Teng2017

Accepted Manuscript Red raspberry and its anthocyanins: Bioactivity beyond antioxidant capacity Hui Teng, Ting Fang, Qiyang Lin, Hongbo Song, Bin Liu, Lei Chen

PII:

S0924-2244(16)30533-7

DOI:

10.1016/j.tifs.2017.05.015

Reference:

TIFS 2017

To appear in:

Trends in Food Science & Technology

Received Date: 18 November 2016 Revised Date:

28 February 2017

Accepted Date: 23 May 2017

Please cite this article as: Teng, H., Fang, T., Lin, Q., Song, H., Liu, B., Chen, L., Red raspberry and its anthocyanins: Bioactivity beyond antioxidant capacity, Trends in Food Science & Technology (2017), doi: 10.1016/j.tifs.2017.05.015.

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ACCEPTED MANUSCRIPT

Red raspberry and its anthocyanins: bioactivity beyond antioxidant

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capacity

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Hui Teng#, Ting Fang, Qiyang Lin, Hongbo Song*, Bin Liu*, Lei Chen#*

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College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian

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350002, China

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#

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*Corresponding author: L. Chen: chenlei841114@hotmail.com; H. Song:

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sghgbode@163.com; B. Liu: liubin618@hotmail.com

Authors contributed equally to this work

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Abstract

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Background: Known as the "golden fruit�, red raspberry is rich in anthocyanins with

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documented biological activities, many of which were systematically investigated.

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Nowadays, raspberry anthocyanins’ importance for Food and Pharmaceutical

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industries is mainly based on the existed scientific works evidencing their potential

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effects on chemoprevention, inflammation, and immune-regulation. Although, much

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of the work in these respective areas which has been conducted in cell culture systems,

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animal and human studies have been steadily rising.

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Scope and approach: In this review, We review and summarize the latest and

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available literature that assesses the health-promoting potential of red raspberries and

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their anthocyanin components in modulating metabolic disease risk, especially

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cardiovascular disease, cancer, all of which share critical metabolic, oxidative, and

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inflammatory links.

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Key findings and conclusions: We also suggested a better evaluation of the

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pharmacological profile of raspberry and its anthocyanins with a clear-cut choice of

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possible human pathologies. Future studies aimed at enhancing the absorption of

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anthocyanins or their metabolites are likely to be necessary for their ultimate use for

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chemoprevention and anti-inflammation.

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Keywords:

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anti-inflammation

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Abbreviations

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AP-1 Activation protein-1

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Bcl-2 B cell lymphoma-2

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Cdk Cyclin dependent kinase

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COX Cyclooxygenase

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COX- 2Cycloxygenase-2

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ERK Extracellular signal-related kinase

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GAPDH, Glyceraldehyde 3-phosphate dehydrogenase;

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HIF-1α Hypoxia-inducible factor-1α

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HO-1, Heme oxygenase-1;

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IL Interleukin

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IL-1β Interleukin-1 β;

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IL-6 Interleukin-6;

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IκB-α Inhibitor of κB-α;

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IKK inhibitor of κB-α kinase;

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iNOS inducible nitric oxide synthase;

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JAK Janus kinase

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JNK c-Jun NH2-terminal kinase;

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LPS Lipopolysaccharide;

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LXRα liver X receptor α

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MAPKs Mitogen-activated protein kinases;

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MAP2K MAPK kinase 1

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MMP Matrix metalloproteinase

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NF-κB Nuclear factor-kappa B

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NO Nitric oxide

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Nrf2 Nuclear transcription factor-E2-related factor 2

antioxidant;

chemoprevention;

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anthocyanin;

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Raspberry;

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ORAC Oxygen radical absorbance capacities

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PDGF Platelet-derived growth factor

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PGE2 Prostaglandin E2

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PI3K, Pphosphatidylinositol 3-kinase

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PKC Protein kinase C

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PIP2 Phosphatidylinostol (3, 4)-bisphosphate

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PIP3 Phosphatidylinostol (3, 4, 5)-triphosphate

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PPARγ Peroxisome proliferator-activated receptor γ

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ROS Reactive oxygen species;

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SOD Superoxide dismutase

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TNF-α Tumor necrosis factor-alpha

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VEGF Endothelial growth factor

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VEGFR-2 VEGF receptor-2

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1. Introduction

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Known as the "golden fruit” (Figure 1), red raspberries are becoming increasingly

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appreciated for their culinary versatility and other applications. The harmless natural

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edible pigment of red raspberry was conspicuous to topic in the pharmaceutical and

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food scientific researches. Red raspberries possess a unique polyphenol profile that is

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characterized primarily by their anthocyanins. The anthocyanins are important natural

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organic compound, besides of being taken as the edible pigment, it also has the vital

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significance to prevent diseases, such as tumor, senile and cardiovascular of humanity,

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as well as the effects on oxidative stress. Since anthocyanins, which are

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sub-categorized into the flavonoids, have strong anti-oxidative activities, they can

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safeguard cells and body away from oxidation by scavenging free radicals (Lei Chen

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& Kang, 2013; L. Chen & Kang, 2014; Wei Chen, Xu, Zhang, Li, & Zheng, 2016).

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Raspberries are widely distributed and cultivated in China. However, industrial

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development of raspberry products got restrictions because of differences in the

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ACCEPTED MANUSCRIPT variety and cultivation environment, as well as the limitation in preservation and

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processing techniques, and etc. In recent years, other berry fruits, such as the

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strawberry, blueberry, cranberry and black raspberry, have been studied for their

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beneficial effects on health. These health benefits include prevention of certain types

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of cancer, cardiovascular diseases, neurodegenerative diseases associated with oxidant

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damage. Comparatively, little work has been done on red raspberries. Therefore, in

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order to provide a theoretical basis for using and developing raspberry resource, the

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major objective of this review is to summarize the latest developments on the

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antioxidant activities of anthocyanin-rich raspberry in cell culture models to discuss

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their underlying molecular mechanisms which drives chemo-preventative and

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anti-inflammatory effects.

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2.

Methods

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This review is a descriptive review of literatures. Keywords such as “raspberry”,

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“anthocyanin”, “extract”, were searched separately or combined in national databases

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such as CNKI, and international databases including Science direct, Pubmed, Springer

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and Scopus. The searched languages were limited to Chinese and English articles. The

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search period was limited from 2006 to June 2016. Overall, 382 articles were

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collected in the first step. Then unrelated articles were excluded according to title and

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abstract evaluations. Articles with incomplete data along with congress and

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conference’s proceedings were excluded. All of reviewed studies were clinical trial or

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experimental researches. Finally, 89 studies got inclusion criteria and were included

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in the study.

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3. Extraction

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Soaking has been extensively used for anthocyanins extraction. In the wine industry,

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it is the most recurrent method that consists in grinding the fresh fruit and putting the

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grape juice in contact with skins to extract the pigments (Ella, Guyot, & Renard,

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2003). According to the extraction methods reported in literatures, the most frequently

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used solvents for anthocyanins extraction from raspberry are methanol, ethanol, and 4

ACCEPTED MANUSCRIPT acetone (Kahkonen, 2003). Meanwhile, using weak acid media (0.1% formic or acetic

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acid) into organic solvent to extract anthocyanins could avoid their hydrolysis (Dai &

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Mumper, 2010). Acidified methanol is widely considered to be the most efficient

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(Kapasakalidis, And, & Gordon, 2006). For example, anthocyanin extractions with

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acidified methanol is 73% more effective than pure water, and 20% and 200% more

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efficient than those extracted with acidified ethanol (Metivier, Francis, & Clydesdale,

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1980) and acetone (J. M. Lee & Johnson, 2004), respectively. However, ethanol is

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preferred due to its non-toxicity in food industry. Nevertheless, under these conditions,

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it is impossible to know whether the hydrolysis of aglycons occurred or not during the

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extraction

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acidified methanol as the extractant has been claimed, special care should be taken to

