Trends in Food Science & Technology 72 (2018) 13–24

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Trends in Food Science & Technology

journal homepage: www.elsevier.com/locate/tifs

Review

Recent advances in understanding the anti-obesity activity of anthocyanins T

and their biosynthesis in microorganisms
Lianghua Xiea, Hongming Sua, Chongde Sunb, Xiaodong Zhenga, Wei Chena,∗
a
Department of Food Science and Nutrition, National Engineering Laboratory of Intelligent Food Technology and Equipment, Key Laboratory for Agro-Products Postharvest
Handling of Ministry of Agriculture, Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, China
b
Laboratory of Fruit Quality Biology, Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, The State Agriculture Ministry Laboratory of
Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Hangzhou 310058, China

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

Keywords: Background: Obesity is a serious health problem and the cause for social and economic burdens. Currently, there
Anthocyanins is still no cure for obesity, while the investment of time and money for one is huge. Recent years, the possibility
Anti-obesity of developing natural products from fruits and vegetables with bioactivities into anti-disease agents has become
Lipid metabolism a hot spot in research. Thus, anthocyanins are increasingly causing more attention, as they have been proved to
Biosynthesis
show anti-obesity effects. Furthermore, recent advances in biosynthesis of anthocyanins in microorganisms have
illustrated a promising way in producing these valuable compounds in large scales.
Scope and approach: Anthocyanins have great importance in developing a cure for obesity and biosynthesis in
microorganisms has high potential in their massive production. This review therefore highlights the recent
advances in the anti-obesity effects of anthocyanins and their biosynthesis in microorganisms. We have com-
prehensively discussed the molecular mechanisms involved in the anti-obesity effects of anthocyanins, the
physicochemical and physiological properties of anthocyanins, the suitability of anthocyanins in anti-obesity
therapies as well as the possibility of biosynthesis in microorganisms in future application.
Key findings and conclusions: Anthocyanins have shown anti-obesity effects through multiple mechanisms, and
biosynthesis of anthocyanins in microorganisms could have extensive applications. Inhibiting lipid absorption,
regulating lipid metabolism, increasing energy expenditure, suppressing food intake and regulating gut micro-
biota are major mechanisms involved. Moreover, anthocyanins are promising candidates in developing anti-
obesity therapies. Further studies are required to explore therapeutic uses of anthocyanins in treating obesity and
application of biosynthesis of anthocyanins in microorganisms in industries.

1. Introduction types of cancer, according to a Lancet commission on obesity

Obesity has become a serious problem that is causing a heavy
burden to the economy and negative effects on people's longevity and
life quality worldwide. Obesity is defined as a body mass index (BMI)
equal to or more than 30 by World Health Organization (WHO), while
overweight is defined as a BMI equal to or more than 25. BMI is cal-
culated through the weight in kilograms divided by the square of the
height in meters. According to a report from WHO, in 2014, about 39%
of adults aged 18 years and over were overweight (38% of men and
40% of women), more than 1.9 billion, and 13% of the world's adult
population (11% of men and 15% of women) were obese. This number
has more than doubled between 1980 and 2014. In 2013, 42 million
children under the age of 5 were overweight or obese. Obesity is driving
global increases in type 2 diabetes, cardiovascular diseases, and several
(Swinburn, Dietz, & Kleinert, 2015). Obesity is also a prerequisite for
metabolic syndrome, which is defined as a clustering of risk factors
including central obesity, insulin resistance, dyslipidemia and hy-
pertension (Carr & Brunzell, 2004; Nguyen, Magno, Lane, Hinojosa, &
Lane, 2008). It is a physiological status as a result of an imbalance
between energy consumption and energy expenditure. Thus, regulating
the energy imbalance either through decreasing energy intake or in-
creasing energy use is a promising strategy for the prevention and
treatment of obesity.
Anthocyanins are anthocyanidins with sugar groups. Anthocyanidin
is a subgroup of flavonoids that shows high bioactivities such as anti-
oxidant effects. Basic structure of flavonoids is called C6-C3-C6, which
is a skeleton of diphenylpropane formed by two benzene rings (ring A
and B) with the linkage of a three-carbon chain that forms a closed

∗
Corresponding author. Department of Food Science and Nutrition, Zhejiang University, 866 Yuhangtang Road, Xihu District, Hangzhou 310058, China.
E-mail address: zjuchenwei@zju.edu.cn (W. Chen).

