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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


(Swinburn, Dietz, & Kleinert, 2015). Obesity is also a prerequisite for
Obesity has become a serious problem that is causing a heavy metabolic syndrome, which is defined as a clustering of risk factors
burden to the economy and negative effects on people's longevity and including central obesity, insulin resistance, dyslipidemia and hy-
life quality worldwide. Obesity is defined as a body mass index (BMI) pertension (Carr & Brunzell, 2004; Nguyen, Magno, Lane, Hinojosa, &
equal to or more than 30 by World Health Organization (WHO), while Lane, 2008). It is a physiological status as a result of an imbalance
overweight is defined as a BMI equal to or more than 25. BMI is cal- between energy consumption and energy expenditure. Thus, regulating
culated through the weight in kilograms divided by the square of the the energy imbalance either through decreasing energy intake or in-
height in meters. According to a report from WHO, in 2014, about 39% creasing energy use is a promising strategy for the prevention and
of adults aged 18 years and over were overweight (38% of men and treatment of obesity.
40% of women), more than 1.9 billion, and 13% of the world's adult Anthocyanins are anthocyanidins with sugar groups. Anthocyanidin
population (11% of men and 15% of women) were obese. This number is a subgroup of flavonoids that shows high bioactivities such as anti-
has more than doubled between 1980 and 2014. In 2013, 42 million oxidant effects. Basic structure of flavonoids is called C6-C3-C6, which
children under the age of 5 were overweight or obese. Obesity is driving is a skeleton of diphenylpropane formed by two benzene rings (ring A
global increases in type 2 diabetes, cardiovascular diseases, and several 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. Trends in Food Science & Technology 72 (2018) 13–24

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

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

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

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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.

galactoside. CA supplemented at 1 g/kg of diet induced 24% decrease of is produced by adipose tissue and modulates the activity of these cells.
body weight gain and decreased liver lipid and triacylglycerol accu- POMC neurons secrete anorexic neuropeptides like cocaine-and-am-
mulation in mice (Jayaprakasam, Olson, Schutzki, Tai, & Nair, 2006). phetamine-regulated transcript (CART) and POMC that reduces appe-
Sweet cherry anthocyanins (CACN) consist of cyanidin 3-(2G-gluco- tite while AgRP neurons are inhibited in response to leptin. However,
sylrutinoside), cyanidin 3-rutinoside and pelargonidin 3-rutinoside. 40 leptin resistance and disorder in other adipocytokines, such as adipo-
and 200 mg/kg CACN were added to diet and induced 5.2% and 11.2% nectin and ghrelin, are quite common for obesity state (Saltiel, 2016).
decrease in body weight gain in mice. CACN attenuated the size of Thus, targeting leptin resistance may be a promising way to develop
epididymal adipocytes and reduced serum lipids. In 3T3-L1 cells, anti-obesity therapies.
200 μg/mL CACN reduced lipid accumulation by 30.7% (Wu, Tang, Yu, Black bean seed coat anthocyanins contain cyanidin 3-glucoside,
Gao, Hu, Chen, et al., 2014). Blueberry anthocyanins (BA) are com- petunidin 3-glucoside and delphinidin 3-glucoside. 24 mg/kg daily in-
posed of 9 different structures, such as 24.4% petunidin 3-arabinoside, take of black bean seed coat anthocyanins reduced the expression of
16.4% delphinidin 3-glucoside and 12.5% cyanidin 3-galactoside. neuropeptide Y (NPY) and induced an increase of γ-amino butyric acid
Consumption of BA at 200 mg/kg reduced 19.4% body weight gain in receptor (GABAB1R) in hypothalamus. Moreover, decreased expression
mice. BA could effectively attenuate epididymal adipocytes, improve of GABAB1R downstream signaling molecules, such as protein kinase A-
lipid profiles, and down-regulate the expression levels of FAS genes α (PKA-α) and phosphorylated cAMP-response element binding protein
(Wu, Jiang, Yin, Long, & Zheng, 2016a). BCAO increased gene ex- (p-CREB) in hypothalamus followed such effect (Badshah et al., 2013).
pressions of carnitine palmitoyltransferase I (CPT-1) and PPAR-α in- Purified cyanidin 3-glucoside enhanced adipocytokines (leptin and
volved in fatty acid oxidation and decreased mRNA expressions of FAS adiponectin) secretion in isolated rat adipocytes (Tsuda et al., 2004).
and SREBP-1c associated with fatty acid synthesis (Park et al., 2015). CACN, MACN, HA reduced leptin secretion in mice. HA and CBE also
Mulberry anthocyanins (MACN) consist of cyanidin 3-glucoside, cya- increased serum adiponectin levels (Qin & Anderson, 2012; Wu et al.,
nidin 3-rutinoside and pelargonidin 3-glucoside. Male C57BL/6 mice 2013b; Wu et al., 2014; Wu, Yin, Zhang, Long, & Zheng, 2016b; Wu
were divided into four groups and given free access to control diet, HFD et al., 2013c).
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 2.5. Regulate gut microbiota
attenuated lipid accumulation and lowered the size of adipocytes (Wu
et al., 2013b). 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
2.4. Suppress food intake the interaction among diet, obesity and gut microbiota. Diet-gut mi-
crobiota interactions could moderate human metabolism. Germ-free
The control center of appetite lies in part of the brain called hy- mice fed a westernized diet (such as high-fat diet) were protected from
pothalamus. Neurons in hypothalamus detect and organize signals of diet-induced obesity. Lean mice that received a microbiota transplant
body energy status to control food intake and energy expenditure. from genetically obese mice had increased adiposity. Lard intake al-
Proopiomelanocortin (POMC) and agouti-related peptide (AgRP) cells tered gut microbiota composition in mice, which promoted obesity and
are the key cell types in this process, which generate peptides compe- white adipose tissue inflammation through Toll-like receptors signaling
titively binding to the melanocortin receptors MC3R and MC4R. Leptin and chemokine CCL2 in mice. But consumption of fish oil that is rich in