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avoid degradation of anthocyanins (hydrolysis reaction) under strong acid media,

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meanwhile, in the case of 3-monoside anthocyanins, the glycoside bonds could also

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be destroyed (Kapasakalidis, et al., 2006). Besides of the acidified extractant, sulfured

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water by using aqueous SO2 solution (HSO3) has also been reported for anthocyanin

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extraction (Cacace & Mazza, 2002). Bisulfite solution can react with anthocyanins

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causing a nucleophilic attack in the molecule (Mazza & Brouillard, 1990) and causing

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a decolorization of monomeric anthocyanins (BerkÊ, Chèze, Vercauteren, & Deffieux,

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1998). In the anthocyanin extractions from black currants, an aqueous solution of 80%

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EtOH saturated with SO2 was used as extractant, and it was observed that the type of

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solvent, SO2 concentration, and the temperature affected the extraction process, and

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the mass transfer rate was higher in sulphurated water than that in ethanol solution

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(Cacace & Mazza, 2002). Thus, it can be concluded that weak acidic media might

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good for improving anthocyanin extraction yield, but hydrolysis of anthocyanins

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during the process was unclear.

raspberry

anthocyanins.

Despite

extensive

use

of

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4. Isolation and identification of active anthocyanins

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The isolation and identification of active compounds have a critical role in the quality

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evaluation of raspberry fruit and its processed food. Because of anthocyanins spectral

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characteristics provide very useful qualitative and quantitative information; actually

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mass spectrometry (MS) and nuclear magnetic resonance (NMR) of 1H and

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become the preferred techniques for anthocyanins identification (Castaneda-Ovando

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et al., 2009). As reviewed by Giusti and Wrolstad (2003), the main methods used in

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the characterization and quantification of anthocyanins is UV–Vis. HPLC with PDA

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detector has been also used in the anthocyanins identification and quantification

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(Kong et al., 2003; Teng, Lee, & Choi, 2013; Teng, Lee, & Choi, 2014), but the

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difficulty to obtain reference compounds and the spectral similarities of the

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anthocyanins represent an important drawback.

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153 5. Chemistry

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Anthocyanins, belong to polyphenol compound, are water-soluble pigments in plants

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which contribute to the brilliant colors of blue, red, and mauve in flowers, fruits and

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leaves. The ionic nature of anthocyanins enables changes of the molecular structure

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according to the prevailing pH, resulting in different colors and hues at different pH

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values. It occurs principally when glycosides of their respective aglycone

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anthocyanidin chromophores generally attached at the 3-position on the C-ring

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(3-monoglycosides) or the 5-position on the A-ring (3, 5-diglycosides) (Prior & Wu,

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2007). Anthocyanins are classified according to the number and position of hydroxyl

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groups on the flavan nucleus which named: cyanidin, delphinidin, petunidin, peonidin,

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malvidin and pelargonidin (Figure 2). The most common sugar of anthocyanidin

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glycosides is glucose, nevertheless, rhamnose, xylose, galactose, arabinose, and

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rutinose (6-O-L-rhamnosyl-D-glucose) can also present (Horbowicz, Kosson,

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Grzesiuk, & Debski, 2008). Although very rare, glycosylation at the 3’, 4’, or 5’

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positions of the B ring is also possible (Wu & Prior, 2005). The sugar moiety may be

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acylated by aromatic acids, general hydroxycinnamic acids (caffeic, ferulic,

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ACCEPTED MANUSCRIPT p-coumaric or sinapic acids) and sometimes by aliphatic acids, namely succinic, malic,

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malonic, oxalic and acetic acids. The acyl moieties are normally linked to the sugar at

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C-3. For anthocyanin biosynthesis, some important structural genes and regulatory

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elements are required. As shown in Figure 3, Holton and Cornish (1995) described

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that malonyl-COA and p-coumaroyl-COA are necessary for the synthesis of

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anthocyanins. Three acetate units were catalyzed and condensed step by step from

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malonyl-COA (with p-coumaroyl-COA) by chalcone synthase to generate

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naringeninchalcone and eriodictyolchalcone. Then, the stereospecific isornerization of

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tetrahydroxychalcone (yellow) could be catalyzed to naringenin (colorless) by

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chalcone isomerase. Naringenin is transformed into dihydrokaempferol by flavanone

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3-hydroxylase, and subsequently, dihydrokaempferol could be hydroxylated by

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flavonoid 3'-hydroxylase to produce dihydroquercetin or to produce dihydromyricetin

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by flavonoid 3’, 5'-hydroxylase. During the process, at least 3 enzymes are needed for

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changing the dihydroflavonols (colorless) into anthocyanins. The first of these

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enzymatic conversions is the reduction of dihydroflavonols to leucoanthocyanidins by

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dihydroflavonol 4-reductase. Further oxidation, dehydration, and glycosylation of the

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different leucoanthocyanidins produce the corresponding brick-red pelargonidin, red

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cyanidin, and blue delphinidin pigments. Anthocyanidin-3-glucosides may be

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modified further into many species by glycosylation, methylation, and acylation.

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The glycosides of the three non-methylated anthocyanidins (delphinidin, cyaniding

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and pelargonidin) are the most abundant in nature, which represent 80% of leaf

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pigments, 69% in fruits and 50% in flowers. The most common anthocyanidins found

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in the edible parts of plants is cyanidin, pelargonidin, peonidin, delphinidin, petunidin,

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and malvidin (Kong, Chia, Goh, Chia, & Brouillard, 2003). The red color of the

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raspberry fruit is related to its anthocyanin composition. Profiles of the anthocyanins

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of

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3-O-glucorutinoside, and cyanidin 3-O-glucoside (De, IbaĂąez, Reglero, & Cano,

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2000). A recent work (Ludwig, et al., 2015) characterized anthocyanins content of

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red

raspberry

mainly

consist of

cyanidin

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3-O-sophoroside,

cyaniding

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commercial

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cyanidin-3-O-(2”-O-glucosyl)

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cyanidin-3-O-rutinoside were maintained with amount of 175, 56, 37, and 20 µM/300

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g raspberries ((Ludwig, et al., 2015), Table 1), respectively. The relative composition

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was cyanidin-3-sophoroside > cyanidin-3-glucorutinoside > cyanidin-3-glucoside >

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cyanidin-3-rutinoside > all pelargonidin glucosides combined (Wu & Prior, 2005). All

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these anthocyanins have also been detected in red raspberries previously, though not

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in a single cultivar (De, et al., 2000). The different anthocyanin contents found in a

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single cultivar between the different papers may be dedicated to the characterization

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of the fruit stages of ripeness, processing, and environmental factors ( Zafrilla et al.,

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2001; Perkins-Veazie, Collins, & Howard, 2008).

raspberry

and

found

rutinoside,

that

cyanidin-3-O-sophoroside,

cyanidin-3-O-glucoside,

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6. Antioxidant capacity

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Raspberries are known to contain the highest antioxidant levels among the fruits.