https://doi.org/10.1016/j.tifs.2017.12.002
Received 26 September 2017; Received in revised form 21 November 2017; Accepted 4 December 2017
Available online 07 December 2017
0924-2244/ © 2017 Elsevier Ltd. All rights reserved.
L. Xie et al.
pyran ring (C ring) with A ring. Flavonoids can be subdivided into
different subgroups depending on the carbon of C ring to which B ring is
attached, as well as the degree of unsaturation and oxidation of C ring.
General subgroups are flavones, flavanones, isoflavones, flavonols,
flavanonols, flavan-3-ols and anthocyanidins (Manach, Scalbert,
Morand, Rémésy, & Jiménez, 2004). Anthocyanidins are characterized
by having an oxonium, namely 2-phenylbenzopyrylium (Hendry &
Houghton, 1996). There are more than 20 different anthocyanidins in
total, while 6 of them are reported most frequently to be bioactive:
cyanidin, delphinidin, pelargonidin, malvidin, peonidin and petunidin.
The most widespread anthocyanins in nature are the glycosides of cy-
anidin, delphinidin and pelargonidin. (Kong, Chia, Goh, Chia, &
Brouillard, 2003). Our group has recently reported that extracts of
anthocyanins-rich raspberry, blackberry, mulberry and bayberry show
antioxidant activity (Bao et al., 2016; Chen et al., 2016c; Xu, Hu, Bao,
Xie, & Chen, 2017), protective effects such as protection against acry-
lamide-induced oxidative stress (Chen, Su, Xu, & Jin, 2017a; Chen, Su,
Xu, Bao, & Zheng, 2016a; Li, Bao, & Chen, 2018), and ethyl carbamate-
induced cytotoxicity (Chen, Xu, Zhang, Su, & Zheng, 2016b; Chen, Li,
Bao, & Gowd, 2017b; Zhang et al., 2017; Bao et al., 2018). During the
past decade, the anti-obesity effects of anthocyanins have gained in-
creasing attention. Various anthocyanins extracts and purified antho-
cyanins are reported to have anti-obesity effects, such as preventing
body weight gain and inhibiting lipid accumulation in adipose tissue.
This review summarizes recent advances on the anti-obesity effects
of anthocyanins and the mechanisms involved. We first discussed
characteristics of anthocyanins, such as physicochemical properties,
metabolism and bioavailability, as well as their therapeutic use. We also
summarized the effects of anthocyanins on obesity associated oxidative
stress and inflammation. Anthocyanins are generally extracted from
natural sources. This method is troubled with problems including high
expenses, low efficiency and complex processes. In contrast, biosynth-
esis in microorganisms is a promising way to achieve massive produc-
tion of anthocyanins. In this review, the recent advances in the bio-
synthesis of anthocyanins in microorganisms are also addressed at
length to provide future perspectives on the production and application
of anthocyanins.
2. Anthocyanins with anti-obesity effects and the major
mechanisms involved
Anthocyanins widely exist in nature, such as fruits, seeds and
flowers, giving them attractive colors. Extraction of anthocyanins from
natural sources has been conducted by research groups all over the
world. Many anthocyanins extracts and purified anthocyanins are re-
ported to have anti-obesity effects in in vitro experiments, animal stu-
dies and clinical trials. Anthocyanins may exert beneficial effects in
obesity by 1.) inhibiting lipid absorption, 2.) increasing energy ex-
penditure, 3.) regulating lipid metabolism, 4.) suppressing food intake,
and 5.) regulating gut microbiota (as shown in Table 1 and Fig. 1).
2.1. Inhibit lipid absorption
Inhibiting digestion and absorption of nutrients is a strategy focused
on gastrointestinal mechanism to reduce energy intake. Excessive
dietary fat intake, especially of saturated fat often results in hyperlipi-
demia and increased weight of adipose tissue. As dietary fat could not
be directly absorbed without the action of pancreatic lipase, developing
pancreatic lipase inhibitors is a rather appealing way to attenuate the
status of obesity (Birari & Bhutani, 2007).
Litchi flower-water extracts (LFWEs) that contain some anthocya-
nins showed a suppressive effect on in vitro lipase activity (Wu et al.,
2013a). Inhibitory effects on pancreatic lipase of extracts from samples
commonly used in Mediterranean diet were reported along with their
anthocyanins contents. Samples include fruits (blackberry, mulberry,
sumac drupe, blood orange and pomegranate), vegetables (red cabbage
14
Trends in Food Science & Technology 72 (2018) 13–24
and violet cauliflower), cereals (black rice); citrus vegetative tissues
(young lemon shoots), legume seeds (black bean); citrus by-products
(blood orange peel) and cereal processing by-products (black rice hull).
The results showed a wide variation of anthocyanins contents in the
extracts. Blood orange and pomegranate juice had the highest antho-
cyanins contents and showed the highest effect in inhibiting pancreatic
lipase activity (Fabroni, Ballistreri, Amenta, Romeo, & Rapisarda,
2016).
2.2. Increase energy expenditure
Energy expenditure is closely related to mitochondrial function. The
AMP-activated protein kinase (AMPK) is a key mediator of signals in
energy balance (Steinberg & Kemp, 2009). AMPK increases expression
of the gene Ppargc1α, which encodes the regulator of mitochondrial
biogenesis peroxisome-proliferator-activated receptor γ coactivator 1 α
(PGC1α) (Luo, Saha, Xiang, & Ruderman, 2005; Wu, Puigserver,
Andersson, Zhang, Adelmant, Mootha, et al., 1999). AMPK inhibits
cellular lipid metabolism upon activation of stimuli such as metabolic
stress and cytokines derived from adipocyte such as leptin and adipo-
nectin. AMPK also inhibits triglyceride synthesis and stimulate fatty
acid oxidation and mitochondrial biogenesis. Thus the activation of
AMPK will potentially reduce hypertriglyceridemia and elevate trigly-
cerides storage in muscle and liver (Hardie, 2008). Also, in adipose
tissues, a group of mitochondrial proteins called uncoupling proteins
mediate thermogenesis (Cannon & Nedergaard, 2004; Spiegelman &
Flier, 2001).
Consumption of chokeberry extracts (CBE) that contain at least 10%
anthocyanins up-regulated peroxisome-proliferator-activated receptor γ
(Pparγ) mRNA level (Qin & Anderson, 2012). Ovariectomized female
rats were fed four different high fat diets with 2% dextrin (OVX con-
trol), 2% BC (black carrot extracts), 2% BCLP (BC fermented with
Lactobacillus plantarum) or 2% BCAO (BC fermented with Aspergillus
oryzae) for 12 weeks, with 10 rats in each group. Another 10 rats were
given sham operation and fed a high fat diet with 2% dextrin and served
as control. BC, BCLP and BCAO all decreased fat mass and weight gain
as well as prevented increase in total serum and LDL cholesterol and
triglycerides compared with OVX control, with the effects in ascending
orders. BCAO had lower cyanidin 3-rutinoside, malvidin 3, 5-diglyco-
side and delphinidin 3-glucoside contents as well as much higher cya-
nidin and malvidin contents compared with BC. The mechanism was
suggested to be through phosphorylated AMPK (pAMPK) to phos-
phorylated Acetyl CoA carboxylase (pACC) (Park et al., 2015). Black
soybean seed coat extract (BE) containing 9.2% cyanidin 3-glucoside,
6.2% epicatechin and 39.8% procyanidins were fed to mice for 14
weeks. BE suppressed fat accumulation in the mesenteric adipose tissue
by up-regulating uncoupling proteins (UCPs). The gene and protein
expression levels of UCP-1 in brown adipose tissue and UCP-2 in white
adipose tissue were up-regulated (Kanamoto et al., 2011). Anthocya-
nins fraction (AF) from purple sweet potato was supplemented at
200 mg/kg per day to mice and reduced weight gain. AF also improved
serum lipid parameters and inhibited hepatic triglyceride accumula-
tion. AF increased AMPK phosphorylation and down-regulated the ex-
pression level of SREBP-1, thus reducing ACC and FAS expression
(Hwang et al., 2011).
2.3. Regulate lipid metabolism
Dyslipidemia is a common feature of obesity. Decreasing lipogenesis
and increasing lipolysis are two aspects to regulate lipid metabolism.
The strategy is stimulating triglyceride hydrolysis to reduce fat storage,
which requires oxidation of newly released fatty acids. Regulators of
fatty acid acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS)
are target in this process (Luo et al., 2005). Sterol regulatory element-
binding proteins (SREBPs) family are transcription factors associated
with fatty acid synthesis. SREBP-1a, SREBP-1c and SREBP-2 are
L. Xie et al. Trends in Food Science & Technology 72 (2018) 13–24

Table 1
Anthocyanins extracts and purified anthocyanins with anti-obesity effects and mechanisms.

Source Anthocyanins Composition Experimental methods (treated Major activity Mechanism of action
dose, subjects, duration of
treatment)

(Litchi chinensis Sonn.) Crude water extract
(flower)
Aronia melanocarpa Crude ethanol/water extract
Daucus carota L. Cyanidin, malvidin,
cyanidin 3-rutinoside,
malvidin 3,5-diglycoside,
and delphinidin 3-glucoside
Glycine max (seed coat) Crude ethanol/water extract
Ipomoea batatas 90% cyanidin and peonidin
acyl glucosides
Lactuca sativa L. Crude water extract
Prunus salicina Lindl. Purified cyanidin 3-glucoside and
crude extract
Zea mays indurata Mostly cyanidin 3-glucoside
Lonicera Caerulea, Mostly cyanidin 3-glucoside
Cornus mas Cyanidin galactoside,
pelargonidin galactoside,
and delphinidin galactoside
Prunus auiun L. Cyanidin 3-(2G-
glucosylrutinoside),
cyanidin 3-rutinoside,
and pelargonidin 3-rutinoside
Vaccinium corymbosum Petunidin 3-arabinoside,
delphinidin 3-glucoside
and cyanidin 3-galactoside
Morus australis P. cyanidin 3-glucoside,
cyanidin 3-rutinoside
and pelargonidin 3-glucoside
Glycine max (seed coat) Cyanidin 3-glucoside,
petunidin 3-glucoside
and delphinidin 3-glucoside
2.5%/5%,
Wistar rats with HCD, 9 weeks
100/200 mg/kg,
Wistar rats, 6 weeks
2%, OVX rats, 12 weeks
0.2%/1%/2%,
C57BL/6 mice with HFD, 14 weeks
200 mg/kg,
ICR mice with HFD, 4 weeks
100/300 mg/kg,
C57BL/6 mice, 28 days
Both 8 mg/kg,
Wistar rats with HFD, 8 weeks
2 g/kg diet,
C57BL/6J mice with HFD, 12
weeks
50/100/200 mg/kg diet,
C57BL/6 mice with HFD, 8 weeks
1 g/kg diet,
C57BL/6 mice with HFD, 8 weeks
200 μg/mL, 3T3-L1 cells;
40/200 mg/kg and 200 mg/kg,
C57BL/6 mice with HFD,
12 weeks and 8 weeks
50, 100 and 200 mg/kg,
C57BL/6 mice with HFD, 8 weeks
40/200 mg/kg and 200 mg/kg,
C57BL/6 mice with HFD,
12 weeks and 8 weeks
6/24 mg/kg,
Sprague-Dawley rats, 40 days
6%/13% decrease Inhibit lipid absorption,
in body weight gain regulate lipid metabolism
Modulate adipogenesis associated Increase energy expenditure,
pathways regulate lipid metabolism, suppress
food intake
13% decrease Increase energy expenditure,
in body weight gain regulate lipid metabolism
16%/30%/21% decrease Increase energy expenditure
in body weight gain
Decrease hepatic triglyceride Increase energy expenditure
accumulation
Decrease total liver lipids Regulate lipid metabolism
Both 9% decrease Regulate lipid metabolism
in body weight gain
Normalize hyperglycemia Regulate lipid metabolism
and hyper leptinemia
Notsignificant/17.1%/24.1% Regulate lipid metabolism, suppress
decrease in body weight gain food intake
24% decrease in weight gain Regulate lipid metabolism
30.7% decrease in lipid Regulate lipid metabolism, suppress
accumulation; 5.2%/11.2% and food intake
29.6% decrease
in body weight gain
19.4% decrease in body weight gain Regulate lipid metabolism, resolve
inflammation
11.8%/21.4% and 32.7% decrease in Regulate lipid metabolism, suppress
body weight gain food intake
Lower body weight Suppress food intake
and food intake

