Journal of Food Bioactives

Volume 9 March 2020

An Official Scientific Publication of the

International Society for Nutraceuticals and Functional Foods (ISNFF)

Editor-in-Chief Fereidoon Shahidi

Co-Editor-in-Chief (Honorary) Beiwei Zhu

ISNFF & ASSOCIATES Publishing Company, Inc. Journal of Food Bioactives

Bring together the results of fundamental and applied research on food bioactives, functional food ingredients, nutraceuticals and natural health products that are known to possess or perceived to have health-promoting properties

www.isnff-jfb.com

Editorial Board

Editor-in-Chief Colin Barrow (Australia) Min-Hsiung Pan (Taiwan) Adriano Costa de Camargo (Chile) Ronald B. Pegg (USA) Fereidoon Shahidi (Canada) Rong Cao (Tsao) (Canada) Kenji Sato (Japan) Richard FitzGerald (Ireland) Angela Shaw (USA) Co-editor-in-Chief (Honorary) Bruce Hamaker (USA) Young-Joon Surh (South Korea) Chi-Tang Ho (USA) Francisco Tomas-Barberan (Spain) Beiwei Zhu (China) Farah Hosseinian (Canada) Petras R. Venskutonis (Lithuania) Charles C. Hu (USA) Chin-Kun Wang (Taiwan) Editorial Board Members Amin Ismail (Malaysia) Dao-Ying Wang (China) You-Jin Jeon (South Korea) Mingfu Wang (Hong Kong) Cesarettin Alasalvar (Turkey) Bo Jiang (China) Yu Wang (USA) Rotimi Aluko (Canada) Hitomi Kumagai (Japan) Hanny C. Wijaya (Indonesia) Emilio Alvarez (Mexico) Shiming Li (USA) Hang Xiao (USA) Ryszard Amarowicz (Poland) Jack Losso (USA) Wallace Yokoyama (USA) Anna Arnoldi (Italy) Kazuo Miyashita (Japan) Hui Zhao (China) Joseph Banoub (Canada) Ganiyu Oboh (Nigeria) Da-Yong Zhou (China)

Instructions for Authors

Journal of Food Bioactives with open access option is an official scientific publication of the International Society for Functional Foods and Nutraceuticals (ISNFF), a not-for-profit Disciplinary Interest Group of the International Union of Food Science and Technology (IUFoST) that was founded in 2007. It publishes original research articles, short communications/research notes, opinion pieces and review articles.

ISSN 2637-8752 Print ISSN 2637-8779 Online

ISNFF & ASSOCIATES Publishing Company, Inc. Connecticut, USA Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Meeting Report J. Food Bioact. 2020;9:1–3

Does COVID-19 affect food safety and security? A summary report on the Extraordinary Scientific Roundtable of IUFoST-CIFST on March 21, 2020

Abstract

The goal of this contribution is to provide a summary report on the Extraordinary Scientific Roundtable on -COV ID-19 and Food Safety co-organized by the International Union of Food Science and Technology (IUFoST) and the Chinese Institute of Food Science and Technology (CIFST). The meeting provided a valuable recount of this corona- virus, particularly that the virus is not transmitted by food. On this basis, so far there is no evidence that COVID-19 has any effect on food safety and security as well as on food bioactives. The challenges faced by the food industry during the pandemic period as well as potential post-pandemic time opportunities were discussed.

This brief write-up takes advantage of a recent Extraordinary Sci- diseases was emphasized by WHO beginning with the One Health entific Roundtable held on March 21, 2020 under the auspices of initiative, although most of these diseases are not prioritized by the International Union of Food Science and Technology (IUFoST), health systems internationally or by most countries. The experi- which is the global scientific organization representing national ence of China to bring the COVID-19 a successful level of control societies and institutes from many countries with membership provides a message of hope for the rest of the world, showing that of food scientists, technologists and engineers. The roundtable, this outbreak can be brought under control even though the eco- which was convened in cooperation with the Chinese Institute of nomic and social costs are high. Several other countries, notably Food Science and Technology (CIFST) included a number of sci- South Korea and Singapore, have also shown that it can be con- entists from international and national emergency response teams trolled if the right strategies are devised. as well as a representative of the food industry. The meeting was It is important to note that there have been no reports of trans- chaired by Dr. Fereidoon Shahidi who is the Chair of the Scientific mission of COVID-19 through food. Therefore, based on current Council of IUFoST and co-chaired by Dr. Junshi Chen, Chief Ad- epidemiological support, this virus is not foodborne. It was noted viser, China National Center for Food Safety Risk Assessment and that the United States Centre for Disease Control and European Dr. Pingfan Rao, IUFoST Past President and Professor and found- Centre for Disease Control have stated that there is also no evi- ing Director of CAS.SIBS-Zhejiang Gongshang University Joint dence that food items imported from China, in accordance with Center for Food and Nutrition Research in Hangzhou, China. The related regulations, will pose any risk of spreading COVID-19 in panel members were Dr. Peter Ben Embarek, Unit Head, Monitor- their countries. In addition, it was emphasized that since, in gener- ing of Nutrition and Food Safety Events, International Food Safety al, no viable viruses survive heat treatment, all cooked food should Authorities Network (INFOSAN), World Health Organization be safe to eat. However, it is most important to refrain from con- (WHO), Dr. Ding Gangqiang, Vice President of CIFST and Direc- suming raw or undercooked animal products. tor of Nutrition and Health Department within the China Centers The WHO has also stated that the COVID-19 virus is trans- of Disease Control, who was deployed in Wuhan, Dr. Patrick Wall, mitted through droplets, or small bits of liquid, primarily through member of the Ireland COVID-19 National Emergency Response sneezing and coughing that produce 3,000–10,000 droplets that and Professor of Public Health at University College, Dublin, Dr. cause “droplet spread”. This conclusion has been supported by Luo Yunbo, Honorary Vice President of CIFST and Director of the other food safety and health authorities. The possible presence Research Centre for Special Food, China Agricultural University, of the virus on food packaging material, as such, is not currently and Ms. Liz Duffy, Vice President for Omnichannel Compliance considered to be causing infection. It might, however, be a func- for Walmart Global eCommerce. tion of the pore size of the material, which has not yet been fully The WHO has stated that COVID-19 appeared to be a zoonotic evaluated (this is a personal opinion of the author and not ex- virus originating in bats but is likely to have gone through an in- pressed by members of the panel). The panel members stated that termediate species to infect humans. Zoonotic diseases are infec- the effective methodology used in Wuhan to control the disease tious ailments caused by bacteria, viruses and parasites transmitted should be employed as much as possible, namely, to identify, directly from animals to humans. Some 75% of emerging infec- admit and isolate confirmed COVID-19 cases and to follow up tious agents are zoonotic. The importance of controlling zoonotic with all their known contacts. In such cases, it is necessary to

Copyright: © 2020 International Society for Nutraceuticals and Functional Foods. 1 All rights reserved. Does COVID-19 affect food safety and security? Shahidi have adequate capacity to isolate and treat severe cases. Here, it is consumers will solely or primarily shop online and may spend a again the personal view of the author that, in the case of the fight significant amount of time online. They are getting inundated with against this virus, personal freedoms must take a lower priority information around the outbreak and unfortunately, a lot of that compared to the welfare of society at large. In the case of Wuhan, information is not founded on science. They are looking at social hotels were used to isolate people who were at risk of infecting media sites and other information sources to gain knowledge about others. The nutritional impact of isolation of individuals indicated the outbreak, and so it is important that consumers get the most that while their energy intake remained unchanged, their intake accurate and scientifically-based information from academic, in- of vitamins, minerals and plant-based protein and fatty acids was dustry and government sources. compromised. In addition, the lack of physical activity may cause What is evident to all, and was agreed by the panel members, obesity and non-communicable diseases (NCD) in the long term. is the necessity to educate consumers and provide them with ac- Thus, the elderly population needs to pay greater attention to diet- curate information so that they can make sound decisions. This related and other risk factors. In Wuhan, providing safe and nu- must be done in a timely manner as we are dealing with a rapidly tritionally balanced meals to doctors, nurses and other healthcare evolving situation. The food industry must ensure the availabil- professionals and also ambulance drivers were considered essen- ity of safe food with adequate wholesomeness and nutritional at- tial. Cooked meals were continuously made available to them in tributes to the global community, especially because certain raw the areas they were serving during the crisis in spite of the dif- materials and ingredients may need to be substituted and process- ficulties encountered. ing conditions changed or modified. This is particularly essential With respect to food availability, it was noted that primary for large cities and urban populations in developing countries. production may not be severely affected because production- ar Meanwhile, it is essential that the personnel working in the food eas are often geographically distant from urban population densi- industry and allied areas of food supply chain remain in good ties. However, the pandemic may affect harvesting, transportation health with consideration of physical distancing in processing and distribution. In this regard, animal feed and ingredients for and preparation plants. This might be provided by temporary food product preparation may be adversely affected, especially if zoning, which may not be possible in all cases. However, where they need to be imported. This is particularly true as many of the such an option exists, only a small number of people are affected primary ingredients originate from China and India for both food and removed if a member tests positive for the virus. Person- and non-food items, such as pharmaceuticals. Thus, the present nel who contract the virus must be given the ability to immedi- reliance on specific markets, products and distribution channels is ately report it to their supervisors and remain isolated at home. probably insufficient or inadequate to respond to anticipated dis- This may result in labor shortages and arrangements should be in ruptions cause by the pandemic. In order to fight this virus, travel, place by all means, including e-technology, to recruit and train conferences, conventions and sporting events have been cancelled new staff. Workers from commercial catering, tourism and hos- and food service providers as well as the entire tourism industry pitality industries with food experience can be redeployed into have been decimated due to the required social distancing. All of other parts of the food industry. The food industry needs to ad- these have consequences for human resources and the economy. dress the anxiety of the consumers via clear, science-based and The food industry needs to keep its staff healthy if it is to func- user-friendly messages that do not make any false claims. The tion successfully. Consumers may also resort to over-stocking and current food safety protocols, including food recalls, must be put hoarding, which adversely affects the supply chain. This happens in place, if and when needed. mostly due to believing rumors and false information from unau- It was also noted that the post-pandemic phase may result in thorized sources. major reviews of food systems with special emphasis on resilience. In order to keep our food supply safe, it is imperative that the Thus, the agri-food systems may take advantage of the opportuni- real importance of food safety with respect to good hygiene prac- ties to make changes, such as making more use of locally produced tices by all be emphasized. This is not just restricted to the food food. This would remove dependence on long-distance transporta- industry and must be practiced by everyone, including interme- tion and distribution by third parties with major carbon footprints. diaries from the food industry to the consumer. All must be re- A paradigm shift for safe food practices should be reinforced by minded to follow the WHO Five Keys to Safer Food (https:// promoting safety habits that were developed and acquired during www.who.int/foodsafety/areas_work/foodhygiene/en/), particu- the pandemic. The food science and technology community will larly handwashing. The disruption of the food supply chain may be in a position to strategically plan and contribute to the recovery also create unexpected food safety risks, particularly for perish- of the food sector in collaboration with other allied disciplines and able foods and vegetables. These have often been a source of stakeholders. The role of food scientists and technologists in shap- concern due to inadequate washing and packaging. It is also nec- ing the policies of governments and in decision-making strategies essary to communicate food safety information in a simple and must be considered in order to ensure the readiness of the food understandable manner. This should be done in a scientifically supply chain in responding to any future pandemic. sound and thoughtful way with respect to those involved in the In summary, it was mentioned that the pandemic of COVID-19 food chain, from producers to intermediaries and consumers, par- posed unprecedented challenges to the food sector, especially ticularly in these uncertain times. Therefore, the development of when many people may need to be quarantined for at least a two- safe and effective foods to promote immune function should be week period. However, it was emphasized that the food industry a priority for the food industry and governments. This is a new with the help of food scientists and technologists can overcome role for functional foods and may include special medical foods these challenges and succeed in providing safe, nutritious and suf- for the elderly as well as immune-compromised individuals and ficient food to the global population. In the future, emphasis may groups. also shift to providing immune-enhancing functional foods, which In view of the popularity of e-commerce in food, especially indicates the important role of food bioactive compounds in reduc- during the current situation with lockdowns and social distancing ing disease risk. in effect, tremendous growth opportunities exist for the industry Finally, it is important to emphasize here the commitment of to cater and respond to the needs of consumers who do not want IUFoST and its national affiliates in assisting the food industry in to shop in person. Thus, in the e-commerce space, in particular, its essential role in society. The International Society for Nutraceu-

2 Journal of Food Bioactives | www.isnff-jfb.com Shahidi Does COVID-19 affect food safety and security? ticals and Functional Foods (ISNFF) www.isnff.org, a disciplinary well as food supply problems in the developing countries. In this interest group of the IUFoST, and its current main publication, the regard, a Task Force was established by the IUFoST to share the Journal of Food Bioactives (JFB) www.isnff-jfb.com are proud to findings and experiences of the food industry in one place with take all measures needed to be a part of this effort to fulfilling its all. For further information, interested parties may contact secre- workplan with regard to addressing food safety and security as [email protected] or the author at [email protected]. Fereidoon Shahidi Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9

Journal of Food Bioactives | www.isnff-jfb.com 3 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Viewpoint J. Food Bioact. 2020;9:4–9

Is there a role of gut microbiota in mental health?

Klaus W. Langea*, Katharina M. Langea, Yukiko Nakamuraa and Shigehiko Kanayab aDepartment of Experimental Psychology, University of Regensburg, Germany bGraduate School of Information Science, Nara Institute of Science and Technology, Japan *Corresponding author: Klaus W. Lange, Institute of Psychology, University of Regensburg, 93040 Regensburg, Germany. Tel: +49 941 9433815; Fax: +49 941 9434496; E-mail: [email protected] DOI: 10.31665/JFB.2020.9213 Received: March 27, 2020; Revised received & accepted: March 31, 2020 Citation: Lange, K.W., Lange, K.M., Nakamura, Y., and Kanaya, S. (2020). Is there a role of gut microbiota in mental health? J. Food Bioact. 9: 4–9.

Abstract

Research on the interaction between gut microbiota and the brain may have implications for our understanding of brain function, cognition, behavior and mental health. The literature on gut microbiota and its role in the pathophysiology and potential treatment of mental disorders has proliferated in recent years. Several neurodevelopmental disorders, including autism spectrum disorders, schizophrenia and attention-deficit/hyperactivity disorder, have been linked to the gut microbiota. The present perspective discusses the promise and pitfalls of gut microbiota research in relation to mental health. The manipulation of intestinal microbes in animals has revealed connections between gut microbiota and both normal and pathological brain functions. The hope fueling this research is that gut microbiota could be harnessed to prevent and treat mental disorders. The links observed between an imbalance of gut microbiota and impaired behavioral and mental states in humans are correlational. It is therefore essential to establish cause and effect relationships. No distinct gut microbiota patterns linked to different mental disorders have yet been identified. Large-scale, longitudinal trials need to examine whether the gut microbiota is a valid therapeutic target for mental disorders and whether pre-clinical findings and initial results of intervention trials (e.g., administration of probiotics) are of clinical relevance.

Keywords: Gut microbiota; Brain behavior research; Probiotics; Mental health; Psychiatry.

