Food and Agricultural Immunology

ISSN: 0954-0105 (Print) 1465-3443 (Online) Journal homepage: https://www.tandfonline.com/loi/cfai20

Chemical composition and immunostimulatory

properties of green alga Caulerpa racemosa var
peltata

Huili Hao, Manqin Fu, Ru Yan, Baolin He, Meiying Li, Qiabiao Liu, Yimian Cai,
Xiaoyong Zhang & Riming Huang

To cite this article: Huili Hao, Manqin Fu, Ru Yan, Baolin He, Meiying Li, Qiabiao Liu, Yimian Cai,
Xiaoyong Zhang & Riming Huang (2019) Chemical composition and immunostimulatory properties
of green alga Caulerpa�racemosa var peltata, Food and Agricultural Immunology, 30:1, 937-954,
DOI: 10.1080/09540105.2019.1646216

To link to this article: https://doi.org/10.1080/09540105.2019.1646216

© 2019 The Author(s). Published by Informa Published online: 26 Jul 2019.

UK Limited, trading as Taylor & Francis
Group

Submit your article to this journal Article views: 3760

View related articles View Crossmark data

Citing articles: 9 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=cfai20
FOOD AND AGRICULTURAL IMMUNOLOGY
2019, VOL. 30, NO. 1, 937–954
https://doi.org/10.1080/09540105.2019.1646216

Chemical composition and immunostimulatory properties of

green alga Caulerpa racemosa var peltata
Huili Haoa, Manqin Fub, Ru Yanc, Baolin Hea, Meiying Lia, Qiabiao Liua, Yimian Caia,
Xiaoyong Zhangd and Riming Huanga
a
Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China
Agricultural University, Guangzhou, People’s Republic of China; bSericultural & Agri-Food Research Institute
Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture/
Guangdong Key Laboratory of Agricultural Products Processing, Guangzhou, People’s Republic of China;
c
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences,
University of Macau, Macao, People’s Republic of China; dJoint Laboratory of Guangdong Province and Hong
Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China
Agricultural University, Guangzhou, People’s Republic of China

ABSTRACT ARTICLE HISTORY

This work aimed to evaluate the nutritional composition and Received 29 April 2019
immunostimulatory activity of an edible green alga Caulerpa Accepted 14 July 2019
racemosa var peltata collected from South China Sea. The results
KEYWORDS
indicated that this alga contained the total carbohydrates (71.67%, Caulerpa racemosa var
DW), crude protein (11.39%, DW), and crude lipid (1.03%, DW), peltata; chemical
and was rich in taurine (7.71 mg/100 g, DW), essential amino composition; RAW264.7;
acids (40.07%), monounsaturated fatty acids (25.72%), and immunostimulatory activity
polyunsaturated fatty acids (26.33%). Moreover, water extraction
of this alga (WEC) increased NO, ROS, TNF-α, and IL-6 in a dose-
dependent manner in RAW264.7 macrophage cells. Furthermore,
WEC contained the total sugar (34.19 ± 0.46 mg/g), total protein
(6.76 ± 0.32 mg/g), total phenolic (1.93 ± 0.04 mg/g), and total
flavonoid (4.80 ± 0.10 mg/g). In addition, the MSn analysis of WEC
indicated that the presence of 19 compounds. Thus, these
findings suggest that this alga may have a potential nutraceutical
value.

Introduction
Seaweeds have been used as food in Asian countries like China, Japan and Korea for a long
history, not only tasty but also healthy (Tanna & Mishra, 2018). Besides nutritional value,
they also have different physiological effects on health and disease (Koirala, Jung, & Choi,
2017; Wijesinghe & Jeon, 2012), due to their abundant and diverse primary and secondary
metabolites. These metabolites have been applied for many biological and functional needs

CONTACT Xiaoyong Zhang zhangxiaoyong@scau.edu.cn Joint Laboratory of Guangdong Province and Hong Kong
Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural Uni-
versity, Guangzhou 510642, People’s Republic of China; Riming Huang huangriming@scau.edu.cn Guangdong
Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University,
Guangzhou 510642, People’s Republic of China
© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License
(http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any
medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
938 H. HAO ET AL.

(Pires, Dias, Barros, & Ferreira, 2017; Rico et al., 2018), including immunostimulatory (Di
et al., 2017), antioxidant, antimicrobial, and anticancer activities (Omar, Al-Judaibiand, &
El-Gendy, 2018).
Edible green seaweeds contain a varied profile of many nutrients, such as carbo-
hydrates, essential amino acids (Vieira et al., 2018), polyunsaturated fatty acids (Paiva,
Lima, Neto, Marcone, & Baptista, 2017) and minerals. Green seaweeds have attracted a
lot of attentions due to their nutritional value, as also biological activities, such as antiox-
idant effect (Bourguiba, Zahlila, Bouaicha, Amri, & Mezghani, 2017), antimicrobial
activity (Xu et al., 2018) and anticancer effect (Lakmal et al., 2014). Thus, the reason
why it has been investigated and applied in many parts of the world, including Japan,
Korea, Philippines, and some other countries in southeast Asia, is mainly due to its nutri-
tional value and functional benefits. The green seaweed Caulerpa racemosa var peltata,
belong to genus Caulerpa, family Caulerpaceae, is wildly distributed in at tropical and
semi-tropical region, such as in South China Sea. These seaweeds of Caulerpaceae are
also considered as the valuable source of bioactive components. Previous chemical inves-
tigation of Caulerpa alga have exhibited the chemical composition such as rich in proteins,
lipids (especially polyunsaturated fatty acids), vitamins, pigments, and minerals (Hong,
Hien, & Son, 2007; Kumar, Gupta, Kumari, Reddy, & Jha, 2011) and biological properties
such as antiproliferative (Cavas, Baskin, Yurdakoc, & Olgun, 2006), antibacterial (Chan
et al., 2018) and immunostimulatory activities (Shen, Wang, Guo, & Tuo, 2008).
However, there is still a lack of accurate data about proximate and nutritional composition
of C. racemosa var peltata. Thus, it is necessary to investigate the chemical and nutritional
composition before in-depth pharmacological investigation.
The aim of the present study was to determine the chemical and nutritional compo-
sition regarding crude protein, crude fat, total carbohydrates, amino acids, taurine, fatty
acids and minerals. Moreover, the current experiments were to investigate the immunos-
timulatory activity of C. racemosa var peltata on the murine macrophage RAW264.7 cells,
by evaluating the effect on the proliferation of RAW264.7 cells, the production of nitric
oxide (NO), intracellular reactive oxygen species (ROS), TNF-α, and IL-6 on
RAW264.7 cells. The results of this study might supply useful information to further
investigation of bioactive components from C. racemosa var peltata and their mechanism
of action.

2. Materials and methods

2.1. Materials and reagents
The green alga C. racemosa var peltata was collected in April 2017, off the coast of
Naozhou Island, South China Sea. A voucher specimen (No. 20170408) was deposited
in Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agri-
cultural University, China. Glucose, gallic acid, and rutin standards were obtained from
Aladdin Industrial Corporation (Shanghai, China). Murine RAW264.7 macrophage
cells were obtained from Jinan University (Guangzhou, China). Lipopolysaccharide
(LPS) and macroporous resin (XAD-7) were purchased from Shanghai Yuanye Bio-Tech-
nology Co. LTD (Shanghai, China). Dulbecco Modified Eagle Medium (DMEM), phos-
phate buffer solution (PBS), fetal bovine serum (FBS) and penicillin–streptomycin were
 FOOD AND AGRICULTURAL IMMUNOLOGY 939

purchased from Gibco Life Technologies (Grand Island, NY). Cell Counting kit-8 (CCK-8
kit) was purchased from Dongren Chemical Technology Co. LTD. Nitric Oxide (NO) kit
and Reactive Oxygen Species (ROS) assay kit were purchased from Beyotime Biotechnol-
ogy Co. LTD. Mouse TNF-α, and IL-6 ELISA kits were purchased from NeoBioscience
Biotechnology Co. LTD. Other standards were purchased from Sigma Co. (MO, USA).
All other reagents used in this study were of analytical grade.

