol/mPOD mixture. After a 10 s delay for mixing the sample solution, luminescence intensity was measured for 10 s with a luminescence reader (AccuFLEX Lumi 400, Hitachi, Ltd., Tokyo, Japan). For the control condition, distilled water was added to the reaction mixture instead of the sample. Subsequently, the hydroxyl radical scavenging activity was calculated using the following equation (Eq. 1):
 |
where As and Ac correspond to the luminescence intensity in the presence and absence of antioxidant, respectively. IC50 (inhibitory concentration 50%) values, defined as the concentration of sample that is required to scavenge 50% of the hydroxyl radicals, were generated by plotting scavenging activity against the serially diluted sample concentrations. The IC50 values were expressed as total amino acid contents that are commonly found in PL, PL (-), 18 a.a., and EPE. Lower IC50 values indicate higher antioxidant activity.
Ultraviolet C irradiation Ultraviolet C (UVC) irradiation (254 nm) of samples was used as a source of oxidative stress and was performed with an ultraviolet irradiator (FUNA-UV-LINKER, FS-1500, Funakoshi Co., Ltd., Tokyo, Japan). The UVC dose was based on the typical daily dose received in July in a region (Okinawa, Japan) that experiences the highest annual UVB dose (3.5 J/cm2) in Japan (Ministry of the Environment, Japan. 2015, URL cited:i)). Samples (1.5 mL) were placed in 42-mm-diameter petri dishes and exposed to UVC at 0.08, 0.27, and 3.06 J/cm2; the samples were then immediately cooled on ice for 5 min. Subsequently, the samples were diluted 1:10 with distilled water just before hydroxyl radical scavenging activity was measured, as described above, at 0, 5, and 24 h after irradiation.
Antioxidant activity of HA toward hydroxyl radical-induced DNA degradation The protective effect of HA against hydroxyl radical-induced λ DNA degradation was evaluated according to the method reported by Takahashi et al., (2011) with slight modification, as described below.
a) Preparation of λ DNA Five microliters of 3 mol/L sodium acetate (pH 5.5) and 210 µL of ethanol were added to a 100 µL sample of 0.35 µg/µL λ DNA; the solution was subsequently centrifuged (5430R, Eppendorf, Hamburg, Germany) for 10 min at 25 826 g at 10 °C. After centrifugation, the supernatant was removed and discarded, and the pellet was resuspended in 500 µL of ice-cold 70% ethanol and centrifuged for 5 min at 25 826 g at 10 °C. This step was repeated twice. Subsequently, the supernatant was removed, and the DNA pellet was dried with a centrifugal evaporator (CVE-1100, TOKYO RIKAKIKAI Co., Ltd., Tokyo, Japan) for 10 min and then dissolved in PBS to a concentration of 0.5 µg/µL.
b) Preparation of hydroxyl radical solution The hydroxyl radical solution was prepared according to the method reported by Halliwell et al. (Halliwell et al., 1987; Takahashi et al., 2011; Yanai et al., 2008). Briefly, 100 µL each of 0.1 mol/L FeCl3·6H2O, 0.1 mol/L AsA, and 0.1 mol/L EDTA were mixed, and then 1 mL of 0.13 mol/L hydrogen peroxide was added; the mixture was then incubated at 37 °C for 30 min to generate hydroxyl radicals.
c) DNA degradation assay Sample solutions (22.5 µL) containing 1.5 µg of λ DNA were allowed to stand at room temperature for 10 min, and 2.5 µL of the hydroxyl radical solution was added (final volume of 25 µL) prior to incubation at 37 °C for 60 min (the final concentration of hydrogen peroxide in the reaction mixture was 10 mmol/L). For the control, distilled water was added instead of the hydroxyl radical solution. Twenty microliters of each reaction product were subsequently applied to a 0.8% agarose gel containing 0.5 µg/mL ethidium bromide, and electrophoresis was conducted for 30 min at 100 V in TAE buffer. After electrophoresis, DNA was detected with an UV transilluminator system (WUV-M20, WSE-6100, ATTO CORPORATION, Tokyo, Japan).
