Wayne State University

Wayne State University Theses

1-1-2016

In Vitro Gastrointestinal Digestion Model ToMonitor The Antioxidant Properties AndBioaccessbility Of Phenolic Extracts FromElderberry (sambucus Nigra L.)Ninghui ZhouWayne State University,

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Recommended CitationZhou, Ninghui, "In Vitro Gastrointestinal Digestion Model To Monitor The Antioxidant Properties And Bioaccessbility Of PhenolicExtracts From Elderberry (sambucus Nigra L.)" (2016). Wayne State University Theses. 515.https://digitalcommons.wayne.edu/oa_theses/515

IN VITRO GASTROINTESTINAL DIGESTION MODEL TO MONITOR

THE ANTIOXIDANT PROPERTIES AND BIOACCESSBILITY OF

PHENOLIC EXTRACTS FROM ELDERBERRY (SAMBUCUS NIGRA

L.)

by

NINGHUI ZHOU

THESIS

Submitted to the Graduate School

of Wayne State University,

Detroit, Michigan

in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

2016

MAJOR: NUTRITION AND FOOD SCIENCE

Approved by:

___________________________________

Advisor Date

i

DEDICATION

This thesis is dedicated to my family, Shuiqin Lin, Kewu, sister Yulin and brother

Zhihao who support and love me forever. To my undergraduate instructors, Liushui Yan,

Fang Deng, and Xubiao Liu, encourage me tirelessly. My achievement is also dedicated

to my co-workers and my friends, Wenjun, Fei who gives me confidence; to Dr. Sun for

his constant instruction and help.

ii

ACKNOWLEDGEMENTS

I would like to acknowledge and thank my advisor Dr. Kequan Zhou for his

patience and kind guidance during my graduate studies. Acknowledge to Dr. Ahmad

Heydari and Dr. Paul Burghardt, who are willing to serve in my committees. Dr. Shi Sun

helped me with experiments preparation and understanding. My co-worker, Wenjun and

Fei, assisted me with thesis data analysis and critical thinking.

iii

TABLE OF CONTENTS

Dedication·································································································································· i

Acknowledgements ················································································································· ii

List of Figures ························································································································· iv

Chapter 1: Introduction ··········································································································· 1

Chapter 2: Objectives of the Study······················································································· 5

Chapter 3: Materials and Methods ······················································································· 6

Chapter 4: Results and discussion ·····················································································10

Chapter 5: Conclusion ··········································································································13

References ·····························································································································18

Abstract ···································································································································26

Autobiographical Statement ································································································28

iv

LIST OF FIGURES

Figure 1: The process of in vitro digestion ········································································14

Figure 2: Total phenolic content of elderberry digested by heat-inactivated (inactive) and

active digestive enzymes ·····································································································15

Figure 3: Oxygen radical absorbance capacity of elderberry digested by heat-inactivated

(inactive) and active digestive enzymes expressed in μmols TE/ g dry weight treated

mixture·····································································································································16

Figure 4: DPPH radical scavenging capacity of elderberry digested by heat-inactivated

(inactive) and active digestive enzymes expressed in μmols TE/ g dry weight treated

mixture………………………………………………………………………………………17

1

INTRODUCTION

The elderberry fruit (Sambucus nigra L.) is a rich source of anthocyanin

compounds1, 2. Evidence is accumulating which supports the health promoting quality of

anthocyanins3 as a consequence of their inherent phenolic moiety and participation in

redox reactions ubiquitously occurring in biological milieus, ranging from plasma, tissue,

organs and gastrointestinal environments 4-6. Consequently anthocyanin compounds

are proposed to be potent antioxidants7 which may facilitate homeostasis of oxidative

stress. However, a limiting factor of the health promoting quality anthocyanins is their

stability, poor bioavailibity, and degradation as compared to other dietary polyphenolic

compounds such as isoflavones found in soy products. The bioavailability of

anthocyanin derived metabolites from elderberry in humans has been detected8, albeit

in low concentrations 5, 9, 10. With the heightened interest in health promoting role that

bioactive compounds play, screening the potential of candidate compounds efficiently

and cost-effectively with an in vitro digestive model7 which mimics biological digestive

system is a useful tool for determining the fate of the compounds in the preliminary

stage of research 11, 12. To this end, the goal of this study was to investigate the

modification of elderberry phenolic antioxidant compounds in regards to their metabolic

fate including: chemical structure, stability, antioxidant activity, and membrane transport

using an in vitro gastrointestinal digestion and membrane model.