1. Introduction which has been estimated to be of the same order as the number of human cells in the body (Sender et al., 2016). The gut microbiota of humans is determined by genetic, epigenetic and dietary The study of brain functions using neurophysiological, neuropsy- factors (Yadav et al., 2018). Early research studies in the 1980s chological, neurochemical and neuroimaging techniques has long began the mechanistical investigation of the influence of specific been the central approach to understanding the regulation of nor- intestinal microorganisms on brain states in mammals (e.g., Brown mal behavior as well as of altered behavioral states seen in mental et al., 1990). Findings of these studies revealed that gut microbes disorders. However, emerging evidence suggests that researchers (microbiota) appear to affect a wide range of their host’s physi- in this field have often neglected another important system in- ological processes, including cell differentiation and proliferation, volved in normal and abnormal brain functioning, the gastrointes- metabolic and immune functions, as well as behavior (Heiss and tinal system. This relationship seems to be an important one. The Olofsson, 2018; Nicholson et al., 2012; Rooks and Garrett, 2016; gut has a large, independent nervous system, and it communicates Sharon et al., 2016). Microbiota may therefore contribute to vari- closely with the brain through the gut-brain axis. The human gut ous diseases, such as inflammatory diseases, metabolic ailments, is colonized by bacteria, archaea and eukaryotes, the number of cancer and mental disorders. In particular, the gut microbiota plays

4 Copyright: © 2020 International Society for Nutraceuticals and Functional Foods. All rights reserved. Lange et al. Gut microbiota and mental health an important role in modulating developmental processes of the turbations of gut microbiota in individuals with ASD (Son et al., central and enteric nervous systems and is involved in neurogen- 2015). Moreover, alterations in gut microbial communities, which esis, myelination, glial cell function, synaptic pruning and blood- are frequently accompanied by strong food preferences, such as brain barrier permeability (Heiss and Olofsson, 2019). Alterations for starchy, processed foods, might be associated with behavioral in the bi-directional communication pathways between gut micro- problems in children with ASD (McElhanon et al., 2014). Reverse biota and brain may eventually lead to a disrupted neurotransmitter causation, i.e. behavioral changes leading to microbiota altera- balance, increased chronic inflammation and heightened activity tions, may explain the associations observed. of the hypothalamic-pituitary-adrenal axis. Our current under- Schizophrenia is a complex, heterogeneous behavioral and cog- standing of the complex communication systems involved in the nitive syndrome, characterized by diverse symptoms, including gut microbiota-brain axis is limited. cognitive dysfunction, positive symptoms, such as delusions and hallucinations, and negative symptoms, such as impaired motiva- tion and social withdrawal (Owen et al., 2016). Few studies have 2. Gut microbiota in mental disorders investigated the microbiota composition in relation to schizophrenia. Individuals with first-episode psychosis have been reported Research on the interaction between complex communities of gut to show increased numbers of Lactobacillus gut bacteria, which microbes (microbiota) and the brain may have implications for our correlate with the severity of symptoms (Schwarz et al., 2017). In understanding of brain function, cognition, behavior and mental addition, a subgroup of these individuals, who showed the most health. Intestinal dysbiosis has been demonstrated to be linked to profound alterations in microbiota composition, were less likely behavioral impairment (Bruce-Keller et al., 2015; Diaz Heijtz et to respond to therapy (Schwarz et al., 2017). A potential involve- al., 2011; Douglas-Escobar et al. 2013; Tillisch 2014), and this has ment of the microbiota in schizophrenia has been suggested by given rise to investigations of the role of gut microbiota in mental findings showing that the administration of the antibiotic minocy- health. cline, which can induce changes in microbiota composition, po- The findings of studies in germ-free mice, which lack intesti- tentiates the effect of the antipsychotic drug risperidone in people nal and other microbiota, suggest that gut microbiota are essential with chronic schizophrenia (Khodaie-Ardakani et al., 2014). for the development of neuronal systems involved in motor con- ADHD is a common psychiatric diagnosis in childhood and trol, social activity and anxiety behavior (Desbonnet et al., 2014; adolescence and is characterized by age-inappropriate levels of Diaz Heijtz et al., 2011). Furthermore, exaggerated caution and hyperactivity, inattention, and impulsivity. Individuals diagnosed disturbed social behavior displayed by germ-free BALB/c mice with ADHD display long-term social, academic and mental health can be normalized by microbiome transplants from NIH Swiss problems (Lange et al., 2010; Thapar and Cooper, 2016). While mice, which do not show impaired exploratory and social behav- the etiology of ADHD appears to be multifactorial, definite causes ior (Brinks et al., 2007). Conversely, NIH Swiss mice receiving remain unknown. Doubts regarding the effectiveness of commonly BALB/c microbiota transplants presented with marked hesitancy used ADHD therapies on clinically relevant outcome measures, and caution (Bercik et al., 2011). the unproven long-term efficacy of treatment and concerns- sur It has been demonstrated that neurodevelopmental mental dis- rounding potentially serious adverse effects of medication have led orders, including autism spectrum disorders (ASD), schizophrenia to a search for alternative treatment options (Lange, 2017; Lange, and attention-deficit/hyperactivity disorder (ADHD), are com- 2018a; Lange, 2020b), including micro- and macronutrients and monly associated with gastrointestinal symptoms, such as diarrhea diets (Lange, 2018b; Lange, 2018c). Recent findings suggest that and constipation (e.g., McElhanon et al., 2014; Severance et al., the gut microbiome dysbiosis associated with dietary patterns may 2016). Furthermore, alterations in gut microbial composition have be linked to a susceptibility to ADHD (Wang et al., 2020). How- been observed in these conditions (Sharon et al., 2016), suggest- ever, a causal relationship has not been established. ing that microbial dysbiosis may be involved in their pathogenesis. To further our understanding of the effects of gut microbiota The role of gut microbiota has raised high expectations in men- on mental disorders, future studies should also attempt to eluci- tal disorders including the heterogeneous family of neurodevelop- date the mechanisms underlying the influence of microbiota on the mental ASD, which are characterized by early-appearing deficits health of the host. Potential mechanisms include direct activation in cognitive, communicative and social skills and by repetitive of neuronal pathways (vagal afferents), microbial metabolism of sensory-motor behaviors (Lord et al., 2018). Children with ASD nutrients and production of circulating mediators, and immune ac- present with more gastrointestinal problems than healthy controls tivation and circulating inflammatory mediators (Bruce-Keller et (McElhanon et al., 2014). Several dietary interventions, including al., 2018). gluten-free and casein-free diets (Lange et al., 2015; Reissmann et al., 2020) and omega-3 fatty acid supplementation (Lange, 2020a), have been suggested as complementary and alternative medicine 3. Therapeutic modulation of gut microbioata in mental disor- treatments in ASD. However, in view of the less than convincing ders evidence in support of these dietary approaches, it would be premature to recommend them as alternatives in the management of Tools of potential value in the modulation of gut microbiota as a ASD (Lange, 2020d; Reissmann et al., 2020). treatment approach in mental disorders include fecal microbiota ASD has been linked to an abnormal gut microbiota compo- transplantation and dietary changes, such as the administration sition, with distinct increases or decreases in certain microbial of probiotics. The success of microbiota transplantation in the groups (Mulle et al., 2013; O’Mahony et al., 2015). Elevated lev- treatment of refractory Clostridium difficile infection (Mattila et els of Clostridia, Desulfovibrio (Finegold, 2011), Sutterella (Wang al., 2012) has excited some publicity. However, little informa- et al., 2013) and Bacteroidetes (Tomova et al., 2015) and de- tion concerning the potential of this approach in mental disorders creased concentrations of Firmicutes, Prevotella, and Bifidobacter in humans is currently available. An open-label trial in children have been found in the stool of children with autism (Rosenfeld, with autism has shown improved gastrointestinal and behavioral 2015). However, other studies have found no compositional per- functions following fecal microbiota transplantation (Kang et al.

Journal of Food Bioactives | www.isnff-jfb.com 5 Gut microbiota and mental health Lange et al.

2017). However, this trial was not randomized, placebo-controlled search findings in the field. or blinded. In humans, the links observed between an imbalance of gut The composition of gut microbiota is determined in part by the microbiota and impaired behavioral and mental states are correla- diet. While an individual’s gut microbiota is relatively stable over tional. Therefore, it is essential to establish cause and effect rela- time, it responds quickly to dietary changes (David et al, 2014a, tionships. Even if intestinal dysbiosis can causally facilitate mental b). With respect to mental health, highly processed foods, which disorders, the reciprocal effects of impaired brain neurophysiology reduce intestinal microbial diversity, have been shown to be as- on gut microbiota are unclear. Whether differences in gut micro- sociated with an elevated risk of mental disorders (Dawson et al., biota cause disease or whether they are caused by the disease is 2016). Furthermore, several studies have revealed significant in- often impossible to determine. Psychopathological symptoms and verse associations between metrics of diet quality and disturbances behavioral abnormalities may affect food preferences, for exam- of mental health (Flórez et al., 2015; Huddy et al., 2016; Mihrshahi ple, which in turn lead to gut dysbiosis. It is therefore important to et al., 2015; Rienks et al., 2013). However, the findings in regard to ascertain whether gut dysbiosis is simply accompanying a disease these relationships are mixed (Lai et al., 2016; Martínez-González or whether it is causing it. and Sánchez-Villegas, 2016) and call for further controlled trials. Moreover, whether microbes mediate brain pathophysiology as The administration of probiotics could potentially counterbal- well as mental health and disease, and, if so, whether individual or ance the effects of Western diets on mental health. In one study, groups of different microbes are involved should be established. germ-free mice displayed exaggerated and maladaptive responses No distinct microbiota profile has as yet been related to any mental to stress, which were normalized by intestinal recolonization us- disorder. Since the exact alterations in gut microbiota underlying ing probiotics (Sudo et al., 2004). Other studies have shown that pathological changes are unknown even in animals, the diagnostic probiotics improve mood, anxiety and cognition in animal models utility of microbiome analyses is currently limited in clinical prac- (Bravo et al., 2011; Desbonnet et al., 2010; Smith et al., 2014; tice. In addition, while there is some evidence suggesting in both Sudo et al., 2004). In humans, randomized trials have revealed animals and humans that exposure to transient microbes can influ- beneficial effects of probiotics on mood (Messaoudi, 2011; Steen- ence brain physiology and behavior, there is no proof that chang- bergen, 2015). In children with autism, probiotics have been re- ing the resident gut microbiota has therapeutic benefits. A detailed ported to be beneficial in normalizing the Bacteroidetes/Firmicutes analysis of the interaction of the gut microbiota and nutritional balance and in elevating Desulfovibrio spp. levels (Tomova et al., modification at different neurodevelopmental stages may provide 2015). More research is necessary to assess the amelioration of preventative strategies for neurodevelopmental disorders, includ- ASD symptoms following manipulation of the gut microbiota. A ing ASD, schizophrenia and ADHD. placebo-controlled trial has demonstrated that Lactobacillus casei An important aspect in relation to the difficulty in establish- reduces physiological responses to stress while increasing gut mi- ing distinct microbiological patterns concerns the phenotyping of crobiota diversity (Kato-Kataoka, 2016). However, other studies psychiatric disorders. Given that the biological and environmental found no benefits of probiotics versus placebo on mood, anxiety, mechanisms involved in mental disorders are multifactorial, het- stress or sleep quality in healthy volunteers (Kelly et al., 2017). erogeneous and complex, it is unrealistic to expect a one-fits-all In addition, the remarkable degree of inter-personal variability in solution in regard to gut microbiota. An examination of neuropsy- human microbiota needs to be considered (Eckburg et al., 2005). chiatric symptom dimensions may be of greater value than an in- To sum up, the available evidence in support of the use of pro- vestigation of specific disorders. biotics in the field of mental health is suggestive but not sufficient. Five questions for researchers have been proposed as a meas- Future trials evaluating probiotic use in various mental disorders ure to guard against hype in microbiome research (Hanage, 2014). are required in order to determine efficacy and to address several These are: (1) can experiments detect differences that matter? (2) key problems, such as beneficial strains, dosage, method of ad- does the study show causation or just correlation? (3) what is the mechanism? (4) how much do experiments reflect reality? (5) ministration and treatment duration (Wallace and Milev, 2017). could anything else explain the results? In respect of a possible Dietary fiber and prebiotics consistently modulate gut microbiota role of gut microbiota in mental health, none of these questions can composition and function and could therefore become a valuable be answered in the affirmative: the experiments performed have tool in research on the interaction between microbiota and brain been unable to detect differences that matter (no distinct micro- (Dalile et al, 2019). biota patterns related to different mental disorders); the available studies show correlation but no causation (see above); the (animal) experiments do not reflect reality (germ-free mice etc.); the under- 4. Conclusions lying mechanisms remain unknown; other factors could explain the results (e.g., food choices as a consequence of psychopathol- The experimental methods employed in seeking to establish rela- ogy. tionships between gut microbes and behavior include alteration of In summary, gut microbiota-brain research has recently attract- the microbiota in animals and comparison of various behaviors of ed a great deal of attention. Published research and review papers animals with and without microbiota. The manipulation of intesti- in the field have frequently presented misinterpretations of find- nal microbes has revealed connections between gut microbiota and ings as well as speculation and overblown claims, which in turn both normal (emotion and cognition) and pathological (neurode- have been propagated by the media. The interpretations of find- velopmental disorders, anxiety, mood) brain functions. The exist- ings on the interactions between gut microbiota and behavior offer ing scientific literature has engendered hopes that gut microbiota potential avenues for the treatment of mental disorders or even for could be harnessed in the prevention and treatment of mental dis- the enhancement of normal cognition. However, the sobering news orders. If a direct relationship between gut dysbiosis and distinct is that the radical claims made respecting causal relationships, in psychopathological phenotypes could be established, this might particular the extent to which the microbiota may control human lead to novel microbiome-based therapeutic strategies in psychia- behavior, are not justified. Pseudoscientific commercialization, es- try. However, the hype created by some researchers and the media pecially claims by various companies that their analyses of indi- seldom considers the pitfalls inherent in the interpretation of re- vidual fecal microbial content could form the basis of microbiome

6 Journal of Food Bioactives | www.isnff-jfb.com Lange et al. Gut microbiota and mental health

Table 1. Gut microbiota and mental health The gut has a large, independent nervous system, which communicates bi-directionally with the brain through the gut-brain axis Connections between gut microbiota and both normal and pathological brain functions have been revealed Gut microbiota affect a wide range of their host’s physiological processes, including behavior Gut microbiota are essential for the development of neuronal systems involved in social activity and anxiety behavior in rodents Intestinal dysbiosis is linked to behavioral impairment: Role of gut microbiota in mental health Highly processed foods, which reduce intestinal microbial diversity, are associated with an elevated risk of mental disorders Altered gut microbial composition in neurodevelopmental disorders, including ASD, schizophrenia and ADHD, has been found: Involvement of microbial dysbiosis in their pathogenesis The links observed between an imbalance of gut microbiota and behavioral and mental impairment in humans are correlational: Relationship of cause and effect? Reciprocal effects of impaired brain neurophysiology on gut microbiota No distinct gut microbiota patterns have been linked to different mental disorders Treatment approaches modulating gut microbiota in mental disorders include dietary changes (e.g. probiotics) and fecal microbiota transplantation The potential of fecal microbiota transplantation in human mental disorders is unknown Probiotics have been shown to improve mood, anxiety and cognition in animal models Randomized trials have revealed beneficial effects of probiotics on mood in humans Evidence in support of the use of probiotics in mental health is insufficient: beneficial strains, dosage, method of administration and treatment duration

Summary: Gut microbiota as a one-fits-all solution in mental health is unrealistic, since the pathogenesis of mental disorders is multifactorial and complex. Exact alterations in gut microbiota underlying mental disorders are unknown and microbiome analyses are of limited diagnostic use in clinical practice. Large-scale, longitudinal trials need to examine whether the gut microbiota is a valid therapeutic target in mental disorders. cleansing or of personalized (mental) health interventions, are light ing the gut microbiota: The potential of prebiotics in microbiotagut- on substance and heavy on salesmanship. Gut microbiota research brain axis research. Behav. Brain Sci. 42: e69. can have no place in psychiatric practice until future large-scale, David, L.A., Materna, A.C., Friedman, J., Campos-Baptista, M.I., Blackburn, longitudinal trials have established whether the gut microbiota is a M.C., Perrotta, A., Erdman, S.E., and Alm, E.J. (2014a). Host lifestyle valid therapeutic target for mental disorders and whether the cur- affects human microbiota on daily timescales. Genome Biol. 15: R89. David, L.A., Maurice, C.F., Carmody, R.N., Gootenberg, D.B., Button, J.E., rently available pre-clinical findings and initial results of interven- Wolfe, B.E., Ling, A.V., Devlin, A.S., Varma, Y., Fischbach, M.A., Bid- tion trials are of clinical relevance (see Table 1). dinger, S.B., Dutton, R.J., and Turnbaugh, P.J. (2014b). Diet rapidly and reproducibly alters the human gut microbiome. Nature 505: 559–563. References Dawson, S.L., Dash, S.R., and Jacka, F.N. (2016). The importance of diet and gut health to the treatment and prevention of mental disorders. Bercik, P., Denou, E., Collins, J., Jackson, W., Lu, J., Jury, J., Deng, Y., Blen- Int. Rev. Neurobiol. 131: 325–346. nerhassett, P., Macri, J., McCoy, K.D., Verdu, E.F., and Collins, S.M. Desbonnet, L., Clarke, G., Shanahan, F., Dinan, T.G., and Cryan, J.F. (2014). (2011). The intestinal microbiota affect central levels of brain-de- Microbiota is essential for social development in the mouse. Mol. rived neurotropic factor and behavior in mice. Gastroenterology 141: Psychiatry 19: 146–148. 599–609. Desbonnet, L., Garrett, L., Clarke, G., Kiely, B., Cryan, J.F., and Dinan, T.G. Bravo, J.A., Forsythe, P., Chew, M.V., Escaravage, E., Savignac, H.M., Dinan, (2010). Effects of the probiotic Bifidobacterium infantis in the ma- T.G., Bienenstock, J., and Cryan, J.F. (2011). Ingestion of Lactobacil- ternal separation model of depression. Neurosci. 170: 1179–1188. lus strain regulates emotional behavior and central GABA receptor Diaz Heijtz, R., Wang, S., Anuar, F., Qian, Y., Björkholm, B., Samuelsson, A., expression in a mouse via the vagus nerve. Proc. Nat. Acad. Sci. U. S. Hibberd, M.L., Forssberg, H., and Pettersson, S. (2011). Normal gut A. 108: 16050–16055. microbiota modulates brain development and behavior. Proc. Nat. Brinks, V., van der Mark, M., de Kloet, R., and Oitzl, M. (2007). Emotion Acad. Sci. U. S. A. 108: 3047–3052. and cognition in high and low stress sensitive mouse strains: A com- Douglas-Escobar, M., Elliott, E., and Neu, J. (2013). Effect of intestinal mi- bined neuroendocrine and behavioral study in BALB/c and C57BL/6J crobial ecology on the developing brain. JAMA Pediatr. 167: 374–379. mice. Front. Behav. Neurosci. 1: 8. Eckburg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E., Dethlefsen, L., Sar- Brown, R., Price, R.J., King, M.G., and Husband, A.J. (1990). Are antibiotic gent, M., Gill, S.R., Nelson, K.E., and Relman, D.A. (2005). Diversity of effects on sleep behavior in the rat due to modulation of gut bacte- the human intestinal microbial flora. Science 308: 1635–1638. ria? Physiol. Behav. 48: 561–565. Finegold, S.M. (2011). Desulfovibrio species are potentially important in Bruce-Keller, A.J., Salbaum, J.M., and Berthoud, H.R. (2018). Harnessing regressive autism. Med. Hypotheses 77: 270–274. gut microbes for mental health: Getting from here to there. Biol. Psy- Flórez, K.R., Dubowitz, T., Ghosh-Dastidar, M.B., Beckman, R., and Collins, chiatry 83: 214–223. R.L. (2015). Associations between depressive symptomatology, diet, Bruce-Keller, A.J., Salbaum, J.M., Luo, M., Blanchard, E., Taylor, C.M., and body mass index among participants in the supplemental nutri- Welsh, D.A., and Berthoud, H.R. (2015). Obese-type gut microbiota tion assistance program. J. Acad. Nutr. Diet 115: 1102–1108. induce neurobehavioral changes in the absence of obesity. Biol. Psy- Hanage, W.P. (2014). Microbiome science needs a healthy dose of skepti- chiatry 77: 607–615. cism. Nature 512: 247–248. Dalile, B., Verbeke, K., Van Oudenhove, L., and Vervliet, B. (2019). Nourish- Heiss, C.N., and Olofsson, L.E. (2019). The role of the gut microbiota in de-

