CHARACTERIZATION OF PHENOLIC COMPOUNDS FROM PECAN

KERNELS AND THEIR BIOLOGICAL EFFECTS ON ADIPOGENESIS

AND INFLAMMATION

A Thesis

by

ANA GABRIELA ORTIZ QUEZADA

Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2010

Major Subject: Food Science and Technology

Characterization of Phenolic Compounds from Pecan Kernels and Their Biological

Effects on Adipogenesis and Inflammation

Copyright 2010 Ana Gabriela Ortiz Quezada

CHARACTERIZATION OF PHENOLIC COMPOUNDS FROM PECAN

KERNELS AND THEIR BIOLOGICAL EFFECTS ON ADIPOGENESIS

AND INFLAMMATION

A Thesis

by

ANA GABRIELA ORTIZ QUEZADA

Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Approved by:

Co-Chairs of Committee, Luis Cisneros-Zevallos Leonardo Lombardini Committee Members, Joseph Awika Chaodong Wu Chair of Food Science and Technology Faculty, Jimmy T. Keeton

August 2010

Major Subject: Food Science and Technology iii

ABSTRACT

Characterization of Phenolic Compounds from Pecan Kernels and Their Biological

Effects on Adipogenesis and Inflammation. (August 2010)

Ana Gabriela Ortiz Quezada, B. S., Instituto Tecnológico y de Estudios Superiores de

Monterrey, Monterrey Campus

Co-Chairs of Advisory Committee: Dr. Luis Cisneros-Zevallos Dr. Leonardo Lombardini

Pecan [Carya illinoinensis (Wangenh.) K. Koch] kernels from four cultivars were screened for their phenolic content and antioxidant capacity. Ellagitannin content was quantified by HPLC after hydrolysis as free ellagic acid. Characterization of phenolic compounds present in defatted pecan was conducted by liquid chromatography- mass spectrometry. In addition, ellagic acid metabolites, urolithin A and urolithin B, were evaluated for their anti-adipogenic and anti-inflammatory effects in 3T3-L1 preadiadipocyte cells by protein and gene expression.

Variations among cultivars were observed. „GraCross‟ and „Desirable‟ showed the highest total phenolic content; in the same way, „GraCross‟ had the highest values for condensed tannin content, antioxidant capacity using the 2, 2-diphenyl-1-picryl- hydrazyl (DPPH) assay and the oxygen radical absorbance capacity (ORAC). According to these results, the cultivar with the highest condensed tannin content had the highest antioxidant capacity using the DPPH and ORAC assays. „Desirable‟ and „Kiowa‟ had the highest ellagitannin content. Even though ellagitannins comprise a small percentage iv

compared to condensed tannins, the latter have restricted bioavailability and up to trimers can be absorbed. Eleven ellagitannins were identified for the first time in pecans.

Their masses ranged from 433 to 1207 m/z. Condensed tannins up to pentamers were detected. Other compounds included gallic acid, ellagic acid, and isorhamnetin in the glucoside, pentosyl and galloyl pentosyl forms.

Ellagitannins are metabolized in vivo yielding urolithins which are absorbed by the gut. Urolithins are derivatives from ellagic acid. These compounds, urolithin A and urolithin B, were used to treat 3T3-L1 preadypocytes as an obesity model. Results of the adipocytes‟ protein expression immediately after differentiation indicated no inhibition of differentiation of the preadipocytes after treating cells with urolithins and ellagic acid.

Results on the mature adipocytes indicated that treatments with 25 µM of urolithin A, urolithin B, and ellagic acid during differentiation did not significantly reduce their lipid content.

Inflammatory effects in 3T3-L1 adipocytes were evaluated by the LPS model through protein and gene expression. Urolithin A and ellagic acid reduced p-NF-κB protein expression. Urolithin A and urolithin B reduced TNF-α mRNA basal expression, a protective effect in a non-inflammatory state. Interleukin-6 (IL-6) expression was increased by urolithins and lipopolysaccharide (LPS), probablly due to a synergistic inflammatory effect coming from different pathways. v

DEDICATION

To my parents, José Antonio and Socorro, for giving me life, love, support, and guidance. To my brothers Gustavo and Mauricio, who have walked next to me in life. To my boyfriend Rolando, for his love, support, endless patience, for making me laugh and enjoy life. And to God. vi

ACKNOWLEDGEMENTS

I would like to thank my committee co-chairs, Dr. Cisneros and Dr. Lombardini, for the opportunity given to me to do my degree, for trusting me and for their support.

Thanks to my committee members, Dr. Awika, for his support and guidance and Dr. Wu, for letting me work in his lab, for helping me understand adypocytes and for his guidance, support and patience throughout the course of this research.

Thanks to Dr. Morgan, who listened and guided me and to Dr. Sturino, for letting me work in his lab.

My gratitude to my friends and colleagues from food science and horticulture:

Gabriela Angel, Alex Puerta, Nenge Njongmeta, Congmei Cao, Judith Turlington.

Special thanks to the biochemists, Freddy Ibañez and Paula Castillo, for their technical support, for giving me hope and for the good times in the lab. Thanks to colleagues from

CEBAS in Murcia, Spain, Dr. Francisco Tomás-Barberán, for letting me work in his lab, and to Dr. Rocío González-Barrio, for her support no matter where we were.

This project was supported thanks to the financial support of the Vegetable and

Fruit Improvement Center (VFIC), Texas Pecan Board, and the Texas Pecan Growers

Association.

Thanks to my parents, for their continuous encouragement; my brothers, for being there for me, and to my beloved boyfriend, for his love.

vii

TABLE OF CONTENTS

Page

ABSTRACT ...... iii

DEDICATION ...... v

ACKNOWLEDGEMENTS ...... vi

TABLE OF CONTENTS ...... vii

LIST OF FIGURES ...... ix

LIST OF TABLES ...... xii

CHAPTER

I INTRODUCTION AND LITERATURE REVIEW ...... 1

Tannins ...... 3 Tannins bioavailability ...... 6 Health benefits ...... 7 Obesity overview ...... 8 Adipose tissue and gene expression ...... 10 Objectives ...... 13

II PECAN CULTIVAR SCREENING FOR PHENOLICS, ANTIOXIDANT CAPACITY, AND POLYPHENOLIC CHARACTERIZATION BY LC-MS ...... 14

Summary ...... 14 Introduction ...... 14 Materials and methods ...... 16 Results and discussion ...... 23 Conclusions ...... 51

viii

CHAPTER Page

III EFFECTS OF UROLITHINS A AND B AS METABOLITES FROM PECAN ELLAGITANNINS ON ADIPOGENESIS IN 3T3-L1 CELLS ...... 52

Summary ...... 52 Introduction ...... 53 Materials and methods ...... 55 Results and discussion ...... 59 Conclusions ...... 74

IV EFFECTS OF UROLITHINS A AND B IN INFLAMMATION WITH 3T3-L1 CELLS ...... 75

Summary ...... 75 Introduction ...... 75 Materials and methods ...... 77 Results and discussion ...... 82 Conclusions ...... 94

V CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER RESEARCH ...... 95

REFERENCES ...... 97

VITA ...... 120

ix

LIST OF FIGURES

Page

Figure 1 Examples of a condensed (A) and a hydrolyzable (B) tannin ...... 5

Figure 2 Condensed tannins versus ellagitannins from four pecan cultivars ...... 27

Figure 3 LC chromatogram of polyphenolics present in „GraCross‟ pecan kernel at 360 nm ...... 29

Figure 4 LC chromatogram of polyphenolics present in „GraCross‟ pecan kernel at 280 nm ...... 30

Figure 5 Characteristic UV-spectra of ellagitannins from „GraCross‟ ...... 34

Figure 6 UV-spectra of pedunculagin isomers (m/z 783) as they elute at increasing retention times, for „GraCross‟ ...... 34

Figure 7 Extracted ion chromatogram for m/z- 951, from „GraCross‟ ...... 36

Figure 8 Extracted ion chromatogram for m/z 577, from defatted pecan nut extract ...... 36

Figure 9 Two different types of linkages between flavan-3-ols to form condensed tannins ...... 37

Figure 10 Extracted ion chromatogram (EIC) of m/z- 603, 1207, 289, and 865, for „GraCross‟ ...... 37

Figure 11 MS spectra of [M – H]- m/z 1207 and [M – 2H]2- m/z 603, tentatively identified as catechin derivative of stachyurin ...... 38

Figure 12 Proanthocyanidin trimer showing the T-unit ...... 38

Figure 13 Extracted ion chromatogram of m/z- 785 isomers of digalloyl-HHDP glucose ...... 41

Figure 14 Structures of castalagin, vescalagin, and glausrin C ellagitannins with the same MW of 934 ...... 41

x

Page

Figure 15 Extracted ion chromatogram for procyanidin monomer, dimer, trimer, tetramer and pentamer found in „GraCross‟ pecan kernels ...... 44

Figure 16 Structure of the flavonol isorhamnetin glucoside...... 44

Figure 17 HPLC-UV-DAD chromatogram of „Desirable‟ recorded at 280 nm following acid-catalysis in the presence of ascorbic acid (1) and phloroglucinol (2) ...... 47

Figure 18 Mass spectra of proanthocyanidin cleavage products (extension units). .. 48

Figure 19 Mass chromatogram of two procyanidin monomers (terminal units), epigallocatechin (B) and catechin (D), after phloroglucinolysis from „Desirable‟ recorded at 280nm ...... 49

Figure 20 3T3-L1 preadipocytes cell viability after 24 h exposure to treatments at 5-50 µM. Treatments were urolithin A, urolithin B, ellagic acid (urolithin precursor), rutin (positive control), and vehicle (< 0.083% DMSO)...... 60

Figure 21 3T3-L1 preadipocyte cell proliferation after 48 h exposure to treatments at 5-50 µM ...... 61

Figure 22 Intracellular lipid accumulation in 3T3-L1 mature adipocytes after addition of 25 µM urolithin A, urolithin B, ellagic acid (urolithin precursor), rutin (positive control) or the equivalent of vehicle (0.04% DMSO) ...... 63

Figure 23 Expression of the transcription factor PPARγ at the protein level in adipocytes treated with 25 µM of compounds after 3 d differentiation .... 64

Figure 24 Expression of the transcription factor C/EBPα at the protein level in adipocytes treated with 25 µM of compounds after 3 d differentiation .... 65

Figure 25 Expression of the transcription factor PPARα at the protein level in adipocytes treated with 25 µM of compounds after 3 d differentiation .... 66

Figure 26 Expression of the transcription factor PPARγ at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation ...... 69

Figure 27 Expression of the transcription factor C/EBPα at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation ...... 70 xi

Page

Figure 28 Expression of the transcription factor PPARα at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation ...... 71

Figure 29 Expression of the adipokine leptin at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation ...... 72

Figure 30 Expression of the adipokine adiponectin at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation ...... 73

Figure 31 Expression of the transcription factor p-NF-κB p65 at the protein level in mature adipocytes, treated with 25 µM of compounds and LPS . 84

Figure 32 Results obtained for protein abundance of p-NF-κB p65, NF- κB p65, and β-actin for 25 µM treatments by western blot ...... 85

Figure 33 Agarose gel electrophoresis of RNA collected from 3T3-L1 mature adipocytes ...... 85

Figure 34 Relative RNA expression of the proinflammatory cytokine TNF-α in mature adipocytes ...... 88

Figure 35 Relative RNA expression of the proinflammatory cytokine IL-6 in mature adipocytes ...... 89

Figure 36 Relative RNA expression of the transcription factor PPARγ in mature adipocytes ...... 90

Figure 37 Relative RNA expression of the secretory adipokine adiponectin in mature adipocytes ...... 91

xii

LIST OF TABLES

Page

Table 1 Phenolics and antioxidant capacity screening of four pecan cultivars extracted with 70% aqueous acetone ...... 24

Table 2 Characterization of phenolic compounds in „GraCross‟ pecan using HPLC-DAD-ESI-MSn detection ...... 31

Table 3 Contents of proanthocyanidin cleavage products after phloroglucinolysis in four different cultivars of pecan (mg/100 g FW) ...... 50

Table 4 Sequences of primers used in this study ...... 83

1

CHAPTER I

INTRODUCTION AND

LITERATURE REVIEW

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is a species native to a number of states in the southern and midwestern United States and some parts of Mexico (1-2).

United States, Mexico, Israel, South Africa, and Australia are the main pecan producers

(2). In the United States, native trees are concentrated mainly in Texas, Oklahoma, and

Louisiana; whereas most of the pecan trees found east of the Mississippi river constitute imported varieties (3). The pecan tree belongs to the Juglandaceae family–which also includes hickories and walnuts–and that can reach up to 60 m height. It is a wind- pollinated, monoecious (i. e., unisexual flowers are present on the same individual) tree- nut crop that exhibits heterodichogamy, meaning unisexual flowers, and each plant may have only male or female flowers (on certain trees female flowers mature first while on other trees male flowers mature first) (4). Pecan trees tend to bear big crops and light crops in successive years, phenomenon which is also known as alternate bearing. It takes from 6 to 10 years for a pecan tree to bear fruit; at about 8 to 10 years the yields are between 10 and 30 kg of in-shell pecans/tree, increasing to 35-50 kg/tree in the next 5 years, and above 75 kg/tree after 16 years (1, 5). Harvesting starts after the hulls split apart.

______This thesis follows the style of the Journal of Agriculture and Food Chemistry. 2

For mechanical harvesting, the trunk is mechanically shaken and the nuts are collected from the ground by mechanical harvesters (5).

Pecan is the most native nut crop in the United States. The value of the 2009 utilized pecan crop was $398 million. Texas is the third largest pecan producer of commercial varieties in the United States after Georgia and New Mexico, and it has the largest native and seedling production (6).

Pecan is commonly used as an ingredient for desserts, ice cream, candies, or as a snack; it is a good source of protein, dietary fiber, vitamins, minerals and many other bioactive substances which provide health benefits (7-9). According to the USDA

Nutrient Database (10), pecan nuts (kernels) are composed of 72% oil, 87% of which is unsaturated. These fatty acids are protected against oxidation by the high concentrations of γ-tocopherol and polymeric flavanols, also referred to as condensed tannins (10-11).

Clinical studies with human subjects have shown pecans may play an important role in reducing the risk of heart disease by improving the serum lipid profile (11-13). These benefits are mainly due to their high unsaturated fatty acid content (14).

Approximately 97% of the total antioxidant capacity (TAC) of pecan kernels measured by ORAC comes from the hydrophilic portion (15). When pecans where screened together with nine other tree nuts, they presented the highest antioxidant capacity by ORAC (179.40 μmol TE/g fresh weight) and total phenolics measured as gallic acid equivalents (20.16 mg of GAE/g) (15). In another study (16), 98 common foods were screened for their proanthocyanidin content or condensed tannins. It was found that among the nut group, pecans had the second highest content (494 mg/100 g 3 fresh weight), after hazelnuts (500mg/100g fresh weight). Apart from the antioxidant capacity, proanthocyanidins and total phenolics content, little information is available about the phytochemicals contained in the non-fatty portion of pecan kernels, which is believed to protect the oil from oxidation.

A previous study reported that, among the six pecan cultivars analyzed, a large portion of the antioxidant capacity was attributable to the total phenolics and, especially, to the condensed tannins (9). Results showed differences among cultivars and, occasionally, among growing locations. The pecan crude extract could not be analyzed by

HPLC due to the complexity of the chromatogram, therefore, basic and acid hydrolysis of defatted pecans yielded catechin, epicatechin, gallic acid, ellagic acid and an ellagic acid derivative (9). These results confirmed the potential presence of both condensed and hydrolyzable tannins.

