Hydrolysis of Dicaffeoylquinic Acids from Ilex kudingcha Happens in

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Hydrolysis of Dicaffeoylquinic Acids from Ilex Kudingcha Happens in Colon by Intestinal Microbiota Minhao Xie, Guijie Chen, Bing Hu, Li Zhou, Shiyi Ou, Xiaoxiong Zeng, and Yi Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04710 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Journal of Agricultural and Food Chemistry

Hydrolysis of Dicaffeoylquinic Acids from Ilex Kudingcha Happens in Colon by Intestinal Microbiota Minhao Xie,† Guijie Chen,† Bing Hu,† Li Zhou,† Shiyi Ou,‡ Xiaoxiong Zeng ,†,* Yi Sun†,* †

College of Food Science and Technology, Nanjing Agricultural University, Nanjing

210095, China ‡

Department of Food Science and Engineering, Jinan University, Guangzhou 510632,

Guangdong, China

*

To whom correspondence should be addressed. Tel.: +86-25-84396791, E-mail: [email protected] (X. Zeng), [email protected] (Y. Sun) 1

ACS Paragon Plus Environment


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ABSTRACT

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Monocaffeoylquinic acids (mono-CQAs) can be hydrolyzed or metabolized by

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pancreatin, intestinal brush border esterase and microbiota in colon. Data about

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conversion of dicaffeoylquinic acids (diCQAs) in digestion is scarce. DiCQAs-rich

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fraction including 3,4-, 3,5- and 4,5-diCQAs was prepared from Ilex kudingcha and

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the conversion in simulated gastricintestine was investigated. Artificial saliva, gastric

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and pancreatic fluids, Caco-2 monolayer cell and anaerobic fermentation model were

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utilized to mimic digestions of oral cavity, stomach, small intestine and colon in vitro.

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The results revealed that diCQAs remained intact in simulated saliva, gastric and

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pancreatic fluids and within Caco-2 cells. In anaerobic fermentation with human fecal

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slurry, diCQAs were hydrolyzed to mono-CQAs and caffeic acid which were further

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metabolized to caffeic acid and dihydrocaffeic acid, respectively. The hydrolysis of

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diCQAs depended on the chemical structures, carbohydrates in culture medium and

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microbial compositions. Our research demonstrated that hydrolysis of diCQAs

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happened in colon by intestinal microbiota.

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Keyword: Kudingcha; Dicaffeoylquinic acid; Digestion in vitro; Intestinal microbiota

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INTRODUCTION

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Kudingcha, mainly made from the leaves of Ilex genus plants including I. kudingcha

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C.J. Tseng, I. latifolia Thunb and I. cornuta Lindl. ex Paxt, is a popular herb beverage

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in China and some other countries of Southeastern Asia (e.g., Singapore, Malaysia

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and Vietnam).1,2 It is widely consumed as an alternative to green tea made from the

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leaves and buds of Camellia sinensis.1 Kudingcha has the reputation to disperse

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wind-heat, clear toxins from blood, refresh mentalities, prevent deterioration of brain

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and heart functions, and maintain proper body weight in traditional Chinese

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medicine.1,3 Furthermore, kudingcha has been reported recently to possess functions

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of antioxidant, hepatoprotective, antiinflammatory, antidiabetic, antimetastatic,

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anticancer activities and prevention of gastric injury, neuronal damage and metabolic

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disorders.2-10 The diverse biological activities depend on the active components in

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kudingcha, such as triterpenoids, alkaloids, polysaccharides, essential oils, flavonoids,

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and phenolic acids.3,11

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Kudingcha polyphenols consist of coumaroylquinic acids, quercetin 3-rutinoside

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(rutin), kaempferol 3-rutinoside (nicotiflorin), caffeoylquinic acids (CQAs) and CQA

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methyl esters.1,12,13 It has also been reported that there are coumaroylquinic acids

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(CoQAs), feruloylquinic acids (FQAs), caffeoylferuloylquinic acids (CFQAs),

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caffeoyl-sinapoylquinic acids, caffeoylshikimates and caffeoylquinolactones (CQLs)

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in I. paraguariensis (Mate tea).14-16 CQA derivatives, 3-CQA, 4-CQA, 5-CQA,

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3,4-di-O-caffeoylquinic acid (3,4-diCQA), 3,5-diCQA, and 4,5-diCQA, are the