members of this family.
Various researches have shown that anthocyanins could directly
affect lipid metabolism. 150 mg Black soybean (BS) extract containing
10 mg cyanidin 3-glucoside per gram was administrated to high-fat fed
(HFF) rats daily for 6 weeks. Compared with HFF rats, BS affected fatty
acid composition in subcutaneous fat, such as several saturated,
monounsaturated and n-6 polyunsaturated fatty acid. Such reduction in
fatty acid contents may have implications in suppressing inflammation
(Sato et al., 2015). Adzuki beans were suggested to be a health-bene-
ficial food that contained anthocyanin, adzukisaponin and catechin.
Intake of 10% and 20% adzuki bean with high-fat diet (HCD) for 10
weeks was reported to ameliorate serum and hepatic triglyceride levels
in C57BL/6 male rice (Kim, Hong, Jeon, & Kim, 2016). Anthocyanin-
rich Aronia melanocarpa (AM) extract was reported to inhibit hepatic
lipid accumulation through down-regulating PPARγ2 expression both in
vitro and in vivo. C57BL/6N mice given high fat diet (HFD) and
FL83 cells under treatment of free fatty acid (FFA) were used in the
study (Park et al., 2017). Strawberry fraction enriched with anthocya-
nins was shown to have larger impact on decreasing LDL-cholesterol
and triglycerides contents than untreated extract in human hepatocel-
lular carcinoma (HepG2) cells (Forbes-Hernández et al., 2017).
There are also adequate researches that report anthocyanins to play
essential roles in regulating lipid metabolism instead of direct effects.
BCAO decreased hepatic triglyceride, up-regulated gene expression in
fatty acid oxidation, and down-regulated mRNA expression of FAS and
SREBP-1 genes associated with fatty acid synthesis (Park et al., 2015).
Rutgers scarlet lettuce extract (RSLE) contains 43.0 mg/g cyanidin 3-
glucoside equivalents. RSLE treated groups at 100 or 300 mg/kg had a
lower ratio of liver weight to body weight as well as decreased total
liver lipids compared to control group after 28 days of treatment
(Cheng et al., 2014). One study supplemented mice with purified cya-
nidin 3-glucoside or Queen Garnet plum juice (QG) that contains up to
275 mg/100 g fresh fruit (200 mg/100 mL cyanidin 3-glucoside, 30
mg/100 mL cyanidin 3-rutinoside). The result showed that cyanidin 3-
glucoside and QG both reversed metabolic signs induced by high-fat
diet (Bhaswant et al., 2015). Drinking LFWE decreased sizes of livers as
well as perirenal and epididymal adipose tissues, and cell sizes of epi-
didymal adipose tissues in test subjects (Wu et al., 2013a). Consump-
tion of CBE at doses of 100 and 200 mg/kg body weight per day re-
duced epididymal fat and triacylglycerol. CBE also regulated gene
expression in pathways involved in adipogenesis. Expression of genes
Fabp4, Fas and Lpl were inhibited (Qin & Anderson, 2012). Cyanidin 3-
glucoside is shown to regulate lipid metabolism in in vivo and in vitro
studies. Cyanidin 3-glucoside rich purple corn color (PCC) consumed in
2 g/kg diet prevented body weight gain and ameliorated diet-induced
adipocytes hypertrophy in mice. PCC blocked lipid accumulation,
suppressed the expression of genes involved in fatty acid and tria-
cylglycerol synthesis and lowered SREBP-1 mRNA level (Tsuda, Horio,
Uchida, Aoki, & Osawa, 2003). Cyanidin 3-glucoside rich honeysuckle
anthocyanins (HA) supplemented at 100 or 200 mg/kg suppressed body
weight gain. HA reduced serum and liver lipid profiles (Wu et al.,
2013b). Anthocyanins purified from various species are also reported to
regulate lipid metabolism. Cornelian cherry anthocyanins (CA) consist
of cyanidin 3-galactoside, pelargonidin 3-galactoside and delphinidin 3-

15
L. Xie et al.
galactoside. CA supplemented at 1 g/kg of diet induced 24% decrease of
body weight gain and decreased liver lipid and triacylglycerol accu-
mulation in mice (Jayaprakasam, Olson, Schutzki, Tai, & Nair, 2006).
Sweet cherry anthocyanins (CACN) consist of cyanidin 3-(2G-gluco-
sylrutinoside), cyanidin 3-rutinoside and pelargonidin 3-rutinoside. 40
and 200 mg/kg CACN were added to diet and induced 5.2% and 11.2%
decrease in body weight gain in mice. CACN attenuated the size of
epididymal adipocytes and reduced serum lipids. In 3T3-L1 cells,
200 μg/mL CACN reduced lipid accumulation by 30.7% (Wu, Tang, Yu,
Gao, Hu, Chen, et al., 2014). Blueberry anthocyanins (BA) are com-
posed of 9 different structures, such as 24.4% petunidin 3-arabinoside,
16.4% delphinidin 3-glucoside and 12.5% cyanidin 3-galactoside.
Consumption of BA at 200 mg/kg reduced 19.4% body weight gain in
mice. BA could effectively attenuate epididymal adipocytes, improve
lipid profiles, and down-regulate the expression levels of FAS genes
(Wu, Jiang, Yin, Long, & Zheng, 2016a). BCAO increased gene ex-
pressions of carnitine palmitoyltransferase I (CPT-1) and PPAR-α in-
volved in fatty acid oxidation and decreased mRNA expressions of FAS
and SREBP-1c associated with fatty acid synthesis (Park et al., 2015).
Mulberry anthocyanins (MACN) consist of cyanidin 3-glucoside, cya-
nidin 3-rutinoside and pelargonidin 3-glucoside. Male C57BL/6 mice
were divided into four groups and given free access to control diet, HFD
with 45% calories from fat, HFD added with 40 mg/kg diet MACN and
HFD added with 200 mg/kg diet MACN. MACN at 40 mg/kg and
200 mg/kg inhibited 11.8% and 21.4% body weight gain as well as
attenuated lipid accumulation and lowered the size of adipocytes (Wu
et al., 2013b).
2.4. Suppress food intake
The control center of appetite lies in part of the brain called hy-
pothalamus. Neurons in hypothalamus detect and organize signals of
body energy status to control food intake and energy expenditure.
Proopiomelanocortin (POMC) and agouti-related peptide (AgRP) cells
are the key cell types in this process, which generate peptides compe-
titively binding to the melanocortin receptors MC3R and MC4R. Leptin
16
Trends in Food Science & Technology 72 (2018) 13–24
Fig. 1. Molecular mechanisms of the anti-obesity effects of antho-
cyanins. a. Various anthocyanins-rich extracts are potent pancreatic
lipase inhibitors. b. Anthocyanins up-regulate AMPK expression.
AMPK activation increase expression of PGC1α, up-regulated ex-
pression of UCPs and increase excretion of adipocytokines. c.
Anthocyanins inhibit fatty acid synthesis through down-regulating
expression of ACC, FAS and SREBPs, as well as up-regulating fatty
acid oxidation through increasing expression of PPARs and CPT-1.
d. Anthocyanins reduce the expression of NPY, GABAB1R and de-
crease expression of PKA-α and p-CREB. e. Anthocyanins inhibit
pathogen growth, such as Enterococcus spp. and Clostridium per-
fringens, as well as exerting prebiotic effects, such as enhancing
growth of Lactobacillus spp. and Bifidobacterium spp. f.
Anthocyanins ameliorate oxidative stress by reducing ROS pro-
duction, inhibiting expression of NADPH oxidase and GPx, as well
as reducing expression of CYP2E1. Anthocyanins could also resolve
inflammation through decreasing levels of hs-CRP, MCP-1, TNF-α
and IL-6.
is produced by adipose tissue and modulates the activity of these cells.
POMC neurons secrete anorexic neuropeptides like cocaine-and-am-
phetamine-regulated transcript (CART) and POMC that reduces appe-
tite while AgRP neurons are inhibited in response to leptin. However,
leptin resistance and disorder in other adipocytokines, such as adipo-
nectin and ghrelin, are quite common for obesity state (Saltiel, 2016).
Thus, targeting leptin resistance may be a promising way to develop
anti-obesity therapies.
Black bean seed coat anthocyanins contain cyanidin 3-glucoside,
petunidin 3-glucoside and delphinidin 3-glucoside. 24 mg/kg daily in-
take of black bean seed coat anthocyanins reduced the expression of
neuropeptide Y (NPY) and induced an increase of γ-amino butyric acid
receptor (GABAB1R) in hypothalamus. Moreover, decreased expression
of GABAB1R downstream signaling molecules, such as protein kinase A-
α (PKA-α) and phosphorylated cAMP-response element binding protein
(p-CREB) in hypothalamus followed such effect (Badshah et al., 2013).
Purified cyanidin 3-glucoside enhanced adipocytokines (leptin and
adiponectin) secretion in isolated rat adipocytes (Tsuda et al., 2004).
CACN, MACN, HA reduced leptin secretion in mice. HA and CBE also
increased serum adiponectin levels (Qin & Anderson, 2012; Wu et al.,
2013b; Wu et al., 2014; Wu, Yin, Zhang, Long, & Zheng, 2016b; Wu
et al., 2013c).
2.5. Regulate gut microbiota
The role of gut microbiota in human health has been recognized as a
hot spot of research. Various studies have been reported on the asso-
ciation of obesity with altered composition of gut microbiota, as well as
the interaction among diet, obesity and gut microbiota. Diet-gut mi-
crobiota interactions could moderate human metabolism. Germ-free
mice fed a westernized diet (such as high-fat diet) were protected from
diet-induced obesity. Lean mice that received a microbiota transplant
from genetically obese mice had increased adiposity. Lard intake al-
tered gut microbiota composition in mice, which promoted obesity and
white adipose tissue inflammation through Toll-like receptors signaling
and chemokine CCL2 in mice. But consumption of fish oil that is rich in
L. Xie et al. Trends in Food Science & Technology 72 (2018) 13–24