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Journal of Food Bioactives | www.isnff-jfb.com 9 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Review J. Food Bioact. 2020;9:10–22

Pomegranate as a natural source of phenolic antioxidants: a review

Fellipe Lopes de Oliveiraa, Thaise Yanka Portes Arrudaa, Renan da Silva Limab, Sabrina Neves Casarottic and Maressa Caldeira Morzellea* aDepartamento de Alimentos e Nutrição, Faculdade de Nutrição, Universidade Federal do Mato Grosso (FANUT/UFMT), Rua Fernando Corrêa da Costa, 2367, Boa Esperança, 78060-900, Cuiabá, MT, Brazil bDepartment of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, A1B 3X9, Canada cInstituto de Ciências Exatas e Naturais, Universidade Federal de Rondonópolis (ICEN/UFR), Av. dos Estudantes, 5055, Cidade Universi- tária, 78736-900, Rondonópolis, MT, Brazil *Corresponding author: Maressa Caldeira Morzelle, Departamento de Alimentos e Nutrição, Faculdade de Nutrição, Universidade Fed- eral do Mato Grosso (FANUT/UFMT), Rua Fernando Corrêa da Costa, 2367, Boa Esperança, 78060-900, Cuiabá, MT, Brazil. Tel: +55 65 36153312; E-mail: [email protected] DOI: 10.31665/JFB.2020.9214 Received: March 25, 2020; Revised received & accepted: March 31, 2020 Citation: de Oliveira, F.L, Arruda, T.Y.P, da Silva Lima, R., Casarotti, S.N, and Morzelle, M.C. (2020). Pomegranate as a natural source of phenolic antioxidants: a review.. J. Food Bioact. 9: 10–22.

Abstract

Pomegranate, a recognized source of phenolic compounds, has been associated with health-promoting benefits, mostly due to its antioxidant activity. Ellagic and gallic acids, anthocyanins, and ellagitannins are the main phenolics in pomegranate, showing antioxidant activity. For this reason, pomegranate has been used in foods, such as meat products, as an attempt to retard lipid oxidation and increase shelf-life. In recent years,in vitro, in vivo, and human studies reported the antioxidant activity of pomegranate, especially its peels, with reduced incidence of chronic diseases (e.g., cardiovascular ailments, cancer, neurodegenerative disease, type 2 diabetes, chronic kidney disease). This review aims to present the main antioxidant compounds on pomegranate and their biological effects, the antioxidant activity of pomegranate-based foods, the application of pomegranate as a natural antioxidant food additive, the role of pomegranate in the prevention and management of chronic diseases, as well as the trends and prospects regarding the application of pomegranate in innovative food and health.

Keywords: Ellagitannins; Anthocyanin; Antioxidant; Polyphenols.

1. Introduction such pathological conditions (Wilcox et al., 2004). In order to diminish oxidative stress, research has focused on foods and natural compounds displaying antioxidant activity (AA) Existing data suggest that between 16–57% of adults in developed that can contribute to delay or prevent oxidation. With that being countries develop two or more chronic non-communicable dis- said, epidemiological studies point out a significant inverse rela- eases simultaneously. The increasing incidence of these conditions tionship between regular consumption of fruits and vegetables and has encouraged scientific research in this field (Hajat and Stein, a decrease in the incidence of chronic non-communicable diseases. 2018). Reports have shown that the accumulation of free radicals This correlation is a result of the biologically active antioxidants (FR) in the human body as a result of environmental, lifestyle, and present in these foods carrying the potential to protect cells and tis- pathological conditions contribute to an increased risk of chronic sues against damage caused by FR (Magrone et al., 2012; Frozza non-communicable diseases such as type 2 diabetes mellitus, Alz- et al., 2013; Kandylis and Kokkinomagoulos, 2020). heimer’s, cancer, and cardiovascular diseases. This accumulation Among the sources of bioactive compounds, pomegranate (Pu- often results in an imbalance of reduction-oxidation homeostasis nica granatum L.), one of the most traditionally consumed fruits that is likely one of the processes that regulate gene expression in worldwide, stands out for being associated with health-promoting

10 Copyright: © 2020 International Society for Nutraceuticals and Functional Foods. All rights reserved. de Oliveira et al. Pomegranate as a natural source of phenolic antioxidants benefits linked to its consumption. These biological properties 2020;). The approximate ratio of peel, arils, and seeds is described have been attributed mainly to the phenolic compounds (PC) pre- to be 50:40:10 (Andrade et al., 2019). sent in this fruit (Ismail et al., 2017). Given that oxidative stress is a biomarker for numerous chronic non-communicable diseases, the high in vitro AA of pomegranate has stimulated studies aiming 3. Pomegranate as functional food at investigating its effects on human health Islam,( 2016). Pomegranate is widely promoted, with or without scientific sup- The biological role of pomegranate has been attributed, at least in port, to consumers as a superfood capable of fighting a variety of part, to the presence of PC, mainly gallic acid (GA), ellagic acid diseases. This fruit, which has been consumed and used as a func- (EA), hydrolysable and condensed tannins as well as anthocya- tional food in the Middle East for thousands of years, has recently nins, potent antioxidants (Ambigaipalan et al., 2016; 2017). These gained global popularity (Johanningsmeier and Harris, 2011). compounds have been related to numerous health effects, includ- Pomegranate is usually consumed fresh, with the skin being ing the prevention of Alzheimer’s disease, cancer, cardiovascular discarded as waste. Pomegranate peel (PP) corresponds to about disease, and diabetes (Johanningsmeier and Harris, 2011; Salgado 50% of the total weight of the fruit, and it is a source of PC, such et al., 2012; Shahidi et al., 2019). They will be presented and dis- as ellagic acid (EA) and its derivatives, and ellagitannins, such cussed below. as punicalin and punicalagin (Gullon et al., 2016). Moreover, PP presents up to 10 times more bioactive compounds than pulp and seeds (Li et al., 2006). Therefore, pomegranate extracts are used as 3.1. Phenolic compounds (PC) food additives on meat products, typical Brazilian pastry product and others (Veloso et al., 2020). PC are a large group of natural antioxidants commonly found in Therefore, this review aims to highlight the current state-of- plant material, especially fruits. They can modulate gene expres- the-art concerning the phenolic composition of pomegranate, the sion, inflammation, antioxidant, and immune functions, exerting antioxidant potential of pomegranate-based foods, the use of PP a strong influence on human health (Tota et al., 2010; Kang et as a natural antioxidant, its biological effect on the prevention of al., 2011). PC are secondary plant metabolites derived from the chronic diseases, as well as to suggest future trends and prospects shikimic and malonic acid pathways. In foods, they can occur as about the application of pomegranate in new functional foods. soluble (free or esterified to fatty acids) or insoluble-bound (covalently bound to proteins, cellulose, pectin, among others) compounds. Structurally, they present at least one aromatic ring substi- 2. Pomegranate tuted with one or more hydroxyl groups, and their AA is related to the number and position/distribution of such groups (Giada, 2013; Shahidi and Ambigaipalan, 2015). The antioxidant action of phe- Pomegranate has been used in traditional medicine since ancient nolics comes from their ability to scavenge FR through the dona- times. This species was described by Linnaeus in 1758, who sug- tion of a hydrogen atom, which derives from the hydroxyl groups. gested the following classification: Kingdom: Plantae, Order: In general, the higher the number of hydroxyl groups with no steric Myrtales, Family: Lythraceae, Genus: Punica Species: Punica hindrance in the molecule, the greater the antioxidant potential. granatum. It is a small tree or shrub originated in the region en- Besides, based on structural features, phenolics can be grouped compassing Iran to Afghanistan, from where it spread to India and into several sub-classes (e.g., flavonols, phenolic acids, anthocya- the Mediterranean and can grow up to 8 m tall (Andrade et al., nins, proanthocyanidins, hydrolysable tannins), yielding different 2019; Kyriacou et al., 2020). properties (Shahidi and Ambigaipalan, 2015). Currently, there are more than 500 cultivars of pomegranate all The concentration of phenolics changes according to pre-har- over the world (Kandylis and Kokkinomagoulos, 2020), grown in vest (cultivation, harvesting, and weather conditions) and post- Tunisia, Turkey, Spain, Egypt, Morocco, USA, China, India, Ar- harvest (storage and transport) conditions (McCune et al., 2011). gentina, Israel, and South Africa (Singh et al., 2018), as well as Derakhshan et al. (2018) evaluated pomegranate seed, peel and Portugal and Brazil at a less extent. The increased production and pulp from different regions of Iran (Natanz, Shahreza and Doorak), consumption of pomegranate fruit may be related to the mounting and found that PP of Doorak has 50% more AA compared with the evidence of its numerous benefits to human health, especially in other regions. Likewise, the characteristics of the solvent used to what comes to the prevention and/or reduction of risk factors for extract the compounds, as well as the operational conditions used chronic diseases (Akhtar et al., 2015; Yang et al., 2016; Singh et to concentrated fruit-based extracts, have a strong influence on the al., 2018), such as cancer (Hertog et al., 1997), atherosclerosis (Al- nature of the obtained compounds. Jarallah et al., 2013) and Alzheimer’s (Subash et al., 2015; Mor- The main PC found in pomegranate are shown in Table 1. zelle et al., 2016). The pomegranate tree thrives under arid and semi-arid conditions (Robert et al., 2010). In Asia, pomegranate has economic and 3.1.1. Phenolic acids cultural importance due to its high profitability and easy adaptation to various agroclimatic conditions, being commercially cultivated GA and its dimer derivative EA are phenolic acids belonging to in the subtropical, tropical, and temperate regions of the continent the derivatives of benzoic acid class (Shahidi et al., 2019). GA (Bhatia and Asrey, 2019). Similarly to the majority of fruits, the has a low molecular weight, and it is formed by an aromatic ring chemical composition of pomegranate fruit varies according to soil carrying three hydroxyl groups and a carboxylic acid group. It has and climate conditions where the plant is grown, as well as the antioxidant, anticarcinogenic, and antimicrobial activity, as well fruit’s maturation stage at harvest period (Andrade et al., 2019). as protection of cells against oxidative stress, being one of the pri- Pomegranate is a round-shaped edible fruit of 5–12 cm in diam- mary phenolic acids in vegetables and fruits, such as pomegran- eter, with thick skin, usually pink or red. The core of the fruit has a ates. The concentration of GA in pomegranate juice is significantly spongy white tissue that creates spaces full of edible bags, known lower when compared with the peel (Fernandes and Salgado 2016; as arils (Christaki et al., 2011; Kandylis and Kokkinomagoulos, Dludla et al., 2018; Choubey et al., 2018).

Journal of Food Bioactives | www.isnff-jfb.com 11 Pomegranate as a natural source of phenolic antioxidants de Oliveira et al.

Table 1. Phenolic compounds (mg·g−1) of pomegranate pulp and peel Pulp Peel Reference Anthocyanin Delphinidin-3,5-diglucoside 9.43 50.64 Morzelle et al., 2019 cyanidin-3,5-diglucoside 5.57 0.021–23.57 Morzelle et al., 2019, Mehrizi et al., 2017 cyanidin-3-glucoside 0.76 0.007–22.83 Morzelle et al., 2019; Mehrizi et al., 2017 pelargonidin-3,5-diglucoside 0.87 0.005–8.05 Morzelle et al., 2019, Mehrizi et al., 2017 Hydrolyzable tannins Punicalagin A 0.063 1.48–7.5 Morzelle et al., 2019, Rahnemoon et al., 2018 Punicalagin B 0.066 2.38–6.24 Morzelle et al., 2019, Rahnemoon et al., 2018 Phenolic acids Gallic acid 0.07–0.19 0.025–1.01 Morzelle et al., 2019, Li et al., 2016, Song et al., 2016 Ellagic acid 0.54–2.11 0.029–7.07 Li et al., 2016, Song et al., 2016 Chlorogenic acid – 0.004 Song et al., 2016 p-coumaric acid 0.006 0.023 Morzelle et al., 2019 Flavonoids Catechin – 12.8 Ambigaipalan et al., 2016 Epicatechin 0.019 0.010–0.198 Morzelle et al., 2019; Song et al., 2016 Soluble Procyanidins procyandin dimer 42.1 – Ambigaipalan et al., 2016 procyanidin dimer B1 9.09 – Ambigaipalan et al., 2016 procyanidin dimer B2 27.8 – Ambigaipalan et al., 2016 procyanidin dimer B3 37.9 – Ambigaipalan et al., 2016

The biological effect of GA has been previously studied.Liu et Other authors (Bai et al., 2020; Panghal et al., 2020; Trivedi et al. (2020a) investigate the neuroprotective effect of daily orally al., 2020; Abdel-Moneim et al., 2017) also confirmed the antioxi- administered GA. Adult male Sprague Dawley rats (250–350 g) dant potential of GA. were randomly divided into three groups (n = 7/group) treated EA can be found in the free form (less frequent), glycosylated, either with saline solution (control group) or orally administrated or as an ellagitannin. This phenolic acid can be encountered in var- GA at 50 mg/kg or 100 mg/kg via an intragastric needle 1 h prior ious fruits, such as strawberry, red guava, persimmon, raspberry, to an intranigral infusion of lipopolysaccharides (LPS), 4µg/µL, plum, and pomegranate. EA has four rings in its structure, consist- to induce neuroinflammation. The animals continued to receive ing of two phenols with two hydroxyl groups each. Studies have GA daily for another seven days, and after this period, they were demonstrated its anti-inflammatory and AA, which results in the sacrificed by decapitation. Administration of GA (100 mg/kg) prevention of several chronic diseases (Ismail et al., 2012; Shakeri significantly reduced the effects caused by the LPS-infused in et al., 2018; Lima et al., 2019). the substantia nigra of rat brain. More specifically, GA attenu- Allam et al. (2016) verified the protective effect of EA in male ated LPS effects in glial fibrillary acidic protein (a biomarker of MF1 rats (n = 15/group) with induced friction through the subcu- activated astrocytes), ED-1 (a biomarker of activated microglia), taneous application of 0.02 mL of Complete Freund’s Adjuvant inducible NO synthase (a pro-inflammatory enzyme) and inter- (CFA) containing heat-killed Mycobacterium tuberculosis in a leukin 1β (IL-1β) (a pro-inflammatory cytokine). The results also concentration of 5 mg/mL. For this evaluation, the rats were di- showed that GA was capable of inhibiting LPS-induced oxida- vided into four groups: normal control group (treated with 2% di- tive stress and protein conjugation since it attenuated LPS-in- methyl sulfoxide-DMSO), normal treatment group (treated with duced elevation in heme oxygenase-1 level (a redox-regulated EA), control group with arthritis (2% DMSO + CFA) and arthritis protein) and α-synuclein aggregation (a hallmark of central treatment group (CFA + EA). The animals received EA (700 mg/ nervous system neurodegeneration). Furthermore, GA inhibited kg) intraperitoneally, divided into three injections a week (58.33 LPS-induced apoptosis and necroptosis in the nigrostriatal dopa- mg/kg each) for four weeks. The treatment began one week before minergic system of rat brain by avoiding LPS-induced caspase 3 the induction of arthritis by CFA and continued for three weeks activation (a biomarker of programmed cell death) and LPS-in- after the induction of arthritis. At the end of the experiment, the duced increases in receptor-interacting protein kinase (RIPK)-1 animals were sacrificed, and serum levels of IL-1β, interleukin 10 and RIPK-3 levels (biomarkers of necroptosis). Therefore, these (IL-10), interleukin 17 (IL-17), tumor necrosis factor α (TNF-α), outcomes suggest that GA at 100 mg/kg contributed to the reduc- interferon-gama (IFN-γ), and transforming growth factor beta tion in oxidative stress and the inhibition of neuroinflammation. (TGF-β) were measured. EA contributed to the downregulation of

