International Journal of Biological Macromolecules 162 (2020) 1897–1905

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Review

Extraction, structure and bioactivities of the polysaccharides from Ginkgo

biloba: A review
Jiacheng Fang a, Zheng Wang b, Pei Wang c, Ming Wang a,⁎
a
College of Food Science & Engineering, Northwest University, No. 229 Taibai North Road, Xi'an, Shaanxi 710069, China
b
Shaanxi Collaborative Innovation Center of Chinese Medicine Resources Industrialization, Shaanxi University of Chinese Medicine, Xian Yang, Shaanxi 712046, China
c
Department of Neurosurgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, China

a r t i c l e i n f o a b s t r a c t

Article history: Ginkgo biloba L. is distinguished as source of highly promising food and traditional herbal for thousands of years.
Received 14 June 2020 Modern phytochemistry studies have demonstrated that polysaccharide is one of the important biologically-
Received in revised form 15 August 2020 active components of G. biloba. Over the past two decades, the isolation, chemical properties and bioactivities
Accepted 17 August 2020
of polysaccharides from leaves, sarcotesta and seeds of G. biloba have been drawing much attention from scholars
Available online 19 August 2020
around the world. It has been demonstrated that G. biloba polysaccharides have various remarkable biological ac-
Keywords:
tivities, including antioxidant, antiviral, anti-tumor, anti-inflammation, immunomodulatory, and
Ginkgo biloba polysaccharide hepatoprotection effects. Moreover, different materials and extracting methods result in the difference in struc-
Extraction methods ture and bioactivity of G. biloba polysaccharides. The purpose of this review is aimed at providing systematical
Structural characterization and current information on the isolation methods, structural features and bioactivities of G. biloba polysaccha-
Biological activity rides to support their further application as therapeutic agents and functional foods.
Application © 2020 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1897
2. Extraction and purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1898
3. Physiochemical and structural features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1899
4. Biological activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1901
4.1. Antioxidant activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1901
4.2. Antitumor activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1901
4.3. Anti-inflammation activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1902
4.4. Immunostimulatory activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1902
4.5. Antidepressant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1902
4.6. Other biological activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1902
5. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1902
6. Conclusion and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1903
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1903
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1903
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1903

1. Introduction Ginkgoaceae family and also a traditional Chinese herb used for many
years. As a “living fossil”, G. biloba has been around for 250 million
Ginkgo biloba L., also known as yinhsing (Chinese), ginkyo (Japanese), years and is one of the most ancient medicinal tree species widely dis-
maidenhair-tree and silver apricot, is a perennial plant in the tributed in China, Japan, and Korea [1–3]. Nowadays, it is extensively
cultivated worldwide with useful applications of its leaves and seeds
⁎ Corresponding author. in food, health and supplements [4,5]. The seeds of G. biloba have been
E-mail address: wangming@nwu.edu.cn (M. Wang).

https://doi.org/10.1016/j.ijbiomac.2020.08.141
0141-8130/© 2020 Elsevier B.V. All rights reserved.
1898 J. Fang et al. / International Journal of Biological Macromolecules 162 (2020) 1897–1905
consumed as a food and versatile medicine in Chinese, Japanese and
Indonesian traditional medicine owing to its reliable activities to treat
asthma, cough and pyogenic skin infections [6,7]. G. biloba leaf extracts,
especially EGb761, emerged as a source to counteract age-related neu-
rological disorders in the last century [8,9]. Moreover, sarcotesta of
G. biloba revealed potent anti-bacterial activity against vancomycin-
resistant Enterococcus, which also contributes to the environmental
problem caused by excess waste material [10]. Relevant pictures about
the leaves, sarcotesta and seeds of G. biloba are shown in Fig. 1. Modern
pharmacological studies have showed that G. biloba has many other
beneficial bioactivities, such as anti-tumor [11], anti-oxidant [12], anti-
bacterial [13], anti-inflammatory [14], anti-depressant [15],
immunostimulating [16], hepatoprotective effects [17], treatment of is-
chemia/reperfusion (I/R) injury [18], retinal diseases [19], and neurode-
generative diseases [20].
G. biloba is a famous botanical dietary supplement worldwide and
contains hundreds of different bioactive compounds, including terpene
trilactones, flavonoids, fatty acids, proanthocyanidins, and polysaccha-
rides [21,22]. The dominant active components in G. biloba are terpene
trilactones and flavonoids [23,24], which reveal significant free-radical
scavenging activities and have been generally applied in treatment of
cognitive impairment and dementia [25]. Currently, polysaccharides
from G. biloba and their beneficial effects have gained growing attention
from scholars around the world. A variety of extraction methods such as
hot water-extracted, ultrasonic-assisted and enzymatic extraction, as
well as numerous purification methods including ion-exchange chro-
matography and gel filtration, have been used to get the purified
G. biloba polysaccharides. As a result, a large amount of structurally di-
verse polysaccharides have been isolated and purified from the leaves,
sarcotesta and seeds of G. biloba [7,26,27]. These studies provided a se-
ries of bioactive macromolecules, with a wide variety of remarkable
physiological functions, including anti-tumor, anti-oxidant, anti-
inflammation, antiviral, treatment of I/R injury, hepatoprotection and
immunomodulatory effects, which were related to the different struc-
ture features. Diverse biomacromolecule structures were obtained
using different raw materials and purification methods. The seed of
G. biloba is a rich source of mannose (Man), and the sarcotesta contains
more galactose (Gal) and glucose (Glu) compared with the leaves. Re-
markably, the molecular weight of polysaccharides of G. biloba shows
diverse distribution, ranging from 1.0 kDa to 5679 kDa.
As far as we know, there is not any available review concerning
G. biloba polysaccharides, though it has been sufficiently studied by
many scholars. In this review, we aim to systematically summarize the
research findings of the past two decades and to present a comprehen-
sive understanding about the structural characteristics, extraction and
purification techniques of G. biloba polysaccharides, and to attract
Fig. 1. The leaves, sarcotesta and seeds of G. biloba (A: leaves, B: sarcotesta, C: seeds).
more researchers' attention on their remarkable and reliable biological
activities for the better utilization.
2. Extraction and purification methods
In recent years, various methods for extraction, isolation and purifi-
cation of structurally diverse polysaccharides from G. biloba have been
reported. In brief, the G. biloba leaves, sarcotesta and seeds were
washed, drying at 50–65 °C and grinding to a powder. Next, the leaves
powder was then suspended into 80%–95% ethanol at 70 °C for hours,
while the sarcotesta powder was placed into a Soxhlet apparatus with
petroleum at 80 °C to remove the small molecules such as fats and pig-
ments [27,28]. After filtration, the residues were dried and extracted at
70–80 °C over 3 h with hot water (1:10, w/v) for three times [27,29].
The mixture was then centrifuged to separate residue and supernatant,
extraction solutions were then pooled and concentrated under reduced
pressure using a rotary evaporator at 40 °C [27]. The polysaccharides
would be precipitated after adding a three-fold volume of 95% ethanol
to the extracts and storing mixture at 4 °C overnight [30,31]. After wash-
ing with ethanol, acetone and ether successively, the separated precip-
itate was lyophilized. Using the optimized extraction methods, the
productive ratio of G. biloba seed polysaccharides was increased to
4.87% [32], and crude G. biloba leaf polysaccharides were obtained
with a yield of 10%–10.87% [33,34], while the crude polysaccharides
from waste sarcotesta were obtained with a yield of 23% [28].
Extraction conditions, including extraction solvent, pH, the ratio
of raw material to solvent, temperature, and time all have great in-
fluence on the extraction rate of polysaccharides. Jiang et al. used
the orthogonal experiment to improve the extraction yield of poly-
saccharides from G. biloba leaves [35]. The optimized condition was
water to sample as 30:1 (v/w), at 80 °C for 3.5 h. Other techniques,
including ultrasonic-assisted and enzymatic extraction, which
could make the cell wall broken and effectively promote the disso-
lution of the compositions, have also been applied to increase the
extraction rate of polysaccharides. These methods possess the ad-
vantage of being highly efficient, environmentally friendly, and
easily operated owing to the relatively mild reaction conditions.