Table 2
Anthocyanins extracts and purified anthocyanins with effects on obesity associated oxidative stress and inflammation.

Source Anthocyanins Composition Experimental methods (treated Major activity Mechanism of action
dose, subjects, duration of
treatment)

Vaccinium Whole blueberry with defined
corymbosum anthocyanins content
Aronia melanocarpa Extract: at least 10% anthocyanins
Morus australis P. Cyanidin 3-glucoside,
cyanidin 3-rutinoside and
pelargonidin 3-glucoside
Prunus auiun Cyanidin 3-(2G-
L. glucosylrutinoside),
cyanidin 3-rutinoside and
pelargonidin 3-rutinoside
Ipomoea batatas Color: 90% cyanidin and peonidin
acyl glucosides
Prunus cerasus Extract: 12.5–25.0 mg/100 g; pure
cyanidin 3-glucoside
Fragaria vesca Total anthocyanins:
81.65 mg/10 g,
such as pelargonidin 3-glucoside;
pelargonidin 3-malonylglucoside
and pelargonidin 3-rutinoside
Glycine max (seed Extract: 9.2%
coat)
Rubus fruticosus 87% cyanidin 3-glucoside
129/258/310/517/724 mg total
anthocyanins, healthy men,
measured 1, 2, 4, 6 h after intake
100/200 mg/kg body weight,
daily, Wistar rats, 6 weeks
200 mg/kg food,
C57BL/6 mice with HFD, 8 weeks
40/200 mg/kg and 200 mg/kg,
male C57BL/6 mice,
12 weeks and 8 weeks
700 mg/kg/day,
male ICR mice, 20 weeks
150 g/mL; 25μg,
adipose stem cells, 4 h
10 g, overweight adults, 6 h
0.2%/1%/2%,
male C57BL/6 mice, 14 weeks
5%/10%, OVX rats, 100 days
Improve vascular function in an intake- and time- Ameliorate oxidative stress
dependent manner
Inhibit plasma TNF-α and IL6 Ameliorate oxidative stress,
resolve inflammation
Increase the activity of SOD and GPx, and down- Ameliorate oxidative stress,
regulate gene expression of the TNF, IL-6, iNOS, resolve inflammation
and NF-кB
Increase the activity of SOD and GPx, reduce gene Ameliorate oxidative stress,
expression levels of IL-6 and TNFα and also resolve inflammation
reduce gene expression of iNOS
and NF-кB
Suppress ROS production and restore glutathione Ameliorate oxidative stress,
(GSH) content and antioxidant enzymes activities resolve inflammation
Reduce LPS-induced IL-6 secretion Resolve inflammation
attenuate postprandial inflammatory response Resolve inflammation
measured by hsCRP and IL-6
Down-regulate Resolve inflammation
inflammatory cytokines
decrease hepatic NFκB and COX-2 expression Resolve inflammation
levels

polyunsaturated fatty acids could protect mice against such effect
(Caesar, Tremaroli, Kovatcheva-Datchary, Cani, & Backhed, 2015).
Obese people are also reported to have altered gut microbiota. Thus,
targeting gut microbiota through dietary intervention is a promising
way to resolve the obesity state.
Investigation of the interaction among obesity traits, global gene
expression and gut microbiota association using genome-wide associa-
tion study in mice fed with high-fat/high-sucrose diet showed strong
association between genotype and gut microbiota plasticity and iden-
tified obesity related gut microbiota phylotypes (Parks et al., 2013).
Gastrointestinal microbiome modulator (GIMM) consumption increased
satiety and induced an increase in plasma satiety hormones and fecal
short chain fatty acid concentrations compared with placebo group in a
clinical trial (Rebello, Burton, Heiman, & Greenway, 2015). Also,
whether improvement of gut microbiota through consuming antho-
cyanin-rich food could be a novel mechanism to treat obesity has at-
tracted lots of attention.
It is widely recognized that anthocyanins could change growth of
colonic bacteria and act like prebiotics. Such effect may have a certain
impact on the development of obesity (Jamar, Estadella, & Pisani,
2017). Raphanus sativus Sango sprout juice (SSJ) was rich in cyanidin
based-anthocyanins (270 mg/100 g fresh weight) and isothiocyanates.
In a study that showed supplementation of SSJ in obese rats could have
better benefits on prostate, liver and ileum compared with just
switching feed from high-fat diet (HFD) to the regular one (RD),
changes in characteristics of caecal chime microbiota were also as-
sessed. As a result, the researchers found the caecal Enterococcus spp.
concentrations dramatically decreased in obese animals, and SSJ sup-
plementation along with feed switch show better effect in resolving
such reduction and reversing the tendency of an increase in Clostridium
perfringens concentrations. These results indicated that SSJ had a ben-
eficial effect against possible gut pathogens (Vivarelli et al., 2017). An
in vitro study that introduced a fermentation system to investigate on
the metabolism of anthocyanins with fecal bacteria showed that an-
thocyanins along with their metabolites enhanced the growth of Lac-
tobacillus-Enterococcus spp. and Bifidobacterium spp. (Hidalgo et al.,
2012).
An interesting perspective is that the intestinal metabolites of an-
thocyanins are reported to have potential bioactive effects. A study
recruited ten healthy subjects (women) to investigate the constituents
and in vitro antiproliferative effect of their urine samples after acute
intake of anthocyanins and ellagitannins-rich grumixama fruit (Eugenia
brasiliensis Lam.). Their results showed that the majority of circulating
and excreted metabolites in urine were phenolic acids and conjugates of
urolithin, and the urine samples could inhibit proliferation of MDA-MB-
231 human breast cancer cells as a possible result of synergistic effects
of anthocyanins and ellagitannins metabolites (Teixeira et al., 2017).
One clinical study investigated the association of changes in urinary
polyphenol metabolites and changes of fecal microbiota with wine in-
terventions in nine healthy subjects. The trial was designed to be ran-
domized, controlled interventional and crossover, in which the parti-
cipants were given dealcoholized red wine, red wine or gin for 20 days
separately after an initial washout period. As a result, categorization of
participants into tertiles on the basis of fecal bacteria changes showed
that those in tertiles with highest fecal concentration of Bifidobacteria
also had higher changes in urinary concentration of typical anthocyanin
metabolites, such as homovanillic acid, 4-hydroxybenzoic acid and p-
coumaric acid compared with subjects in the lowest tertile. Also, there
was a positive correlation between changes in Bifidobacteria and
changes of these anthocyanins metabolites. Moreover, the researchers
found that changes in 4-hydroxybenzoic acid and syringic acid could
predict about 68.5% of the changes in Bifidobacteria. Thus, this study
demonstrated a correlation between increase in wine anthocyanins-
derived microbial metabolites with Bifidobacteria increase (Boto-
Ordóñez et al., 2014).
These studies shed lights on the prebiotic effects of anthocyanins
and bioactivities of gut microbiome-derived anthocyanins metabolites.
Due to the lack of systemic characterization of gut microbiota and de-
tailed metabolomics studies, the mechanisms of such effects remain
unclear. Still, the interaction between anthocyanins and gut microbiota
could be a novel mechanism to treat obesity.