2.2. Proximate composition analysis

The samples were analysed for ash, crude protein, crude fat, and total carbohydrates using
AOAC official methods (AOAC, 2016; Ziani et al., 2019). The ash content was quantified
by incineration at 550°C. The content of crude protein (N × 6.25) was estimated by the
macro-Kjeldahl method. The content of crude fat was estimated by the Soxhlet extraction
method, briefly, the sample was extracted with petroleum ether and evaporated the extrac-
tion to dryness with constant weight. The total carbohydrate content of the sample was
determined by difference. The moisture content was determined by adequately drying
sample at 105°C until a constant weight was kept.

2.3. Nutritional composition analysis

2.3.1. Amino acid profile analysis
Amino acids were quantified by an automatic amino acid analyzer which contained ion-
exchange chromatography with post-column ninhydrin derivation and spectrophoto-
metric detection (wavelength 280 nm for tryptophan, 440 nm for proline and 570 nm
for other amino acids) (Misurcova et al., 2014). An alkaline hydrolysis was used for trypto-
phan (Shen et al., 2018) and other amino acids were disposed by acidic hydrolysis.
For tryptophan analysis, 100 mg sample was hydrolysed by NaOH 4 M at 110°C for
20 h under nitrogen gas. The hydrolysate was neutralized by HCl and added sodium
citrate buffers to reach the constant volume for injecting into the analyser.
For other amino acid analysis, alga powder (100 mg) was hydrolysed with 6 M hydro-
chloric acid at 110°C for 24 h under a nitrogen atmosphere. The hydrolysed sample was
repeatedly dried in a rotary evaporator. The resident was dissolved in 1 mL of sodium
citrate buffers and subjected to the automatic amino acid analyzer.

2.3.2. Taurine analysis

Taurine was determined using high-performance liquid chromatography (HPLC) with
fluorescence detection. 3 g of alga sample was transferred to conical flask and dissolved
into 20 mL of deionized water, and sonicated for 10 min. The solution was mixed with
50 mL of 0.375 M metaphosphoric acid under ultrasonic treatment for 15 min, then the
sample was centrifuged at 3220 g for 10 min. 20 μL of the concentrated supernatant
was injected into the HPLC equipped with sodium ion column (25 cm × 4.6 mm). The
mobile phase consisted of trisodium citrate solution which delivered to the column at a
flow rate of 0.4 mL/min.
940 H. HAO ET AL.

2.3.3. Fatty acid profile analysis

The fatty acid profile was obtained by gas chromatography (GC) with a flame ionization
detector (FID) (Shi, Yao, Zhu, & Ren, 2017). Alga powder (60.0 mg) was transferred to an
ampoule and was accurately mixed with 2.0 mL of internal standard solution (5.00 mg/
mL), four mL of 2,2,4-trimethylpentane and 200 μL of 0.5 M potassium hydroxide metha-
nol solution. After shaking vigorously for 30 s, the solution was added with 1 g sodium
bisulphate to neutralize the potassium hydroxide. The suspension of static settlement
was transferred to insert vials for further analysis of GC.
The fatty acid composition was analysed using GC with a fused silica capillary column
(100 m × 0.25 mm ID × 0.2 μm film thickness). Purified nitrogen was used as a carrier gas.
A split injection system was used with an auto injector with a split ratio of 100:1 and an
injector temperature of 270°C and the detector temperature was set at 280°C. Sample of
1.0 μL was injected at an initial column temperature of 100°C for 13 min. The temperature
was then raised at 10°C min−1 to –180°C (held for 6 min), and then increased at 1°C
min−1–200°C (held for 20 min). Finally, the temperature was increased to 230°C (held
for 10.5 min) at a rate of 4°C min−1, thus giving a final gradient of 85 min total
runtime. A calibration mixture of fatty acid standards was processed in the same method.

2.3.4. Mineral elements analysis

The mineral elements were measured by inductively coupled plasma atomic emission
spectrometry (ICP-AES). Alga sample (3 g) was placed in a porcelain crucible and
ashed in a muffle furnace at 550°C for 8 h. After cooling, the material was digested in
10 mL of concentrated HNO3 and then diluted with distilled water to 25 mL. This solution
was filtered before storage. A blank digest was also carried out in the same way.

2.4. Extraction procedure

The C. racemosa var peltata powder (50.0 g) was extracted in 1000 mL of water at 85°C for
3 h. The extractive was further filtrated through a gauze (200 mesh) and concentrated to
5% of the original volume by a rotary evaporator at 65°C, and then lyophilized by freeze-
drying to obtain water extraction of C. racemosa var peltata (WEC) for immunostimula-
tory experimentation.

2.5. Immunostimulatory activity

2.5.1. Cell line culture
The murine macrophage RAW264.7 cells were cultured in DMEM supplemented with
10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin at 37°C in a
humidified incubator with 5% CO2. The cells were cultivated in culture flasks and subcul-
tured for experimentation.

2.5.2. Assay of cell viability

RAW264.7 cells were seeded at a density of 3.0 × 104 cells/mL in 96-well plates and cul-
tured for 24 h at 37°C with 5% CO2. WEC (100 μL) were added at the final concentrations
of 0, 20, 100, 200, 400, 800 and 1600 μg/mL. After 24 h of incubation in the incubator at
 FOOD AND AGRICULTURAL IMMUNOLOGY 941

37°C with 5% CO2, 10 μL of CCK-8 solution was added to every well and then absorbance
was recorded at 450 nm using an ELISA plate reader after 2 h of incubation.

2.5.3. Assay of NO and ROS production

The immunostimulatory activity of WEC was determined by quantifying the NO and ROS
levels released in the supernatant of RAW264.7 macrophages, according to the reported
method (Dong et al., 2018). The levels of NO were evaluated by Nitric Oxide kit according
to the manufacturer’s protocols. 500μL/well of RAW264.7 cells were cultured in 24-well
plates at a density of 3.0 × 105 cells/mL and exposed to WEC with different concentrations
(20, 100, 200 and 400 µg/mL) for 24 h. LPS (5 µg/mL) was used as a positive control.
Absorbance at 540 nm was measured in an ELISA plate reader.
The levels of intracellular ROS were evaluated by quantifying the fluorescence emitted
by DCFH-DA. 1 mL/well of RAW264.7 macrophages were cultured in 12-well plates at a
density of 5.0 × 105 cells/mL and exposed to WEC with different concentrations (20, 100,
200 and 400 µg/mL) for 24 h. LPS (5 µg/mL) was used as a positive control. At the end of
the treatment period, the supernatant was removed, cells were washed with phosphate
buffered saline (PBS) and 10 µM DCFH-DA in DMEM without FBS were added, and
were subsequently incubated at 37°C for 30 min. DCFH-DA was then removed, cells
were washed three times with DMEM without FBS, and the emitted fluorescence was
quantified on a flow cytometer (Beckman Coulter, CYTOFLEX, USA). Similarly,
1 mL/well of RAW264.7 macrophages were cultured in laser confocal dish at a
density of 1.0 × 106 cells/mL, dealt with the same procedure, and determined by laser
scanning confocal microscope (Zeiss, LSM 7DUO, Germany). The DCF fluorescent
images at excitation wavelength of 488 nm/535 nm emission filter were elaborated as
arbitrary units/pixel by the LSCM. Four fields were randomly selected to observe and
images were taken.