Dietary polyphenols are reducing agents derived from plants and serve as an

additional source of antioxidants that protect against oxidative stress13. Structurally

anthocyanins are a type of polyphenols which are positively charged oxonium

2

compounds with antioxidant properties. There are many studies demonstrating the

bioactivity of anthocyanins regarding their protection against oxidative stress associated

diseases14 15-20. The antioxidant activities of anthocyanins on a variety of fruit derived

sources have been assessed 21, 22. For instance, the elderberry fruit is comprised of 3

major anthocyanins: 1) cyanidin 3-glucoside (Cy 3-glu) which accounts for the majority

of anthocyanin (54%), 2) cyanidin 3-sambubioside (Cy 3-sam, 39.7%), and 3) cyanidin

3-sambubioside-5-glucoside (Cy 3-sam-5-glc, 6%) 21. The Cy 3-sam and Cy 3-glc are

the two main elderberry derived cyanidins which have received the most attention in

regards to their bioavailability in humans 4, 23-27. Biological activities such as antioxidant

capacity of these anthocyanin derived compounds and other metabolites may play a

role in the prevention of diseases. As such, in vitro digestion models that assess the

modification of phenols and antioxidant capacity provides a tool to rapidly and more

cost-effectively, as compared to animal and human models, screen and identify

potential candidates that may contribute to the prevention of oxidative stress.

Consensus on the metabolic fate of anthocyanins and anthocyanin derived

metabolites is lacking in regards to the mechanisms involved in the modification on

chemical structure, stability, absorption, bioavailability, impact on oxidative status,

distribution to target tissues, and role in promoting health 24, 25, 28-30. It is clear that the

potential of ameliorative bioactive properties of food derived polyphenolic compounds

as well as their active metabolites depend on their bioavailability both in terms of

exposure to resident cells and transport in the circulatory system and subsequent target

organs 29, 31, 32. For example, improved oxidative stress status may be a consequence of

anthocyanin derived compounds having a direct effect on the intestinal mucosa by

3

existing as a potent antioxidant. The importance of better understanding the antioxidant

status of phenolic derived compounds post digestion would clarify their impact on the

gastrointestinal system and other biologically relevant target tissues such as their

impact on oxidative stress. They could provide antioxidant protection in the

gastrointestinal milieu locally. Direct evidence on the bioavailability of select phenolic

compounds has been obtained by measuring their concentrations in plasma and urine

after consumption. Indirect evidence of their absorption through gastrointestinal

membranes has been detected from the observation of an increase in the antioxidant

capacity of the plasma after the consumption of phenolic rich food sources. Additionally,

it is important to understand the modification to chemical structures of the digested

dietary phenols which are an additional factor that governs the rate and extent of

absorption and the chemical forms of the biological metabolites.

In the present work an in vitro digestive model, similar to other methods previously

used on phenolic compounds 33-35, was designed which provides a rapid, inexpensive

preliminary strategy to investigate the elderberry and its associated bioactive

compounds7. In vitro gastrointestinal models are useful because they can be

customized to mimic or simulate biological digestive processes, useful for elucidating

and monitoring the biofunctional status and bioavailability of specific components 33, 36.

During digestion anthocyanins are exposed to a wide range of pH conditions in the

gastric and intestinal tract, both of which may affect their bioavailability 33. The stomach

is one of the metabolic sites of digestion of anthocyanins as well as a site were cyanins

are absorbed through the gastric membrane for transport in the plasma. Anthocyanins

are exposed to pepsin in the acidic gastric milieu. The pancreatin digestion occurs in the

4

small intestine which is the site of the majority of cyanin absorption. Understanding the

instability and degradation of anthocyanins within the gastrointestinal tract is important

for clarifying factors affecting anthocyanin bioavailibity37 38, 39. For example, it has been

hypothesized that the cyanin ionic form existing at neutral pH in the small intestine are

less stable and degrade rapidly into unknown metabolites 40. On the contrary,

anthocyanins are stable in their acidic form typically present in the gastric milieu 33, 41.