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

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

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L. Xie et al. Trends in Food Science & Technology 72 (2018) 13–24

3. Characteristics and therapeutic use evaluate its effect upon ethanol-induced hepatic injury. The results
indicated that both doses were able to reverse the increase in CYP2E1
Anthocyanins have also been reported to have active effects on induced by chronic ethanol intake in test mice (Chao, Liu, Wu, & Yin,
oxidative stress and inflammation that are associated with obesity be- 2015; Yin, Wang, Liu, & Mong, 2017). Administration of anthocyanin
sides their anti-obesity activity. In this section, we have first summar- rich extract of Hibiscus sabdariffa calyces (HSARE) at 100 mg/kg/d for 4
ized anthocyanins with such activities, as shown in Fig. 1 and Table 2, weeks was found to decrease the immunopositivity of CYP2E1 in liver
then discussed their physicochemical characteristics as well as ab- tissue of test rats (Ezzat et al., 2016). Cyanidin 3-glucoside pretreat-
sorption and metabolism, ended up with commenting on studies of ment was also reported to inhibit expression of CYP2E1 in MDA-MB-
anthocyanins’ therapeutic use. 231 cells with AA intervention (Song et al., 2013).

3.1. Anthocyanins with effects on obesity associated oxidative stress and


inflammation 3.1.2. Resolve inflammation
Obesity is characteristic with chronic systemic low-grade in-
3.1.1. Ameliorate oxidative stress flammation. Adipocytes secrete factors such as monocyte chemoat-
Fat accumulation is associated with systemic oxidative stress as tractant protein-1 (MCP-1) that induce monocytes differentiation into
confirmed by human and mice studies. In adipose tissue of obese mice, macrophages. Adipocytes and macrophages interact and increase cir-
reactive oxygen species (ROS) production increased, followed by aug- culating proinflammatory cytokines, such as TNF-α, IL-6 that promote
mented expression of NADPH oxidase and decreased expression of an- inflammation response. Circulating TNF-α and IL-6 promote liver pro-
tioxidative enzymes. In addition, elevated levels of fatty acids increased duction of an acute-phase reactant C-reactive protein (CRP) (Bastard
oxidative stress through NADPH oxidase activation in cultured adipo- et al., 2006; Lee & Pratley, 2005). Resolving inflammation, such as
cytes. Oxidative stress caused dysregulated production of adipocyto- ameliorating proinflammatory cytokines could improve obesity state.
kines such as adiponection. Targeting oxidative stress such as inhibiting Strawberry beverage containing 81.65 mg/10 g anthocyanins in
NADPH oxidase could regulate adipocytokines production and ame- total, (including 67.99 mg/10 g pelargonidin 3-glucoside; 9.48 mg/10 g
liorate hyperlipidemia (as shown in Table 2) (Furukawa et al., 2004). pelargonidin 3-malonylglucoside and 2.35 mg/10 g pelargonidin 3-ru-
Most anthocyanins and their aglycones are strong antioxidants with tinoside) were tested for the association between strawberry antho-
activities comparable to widely used antioxidants such as Trolox, ca- cyanins and postprandial inflammation in a clinical trial. The straw-
techin and quercetin in systems like emulsion and LDL (Kähkönen & berry beverage significantly attenuated the postprandial inflammatory
Heinonen, 2003). Blueberry intake with interventions containing 129, response as measured by high-sensitivity C-reactive protein (hs-CRP)
258, 310, 517 and 724 mg total anthocyanins improved vascular and IL-6 induced by the HCFM (Edirisinghe et al., 2011). CBE lowered
function in healthy men in an intake-dependent and time-dependent gene expression levels of inflammatory cytokines, such as Il1β, Il6 and
manner. The effects of phenolic metabolites on neutrophil NADPH Tnfα (Qin & Anderson, 2012). PSPC suppressed the c-Jun-N-terminal
oxidase activity were suggested to be the mechanism (Rodriguez- kinase 1 (c-JNK1) and I kappa B kinase β (IKKβ) activation and nuclear