2.5.4. Assay of cytokine production

500 μL/well of RAW264.7 cells were adjusted at a density of 3.0 ×105 cells/mL in 24-well
plates and cultured for 24 h at 37°C in a humidified incubator with 5% CO2. Further,
RAW264.7 cells were treated with WEC (20, 100, 200 and 400 µg/mL) or LPS (5 μg/
mL) in DMEM medium. The treated cells were then incubated for an additional 24 h.
The levels of TNF-α and IL-6 in the supernatants of the RAW264.7 cells from each
group were determined using commercial kits in accordance with the manufacturer’s
instructions.

2.6. Proximate composition analysis of WEC

The total sugar content was determined by the phenol-sulfuric acid method (Masuko et al.,
2005) using mannose as the reference. The protein content was determined by the Brad-
ford’s method (Bradford, 1976) using bovine serum albumin as the standard. The total
phenolic content (TPC) was measured by the Folin–Ciocalteu colorimetric method
(Song et al., 2018) using gallic acid as a reference. The total flavonoid content (TFC)
was measured by aluminium nitrate colorimetric method (Song et al., 2018) using rutin
as a standard. These results were expressed as mg/g of extracts.
942 H. HAO ET AL.

2.7. Compounds analysis of WEC by HPLC-ESI-QTOF-MS

WEC (600 mg) was subjected to a macroporous resin (XAD-7) column chromatography
with water and 80% methanol as eluent. The 80% methanol eluted fraction was collected,
dried, redissolved with methanol, and filtered through a 0.22 µm syringe filter for further
MS analysis. MS was performed with a Bruker Maxis QTOF-MS with an ESI (Bruker Dal-
tonics, Germany) coupled to a HPLC (Agilent 1260, Germany) (Ju et al., 2019; Xu et al.,
2019). Redissolved extract (15 µL) was injected with a split ratio of 15:1 into the system for
analysis. Chromatographic separation was carried out at 30°C in a reversed phase Luna
C18 column (5 μm, 150 × 4.6 mm, Phenomenex, USA) with mobile phases A (0.1%
formic acid in water) and B (methanol). The flow rate was set at 1 mL/min. The gradient
profile was as follows: 0–20 min, 5–10% B; 20–30 min, 10–20% B; 30–40 min, 20–40% B;
40–90 min, 40–100% B. The main working parameters for the mass spectrometers were
set as follows: capillary voltage, 3.8 kV; nebulizer voltage, 1.0 bar; dry gas flow rate, 6.0
L/min; dry heater temperature, 180°C; MS scan in positive mode; scan range 100–
2000 m/z.

2.8. Statistical analysis

Data are expressed as the mean ± standard deviation (SD) of three replicates except fatty
acids analysis. Significant differences between the means of parameters were calculated by
one-way ANOVA using SPSS 19.0 software. P < 0.05 was considered statistically
significant.

3. Results and discussion

3.1. Proximate composition of C. racemosa var peltata
The proximate composition of C. racemosa var peltata, based on dry weight, is illustrated
in Table 1. The contents measured, in diminishing order, were carbohydrates (71.67% ±
0.36%), crude protein (11.39% ± 0.32%), ash (7.97% ± 0.46%) and crude lipid (1.03% ±
0.01%). Seaweeds are degenerative rapidly within a few days after harvesting and decreas-
ing their free moisture are an essential way to prevent it. Moisture content was also
detected in air-dried C. racemosa var peltata and was only 7.94% ± 0.07% which was

Table 1. Proximate composition of C. racemosa var peltata and comparison with different species of
seaweeds.
Protein Lipid Carbohydrates
Species Ash (% DW) (% DW) (% DW) (% DW) References
C. racemosa var peltata 7.97 ± 0.46 11.39 ± 0.32 1.03 ± 0.01 71.67 ± 0.36
C. racemosa var lv. a 26.74 ± 2.17 17.28 ± 0.63 2.11 ± 0.04 53.37 Nagappan and Vairappan (2014)
C. racemosa var cm. a 23.81 ± 1.35 17.36 ± 0.72 2.21 ± 0.05 55.92 Nagappan and Vairappan (2014)
C. lentillifera a 22.20 ± 0.27 9.26 ± 0.03 1.57 ± 0.02 66.97 Nguyen et al. (2011)
Grateloupia turuturu b 20.52 ± 0.01 22.5 ± 0.3 2.2 ± 0.1 43.2 Rodrigues et al. (2015)
S. muticum c 22.94 ± 0.06 16.9 ± 0.2 1.45 ± 0.07 49.3 Rodrigues et al. (2015)
a
green alga, Caulerpaceae.
b
red alga.
c
brown alga.
(A) C. racemosa var lv.: Caulerpa racemosa var laetevirens; (B) C. racemosa var cm.: Caulerpa racemosa var clavifera
f. microphysa.
 FOOD AND AGRICULTURAL IMMUNOLOGY 943

Table 2. Amino acids composition of C. racemosa var peltata.

Amino acids Contenta,b Percentage of TAA (%)
Non-essential amino acids
Glu 5.17 ± 0.05 15.27
Asp 1.23 ± 0.05 3.63
Ser 2.23 ± 0.05 6.59
Gly 2.47 ± 0.05 7.29
Ala 2.63 ± 0.05 7.77
Tyr 1.13 ± 0.05 3.34
Pro 2.30 ± 0.16 6.79
His 1.13 ± 0.05 3.34
Arg 2.00 ± 0.00 5.91
Essential amino acids
Thr 1.70 ± 0.00 5.02
Val 2.30 ± 0.00 6.79
Met 0.86 ± 0.01 2.54
Ile 1.63 ± 0.05 4.81
Leu 2.83 ± 0.05 8.36
Phe 1.90 ± 0.08 5.61
Lys 1.90 ± 0.00 5.61
Trp 0.45 ± 0.01 1.33
EAA 13.57 40.07
NEAA 20.29 59.93
EAAs / NEAAs 0.67 –
a
Values expressed in mean values ± standard deviation.
b
(mg/g of C. racemosa var peltata).
TAA: total amino acids. EAA: essential amino acids. NEAA: non-essential amino acids. EAAs
/ NEAAs: the ratio of total essential amino acids to total non-essential amino acids.

comparative with Gracilaria gracilis (7.99% ± 0.02%) and Codium tomentosum (9.0% ±
0.2%) (Rodrigues et al., 2015). Carbohydrates with the content of 71.67% were the predo-
minate component of C. racemosa var peltata. Moreover, carbohydrate content of C. race-
mosa var peltata was also the most abundant compared with the reported data of the genus
Caulerpa, red alga Grateloupia turuturu and brown alga Sargassum muticum showed in
Table 1. Lipid content was extremely low, which is consistent with previous reports
(Nguyen, Ueng, & Tsai, 2011; Rodrigues et al., 2015). C. racemosa var peltata with low
lipid and rich in carbohydrates could be considered as low calories from a nutritional per-
spective (Holdt & Kraan, 2011). Additionally, the crude protein of C. racemosa var peltata
(11.39%) had a comparative level of that in C. lentillifera (9.26%) (Nguyen et al., 2011). In
contrast, the ash content (7.97%, DW) of C. racemosa var peltata was less than the content
of the other reported species (Table 1), not only red alga G. turuturu and brown alga
S. muticum but also the genus Caulerpa listed in Table 1.