12 Journal of Food Bioactives | www.isnff-jfb.com de Oliveira et al. Pomegranate as a natural source of phenolic antioxidants pro-inflammatory cytokines and upregulation of anti-inflammatory addition, there was an improvement in mitochondrial function due cytokines. Serum levels of IL-1β, TNF-α, and IL-17, which are to high levels of complex proteins I, II, and IV, and an increase in pro-inflammatory cytokines, were reduced. However, serum levels the expression of forkhead box O1 (Fox 01), different from the of IL-10 and IFN-γ significantly increased, while serum levels of P407 group. TGF-β did not significantly alter with EA treatment. Other recent The antioxidant potential of punicalagin has already been re- studies (Chen et al., 2016; Baluchnejadmojarad et al., 2017; Liu et ported by many researchers such as Clementi et al. (2018), Kim et al., 2020b;) have confirmed the antioxidant and anti-inflammatory al. (2017), Pathakoti et al. (2017) and Berköz and Allahverdiyev potential of EA. (2017) among others, which contributes to continuous and increasing interest in this compound. Taking into consideration the reported evidence, supplements containing punicalagin represent a 3.1.2. Hydrolysable tannins promising intervention tool for the prevention and/or treatment of human cardiovascular diseases. Hydrolyzable tannins comprise intermediate to high-molecular- weight phenolics, weighing up to 30,000 Da. Their structure is 3.1.3. Anthocyanins composed of esters of GA (gallotannins) or EA (ellagitannins) with a glucose core, being readily hydrolyzed by acids or enzymes into monomeric products (Shahidi et al., 2019). Their gastrointestinal Anthocyanins are PC belonging to the subgroup of flavonoids. (GI) tract absorption is slow, and they are hydrolyzed into several Their chemical structures are composed of glycosides whose agly- other compounds, such as EA and its derivatives (e.g., urolithins). cones are derived from polyhydroxy or polymethoxy of 2-phe- Hydrolyzable tannins occur naturally in fruits, such as strawber- nylbenzopyrryl salts. They are water-soluble vegetable pigments ries, raspberries, blackberries, and pomegranates. Ellagitanins and responsible for the color blue, purple, and red in fruits and flowers gallotannins are heavily present in seeds, pericarp, and peel of (Bendokas et al., 2019). Due to their ability to reproduce colors pomegranate (Rodrigues et al., 2019; Kandylis and Kokkinoma- and low toxicity, anthocyanins can have several applications in the goulos, 2020). food industry (Shen et al., 2020). Punicalagin belongs to the ellagitannins group and gives the Among the compounds identified as anthocyanins, delphinidin- pomegranate skin its yellow color. This compound can also end up 3-glucoside is present in greater quantity compared to other antho- in pomegranate juice during the fruit’s processing. Studies report cyanins in fruits. Delphinidins are the anthocyanins with highest anti-cancer, anti-inflammatory, and antioxidant properties (Cerdá antioxidant power due to the high number of hydroxyl groups in et al., 2003; Berköz and Allahverdiyev, 2017; Liu et al., 2019) for the B ring (Lee et al., 2015). Cyanidin and delphinidin have anti- this compound. oxidant activity superior to vitamin C (Kim and Lee, 2004). The antioxidant capacity of punicalagin was observed by Chu Over the past two decades, the attention has shifted to the car- and Han (2018) in an in vitro model of Parkinson’s disease (neu- dioprotective effects displayed by anthocyanins. Reis et al. (2016) roblastoma cells SH-SY5Y treated with 6-hydroxydopamine concluded through a systematic review that anthocyanin is a poten- (6-OHDA). At 2 h prior to 6-OHDA treatment, SH-SY5Y cells tially favorable agent in the prevention of cardiovascular diseases, were pre-treated with punicalagin at different concentrations (0, as it inhibits inflammatory processes, endothelial dysfunction, and 50, 100, and 200 µM) or dimethyl sulfoxide (DMSO, 0.1%, v/v). vasodilators production. Its mechanism of action involves antioxi- The following parameters were analyzed: cell viability using the dant activity, scavenging of FR and/or anions, inhibiting xanthine 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bro- oxidase (XO), ion metal chelation, targeting arachidonic acid, and mide assay; cell damage by the presence of lactate dehydroge- nuclear factor κ light-chain enhancer of activated B cells (NF-κB), nase (LDH); intracellular production of Reactive Oxygen Species TNF-α, and adhesion molecules, and suppression of heme oxyga- (ROS) with a 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) nase-1. According to the authors, there are still positive effects on probe; and the superoxide dismutase (SOD) activity. All tested the oxidation of low density lipoprotein (LDL), very low-density concentrations of punicalagin exhibited protective action, con- lipoproteins (VLDL), high-sensitive C-reactive protein (CRP), and trarily to the control treatment (cells treated with DMSO): cell total triacylglycerols, thus improving the clinical status of patients viability increased, released LDH was significantly eliminated, with cardiovascular diseases. intracellular ROS values were restored, and there was a rise in Krga and Milenkovic (2019) reported that anthocyanins and its SOD activity. metabolites seem to regulate different cellular processes involved Liu et al. (2019) observed the antioxidant potential of punica- in the development of cardiovascular diseases, controlling the ac- lagin on the acute hyperlipidemic mouse model (male C57BL6 rats tivity of cell signaling proteins and transcription factors, modu- from 6 to 8 weeks of age). Animals were divided into four groups: lating the expression of genes and miRNA, also suggesting that control group (n = 8), hyperlipidemia group (n = 7, P407 group), the modulation of inflammatory responses and improvements in low dose group (50 mg punicagalin/kg/day), high dose group (200 endothelial function are probably the most important actions of mg of punicagalin/kg/day). The intervention lasted nine days and these compounds. Other evidence, such as the increase in high was performed by oral gavage. On the tenth day, rats were induced density lipoprotein (HDL), cholesterol levels in prehypertensive to have acute hyperlipidemia by applying 0.5 g/kg of poloxamer and non-dyslipidemic men, were observed by Hassellund et al. 407 (P407) via intraperitoneal injection, whereas animals in the (2013). control group received the same volume of sterile saline. The dose Despite the rising evidence based on epidemiological, clinical, of 200 mg/kg was chosen for later trials because it partially nor- and in vitro studies about the benefits of dietary anthocyanins in malized the levels of triacylglycerols and cholesterol in the serum, assisting the prevention of cardiovascular diseases, its mode of as well as ROS in the thoracic aorta compared to the P407 group, action is still not entirely clear. Thereupon, further well-designed which had high levels of these compounds. The structure of the research is essential to thoroughly explain the molecular mecha- artery endothelial cells was checked by H&E staining. The group nisms through which anthocyanins ameliorate cardiovascular dis- treated with punicalagin showed positive results, with no rupture eases. Also, research is needed in order to possibilitate the use of of the elastic membrane, thus resulting in a normal structure. In these compounds in the clinical management of such conditions,

Journal of Food Bioactives | www.isnff-jfb.com 13 Pomegranate as a natural source of phenolic antioxidants de Oliveira et al.

Table 2. Phenolic compounds in pomegranate based-foods Pomegrante-based products GA EA Punicalagin Wine (mg/L) 80.4–108.8 – – (Akalin et al., 2018) Fermented milk (μg/g) PP 150 mg/La 152.4–167 9.62–15.4 111.2–162.5 (Al-Hindi and El Ghani., 2020) PP 300 mg/La 182.1–195.7 13.2–19.6 190.8–211.6 Juice Conventional Organic (mg/L) – Tr 201 (Cano-Lamadrid et al., 2016) – Tr 104 Juice commercial (mg/L) 1.1–10.72 2.1–37.9 1259.8 (Gil et al., 2000; Fischer et al., 2011; Özgüvem et al., 2019) Juice commercial (mg/g) 0.05–0.14 0.02 0.01–0.70 (Hmid et al., 2017) Juice concentrate (mg/L) – 172.8 1,353.1 (Gil et al., 2000) Juice with arils (mg/L) 12.42–88.51 8.7–95.02 22.8–25.5 (Gil et al., 2000; Hmid et at., 2017) aSuplemented with pomegranate peel extract; bImpure montmorillonite and extract of pomegranate fruit waste (%). GA: Gallic acid; EA: Ellagic acid. in addition to assessing a suitable effective dose, duration of treat- alternative to improve quality, limit the growth of undesirable ment, and lasting effects. microorganisms, and decrease lipid oxidation (Derakhshan et al., 2018). In several studies, pomegranate extracts and juices have demonstrated superior antioxidant potential than foods popular for 3.2. Antioxidant activity of pomegranate juice, pulp, and peel their antioxidant properties, with values up to three times more antioxidant activity than red wine and green tea (Gil et al., 2000; Pomegranate juice has more than 30 nutrients and 79 bioactive Johanningsmeier and Harris, 2011). In vitro assays of antioxidant compounds, mainly phenolics, such as flavonoids, phenolic acids, activity of pomegranate pulp, peel, and juice are shown in Table 3. and hydrolyzable tannins (Ambigaipalan et al., 2016; Khomich et The processing to obtain industrialized pomegranate juice af- al., 2019), as can be observed in Table 2. Antioxidant phenolics of fects the product’s phenolic composition, especially when com- pomegranate-based foods are shown in Table 2. pared with homemade juice, which will consequently impact the Due to its high antioxidant activity, pomegranate has been ap- antioxidant activity and availability of PC in this source. Dżugan plied in different types of food in order to avoid loss of bioactive et al. (2018) characterized and compared industrialized and home- components. Moreover, because of the growing interest in using made pomegranate juices, suggesting that industrialized juices natural preservatives in foods, pomegranate could be a feasible have an antioxidant capacity superior to that found in homemade

Table 3. In vitro assays of antioxidant activity of pomegranate peel, pulp, seed and juice In vitro assay Extract Origin Range Reference Peel β-carotene bleaching test ethanol Iran 45–58 Derakhshan et al., 2018 (Antioxidant activity %) Seed β-carotene bleaching test ethanol Iran 34–54 Derakhshan et al., 2018 (Antioxidant activity %) Juice β-carotene bleaching test ethanol Iran 9–10 Derakhshan et al., 2018 (Antioxidant activity %) Juicea FRAP (mmol TE/L of juice) Aqueous India 22.09–25.68 Dżugan et al., 2018 Juiceb FRAP (mmol TE/L of juice) Aqueous Turkey 57.17 Dżugan et al., 2018 Israel Azerbaijan 30.86 Russia 70.33 Azerbaijan 8.23 47.96 Extractc Scavenger Effect on Superoxide ethanol n.i. 95 Sorrenti et al., 2019 Anion (% of inhibition) Peel ORAC (μmol TE/g DW) ethanol United States 7423.0 Morzelle et al., 2019 Pulp ORAC (μmol TE/g DW) ethanol United States 323.8 Morzelle et al., 2019 aHomemade juice; bCommercial juice; c0.028 mg·mL−1. n.i.: not identified.

14 Journal of Food Bioactives | www.isnff-jfb.com de Oliveira et al. Pomegranate as a natural source of phenolic antioxidants

Table 4. Use of pomegranate as a natural antioxidant in foods Reference Type of food Results Ahmed et al., 2017 broiler meat improved nutritional quality, fatty acid profile, and shelf life Ahmed et al., 2015 broiler meat improved fatty acid profile and reduced lipid oxidation Berizi et al., 2018 rainbow trout prevented the oxidation of fats and proteins and antimicrobial activity Devatkal et al., 2010 goat meat patties reduced lipid oxidation (TBARS) Devatkal et al., 2011 salted chicken patties reduced lipid oxidation (TBARS) Dua et al., 2016 fat rich meat lower TBARS values Gomalkani et al., 2020 Linseed oil improved oxidative stability Ismail et al., 2019 Minced Shrimps inhibited TBARS production during 28 days of refrigerated storage Martínez et al., 2019 Fish Patties delayed lipid oxidation, measured as volatile compounds, and the microbiological spoilage Morsy et al., 2018 meatballs Reduced contents of peroxide, TBARS, and total volatile base nitrogen (TVB-N) Natalello et al., 2020 lamb meat reduced lipid oxidation, greater concentration of vitamin E and polyunsaturated fatty acids Naveena et al., 2008b chicken patties inhibited lipid oxidation Qin et al., 2013 Pork Meat reduced lipid oxidation juices. According to the authors, this outcome can be explained by pid oxidation, measured by the formation of volatile compounds the fact that usually, the fruit is used entirely by the industry, in- (1-penten-3-ol, hexanal, 2-nonanone, 1,6-octadien-3-ol, nonanal cluding its the peels, which have higher phenolic content than the and pentadecane). pulp. On the other hand, in handmade juice production, the peels Studies conducted by Naveena et al. (2008a, 2008b) showed are usually discarded. Despite that, the content of anthocyanins in that PP (5 to 20 mg tannic acid equivalents/100 g meat) was able homemade juices was higher than their industrialized counterparts. to inhibit lipid oxidation of chicken patties (cooked to an inter- The scientific community has studied the composition of agro- nal temperature of 80 °C, and stored in low-density polyethylene industrial by-products in order to ensure that they are properly pouches for 15 days at 4 °C) to a much greater extent than syn- used, avoiding an excessive accumulation of waste in the environ- thetic antioxidant (BHT). Moreover, PP did not have a significant ment. In addition, a large proportion of consumers demand natural impact on the overall sensory attributes of the finished product. ingredients due to their health claims, especially the ones obtained In another study (Ismail et al., 2019), the effect of hydro-al- from sustainable sources (Melo et al., 2015; Gómez et al., 2016). It coholic extracts of PP on the control of lipid oxidation of shrimp should be pointed out that agro-industrial waste has considerable meat was investigated. Minced shrimp meat was treated with the amounts of bioactive substances recognized for their health-pro- extract at different concentrations (0,5%, 1.0%, and 2.0%), and moting properties and technological application as antioxidants, thiobarbituric reactive substances (TBARS) were measured from potentially prebiotic ingredients, or even as food dyes (Vásquez- day 0 to day 28 of storage at 4 °C. The rate of TBARS production Olivo et al., 2018). was significantly lower in samples marinated with 1.0% and 2.0% Pomegranate by-products have the potential to be reused. Ac- of the extract in comparison to samples treated with a synthetic cording to the literature, 37 kg of waste is generated for every 100 antioxidant (BHT, 0.05%). Moreover, a slight increase (10%) in kg of processed pomegranate. Of this total, 23 kg corresponds to TBARS in shrimp samples treated with 2.0% extracts of PP from the peel and 14 kg to the seeds. From these residues, 180 g of mi- day 0 to day 28 of storage was identified. Thereupon, hydro-alco- croencapsulated phenolics can be recovered from the peels (Gul- holic extract of PP was found to protect shrimp meat against lipid lon et al., 2015). peroxidation. PP is widely known for its phytochemicals levels, with com- The use of pomegranate as a natural antioxidant in foods is pounds that carry medicinal and nutritional significance, as well as shown in Table 4. a higher antioxidant activity than pulp and seeds (Morzelle et al., Pomegranate can increase the antioxidant activity of juice, tea, 2019). Due to its high antioxidant power, pomegranate extract is an and other beverages. Lyophilized PPs were added to tomato and excellent alternative to be used as a food preservative, contributing orange juices with strawberry at different concentrations (0.5, 1.0, to extend shelf life. Lipid oxidation is among the major causes of 1.5, and 2.0%). Orange and tomato juice samples enriched with deterioration in meat, ultimately causing undesirable sensory, nu- higher dried extract concentration (2.0%) showed an increase of tritional, and physicochemical changes (Horbańczuk et al., 2019). over 30 and 25 times, respectively, in antioxidant activity as com- With that being said, and taking into consideration the cost-effec- pared to the juice control (without extract). However, although tive aspect, studies concerning the use of PP as natural antioxidants both flavors of enriched juices had high levels of antioxidants, have grown over the years (Smaoui et al., 2020). orange juice with a concentration of 2% of the dry extract was Martínez et al. (2019) carried out in vitro tests (FRAP, ORAC) rejected in the sensory analysis due to the astringent flavor of the to measure the antioxidant activity of PP and evaluate the ability PP (Salgado et al., 2012). of the extracts (concentration of 200 ppm) to prevent oxidation González-Molina et al. (2009) produced a polyphenol-rich of fish burgers stored for 11 days at 4 °C. PP extract delayed li- drink based on lemon and pomegranate juice in different propor-

