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Process Biochemistry 48 (2013) 1387–1394

Contents lists available at ScienceDirect

Process Biochemistry

jo ur nal home p age: www.elsev ier .com/ locate /procbio

etrameric peptide purified from hydrolysates of biodiesel byproductsf Nannochloropsis oculata induces osteoblastic differentiationhrough MAPK and Smad pathway on MG-63 and D1 cells

inh Hong Thi Nguyena,1, Zhong-Ji Qianb,1, Van-Tinh Nguyenb, Il-Whan Choic,oo-Jin Heod, Chul Hong Ohd, Do-Hyung Kangd, Geun Hyung Kime,∗∗, Won-Kyo Jungb,∗

Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, VietnamDepartment of Marine Life Science, and Marine Life Research and Education Center, Chosun University, Gwangju 501-759, Republic of KoreaDepartment of Microbiology, Inje University College of Medicine, Busan 614-735, Republic of KoreaGlobal Bioresources Research Center, Korea Institute of Ocean Science & Technology, Ansan 426-744, Republic of KoreaDepartment of Bio-Mechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea

r t i c l e i n f o

rticle history:eceived 23 March 2013eceived in revised form 31 May 2013ccepted 28 June 2013vailable online 6 July 2013

eywords:arine microalgaeannochloropsis oculata

a b s t r a c t

Ongoing efforts to search for bioactive substances for bone diseases have led to the discovery of naturalproducts with substantial bioactive properties. In this present study, an osteoblast activating-peptidewas isolated from biodiesel by-products of microalgae, Nannochloropsis oculata. To utilize biodiesel by-products of N. oculata and evaluate their beneficial effects, enzymatic hydrolysis was carried out usingcommercial enzymes such as alcalase, flavourzyme, neutrase, trypsin (PTNTM), protamex and alcalasehydrolysate exhibited the highest osteoblastic differentiation activity. Using consecutive purification byliquid chromatographic techniques, an osteoblast-differentiatory peptide was purified and identified tobe a peptide (MPDW, 529.2 Da) by the tandem MS analysis. The results showed that purified peptide

iodiesel byproductssteoblast differentiationurified peptide

promotes osteoblast differentiation by increasing expression of several osteoblast phenotype markerssuch as alkaline phosphatase (ALP), osteocalcin, collagen type I, BMP-2, BMP2/4 and bone mineralizationin both human osteoblastic cell (MG-63) and murine mesenchymal stem cell (D1). In addition, the purifiedpeptide induced phosphorylation of MAPK and Smad pathway in both cells. These results suggest thatpeptide possesses positive effects on osteoblast differentiation and may provide possibility for treatingbone diseases.

© 2013 Elsevier Ltd. All rights reserved.

. Introduction

Osteoporosis is a systemic skeletal disease characterized by lowone mineral density and micro-architectural deterioration of boneissue, consequently resulting in increases in bone fragility andusceptibility to fracture. According to the World Health Organiza-ion (WHO) definition, osteoporosis affects 30% of post-menopausalomen, a proportion that rises to 70% in women aged over 80

ears [1]. In addition, some drugs routinely used for treating ofultiple diseases have detrimental effects on either skeleton or

nduce osteoporosis disease [2]. Osteoporosis, that bone resorption

∗ Corresponding author. Tel.: +82 62 230 6657; fax: +82 62 230 6657.∗∗ Corresponding author. Tel.: +82 31 290 7828.

E-mail addresses: gkimbme@skku.edu (G.H. Kim), wkjung@chosun.ac.krW.-K. Jung).

1 These authors contributed equally to this study.

359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.procbio.2013.06.031

is greater than bone formation, has becoming an important publichealth problem leading to an increased risk of developing spon-taneous and traumatic fractures. This disease can be treated viaosteoclast and/or osteoblast action [3]. Therefore, drugs that wouldact via promoting bone formation (osteoblast) and/or inhibitingbone resorption (osteoclast) could be a tool for desirable ther-apy.