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There are numerous reports on oxygen radical absorbing assay to measure antioxidant

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capacity of anthocyanins from raspberry fruits (Liu, et al., 2002; Pantelidis,

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Vasilakakis, Manganaris, & Diamantidis, 2007; S. Y. Wang & Lin, 1999). An

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evidence presented by Liu, et al. (2002) further suggested that darker colored

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raspberries have more effective antioxidant activity. The antioxidant activity of

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raspberries is primarily constituted by anthocyanins and ellagitannins which

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contributed about 25% and 52% to the total antioxidant activity (Beekwilder, et al.,

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2005). According to a study (Kahkonen, 2003) in which antioxidant activity of

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anthocyanins and their aglycons were evaluated, anthocyanins lacking of the

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O-diphenyl structure in the B ring (malvidin, pelargonidin, petunidin, and peonidin)

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had a lower efficiency toward the DPPH radical as compared to cyanidin and

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delphinidin. Peonidin, having a CH3 group in the 3′-position in addition to an OH

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group in the 4′-position, was more active than pelargonidin (Kahkonen, 2003). As

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reported by Fukumoto and Mazza (2000)， the third hydroxyl group in the B ring

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ACCEPTED MANUSCRIPT enhanced the activity, as delphinidin with hydroxyl groups in the 3′-, 4′-, and

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5′-positions was significantly more effective than cyanidin with hydroxyl groups only

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in 3′- and 4′-positions. In addition, H. Wang, Guohua Cao, and † (1997) observed that

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glycosylation of anthocyanins affected the antioxidant capacity. In fact, glycosylation

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of cyanidin to cyaniding-3-glucoside increased the activity, but glycosylation of

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malvidin to the corresponding malvidin-3-glucoside decreased the activity, and

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pelargonin and its pelargonin-3-glucoside had similar responses (H. Wang, et al.,

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1997). In the DPPH assay, antioxidant activity for the monoglucosides of cyanidin,

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pelargonidin, and peonidin were lower than their aglycon forms, whereas in the

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β-carotene bleaching method the monoglycosides of malvidin, pelargonidin, and

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peonidin showed higher activities than the aglycons (Fukumoto & Mazza, 2000). In

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fact, results of antioxidant activity obtained by Seeram, et al. (2006) revealed that the

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pure cyanidin glycosides increased antioxidant activity with a decreasing number of

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sugar

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cyanidin-3-rutinoside showed a better activity than cyanidin-3-glucosylrutinoside, and

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the aglycone cyanidin showed the strongest activity at much lower concentrations. To

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date, anthocyanins are demonstrated to have strong antioxidant effects in vitro assays.

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The double bonds present in the phenolic ring, the hydroxyl side chains, and even the

244

glycosylation all contribute to the free radical scavenging activity. In summary,

245

anthocyanidin are potent antioxidants, as discussed above, the change in the

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antioxidant action that results from the differences in glycosylation is very much

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dependent on the assay used. Since anthocyanins have a strong anti-oxidative activity

248

and safeguard cells and body away from oxidation, and mechanism in the cellular

249

defense against oxidative were discussed. It is well known that the NF-E2-related

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factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway plays an

251

important role in cellular defense. ARE is a cis-acting regulatory element of genes

252

encoding phase II detoxification enzymes and antioxidant proteins, such as NADPH:

253

quinone oxidoreductase, glutathione-transferases, and glutamate-cysteine ligase.

(Seeram,

Momin,

Nair,

&

Bourquin,

2001).

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ACCEPTED MANUSCRIPT Interestingly, it has been reported that Nrf2 regulates a wide array of ARE-driven

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genes in various cell types (Element, 2004). Likewise, raspberry anthocyanins

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inhibited the activation of NF-ÎşB, possibly due to their inhibitory effect on the

257

expression of NF-ÎşB-dependent NOX-1 (S. G. Lee, et al., 2014). As shown in Table 2,

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several in vitro cell culture, chemical assay, and enzyme activity studies have been

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used to assess the possible effects of red raspberry extracts/fractions on indicators of

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oxidative stress. Anthocyanins, presented in red raspberry, particularly cyanidin

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glycosides, have been found to possess the effect on oxygen species (ROS)-dependent

262

activation of p38 MAPK and JNK (W. Chen, Su, Xu, Bao, & Zheng, 2016b; Jiang,

263

Tang, Zhang, Liu, & Guo, 2014). Feng and co-authors (Feng, et al., 2007) have

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demonstrated that peroxides but not superoxides increased in leukemia cells after

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being treated with cyanidin-3-rutinoside. This result suggested that the possible

266

mechanisms for antioxidant activity of cyanidin-3-rutinoside could rather be an

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interference with the glutathione antioxidant system, which is involved in peroxide

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scavenging. Further study has also suggested that cyanidin-3-rutinoside could

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modulate the glutathione activity, including regulation of GSH (reducing glutathione

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content) intracellular levels (Solomon, et al., 2010) or inhibition of glutathione

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enzyme activity (Cvorovic, et al., 2010). In addition, cyanidin-mediated antioxidant

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enzyme expression involved the ERK and JNK pathways, but not p38 MAPKs (Shih,

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Hwang, Yeh, & Yen, 2012; ). Besides, cyanidin has also been proven to act as

274

Nrf2-ARE signaling transmitter, showing a strong effect on PPAR-Nrf2 activation (S.

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G. Lee, et al., 2014). Nevertheless, cyanidin-3-O-glucoside treatment of HepG2 cells

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increased Gclc expression results in a decrease in hepatic ROS levels and

277

MKK4-JNK-Fas proapoptotic signaling (Zhu, Jia, Wang, Zhang, & Xia, 2012). The

278

protective role of cyaniding-3-O-glucoside also appears to be due to the capacity of

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eliciting cell adaptive response, through the modulation of the Nrf2/NF-kB cellular

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pathway (Anwar, et al., 2014). Likewise, cyaniding-3-O-glucoside treatment of

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HepG2 cells enhances total cellular GSH levels and dramatically increases the

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ACCEPTED MANUSCRIPT GSH/GSSG ratio, which is considered as an indicator of oxidative stress (Anwar, et

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al., 2014). Interestingly, another research group found that delphenidin could

284

effectively inhibit EGF-induced auto-phosphorylation of EGFR, activation of PI3K,

285

phosphorylation of AKT and MAPK (Bei, et al., 2009). Participation of MAPKs in

286

ARE regulation in an Nrf2-dependent manner has been described (Mandlekar, Hong,

287

& Kong, 2006). Altogether, it was suggested that raspberry and its anthocyanins

288

played an important role in antioxidant to struggle with oxidant induced injury, and

289

thus they could serve as chemopreventive agents, which might closely related to their

290

chemical structure and glycosylation degree.

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7. Chemopreventative potential

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Overwhelming evidences suggest that red raspberry fruits have chemo-preventive and

294

chemo-therapeutic activities through the modulation of multiple molecular targets

295

making them ideal for the prevention/treatment of cancer (Lala, et al., 2006). The

296

anticancer potential of raspberries has been related, at least in part, to a multitude of

297

anti-oxidative anthocyanins. In the intrinsic pathway, anthocyanin treatment of cancer

298

cells results in an increase in mitochondrial membrane potential, cytochrome c release

299

and modulation of caspase-dependent anti and pro-apoptotic proteins. In the extrinsic

300

pathway, anthocyanins modulate the expression of FAS and FASL (FAS ligand) in

301

cancer cells resulting in apoptosis (L. S. Wang & Stoner, 2008). As shown in Table 3,

302

there are several reports focused on the effect of anthocyanins on cancer treatments

303

(Jung, et al., 2009; Nichenametla, Taruscio, Barney, & Exon, 2006; Seeram, et al.,

304

2006). They revealed that anthocyanins can affect basic cell functions related to

305

cancer development and inhibit the growth of tumors by induction of cell cycle arrest

306

and apoptosis. For example, Zhang, Vareed, and Nair (2005) found that cyanidin,

307

delphinidin, pelargonidin, petunidin, malvidin and their glycosides inhibited cell

308

proliferation by blocking cell cycle regulator proteins (e.g., p53, p21, p27, cyclin D1,

309

cyclin A, and etc.). Cyanidin, delphinidin, and petunidin, meanwhile, also inhibit the

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ACCEPTED MANUSCRIPT phosphorylation of JNK1/2, MAPK1/2, ERK1/2, the kinases Raf1, and MAP2K1, and

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subsequently hinder the activation of AP-1 and NF-κB, accordingly suppressing the

312

secretion of prostaglandin E2 and the expression of COX-2 in multifarious cell types

313

(P. N. Chen, et al., 2006; Hou, et al., 2004; Shih, Yeh, & Yen, 2005). The binding of

314

cyanidin, delphinidin, and petunidin to kinases Raf1 and MAP2K1 have also revealed