Journal of Food Bioactives | www.isnff-jfb.com 15 Pomegranate as a natural source of phenolic antioxidants de Oliveira et al. tions (25%, 50%, and 75% for both juices) and the results suggest- upon pomegranate consumption. Casedas et al. (2020) evalu- ed that the formulation of 75% pomegranate juice and 25% lemon ated the cytoprotective effect of this metabolite on neuronal cells juice presents the potential for the development of new functional (Neuro-2a) subjected to oxidative stress through treatment with drinks, emphasized by its high antioxidant capacity. However, a hydrogen peroxide (H2O2), as well as its direct antioxidant activ- sensory analysis was not performed in order to detect the accept- ity and inhibitory properties against oxidases. Cells treated with ability of this product. The phenolic-driven sensory changes in urolithin A (0.5 and 1 µM) and H2O2 showed a more effective re- functional foods has been discussed by de Camargo and Schwem- sponse to oxidative stress than the control. Mitochondrial activity ber (2019). (MTT assay), redox state (ROS formation and lipid peroxidation), Despite the feasibility of using PP as a natural antioxidant, ex- and the activity of antioxidant enzymes (CAT, SOD, GPx) were traction methods should be improved, aiming at sustainable and significantly ameliorated. Additionally, urolithin A enhanced the cost-effective approaches. Similarly, their biological activities expression of cytoprotective peroxiredoxins 1 and 3. Finally, the should always be examined in comparison with a synthetic anti- inhibition of oxidizing enzymes, such as monoamine oxidase A oxidant, as well as their efficacy to prevent lipid oxidation in dif- and tyrosinase, was also detected in a dose-dependent manner. ferent foods. Also, in vitro assays and pre-clinical and clinical tri- Fazio et al. (2018) treated cell culture of murine fibroblasts als should be carried out to address their potential toxic effects on (3T3–L1) and human embryonic renal epithelium (Hek-293) with human health in order to mitigate risks to the consumers (Andrade pomegranate acetonic and methanolic extracts (15, 30, 60 and 120 et al., 2019). μg/mL). The cells had previously been induced to oxidative stress with menadione. As a result, both extracts showed potential ROS scavenging activity, in addition to being able to contribute to the 4. Health effects of pomegranate phenolics antitumor function.

4.1. Cellular antioxidant activity 4.2. Type 2 diabetes

Oxygen plays a vital role in the human organism, being part of Studies have indicated that oxidative stress plays an essential role metabolic processes. Under normal circumstances, a small per- in the pathogenesis of type 2 diabetes. Overload of glucose and ox- centage of the electrons passing through the electron transport idative phosphorylation enhances the generation of ROS through chain leaks out of the mitochondria, combines with molecular various tissues and metabolic processes in the mitochondria (Bur- oxygen, and forms ROS (Valko et al., 2007). These radical and gos-Moron et al., 2019). non-radical chemical species can be harmful to the organism if Katz et al. (2007) discussed the relationship between pomegran- produced in excess, causing cellular damage. In order to prevent ate extracts or juice and type 2 diabetes. The authors stated that the this, cells contain endogenous antioxidant defenses, such as the mechanism was not clear, with the studies showing that the anti- enzymes SOD, glutathione peroxidase (GPx), and catalase (CAT) oxidant activity of pomegranate may be involved in the process. (Niederländer et al., 2008). However, Ambigaipalan et al. (2016,) demonstrated that phenolics External factors (e.g., diseases, drugs, pollution, poor eating from pomegranate inhibit the activity of alpha-glucosidase, a car- habits) can contribute to increased production of ROS, leading to bohydrate-hydrolysing enzyme present in the small intestinal brush the biological condition known as oxidative stress. Besides, inter- border that participates in the breakdown of complex carbohydrates nal factors such as enzymes from the P450 complex, XO, and nico- and enables their absorption. Other studies have shown the effects tinamide adenine dinucleotide (NADPH) are sources of oxidative of PP and other fractions on metabolic variables associated with the stress (Sosa et al., 2013). pathologic markers of type 2 diabetes (Medjakovic and Jungbauer, The oxidative stress condition caused by the prevalence of ROS 2013; Banihani et al., 2013; Chukwuma et al., 2020). and reactive nitrogen species (RNS) is detrimental to cellular func- Pomegranate affects type 2 diabetes by reducing oxidative tions since they can damage proteins, lipids, and genetic material stress and lipid peroxidation. This reduction may occur by directly (DNA and RNA) (Brigelius-Flohe, 1999; Islam, 2016). Several scavenging FR, increasing the activity of antioxidant enzymes, degenerative and cardiovascular diseases, cancer, diabetes, and a metal chelation, reducing resistin formation, influencing NO pro- decline in the immune system involve cellular damage caused by duction and modulating selected transcriptional factors, such as oxidative stress. This association shows that the incidence of these NF-κB (Katz et al., 2007; Makino-Wakagi et al., 2012; Banihani ailments is closely related to the prevalence of reactive substances et al., 2013). Also, pomegranate enhances peroxisome proliferator- in the body (Rauter et al., 2012; Sosa et al., 2013; Islam, 2016) activated receptor-gamma (PPAR-γ), a transcriptional factor key to There is mounting evidence about the function of dietary an- carbohydrate metabolism (Huang et al., 2005). tioxidants in human health. Antioxidant compounds from fruits, Polar and non-polar extracts of PP, the fruit’s edible parts, arils, such as pomegranate, are able to neutralize FR present in the and seeds reduced lipid peroxidation and modulated antioxidant body, thus helping to protect cells and tissues. Consequently, this status of diabetic and oxidative stress-induced rats. Besides, pome- mechanism of action contributes to the prevention of aging and the granate juice, extracts, or their polyphenols reduce blood glucose, increased incidence of chronic non-communicable diseases (Mc- increase glycogen on liver and insulin secretion, modulate insulin Cune et al., 2011; Pereira et al., 2012). terminating factors, improve lipid profile, and glucose tolerance Numerous in vitro and in vivo studies have been carried out (Banihani et al., 2013) . to understand the mechanisms through which pomegranate could Huang et al. (2005) showed that pomegranate flower extracts, improve oxidative stress and prevent chronic non-communicable with a high concentration of GA, enhanced PPAR-γ mRNA in hu- diseases. Most studies were done with the bioactive compounds man THP-1-differentiated macrophage cells. alone or together with the food matrix. However, studies must be Chukwuma et al. (2020) described that acetonic extract of PP conducted to show the biological effect, focusing on the mecha- has 3.5 times greater α-amylase inhibitory activity than aqueous nism of action by biotransformed forms (de Camargo et al., 2018). extracts. The inhibitory effect of α-amylase promoted by PP was Urolithin A is a metabolite generated from ellagitannins and EA associated with the presence of ferulic acid, known for its phar-

16 Journal of Food Bioactives | www.isnff-jfb.com de Oliveira et al. Pomegranate as a natural source of phenolic antioxidants macological potential of inhibiting this metabolic enzyme. The controlled double-blind study to investigate the effect of ingesting authors also found that the acetone extract of PP possesses com- pomegranate juice three times a week for one year on oxidative pounds with antidiabetic and antioxidant effects, with minimal stress and inflammatory processes of hemodialysis patients. Pa- toxicity. tients were randomly assigned to treatment group (n = 66) receiv- Clinical trials have tried to establish a connection between ing 100 mLof pomegranate juice, or control group (n = 35) receiv- pomegranate consumption and reduced diabetes risk, mainly using ing 100 mLof placebo juice (similar to pomegranate juice in color pomegranate juice. Pomegranate juice (50 mL/d, 3 months) has and taste). The primary outcomes were the levels of oxidative been reported to oxidize a N-linoleoyl tyrosine, synthetic oxidative stress and inflammation biomarkers, whereas the secondary out- stress marker, on diabetic patients (Szuchman et al., 2008). comes were hospitalizations due to infections and the progression Studies with pomegranate extracts, polar or non-polar, can of the atherosclerotic process. The results indicated that the intake give the best results for those in which pomegranate juice is used of pomegranate juice resulted in a significant reduction of protein (Jelodar et al., 2007). oxidation, lipid oxidation, and inflammatory biomarker levels. Additionally, the intake of pomegranate juice resulted in a significantly lower incidence of hospitalization due to infections. The 4.3. Cardiovascular diseases beneficial effects lasted up to three months postintervention. Fur- thermore, 25% of patients receiving pomegranate juice improved, Cardiovascular diseases are a major cause of death worldwide, and only 5% progressed in the atherosclerotic process. Conversely, with oxidative stress playing a key role in the development of these more than 50% of patients in the group receiving placebo showed ailments. Plant-based bioactive compounds have strong antioxi- progression, and none showed any improvement in the atheroscle- dant and anti-inflammatory properties, exhibiting cardioprotective rotic process. The authors concluded that the ingestion of pome- effects. granate juice for a prolonged period improved non-traditional risk Evidence suggests that pomegranate may be included in a heart- factors for cardiovascular disease, attenuated the progression of healthy diet. Cardioprotective effects of pomegranate polyphenols the atherosclerotic process, strengthened innate immunity, and, include decreased serum cholesterol, reduced lipid peroxidation therefore, may contribute to reducing morbidity among patients levels, reduced intima-media thickness, diminished levels of NO, undergoing hemodialysis. reduced blood pressure and angiotensin-converting enzyme (ACE) Another study demonstrated in a crossover trial that the con- activity, inhibition of LDL oxidation, reduced TNF-α, IL-6 and sumption of 100 mL pomegranate juice immediately after hemodi- CRP (Basu and Penugonda, 2009; Wang et al., 2020). alysis three times a week during eight weeks promoted significant Atherosclerosis is a degenerative artery disease, where the reduced levels of oxidative stress of patients (n = 41), which was role of oxidative stress on its initiation and progression is well measured by the total antioxidant capacity and malondialdehyde established. Pomegranate reduced atherosclerotic lesion areas in levels (Boldaji et al., 2020). immune-deficient mice (Basu and Penugonda, 2009). Aviram et Although the beneficial effects of consuming pomegranate juice al (2008) evaluated in vivo and in vitro antiatherogenic effects of are observed in patients, studies indicate that supplementation with phenolics from PPs, arils, seed, and flowers. Thein vivo study was isolated polyphenolic compounds extracted from the fruit would conducted with atherosclerotic apolipoprotein E-deficient mice, not have the same beneficial action. Patients undergoing hemo- which consumed pomegranate extracts for three months. Pome- dialysis consumed a food supplement with 1,000 mg of purified granate phenolics reduced 70% atherosclerotic lesion area (except pomegranate polyphenolic extract for six months, and no signifi- for pomegranate seed), 15% oxidized LDL, and 53% peroxide cant effects were observed on oxidative stress markers (Wu et al., content. Pomegranate, especially pomegranate juice and flowers, 2015). reduced serum lipids, and glucose levels. Punicalagin is able to activate FoxO1 (forkhead box O1), the 4.5. Neurodegenerative disease main regulator of enzyme antioxidant defense, and through this mechanism, punicalagin can prevent vascular dysfunction and promote mitochondrial biogenesis and increased cellular paraoxonase Excessive production of ROS and nitrogen species (RNS) has also 2 (PON2) activity (Liu et al., 2019). been linked to neurodegenerative diseases, including Alzheimer’s Furthermore, pomegranate flower extract decreased cardiac fi- diseases (AD), Amyotrophic lateral sclerosis, Huntington’s dis- brosis in rats through the modulation of the NF-κB pathway and ease, Multiple sclerosis, and Parkinson’s diseases (Islam, 2016). cardiac endothelin-1, a protein involved in blood vessel constric- Functional foods to prevent and/or treat neurodegenerative diseas- tion and increased blood pressure (Huang et al., 2005). es represent a promising field of study currently gaining popularity (Morzelle et al., 2016). Loren et al. (2005) evaluated if neonatal protection against hy- 4.4. Chronic kidney disease poxic-ischemic encephalopathy could be achieved by supplement- ing the maternal diet with pomegranate juice. Hypoxic-ischemic Increases in both systemic inflammation and oxidative stress are encephalopathy is an important cause of morbidity and mortality, established as non-traditional critical elements involved in the im- requiring effective therapies for prevention and treatment. Results mune dysregulation of patients undergoing hemodialysis. Patients have shown that pomegranate juice in the maternal diet resulted suffering from chronic kidney disease (CKD) have low levels of in a decreased loss of brain tissue (>60%) and inhibition of cas- antioxidants, such as glutathione and superoxide, and high levels pase-3. Pomegranate juice, when included in maternal diet pos- of prooxidants substances circulating in their bodies. sibly has a neuroprotective effect on the neonatal brain. Given the connection between oxidative stress and many he- The neurodegenerative disease has an accumulation of specific modialysis comorbidities, antioxidant consumption could be a proteins such as PrPSc príons in Creutzfeldt Jacob’s disease and low-cost non-drug strategy, and probably an effective therapy to β-amyloid in AD, and share common characteristics such as neu- attenuate the decline of antioxidant defense in patients on dialysis. ronal death and oxidative damage. Mizhari et al. (2014) evaluated Shema-didi et al. (2012) conducted a randomized placebo- whether the reduction in oxidation through the consumption of

