Food Chemistry 245 (2018) 899–910

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Review

Structures, bioactivities and future prospective of polysaccharides from T

Morus alba (white mulberry): A review
⁎
Xirui Hea,b,1, , Jiacheng Fangb,1, Yinlan Ruanc,d, Xiaoxiao Wangb, Yin Sunb, Ni Wub,
⁎
Zefeng Zhaob, Yu Changa, Ning Ninga, Hao Guoa, Linhong Huanga,
a
Honghui Hospital, Xi’an Jiaotong University, Xi’an 710054, PR China
b
Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education, Xi’an 710069, PR China
c
ARC Centre of Excellence for Nanoscale BioPhotonics, Adelaide, SA 5005, Australia
d
Institute for Photonics and Advanced Sensing, The University of Adelaide, Adelaide, SA 5005, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Morus alba L. (family Moreaceae), also known as white mulberry, is distinguished as a source of highly promising
Medicinal and edible traditional medicines (including Mori Folium, Mori Fructus, Mori Ramulus and Mori Cortex) and also functional
Mori Folium foods. Over the past two decades, the vast majority of the studies with regard to the isolation and bioactivities of
Mori Fructus M. alba polysaccharides have mainly focused on its leaves and fruits, which are both medicinal and edible. The
Mori Ramulus
tender M. alba leaf is edible and can be used to make tea, the mature M. alba fruit is sweet and juicy. M. alba
Mori Cortex
fruits and leaves contain rich bioactive polysaccharides, which are shown to possess various promising bioac-
tivities, mainly including antidiabetic, immunomodulation, anti-inflammation, antioxidation, anti-obesity, he-
patoprotection and renoprotection. The main purpose of this review is to provide systematically reorganized
information on structural characteristics and bioactivities of M. alba polysaccharides to support their further
therapeutic potentials and sanitarian functions.

1. Introduction meanwhile widely cultivated and naturalized elsewhere. Nowadays, M.

Morus alba L (family Moreaceae), also known as white mulberry
(English), 桑树 (Chinese), witte moerbei (Dutch), valkomulperi
(Finnish), mûrier blanc (French), weiβe maulbeere (German), tut
(Hindi), gelso bianco (Italian), kuwa (Japanese), mulbari (Malayalam),
amoreira-branca (Portuguese), dud alb (Romanian), šelkovica belaja
(Russian) (Encyclopedia of Life, 2017), is a short-lived, fast-growing,
small to medium sized tree. The genus Morus contains approximately
nineteen members (The Plant List, 2017), and the most commonly
grown species are white mulberry (M. alba L.), black mulberry (M. nigra
L.), and red mulberry (M. rubra L.) (Ercisli & Orhan, 2007). Among the
three species, M. alba is the dominant one, and it is native to China and
alba is mainly distributed in the temperate and tropical regions, in-
cluding South Europe, North America, East and South East Asia, and
southeastern Australia, and also some parts of Africa (Encyclopedia of
Life, 2017).
Herein, this review summarizes four parts of M. alba, and the vast
majority of the content focuses on its leaves and fruits, which are
medicinal and edible. M. alba leaves can be used to make tea, and are
also the diet for the silkworm (Bombyx mori) employed in the com-
mercial production of silk (Katayama, Takano, & Sugimura, 2008). M.
alba fruits have a dominant taste of sweet and usually somewhat bland,
but a richer flavor develops after drying. In recent years, with con-
siderable work done on M. alba cultivation under various conditions,

Abbreviations: ABTS, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid); AG II, arabinogalactans type II; Akt, protein kinase B; Bcl-2, B-cell lymphoma-2; BMDC, bone-marrow-derived
dendritic cell; cAMP, cyclic adenosine monophosphate; CAT, catalase; CMPS, cationic M. alba leaf polysaccharides; DCs, dendritic cells; ERK, extracellular signal-related kinases; FBG,
fasting blood glucose; FRAP, ferric-reducing antioxidant power; GLUT4, glucose transporter type 4; GPx, glutathione peroxidase; GSH-Rd, glutathione reductase; GSP, glycosylated serum
protein; HDL-C, high-density lipoprotein cholesterol; IFN-β, interferon-β; IFN-γ, interferon-γ; IL, interleukin; iNOS, inducible nitric oxide synthase; IRSs, insulin receptor substrates; JNK,
c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; MnSOD, manganese superoxide dismutase; MLPs, M. alba leaf
polysaccharides; MFPs, M. alba fruit polysaccharides; MP, an isolated M. alba fruit glycoprotein; MRPs, M. alba branch polysaccharides; MCPs, M. alba root bark polysaccharides; MHC,
major histocompatibility complex; NK, natural killer; NO, nitric oxide; PARP, poly (ADP-ribose) polymerase; PDX-1, pancreatic duodenal homeobox-1; p-ERK, phosphor-extracellular
regulated protein kinases; pTGF-β1, plasmid transforming growth factor β1; RG-I, rhamnogalacturonan type I; PI3K, phosphatidylinositol 3-kinase; STZ, streptozocin; T2DM, type 2
diabetes mellitus; TC, total cholesterol; TG, triglyceride; TNF-α, tumor necrosis factor-α
⁎
Corresponding authors at: Honghui Hospital, Xi’an Jiaotong University, Xi’an 710054, PR China (X. He).
E-mail addresses: hxrhist@163.com (X. He), huanglhlab@163.com (L. Huang).
1
Equally first authors.