Mateos et al., 2013). CBE consumption increased plasma adiponectin factor-kappa B (NF-κB) p65 nuclear translocation (Zhang et al., 2013).
levels, decreased plasma tumor necrosis factor-α (TNF-α) and inter- Intake of 10% blackberries containing 87% cyanidin 3-glucoside could
leukin-6 (IL-6) levels as well as and inhibited Fabp4, Fas and Lpl mRNA induce the protective effect against weight gain and inflammation in
levels (Qin & Anderson, 2012). Purple Sweet Potato Color (PSPC) rats associated with ovariectomy-induced menopause. Addition of tart
suppressed ROS production and restored glutathione (GSH) content and cherry extract with 150 g/mL anthocyanins content and purified cya-
activities of antioxidant enzymes. PSPC also prevented the oxidative nidin 3-glucoside resulted in LPS-induced secretion of IL-6 in adipose
stress mediated endoplasmic reticulum (ER) stress in mice liver (Zhang stem cell in a dose-dependent manner (Kaume, Gilbert, Brownmiller,
et al., 2013). Consumption of CACN and MACN at 200 mg/kg diet re- Howard, & Devareddy, 2012). BE suppressed gene expression levels of
duced MDA production and increased SOD and glutathione peroxidase major inflammatory cytokines, TNF-α and MCP-1 in white adipose
(GPx) activities (Wu et al., 2016b). tissue (Kanamoto et al., 2011). BA down-regulated the expression levels
Obesity along with chronic over-nutrition is known to be the key of TNF-α, IL-6 and PPARγ (Wu et al., 2016a). Also, CACN and MACN
risk factor for nonalcoholic fatty liver disease (NAFLD) development, down-regulated the gene expression of TNF-α, IL-6, iNOS, and NF-кB
which is characteristic of fat accumulation (steatosis). NAFLD is also genes (Wu et al., 2016b).
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, 3.2. Characteristics of anthocyanins
such as oxidative stress, thus resulting in development of nonalcoholic
steatohepatitis (NASH). Among various sources of oxidative stress, cy- 3.2.1. Physicochemical properties of anthocyanins
tochrome P450 2E1 (CYP2E1) was reported to be closely related with Acetone, methanol, ethanol and water were commonly used to ex-
NASH development (Abdelmegeed et al., 2012). The role CYP2E1 plays tract anthocyanins due to similarity in their polarity, though acetone
in metabolism of fatty acid, which leads to an increase in toxic lipid and methanol were forbidden to use when preparing extracts for food
peroxides levels along with its possibly increase of expression in NASH processing. Extraction of anthocyanins generally requires acids, for the
makes it a likely candidate as a genetical risk factor for both NAFLD and use of stabilizing anthocyanins in the flavylium cation form that ap-
NASH (Dey, 2013). Interestingly, anthocyanins are reported to inhibit peared red at low pH. Also, as solutions containing hydrochloric acid
CYP2E1 activity. Thus, the possible effects of anthocyanins on attenu- may hydrolyze acylated anthocyanins, using organic acids, such as ci-
ating obesity, NAFLD/NASH development and associated oxidative tric acids may be a better choice (Kırca, Özkan, & Cemeroğlu, 2007).
stress are worth attention. The number and position of hydroxyl and methoxy substituents as
Blueberry anthocyanins extract (BAE) with a total anthocyanins electron donating groups along with glycosylation parameters like site
content of 257.6 mg/g (over 90% being cyanidin 3-glucoside) was re- and number of glycosylation and sugar type have great impact on the
ported to ameliorate ROS overproduction and GSH depletion induced activities of anthocyanins (Kähkönen & Heinonen, 2003). Chemical
by the acrylamide (AA) as well as inhibiting protein expression of modification in sugar groups can change the bioavailability of antho-
CYP2E1 in a mice model (Zhao et al., 2015). Gynura bicolor leaf aqueous cyanins. Acylation of anthocyanins generally results in reduced ab-
extract (GAE) with a total anthocyanins content of 2135 mg per 100 g sorption. Furthermore, anthocyanins with diglycosides such as sambu-
dry weight was added to feed of mice at 0.25% or 0.5% in a study to bioside or rutinoside show increased stability (Prior & Wu, 2006).