17
L. Xie et al.
3. Characteristics and therapeutic use
Anthocyanins have also been reported to have active effects on
oxidative stress and inflammation that are associated with obesity be-
sides their anti-obesity activity. In this section, we have first summar-
ized anthocyanins with such activities, as shown in Fig. 1 and Table 2,
then discussed their physicochemical characteristics as well as ab-
sorption and metabolism, ended up with commenting on studies of
anthocyanins’ therapeutic use.
3.1. Anthocyanins with effects on obesity associated oxidative stress and
inflammation
3.1.1. Ameliorate oxidative stress
Fat accumulation is associated with systemic oxidative stress as
confirmed by human and mice studies. In adipose tissue of obese mice,
reactive oxygen species (ROS) production increased, followed by aug-
mented expression of NADPH oxidase and decreased expression of an-
tioxidative enzymes. In addition, elevated levels of fatty acids increased
oxidative stress through NADPH oxidase activation in cultured adipo-
cytes. Oxidative stress caused dysregulated production of adipocyto-
kines such as adiponection. Targeting oxidative stress such as inhibiting
NADPH oxidase could regulate adipocytokines production and ame-
liorate hyperlipidemia (as shown in Table 2) (Furukawa et al., 2004).
Most anthocyanins and their aglycones are strong antioxidants with
activities comparable to widely used antioxidants such as Trolox, ca-
techin and quercetin in systems like emulsion and LDL (Kähkönen &
Heinonen, 2003). Blueberry intake with interventions containing 129,
258, 310, 517 and 724 mg total anthocyanins improved vascular
function in healthy men in an intake-dependent and time-dependent
manner. The effects of phenolic metabolites on neutrophil NADPH
oxidase activity were suggested to be the mechanism (Rodriguez-
Mateos et al., 2013). CBE consumption increased plasma adiponectin
levels, decreased plasma tumor necrosis factor-α (TNF-α) and inter-
leukin-6 (IL-6) levels as well as and inhibited Fabp4, Fas and Lpl mRNA
levels (Qin & Anderson, 2012). Purple Sweet Potato Color (PSPC)
suppressed ROS production and restored glutathione (GSH) content and
activities of antioxidant enzymes. PSPC also prevented the oxidative
stress mediated endoplasmic reticulum (ER) stress in mice liver (Zhang
et al., 2013). Consumption of CACN and MACN at 200 mg/kg diet re-
duced MDA production and increased SOD and glutathione peroxidase
(GPx) activities (Wu et al., 2016b).
Obesity along with chronic over-nutrition is known to be the key
risk factor for nonalcoholic fatty liver disease (NAFLD) development,
which is characteristic of fat accumulation (steatosis). NAFLD is also
known as hepatic dysfunction in metabolic syndrome. Based on the
hypothesis called “two-hit” in pathophysiology, the primary insult
(steatosis) could make the liver vulnerable to the secondary insults,
such as oxidative stress, thus resulting in development of nonalcoholic
steatohepatitis (NASH). Among various sources of oxidative stress, cy-
tochrome P450 2E1 (CYP2E1) was reported to be closely related with
NASH development (Abdelmegeed et al., 2012). The role CYP2E1 plays
in metabolism of fatty acid, which leads to an increase in toxic lipid
peroxides levels along with its possibly increase of expression in NASH
makes it a likely candidate as a genetical risk factor for both NAFLD and
NASH (Dey, 2013). Interestingly, anthocyanins are reported to inhibit
CYP2E1 activity. Thus, the possible effects of anthocyanins on attenu-
ating obesity, NAFLD/NASH development and associated oxidative
stress are worth attention.
Blueberry anthocyanins extract (BAE) with a total anthocyanins
content of 257.6 mg/g (over 90% being cyanidin 3-glucoside) was re-
ported to ameliorate ROS overproduction and GSH depletion induced
by the acrylamide (AA) as well as inhibiting protein expression of
CYP2E1 in a mice model (Zhao et al., 2015). Gynura bicolor leaf aqueous
extract (GAE) with a total anthocyanins content of 2135 mg per 100 g
dry weight was added to feed of mice at 0.25% or 0.5% in a study to
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Trends in Food Science & Technology 72 (2018) 13–24
evaluate its effect upon ethanol-induced hepatic injury. The results
indicated that both doses were able to reverse the increase in CYP2E1
induced by chronic ethanol intake in test mice (Chao, Liu, Wu, & Yin,
2015; Yin, Wang, Liu, & Mong, 2017). Administration of anthocyanin
rich extract of Hibiscus sabdariffa calyces (HSARE) at 100 mg/kg/d for 4
weeks was found to decrease the immunopositivity of CYP2E1 in liver
tissue of test rats (Ezzat et al., 2016). Cyanidin 3-glucoside pretreat-
ment was also reported to inhibit expression of CYP2E1 in MDA-MB-
231 cells with AA intervention (Song et al., 2013).
3.1.2. Resolve inflammation
Obesity is characteristic with chronic systemic low-grade in-
flammation. Adipocytes secrete factors such as monocyte chemoat-
tractant protein-1 (MCP-1) that induce monocytes differentiation into
macrophages. Adipocytes and macrophages interact and increase cir-
culating proinflammatory cytokines, such as TNF-α, IL-6 that promote
inflammation response. Circulating TNF-α and IL-6 promote liver pro-
duction of an acute-phase reactant C-reactive protein (CRP) (Bastard
et al., 2006; Lee & Pratley, 2005). Resolving inflammation, such as
ameliorating proinflammatory cytokines could improve obesity state.
Strawberry beverage containing 81.65 mg/10 g anthocyanins in
total, (including 67.99 mg/10 g pelargonidin 3-glucoside; 9.48 mg/10 g
pelargonidin 3-malonylglucoside and 2.35 mg/10 g pelargonidin 3-ru-
tinoside) were tested for the association between strawberry antho-
cyanins and postprandial inflammation in a clinical trial. The straw-
berry beverage significantly attenuated the postprandial inflammatory
response as measured by high-sensitivity C-reactive protein (hs-CRP)
and IL-6 induced by the HCFM (Edirisinghe et al., 2011). CBE lowered
gene expression levels of inflammatory cytokines, such as Il1β, Il6 and
Tnfα (Qin & Anderson, 2012). PSPC suppressed the c-Jun-N-terminal
kinase 1 (c-JNK1) and I kappa B kinase β (IKKβ) activation and nuclear
factor-kappa B (NF-κB) p65 nuclear translocation (Zhang et al., 2013).
Intake of 10% blackberries containing 87% cyanidin 3-glucoside could
induce the protective effect against weight gain and inflammation in
rats associated with ovariectomy-induced menopause. Addition of tart
cherry extract with 150 g/mL anthocyanins content and purified cya-
nidin 3-glucoside resulted in LPS-induced secretion of IL-6 in adipose
stem cell in a dose-dependent manner (Kaume, Gilbert, Brownmiller,
Howard, & Devareddy, 2012). BE suppressed gene expression levels of
major inflammatory cytokines, TNF-α and MCP-1 in white adipose
tissue (Kanamoto et al., 2011). BA down-regulated the expression levels
of TNF-α, IL-6 and PPARγ (Wu et al., 2016a). Also, CACN and MACN
down-regulated the gene expression of TNF-α, IL-6, iNOS, and NF-кB
genes (Wu et al., 2016b).
3.2. Characteristics of anthocyanins
3.2.1. Physicochemical properties of anthocyanins
Acetone, methanol, ethanol and water were commonly used to ex-
tract anthocyanins due to similarity in their polarity, though acetone
and methanol were forbidden to use when preparing extracts for food
processing. Extraction of anthocyanins generally requires acids, for the
use of stabilizing anthocyanins in the flavylium cation form that ap-
peared red at low pH. Also, as solutions containing hydrochloric acid
may hydrolyze acylated anthocyanins, using organic acids, such as ci-
tric acids may be a better choice (Kırca, Özkan, & Cemeroğlu, 2007).
The number and position of hydroxyl and methoxy substituents as
electron donating groups along with glycosylation parameters like site
and number of glycosylation and sugar type have great impact on the
activities of anthocyanins (Kähkönen & Heinonen, 2003). Chemical
modification in sugar groups can change the bioavailability of antho-
cyanins. Acylation of anthocyanins generally results in reduced ab-
sorption. Furthermore, anthocyanins with diglycosides such as sambu-
bioside or rutinoside show increased stability (Prior & Wu, 2006).
L. Xie et al.
3.2.2. Anthocyanins absorption and metabolism
Poor absorption of anthocyanins has been a real trouble in clinical
trials. For the berry species, various berries other than cranberry an-
