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The anti-aging properties of a human placental hydrolysate combined with dieckol isolated from Ecklonia cava
- Su Kil Jang†1,
- Do Ik Lee†2,
- Seung Tae Kim1,
- Gwang Hoon Kim1,
- Da Woon Park1,
- Jung Youl Park3,
- Daehee Han1,
- Jae Kwon Choi4,
- Yoon-bok Lee4,
- Nam-Soo Han5,
- Yun Bae Kim5,
- Jeongsu Han6 and
- Seong Soo Joo1Email author
© Jang et al. 2015
Received: 7 July 2015
Accepted: 23 September 2015
Published: 5 October 2015
In the present study, we aimed to examine the anti-aging properties of human placental hydrolysate (HPE) and dieckol (DE) from Ecklonia cava against free radical scavenging, muscle hypertrophy-related follistatin mRNA expression, amelioration of cognition-related genes and proteins, inhibition of collagenase-regulating genes, and elastinase activity.
The anti-aging effects were examined in human fibroblast (CCD986sk), mouse myoblast (C2C12), and neuroblastoma (N2a) cell models, by employing various assays such as 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) scavenging, hydroxyl radical-mediated oxidation, quantitative real-time polymerase chain reaction, enzyme activity, and immunocytochemistry observation.
Our results show that HPE combined with DE (HPE:DE) strongly scavenged DPPH radicals and protected proteins against degradation by hydroxyl radical attack. HPE:DE effectively inhibited matrix metalloproteinase-1 expression, protein kinase C alpha expression, and elastinase activity. Furthermore, HPE:DE improved the expression of cognition-related genes (choline acetyltransferase and vesicular acetylcholine transporter). These events may proactively contribute to retard the aging processes and the abrupt physiological changes probably induced by mitochondrial dysfunction with aging.
Based on these findings, we conclude that the combined treatment of HPE:DE may be useful for anti-aging therapy in which the accumulation of oxidative damage is the main driving force.
KeywordsHuman placental hydrolysate Dieckol Muscle Cognition Collagenase Mitochondria
Aging is a series of biological changes that follow a natural progression from birth to death and is a multidimensional process of physical, psychological, and social changes. Identifying the major contributing factors to aging and increasing longevity without age-related illness is a cherished desire for human beings. Although much scientific knowledge has accumulated, preventing aging and prolonging lifespan continue to be a focus of attention. Aging-associated diseases that are not age-specific include atherosclerosis and cardiovascular disease, cancers, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, and Alzheimer’s disease . Excess production of free radicals may cause age-related impairment through oxidative damage to biomolecules, and mitochondria are the main target of free radical attack [2–4]. In addition, age-associated cognitive decline and neurogenic impairment, which may be caused by reduced superoxide dismutase and increased oxidative stress during aging, are important during aging but not fully understood [5, 6]. Human placenta, which includes diverse bioactive molecules, has attracted attention for managing the aging process [7, 8]. The placenta also possesses anti-oxidative, anti-inflammatory, anti-melanogenic, and collage-synthesizing properties that are effective anti-aging agents and rejuvenating to the body [9–11]. Dieckol (DE) was recently isolated from Ecklonia species, and this oligomeric polyphenol of phloroglucinols  has been reported to have diverse biological activities, such as antioxidant , anti-plasmin inhibitory , anti-mutagenic, anti-bacterial , anti-viral , tyrosinase inhibitory , anti-adipogenic , and matrix metalloproteinase-1 (MMP-1) inhibitory activities . Thus, we hypothesized that increased free radical production may play a central role in aging and cause muscle and neuronal damage. In this study, we report the optimal effects of a human placental hydrolysate (HPE) combined with DE by focusing on the enhancement of aging-related indices, such as oxidative stress and muscle and cognitive impairment.