Process of osteoblast differentiation is necessary for bonestrength and remodeling, and can be subdivided in three sub-sequent stages including proliferation stage, extracellular matrixsynthesis and maturation stage, and mineralization stage [4]. Dur-ing osteoblast differentiation, phenotype markers such as alkalinephosphatase (ALP), collagen type I, and osteocalcin concomi-tantly with mineralization are highly expressed, and osteoblast

differentiation is regulated by various signaling pathways suchas BMP-Smads [5,6], mitogen-activated protein kinases (MAPKs),and nuclear factor kappa-light-chain-enhancer of activated B cells(NF-�B) [7,8]. MAPKs families play an important role in complex

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388 M.H.T. Nguyen et al. / Process

ellular programs like proliferation, differentiation, development,ransformation, and apoptosis [9]. During the bone differentia-ion of osteogenic cells, the MAPK signaling cascade is activatedlong with the expression of various osteomarkers [10]. Smadslay important roles in osteoblast differentiation. The BMP-specific-Smads, Smad1, 5, and 8, transiently and directly interact withctivated BMPRs (BMP receptors) and become phosphorylated.mad1/5/8 then form heteromeric complexes with Smad4 (Co-mad) and translocate into the nucleus where they regulateranscription of various target genes. The Smad pathway is a well-haracterized BMP signaling pathway. However, BMPs also initiateon-Smad interacellular signaling pathways. Several lines of evi-ence suggested that BMPs activate the MAPK family of signalingolecules, i.e., ERK1/2, p38, and stress-activated protein kinase/Jun-terminal kinase [6].

Marine microalgae, or phytoplankton, are mostly representedn ocean populations, and the best known are the diatomsBacillariophyta), the dinoflagellates (Dinophyta), the green algaeChlorophyta) and the blue-green algae (Cyanophyta). The uni-ellular marine microalgae were considered to be an aboundingesource for carotenoids, lipids, and polysaccharides, and wereidely investigated in the fields of food supplements and bio-fuelroduction [11]. While terrestrial plants in temperate climates canchieve a photoconversion efficiency of only below 1%, microalgaean convert up to 5% of the solar energy into chemical energy [12].icroalgae are efficient in converting solar energy into metabolites,

uch as lipids, proteins, carbohydrates, pigments and vitamins.oreover, microalgae can provide various types of renewable

nergy sources, such as methane, bio-hydrogen, and biodiesel [13].hus, they can apply in food, feed, natural compounds, or aquacul-ures field as well as in biofuel. Nanochloropsis oculata (N. oculata) is

icroalgae that mostly been known from the marine environmentut also occur in fresh and brackish water.

N. oculata is able to build up a high concentration of a range ofigments such as astaxanthin, zeaxanthin and canthaxanthin, andas been shown to be suitable for algal biofuel production due to itsase of growth and high oil content (28.7% of dry weight), mainlynsaturated fatty acids and a significant percentage of palmitic acid.

t also contains enough unsaturated fatty acid, linolenic acid andolyunsaturated acid for a quality biodiesel. Morever, N. oculataas a high rate of protein as well as carbohydrate [14]. Thus, these of N. oculata has been recognized for human diets and biomass,nd the beneficial effect of reducing blood pressure in hyperten-ive rats have demonstrates by feeding experiments [15]. Manytudies have been reported that marine bioactive peptides can besed as functional foods, nutraceuticals, or pharmaceuticals due toheir therapeutic potential in the treatment or prevention of vari-us diseases [16]. Peptides-derived from marine microalgae haveotential activity on antioxidant, antihypertensive, anticancer [17].. oculata possess a numerous of protein which break down to pep-

ide, amino acid, or protein fractions by protease hydrolysis. In thistudy, a peptide purified from biodiesel by-products of N. oculatafter applying to enzymatic hydrolysis and induced osteoblast dif-erentiation in osteoblastic (MG-63) and mesenchymal stem cellsD1) by activation of mitogen-activated protein kinases (MAPKs)nd Smads pathways.

. Materials and methods

.1. Materials

Human osteosarcoma (MG-63) was obtained from American Tissue Cultureollection (ATCC). Murine mesenchymal stem cells (D1) were purchased from

TCC (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and

etal bovine serum (FBS) was obtained from Gibco-BRL, Life Technologies (Grandsland, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM), trypsin–EDTA,enicillin/streptomycin/amphotericin (10,000 U/ml, 10,000 �g/ml, and 2500 �g/ml,espectively), and fetal bovine serum (FBS) were obtained from Gibco BRL, Life

mistry 48 (2013) 1387–1394

Technologies (USA). MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide)reagent, Alizarin red-S, p-nitrophenyl phosphate (p-NPP), cetylpyridinium chlo-ride monohydrate and dexamethasone were purchased from Sigma Chemical Co.(St. Louis, MO, USA). Digestive proteases (Alcalase, Neutrase, Flavourzyme, TrypsinNovo (PTNTM, 6.0S), and Protamex) were purchased from Novozymes (Novo Nordisk,Denmark). RT-PCR reagents were purchased from Promega (Madison, WI, USA).Primary and secondary antibodies for Western blot analysis were purchased fromSanta Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Amersham Pharmacia Bio-sciences (Piscataway, NJ, USA), respectively. All other reagents used in this studywere analytical grade chemicals.