Due to this dynamic environment the stability of cyanins may be the reason for their

limited absorption across gastric or intestinal walls and subsequent metabolic fate.

Information on bioavailability of specific phenolic compounds can be obtained by

conducting human and animal absorption studies 42. However, there remains a need to

take advantage of in vitro gastrointestinal models which can be executed more rapidly

and are typically an inexpensive approach to assess bioavailability.

For the purposes of this study, both the effect of live and dead pepsin activity in the

stomach on the functional properties of elderberry were compared, such as their

antioxidant capacity in regards to oxygen radical absorbance capacity (ORAC) and

ability to scavenge reactive free radicals such as (DPPH). Similarly, the small intestinal

pancreatin enzyme in both the dead and live forms was assessed for its influence on

the antioxidant properties of the elderberry. This is important because most of previous

studies for antioxidant evaluation where the important effects of GI digestion on dietary

antioxidant bioaccessibility were missed.

5

Objective of the Study

The objective of this study was to investigate the antioxidant properties of

elderberry as affected by simulated gastrointestinal digestion. Antioxidant properties of

the samples before and after GI digestion were evaluated by total phenolic content

(TPC) assay, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and

Oxygen radical absorbance capacity (ORAC) assay.

6

MATERIALS AND METHODS

Materials

Pancreatin, pepsin, gallic acid, folin-ciocalteu’s phenol reagent, were purchased

from Sigma-Aldrich (St. Louis, MO, USA). Formic acid was obtained from Fisher (Fair

Lawn, NJ, USA). Bile salts, fluorescein, were acquired from Fluka (Buchs, Switzerland).

2, 2-diphenyl-1-picrylhydrazyl (DPPH) and pelargonidin chloride were purchased from

Aldrich (Milwaukee, WI). Trolox was purchased from ACROS (Geel, Belgium). 2, 2’ -

Azobis (2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako

(Richmond, VA, USA).

Sample and Standard Preparation

Samples of organic elderberries (Sambucus Nigra L.) were obtained from Starwest

Botanicals, Inc. the elderberry samples were frozen and chopped into small pieces,

stored overnight at -80 ˚C and lyophilized with a freeze-dryer (Labconco Freeze Dry

System Lyph Lock 6, Kansas City, MO, USA). The samples were milled into fine powder

and were then used for the in vitro digestion.

In vitro gastrointestinal digestion simulation

An overview of the in vitro digestion process utilized is presented in Figure 1. In

vitro digestion was performed in a shaking water bath (Pegasus scientific, Rockville,

MD, USA). The procedure was comprised of a pepsin-HCl digestion for 2 hours at 37 ˚C

(to simulate gastric digestion) and a pancreatin digestion with pancreatin and bile salts

for 2 hours at 37 ˚C (to simulate small intestinal digestion) 43. One gram of pepsin was

dissolved in 100 mL distilled water to make the pepsin solution, whereas 0.4 g of

7

pancreatin and 2.5 g of bile salts were dissolved in 100 mL distilled water to make the

pancreatin-bile salt solution. The Control (treated with inactive enzymes) and Treatment

(treated with active enzymes) samples groups were treated with these solutions as

indicated above. As the control group, the solutions were boiled then cooled down to

room temperature before treatment, thus deactivating the digestive enzymes. The

procedure was as follows: 1 g dried fruit powder was mixed with 2 mL pepsin solution

(inactive for Control, active for Treatment) and 17 mL distilled water. The mixture was

adjusted to pH 1.7~2.0 with HCl and incubated at 37 ˚C for 2 hours in a water bath

under shaking. After incubation, Pipet 2 ml from each digested mixture into a 15 ml

centrifuge tube and stored in a freezer (the mixture fractions were then extracted by

aqueous acetone for antioxidant evaluation). The remaining mixture will go to next

pancreatin/bile salts digestion where the mixture was adjusted to pH 8.0 with 1N NaOH

and 2 mL pancreatin-bile salts solution was added. The mixtures were digested in the

water bath shaker for 2 hours under shaking. Following the digestion process, the

digested mixture were stored at -80 ˚C overnight then freeze-dried. The lyophilized

mixture were then extracted with aqueous acetone (1:1, v/v) and vortexed 3 times for 5

minutes each. The extracts were then filtered using 0.45 μm Whatman filter paper and

diluted with aqueous acetone to a concentration of 50 mg/mL. These samples were

then used for antioxidant evaluation including TPC assay, ESR, DPPH radical

scavenging assay, and ORAC assay.