315

to be noncompetitive with ATP (Kang, et al., 2008). Moreover, malvidin, delphinidin,

316

and their glycosides have also been reported to induce apoptosis through the

317

inhibition of NF-κB, and down-regulation of Bcl-2, and consequently, the expression

318

of Bax was increased, and caspases 3 and 9 were activated (Choung, Lim, & Choung,

319

2012; Feng, et al., 2007; Mulabagal, Lang, Dewitt, Dalavoy, & Nair, 2009; Chen et al.,

320

2016). For cyanidin-glycosides, the cyanidin-3-glucoside, cyanidin-3-rutinoside,

321

cyanidin-3-(2G-xylosylrutinoside), and cyanidin-3-O-sophoroside are proved to be

322

potent anti-inflammatory, anti-oxidant, and pro-apoptotic agents (Choung, et al., 2012;

323

Feng, et al., 2007). Moreover, pelargonidin-3-O-glucoside and cyanidin-3-rutinoside

324

inhibit the activation of NF-κB and AP-1 by aiming at the PI3K/Akt, MAPK, and

325

JNK signaling cascades (Duncan, 2009; Feng, et al., 2007; Wyzgoski, et al., 2010). In

326

this line, treatment with mixture of these anthocyanin-glycosides at various

327

concentrations, B16-F1 cell metastasis was found to be suppressed by reduction of

328

MMP-2, MMP-9, and NF-κB p65 expressions through the suppression of PI3K/Akt

329

pathway and inhibition of NF-κB levels. Additionally, anthocyanins-rich extract of

330

red raspberry was also found to induce cell cycle block at G1/G0 and G2/M phases

331

and inhibited tumor angiogenesis through down-regulation of VEGF, VEGFR-2,

332

PDGF, PDGFR, HIF-1α, and MMPs, as well as inhibition of phosphorylation of

333

EGFR, VEGFR and PDGFR (Bei, et al., 2009; Hsieh, et al., 2013; Huang, Shih,

334

Chang, Hung, & Wang, 2008). More recently, a group of researchers investigated

335

raspberry extracts for their ability to inhibit the growth of HT-29, Caco-2, MRC5,

336

A549, and H1299 tumor cell lines, indicating that the extract modulated the

337

expression and activation of multiple genes associated with these cellular functions,

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ACCEPTED MANUSCRIPT including genes involved in the PI3K/Akt, ERK, JNK, and MAPK pathways (Hsieh,

339

et al., 2013). However, observations from Cvorovic, et al. (2010) suggested that

340

anthocyanin played a dual role in anticancer effect, which depends on cell type: cells

341

with low basal metabolic rates acted as free radical scavengers and protected them

342

from oxidative stress; in malignant cells, they acted as pro-oxidants by both

343

scavenging ROS and triggering mitochondrial apoptotic pathway (Cvorovic, et al.,

344

2010).

RI PT

338

To date, potential chemopreventive effect of raspberry extract and its

346

anthocyanins have been proved by in vitro cell culture models, animal studies, and

347

even on humans. As researches continue, data including radical scavenging activity,

348

stimulation of phase II detoxifying enzymes, anti-proliferation, anti-inflammation,

349

anti-angiogenesis, anti-invasiveness, and induction of apoptosis and differentiation of

350

raspberry are also constantly emerging. The raspberry anthocyanins modulate the

351

expression and activation of multiple genes involved in the Nfr2-ARE, PI3K/Akt,

352

ERK, JNK, and MAPK pathways, affecting cellular functions. However, in order to

353

predict the chemo-preventive effects of dietary raspberry and its active anthocyanins

354

on different organ sites, tissue-bound anthocyanins should be measured and the role

355

of gut bacteria in the metabolism and uptake of anthocyanins should also be

356

investigated. Finally, more studies should be undertaken to determine if the

357

anti-cancer effects of raspberry are connected with the parent compounds and/or their

358

metabolites.

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360

8. Anti-inflammation potential in vitro

361

Inflammation is usually a protective action by the organism to initiate the healing

362

process and to remove the injurious stimuli. Always, wounds and infections will be

363

cleaned by the defense system of host. But in some pathological situations, chronic

364

inflammation can also lead to diseases of hosts, such as rheumatoid arthritis, hay fever,

365

atherosclerosis, glomerulonephritis, gastroenteritis and etc., sometimes even promotes 13

ACCEPTED MANUSCRIPT the progression of cancer. For that reason, inflammation is normally closely regulated

367

by the body, including the regulation of inflammatory cytokines and mediator

368

secretion. Since those factors can significantly promote the progression of

369

inflammation, it is wildly believed that down-regulated secretion of factors could be

370

useful for the therapy of inflammation-related diseases. With the secretion of

371

inflammatory mediators, such as histamine, leukotriene, PGE2, bradykinin,

372

complement system, thrombin and etc., vascular system got prominent variations,

373

including increased permeability, vasodilation, and slowing of blood flow rate, thus,

374

finally leading to the recruitment and extravasation of leukocytes. Leukocytes,

375

especially granulocytes secrete cytokines of TNF-α, IL-6, IL-1β, promoting the

376

secretions of others inflammatory mediators and recruiting the macrophage to

377

inflammatory site, and then making disease worse. As discussed above, raspberry

378

anthocyanins can act as pro-oxidants to change cellular redox status, resulting in the

379

stimulation of endogenous antioxidant defense systems through Nrf2-ARE pathway.

380

Although Nrf2 is well known for its critical role in endogenous antioxidant defense

381

mechanism, Nrf2 also plays an important role in the regulation of inflammatory

382

pathways. Nrf2-ARE-regulated genes contribute to cellular protection against

383

oxidative stresses and potentiation of antioxidant defense capacity in cells, and

384

modulation of Nrf2-ARE signaling may also have profound effects on the

385

redox-sensitive inflammation-regulating factors, such as NF-κB and AP-1. On the

386

other hand, NF-κB controls various gene expressions when inflammatory responses

387

occur (Q. Li & Verma, 2002). The NF-κB family includes NF-κB1 (p50/p105),

388

NF-κB2 (p52/p100), p65 (RelA), RelB, and c-Rel. In RAW 264.7 cell models, NF-κB

389

is found to be sequestered in the cytosol as a latent form bound to inhibitory proteins,

390

and a set of inhibitors (IκBs) are phosphorylated by IKK after LPS stimulation

391

(Kanarek & Ben ‐ Neriah, 2012). Once phosphorylated, IκB is targeted for

392

degradation by the 26S proteasome, and the free or activated transcription factor of

393

NF-κB is able to translocate into the nucleus and to induce the transcription of

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14

ACCEPTED MANUSCRIPT specific genes, many of which are responsible for inflammation. Pro-inflammatory

395

genes and carcinogens transcribed by NF-κB include IL-6, IL-1β, TNF-α, and cyclin

396

D1. IL-6 and IL-1β induce the expressions of COX2 and iNOS, leading to the

397

productions of PEG2 and NO, respectively (Folmer, et al., 2014). As shown in Table

398

4, several authors have reported that some raspberry anthocyanins showed an

399

inhibitory effect on COX-2 by suppressing C/EBP, AP-1 and NF-κB (Hou, et al.,

400

2004), as well as inhibition of iNOS protein and mRNA expressions in

401

LPS-stimulated RAW 264 cells (Hamalainen, Nieminen, Vuorela, Heinonen, &

402

Moilanen, 2007; Hwang, et al., 2011). Raspberry extract and its anthocyanins in

403

particular, inhibited LPS-induced NF-κB activation and the production of COX-2.

404

Subsequently, the inhibition of COX-2 gene expression could decrease the

405

productions of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α

406

(Muñoz-Espada & Watkins, 2006; J. Wang & Mazza, 2002). Anthocyanin glycoside,

407

cyanidin-3-glucoside was also approved to be able to inhibit iNOS and COX-2

408

expressions by inducing liver X receptor alpha activation in THP-1 macrophages (L.