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principal proportion and account for more than 90% of total polyphenols of

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kudingcha.1,17-19 CQAs consist of a family of esters formed between caffeic acid (CA)

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and quinic acid (QA), and the acylation may be at C-3, C-4 and/or C-5 position of the

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moiety of QA. Due to different substitute degree and position of caffeoyl group,

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CQAs comprise many isomers, analogs and epimers, and the structures (nomenclature

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according to IUPAC17,20) of the compounds mentioned above are illustrated in Figure

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1. Nowadays, a growing body of evidence has demonstrated that CQAs have

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antioxidant, antimicrobial, inhibiting α-glucosidase, attenuating diabetes, anti-obesity

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and anti-inflammatory activities.17,21-25

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Stalmach et al.26 reported that more than 70% of ingested chlorogenic acids and

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their metabolites were found in ileal fluid. It has also been reported that some coffee

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components and chlorogenic acid metabolites, including sulfates of CA, ferulic acid

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and CQLs, reacheded peak plasma concentrations within 1 h in coffee ingested

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subjects, indicating their absorption in small intestine. In contrast, dihydroferulic acid

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and its 4-O-sulfate, dihydrocaffeic acid and its 3-O-sulfate needed a longer time (> 4 h)

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to reach maximum plasma concentration, indicating their absorption in large intestine

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and the possibility of involved catabolism by colonic microbiota.27 CQAs could be

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uptake in stomach and upper digestive tract.28 However, CQAs have so seldom

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bioavailability in their intact forms that lower than 1% for mono-CQAs and trace of

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di-CQAs could be absorbed.29 Therefore, the metabolism and digestive conversion of

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CQAs in gastrointestine may play a crucial role in exerting their bioactivities. In small

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intestine, 5-CQA and 3-CQA could be transformed to CA by pancreatic esterase and

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brush border esterase.30 Mono-CQAs could be also degraded by colonic microbiota

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and be hydrolyzed to QA and CA, and CA could be further converted to

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dihydrocaffeic acid (DHCA) and other metabolites.31 Coffee is the most popular

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beverage in Western countries, and CQAs are abundant in coffee, especially

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mono-CQAs. As diCQAs are limited in coffee, they have caught less attention in

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previous publications and scare data on transformations of diCQAs in gastrointestine

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have been reported. Kudingcha is rich in diCQAs, and it is a good option to be

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utilized to study the hydrolysis of diCQAs in digestive tract. In the present study,

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therefore,

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chromatography of macroporous resin HP-20 column. Then, enzymatic reactions were

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employed to mimic the digesting progress of diCQAs in oral cavity, stomach and

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small intestine. Furthermore, human colon adenocarcinoma (Caco-2) monolayer cell

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model was applied to examine the diCQAs metabolism by bound esterase of epithelial

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cells. Finally, anaerobe fermentation with fecal slurry was used to investigate the

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potential hydrolyzing function of intestinal microbiota on diCQAs.

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MATERIALS AND METHODS

diCQAs-rich fraction

was firstly prepared from kudingcha

by

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Materials and Reagents. α-Amylase, pepsin, pancreatin, CA, DHCA, and methanol

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of high performance liquid chromatography (HPLC) grade were purchased from

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Sigma Chemical Co., Ltd. (St. Louis, MO, USA). Standards of 3-CQA, 4-CQA,

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5-CQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA were obtained from Chengdu

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Biopurify Phytochemicals Ltd. (Chengdu, China). Folin-Ciocalteu reagent and formic

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acid of HPLC grade were purchased from Aladdin Industrial Inc. (Shanghai, China).

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Fructooligosaccharide (FOS) was provided by Quantum Hi-Tech Biological Co., Ltd 5



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(Jiangmen, China). Caco-2 cell line was obtained from Shanghai Cell Bank of

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Chinese Academy of Sciences (Shanghai, China). 12-Well Transwell plates fitted with

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polycarbonate semi-permeable inserts of pore size 0.4 µm and area 1.12 cm2 were

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obtained from Corning Costar Corp. (Corning, NY, USA). Kudingcha made from the

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leaves of I. kudingcha was obtained from Hainan Yexian Bio-Science Co. (Haikou,

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China). All the other chemical reagents were of analytical grade.