3.2. Nutritional composition of C. racemosa var peltata

3.2.1. Amino acids content
The results of amino acids profile presented in Table 2 showed that 17 amino acids
detected in C. racemosa var peltata contain all essential amino acids with a content of
13.57 mg/g and non-essential amino acids with a content of 20.29 mg/g. The EAAs/
NEAAs ratio of the alga was 0.67, which agreed with the value between 0.49 and 0.68
reported in the literature (Astorga-Espana, Rodriguez-Galdon, Rodriguez-Rodriguez, &
Diaz-Romero, 2016). Based on the reference value of 0.6 recommended by the FAO/
WHO (Liu et al., 2016), C. racemosa var peltata could be considered as a nutritive
944 H. HAO ET AL.

source of amino acids. The amino acid composition of C. racemosa var peltata showed glu-
tamic acid was the most abundant amino acid with a content of 5.17 ± 0.05 mg/g, agreed
with the reported data of nine seaweed species (Gaillard et al., 2018). Glutamic acid (5.17
± 0.05 mg/g), alanine (2.63 ± 0.05 mg/g), glycine (2.47 ± 0.05 mg/g) and aspartic acid
(1.23 ± 0.05 mg/g) play an important role in special flavour and taste of seaweeds
(Vieira et al., 2018), these amino acids abundant in C. racemosa var peltata suggest that
the alga has a potential value of flavour enhancers and tasty food.

3.2.2. Taurine content

The taurine content of C. racemosa var peltata was 7.71 ± 0.15 mg/100 g of dry weight,
which was higher than that of different seaweeds (36.65 ± 0.89 μg/g, 33.01 ± 1.53 μg/g,
24.02 ± 1.37 μg/g, 19.15 ± 0.72 μg/g, 6.04 ± 0.98 μg/g) (Cao, Duan, Guo, Guo, & Zhao,
2014). Taurine as a kind of non-protein amino acid largely is present in marine animal
cells and mammalian tissue. It is closely related to the synthesis and metabolism of
sulphur amino acids (methionine, cysteine, cystine). It has been reported that taurine
and its derivatives possessed effective biological activities, such as the protective effect
of taurine against heart attack may be attributed to enhancing endogenous thrombolytic
activity (Ijiri et al., 2013) ameliorating diabetic complications like diabetic neuropathy,
retinopathy, and hematological dysfunctions (Sarkar, Basak, Ghosh, Kundu, & Sil,
2017). Thus, C. racemosa var peltata could be a promising natural source of taurine
with benefits for human health.

3.2.3. Fatty acid content

The analysis of fatty acid composition of C. racemosa var peltata shown in Table 3 revealed
that the saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids
were at the rate of 48%, 25.72% and 26.33%, respectively. Among the saturated fatty acids,

Table 3. Fatty acid composition of C. racemosa var peltata.

Fatty acids Concentration (% w/w) Fatty acids Concentration (% w/w)
C16:1 (n-7) 2.70 C8:0 0.70
C18:1 (n-9) 20.9 C10:0 1.20
C20:1 (n-9) 0.45 C12:0 0.48
C22:1 (n-9) 0.83 C14:0 2.20
C24:1 (n-9) 0.84 C16:0 25.2
C18:2 (n-6) 13.0 C17:0 3.00
C20:2 (n-7) 0.39 C18:0 3.90
C18:3 (n-3) 8.50 C20:0 9.40
C18:4 (n-5) 0.33 C22:0 0.72
C20:4 (n-6) 1.80 C24:0 1.20
C22:4 (n-7) 0.34 SFA 48.00
C20:5 (n-3) 1.40 PUFA/SFA 0.55
C22:6 (n-3) 0.57 FA (n-6) 14.80
MUFA 25.72 FA (n-3) 10.47
PUFA 26.33 n-6/n-3 1.41
Octanoic acid (C8:0), Decanoic Acid (C10:0), Lauric acid (C12:0), Myristic acid (C14:0), Palmitic acid (C16:0), Palmitoleic acid
(C16:1), Heptadecanoic acid (C17:0), Stearic acid (C18:0), Oleic acid (C18:1), Linoleic acid (C18:2), α-Linolenic acid (C18:3),
Octadecatetraenoic acid (C18:4), Arachidic acid (C20:0), Gondoic acid (C20:1), cis-11,14-Eicosadienoic acid (C20:2), Arachi-
donic acid (C20:4), cis-5,8,11,14,17-Eicosapentaenoic acid (C20:5), Behenic acid (C22:0), Erucic acid (C22:1), cis-7,10,13,16-
Docosatetraenoic acid (C22:4), cis-Docosahexaenoic acid (C22:6), Lignoceric acid (C24:0), Selacholeic acid (C24:1).
SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; FA(n-6): total Omega-6
fatty acids; FA(n-3): total Omega-3 fatty acids; n-6/n-3: the ratio of total Omega-6 fatty acids to total Omega-3 fatty acids.
 FOOD AND AGRICULTURAL IMMUNOLOGY 945

palmitic acid (25.2% w/w) was dominant in accordance with a green seaweed Ulva rigida
(Gao, Clare, Chatzidimitriou, Rose, & Caldwell, 2018) and a brown seaweed Sargassum
naozhouense (Peng et al., 2013). Oleic acid as a monounsaturated fatty acid was also abun-
dant with the rate of 20.9%, which exhibited a comparative level of the brown seaweeds
Ascophyllum nodosum and Fucus vesiculosus (Lorenzo et al., 2017). It has been reported
oleic acid have effect on reducing coronary heart disease risk (Nishi et al., 2014), the
oleic acid abundant in C. racemosa var peltata indicated that the alga could be the superior
source of dietary monounsaturated fatty acid. Linoleic acid (13.0%, w/w) was the major
polyunsaturated fatty acid followed by α-linolenic acid (8.50%, w/w), both are essential
fatty acids that were known for accelerating the metabolism of blood cholesterol. Other
ω-6 and ω-3 polyunsaturated fatty acids, arachidonic acid (AA), cis-eicosapentaenoic
acid (EPA) and cis-docosahexaenoic acid (DHA) were with ratios of 1.80%, 1.40% and
0.57%, respectively. Furthermore, the ratio of total ω-6 fatty acids to total ω-3 fatty
acids was measured to be 1.41:1 approximately a suggested 1:1 ratio (Simopoulos,
2006). Many researches have indicated that the consumption of foods containing a low
ω-6: ω-3 PUFA ratio, or one that is supplemented with long chain ω-3 PUFAs, reduces
risk factors for chronic disease (Khadge et al., 2018), including cancer (Berquin,
Edwards, & Chen, 2008), obesity (Mejia-Barradas et al., 2014) and coronary heart
disease (Petropoulos et al., 2018). Moreover, the PUFA/SFA ratio (0.55) was higher
than 0.45 that is an essential value of high nutritional food. Therefore, C. racemosa var
peltata could have a wonderful source of fatty acids and a prospective functional food
for chronic disease.

3.2.4. Mineral contents

Analysis of mineral contents of C. racemosa var peltata indicated that sodium content
(6300 ± 95 mg/100 g) was the highest among the macro elements, followed by calcium
(1990 ± 175 mg/100 g), magnesium (640 ± 10 mg/100 g) and potassium (480 ± 5.8 mg/
100 g). The high Na/K ratio (approximately 13:1) of C. racemosa var peltata due to a
low content of potassium was different from the other Caulerpaceae seaweeds (Kumar
et al., 2011). This result indicates that if C. racemosa var peltata was served as functional
food, it’s necessary to increase the supplement of potassium to balance the Na/K ratio
(Chan & Matanjun, 2017). The analysis of microelements revealed that C. racemosa var
peltata possessed iron (263.3 ± 4.2 mg/100 g), manganese (16.2 ± 0.5 mg/100 g) and zinc
(0.9 ± 0.04 mg/100 g), and the content of copper was less than 0.5 mg/100 g. Iron rich
in C. racemosa var peltata suggests that C. racemosa var peltata could be a suitable
source of microelement for iron supplement in food.

