1. Process Biochemistry 48 (2013) 1387–1394
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Tetrameric peptide purified from hydrolysates of biodiesel byproducts
of Nannochloropsis oculata induces osteoblastic differentiation
through MAPK and Smad pathway on MG-63 and D1 cells
Minh Hong Thi Nguyen a,1 , Zhong-Ji Qian b,1 , Van-Tinh Nguyen b , Il-Whan Choi c ,
Soo-Jin Heo d , Chul Hong Oh d , Do-Hyung Kang d , Geun Hyung Kim e,∗∗ , Won-Kyo Jung b,∗
a
Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Vietnam
Department of Marine Life Science, and Marine Life Research and Education Center, Chosun University, Gwangju 501-759, Republic of Korea
c
Department of Microbiology, Inje University College of Medicine, Busan 614-735, Republic of Korea
d
Global Bioresources Research Center, Korea Institute of Ocean Science & Technology, Ansan 426-744, Republic of Korea
e
Department of Bio-Mechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea
b
a r t i c l e
i n f o
Article history:
Received 23 March 2013
Received in revised form 31 May 2013
Accepted 28 June 2013
Available online 6 July 2013
Keywords:
Marine microalgae
Nannochloropsis oculata
Biodiesel byproducts
Osteoblast differentiation
Purified peptide
a b s t r a c t
Ongoing efforts to search for bioactive substances for bone diseases have led to the discovery of natural
products with substantial bioactive properties. In this present study, an osteoblast activating-peptide
was isolated from biodiesel by-products of microalgae, Nannochloropsis oculata. To utilize biodiesel byproducts of N. oculata and evaluate their beneficial effects, enzymatic hydrolysis was carried out using
commercial enzymes such as alcalase, flavourzyme, neutrase, trypsin (PTNTM ), protamex and alcalase
hydrolysate exhibited the highest osteoblastic differentiation activity. Using consecutive purification by
liquid chromatographic techniques, an osteoblast-differentiatory peptide was purified and identified to
be a peptide (MPDW, 529.2 Da) by the tandem MS analysis. The results showed that purified peptide
promotes osteoblast differentiation by increasing expression of several osteoblast phenotype markers
such as alkaline phosphatase (ALP), osteocalcin, collagen type I, BMP-2, BMP2/4 and bone mineralization
in both human osteoblastic cell (MG-63) and murine mesenchymal stem cell (D1). In addition, the purified
peptide induced phosphorylation of MAPK and Smad pathway in both cells. These results suggest that
peptide possesses positive effects on osteoblast differentiation and may provide possibility for treating
bone diseases.
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Osteoporosis is a systemic skeletal disease characterized by low
bone mineral density and micro-architectural deterioration of bone
tissue, consequently resulting in increases in bone fragility and
susceptibility to fracture. According to the World Health Organization (WHO) definition, osteoporosis affects 30% of post-menopausal
women, a proportion that rises to 70% in women aged over 80
years [1]. In addition, some drugs routinely used for treating of
multiple diseases have detrimental effects on either skeleton or
induce 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.kr
(W.-K. Jung).
1
These authors contributed equally to this study.
1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.06.031
is greater than bone formation, has becoming an important public
health problem leading to an increased risk of developing spontaneous and traumatic fractures. This disease can be treated via
osteoclast and/or osteoblast action [3]. Therefore, drugs that would
act via promoting bone formation (osteoblast) and/or inhibiting
bone resorption (osteoclast) could be a tool for desirable therapy.
Process of osteoblast differentiation is necessary for bone
strength and remodeling, and can be subdivided in three subsequent stages including proliferation stage, extracellular matrix
synthesis and maturation stage, and mineralization stage [4]. During osteoblast differentiation, phenotype markers such as alkaline
phosphatase (ALP), collagen type I, and osteocalcin concomitantly with mineralization are highly expressed, and osteoblast
differentiation is regulated by various signaling pathways such
as 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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M.H.T. Nguyen et al. / Process Biochemistry 48 (2013) 1387–1394
cellular programs like proliferation, differentiation, development,
transformation, and apoptosis [9]. During the bone differentiation of osteogenic cells, the MAPK signaling cascade is activated
along with the expression of various osteomarkers [10]. Smads
play important roles in osteoblast differentiation. The BMP-specific
R-Smads, Smad1, 5, and 8, transiently and directly interact with
activated BMPRs (BMP receptors) and become phosphorylated.