Statistical analysis Results are expressed as the mean ± standard deviation (SD) of three independent experiments (6–9 technical replicates). Tukey's test was used to determine differences between groups with R software (ver. 4.1.0, R Foundation for Statistical Computing, Vienna, Austria) (Fig. 1). Differences between two groups were analyzed by Student's t-test or Welch's t-test (Microsoft Office Excel 2019, Microsoft Corporation, WA, USA). Multiple comparisons of data were analyzed using either two-way ANOVA followed by Tukey's test (Table 2) (R software) or two-way repeated measures ANOVA (Fig. 3) (Microsoft Office Excel 2019). Differences were considered significant when p < 0.05.
Comparison of hydroxyl radical scavenging activity of LHA and PL.
Hydroxyl radical scavenging activity of PL and the same concentration of LHA in PL were assessed by chemiluminescence as described in the Materials and Methods. AsA was used as a control. Data are expressed as mean ± SD of at least three independent experiments (n = 7–9). Different characters in each concentration (0.01, 0.017, 0.03, and 0.1 mg/mL) mean significant differences (p < 0.001).
Hydroxyl radical scavenging activity of PL and HA The hydroxyl radical scavenging activity of PL was assessed using the chemiluminescent method described above. PL scavenged hydroxyl radicals, and a good linear correlation (r = 0.946) between PL concentration (1/100–1/10) and hydroxyl radical scavenging activity (55–90%, Fig. 1) was observed. The well-known antioxidant AsA also exhibited hydroxyl radical scavenging in a concentration-dependent manner, verifying that this method is suitable for assessing the antioxidant activity of PL. Moreover, PL exhibited significantly stronger scavenging activity than the equivalent concentration of LHA in PL (p < 0.001). Interestingly, LHA solution did not exhibit concentration dependence for its hydroxyl radical scavenging activity (average: 18.6% over a 10-fold concentration range). As with LHA, the hydroxyl radical scavenging activity of HHA (high molecular weight HA from rooster combs) also did not exhibit concentration dependence for its hydroxyl radical scavenging activity, and the intensity at each concentration was indistinguishable from that of LHA (average: 20.4%).
Effect of LHA on hydroxyl radical scavenging activity of PL As mentioned above, LHA exhibits hydroxyl radical scavenging activity in vitro. The hydroxyl radical scavenging activity of PL and PL (-) was compared to clarify whether LHA in PL is involved in its activity. Although both PL and PL (-) exhibited hydroxyl radical scavenging activity, the activity of PL was significantly higher than that of PL (-) (Fig. 2, p < 0.05, p < 0.01). In addition, the IC50 values were calculated as total amino acid contents and compared between PL and PL (-). The IC50 value for PL was 6.85 ± 0.92 mg/100 g, whereas the IC50 of PL (-) was 11.67 ± 4.09 mg/100 g (p < 0.05, Table 1). Therefore, it is inferred that LHA contributes to the antioxidant activity of PL.
LHA enhances the antioxidant activity of PL. The hydroxyl radical scavenging activity of PL was compared with the same concentration of PL (-). Each value of PL and PL (-) is represented as the total free amino acid content (mg/100 g). Data are expressed as mean ± SD of at least three independent experiments (n = 9). Asterisks denote values that are statistically different as determined by Student's t-test (*: p < 0.05, **: p < 0.01).
| mean ± SD Total free amino acid content (mg/100 g) |
|
|---|---|
| PL | 6.85 ± 0.92* |
| PL (−) | 11.67 ± 4.09 |
The IC50 values of PL and PL (−) were calculated from the concentration response data presented in Fig. 2 using the equations described in the Materials and Methods. Each value is represented as the total free amino acid content (mg/100 g). Data are expressed as mean ± SD of at least three independent experiments (n = 6). Asterisks denote values that are statistically different as determined by Welch's t-test (*: p < 0.05).