Total Phenolic Content (TPC) Assay

TPC was evaluated using a Beckman DU 640 spectrophotometer (Beckman

Coulter, Fullerton, CA) with Folin-ciocalteu’s phenolic reagents 44. Samples resulting

8

from in vitro digestion were diluted to 25 mg/mL with aqueous acetone. Gallic acid was

used as a standard for preparing the standard curve (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL

in aqueous acetone). All the samples and standards were run in triplicates. Each test

tube contained 25 μL of a sample or standard and 250 μL distilled water. 750 μL Folin-

Ciocalteu’s phenol reagent was then added to each tube and mixed thoroughly using a

vortex mixer. Then, 500 μL of 200 mg/mL sodium carbonate was added to each tube

and vortexed. Samples and standards were incubated for 2 hours at room temperature

in the dark. Absorbance was detected at 765 nm using a spectrophotometer and the

TPC of a sample was expressed as milligrams of gallic acid equivalents (GAE) per 100

g fresh fruit weight.

2, 2-diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay

The protocol followed by Brand-Williams et al. was modified 45. This assay

measures the ability of the samples to quench DPPH radicals. The digested samples

were diluted to 10 mg/mL with pure ethanol and then centrifuged at 6900 g for 20

minutes to eliminate residues. 100 μL of each sample was mixed with 150 μL of DPPH

radical solution in a 96-well microplate and absorbance was measured at room

temperature every 5 minutes for 2 hours at 500 nm, using an HTS 7000 Bio Assay

Reader (Perkin Elmer, Norwalk, CT). All samples were run in triplicates. The percent

scavenging capacity was calculated using the following equation: Scavenging effect

(%)= [Abscontrol －(Abssample－Abssample background)]/ Abscontrol×100.

Oxygen Radical Absorbance Capacity (ORACFL) Assay

9

ORAC was performed as described by Zhou et al. 44. The digested samples were

diluted with aqueous acetone to a concentration of 1.3 mg/mL. All samples and

standards were run in triplicates. The mixture of fluorescein and samples or standards

was placed in a 96-well microplate. The plate was incubated for 15 minutes at 37 ˚C

and 35 μL AAPH was then added to each sample and fluorescence was measured

every 5 minutes for 100 minutes at an excitation wavelength of 485 nm and an emission

wavelength of 520 nm. Results were expressed as μmol/L trolox equivalent (TE)/100

gram fresh fruit weight.

Statistical Analysis

Antioxidant data executed from the above performed assays was analyzed via

SPSS using independent samples t-test. Data for each parameter is reported as mean ±

SD. Outcomes were compared using p ≤ 0.05 as a cutoff point for statistical

significance.

10

RESULTS AND DISCUSSION

Total Phenolic Content (TPC) Assay

Evaluation of TPC in samples is commonly used to determine the amount of

accessible phenolic antioxidants in a sample potentially possesses 46, 47. As shown in

Figure 2. The TPC values of the elderberry powder after first step stomach digestion

simulation with inactive and active pepsin were 34.1 and 32.3 mg gallic acid equivalent

(GAE) per gram (dry weight) of the elderberry mixture, respectively. After sequential

intestinal digestion simulation, the TPC values of the digested mixture were 24.0

(treated with inactive pancreatic enzymes) and 25.2 (treated with active pancreatic

enzymes) per gram, respectively. The TPC contained in our elderberry sample (on dry

weight) are comparable to the previous studies where 24 varieties of American

elderberry samples contained 2.7-5.8 mg GAE per gram fresh weight48, 49. During the

digestion simulation sequentially with pepsin and pancreatic juice, no significant

difference was detected between the TPC of the elderberry mixtures treated with

inactive and active digestive enzymes. Our results suggest that the GI digestion process

have no significant impact on the extractability (accessibility) of the elderberry. This is in

contrast to a previous study showing that GI digestion improves the accessibility of

wolfberry phenolics50. Indeed, studies of different fruits have shown varying results

during the digestion 51-53. Nevertheless, the result indicates that the process of digestion

does not seem to destroy or reduce the amount of accessible phenolic antioxidants in

elderberry.