https://doi.org/10.1016/j.foodchem.2017.11.084
Received 27 June 2017; Received in revised form 16 October 2017; Accepted 22 November 2017
Available online 23 November 2017
0308-8146/ © 2017 Elsevier Ltd. All rights reserved.
X. He et al.
Fig. 1. Fresh leaves of Morus alba L. (a); fresh fruits of Morus alba L. (b); fresh root bark of Morus alba L. (c); fresh branches of Morus alba L. (d); Mori Folium (桑叶) (e); Mori Fructus (桑椹)
(f); Mori Cortex (桑白皮) (g); Mori Ramulus (桑枝) (h).
mulberry fruit juice has been commercially produced as a health bev-
erage, which is popular in East Asian countries, such as China, Japan
and Korea.
M. alba has had all parts of it used in Traditional Chinese Medicine
for thousands of years (Chinese Herbalism Editorial Board, 1999),
particularly its leaves, fruits, branches, and root bark, are all recorded
in the 2015 edition of Chinese Pharmacopoeia (China Pharmacopoeia
Commission, 2015). Mori Folium (桑叶), the dry leaves of M. alba
collected in the autumn, has wind and heat dispelling, lung clearing and
dryness moisturizing, liver clearing and eyesight improving properties;
Mori Fructus (桑椹), the dry ripe aggregate fruit of M. alba, can be used
for liver-kidney yin deficiency syndrome; Mori Ramulus (桑枝), the dry
young branches of M. alba collected in the late spring and early
summer, is a wind-dampness dispelling formula which can be used for
the treatment of arthritis and rheumatism; Mori Cortex (桑白皮), the
dry root bark of M. alba collected in the late autumn and early spring, is
capable of purging the lung to calm panting and inducing diuresis to
alleviate edema (China Pharmacopoeia Commission, 2015; Tang &
Eisenbrand, 1992). Their relevant pictures are listed in Fig. 1. To date,
China Food and Drug Administration (CFDA) has approved patent
drugs, namely Sang Shen Paste/Granule, Sang Ma Pill/Oral Solution,
and Sang Zhi Granule, which contain Mori Fructus, Mori Folium, and
Mori Ramulus as the main medicinal ingredient, respectively (China
Food, 2017).
The main active constituents in M. alba include flavonoids, alka-
loids, stilbenes, diels–alder type adducts, and polysaccharides (Tang &
Eisenbrand, 1992; Yang, Wang, & Li, 2014). To date, it has been re-
ported that M. alba leaf polysaccharides (MLPs), M. alba fruit poly-
saccharides (MFPs), M. alba branch polysaccharides (MRPs), and M.
alba root bark polysaccharides (MCPs) have various bioactivities, but
most currently evidence focuses on the antidiabetic properties of MLPs
and MFPs. According to the available literatures, the monosaccharide
composition of MLPs is predominantly glucose, galactose, arabinose,
fructose, xylose, rhamnose, glucuronic acid, galacturonic acid, and
meanwhile a small quantity of mannose and sorbose (Katayama et al.,
2008; Lin, Tsai, & Lai, 2009; Ouyang, Chen, & Li, 2005; Sanavova &
Rakhimov, 1997), while the monosaccharide composition of MFPs is
mainly glucose, galactose, arabinose, and galacturonic acid (Chen, You,
Abbasi, Fu, & Liu, 2015). Although the vast majority of M. alba poly-
saccharides were shown to be acidic heteropolysaccharides, they are
also a source of glucans with α, β-glycosidic linkages (Tian, 2014; Xia,
Shen, Wang, & Zhang, 2009; Zhang, Liao, Cong, Dong, & Ding, 2013).
Pharmacological results have revealed that some crude M. alba
polysaccharides and other purified fractions possess various promising
bioactivities (Fig. 2), including antidiabetic activity, im-
munomodulatory activity, anti-inflammatory activity, antioxidant ac-
tivity, hepatoprotective and renoprotective activities and anti-obesity
activity. Reviewing the available literatures, no review concerning M.
alba polysaccharides is available. In this review, we intend to system-
atically summarize the research findings in the past twenty years and
provide a comprehensive insight into the structure characteristics,
900
Food Chemistry 245 (2018) 899–910
pharmacological effects, and future prospective of M. alba poly-
saccharides to provide knowledge to people for better utilization of
them.
2. Extraction methods and purification procedures
Briefly, the M. alba leaves and/or fruits were washed, dried and
ground to obtain fine powders with different particle size (10–60 mesh)
(Samavati & Yarmand, 2013), then were immersed in ether or ethanol
overnight (Thirugnanasambandham, Sivakumar, & Maran, 2015; Wei,
Hu, Bo, Huang, & Jiang, 2007) and refluxed with 80–95% ethanol for
hours to remove lipophilic compounds (Chen et al., 2015; Deng, Zhou,
& Chen, 2014; Zhang et al., 2014a), the residues were successively
extracted in a Soxhlet apparatus with 80% ether or 80% ethanol to
remove pigment, oligosaccharides and some small molecule materials
(Deng et al., 2014; Samavati & Yarmand, 2013), the organic solvent was
volatilized and thus the pretreated dry powders were obtained. The list
of the extraction methods of polysaccharides in M. alba leaf and M. alba
fruit obtained from the pretreated dry powders is summarized in
Table 1. Generally, the extraction time has a great influence on the
yield for the conventional water extraction, and the extraction tem-
perature together with the liquid–solid ratios are usually at a range of
70–92 °C and 10–34 (ml/g), respectively (Jia, Zhang, Lan, Yang, & Sun,
2013; Samavati & Yarmand, 2013; Tang et al., 2011; Wei et al., 2007;
Ying, Han, & Li, 2011; Yuan et al., 2015). The basic extraction rule is to
break the cell wall from the outer layer to the inner layer with mild-
strong extraction conditions, and it has been shown that the localization
of mucilaginous polysaccharides in mulberry leaves is specifically in
inner epidermal cells situated in adaxial leaves (Katayama et al., 2008).
According to the recent experimental work, electroporation, ultra-
sound, and enzyme were applied to help break the mulberry leaf and
fruit cell walls to assist polysaccharide dissolution, and enzyme assis-
tance seems to be the most effective way (Chen et al., 2015; Deng et al.,
2014; Ying et al., 2011; Zhang et al., 2016). Response surface metho-
dology (RSM) based on a Box-Behnken design (BBD) or Central Com-
posite Rotatable design (CCRD) was applied to optimize the extraction
conditions, and the resulting extraction conditions were used to obtain
crude MLPs and MFPs through water extraction and ethanol pre-
cipitation (Chen et al., 2015; Deng et al., 2014; Ying et al., 2011). Taken
together, with the application of various technologies, a higher yield
can be obtained, even with shorter extraction time, fewer extraction
times, lower extraction temperature and smaller solid–liquid ratio.
Furthermore, the microwave assistive technology which can reduce
emulsion viscosity and break the outer film was also applied to MLPs
extraction (Thirugnanasambandham et al., 2015; Ying et al., 2011), the
extraction time is further shortened while maintaining a high yield. It is
worth mentioning that a 16-series fluidic system based on the trans-
former concept was built for the induced-electric-field-assisted extrac-
tion of MLPs. Extraction was accelerated by exposing a secondary coil,
made from a flowing mixture of sample grains and extractant, to a
20–80 kHz induced alternating electric field. It was suggested that
X. He et al. Food Chemistry 245 (2018) 899–910

Fig. 2. Recorded biological properties of polysaccharides

from different parts of Morus alba L. (white mulberry).

damaged degrees of the cell membrane increased by electroporation
effect, and the 16-series system is performed optimally with a yield of
8.54% at a liquid–solid ratio of 20.0, pH 2.5, 60 °C, 1000 V excitation
voltage, and 20 kHz frequency (Yang, Zhang, Jin, Jin, & Xu, 2017).
Crude polysaccharides from M. alba can be further purified with a
series of combined techniques, including precipitation with ethanol;
protein removal by Savage reagent; flavonoid and phenol removal
through macroporous resin; decolorization by H2O2, macroporous
resin, or activated carbon; ion-exchange chromatography; gel filtration;
and affinity chromatography (Chen et al., 2015; Jia et al., 2013; Luo,
Wang, & Cao, 2011; Samavati & Yarmand, 2013;
Thirugnanasambandham et al., 2015; Wei et al., 2007; Ying et al., 2011;
Zhang et al., 2016). Generally, after precipitation, deproteinization,
decolorization, dialysis, evaporation, filtration, and lyophilization, the
crude M. alba polysaccharides are obtained. Once again dissolved into
solution, the resultant polysaccharide solution was usually subjected to
different column chromatography mentioned above, and sequentially
eluted with appropriate running buffer, the purified M. alba poly-
saccharides could be ultimately obtained.
3. Physiochemical and structural features
Based on the available literatures, a total of more than fifty-two
polysaccharides were obtained, especially MLPs and MFPs, which oc-
cupy the vast majority. Herein, we list the reported fifty-two M. alba
polysaccharides and provided a comprehensive information with regard
to their molecular weight, monosaccharide composition, structures,
bioactivities, and associated references in Table 2. A summary of the
commonly used chromatography and corresponding columns and elu-
tion conditions for homogeneity and molecular weight determination
and monosaccharide composition analysis of M. alba polysaccharides
has been presented in Table 3.
Early in 1994, a crude acidic polysaccharide consisting of GalA re-
sidues and neutral monosaccharides including Glc, Gal, and Ara was
isolated from the M. alba leaves (Rakhimdzhanov, Sanavova, & Abidov,
1994). Three years later, the water-soluble polysaccharides (WSPSs),
the pectin substances (PcSs), along with the hemicelluloses (HMCs-A
and -B) were isolated from the aqueous extract, the buffer solution, and
the alkaline extract of Morus alba leaves, respectively. The quantita-
tively predominating polysaccharides in the M. alba leaves were shown
to be PcSs which contained mainly Gal, Glc, Ara, Rha, and GalA. The
same as PcSs, WSPSs have practically the same qualitative mono-
saccharide composition, however relatively small proportion of Gal and
Glc. The alkali-soluble polysaccharides (HMCs-A and -B) differ by their
monosaccharide components: the HMCs-A contains mainly Gal, Xyl,
and Glc, and the HMCs-B contains mainly Glc, Rha, Gal, Xyl, and Ara
(Sanavova & Rakhimov, 1997). Recent experimental work showed that
a polysaccharide obtained from aqueous extract of M. alba leaves is
composed of Glc, Rha, Ara, Gal, Xyl, Man, GalA, and GlcA, and the
composition and distribution of monosaccharides is approximately
consistent with the findings in previous studies (Li et al., 2011).
A pectic polysaccharide named SDA was obtained from the boiling
aqueous extract of M. alba leaves, which showed a symmetrical narrow
peak on high-performance gel filtration chromatography (HPGFC). A
combination of sugar composition analysis revealed that it was com-
posed of Rha, Ara, Xyl, Glc, Gal, and GalA units, and its molecular
weight was determined to be 15 kDa by HPGFC. Predominant signals at
δ 99.0, 77.2 were assigned to C-1 and C-4 of (1 → 4)-linked GalA re-
sidues, respectively, a combination of linkage analysis, partial acid
hydrolysis, ESIMS, 1HNMR, and 13C NMR spectral analyses revealed
that SDA has an α-(1 → 4)-galacturonan backbone with branches of
(1 → 2)-linked Rha residues, and with side chains of GalA residues

Table 1
A summary of the extraction of M. alba leaf polysaccharides (MLPs) and M. alba fruit polysaccharides (MFPs).