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L. Xie et al. Trends in Food Science & Technology 72 (2018) 13–24

3.2.2. Anthocyanins absorption and metabolism anthocyanins at 200 mg/kg diet prevented body weight gain by 32.7%
Poor absorption of anthocyanins has been a real trouble in clinical and 29.6%, with the effect of orlistat being 17.1% (Wu et al., 2016b).
trials. For the berry species, various berries other than cranberry an- Clinical trials that use anthocyanins extracts and purified anthocyanins
thocyanins glycosides have been found to be absorbed and excreted into as dietary supplement have also been carried out. Hibiscus sabdariffa
urine unmetabolized by both animals and human beings. Cranberry extracts (HSE) containing 1.43% flavonoids, 2.5% anthocyanins and
showed slightly better absorption, as 5% of the dose consumed within 1.7% phenolic acid were prepared from H. sabdariffa L. 450 mg HSE
24 h were excreted into urine with a maximum excretion period be- and 50 mg starch were combined into one capsule and given to 19
tween 3 and 6 h after consumption (Zhu, Miao, Meng, & Zhong, 2015). subjects (HSE-treated group) for 3 months. 17 subjects were given
In a clinical trial that used a cross-over design, 24 overweight adults placebo capsule with 500 mg starch in one dose and set as control. At
were given high-carbohydrate, moderate-fat meal (HCFM) accom- the end of the study, HSE-treated group had reduced body weight, BMI,
panied by either a strawberry or a placebo beverage. Their postprandial body fat, waist-to-hip ratio, as well as decreased free fatty acid (Chang,
changes in plasma anthocyanins along with physiological status were Peng, Yeh, Kao, & Wang, 2014). Drinking purple tea daily for 1 month
assessed for 6 h, and their postprandial concentrations of pelargonidin was reported to improve obesity parameters including body weight,
sulfate and pelargonidin 3-glucoside were increased when the straw- BMI and body fat mass compared to baseline (Shimoda, Hitoe,
berry beverage was consumed (Edirisinghe et al., 2011). A clinical Nakamura, & Matsuda, 2015).
study aimed at investigating bioavailability of bilberry anthocyanins A particular advantage of anthocyanins is their well-recognized
intake in healthy volunteers with or without colon demonstrated that safety. Acute and chronic studies have been conducted in vivo to eval-
colon was an important site for absorption of anthocyanins as well as uate the oral toxicity of black soybean hull extract (BE) containing 9.2%
their degradation products. In this study, about 30% of consumed an- cyanidin 3-glucoside. LD50 of BE was reported to be more than 2.5 g/kg
thocyanins remained stable, when passing through upper part of in- body weight in test Sprague-Dawley rat and C57BL/6 mice, and no
testine and only 20% of degradants were produced. Moreover, most of significant decrease in body weight or death was recorded in the acute
intact anthocyanin went through absorption in the small intestine, oral toxicity study. In the chronic oral toxicity study, no toxicologically
while larger amounts of degradants reached the circulation in both significant clinical changes or mortality was observed in male and fe-
groups compared with anthocyanins (Mueller et al., 2017). Plasma male C57BL/6 mice, and the estimation of no-observed adverse-effect-
concentrations of anthocyanins were several tens of nanomoles per liter level of BE was 5.0% (Fukuda et al., 2011). Reviewed anthocyanin
after an intake of about 110–200 mg, and urinary excretion percentage human oral consumption trials with doses ranging from 788 mg to
of anthocyanins was 0.005–0.1% of intake (Manach et al., 2004). 883 mg reported no adverse effects (Kay et al., 2005). In 2013, the
Glucuronide and methyl-glucuronide derivatives were the major forms European Food Safety Authority concluded the anthocyanins to be
of primary metabolites when cyanidin 3-glucoside was absorbed and unlikely of safety concern as a result of evaluating toxicologic data
then transported in serum and urine after oral intake of a moderate-to- mainly derived from studies of blackcurrant extracts and grape-skin
high dose. In one clinical study with three subjects, the serum con- (European Food Safety Authority, 2013). China is the first country to
centration of total anthocyanins and their metabolized forms reached define a daily recommended intake of 50 mg/day for anthocyanins in
maximum at 2.8 h, with a maximum urinary excretion rate at 3.72 h adults, but a Tolerable Upper Intake Level remains undefined (Chinese