By aqueous enzymatic extraction, Wang et al. extracted polysac-
charides from the mesosperm of G. biloba and investigated the ef-
fects of solid-liquid ratio, enzyme dosage, temperature, and time
via the single factor and orthogonal tests [36]. Under that optimal
condition, the extracting amount of polysaccharide could reach
up to 103.041 mg/g. Zhang et al. developed a polyethylene glycol
(PEG)-based ultrasound-assisted enzymatic extraction method
and increased G. biloba leaves polysaccharides yield to 7.29 ±
0.21% [37]. The optimal extraction conditions were as follows: a
 J. Fang et al. / International Journal of Biological Macromolecules 162 (2020) 1897–1905
pH of 4.34, an extraction temperature of 51.88 °C and an extraction
time of 37.13 min. Besides, an ultrasonic-assisted enzymatic ex-
traction method was also used and optimized to get the antioxidant
polysaccharides from G. biloba sarcotesta [38]. As a result, optimal
conditions were: a pH of 4.5, an extraction temperature of 60 °C,
an extraction time of 31 min, and a cellulase concentration of
3.2%. A recent study showed that the polysaccharide extracted by
hot water showed a large number of small compact spherical struc-
tures and had the largest molecular weight (4.2 × 105 Da), while
the polysaccharide extracted by ultrasonic-assisted extraction
method exhibited an irregular pleated porous shape and had the
smallest molecular weight (1.34 × 104 Da) [39]. In short, different
extraction methods cause differences in monosaccharide composi-
tion, molar ratio, and activities. Before characterization, the ex-
tracts were usually purified by deproteinization with the Sevage
method (CHCl3/nBuOH = 5:1) to obtain the crude polysaccharides
[27,40].
Crude polysaccharides from G. biloba can be further collected,
dialyzed, concentrated, and lyophilized by a series of adsorbents
and eluents to provide pure polysaccharides (Fig. 2). In brief, ion-
exchange chromatography (DEAE-Sepharose, DEAE-Cellulose,
DEAE-Sephadex) was used in the separation of neutral polysaccha-
ride through gradient salt elution, while gel filtration column chro-
matography (Sephadex G, Sephacryl S, Sepharose CL) was often
applied in the separation of polysaccharides with different molecu-
lar weights [41]. The associated proteins in crude polysaccharides
were removed using trichloracetic acid (TCA) or the Sevag method
[42,43]. Ultimately, pure polysaccharide could be obtained by con-
centration, dialysis, and freeze-drying. Zhang et al. isolated three
polysaccharide monomers (GBP11, GBP22, and GBP33) from
G. biloba leaves using the DEAE-Sepharose Fast Flow column and
Sephadex G-100 column [44]. Chen et al. fractionated G. biloba exo-
carp polysaccharides using DEAE-Sepharose Fast Flow chromatog-
raphy eluting with 0 M, 0.05 M, 0.1 M, 0.15 M, and 0.2 M NaCl,
respectively. The collected fraction was further purified by gel-
filtration chromatography on a Superdex 200 column, with 0.5 M
NaCl as the eluent to get two puried polysaccharides GBEP-NN
and GBEP-AA [40]. Last but not least, it is necessary to choose
proper separation and purification methods according to the char-
acteristics of the polysaccharides [45].
Fig. 2. Schematic representation of the extraction, purification, biological activities and industrial applications of G. biloba polysaccharides.
1899
3. Physiochemical and structural features
Since the 1990s, a large number of literatures on the isolation and
structural identification of G. biloba polysaccharides with different
types has been carried out because of their extensive intensive physio-
logical activities. More than thirty structurally diverse polysaccharides
with a variety of monosaccharide constituents have been extracted
from leaves, seeds and sarcotesta of G. biloba. Their structure
characteristics have been investigated and identified using a combina-
tion of techniques, such as high-performance ion chromatography,
high-performance liquid chromatography (HPLC), gas chromatography
(GC), UV–visible spectroscopy, fourier transform infrared spectra (IR),
nuclear magnetic resonance spectroscopy (NMR) and X-ray photoelec-
tron spectra [46,47]. Herein, we listed the reported G. biloba polysaccha-
rides in the past few years and present integrated information about
concerning their average molecular weight, monosaccharide composi-
tion, primary structures characteristics, bioactivities and corresponding
references in Table 1.
The average molecular weight of G. biloba polysaccharides is influ-
enced by extraction and separation techniques, including the sedimen-
tation method, gel filtration chromatography (with Sepharose CL-6B
analysis), HPLC (with TSK-GEL column) and high pressure gel perme-
ation chromatography (HPGPC) method. Two polysaccharides GBEP-
NN (4.567 × 106 Da) and GBEP-AA (5.679 × 106 Da) were isolated
from G. biloba sarcotesta by using a DEAE Sepharose fast flow anion-
exchange column [40]. Four types of polysaccharides were previously
obtained from G. biloba sarcotesta via cellulose DEAE-52 and Sephadex
G-100 column chromatography with the molecular weights as follows:
BEPP11 (3.4 × 103 Da), GBEPP22 (4.8 × 104 Da), GBEPP33 (3.1 × 105 Da)
and GBEPP44 (9.5 × 105 Da) [28]. GPB, a water-soluble polysaccharide
with a molecular weight of 10 kDa, was extracted from G. biloba leaves
by hot water further purified and detected using Sephadex G75 column
and HPGPC [33]. The range from 103 to 106 Da in the different conditions
of G. biloba polysaccharides was obtained because of different raw ma-
terials and experiment processes.
The bioactivities of polysaccharides are related to their structures,
especially the composition and manner of various monosaccharides. In
most cases, derivatization, hydrolysis, GC and HPLC detection are used
to analyze the monosaccharide compositions [48,49]. Polysaccharides
extracted from leaves, seeds or sarcotesta of G. biloba have obvious
1900 J. Fang et al. / International Journal of Biological Macromolecules 162 (2020) 1897–1905

Table 1
The polysaccharides extracted from G. biloba.

No. Compound Source Mw (Da) Monosaccharide composition Biological activities Ref.