2.2. Preparation of enzyme hydrolysates of biodiesel byproducts from microalgae,N. oculata

The microalgae, N. oculata was originally obtained from the National ResearchInstitute of Aquaculture Fisheries Research Agency (Kanagawa, Japan) and screenedfor growth and biomass production at the Jeju Center of Korea Basic Science Institute,Korea.

Biodiesel containing total lipid was extracted from freshly N. oculata three timeswith a mixture of CHCl3:MeOH (2:1) as previous report with slightly modification.Then the extracts were centrifuged at 3000 × g for 30 min, and the supernatant wasadded to three times volume of 0.9% NaCl solution. The bottom layer of extracts wasused for further experiment to analyze biodiesel qualities.

To extract active peptide from biodiesel byproducts of marine microalgae,enzymatic hydrolysis was performed using various commercial enzymes (alcalase,neutrase, flavourzyme, PTN, and protamex) with each optimal condition. Optimumhydrolysis conditions and characteristics of the enzymes were described in Table 1.The optimum pH of the each reaction mixtures were adjusted with 1 M HCl/NaOH.The hydrolysates method described by Senevirathne et al. [18] was used with slightmodifications to prepare the enzymatic hydrolysates from biodiesel byproducts ofmarine microalgae. At enzyme/substrate ratio of 1/100 (w/w), 1% substrate andenzyme were mixed. Thereafter, the pH of the each hydrolysate was adjusted to pH 7with 1 M HCl/NaOH. The substrates and each enzyme mixture were incubated for 8 hat each optimal temperature with stirring and then heated in a boiling water bathfor 10 min to inactivate the enzyme. Lyophilized hydrolysates were stored under−80 ◦C until use.

2.3. Purification of osteoblast activating peptide

2.3.1. Recyling preparative HPLC (high-performance liquid chromatography)Osteoblast activating peptide was purified from enzymatic hydrolysates using

recycling preparative HPLC on a Jaigel W253 column. The lyophilized protein(20 mg/ml) was loaded onto the column equilibrated with 20 mM phosphate buffer(pH 6.8), and eluted at a flow rate of 3 ml/min. Each fraction was monitoredat 215 nm, and dried using a lyophilizer under −80 ◦C. Osteoblast differentiationactivity was also investigated, and a highest osteoblast activating fraction was deter-mined to purify by chromatography as the next step.

2.3.2. High-performance liquid chromatography (HPLC)The fraction exhibiting osteoblast differentiation activity was further purified

using reversed-phase high-performance liquid chromatography (RP-HPLC) on YMCpack pro C18 column with a linear gradient of acetonitrile (0–60% in 60 min) at aflow rate of 1.0 ml/min. Elution peaks was detected at 215 nm. Potent peaks werecollected, lyophilized, and then evaluated osteoblast differentiation activity. Thefinally purified peptide was analyzed amino acid sequence.

2.4. Determination of amino acid sequence

Accurate molecular mass and amino acid sequence of the purified peptide weredetermined with a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupledto an electrospray ionization (ESI) source. The purified peptide was injected intothe electrospray source following dissolution in methanol/water (1:1, v/v), and itsmolecular mass was determined by a doubly charged (M+2H)2+ state in the massspectrum. Following molecular mass determination, the peptide was automaticallyselected for fragmentation and its sequence information was obtained by tandemmass spectroscopy (MS) analysis.

2.5. Culture of cells and viability determination

Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing5% FBS at 37 ◦C in a 5% CO2 humidified incubator. To induce osteogenic differ-entiation in D1 cells, culture media were changed at 3 days to ODM (DMEMsupplemented with 50 �g/ml ascorbic acid, 10−8 M dexamethasone, and 10 mM �-glycerolphosphate). After culture for another 3 days, one group was cultured onlyfor ODM, while another group was cultured for ODM plus the sample. D1 cells were

then analyzed 24 or 48 h later. Cells were grown in 96-well plates at a density of5 × 103 cells/well in the presence of different concentrations of samples. Cell via-bility was determined by use of the MTT assay. MTT was used as an indicator ofcell viability as determined by mitochondrial-dependent reduction to formazan. Inbrief, the cells were seeded and then treated with various reagents for the indicated

M.H.T. Nguyen et al. / Process Biochemistry 48 (2013) 1387–1394 1389

Table 1Characteristics and optimum hydrolysis conditions of the specific enzymes.