Types Time Solid-liquid ratio Temperature Solvent Other conditions Yied (%) References

Routine extraction
MLPs 90 min 1:24 70 °C water unknown 2.64 Tang et al. (2011)
MLPs 60 min 1:10 70 °C water unknown 2.91 Jia et al. (2013)
MLPs 2 times, 85 min each time 1:17 80 °C water unknown 4.67 Ying et al. (2011)
MLPs 4 times, 300 min each time 1:18 85 °C water unknown 12.00 Samavati and Yarmand (2013)
MLPs 2 times, 210 min each time 1:34 92 °C water unknown 10.00 Yuan et al. (2015)
MFPs 2 times, 240 min each time 1:30 80 °C water unknown 5.71 Wei et al. (2007)

Ultrasound-assisted extraction
MLPs 20 min 1:15 80 °C water ultrasonic power 60 W 10.79 Ying et al. (2011)
MLPs 80 min 1:53 57 °C water ultrasonic power 100 W 6.92 Zhang et al. (2016)
MFPs 75 min 1:40 69 °C water ultrasonic power 190 W 3.13 Chen et al. (2015)

Microwave-assisted extraction
MLPs 11 min 1:25 70 °C water microwave power 436 W 9.53 Ying et al. (2011)
MLPs 10 min unknown unknown water microwave power 170 W 9.41 Thirugnanasambandham et al. (2015)

Enzyme-assisted extraction
MFPs 38 min 1:11 58 °C water ratio of enzyme amount 0.40% 16.16 Deng et al. (2014)

901
 Table 2
Composition of monosaccharides, molecular weight, main structures, and bioactivities of MLPs, MFPs, MRPs, and MCPs.
X. He et al.

No. Name Monosaccharide composition M.W. (Da) Main structure Bioactivity Ref.

M. alba leaf polysaccharides (MLPs)

1 WSPSs Gal, Glc, Man, Xyl, Ara, Rha in a ratio of 6.0:9.7:1.3:1.0:4.4:5.3 ND ND ND Sanavova and
Rakhimov (1997))
2 PcSs Gal, Glc, Man, Xyl, Ara, Rha, GalA in a ratio of ND ND ND Sanavova and
12.6:17.4:1.6:1.0:5.9:4.2:49.3 Rakhimov (1997)
3 HMCs-A Gal, Glc, Man, Xyl, Ara, Rha in a ratio of 13.9:4.9:2.0:5.7:1.1:1.0 ND ND ND Sanavova and
Rakhimov (1997)
4 HMCs-B Gal, Glc, Man, Xyl, Ara, Rha in a ratio of 16.5:23.6:1.0:16.1:13.3:20.6 ND ND ND Sanavova and
Rakhimov (1997)
5 MP11 Rha, Ara, Xyl, Man, Glc, Gal in a ratio of 26:16:3:3:1:20 ND ND ND Ouyang et al. (2005)
6 MP12 Rha, Glc in a ratio of 3:1 ND ND ND Ouyang et al. (2005)
7 MP13 Rha ND ND ND Ouyang et al. (2005)
8 APFM Rha, Ara, Fru, Glc, Gal in a ratio of 8.91:2.71:1.00:3.75:6.04 5.0 × 105 ND ND Jin et al. (2007)
9 MP-3b Rha, Ara, Xyl, Glc, Gal, GalA in a ratio of 0.65:1.00:0.23:0.42:0.98:0.60 8.9 × 104 ND ND Xia et al. (2009)
10 SDA Rha, Ara, Xyl, Glc, Gal, GalA in a ratio of 5:4:1:2:6:38 1.5 × 104 α-(1 → 4)-galacturonan backbone ND Xia et al. (2008)
11 Mp-3 Rha, Gal, Glc, GalA, GlcA in a ratio of 1:0.2:0.5:2.3:1.5 5.5 × 105 rhamnogalacturonan mainly consisting of (1 → ND Katayama et al.
2,3)-Rha, (1 → 4,1 → 3,4)-uronic acid, and terminal (2008)
uronic acid residues
12 SJB Rha, Ara, Glc, Gal, GalA in a ratio of 1.00:1.26:0.66:2.04:0.95 5.4 × 104 backbone composed of (1 → 2,1 → 2,4)-Rhap and PTP1B inhibitory activity Xia et al. (2008)
(1 → 4,1 → 3,4)-GalAp
6
13 Hw Rha, Ara, Gal, Glc, Xyl, Man in a ratio of 6.1 × 10 ND ND Lin et al. (2009)
23.47:20.00:23.13:26.19:7.03:0.23
6
14 Alk Rha, Ara, Gal, Glc, Xyl, Man in a ratio of 6.6 × 10 ND ND Lin et al. (2009)
27.11:21.48:23.62:21.37:6.17:0.22
15 MPL1 Fru, Ara, Rha, Xyl, Glc in a ratio of 58.0:9.9:5.8:5.1:21.2 1.2 × 104 backbone composed of (1 → 3)-linked glycosyl ND Luo and Cao (2010)

902
bonds
16 MPL2 Fru, Ara, Rha, Xyl, Glc in a ratio of 45:6.74:17.2:7.3:24.1 7.6 × 103 backbone composed of (1 → 3)-linked glycosyl ND Luo and Cao (2010)
bonds
4
17 MPS-1 Sor, Ara, Xyl, Glc in a ratio of 3:2:17:110 2.5 × 10 (1 → 3)-branched (1 → 6)-β-glucan ND Ying et al. (2011)
18 MPS-2 Rha, Ara, Xyl, Glc, Gal, Man in a ratio of 13.8:9.7:14.4:9.6:9.3:13.5 6.1 × 104 β-(1 → 6,1 → 2), α-(1 → 2,1 → 3)-branched (1 → 3)- ND Ying et al. (2011)
β-glucan
19 HDP Glc, Rha, Ara, Gal, Xyl, Man, GalA, GlcA in a ratio of 1.9 × 104 ND hypoglycemic activity Li et al. (2011)
1.00:5.39:2.04:5.02:0.20:0.49:5.67:6.14
20 MLP Man, GalA, Rha in a ratio of 1.0:7.2:20.0 5.6 × 105 high-methoxyl pectin ND Jia et al. (2013)
21 MLPII Ara, Xyl, Glc, Rha, Man in a ratio of 1.00:2.13:6.53:1.04:8.73 8.1 × 103 β-glycosidic linkages hypoglycemic activity Zhang et al. (2014b)
22 MLP-3a Man, Rha, GlcA, GalA, Glc, Gal, Ara in a ratio of 8.1 × 104 β-glycosidic linkages antioxidant activity Yuan et al. (2015)
0.77:4.53:0.81:1.21:3.47:12.55:11.14
23 MLP-3b Rha, GlcA, GalA, Gal, Ara in a ratio of 1.57:0.20:6.10:1.27:0.89 3.6 × 103 β-glycosidic linkages antioxidant activity Yuan et al. (2015)
24 MLP3 Ara, Man, Rha, Gal, Glc in a ratio of 5.27:0.75:6.83:1.89:1.72 ND ND antioxidant activity Zhang et al. (2016)
25 MLP Carbohydrate, uronic acid in a ratio of 75.34:15.75 3.6 × 104 β-glycosidic linkages ND Yang et al. (2017)
(CFHE)
26 MLP Carbohydrate, uronic acid in a ratio of 80.43:11.13 3.8 × 104 β-glycosidic linkages ND Yang et al. (2017)
(IEFAE)
3
27 SDC Rha, Ara, Xyl, Gal, GalA in a ratio of 1.0:1.6:1.2:2.0:41.7 8.0 × 10 α-(1 → 4)-galacturonan backbone ND Xia et al. (2009)
28 PMP11 Rha, GalA, GlcA in a ratio of 2.27:1.59:1.00 2.1 × 106 backbone mainly composed of β-(1 → 2,1 → 3)- ND Zhao et al. (2010)
linked Rha
29 PMP12 Rha, Ara, Gal, GlcA in a ratio of 1.00:1.56:1.57:1.08 > 1.0 × 106 backbone mainly composed of β-(1 → 3)-linked Rha ND Zhao et al. (2010)
30 MPA-1 Glc 1.1 × 105 α-(1 → 4)-glucan backbone ND Xia et al. (2009)

M. alba fruit polysaccharides (MFPs)

31 FMAP Gal, Man, Glc in a ratio of 28.6:65.9:5.2 1.3 × 105 backbone composed of (1 → 3)-linked mannan with ND Wei et al. (2007)
branches of galactosyl and glucosyl residues
32 FMAP-2 Glc, Tal, Ara, Gal, Man, Fru in a ratio of 3.0 × 105 ND hypoglycemic activity Hu (2009)
34.8:25.47:22.78:8.60:4.49:3.87
(continued on next page)
Food Chemistry 245 (2018) 899–910
 Table 2 (continued)

No. Name Monosaccharide composition M.W. (Da) Main structure Bioactivity Ref.
X. He et al.