name

1 GBP50S2 Leaves 2.3 × 105 Rha:Gal:Man = 4.2:1:3.8 Antioxidant [31]

2 GPB Leaves 1.0 × 104 Gal:Man:Glc:Ara:Rha:GalA = 6.0:2.4:1.9:2.1:1.9:1.0 – [33]
3 p-PGBL Leaves 1.0 × 104 Gal:Man:Glu:Ara:Rha = 6:2.4:1.7:4.6:6.8:1 Anti-inflammatory [50]
4 GBP11 Leaves 1.944 × 106 Glu:Gal:Ara:Rha = 62.2:1.98:3.84:1.24 Antioxidant [44]
5 GBP22 Leaves 5.167 × 106 Glu:Gal:Ara:Rha = 3.57:68.0:4.67:0.36 Antioxidant
6 GBP33 Leaves 4.598 × 104 Glu:Gal:Ara:Rha = 5.34:48.8:1.70:8.97 Antioxidant
7 GBPw Leaves 2.8 × 104 Ara:Man:Gal:Glu = 8.2:0.5:0.5:0.3 Protective effects on I/R injury [30]
8 GPS Leaves 2.9696 × 105 Man:Rha:Glu:Gal:Ara = 9.73:31.54:24.81:5.72:8.30 Antidepressant activities [29]
9 GBPS-2 Leaves 6.72 × 105 Man:Rha:GlcA:GalA:Glc:Gal:Ara = 0.08:0.12:0.16: 0.06:0.11:1.00:0.32 Antioxidant and immunostimulating [27]
activities
10 GBPS-3 Leaves 7.23 × 105 Man:Rha:GlcA:GalA:Gal:Ara = 0.92:1.00:0.83:0.11:0.42:0.23 Antioxidant and immunostimulating
activities
6
11 GBEP-NN Sarcotesta 4.567 × 10 Rha:Ara:Man:Glu:Gal = 1.88:2.41:1.53:1:2 Antioxidant [40]
12 GBEP-AA Sarcotesta 5.679 × 106 Man:Rha:GluA:GalA:Gal:Glu:Gal:Xyl:Ara:Fuc = 11.85:15.98:1:5.05:1:5.98: Antioxidant
193.52:3.34:14.56:2.67
13 GBEPP11 Sarcotesta 3.4 × 103 Rha:Glu = 1.9:1 Anti-tumor [28]
14 GBEPP22 Sarcotesta 4.8 × 104 Rha:Glu:Gal = 16:10:0.96 Anti-tumor
15 GBEPP33 Sarcotesta 3.1 × 105 Rha:Glu:Gal = 2.64:1:1.43 Anti-tumor
16 GBEPP44 Sarcotesta 9.5 × 105 Rha:Glu:Gal = 37.01:1:8.46 Anti-tumor
17 GBSP3a Sarcotesta 2.15 × 104 Gal:Man:Glc:Ara:Rha:GalA = 3.8:2.2:1.8 0.9:1.5:1.6 Anti-inflammatory [34]
18 GBSP Seeds 1.86 × 105 – Anti-tumor [61]
19 GBPB-W Leaves 2.63 × 104 Rha:Ara:Gal:Glu:Man = 3.48:8.47:3.73:1.76:1 Antioxidant [82]
20 GBPB-S Leaves 1.91 × 104 Rha:Ara:Gal:Glu:Man = 5.34:5.37:5.27:1:1.68 Antioxidant
21 HWE Seeds 4.2 × 105 Man Antioxidant, α-glucosidase inhibitory [39]
activities
4
22 ETE Seeds 1.72 × 10 Man:Glu = 6.41:1.00 Antioxidant, α-glucosidase inhibitory
activities
23 UTE Seeds 1.34 × 104 Man:Rha = 16.87:1.00 Antioxidant, α-glucosidase inhibitory
activities
24 UETE Seeds 2.02 × 104 Man:Rha:Glu = 8.25:1.00:1.53 Antioxidant, α-glucosidase inhibitory
activities
25 GBLPI Leaves 4.1861 × 104 Rha:Ara:Man:Glu:Gal = 1:3.5:18.2:7.4:7.5 Antioxidant [83]
26 GBLPII Leaves 3.61352 × 105 Rha:Ara:Man:Gal = 1.5:1.6:1:2.2 Antioxidant
27 GBLP III Leaves 6.37533 × 105 Rha:Ara:Man:Gal = 1.6:1:1.1:1.7 Antioxidant
28 GBEE Sarcotesta – Man:Rha:GalA:Glu:Gal:Ara = 0.032:0.14:0.296:0.403:0.106:0.046 Anti-tumor [84]
29 GSPS Seeds 1.6 × 106 Rha:Ara:Gal:Glu:Man = 3.5:8.5:3.4:1.8:1 Improve immune function [72]
30 GBLP Leaves 1.2 × 104 Gal:Man:Glu:Ara:Rha:GalA = 32.21:20.82:9.39:6.71:14.76:16.11 Hepatoprotective activity [78]
31 GF1 Leaves 2.3 × 104 Ara:Man:Glu:Gal = 91:3.5:2.5:3 – [52]
32 GF2a Leaves 5.0 × 105 Ara:Rha:Man:Glu:Gal:GluA:GalA = 3:26:29:5.5:1.5:29:7 –
33 GF2b Leaves 2.4 × 104 Ara:Rha:Man:Glu:Gal:GluA:GalA = 9:22:30:8:2:25:4 –
34 GF3 Leaves 4.0 × 104 Ara:Rha:Gal:GluA:GalA = 18:15:16:15:30 –

Note: Mw, molecular weight; Ara, arabinose; Fuc, fucose; Gal, galactose; GalA, galacturonic acid; Glu, glucose; GluA, glucose acid; Man, mannose; Rha, hamnose; Xyl, xylose.

differences in their monosaccharide composition. However, most of
these polysaccharides are composed of rhamnose (Rha), Gal, Man, xy-
lose (Xyl), arabinose (Ara), Glu and fucose (Fuc) with different molar
fractions of the individual components [31,33]. By using Sephadex G-
75 column and GC analysis, a purified polysaccharide was separated
from G. biloba leaves and named as p-PGBL, which consisted of Gal,
Man, Glu, Ara, Rha and galacturonic (Glc) with the ratios of
6:2.4:1.7:4.6:6.8:1 [50]. Interestingly, the uronic acid was detected in
some polysaccharides. Two acidic heteropolysaccharides (GBPS-2 and
GBPS-3), isolated from G. biloba leaves though a diethylaminoethanol
Sepharose Fast Flow column, were analyzed for monosaccharide com-
position. As a result, glucuronic acid (GlcA) and galacturonic acid
(GalA) were determined in GBPS-2 and GBPS-3, which were composed
of Man, Rha, GlcA, GalA, Glc, Gal, and Ara with the molar ratio of
0.08:0.12:0.16:0.06:0.11:1.00:0.32 and Man, Rha, GlcA, GalA, Gal, and
Ara with the molar ratio of 0.92:1.00:0.83:0.11:0.42:0.23, respectively
[27]. The diversity of monosaccharide composition and molar ratio
among these polysaccharides is associated with the raw materials, sep-
aration and purification methods. For instance, Hu et al. extracted four
polysaccharides from G. biloba seeds by hot water-treated, ultrasound-
treated, enzyme-treated, and ultrasound-enzyme treated methods re-
spectively [39]. The four extracted polysaccharides were different in
monosaccharide composition: hot water-treated polysaccharide was
mainly composed of mannose, while ultrasound-enzyme treated ex-
traction was constructed by mannose: glucose: rhamnose with the
molar ratio of 8.25:1.53:1.00. Interestingly, the rearrangement of
atoms might happened and some mannoses might be converted into
rhamnoses by the ultrasound or enzyme treatment [39].
In recent years, many polysaccharides with different structural fea-
tures have been obtained from G. biloba, but only a small amount of
structural information was published. The combination of different
techniques including IR spectroscopy, NMR and GC–MS were applied
to clarify the chemical structure of G. biloba polysaccharides [51]. A neu-
tral polysaccharide (GF1) from G. biloba leaves consisted of 1,5-linked
arabinose residues and three in twelve arabinose molecules were
branched via C-2 or C-3. Also, an acidic G. biloba polysaccharide
(GF2a) was also isolated and determined by GC–MS, 13C NMR, hydroly-
sis, and methylation analysis. The results indicated that GF2a has a back-
bone of 1,2,4-branched mannose which occasionally branches at C-4
and the branches were mainly composed of rhamnose and glucuronic
acid [52]. Yuan et al. isolated a novel G. biloba leaves polysaccharide
named GBP50S2, which were composed of both α- and β-type glyco-
sidic linkages in its structure. Using IR spectroscopy analysis, GBP50S2
was concluded to has a backbone of (1 → 4)-linked α-D-
mannopyranosyl, which occasionally branches at O-3. Moreover, three
branches of GBP50S2 contained β-L-rhamnopyranosyl residues,
(1 → 4)-linked and (1 → 3, 4)-linked α-D-galactopyranosyl terminated
with β-L-rhamnopyranosyl residues, respectively [31]. According to the
results of methylation, NMR and IR analyses, the structures and confor-
mations of a new polysaccharide from leaves of G. biloba (GPB) were
 J. Fang et al. / International Journal of Biological Macromolecules 162 (2020) 1897–1905
identified to be the main linkage types being 1,6-linked Gal and 1,3,6-
linked Gal [33]. The proposed repeating unit of GPB was shown in
Fig. 3. To date, the primary structure of G. biloba polysaccharides has
been studied in many experiments, and further studies should be con-
ducted using advanced techniques to have a deeper understanding of
the structure-bioactivity relationship.
4. Biological activities
In China, G. biloba is effective medicine and herbal dietary supple-
ments. Recent researches have proven that polysaccharides in
G. biloba show various biological activities, such as anti-oxidant, anti-
tumor, anti-inflammatory, hepatoprotective, antidepressant properties,
and so on.