Journal of Food Bioactives | www.isnff-jfb.com 17 Pomegranate as a natural source of phenolic antioxidants de Oliveira et al. natural antioxidants from pomegranate seeds alters the manifesta- duced by βA1–42 (Ansari et al., 2009). tion of Creutzfeldt Jacob’s disease in transgenic mice. The pome- Acetylcholinesterase is a key enzyme in Alzheimer’s disease. granate seed oil has a natural antioxidant compound—a punic High levels of acetylcholinesterase, and consequently low levels acid—a polyunsaturated acid and significantly delayed the onset of the neurotransmitter acetylcholine, are commonly found in of the disease when administered to asymptomatic animals and patients with AD. Drugs used to attenuate the symptoms of AD postponed the worsening of the problem in animals induced to the are anticholinesterasics. Morzelle et al. (2019) evaluated the an- disease. The treatment reduced lipid oxidation and neuronal loss, ticholinesterase effect of PP and methanolic pulp extracts. Phe- which indicates a strong neuroprotective effect. nolics from PP showed inhibition of acetylcholinesterase, which Bookheimer et al. (2013) showed the effect of pomegranate was dependent on the phenolic concentration. PP extract (3 mg/ juice (eight ounces for four weeks) in elderly individuals (n = mL) showed 58% of inhibition. Increasing the options of natural 34) with age-related memory loss. After four weeks, pomegran- compounds bearing acetylcholinesterase could be helpful for the ate juice promoted an increase in plasma Trolox-equivalent an- management of AD. tioxidant capacity (TEAC) and a significant improvement in the Morzelle et al. (2016) demonstrated the effect of PP extract Buschke selective reminding test. (PPE) on biomarkers of oxidative stress (lipid peroxidation and Braidy et al. (2013) evaluated the neuroprotective effect of SOD activity) in a mouse model of neurodegeneration. Male pomegranate on an in vitro model for Parkinson’s disease. Results C57Bl/6 mice were chronically infused for 35 days with amyloid-β indicated that the juice extracted from the pomegranate pulp had peptide 1–42 (Aβ) using mini-osmotic pumps and treated with interesting properties in order to delay age-related neurodegenera- PPE (800 mg/kg/day). The levels of malondialdehyde (MDA) and tion. SOD activity were evaluated on the liver and brain, respectively. Studies have shown pomegranate as neuroprotective towards Lipid peroxidation, probably caused by the generation of FR dur- Alzheimer’s disease (Choi et al., 2001; Hartman et al., 2006; Ro- ing the Aβ deposit, has been linked to AD, and these oxidative janathammanee et al., 2013; Subash et al., 2015). Pomegranate events can lead to neuronal death, contributing to cognitive decline phenolics, mainly punicalagin, may be responsible for this neu- in patients with AD. PP promoted a reduction of lipid peroxida- roprotective effect (Rojanathammanee et al., 2013; Braidy et al., tion in the liver but did not increase the SOD activity in the brain. 2013). Such data suggest that the antioxidant effect of the extract is inde- Injuries caused by FR are precedents of amyloid deposition pendent of the endogenous antioxidant capacity. The intake of PP in the brain, which raises the hypothesis that possibly such stress extract could contribute to neuroprotection as an antioxidant and would be the start for amyloidogenesis (Nunomura et al., 2006). by stabilizing or reverting injury caused by oxidative stress. This Besides, FR are closely related to synaptic dysfunction, cascades antioxidant effect is also related to the high content of PC (mainly of apoptosis, tau protein hyperphosphorylation, which causes im- punicalagin and GA) in the extract (Morzelle et al., 2019). The pairment of cognitive ability (Mattson, 2004). proposed mechanism for antioxidant activity is the capacity of the Transgenic mice model of Alzheimer’s disease supplemented extract to promote hydroxyl radical scavenging. with nanodroplet formulation of pomegranate seed oil showed decreased lipid oxidation and neuronal loss (Mizrahi et al., 2014). The consumption of pomegranate juice promoted benefits, be- 5. Conclusion havioral and neurological, in transgenic mice (APPSW/Tg2576) (Hartman et al., 2006). Results showed that the consumption of 5 Previous studies have shown that pomegranate is a functional fruit mL/day of pomegranate juice promoted a more than 50% decrease with a myriad of benefits on chronic non-communicable diseases, of βA on the hippocampus. such as type 2 diabetes, cardiovascular disease, CKD, and neuro- Neuroprotective potential of pomegranate pulp extract (800 mg/ degenerative disease. Pomegranate pulp, peel, and seed extracts kg/dia for 30 days) on mice after acute infusion with Amyloid-β represent an excellent alternative to be used in the industry as a Peptide were analyzed by Choi et al. (2011). Rojanathammanee preservative, contributing to extend food’s shelf life. The effect of et al. (2013) analyzed the effect of consumption of pomegranate pomegranate on foods and the biological benefits were associated pulp extract for three months in transgenic mice (APP/PS1) mod- with PC, especially anthocyanins and hydrolyzable tannins. How- els of AD. The consumption of pomegranate pulp extract had an ever, the mechanism behind the action of phenolic metabolites anti-inflammatory effect on the brain that could possibly slow the from pomegranate and its by-products deserves further investiga- progression of AD. The PC present in the extract were tested in tion. isolation in cell cultures, and the results showed that the active compounds were punicalagin and ellagic acid. Subash et al. (2015) studied whether transgenic AD rats (APP- References sw / Tg2576) supplemented with pomegranate for 15 months have an improvement in memory, anxiety, and learning. In the experi- Abdel-Moneim, A., Yousef, A.I., El-Twab, S.M.A., Reheim, E.S.A., and Ash- ment, animals with four months of age received a diet containing our, M.B. (2017). Gallic acid and p-coumaric acid attenuate type 2 4% pomegranate inserted directly into the feed (pellets) until they diabetes-induced neurodegeneration in rats. Metab. Brain Dis. 32: were 19 months old. The results suggest that pomegranate dietary 1279–1286. supplementation slowed the progression of cognitive and behavio- Ahmed, S.T., Islam, M.M., Bostami, A.B.M.R., Mun, H., Kim, Y., and Yang, C. ral changes in AD. (2015). Meat composition, fatty acid profile and oxidative stability of meat from broilers supplemented with pomegranate (Punica grana- EA significantly decreased neurotoxicity induced by βA1–42 in a human cell line (SH-SY5Y) (Feng et al., 2009). Moreover, querce- tum L.) by-products. Food Chem. 188: 481–488. Ahmed, S.T., Ko, S., and Yang, C. (2017). Improving the nutritional qual- tin 3-O-glucuronide, also found in pomegranate, significantly re- ity and shelf life of broiler meat by feeding diets supplemented with duced the production of βA peptide in primary neuronal cultures fermented pomegranate (Punica granatum L.) byproducts. Br. Poult. generated from an AD model animal (Ho et al., 2013). Another Sci. 58(6): 694–703. study indicated that pre-treatment of primary cultures of the hip- Akalin, A.C., Bayram, M., and Anli, R.E. (2018). Antioxidant phenolic com- pocampus with quercetin significantly attenuated cytotoxicity in- pounds of pomegranate wines produced by different maceration

18 Journal of Food Bioactives | www.isnff-jfb.com de Oliveira et al. Pomegranate as a natural source of phenolic antioxidants

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Review J. Food Bioact. 2020;9:23–35

Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? A critical review focusing on bioactive molecule screening assays in in vitro and in vivo models

Fereidoon Shahidi* and JuDong Yeo

Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9 *Corresponding author: Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9. Tel: +1 709 864 8552; E-mail: [email protected] DOI: 10.31665/JFB.2020.9215 Received: March 18, 2020; Revised received & accepted: March 31, 2020 Citation: Shahidi, F., and Yeo, J.D. (2020). Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? A critical review focusing on bioactive molecule screening assays in in vitro and in vivo models. J. Food Bioact. 9: 23–35.

Abstract

Increasing evidence has proven the potent antioxidative effectiveness of bioactives in natural products for preventing/suppressing chronic diseases. In this connection, the development of efficient methods that are suitable to screen bioactivesin vitro and in vivo tests has taken place. Thus, a variety of assays have been used in the extraction of bioactives, testing of their antioxidant potential in bothin vitro and in vivo model, and evaluating lipid oxidation are comprehensively discussed here. This review mainly focuses on the principle and the use of individual assays in both in vitro and in vivo models. Given that induvial assays have pros and cons due to the discrepancy in the reaction environment and applied biological system, application to the various assays in order to complement the drawbacks of each assay is highly recommended to obtain reasonable information from experimental trials.

Keywords: Bioactives; Antioxidant activity; Extraction; Lipid oxidation; Cell line; In vivo; Animal model.

1. Introduction to measure antioxidant potential of bioactive compounds including ferrous-ion chelating activity (Carter, 1971), lipid peroxidation Antioxidants play a crucial role in both food and biological sys- inhibition assay (Daker et al., 2008), deoxyribose assay (Chen et tems by retarding oxidative processes and providing health-pro- al., 2005), and superoxide dismutase mimetic activity (Naithani moting activities. Different antioxidant mechanisms are involved et al., 2006), among others. In addition, Folin-Ciocalteu’s phenol in preventing lipid oxidation such as radical scavenging potential reagent, reducing ability, radical scavenging potential using 2, through hydrogen atom transfer (HAT) and single electron transfer 2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis(3-eth- (SET) as well as metal chelation. These mechanisms contribute ylbenzothiazoline-6-sulfonic acid (ABTS) have been widely em- to the stabilization of free radicals generated from lipid oxidation ployed to determine antioxidant potential of bioactive compounds as well as preventing the initiation of autoxidation by chelating (Shahidi and Zhong, 2015). In addition, cell-based assays have prooxidative transition metal ions. In biological systems, anti- also been developed to evaluate antioxidant potential through cel- oxidants are responsible for inhibiting chronic diseases including lular antioxidant activity (CAA), which was first reported byWang cancer, inflammatory diseases, diabetes, cardiovascular and neuro- and Joseph (1999), and is now widely used to measure antioxidant degenerative disorders (Shahidi and Yeo, 2018). Moreover, bioac- activity and bioavailability of bioactives from natural products by tives in foods exhibit strong inhibitory effects against the incidence considering cellular uptake and metabolism. of Parkinson’s and Alzheimer’s diseases (Shahidi and Yeo, 2018). Recently, the above colorimetric assays have been criticized by A variety of analytical methods have been developed and used some researchers due to the lack of standardized procedures and

Copyright: © 2020 International Society for Nutraceuticals and Functional Foods. 23 All rights reserved. Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? Shahidi et al. the weak correlation between antioxidant screening assays and (Nieto et al., 2010). PLE treatment is carried out in a stainless steel biological evaluation methods. However, given that individual as- extraction cell where the solid sample is placed in which a wide says have pros and cons due to the discrepancies in the reaction range of pressure (500–3,000 psi) and temperature (40–200 °C) medium and applied biological systems, the use of various assays are applied to optimize the extraction rate of desired compounds in to address the drawbacks of each would be necessary to acquire samples, leading to the decreased extraction time and the amount realistic and trustworthy data from experimental trials. of solvent required (Garcia-Salas et al., 2010). The proper combi- In this contribution, detailed principles and practical uses of nation of high pressure and temperatures enables the use of a lesser various methodologies including colorimetric antioxidant screen- amount of solvent for the extraction of bioactive molecules. The ing assays, cell lines, and animal tests as well as lipid oxidation remaining solvent beyond their normal boiling point at the opti- measurement assays are thoroughly discussed. mized temperature and pressure not only increases the solubility of analyte and its mass transfer rate, but also decreases the viscosity and the surface tension of solvents (Ibañez et al., 2012). 2. Methodologies for the extraction of bioactives from natural PLE has been widely used to extract bioactive compounds in sources food matrices to improve their extraction efficiency.Erdogan et al. (2011) optimized the extraction condition of PLE to obtain bioactive compounds such as gallocatechin, catechin, epicatechin gal- 2.1. Pulsed-electric field extraction (PEF) late, caffeic acid, chlorogenic acid, and myricetin from Anatolia propolis. Setyaningsih et al. (2016) attempted a number of com- Pulsed electric field (PEF), a non-thermal treatment, uses short puls- binations including solvent composition, extraction temperature, es of electricity to improve the extraction of bioactive compounds in pressure, and static extraction time, among others, to find optimum foods. The basic principle for improvement in the extraction yield combination for the extraction of bioactive compound in rice (Ory- using PEF is to accelerate the disintegration of the cell membranes za sativa) grains, and they found that the extraction temperature, in food by using an electric field that passes through them (Azmir et solvent and static extraction time were the most important factors al., 2013). Briefly, at a critical value of transmembrane potential (>1 in the extraction of bioactives using PLE treatment. The applica- V), repulsion between charge carrying molecules occurs due to the tion of PLE was also extended to the extraction of isoflavones. dipole nature of membrane molecules, which leads to the formation Rostagno et al. (2004) optimized the extraction condition of PLE of pores and increases the permeability of solvent into the intracellu- to isolate isoflavones in soybean. The best condition of PLE was lar space of plant and animal cells (Bryant and Wolfe, 1987). The ef- 0.1 g of sample, 100 °C, and three (7 min) static extraction cy- ficiency of PEF depends on many parameters such as field strength, cles. They also reported that malonyl glucoside was degraded at energy input, pulse number, temperature and physical and chemical temperatures higher than 100 °C. Luthria (2008) emphasized that properties of the samples (Heinz et al., 2003). temperature, pressure, solid-to-solvent ratio, and particle size were PEF has been extensively used to enhance the efficiency of ex- the significant parameters in the use of PLE for the extraction of traction yield of foods. Guderjan et al. (2005) reported that the phenolics from parsley (Petroselinum crispum) flakes. application of PEF increased the extraction yield of phytosterol from maize and genistein and daidzein from soybeans by up to 2.3. Microwave assisted extraction (MAE) 32.4 and 21.0%, respectively. The PEF treatment also increased the extraction rate of phenolics such as anthocyanins. For example, Corralesa et al. (2008) found that PEF treatment provided useful Microwave is a wavelength range from 1 mm to 1 m along with means to extract anthocyanin monoglucosides in grape by-prod- frequencies from 300 MHz to 300 GHz. This long-wavelength ucts. This treatment also improved the extraction of polyphenols consists of two oscillating fields such as electric field and magnetic and anthocyanins in Merlot skin (Delsart et al., 2012). Liu et al. field, which are perpendicular (Azmir et al., 2013). The generation (2018) studied the effect of PEF treatment on the extraction yield of heat by microwave is caused by the strong rotation of the dipole of water-soluble phenolics in onion and found that PEF signifi- molecule upon the absorption of microwave (Jain, 2009). In addi- cantly elevated the extraction yield of phenolics and this was well tion, heat is also generated by the collision of molecules due to the reflected in their antioxidant potential. Moreover, PEF treatment frequent changes in the direction of the field sign.Alupului (2012) increased the extraction yield of polyphenols in tea leaves by two described three main steps regarding the MAE applied extraction times compared to the conventional thermal methods without any of bioactive molecules from food matrices; the isolation of bioac- significant changes in phenolic composition as evidenced by mass tives from the active sites of food matrices at high pressure and spectrometry analysis (Liu et al., 2019). Bozinou et al. (2019) re- temperature, the diffusion of solvents to the sample matrices, and ported that the highest extraction yield in PEF treatment was found the liberation of bioactive compounds to the solvents. This mecha- at a field strength of 7 kV/cm for 40 min in the extraction of phe- nism facilitates the extraction of desired compounds from samples nolics from freeze-dried leaves of Moringa oleifera. and increases the extraction efficiency. The MAE is a well proven efficient method for the extraction of bioactive compounds from foods. Lovrić et al. (2017) tested the 2.2. Pressurized liquid extraction (PLE) effectiveness of microwave-assisted extraction in the isolation of phenolics in blackthorn flowers in which they used three different Pressurized liquid extraction (PLE), which utilizes high pressure microwave powers of 100, 200 and 300 W. High microwave power and temperature, is an advanced technique to isolate analytes from showed stronger effectiveness in the extraction yield of phenolics, natural products by the residual or remaining solvent beyond their and this was well reflected in their antioxidant potential using the normal boiling point (Klejdusa et al. 2009). Pressurized liquid 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity assay. extraction was first described by Richter et al. (1996), but since However, long extraction time (>5 min) in microwave treatment then this method has also been referred to as high pressure solvent caused the degradation of phenolics, which led to a decrease in extraction (HSPE), pressurized fluid extraction (PFE), enhanced their antioxidant capacity. Pan et al. (2003) tested the efficiency of solvent extraction (ESE), and accelerated fluid extraction (ASE) MAE treatment in the extraction of phenolics from green tea leaves

24 Journal of Food Bioactives | www.isnff-jfb.com Shahidi et al. Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? by comparison with the conventional extraction methods (without plant- and animal-based foods by 6 to 35%. The advantages of microwave treatment). This strongly supports the effectiveness of UAE include reducing extraction time, energy, and use of solvent. MAE treatment in the extraction of bioactive compounds. A higher Moreover, this approach increases the stability of bioactive com- extraction rate was also found in the isolation of flavolignin and pounds during extraction compared to other extraction methods silybinin from Silybum marianum by MAE treatment (Dhobi et that use high temperature and pressure (Chemat et al., 2017). al., 2009). In another study, the usefulness of MAE was confirmed UAE was also applied to isolate alkaloids and phenolic acids from in the liberation of insoluble-bound phenolic acids from bran and natural products. Yang et al. (2011) employed ionic liquid-based flour fractions of sorghum and maize Chiremba( et al. 2012). UAE to extract three alkaloids, namely vindoline, catharanthine and vinblastine from Catharanthus roseus. Zu et al. (2012) also utilized ionic liquid-based UAE to extract phenolcarboxylic acids, 2.4. Supercritical fluid extraction (SFE) carnosic acid and rosmarinic acid from Rosmarinus officinalis.