Fresh E. cava was collected from the Jeju Island coast of South Korea in February 2013. A voucher specimen (NIBRAL0000145247) was authenticated by Prof. Joo (Biopharmaceutical Lab, College of Life Science, Gangneung National University, Republic of Korea), and deposited at the National Institute of Biological Resources, Incheon, Republic of Korea. Epiphytes, salt, and sand were completely removed with tap water. The samples were sanitized with 70 % ethanol, rinsed with deionized water, and freeze-dried. Finely ground E. cava (100 g) was steeped in 1 L of 80 % aqueous ethanol for 24 h repeatedly for 3 days at room temperature. The ethanol hydrolysates were combined, filtered through filter paper (Whatmann International Ltd., Maidstone, UK), evaporated, and dried completely. After the hydrolysate was suspended on 1 L distilled water, the organic soluble fraction was obtained with ethyl acetate. Finally, DE was obtained by purifying the polar fraction using the Prep-LC (LC-9104, JAI) system equipped with an ODS column in methanol solvent as described previously . The HPE (Laennec, human placenta hydrolysate) was obtained from Japan Bioproducts Industry Co., Ltd. (Tokyo, Japan).
Amino acid analysis
Amino acid concentrations were measured with an automatic amino acid analyzer (L-8800; Hitachi, Tokyo, Japan). Sample aliquots containing 8–12 mg protein were placed in a 20-mL cuvette and mixed with 9 mL of 6 M HCl. After sealing the cuvette, the samples were hydrolyzed at 110 °C for 24 h under N2. The hydrolysates were transferred to a 100 mL volumetric flask, mixed with 9 mL 6 M NaOH, and diluted with 0.02 N HCl. Then, all samples were filtered and loaded in a Hitachi L-8800 amino acid analyzer for the analysis.
Radical scavenging and protein protection assays
The 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) radical is one of the few stable organic nitrogen radicals and has a deep-purple color. Fractions were reacted with the DPPH solution to evaluate free radical scavenging activity. Each lyophilized fraction was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) as a stock solution (100 mg/mL), and each fraction was reacted with 0.3 mM DPPH in methanol. Various concentrations of HPE or DE (0.01–100 μg/mL) were reacted with the DPPH radical solution for 20 min at room temperature, and absorbance was measured at 517 nm. DPPH free radical scavenging activity was calculated using the following equation: DPPH scavenging activity (%) = [Ac – (A – As)]/Ac × 100, where Ac is the absorbance of the control DPPH solution, A is absorbance of the sample with the DPPH solution, and As is absorbance of the sample. Hydroxyl radical-mediated oxidation experiments were performed for the protein protection assay using a metal-catalyzed reaction, as described previously with some modifications . The target protein, bovine serum albumin (BSA), was dissolved in a 150 mM phosphate buffer (pH 7.3) to a final concentration of 0.5 mg/mL. The BSA solution was incubated with and without 100 μM copper (Cu2+) and 2.5 mM H2O2 in the presence and absence of the samples. The control antioxidant was 0.1 mM ascorbate, which was directly dissolved in PBS. The reactions were carried out in open tubes and placed in a shaking water bath maintained at 37 °C. After the reaction was complete, each mixture was separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with 0.1 % Coomassie Blue Brilliant solution.
Elastase inhibition assay
This assay was performed in 0.2 mM Tris–HCl buffer (pH 8.0) in accordance with a previous study with minor modifications . In brief, porcine pancreatic elastase (Sigma-Aldrich) was dissolved to prepare a 3.33 mg/mL stock solution in sterile water. The N-succinyl-Ala-Ala-Ala-p-nitroanilide substrate was dissolved in buffer to 1.6 mM. The test hydrolysates were incubated with the enzyme for 20 min before adding substrate to begin the reaction. The final reaction mixture (250 μL total volume) contained buffer, 0.8 mM substrate, 1 μg/mL enzyme, and various concentrations of HPE, DE, and HPE:DE, as indicated. Asc (100 μM) was used as the positive control. Absorbance values between 381 and 402 nm were measured immediately following addition of the substrate and then continuously for 20 min using a Spectra Max 340 Microplate Reader in Nunc 96 well microtiter plates. The percent inhibition of elastase was calculated as follows: Inhibition (%) = [(ODcontrol − ODsample)/ODcontrol] × 100.