Oxygen Radical Absorbance Capacity (ORACFL) Assay

11

ORAC assay was used to determine how GI digestion could affect the peroxyl

radical scavenging capacity of elderberry. Trolox was used as the standard with results

expressed as micromoles of Trolox equivalents (TE) per gram dry weight of the

digested mixture. As presented in Figure 3, The ORAC values of the elderberry powder

after sequential gastric and intestinal digestion simulation were 712.8 (G-) versus 661.8

(G+) and 581.2 (GP-) versus 587.1 (GP+) μmol TE per gram of the digested mixture,

respectively. Similar to the effect of GI digestion on TPC of elderberry, we found no

significant difference between the ORAC of the elderberry mixtures treated with inactive

and active digestive enzymes. Although many studies have shown positive correlation

between TPC and ORAC on various fruits or vegetables, higher total phenolic content

does not always correspond to higher ORAC 54, 55. In our case of elderberry, it appears

that the TPC and ORAC of elderberry are positively correlated and both are not

significantly affected by GI digestions.

DPPH Radical Scavenging Assay

Antioxidant capacity of the digested elderberry was further determined by DPPH

radical scavenging assay which also use Trolox as antioxidant standard and expressed

as μmol TE per gram (dry weight) of the digested mixture. As shown in Figure 4, the

DPPH radical scavenging activity of the elderberry powder after sequential gastric and

intestinal digestion simulation were 276.1 (G-) versus 166.5 (G+) and 297.8 (GP-)

versus 98.5 (GP+) μmol TE per gram of the digested mixture, respectively. The DPPH

radical scavenging activity of the elderberry was reduced by 39.7% (G- versus G+,

P<0.05) after gastric digestion and by 66.9% (GP- versus GP+, P<0.05) after GI

digestion. Our result showed that GI digestion significantly reduced DPPH radical

12

scavenging activity of the elderberry. This is in contrast to the results for TPC and

ORAC where GI digestion had no significant effect. Our result also is in contrast to the

result a previous study found that digestion process increased DPPH scavenging

activity on 25 commercially available fruit juices 56. DPPH radical scavenging assay has

been commonly used for antioxidant activity evaluation 57. However, the assay which

differs from ORAC assay by the type of radical produced, scavenging method and

measurement. It is usual that DPPH radical scavenging activity is not positively

correlated with TPC or ORAC for an antioxidant sample, especially when the sample

contains a variety of antioxidants that have different mechanisms of action 58. Previous

studies have shown varying effects of digestion process on DPPH radical scavenging

capacities of various fruits59 53, 60, 61. The results indicate that the digestion process may

change the composition of phenolic compounds but not the overall phenolic content

(TPC) in elderberry.

13

CONCLUSION

In vitro models provide a tool for rapidly assessment of impact of digestion on

antioxidant bioaccessibility of antioxidant nutrients which can be expected to vary in this

dynamic environment. Overall, the in vitro findings obtained in this study have

demonstrated that digestion process may reduce antioxidant activity (DPPH radical

scavenging activity) of elderberry. However it is well appreciated that true physiological

impact and efficacy of antioxidants may only be speculated pending in vivo studies; the

results of this study indicate that such studies are warranted. In the future, it will be

important to clarify the antioxidant metabolism based on whole foods consumption as

well as analyzing factors affecting bioavailability, including interaction with other dietary

compounds and metabolized metabolites.