4.1. Antioxidant activities
As one of the primary factors, oxidation is connected with the devel-
opment of many chronic degenerative diseases [53]. Polysaccharides
from G. biloba are reported as a kind of effective and highly promising
source of antioxidants [54,55]. By measuring the ability to scavenge
2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, superoxide radicals,
and ABTS radicals in vitro, the combination of assays have demonstrated
that G. biloba leaves polysaccharides (GBPSs) possess obvious antioxi-
dant activity [27]. Ren et al. isolated two polysaccharides from leaves
of G. biloba and named as GBPS-2 and GBPS-3, which exhibited notice-
able ability to scavenge ABTS radicals. At a test concentration of
4000 μg/mL, ABTS radical scavenging activities of GBPS-2 and GBPS-3
were 74.34% and 82.01%, respectively [27]. Chen et al. proved that
crude polysaccharide extracted from G. biloba sarcotesta (GBEP) prov-
ince dose-dependently scavenged DPPH and hydroxyl radicals in a
concentration-dependent manner. At a dose of 5 mg/mL, the hydroxyl
radical scavenging effect of GBEP was 90.52%, which was stronger
than ascorbic acid [40].
Fig. 3. Proposed repeating unit of GF1, GPB and GBP50S2 [31,33,52].
1901
G. biloba leaves polysaccharide GBP50S2 possessed DPPH radical-
scavenging activity with an IC50 value of 0.412 mg/mL and showed hy-
droxyl radical-scavenging activity with an IC50 value of 0.482 mg/mL
[31]. Another water-soluble polysaccharide from G. biloba leaves
showed a better scavenging activity against hydroxyl, DPPH, and super-
oxide radicals than vitamin C [35]. Zhang et al. found that three polysac-
charides GBP11, GBP22 and GBP33 demonstrate free radical scavenging
activity on DPPH, ABTS, and superoxide anion [44]. These results indi-
cated that G. biloba polysaccharides seemed to be an efficacious antiox-
idant and could prevent the food oxidative deterioration. However, the
relationships between antioxidant activities of G. biloba polysaccharides
and their structural features are not comprehensively elucidated, and
in vivo studies are less fully described.
4.2. Antitumor activity
Many polysaccharides isolated from plants have exhibited promis-
ing antitumor activities and non-toxic [56,57]. Polysaccharides from
both G. biloba leaves and exocarp significantly inhibited the prolifera-
tion of 4T1 breast cancer cells and human endometrial cancer cell
HEC-1B in a dose-dependent manner [58,59]. Wu et al. evaluated the
antitumor activity of the seven polysaccharide samples and found that
all samples exerted remarkable antitumor activity against U937 and
AGS, whereas weak antitumor activity on SGC. Among the polysaccha-
rides, the activity of GBEPP11 was the highest, whose inhibitory rate
was up to 69.2% at the concentration of 640 μg/mL [28]. Subsequent
structure-activity relationship analysis indicated that the antitumor ac-
tivity of polysaccharides increased as their MW decreased, and the acet-
ylation contributed to its antitumor activity.
Xu et al. treated thirty gastric cancer patients by using G. biloba exo-
carp polysaccharides (GBEP) capsules with a 73.4% effective rate. GBEP
not only elevated the apoptosis rate of human gastric cancer SGC-
7901 cells in vitro but also induced tumor cells apoptosis and differenti-
ation in patients. The expression of the c-fos gene was effectively up-
1902 J. Fang et al. / International Journal of Biological Macromolecules 162 (2020) 1897–1905
regulated, while the gene expression of c-myc and bcl-2 were down-
regulated by GBEP in a dose-dependent manner [60]. GBSP, a high-
purity polysaccharide isolated and purified by G. biloba seeds, effectively
induced the hepatoma cell line SMMC-7721 apoptosis, and significantly
inhibited the SMMC-7721 cells division [61].
A selenium-containing polysaccharide (Se-GBLP) from the leaves of
G. biloba has also been evaluated because of the excellent antitumor ac-
tivity against human bladder cancer T24 cells. The antitumor activity of
Se-GBLP may be associated with its inhibitory effect on anti-apoptotic
Bcl-2 expression, and changes in mitochondrial membrane potential,
suggesting that Se-GBLP-induced apoptosis occurs via the
mitochondria-dependent pathway [62].
4.3. Anti-inflammation activity
The pathogenesis of many diseases is caused by inflammation, in-
cluding liver diseases [63], cancer [64], and diabetes [65]. A large num-
ber of studies have shown that natural polysaccharides including
G. biloba polysaccharides have significant anti-inflammation activities.
Polysaccharides derived from G. biloba leaves (PGBL) was reported to
decrease the sensitivity of monocytes to lipopolysaccharide (LPS), and
inhibited the RAW 264.7 cells to express inflammatory factor [66]. Ye
et al. revealed the effect of GBSP3a (a water-soluble G. biloba sarcotesta
polysaccharide) on LPS-induced RAW264.7 macrophages. The secretion
of nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin-6
(IL-6) and interleukin-1 beta (IL-1β) were remarkably inhibited by
GBSP3a. GBSP3a also inhibited excessive mRNA and protein expression
levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2
(COX-2) in a dose-dependent manner. Besides, the anti-inflammation
mechanisms of GBSP3a could be summarized as suppression of nuclear
factor-κB (NF-κB) and MAPK signaling pathways [34]. Zhou et al. evalu-
ated the anti-inflammatory properties of G. biloba leaves polysaccha-
rides (P-PGBL) in vitro, and the result showed that P-PGBL treatment
significantly inhibited the secretion of pro-inflammatory mediators
and cytokines such as IL-1β and IL-6 in LPS-induced RAW264.7 macro-
phage cells. It was also found that P-PGBL treatment down-regulated
the mRNA and protein expression of Toll-like receptor 4 (TLR4) and
NF-κB p65 in a dose-dependent manner, indicating that P-PGBL pos-
sesses potential anti-inflammation effect though inhibiting TLR4/NF-
κB signaling pathway [67].
The anti-inflammation activity and mechanism of G. biloba polysac-
charides have also been investigated in vivo. Fei et al. investigated the
anti-inflammation function of a purified polysaccharide of G. biloba
leaves (p-PGBL) using a xylol induced ear edema and acute peritonitis
mice model. The results suggested that p-PGBL could effectively inhibit
the inflammatory process by interfering with the interaction between
P-selectin and its ligands in mice, and by interfering with HL-60 cells ad-
hesion [50]. Moreover, another G. biloba leaves polysaccharide (GBPw)
was reported to have anti-inflammation effects on rats. GBPw treatment
significantly reduced NO overproduction, increased the levels of anti-
inflammatory cytokines such as IL-10, and decreased the levels of pro-
inflammatory cytokines such as TNF-α and IL-1β [30].
4.4. Immunostimulatory activity
Immunostimulation is an important body's defense strategy for
fighting infections, and macrophage viability is regarded as the critical
indicator of immune activation [68]. Polysaccharides from G. biloba
have been used to improving immune function by activating macro-
phages. Ren et al. have demonstrated that GBPS-2 significantly pro-
motes macrophage proliferation at a concentration of 200 μg/mL. Both
GBPS-2 and GBPS-3 exhibited immunostimulating effects by promoting
NO and cytokine secretion in murine macrophage cells RAW264.7 [27].
Interestingly, in contrast to the result of their antioxidant activities,
GBPS-2 showed higher immunostimulatory activity than GBPS-3, indi-
cating that fragments rich in galactose may exert higher bioactivity.