Enzyme Sources Activity Optimize condition

pH T (◦C)

Alcalase Bacteria-Bacillus licheniformis 2.4 AU/g 8.0 50Flavourzyme Fungus-Aspergillus oryzae 500 APU/g 7.0 50

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2

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Neutrase Bacteria-Bacillus amyloliquefaciens

Trypsin (PTNTM) Porcine pancreasProtamex Bacteria-Bacillus

ime periods. After various treatments, the medium was removed and the cells werencubated with a solution of 0.5 mg/ml MTT. After incubation for 3 h at 37 ◦C and 5%O2, the supernatant was removed and the formation of formazan was observed byonitoring the signal at 540 nm using a microplate reader (PowerWave XS model;

ioTek Instruments, Inc., Winooski, VT, USA).

.6. Alkaline phosphatase (ALP) activity

Cells were seeded into 96-well plates at a density of 5 × 103 cells/well andultured for 24 h. The agent to be tested was added to the wells, and incubationontinued for 2 days. The cells were then washed three times with physiologicalaline, and cellular protein concentration was determined by incubation in B (bicin-honinic acid) protein assay reagent containing 0.1% Triton X-100 for 1 h at 37 ◦C. Theeaction was stopped by adding 1 M NaOH, and the absorbance measured at 560 nm.lkaline phosphatase activity in the cells was assayed after appropriate treatmenteriods by washing the cells three times with physiological saline, then measuringellular activity by incubation for 1 h at 37 ◦C in 0.1 M NaHCO3–Na2CO3 buffer, pH0, containing 0.1% Triton X-100, 1.5 mM MgCl2 and 15 mM p-nitrophenyl phos-hate (p-NPP). In the presence of ALP, p-NPP is transformed to para-nitrophenolnd inorganic phosphate. The ALP activity of the samples was determined fromhe absorbance at 405 nm using a spectrophotometer. ALP activity was calculatedollowing equation in which A and A0 were relative absorbance with and withoutample, respectively.

LP activity (%) = A − A0

A0× 100

.7. Mineralization

The degree of mineralization was determined in the 24-well plates using Alizarined S staining after 7 days treatment. Briefly, cells were fixed with 70% (v/v)thanol for 1 h and were then stained with 40 mM Alizarin Red S in deionizedater (pH = 4.2) for 15 min at room temperature. After removing Alizarin Red S

olution by aspiration, cells were incubated in PBS for 15 min at room temperaturen an orbital rotator. Then the cells were rinsed once with fresh PBS, and subse-uently destained for 15 min with 10% (w/v) cetylpyridinium chloride in 10 mModium phosphate (pH = 7.0). The extracted stain was then transferred to a 96-ell plate, and the absorbance at 562 nm was measured using a microplate reader

Tecan Austria GmbH, Austria). Activity was calculated following equation in which and A0 were relative absorbance with and without sample, respectively. Theineralization activities of peptide-treated groups were compared with that of

lank (not treated sample, but vitamin C (50 �g/ml/�-glycerophosphate (10 mM))reated).Mineralization level(%) = A−A0

A0× 100

.8. RT-PCR analysis

RNA was isolated with TRIzol reagent, and 1 �g of total RNA were reverse tran-cribed to cDNA using AMV reverse transcriptase. The oligonucleotides used forCR were: 5′-CCACGTCTTCACATTTGGTG-3′ (forward primer) and 5′-AGACTGCGCC-AGTAGTTGT-3′ (reverse primer) for human ALP; 5′-TTCACCACCACCATGGAG-AGGC-3′ (forward primer) and 5′-GGCATGGACTGTGGTCATGA-3′ (reverse primer)

or human GAPDH; 5′-ATGAGAGCCCTCACACTCCTC-3′ (forward primer) and′-GCCGTAGAAGCGCCGATAGGC-3′ (reverse primer) for osteocalcin; and 5′-CAGATTGAGACCCTCCTCA-3′ (forward primer) and 5′-ATGCAATGCTGTTCTTGCAG-′ (reverse primer) for collagen type I. PCR products were detected by 1.2% agaroseel electrophoresis and photographed.