Identification of the PL component involved in enhancing antioxidant activity in the presence of LHA In addition to LHA, PL is composed of multiple antioxidants including EPE, which is primarily composed of 18 types of free amino acids. To clarify the involvement of EPE or its constituent amino acids in enhancing the effects of LHA on hydroxyl radical scavenging activity, EPE and 18 a.a. solutions were prepared and assessed as follows. LHA was added to the EPE or 18 a.a. solutions, and the radical scavenging activities were determined. The resultant IC50 values were analyzed by two-way ANOVA to evaluate the interaction in the presence of LHA, in the combined solutions (18 a.a. or EPE), and their effects on radical scavenging activity. The results showed a significant difference in the presence of LHA [F (1, 20) = 10.37, p < 0.01], between the solutions [F (1, 20) = 16.25, p < 0.01], and for the combined solution [F (1, 20) = 12.32, p < 0.01]. However, the results of subsequent Tukey's tests showed no significant difference between the IC50 values for 18 a.a. in the absence and presence of LHA. In contrast, the IC50 value for the combination of EPE and LHA was significantly lower than for EPE alone or the 18 a.a. group (p < 0.01, Table 2). These results clarify that LHA synergistically enhances hydroxyl radical scavenging activity in the presence of EPE. Similar results were obtained with HHA. Moreover, this result suggests that an EPE component other than the amino acids is involved in enhancing antioxidant activity in the presence of LHA.
The effect of UVC irradiation on the hydroxyl radical scavenging activity of PL and its relevance to LHA PL and PL (-) were UVC irradiated (0.08, 0.27, and 3.06 J/cm2) as an oxidative stress, and temporal changes in hydroxyl radical scavenging activity were assessed at 0, 5, and 24 h post-irradiation (T0, T5, and T24). The results are shown in Fig. 3A and 3B. These data exhibited a normal distribution, except for the samples that were exposed to high doses of UVC (3.06 J/cm2). Therefore, low doses (0.08 and 0.27 J/cm2) of UVC were used to irradiate PL and PL (-), which had a normal distribution, and the following were evaluated: 1) the relationship between UVC dose and hydroxyl radical scavenging activity in UVC-irradiated samples, 2) the relationship between the time after UVC irradiation and hydroxyl radical scavenging activity, 3) whether the hydroxyl radical scavenging activity of UVC-irradiated samples was restored to the same levels as the unirradiated samples over time, and 4) whether LHA affected the restoration of hydroxyl radical scavenging activity of irradiated samples. As a result, 1) when PL and PL (-) were exposed to low doses of UVC (0.08 and 0.27 J/cm2), the hydroxyl radical scavenging activities were attenuated in a UVC dose-dependent manner, as shown in Fig. 3A and Fig. 3B (0 h (†)), which indicated a negative correlation (PL: r = −0.996, PL (-) = −0.994). 2) When comparing the hydroxyl radical scavenging activity of UVC-irradiated samples over time at each dose (Fig. 3A and Fig. 3B, #1–#4), there was a positive correlation between the elapsed time after irradiation and hydroxyl radical scavenging activity, as shown in Fig. 4 [PL: r = 0.827, PL (-): r = 0.941 at 0.08 J/cm2 (Fig. 4A); PL: r = 0.854, PL (-): r = 0.951 at 0.27 J/cm2 (Fig. 4B)]. Furthermore, there was considerable variation in the measured values of PL (-) at 24 h post-irradiation compared to PL. 3) The hydroxyl radical scavenging activities of UVC-irradiated PL and PL (-) were individually analyzed by a multiple comparison procedure (Tukey's test) at each UVC dose (#1–#4), including unirradiated [UVC (-), T 0] samples, to evaluate whether there was a statistically significant difference at each time. Although the hydroxyl radical scavenging activities of PL and PL (-) were significantly attenuated (p < 0.05) in a UVC dose-dependent manner immediately after UVC treatment as mentioned above, subsequently, these activities were restored to the same level as pre-irradiation 24 h after exposure to UVC, except for the exposure at 3.06 J/cm2 [Fig. 3A and Fig. 3B, a–c). 4] The measurement data from UVC-irradiated PL and PL (-) were analyzed by two-way repeated measures ANOVA (#1 vs. #3, #2 vs. #4) to evaluate the interaction between the two factors (i.e., the presence of LHA and elapsed time) to assess whether LHA controls the restoration of hydroxyl radical scavenging activity in UVC-irradiated samples at each UVC dose. As a result, the effect of LHA addition [0.08 J/cm2: F (1, 30) = 56.30, p < 0.01; 0.27 J/cm2: F (1, 30) = 24.38, p < 0.01] and elapsed time [0.08 J/cm2: F (2, 30) = 4.52, p < 0.05; 0.27 J/cm2: F (2, 30) = 31.64, p < 0.01] were statistically significant, while the interactions were not significant at any UVC dose [0.08 J/cm2: (F2, 30) = 0.609, p = 0.55; 0.27 J/cm2: F (2, 30) = 2.04, p = 0.14]. These results suggest that the hydroxyl radical scavenging activity of UVC-irradiated PL was temporarily attenuated depending on the UVC dose, while it recovered in an LHA-dependent process.