Smad1/5/8 then form heteromeric complexes with Smad4 (CoSmad) and translocate into the nucleus where they regulate
transcription of various target genes. The Smad pathway is a wellcharacterized BMP signaling pathway. However, BMPs also initiate
non-Smad interacellular signaling pathways. Several lines of evidence suggested that BMPs activate the MAPK family of signaling
molecules, i.e., ERK1/2, p38, and stress-activated protein kinase/Jun
N-terminal kinase [6].
Marine microalgae, or phytoplankton, are mostly represented
in ocean populations, and the best known are the diatoms
(Bacillariophyta), the dinoflagellates (Dinophyta), the green algae
(Chlorophyta) and the blue-green algae (Cyanophyta). The unicellular marine microalgae were considered to be an abounding
resource for carotenoids, lipids, and polysaccharides, and were
widely investigated in the fields of food supplements and bio-fuel
production [11]. While terrestrial plants in temperate climates can
achieve a photoconversion efficiency of only below 1%, microalgae
can convert up to 5% of the solar energy into chemical energy [12].
Microalgae are efficient in converting solar energy into metabolites,
such as lipids, proteins, carbohydrates, pigments and vitamins.
Moreover, microalgae can provide various types of renewable
energy sources, such as methane, bio-hydrogen, and biodiesel [13].
Thus, they can apply in food, feed, natural compounds, or aquacultures field as well as in biofuel. Nanochloropsis oculata (N. oculata) is
microalgae that mostly been known from the marine environment
but also occur in fresh and brackish water.
N. oculata is able to build up a high concentration of a range of
pigments such as astaxanthin, zeaxanthin and canthaxanthin, and
has been shown to be suitable for algal biofuel production due to its
ease of growth and high oil content (28.7% of dry weight), mainly
unsaturated fatty acids and a significant percentage of palmitic acid.
It also contains enough unsaturated fatty acid, linolenic acid and
polyunsaturated acid for a quality biodiesel. Morever, N. oculata
has a high rate of protein as well as carbohydrate [14]. Thus, the
use of N. oculata has been recognized for human diets and biomass,
and the beneficial effect of reducing blood pressure in hypertensive rats have demonstrates by feeding experiments [15]. Many
studies have been reported that marine bioactive peptides can be
used as functional foods, nutraceuticals, or pharmaceuticals due to
their therapeutic potential in the treatment or prevention of various diseases [16]. Peptides-derived from marine microalgae have
potential activity on antioxidant, antihypertensive, anticancer [17].
N. oculata possess a numerous of protein which break down to peptide, amino acid, or protein fractions by protease hydrolysis. In this
study, a peptide purified from biodiesel by-products of N. oculata
after applying to enzymatic hydrolysis and induced osteoblast differentiation in osteoblastic (MG-63) and mesenchymal stem cells
(D1) by activation of mitogen-activated protein kinases (MAPKs)
and Smads pathways.
Technologies (USA). MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide)
reagent, Alizarin red-S, p-nitrophenyl phosphate (p-NPP), cetylpyridinium chloride monohydrate and dexamethasone were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). Digestive proteases (Alcalase, Neutrase, Flavourzyme, Trypsin
Novo (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 from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Amersham Pharmacia Biosciences (Piscataway, NJ, USA), respectively. All other reagents used in this study
were analytical grade chemicals.