The use of supercritical fluid was first reported by Zosel (1964) who applied it for the decaffeination of coffee. Subsequently, SFE 3. In vitro assays for measuring antioxidant capacity has been widely used in other fields such as environmental, pharmaceutical and polymer applications and food analysis (Zougagh et al., 2004). A substance reaches the supercritical state once it is 3.1. Semi-quantification of phenolics by total phenolic content present at the temperature and pressure beyond its critical point (TPC) assay (Inczedy et al., 1998). Supercritical fluid has gas-like properties in diffusion, viscosity, and surface tension, and liquid-like density, TPC is the most common and practical assay to determine the level which leads to the reduced extraction time and increased extraction of phenolics in natural sources using Folin-Ciocalteu reagent con- yield (Sihvonen et al., 1999). taining phosphomolybdic/ phosphotungstic acid complexes. The Supercritical carbon dioxide (SC-CO2) is the most common sol- basic principle of the TPC assay is based on the electron-transfer vent in the utilization of SFE due to its critical conditions such as reaction of antioxidants to form phosphomolybdic/phosphotung- temperature (31.1 °C) and pressure (73.8 MPa) (Garcia-Salas et stic acid complexes in an alkaline medium. This reaction produces al., 2010). However, it has a limitation in the extraction of polar a blue-colored chromophore that is detectable at 700 nm with a compounds such as phenolics by the non-polar nature of SC-CO2. UV/visible-spectrophotometer. The exact mechanism for the for- In order to address this drawback, chemical modifiers have been mation of the complex so formed has not yet been established, widely used. For example, a small amount of dichloromethane however it is assumed that the reaction might involve a reversible (CH2Cl2) significantly enhanced the polarity of carbon dioxide one- or two-electron reduction reaction (Huang et al., 2005; Sin- (Hawthorne et al., 1994). gleton et al., 1999; Ainsworth and Gillespie, 2007). Supercritical fluid extraction has been extensively used to extract Despite the excellent performance of TPC assay in measur- bioactive compounds in natural products. SFE method was em- ing the tentative quantification of phenolics in natural sources, ployed to extract naringin from citrus paradise, and the use of etha- there are criticisms for the utilization of TPC assay due to the nol (15 wt.%) as a modifier or co-solvent significantly increased the poor selectivity of Folin-Ciocalteu reagent in the reaction me- extraction yield of naringin at 9.5 MPa and 58.6 °C (Giannuzzo et dium; namely, this reagent can also react with some amino acids al., 2003). Khorassani and Taylor (2004) employed SFE method to in addition to phenolic compounds. In this sense, Harnly (2017) extract catechin and procyanidins from grape seed. The inclusion of argued that “Only state-of-the-art techniques such as liquid chro- methanol as a modifier elevated the extraction yield of catechin and matography-mass spectroscopy (LC-MS) to identify and quantify procyanidins by more than 79%. Alkaloids such as theobromine and phenolics should be used in scientific research”. Without a doubt, theophylline were extracted from Ilex paraguaryensis (herbal mate the advanced high techniques such as HPLC and HPLC-MS or tea) using SFE along with a wide range of temperature (13–343 °C) (MS/MS) have an excellent performance in the identification and and pressure (14–24 MPa) (Saldaña et al., 1999). Verma et al. (2008) quantification of molecules in samples along with extremely high utilized SFE to extract indole alkaloids such as catharanthine from accuracy and sensitivity. However, HPLC system scans relatively Catharanthus roseus leaves and reported that the optimum condition a narrow range of compounds, compared to TPC, due to the speci- for the extraction of catharanthine was at 25 MPa and 80 °C using fied analysis conditions such as wavelength, packing material of 6.6% methanol as a modifier. Thus, supercritical fluid extraction has column, and composition of mobile phase, among others, which is been well proven as an excellent means for the extraction of bioac- optimized to detect targeted compounds. This indicates that some tive compounds from natural products. compounds may not be detected in HPLC analysis under certain analysis condition. Here, TPC assay may provide a useful means to complement the aforementioned flaw of HPLC analysis by detect- 2.5. Ultrasound-assisted extraction (UAE) ing all molecules that react with Folin-Ciocalteu reagent. Thus, it is important to reconcile two different assays to address the disad- Ultrasound is a special type of sound wave with frequencies be- vantages of each method. yond the upper audible limit of human hearing, which passes through a medium by generating compression and expansion. The 3.2. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ultrasound energy facilitates the release of bioactive compounds by assisting the access of solvent to the food matrices in which assay and Trolox equivalent antioxidant capacity (TEAC) assay ultrasound energy enhances the diffusion of solvent across the cell wall and rinsing the cell components upon the disintegration of The practical use of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radi- cells (Herrera and Luque de Castro, 2005). cal to measure the antioxidant potential of bioactive compounds UAE has been widely used to improve the extraction efficiency was proposed more than 50 years ago (Blois, 1958; Papariello and of bioactive compounds in food matrices. Vilkhu et al (2008) re- Janish, 1966). The outstanding stability of this free radical, com- ported that UAE improved the extraction yield of bioactives from pared to other free radicals such as hydroxyl (HO•), lipid alkyl

Journal of Food Bioactives | www.isnff-jfb.com 25 Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? Shahidi et al.

(L•), alkoxyl (LO•), lipid peroxyl (LOO•) radicals, allows its com- radical scavenging ability was also employed to determine the an- mon use in assessing hydrogen atom or electron donating potential tioxidant potential of lentil hulls (Yeo and Shahidi, 2020). of antioxidants. The reaction mechanism of this assay is based on the neutralization of DPPH radical by receiving a hydrogen atom 3.4. Inhibitory activity against the oxidation of LDL cholesterol or an electron from antioxidants followed by a color change from purple to yellow. The alteration is detectable by ultraviolet-visible spectrophotometry at λmax 517 nm. The broad utilization of DPPH LDL cholesterol transports lipophilic compounds such as fatty ac- assay in the related fields such as food, medicine, and cosmetic, ids, triacylglycerols, cholesterol, and cholesterol esters to the cell demonstrating its excellent performance in testing radical scaveng- intracellular space, playing a crucial role in the growth and main- ing potential of bioactive molecules (Halliwell, 1997; Liu, 2003; tenance of cells. Nevertheless, they are referred to as “bad choles- Sánchez-Moreno, 2002). Moreover, the long history of DPPH as- terol”, since they are responsible for causing cardiovascular dis- say has developed DPPH chemistry such as stoichiometry factor ease; briefly, once they are oxidized, they accumulate at the arterial (n), defined as “number of electron or hydrogen atoms that anti- wall, leading to a narrowing of blood vessels. Thus, the inhibitory oxidants can donate to the free radicals”, and their mechanisms un- activity against the oxidation of LDL cholesterol is important for der versatile reaction environments such as temperature, pH, and preventing possible vascular diseases. polarity of the solvent (Yeo and Shahidi, 2019a; Yeo and Shahidi, The determination of inhibitory activity against the oxidation of 2019b; McGowan et al., 1959; Dawidowicz et al., 2012). LDL cholesterol starts with the dialysis treatment to remove eth- Miller et al. (1993) introduced TEAC assay to assess the electron ylenediaminetetraacetic acid (EDTA) which is added to prevent donating potential of antioxidants using 2,2′-azinobis (3-ethylbenzo- possible oxidation of LDL cholesterol during storage. The purified thiazoline-6-sulfonic acid (ABTS•+). The chemical reaction between LDL cholesterol is subjected to the oxidation process in the pres- ABTS and the strong oxidizing agent potassium persulfate produces ence of a transitional metal ion such as copper ion (Cu2+) to induce + the blue/green ABTS• chromophore, having maximum absorption lipid oxidation. Peroxyl radical is also used to induce the oxidation at 645, 734, and 815 nm (Miller et al., 1993). The addition of antiox- of LDL cholesterol by the inclusion of 2,2′-azobis (2-methylpropi- + + idants to the solution of ABTS• chromophore neutralizes ABTS• onamidine) dihydrochloride (AAPH). The mixture of LDL choles- via electron or hydrogen atom donation, which displays the decol- terol and prooxidant is incubated at the physiological temperature orization of the radical cation, leading to the absorption decrease at + of 37 °C, and the degree of LDL cholesterol oxidation is measured 645, 734, and 815 nm (Shahidi and Zhong, 2015). ABTS• is appli- by monitoring the formation of conjugated dienes of phospholipids cable in a wide range of solvent system due to its high solubility in or triacylglycerols of LDL cholesterol at 234 nm using UV-visible both hydrophilic and lipophilic media (Arnao, 2000). spectrophotometry. The inhibitory activity against the oxidation of Despite the great performance of DPPH radical scavenging ca- LDL cholesterol has been widely employed by many researchers pacity and TEAC assays, it has been a debate as to whether these to evaluate antioxidant capacity and potential anti-atherogenic ef- two assays are appropriate means to measure electron or hydro- fect of extracts of natural products (Chandrasekara and Shahidi, gen atom donating ability of antioxidants due to the discrimination 2011; Madhujith and Shahidi, 2007; Zhong and Shahidi, 2012). with reactive oxygen species (ROS) which are found in real food and biological systems (Benzie and Strain, 1999). This criticism has led to the application of more relevant radicals such as hydrox- 3.5. Inhibitory activity against DNA oxidation yl radical to evaluate the scavenging potential of test materials and to complement the drawback of DPPH and TEAC assays. The oxidation and damage to DNA strand result in the mutation of cells, which leads to their dysfunction. Thus, suppression of DNA 3.3. Hydroxyl radical scavenging potential oxidation is vital to maintain the health of cells. In this assay, the oxidation of DNA strand is induced by hydroxyl or peroxyl radicals to determine the inhibitory activity of antioxidants against the Hydroxyl radical is produced from O .− and H O in the presence 2 2 2 oxidation of a supercoiled plasmid pBR322 DNA (Chandrasekara of transitional metal ions during Fenton reaction. The excessive and Shahidi, 2011; Zhong and Shahidi, 2012). The scission of production of hydroxyl radicals causes oxidative stress, leading to DNA strand due to oxidation is determined by gel electrophoresis, the damage of cells. For instance, Rojanasakul et al. (1993) re- and bands are analyzed after visualization. The level of intact su- ported that hydroxyl radicals cause a cytotoxic effect in alveolar percoiled DNA strand and nicked DNA fractions is measured by macrophages via the elevation of [Ca2+] concentration, which fur- densitometry as indicated by the intensity. The inhibitory activi- ther interferes with homeostasis and cell death. Moreover, hydrox- ties of phenolics in barley (Hordeum vulgarae L.) and different oat yl radical is responsible for the endogenous oxidation of cellular DNA (Cadet et al., 1999). Thus, the prevention of the excessive (Avena sativa) cultivars against the oxidation of DNA strand were level of hydroxyl radical in cellular space is crucial, and antioxi- determined, and they confirmed the high effectiveness of phenolics dants (free radical scavengers) have been well proven for control- in preventing the scission of supercoiled DNA strand by hydroxyl ling the level of hydroxyl radicals. Alshikh et al. (2015) measured radical (Madhujith and Shahidi, 2007; Singh et al., 2019). Alshikh hydroxyl radical scavenging ability of five lentil cultivars, namely et al. (2015) measured the inhibitory activity of phenolics in lentil CDC SB-2, 3494-6, CDC invincible, CDC green land, and Maxim cultivars in the formation of nicked or linear form of DNA strand using electron paramagnetic resonance (EPR). Among the three upon oxidation. The inhibition of the DNA strand scission of phe- free, esterified, and insoluble-bound fractions, the esterified frac- nolics in six lentil cultivars ranged from 28.1 to 91.7%. tion showed a better hydroxyl radical scavenging ability than the others, and they concluded that the result might be related to the in vivo high content of flavonoids in the esterified fraction. In addition, 4. Measuring the absorption rate of antioxidants in hydroxyl radical scavenging ability had a high correlation coeffi- system cient with total phenolic content (TPC) and total flavonoid content (TFC), as well as the condensed tannin content (CTC). Hydroxyl The absorption of antioxidants takes place mainly at the lumen of

26 Journal of Food Bioactives | www.isnff-jfb.com Shahidi et al. Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned?

Figure 1. Absorption mechanism of flavonoid glucosides in the small intestine. small intestine, followed by their circulation in bloodstream and of human test by observing the level of their metabolites in plasma further phase II metabolism in liver. In this contribution, flavo- after the ingestion of lightly fried onions. Five metabolites such noid glucoside was selected as an example to explain the absorp- as quercetin-3-O-glucuronide, quercetin-3-sulfate, isorhamnetin- tion mechanism of antioxidants in the small intestine. The detailed 3-O-glucuronide, quercetin-O-glucuronide-sulphate, and querce- mechanism of the absorption of flavonoid glucoside in the small tin-O-diglucuronide were found in plasma, and they concluded intestine and into the bloodstream is described in Figure 1. Briefly, that the absorption of quercetin and isorhamnetin derivatives may flavonoid glucosides that are introduced in the small intestine after occur in the proximal part of the small intestine. Ottaviani et al. the ingestion of food undergo deglucosylation by lactase phlorizin (2012) reported that (−)-epicatechin, (+)-catechin, and (−)-cat- hydrolase (LPH) in the brush-border of small intestine. This pro- echin ingested as the cocoa-based drink were metabolized into cess increases the lipophilicity of flavonoids by removing the hy- (−)-epicatchin-3′-O-glucuronide and (−)-epicatchin-3′-sulfate in drophilic sugar moiety of the glucoside form, facilitating the pas- plasm in human body. Actis-Goreta et al. (2012) also observed sive diffusion of them in the membrane of the epithelial cells. The the absorption of (−)-epicatechin, (+)-catechin, and (−)-catechin flavonoid aglycone in the epithelial cells undergoes sulfation, glu- after the ingestion of dark chocolate containing (−)-epicatechin, curonidation, and methylation by the enzymes such as sulfotrans- (+)-catechin, and (−)-catechin in human system. They identified ferase, uridine diphosphate glucuronosyl transferase (UGT), and same metabolites such as (−)-epicatchin-3′-O-glucuronide and catechol-O-methyltransferases, leading to the formation of flavo- (−)-epicatchin-3′-sulfate in plasma as well as their additional work noid conjugates. These processes again enhance the hydrophilicity showed the approximately 20% of urinary recovery of tested com- of the flavonoids, followed by the improvement of their solubility pounds (Ottaviani et al., 2012). Prior (2012) studied the absorption in the plasma. Once introduced into the bloodstream, they are sub- of anthocyanins in raspberries such as cyanidin-3-O-sophoroside, jected to phase II metabolism in the liver before urinary excretion. cyanidin-3-O-(2′-O-glucosyl) rutinoside, cyanidin-3-O-glucoside, Many studies have so far been conducted on the absorption and cyanidin-3-O-rutinoside and found only two corresponding of antioxidants in rat and human trials (Table 1). Chesson et al. metabolites such as cyanidin-3-O-glucoside and cyanidin-O-glu- (1999) investigated the absorption rate of hydroxycinnamates in curonide in plasma along with only 0.007% of urinary recovery. a rat model system and found that 25% of consumed 14C-labeled In short, most of the antioxidants were absorbed in the small in- hydroxycinnamates were observed in body tissue after 2 hours. testine, and subsequently changed their structures via enzymatic Adam et al. (2002) determined the absorption rate of ferulic acid in rat system by feeding ferulic acid-enriched semi-purified di- reaction at the sulfatase and β-glucuronidase in the small intestine, ets. The result showed that approximately 50% of ingested feru- liver, and kidney. This process increases the polarity of antioxidant lic acid was found in the urine; they suggested the absorption of metabolites, thus facilitating their solubility in blood and then cir- the compounds through the passive diffusion or + Na /dependent culation in whole body. carrier-mediated transport process in the small intestine. A large proportion of ferulic acid was found in the conjugated form such as sulfated metabolites (58%) and glucuronidated metabolites 5. Application of cell lines for measuring antioxidant potential (18%) in plasma, which may occur in the small intestine, liver, and kidney by the enzymatic reaction of sulfatase or β-glucuronidase The chemical methods such as radical scavenging ability, reduc- (Karakaya, 2004). Wolffram et al. (1995) studied the mechanism ing power, and TPC provide important preliminary information

Journal of Food Bioactives | www.isnff-jfb.com 27 Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? Shahidi et al.

Table 1. Methods for measuring the absorption of antioxidants and their metabolites in rat and human systems Antioxidant Method Result Mechanism Reference Hydroxycinnamic Feeding 14C-labeled Observation of 25% of – Chesson et al. (1999) acid hydroxycinnamates (Rat) labeled hydroxycinnamates in body tissue after 2 h Ferulic acid Feeding FA-enriched Recovery of <50% the Passive diffusion Adam et al. (2002) sem-purified diets (Rat) ingested dose in urine or Na+/dependent carrier-mediated transport Cinnamic acid Mucosal uptake - Na+/dependent Wolffram et al. (1995) technique (in vitro) carrier-mediated transport Quercetin and Ingestion of 270 g of Detection of five metabolites Proximal part of Mullen et al. (2006) isorhamnetin fried onions containing after sulfation and the small intestine glucosides quercetin and isorhamnetin glucuronidation in plasma glucosides (Human) (−)-Epicatechin, Ingestion cocoa-based drink Detection of trace amounts of Absorption in Ottaviani et al. (2012) (+)-catechin, and containing epicatechin metabolites such as (−)-epicatchin- the small intestine (−)-catechin and catechin (Human) 3′-O-glucuronide and (−)-epicatchin- 3′-sulfate in plasma (−)-Epicatechin, Ingestion of dark Detection of metabolites such as – Actis-Goreta et (+)-catechin, and chocolate and measuring (−)-epicatchin-3′-O-glucuronide and al. (2012) (−)-catechin metabolites in plasma (−)-epicatchin-3′-sulfate in plasma and and urine (Human) approximately 20% of urinary recovery Anthocyanins Ingestion of raspberries Detection of two metabolites such – Prior, (2012) containing cyanidin-based as cyanidin-3-O-glucoside and a anthocyanins (Human) cyanidin-O-glucuronide in plasma and 0.007% of urinary recovery in the determination of the antioxidant potential of test materials. Chang, 2010; Ziberna et al., 2010; Roy et al., 2009). However, However, given the complex pathways for the suppression of oxi- CAA revealed a limitation by displaying a substantial variation dative stress in biological systems including up-regulation of anin the different cell lines. Myricetin showed a similar CAA with tioxidant enzymes, modulation of gene expression, and changing kaempferol in red blood cells (RBD) by displaying 82.2 and 80.4 cell signalling, the use of chemical assays is insufficient to pre- μmol of QE/100 μmol, whereas in HepG2 cells, more than seven dict the effectiveness of antioxidants in biological systems (Jones, times lower CAA of myricetin was found compared to kaempferol 2006). The occasional lack of correspondence between the bio- (Wolfe et al., 2008; Blasa et al., 2011). Moreover, a high CAA of logical system and the aforementioned chemical assays has led to catechin in Caco-2 cells showed contrary results in HepG2 and red the development of cellular antioxidant assays (CAA) (Wang and blood cells by showing only a minor effect. In addition, Kellett et Joseph (1999). The central principle of the CAA is based on the al. (2018) compared CAA of catechin and quercetin in different combination of chemical assays and cellular system to predict the cell lines; namely, catechin did not show cellular antioxidant activ- inhibition capacity of antioxidants against reactive oxygen species ity in HepG2 cells, whereas it suppressed 54.1 % of oxidation in (ROS) generated in cultured cell lines. Briefly, the 2′,7′-dichloro- Caco-2 cells. Moreover, quercetin had a ten times higher inhibition fluorescin diacetate (DCFH-DA) introduced to the cellular system in Caco-2 cells compared to HepG2 cells. They assumed that the is deacylated by the cellular esterases, then the DCFH is oxidized discrepancy might be due to the difference of the active membrane by ROS and reactive nitrogen species (RNS) such as nitric oxide transport of those flavonoids in the two cell lines. and peroxynitrite, followed by alteration into DCF (Wolfe et al., 2008; Wolfe and Liu, 2007). The intensity of DCF is recorded using a fluorescence spectrometer by exploiting its fluorescent prop- 6. Measuring antioxidant potential in animal models erty (λexc = 485 nm, λem = 535 nm). Many experts have employed CAA to assess the antioxidative potential of bioactive compounds from natural sources such as 6.1. Zebrafish model fruits, vegetables, and legumes (Song et al., 2010; Wolfe et al., 2008; Wolfe and Liu, 2007). Blasa et al. (2011) measured the cel- Zebrafish has been widely used as a vertebrate model organism in lular antioxidant activity of botanical extracts using red blood cells scientific research such as ecotoxicology and developmental bi- (CAA-RBC). Aside from those aforementioned reports, cellular ology. Due to its convenience in use and cost-effectiveness, the antioxidant activity assay was also conducted in the different cell application of zebrafish model has been largely extended to other lines such as human gastric adenocarcinoma cell line, vascular en- fields such as medicine, neurosciences and cancer research and dothelial cells EA.hy926, human macrophage cell line U937, and food science as well as testing of cosmetics where the use of other human lung fibroblasts(WI38, IMR-90) (Sessa et al., 2011; Xu and animal models is not considered acceptable to some consumers

28 Journal of Food Bioactives | www.isnff-jfb.com Shahidi et al. Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned?