This finding is similar to the structure-immunostimulatory activity rela-
tionship of Sutherlandia frutescens polysaccharides [69]. Besides, a crude
polysaccharide from the leaves of G. biloba was investigated by testing
the phagocytosis capacity of PMN cells, serum complement function,
and splenic T-lymphocytes activity. The result showed that
immunostimulatory activity G. biloba polysaccharide is equal or higher
to positive medicine Levamisole, indicating the potential application of
G. biloba as functional food supplements [70]. Another G. biloba leaves
polysaccharide also exerted immunogenicity and protection on the
vvIBDV inactivated vaccine at the concentration of 20 g/L [71]. In addi-
tion, a water-soluble polysaccharide from G. biloba seeds (GSPs) was re-
ported to exert positive immune modulation on animals by enhancing
IL-2 and TNF-a secretion. It also has been reported that GSPs efficiently
induce the maturation and functions of bonemarrow-derived dendritic
cells [72].
4.5. Antidepressant activity
Depression is a common and long-lasting mental illness affecting the
mood and emotions, generating worldwide economic losses [73]. Gut
microbiota especially probiotics have been reported to affect the de-
pression development and further treatment, and polysaccharides ex-
hibited anti-depressive effects via regulating microbiota-gut-brain axis
[74,75]. Chen et al. extracted and separated a water-soluble neutral
polysaccharide from Ginkgo leave powder (GPS), which was composed
of rhamnose, mannose, galactose, glucuronic acid, and arabinose, with a
molar ratio of 31.54:29.73:5.72:24.81:8.30. Experimental studies per-
formed in unpredictable chronic mild stress treatment mice have sug-
gested that GPS reduced the immobility times and anxiety-like
behavior in the tail suspension test and open field test respectively,
which was similar to the antidepressant paroxetine. Additional studies
further illuminated that GPS not only alleviated the stress-induced re-
duction in the density of serotonin and dopamine-positive cells, but
also increased the richness of Lactobacillus and reversed depression-
associated gut dysbiosis [29].
4.6. Other biological activities
Other biological activities of G. biloba polysaccharides have also been
evaluated except for the above-mentioned activities. In traditional Chi-
nese medicine, G. biloba was considered as an active resource for regu-
lating the cardiovascular system [76,77]. Yang et al. observed that
G. biloba polysaccharides can protect the focal I/R injury in rat brain
[30]. Yan et al. explored the hepatoprotective effect and underlying
mechanism of a water-soluble polysaccharide from G. biloba leaves
(GBLP) on nonalcoholic fatty liver disease rats. GBLP significantly re-
duced lipid peroxidation, enhanced antioxidant defense system and
liver function. Moreover, GBLP showed a beneficial effect on obesity-
associated insulin resistance by reducing serum glucose and insulin
levels [78]. Polysaccharide from G. biloba exocarp exhibited potent anti-
viral activity against porcine epidemic diarrhea virus in time-, dose- and
temperature-dependent manners. In addition, G. biloba polysaccharide
possessed an obvious inhibiting effect on viral attachment and entry
steps of the virus life cycle, showing its potential candidate for antivirals
discovery [26]. Combined with aerobic exercise, G. ginkgo polysaccha-
ride promoted insulin secretion, controlled the weight and blood glu-
cose in diabetic rats [79].
5. Applications
G. biloba is one of the most widely sold botanical dietary supple-
ments worldwide [80]. Some preparations that contain G. biloba poly-
saccharides as the medicinal ingredient such as Xinkang capsule have
penetrated into many fields for improving human health. Besides,
some functional beverage, candy, and biological fresh-keeping agents
 J. Fang et al. / International Journal of Biological Macromolecules 162 (2020) 1897–1905
Table 2
Patents list of products containing G. biloba polysaccharides and their claimed pharmacological properties.
Application Main composition
Functional beverage Polysaccharides of G. biloba leaves, honey
Functional candy Polysaccharides of G. biloba seeds, isomaltooligosaccharide, lactose
Functional beverage Polysaccharides of G. biloba leaves
Health product Polysaccharides of G. biloba leaves, procyanidins
Humectants Polysaccharides of G. biloba seeds, glycerinum, propylene glycol, sorbitol
Biological fresh-keeping agent Polysaccharides of G. bilobaleaves, Perilla frutescens essential oil
Biological fresh-keeping agent Polysaccharides of G. biloba sarcotesta, gingko ketone, ginkgol
Pharmaceutical Polysaccharides of G. biloba sarcotesta, polyethylene glycol, mannitol
containing G. biloba polysaccharides that improve health are shown in
Table 2.
G. biloba leaf polysaccharide (GBLP) has also been used as the reduc-
ing and stabilizing agent in preparing stable and biocompatible palla-
dium nanoparticles (Pd-n-GBLP NPs). Pd-n-GBLP NPs, with narrow
size distribution from 7.61 to 13.13 nm, were highly biocompatible
with HeLa cells and would be applied to sensitively detect the glucose
concentration in blood. G. biloba leave polysaccharide stabilized palla-
dium nanoparticles have great potential in biomedical detection in the
future [46]. In addition, producing areas, growing years, as well as natu-
ral environments, have a large impact on the content and quality of
G. biloba leaf polysaccharide. Wang et al. detected 16 groups of polysac-
charides extracted from G. biloba leaf in different groups, and the results
indicated that the content of crude polysaccharides was the highest in
the G. biloba from Bozhou in Anhui province [81]. These findings may
contribute to the better application of G. biloba polysaccharides.
6. Conclusion and future prospects
G. biloba, one of the oldest plants in the world, has fascinated
humans for centuries by its highly promising application in healthy
food and medicine. In recent years, especially in the past two decades,
phytochemical investigations have isolated from the leaves, seeds, and
sarcotesta of G. biloba and identified several important bioactive poly-
saccharides, which have been proven one of its main active ingredients
in G. biloba. The studies on polysaccharides of G. biloba mainly focused
on the extraction, purification, structural analysis, and bioactivities.
G. biloba polysaccharides can be isolated and purified via several extrac-
tion methods and among them ultrasound-enzyme treated extraction
method would be more efficient. G. biloba polysaccharides have a
variety of biological activities including anti-oxidant, anti-tumor, anti-
inflammatory, hepatoprotective properties, immunostimulatory activ-
ity, anti-depressant properties, and so on. The polysaccharides from dif-
ferent raw materials can result in the differentiation of chemical
properties, structure characteristics and bioactivities. In brief, the con-
tent of mannose is higher in seeds of G. biloba, while the content of ga-
lactose and glucose are higher in their sarcotesta. Most of the
polysaccharides from G. biloba leaves exhibit remarkable anti-oxidant
activities, while polysaccharides from G. biloba sarcotesta show greater
anti-tumor activities. Furthermore, ultrasound-enzyme treated extrac-
tion polysaccharide exhibited the highest α-glucosidase inhibitory ac-
tivity and antioxidant activities. However, most of the studies have
only reported the pharmacological activity of G. biloba polysaccharides
in vitro or in vivo, and their mechanisms lack depth. In addition, explo-
ration of the relationship between the structure and bioactivity of
G. biloba polysaccharides is rare. Therefore, to better evaluate the health
effects of G. biloba polysaccharides in human health, the observations
in vivo and clinical studies should be carried out. Moreover, further in-
sights into the structure-activity relationship of G. biloba polysaccha-
rides are required to offer a comprehensive and profound basis for the
extensive development of G. biloba polysaccharides.
1903
Pharmacological properties Publish number
Immune regulation, anti-aging CN108391772A
Immune regulation, regulate blood sugar level CN110122646A
Immune regulation CN107988030A
Enhancing immunity function CN106389180A
Reduce the irritation of cigarettes CN104031160A
Bacteriostasis and antiseptic action CN109294191A
Prevent bacterial growth CN101695315A
Antineoplastic CN101036674
Declaration of competing interest
All authors have no personal or financial conflicts of interest.
Acknowledgements
The present work was supported by the program for the National
Natural Science Foundation of China (No. 81973687).
References
[1] B. Singh, P. Kaur, Gopichand, R.D. Singh, P.S. Ahuja, Biology and chemistry of Ginkgo
biloba, Fitoterapia 79 (6) (2008) 401–418.
[2] K. Nakanishi, Terpene trilactones from Gingko biloba: from ancient times to the 21st
century, Bioorg. Med. Chem. 13 (17) (2005) 4987–5000.