Tannins

Tannins are secondary plant metabolites that can be found in different tissues of the plant, such as the bark, wood, leaves, fruit, roots, and seeds. Their function is to protect plants against attacks by pathogens, insects, or herbivores. Tannins have long been used to tan animal skins (“tanning”) to form leather, tanning refers to the cross- linking of the skin‟s collagen chains. Tannins are water-soluble phenolic compounds of high molecular weight that have the ability to precipitate proteins and alkaloids. Tannin- containing plant extracts have many medicinal uses in China and Japan, as astringents, anti-diarrhea, as diuretics (17-18), against stomach and duodenal tumors (19), and as 4 antiseptic and anti-inflammatory pharmaceuticals. They are used in the food industry as clarifying agents for beer, wine, and fruit juices (20). Tannins are classified as condensed or hydrolyzable tannins (Figure 1). Condensed tannins, also known as proanthocyanidins, are oligomers of flavan-3-ols linked together by carbon-carbon bonds (B-type with 4-6 and 4-8 linkages, while A-type condensed tannins have an additional ether bond); they are the second most abundant natural phenolics after lignin. The most common flavan-3- ols in proanthocyanidins are afzelechin, epiafzelechin, catechin, epicatechin, gallocatechin, and epigallocatechin; they can also be esterified with gallic acid to form 3-

O-gallates (21). These compounds have been found in tea, grape seeds and skin, and cocoa (22). Hydrolyzable tannins are mainly glucose esters of gallic acid, and its dimer, hexahydroxydiphenic acid (HHDP), with different combinations; they are classified into two types: ellagitannins and gallotannins, depending on the product they yield after hydrolysis (ellagic and gallic acid, respectively) (22-23). Hydrolyzable tannins have been analyzed by HPLC-ESI-MSn for many fruits and vegetables, such as raspberry (24-25), strawberry (26), acorns (27-28), muscadine grape (29).

Analysis of tannins has been done using spectrophotometric techniques (30-31), thin layer chromatography (TLC), fractionation and HPLC-DAD (32-33). Nuclear magnetic resonance (NMR) and mass spectrometry have been applied for identification of individual compounds (34). In recent years, HPLC-DAD-ESI-MSn has become a common and valuable tool that allows the identification of compounds by providing information about the molecular mass and fragmentation patterns present in the analyte,eliminating the need to hydrolyze and have authentic standards (35). 5

OH A HO OH B OH B O OH O HO O O O O OH O HO O H OH OH O OH O OH OH HO H O OH O HO O OH HO OH OH OH HO OH HO OH Figure 1. Examples of a condensed (A) and a hydrolyzable (B) tannin. Glausrin C 6

Electrospray ionization-MS has been employed for structure elucidation even in crude tannin mixtures. Since these polyphenols are weakly acidic, negative ionization is preferred over protonation; both positive and negative ion modes have been used for analysis of condensed tannins whereas negative ionization has been successful for the hydrolyzable tannins (27-29, 34, 36-39). The advantage of tandem mass spectrometry

(MSn) over MS is that the mass can be further fragmented, until the characteristic daughter ions determine the type of compound. This was true for an ellagitannin study which discerned between the same mass m/z- 301 among ellagic acid from quercetin derivatives (38).

Tannins bioavailability

Recent studies regarding the bioavailability and metabolism of ellagitannins have shown that they are hydrolyzed in the human digestive tract, releasing ellagic acid, which is then metabolized by the gut microflora to form urolithins A, B, C or D. When urolithins are absorbed they are further metabolized by phase II enzymes in the liver yielding urolithin-glucuronide, urolithin sulfatase and methyl urolithin (40). These compounds have been found in the urine and blood of human subjects after consumption of pomegranate juice, raspberry, strawberry, oak-aged red wine, and walnuts (40-42). A model using an Iberian pig analyzed the fate of urolithins after consumption of acorns

(43).

On the other hand, condensed tannins up to trimers have been reported to be absorbed by the intestinal cell monolayer model using Caco-2 (44); nevertheless, this 7 study analyzed only monomers through trimers and a hexamer, leaving doubts for tetramers and pentamers of procyanidin. A review by Awika and Rooney (45), mentioned that monomers and dimers of procyanidin may be directly bioavailable in humans, whereas the rest of the oligomers may be degraded later by the colon microflora into simpler phenolic acids that could then be absorbed. Nevertheless, non-absorbed consensed tannins have been proven to favorably alter the gastrointestinal (GI) flora as they exhibited antimicrobial activity against pathogenic bacteria (46). The potential presence of condensed and hydrolyzable tannins in pecan has been postulated (9). Further research is needed to assess the type of hydrolyzable tannins and the medium degree of polymerization present in pecan kernels.

Health benefits

Tannins are generally known to have certain health benefits such as antioxidant, anti-allergenic, anti-hypertensive, and antitumorigenic, as well as antimicrobial activities

(47). Condensed tannins are present in foods of high antioxidant capacity, such as wine, cocoa, and green tea (21). They are associated with a lower incidence of cardiovascular disease, prevention of LDL oxidation, reduction of thrombosis, lessening of the inflammatory process in atherosclerosis, etc. (48). Hydrolyzable tannins have been identified in several fruits and nuts, such as pomegranate juice, acorns, blackberries, strawberries, raspberries, walnuts, muscadine grapes (24, 26-29, 38, 49-50).

Ellagitannins have recently attracted attention because of their antioxidant capacity (49, 51), antiproliferative activity against prostate cancer (52-53), antibacterial 8 properties (54), and as apoptosis inducers in colon cancer (55), among others. Upon hydrolysis, ellagitannins release ellagic acid, which is also of particular interest as it, reportedly, has antiviral (56) and anti-inflammatory properties (57), and provides protection against cancers of the colon, lung and esophagus (58-59).

Urolithins have proven beneficial effects against prostate and colon cancer and exert some proestrogenic/antiestrogenic effects (52, 55, 60), as well as anti-inflammatory activity in colon fibroblasts and protection against oxidative stress (61-62).

Pecan kernels exhibit high antioxidant activity, yet there are no studies related to the bioactivities of defatted pecans. There have been studies to test the bioactivities of ellagitannins (53, 63); however, these studies do not consider the fact that ellagitannins are hydrolyzed into ellagic acid which is then metabolized by the gut microflora into urolithins. Most ellagitannins present in foodstuff are transformed to ellagic acid during the digestion in the first part of the gastrointestinal tract, meaning also that bacteria present in the jejunum are able to metabolize ellagic acid into a number of degradation metabolites (55).

Obesity overview

Many studies have focused on the anticarcinogenic, antimutagenic, anti- inflammatory, or chemopreventive activities of phytochemicals; whereas effects of these compounds as potential anti-obesity treatments have started to acquire attention in recent years. Obesity is a global epidemic that started over 20 years ago and is defined as

“abnormal or excessive fat accumulation that may impair health” by WHO (64). Obesity 9 is present in both developed and developing countries and it is characterized by several metabolic disorders, most of them related to glucose homeostasis and to the development of cardiovascular diseases (65). In 2006, WHO reported that more than 1 billion adults (>

15 years of age) were overweight, that at least 400 million adults were obese and projected that, by year 2015, about 2.3 billion adults will be overweight and more than

700 million will be obese (64). An estimated 22 million children worldwide, under age 5, are estimated to be overweight, which increases risk of premature death and disability during adulthood (64). Obesity is a complex disorder with multiple causes including both genetic and environmental factors, where adverse health effects are seen after prolonged periods of positive energy balance, a high consumption of energy-dense foods and reduced physical activity, and is mostly related to a high-fat diet and sugars ingestion in

Western countries (66). Among obesity causal factors are the abundance of food, increase of snacking and eating late at night, the decrease in physical activity due to sedentary work, changing modes of transportation and urbanization (67).

Distribution of body fat is relevant from the clinical point of view, as it has been proven that visceral fat obesity–where fat accumulation is predominant in the intra- abdominal cavity–is accompanied with more frequent disorders of glucose and lipid metabolism, and hypertension, compared to subcutaneous fat obesity (68). Obesity is associated with the risk of insulin resistance (type 2 diabetes), fatty liver disease, hypertension, atherosclerosis, and chronic inflammation (66, 69).

10

Adipose tissue and gene expression

Adipose tissue had been considered as inert, having the sole function of energy storage; however, the increase in knowledge concerning its role in energy balance along with the increased occurrence of obesity and metabolic disorders, made the scientific community focus on this tissue. Adipose tissue is classified into brown (BAT) and white adipose tissue (WAT). BAT is present in significant amounts in infants, but it is negligible in adults; it plays an important role in nonshivering thermogenesis and produces heat whenever the organism is in need for extra heat, e.g., posnatally (70).

WAT is the main site for energy storage in the form of triacylglycerols and provides thermal and mechanical insulation. In the past decade, WAT has been recognized as an endocrine organ, and its adipocytes secrete major hormones, mainly leptin and adiponectin, together with an array of protein signals and transcription factors, called adipokines, that regulate homeostasis, blood pressure, immune function, angiogenesis and energy balance (71-72). Obesity results in an excess of WAT; it alters adipose tissue metabolic and endocrine function by increasing the release of fatty acids, hormones, and proinflammatory molecules that enhance obesity associated complications (73).

Obesity is characterized by adipose tissue growth, where adipocytes increase in size (hyperthrophy) and new adipocytes from precursor cells are formed (hyperplasia).

The adipocyte life cycle involves the transition from undifferentiated fibroblast-like preadipocytes into mature adipocytes; treatments that regulate both size and number of adipocytes may provide a better therapeutic approach for treating obesity (74). 11

Over 50 adipokines have been discovered to date; among the most studied ones are leptin and adiponectin. Interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) are regarded as proinflammatory cytokines. Leptin regulates appetite, food intake and energy expenditure; weight gain results in an increase plasma leptin level (75). Adiponectin plays a role in energy homeostasis and insulin sensitivity by protecting against inflammation and obesity-liked insulin resistance (76). The excessive production of proinflammatory adipokines has been connected to the metabolic complications of obesity (73). TNF-α is involved in inflammatory responses and is a key regulator of the synthesis of IL-6. A relationship between plasma TNF-α system and indices of obesity has been reported (71). IL-6 regulates immune response, hematopoiesis, acute phase response, and inflammation; it also plays a role in the pathogenesis of insulin resistance

(77). Macrophages are responsible for most adipose tissue TNF-α expression and significant amounts of IL-6; their numbers increase in obesity and participate in inflammatory pathways that are activated in adipose tissue of obese individuals (73).

Nevertheless, inflammatory status of macrophages is more relevant than the number of macrophages in terms of controlling the production of these pro-inflammatory adipokines

(78).

In mature adipocytes, expression of the above-mentioned adipokines and their response to molecules, such as TNF-α, is closely regulated by different transcription factors, whose function is to modulate gene expression in order to maintain homeostasis.

Some important transcription factors involved in the adipocyte differentiation process and their response to some pro-inflammatory cytokines are PPARγ, C/EBPα and NF-κB. 12

In mammalian cells, the NF-κB family of transcription factors consist of four proteins: p65 (RelA), RelB, p105/p50 and p100/p52, which are expressed in 3T3-L1 adipocytes

(79). The NF-κB pathway is a central regulator of cellular responses in a healthy state and in disease. When cells are in a resting state, NF-κB dimers are normally kept inactive by association with proteins of the inhibitor of NF-κB (IκB) family. But NF-κB is rapidly activated in response to stress-inducing stimuli, providing the cell with a sensitive and quick detection system for potential threats. NF-κB also determines cellular responses by translating upstream signals into a rapid reprogramming of gene expression. In addition,

NF-κB has been implicated in the pathogenesis of many inflammation-related diseases since it promotes the expression of pro-inflammatory genes (80). Moreover, the TNF-α stimulus promotes the inhibition of the transcriptional activity of PPARγ, probably in a manner-dependent by NFκB p65. It has been reported that NF-κB (p65) inhibited

PPARγ-responsive reporter gene expression in a dose-dependent manner (81).

For the past 30 years, in vitro systems have been used to study adipocyte differentiation; 3T3-L1 being the most frequently employed cell line (82). During the growth phase, preadipocytes are morphologically similar to fibroblasts; at confluence, they enter a growth arrest phase before undergoing the differentiation process. Induction of differentiation converts the preadipocyte to an adipocyte, it aquires a spherical shape, lipid droplets begin to accumulate, and it starts to get the morphological and biochemical characteristics of a mature white adipocyte (83). Adipocyte differentiation involves a set of gene-expression events, where the peroxisome proliferator-activated receptor-γ

(PPARγ) and the CCAATT/enhancer binding protein-α (C/EBP-α) play a crucial role as 13 transcription factors. PPARγ is a member of the nuclear–receptor superfamily; its expression induces growth arrest and initiates adipogenesis in fibroblasts. It is also required for maintenance of the differentiated state (84). C/EBP-α is expressed just before the transcription of most adipocyte-specific genes; it works together with PPARγ, and is necessary for the acquisition of insulin sensitivity (84-85). PPARα belongs to the nuclear- receptor superfamily; it has been proven to induce gene expression involved with free fatty acid (FFA) uptake and mitochondrial β-oxidation (86-87). Obesity, type 2 diabetes and insulin resistance are closely associated to chronic inflammation that is characterized by an increase in pro-inflammatory cytokine production, such as TNF-α and IL-6 (88).

Objectives

1. To screen four pecan cultivars for their total phenolics, condensed tannins,

ellagitannins, and antioxidant capacity by DPPH and ORAC.

2. To characterize the defatted pecan kernel‟s phytochemicals by liquid

chromatography-mass spectrometry (LC-MS).

3. To evaluate the effects of the metabolites urolithin A and urolithin B, and the

ellagitannin precursor, ellagic acid, during adipogenesis in 3T3-L1 cells.

4. To evaluate the effects of the metabolites urolithin A and urolithin B, and the

ellagitannin precursor, ellagic acid, in inflammation by using 3T3-L1 cells. 14

CHAPTER II

PECAN CULTIVAR SCREENING FOR PHENOLICS, ANTIOXIDANT

CAPACITY, AND POLYPHENOLIC CHARACTERIZATION BY LC-MS

Summary

Pecan [Carya illinoinensis (Wangenh.) K. Koch] kernels have been found to have high antioxidant capacity and high condensed tannin content. The majority of the previous studies have focused on pecan oil, while few ones have analyzed the antioxidant exerting part: defatted pecan. The present study screened kernels from four cultivars

(Choctaw, Desirable, GraCross, and Kiowa) of pecan for their total phenolic content, condensed tannin content, ellagitannin content, and antioxidant capacity by DPPH and oxygen radical absorbance capacity (ORAC) assays. The polyphenolic profile of the crude extract was developed by LC-MS. „GraCross‟ had the highest total phenolic content, antioxidant capacity and condensed tannin content, but „Desirable‟ had the highest phenolic content and ellagitannin content. Condensed tannins up to pentamers, twelve ellagitannins, gallic acid, valoneic acid dilactone, ellagic acid, isorhamnetin glucoside, pentosyl, and galloyl pentosyl were found.

Introduction

Pecan is native to the midwestern and southern United States and scattered spots in Mexico. It was originally found along the rivers of Texas, Oklahoma and Louisiana

(3). Health benefits associated with pecan kernels have been mainly related to the lipid 15 portion (11-13), as it comprises about 72% of weight. In a clinical study conducted on healthy adults, consumption of pecan kernels favorably altered the serum lipid profile without increasing body weight (12). When pecans‟ lipophilic and hydrophilic antioxidant capacities were quantified with the ORAC assay, 98% of the total antioxidant capacity was attributed to the hydrophilic portion (15). Another study screened several nuts for their proanthocyanidin content – or condensed tannins – and it revealed that pecans had the second highest content, with no significant difference from the highest value found in hazelnuts (16). A previous study from our lab reported that, after screening defatted kernels from six pecan cultivars, there was a strong correlation between antioxidant capacity by ORAC and DPPH and total phenolic content (9). The study also found that 45% of the total phenolics belonged to the condensed tannins, but it also suggested the potential presence of hydrolyzable tannins after finding that gallic and ellagic acid were present in pecan extract after hydrolysis. It has been acknowledged that tannins are partly responsible for pecan kernel coloration, and that they are mainly located in the pellicle rather than in the meat itself (1). Findings on defatted pecan‟s high antioxidant capacity left a research opportunity to better understand the chemistry of this non-lipid portion.

The objectives of the present study were to screen four pecan cultivars for their total phenolics, condensed tannins, ellagitannins, and antioxidant capacities by ORAC and DPPH. To characterize the polyphenols present in the defatted kernel by liquid chromatography-mass spectrometry and phloroglucinolysis.