409

S. Wang & Stoner, 2008), and to inhibit the LPS-induced IκB-α degradation and

410

NF-κB activation, as well as the translocation of p65 subunit of NF-κB into the

411

nucleus in RAW264.7 cells (Min, Ryu, & Kim, 2010). Cyanidin-3-glucoside as well

412

as its metabolites of cyanidin and protocatechuic acid also significantly inhibited the

413

protein expressions of TNF-α and IL-1β, reducing the productions of PGE2 and NO

414

in LPS-treated RAW 264.7 cells (Min, et al., 2010). In this line, if raspberry

415

cyanidin-3-glucoside is orally administered, its metabolites of cyanidin and

416

protocatechuic acid may also express potent anti-inflammatory effects by regulating

417

NF-κB and MAPK activation. Moreover, previous study reported by Hou, et al. (2004)

418

indicated that delphinidin, but not peonidin, could inhibit the activations of MAPKK

419

(SEK and MEK) and MAPK (ERK and JNK), and sequentially suppress AP-1

420

activation and cell transformation. Likewise, raspberry delphinidin can also exert a

421

significant anti-inflammatory activity by inhibiting the degradation of IκB-α, nuclear

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15

ACCEPTED MANUSCRIPT translocation of p65 and phosphorylation of JNK (Hou, Yanagita, Uto, Masuzaki, &

423

Fujii, 2005). In general, the B-ring ortho-dihydroxyphenyl anthocyanin such as

424

delphinidin and cyanidin showed powerful anti-inflammatory activities (Cespedes,

425

Galindo, & Couso, 2010; Shih, Yeh, & Yen, 2007), while pelargonidin, peonidin and

426

malvidin without ortho-dihydroxyphenyl structure failed to show the above activities

427

(Hou, et al., 2005). Moreover, the number of OH at B-ring seems to be related to a

428

molecular conformation influenced the interactions between raspberry anthocyanins

429

and enzymes such as tyrosine kinase and protein kinase C, which are involved in the

430

transcriptional activity of COX-2 (O'Leary, et al., 2004). Indeed, the ortho-dihydroxy

431

structure of anthocyanidins, which is essential for suppressing COX-2 expression, is

432

very similar to those required for the inhibition of tyrosine kinase and protein kinase.

433

The structure-activity relationship studies indicated that ortho-dihydroxyphenyl

434

structure on the B-ring of anthocyanidins is, at least, required to suppress COX-2

435

expression (Triebel, Trieu, & Richling, 2012; Chen, Teng, Fang, & Xiao, 2016).

436

Additionally, some other signaling cascades, which aside from that involved in

437

anti-inflammatory activities, targeted by raspberry and its anthocyanins are also

438

involved in the anti-inflammatory action including PI-3K/Akt, Ras/MAPK, and

439

JAK/STAT pathway (L. S. Wang & Stoner, 2008). The inhibition of NF-κB by

440

raspberries results from the phosphorylation of IκB-α and the inhibition of the

441

PI-K/Akt pathway (Lu, Li, Zhang, Stoner, & Huang, 2006). Nevertheless, Xia and

442

co-authors (Xia, et al., 2007) declared that single anthocyanin isolated from raspberry

443

effectively up-regulated the signaling pathway of the nuclear receptors, such as LXR

444

α and PPAR γ.

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422

446

9. immunomodulatory potential in vivo

447

Immunomodulatory activity is closely related to antioxidant chemo-prevention. It has

448

been suggested that raspberry and its anthocyanins possessed anti-inflammatory as

449

well as chemo-preventive properties. A recent study (Cuevasrodríguez, et al., 2010) 16

ACCEPTED MANUSCRIPT compared anthocyanin-rich with -poor fractions of raspberries in vivo. And the

451

authors showed that the anthocyanin-rich fraction ameliorated symptoms of acute

452

mouse colitis model, which was in a good agreement with the results obtained in

453

LPS-activated RAW264.7 macrophages (suppressed inflammatory NF-kB and AP1

454

signaling, down-streamed gene expression by the anthocyanin-rich fraction). Juices

455

from anthocyanin-rich raspberry significantly inhibited mutagenesis caused by the

456

direct-acting mutagen methyl methane-sulfonate and the metabolically activated

457

carcinogen benzo (a) pyrene in vivo animal models (Hope Smith, et al., 2004). On

458

some levels, in vivo evidence has not been completed on red raspberries, although

459

many berries have compounds in common with red raspberries. However, studies on

460

the effects of other berry species could give an indication of the potential effects of

461

red raspberries. Administration of vitamin E-deficient with relevant levels of

462

cyanidin-3-glycoside at 100 mg/kg for 12 weeks, showed no effect against lipid

463

peroxidation, ROS generation, or cell membrane damage (Duthie, et al., 2005; Chen

464

et al., 2016b). However, a different study, with the same animal model employing 250

465

mg/kg of a bilberry anthocyanin extract, containing some raspberry anthocyanins,

466

significantly enhanced the plasma antioxidant capacity (Talavera, et al., 2006). As

467

well, giving a combination of the 3-glucopyranoside forms of delphinidin, cyanidin,

468

petunidin, peonidin, and malvidin at a concentration of 1 g/kg diet, markedly

469

decreased elevations in DNA damage and hydroperoxides in liver and increased

470

plasma antioxidant capacity (Ramirez-Tortosa, et al., 2001). Decreased oxidative

471

stress and anti-inflammatory effect were found in response to raspberry treatment in

472

vivo (Hope Smith, et al., 2004). In an adjuvant-induced arthritis rat model, animals

473

were gavaged daily with red raspberry extract at 120 mg/kg for 30 days after adjuvant

474

injection, and it was found that the incidence and severity of arthritis, the degree of

475

bone resorption, soft tissue swelling, and osteophyte formation were significantly

476

reduced, and thereby articular destruction in animals was prevented (Jean-Gilles, et al.,

477

2012). It is also worth noticing that high doses of up to 400 mg/kg of tart cherry

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17

ACCEPTED MANUSCRIPT anthocyanins have been previously used for anti-inflammatory animal studies (Ibold,

479

et al., 2007). In a mouse colitis model, dextran sulfate sodium (DSS)-induced weight

480

loss and histological damage were significantly ameliorated by raspberry extract

481

treatment (L. Li, et al., 2014). On the other hand, in gastritis model, animals treated

482

with ellagitannins at a dose of 20 mg/kg/day increased endogenous antioxidant

483

defenses enzymes such as CAT and SOD (Sangiovanni, et al., 2013; Teng, Chen, &

484

Song, 2016). In humans, limited studies have been reported to investigate the effects

485

of red raspberries on oxidative stress and inflammation. A beverage containing

486

raspberry, black grape, and red currant concentrates was used to investigate

487

exercise-induced oxidative stress in cyclists (Morillasruiz, et al., 2005). Results turned

488

out that compared to pre-exercise and control levels, post-exercise levels of protein

489

and DNA oxidation were significantly decreased in the treatment group receiving the

490

antioxidant-rich beverage. Another study (Ramirez-Tortosa, et al., 2001) by using

491

concentrated juices of grape, cherry, and raspberry as a complementary food to

492

adulthood, the intake of this dessert at a dose of 200 g/day for 2 weeks did not change

493

oxidative stress in institutionalized elderly men and women. Although no conclusion

494

can be drawn directly to raspberries, the trials suggest that raspberry may be useful in

495

re-establishing homeostasis from being damaged under stress situations.

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10. Summary of pharmacological activities

498

In vitro studies provided informative evidence for understanding the potential human

499

health implications of plant bioactivity through their targets and mechanisms of action.