[3] K.-i. Hatano, T. Miyakawa, Y. Sawano, M. Tanokura, Chapter 63 - antifungal and lipid
transfer proteins from Ginkgo (Ginkgo biloba) seeds, in: V.R. Preedy, R.R. Watson,
V.B. Patel (Eds.), Nuts and Seeds in Health and Disease Prevention, Academic
Press, San Diego 2011, pp. 527–534.
[4] T.K. Mohanta, Y. Tamboli, P.K. Zubaidha, Phytochemical and medicinal importance
of Ginkgo biloba L, Nat. Prod. Res. 28 (10) (2014) 746–752.
[5] S.F. Omidkhoda, B.M. Razavi, H. Hosseinzadeh, Protective effects of Ginkgo biloba L.
against natural toxins, chemical toxicities, and radiation: a comprehensive review,
Phytother. Res. 33 (11) (2019) 2821–2840.
[6] S. Sandeep Kumar, E.B. George, A. Gjumrakch, E. Valentina, Ginkgo biloba as an alter-
native medicine in the treatment of anxiety in dementia and other psychiatric dis-
orders, Curr. Drug Metab. 18 (2) (2017) 112–119.
[7] H.Y. Wang, Y.Q. Zhang, The main active constituents and detoxification process of
Ginkgo biloba seeds and their potential use in functional health foods, J. Food
Compos. Anal. 83 (2019), 103247.
[8] H.H. Dodge, T. Zitzelberger, B.S. Oken, D. Howieson, J. Kaye, A randomized placebo-
controlled trial of Ginkgo biloba for the prevention of cognitive decline, Neurology
70 (19 Pt 2) (2008) 1809–1817.
[9] M.A. El-Ghazaly, N.A.H. Sadik, E.R. Rashed, A.A. Abd-El-Fattah, Neuroprotective effect
of EGb761 (R) and low-dose whole-body gamma-irradiation in a rat model of
Parkinson's disease, Toxicol. Ind. Health 31 (12) (2015) 1128–1143.
[10] J.G. Choi, S.I. Jeong, C.S. Ku, M. Sathishkumar, J.J. Lee, S.P. Mun, S.M. Kim, Antibacte-
rial activity of hydroxyalkenyl salicylic acids from sarcotesta of Ginkgo biloba
against vancomycin-resistant Enterococcus, Fitoterapia 80 (1) (2009) 18–20.
[11] H.H. Ahmed, H.S. El-Abhar, E.A.K. Hassanin, N.F. Abdelkader, M.B. Shalaby, Ginkgo
biloba L. leaf extract offers multiple mechanisms in bridling N-methylnitrosourea
– mediated experimental colorectal cancer, Biomed. Pharmacother. 95 (2017)
387–393.
[12] E. Pereira, L. Barros, I.C.F.R. Ferreira, Chemical characterization of Ginkgo biloba L. and
antioxidant properties of its extracts and dietary supplements, Ind. Crop. Prod. 51
(2013) 244–248.
[13] N. Zhang, W. Lan, Q. Wang, X. Sun, J. Xie, Antibacterial mechanism of Ginkgo biloba
leaf extract when applied to Shewanella putrefaciens and Saprophytic staphylococ-
cus, Aquacult. Fish. 3 (4) (2018) 163–169.
[14] B. Gargouri, J. Carstensen, H.S. Bhatia, M. Huell, G.P.H. Dietz, B.L. Fiebich, Anti-
neuroinflammatory effects of Ginkgo biloba extract EGb761 in LPS-activated pri-
mary microglial cells, Phytomedicine 44 (2018) 45–55.
[15] C.X. Dai, C.C. Hu, Y.S. Shang, J. Xie, Role of Ginkgo biloba extract as an adjunctive
treatment of elderly patients with depression and on the expression of serum
S100B, Medicine 97 (39) (2018), e12421.
[16] S. Hajirezaee, A. Rafieepour, S. Shafiei, R. Rahimi, Immunostimulating effects of
Ginkgo biloba extract against toxicity induced by organophosphate pesticide, diazi-
non in rainbow trout, Oncorhynchus mykiss: innate immunity components and
immune-related genes, Environ. Sci. Pollut. Res. 26 (9) (2019) 8798–8807.
[17] L. Yang, C.Z. Wang, J.Z. Ye, H.T. Li, Hepatoprotective effects of polyprenols from
Ginkgo biloba L. leaves on CCl4-induced hepatotoxicity in rats, Fitoterapia 82 (6)
(2011) 834–840.
[18] M. Yan, M. Li, S.L. Gu, Z. Sun, T.F. Ma, X. Ma, Ginkgo biloba extract protects diabetic
rats against cerebral ischemia-reperfusion injury by suppressing oxidative stress
and upregulating the expression of glutamate transporter 1, Mol. Med. Rep. 21 (4)
(2020) 1809–1818.
1904 J. Fang et al. / International Journal of Biological Macromolecules 162 (2020) 1897–1905
[19] I. Martinez-Solis, N. Acero, F. Bosch-Morell, E. Castillo, M.E. Gonzalez-Rosende, D.
Munoz-Mingarro, T. Ortega, M.A. Sanahuja, V. Villagrasa, Neuroprotective potential
of Ginkgo biloba in retinal diseases, Planta Med. 85 (17) (2019) 1292–1303.
[20] R. Ihl, Effects of Ginkgo biloba extract EGb 761® in dementia with neuropsychiatric
features: review of recently completed randomised, controlled trials, Int. J. Psychia-
try Clin. Pract. 17 (sup1) (2013) 8–14.
[21] G.L. Ma, J. Xiong, G.X. Yang, L.L. Pan, C.L. Hu, W. Wang, H. Fan, Q.H. Zhao, H.Y. Zhang,
J.F. Hu, Biginkgosides A–I, unexpected minor dimeric flavonol diglycosidic truxinate
and truxillate esters from Ginkgo biloba leaves and their antineuroinflammatory and
neuroprotective activities, J. Nat. Prod. 79 (5) (2016) 1354–1364.
[22] S. Beck, J. Stengel, Mass spectrometric imaging of flavonoid glycosides and
biflavonoids in Ginkgo biloba L, Phytochemistry 130 (2016) 201–206.
[23] X.G. Liu, S.Q. Wu, P. Li, H. Yang, Advancement in the chemical analysis and quality
control of flavonoid in Ginkgo biloba, J. Pharm. Biomed. Anal. 113 (2015) 212–225.
[24] E. Maltas, H.C. Vural, S. Yildiz, Extraction of genomic DNA from polysaccharide- and
phenolics-rich Ginkgo biloba, J. Med. Plants Res 5 (3) (2011) 332–339.
[25] J. Birks, J. Grimley Evans, Ginkgo biloba for cognitive impairment and dementia,
Cochrane Db. Syst. Rev. 1 (2) (2009), CD003120.
[26] J.H. Lee, J.S. Park, S.W. Lee, S.Y. Hwang, B.E. Young, H.J. Choi, Porcine epidemic diar-
rhea virus infection: inhibition by polysaccharide from Ginkgo biloba exocarp and
mode of its action, Virus Res. 195 (2015) 148–152.
[27] Q. Ren, J. Chen, Y. Ding, J.H. Cheng, S. Yang, Z.H. Ding, Q.Y. Dai, Z.E. Ding, In vitro an-
tioxidant and immunostimulating activities of polysaccharides from Ginkgo biloba
leaves, Int. J. Biol. Macromol. 124 (2019) 972–980.
[28] X.Y. Wu, G.H. Mao, T. Zhao, J.L. Zhao, F. Li, L.H. Liang, L.Q. Yang, Isolation, purification
and in vitro anti-tumor activity of polysaccharide from Ginkgo biloba sarcotesta,
Carbohydr. Polym. 86 (2) (2011) 1073–1076.
[29] P. Chen, M.F. Hei, L.L. Kong, Y.Y. Liu, Y. Yang, H.B. Mu, X.Y. Zhang, S.T. Zhao, J.Y. Duan,
One water-soluble polysaccharide from Ginkgo biloba leaves with antidepressant ac-
tivities via modulation of the gut microbiome, Food Funct. 10 (12) (2019)
8161–8171.