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Preparation of diCQAs-rich Fraction from Kudingcha. The diCQAs-rich fraction

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was prepared from kudingcha as described previously.12 Kudingcha was soaked in hot

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water with a solid-liquid ratio of 1:10 (w/v) at 96 ℃ for 30 min, and then the extract

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was filtered and centrifuged. The resulting supernatant was freeze-dried to afford

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crude kudingcha extract. The extract was dissolved in water and loaded onto a column

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of macroporous HP-20 resin (Mitsubishi Chemical Corp., Tokyo, Japan). The column

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was eluted with pure water for at least 4 bed volumes (BV). After that, ethanol

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solution (70%, v/v) was employed to elute down the target kudingcha diCQAs, and

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the eluted fraction solution was rotary evaporated under vacuum and freeze-dried,

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affording the diCQAs-rich fraction. The chromatography procedure was monitored

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with a C-635 UV photometer (BUCHI, Switzerland) at wavelength of 325 nm.

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Determination of Total Polyphenols Content. The total polyphenols content of

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diCQAs-rich fraction was measured by Folin-Ciocalteu method as previously

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reported.19 Briefly, the sample of diCQAs-rich fraction was dissolved in water to a

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concentration of 0.1 mg/mL. An aliquot of sample of 0.5 mL was mixed with 1.0 mL

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of Folin-Ciocalteu reagent solution and kept at 30 ℃ for 5 min in dark. After that,

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2.0 mL of Na2CO3 solution (200.0 g/L) was added, and the mixture was shaken gently

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for 1 h before the absorbance at 747 nm was detected with a V-1200

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spectrophotometer (Mapada Instruments, Shanghai, China). A calibration curve was

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established with 3,5-diCQA as the standard.

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Simulated Saliva Digestion. Saliva digestion in vitro was performed according to

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the reported method32 with some modifications. The simulated saliva solution

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consisted of sodium chloride (NaCl, 0.12 g/L), potassium chloride (KCl, 0.15 g/L),

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mucin (1.0 g/L) and α-amylase (2.0 g/L). The diCQAs-rich fraction was dissolved in

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the simulated saliva solution to a final concentration of 1.0 mg/mL. The digesting

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system was kept at 37 ℃ in triplicate with a shake at 60 rpm. An aliquot for further

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analysis was collected at 0, 10, 20, 30 and 60 min after incubation. A saliva fluid with

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boiling inactivated enzyme was used as control.

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Gastric Digestion in vitro. The gastric fluid was prepared as described.32 The

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diCQAs-rich fraction was introduced to the simulated gastric solution (NaCl 4.5 g/L,

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mucin 1.5 g/L and pepsin 2.5 g/L, pH was adjusted to 1.8 with 1.0 M HCl solution),

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and the gastric digestion was carried out as described above in saliva digestion. An

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aliquot was collected every 30 min for further analysis. A pepsin-inactivated gastric

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fluid was also used in the experimental section to eliminate the hydrolysis effect of

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the pH.

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Simulated Pancreatic Digestion. The simulated pancreatin solution, consisting of

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NaCl 5.4 g/L, calcium chloride (CaCl2) 0.33 g/L, KCl 0.65 g/L, phosphate buffer

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saline (PBS, pH 7.0) 5 mM, bile salt 10.0 g/L and porcine pancreatin 35.0 g/L, was

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prepared according to reported method with some modifications.30,33 The pancreatic

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digestion was carried out at 37 ℃ in triplicate, and an aliquot was collected every 30

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min. A simulated pancreatic solution was boiled to inactivate the digestive enzymes

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and used as control.

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Caco-2 Cell Culture and Metabolism. The Caco-2 cell metabolism experiments

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were performed as previously published literatures.30,34 The Caco-2 cells were

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cultured in dulbecco's modified eagle medium (DMEM) supplied with 10% fetal

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bovine serum, 100 IU penicillin and 100 mg/L streptomycin at 37 ℃ under 5%

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CO2-humidified atmosphere. Cell viability test was performed by using the

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methylthiazolyldiphenyl-tetrazolium bromide (MTT) method to examine the cytotoxic

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effects of 3,4-diCQA, 3,5-diCQA and 4,5-diCQA to Caco-2 cells. The Caco-2 cells

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were seeded into Transwell inserts at a density of 5000 cells/well. After incubation for

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22 days, a Millicell ERS volt-ohm meter fitted with a chopstick probe (Millipore,

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USA) was used to record the trans-epithelial electrical resistance (TEER) to confirm

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the integrity of the Caco-2 monolayer cell membrane. Before metabolism study,

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DMEM medium was replaced with transport buffer (Hank’s balanced salts solution

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supplied with 1.8 mM CaCl2 (modified HBSS), pH 7.2) in the apical (0.5 mL) and

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basal (1.5 mL) compartments, and the plates were incubated in CO2 cell incubator for

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15 min to allow equilibration. Apical and basal solutions were carefully removed and

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0.5 mL of sample solution (3,4-, 3,5- or 4,5-diCQA dispersed in modified HBSS) was

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added to the apical compartment, while the basal solutions was transport buffer only.