thocyanins glycosides have been found to be absorbed and excreted into
urine unmetabolized by both animals and human beings. Cranberry
showed slightly better absorption, as 5% of the dose consumed within
24 h were excreted into urine with a maximum excretion period be-
tween 3 and 6 h after consumption (Zhu, Miao, Meng, & Zhong, 2015).
In a clinical trial that used a cross-over design, 24 overweight adults
were given high-carbohydrate, moderate-fat meal (HCFM) accom-
panied by either a strawberry or a placebo beverage. Their postprandial
changes in plasma anthocyanins along with physiological status were
assessed for 6 h, and their postprandial concentrations of pelargonidin
sulfate and pelargonidin 3-glucoside were increased when the straw-
berry beverage was consumed (Edirisinghe et al., 2011). A clinical
study aimed at investigating bioavailability of bilberry anthocyanins
intake in healthy volunteers with or without colon demonstrated that
colon was an important site for absorption of anthocyanins as well as
their degradation products. In this study, about 30% of consumed an-
thocyanins remained stable, when passing through upper part of in-
testine and only 20% of degradants were produced. Moreover, most of
intact anthocyanin went through absorption in the small intestine,
while larger amounts of degradants reached the circulation in both
groups compared with anthocyanins (Mueller et al., 2017). Plasma
concentrations of anthocyanins were several tens of nanomoles per liter
after an intake of about 110–200 mg, and urinary excretion percentage
of anthocyanins was 0.005–0.1% of intake (Manach et al., 2004).
Glucuronide and methyl-glucuronide derivatives were the major forms
of primary metabolites when cyanidin 3-glucoside was absorbed and
then transported in serum and urine after oral intake of a moderate-to-
high dose. In one clinical study with three subjects, the serum con-
centration of total anthocyanins and their metabolized forms reached
maximum at 2.8 h, with a maximum urinary excretion rate at 3.72 h
and a half-life of elimination at 4.12 h (Kay, Mazza, & Holub, 2005).
13
C-labelled study showed that seventeen 13C-labelled compounds in-
cluding 13C5-C3G and its degradation products, phloroglucinaldehyde
(PGA) and protocatechuic acid (PCA) could be identified in the serum
after oral intake of 500 mg 6,8,10,3′,5′-13C5-C3G in eight subjects. The
phenolic metabolites had ranged maximal concentrations from 10 to
2000 nm, post-consumption between 2 and 30 h and ranged elimina-
tion half-lives from 0.5 to 96 h. Hippuric acid and ferulic acid were
identified as the major phenolic metabolites, with peaks at about 16
and 8 h (de Ferrars et al., 2014).
3.3. Anthocyanins in anti-obesity therapy
Anthocyanins are reported to show anti-obesity effects through in-
hibiting lipid absorption and suppressing food intake. Both mechanisms
are appealing for therapeutic use. Currently there are seven anti-obesity
drugs approved by U.S. Food and Drug Administration (FDA). Most of
these drugs target central nervous system (CNS) to affect energy intake,
including phentermine (prescribed alone or combined with topir-
amate), benzphetamine and phendimetrazine, diethylpropion, lorca-
serin, liraglutide and naltrexone combined with bupropion. However,
use of these drugs is accompanied with side effects, such as hyperten-
sion, cardiac valve disorders, nausea, diarrhea and vomiting. Orlistat is
an exception that works on digestive system and has side effects like
oily stools and fecal inconsistency (Adan, 2013; Gadde, 2014; Wong,
Sullivan, & Heap, 2012).
According to animal studies, anthocyanins have weight reducing
effects in comparable to current drugs. Supplementation of honeysuckle
anthocyanins for 100 mg/kg diet reduced body weight gain by 17.1%
compared with control group, which was similar to 16.9% reduction in
orlisat group (Wu et al., 2013c). Some anthocyanins prevented weight
gain to a greater extent. Supplementation at 200 mg/kg of diet resulted
in a higher reduction of 24.1%. Intake of mulberry and sweet cherry
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Trends in Food Science & Technology 72 (2018) 13–24
anthocyanins at 200 mg/kg diet prevented body weight gain by 32.7%
and 29.6%, with the effect of orlistat being 17.1% (Wu et al., 2016b).
Clinical trials that use anthocyanins extracts and purified anthocyanins
as dietary supplement have also been carried out. Hibiscus sabdariffa
extracts (HSE) containing 1.43% flavonoids, 2.5% anthocyanins and
1.7% phenolic acid were prepared from H. sabdariffa L. 450 mg HSE
and 50 mg starch were combined into one capsule and given to 19
subjects (HSE-treated group) for 3 months. 17 subjects were given
placebo capsule with 500 mg starch in one dose and set as control. At
the end of the study, HSE-treated group had reduced body weight, BMI,
body fat, waist-to-hip ratio, as well as decreased free fatty acid (Chang,
Peng, Yeh, Kao, & Wang, 2014). Drinking purple tea daily for 1 month
was reported to improve obesity parameters including body weight,
BMI and body fat mass compared to baseline (Shimoda, Hitoe,
Nakamura, & Matsuda, 2015).
A particular advantage of anthocyanins is their well-recognized
safety. Acute and chronic studies have been conducted in vivo to eval-
uate the oral toxicity of black soybean hull extract (BE) containing 9.2%
cyanidin 3-glucoside. LD50 of BE was reported to be more than 2.5 g/kg
body weight in test Sprague-Dawley rat and C57BL/6 mice, and no
significant decrease in body weight or death was recorded in the acute
oral toxicity study. In the chronic oral toxicity study, no toxicologically
significant clinical changes or mortality was observed in male and fe-
male C57BL/6 mice, and the estimation of no-observed adverse-effect-
level of BE was 5.0% (Fukuda et al., 2011). Reviewed anthocyanin
human oral consumption trials with doses ranging from 788 mg to
883 mg reported no adverse effects (Kay et al., 2005). In 2013, the
European Food Safety Authority concluded the anthocyanins to be
unlikely of safety concern as a result of evaluating toxicologic data
mainly derived from studies of blackcurrant extracts and grape-skin
(European Food Safety Authority, 2013). China is the first country to
define a daily recommended intake of 50 mg/day for anthocyanins in
adults, but a Tolerable Upper Intake Level remains undefined (Chinese
Nutrition Society, 2013). Based on a report published in 2015, there has
been no indication of anthocyanin toxicity in published studies of
human intervention. Noticeably, an acceptable daily intake specifically
for grape-skin extracts-derived anthocyanins at 2.5 mg/kg per day has
been established by the Joint FAO/WHO Expert Committee on Food
Additives (Wallace & Giusti, 2015).
4. Biosynthesis of anthocyanins in microorganisms
Anthocyanins are currently obtained from extraction and purifica-
tion from natural sources. Such methods are characteristic of high ex-
pense, low production rate and rather complex processes. Moreover,
there is still limited progress in chemical synthesis of anthocyanins for
the lack of understanding of mechanisms on related key reactions. As a
result, purified anthocyanins are quite expensive. For future research
such as therapeutic use for obesity and association between structure
and activity of anthocyanins, it's quite necessary to develop a cheap and
efficient way to produce anthocyanins (Prior & Wu, 2006; Smeriglio,
Barreca, Bellocco, & Trombetta, 2016; Yun, 2010). Anthocyanins bio-
synthesis in microorganisms is thus a rather promising way to achieve
such purpose. In this section, biosynthesis in microorganisms refers to
production of anthocyanins from genetically engineered microorgan-
isms that express plant anthocyanins biosynthetic pathways.
4.1. Biological synthesis of anthocyanins in plants
Biological synthesis of anthocyanins in plants has been researched
in detail, especially in the model plant arabidopsis thaliana and berries.
Anthocyanins accumulated in plants during ripening, with its compo-
sition reaching a peak at fruit maturity. Procyanidins and quercetin
were the major flavonoids at early stages of berry development, but
their levels decreased during the ripening progress. In the meantime,
concentration of anthocyanins increased dramatically during later
L. Xie et al. Trends in Food Science & Technology 72 (2018) 13–24