and a half-life of elimination at 4.12 h (Kay, Mazza, & Holub, 2005). Nutrition Society, 2013). Based on a report published in 2015, there has
13
C-labelled study showed that seventeen 13C-labelled compounds in- been no indication of anthocyanin toxicity in published studies of
cluding 13C5-C3G and its degradation products, phloroglucinaldehyde human intervention. Noticeably, an acceptable daily intake specifically
(PGA) and protocatechuic acid (PCA) could be identified in the serum for grape-skin extracts-derived anthocyanins at 2.5 mg/kg per day has
after oral intake of 500 mg 6,8,10,3′,5′-13C5-C3G in eight subjects. The been established by the Joint FAO/WHO Expert Committee on Food
phenolic metabolites had ranged maximal concentrations from 10 to Additives (Wallace & Giusti, 2015).
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 4. Biosynthesis of anthocyanins in microorganisms
identified as the major phenolic metabolites, with peaks at about 16
and 8 h (de Ferrars et al., 2014). Anthocyanins are currently obtained from extraction and purifica-
tion from natural sources. Such methods are characteristic of high ex-
3.3. Anthocyanins in anti-obesity therapy pense, low production rate and rather complex processes. Moreover,
there is still limited progress in chemical synthesis of anthocyanins for
Anthocyanins are reported to show anti-obesity effects through in- the lack of understanding of mechanisms on related key reactions. As a
hibiting lipid absorption and suppressing food intake. Both mechanisms result, purified anthocyanins are quite expensive. For future research
are appealing for therapeutic use. Currently there are seven anti-obesity such as therapeutic use for obesity and association between structure
drugs approved by U.S. Food and Drug Administration (FDA). Most of and activity of anthocyanins, it's quite necessary to develop a cheap and
these drugs target central nervous system (CNS) to affect energy intake, efficient way to produce anthocyanins (Prior & Wu, 2006; Smeriglio,
including phentermine (prescribed alone or combined with topir- Barreca, Bellocco, & Trombetta, 2016; Yun, 2010). Anthocyanins bio-
amate), benzphetamine and phendimetrazine, diethylpropion, lorca- synthesis in microorganisms is thus a rather promising way to achieve
serin, liraglutide and naltrexone combined with bupropion. However, such purpose. In this section, biosynthesis in microorganisms refers to
use of these drugs is accompanied with side effects, such as hyperten- production of anthocyanins from genetically engineered microorgan-
sion, cardiac valve disorders, nausea, diarrhea and vomiting. Orlistat is isms that express plant anthocyanins biosynthetic pathways.
an exception that works on digestive system and has side effects like
oily stools and fecal inconsistency (Adan, 2013; Gadde, 2014; Wong, 4.1. Biological synthesis of anthocyanins in plants
Sullivan, & Heap, 2012).
According to animal studies, anthocyanins have weight reducing Biological synthesis of anthocyanins in plants has been researched
effects in comparable to current drugs. Supplementation of honeysuckle in detail, especially in the model plant arabidopsis thaliana and berries.
anthocyanins for 100 mg/kg diet reduced body weight gain by 17.1% Anthocyanins accumulated in plants during ripening, with its compo-
compared with control group, which was similar to 16.9% reduction in sition reaching a peak at fruit maturity. Procyanidins and quercetin
orlisat group (Wu et al., 2013c). Some anthocyanins prevented weight were the major flavonoids at early stages of berry development, but
gain to a greater extent. Supplementation at 200 mg/kg of diet resulted their levels decreased during the ripening progress. In the meantime,
in a higher reduction of 24.1%. Intake of mulberry and sweet cherry concentration of anthocyanins increased dramatically during later

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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.

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L. Xie et al. Trends in Food Science & Technology 72 (2018) 13–24

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

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Table 3
Anthocyanins biosynthesis in microorganisms.

Precursor Recombinant enzymes Microorganism Anthocyanins Composition


and Production rate

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

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