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An aliquot for further analysis was collected at 0, 50, and 100 min of incubation,

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respectively.

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Intestinal Microbiota Conversion in vitro. The intestinal microbiota metabolism of

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diCQAs-rich fraction was mimicked in an anaerobic fermentation model with fecal

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microbe as reported with some modifications.35,36 The fecal samples were obtained

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from 2 healthy volunteers (2 males, 22 or 28 years old) who did not have any

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gastrointestinal disorders or get any antibiotics over the last 3 months. The fecal slurry

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was prepared by mixing fresh fecal samples with autoclaved modified physiological

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saline solution (NaCl 9.0 g/L, cysteine-HCl 0.5 g/L) to yield 10% (w/v) suspensions.

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The diCQAs-rich fraction was dissolved in autoclaved basal nutrient growth medium

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(peptone 2.0 g/L, yeast extract 2.0 g/L, NaCl 0.1 g/L, K2HPO4 0.04 g/L, KH2PO4 0.04

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g/L, MgSO4 0.01g/L, CaCl2 0.01 g/L, NaHCO3 2.0 g/L, hemin 0.02 g/L, cysteine-HCl

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0.5 g/L, bile salts 0.5 g/L, resazurin 1.0 mg/L, Tween 80 2.0 mL/L, vitamin K1 10

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µL/L, pH adjusted to 7.0 with HCl solution) supplied with different carbon sources

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(glucose, sucrose, FOS or inulin, 10.0 g/L). The intestinal microbiota digestion

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reactions in flasks contained 2.5 mL of the fecal slurry and 22.5 mL of nutrient growth

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medium. After mixed well, the flasks were settled in a MGC AnaeroPack®-Anaero

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system (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) immediately and

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incubated at 37 ℃, and an oxygen indicator (Mitsubishi) was utilized to confirm that

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the oxygen content in the sealed box was lower than 0.1%. During the fermentation,

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gentle shaking was done every 6 h in order to make the fermentation system

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homogenous as possible. After fermentation of 24 h, an aliquot was collected for

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following analysis.

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Analysis of diCQAs and Metabolites. Analysis of the metabolites resulting from

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different digestion steps was performed with a HPLC system (Shimadzu Corp., Kyoto,

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Japan).12 The HPLC systems consisted of a DGU-20A degasser, LC-20AD pump, a

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CTO-20A column oven, a SIL-20A auto sampler and a SPD-M20A diode array

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detector. Chromatographic separation was achieved on a TSKgel ODS-80 TsQA

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column (4.6 × 250 mm, 5 µm, TOSOH, Japan) using a 42-min gradient of solvent A

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(water), B (methanol), and C (1% formic acid) based on a previously established

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method as follows: 0-24 min, A from 60% to 35%, B from 20% to 45%, C kept at

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20%; 24-42 min, A, B, and C kept at 35%, 45% and 20%, respectively. The flow rate

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was 0.5 mL/min, and the column oven was set at 40 ℃. The detecting wavelengths

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were set at 280 and 325 nm. The quantity of diCQA was determined according to its

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peak area at wavelength of 325 nm, and the retention ratio was calculated as the value

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of the content of diCQA after digestion to its initial content.

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Statistical Analysis. Data were expressed as the mean ± standard deviation (SD) of

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triplicates. The least significant difference (LSD), Duncan’s multiple range test, and

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one-way analysis of variance (ANOVA) were used for multiple comparisons. P < 0.05

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was considered to be of statistically significant difference.