2. Materials and methods
Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing
5% FBS at 37 ◦ C in a 5% CO2 humidified incubator. To induce osteogenic differentiation in D1 cells, culture media were changed at 3 days to ODM (DMEM
supplemented with 50 ␮g/ml ascorbic acid, 10−8 M dexamethasone, and 10 mM ␤glycerolphosphate). After culture for another 3 days, one group was cultured only
for 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 of
5 × 103 cells/well in the presence of different concentrations of samples. Cell viability was determined by use of the MTT assay. MTT was used as an indicator of
cell viability as determined by mitochondrial-dependent reduction to formazan. In
brief, the cells were seeded and then treated with various reagents for the indicated
2.1. Materials
Human osteosarcoma (MG-63) was obtained from American Tissue Culture
Collection (ATCC). Murine mesenchymal stem cells (D1) were purchased from
ATCC (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and
fetal bovine serum (FBS) was obtained from Gibco-BRL, Life Technologies (Grand
Island, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM), trypsin–EDTA,
penicillin/streptomycin/amphotericin (10,000 U/ml, 10,000 ␮g/ml, and 2500 ␮g/ml,
respectively), and fetal bovine serum (FBS) were obtained from Gibco BRL, Life
2.2. Preparation of enzyme hydrolysates of biodiesel byproducts from microalgae,
N. oculata
The microalgae, N. oculata was originally obtained from the National Research
Institute of Aquaculture Fisheries Research Agency (Kanagawa, Japan) and screened
for 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 times
with 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 was
added to three times volume of 0.9% NaCl solution. The bottom layer of extracts was
used 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. Optimum
hydrolysis 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 slight
modifications to prepare the enzymatic hydrolysates from biodiesel byproducts of
marine microalgae. At enzyme/substrate ratio of 1/100 (w/w), 1% substrate and
enzyme were mixed. Thereafter, the pH of the each hydrolysate was adjusted to pH 7
with 1 M HCl/NaOH. The substrates and each enzyme mixture were incubated for 8 h
at each optimal temperature with stirring and then heated in a boiling water bath
for 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 monitored
at 215 nm, and dried using a lyophilizer under −80 ◦ C. Osteoblast differentiation
activity was also investigated, and a highest osteoblast activating fraction was determined 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 YMC
pack pro C18 column with a linear gradient of acetonitrile (0–60% in 60 min) at a
flow rate of 1.0 ml/min. Elution peaks was detected at 215 nm. Potent peaks were
collected, lyophilized, and then evaluated osteoblast differentiation activity. The
finally 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 were
determined with a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled
to an electrospray ionization (ESI) source. The purified peptide was injected into
the electrospray source following dissolution in methanol/water (1:1, v/v), and its
molecular mass was determined by a doubly charged (M+2H)2+ state in the mass
spectrum. Following molecular mass determination, the peptide was automatically
selected for fragmentation and its sequence information was obtained by tandem
mass spectroscopy (MS) analysis.
2.5. Culture of cells and viability determination

3. M.H.T. Nguyen et al. / Process Biochemistry 48 (2013) 1387–1394
1389
Table 1
Characteristics and optimum hydrolysis conditions of the specific enzymes.
Enzyme
Sources
Activity
Optimize condition
pH
Alcalase
Flavourzyme
Neutrase
Trypsin (PTNTM )
Protamex
Bacteria-Bacillus licheniformis
Fungus-Aspergillus oryzae
Bacteria-Bacillus amyloliquefaciens
Porcine pancreas
Bacteria-Bacillus
time periods. After various treatments, the medium was removed and the cells were
incubated with a solution of 0.5 mg/ml MTT. After incubation for 3 h at 37 ◦ C and 5%
CO2 , the supernatant was removed and the formation of formazan was observed by
monitoring the signal at 540 nm using a microplate reader (PowerWave XS model;
BioTek Instruments, Inc., Winooski, VT, USA).
2.6. Alkaline phosphatase (ALP) activity
Cells were seeded into 96-well plates at a density of 5 × 103 cells/well and
cultured for 24 h. The agent to be tested was added to the wells, and incubation
continued for 2 days. The cells were then washed three times with physiological
saline, and cellular protein concentration was determined by incubation in B (bicinchoninic acid) protein assay reagent containing 0.1% Triton X-100 for 1 h at 37 ◦ C. The
reaction was stopped by adding 1 M NaOH, and the absorbance measured at 560 nm.
Alkaline phosphatase activity in the cells was assayed after appropriate treatment
periods by washing the cells three times with physiological saline, then measuring
cellular activity by incubation for 1 h at 37 ◦ C in 0.1 M NaHCO3 –Na2 CO3 buffer, pH
10, containing 0.1% Triton X-100, 1.5 mM MgCl2 and 15 mM p-nitrophenyl phosphate (p-NPP). In the presence of ALP, p-NPP is transformed to para-nitrophenol
and inorganic phosphate. The ALP activity of the samples was determined from
the absorbance at 405 nm using a spectrophotometer. ALP activity was calculated
following equation in which A and A0 were relative absorbance with and without
sample, respectively.