3.3. Immunostimulatory activity

3.3.1. Effects of WEC on RAW264.7 cells viability
The viability of macrophages can be regarded as an indicator of immune activation (Yuan
et al., 2015). In this study, viability of RAW264.7 cells treated by WEC was measured by
CCK-8 assay. As illustrated in Figure 1, the viability of control (without sample) was set as
100%, and the viability ratio of macrophages was obviously promoted by samples in
general. WEC significantly increased the viability of RAW264.7 cells in a dose-dependent
manner from 20 to 400 μg/mL. When the concentration of WEC was up to 400 μg/mL, the
946 H. HAO ET AL.

Figure 1. Effect of WEC on the proliferation.

viability showed a slight decline, but the value is still higher than the control, which may be
due to the accumulation of metabolites or competition of nutrient. Thus, we had chosen
the concentrations that were not higher than 400 μg/mL for further investigation.

3.3.2. Effects of WEC on NO and intracellular ROS production

Macrophages can defend against pathogen by generating cytotoxic and inflammatory mol-
ecules like NO and intracellular ROS that are valuable index to evaluate activation effects
of WEC on macrophages (Dong et al., 2018). In this study, NO production was examined
using the Griess reagent. As shown in Figure 2(A), compared with the control group, WEC
showed no obvious effect on NO production at concentration of 20 μg/mL, but WEC pro-
moted the production of NO in a dose-dependent manner at concentrations between 20
and 400 μg/mL. Especially, the production of NO at concentration of 400 μg/mL showed a
comparative level of the LPS-treated group.

Figure 2. (A) Production of nitric oxide (NO). (B) Production of reactive oxygen species (ROS). *P < 0.05,
**P < 0.01, vs. the control group.
 FOOD AND AGRICULTURAL IMMUNOLOGY 947

Figure 3. Production of ROS in RAW264.7 cells treated with WEC analysis by LSCM (A) control (B) LPS
(C) 20 μg/mL (D) 100 μg/mL (E) 200 μg/mL (F) 400 μg/mL.

ROS generation was measured using the DCFH-DA. Consistent with the effect of WEC
on the NO release, ROS generation showed no significant effect on low concentration of 20
μg/mL, but enhanced in a dose-dependent manner illustrated in Figure 2(B). This result
may be explained with the reason that the surplus NO competed with oxygen causing a
limitation of transfer in the electron transport chain which result in the generation of
ROS (Ma et al., 2014). The ROS generation was further observed by LSCM (Ran,
Zhang, Wen, & Ma, 2019; Yang, Wu, Tang, Kong, & Lu, 2009). As shown in Figure 3,
ROS are widely distributed equably throughout the cells, not just in some parts of the
cells. Untreated RAW264.7 cells hardly secreted ROS showed in Figure 3(A). However,
the positive control (Figure 3(B)) can significantly promote the generation of ROS. As
illustrated in Figure 3(C–F), with the treatment of increasing concentration of WEC,
the number of cells with fluorescence increased, indicating WEC had the ability to facili-
tate the generation of ROS in a dose-dependent manner. Analysis of ROS determined by
FCM and LSCM were basically consistent. Generally, these results suggest that WEC could
promote the secretion of NO and ROS in activated RAW264.7 cells.

3.3.3. Effects of WEC on cytokines production

Activated macrophages generate proinflammatory cytokines such as TNF-α and IL-6 that
play an important role in inhibiting pathogens (Lee & Hong, 2011). To evaluate the effects
of WEC on activating macrophages, cytokines (TNF-α and IL-6) production of RAW264.7
cells treated with WEC were further measured in this study. As illustrated in Figure 4(A),
WEC exhibited an inapparent promoting effect at the dosage of 20 μg/mL. However, when
treating RAW264.7 cells at higher dosages of 100 and 400 μg/mL, WEC exhibited a
948 H. HAO ET AL.

Figure 4. Effect of WEC on TNF-α (A) and IL-6 (B) secretion of RAW264.7 macrophages. *P < 0.05, ** P <
0.01, vs the control group.

significant enhancement in TNF-α secretion compared with the control group. The pro-
duction of IL-6 in RAW264.7 cells was enhanced in a dose-dependent manner showed in
Figure 4(B). Compared with the control group, WEC significantly increased the pro-
duction of IL-6 at the concentration ranging from 100 to 400 μg/mL (P < 0.01). Consistent
with the NO and ROS production, the secretion of cytokines (TNF-α and IL-6) showed no
difference with the untreated group when treating RAW264.7 cells at dosage of 20 μg/mL.
These results indicate that WEC (100, 200 and 400 μg/mL) can promote the secretion of
proinflammatory cytokines in activated RAW264.7 cells.

3.4. Proximate composition of WEC

In this study, the total sugar content, total protein content, TPC, and TFC of WEC were
determined, and listed as followed: the total sugar content of WEC was 34.19 ± 0.46 mg/g,
the total protein content was 6.76 ± 0.32 mg/g, the TPC was 1.93 ± 0.04 mg/g, and the TFC
was 4.80 ± 0.10 mg/g. In general, total sugars were the abundant components compared to
protein, TPC, and TFC. Previous investigation revealed that polysaccharides from marine
algae possessed significant immunostimulatory activities, such as C. cupressoides var
flabellata (Barbosa et al., 2019), C. lentillifera (Maeda, Ida, Inara, & Sakamoto, 2012).
Phenols and flavonoids have been reported with immunostimulatory effects (Llaurado
et al., 2013), but there are still no reports on phenols and flavonoids’ immunostimulatory
activities of Caulerpaceae. Thus, in consideration of the reported data of total sugar
content and polysaccharides of Caulerpaceae, the proposed main immunostimulatory
composition in WEC may be polysaccharides.

3.5. Compound profile of WEC measured by HPLC-ESI-QTOF-MS

The HPLC-ESI-QTOF-MS was used to further analyse the compound profile of WEC. A
list with the molecular ions ([M+H]+) found in the WEC and the proposed tentative
identification is given in Table 4. A total of 19 compounds were detected in the WEC.
Among them, twelve compounds from WEC remained unknown, and these small mol-
ecules should be needed further investigation of their structures that were elucidated
 FOOD AND AGRICULTURAL IMMUNOLOGY 949

Table 4. Proposed tentative identification of the compounds in WEC by HPLC-ESI-QTOF-MS.

TR Molecular
Compounds (min) [M+H]+ (m/z abundance) Proposed compounds formula References
1 7.9 MS: 413.2565; MS/MS: Fucosterol C29H48O Maximo, Ferreira,
301.1448, 413.2565 Branco, Lima, and
Lourenco (2018)
2 8.7 MS: 552.1834; MS/MS: α-tocoxylenoxy C37H58O3 Yang et al. (2015)
152.0578, 284.1063
3 11.1 MS: 391.2786; MS/MS: 10,11- C21H26O7 Guerriero, Meinesz,
149.0237 epoxycaulerpenyne D’Ambrosio, and
Pietra (1992)
4 15.1 MS: 485.1910; MS/MS: Unknown
279.0136
5 39.9 MS: 619.2803; MS/MS: Unknown
191.0796, 321.1394,
387.1379
6 47.6 MS: 356.1148; MS/MS: Unknown
190.0957, 321.0731
7 49.9 MS: 449.1087; MS/MS: Unknown
251.1672, 447.2661
8 51.8 MS: 696.2171; MS/MS: Unknown
341.2513, 679.5269
9 58.2 MS: 313.1127; MS/MS: Phytene-3(20)-1,2-diol C20H40O2 Mao and Guo (2010)
295.0969
10 61.1 MS: 399.1745; MS/MS: Caulerpin C24H18N2O4 Liu et al. (2013)
295.1209, 399.1745
11 64.4 MS: 391.2147; MS/MS: Taxifolial C C21H26O7 Guerriero et al. (1992)
155.0746, 313.1372,
393.2116
12 67.8 MS: 385.1245; MS/MS: Monomethyl C23H16N2O4 Cheng, Zhou, Wu, and
309.1080 caulerpinate Zou (2008)
13 71.4 MS: 647.3315; MS/MS: Unknown
335.1912
14 72.5 MS:377.2412; MS/MS: Unknown
232.9658, 377.2412
15 77.3 MS: 369.1863; MS/MS: Unknown
234.1927, 333.2187
16 79.7 MS: 301.1476; MS/MS: Unknown
173.1435
17 81.8 MS: 363.2244; MS/MS: Unknown
226.1547, 363.2244
18 83.3 MS: 969.7086; MS/MS: Unknown
281.0452, 517.3440,
907.5530
19 89.0 MS: 908.3401; MS/MS: Unknown
233.1806, 389.2517,
563.4004, 732.4989,
907.7502