500

However, caution should be exercised in interpreting results from in vitro studies

501

because parent compounds are often applied (instead of a mixture of parent and

502

metabolites as expected in vivo) and often at concentrations that far exceed

503

physiological concentrations. Nonetheless, in vivo animal data have supported many

504

of the in vitro findings, suggesting that red raspberry fruit and its individual

505

anthocyanin have various metabolic-stabilizing activities which were associated with

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ACCEPTED MANUSCRIPT 506

improvements on antioxidant effect. Indeed, these preclinical data warranted

507

follow-up research in humans.

508 11. Absorption, metabolism, and bioavailability

510

It is well known that to achieve any effect in specific tissues or organs, these bioactive

511

compounds must be bioavailable, i.e., effectively absorbed from the gut into the

512

circulation and delivered to the appropriate location within the body. Because

513

anthocyanins belong to polar compounds, generally, it is difficult to be absorbed by

514

the digestive tract. Current absorption, distribution, metabolism, and bioavailability

515

data from human studies regarded plasma max concentration of anthocyanin and its

516

glycoside by 0.5-4 h are reached at amounts between 1.4 and 592 nM after doses of

517

60-1300 mg anthocyanins (Czank, et al., 2013). Average urinary excretion is reported

518

between 0.03 and 4 % of the ingested dose, having elimination half-lives of 1.5-3 h

519

(Kay, 2006). For single anthocyanin bioavailability, the serum excretion rate had a

520

max of cyanidin-3-glucoside after 1.8 h, which was similar with cyanidin-3-glucosyl

521

rutinoside (Seymour, et al., 2014). Interestingly, serum cyanidin-3-glucoside was back

522

to baseline at hour 6, faster than cyanidin-3-glucosyl rutinoside. The most abundant

523

raspberry anthocyanins have less than 6% bioavailability of the initial dose ingested

524

(Mcdougall, Dobson, Smith, Alison Blake, & Stewart, 2005; McGhie, Ainge, Barnett,

525

Cooney, & Jensen, 2003; Tian, Giusti, Stoner, & Schwartz, 2006). However, more

526

recently, a pharmacokinetic study using 13C-tracer on the B ring of anthocyanin found

527

that the bioavailability cyanidin-3-glucoside was 12.38 % (with 5.37% excreted in

528

urine and 6.91% in breath), which are more bioavailable than previously perceived in

529

humans (Czank, et al., 2013). When consider colonic metabolites, the bioavailability

530

of anthocyanins and proanthocyanidins of raspberry may range from 12% to 18%

531

(Czank, et al., 2013; Gonthier, et al., 2003). These compounds can be absorbed in the

532

intestine, and further subjected to phase II metabolism in the gut or liver (Kay, 2006;

533

Wu, Cao, & Prior, 2002). Also, spontaneous degradation or microbial catabolism of

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19

ACCEPTED MANUSCRIPT 534

anthocyanins may lead to the formation of phenolic acids, such as protocatechuic acid

535

(in Fig. 3, (Czank, et al., 2013)). Evidence based on in vivo colonic catabolism

536

revealed that, after consumption of berry juice and extract, hippuric acid accounted

537

for

538

3,4-dihydroxyphenylacetic

539

3-(4-hydroxyphenyl) propionic acid (0.09%) in the urinary metabolites (Xie, et al.,

540

2016). Understanding the structural factors that influence bioavailability, absorption

541

and metabolism are essential to determine if the anthocyanins are better absorbed or

542

known active metabolites are formed. The mechanisms of absorption differ from site

543

to site, and they depend on the structure of the molecules being absorbed. Evidence

544

based on in vitro gastric (He, Wallace, Keatley, Failla, & Giusti, 2009) and microbial

545

fermentation studies (SĂĄnchez-PatĂĄn, et al., 2012; Teng, Chen, Fang, Yuan, & Lin,

546

2017; ) suggested that, after ingestion, anthocyanins were likely to be broken down

547

into phenolic degradation products and then further metabolized. Anthocyanin’s intact

548

form, methyl, sulfate and glucuronyl conjugates, or its corresponding phenolic acids

549

can be found in plasma ((McGhie, et al., 2003), Figure 4). Although aglycones can

550

exist in plasma for a short time, they are prone to degrade due to their instability;

551

however, binding with proteins might preserve their intact structures (Hribar & Ulrih,

552

2014). Although anthocyanins can cross the blood-brain barrier, the plasma

553

concentrations of anthocyanins are low, and efficient transport is crucial for their

554

accessibility to tissues. However, besides their reduction of oxidative stress, the

555

mechanisms behind their influence on neuronal activity are not completely

556

understood.

the

total

phenolics,

acid

followed

(0.46%),

by

ferulic

protocatechuic

acid

acid

(0.80%),

(0.15%),

and

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of

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557

98.5%

558

12. Critical considerations and perspective

559

It is worth noticing that a large proportion of published studies were conducted in

560

vitro trials, but as we known that evaluation of antioxidant activity in vitro is

561

significantly up to the assay applied and some assays showed no correlation to any 20

ACCEPTED MANUSCRIPT biological system. Moreover, a portion of in vivo researches employed on raspberry

563

anthocyanin are completed by quite outdated test systems (lipid peroxidation

564

measurement by TBARS in vivo). Therefore, confirmation of in vivo models are

565

indispensable. As for the antioxidative mechanism of raspberry and its anthocyanin,

566

undoubtedly, it is mainly dependent on their redox attributions, which play an

567

important part in neutralization of ROS. Another conceivable mechanism of human

568

self-protection induced by free radicals is likely manifested by binding of metal ions,

569

particularly for Cu (II). Nevertheless, most of studies are likely to disregard the role

570

that raspberry and its anthocyanins could have antioxidant capacity by suppressing

571

radical- generating enzymes or by detoxifying enzymes (such as SOD, GSH, GR and

572

etc.).

573

Another question depends on the concentration levels of sample used in vitro, which

574

are usually several folds above the plasma concentration. Such analogous inaccuracies

575

also exist in some in vivo works on bioavailability evaluation and metabolism study,

576

in which given doses exceeded the average recommended dietary intake.

577

There are other concerns and uncertainties since the cited works on raspberry

578

anthocyanins considered them as both natural chemicals and edible extracts. Under

579

some circumstances, rare or no message was referred about i) species varieties,

580

cultivation information, and purity of the sample; ii) quantitative determinations of the

581

anthocyanins; iii) arrays used for the analysis and, iv) extraction method and

582

afterwards processing. Apparently, the above information is critical for other

583

researchers reproducing the trials and to compare their works. On the other hand,

584

single or few outdated studies could be available involving anthocyanin-rich extracts

585

on the anti-carcinogenic activity, but rarely can find related works on other proposed

586

biological activity such as anti-inflammatory, gastric-protection, and antiviral activity,

587

which are certainly necessary. Hence, in order to find out whether anthocyanins from

588

raspberry are really enable to positively affect the incidence rate and progression of

589

many chronic diseases, vast works in different areas are still required. These involves

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21

ACCEPTED MANUSCRIPT i) extensive studies on detailed information for metabolism of anthocyanin-glycosides

591

in body system; ii) analysis of factors might influence bioavailability of anthocyanins,

592

and if there is any interactions occurred with other dietary compounds (i.e., other

593

polyphenol, protein, fat and etc.); iii) epidemiological studies to assess the

594

relationship between the consumption of anthocyanin-glycoside enriched dishes and

595

pathology incidences. The listed suggestions may help consumers better understand

596

anthocyanin-glycosides contributions to human nutrition. When consider a possible

597

remedial usage of anthocyanin-based functional foods or drugs, relative studies on

598

metabolism, absorption, distribution, and excretion of anthocyanin-glycosides taken

599

by main possible ways (by oral, intravenous, intraperitoneal, or intrathecal) are largely

600

insufficient but indispensable. Better assessment of the pharmacological information

601

on anthocyanin-glycosides, with a distinctive option for possible human diseases to be

602

taken care in the future with anthocyanin-glycoside-based functional foods or drugs,

603

is certainly necessary.