16

Materials and methods

Chemicals. The following chemicals were used in the experiments: Folin-

Ciocalteau‟s phenol reagent, 2, 2-diphenyl-1-picryl-hydrazyl (DPPH), Trolox (6- hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid), (+)-catechin hydrate, and fluorescein sodium salt were purchased from Sigma (St. Louis, MO). Sodium carbonate was sourced from LabChem (Pittsburg, PA). Chlorogenic acid and epicatechin were acquired from Fluka (Milwakee, WI). Vanillin was from Acros Organics (Fair Lawn,

NJ). 2, 2‟-Azobis (2-amidino-propane) dihydrochloride (AAPH) from Wako Chemicals

(Richmond, VI). Potassium phosphate monobasic was from J. T. Baker (Phillipsburg,

NJ). Potassium phosphate dibasic from Fisher BioReagents (Fair Lawn, NJ). Hexanes, diethyl ether, methanol and acetone HPLC grade from EMD Chemicals (Gibbstown, NJ).

Formic acid, hydrochloric acid, and acetonitrile HPLC grade were from Mallinckrodt

Chemicals (Phillipsburg, NJ). Water used was nanopure grade.

Pecan samples. Pecan fruits were mechanically harvested in Fall 2007 at a commercial orchard located in Mumford, Texas. After the frutis were cracked and manually shelled, kernels were stored at -20 ºC. The pecan kernels were defatted according to the method of Villarreal-Lozoya et al. (89). In brief, whole kernels were chopped in a food processor and defatted three times with hexane (1:20; w/v) in an

Ultraturrax T25 homogenizer (IKA Works, Willmington, NC). The homogenate was filtered and the defatted powder was left in the hood overnight for residual hexane evaporation. The powder was then crushed with a mortar and pestle and flushed with 17 nitrogen. Samples were stored at -20 ºC until further analysis and six replicates were done per cultivar.

A 1-g aliquot of defatted pecan powder was homogenized with 20 mL of 70% aqueous acetone and left overnight in an Eberbach shaker (Ann Arbor, MI) in dark at 5

ºC. The tubes were centrifuged at 18 000 g for 12 min and 11 mL of supernatant was collected. These extracts were used to measure total phenolic content, antioxidant capacity with DPPH and ORAC, and for identification of hydrolyzable tannins.

Total phenolic content. Total phenolic content was measured in a method adapted from Swain and Hillis (90), where 500 μL of pecan extract were mixed with 8 mL of water, then 500 µL of 0.25N Folin-Ciocalteau reagent was added, and, after 3 min,

1 mL of sodium carbonate was added. The reaction was left in dark at room temperature for 2 h, and then 300 µL from each tube was transferred to a clear 96-well plate

(Fisherbrand, Thermo Fisher Scientific, Pittsburgh, PA). Absorbance was measured at

725 nm in a plate reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski, VT). A blank sample was also run with water. Finally, a standard curve was developed with chlorogenic acid in water. Total phenolic content was reported as mg chlorogenic acid equivalents/g defatted pecan.

Condensed tannins. Condensed tannins, or proanthocyanidins, were measured according to the method of Price et al. (31) and adapted for pecans by Villarreal-Lozoya et al. (9). In brief, 0.5 g of defatted pecan powder was mixed with 15 mL 1% HCl in methanol, vortexed and left in a water bath at 30 ºC for 20 min. Tubes were vortexed again at the middle of the incubation time and centrifuged at 7740g for 15 min. 18

Supernatant was collected and 1 mL was used for the sample measurement and another 1 mL for the blank. Both tubes were placed in the water bath at 30 ºC. For the sample tube,

5 mL of vanillin reagent (0.5 g vanillin/4% HCl in methanol) was added, whereas 4%

HCl in methanol was added to the blank tube. After 20 min, absorbance of each sample and the blanks were read at 500 nm in a UV-Vis spectrophotometer (UV-1201S,

Shimadzu Corporation, Kyoto, Japan). A standard curve was run with catechin before and after the samples were run. Condensed tannins were reported as mg catechin equivalents/g defatted pecan.

Quantification of ellagitannins by HPLC. Since quantification of ellagitannins cannot be done as such due to the lack of standards (42, 91), they were quantified as ellagic acid equivalents after hydrolysis. Free ellagic acid naturally contained in the crude extract was subtracted from the hydrolysis value. Hydrolysis was carried out based on the method by Cerdá and others (42), with some modifications. In brief, 5 mL of 2 M HCl was added to 1 mL of the reconstituted aqueous kernel extract and left in an Isotemp

145D block heater (Fisher Scientific, Pittsburgh, PA) at 85 ºC for 20 h. The hydrolyzate was washed 3x with diethyl ether, and the organic phases were pooled together and evaporated in a SpeedVac concentrator (Thermo, Marietta, OH) at 35 ºC until dryness.

Samples were reconstituted with 1 mL of methanol, filtered with a 0.45µm PTFE filter and 50 µL were injected into the HPLC.

The HPLC system consisted of two Waters 515 gradient pumps, Waters 717 autosampler and a PDA detector (Waters Corp., Milford, MA). Mobile phase consisted of

5% formic acid in water (A) and acetonitrile (B). Flow rate was 1 mL/min. Sample was 19 passed through a 4.6 x 150mm Atlantis dC18 5 µm column (Waters, Milford, MA) coupled with a guard column of the same chemistry. Gradient was the same as with the

LC-MS analysis. An ellagic acid standard curve was developed to quantify the ellagic acid before and after hydrolysis.

Antioxidant activity with DPPH. Total antioxidant activity was measured with the DPPH radical, adapted from Brand-Williams et al. (92). Pecan extract (150 µl) was mixed with 2.85 mL of DPPH diluted solution. Tubes were covered, left overnight in a shaker (Eberbach, Ann Arbor, MI) at room temperature. An aliquot of 300 μL of each sample was deposited in a clear 96-well plate (Fisherbrand, Thermo Fisher Scientific,

Pittsburgh, PA) and absorbance was read at 515 nm in a plate reader (Synergy HT, Bio-

Tek Instruments, Inc., Winooski, VT). Blanks were run with water and a standard curve was run for trolox. The decrease in absorbance due to antioxidants was obtained by subtracting the sample‟s absorbance from the DPPH solution‟s absorbance. Antioxidant activity was reported as mg trolox equivalent/g defatted pecan.

Antioxidant capacity with ORAC. Antioxidant capacity by ORAC was done according to the methods previously described (93-94). Solutions for this assay were prepared with 75 mM phosphate buffer (pH 7.4). Fluorescein sodium salt (112.5 mg) was suspended in 50 mL of PBS (FL1) and a second solution (FL2) was made by diluting

(FL1) in PBS 1:100; these solutions were stored at 5ºC. A dark 96-well plate (Costar,

Corning Inc., Corning, NY) was loaded with 25 μL of diluted pecan extract and incubated at 45 ºC for 45 min. A third fluorescein solution [FL3] was made fresh by diluting 40 μL

[FL2] in 25 mL PBS. 2, 2‟-Azobis (2-amidino-propane) dihydrochloride (AAPH) reagent 20 was prepared with PBS. The plate was inserted into a plate reader (Synergy HT, Bio-Tek

Instruments, Inc., Winooski, VT), where 200 μL (FL3) and 75μL AAPH were automatically dispensed into each well. Fluorescence readings were taken for excitation

(485 nm) and emission (520 nm) at 1 min intervals for each well during 50 min and blanks were run with PBS. A standard curve with trolox was run to calculate the antioxidant capacity as mol trolox equivalents/g defatted pecan. Results were calculated as the area under the curve.

LC-MS analysis of pecan phenolics. Acetone from the pecan extracts was evaporated using a Speed Vac concentrator (Thermo, Marietta, OH) under vacuum at 35

ºC. The remaining aqueous extract was filtered with a 0.45 µm PTFE filter and freeze- dried (FTS Systems, Inc., Stone Ridge, NY) for 48 h. Liophilizates were nitrogen-flushed and stored at -20 ºC until further analysis. Extracts were reconstituted in 5 mL of water, diluted for analysis, passed through a 0.45m PTFE filter and a volume of 20 μL was injected into the LC-MS.

The LC-MS system consisted of a triple quadruple ion trap LCQ Deca XP Max

(Thermo Finnigan, San Jose, CA), a Surveyor P2000 quaternary pump, autosampler, and a UV 2000 PDA detector (Thermo Finnigan, San Jose, CA). The mobile phase consisted of 1% formic acid in water (solvent A) and acetonitrile (solvent B). Solvent gradient started with 1% B, increasing linearly to 25% at 20 min, reaching 55% at 30 min, 90% B at 31 min for 5 min. Column was equilibrated for 10 min in between samples. A 150 x

2.00 mm Synergi 4µm Hydro RP 80A column (Phenomenex, Torrance, CA) was used with a guard column of the same chemistry. Sample was delivered to the MS by 21 electrospray ionization. MS analysis was done in the negative ion mode; mass range was

80-1500 m/z. The MS conditions were as follows: 50 arb units sheath gas, 0 arb units auxiliary gas, temperature 275 ºC, spray voltage 4 kV, capillary voltage -21 V, tube lens offset -60 V. Nitrogen was used as the sheath gas and helium as the collision gas. MS/MS and MS3 analysis were used during the identification. Collision energies of 50% and 35% were used for the MSn analysis.

Phloroglucinolysis for qualitative and quantitative analysis of pecan condesed tannins after phloroglucinolysis by HPLC-MS/MS. The procedure used was adapted from the manuscript previously published by Kennedy & Jones (95). A 0.1 N

HCl methanol solution of phloroglucinol (50 g/L) and ascorbic acid (10 g/L) was prepared. The lyophilized samples were reacted in this solution (62.5 g/L) and vortexed for complete solubilization of the powder and then incubated at 50 ºC for 20 min.

Reaction was stopped by placing samples in an ice bath and by diluting the reaction medium with 1 mL of a 40 mM sodium acetate solution. The procedure used for the calculation of the apparent mean degree of polymerization (mDP) was previously described (95). Samples (10 L) were analyzed by reversed phase using a HPLC-DAD-

MS/MS system (Agilent Technologies, Waldbronn, Germany) equipped with a binary pump G1312A, autosampler G1313 A, photodiode array detector G1315B, controlled by

Agilent software v. A.08.03 and degasser G1322A). The mass detector was an ion-trap mass spectrometer equipped with an ESI system (capillary voltage, 5 kV; dry temperature, 350 °C). Mass scan (MS) and MS/MS daughter spectra were measured from m/z 200 to 2200 using the ultra scan mode (13,000 m/z per s). Collision-induced 22 fragmentation experiments were also performed using helium as collision gas, and the collision energy was set at 50%. Mass spectrometry data were acquired in the negative ionization mode. Separations were achieved on an Atlantis dC18 column (particle size 5

µm, 250 x 4.6 mm) purchased from Waters (Barcelona, Spain). The mobile phase was water-formic acid (98:2 v/v) (A) and methanol (B). The flow rate was 1 mL/min and a linear gradient, starting with 5 % B at time 5, 8 % B at 10 min, 13 % B at 15 min to reach

15 % B at 19 min, 40 % B at 47 min, 65 % B at 64 min and 98 % B at 69 min for 5 min and returning to the initial conditions (5 % B). The different flavonoids were identified by their UV spectrum, molecular mass: (epi)gallocatechin [M – H]- m/z 305; catechin [M

– H]- m/z 289, daughter ions and fragmentation pattern: (epi)gallocatechin-phloroglucinol

[M – H]- m/z 429, 305; catechin-phloroglucinol [M – H]- m/z 413, 289; epicatechin 3-O- gallate-phloroglucinol [M – H]- m/z 565, 413, 289. Quantification was performed using

UV detection at 280 nm with epicatechin and catechin as external standards. Results were expressed as mg/100 g of fresh weight (FW).

Statistical analysis. Data represent the mean of 6 replicate analyses for the total phenolics, condensed tannins, ORAC and DPPH; whereas triplicates were performed for ellagitannin quantification and condensed tannins quantification by phloroglucinolysis.

Analysis of means separation (Tukey HSD for total phenolics, condensed tannins,

ORAC, DPPH, and ellagitannins and Duncan post-hoc test for phloroglucinolysis, P <

0.05) were conducted using SPSS 15.0 (SPSS Inc., Chicago, IL).

23

Results and discussion

Total phenolics and tannins. The total phenolic content (TP) of the four pecan cultivars ranged from 76 to 121 mg CAE/g defatted pecan (Table 1). It was observed that

„GraCross‟ and „Desirable‟ had significantly higher values than „Choctaw‟ and „Kiowa‟, with the following trend: „GraCross‟ ≈ „Desirable‟ > „Choctaw‟ ≈ „Kiowa‟. It has been reported that total phenolics differ among cultivars, cultivation practices (organic vs. conventional), and growing locations (89, 96). „Desirable‟ and „Koiwa‟ had been screened in a previous study with significant differences obtained as „Kiowa‟ had 76 ±

2.5 CAE/g and „Desirable‟ from two growing locations had 70 ± 2.0 CAE/g and 62 ± 2.3

CAE/g (89). Another study compared the cultivation methods (organic versus conventional) of three pecan cultivars „Cheyenne‟, „Desirable‟, and „Wichita‟ (96).

Phenolics were quantified after LH-20 chromatography by HPLC of aqueous methanolic extractions; no differences in ellagic acid, catechin or gallic acid content were seen for

„Cheyenne‟ grown either with organic or conventional practices (96). For „Desirable‟, the organic method doubled the conventional in terms of catechin content (90.00 g/kg versus

48.21 g/kg, respectively), whereas for ellagic acid content, the same trend was observed with more than 4-fold the content for organic than conventional (86.21 g/kg versus 20.96 g/kg, respectively) (96). Gallic acid content was higher in conventionally grown

„Wichita‟ than in those grown organically. A larger difference was observed for ellagic acid, as kernels grown with organic methods had a higher value than those grown with conventional methods (96). 24

Table 1. Phenolics and antioxidant capacity screening of four pecan cultivars extracted with 70% aqueous acetone.

ETc TPa CTb AC ORACd AC DPPHe (mg EA/100 g (mg CAE/g) (mg CE/g) (µmol TE/g) (mg TE/g) pecan FW)

Choctaw 86 ±4.5 bf 68±3.9 b 46±3.2 ab 658±40.4 b 117±19.7 b Desirable 111±5.6 a 65±5.9 b 52±5.4 a 702±42.6 b 116±19.7 b GraCross 121±13.2 a 94±15.0 a 42±2.5 b 858±89.8 a 163±18.9 a Kiowa 76±8.4 b 43±6.8 c 50±4.5 a 507±52.9 c 78±16.9 c Averageg 98±20.4 68±1.3 47±5.3 681±56.4 118±6.7 a Total phenolic content, milligrams of chlorogenic acid equivalents per gram of defatted kernel; b Condensed tannin content, milligrams of catechin equivalents per gram of defatted kernel; c Ellagitannin content, milligrams of ellagic acid per 100 grams of pecan kernel fresh weight; d Antioxidant capacity by ORAC, micromoles of Trolox equivalents per gram defatted kernel; e Antioxidant capacity by DPPH, milligrams Trolox equivalents per gram defatted pecan kernel; f Values with similar letters are not significantly different (Tukey HSD, P < 0.05); gMean values of cultivars analyzed ± S. D.

25

In the present study, the condensed tannins content, quantified by the vanillin assay as catechin equivalents, ranged from 43 to 94 mg CE/g defatted pecan, where

„GraCross‟ had the highest value, followed by „Desirable‟ and „Choctaw‟, and then

„Kiowa‟ (Table 1). The average of the four cultivars was 68 mg CE/g, which is 2-fold higher than reported for the six pecan varieties that were tested previously (89). Tannin content ranged from 1.1-2.3% of kernel weight (considering 71.97% oil and 3.5% water content in pecan (10)). These obtained values are comparable to those previously reported by different varieties, with the exception of „GraCross‟ that had not been evaluated (89, 97). Other values for condensed tannins, or proanthocyanidins, among nuts have been found to be 0.4%, 0.5%, and 0.2% of kernel weight for pecans, hazelnuts, and pistachios, respectively (16).

On the other hand, hydrolyzable tannins, or ellagitannins quantified as ellagic acid equivalents by HPLC after acid hydroysis, showed an inverse trend as the one seen for condensed tannins, because „Desirable‟ and „Kiowa‟ presented the highest content, followed by „Choctaw‟ and „GraCross‟ (Table 1). The ellagitannin content ranged from

42 to 52 mg EA/100 g fresh weight; these values are similar to those reported for strawberries (40 mg EA/100 g FW), but lower from red raspberry (70 EA/100 g FW)

(91).