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RESULTS AND DISCUSSION

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Stability of diCQA in Stimulated Saliva, Gastric and Pancreatic Digestion. In the

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present study, HP-20 column chromatography was applied to prepare the targeted

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diCQAs-rich fraction, and Folin-Ciocalteu reaction and HPLC analysis showed that

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diCQAs were the dominant components of the diCQAs-rich fraction. The obtained 10


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diCQAs-rich fraction promised 3,4-, 3,5- and 4,5-diCQAs, but the other diCQAs and

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derivatives such as 1,3- and 1,5-diCQAs and 3,5-dicaffeoyl-epi-QA n-butyl ester,

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which had been detected in Ilex spp,15,16,37 were not detected. Furthermore, the

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fraction was free of mono-CQAs and CA, and it would not interfere in the further

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experiments. Thus, it was utilized in the following experiments.

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The digestive tract is filled with diverse hydrolytic enzymes responsible for

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digestion of food components, such as proteases, glycoside hydrolases and esterases.

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Considering the chemical structures of diCQAs, they could be potentially hydrolyzed

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by digestive enzymes, especially the pancreatic enzymes as they consist of various

202

lipases, peptidases, amylases and carboxylesterases. It has already been reported that

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pancreatic esterases are able to catalyze the hydrolysis of 5-CQA to CA and QA.30

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The retentions of diCQAs in stimulated saliva, gastric and small intestine fluid

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incubated for different periods were summarized as shown in Table 1, and the HPLC

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profile is showed in Figure 2. The three diCQAs compounds, 3,4-diCQA (peak 1),

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3,5-diCQA (peak 2) and 4,5-diCQA (peak 3), remained about 100% after simulated

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digestions in saliva, gastric and pancreatic fluids. No significance was observed

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between the initial amount and that for stimulated digestion of 1 or 3 h of diCQAs.

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There were also no new chromatographic peaks observed in treatments by stimulated

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digestions, especially peaks of mono-diCQAs and CA. The results indicated that

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diCQAs could remain undigested and intact in saliva, gastric and pancreatic fuilds,

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and α-amylase, pepsin, and pancreatic enzymes were not able to hydrolyze diCQAs.

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It has been reported that no metabolites are detected in stomach and small

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intestine compartments after digestion of a mixture of 5-CQA, CA, ferulic acid, and

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rutin in a multi-reactor gastrointestinal model for 24 h.38 Our present results share the

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similar tendency with the reported data. 5-CQA, however, can be hydrolyzed to CA

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by pancreatic esterases.30 Galloylshikimic and galloylqunic acids including tetra-, tri-,

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di- and mono-galloylquinic acids would be hydrolyzed in stomach and small

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intestine.39 Compared to mono-CQAs, diCQAs have one more caffeoyl group, which

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makes diCQAs larger space size and alters the interaction between the phenolic

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molecules and proteins. The additional caffeoyl group may affect diCQAs as the

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substrates of the digestive enzymes. In addition, diCQAs exhibit higher affinity to

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proteins, resulting in more possibility to interact with proteins. CQAs can bind to

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α-glucosidase and pancreatic lipase, and noncompetitively and competitively inhibit

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their activities, respectively.17,40 The diCQAs could be docked into the catalytic triad

227

of pancreatic lipase, but the lipase could not hydrolyze the ester bonds of diCQAs. In

228

our experiments, the proteins present in simulated digestions exhibited little impact on

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the results, but in some cases the different proteins present in study might affect the

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digestion. It has been reported that the characteristic proline-rich proteins were able to

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bind phenolic compounds including tannins and CQAs,41 and the binding might be

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expected to impair enzymatic modifications.

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The phenomena of acyl migrations, degradations and conjugate addition of water

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in CQAs have been reported in coffee brewing or boiling water bath process, but they

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had not been observed in the present study. CQAs undergo reversible acyl migration

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in aqueous acidic and basic conditions, and the acyl migration process is strongly pH

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dependent.42 Both mono- and di-CQAs will be reduced at 100 ℃, and their thermal

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stabilities are different from that in alkaline aqueous condition.43 During coffee

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brewing or CQAs heated, water can be conjugate added to the olefinic moiety of

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CQAs to form the water adducts, hydroxydihydrocaffeoylquinic acids.44 The acyl

241

migration and hydrolysis are pH and temperature dependent actually.45 In our study,

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the temperature (37 ℃) was much lower than 100 ℃, and pH was neutral or close to

243

neutral except simulated gastric condition with the pH value at 2. CQAs would be

244

stable in the moderate conditions, and the mild conditions were not beneficial to acyl

245

migration or hydrolysis. Our tests were performed with a combination of three

246

diCQAs, and the simultaneous presence of the three compounds would inhibit their

247

conversion to each other with equilibrium to some extent. In addition, the interactions

248

between diCQAs and proteins in the conditions could also contribute to restrain acyl

249

migration.