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604 Acknowledgements

606

This research was supported by Major Projects of Science and Technology of Fujian

607

Province (2014NZ0002-1), and the construction project of top university at Fujian

608

agriculture and forestry university of China (Grant No. 612014042 and Grant No.

609

612014043).

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Conflict of interest

612

The authors declare that there are no conflicts of interest.

613 614

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1028

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1033

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Table legends

1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077

Table 1. Anthocyanin composition identified and quantified in raspberries by Ludwig et al. (2015) and Paterson et al., (2009).

Table 2. Raspberry extract and its anthocyanins modulate proteins that involved in the antioxidant effect in cultured cell. The arrow indicates an increase (↑) or decrease (↓) in the levels or activity of the different parameters analyzed.

Table 3. Raspberry extract and its anthocyanins modulate proteins that involved in the anti-cancer effect in cultured cells. The arrow indicates an increase (↑) or decrease (↓) in the levels or activity of the different parameters analyzed.

Table 4. Raspberry extract and its anthocyanins modulate proteins that involved in the anti-inflammatory effect in cultured cells. The arrow indicates an increase (↑) or decrease (↓) in the levels or activity of the different parameters analyzed.

M AN U

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Figure legends Figure 1. The appearance of red raspberry fruit .

Figure 2. Chemical structures of anthicyanins in raspeberry fruit.

Figure 3. Schematic presentation of the biosynthesis of anthocyanins in raspberry.

First phenylalanine reacts with malonyl CoA to produce 4-hydroxycinnamoyl CoA. Under the catalytic control of chalcone synthase 4-hydroxycinnamoyl CoA condenses with three molecules of malonyl CoA to form a chalcone. Chalcone isomerase closes the heterocyclic ring to form naringenin. The B-ring is moved from the 2-position to the 3-position by isoflavone synthase. Isoflavone dehydratase removes water to generate the C-C3 double bond in the heterocyclic ring.

Figure 4. Diagram of the proposed metabolic pathway of cyaniding-base anthocyanins in raspberry as reviewed in this study (Monagas et al., 2010, Wu et al., 2002).

39

ACCEPTED MANUSCRIPT Table 1. Anthocyanin composition identified and quantified in raspberries by Ludwig, et al. (2015) and Paterson, et al. (2013).

Compounds µM/300 g raspberries mg/100 g (Ludwig, et al., 2015) (Paterson, et al., 2013) Total anthocyanins 292±10 Cyanidin-3-O-sophoroside 175±6 25.4 G 7.2 Cyanidin-3-O-(2 -O-glucosyl)rutinoside 56±2 Cyanidin-3-O-glucoside 37±1 3.9 Cyanidin-3-O-rutinoside 20±1 2.3 G Cyanidin-3-O-(2 -O-xylosyl)rutinoside 2.7±0.1 0.06 Cyanidin-3,5-O-diglucoside Pelargonidin-3-O-sophoroside 1.2±0.0 0.1 Pelargonidin-3-O-glucoside 1.1±0.0 0.12

M AN U TE D EP AC C

1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091

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1078 1079

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Cyanidin

50 µM, 24 h

Hepa1c1c7, BPRc

Delphinidin

50 µg/µM, 24 h

HepG2, LS174T

Petunidin

50 µM, 48 h

HepG2, LS174T

Peonidin

HepG2, LS174T

Malvidin

HepG2, LS174T

Pelargonidin

HepG2, HUVECs RAW264.7

Cyanidin-3-O-glucoside

↑GSH, ↓ROS, ↑mitochondrial membrane potential ↑CAT, ↑SOD, ↑Nrf2 siRNA, ↑PPARγ, ↑peroxisomal β-oxidation, ↑the peroxisomal Membrane, ↓ROS, act as Nrf2-ARE signaling transmitter ↑QR activity, ↑HO-1, ↑GSH, ↑GST, ↑γ-GCS, ↑GR ↑luciferase activity, ↑AhR, ↑CYP1A1 mRNA, ↑CYP1A2 mRNAs ↑luciferase activity, ↑AhR, ↑CYP1A1 mRNA, ↑CYP1A2 mRNAs ↑luciferase activity, ↑AhR, ↑CYP1A1 mRNA, ↑CYP1A2 mRNAs ↑luciferase activity, ↑AhR, ↑CYP1A1 mRNA, ↑CYP1A2 mRNAs ↓ROS, ↑glutamate–cysteine ligase catalytic subunit, ↑p-cAMP-response element binding protein (CREB), ↑protein kinase A (PKA) activation, ↓MKK4–JNK, ↓MKK4–JNK–Fas,

M AN U

50 µM, 48 h

EP

50 µM, 48 h

10-50 µM, 48 h

AC C

，

Effect

SC

HepG2

TE D

Cell culture or in Crude extract, pure anthocyanins Treatment vivo model or its fraction Caco-2 Raspberry extract 2 mg/mL, 24 h

RI PT

Table 2. Raspberry extract and its anthocyanins modulate proteins that involved in the antioxidant effect in cultured cells

1-100 µM, 48 h

41

Reference (W. Chen, Su, Xu, Bao, & Zheng, 2016a) (J. M. Lee & Johnson, 2004)

(Jiang, et al., 2014) (Kamenickova, et al., 2013) (Kamenickova, et al., 2013) (Kamenickova, et al., 2013) (Kamenickova, et al., 2013) (Anwar, et al., 2014; A Speciale, et al., 2013; Antonio Speciale, et al., 2010; Zhu, et al., 2012)

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Cyanidin-3-O-(2G-O-glucosyl)

HL-60, CCRF-CEM

Cyanidin-3-O-rutinoside

CGNs (Sprague–Dawley rat)

Pelargonidin-3-O-glucoside

RI PT

HepG2

SC

Cyanidin-3-O-sophoroside

AC C

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HepG2

↑GSH, ↑GSH/GSSG, ↑GCL, ↑Nrf2/ARE pathway, ↑NF-κB p65, ↑SOD, ↑HIF-1α, ↑MMPs, ↑HO-1 50-200 µg/mL, ↑SOD, ↓ROS, ↑Nrf2, ↑P13K, ↓p38, 24 h ↓JNK, ↓ERK, 50-200 µg/mL, ↑SOD, ↓ROS, ↑Nrf2, ↑P13K, ↓p38, 24 h ↓JNK, ↓ERK, 120 µM, 1 h ↓ROS, ↓p38MAP, ↓ASK1 ↓p-MAPKs, ↓Bcl-2, ↑Bax ↓Bcl-xL, ↓JNK, ↓BimEL 100-400 µM, ↓Bcl-2, ↑GSH, ↓lipid peroxidation, 12 days ↓cardiolipin oxidation, ↓Mitochondrial fragmentation, ↓OPA1 cleavage, ↑GSHpx, ↓ERK1/2

42

(Cho, et al., 2015) (Cho, et al., 2015) (Feng, et al., 2007)

(Kelsey, Hulick, Winter, Ross, & Linseman, 2011)

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Cell type

Crude extract, pure Treatment anthocyanins, or its fraction MRC5, Raspberry extract 100 µg/mL, 24 h

Effect

Reference

↓proliferation in the G1 phase,↓MMP-2, ↓u-PA, ↓calpain-2, ↓p-FAK, ↓t-FAK, ↓pSrc,↓ t-Src, ↓p-paxillin, ↓ t-paxillin, f ↓ERK1/2, ↓AP-1 binding activity ↓Cell proliferation, ↓H2O2-induced DNA damage Induction of apoptosis and arrest of cells in G 2 -M Phase, ↓IkB kinase γ (NEMO), ↓p-p65, ↓p-p50, ↓NF-kB DNA binding activity, ↑ caspase activity, ↓NF-kB/p65, ↓Bcl2, ↓Ki67, ↓PCNA, ↓p-ERK, ↓p-JNK, ↓MEK, ↓SEK, ↓AP-1 activity, ↓CYP2A6 ↓Cell proliferation ↓AP-1 activity ↓CYP2C9 enzyme, ↓cytochromes P450 ↓CYP2A6, ↓CYP2B6,