6
33 JS-MP-1 Gal, Ara, Rha, Glc, Xyl, Man, Fuc, GalA, GlcA in a ratio of 1.6 × 10 backbone composed of rhamnogalacturonan with immunomodulating and anti- Choi et al. (2016)
37.6:36.3:18.4:3.1:1.7:1.6:1.3:9.00:2.25 (1 → 4)-α-D-GalAp and (1 → 2)-α-L-Rhap obesity activities and Lee et al. (2013)
34 MFP-1 Ara, Gal, Glc, GalA in a ratio of 7.19:6.33:86.48:12.59 ND ND antioxidant and hypoglycemic Chen et al. (2015)
activities
Ara, Gal, Glc, Xyl, Man, GalA in a ratio of 9.5 × 103 (8.39%), 7.9 × 103 ND antioxidant and hypoglycemic Chen et al. (2016a)
19.19:31.4:26.31:5.98:7.12:1.57 (36.32%), 1.0 × 103 (5.89%), activities
0.7 × 103 (33.31%)
Gal, Glc, Ara, Rha, Man in a ratio of 8.00:20.40:8.74:5.00:1.00 1.1 × 105 ND effect on alcohol Wang, Deng, Chen,
dehydrogenase and Zhou (2017)
35 MFP-2 Ara, Gal, Glc, GalA in a ratio of 36.01:34.12:29.87:14.52 ND ND antioxidant and hypoglycemic Chen et al. (2015)
activities
Ara, Gal, GalA in a ratio of 44.90:55.20:2.11 149.4 × 103 (28.16%), 9.3 × 103 ND antioxidant and hypoglycemic Chen et al. (2016a)
(20.84%), 2.6 × 103 (3.64%), activities
1.5 × 103 (26.58%)
Ara, Gal, Rha, Glc, GalA in a ratio of 34.20:38.20:8.00:17.50:15.10 1.3 × 105 ND effect on alcohol Wang et al. (2017)
dehydrogenase
3 3
36 MFP-3 Ara, Gal, GalA in a ratio of 55.30:44.70:6.18 166.7 × 10 (45.08%), 5.0 × 10 ND antioxidant and hypoglycemic Chen et al. (2016a)
(20.84%), 1.5 × 103 (27.65%) activities
GalA, Gal, Glc, Rha, Ara in a ratio of 28.30:22.60:20.90:18.60:15.10 2.0 × 105 β-glycosidic linkages effect on alcohol Wang et al. (2017)
dehydrogenase
37 MFP-4 Ara, Gal, Glc, GalA in a ratio of 59.86:27.15:12.99:31.90 184.7 × 103 (7.09%), 64.4 × 103 ND antioxidant and hypoglycemic Chen et al. (2016a)
(5.2%), 1.5 × 103 (56.05%), activities
1.5 × 103 (26.58%)
Glc, Gal, Rha, GalA, Ara, GlcA in a ratio of 1.8 × 105 β-glycosidic linkages effect on alcohol Wang et al. (2017)
47.40:34.90:36.10:33.10:19.90:4.10 dehydrogenase
38 MFP3P Rha, Ara, Gal, Glc, GalA in a ratio of 25.98:21.51:23.1:13.06:16.35 1.4 × 105 (1 → 6)-linked Glc, (1 → 2,1 → 3)-linked Rha, (1 → antioxidant and hypoglycemic Chen et al. (2016)

903
3)-linked Gal activities
39 MFPs Glc, GalA, Rha, Gal in a ratio of 39.79:17.15:15.16:14.48, along with a ranged from 1.1 × 105 to α-glycosidic linkages antioxidant activity Zhou et al. (2017)
small quantity of GlcA, Xyl, Ara, and Man 1.8 × 105
40 PMF1 Xyl, Gal in a ratio of 3.26:96.74 7.2 × 104 (1 → 2), (1 → 3), or (1 → 6)-linked hexapyranose ND Tian (2014)
residues
41 PMF2 Glc 8.4 × 104 (1 → 2), (1 → 3), or (1 → 6)-linked hexapyranose ND Tian (2014)
residues, α, β-glycosidic linkages
42 PMF3 Rha, Fuc, Xyl, Man, Glc, Gal in a ratio of 1.0 × 105 (1 → 2), (1 → 3), or (1 → 6)-linked hexapyranose ND Tian (2014)
9.31:1.76:13.09:14.18:25.49:36.17 residues, α, β-glycosidic linkages

M. alba branch polysaccharides (MRPs)

43 RMP Glc, Gal, Man, Rha, Ara in a ratio of 62.1:13.8:6.7:11.6:5.8 4.6 × 105 ND anti-inflammatory and Guo et al. (2013b)
antioxidant activities
44 RMPs-1 Rha, Ara, Glc, Gal, GalA in a ratio of 1.08:1.00:1.40:1.57:3.80 5.8 × 105 (1 → 2,1 → 4)-linked glycosidic linkages Hela cells inhibitory activity Wang (2010)
45 RMPs-2 Rha, Glc, Gal, GalA in a ratio of 24.0:1.00:1.34:12.61 6.5 × 105 (1 → 2,1 → 4)-linked glycosidic linkages Hela cells inhibitory activity Wang (2010)
46 MBBP-1 Rha, Xyl, Ara, Man, Glc, Gal in a ratio of 1.1 × 105 backbone mainly composed of (1 → 3)-linked Glc antioxidant activity Qiu et al. (2016)
4.53:2.49:4.38:4.67:17.85:5.88
47 MBBP-2 Rha, Xyl, Ara, Man, Glc, Gal, GalA in a ratio of 1.2 × 105 backbone mainly composed of (1 → 3)-linked Rha antioxidant activity Qiu et al. (2016)
26.85:13.8:3.14:4.4:6.1:3.19:4.9 and (1 → 2,4)-linked Xyl

M. alba root bark polysaccharides (MCPs)

48 CMA-a-1 Glc 2.2 × 105 amylopectin-like polysaccharide with an average ND Zhang et al. (2013)
chain length of 23
49 CMA-a-5 Glc 1.6 × 104 amylose fraction with an average chain length of 30 ND Zhang et al. (2013)
50 CMA-b1-1 Rha, Ara, Xyl, Gal, GalA, Glc in a ratio of 5.4 × 104 backbone composed of alternating (1 → 4)-linked α- SMMC7721 hepatoma cells Zhang et al. (2013)
8.6:31.3:11.0:24.6:16.9:7.4 D-GalA and (1 → 2)-linked α-L-Rha inhibitory activity

Abbreviations: Ara, arabinose; Xyl, xylose; Man, mannose; Glc, glucose; Gal, galactose; Fuc, fucose; Fru, fructose; Rha, rhamnose; Sor, sorbose; Tal, talose; GlcA, glucuronic acid; GalA, galacturonic acid. ND, not detected.
Food Chemistry 245 (2018) 899–910
X. He et al. Food Chemistry 245 (2018) 899–910

Table 3
A summary of the commonly used chromatography and corresponding columns and elution conditions for homogeneity and molecular weight determination and monosaccharide
composition analysis of M. alba polysaccharides.

No. Chromatography Column Elution condition Ref.

Homogeneity and molecular weight determination

1 HPLC Shodex OHpak SB-806HQ (8.0 × 300 mm) deionized water, 0.8 ml/min Lee et al. (2013)
2 HPLC TSK G-3000 SW (7.5 × 300 mm) phosphate buffer (pH 6.0), 1.0 ml/min Wei et al. (2007)
3 HPLC TSK G4000PWXL (7.8 × 300 mm, 10 μm) deionized water, 0.6 ml/min Qiu et al. (2016)
4 HPLC TSK-GEL GMPWXL (7.8 × 300 mm) deionized water, 0.6 ml/min Jin et al. (2007)
5 HPGFC TSK PWG4000 0.1 mol/L NaNO3, 0.5 ml/min Xia et al. (2009)
6 HPGFC TSK-GEL G4000PW 0.003 mol/L CH3COONa, 0.5 ml/min Zhao et al. (2010)
7 HPGPC Shodex SB-803 (8.0 × 300 mm) 0.1 mol/L NaCl, 0.5 ml/min Luo and Cao (2010)
8 HPGPC Shodex OH-pakSB-804 HQ (8.0 × 300 mm) 0.2 mol/L NaCl, 0.5 ml/min Yuan et al. (2015)
9 HPGPC TSK-GEL G-5000 PWXL (7.8 × 300 mm, 10 μm) and TSK-GEL G-3000 0.02 mol/L KH2PO4, 0.6 ml/min Chen et al. (2016)
PWXL (7.8 × 300 mm, 5 μm)
10 HPGPC TSK-GEL Guard PWXL (6.0 × 40 mm), TSK-GEL5000K gel PWXL 0.02 mol/L KH2PO4, 0.6 ml/min Chen et al. (2016a)
(7.8 × 300 mm), and TSK-GEL3500K gel PWXL (7.8 × 300 mm)
11 HPSEC TSK-GEL G4000PWXL (7.8 × 300 mm, 5 μm) deionized water, 0.6 ml/min Zhang et al. (2016) and Zhu
et al. (2010)