[30] Y.M. Yang, P.F. Liu, L.X. Chen, Z.J. Liu, H.X. Zhang, J.J. Wang, X.S. Sun, W.Q. Zhong, N.
Wang, K. Tian, J.S. Zhao, Therapeutic effect of Ginkgo biloba polysaccharide in rats
with focal cerebral ischemia/reperfusion (I/R) injury, Carbohydr. Polym. 98 (2)
(2013) 1383–1388.
[31] F. Yuan, R.M. Yu, Y. Yin, J.R. Shen, Q.F. Dong, L. Zhong, L.Y. Song, Structure character-
ization and antioxidant activity of a novel polysaccharide isolated from Ginkgo
biloba, Int. J. Biol. Macromol. 46 (4) (2010) 436–439.
[32] X.H. Li, Q. Yang, L. Wang, Microwave assisted extraction of polysaccharides of Ginkgo
biloba, Food Sci. Technol 37 (2012) 160–163.
[33] J.F. Yang, D.Y. Zhou, Z.Y. Liang, A new polysaccharide from leaf of Ginkgo biloba L,
Fitoterapia 80 (1) (2009) 43–47.
[34] J. Ye, C.Q. Ye, Y.Y. Huang, N. Zhang, X.Q. Zhang, M.T. Xiao, Ginkgo biloba sarcotesta
polysaccharide inhibits inflammatory responses through suppressing both NF-
kappa B and MAPK signaling pathway, J. Sci. Food Agric. 99 (5) (2019) 2329–2339.
[35] B. Jiang, H.Y. Zhang, C.J. Liu, Y.Y. Wang, S.D. Fan, Extraction of water-soluble polysac-
charide and the antioxidant activity from Ginkgo biloba leaves, Med. Chem. Res. 19
(3) (2010) 262–270.
[36] G.X. Wang, H.Y. Huang, Y.Z. Yang, C.G. Li, Q. Luo, D. Song, Extraction of polysaccha-
rides from the mesosperm of Ginkgo biloba L. byaqueous enzymatic extraction,
Bangladesh, J. Bot 48 (3) (2019) 711–717.
[37] L.J. Zhang, S.S. Guo, M.S. Wang, L. He, PEG-based ultrasound-assisted enzymatic ex-
traction of polysaccharides from Ginkgo biloba leaves, Int. J. Biol. Macromol. 80
(2015) 644–650.
[38] G.S. Zhou, Y.P. Tang, Y. Li, J.A. Duan, Multi-response optimization of ultrasonic
assisted enzymatic extraction of antioxidant polysaccharides from waste Ginkgo
biloba sarcotesta, Chiang Mai J. Sci. 47 (3) (2020) 499–512.
[39] J.L. Hu, Y. Liu, L.M. Cheng, R.J. Shi, A. Qayum, A. Bilawal, G. Munkh-Amgalan, M.A.
Hussain, Z.M. Jiang, B. Tian, Comparison in bioactivity and characteristics of Ginkgo
biloba seed polysaccharides from four extract pathways, Int. J. Biol. Macromol. 159
(2020) 1156–1164.
[40] J.J. Chen, T. Zhang, B. Jiang, W.M. Mu, M. Miao, Characterization and antioxidant ac-
tivity of Ginkgo biloba exocarp polysaccharides, Carbohydr. Polym. 87 (1) (2012)
40–45.
[41] Y.P. Zhu, L. Yang, C.N. Zhang, Y.L. Tian, F.F. Zhang, X.T. Li, Structural and functional
analyses of three purified polysaccharides isolated from Chinese Huaishan-yams,
Int. J. Biol. Macromol. 120 (2018) 693–701.
[42] X.Y. Long, Q. Yan, L.J. Cai, G.Y. Li, X.G. Luo, Box-Behnken design-based optimization
for deproteinization of crude polysaccharides in Lycium barbarum berry residue
using the Sevag method, Heliyon 6 (5) (2020), e03888.
[43] E.E. Woodside, C.A. Frick, W. Kocholaty, Trichloracetic acid soluble polysaccharide-
protein complexes, Proc. Soc. Exp. Biol. Med. 119 (1965) 327–331.
[44] C.W. Zhang, C.Z. Wang, R. Tao, Characterization and antioxidant activities of polysac-
charides extracted from enzymatic hydrolysate of Ginkgo biloba leaves, J. Food
Biochem. 41 (3) (2017), e12352.
[45] L. Shi, Bioactivities, isolation and purification methods of polysaccharides from nat-
ural products: a review, Int. J. Biol. Macromol. 92 (2016) 37–48.
[46] Y.S. Cui, X. Lai, K. Liu, B. Liang, G.L. Ma, L.G. Wang, Ginkgo biloba leaf polysaccharide
stabilized palladium nanoparticles with enhanced peroxidase-like property for the
colorimetric detection of glucose, RSC Adv. 10 (12) (2020) 7012–7018.
[47] G.C. Deng, J.Y. Sun, G.F. Lu, Z.N. Ju, L.L. Ye, Study on selenium and polysaccharide dis-
tribution in different time Ginkgo leaf, Spectrosc. Spectr. Anal. 31 (2) (2011)
543–546.
[48] B.L. Fan, T.T. Li, X.F. Song, C. Wu, C.Q. Qian, A rapid, accurate and sensitive method for
determination of monosaccharides in different varieties of Osmanthus fragrans Lour
by pre-column derivatization with HPLC-MS/MS, Int. J. Biol. Macromol. 125 (2019)
221–231.
[49] C.Y. Cao, L.L. Wang, L.L. Wang, Y. Gong, C.Q. Ai, C.R. Wen, S. Song, A strategy to iden-
tify mixed polysaccharides through analyzing the monosaccharide composition of
disaccharides released by graded acid hydrolysis, Carbohydr. Polym. 223 (2019),
115046.
[50] R. Fei, Y. Fei, S. Zheng, Y.G. Gao, H.X. Sun, X.L. Zeng, Purified polysaccharide from
Ginkgo biloba leaves inhibits P-selectin-mediated leucocyte adhesion and inflamma-
tion, Acta Pharmacol. Sin. 29 (4) (2008) 499–506.
[51] B. Jiang, Y. Zhang, T.Y. Guo, H.F. Zhao, Y.C. Jin, Structural characterization of lignin
and lignin-carbohydrate complex (LCC) from Ginkgo shells (Ginkgo biloba L.) by
comprehensive NMR spectroscopy, Polymers 10 (7) (2018) 736.
[52] J. Kraus, Water-soluble polysaccharides from Ginkgo biloba leaves, Phytochemistry
30 (9) (1991) 3017–3020.
[53] I.F.F. Benzie, Evolution of antioxidant defence mechanisms, Eur. J. Nutr. 39 (2)
(2000) 53–61.
[54] J.Q. Wang, S.Z. Hu, S.P. Nie, Q. Yu, M.Y. Xie, Reviews on mechanisms of in vitro anti-
oxidant activity of polysaccharides, Oxidative Med. Cell. Longev. 64 (2016) 1–13.
[55] R. Jiao, Y.X. Liu, H. Gao, J. Xiao, K.F. So, The anti-oxidant and antitumor aroperties of
plant polysaccharides, Am. J. Chin. Med. 44 (3) (2016) 463–488.
[56] C.X. Jiang, Q.P. Xiong, S.L. Li, X.R. Zhao, X.X. Zeng, Structural characterization,
sulfation and antitumor activity of a polysaccharide fraction from Cyclina sinensis,
Carbohydr. Polym. 115 (2015) 200–206.
[57] X. Meng, H.B. Liang, L.X. Luo, Antitumor polysaccharides from mushrooms: a review
on the structural characteristics, antitumor mechanisms and immunomodulating
activities, Carbohydr. Res. 424 (2016) 30–41.
[58] P. Chang, Y. Xu, D.Y. Zhou, J.D. Wu, S.L. Ma, Effects of polysaccharides from Ginkgo
biloba on proliferation of 4T1 breast cancer cells and expression of GLUT family
genes, Chin. Pharmacol. Bull. 34 (9) (2018) 1301–1307.