Human fibroblast (CCD986sk), mouse myoblast (C2C12), and neuroblastoma cell lines (N2a) (Korean Cell Line Bank, Seoul, Republic of Korea) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone. Logan, UT, USA.) supplemented with 10 % fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA.). The cultures were maintained under 5 % CO2 at 37 °C in tissue culture flasks. The cells were grown to > 90 % confluency and subjected to no more than 20 cell passages. Media were changed every 2–3 days. Subconfluent cells were harvested and seeded at a density of 5 × 105 cells or 1.5 × 106 cells into poly-L-lysine-coated 35-mm or 60-mm culture plates. After plating for 24 h, the medium was replaced with serum-free DMEM, washed once with phosphate-buffered saline (PBS), and treated with HPE, DE, or the positive controls of phorbol myristic acetate (PMA), and ascorbic acid (Asc) (Sigma-Aldrich, St. Louis, MO, USA.).
Cell viability in response to HPE and DE stimulation was investigated in 96-well microtiter plates (2 × 104 cells/mL) following a 24-h culture using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). This system uses WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], which produces water-soluble colored formazan upon bioreduction in the presence of the electron carrier, 1-methoxy-5-methylphenazinium methylsulfate. The plates were measured at 450 nm (Spectra Max 340, Molecular Devices, Sunnyvale, CA, USA.), and data from triplicate cultures are expressed as percent viability vs. the control.
Quantitative real-time polymerase chain reaction (PCR) assay
Primer sequences used for the real-time polymerase chain reaction analysis
Amino acid sequence
Product size (bp)
Immunocytochemistry (ICC) and microscopic observations
Cultured N2a cells were fixed in 4 % paraformaldehyde in PBS for 15 min, washed twice with PBS supplemented with 100 mM glycine for 5 min, and incubated with permeabilization buffer consisting of 0.1 % Triton X-100 (Sigma-Aldrich) in PBS for 30 min at room temperature. Blocking was performed with 1 % BSA for 30 min at room temperature as previously described . Then, choline acetyltransferase (ChAT) or vesicular acetylcholine transporter (VAChT) mouse monoclonal antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA.) was added to 1 % BSA in PBS with Tween 20 and incubated for 2 h at room temperature. The cells were washed three times with PBS before fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (1:200; Cell Signaling Technology, Danvers, MA, USA.) was added to 1 % BSA for 1 h at room temperature. The cells were rinsed and counterstained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich) for 10 min, followed by two PBS washes. The cultures were visualized with an inverted fluorescent microscope system (Eclipse Ti-S; Nikon, Tokyo, Japan) at a magnification of × 600.
Statistical comparisons between groups were performed using one-way analysis of variance with Dunnet’s post-hoc test and SPSS v. 17 software (SPSS, Inc., Chicago, IL, USA.). A p < 0.05 was considered significant.
Results and discussion
Among many the age-related changes that begins in adulthood, muscle weakness, cognitive decline, and the accumulation of reactive oxygen species (ROS) are closely related because ROS are major causative factors of aging through their oxidative deteriorating effects [24, 25]. Neurodegenerative diseases and the degenerative loss of skeletal muscle mass (sarcopenia) during aging are critically linked to mitochondrial dysfunction, which cannot functionally regulate or scavenge ROS via antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [26, 27]. In addition, the main amino acid reservoir in the body is skeletal muscle, which contains approximately 75 % of all protein and progressively loses muscle mass and function during aging .
Amino acid composition
Sulphuric amino acids
Aromatic amino acids
Essential amino acids
Nonessential amino acids
The HPE:DE combination effectively improved free radical scavenging, muscle hypertrophy-related FSTN mRNA expression, ameliorated cognition-related genes (ChAT and VAChT) and proteins, and inhibited MMP1/PKCα expression and elastinase activity, suggesting that the combined treatment of HPE:DE may be useful for anti-aging therapy in which the accumulation of oxidative damage is the main driving force.
This research was supported by High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (MAFRA; grant number 113034–3).
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