Monosaccharide composition analysis

1 HPLC Zorbax NH2 (4.6 × 250 mm, 5 μm) acetonitrile/water (85:15, v/v), 1.0 ml/min Jin et al. (2007)
2 HPLC Waters X bridge amid (4.6 × 250 mm) acetonitrile/water (70:30, v/v), 0.6 ml/min Qiu et al. (2016)
4 HPLC Eclipse Plus C18 (4.6 × 250 mm, 5 μm) acetonitrile/phosphate buffered saline (pH Yuan et al. (2015)
6.7) (17:83, v/v), 1.0 ml/min
5 IEC CarboPac PA1 analytic column (4.0 × 250 mm) gradient elution, NaOH aqueous solution, Chen et al. (2016)
1.0 ml/min
6 IEC CarboPac PA1 analytic column (4.0 × 250 mm) and CarboPac PA1 guard gradient elution, NaOH aqueous solution, Chen et al. (2016a) and
column (4.0 × 50 mm) 0.6 ml/min Liao et al. (2015)
7 HPAEC CarboPac PA-1 (4.0 × 250 mm) 0.2 mol/L NaOH and 9% mobile phase B, Lee et al. (2013)
1.0 ml/min

Abbreviations: HPAEC, high-performance anion exchange chromatography; HPGFC, high-performance gel filtration chromatography; HPGPC, high-performance gel permeation chro-
matography; HPLC, high-performance liquid chromatography; HPSEC, high-performance size-exclusion chromatography; IEC, ion-exchange chromatography.

attached to the O-3 and O-2, or (1 → 2)-linked Rha residues attached to
the O-4. The side chains were shown to contain (1 → 5)-linked arabinan
with branches of Ara residues attached to the O-3, branched (1 → 3)-
linked rhamman, linear (1 → 4)-linked xylan, linear (1 → 4)-linked
glucan, and linear (1 → 2)-linked galactan (Xia, Liu, Zhang, & Luo,
2008). Another acidic polysaccharide named Mp-3b obtained from the
boiling aqueous extract of M. alba leaves possesses the same qualitative
monosaccharide composition as SDA, however the molecular weight is
six times that of SDA (Xia, Liu, Luo, & Zhang, 2009). Basically con-
sistent in the qualitative monosaccharide composition of Mp-3b and
SDA, a pectic polysaccharide named SJB obtained from the alkali ex-
tract of M. alba leaves was shown to possess a backbone composed of
(1 → 2,1 → 2,4)-Rhap and (1 → 4,1 → 3,4)-GalAp with side chains at-
tached to the O-4 of Rhap residues and the O-3 of GalAp residues, in-
cluding terminal (1 → 5,1 → 3,5)-linked Araf, terminal (1 → 4)-linked
Glcp, and terminal (1 → 4,1 → 3,1 → 6)-linked Galp (Xia, Luo, & Zhang,
2008). Another acidic polysaccharide named Mp-3 separated from
water-soluble mucilage was structurally analyzed, it was shown to
mainly contain Rha, Gal, Glc, GalA, and GlcA. Methylation and gas
chromatography-mass spectrometry analysis indicated that Mp-3 is a
rhamnogalacturonan mainly consisting of (1 → 2,3)-linked Rha re-
sidues, (1 → 3,4)- and (1 → 4)-linked GalA and GlcA residues, and
nonreducing terminal uronic acid residues (Katayama et al., 2008). Two
polysaccharide fractions named MPL1 and MLP2 (Fig. 3) with unusual
monosaccharide composition were also obtained from M. alba leaves,
MPL1 and MPL2 have dominant proportion of Flu and Glc, which are
different from the above mentioned MLPs. A combination of periodate
oxidation and Smith degradation reaction revealed that the backbones
of MPL1 and MPL2 consist of (1 → 3)-linked glycosidic linkages and the
side chains consist of (1 → 2)-linked glycosidic linkages (Luo & Cao,
2010). β-glycosidic linkages were found widespread in MLPs, as evi-
denced by the fact that a FT-IR spectrum characteristic peak at around
890 cm−1 frequently appeared (Yang et al., 2017; Yuan et al., 2015;
Zhang et al., 2014a). Furthermore, two MLPs (PMP11 and PMP12) were
shown to possess a backbone mainly composed of β-(1 → 2,1 → 3)-
linked Rha and β-(1 → 3)-linked Rha (Zhao, Chang, Wang, Zhang, &
Ouyang, 2010; Zhao, Wang, Yu, Xu, & Ouyang, 2010), other two MLPs
(SDA and SDC) were shown to possess an α-(1 → 4)-galacturonan
backbone (Xia, Liu, Shen, Zhang, & Luo, 2009; Xia et al., 2008), and
MPA-1 was shown to be an α-(1 → 4)-glucan with branches of (1 → 6)-
linked Glc residues attached to the O-3 and O-2 (Xia et al., 2009).
A water-soluble M. alba fruit polysaccharide named FMAP (Fig. 4)
was found to be composed of Gal, Man, and Glc. Absorption at 890 and
1045 cm−1 in IR indicated that FMAP had a β-pyranosidic linkages, and
the backbone of the polysaccharide was shown to be composed of (1 →
3)-linked mannan with branches of galactosyl and glucosyl residues
attached to the O-6 (Wei, Zhou, Zang, & Jiang, 2007). An important
pectic M. alba fruit polysaccharide named JS-MP-1 (Fig. 5) was isolated
and purified by crushing the fresh fruits then performing ethanol pre-
cipitation and DEAE-cellulose ion exchange chromatography (Lee et al.,

Fig. 3. The hypothetic structure of MPL1 and MPL2 (Luo & Cao, 2010).

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X. He et al. Food Chemistry 245 (2018) 899–910

from 4 to 5 ppm were assigned to the β-glycopyranosidic ring. The

signals at 3.80, 3.90, 4.31 and 5.15 ppm were assigned to H-1 to H-4 of
(1 → 4) α-galacturonic acid residues, respectively (Ye et al., 2011). The
13
C NMR spectra at 97.5, 98.5, 99.0, 107.5 and 109.3 ppm corre-
sponded to C-1 of the (1 → 6)-linked α-D-Glc unit, C-1 of the (1 → 2)-
linked α-L-Rha unit, C-1 of the (1 → 3)-linked α-D-Gal unit, C-1 of the
(1 → 3)-linked β-L-Rha unit, and C-1 of the (1→)-linked α-L-Ara unit,
respectively (Chen et al., 2016). The other three MFPs (PMF1, PMF2,
Fig. 4. The hypothetic structure of FMAP (Wei et al., 2007). PMF3) were shown to possess (1 → 2), (1 → 3), or (1 → 6)-linked hex-
apyranose residues, and α, β-glycosidic linkages (Tian, 2014).
Two water-soluble MRPs named MBBP-1 and MBBP-2 were shown
2013). JS-MP-1 was revealed to be mainly composed of Gal (37.6%),
to be mainly composed of Rha, Xyl, Ara, Man, Glc, and Gal, and
Ara (36.3%), and Rha (18.4%), while other major sugars, including Glc,
meanwhile MBBP-2 possesses GalA. Absorptions between 1300 and
Xyl, Man, and Fuc, act as minor components, the mole ratio of GalA and
1000 cm−1 were characteristic of the stretching vibrations of pyranose
GlcA is at approximately 4:1 (Lee et al., 2013). Strong absorption at
ring (Liu, Wang, Xu, & Wang, 2007; Liu et al., 2007; Sun & Tomkinson,
1621 and 1416 cm−1 corresponded to anti-symmetric and symmetric
2002). The absorption at 897 cm−1 corresponded to β-glycosidic lin-
stretching vibrations of COO−. Intense and highly overlapping bands in
kages. Smith degradation, methylation analysis, and NMR spectroscopy
the region of 948–1150 cm−1 indicated the presence of COC, CO and
further revealed that MBBP-1 has a backbone mainly consisting of (1 →
CC stretching vibrations in JS-MP-1 (1150 and 948 cm−1 for RG1, 1093
3)-linked Glc, and MBBP-2 has a backbone mainly consisting of (1 → 3)-
and 1045 cm−1 for arabinan, 1074 cm−1 for galactan) (Kacurakova,
linked Rha and (1 → 2,4)-linked Xyl (Qiu, He, & Zhang, 2016). Three
Capek, Sasinkova, Wellner, & Ebringerova, 2000; Lee et al., 2013).
homogeneous MCPs named CMA-a-1, CMA-a-5, and CMA-b1-1 were
Absorption at 894 cm−1 corresponded to β-anomeric sugar units. JS-
also obtained. A combination of sugar analyses, methylation analyses,
MP-1 is an acidic heteropolysaccharide, most likely a variant of bran-
IR, and NMR spectroscopic methods revealed that CMA-a-1 is an
ched RG-I pectic polysaccharide, containing no, or negligible, levels of
amylopectin-like polysaccharide with an average chain length of 23,
sulfate esters and proteins (Lee et al., 2013). Another recent study
containing (1 → 4)-linked α-D-Glcp backbone with branches of (1 → 6)-
further demonstrated that the neutral sugar side chains in JS-MP-1 are
linked Glc attached to the O-6, and CMA-a-5 is mainly an amylose
(1 → 5)-α-L-arabinan and branched arabinogalactans type II (AG II)
fraction with an average chain length of 30. CMA-b1-1 was character-
having the (1 → 6)-β-D-galactan core with 3-O-bound terminal α-L-Araf.
ized as a RG-I type pectic polysaccharide, containing a backbone
The arabinan chains are bound to the RG I backbone at the O-4 position
composed of alternating (1 → 4)-linked α-D-GalA and (1 → 2)-linked α-
of some α-L-Rhap units. However, the mode of linkage between RG I
L-Rha residues, with various branches of T-, (1 → 3)-, (1 → 5)-, (1 →
and AG II chains is unclear (Choi et al., 2016). In addition, there were
3,5)-linked Araf, T-, (1 → 6)-, (1 → 3,6)-linked Galp, (1 → 4)-linked
also several other acidic heteropolysaccharides isolated from M. alba
Xylp, and (1 → 4)-linked Glcp attached to the O-4 of Rha and the O-6 of
fruits, such as MFP-1, MFP-2, MFP-3, MFP-4 and MFP3P (Chen, Zhang,
GalA (Zhang et al., 2013).
Fu, & Liu, 2016; Chen et al., 2015, 2016a). As for MFP3P, the 1H NMR
spectra from 3.5 to 4.15 ppm were assigned to the glycosidic ring, and