ALP activity (%) =
A − A0
× 100
A0
2.7. Mineralization
The degree of mineralization was determined in the 24-well plates using Alizarin
Red S staining after 7 days treatment. Briefly, cells were fixed with 70% (v/v)
ethanol for 1 h and were then stained with 40 mM Alizarin Red S in deionized
water (pH = 4.2) for 15 min at room temperature. After removing Alizarin Red S
solution by aspiration, cells were incubated in PBS for 15 min at room temperature
on an orbital rotator. Then the cells were rinsed once with fresh PBS, and subsequently destained for 15 min with 10% (w/v) cetylpyridinium chloride in 10 mM
sodium phosphate (pH = 7.0). The extracted stain was then transferred to a 96well plate, and the absorbance at 562 nm was measured using a microplate reader
(Tecan Austria GmbH, Austria). Activity was calculated following equation in which
A and A0 were relative absorbance with and without sample, respectively. The
mineralization activities of peptide-treated groups were compared with that of
blank (not treated sample, but vitamin C (50 ␮g/ml/␤-glycerophosphate (10 mM))
A−A
treated).Mineralization level(%) = A 0 × 100
0
2.8. RT-PCR analysis
RNA was isolated with TRIzol reagent, and 1 ␮g of total RNA were reverse transcribed to cDNA using AMV reverse transcriptase. The oligonucleotides used for
PCR were: 5 -CCACGTCTTCACATTTGGTG-3 (forward primer) and 5 -AGACTGCGCCTAGTAGTTGT-3 (reverse primer) for human ALP; 5 -TTCACCACCACCATGGAGAAGGC-3 (forward primer) and 5 -GGCATGGACTGTGGTCATGA-3 (reverse primer)
for human GAPDH; 5 -ATGAGAGCCCTCACACTCCTC-3 (forward primer) and
5 -GCCGTAGAAGCGCCGATAGGC-3 (reverse primer) for osteocalcin; and 5 CCAGATTGAGACCCTCCTCA-3 (forward primer) and 5 -ATGCAATGCTGTTCTTGCAG3 (reverse primer) for collagen type I. PCR products were detected by 1.2% agarose
gel electrophoresis and photographed.
2.9. Western blot analysis
Western blotting was performed according to standard procedures. Briefly, cells
were lysed in RIPA buffer containing containing 50 mM Tris–HCl (pH 7.5), 0.4%
Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2 , 2 mM phenylmethylsulfonyl fluoride,
80 ␮g/ml of leupeptin, 3 mM NaF and 1 mM DTT at 4 ◦ C for 30 min. Cell lysates
(25 ␮g) were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech.,
England, UK), blocked with 5% skim milk, and hybridized with primary antibodies
2.4 AU/g
500 APU/g
0.8 AU/g
1.35 AU/g
1.5 AU/g
T (◦ C)
8.0
7.0
6.0
8.0
6.0
50
50
50
40
40
(diluted 1:1000). After incubation with horseradish-peroxidase-conjugated secondary antibodies (diluted 1:5000) at room temperature, immune-reactive proteins
were detected using a chemiluminescent ECL assay kit (Amersham Pharmacia Biosciences, England, UK) according to the manufacturer’s instructions. Western blots
were visualized using an LAS3000® Luminescent image analyzer and protein expression 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 based
on the data taken from at least three independent experiments conducted on separate days using freshly prepared reagents. Statistical analyses were performed using
student’s t-test. The statistical significances were achieved when P < 0.05.
3. Results
3.1. Effects of enzymatic hydrolysates on cell viability and
alkaline phosphatase activity
To produce ALP activity peptide, biodiesel byproduct of N. oculata was separately hydrolyzed using various commercial digestive
enzymes. In this experiment, five proteolytic enzymes such as
alcalase, flavourzyme, neutrase, PTN, and protamex, were selected
to evaluate their effectiveness on degradation of biodiesel byproduct of N. oculata for ALP activity. First, the cytotoxic effect of various
concentrations of hydrolysates (BD-A, BD-F, BD-N, BD-PT, and BDPr) on human osteoblastic (MG-63) and murine mesenchymal stem
(D1) cells were determined by MTT assay. As shown in Fig. 1A and
B, five hydrolysates exhibited no significant effects on cell proliferation at the concentrations (10–1000 ␮g/ml) after treatment
to MG-63 and D1 cells for 24 h. So hydrolysate concentrations of
20–500 ␮g/ml were used to examine their effects on osteoblastic
cells differentiation.