14

Adjust pH of the remaining digestive mixture

(~18 mL) to 8.0

+

2 mL inactive pancreatin-bile salts solution

Figure 1-The process of in vitro digestion

Antioxidant properties

Total Phenolic Content (TPC) assay

2, 2-diphenyl-1-picrylhydrazyl (DPPH)

Radical Scavenging assay

Oxygen Radical Absorbance Capacity

(ORAC) assay

Lyophilize the digested mixture (PP+)

Extracted with 50% acetone and vortex 3 times for 5 minutes. Filter sample

solutions with 0.45 μm filter paper

Lyophilize the digested mixture (PP-)

Incubate at 37 ˚C for 2 hours in a water bath at 100 rpm

Adjust pH of sample solutions to 1.7~2.0

+

Incubate at 37 ˚C for 2 hours in a water bath at 100 rpm

Treatment

1g dried fruit powder

+

2 mL active pepsin solution

+

17 mL distilled water

Control

1g dried fruit powder

+

2 mL inactive pepsin solution

+

17 mL distilled water

Lyophilized elderberries

Take out ~2 ml digested mixture

and lyophilized (P+) for testing

antioxidant properties

Take out ~2 ml digested mixture

and lyophilized (P-) for testing

antioxidant properties

Adjust pH of the remaining digestive

mixture (~18 mL) to 8.0

+

2 mL active pancreatin-bile salts solution

15

Figure 2. Total phenolic content of elderberry digested by heat-inactivated (inactive) and active digestive enzymes (mg GAE/g dry weight treated mixture).

TPC of the samples was determined in triplicate. Bars with different superscripts indicate significant difference (p<0.05).

16

Figure 3. Oxygen radical absorbance capacity of elderberry digested by heat-inactivated (inactive) and active digestive enzymes expressed in μmols TE/ g dry weight treated mixture. ORAC of the samples were determined in triplicate. Bars with

different superscripts indicate significant difference (p<0.05).

17

Figure 4. DPPH radical scavenging capacity of elderberry digested by heat-inactivated (inactive) and active digestive enzymes expressed in μmols TE/ g dry weight treated mixture. Bars with different superscripts indicate significant difference

(p<0.05).

18

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26

ABSTRACT

IN VITRO GASTROINTESTINAL DIGESTION MODEL TO MONITOR THE ANTIOXIDANT PROPERTIES AND BIOACCESSBILITY OF PHENOLIC

EXTRACTS FROM ELDERBERRY (SAMBUCUS NIGRA L.)

by

NINGHUI ZHOU

May 2016

Advisor: Dr. Kequan Zhou

Major: Nutrition and Food Science

Degree: Master of Science

This study performed an in vitro gastrointestinal digestion model to monitor the

antioxidant properties of phenolic extracts derived from the elderberry (Sambucus nigra

L.). The stability and antioxidant qualities of phenolic compounds from elderberry were

assessed by an in vitro gastrointestinal digestion model involving pepsin digestion (to

simulate gastric digestion) and pancreatin digestion (to simulate small intestine

conditions). Digested and undigested elderberry were subject to antioxidant assays

including total phenolic content (TPC) assay, DPPH radical scavenging assay, and

oxygen radical absorbance capacity (ORAC) assay. Our result showed that digestion

process significantly reduced DPPH radical scavenging activity but had no significant

effect on TPC and ORAC of elderberry. This indicates a need to investigate phenolic

27

compounds in digestion that mimic biological gastrointestinal environments and to

monitor these compounds for changes individually if possible.

KEYWORDS. Elderberry (Sambucus nigra L.), antioxidant, in vitro, gastrointestinal

digestion model, anthocyanins, bioavailability, pepsin, pancreatin

28

AUTOBIOGRAPHICAL STATEMENT

Ninghui Zhou received her bachelor of Applied Chemistry from Nanchang

University of Aeronautics, Jiangxi, China in 2013. In 2013, she joined Wayne State

University (WSU) and is currently completing her graduate studies towards the

accomplishment of a master of Nutrition and Food Science degree.

During her graduate studies, Ninghui was a member of Institute of Food

Technology from 2014 to 2016 as well as a membership in the “Nutrition and Food

Science Graduate Students Organization” at Wayne State University from 2013 to 2016.

She also received the WSU Graduate Professional Scholarship throughout her course

of study for the academic year (Sep. 2013).