Fig. 5. The hypothetic structure of JS-MP-1 (Choi et al.,

2016).

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4. Biological activities
4.1. Antidiabetic activity
There is cumulative evidence to suggest that MLPs, MRPs and MFPs
are capable of significantly decreasing fasting blood glucose (FBG) and
glycosylated serum protein (GSP) levels in diabetic rats, which provide
direct evidence of the hypoglycemic function of polysaccharides from
Morus alba (Guo et al., 2013b; Jiao et al., 2017; Zhang et al., 2014a,b).
It was shown that MFPs and MRPs can increase the level of insulin, thus
resulting in the enhancement of insulin sensitivity (Chen, Huang, Li, &
Fu, 2017; Guo et al., 2013b). MFP3P-Se, a selenide of MFPs, showed
more significant hypoglycemic effects than MFP3P via promoting pan-
creatic cell proliferation and increasing glucose metabolism and insulin
secretion (Chen et al., 2016). MLPs were reported to ameliorate insulin
secretory capacity (Zhang et al., 2014b), since increased levels of
fasting insulin and C-peptide were found in diabetic rats after MLPs
treatment (Zhang et al., 2014a). More importantly, according to ex-
perimental findings summarized below, MLPs, MFPs and MRPs can
inhibit β-cell apoptosis and increase β-cell counts, target β-cell dys-
function with anti-inflammatory and antioxidant effects, inhibit α-
amylase and α-glycosidase, restore glucose metabolism in insulin-de-
pendent and non-insulin-dependent pathways.
Insulin signaling pathway plays a vital important role in the pa-
thogenesis of type 2 diabetes mellitus (T2DM), and insulin receptor
substrates (IRSs) play key roles in the insulin signaling pathway
(Withers et al., 1998, 1999). PI3K/AKT pathway occurs after the IRS-2
activation, which plays an extremely important role in insulin re-
sistance (Datta et al., 1995; Rondinone et al., 1997), and PI3K is pri-
marily involved in insulin signaling, and AKT ultimately plays a critical
role in insulin sensitivity (George et al., 2004). Based on the above
findings, Ren and colleagues demonstrated that MLPII treatment
(200 mg/kg, i.g.) can effectively normalize hepatic glucose metabolism
and insulin signaling via inhibiting the expression of protein-tyrosine
phosphatase 1B, activating the PI3K/AKT pathway (Ren et al., 2015).
Two other MFPs fractions (MFP50 and MFP90) (400 mg/kg, i.g.) can
significantly upregulate the expression of IRS-2 and AKT, and mean-
while upregulate the expression of glucose transporter type 4 (GLUT4),
which is an insulin-regulated glucose transporter and serves the rate-
limiting step of glucose metabolism (Jiao et al., 2017).
Moreover, MLPs were shown to target glucose metabolism in non-
insulin-dependent pathway. It was demonstrated that oral administra-
tion of MLPII (200 mg/kg) to diabetic rats ameliorates insulin secretory
capacity and increases insulin content in pancreatic β-cells via in-
creasing pancreatic duodenal homeobox-1 (PDX-1) expression and re-
storing PDX-1 nuclear localization, thereby trans-activating the ex-
pression of insulin, glucokinase, and glucose transporter 2 (GLUT2)
(Zhang et al., 2014b). The other non-insulin-dependent pathway with
regard to MLPII targeting glucose metabolism is about the liver. It is
well-known that excessive hepatic gluconeogenesis and glucose pro-
duction are important factors to diabetic hyperglycemia. Evidence
suggested that the addition of MLPII (200 mg/kg) to the diet lowers
endogenous glucose production by elevating the liver glycogen level
and liver glucokinase activity (Zhang et al., 2014a), and oral adminis-
tration of MFP50 and MFP90 (400 mg/kg) improves glucose metabo-
lism (Jiao et al., 2017). MFP50 and MFP90 can also significantly de-
crease serum alanine transaminase levels in T2DM rats, indicating that
MFP50 and MFP90 impart a potential ameliorative effect on hepatic
function (Jiao et al., 2017). However, the experimental results showed
that MFP50 and MFP90 cannot increase insulin secretion (Jiao et al.,
2017), which is a contradictory finding different from another work in
this area (Chen et al., 2017).
Chronic inflammation is widely found in diabetes, and blocking
inflammatory factors in pancreas is beneficial to preventing against islet
damage and controlling diabetes developments. Manganese superoxide
dismutase (MnSOD) and glutathione reductase (GSH-Rd) may
906
Food Chemistry 245 (2018) 899–910
effectively relieve peroxide damage and maintain the balance between
free radical production and antioxidant defense ability (Djordjevic,
Spasic, Jovanovic-Galovic, Djordjevic, & Grubor-Lajsic, 2004). Mal-
onaldehyde (MDA) can cumulate and damage the structure of cellular
membrane, resulting in cell dysbolism and cytotoxicity (Esterbauer,
1993). Based on the above findings, it was suggested that administra-
tion of MFPs (1000 mg/kg, i.g.) can not only increase the activities of
antioxidant enzymes superoxide dismutase (SOD), glutathione perox-
idase (GPx) and catalase (CAT) in the liver in STZ-induced diabetic
mice, but also effectively decrease the level of MDA. (Chen et al., 2017).
Evidence suggested that administration of MRPs (600 mg/kg, i.g.) can
effectively reverse the up-regulated TNF-α, IL-8, IL-6 and COX-2 in the
STZ-induced diabetic mice, and meanwhile MRPs can ward off the
abnormalities of MnSOD and GSH-Rd and reduce the MDA content
(Guo et al., 2013b; Liu, Ma, & Zhang, 2017). It was also suggested that
the addition of MLPII (200 mg/kg) to the diet can significantly reduce
8-hydroxy-2-deoxyguanosine and MDA levels, and restore the cellular
defense functions of antioxidant enzymes in diabetic rats (Ren et al.,
2015).
Diabetes mellitus is often accompanied by lipid abnormalities,
which can cause β-cell dysfunction. A high-purity MLPs named MLP
(200 mg/kg, i.g.) was shown to improve blood lipid profiles by reducing
serum total cholesterol (TC) and serum triglyceride (TG) levels, and
increasing serum high-density lipoprotein cholesterol (HDL-C) level
(Zhang et al., 2014a). Similar results were also gained from MFP50 and
MFP90 (400 mg/kg, i.g.) (Jiao et al., 2017). In addition, MLP was also
shown to decrease the excessive fat accumulation, which is associated
with the insulin sensitivity of liver cells (Zhang et al., 2014a).
The destruction of pancreatic β-cells plays a primary role in the
impairment of insulin secretion in diabetes, and besides insulin secre-
tion insufficient and insulin resistance, T2DM is also characterized by β-
cell apoptosis. Evidence suggested that MLPs treatment can markedly
alleviate the pathological changes in pancreatic islets and recover the
number of pancreatic β-cells in diabetic rats (Zhang et al., 2014a,b).
Similarly, other recent studies also demonstrated that the MRPs
(600 mg/kg, i.g.) treatment contributes to the architectural ameliora-
tion of islet structure and a gradual increase in β-cell counts (Xu, Yang,
Wang, Huang, & Huang, 2015). Moreover, oral administration of MLPII
(200 mg/kg) was shown to significantly inhibit pancreatic islet cell
apoptosis in diabetic rats, and MLPII treatment ameliorates the ultra-
structural changes of pancreatic β-cells (Zhang et al., 2014b). Further
in-depth research demonstrated that one of the underlying molecular
mechanisms of MLPII-inhibited and MRPs-inhibited β-cell apoptosis
involves the elevation of Bcl-2/Bax ratio (Xu et al., 2015; Zhang et al.,
2014b), and MRPs exert pronounced hypoglycemic effect via regulating
the intrapancreatic JNK/p38 pathway (Xu et al., 2015).
4.2. Immunomodulatory activity
There is cumulative evidence to suggest that MLPs, MFPs, MRPs,
and MCPs have immunomodulating activity, which has been assessed
on natural killer (NK) cells, dendritic cells (DCs), T-cells, B-cells, and
macrophage-dependent immune system responses.
MRPs (600 mg/kg, i.g.) were suggested to be able to enhance the
cellular immune function, humoral immunity, and nonspecific im-
munity (Hong, Zhang, Jiang, & Shi, 2011), and increase NK cell activity
in normal mouse (Fei, Zhao, Zhan, Shi, & Zhu, 2009). MLPs (125, 250,