Fig. 2. Major steps of anthocyanins biosynthetic pathway. PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl CoA ligase; CHS, chalcone synthase;
CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase; ANS,
anthocyanidin synthase.

20
L. Xie et al.
stages of ripening. As a result, anthocyanins became the most abundant
flavonoids in ripe berries. Anthocyanins accumulation is correlated
with the expression of flavonoids pathway genes and controlled by
anthocyanins gene expression that occurred in two stages during berry
development. One was early in the development, when most genes
involved in this pathway were expressed and berry color started to
develop, the other is after veraison. The process coincided with in-
creased expression of the 11 genes involved in anthocyanins biosyn-
thetic pathway. In white color mutants, the expression of flavonoid
pathway genes was reduced (Jaakola et al., 2002; Zifkin et al., 2012).
4.1.1. Anthocyanins biosynthetic pathways
The anthocyanins biosynthetic pathways start with phenylalanine
and consists of three steps: beginning steps from phenylalanine to 4-
coumaroyl CoA, early steps from 4-coumaroyl CoA and Malonyl CoA to
dihydroflavonol and late steps from dihydroflavonol to anthocyanins,
as shown in Fig. 2 (Shi & Xie, 2014). Beginning steps consist of three
consecutive processes: from phenylalanine to cinnamic acid, then
through coumaric acid to 4-coumaroyl CoA, respectively catalyzed by
phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H)
and 4-coumaroyl CoA ligase (4CL) (Winkel-Shirley, 2001). During these
processes, hydroxycinnamic acid derivatives like monolignols and si-
napate are also produced. Early steps are the following steps from 4-
coumaroyl CoA via chalcone and naringenin to dihydrokaempferol,
which are catalyzed by chalcone synthase (CHS), chalcone isomerase
(CHI) and flavanone 3-hydroxylase (F3H) respectively. Dihy-
drokaempferol is subsequently hydroxylated to synthesize dihy-
droquercetin, which is catalyzed by the flavonoid 3′- hydroxylase
(F3′H). Dihydroflavonols could be dehydroxylated to flavonols under
catalysis of flavonol synthase (FLS). The chemical structures and pig-
mentation of anthocyanins are determined by enzymes monooxygenase
flavonoid 3′-hydroxylase and flavonoid 3′5′-hydroxylase (F3′5′H). The
last steps are composed of processes from dihydroflavonols through
leucoanthocyanidins to anthocyanidins along with further modifica-
tions such as glycation and acylation that converts anthocyanidins to
diverse anthocyanins. These steps are consecutively catalyzed by di-
hydroflavonol reductase (DFR) and anthocyanidin synthase (ANS,
whose other name is leucoanthocyanidin dioxygenase, LDOX) (Saito
et al., 2013; Shi & Xie, 2014).
4.1.2. Regulation of anthocyanins biosynthesis
In anthocyanins biosynthetic pathway, genes encoding each enzyme
are well understood. For the beginning steps, PAL isomers are encoded
by four genes, with PAL1 and PAL2 involved in this pathway (Huang
et al., 2010). C4H is encoded by only one gene, while 4CL is reported to
be encoded by a small family of genes, with 4CL3 preferably involved in
flavonoid pathway and 4CL1 and 4CL2 associated with hydro-
xycinnamic acid derivatives. Genetic and biochemical characterization
of genes encoding early steps enzymes have been reported, and trans-
parent testa due to lack of production of anthocyanins and proantho-
cyanidins were reported in knockout mutation of these genes. The two
enzymes DFR and ANS involved in last steps are encoded by only one
gene respectively, and the knockout mutants of either of these two
genes lead to phenotypes of transparent testa in seeds (Saito et al.,
2013; Shi & Xie, 2014).
Regulation of anthocyanins biosynthetic pathway has been widely
researched. Pathway genes involved in flavonoid biosynthesis were
reported to be co-regulated. Studies on mutants, profiling of gene ex-
pression and interactions of protein-DNA and protein-protein show
anthocyanins biosynthetic pathway genes are generally regulated by a
ternary WD40-bHLH-MYB (WBM) complex which is composed of the
three transcription factors WD40, bHLH and MYB. There are four
comparatively similar MYB transcription factors that control bio-
synthesis of anthocyanins in vegetative tissues, including PAP1/
MYB75, PAP2/MYB90, MYB 113 and MYB 114. These transcription
factors are all R2R3-MYB with two imperfect repeats in the domain of
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Trends in Food Science & Technology 72 (2018) 13–24
MYB. Among them, PAP1/MYB75 is a major regulator of anthocyanins
biosynthesis, as the expression level of PAP1 is the highest compared
with its homologs. Also, activation of PAP1 alone could lead to acti-
vation of anthocyanins production, especially the two enzymes of late
steps in anthocyanins biosynthetic pathway. Moreover, overexpression
of PAP1 has been reported to cause a sudden increase in anthocyanins
levels (Cheynier, Comte, Davies, Lattanzio, & Martens, 2013; He,
Zhang, & Zhang, 2016; Verdier et al., 2011).
The biosynthesis of anthocyanins is also influenced by various
abiotic factors, such as high light, low temperature, sucrose, nutrient
depletion and phytohormones. Studies have shown expression of all
anthocyanins biosynthetic pathway genes under high light condition,
with an increase in the level of anthocyanins. Also, production of an-
thocyanins can be enhanced by treatment with sucrose (Teng,
Keurentjes, Bentsink, Koornneef, & Smeekens, 2005). Low concentra-
tion of nitrogen will also result in increased anthocyanins level. The
elicitor and signal molecule Jasmine (JA) could strongly increase an-
thocyanins biosynthesis. These abiotic factors regulate the expression of
the pathways through induction of PAP1. Cell lines of anthocyanins-
producing cells through tissue culture were used for studying the me-
chanism of how different factors control the activities of MYB. Cells
were cultured in modified MS medium (without NH4NO3 and with half
concentration of KNO3) supplemented with 0.1 mg/L 2, 4-di-
chlorophenoxyacetic acid (2, 4-D) and 0.25 mg/L kinetin. As compar-
ison, cells without anthocyanins-producing capacity were also main-
tained (Park et al., 2013; Primetta, Karppinen, Riihinen, & Jaakola,
2015).
4.2. Anthocyanins biosynthesis in microorganisms
Research on microorganisms successfully modified to biosynthesize
anthocyanins has been making steady progress, especially in E. coli, as
shown in Table 3. Recombination technique was widely used to express
key step enzymes in anthocyanins biosynthetic pathway both in vitro
and in vivo. Recombinant ANSs from snapdragon, petunia, torenia, and
maize was used in vitro to catalyze anthocyanidin formation from a 2-
oxoglutarate-dependent oxidation of leucoanthocyanidin (Nakajima,
Tanaka, Yamazaki, & Saito, 2001; Yan, Chemler, Huang, Martens, &
Koffas, 2005). A recombinant complex of four-step metabolic pathway
was constructed in engineered E. coli to produce anthocyanins from
cheap precursors such as flavanones and flavan-3-ols (Yan, Huang, &
Koffas, 2007). Such complex consisted of heterologous originated plant
genes: F3H and ANS from Malus domestica, D4R from Anthurium an-
draenum and 3-GT from Petunia hybrida. The researchers managed to
acquire two kinds of anthocyanins: pelargonidin 3-glucoside (0.98 mg/
L) and cyanidin 3-glucoside (2.07 mg/L) from their perspective flava-
none precursors, naringenin and eriodictyol in amount of minigrams.
Cyanidin 3-glucoside was produced from its flavan-3-ol, (+)-catechin
precursor at higher yields (16.1 mg/L). On that basis, they conducted
further study and concluded glucosyl donor, UDP-glucose was the key
metabolic limitation. Enhanced intracellular UDP-glucose production
results in elevated production of cyanidin 3-glucoside at 70.7 mg/L and
pelargonidin 3-glucoside at 78.9 mg/L from their precursor flavan-3-ols
without extracellular supplementation of UDP-glucose (Yan, Li, &
Koffas, 2008). Moreover, with optimization of associated pathway, such
as engineering of an alternative carbon assimilation pathway and the
inhibition of competitive reaction pathways the authors achieved the
production of plant-specific flavanones up to 700 mg/L and anthocya-
nins up to 113 mg/L from phenylpropanoic acid and flavan-3-ol pre-
cursors (Leonard et al., 2008). On this basis, the same research group
carried out a study to further improve cyanidin 3-glucoside production
in a systematic way. They reported enhancement of (+)-catechin and
UDP-glucose availability, as well as improvement of the expression of
ANS and 3GT. Final titer of cyanidin 3-glucoside was achieved as
350 mg/L (Lim et al., 2015).
Recently, a new method called microbial co-culture was used by this
L. Xie et al. Trends in Food Science & Technology 72 (2018) 13–24