Although the previous study showed a condensed tannin content 50% lower than the current work, the ellagitannin content was 2-fold than the current work (89). This may indicate a possible inverse correlation among the condensed and hydrolyzable tannins, but the latter were present in a very small quantity (0.04-0.05% kernel weight) compared 26 to condensed tannins (1.1-2.3% kernel weight) (Fig. 2). Others have reported values for condensed tannins in pecan of known cultivars of 0.7-1.7% and 0.4% of unknown cultivar(s) (9, 97-98).

A strong correlation was reported for condensed tannins and antioxidant capacity of pecan kernels; which indicated that condensed tannins play a major role in the antioxidant capacity of pecan in vitro. Bioavailability up to trimers has been proven for condensed tannins with a Caco-2 cell model (44); therefore, around 17% of total condensed tannins (from monomers through trimers) could be absorbed by the gut (16), which translates to 0.12-0.39% from the condensed tannins herein reported.

Antioxidant capacity by DPPH and ORAC. The antiradical activity measured by the DPPH scavenging capacity assay ranged from 78 ± 16.9 to 163 ± 18.9 mg TE/g defatted pecan. The highest value was achieved by „GraCross‟, followed by „Desirable‟ and „Choctaw‟, and then „Kiowa‟. The average of the four cultivars was 118 ± 16.9 mg

TE/g defatted pecan, which is similar to 97 ± 6.7 mg TE/g defatted pecan, reported for six cultivars (89).

Differences among cultivars were observed in ORAC activity as „GraCross‟ had the highest value (858 ± 89.8 µmol TE/g defatted pecan), followed by „Desirable‟ and

„Choctaw‟ (702 ± 42.6 and 658 ± 40.4 µmol TE/g defatted pecan, respectively), and

„Kiowa‟ (507 ± 52.9 µmol TE/g defatted pecan). The mean was 681 ± 56.4 µmol TE/g defatted pecan, a slightly higher value than the previously reported of 583 ± 32 µmol

TE/g defatted pecan (89). 27

Figure 2. Condensed tannins versus ellagitannins from four pecan cultivars. Oil from the kernel was extracted; condensed tannins were evaluated with the vanillin assay and ellagitannins were quantified as ellagic acid equivalent after 2 M HCl hydrolysis by HPLC-DAD. For condensed tannins, n = 6 for each average mean ± S.D., while for ellagitannins n = 3 for each average ± S.D.

28

ORAC values reported for nuts (15) are 584 µmol TE/g d.w. for pecans, 435 µmol TE/g d.w. for walnuts, 309 µmol TE/g d.w. for hazelnuts, and 252 µmol TE/g d.w. for pistachios.

The same trend of „GraCross‟ > „Desirable‟ ≈ „Choctaw‟ > „Kiowa‟ was observed for both antioxidant capacities and condensed tannins with the vanillin assay.

It could be inferred that condensed tannins have a greater effect on antioxidant capacity than hydrolyzable tannins. A strong correlation of r2 = 0.75 was found between ORAC and condensed tannins for the previous study (89), and r2 = 0.96 for the present study.

LC-MS analysis of phenolics. The main polyphenolic compounds (Figures 3 and

4; Table 2) present in pecan kernels after removing the oil were identified based on their retention time, UV-spectra, mass spectra, and fragmentation patterns. Most of the peaks were visible at 360 and 280 nm. Data is presentd for the GraCross cultivar; all four cultivars presented the same phenolic profile, but with differences in some compounds.

- Peak 1 (tR = 2.95 min; λmax 248 nm) had a [M – H] at m/z 481 and its MS/MS yielded an ion at m/z 301 [M – 180]-, loss of a glucose unit. It was identified as HHDP- glucose (29). Since m/z 301 was the most abundant, it was further fragmented (MS3) and m/z 229, 257 resulted as the daughter ions, which are characteristic of ellagic acid (38).

Figure 5 shows the different UV spectra seen for the ellagitannins, which acquire an ellagitannin-like spectra as they elute. This peak gives a more intense signal when observed at 280 nm. 29

Figure 3. LC chromatogram of polyphenolics present in „GraCross‟ pecan kernel at 360 nm. Pecan extract was obtained by mixing defatted pecan powder and 70% aqueous acetone (1:20; w:v). Peak assignments are listed in Table 2.

29

30

Figure 4. LC chromatogram of polyphenolics present in „GraCross‟ pecan kernel at 280 nm. Pecan extract was obtained by mixing defatted pecan powder and 70% aqueous acetone (1:20; w:v). Peak assignments are listed in Table 2.

30

31

Table 2. Characterization of phenolic compounds in „GraCross‟ pecan using HPLC-DAD-ESI-MSn detection.

Peak t λmax [M – H]- MS/MS MS3 R Tentative identification No. (min) (nm) (m/z) (m/z)a (m/z)a 1 2.95 HHDP-glucose 248 481 301, 275, 257 257, 229 2 4.82 Gallic acid 280 169 125 - 3 8.08 Pedunculagin isomer 246 783 481, 301, 275, 257 257, 229 4 10.59 Pedunculagin isomer 246, 357, 371, 378 783 481, 301, 275, 231 257, 229 5 10.93 Pedunculagin isomer 246, 371 783 481, 301, 275, 231 257, 229 6 11.18 Pedunculagin isomer 247, 371 783 481, 301, 275, 257 257, 229 7 11.78 Galloyl pedunculagin 245 951 933, 799, 649, 301 - 8 11.99 Galloyl pedunculagin 245 951 933, 799, 649, 301 - 9 12.91 Procyanidin dimer 278 577 451, 407, 425, 289, 245 245, 243 10 13.72 Catechin 275 289 245 - Catechin deriv of stachyurin 275 1207 603, 458, 289 - 11 13.99 HHDP galloyl glucose 273 633 481, 301, 257, 229 257, 229 Strictinin/isostrictinin 12 14.4 Galloyl-bis-HHDP glucose 275 935 917, 783, 633, 481, 301 - Casuarictin/casuarinin 13 14.87 Procyanidin tetramer 272 1153 1135, 1027, 983, 865, 577 - 14 15.28 Digalloyl HHDP glucose 276, 337 785 633, 483, 301 - 15 15.71 Castalagin/Vescalagin 276, 337 933 631, 451, 301 257, 229 543, 525, 405, 363, Procyanidin trimer 276, 337 865 695, 577, 451, 407, 287 289, 243 16 16.64 Castalagin/Vescalagin 273 933 631, 451, 301 257, 229 17 16.99 Castalagin/Vescalagin 274, 346, 363 933 631, 451, 301 257, 229 Valoneic acid dilactone 274, 346, 363 469 425, 301 407, 301, 245, 243 18 17.64 Unkown 247, 362 551 529 - 19 17.83 Procyanidin trimer 275 865 695, 577, 451, 407, 287 - Procyanidin tetramer 275 1153 1135, 1027, 983, 865, 577 -

a Ions in boldface indicate the most intense product ion, which was chosen for MS3.

31

32

Table 2. (Continued).

Peak t λmax [M – H]- MS/MS MS3 R Tentative identification No. (min) (nm) (m/z) (m/z)a (m/z)a 20 18.28 Procyanidin trimer 275, 362 865 695, 577, 451, 407, 287 - Procyanidin tetramer 275, 362 1153 1135, 1027, 983, 865, 577 - Procyanidin pentamer 275, 362 1441 1153, 865, 575 - 21 18.63 Procyanidin trimer 277 865 695, 577, 451, 407, 287 - Procyanidin tetramer 277 1153 1135, 1027, 983, 865, 577 - Procyanidin pentamer 277 1441 1153, 865, 575 - 22 18.87 Procyanidin trimer 277 865 695, 577, 451, 407, 287 - Procyanidin tetramer 277 1153 1135, 1027, 983, 865, 577 - Procyanidin pentamer 277 1441 1153, 865, 575 - Ellagic acid pentose 23 19.39 250, 360 433 301 257, 229 conjugate 24 20.02 Isorhamnetin glucoside 247, 359 477 315, 300 244 25 20.46 Ellagic acid 248, 367 301 257, 229 - Glausrin C + gallic acid 248, 367 1085 933, 783, 633, 481, 451 301 26 22.31 Isorhamnetin pentosyl 247, 359 447 315, 300 300, 244, 216, 200 27 22.55 Isorhamnetin pentosyl 247, 363 447 315, 300 300, 244, 216, 200 Ellagic acid galloyl pentose 247, 363 585 433, 415, 301 257, 229 conjugate Ellagic acid galloyl pentose 28 23.57 246, 360 585 433, 415, 301 257, 229 conjugate Isorhamnetin galloyl 29 25.34 246, 268, 353, 365 599 447, 315, 300 315, 300, 244, 216 pentosyl Isorhamnetin galloyl 30 25.59 246, 268, 353, 365 599 447, 315, 300 300, 244, 216 pentosyl a Ions in boldface indicate the most intense product ion, which was chosen for MS3.

32

33

- Peak 2 (tR = 4.82 min; λmax 270 nm) had a [M – H] at m/z 169, corresponding to gallic acid (27). The MS/MS yielded the characteristic ion at m/z 125, with a carboxyl loss (44 amu).

Peaks 3 (tR = 8.08 min; λmax 246 nm), 4 (tR = 10.59 min; λmax 246, 371 nm), 5 (tR

- = 10.93 min; λmax 246, 371 nm), and 6 (tR = 11.18 min; λmax 247, 371 nm) had a [M - H] at m/z 783 and were identified as pedunculagin isomers, as it has been previously reported (24, 99). At different retention times, this ion presented the same mass and fragmentation pattern m/z 481, 301, indicating loss of a HHDP and a glucose moiety, respectively. When HHDP is released from a molecule (MS/MS fragmentation or by hydrolysis) it yields ellagic acid, which is the dilactone of HHDP (30, 91). Since the most abundant daughter ion was at m/z 301; it was further fragmented with MS3 ions at m/z 257 and 229, characteristic of ellagic acid. Pedunculagin isomers have also been found in walnuts, blackberries, and strawberries (24, 50, 99). These peaks gave a more intense signal when observed at 280 nm. Figure 6 shows the different UV spectra found for this compound at different times. It is observed that absorbance at 360 nm increases as compounds eluted.

Peaks 7 (tR = 11.78 min; λmax 245 nm) and 8 (tR = 11.99 min; λmax 247, 371 nm) had a [M – H]- at m/z 951 and was identified as an ellagitannin due to its fragmentation pattern (m/z 907, 783, 481, 451, 301). MS3 of ion m/z 301 could not be achieved due to a low signal of this ion at the MS2. This compound has been identified as tris galloyl

HHDP glucose (100); while another study considered this compound as an ellagitannin and found it eluted at different times for blackberries (24). 34

Figure 5. Characteristic UV-spectra of ellagitannins from „GraCross‟. These ellagitannins were eluted during the first 14 min (A), and the ones eluted at around 20 minutes (B).

Figure 6. UV-spectra of pedunculagin isomers (m/z 783) as they elute at increasing retention times, for „GraCross‟. Their start to acquire an ellagitannin typical spectra.

35

The ion at m/z 907 resulted from the loss of a CO2 (44 amu). A loss of gallic acid

(168 amu) is observed from the daughter ion at m/z 783; whereas a loss of an HHDP was seen between m/z 783 and 481, and a glucose between m/z 481 and 301. Lastly, this molecule seems to be formed by 2 HHDP, a gallic acid and glucose; it is similar to pedunculagin at m/z 783, but with an additional galloyl moiety. This compound eluted at different times (Figure 7).

Peak 9 (tR = 12.91 min; λmax 278 nm) was identified as a procyanidin dimer due to its catechin-like UV spectrum and [M – H]- at m/z 577. Different procyanidin dimer combinations and their fragmentation patterns were described (21, 37), the results obtained from this study match the description of the dimer composed of (epi)catechin-

(epi)catechin, as it presents a fragmentation pattern of m/z 451, 425, 407, 289, 245. It is observed from this fragmentation that the major fragment ion is due to the cleavage of the interflavonoid C-C linkage with a loss of a catechin unit (101). The loss at m/z 451 yielded a gallic acid loss (126 amu); whereas the ion at m/z 425 resulted from a Retro

Diels-Alder (RDA) reaction in the terminal unit.

It has been documented that RDA is the most important fragmentation for structure elucidation of dimers (37). The m/z 407 results from the loss of H2O from the ion at m/z 425 and the ion at m/z 451 results from a loss of a gallic acid (126 amu).

Figure 8 presents the extracted ion chromatogram for this mass; where 2 major and 2 minor peaks were observed. All these peaks, which mean, when this ion is present, have the same fragmentation pattern. The stereoisomers cannot be determined by mass spectrometry, as it has been pointed out before (102). 36

Figure 7. Extracted ion chromatogram for m/z- 951, from „GraCross‟.

Figure 8. Extracted ion chromatogram for m/z 577, from defatted pecan nut extract. It was identified as a procyanidin dimer, from „GraCross‟. 37

A OH B OH HO O OH HO O O OH OH OH OH OH OH O OH HO O OH OH HO OH OH OH

Figure 9. Two different types of linkages between flavan-3-ols to form condensed tannins.

Figure 10. Extracted ion chromatogram (EIC) of m/z- 603, 1207, 289, and 865, for „GraCross‟. These ions elute at similar times. 38

06130909 #677-706 RT: 13.71-14.27 AV: 15 NL: 3.88E7 T: - c ESI Full ms [80.00-1500.00] 865.08

35000000

30000000

25000000 1206.90

20000000 Intensity 15000000 90.77 334.80 603.20 136.57 10000000 747.91 1414.94 1066.90 289.06 5000000 402.75 1265.60 224.81 518.91 0 200 400 600 800 1000 1200 1400 m/z

Figure 11. MS spectra of [M – H]- m/z 1207 and [M – 2H]2- m/z 603, tentatively identified as catechin derivative of stachyurin.

OH OH

HO O OH

T-Unit OH OH OH OH

HO O OH M-Unit OH OH OH OH

HO O

B-Unit OH OH

Figure 12. Proanthocyanidin trimer showing the T-unit.

39

A study where 88 foods where screened for their condensed tannins, found that pecan had the B-type linkage (21), the ion at m/z 289 resulting from the MS/MS of m/z 577 characterizes the B-type linkage (37). Figure 9 shows the two different types of linkages between flavan-3-ols to form condensed tannins.

Peak 10 (tR = 13.72 min; λmax 275 nm) showed the presence of three compounds which had [M – H]- at m/z 289, 865 and 1207, respectively (Figure 10). The first was tentatively identified as a catechin derivative of stachyurin (103); the [M – H]- at m/z

1207 could not be further fragmented, since the daughter ions varied considerably. In the

MS spectra (Figure 11), a [M – 2H]2- was noticed at m/z 603, and its MS/MS yielded as m/z 458 and 289, which correspond to [M – 2H – catechin]2- and catechin. No MS3 could be performed as the signal for this ion was too weak.

The second compound was identified as a (+)-catechin, due to its retention time,

UV spectrum and mass of m/z 289. The product ion of MS/MS analysis was m/z 245, corresponding to the loss of carbon dioxide (44 amu), as it has been reported to correspond to this flavan-3-ol (99). An authentic reference standard of (+)-catechin was run and matched the above mentioned parameters.

The third compound was identified as a procyanidin trimer (Figure 12), as it has been widely reported for this mass with the fragmentation pattern characterized by the losses of (epi)catechin units (288 amu) (37, 101, 104-105).

- Peak 11 (tR = 13.99 min; λmax 273 nm) had a [M – H] at m/z 633 and was identified as HHDP galloyl glucose (24, 100); other authors refer to this compound as strictinin or isostrictinin (50, 106). The latter compounds vary only in the positions of 40

HHDP and the galloyl moiety in the glucose core, they were first found in tea (33). The

MS/MS of m/z 481 resulted in a loss of a galloyl moiety (152 amu), followed by a subsequent loss of glucose (m/z 301). The mass m/z 301 was further fragmented and yielded the characteristic daughter ions of ellagic acid, m/z 229 and 257.