| 18 a.a. Sol. | EPE | |
|---|---|---|
| Total free amino acid content (mg/100 g) | ||
| HA (−) | 20.21 ± 2.94a | 19.74 ± 2.91a |
| LHA (+) | 20.46 ± 1.26a | 13.77 ± 0.48b |
| HHA (+) | 22.97 ± 2.94a | 12.94 ± 3.21b |
LHA was added to 18 a.a. or EPE, and the resulting solutions were subsequently subjected to serial dilutions up to 1/100 with distilled water. Luminescence intensity was measured, and IC50 values were calculated as described in the Materials and Methods. Each IC50 value is represented as total free amino acid content (mg/100 g). Data are expressed as mean ± SD of at least three independent experiments (n = 6). Statistically significant differences between groups were determined by two-way ANOVA followed by Tukey's test. A value of p < 0.01 (b) compared with the other groups (a) was considered statistically significant.
Effect of UVC irradiation on radical scavenging activity of PL containing LHA (PL) and lacking LHA [(PL (-)].
PL and PL (-) were UVC irradiated, and subsequently their hydroxyl radical scavenging activities were assessed at 0, 5, and 24 h (T 0, T 5, and T24) [(A: PL, B: PL (-)]. The samples were subjected to 1/10 dilutions with distilled water just before measurement of hydroxyl radical scavenging activity. Data are expressed as mean ± SD of at least three independent experiments (n = 6). †: The relationships between the UVC irradiation dose and hydroxyl radical scavenging activity immediately after UVC irradiation (T 0) of PL and PL (-) were assessed. Different letters on the box plot indicate significant differences (Tukey's test, p < 0.05) within each group at #1 to #4, including UVC (-). The data from samples exposed to high-dose UVC (3.06 J/cm2) are also presented as a reference.
Responsiveness of LHA to DNA degradation by hydroxyl radicals DNA is a biomolecule that is a primary target of ROS. The ability of PL to prevent DNA degradation by hydroxyl radicals was evaluated, and the role of LHA assessed. Preliminary experiments were conducted with the well-known hydroxyl radical scavengers edaravone (1.5 mg/mL) and AsA (0.88 mg/mL) to confirm the validity of the assay. Edaravone (Radicut®) is a main component of a medication approved for use in Japan to treat patients with acute cerebral infarction (Kikuchi et al., 2013; Watanabe et al., 2004). Fig. 5 shows the electrophoresis pattern of λ DNA with or without exposure to hydroxyl radicals. λ DNA is 48.5 kbp and is detected as a single band (23.1 kbp) in the absence of exposure to hydroxyl radicals (lane 1, Fig. 5A). In contrast, the sample resolves as a smear when DNA is exposed to hydroxyl radicals, as shown in lane 2. λ DNA was completely degraded when dissolved in AsA (data not shown) and exposed to hydroxyl radicals. This indicates that AsA did not protect DNA against oxidative stress, which is consistent with the results of a previous report (Takahashi et al., 2011). In contrast, when λ DNA was dissolved in edaravone and exposed to hydroxyl radicals, intact λ DNA was detected (data not shown), as expected. These results correspond with previous reports, and it can be concluded that the method used in this study was valid, and optimal conditions for assessment were utilized.
Positive correlation between hydroxyl radical scavenging activity of PL and PL (-) and elapsed time after UVC irradiation.
The graphs above show the hydroxyl radical scavenging activity of UVC-irradiated PL and PL (-) by each dose [(A: 0.08 J/cm2 (#1 and #3 at Fig. 3A and 3B); B: 0.27 J/cm2 (#2 and #4 at Fig. 3A and 3B)] with time.