In ALP activity assay (Fig. 1C and D), alcalase-proteolytic
hydrolysate (BD-A) which showed the highest ALP activity among
the tested hydrolysates (Neutrase, Flavourzyme, PTN, and Protamex) at a dose-dependent manner (20–500 ␮g/ml) after 48 h of
treatment in MG-63 and D-1 cells, but it has reached to peak at
concentration of BD-A 20 ␮g/ml in D1 cells (Fig. 1D). Therefore, we
selected 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 peaks
were monitored at 215 nm, and the signals were determined in
UV and RI (refractive index) detector (Fig. 2A). The UV detector
selectively detects substances that readily absorb UV, while the RI
detector is versatile because of their high sensitivity even for substances having no ability to absorb UV. The combination of these
two detectors enables the analysis and preparation for a wide range
of substances. Thus, four fractions were collected for next purification steps as in Fig. 2A. Each fraction was pooled, lyophilized,
and its ALP activity was measured. Fraction II exhibited the highest osteoblast differentiation by increasing ALP activity in both of
MG-63 and D1 cells compared with other fractions (Fig. 2B and C).

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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
(10–1000 ␮g/ml) of hydrolysates (BD-A, BD-F, BD-N, BD-PT, and BD-Pr) on MG-63 (A) and D1 (B) cells were determined by MTT assay. (C and D) Alkaline phosphatase
(ALP) activity of hydrolysates. The MG-63 (C) and D 1 (D) cells were treated with various concentrations (20–500 ␮g/ml) of hydrolysates for 48 h. Each value is the average
of triplicate cultures, and each bar indicates means ± S.D.
The lyophilized active fraction II was further separated by RP-HPLC
on YMC pack pro C18 column with a linear gradient of acetonitrile (0–60%), and one clear fraction was collected (Fig. 2D). Finally,
one peptide having potent activities potencies was purified. The
amino acid sequence of purified peptide was determined to be MetPro-Asp-Trp (529.2 Da, Fig. 2E). The peptide determined by ESI/MS
spectroscopy was in excellent agreement with the theoretical mass
calculated from the sequence.
3.3. Effect of purified peptide on mineralization in MG-63 and D1
cells
ALP known as a marker for early stage of osteoblast differentiation, bone mineralization has to be known as terminal stage. In the
present study, bone mineralization, process by which organic tissue becomes hardened by the physiologic deposit of calcium salts,
was quantification by optical density measurement of Alizarin RedS extracted from stained culture cells in the presence of peptide.
Fig. 3 illustrates that both of MG-63 and D1 cells showed clearly red
color depend on increased concentrations (37.5–150 ␮M) of NOP.
However, mineralization signals are more strength in D1 cells than
in MG-63 cells in the presence of NOP. The results indicated that
peptide might induce osteoblastic cells differentiation in both early
and terminal stages.
3.4. Effect of purified peptide on expression of several specific
genes in MG-63 and D1 cells
Some of the most frequently used markers of osteoblast differentiation process are alkaline phosphatase (ALP), collagen type
I, and osteocalcin. Type I collagen is the most abundant protein
of the bone matrix. Collagenase plays a critical function in bone
remodeling. Osteocalcin and alkaline phosphatase (ALP) have to be
known as terminal and early marker of osteoblast differentiation
process, respectively. Thus, we examined gene expression of these
markers in MG-63 and D1 cells. The results shown that purified
peptide resulted in increase of mRNA expression of all these genes
in both cells in a dose-dependent manner (Fig. 4A). In addition,
the levels of type I collagen mRNA were more sensitive to peptide
than were the levels of ALP or osteocalcin mRNA. Thus, the results
might suggest positive effects of purified peptide on osteoblastic
cells differentiation.

5. 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 mobile
phase, 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
(20–500 ␮g/ml) of for 48 h. (C) ALP activity of HPLC fractions in D1 cell was treated with various concentrations (20–500 ␮g/ml) of for 48 h. (D) Reversed-phase HPLC pattern
on YMC pack pro C18 column of active fraction (FrVI) with 60% acetonitrile containing 0.1% TFA at a flow rate of 1 ml/min, using UV detector at 215 nm. (E) Identification of
the molecular mass and amino acid sequence of the purified peptide using ESI/MS spectroscopy.
Binding of BMPs to preformed heteromeric receptor complexes,
two major types of membrane-bound serine/threonine kinase
receptors subsequently known as the type-I and type-II receptors,
results in activation of MAPKs and Smad pathway. Thus, we firstly
examined the expression of BMP-2 protein and mRNA in MG-63
and D1 cells in the presence of the purified peptide. As shown in
Fig. 4B, the peptide resulted in significantly increase both of protein
and gene expression.