- Peak 12 (tR = 14.4 min; λmax 275 nm) had a [M – H] at m/z 935 and was tentatively identified as galloyl-bis-HHDP glucose (24); other authors refer to it as casuarictin or casuarinin, the only difference is that the first one has a glucose molecule and the latter one, a polyol (32, 50). The MS/MS m/z 917 results in a hydroxyl group loss in the form of a water molecule (18 amu); whereas the rest of the daughter ions found at m/z 633, 481, 301, represent a typical ellagitannin fragmentation pattern: [M –

302]- loss of a HHDP, and m/z 481 loss of a galloyl moiety (152 amu).

Peaks 13 (tR = 14.87 min; λmax 272 nm); 19 (tR = 17.83 min; λmax 275 nm); 20 (tR

= 18.28 min; λmax 275, 362 nm); 21 (tR = 18.63 min; λmax 277 nm); and 22 (tR = 18.87

- min; λmax 277 nm) had a [M – H] at m/z 1153 and were identified as a procyanidin tetramer (28, 36, 105, 107). The MS/MS yielded [M – H]- at m/z 1135, 1027, 983, 865,

577, which correspond to losses of H2O (18 amu), gallic acid (126 amu), (epi)catechin

(288 amu), and procyanidin dimer (576 amu), respectively.

- Peak 14 (tR = 15.28 min; λmax 276, 337 nm) had a [M – H] at m/z 785 and was identified as digalloyl-HHDP glucose. The MS/MS fragments at m/z 633, 483, 301 showed the loss of a galloyl moiety at m/z 633; loss of HHDP at m/z 483; coincided with those previously reported for these isomers (24, 27, 50). 41

Figure 13. Extracted ion chromatogram of m/z- 785 isomers of digalloyl-HHDP glucose. They were identified from defatted pecan nut extract from GraCross cultivar.

HO OH HO OH OH

HO OH HO OH OH O O O O O O R1 O O O O HO O H OH O O O R2 O HO OH O OH O O O HO H O HO OH OH O OH OH HO OH HO OH OH HO OH HO OH Castalagin R1 = H, R2 = OH Vescalagin R1 = OH, R2 = H Glausrin C

Figure 14. Structures of castalagin, vescalagin, and glausrin C ellagitannins with the - same MW of 934. This molecular weight corresponds to the m/z 933 found at tR = 15.71, 16.64, 16.99 min. 42

When extracting the ion chromatogram (Figure 13), four peaks were seen, but the most intense peak shows at this retention time.

Peaks 15 (tR = 15.71 min; λmax 276, 337 nm), 16 (tR = 16.64 min; λmax 273 nm),

- and 17 (tR = 16.99 min; λmax 274, 346, 363 nm) had a [M – H] at m/z 933 and were tentatively identified as castalagin/vescalagin isomer as it has been reported for heartwood and chestnut wood, raspberries (24, 101, 108). The difference between castalagin and vescalagin relies on their stereochemistry, in the position C6 of the glucose core (Figure 14). The MS/MS fragments yielded a HHDP loss (302 amu) at m/z

631, a subsequent glucose loss (180 amu) at m/z 451, and a loss of gallic acid (150 amu) at m/z 301. Another ellagitannin found in walnut with the same m/z 933 has been named

Glausrin C (50). It was characterized as a C-glucosidic ellagitannin, since gallic acid was located as a tergalloyl form after running 1H NMR.

- At the same retention time as peak 15 (tR = 15.71 min) a [M – H] at m/z 865, was identified as procyanidin trimer. It had the same fragmentation pattern as for the one described earlier.

Peak 17 (tR = 16.99 min; λmax 274, 346, 363 nm) had 2 compounds coeluting at m/z 933 and at m/z 469. The first had been previously identified as castalagin/vescalagin or Glausrin C (24, 50, 101, 108). The latter was identified as valoneic acid dilactone due

- - to its mass to charge ratio, [M – 44] and [M – 168] losses of CO2 and gallic acid, respectively. Valoneic acid dilactone has been identified in acorns and walnuts (27, 109).

Peak 18 (tR = 17.64 min; λmax 247, 362 nm) was originally thought to be a mass of m/z 1103, but the full MS also showed a mass half this value, m/z 551. To discern 43

between both ions, a MS/MS was run with lower collision energy (35%). Only m/z 551 could be fragmented, whereas no signal was found for any m/z 1103 daughter ions. Since

35% collision energy was entirely breaking this mass, CE was reduced to 25%; a low signal for m/z 551 was seen along with the daughter ion m/z 529. This ion remains unknown, since no MS3 data could be obtained due to low signal values for the MS/MS.

- Peak 19 (tR = 17.83 min; λmax 275 nm) presented [M –H] at m/z 865 and 1153, corresponding to procyanidin trimer and tetramer, respectively. Condensed tannins had a significant presence through these pecan extracts; this could be confirmed by the extracted ion chromatogram (EIC) of the respective masses from monomer to pentamer

(Figure 15).

Peaks 20 (tR = 18.28 min; λmax 275, 362 nm); 21 (tR = 18.63 min; λmax 275, 362 nm); and 22 (tR = 18.87 min; λmax 275, 362 nm); presented procyanidin trimers, tetramers, and pentamers. These masses were found at different times due to the reversed phase separation method applied. On this matter, several publications have proposed a normal phase separation method for proanthocyanidins (36, 107, 110) which separates them on their oligomers; there have also been work using LC-MS reversed-phase to identify the oligomers present (37, 101, 105).

- Peak 23 (tR = 19.39 min; λmax 250, 360 nm) had a [M – H] at m/z 433 and was identified as ellagic acid pentose conjugate (27, 38). The UV spectrum is similar to that of ellagic acid and was eluted before ellagic acid. A pentose loss (132 amu) is observed at m/z 301, this loss is characteristic of 5-carbon sugars. The MS/MS analysis of m/z 301 yielded ions at m/z 257 and 229, corresponding to ellagic acid. 44

Figure 15. Extracted ion chromatogram for procyanidin monomer, dimer, trimer, tetramer and pentamer found in „GraCross‟ pecan kernels.

OCH3

HO O OH

OGlu OH O

Figure 16. Structure of the flavonol isorhamnetin glucoside. 45

Peak 24 (tR = 20.02 min; λmax 247, 359 nm) was identified as isorhmamnetin glucoside, with a m/z 477 (111-113). This compound is classified as a flavonol, its structure is shown in Figure 16; and its MS/MS showed a loss of glucose (162 amu) at m/z 315 and a subsequent loss of a methyl group (15 amu) at m/z 300.

Peak 25 (tR = 20.46 min; λmax 248, 367 nm) was identified as ellagic acid (m/z

301). Retention time, UV spectrum, ion mass and fragmentation pattern (m/z 257, 229) matched the authentic standard.

At this same time, a [M – H]- at m/z 1085 was also detected, tentatively identified as a Glausrin C/Castalagin/Vescalagin isomer plus a galloyl moiety, observed by the loss of 152 amu. The fragmentation pattern matched the one for m/z 933, which was described for peaks 12-14.

Peaks 26 (tR = 22.31 min; λmax 247, 359 nm) and 27 (tR = 22.55 min; λmax 248,

363 nm) with a [M – H]- at m/z 447, were tentatively identified as isorhamentin pentosyl.

Their UV spectra was similar to those found in ellagitannins, but their fragmentation pattern showed m/z 315 and 300, which could be mistaken for ellagic acid or quercetin

(m/z 301). The MS/MS daughter ions indicated a loss of a pentose (132 amu) and a methyl moiety (15 amu). Isorhamnetin‟s fragmentation pattern has been described as ions at m/z 315  m/z 300. MS3 fragments at m/z 300 yielded m/z 300, 244, 216 and

200, which matched those reported for isorhamnetin found in mango, onions, and apples

(111, 113-114).

- Peak 28 (tR = 23.57 min; λmax 246, 360 nm) had a [M – H] at m/z 585 and was tentatively identified as an ellagic acid galloyl pentose conjugate. The MS/MS showed 46

ion signals at m/z 433 and 301, which refer losses of galloyl (152 amu) and pentosyl units (132 amu). The MS3 at m/z 301 yielded ellagic acid with its characteristic fragmentation pattern (m/z 257 and 229).

Peaks 29 (tR = 25.34 min; λmax 246, 268, 353, 365 nm) and 30 (tR = 25.59 min;

λmax 246, 268, 353, 365 nm) were tentatively identified as isorhamnetin pentosyl isomers with a galloyl unit ([M – H]- at m/z 599). Their MS/MS resulted in a loss of a galloyl moiety, 152 amu, as observed at m/z 447. The ion at m/z 447 had the same mass as peaks

23 and 24. A further loss of a pentosyl unit (132 amu) at m/z 315 was observed leaving the aglycon for isorhamnetin (m/z 315), which fragmented at m/z 300.

Phloroglucinolysis for condensed tannins by HPLC-DAD-MS/MS. In the crude pecan extract, monomers through pentamers of proanthocyanidins were found. In order to confirm the predominance of monomers of catechin and epicatechin as cleavage products of proanthocyanidins, an additional analysis of acid catalysis in the presence of excess phloroglucinol (phloroglucinolysis) was done as (95) previously described.

Phloroglucinolysis-HPLC chromatogram recorded at 280 nm of „Desirable‟ pecan is shown in Figure 17. The chromatogram presented two peaks at the beginning of the run, corresponding to ascorbic acid and phloroglucinol followed by the presence of adducts and monomers of procyanidins. Peaks A, C and E (Figure 18) were identified by the

MS/MS spectra as phloroglucinol adduct of (epi)gallocatechin (peak A; m/z 429 and aglycone fragment at m/z 305), catechin-phloroglucinol adduct (peak C m/z 413 and aglycone fragment at m/z 289) and epicatechin 3-O-gallate-phloroglucinol (peak E) which gave a rise to m/z 565 releasing a gallate group of m/z 152 and giving a fragment 47

Figure 17. HPLC-UV-DAD chromatogram of „Desirable‟ recorded at 280 nm following acid-catalysis in the presence of ascorbic acid (1) and phloroglucinol (2). Abbreviations of extension units: A: (epi)gallocatechin-phloroglucinol; C: catechin-phloroglucinol; E: epicatechin 3-O-gallate-phloroglucinol. Abbreviations of terminal units: B: epigallocatechin; D: catechin.

48

Intens. -MS2 (429.0), 6.9min #195 3000 (A)

2500

2000

1500 304.9

1000

500

(C) -MS2 (413.0), 10.4min #252 125 288.9 100

75

50

25

0

(E) -MS2 (565.1), 16.1min #385 6000 413.0 5000

4000

3000

2000 288.9 1000

0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

Figure 18. Mass spectra of proanthocyanidin cleavage products (extension units). (A) epigallocatechin-phloroglucinol, (C) catechin-phloroglucinol, and (E) epicatechin 3-O- gallate-phloroglucinol after phloroglucinolysis from „Desirable‟ recorded at 280nm.

49

Intens. 800 (B) -MS, 8.8min #215

304.9 600

400

200

0

6000 (D) -MS, 13.7min #331

5000 288.9

4000

3000

2000

1000

0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

Figure 19. Mass chromatogram of two procyanidin monomers (terminal units), epigallocatechin (B) and catechin (D), after phloroglucinolysis from „Desirable‟ recorded at 280nm. 50

Table 3. Contents of proanthocyanidin cleavage products after phloroglucinolysis in four different cultivars of pecan (mg/100 g FW).

Phloroglucinolysis Cultivar Flavan-3-ols mDP b mg/100 g FWa

Choctaw Catechin 741.9 ± 53.0 ac 3.4 Choctaw Epicatechin 98.6 ± 9.2 b - Choctaw (Epi)gallocatechin 104.6 ± 31.3 b 1.8 Total Choctawd 945.1

Desirable Catechin 743.6 ± 9.2 a 2.9 Desirable Epicatechin 131.3 ± 2.5 b - Desirable (Epi)gallocatechin 115.8 ± 0.9 c 1.7 Total Desirable 990.7

GraCross Catechin 1323.6 ± 18.2 a 3.6 GraCross Epicatechin 154.5 ± 6.2 b - GraCross (Epi)gallocatechin 164.3 ± 3.3 b 1.4 Total GraCross 1642.5

Kiowa Catechin 669.0 ± 17.4 a 3.0 Kiowa Epicatechin 164.2 ± 6.2 b - Kiowa (Epi)gallocatechin 102.8 ± 5.0 c 1.6 Total Kiowa 936.0 a Phloroglucinolysis, milligrams per 100 grams of pecan kernel fresh weight; b Mean degree of polymerization; c means followed by the same letter are not different according to Duncan test (P < 0.05). d Data are expressed as means ± S.D. (n=3).

51

ion of m/z 413 and aglycone fragment at m/z 289. Also, HPLC-DAD-MS analysis after phloroglucinolysis were identified both terminal units, epigallocatechin (peak B; m/z

305) and catechin (peak D; m/z 289) monomers respectively (Figure 19). Moreover, the mean degree of polymerization (mDP) of pecan proanthocyanidins ranged between 1.4

(minimum value) and 3.6 (maximum value) (Table 3), indicating that these flavan-3-ol molecules are lowly to moderately polymerized. After phloroglucinolysis, the highest proanthocyanidin values reached up to 1642.5 mg/100 g FW for „GraCross‟.

Phloroglucinolysis followed by HPLC-DAD-MS/MS-analysis was performed in order to verify the presence of proanthocyanidins and to determine their structural composition. Thus, this method definitively increased the quantitative conversion of proanthocyanins into their constitutive subunits and higher selectivity in the formation of adducts (95).

Conclusions

Differences among cultivars were found as previously reported, with emphasis in the fact that the cultivar with the highest condensed tannin content had the highest antioxidant capacity by both DPPH and ORAC assays. The four cultivars presented the same polyphenolic profile by LC-MS, with variations in the quantities of compounds.

Twelve ellagitannins were identified for the first time in pecans, along with gallic acid, valoneic acid dilactone, ellagic acid, isorhamnetin in the glucoside, pentosyl and galloyl pentosyl forms, and condensed tannins up to pentamers. 52

CHAPTER III

EFFECTS OF UROLITHINS A AND B AS METABOLITES FROM PECAN

ELLAGITANNINS ON ADIPOGENESIS IN 3T3-L1 CELLS

Summary

The increasing prevalence of obesity and its association to metabolic disorders are a major concern; treatments that may prevent or reduce obesity are being researched.

Phytochemicals in foods have been linked to several health benefits, including anti obesity. Pecan contains ellagitannins, which are metabolized in vivo by the gut microflora into urolithins. The present study evaluated the effects of urolithins A and B, and their precursor ellagic acid, during adipogenesis in 3T3-L1 preadipocytes. Cell cytotoxicity and proliferation were determined in preadipocytes and from these results

25 µM concentration was selected. For the consecutive studies, cells were induced to differentiation and treated from days 0-3 with 25µM of these compounds and were matured. Protein expression was evaluated by western blots and expression of transcription factors PPARγ, C/EBPα, and PPARα was evaluated at day 3 and at maturity (day 13); expressions of adipokines leptin and adiponectin were tested at day

13. Intracellular lipid accumulation was measured at maturity (day 13). Results showed no cytotoxic effects up to 50 µM of urolithins A and B, and ellagic acid. Cell proliferation rate was reduced by ellagic acid at ≤ 10 µM. No changes in protein expression for PPARγ, C/EBPα, and PPARα were seen at 25 µM. At maturity, no significant changes were detected for PPARγ and PPARα expression, whereas C/EBPα 53

expression was reduced by all treatments. Effects on intracellular lipid accumulation were not observed for the treatments. The present results suggest that urolithins A and B, and ellagic acid did not significantly reduced intracellular lipid accumulation and adipogenic regulatory protein expression levels.

Introduction

Obesity is a complex chronic disease that involves insulin resistance, hyperglycemia, hypertension, low HDL-cholesterol. This multiple condition characterized by the presence of excess visceral fat (65) is considered as a global epidemic by the World Health Organization (64), and involves more than 1billion overweight adults and over 400 million obese. Obesity is induced by increase in size of adipocytes and by the formation of new adipocytes from precursor cells. The number of adipocytes present in a person is mainly determined by the differentiation process.