Table 3
Anthocyanins biosynthesis in microorganisms.

Precursor Recombinant enzymes Microorganism Anthocyanins Composition

and Production rate

Leucoanthocyanidin
Naringenin or eriodictyol
Naringenin or eriodictyol;
(+) catechin
Phenylpropanoic acid and
flavan-3-ol
(+)-catechin
Petunia ANS and 3-GT
Combinational plasmid encoding a four-step metabolic
pathway: FHT from Malus domestica, DFR from Anthurium
andraeanum, ANS from M. domestica
and 3-GT from Petunia hybrida
Combinational plasmid as described above; integration of
LAR in anthocyanins biosynthetic pathwy
when precursor is (+)-catechin;
Plant-specific 4CL, CHS, CHI, strain capable of malonate
assimilation (E2M) and disruption of udg gene encoding
for UDP-glucose dehydrogenase
Combinational plasmid encoding FHT, DFR, ANS, 3-GT
and LAR
in vitro, Cyanidin 3-glucoside, enzyme activities of petunia ANS and
E. coli Extracts maize ANS: 0.972 and 0.215 mircokatal/kg
E. coli Cyanidin 3-glucoside 6.0 μg/L,
pelargonidin 3-glucoside 5.6 μg/L
E. coli Cyanidin 3-glucoside 2.07 mg/L, pelargonidin 3-glucoside
0.98 mg/L from precursors naringenin and eriodictyol; Cyanidin
3-glucoside 16.1 mg/L from (+)-catechin precursor; Cyanidin 3-
glucoside 70.7 mg/L, pelargonidin 3-glucoside 78.9 mg/L after
optimization
E. coli plant-specific flavanones up to 700 mg/L and anthocyanins up to
113 mg/L
E. coli Cyanidin 3-glucoside up to 350 mg/L

group to biosynthesize anthocyanins in E. coli. They first reported to
apply E. coli co-culture to improve the titer of flavan-3-ols by 970 folds
compared with previous publications of monoculture production. The
idea was to divide flavonoid pathway into different co-culture modules
and carefully optimize each module and parameters of the experiment.
Flavan-3-ol titer of 40.7 mg/L was achieved (Jones et al., 2016). Then,
they introduce this method to biosynthesize anthocyanins and reported
complete anthocyanins biosynthesis in E. coli. Anthocyanins biosyn-
thetic pathways were divided into four different modules. Together,
this E. coli co-culture resulted in a titer of 9.5 mg/L pelargonidin 3-
glucoside with glucose, glycerol and xylose as substrates (Jones et al.,
2017). These studies take advantage of compartmentation of biosyn-
thetic pathways and thus resolving metabolic burden as well as al-
lowing for module-specific strain optimization independently. With a
clear understanding of the pathway and mechanisms of regulation, the
biosynthesis of anthocyanins in microorganisms is indeed a promising
field of research (as shown in Table 3).
5. Conclusion
Obesity is one of the major health concerns over the world, and its
increased rate announcing the world for present and future challenges.
There are still quite limited choices to prevent or resolve the situation of
obesity. The available drugs for weight management are of low efficacy
and troublesome side effects such as cardiovascular risks. Anthocyanins
are a group of flavonoids that show anti-obesity effects through various
mechanisms: inhibit lipid absorption, regulate lipid metabolism, in-
crease energy expenditure and suppress food intake. With the emer-
gence of translational studies, regulation of gut microbiota could also
be a potential mechanism. Current anti-obesity drugs mostly aim at
central nervous system with orlistat exceptionally works on gastro-
intestinal tract. Natural products, especially flavonoids have always
been a rich and especially important resource for drug discovery and
food supplement development. Among them, anthocyanins stand out to
show anti-obesity effects that have been widely proved by animal stu-
dies and clinical trials. Of the reported anthocyanins, cyanidin-3-glu-
coside is the most active molecule of this family with strong free radical
scavenging ability. Its major mechanism of anti-obesity effects is to
decrease lipogenesis. Moreover, anthocyanins are reported to prevent
body weight gain of mice to a greater extent than orlistat in several
studies. As anthocyanins are reported to target obesity through sup-
pressing food intake and inhibiting lipid absorption, therapeutic use of
these anthocyanins extracts and purified anthocyanins are quite pro-
mising. Anthocyanins are widely involved in our diet worldwide with
their consumption being the highest among flavonoids, people will find
them more acceptable and less painful to take compared with drugs.
Included in daily intake as part of the diet, it's more likely for the pa-
tients to persist with the treatment.
Studies have reported anthocyanins extracts and purified antho-
cyanins from different sources to have anti-obesity effects, but whether
anthocyanins play the decisive role in the extracts and their structure-
activity relationship remains to be poorly understood. These are points
with potential of future research. Furthermore, the high expenses to
obtain purified form and no available way for chemical synthesis have
made anthocyanins quite expensive despite their wide existence in
nature. Thus, biosynthesis in microorganisms is an effective way to deal
with this situation. Consecutive research on construction of anthocya-
nins biosynthetic pathway in Escherichia coli succeeded in producing
basic anthocyanins such as cyanidin 3-glucoside and pelargonidin 3-
glucoside from their flavonol-3-ol precursors with high yields.
Application of E. coli co-culture enabled complete biosynthesis of pe-
largonidin 3-glucoside from glucose, glycerol and xylose. Still, more
studies are needed to biosynthesize anthocyanins with more complex
structures, especially those reported to show anti-obesity effects.
Systematic pathway design and optimization of combinational plasmid
technique are plausible ways to consider. Eukaryotic systems such as
Saccharomyces cerevisiae could be potential of biosynthesizing some
anthocyanins varieties. In general, anthocyanins have a large potential
to be developed as part of combined therapy for obesity, whose massive
production can be achieved through biosynthesis in microorganisms.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by Grants from Zhejiang Provincial
Natural Science Foundation of China (LR18C200002), National Key
Technology R&D Program of China (2016YFD0401201), and
Fundamental Research Funds for the Central Universities
(2017QNA6006).
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