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Metabolism in Caco-2 Cells. In order to investigate the cellular hydrolysis of

251

diCQAs, Caco-2 cells were seeded in transwell plates to form cell monolayer. The

252

final monolayer resistances corrected for cell-free membrane resistance were detected

253

to validate the monolayer integrity. As results, the TEER values of test wells were 452

254

± 46 Ω·cm2 (> 400 Ω·cm2) after incubation for 22 days, which was indicative of an

255

integral monolayer with established tight junctions and a well-developed apical-brush

256

border.34 In addition to hydrolysis, the Caco-2 transwell model was also used to check

257

the trans-monolayer transportation of diCQAs.

258

As shown in Table 2, the concentrations of diCQAs in the apical sides after

13



259

incubated for 50 and 100 min remained constant compared to original conditions. No

260

diCQAs were detected in the basal compartments. The data demonstrated that Caco-2

261

cells were not able to hydrolyze diCQAs in 100 min, and the brush border esterase

262

had no potential to catalyze the hydrolysis of diCQAs. Slight part of 3-CQA has been

263

reported to be hydrolyzed to CA by Caco-2 brush border esterase,30 while in our case

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no hydrolyte CA had been detected in all the test wells. Besides, diCQAs were seldom

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transported across Caco-2 monolayer. Similarly, other six dietary polyphenols (CA,

266

chrysin, gallic acid, quercetin, resveratrol and rutin) are poorly transported across

267

Caco-2 cells.46

268

Hydrolysis and Metabolism of diCQAs by Colonic Microbiota in vitro.

269

Anaerobic fermentation in vitro was utilized to mimic the degradation of diCQAs by

270

colonic microbial species. The hydrolysis ratios of diCQAs by fecal microbiota from

271

the two volunteers are shown in Table 3. Obviously, diCQAs were able to be

272

hydrolyzed by fecal microbiota as retention of diCQAs decreased significantly. When

273

cultured with sucrose, FOS or inulin, about 40% 3,4-diCQA and near 20% of

274

4,5-diCQA were hydrolyzed by the microorganisms from volunteer 1 compared to

275

control (anaerobic medium without fecal slurry). 3,5-DiCQA, however, was seldom

276

hydrolyzed except when it was incubated in medium with sucrose, only 5 % of

277

3,5-diCQA was degraded in such condition. Considering the results of simulated

278

saliva, gastric, small intestine digestions, it meant that colon was the main site of

279

hydrolysis of diCQAs, and the metabolism was attributed to colonic microbiota. The

280

hydrolysis rates for the three diCQA compounds were different, and hydrolysis rates

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of diCQAs decreased in order of 3,4-diCQA > 4,5-diCQA > 3,5-diCQA. It showed

282

that the abilities of intestinal microbiota to catalyze diCQAs were not the same. The

283

ester bonds between QA and CA of diCQAs could be broken down by colonic

284

microbiota, and the breakage depended on the caffeoyl positions. The hydrolysis rates

285

are associated with phenolic membrane transporters and bacterial intracellular

286

enzymes. The difference could depend on the composition of microbial community.

287

The chromatographic profiles of microbial metabolites are exhibited in Figure 3 (data

288

for volunteer 2 not shown). As illustrated in the figure, the peaks of diCQAs (1, 2, and

289

3) were reduced, and peaks of esterase-catalyzing products of diCQAs, mono-CQAs

290

(4, 5, and 6) and CA (8), appeared. As previously reported, mono-CQAs can

291

converted to CA and QA, and CA can be further metabolized to DHCA and following

292

metabolites.31 The appearance of DHCA (peak 7) confirmed the hydrolysis of diCQAs

293

and explained why the peak areas of mono-CQAs and CA were so small. Furthermore,

294

the hydrolyzing abilities of the colonic microbiota from the two volunteers were

295

different, and microbiota from volunteer 1 exhibited more powerful metabolizing

296

potential than that from volunteer 2 (Table 3). It meant that individuals would have

297

different potential to catalyze polyphenols in their colons due to diverse gut

298

microbiota community compositions. The results of fermentation with different

299

carbohydrates were distinct, indicating that the carbon resource significantly affected

300

the polyphenol-metabolizing abilities of colonic microbiota. Thus, the dietary patterns

301

would have impact on degradation of polyphenols in colon since foods is the main

302

resource of carbohydrates for gut microbiota. Distinct carbohydrates may activate

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different metabolism pathways and affect the secondary metabolism. In addition,

304

dietary patterns shape the gut microbiome,47 which is the pool for metabolizing genes

305

and enzymes, and this would also be the reason of the effect of carbohydrates.