and 500 mg/kg, i.g.) were shown to significantly improve carbon
clearance capacity, serum hemolysin level, and spleen lymphocyte
transformation capability induced by ConA, and meanwhile low dose of
MLPs (125 mg/kg, i.g.) could significantly increase the thymus index
(Hou, Liao, Liu, Zhou, & Deng, 2011). In addition, it was suggested that
a M. alba fruit glycoprotein named MP (125–2000 μg/ml) can sig-
nificantly increase B and/or T cells proliferation (Liu & Lin, 2014).
While in spite of the strong induction on B lymphocyte proliferation,
MCPs were shown to reduce IgM antibody production from B cells (Kim
X. He et al.
et al., 2000). In vitro experimentation showed that high concentration of
MP in the absence of LPS can significantly protect murine primary
immune cells from apoptotic cell death via modulating the Bcl-2/Bak
ratio (Liu & Lin, 2014). However, another study demonstrated contra-
dictory findings with regard to the enhancing immunoactivity of
polysaccharide from M. alba branches (Wang, Wen, & Hu, 1995).
Dendritic cells (DCs) are the most potent antigen-presenting cells
that initiate the majority of immune responses, including tumor-specific
T cell responses. Maturation of dendritic cells (DCs) is usually atte-
nuated in the tumor microenvironment, which is an important im-
munological problem in DC-based immunotherapy of cancer.
Nowadays, plant and fungal-derived polysaccharides have been used as
inducers of DC maturation, especially polysaccharides from Grifola
frondosa have been assessed in clinical trials of cancer immunotherapy
(He et al., 2017). Based on recent experimental findings, MFPs were
shown to be capable of inducing phenotypic and functional maturation
of DCs, as evidenced by several changes in MFP-treated DCs (Shin et al.,
2013). MFPs were confirmed to induce phenotypic and functional
maturation of DCs at least in part via TLR4, since DCs having mutated
TLR4 are hyporesponsive to MFPs compared to DCs having normal
TLR4 (Shin et al., 2013). In vitro experimentation further showed that
MFPs can activate mitogen-activated protein kinase (MAPK) and the
NF-κB signaling pathways, which regulate phenotypic and functional
maturation of DCs, and regulate survival and migration of DCs via ac-
tivating ERK and p38, respectively (Shin et al., 2013). Another recent
study also demonstrated similar results with regard to MLPs acting on
murine bone-marrow-derived dendritic cell (BMDC) maturation (Xue
et al., 2015).
Furthermore, In vitro experimentation has revealed that JS-MP-1 has
immunostimulating effects on murine RAW264.7 macrophage cells (Lee
et al., 2013), as evidenced by the fact that: (i) the protein levels of both
TNF-α and IL-6 in the culture supernatants of RAW264.7 macrophages
increased in a concentration-dependent manner, especially at the con-
centration of 50 μg/ml, the JS-MP-1 treatment stimulated the secretion
of these cytokines approximately 90-times and 20-times greater, re-
spectively, than the absence of any treatment; (ii) the treatment of
RAW264.7 cells with varying concentrations of JS-MP-1 markedly in-
creased the production of chemokines (RANTES and MIP-1α) in a dose-
dependent manner; (iii) iNOS and COX-2 expression dose-dependently
increased upon treatment of cells with various doses of JS-MP-1
(0–50 μg/ml), and the treatment of cells with JS-MP-1 resulted in a
dose-dependent increase in NO production, which is produced as a
result of the stimulation of iNOS.
4.3. Anti-inflammatory activity
There is cumulative evidence to suggest that MP has anti-in-
flammation potential via modulating pro-/anti-inflammatory cytokine
secretion profiles. Pro-/anti-inflammatory cytokine levels secreted by
LPS-stimulated macrophages cultured with MP (500 μg/ml) for 48 h
were determined using ELISA method. The corresponding results
showed that MP treatment can significantly decrease pro-inflammatory
cytokines including IL-1β and IL-6, and markedly increase the anti-in-
flammatory cytokine IL-10 (Liu & Lin, 2012). Two years later, it was
suggested that MP treatment (125–2000 μg/ml) can concentration-de-
pendently increase IL-4, IL-5, IL-10, IL-12, and TNF-α secretion levels,
and meanwhile MP has Th2 inclination and anti-inflammation potential
in the absence of LPS, as evidenced by the fact that (IL-2 + TNF-α)/(IL-
4 + IL-5 + IL-10) (Th1/Th2) cytokine secretion ratios significantly
decreased (Liu & Lin, 2014). Furthermore, evidence suggested that
administration of MRPs (600 mg/kg, i.g.) can effectively reverse the up-
regulated TNF-α, IL-8, IL-6 and COX-2 in the STZ-induced diabetic mice
(Guo et al., 2013b; Liu et al., 2017).
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Food Chemistry 245 (2018) 899–910
4.4. Antioxidant activity
There is cumulative evidence to suggest that MLPs, MFPs and MRPs
possess antioxidant activity, which are mentioned before in section 4.1
Antidiabetic activity (Chen et al., 2017; Deng et al., 2014; Guo et al.,
2013b; Liu et al., 2017; Ren et al., 2015). However, another study de-
monstrate contradictory findings with regard to the antioxidant activity
of MLPs. It was suggested that the antioxidant activities of MLPs de-
crease with increasing polysaccharide purity (Zhang et al., 2016), and
the FRAP value and ABTS radical scavenging activity of a MLPs named
MLP3 changed slightly with an increasing concentration
(15.625–1000 μg/ml), suggesting that polysaccharides may not serve
the antioxidant activity (Zhang et al., 2016). However, although MLPs
have very little antioxidant activity alone, they can strongly enhance
the antioxidant activity of flavonoids. It was suggested that the FRAP
value of a combination of quercetin (10 μg/ml) and MLP3 (15.625 μg/
ml) was 0.168 μM Fe2+/mg (the FRAP values for 10 μg/ml quercetin
and 15.625 μg/ml MLP3 were 0.124 μM and 0.007 μM Fe2+/mg, re-
spectively), and the synergistic effect between MLP3 and quercetin
gradually enhanced with the increase of MLP3 concentration. Similar
results were also gained from the ABTS test (Zhang et al., 2016). Similar
other studies also showed that the crude MLPs exhibited relative higher
activity on Fe2+ chelating and also DPPH, hydroxyl, superoxide and
ABTS radicals scavenging than purified MLPs (Samavati & Yarmand,
2013; Yuan et al., 2015). In addition, it was shown that the antioxidant
activities of the sulfated, phosphorylated and acetylated derivatives are
stronger than that of the unmodified MRPs (Zhang, Jin, & Shi, 2008).
4.5. Hepatoprotective and renoprotective activities
A study carried out to evaluate the hepatoprotective activity of
MFPs in mice with acute and subacute alcoholic-induced liver injury
upon oral administration of MFPs resulted in the improved serological
indexes (AST and ALT) and hepatic indicators (GSH, SOD, GSH-Px, and
MDA). The hepatic histopathology results also showed a prominent
lipid degeneration and microvesicular steatosis attenuation in the MFPs
groups, and hepatocyte regeneration and no necrotic cells were ob-
served. These results indicated that the outstanding hepatic protecting
activity of MFPs might be related to the activation on ethanol dehy-
drogenase, the elimination of free radicals, and/or the inhibition on
lipid peroxidation capacities (Deng et al., 2014; Zhou et al., 2017).
Similar results were also gained from CCl4-induced liver injury in rats
upon intragastric administration of MFPs (100 and 200 mg/kg), and the
underlying mechanisms were considered to be related to the anti-
oxidant and anti-inflammatory effects of MFPs, as evidenced by the fact
that a significant decrease in the contents of MDA, IFN-γ, and TNF-α in
liver, and a significant increase in the IL-10 content and activities of
SOD, CAT, and GSH-Px were observed (Xia, Chen, Tao, & Tuerhong,
2015).
MRPs possess renoprotective effect, and the underlying mechanism
is related to the regulation on serum renal function markers and the
inhibition on the intrarenal IL-1/NF-κB pathway, which were drawn
from a study carried out on STZ-induced diabetic nephropathy mice