based on extensive spectroscopic analysis (NMR, MS, CD, X-ray) and by comparison of
the data with those of related secondary metabolites. Seven compounds could be charac-
terized based on their MS data and by comparison with those of reported data. The 7 pro-
posed compounds were fucosterol, α-tocoxylenoxy, 10,11-epoxycaulerpenyne, phytene-3
(20)-1,2-diol, caulerpin, taxifolial C, monomethyl caulerpinate, respectively. Among
them, fucosterol from Hizikia fusiforme (a brown seaweed) with immunostimulatory
activity have been reported (Park et al., 2017). Therefore, fucosterol in WEC may be
other efficient component of immunostimulatory effect.
950 H. HAO ET AL.

4. Conclusion
In order to take full advantage of an edible green alga C. racemosa var peltata collected
from South China Sea. This study has characterized the chemical composition and nutri-
tional properties of this edible C. racemosa var peltata, and explored the immunostimula-
tory activity of WEC. Preliminary phytochemical analysis revealed polysaccharides or
fucosterol may be the efficient components of WEC’s immunostimulatory effect. Accord-
ing to the obtained results, C. racemosa var peltata could be considered as a superior
source of functional components including essential amino acids, taurine, polyunsaturated
fatty acids and minerals. Moreover, the significant immunostimulatory activity of WEC
indicated that it could be proposed as new food ingredients with functional properties.
However, it is worthwhile to further identify the active components of WEC and evaluate
their activities in vivo.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Natural Science Foundation of Guangdong Province: [Grant Number
2016A030313151]; Guangdong Provincial Key Laboratory of Applied Botany, South China Botani-
cal Garden, Chinese Academy of Sciences: [Grant Number AB2018004]; Guangzhou Planned
Program in Science and Technology: [Grant Number 201803020003]; Guangdong Provincial
Special Fund For Modern Agriculture Industry Technology Innovation Teams: [Grant Number
NO.2019KJ122]; Science and Technology Planning Project of Guangdong Province: [Grant
Number 2017A020217002]; Program of Department of Ocean and Fisheries of Guangdong Pro-
vince: [Grant Number GDME2018C014].

Ethics statements
The work did not include any human subjects and animal experiments.

References
AOAC. (2016). The association of official analytical chemists international. Official Methods of
Analysis, 38(8), 431.
Astorga-Espana, M. S., Rodriguez-Galdon, B., Rodriguez-Rodriguez, E. M., & Diaz-Romero, C.
(2016). Amino acid content in seaweeds from the Magellan Straits (Chile). Journal of Food
Composition and Analysis, 53, 77–84. doi:10.1016/j.jfca.2016.09.004
Barbosa, J. D., Costa, M. S. S. P., de Melo, L. F. M., de Medeiros, M. J. C., Pontes, D. D., Scortecci, K.
C., & Rocha, H. A. O. (2019). In vitro immunostimulating activity of sulfated polysaccharides
from Caulerpa cupressoides Var. Flabellata. Marine Drugs, 17(2), 105. doi:10.3390/md17020105
Berquin, I. M., Edwards, I. J., & Chen, Y. Q. (2008). Multi-targeted therapy of cancer by omega-3
fatty acids. Cancer Letters, 269(2), 363–377. doi:10.1016/j.canlet.2008.03.044
Bourguiba, I., Zahlila, A., Bouaicha, N., Amri, M., & Mezghani, S. (2017). Antioxidant effect of the
marine green alga Ulva rigida ethanolic precipitate in yeast cells and zebrafish embryos. South
African Journal of Botany, 113, 253–260. doi:10.1016/j.sajb.2017.09.001
 FOOD AND AGRICULTURAL IMMUNOLOGY 951

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quan-
tities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72,
248–254.
Cao, Y., Duan, J. A., Guo, J. M., Guo, S., & Zhao, J. L. (2014). Rapid determination of nucleosides,
nucleobases and free amino acids in brown seaweeds using ultra-performance liquid chromato-
graphy coupled with triple quadrupole mass spectrometry. Journal of Applied Phycology, 26(1),
675–686. doi:10.1007/s10811-013-0079-3
Cavas, L., Baskin, Y., Yurdakoc, K., & Olgun, N. (2006). Antiproliferative and newly attributed
apoptotic activities from an invasive marine alga: Caulerpa racemosa var. cylindracea.
Journal of Experimental Marine Biology and Ecology, 339(1), 111–119. doi:10.1016/j.jembe.
2006.07.019
Chan, P. T., & Matanjun, P. (2017). Chemical composition and physicochemical properties of tro-
pical red seaweed, Gracilaria changii. Food Chemistry, 221, 302–310. doi:10.1016/j.foodchem.
2016.10.066
Chan, Y. S., Ong, C. W., Chuah, B. L., Khoo, K. S., Chye, F. Y., & Sit, N. W. (2018). Antimicrobial,
antiviral and cytotoxic activities of selected marine organisms collected from the coastal areas of
Malaysia. Journal of Marine Science and Technology-Taiwan, 26(1), 128–136. doi:10.6119/Jmst.
2018.02_(1).0012
Cheng, F., Zhou, Y., Wu, J., & Zou, K. (2008). Chemical constituents of Caulerpa peltata. Shizhen
Guoyi Guoyao, 19(4), 856–857.
Di, T., Chen, G. J., Sun, Y., Ou, S. Y., Zeng, X. X., & Ye, H. (2017). Antioxidant and immunostimu-
lating activities in vitro of sulfated polysaccharides isolated from Gracilaria rubra. Journal of
Functional Foods, 28, 64–75. doi:10.1016/j.jff.2016.11.005
Dong, Z. P., Li, C., Huang, Q., Zhang, B., Fu, X., & Liu, R. H. (2018). Characterization of a novel
polysaccharide from the leaves of Moringa oleifera and its immunostimulatory activity. Journal
of Functional Foods, 49, 391–400. doi:10.1016/j.jff.2018.09.002
Gaillard, C., Bhatti, H. S., Novoa-Garrido, M., Lind, V., Roleda, M. Y., & Weisbjerg, M. R. (2018).
Amino acid profiles of nine seaweed species and their in situ degradability in dairy cows. Animal
Feed Science and Technology, 241, 210–222. doi:10.1016/j.anifeedsci.2018.05.003
Gao, G., Clare, A. S., Chatzidimitriou, E., Rose, C., & Caldwell, G. (2018). Effects of ocean
warming and acidification, combined with nutrient enrichment, on chemical composition and
functional properties of Ulva rigida. Food Chemistry, 258, 71–78. doi:10.1016/j.foodchem.2018.
03.040
Guerriero, A., Meinesz, A., D’Ambrosio, M., & Pietra, F. (1992). Isolation of toxic and potentially
toxic sesqui- and monoterpenes from the tropical green seaweed Caulerpa taxifolia which has
invaded the region of Cap Martin and Monaco. Helvetica Chimica Acta, 75(3), 689–695.
doi:10.1002/hlca.19920750303
Holdt, S. L., & Kraan, S. (2011). Bioactive compounds in seaweed: Functional food applications and
legislation. Journal of Applied Phycology, 23(3), 543–597. doi:10.1007/s10811-010-9632-5
Hong, D. D., Hien, H. M., & Son, P. N. (2007). Seaweeds from Vietnam used for functional food,
medicine and biofertilizer. Journal of Applied Phycology, 19(6), 817–826. doi:10.1007/s10811-
007-9228-x
Ijiri, Y., Ikarugi, H., Tamura, Y., Ura, M., Morishita, M., Hamada, A., … Yamamoto, J. (2013).
Antithrombotic effect of taurine in healthy Japanese people may be related to an increased
endogenous thrombolytic activity. Thrombosis Research, 131(2), 158–161. doi:10.1016/j.
thromres.2012.09.021
Ju, J., Xie, Y. F., Guo, Y. H., Cheng, Y. L., Qian, H., & Yao, W. R. (2019). Antibacterial activities of
bayberry extract on foodborne pathogens and identification of its active components. Food and
Agricultural Immunology, 30(1), 385–397. doi:10.1080/09540105.2019.1589427
Khadge, S., Sharp, J. G., Thiele, G. M., McGuire, T. R., Klassen, L. W., Duryee, M. J., … Talmadge, J.
(2018). Dietary omega-3 and omega-6 polyunsaturated fatty acids modulate hepatic pathology.
Journal of Nutritional Biochemistry, 52, 92–102. doi:10.1016/j.jnutbio.2017.09.017
952 H. HAO ET AL.