[59] W. Liu, X.L. Xie, X. Liu, Z.X. Wang, The effects of Ginkgo biloba exocarp polysaccha-
rides on proliferation of human endometrial cancer cell HEC-1B, Chin. J. Biochem.
Pharm. 33 (6) (2012) 841–843.
[60] A.H. Xu, H.S. Chen, B.C. Sun, X.R. Xiang, Y.F. Chu, F. Zhai, L.C. Jia, Therapeutic mech-
anism of Ginkgo biloba exocarp polysaccharides on gastric cancer, World J.
Gastroenterol. 9 (11) (2003) 2424–2427.
[61] Q. Chen, G.W. Yang, L.G. An, Apoptosis of hepatoma cells SMMC-7721 induced by
Ginkgo biloba seed polysaccharide, World J. Gastroenterol. 8 (5) (2002) 832–836.
[62] D. Chen, S. Sun, D. Cai, G. Kong, Induction of mitochondrial-dependent apoptosis in
T24 cells by a selenium (Se)-containing polysaccharide from Ginkgo biloba L. leaves,
Int. J. Biol. Macromol. 101 (2017) 126–130.
[63] P. Lam, F. Cheung, H.Y. Tan, N. Wang, M.F. Yuen, Y.B. Feng, Hepatoprotective effects
of Chinese medicinal herbs: a focus on anti-inflammatory and anti-oxidative activ-
ities, Int. J. Mol. Sci. 17 (4) (2016) 37.
[64] L. Coussens, Z. Werb, Inflammation and cancer, Nature 420 (6917) (2020) 860.
[65] K.E. Wellen, G.S. Hotamisligil, Inflammation, stress, and diabetes, J. Clin. Invest. 115
(5) (2005) 1111–1119.
[66] C.M. Zhang, L.H. Lin, G.R. Li, J.A. Ma, X. Han, R. Fei, PGBL inhibits the RAW 264.7 cells
to express inflammatory factor, Bio-Med. Mater. Eng. 26 (2015) S2069–S2075.
[67] X.L. Zhou, M. Yang, B.G. Xue, H.T. He, C.M. Zhang, Anti-inflammatory action of Ginkgo
biloba leaf polysaccharide via TLR4/NF-kappa b signaling suppression, Biomed. Res.
25 (4) (2014) 449–454.
[68] Q.X. Yuan, L.Y. Zhao, Q.Q. Cha, Y. Sun, H. Ye, X.X. Zeng, Structural characterization
and immunostimulatory activity of a homogeneous polysaccharide from
Sinonovacula constricta, J. Agric. Food Chem. 63 (36) (2015) 7986–7994.
[69] B.Z. Zhang, W.K. Leung, Y.F. Zou, W. Mabusela, Q. Johnson, T.E. Michaelsen, B.S.
Paulsen, Immunomodulating polysaccharides from Lessertia frutescens leaves: isola-
tion, characterization and structure activity relationship, J. Ethnopharmacol. 152 (2)
(2014) 340–348.
[70] M. Hancianu, M. Pavelescuz, A. Miron, A.C. Aprotosoaie, E. Grigorescu, C. Lupusoru,
U. Stanescu, Polysaccharides fraction isolated from Ginkgo biloba folium:
immunopharmacological properties, Rev. Med. Chir. 111 (4) (2007) 1070–1073.
[71] X.Y. Meng, Z.F. Chu, J.J. Zhang, H. Zhang, S.J. Wang, K. Wei, R.L. Zhu, Immunomodu-
latory functions of Ginkgo biloba leaves polysaccharide on vvIBDV vaccine, Chin. J.
Vet. Sci. 39 (4) (2019) 640–645.
[72] Y.H. Chen, Y.M. Meng, Y. Cao, H. Wen, H. Luo, X.H. Gao, F.P. Shan, Novel analysis of
maturation of murine bone-marrow-derived dendritic cells induced by Ginkgo
seed polysaccharides, Hum. Vaccines Immunother. 11 (6) (2015) 1387–1393.
[73] R. Matraszek-Gawron, M. Chwil, P. Terlecka, M.M. Skoczylas, Recent studies on anti-
depressant bioactive substances in selected species from the genera hemerocallis
and gladiolus: a systematic review, Pharmaceuticals 12 (4) (2019) 172.
[74] E. Vaghef-Mehrabany, V. Maleki, M. Behrooz, F. Ranjbar, M. Ebrahimi-Mameghani,
Can psychobiotics “mood” ify gut? An update systematic review of randomized con-
trolled trials in healthy and clinical subjects, on anti-depressant effects of probiotics,
prebiotics, and synbiotics, Clin. Nutr. 39 (5) (2019) 1395–1410.
[75] T.X. Yan, T.T. Nian, Z.Z. Liao, F. Xiao, B. Wu, K.S. Bi, B.S. He, Y. Jia, Antidepressant ef-
fects of a polysaccharide from okra (Abelmoschus esculentus (L) Moench) by anti-
inflammation and rebalancing the gut microbiota, Int. J. Biol. Macromol. 144
(2020) 427–440.
[76] Z.M. Shu, A.H. Shar, M. Shahen, H. Wang, M. Alagawany, M.E. Abd El-Hack, S.A.
Kalhoro, M. Rashid, P.A. Shar, Pharmacological uses of Ginkgo biloba extracts for car-
diovascular disease and coronary heart diseases, Int. J. Pharmacol. 15 (1) (2019)
1–9.
[77] D.O.L. Teik, X.S.A. Lee, C.J. Lim, C.M. Low, M. Muslima, L. Aquili, Ginseng and Ginkgo
biloba effects on cognition as modulated by cardiovascular reactivity: a randomised
trial, PLoS One 11 (3) (2016), e0150447.
 J. Fang et al. / International Journal of Biological Macromolecules 162 (2020) 1897–1905
[78] Z.G. Yan, R.F. Fan, S.J. Yin, X.N. Zhao, J.Z. Liu, L.H. Li, W.Q. Zhang, L.J. Ge, Protective ef-
fects of Ginkgo biloba leaf polysaccharide on nonalcoholic fatty liver disease and its
mechanisms, Int. J. Biol. Macromol. 80 (2015) 573–580.
[79] R.K. Zhu, J. Xie, Effect of aerobic exercise combined with ginkgo polysaccharide on
weight, blood glucose and glycosylated serum protein in diabetic rats, Pak. J.
Pharm. Sci. 31 (3) (2018) 1045–1050.
[80] N. Mei, X. Guo, Z. Ren, D. Kobayashi, K. Wada, L. Guo, Review of Ginkgo biloba-
induced toxicity, from experimental studies to human case reports, J. Environ. Sci.
Health, Part C: Environ. Carcinog. Ecotoxicol. Rev. 35 (1) (2017) 1–28.
[81] X.H. Wang, Y.L. Sun, W.Z. Li, D. Kong, Effects of producing area and growing years on
the content of polysaccharide and flavone in Ginkgo biloba leaves, J. Chin. Med.
Mater. 39 (6) (2016) 1341–1342.
1905
[82] X.Q. Hu, F. Yuan, C.Y. Yan, Y.Y. Huang, R.M. Yu, The purification and characterization
of polysaccharides isolated from Ginkgo biloba and their in vitro antioxidant activi-
ties, J. Chin. Med. Mater. 34 (12) (2011) 1950–1953.
[83] G. He, W. Liu, H.P. Li, L. Lang, X.Q. Guo, J. Yan, X.J. Gou, Isolation and purification,
structure identification and antioxidant activity of polysaccharides of Ginkgo biloba
leaf, Sci. Technol. Food Ind. 36 (22) (2015) 81–86.
[84] L.L. Mao, Y. Chen, B.Y. Hu, A.H. Xu, Analysis of monosaccharide compositions in poly-
saccharides from exopleura of Ginkgo biloba, China J. Chin. Mater. Med. 39 (2)
(2014) 262–266.