3.5. Effect of purified peptide on MAPK and Smad pathway
The common mechanism of osteoblast activating is seemly
mediated by serine–threonine kinases of the mitogen-activated
protein kinase (MAPK) and Smad family. Therefore, investigation
the effects of peptide on expression of Smad1/5/8 and three different MAPKs such as ERK, JNK, and p38 were performed. The result
showed that phosphorylation of ERK, JNK, and p38 expressions are
significantly increased in the presence of peptide in both MG-63
and D1 cells (Fig. 5A and B). The expressions of the phosphorylation of these kinases were increased in a dose-dependent manner in
MG-63 cells (Fig. 5A). However, they were reached to peak at concentration 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 significantly increase the expression of phosphorylation of Smad1/5/8
in both MG-63 and D1 cells (Fig. 5B). Hence, it could be suggested
that the activation of osteoblastic differentiation was resulted from
MAPK and Smad mediated up-regulation.
4. Discussion
Although marine organisms are enormous promising resource
of bioactive substances and natural products, there are limited
reports on them. Marine organisms represent a valuable source of

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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-S
staining 7 days after cell culture; photography of representative stained cell culture.
The production of mineralization was increased by peptide in a dose dependent
manner (37.5–150 ␮M). Each value is the average of triplicate cultures, and each
bar indicates means ± S.D.
new compounds. In this study, we found out bioactive peptide from
microalgae N. oculata that induced osteoblast differentiation. The
discovery of the bio-regulatory role of different endogenous peptides in the organism as well as the understanding of the molecular
mechanisms of action of some new bioactive peptides obtained
from natural sources on specific cellular targets, contributed to
consider peptides also as promising lead drug candidates [19].
Functional peptides, can be purified from enzymatic hydrolysis of
various proteins, may act as potential physiological modulators
of metabolism during intestinal digestion of nutrients [20]. These
peptides exhibited various bioactivities such as antioxidative [16],
antihypertensive [21] and antimicrobial [22]. Bioactive peptides
from sponges, ascidians, mollusks, sea anemones and seaweeds
with their pharmacological properties and obtainment methods
are reviewed by Aneiros and Garateix [19]. Thus marine organisms
contain potential of natural products for industry or pharmacy, and
a great number of them are undiscovered.
Protein can be broken down to their constituent amino acids
by a variety of methods. Enzymatic hydrolysis is widely utilized to
meliorate their functional and nutritional properties [23]. Bioactive
peptides can be obtained from organisms proteins by enzymatic
hydrolysis of proteins [24]. In our study, biodiesel by-products
of microalgae N. oculata were hydrolyzed with various enzymes
(alcalase, flavourzyme, neutrase, PTN, and protamex) to extract
osteoblast activating peptide. Among these enzyme hydrolysates,
alcalase hydrolysate expressed the highest ALP activity and further
used for purification. Alcalase has been used in the past for the
production of biological peptides and it has endo-peptidase characteristics. Bioactive peptides produced by alcalase are resistant
to digestive enzymes such as pepsin, trypsin and ␣-chymotrypsin,
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 and
D1 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 RNA
used for cDNA synthesis. (B) Expression of BMPs (BMP-2 and BMP2/4) gene and
protein in MG-63 and D1 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 RNA used for cDNA synthesis. ␤-Actin was used as protein
expressions control.
which would allow for absorption of peptides contained in the
sort of hydrolysates. Moreover, alcalase produce shorter peptide
sequences as well as terminal amino acid sequences responsible
for various bioactivities [20].
Alcalase hydrolysate was subjected to purification using consecutive HPLC. Amino acid sequence was identified using ESI/MS and
shown to be Met-Pro-Asp-Trp (529.2 Da). Methionine, one of the
two sulfur-containing amino acids, has a non-polar thio ether group
in its side chain. Proline has an aliphatic side chain with a distinctive cyclic structure. The secondary amino group of proline residues
is held in a rigid conformation that reduces the structural flexibility of polypeptide regions containing proline. Proline is known to
be prevented digestion of enzymes and may pass from the capillary into the circulation of blood in the sequence of short peptides
[25,26]. Aspartic acid is an acidic amino acid, which has a second
carboxyl group. Tryptophan, with its aromatic side chains, is relatively nonpolar (hydrophobic). It can participate in hydrophobic
interactions. Tryptophan is significantly more polar than phenylalanine because of the nitrogen of the tryptophan indole ring. The
diverse biological activities of functional peptides depend on structural properties and molecular size of precursor proteins, amino
acid compositions, or free amino acids [27]. The sequence of purified peptide is a short peptide (oligopeptide) and has three per
fourth hydrophobic amino acids. Recent research has indicated that
high levels of hydrophobic and aromatic amino acids may aid in
healing after multiple trauma. Oligopeptides are frequently synthesized, and their biological activity is assessed. Short peptides

7. M.H.T. Nguyen et al. / Process Biochemistry 48 (2013) 1387–1394
1393
in normal cell (D1) than in tumor cell (MG-63) as the results of ALP
activity and Western blot analysis for MAPKs and Smads.