Research in gene and protein expression related to adipogenesis has been done in the murine 3T3-L1 cell line. The expression of nuclear transcription factors, such as

CCAAT/enhancer binding protein (C/EBP) and peroxisome proliferator-activated receptor (PPAR) have been shown to be the most important adipogenic regulators. After induction of differentiation, C/EBPβ and C/EBPδ are expressed within 24 h, whereas

C/EBPα and PPARγ are expressed after 36-48 h (115-116). Another transcription factor from the PPAR family, PPARα, is involved in stimulating beta-oxidation of fatty acids

(117). Among the white adipose tissue secretory hormones (adipokines) herein studied are leptin and adiponectin. Leptin is a protein secreted exclusively by adipocytes and 54

regulates appetite, food intake and energy expenditure; weight gain results in an increase plasma leptin level (75). Adiponectin plays a role in energy homeostasis and insulin sensitivity, by protecting against inflammation and obesity-liked insulin resistance (76).

Research conducted on the effects of certain phenolic acids and flavonoids on adipogenesis using 3T3-L1 cell model demonstrated that phytochemicals like quercetin, genistein, resveratrol, and rutin, inhibited lipid accumulation in mature 3T3-L1 adipocytes (118-122). During these experiments, cells were treated with compounds during differentiation through maturation or when adipocytes were mature. In the same way, ellagitannins from Lagerstroemia speciosa leaves suppressed adipocyte differentiation (63) and extracts from Lindera obtusiloba inhibited adipogenesis and exerted anti-inflammatory effects in 3T3-L1 preadipocytes (123).

Ellagitannins are large molecules with high molecular weights and belong to the hydrolyzable tannins family. It has been proven that these compounds hydrolyze and that ellagic acid is metabolized by gut microflora and is absorbed by the epithelium in the form of urolithin (41). Urolithins have proven beneficial effects against prostate and colon cancer; they exert some proestrogenic/antiestrogenic effects (52, 55, 60), as well as anti-inflammatory activity in colon fibroblasts and protection against oxidative stress

(61-62).

The objective of this study was to evaluate the ability of urolithins A and B, and the urolithin-precursor ellagic acid, to inhibit adipocyte differentiation and/or reduce mature adipocyte growth.

55

Materials and methods

Chemicals. The following chemicals were used in the experiments: 3-isobutyl-1- methylxanthine, dexamethasone, insulin, rutin, and Dulbecco‟s Modified Eagle‟s

Medium, and protease inhibitor cocktail were purchased from Sigma (St. Louis, MO).

Glucose was from Acros Organics (Fair Lawn, NJ). Sodium bicarbonate was from

Mallinckrodt Chemicals (Phillipsburg, NJ). Murine 3T3-L1 preadipocytes, fetal bovine serum (FBS), trypsin EDTA, and DMSO were acquired from the American Type

Culture Collection (ATCC) (Manassas, VA). Penicillin-Streptomycin was bought from

Invitrogen (Carslbad, CA). Urolithins A and B were manufactured by Kylolab (Ceuti,

Spain). Ellagic acid was from MP Biomedicals (Solon, OH). Cell lysis buffer was obtained from Cell Signalling Technology (Danvers, MA). 10% sodium dodecyl sulfate solution, 30% acrylamide/bisacrylamide solution, N, N, N', N'- tetramethylethylenediamine (TEMED), ammonium persulfate (APS), Tween 20, and

Precision Plus Protein marker were from Bio-Rad Laboratories (Hercules, CA).

Laemmli‟s loading buffer was acquired from Fermentas Inc. (Glen Burnie, MD).

Polyvinylidene fluoride (PVDF) membranes were from Millipore Corp. (Billerica, MA).

Antibodies for PPARγ (sc-7196), C/EBP α (sc-61), PPARα (sc-9000), β-actin (sc-

47778), and leptin (sc-842), were purchased from Santa Cruz Biotechnology (Santa

Cruz, CA). Adiponectin primary antibody (5903-50) was obtained from BioVision

(Mountain View, CA). Goat anti-rabbit-HRP polyclonal secondary antibody (A120-

101P) was from Bethyl Laboratories (Montgomery, TX).Water used was nanopure grade. 56

Cell culture. 3T3-L1 preadipocytes were cultured in high glucose Dulbecco‟s

Modified Eagle‟s Medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, and kept under humidified atmosphere at 37 ºC and 5%

CO2. Medium was replaced every other day until they reached 100% confluency.

Cell viability and proliferation assay. Cytotoxicity effects on preadipocytes were determined with the MTS assay (Promega Corp., Madison, WI), according to the manufacturer‟s instructions. The MTS consisted of a [3-(4,5dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt which converts into soluble formazan by dehydrogenase enzymes found in metabolically active cells.

The quantity of formazan product is measured at 490 nm and is directly proportional to the number of living cells in culture. 3T3-L1 cells were seeded at a density of 7500 cells/well in a 96-well plate and left with 10% FBS/DMEM medium for 24 h.

Treatments of urolithin A, urolithin B, ellagic acid (urolithin precursor), and rutin (as positive control) were added at concentrations of 5, 10, 25, 40, and 50 µM in DMSO.

Final DMSO concentration in medium was < 0.083%; DMSO was added at this percentage as a control. Cell viability was measured at 6, 12, 24, and 48 h; absorbance was recorded at 490 nm in a plate reader (Synergy HT, Bio-Tek Instruments, Inc.,

Winooski, VT).

Cell differentiation and treatments for adipogenesis assays. 3T3-L1 cells were seeded at a density of 10,000 cells/well in 6-well plates. Preadipocytes were induced to differentiation 2 days after they reached 100% confluency, which will be referred to as day 0. Growth medium was supplemented with 0.5 mM 3-isobutyl-1- 57

methylxanthine (IBMX), 1 μM dexamethasone, and 10 μg/mL insulin, for 48 h (78).

Also on day 0, treatments of urolithin A, urolithin B, ellagic acid and rutin (positive control) were added to reach a final concentration of 25µM and diluted in DMSO

(0.04% in medium concentration). A control containing 0.04% DMSO was also run. At day 2, medium was supplemented with insulin (10 μg/mL) for two additional days, until day 4. After day 4, 10% FBS/DMEM medium was replaced every other day to let the cells mature. Protein was collected at day 3, to see immediate effects on differentiation, and later at day 13, when adipocytes had lipid droplets to see effects during maturation.

Quantification of lipid content. Intracellular lipid content was measured using a commercially available kit (AdipoRed, Lonza Wakersville, Inc., Wakersville, MD).

AdipoRed is a solution of the Nile Red stain, which fluoresces and enables the quantification of intracellular lipid droplets. Preadipocytes were seeded at a density of

10,000 cells/well in 6-well plates, differentiated after 2 days of 100% confluency and treated with urolithin A, urolithin B, ellagic acid, and rutin (positive control) at 25 µM in

DMSO (0.04%). A control containing DMSO (0.04%) was also run. On day 13, cells were washed with 2 mL PBS (pH 7.4), then 5 mL PBS were left per well and 140 μL

AdipoRed were added, left the plate at room temperature for 10 min and fluorescence readings were measured by well scanning with excitation at 485 nm and emission at 560 nm in a plate reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski, VT).

Measurements were expressed as relative fluorescence units (RFU).

SDS-PAGE. Protein from cells was collected at days 3 and 13. Cells were harvested with a cell lysis buffer, following the manufacturer‟s instructions. Briefly, 58

each well was washed twice with ice cold PBS, added 100 μL lysis buffer, containing protease inhibitor cocktail. Cells were then scraped, left at -80 ºC overnight, centrifuged at 14,000 rpm at 4 ºC and supernatant was stored at -80 ºC. The total protein was quantified using the BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific, Inc.,

Rockford, IL) and 25 µg of protein equivalent volume were mixed with Laemmli‟s loading buffer, boiled, and loaded on a gel with a pre-stained, broad-range, molecular weight protein marker. Proteins were fractionated by electrophoresis using 10% polyacrylamide gels; the stacking gel was run at 60 V for 30 min and protein fractionation (resolving gel) was run for 1.5 h at 100 V. The gels were transferred by wet blotting onto PVDF membranes at 100 V for 1 h at 4 °C. The membranes were then blocked with 5% skim milk in tris buffered saline with 1% Tween-20 (TBS-T) for 1 h with gentle shaking; three 5-min washes with TBST were performed consecutively.

Membranes were then incubated with a specific primary antibody against PPARγ

(1:200), PPARα (1:200), C/EBPα (1:200), leptin (1:200), or adiponectin (0.75 µg/mL), according to the manufacturer‟s instructions. For internal control, membranes were incubated with a conjugated HRP primary antibody against β-actin at a dilution of

1:1,500. The membranes were washed three times with TBS-T and incubated for 1 h with the secondary antibody conjugated with horseradish peroxidase (HRP) at 1:6,000 dilution. Specific bands were developed using a SuperSignal West Femto enhanced chemiluminescence (ECL) Western blotting detection kit (Pierce, Thermo Fisher

Scientific, Inc., Rockford, IL) after 30 s incubation and signals were captured by CCD

Camera (Cascade II:512, Photometrics,Tucson, AZ) using the WinView/32 software 59

(Version 2.5, Princeton Instruments, Trento, NJ). Bands were measured using densitometry with ImageJ software (NIH, Bethesda, MD).

Statistical analysis. Data represent the mean of triplicates ± S.D. for cell viability and proliferation assays, for the rest of the assays mean of triplicates ± S.E.

(standard error) was presented. Statistical significance was assessed by t-test.

Differences were considered significant when P ≤ 0.05. Tests were conducted using

SPSS 15.0 (SPSS Inc., Chicago, IL).

Results and discussion

Cell viability and proliferation. None of the tested compounds affected the cytotoxicity at concentrations tested (no significant differences P ≤ 0.05) (Figure 20).

Similar results were found in human colonic fibroblasts by (62) for urolithin A, urolithin

B, and ellagic acid at concentrations of 1 and 10 μM.

Since no changes were observed in cell viability, the cell proliferation was determined at the same concentrations and measurements were performed at 6, 12, 24, and 48 h (Figure 21). At the tested concentrations, cell proliferation was not affected when urolithin A, urolithin B, and rutin were present; however, ellagic acid maintained the cell number constant. This could be due to inhibition of proliferation as a consequence of arrest at any specific point of the cell cycle or due to a similar proliferation/apoptosis rate. Based on our results in cell viability and proliferation, the 25

μM concentration was chosen because levels of 18.6 μM have been detected in human plasma (41). 60 3T3-L1 preadipocytes cell cytotoxicity at 24 h

140

120

100

80

60

% of control survival control of % 40 Urolithin A Urolithin B 20 Ellagic acid Rutin

0 0 10 20 30 40 50

Concentration ( M)

Figure 20. 3T3-L1 preadipocytes cell viability after 24 h exposure to treatments at 5-50 µM. Treatments were urolithin A, urolithin B, ellagic acid (urolithin precursor), rutin (positive control), and vehicle (< 0.083% DMSO). Values are mean ± S.D. and n = 3. 61

35000 30000 5 M 10 M 30000 A 25000 B

25000 5 µM 10 µM 20000 20000 15000 15000 10000 10000

Proliferation number) (cell Proliferation (cell number) (cell Proliferation 5000 5000

0 0 0 10 20 30 40 50 0 10 20 30 40 50 Time (h) Time (h)

35000 30000 25 M 40 M 30000 C 25000 D

25000 25 µM 40 µM 20000 20000 15000 15000 10000 10000

Proliferation (cell number) Proliferation (cell 5000 Proliferation number) (cell 5000

0 0 0 10 20 30 40 50 0 10 20 30 40 50 Time (h) Time (h) 30000 50 M 25000 E 50 µM 20000 Urolithin A Urolithin B 15000 Ellagic acid Rutin (+ control) 10000 Control

Proliferation number) (cell 5000

0 0 10 20 30 40 50 Time (h)

Figure 21. 3T3-L1 preadipocyte cell proliferation after 48 h exposure to treatments at 5-50 µM. Treatments consisted of urolithin A, urolithin B, ellagic acid (urolithin precursor), rutin (positive control), and vehicle (< 0.083% DMSO). Measurements were done at 6, 12, 24, and 48 h. Values are mean ± S.D. and n = 3. 62

Intracellular lipid quantification. The results of this test are presented in Figure

22. These compounds did not have an effect in the inhibition of the intracellular lipid content when added during the first 3 days of differentiation. Only the positive control, rutin, demonstrated a lesser accumulation of lipids. Similar results were detected by Hsu et al. (119) who found that rutin inhibited triglyceride content in 3T3-L1 mature adipocytes by 83% when treated with 50-250 µM for 72 h at maturity.

Effects of urolithins A and B on adipogenesis’ protein expression.

Day 3. The transcription factors PPARγ and C/EBPα were not significantly (P ≤

0.05) affected by any of the treatments (Figures 23 and 24). In the case of PPARγ, a minimal reduction in expression was observed in presence of urolithin B, ellagic acid, and rutin, compared to the control. On the other hand, expression of the transcription factor related to trigger genes involved in fatty acid oxidation, PPARα, was not affected significantly (P ≤ 0.05) in the presence of urolithin A, ellagic acid, and rutin (Figure 25).

At the early stage of adipogenesis, the transcription of PPARγ and C/EBPα can be mediated by C/EBPδ and C/EBPβ; subsequently, PPARγ and C/EBPα can cross- regulate each other independently (124). PPARγ and C/EBPα alone or together induce and maintain the differentiated phenotype of adipocytes (83). The results obtained indicate that 3T3-L1 cells were differentiated with no effects on adipogenic markers by the treatments at 25 µM.

63

60000

50000 b b b b bc

40000

30000

20000

Relative Fluorescence Units (RFU) Units Fluorescence Relative 10000 a

0 Preadipocytes Control Urolithin A Urolithin B Ellagic acid Rutin

Figure 22. Intracellular lipid accumulation in 3T3-L1 mature adipocytes after addition of 25 µM urolithin A, urolithin B, ellagic acid (urolithin precursor), rutin (positive control) or the equivalent of vehicle (0.04% DMSO). Cells were treated from day 0 to day 3, and were evaluated when mature at day 13. Data are means ± S. E. (n = 3) and different letters denote significance among treatments (P ≤ 0.05) performed by t-test.

64

1.4

1.2 a a 1.0 a a 0.8 a

0.6

0.4

Relative protein expression Relative 0.2

0.0 Control UroA UroB EA Rutin PPAR -actin

Figure 23. Expression of the transcription factor PPARγ at the protein level in adipocytes treated with 25 µM of compounds after 3 d differentiation. Treatments consisted of urolithin A (UroA), urolithin B (UroB), ellagic acid (urolithin precursor) (EA), rutin (positive control) or the equivalent of vehicle (0.04% DMSO) for 72 h. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) performed by t-test. Western blot images are representative of three independent experiments.

65

1.4 a 1.2 a a a a 1.0

0.8

0.6

0.4

Relative proteinRelative expression 0.2

0.0 Control UroA UroB EA Rutin C/EBP -actin

Figure 24. Expression of the transcription factor C/EBPα at the protein level in adipocytes treated with 25 µM of compounds after 3 d differentiation. Treatments consisted of urolithin A (UroA), urolithin B (UroB), ellagic acid (urolithin precursor) (EA), rutin (positive control) or the equivalent of vehicle (0.04% DMSO) for 72 h. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) performed by t-test. Western blot images are representative of three independent experiments.

66

1.4

1.2 a a 1.0

0.8 a a a 0.6

0.4

Relative protein expressionRelative 0.2

0.0 Control UroA UroB EA Rutin PPAR -actin

Figure 25. Expression of the transcription factor PPARα at the protein level in adipocytes treated with 25 µM of compounds after 3 d differentiation. Treatments consisted of urolithin A (UroA), urolithin B (UroB), ellagic acid (urolithin precursor) (EA), rutin (positive control) or the equivalent of vehicle (0.04% DMSO) for 72 h. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) performed by t-test. Western blot images are representative of three independent experiments. 67

Day 13. In mature adipocytes, which were treated at the early stage of differentiation with the compounds, the transcription factor PPARγ did not show any significant differences (P ≤ 0.05) compared to the control (Figure 26); a decrease for urolithin A treatment was observed but this was not significant. The transcription factor

C/EBPα was significantly (P ≤ 0.05) down-regulated by urolithin A, urolithin B, ellagic acid, and rutin (Figure 27). However, no significant effects (P ≤ 0.05) on PPARα expression were observed for any of the treatments (Figure 28); a small but no significant increase was observed for rutin. This small increase could be associated to the results obtained on intracellular lipid accumulation (Figure 22), since this transcription factor is involved with fatty acid oxidation genes (87).