306

A major part of ingested dietary polyphenols arrives at colon in intact form, where

307

they undergo further metabolism to metabolites by the resident microbiota.48 The

308

microbial conversions incorporate depolymerization, hydrolysis, reduction, oxidation,

309

dehydrogenation, dehydroxylation and ring-fission.49 Tea catechins are typically

310

metabolyzed to specific hydroxyphenyl-γ-valerolactones.50 The mixture of 5-CQA,

311

CA, ferulic acid, and rutin are transformed to metabolites including dihydrocaffeic,

312

phenylpropionic, phenylacetic and vanillic acids in colonic vessels.38 The polyphenols

313

of

314

3-(4-hydroxyphenyl)propanoic acid, and several undefined compounds are degraded

315

rapidly within 10 h by microbial metabolism.51 Chebulic ellagitannins are metabolized

316

to urolithin during microbial transformation. Polyphenol degradation patterns are

317

different in ascending colon, transverse colon and descending colon, due to their

318

dependence on microbial communities.52 5-CQA could be degraded to CA, and CA

319

would be metabolized to DHCA, 3-(3’-hydroxyphenyl)propionic acid and further

320

metabolites successively.31 In our present study, diCQAs were firstly degraded to

321

mono-CQAs, and then they could shared the similar transformation pathway with

322

5-CQA. Notably, phenolic compounds are reported to be catalyzed to metabolite

323

rapidly in 6 or 10 h by gut microbiota, but small portion of diCQAs had been

324

converted in 24 h in present experiments. The difference can be associated with the

mango

and

banana

containing

catechins,

4-hydroxyphenylacetic

16


acid,

Page 17 of 34


325

microbial profiles. CQAs are widely dispersed in daily vegetable foods and beverage,

326

especially coffee, and they are ingested every day. They reach human colon, and

327

enrich CQAs-metabolizing microbial species. The diCQAs are much less consumed

328

than the polyphenols that are rich in daily foods and less amount of

329

diCQAs-catalyzing microorganisms inhabit in gut relevantly.

330

In conclusion, the digestive process of diCQAs in vitro is concluded in Figure 4.

331

The in vitro experiments revealed that diCQAs from I. kudingcha could not be

332

hydrolyzed by saliva fluid, gastric solution, pancreatic digestive enzymes, or small

333

intestinal epithelial cells. The results indicated that diCQAs almost arrived at colon in

334

intact forms. The diverse microbial species inhabiting there had the ability to

335

metabolize diCQAs to hydrolytes mono-CQAs and CA, and they could be converted

336

to further metabolites including DHCA. The microbial converting ability depended on

337

individuals and diet customs. The real digestive process of diCQAs in vivo may have

338

some differences from the results in vitro, and it still needs to be investigated.

339

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Microbial biotransformation of polyphenols during in vitro colonic fermentation

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of masticated mango and banana. Food Chem. 2016, 207, 214-222.

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SIMulator of the GastroIntestinal tract SIMGI. LWT-Food Sci. Technol. 2015, 61,

511

283-289.

512 513

Note: This work was supported by Grants-in-Aid for scientific research from the

514

National Natural Science Foundation of China (31171666) and a project funded by the

515

Priority Academic Program Development of Jiangsu Higher Education Institutions

516

(PAPD).

25



517

Figure Caption

518

Figure 1. Chemical structures of caffeoylquinic acid derivatives (IUPAC numbering

519

system).

520

Figure 2. HPLC profiles of digestion of diCQAs-rich fraction in saliva (60 min),

521

gastric and pancreatic fluids for 180 min (recorded at 325 nm, 1, 3,4-diCQA; 2,

522

3,4-diCQA; 3, 4,5-diCQA).