Koirala, P., Jung, H. A., & Choi, J. S. (2017). Recent advances in pharmacological research on
Ecklonia species: A review. Archives of Pharmacal Research, 40(9), 981–1005. doi:10.1007/
s12272-017-0948-4
Kumar, M., Gupta, V., Kumari, P., Reddy, C. R. K., & Jha, B. (2011). Assessment of nutrient com-
position and antioxidant potential of Caulerpaceae seaweeds. Journal of Food Composition and
Analysis, 24(2), 270–278. doi:10.1016/j.jfca.2010.07.007
Lakmal, H. H. C., Samarakoon, K. W., Lee, W., Lee, J. H., Abeytunga, D. T. U., Lee, H. S., & Jeon, Y.
J. (2014). Anticancer and antioxidant effects of selected Sri Lankan marine algae. Journal of the
National Science Foundation of Sri Lanka, 42(4), 315–323. doi:10.4038/jnsfsr.v42i4.7730
Lee, J. S., & Hong, E. K. (2011). Immunostimulating activity of the polysaccharides isolated from
Cordyceps militaris. International Immunopharmacology, 11(9), 1226–1233. doi:10.1016/j.
intimp.2011.04.001
Liu, Y. T., Chen, D., You, Y. X., Zeng, S. Q., Li, Y. W., Tang, Q. Q., … Chen, D. W. (2016).
Nutritional composition of boletus mushrooms from Southwest China and their antihyperglyce-
mic and antioxidant activities. Food Chemistry, 211, 83–91. doi:10.1016/j.foodchem.2016.05.032
Liu, D. Q., Mao, S. C., Zhang, H. Y., Yu, X. Q., Feng, M. T., Wang, B., … Guo, Y. W. (2013).
Racemosins A and B, two novel bisindole alkaloids from the green alga Caulerpa racemosa.
Fitoterapia, 91, 15–20. doi:10.1016/j.fitote.2013.08.014
Llaurado, G., Morris, H. J., Lebeque, Y., Gutierrez, A., Fontaine, R., Bermudez, R. C., & Perraud-
Gaime, I. (2013). Phytochemical screening and effects on cell-mediated immune response of
Pleurotus fruiting bodies powder. Food and Agricultural Immunology, 24(3), 295–304. doi:10.
1080/09540105.2012.686988
Lorenzo, J. M., Agregan, R., Munekata, P. E. S., Franco, D., Carballo, J., Sahin, S., … Barba, F. J.
(2017). Proximate composition and nutritional value of three macroalgae: Ascophyllum
nodosum, Fucus vesiculosus and Bifurcaria bifurcata. Marine Drugs, 15(11), 360. doi:10.3390/
md15110360
Ma, Y. H., Xing, Y. Y., Mi, H. W., Guo, Z. Q., Lu, Y. Y., & Xi, T. (2014). Extraction, preliminary
characterization and immunostimulatory activity in vitro of a polysaccharide isolated from
Strongylocentrotus nudus eggs. Carbohydrate Polymers, 111, 576–583. doi:10.1016/j.carbpol.
2014.04.010
Maeda, R., Ida, T., Inara, H., & Sakamoto, T. (2012). Immunostimulatory activity of polysacchar-
ides isolated from Caulerpa lentillifera on macrophage cells. Bioscience Biotechnology and
Biochemistry, 76(3), 501–505. doi:10.1271/bbb.110813
Mao, S., & Guo, Y. (2010). Diterpenoids of the green alga Caulerpa taxifolia from the east China sea.
Zhongguo Haiyang Daxue Xuebao, Ziran Kexueban, 40(5), 79–82.
Masuko, T., Minami, A., Iwasaki, N., Majima, T., Nishimura, S. I., & Lee, Y. C. (2005).
Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Analytical
Biochemistry, 339(1), 69–72. doi:10.1016/j.ab.2004.12.001
Maximo, P., Ferreira, L. M., Branco, P., Lima, P., & Lourenco, A. (2018). Secondary metabolites and
biological activity of invasive macroalgae of Southern Europe. Marine Drugs, 16(8), 265. doi:10.
3390/md16080265
Mejia-Barradas, C. M., Del-Rio-Navarro, B. E., Dominguez-Lopez, A., Campos-Rodriguez, R.,
Martinez-Godinez, M. D., Rojas-Hernandez, S., … Miliar-Garcia, A. (2014). The consumption
of n-3 polyunsaturated fatty acids differentially modulates gene expression of peroxisome prolif-
erator-activated receptor alpha and gamma and hypoxia-inducible factor 1 alpha in subcu-
taneous adipose tissue of obese adolescents. Endocrine, 45(1), 98–105. doi:10.1007/s12020-
013-9941-y
Misurcova, L., Bunka, F., Ambrozova, J. V., Machu, L., Samek, D., & Kracmar, S. (2014). Amino
acid composition of algal products and its contribution to RDI. Food Chemistry, 151, 120–
125. doi:10.1016/j.foodchem.2013.11.040
Nagappan, T., & Vairappan, C. S. (2014). Nutritional and bioactive properties of three edible species
of green algae, genus Caulerpa (Caulerpaceae). Journal of Applied Phycology, 26(2), 1019–1027.
doi:10.1007/s10811-013-0147-8
 FOOD AND AGRICULTURAL IMMUNOLOGY 953