The secretory hormones/adipokines, leptin and adiponectin were measured from the intracellular protein. For leptin, an adipokine known to influence insulin resistance, an increase for the ellagic acid treatment was observed at day 13 (Figure 29). This is an undesirable change since an increase in plasma leptin levels influence insulin resistance

(125), and is associated to weight gain (75). Nevertheless, rutin, the positive control, decreases the leptin protein quantity produced in the cell, which makes this compound healthier for the organism (126).

Adiponectin, a peptide expressed specifically and abundantly in adipose tissue, plays a role in energy homeostasis and insulin sensitivity, by protecting against inflammation and obesity-liked insulin resistance (76) was significantly (P ≤ 0.05) down-regulated by urolithin A and rutin (Figure 30). These results were not expected as they contradict the reference (119), where it states that rutin concentrations of 50–250 68

µM increased the expression of adiponectin after treating mature 3T3-L1 for 24 h.

Nonetheless, these experiments were conducted differently and cannot be compared to the results obtained in this study. The decrease in adiponectin for urolithin A and rutin treatments during the differentiation stage could produce an adipose tissue dysfunction making it less sensitive to glucose.

To summarize, this study was focused on the bioactivity of ellagic acid, urolithin

A, and urolithin B present in pecan extract and their role as metabolites, as possible differentiation inhibitors of 3T3-L1 cells. It was hypothesized that the transcription factors PPARγ and C/EBPα would be decreased and that PPARα transcription factor, which participates in aspects of lipid catabolism such as fatty acid oxidation, would be increased.

Additionally, it is necessary to test these compounds in an in vivo system, since in vitro cell models do not necessarily reflect what happens in vivo. Results showed that when these compounds were added at differentiation and for 3 days only, no significant changes in intracellular lipid accumulation and adipogenic regulatory protein expression levels were observed both at day 3 and at day 13. Other reports observed significant changes in intracellular lipid accumulation and regulatory proteins when 3T3-L1 cells were cultured with polyphenols or plant extracts during differentiation and maturation

(74, 118, 121-122, 127-128). The positive results obtained by these studies make us restate that a longer exposure time (6 to 8 days, instead of 3 days) of any of the compounds used in this study on 3T3-L1 cells may produce a positive effect on the cells. 69

1.4 a a a a 1.2 a 1.0

0.8

0.6

0.4

Relative protein expressionRelative 0.2

0.0 Control UroA UroB EA Ru PPAR -actin

Figure 26. Expression of the transcription factor PPARγ at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation. Treatments consisted of urolithin A (UroA), urolithin B (UroB), ellagic acid (urolithin precursor) (EA), rutin (positive control) or the equivalent of vehicle (0.04% DMSO) for 72 h at day 0. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) performed by t-test. Western blot images are representative of three independent experiments.

70

1.2 a 1.0 b b 0.8 b b

0.6

0.4

Relative protein expression Relative 0.2

0.0 Control UroA UroB EA Ru C/EBP -actin

Figure 27. Expression of the transcription factor C/EBPα at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation. Treatments consisted of urolithin A (UroA), urolithin B (UroB), ellagic acid (urolithin precursor) (EA), rutin (positive control) or the equivalent of vehicle (0.04% DMSO) for 72 h at day 0. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) performed by t-test. Western blot images are representative of three independent experiments.

71

1.2 a a a a 1.0 a 0.8

0.6

0.4

Relative protein expression protein Relative 0.2

0.0 Control UroA UroB EA Ru

PPAR -actin

Figure 28. Expression of the transcription factor PPARα at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation. Treatments consisted of urolithin A (UroA), urolithin B (UroB), ellagic acid (urolithin precursor) (EA), rutin (positive control) or the equivalent of vehicle (0.04% DMSO) for 72 h at day 0. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) performed by t-test. Western blot images are representative of three independent experiments.

72

1.4 b 1.2 abc a abc 1.0

0.8 c

0.6

0.4

Relative proteinRelative expression 0.2

0.0 Control UroA UroB EA Ru Leptin -actin

Figure 29. Expression of the adipokine leptin at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation. Treatments consisted of urolithin A (UroA), urolithin B (UroB), ellagic acid (urolithin precursor) (EA), rutin (positive control) or the equivalent of vehicle (0.04% DMSO) for 72 h at day 0. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) performed by t-test. Western blot images are representative of three independent experiments.

73

1.2 a a a 1.0 a 0.8 b

0.6

0.4

Relative protein expressionRelative 0.2

0.0 Control UroA UroB EA Ru

Adiponectin -actin

Figure 30. Expression of the adipokine adiponectin at the protein level in adipocytes treated with 25 µM compounds after 13 d differentiation. Treatments consisted of urolithin A (UroA), urolithin B (UroB), ellagic acid (urolithin precursor) (EA), rutin (positive control) or the equivalent of vehicle (0.04% DMSO) for 72 h at day 0. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) performed by t-test. Western blot images are representative of three independent experiments.

74

Conclusions

In this study, urolithin A and urolithin B, and their precursor, ellagic acid, did not present significant cytotoxic activities up to a 50 µM concentration in 3T3-L1 preadypocytes. Cell proliferation was not affected by these compounds when treated up to 40 µM, only ellagic acid diminished the proliferation rate from 10 µM. 3T3-L1 cells were induced to differentiation and treated at the same time with 25µM compounds, and no immediate effects on the inhibition of adipogenesis were observed, as the expression of differentiation markers, PPARγ and C/EBPα was slightly decreased but not significantly. Intracellular lipid accumulation was measured with mature adipocytes at day 13 and no differences were observed compared to the control. The positive control, rutin, had significantly less intracellular lipid content. In the long term, effects in the protein level of adipogenic markers were not detected, except for C/EBPα, which was significantly decreased by all treatments. Therefore, the compounds urolithins A and B, and ellagic acid did not affect significantly adipogenesis or lipid accumulation

(measured at day13) when 3T3-L1 cells were exposed for three days during differentiation. 75

CHAPTER IV

EFFECTS OF UROLITHINS A AND B IN INFLAMMATION WITH 3T3-L1

CELLS

Summary

Chronic inflammation has been linked to obesity, type 2 diabetes, and suppression of functional adipokines, such as adiponectin, among others. The present study evaluated the effects of ellagitannins metabolites, urolithin A and B, and their precursor, ellagic acid, on inflammation induced by lipopolysaccharide (LPS) addition to mature 3T3-L1 adipocytes. Protein expression of the phosphorylated state of NF-κB p65 was evaluated by western blot and the gene expression of the pro-inflammatory cytokines TNF-α and IL-6, transcription factor PPARγ and adipokine adiponectin was also tested by quantitative PCR. Results showed that urolithins A and B may decrease inflammation by LPS, but further work is needed to confirm the findings.

Introduction

Fifteen years ago, a link between chronic inflammation and obesity has been reported when messenger RNA expression of tumor necrosis factor-α (TNF-α) was observed in different rodent models of obesity and diabetes (129). Another adipocyte secretory product, interleukin-6 (IL-6), is increased in obese subjects (130). Obesity and the metabolic conditions linked to it have been associated with a chronic inflammatory response, which involves abnormal cytokine production and activation of inflammatory 76

signaling pathways (131). The 3T3-L1 cell line secretes these pro-inflammatory cytokines, TNF-α and IL-6, the latter one impairs insulin signaling, which induces insulin resistance (132). The upregulation of the adipokine adiponectin is viewed as a positive effect, as it improves glucose and lipid metabolism, and exhibits anti- inflammatory properties (133-134). The activation of the nuclear transcription factor

PPARγ in adipocytes has been shown to reverse the inflammatory signaling and proinflammatory cytokine expression, as well as to restore insulin signaling, allowing the adipocytes to function properly (135). The nuclear factor-κB (NF-κB) activates rapidly in response to stress-inducing stimuli, providing the cell with a sensitive and quick detection system for potential threats. In addition, NF-κB has been implicated in the pathogenesis of many inflammation-related diseases since it promotes the expression of pro-inflammatory genes (80). Therefore, prevention and treatment of obesity should combine anti-obesity and anti-inflammatory effects.

Phytochemicals contained in foods are being sought after for their possible health benefits, since most studies use synthetic drugs and may have side effects. Abietic acid, a diterpenoid found in conifer species, has been found to have anti-inflammatory effects on macrophages, which is mediated by PPARγ activation (136). Abscisic acid, a plant hormone, was shown to be anti-inflammatory when fed to mice, as the TNF-α gene expression was down-regulated (137). The garlic-derived organosulfur 1,2-vinyldithiin, also reduced the secretion of IL-6 (138).

Ellagitannins found in pecan kernels belong to the hydrolyzable tannin family. It has been proven that these compounds hydrolyze upon ingestion and that ellagic acid is 77

metabolized by gut microflora and is absorbed by the epithelium in the form of urolithin

(41). Urolithins have proven beneficial effects against prostate (52) and colon cancer

(55); they exert some proestrogenic/antiestrogenic effects (60) and protection against oxidative stress (61). Anti-inflammatory activity of urolithins was reported in human colonic fibroblasts after inflammation was induced with interleukin-1β (IL-1β). In this study, it was observed that urolithins A and B inhibited NF-κB translocation to nucleus affecting transcription of pro-inflammatory proteins (62).

The objective of this study was to evaluate the anti-inflammatory effects of urolithins A and B by analysis of gene expression of pro-inflammatory cytokines: TNF-α and IL-6, the transcription factor PPARγ, and adiponectin. Phosphorylation state of the

NF-κB p65 after addition of an inflammatory inducer will be evaluated by protein expression.

Materials and methods

Chemicals. The following chemicals were used in the experiments: 3-isobutyl-1- methylxanthine, dexamethasone, insulin, trypsin, Dulbecco‟s Modified Eagle‟s Medium, and lipopolysaccharide from E. coli ser. 0111:B4 were purchased from Sigma (St. Louis,

MO). Glucose was from Acros Organics (Fair Lawn, NJ). Sodium bicarbonate was from

Mallinckrodt Chemicals (Phillipsburg, NJ). Murine 3T3-L1 preadipocytes, fetal bovine serum (FBS), and DMSO were acquired from the American Type Culture Collection

(ATCC) (Manassas, VA). Penicillin-Streptomycin was bought from Invitrogen

(Carslbad, CA). Urolithins A and B were manufactured by Kylolab (Ceuti, Spain). 78

Ellagic acid was from MP Biomedicals (Solon, OH). 10% sodium dodecyl sulfate solution, 30% acrylamide/bisacrylamide solution, N, N, N', N'- tetramethylethylenediamine (TEMED), ammonium persulfate (APS), Tween 20, and

Precision Plus Protein marker were from Bio-Rad Laboratories (Hercules, CA).

Laemmli‟s loading buffer was acquired from Fermentas Inc. (Glen Burnie, MD).

Polyvinylidene fluoride (PVDF) membranes were from Millipore Corp. (Billerica, MA).

Antibodies for p-NF-κB p65 (sc-33039) and β-actin (sc-47778), and secondary rabbit anti-goat-HRP (sc-2768) were purchased from Santa Cruz Biotechnology (Santa Cruz,

CA). Antibody for NF-κB p65 (C22B4) was obtained from Cell Signalling Technology

(Danvers, MA). Adiponectin primary antibody (5903-50) was obtained from BioVision

(Mountain View, CA). Goat anti-rabbit-HRP polyclonal secondary antibody (A120-

101P) was from Bethyl Laboratories (Montgomery, TX). Primers and oligo dT were from Integrated DNA Technologies (Coralville, IA). dNTPs were from Promega

(Madison, WI). Water used was nanopure grade.

Cell culture. 3T3-L1 preadipocytes were cultured in high glucose Dulbecco‟s

Modified Eagle‟s Medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, and kept under humidified atmospheres at 37 ºC and 5%

CO2. Medium was replaced every other day until cells reached 100% confluency.

Cell differentiation and treatments for inflammation assays. Preadipocytes were seeded in 6-well plates and allowed to reach confluency; differentiation was induced 2 days after 100% confluency (day 0). On day 0, growth medium was supplemented with 10 μg/mL insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1- 79

methylxanthine (IBMX) for 48 h (78). At day 2, medium was supplemented with insulin

(10 μg/mL) for two additional days. After day 4, 10% FBS/DMEM medium was replaced every other day to let the cells mature.

At day 8, 25µM of urolithin A, urolithin B, and ellagic acid were added for 24 h in DMSO as the carrier (0.04%). A control containing 0.04% DMSO was also run.

Concentrations of 25 µM were used according to the results obtained in Chapter III.

Lipopolysaccharide (LPS), an acute inflammation inducer, was added to the cells at a concentration of 100 ng/mL for the last 1 hour of treatment exposure.

SDS-PAGE. Cells were harvested after LPS addition for 1 h with cell lysis buffer, following the manufacturer instructions. Briefly, each well was washed twice with ice cold PBS, and 100 μL of ice-cold lysis buffer (50 mM HEPES, 150 mM NaCl,

10 mM sodium pyrophosphate, 100 mM NaF, 1.5 mM MgCl2, 1 mM EGTA, 100 µM sodium orthovanate, 1% TritonX-100, 1 mM PMSF, 1 µg/mL leupeptin) (pH 7.5) was added. Cells were then scraped and left at -80 ºC overnight, and centrifuged at 14,000 rpm at 4 ºC. Supernatant was collected and stored at -80 ºC. The total protein was quantified using the BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific, Inc.,

Rockford, IL) and 25 µg of protein equivalent volume were mixed with Laemmli‟s loading buffer, boiled, and loaded on a gel with a pre-stained, broad-range, molecular weight protein marker. Proteins were fractionated by electrophoresis using 10% polyacrylamide gels; the stacking gel was run at 60 V for 30 min and protein fractionation (resolving gel) was run for 1.5 h at 100 V. The gels were transferred by wet blotting onto PVDF membranes at 100 V for 1 h at 4 °C. The membranes were then 80

blocked with 5% skim milk in tris buffered saline with 1% Tween-20 (TBS-T) for 1 h with gentle shaking; three 5-min washes with TBST were performed consecutively.

Membranes were then incubated with a specific primary antibody against p-NF-κB p65

(1:200), according to the manufacturer‟s instructions. For internal control, membranes were incubated with conjugated HRP primary antibody against β-actin at a dilution of

1:1,500. The membranes were washed with TBS-T and incubated for 1 h with the secondary antibody conjugated with horseradish peroxidase (HRP) at 1:6,000 dilution.

Specific bands were developed using a SuperSignal West Femto enhanced chemiluminescence (ECL) Western blotting detection kit (Pierce, Thermo Fisher

Scientific, Inc., Rockford, IL) after 30 s incubation and signals were captured by CCD

Camera (Cascade II:512, Photometrics,Tucson, AZ) using the WinView/32 software

(Version 2.5, Princeton Instruments, Trento, NJ). Bands were measured using densitometry with ImageJ software (NIH, Bethesda, MD).

RNA extraction and reverse transcription. Total cellular RNA was extracted from 3T3-L1 mature adipocytes after treatment for 24 h and LPS addition for the last 1 h, using the RNeasy mini kit (Qiagen, Valencia, CA). RNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Willmington,

DE), and volume was adjusted to 1µg RNA. Equal aliquots were loaded on 1% agarose gels, stained with ethidium bromide, to check for RNA integrity. For cDNA synthesis, 1

µg RNA was reverse-transcribed into cDNA using the SuperScript III first-strand synthesis supermix (Cat. No. 18080-004, Invitrogen, Carlsbad, CA), following the manufacturers protocol. Briefly, oligo dT (300 ng), 10 mM dNTPs mix (500 µM), and 81

PCR grade water were mixed with 1 µg RNA. Mixture was heated to 65 °C, for 5 min followed by ice incubation for at least 1 min. Added first strand buffer, 5mM dithiotreitol (DTT), 40 units RNAse out, and 200 units SuperScript III and incubated at

55 °C for 60 min, and reaction was inactivated by heating to 70 °C for 15 min. cDNA samples were stored at -20°C until further analysis.