.9. Western blot analysis

Western blotting was performed according to standard procedures. Briefly, cellsere lysed in RIPA buffer containing containing 50 mM Tris–HCl (pH 7.5), 0.4%

onidet P-40, 120 mM NaCl, 1.5 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride,0 �g/ml of leupeptin, 3 mM NaF and 1 mM DTT at 4 ◦C for 30 min. Cell lysates25 �g) were separated by 10% SDS-polyacrylamide gel electrophoresis, trans-erred onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech.,ngland, UK), blocked with 5% skim milk, and hybridized with primary antibodies

0.8 AU/g 6.0 501.35 AU/g 8.0 401.5 AU/g 6.0 40

(diluted 1:1000). After incubation with horseradish-peroxidase-conjugated sec-ondary antibodies (diluted 1:5000) at room temperature, immune-reactive proteinswere detected using a chemiluminescent ECL assay kit (Amersham Pharmacia Bio-sciences, England, UK) according to the manufacturer’s instructions. Western blotswere visualized using an LAS3000® Luminescent image analyzer and protein expres-sion was quantified by MULTI GAUGE V3.0 software (Fujifilm Life Science, Tokyo,Japan).

2.10. Statistical analysis

All data are presented as means ± SD. The mean values were calculated basedon the data taken from at least three independent experiments conducted on sepa-rate days using freshly prepared reagents. Statistical analyses were performed usingstudent’s t-test. The statistical significances were achieved when P < 0.05.

3. Results

3.1. Effects of enzymatic hydrolysates on cell viability andalkaline phosphatase activity

To produce ALP activity peptide, biodiesel byproduct of N. ocu-lata was separately hydrolyzed using various commercial digestiveenzymes. In this experiment, five proteolytic enzymes such asalcalase, flavourzyme, neutrase, PTN, and protamex, were selectedto evaluate their effectiveness on degradation of biodiesel byprod-uct of N. oculata for ALP activity. First, the cytotoxic effect of variousconcentrations of hydrolysates (BD-A, BD-F, BD-N, BD-PT, and BD-Pr) on human osteoblastic (MG-63) and murine mesenchymal stem(D1) cells were determined by MTT assay. As shown in Fig. 1A andB, five hydrolysates exhibited no significant effects on cell pro-liferation at the concentrations (10–1000 �g/ml) after treatmentto MG-63 and D1 cells for 24 h. So hydrolysate concentrations of20–500 �g/ml were used to examine their effects on osteoblasticcells differentiation.

In ALP activity assay (Fig. 1C and D), alcalase-proteolytichydrolysate (BD-A) which showed the highest ALP activity amongthe tested hydrolysates (Neutrase, Flavourzyme, PTN, and Pro-tamex) at a dose-dependent manner (20–500 �g/ml) after 48 h oftreatment in MG-63 and D-1 cells, but it has reached to peak atconcentration of BD-A 20 �g/ml in D1 cells (Fig. 1D). Therefore, weselected BD-A for the purification of the ALP activity peptide.

3.2. Purification profiles of ALP activity peptide from BD-A

The lyophilized BD-A was dissolved in 20 mM phosphate buffer(pH 6.8), and loaded onto a JAIGEL-W253 column. Elution peakswere monitored at 215 nm, and the signals were determined inUV and RI (refractive index) detector (Fig. 2A). The UV detectorselectively detects substances that readily absorb UV, while the RIdetector is versatile because of their high sensitivity even for sub-stances having no ability to absorb UV. The combination of thesetwo detectors enables the analysis and preparation for a wide rangeof substances. Thus, four fractions were collected for next purifi-

cation steps as in Fig. 2A. Each fraction was pooled, lyophilized,and its ALP activity was measured. Fraction II exhibited the high-est osteoblast differentiation by increasing ALP activity in both ofMG-63 and D1 cells compared with other fractions (Fig. 2B and C).

1390 M.H.T. Nguyen et al. / Process Biochemistry 48 (2013) 1387–1394

Fig. 1. Effects of hydrolysates on cell viability and alkaline phosphatase (ALP) activity in MG-63 and D1 cells. (A and B) Cytotoxic effect of various concentrations( 3 (A)

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10–1000 �g/ml) of hydrolysates (BD-A, BD-F, BD-N, BD-PT, and BD-Pr) on MG-6ALP) activity of hydrolysates. The MG-63 (C) and D 1 (D) cells were treated with vf triplicate cultures, and each bar indicates means ± S.D.

he lyophilized active fraction II was further separated by RP-HPLCn YMC pack pro C18 column with a linear gradient of acetoni-rile (0–60%), and one clear fraction was collected (Fig. 2D). Finally,ne peptide having potent activities potencies was purified. Themino acid sequence of purified peptide was determined to be Met-ro-Asp-Trp (529.2 Da, Fig. 2E). The peptide determined by ESI/MSpectroscopy was in excellent agreement with the theoretical massalculated from the sequence.