(Guo et al., 2013a). Another recent study demonstrated that the re-
noprotective effect of MRPs might be associated with the inhibition on
the activation of the NF-κB/TGF-β1 pathway in high-fat diet/STZ-in-
duced diabetic nephropathy rats (Li et al., 2015).
4.6. Anti-obesity activity
In vitro evidence suggested that JS-MP-1 (500 μg/ml) is able to ef-
fectively inhibit the proliferation of pre-adipocyte cells. Further ex-
perimental work performed in vitro with 3T3-L1 cells suggested that the
underlying mechanisms of the anti-proliferative effect of JS-MP-1 on
pre-adipocyte cells are associated with an induction of DNA fragmen-
tation and also an induction of apoptotic cell death, as evidenced by the
X. He et al.
facts that the expression of apoptosis-related proteins, such as caspase-3
and -9 and poly (ADP-ribose) polymerase (PARP) significantly in-
creased upon JS-MP-1 treatment. In addition, mitochondrial dysfunc-
tion that initiates caspase cleavage is also an important character in
cellular apoptosis. It was found that JS-MP-1 can induce mitochondrial
membrane depolarization. On the other hand, it was shown that JS-MP-
1 dose-dependently reduced Bcl-2 expression and increased Bax ex-
pression. The Bcl-2/Bax ratio significantly decreased with approxi-
mately 90% reduction upon JS-MP-1 treatment (200 μg/ml).
Furthermore, the treatment of 3T3-L1 cells with JS-MP-1 (500 μg/ml)
was found to significantly reduce the p-ERK level and markedly sti-
mulate the p38 phosphorylation. Taken together, these results de-
monstrated that JS-MP-1 can inhibit the proliferation pre-adipocyte
cells through induction of cell apoptosis, which in turn reduce fat cell
numbers and thus adipose mass by inducing the apoptosis of pre-adi-
pocyte cells through the regulation of the MAPKs signaling pathway,
the induction of mitochondrial dysfunction and DNA fragmentation in
3T3-L1 pre-adipocyte cells (Choi et al., 2016).
4.7. Effect on gut microbiota
MFPs could be beneficial to obesity and diabetes by regulating the
gut microbiota. Dietary addition of MLPs can improve the ecology of
gut microbiota. It was shown that MFPs have a potential prebiotic ef-
fect, thereby modulating colonic bacterial composition by increasing
the beneficial bacterial population (Bacteroidetes) and reducing the
harmful bacterial population (Firmicutes) (Chen, Huang, Fu, & Liu,
2016). The overall effects of MLPs treatment in inhibiting gut Escher-
ichia coli and promoting gut lactobacilli and bifidobacteria were superior
to those of the control treatment and chlortetracycline treatment. The
MLPs treatment (900 mg/kg) could significantly inhibit gut E. coli and
promote gut-beneficial bacteria (Zhao et al., 2015).
4.8. Effect on lipolysis
The effect of MFPs on lipid digestion in simulated saliva, gastric and
intestine digestion was investigated, it was suggested that the lipid
emulsion digestion mainly occurred in the simulated intestine phase.
MFPs could significantly decrease both rate and extent of lipid digestion
compared to the control. Experimental work also revealed that the in-
itial molecular weight and linkage type of polysaccharides had great
impact on the polysaccharide-lipid emulsion digestion (Chen, Huang,
You, & Fu, 2017). In the vitro digestion model, the saliva had no effect
on the crude polysaccharides obtained from M. nigra fruits, while the
crude polysaccharides could be digested by gastric and intestinal fluid
through the breakdown of glycosidic bonds. In addition, the crude
polysaccharides were shown to have a greater inhibitory impact on
lipid digestion when pass through the simulated saliva, gastric and in-
testinal tract (Chen et al., 2016b).
5. Conclusions and future perspectives
Morus alba is distinguished as a source of highly promising tradi-
tional medicines (including Mori Folium, Mori Fructus, Mori Ramulus,
and Mori Cortex) and also functional foods (especially its fruits and
leaves), thus has gained much attention. Over the past twenty years,
phytochemical investigations have led to the isolation of a large
number of bioactive M. alba polysaccharides and other purified frac-
tions which were shown to possess various bioactivities, including an-
tidiabetic activity, immunomodulatory activity, anti-inflammatory ac-
tivity, antioxidant activity, anti-obesity activity, hepatoprotective and
renoprotective activities. The antidiabetic activity is the most in-
tensively studied subject. Taken together, M. alba polysaccharides can
inhibit pancreatic β-cell apoptosis and increase pancreatic β-cell counts,
target pancreatic β-cell dysfunction with anti-inflammatory and anti-
oxidant effects, inhibit α-amylase and α-glycosidase, restore glucose
908
Food Chemistry 245 (2018) 899–910
metabolism in insulin-dependent and non-insulin-dependent pathways.
Surprisingly, there is a wealth of evidence obtained from rodent to
suggest Morus alba polysaccharides (especially MFPs and MLPs) effec-
tive in lowering blood sugar regardless of the previous state including
streptozotocin-induced diabetic, alloxan-induced diabetic, diet-induced
diabetic, or normal rodents, yet no sufficient human evidence suggests
how potent hypoglycemic health benefits of MFPs and MLPs are in
humans. Furthermore, many other possible hypoglycemic mechanisms
also have not been clarified, including: (1) whether MFPs and MLPs
target insulin enhancement via the MAPK pathway modulators; (2)
whether MFPs and MLPs target glucose metabolism in non-insulin-de-
pendent pathways, such as inhibiting glucose uptake or 5′-AMP-acti-
vated protein kinase activators; (3) whether MFPs and MLPs target the
cAMP-PKA pathway or the ubiquitin–proteasome pathway. More im-
portantly, considering the facts that T2DM is currently a global health
problem and various synthetic anti-diabetic drugs have severe side ef-
fects, such as hypoglycemia, weight gain, gastrointestinal reaction, and
edema. Natural plants could be sources of new drugs for the treatment
of T2DM, and Morus alba polysaccharides as excellent representatives
deserve further efforts on human test due to their good safety and
promising efficacy on experimental animals.
TLR4 has been confirmed to be involved in the MFPs-induced DC
maturation through MAPKs and NF-κB activation, however, other re-
ceptor candidates including Dectin-1 and complement receptor 3 (CR3),
especially Dectin-1 the major β-glucan receptor on macrophages and
DCs is also likely to be involved in, thus further research on receptors
will be helpful in elucidating the mechanisms underlying immune
modulation by MFPs. Furthermore, considering the improvements in
circulating lipids and cholesterol and the fairly potent reductions in
atherosclerotic plaque buildup, M. alba is also likely to benefit cardio-
vascular health.
Interestingly enough, a particle-size observation indicates that the
cationic M. alba leaf polysaccharides (CMPS) can efficiently combine
with plasmid transforming growth factor β1 (pTGF-β1) to form na-
noscaled particles, and a series of comprehensive investigations suggest
that the CMPS/pTGF-β1 nanoparticles can potentially be developed
into a promising nonviral gene vector (Deng et al., 2012). This kind of
research will contribute to the promotion of the effective application of
traditional Chinese medicine to the area of gene therapy.
Last but not least, there is, however, a need to further elucidate the
structures of M. alba polysaccharides, and future research is also re-
quired to determine the relationship between the three-dimensional
structures and functions of M. alba polysaccharides. This knowledge
will help scientists to design more potential health promoting phar-
maceuticals and functional foods based on M. alba polysaccharides by
means of structural modification.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgment
This work was financially supported by Natural Science Foundation
of Shaanxi Province, China (Grant No. 2017JM8054).
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