Antioxidant and protective effects of PL and PL components on hydroxyl radical-induced DNA degradation. Sample preparation and subsequent electrophoresis were performed as described in the Materials and Methods. M: DNA size maker (λ-Hind III digest), lanes 1 and 2 are λ DNA without antioxidant (DDW) and no ROS (1; −) or with ROS (2; +), respectively. Lanes 3 to 22 are λ DNA with test substances (3–4, PL; 5–6, PL (-); 7–8, 18 a.a.; 9–10, EPE; 11–12, LHA; 13–14, 18 a.a. + LHA; 15–16, EPE + LHA; 17–18, HHA; 19–20, 18 a.a. + HHA; 21–22, EPE + HHA, including no ROS (3, 5, 7, 9, 11, 13, 15, 17, 19, and 21) or with ROS (4, 6, 8, 10, 12, 14, 16, 18, 20, and 22), respectively.
When λ DNA was dissolved in PL or PL (-) and exposed to hydroxyl radicals, the bands remained visible and were detected at the expected position for intact λ DNA (lane 1) (Fig. 5A, lanes 3–6). Subsequent experiments aimed to identify the antioxidant in PL that accounts for the protection of DNA from oxidative stress. λ DNA was dissolved in LHA and other PL constituents, such as 18 a.a., EPE, and a mixture of the two (18 a.a. + EPE). Smears were observed when λ DNA was dissolved in 18 a.a. and 18 a.a. + LHA with exposure to hydroxyl radicals (Fig. 5B, lanes 8 and 14). In the case of λ DNA, when it was dissolved in LHA and exposed to hydroxyl radicals, only a faint smear was visible (Fig. 5B, lane 12). In contrast, intact bands were detected when λ DNA was dissolved in EPE or LHA + EPE prior to exposure to hydroxyl radicals (Fig. 5B, lanes 10 and 16). While EPE prevents oxidative DNA damage by itself, its antioxidant activity is synergistically enhanced in the presence of LHA, as shown in Table 2. In addition, similar results were obtained with HHA (Fig. 5C). Accordingly, these results suggest that although LHA could not prevent DNA from oxidative damage by itself, its antioxidative activity is enabled in the presence of EPE.
HA is a readily water soluble, colorless, and transparent substance that exhibits unique physicochemical properties, such as high viscosity and exceptional water retention. Therefore, HA has been widely used as an ingredient in cosmetics, dietary supplements, and medicines. In cosmetic products, HA is used mainly in moisturizing creams, lotions, and similar products because of its ability to hydrate and plump the skin. In addition, HA is included in oral supplements with the aim of imparting a cutaneous anti-aging effect and improving skin conditions. These HA products possess desirable functions and enable differentiation from competing products. Recently, it has become clear that the beneficial effects of HA on the physiological function of biomolecules are attributable to its radical scavenging activity. The aim of the present study was to evaluate the radical scavenging activity of LHA in the context of interactions with other components of PL. The results of chemiluminescence-based radical scavenging assays indicated that LHA scavenged hydroxyl radicals in a concentration-independent manner, as shown in Fig. 1. In contrast, when LHA and individual antioxidant constituents of PL (18 a.a., EPE) were combined and continuously diluted to measure the radical scavenging activity, concentration dependence was obtained, which enabled IC50 calculations and comparisons (Table 2). The IC50 was significantly enhanced when LHA and EPE were combined, reaching levels comparable to PL. Previous reports have suggested that the mechanism for hydroxyl radical scavenging of HA is as follows: 1) The carboxylic groups present in HA function as metal chelators, thereby preventing hydroxyl radical generation via the Fenton reaction, 2) HA might directly scavenge hydroxyl radicals (Campo et al., 2003b; Halicka et al., 2009; Zhao et al., 2008). The results of the chemiluminescence experiments support the latter mechanism, where LHA scavenges hydroxyl radicals. In addition, the results of the UVC irradiation and DNA degradation assays proved that the chelating properties of LHA are not involved in its hydroxyl radical scavenging activity. Thus, LHA is thought to exhibit the following distinctive properties: 1) LHA exhibits weak hydroxyl radical scavenging activity by itself, 2) the radical scavenging activity is improved when LHA is mixed with other antioxidants such as EPE, and 3) a constant concentration of LHA coexists with other antioxidants. These properties were confirmed for HHA, and in contrast to a previous report (Ke et al., 2011), the intensity of hydroxyl radical scavenging activity was equivalent to that of LHA. This observation is presumed to be due to the analytes being present at low concentrations (≤ 100 µg/mL). Therefore, it is thought that the intensity of hydroxyl radical scavenging activity of HA is not dependent on the molecular weight at low concentrations.