.3. Effect of purified peptide on mineralization in MG-63 and D1ells

ALP known as a marker for early stage of osteoblast differentia-ion, bone mineralization has to be known as terminal stage. In theresent study, bone mineralization, process by which organic tis-ue becomes hardened by the physiologic deposit of calcium salts,as quantification by optical density measurement of Alizarin Red-

extracted from stained culture cells in the presence of peptide.ig. 3 illustrates that both of MG-63 and D1 cells showed clearly redolor depend on increased concentrations (37.5–150 �M) of NOP.owever, mineralization signals are more strength in D1 cells than

and D1 (B) cells were determined by MTT assay. (C and D) Alkaline phosphatase concentrations (20–500 �g/ml) of hydrolysates for 48 h. Each value is the average

in MG-63 cells in the presence of NOP. The results indicated thatpeptide might induce osteoblastic cells differentiation in both earlyand terminal stages.

3.4. Effect of purified peptide on expression of several specificgenes in MG-63 and D1 cells

Some of the most frequently used markers of osteoblast dif-ferentiation process are alkaline phosphatase (ALP), collagen typeI, and osteocalcin. Type I collagen is the most abundant proteinof the bone matrix. Collagenase plays a critical function in boneremodeling. Osteocalcin and alkaline phosphatase (ALP) have to beknown as terminal and early marker of osteoblast differentiationprocess, respectively. Thus, we examined gene expression of thesemarkers in MG-63 and D1 cells. The results shown that purifiedpeptide resulted in increase of mRNA expression of all these genesin both cells in a dose-dependent manner (Fig. 4A). In addition,

the levels of type I collagen mRNA were more sensitive to peptidethan were the levels of ALP or osteocalcin mRNA. Thus, the resultsmight suggest positive effects of purified peptide on osteoblasticcells differentiation.

M.H.T. Nguyen et al. / Process Biochemistry 48 (2013) 1387–1394 1391

Fig. 2. Purification profiles of ALP activity peptide. (A) Recycling preparative HPLC pattern on a Jaigel W253 column of BD-A carried out with distilled water as the mobilephase, at a 2.5 ml/min flow rate, using refractive index (RI) and UV detector. (B) ALP activity of HPLC fractions in MG-63 cell was treated with various concentrations( ith vo ing 0.1t pectr

trreaFa

3

mptes

20–500 �g/ml) of for 48 h. (C) ALP activity of HPLC fractions in D1 cell was treated wn YMC pack pro C18 column of active fraction (FrVI) with 60% acetonitrile containhe molecular mass and amino acid sequence of the purified peptide using ESI/MS s

Binding of BMPs to preformed heteromeric receptor complexes,wo major types of membrane-bound serine/threonine kinaseeceptors subsequently known as the type-I and type-II receptors,esults in activation of MAPKs and Smad pathway. Thus, we firstlyxamined the expression of BMP-2 protein and mRNA in MG-63nd D1 cells in the presence of the purified peptide. As shown inig. 4B, the peptide resulted in significantly increase both of proteinnd gene expression.

.5. Effect of purified peptide on MAPK and Smad pathway

The common mechanism of osteoblast activating is seemlyediated by serine–threonine kinases of the mitogen-activated

rotein kinase (MAPK) and Smad family. Therefore, investigationhe effects of peptide on expression of Smad1/5/8 and three differ-nt MAPKs such as ERK, JNK, and p38 were performed. The resulthowed that phosphorylation of ERK, JNK, and p38 expressions are

arious concentrations (20–500 �g/ml) of for 48 h. (D) Reversed-phase HPLC pattern% TFA at a flow rate of 1 ml/min, using UV detector at 215 nm. (E) Identification of

oscopy.

significantly increased in the presence of peptide in both MG-63and D1 cells (Fig. 5A and B). The expressions of the phosphoryla-tion of these kinases were increased in a dose-dependent manner inMG-63 cells (Fig. 5A). However, they were reached to peak at con-centration 20 �g/ml and then decreased but still higher than blank(untreated) in case of phosphorylation of ERK and p38 in D1 cells(Fig. 5B). In addition, the presence of peptide was resulted in sig-nificantly increase the expression of phosphorylation of Smad1/5/8in both MG-63 and D1 cells (Fig. 5B). Hence, it could be suggestedthat the activation of osteoblastic differentiation was resulted fromMAPK and Smad mediated up-regulation.