Real-time quantitative RT-PCR. Gene expression was quantified by real-time quantitative RT-PCR using Power SYBR Green PCR Master Mix (Applied Biosystems,

Foster City, CA), following the manufacturer‟s instructions. DNA amplification was carried out using a 7900 HT Sequence Detection System (Applied Biosystems, Foster

City, CA), and the detection was performed by measuring the binding of the fluorescence dye SYBR Green to double-stranded DNA. Reactions were performed in a

20 μL volume containing 25 ng cDNA with 0.3 µM primers; all the primer sets are listed in table 4 and were provided by Integrated DNA Technologies (IDT, Coralville, IA).

After 10 min polymerase activation at 95 °C, 40 cycles with 95 °C for 15 s

(denaturation), 60 °C for 1 min (annealing/extension) were performed. Fluorescence was measured at the end of the 60 °C extension period. To confirm amplification specificity, the PCR products were evaluated by a melting curve analysis to exclude primer dimers and non-specific amplification. The relative expression of each gene was normalized by

β-actin, and was calculated following the comparative Ct method (ΔΔCt), also known as

- ΔΔCt the 2 method (139). The relative expression of each gene or sample presented in this study was compared to 3T3-L1 cells control (0.04% DMSO) to which LPS was not added. 82

The following equation was used to calculate the relative mRNA expression.

ΔΔCt = ΔCt, sample – ΔCt, reference

Here, ΔCt, sample is the Ct value for any sample normalized to the internal housekeeping gene; whereas ΔCt, reference is the Ct value for the calibrator, or control, also normalized to the internal housekeeping gene. For the ΔΔCt calculation to be valid, the amplification efficiencies of the target and the internal reference were approximately equal. The fold determination of the relative mRNA expression for each target gene was calculated according to the following equation:

Fold determination = 2-ΔΔCt

Statistical analysis. Data represent the mean of triplicates ± S.E. (standard error)

(n = 3). Statistical significance was assessed by t-test (P ≤ 0.05). Tests were conducted using SPSS 15.0 (SPSS Inc., Chicago, IL).

Results and discussion

Effects of urolithin A and B on NF-κB p65 protein expression. Results from the relative expression of the protein p-NF-κB p65 after its exposure to treatments are shown in Figures 31 and 32. On the non-LPS treatments of urolithin A, urolithin B, and ellagic acid, a significant protective effect was shown only by urolithin B by a reduction in its expression, while urolithin A remained constant, and ellagic acid increased expression of p-NF-κB p65 significantly. When cells were induced to inflammation by

LPS addition, a significant decrease in expression was observed by urolithin A (+ LPS) and ellagic acid (+ LPS), whereas urolithin B (+ LPS) effect was not significant. 83

Table 4. Sequences of primers used in this study.

Primer Sequence PPARγ-F 5‟- GAT GCA CTG CCT ATG AGC ACT T -3‟ PPARγ-R 5‟- AGA GGT CCA CAG AGCTGA TTC C -3‟ TNF-α-F 5‟- ACT GGC AGA AGA GGC ACT CC -3‟ TNF-α-R 5‟- CGA TCA CCC CGA AGT TCA -3‟ IL-6-F 5‟- TGA CAA CCA CGG CCT TCC CT -3‟ IL-6-R 5‟- AGC CTC CGA CTT GTG AAG TGG T -3‟ β-actin-F 5‟- CCC AGG CAT TGC TGA CAG G -3‟ β-actin-R 5‟-TGG AAG GTG GAC AGT GAG GC -3‟

84

2.5 A 2.0 c 1.5 a a - LPS 1.0 b

0.5

0.0 2.5 * a B 2.0 ab * b B p65 relativeprotein expression b  1.5 + LPS 1.0

p-NF-

0.5

0.0

Control Urolithin A Urolithin BEllagic acid

Figure 31. Expression of the transcription factor p-NF-κB p65 at the protein level in mature adipocytes, treated with 25 µM of compounds and LPS. Treatments consisted of urolithin A, urolithin B, ellagic acid or the equivalent of vehicle (0.04% DMSO) for 24 h. (+) indicates addition of LPS, while (-) no LPS. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) and * denotes significant differences (P ≤ 0.05) within same treatment with and without LPS performed by t-test.

85

p-NF-κB p65 NF-κB p65

β-actin

Figure 32. Results obtained for protein abundance of p-NF-κB p65, NF- κB p65, and β- actin for 25 µM treatments by western blot. Treatments consisted of urolithin A, urolithin B, ellagic acid or the equivalent of vehicle (0.04% DMSO) for 24 h. (+) indicates addition of LPS, while (-) no LPS. Western blot images are representative of 3 independent experiments.

Figure 33. Agarose gel electrophoresis of RNA collected from 3T3-L1 mature adipocytes.

86

When comparing among treatments of urolithin A (+ LPS) and (- LPS), a significant decrease in the expression levels of p-NF-κB p65 was observed, suggesting a possible protective effect against inflammation for this compound.

Although the action pathway of these urolithins was not studied, up to date it is known that other compounds are able to down-regulate the NF-κB expression through several pathways, including the reduction of the endoplasmic reticulum stress (140), by decreasing reactive oxygen species (ROS) production, and inhibiting the phosporylation of IκB-α (141).

Effects of urolithin A and B on tumor necrosis factor-α (TNF-α) gene expression. Analysis of gene expression by quantitative PCR was performed for genes involved in inflammation and insulin resistance: tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), peroxisome proliferator-activated receptor γ (PPARγ), and adiponectin. A previous assessment of RNA integrity from the recovered cell samples was done by 1% agarose gel electrophoresis (Figure 33).

The obtained results for TNF-α, a NF-κB transcription‟s target gene (142), showed a significant decrease (P ≤ 0.05) for urolithin A (- LPS), urolithin B (- LPS), and ellagic acid (- LPS) treatments compared to the control (Figure 34A). Treatments challenged with LPS (+ LPS) did not show significant differences among them and the control; however, when comparing between (+LPS) and (- LPS) treatments, all of those who were challegened with LPS presented a significant increase in TNF-α expression, denoted by a (*) from Figure 34B. 87

Effects of urolithin A and B on interleukin-6 (IL-6) gene expression. Results on the expression of the mRNA IL-6 after treatments with urolithin A, urolithin B, and ellagic acid are shown in Figure 35. A significant increase (P ≤ 0.05) in the transcriptional level of IL-6 was detected for urolithin A (- LPS) and urolithin B (- LPS) treatments (Figure 35A); these results may suggest that these metabolites could activate the signal transducer and activator of transcription 3 (STAT3) pathway (143) and may not be LPS dependent, because LPS activates the toll-like receptor 4 (TLR4) pathway.

While these effects were exclusive of the urolithins, ellagic acid (- LPS) produced a significant decrease in the transcript of IL-6 at the basal level. When mature adipocytes were treated with urolithins A and B and ellagic acid, independently, plus LPS addition

(Figure 35B), a significant increase in IL-6 levels was observed due to the fact that the dependent pathways STAT3 and TLR4 produce an augmentation in the levels of this cytokine. For the ellagic acid (+ LPS) treatment, similar levels of IL-6 were found compared to those of the control (+ LPS), being these compounds independent of the

STAT3 pathway. Significant differences were observed for all treatments co-incubated with LPS and their counterparts with no LPS.

Effects of urolithin A and B on the peroxisome proliferator-activated receptor γ (PPARγ) gene expression. In an inflammatory state of adipocytes, it is desirable that expression of PPARγ increases, as it has been proposed to reduce cytokine

(TNF-α, IL-6) production by inhibiting the activity of the pro-inflammatory transcription factor NF-κB (86). 88

5 A 4

3 - LPS 2 a 1 b -actin b b

 0 5 a*

TNF- B 4 *

Relative mRNA expression RelativemRNA a 3 * * a a + LPS 2

1

0

Control Urolithin A Urolithin B Ellagic acid

Figure 34. Relative RNA expression of the proinflammatory cytokine TNF-α in mature adipocytes. Treatments consisted in 25 µM urolithin A, urolithin B, ellagic acid or vehicle (0.04% DMSO) for 24 h and LPS (100 ng/mL) for the last 1 h. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) and * denotes significant differences (P ≤ 0.05) within same treatment with and without LPS performed by t-test.

89

8 A 6

4 - LPS b 2 bc a d

-actin 0 8  b * B

IL-6 6 * bc

Relative mRNA expression mRNA Relative 4 + LPS * a ad* 2

0

Control Urolithin A Urolithin B Ellagic acid Figure 35. Relative RNA expression of the proinflammatory cytokine IL-6 in mature adipocytes. Treatments consisted in 25 µM urolithin A urolithin B, ellagic acid or vehicle (0.04% DMSO) for 24 h and LPS (100 ng/mL) for the last 1 h. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) and * denotes significant differences (P ≤ 0.05) within same treatment with and without LPS performed by t-test.

90

1.2 a A 1.0

0.8 b bc 0.6 - LPS 0.4 bd

0.2

-actin 0.0

 1.2 B 1.0 PPAR *a 0.8 Relative mRNA expression mRNA Relative a a 0.6 a + LPS 0.4

0.2

0.0

Control Urolithin A Urolithin B Ellagic acid

Figure 36. Relative RNA expression of the transcription factor PPARγ in mature adipocytes. Treatments consisted in 25 µM urolithin A, urolithin B, ellagic acid or vehicle (0.04% DMSO) for 24 h and LPS (100 ng/mL) for the last 1 h. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) and * denotes significant differences (P ≤ 0.05) within same treatment with and without LPS performed by t-test. 91

2.0 a A 1.5 a 1.0 a - LPS b 0.5

-actin

 0.0 2.0 B

1.5

Adiponectin b *

Relative mRNA expression mRNA Relative a ab 1.0 ab + LPS

0.5

0.0

Control Urolithin A Urolithin B Ellagic acid Figure 37. Relative RNA expression of the secretory adipokine adiponectin in mature adipocytes. Treatments consisted in 25 µM urolithin A, urolithin B, ellagic acid or vehicle (0.04% DMSO) for 24 h and LPS (100 ng/mL) for the last 1 h. Data are means ± S.E. (n = 3), different letters denote significant differences among treatments (P ≤ 0.05) and * denotes significant differences (P ≤ 0.05) within same treatment with and without LPS performed by t-test.

92

Results showed that PPARγ expression was significantly decreased (P ≤ 0.05) for urolithin A (- LPS), urolithin B (- LPS), and ellagic acid (- LPS) (Figure 36A), whereas no significant differences were shown by the treatments with LPS (Figure 36B).A significant decrease was observed for control (+ LPS) compared to control (-LPS). An increase in the expression of PPARγ was expected, because in this way, inflammation produced by increased expression of p-NF-κB p65 in treatments with LPS could be reverted.

Effects of urolithin A and B on adiponectin gene expression. An increment in adiponectin gene expression was desirable since it attenuates excessive inflammatory responses in adipocytes (144) and is related to insulin resistance (76, 133). Results showed a significant decrease for ellagic acid treatment (- LPS), whereas urolithins A and B did not present an effect compared to the control (- LPS) (Figure 37A). When LPS was present (+ LPS) (Figure 37B), urolithin A showed an increase in adiponectin expression, but no significant changes were observed for the other treatments. A significant decrease (*) in adiponectin expression between control (- LPS) and control (+

LPS) was found, which could be related to the observed increase in expression levels of

TNF-α and IL-6. This behavior has been observed in previous reports (145-148).

To recapitulate, the present study focused in the possible bioactivities of urolithin

A and urolithin B as ellagitannins metabolites, and ellagic acid, as an ellagitannin component and precursor of urolithins, on the reduction of inflammation using 3T3-L1 adipocytes. An inflammatory state was achieved by the addition of LPS, this was confirmed by the controls, where LPS increased p-NF-κB p65 protein expression and 93

TNF-α and IL-6 mRNA expression by roughly 2-fold. A significant decrease was observed for mRNA expression of PPARγ and adiponectin in the control with LPS compared to the control without LPS. In this inflammatory condition, urolithin A favorably decreased p-NF-κB p65, but increased IL-6 expression probably due to synergistic effects by activating STAT3 and TLR4 pathways. No effect was observed for

TNF-α, but adiponectin expression was increased significantly. Treatment with urolithin

B did not have a significant effect on p-NF-κB p65 and adiponectin; nonetheless, it positively decreased TNF-α expression (in LPS absence). An increased expression of IL-

6 and no effect over PPARγ expression were observed for both urolithins. Ellagic acid treatment positively decreased p-NF-κB p65, still no effect on TNF-α, IL-6, and adiponectin was found; a decreased PPARγ expression was also obtained (without LPS).

When no inflammation was induced (- LPS), effects by the compounds on the basal level were also relevant. Urolithin A maintained similar expression of p-NF-κB p65 compared to the control, while for TNF-α mRNA expression; it had a protective effect by lowering it significantly. Interleukin-6 (IL-6) expression was increased by the addition of urolithin A and no significant change in expression of PPARγ. No significant effect on adiponectin expression was detected. Urolithin B treatment favorably decreased p-NF-κB p65 and TNF-α expression, but unfavorably increased IL-6 and decreased PPARγ. No change in adiponectin expression was noticed. Notably, ellagic acid increased p-NF-κB p65; but a significant decrease was seen for mRNA expression of TNF-α, IL-6, PPARγ and adiponectin.

94

Conclusions

The metabolites used in this study, urolithin A and urolithin B, decreased the phosphorylation state of the transcription factor NF-κB p65 in an inflammatory and non inflammatory state, respectively, which suggest that these compounds may have a protective effect against inflammation in mature adipocytes. At basal conditions, urolithin A, urolithin B, and ellagic acid decreased TNF-α mRNA expression significantly, indicating a protective effect; however, when LPS was added, a significant increase in this gene was presented. The protective effect observed at the basal level in

TNF-α was not strong enough to overcome LPS inflammation induction. Larger concentrations of these compounds may induce the same behavior at the basal level.

The transcription factor PPARγ basal level was down-regulated by all treatments, but this effect was not maintained when adipocytes were challenged with LPS. In an inflammatory state, urolithin B and ellagic acid did not present effects in adiponectin mRNA expression, but urolithin A induced the expression of this gene significantly, suggesting an improvement in adipocyte functionality by reducing insulin resistance. 95

CHAPTER V

CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH

Pecan nut is a regional crop from the southern United States that has many research areas to be explored, especially in relation to its nutraceutical content. Previous reports have stated that pecan kernels exhibit the highest antioxidant activity among the top commercial nuts. This property comes from the hydrophilic portion that is believed to protect the oil. In the present study, variations in total phenolic content, condensed tannins, ellagitannins, and antioxidant activities were found among four pecan cultivars.

Pecan polyphenols comprised gallic acid, catechin, ellagic acid, ellagitannins, condensed tannins, and different forms of isorhamnetin.

Since pecan ellagitannins are metabolized in vivo, cell culture work was done with their metabolites urolithin A and urolithin B at concentrations that mimic what has been found in human plasma. These metabolites presented no cytotoxic effects in 3T3-

L1 preadipocytes, as well as no alterations on cell proliferation up to 40 µM. The compounds urolithins A and B, and ellagic acid did not affect significantly adipogenesis or lipid accumulation (measured at day13) when 3T3-L1 cells were exposed for three days during differentiation.

Results from the inflammation study showed that urolithin A and urolithin B decreased the phosphorylation state of the transcription factor NF-κB p65 under inflammatory and non-inflammatory conditions, respectively. Suggesting that these compounds may have a protective effect against inflammation in mature adipocytes. 96

In future studies, it is recommended to test effects of catechin, epicatechin, procyanidin dimers and trimers in adipogenesis and/or inflammation. Longer exposure time of the test compounds with the cells could be performed for adipogenesis. Both type of studies should follow specific pathways.

97

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120

VITA

Ana Gabriela Ortiz Quezada received her Bachelor of Science degree in food industry engineering from Instituto Tecnológico y de Estudios Superiores de Monterrey,

Monterrey Campus in 2006. She entered the food science and technology program at

Texas A&M University in January 2008 and received her Master of Science degree in

August 2010. Her research interests include food chemistry, food analysis, food processing, nutraceuticals and food engineering.

Ms. Ortiz Quezada may be reached at the Department of Horticultural sciences,

Rm 202 HFSB, College Station, TX, 77843-2133. Her email is [email protected].