MAPKs and Smad play important roles in regulation of
osteoblast differentiation. MAPKs families play an important role in
complex cellular programs like proliferation, differentiation, development, transformation, and apoptosis. MAPK signaling also has a
critical role in osteoblast differentiation. During the bone differentiation of osteogenic cells, the MAPK signaling cascade is activated
along with the expression of various osteomarkers. Some publications have reported the critical role of MAPKs in osteogenesis, such
as activation of phosphorylation of ERK (p-ERK), p-p38, and p-JNK
are required for osteoblast differentiation [31]. Various osteogenic
stimuli, including BMP-2, are known to induce the expression of
osteomarkers such as alkaline phosphatase (ALP), osteocalcin along
with the activation of MAPK signaling molecules [32].
Smad play important roles in osteoblast differentiation. The
BMP-specific R-Smads, Smad 1, 5, and 8, transiently and directly
interact with activated BMPR-Is and become phosphorylated at
SSXS motifs at their C termini. The phosphorylation of Smad1/5/8,
which is usually triggered by the BMP-dependent oligomerization
of BMP receptors type I (BMP-R1) and type II (BMP-R2), are necessary for osteoblast differentiation [33,34]. The Smad pathway
is a well-characterized BMP signaling pathway. However, BMPs
also initiate non-Smad interacellular signaling pathways. Hence,
it could be suggested that the inducement of osteoblast differentiation was resulted from phosphorylation of MAPKs (p-ERK, p-JNK,
p-p38) and Smads mediated up-regulation.
5. Conclusion
Fig. 5. Effects of peptide on MAPK and Smad pathway in MG-63 and D1 cells. Cells
were 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 to
Western 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 subjected
to Western blotting with antibodies specific to Smad1/5/8 and phosphorylation of
Smad 1/5/8. ␤-actin was used as control.
are likely to be extremely dynamic in solution. They were modeled
on the cytoplasmic regions of transmembrane proteins and were
identified from an evolutionary sequence analysis of specificitydetermining and conserved residues [28].
Mesenchymal stem cells, or MSCs, are multipotent stem cells
that can differentiate into cells of the mesodermal lineage, such as
adipocytes, osteocytes and chondrocytes, as well as cells of other
embryonic lineages. Mesenchymal stem cells derived from bone
marrow have been used to repair skeletal bone and hard tissues
[29,30]. In this study, we differentiated murine mesenchymal stem
cells (D1 cells) into osteoblast cells and used to investigate the
effects of the purified peptide from biodiesel by-products of N. oculata on osteoblast differentiation, together with osteoblastic cells
(MG-63). The results indicated that peptide resulted in activation of
osteoblast differentiation in both of these cells. In addition, peptide
might effect on osteoblast differentiation at lower concentrations
In our study, we sought to investigate the effects of purified
peptide derived from microalgae N. oculata on osteoblast differentiation in human osteoblast (MG-63) and murine mesenchymal stem
(D1) cells. The presence of peptide significantly induced osteoblastic cell differentiation in both of early and terminal stage by increase
the levels of alkaline phosphatase, collagen type I, osteocalcin, BMP2, 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, in
order to take more evidences to evaluate positively for its application in bone health supplement, its bioavailability will be examined
by further in vivo studies.
Acknowledgements
This research was supported by Fishery Commercialization
Technology Development Program, Ministry of Agriculture, Food
and Rural Affairs, and Basic Science Research Program through
the National Research Foundation of Korea (NRF) funded by the
Ministry of Science, ICT and Future Planning (2013-013577). This
research was also supported by the Korea Institute of Ocean Science
& Technology (PE98931).
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