4. Discussion

Although marine organisms are enormous promising resourceof bioactive substances and natural products, there are limitedreports on them. Marine organisms represent a valuable source of

1392 M.H.T. Nguyen et al. / Process Biochemistry 48 (2013) 1387–1394

Fig. 3. Effects of peptide on mineralization of MG-63 and D1 cells. Alizarin Red-Sstaining 7 days after cell culture; photography of representative stained cell culture.Tmb

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Fig. 4. (A) Effects of peptide on mRNA expression of several specific markers (ALP,osteocalcin, and collagen type 1) in osteoblast differentiation process in MG-63 andD1 cells. Cells were treated with different concentrations (37.5–150 �M) of peptide.GAPDH mRNA expression levels were used to confirm the equal amounts of RNAused for cDNA synthesis. (B) Expression of BMPs (BMP-2 and BMP2/4) gene andprotein in MG-63 and D1 cells. Cells were treated with different concentrations

he production of mineralization was increased by peptide in a dose dependentanner (37.5–150 �M). Each value is the average of triplicate cultures, and each

ar indicates means ± S.D.

ew compounds. In this study, we found out bioactive peptide fromicroalgae N. oculata that induced osteoblast differentiation. The

iscovery of the bio-regulatory role of different endogenous pep-ides in the organism as well as the understanding of the molecular

echanisms of action of some new bioactive peptides obtainedrom natural sources on specific cellular targets, contributed toonsider peptides also as promising lead drug candidates [19].unctional peptides, can be purified from enzymatic hydrolysis ofarious proteins, may act as potential physiological modulatorsf metabolism during intestinal digestion of nutrients [20]. Theseeptides exhibited various bioactivities such as antioxidative [16],ntihypertensive [21] and antimicrobial [22]. Bioactive peptidesrom sponges, ascidians, mollusks, sea anemones and seaweedsith their pharmacological properties and obtainment methods

re reviewed by Aneiros and Garateix [19]. Thus marine organismsontain potential of natural products for industry or pharmacy, and

great number of them are undiscovered.Protein can be broken down to their constituent amino acids

y a variety of methods. Enzymatic hydrolysis is widely utilized toeliorate their functional and nutritional properties [23]. Bioactive

eptides can be obtained from organisms proteins by enzymaticydrolysis of proteins [24]. In our study, biodiesel by-productsf microalgae N. oculata were hydrolyzed with various enzymesalcalase, flavourzyme, neutrase, PTN, and protamex) to extractsteoblast activating peptide. Among these enzyme hydrolysates,lcalase hydrolysate expressed the highest ALP activity and further

sed for purification. Alcalase has been used in the past for theroduction of biological peptides and it has endo-peptidase char-cteristics. Bioactive peptides produced by alcalase are resistanto digestive enzymes such as pepsin, trypsin and �-chymotrypsin,

(37.5–150 �M) of peptide. GAPDH mRNA expression levels were used to confirmthe equal amounts of RNA used for cDNA synthesis. �-Actin was used as proteinexpressions control.

which would allow for absorption of peptides contained in thesort of hydrolysates. Moreover, alcalase produce shorter peptidesequences as well as terminal amino acid sequences responsiblefor various bioactivities [20].

Alcalase hydrolysate was subjected to purification using consec-utive HPLC. Amino acid sequence was identified using ESI/MS andshown to be Met-Pro-Asp-Trp (529.2 Da). Methionine, one of thetwo sulfur-containing amino acids, has a non-polar thio ether groupin its side chain. Proline has an aliphatic side chain with a distinc-tive cyclic structure. The secondary amino group of proline residuesis held in a rigid conformation that reduces the structural flexibil-ity of polypeptide regions containing proline. Proline is known tobe prevented digestion of enzymes and may pass from the capil-lary into the circulation of blood in the sequence of short peptides[25,26]. Aspartic acid is an acidic amino acid, which has a secondcarboxyl group. Tryptophan, with its aromatic side chains, is rel-atively nonpolar (hydrophobic). It can participate in hydrophobicinteractions. Tryptophan is significantly more polar than phenyl-alanine because of the nitrogen of the tryptophan indole ring. Thediverse biological activities of functional peptides depend on struc-tural properties and molecular size of precursor proteins, aminoacid compositions, or free amino acids [27]. The sequence of puri-fied peptide is a short peptide (oligopeptide) and has three per

fourth hydrophobic amino acids. Recent research has indicated thathigh levels of hydrophobic and aromatic amino acids may aid inhealing after multiple trauma. Oligopeptides are frequently syn-thesized, and their biological activity is assessed. Short peptides