UVC irradiation of samples resulted in the radiation dose-dependent generation of exogenous ROS (data not shown) and subsequent reduction in the hydroxyl radical scavenging activity. The radical scavenging activity of PL and PL (-) was restored to pre-irradiation levels after 24 h following exposure to low doses of UVC (0.08 or 0.27 J/cm2), whereas exposure to a high dose of UVC (3.06 J/cm2) resulted in immediate attenuation of the activity that was only partially restored in PL after 24 h (reference data). This study demonstrated that LHA is significantly involved in the restoration of these activities. The “recovery” phenomenon of hydroxyl radical scavenging activity observed in PL is likely caused by LHA controlling the amount of hydroxyl radical generation post-UVC irradiation via its function as an antioxidant in the presence of other PL components, providing that the radical scavenging capacity of PL is not exceeded. It was also shown that the capability to enhance the antioxidant activity of LHA was maintained even after UVC irradiation.
The results of the DNA degradation assays indicated that the weak antioxidant capacity of LHA cannot prevent oxidative DNA damage due to hydroxyl radicals. This suggested that the chelating property of LHA is not involved in hydroxyl radical scavenging activity. Previous reports have indicated that antioxidants exhibit differences in their scavenging ability depending on the type of ROS. Hydroxyl radical-induced DNA fragmentation was observed in the presence of AsA, as previously reported. The antioxidant properties of AsA are well characterized, and it is known to exhibit weak activity against hydroxyl radicals with strong activity against peroxynitrite (ONOO-) and chlorine monoxide (ClO-) (Takahashi et al., 2011). In addition to hydroxyl radicals, LHA has also been reported to directly scavenge superoxide anion radicals (O2-) (Ke et al., 2011). Thus, it is assumed that LHA also exhibits distinct differences in its scavenging ability among these ROS species. The chemiluminescence measurement results clearly indicated that the hydroxyl radical scavenging activity of LHA was significantly lower than that of PL, which prevents DNA degradation. A comparison of IC50 values for 18 a.a., in whose presence a smear band was observed, and that of 18 a.a. + LHA, indicated that there was no significant difference in their values (Table 2), which were lower than that of PL. In contrast, when LHA coexisted with EPE, the antioxidant activity was significantly enhanced and the IC50 value was similar to that of PL (-), which prevented DNA fragmentation. Therefore, it is clear that the degree of hydroxyl radical scavenging activity exhibited by individual antioxidants is the determining factor for the responsiveness to oxidative stress. In other words, it is necessary that each antioxidant possesses a certain level of antioxidative activity to protect biomolecules from oxidative damage. Moreover, antioxidants that coexist with LHA and synergistically increase its activity have an affinity for LHA; this study revealed that the non-amino acid components of EPE cooperate with LHA. Although the IC50 value of EPE was as low as that of 18 a.a., DNA fragmentation was not observed in the presence of EPE. While the reason for this remains unclear, it is presumed to be due to EPE containing multiple antioxidants. It is thought that the non-amino acid antioxidative components of EPE, such as peptides and nucleic acids, exert different activities in eliminating ROS and preventing DNA damage following exposure to exogenous oxidative stress.
In conclusion, although LHA directly weakly and scavenges hydroxyl radicals, its activity is improved when a constant concentration of LHA coexists with other antioxidants. Consequently, this study revealed that LHA provides beneficial antioxidant activity in PL, which consists of multiple antioxidants and is an excellent functional material.
Acknowledgements I am deeply grateful to Prof. Dr. H. Arakawa (Showa University) for valuable advice and comments on this study and proof reading.
Conflict of interest There are no conflicts of interest to declare.