523

Figure 3. Chromatographic profiles of metabolism of diCQAs by colonic microbiota

524

from volunteer 1 in anaerobic fermentation with different carbohydrates for 24 h

525

(recorded at 280 nm, 1, 3,4-diCQA; 2, 3,4-diCQA; 3, 4,5-diCQA; 4, 3-CQA; 5,

526

4-CQA; 6, 5-CQA; 7, DHCA; 8, CA).

527

Figure 4. Proposed catabolism of diCQAs in digestive tract (A) and chemical

528

structural illustration of conversion of diCQAs in colon by microbiota (B).

26


Page 26 of 34

Page 27 of 34


Table 1 Retention (%) of diCQAs in Saliva, Gastric and Pancreatic Digestion Fluids. 3,4-diCQA

3,5-diCQA

4,5-diCQA

Retention (%) 10 min

98.9±4.6a

99.7±3.0a

98.6±4.1a

30 min

101.9±8.1a

102.3±9.0a

101.1±7.6a

60 min

102.8±8.2a

103.4±7.5a

101.8±6.6a

10 min

99.5±5.0a

100.1±4.7a

100.8±3.7b

30 min

98.9±6.7a

99.5±5.6a

98.6±6.5a

60 min

97.4±5.9a

98.2±6.1a

99.2±5.1a

120 min

97.9±5.1a

98.4±5.3a

98.8±6.2a

180 min

98.5±4.8a

99.1±4.7a

97.6±4.5a

10 min

99.2±1.6a

99.0±2.4a

98.9±5.7a

30 min

99.9±3.3a

100.0±3.0a

99.1±2.8a

60 min

101.1±3.7a

100.6±2.5a

99.9±2.5a

120 min

101.4±2.7a

101.9±4.1a

100.1±2.9a

180 min

101.9±4.2a

100.5±3.6a

100.7±4.0a

Saliva Digestion

Gastric Digestion

Pancreatic Digestion

a

The same letter means no statistical significance was observed in a stimulated

digestion process between different times.

27



Page 28 of 34

Table 2 Retention (µg/mL) of diCQAs Metabolized by Caco-2 Cell Monolayer in Apical and Basal Compartments. 3,4-diCQA

3,5-diCQA

4,5-diCQA

Retention (µg/mL) Apical

400.0±4.2a

400.0±5.6a

400.0±3.8a

Basal

ND

ND

ND

Apical

404.9±13.5a

405.6±12.2a

403.8±12.8a

Basal

ND

ND

ND

Apical

407.5±15.4a

409.6±13.6a

406.7±13.5a

Basal

ND

ND

ND

0 min

50 min

100 min

ND

a

Not detected (lower than the detection limit).

No statistical significance was observed between the values at different times with a

same letter.

28


Page 29 of 34


Table 3 Retention of diCQAs in Anaerobic Fermentation with Different Sugars as Carbohydrate Sources by Intestinal Microbiota from 2 Volunteers. 3,4-diCQA

3,5-diCQA

4,5-diCQA

Retention (%) Glucose

97.5±3.5a

100.8±4.6a

93.2±4.5a

Volunteer

Sucrose

60.4±4.1b

95.0±2.2b

84.3±3.7b

1

FOS

55.5±2.0c

99.4±3.6a

84.9±2.4b

Inulin

53.3±3.2c

99.5±2.9a

82.6±5.0b

Glucose

100.3±2.9a

101.5±4.2a

99.9±4.6a

Volunteer

Sucrose

100.6±5.1a

100.4±3.3a

98.9±5.9a

2

FOS

90.6±4.2b

99.6±4.9a

91.5±6.0b

Inulin

91.9±6.4b

99.1±5.3a

92.0±3.1b

a-c

Means with different letters differ significantly (P < 0.05).

29



Page 30 of 34

Compound

R1

R2

R3

3-CQA 4-CQA 5-CQA 3,4-CQA 3,5-CQA 4,5-CQA

Caffeoyl OH OH Caffeoyl Caffeoyl OH

OH Caffeoyl OH Caffeoyl OH Caffeoyl

OH OH Caffeoyl OH Caffeoyl Caffeoyl

Figure 1

30


Page 31 of 34


Figure 2

31



Figure 3

32


Page 32 of 34

Page 33 of 34


Figure 4

33



Table of Contents (TOC) Graphic

34


Page 34 of 34

Hydrolysis of Dicaffeoylquinic Acids from Ilex kudingcha Happens in

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