M.H.T. Nguyen et al. / Process Bioche

Fig. 5. Effects of peptide on MAPK and Smad pathway in MG-63 and D1 cells. Cellswere treated with different concentrations (37.5–150 �M) of peptide for 24 h. (A)Equal amounts of protein in the cell lysates were electrophoresed and subjected toWestern blotting with antibodies specific to p-ERK, ERK, p-JNK, JNK, p-p38, and p38.(B) Equal amounts of protein in the cell lysates were electrophoresed and subjectedto Western blotting with antibodies specific to Smad1/5/8 and phosphorylation ofS

aoid

taem[cel(om

Wnt, & transforming growth factor-� signaling pathways in bone morphogenicprotein 2-induced osteogenesis. Notch target gene Heyl inhibits mineralization

mad 1/5/8. �-actin was used as control.

re likely to be extremely dynamic in solution. They were modeledn the cytoplasmic regions of transmembrane proteins and weredentified from an evolutionary sequence analysis of specificity-etermining and conserved residues [28].

Mesenchymal stem cells, or MSCs, are multipotent stem cellshat can differentiate into cells of the mesodermal lineage, such asdipocytes, osteocytes and chondrocytes, as well as cells of othermbryonic lineages. Mesenchymal stem cells derived from bonearrow have been used to repair skeletal bone and hard tissues

29,30]. In this study, we differentiated murine mesenchymal stemells (D1 cells) into osteoblast cells and used to investigate theffects of the purified peptide from biodiesel by-products of N. ocu-ata on osteoblast differentiation, together with osteoblastic cells

MG-63). The results indicated that peptide resulted in activation ofsteoblast differentiation in both of these cells. In addition, peptideight effect on osteoblast differentiation at lower concentrations

mistry 48 (2013) 1387–1394 1393

in normal cell (D1) than in tumor cell (MG-63) as the results of ALPactivity and Western blot analysis for MAPKs and Smads.

MAPKs and Smad play important roles in regulation ofosteoblast differentiation. MAPKs families play an important role incomplex cellular programs like proliferation, differentiation, devel-opment, transformation, and apoptosis. MAPK signaling also has acritical role in osteoblast differentiation. During the bone differen-tiation of osteogenic cells, the MAPK signaling cascade is activatedalong with the expression of various osteomarkers. Some publica-tions have reported the critical role of MAPKs in osteogenesis, suchas activation of phosphorylation of ERK (p-ERK), p-p38, and p-JNKare required for osteoblast differentiation [31]. Various osteogenicstimuli, including BMP-2, are known to induce the expression ofosteomarkers such as alkaline phosphatase (ALP), osteocalcin alongwith the activation of MAPK signaling molecules [32].

Smad play important roles in osteoblast differentiation. TheBMP-specific R-Smads, Smad 1, 5, and 8, transiently and directlyinteract with activated BMPR-Is and become phosphorylated atSSXS motifs at their C termini. The phosphorylation of Smad1/5/8,which is usually triggered by the BMP-dependent oligomerizationof BMP receptors type I (BMP-R1) and type II (BMP-R2), are nec-essary for osteoblast differentiation [33,34]. The Smad pathwayis a well-characterized BMP signaling pathway. However, BMPsalso initiate non-Smad interacellular signaling pathways. Hence,it could be suggested that the inducement of osteoblast differenti-ation was resulted from phosphorylation of MAPKs (p-ERK, p-JNK,p-p38) and Smads mediated up-regulation.

5. Conclusion

In our study, we sought to investigate the effects of purifiedpeptide derived from microalgae N. oculata on osteoblast differenti-ation in human osteoblast (MG-63) and murine mesenchymal stem(D1) cells. The presence of peptide significantly induced osteoblas-tic cell differentiation in both of early and terminal stage by increasethe levels of alkaline phosphatase, collagen type I, osteocalcin, BMP-2, BMP2/4 and bone mineralization in both cells. Furthermore,peptide increased phosphorylation of MAP kinases (p-ERK, p-JNK,p-p38) and Smads (p-Smad1/5/8) expression. From the results, inorder to take more evidences to evaluate positively for its applica-tion in bone health supplement, its bioavailability will be examinedby further in vivo studies.

Acknowledgements

This research was supported by Fishery CommercializationTechnology Development Program, Ministry of Agriculture, Foodand Rural Affairs, and Basic Science Research Program throughthe National Research Foundation of Korea (NRF) funded by theMinistry of Science, ICT and Future Planning (2013-013577). Thisresearch was also supported by the Korea Institute of Ocean Science& Technology (PE98931).

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