(Hsieh, et al., 2013)

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12.5-200 µg/mL, 24 h 100 µM, 30 min 0-180 µM, 48 h

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AGS, HCT-116, NCI-H460, Cyanidin MCF-7, SF-268, HT-29 PCa LNCaP, C4-2, 22R N 1, Delphinidin PC3 cells, LH44, LH45, HEP220670

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Table 3. Raspberry extract and its anthocyanins modulation of proteins involved in the anti-cancer effect in cultured cells

MCF-7

Petunidin

0-20 µM, 30 min

LH44, LH45, HEP220670

Peonidin

0-100 µM, 30 min

(Glei, et al., 2003) (Hafeez, et al., 2008; Hou, et al., 2004)

(Hou, et al., 2004; Zhang, et al., 2005) (Srovnalova, et al., 2014)

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In vitro chemical analysis

HL-60, MOLT-4, Daudi

LNCaP, A549

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MCF-7

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RAW 264.7, AGS

↓CYP2C9, ↓CYP3A4 Malvidin 0–100 µM, 30 min, ↓ROS, ↓NF-kB, ↓PARP, 500 µM, 4-16 h ↓MAPK, ↑MKP-1 ↓PI-3K-Akt, ↓AP-1 activity, ↑p38, ↓Erk, ↓p-Erk, ↓p-p38, ↑apoptosis Pelargonidin 0-20 µM, 30 min ↓Cell proliferation ↓AP-1 activity Cyanidin-3-O-glucoside 500 µM, 4-16 h ↓MMP-2, ↓urokinase-type plasminogen activator (u-PA), ↑TIMP-2, ↑PAI, ↓c-JNK, ↓NF-kB, ↑p38, ↓Erk Cyanidin-3-O-sophoroside 0-100 µM, 1 h ↓CYP2C9 enzyme,↓cytochromes P450 ↓CYP2A6, ↓CYP2B6, ↓CYP2C9, ↓CYP3A4 G Cyanidin-3-O-(2 -O-glucos 250 µg/mL, 1 h ↓lipid peroxidation, ↓COX-1, yl) rutinoside containing ↓COX-2 fraction of raspberry Cyanidin-3-O-rutinoside 120 µM, 1 h ↓Caspase-3, ↓caspase-9 activity,↑p-p38, p-MAPK, ↑p-JNK, ↓ Bcl-2, ↓Bcl-xL ROS, ↑Bim, ↑Bax, ↑Bak G Cyanidin-3-O-(2 -O-xylosyl 500 µg/mL, 8 days ↓ROS, ↑IL-6, ↑TNF-α,↓Cell )rutinoside containing proliferation

(Bognar, et al., 2013; Hou, et al., 2004; Meiers, et al., 2001; Shih, et al., 2005) (Hou, et al., 2004; Zhang, et al., 2005) (P. N. Chen, et al., 2006; Shih, et al., 2005) (Srovnalova, et al., 2014)

(Mulabagal, et al., 2009) (Duncan, 2009; Feng, et al., 2007)

(Choung, 2012)

et

al.,

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↓GSH,↓GST,↓GPx, ↓GRd,↓Caspase-3, ↑Nrf2-ARE,↑PI3K

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50 µM , 24 h

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CRL-1439

fraction of raspberry Pelargonidin-3-O-glucoside

(Shih, et al., 2007)

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Table 4. Raspberry extract and its anthocyanins modulate proteins that involved in the anti-inflammatory effect in cultured cells

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Cell culture or in vivo Crude extract, pure anthocyanins, Treatment Effect model or its fraction 50 µg/mL, ↓NO, ↓iNOS, ↓COX-2, RAW264.7, Bovine nasal Raspberry extract ↓IL-1β, explants 69 min ↓IL-6, ↓NF-κB, ↓AP-1, ↓IKK,↓IκBα, ↓p65 ↓JNK, ↓MAPKs RAW 264.7 Cyanidin 5 µM ↓TNF-α, ↓IL-1β, ↓NO, ↓PGE2, 60 min ↓iNOS, ↓COX-2, ↓NF-κB, ↓IκBα HCT116 Delphinidin 30-240 µM ↓Bax, ↑Bcl-2, ↓PARP, ↓IKKα, 48 h ↓IκBα, ↓p-IκBα,↓ p-p65, ↓nuclear translocation of NF-κB/p65, ↓NF-κB/p65 DNA binding activity, ↓NF-κB HaCaT Peonidin 10-200 µM, ↓iNOS activity, ↓iNOS protein 1h expression, ↓IκBα degradation,↓p-ERK1/2, ↓NF-κB, ↓TNF-α, ↓IL-1β, ↓NO, ↓PGE2,↓ ICAM-1, ↓COX-2, ↓p65, ↑p-IκBα RAW264.7 Malvidin 0-100 µM, ↓NF-κB, ↓ROS,↓JNK, ↓ERK1/2, ↓ 30 min p38, ↑MAPK phosphatase-1, ↑Akt, ↑GSK-3ß,↑mitochondrial membrane potential Primary cultures of glial Pelargonidin 0.3 µM, 24 ↓TNF-α, ↓IL-1β, ↓NO, ↓PGE2, cells (astrocytes and h ↓iNOS, ↓COX-2, ↓NF-κB, ↓IκBα, microglia ↓iNOS, ↓p38, ↓STAT-1

Reference (Jean-Gilles, et al., 2012; L. Li, et al., 2014) (Min, et al., 2010) (Yun, Afaq, Khan, & Mukhtar, 2009)

(Min, et al., 2010)

(Bognar, 2013)

et

al.,

(Vafeiadou, et al., 2009)

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Human monocytic cell line, A mixture of 3-O-rutinosides of Clinical trial (61women, 59 cyanidin and delphinidin, and men) 3-O-β-galactosides, 3-O-β-glucosides, and 3-O-β-arabinosides of cyanidin, peonidin, delphinidin, petunidin, and malvidin).

10-80 µM, ↓iNOS activity, ↓iNOS protein 30 min; 5 expression, ↓IκBα µM, 60 min degradation,↓p-ERK1/2, ↓NF-κB, ↓TNF-α, ↓IL-1β, ↓NO, ↓PGE2,↓ ICAM-1, ↓COX-2, ↓p65, ↑p-IκBα 100 µg/mL, ↓NF-κB, ↓IL-13, ↓IL-4, ↓p65 30 min ↓IL-1b, ↓IL-1 receptor antagonist 300 mg (IL-1Ra), ↓IL-2, ↓IL-4, ↓IL-6, ↓IL-8, anthocyanin ↓IL-10, ↓IL-12, ↓IL-13, ↓ IL-17, s/d; 100 g ↓TNFa, ↓IFNa, ↓IFNg, of fresh ↓granolyte/macrophage mixture colony-stimulating factor (GMCSF), ↓macrophage inflammatory protein (MIP)1-α, MIP-1β, immunoprotein ↑(IP)-10, ↓monocyte chemoattractant protein-1 (MCP-1), ↓eotaxin, and regulated upon activation, ↑normal T cell expressed and secreted (RANTES)

RI PT

264.7, Cyanidin-3-O-glucoside

SC

RAW

M AN U

J774, HaCaT

(Kim, et al., 2012; Min, et al., 2010; Pergola, Rossi, Dugo, Cuzzocrea, & Sautebin, 2006) (Karlsen, et al., 2007)

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ACCEPTED MANUSCRIPT Highlights Anthocyanins showed the promissory effects on oxidative-stress related diseases Raspberry fruit is a potential source for anthocyanins, but studies are scarce.

Raspberry anthocyanin is important for Food and Pharmaceutical industries

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Chemo-preventative and anti-inflammatory effects of raspberry were summarized.

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Several factors affect bioavailability and bio-efficacy of anthoxyanins