Nguyen, V. T., Ueng, J. P., & Tsai, G. J. (2011). Proximate composition, total phenolic content, and
antioxidant activity of seagrape (Caulerpa lentillifera). Journal of Food Science, 76(7), C950–
C958. doi:10.1111/j.1750-3841.2011.02289.x
Nishi, S. K., Kendall, C. W., Bazinet, R. P., Bashyam, B., Ireland, C. A., Augustin, L. S., … Jenkins, D.
J. (2014). Nut consumption, serum fatty acid profile and estimated coronary heart disease risk in
type 2 diabetes. Nutrition, Metabolism and Cardiovascular Diseases, 24(8), 845–852. doi:10.1016/
j.numecd.2014.04.001
Omar, H., Al-Judaibiand, A., & El-Gendy, A. (2018). Antimicrobial, antioxidant, anticancer activity
and phytochemical analysis of the red alga, Laurencia papillosa. International Journal of
Pharmacology, 14(4), 572–583. doi:10.3923/ijp.2018.572.583
Paiva, L., Lima, E., Neto, A. I., Marcone, M., & Baptista, J. (2017). Nutritional and functional bioac-
tivity value of selected Azorean macroalgae: Ulva compressa, Ulva rigida, Gelidium microdon,
and Pterocladiella capillacea. Journal of Food Science, 82(7), 1757–1764. doi:10.1111/1750-
3841.13778
Park, S. Y., Hwang, E., Shin, Y. K., Lee, D. G., Yang, J. E., Park, J. H., & Yi, T. H. (2017).
Immunostimulatory effect of enzyme-modified Hizikia fusiformein a mouse model in vitro
and ex vivo. Marine Biotechnology, 19(1), 65–75. doi:10.1007/s10126-017-9727-y
Peng, Y., Xie, E., Zheng, K., Fredimoses, M., Yang, X., Zhou, X., … Liu, Y. (2013). Nutritional and
chemical composition and antiviral activity of cultivated seaweed Sargassum naozhouense Tseng
et Lu. Marine Drugs, 11(1), 20–32. doi:10.3390/md11010020
Petropoulos, S. A., Pereira, C., Ntatsi, G., Danalatos, N., Barros, L., & Ferreira, I. C. F. R. (2018).
Nutritional value and chemical composition of Greek artichoke genotypes. Food Chemistry,
267, 296–302. doi:10.1016/j.foodchem.2017.01.159
Pires, T. C. S. P., Dias, M. I., Barros, L., & Ferreira, I. C. F. R. (2017). Nutritional and chemical
characterization of edible petals and corresponding infusions: Valorization as new food ingredi-
ents. Food Chemistry, 220, 337–343. doi:10.1016/j.foodchem.2016.10.026
Ran, Z. L., Zhang, Y. L., Wen, X. Y., & Ma, J. X. (2019). Curcumin inhibits high glucose-induced
inflammatory injury in human retinal pigment epithelial cells through the ROS-PI3K/AKT/
mTOR signaling pathway. Molecular Medicine Reports, 19(2), 1024–1031. doi:10.3892/mmr.
2018.9749
Rico, D., Diana, A. B. M., Milton-Laskibar, I., Fernandez-Quintela, A., Silvan, J. M., Rai, D. K., …
Martinez-Villaluenga, C. (2018). Characterization and in vitro evaluation of seaweed species as
potential functional ingredients to ameliorate metabolic syndrome. Journal of Functional Foods,
46, 185–194. doi:10.1016/j.jff.2018.05.010
Rodrigues, D., Freitas, A. C., Pereira, L., Rocha-Santos, T. A. P., Vasconcelos, M. W., Roriz, M., …
Duarte, A. C. (2015). Chemical composition of red, brown and green macroalgae from Buarcos
bay in Central West coast of Portugal. Food Chemistry, 183, 197–207. doi:10.1016/j.foodchem.
2015.03.057
Sarkar, P., Basak, P., Ghosh, S., Kundu, M., & Sil, P. C. (2017). Prophylactic role of taurine and its
derivatives against diabetes mellitus and its related complications. Food and Chemical
Toxicology, 110, 109–121. doi:10.1016/j.fct.2017.10.022
Shen, R. L., Ma, Y. L., Jiang, L. B., Dong, J. L., Zhu, Y. Y., & Ren, G. X. (2018). Chemical compo-
sition, antioxidant, and antiproliferative activities of nine Chinese proso millet varieties. Food
and Agricultural Immunology, 29(1), 625–637. doi:10.1080/09540105.2018.1428283
Shen, W. Z., Wang, H., Guo, G. Q., & Tuo, J. J. (2008). Immunomodulatory effects of Caulerpa race-
mosa var peltata polysaccharide and its selenizing product on T lymphocytes and NK cells in
mice. Science in China Series C: Life Sciences, 51(9), 795–801. doi:10.1007/s11427-008-0106-9
Shi, Z. X., Yao, Y., Zhu, Y. Y., & Ren, G. X. (2017). Nutritional composition and biological activities
of 17 Chinese adzuki bean (Vigna angularis) varieties. Food and Agricultural Immunology, 28(1),
78–89. doi:10.1080/09540105.2016.1208152
Simopoulos, A. P. (2006). Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic vari-
ation: Nutritional implications for chronic diseases. Biomedicine & Pharmacotherapy, 60(9),
502–507. doi:10.1016/j.biopha.2006.07.080
954 H. HAO ET AL.

Song, Y., Wei, X. Q., Li, M. Y., Duan, X. W., Sun, Y. M., Yang, R. L., … Wang, H. (2018). Nutritional
composition and antioxidant properties of the fruits of a Chinese wild Passiflora foetida.
Molecules, 23(2), 459. doi:10.3390/molecules23020459
Tanna, B., & Mishra, A. (2018). Metabolites Unravel nutraceutical potential of edible seaweeds: An
emerging source of functional food. Comprehensive Reviews in Food Science and Food Safety, 17
(6), 1613–1624. doi:10.1111/1541-4337.12396
Vieira, E. F., Soares, C., Machado, S., Correia, M., Ramalhosa, M. J., Oliva-teles, M. T., … Delerue-
Matos, C. (2018). Seaweeds from the Portuguese coast as a source of proteinaceous material:
Total and free amino acid composition profile. Food Chemistry, 269, 264–275. doi:10.1016/j.
foodchem.2018.06.145
Wijesinghe, W. A. J. P., & Jeon, Y. J. (2012). Exploiting biological activities of brown seaweed
Ecklonia cava for potential industrial applications: A review. International Journal of Food
Sciences and Nutrition, 63(2), 225–235. doi:10.3109/09637486.2011.619965
Xu, Z., Mao, T. M., Huang, L., Yu, Z. C., Yin, B., Chen, M. L., & Cheng, Y. H. (2019). Purification
and identification immunomodulatory peptide from rice protein hydrolysates. Food and
Agricultural Immunology, 30(1), 150–162. doi:10.1080/09540105.2018.1553938
Xu, P. H., Tan, H. Q., Tin, W. G., Li, Y. F., Santhoshkumar, C., Li, P., & Liu, W. H. (2018).
Antioxidative and antimicrobial activities of intertidal seaweeds and possible effects of abiotic
factors on these bioactivities. Journal of Oceanology and Limnology, 36(6), 2243–2256. doi:10.
1007/s00343-019-7046-z
Yang, P., Liu, D. Q., Liang, T. J., Li, J., Zhang, H. Y., Liu, A. H., … Mao, S. C. (2015). Bioactive con-
stituents from the green alga Caulerpa racemosa. Bioorganic & Medicinal Chemistry, 23(1), 38–
45. doi:10.1016/j.bmc.2014.11.031
Yang, H., Wu, Q. Y., Tang, M., Kong, L., & Lu, Z. H. (2009). Cell membrane injury induced by silica
nanoparticles in mouse macrophage. Journal of Biomedical Nanotechnology, 5(5), 528–535.
doi:10.1166/jbn.2009.1061
Yuan, Q. X., Zhao, L. Y., Cha, Q. Q., Sun, Y., Ye, H., & Zeng, X. X. (2015). Structural characteriz-
ation and immunostirnulatory activity of a homogeneous polysaccharide from Sinonovacula
constricta. Journal of Agricultural and Food Chemistry, 63(36), 7986–7994. doi:10.1021/acs.
jafc.5b03306
Ziani, B. E. C., Rached, W., Bachari, K., Alves, M. J., Calhelha, R. C., Barros, L., & Ferreira, I. C. F. R.
(2019). Detailed chemical composition and functional properties of Ammodaucus leucotrichus
Cross. & Dur. And Moringa oleifera Lamarck. Journal of Functional Foods, 53, 237–247.
doi:10.1016/j.jff.2018.12.023