(Kinth et al., 2013). Zebrafish has similar physiology and anatomy tion of a biomarker relevant to ageing. In addition, other studies on to mammals as well as being present orthologous genes for 70% the stress resistance of the extracts from natural products such as of the human ones. However, the central reasons for the wide use Sophora moorcroftiana and M. Willow bark in Caenorhabditis el- of Zebrafish in research area are the low cost for maintenance and egans model have also been reported (Zhang et al., 2018; Ishikado experimentation, their high fecund and rapid breeding, and the fea- et al., 2013). The above examples strongly support the usefulness sibility of the in vivo visualization of complex responses (Caro et of Caenorhabditis elegans model to screen bioactivities of anti- al., 2016). oxidants. The antioxidants activity of bioactives in zebrafish model can be determined through the direct measurement of reactive oxygen 6.3. Fruit fly model species production and biomarkers produced by oxidative damage under both normal physiological conditions and after treatment with an oxidative stressor (Caro et al., 2016). Among those ap- Fruit fly (Drosophila melanogaster) has been used as a crucial proaches in zebrafish model, the primary method to investigate the model system for exploring the mechanisms regulating essential antioxidant activity of bioactives is to detect the quantity of ROS biological processes as well as understanding the molecular mech- using fluorescence indicators. For that, fluorescent probes such as anisms of development and physiology (Arias, 2008). This further DCFH-DA (dichlorodihydrofluorescein diacetate) and HPF (hy- enabled a deep and comprehensive understanding of human dis- droxyphenyl fluorescein) are utilized in which these fluorescent eases at the molecular level (Botas, 2007). probes emit a fluorescence signal once they are oxidized by ROS. Fruit fly possesses analogous lipid metabolism and transport Thus, this method enables rapid and direct detection of oxidative system to mammalian hepatocytes as well as having a short life stress (Caro et al., 2016). For instance, Yang et al. (2012) used span, which facilitates investigation of the inhibitory activity of DCFH-DA to detect the generation of ROS in zebrafish upon ultra- bioactives against oxidative stress by measuring their lifespan violet (UV) B radiation and to investigate the antioxidant effect of (Gutierrez et al., 2007). Lin et al (2018) investigated the antiaging quercitrin. Moreover, other studies have utilized zebrafish model effect of sea cucumber (Cucumaria frondosa) hydrolysate in a fruit to explore the bioactivity of antioxidants. For example, Shih et al. fly model and reported that these hydrolysates effectively extended (2012) demonstrated the potent antioxidant effect of 3′,4′-diamino- the lifespan of fruit flies. Huangfu et al (2013) employed fruit fly flavones using zebrafish larvae and Chen et al. (2012) also used model system to test the antiaging effects of astaxanthin-rich alga this model to evaluate the antioxidant effect of several flavonoids. (Haematococcus pluvialis) in which it markedly enhanced their Furthermore, the antioxidant activity of bioactives was determined lifespan and reduced the age-related decline in locomotor function. by monitoring oxidative stress-induced cell death in a zebrafish Zou et al. (2017) studied the antiaging effect of riboflavin in fruit model. Yang et al. (2012) and Shih et al. (2012) counted stress- fly model system and found that riboflavin significantly extended induced cell death in zebrafish model to evaluate the antioxidant the lifespan and reproduction of fruit fly by elevating superoxide potential of quercitrin and 3′,4′-diaminoflavones. dismutase 1 (SOD1) and catalase (CAT). Moreover, fruit fly was Lipid peroxidation markers such as malondialdehyde (MDA) used to demonstrate the improvement of methionine sulfoxide re- are also used to measure the antioxidant potential of bioactives in ductase activity under the oxidative stress conditions by feeding zebrafish model. Kim et al (2012) reported the potent antioxidant methyl-cysteine fortified meals Wassef( et al., 2007). activity of acai puree by showing the suppressed the hepatic MDA level in hypercholesterolemic zebrafish after feeding acai puree. 6.4. Mouce model Moreover, MDA was the efficient lipid peroxidation biomarkers in zebrafish model in examining their vitamin C deficiency; zebrafish suffering vitamin C deficiency showed a high MDA level, which Mouce model is the most common system to examine the antioxi- subsequently affected their energy metabolism (Kirkwood et al., dant capacity of natural products. Prior to the testing antioxidant 2012). capacity, mice are treated with oxidative stressors such as 6-hydroxydopamine (6-OHDA), alloxan t-BHP, streptozotocin (STZ), high-fat diets, ethanol, and D-galactose (Zhang et al., 2017), then 6.2. Caenorhabditis elegans model antioxidant capacity is determined by measuring the activity of antioxidant enzymes such as glutathione reductase and catalase Caenorhabditis elegans has been employed as a model system to and malondialdehyde (MDA) as the oxidative stress biomarkers investigate the bioactivities of antioxidants such as anti-inflamma- (Zhang et al., 2017). tory, antioxidant, chemopreventive, and chemotherapeutic activi- Recent literature in the use of mice model to measure the an- ties as well as inhibiting obesity, aging, and Alzheimer’s disease tioxidant capacity of natural products is summarized below. Lin since the early 1960s (Shen et al., 2017). This organism has many et al. (2018) studied the effect of sea cucumber (Cucumaria fron- advantages as a model system in lab research such as convenience dosa) hydrolysate on the age-related neurodegenerative disor- to cultivate in laboratory, short life-cycle (3.5 days from the eggs ders in D-galactose-induced aging mice, and the results showed to adult worms) and high reproducibility (they produce over 1,000 that sea cucumber hydrolysate significantly attenuated oxidative eggs every day), and easy to observe using a microscope due to stress D-galactose-induced aging mice. Costa Barros-Gomes et al. the transparency of their body (Luo et al., 2019). Thus, Caeno- (2018) measured the antioxidant potential of extract from Seaweed rhabditis elegans has been widely used to measure the bioactivi- Gracilaria birdiae (GB) in mice model; GB extract decreased their ties of antioxidants (Shen et al., 2017). Liao et al. (2011) explored weight gain and the level of blood sugar level after feeding 21 the antioxidant potential of curcumin in Caenorhabditis elegans days of meals containing GB extract and enhanced the activity of in which the extended lifespan of the organism was noticed. In antioxidant enzymes such as glutathione reductase and catalase. this, curcumin suppressed the level of intracellular reactive oxy- Aremu et al. (2019) examined antioxidant effect of 70% ethanol- gen species and lipofuscin in Caenorhabditis elegans during ag- water (v/v) extract of Taraxacum officinale in Nω-nitro-L-arginine ing. Cañuelo et al. (2012) found that tyrosol markedly extended the methyl ester (L-NAME)-induced hypertensive rats: The reduced lifespan of Caenorhabditis elegans as well as delaying the genera- MDA levels and increased total antioxidant capacities (TAC)

Journal of Food Bioactives | www.isnff-jfb.com 29 Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? Shahidi et al. were found in rats after feeding Taraxacum officinale extract for of 11 hydroperoxide isomers in oxidized methyl linoleate using 21 days. Diabetic mice were also used to evaluate the antioxidant HPLC system. Browne and Armstrong, (2000) utilized reversed- and anti-diabetic activity of polysaccharides from Guava (Psidium phase HPLC to assess lipid peroxidation in human blood plasma, guajava L., Myrtaceae) leaves (Luo et al., 2019). Diabetic mice and they identified 13 hydroxy- and hydroperoxy polyunsaturated were induced by using streptozotocin combined with a high-fat fatty acids at 236 nm using UV-visible detector (UVD). Morales et diet; they were then fed with Guava leaves extract; the Guava al. (2014) investigated the formation of hydroperoxides from three leaves extract markedly improved TAC and superoxide dismutase sunflower oils with different levels of oleic and linoleic acids at the (SOD) as well as suppressing the damage to liver, kidney, and heating temperature of 40 °C using HPLC system equipped with pancreas. Tert-butyl hydroperoxide (t-BHP) has also been widely two detectors such as UVD and evaporative light scattering detec- used to induce oxidative stress in mice models. Kim et al. (2015) tor (ELSD) in series. This method allowed the detection of oleate utilized t-BHP to induce oxidative stress in mice to explore the hydroperoxides and the simple hydroperoxydienes of linoleate and antioxidant activity of extract from Celosia cristata L.flower. The linolenate. In addition, Zeb, (2012) found epoxy hydroperoxides, Celosia cristata L. flower extract significantly reduced the hepatic epoxy epidioxides, and mono-epoxides as the oxidation products levels of MDA and serum level of triacylglycerol against t-BHP- of camellia seed oil using liquid chromatography with electrospray induced oxidative stress. ionization mass spectrometry (HPLC-ESI-MS).

7.3. Conjugated dienes (CDs) 7. Methods for measuring lipid oxidation for evaluating antioxidant effects Fatty acids containing methylene-interrupted dienes or polyenes such as linoleic, linolenic, and high polyunsaturated fatty acids can 7.1. Lipid oxidation form conjugated double bonds through the shifting of electrons upon lipid oxidation. The resultant conjugated dienes or trienes are Autoxidation, the spontaneous chemical reaction of atmospheric detectable at λmax 233–238 nm using UV-visible spectrophotome- oxygen with lipids, results in extensive alteration in chemical and try. Conjugated dienes include lipid oxidation products having two physical properties of foods as well as biological systems (Shahidi or more double bonds in series such as trans, trans-2,4-alkadien- and Zhong, 2010). Catalysts such as heat, light, enzymes, metals, trans, trans-2,6-nonadienal, and trans, trans-2,4-decadienal. als, metalloproteins and microorganisms are responsible for lipid Hydroperoxides also contain conjugated dienes in their chemical oxidation, resulting in the formation of off-flavors and changes in structure, except hydroperoxides formed from oleic acid having the physical and chemical properties of foods. Reactive oxygen only one double bond. species (ROS) also cause lipid oxidation of cellular membrane in The simple, low cost, procedure for measuring conjugated biological systems and this may lead to the destruction of cell life, dienes enables the easy application of this procedure to a wide followed by the increase in the incidence of chronic diseases such range of samples including food and biological materials. Yeo et as inflammation, atherosclerosis and ageing, among others (Dal- al. (2011) evaluated the velocity of lipid oxidation of lard at four ton et al.,1999; Davies, 2000; Kruidenier and Verspaget, 2012). different temperatures using CDs, showing the increase of the rate Moreover, the formation of polymeric products by oxidation of of lipid oxidation depending on the intensity of heat energy. Inter- phospholipids in cellular membranes deteriorates the membrane estingly, the level of CDs was reduced after the specific oxidation transport and cell signalling capacity as observed in Alzheimer’s time at high temperatures such as 150 and 180 °C. They concluded disease due to the reduced fluidity of the membrane (Lyras et al., that the decrease in CDs might be due to the degradation of hydrop- 1997). eroxides and CDs by high heat energy. Peña-Ramos and Xiong (2003) measured lipid oxidation of pork patties after cooking at 70 °C using CDs; soy protein hydrolysate produced by chymotrypsin 7.2. Using HPLC for the determination of peroxide value (PV) or Flavourzyme significantly inhibited the formation of CDs during cooking and storage at 4 °C for 7 days. Meanwhile, CDs assay Hydroperoxides (ROOH) are the primary products of lipid oxida- has a serious limitation in the application of samples containing tion and do not possess any off-flavour or off-odour by themselves. pigments such as carotenoids and phenolic compounds since these However, once they are broken down into smaller secondary prod- molecules interfere with the absorbance reading at 233–234 nm ucts such as aldehydes, ketones, alcohols, esters, and hydrocar- using UV spectrophotometry. bons by β-scission, the resultant compounds generate off-flavour compounds with low threshold values (Shahidi and Zhong, 2010). Peroxide value is a classical means for the quantitation of hy- 7.4. Thiobarbituric acid (TBA) test droperoxides produced from lipid oxidation using an iodometric method. The basic principle for measuring the level of peroxides The TBA test is an old and classic assay to assess the oxidation of in oxidized lipid relies on the reduction of the hydroperoxide fats and oils. Malondialdehyde (MDA) generated in lipid oxida- group (-OOH) by iodide ion under acidic condition. The amount tion reacts with 2-thiobarbituric acid, forming the pink complex of iodine (I2) released in the above reaction is proportional to the of TBA-MDA adduct being detectable at 530–532 nm. Aside from level of peroxide in the reaction medium. The measurement of the MDA, 2-alkenals and 2,4-alkedienals can also react with TBA, but liberated I2 is carried out by titration using sodium thiosulphate the exact mechanism of the reaction is not yet well understood. solution along with starch solution as indicator. Total amount of Zhou et al. (2019) measured the lipid oxidation of mussels (myt- sodium thiosulphate solution required for the titration is used for ilus edulis) meat during storage at 4 °C for four days using TBA the determination of the level of hydroperoxide in sample. test. The increased TBA value was found with the storage time, High performance liquid chromatography (HPLC) has been and this showed high consistency with other measurements used widely employed for the determination of the level of hydroper- such as volatile basic nitrogen (TVB-N), peroxide value (POV), oxides in oxidized oil/fat. Wang and Joseph (1999) found a total and total oxidation (TOTOX). Liu et al. (2018) determined the lev-

30 Journal of Food Bioactives | www.isnff-jfb.com Shahidi et al. Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? el of lipid oxidation of processed clam (Ruditapes philippinarum) p-anisidine value and K270. Guzmán et al. (2011) reported the use by hot-air drying treatment using different assays such as TBA test, of low-resolution Raman spectroscopy to propose a new approach peroxide value (POV), total oxidation value (TOTOX), and oxida- for the analysis of oxidized olive oil. They monitored well-defined tion test (OXITEST). In results, TBA test showed a similar trend bands at 1,267, 1,302, 1,655, and 1,747 cm−1 corresponding to sym- with POV and TOTOX, whereas OXITEST displayed a contrary metric double bond in cis δ (=CH), in-phase twist methylene, cis result compared to other assays. Wanasundara and Shahidi (1995) double bonds stretching (C=C), and ester stretching (C=O), respec- also assessed the lipid oxidation of canola and soybean oils during tively. A high correlation coefficient with other parameters proved storage at 65 °C for 30 days and found an excellent correlation the usefulness of low-resolution Raman spectroscopy as a method coefficient with TBA test and CDs. Shahidi et al. (1987) reported to assess oxidation of oil/fat along with a simple, non-destructive, that a high correlation was found between the hexanal content and analytical procedure. Vaskova and Buckova (2015) investigated the TBA values in cooked ground pork systems. the alteration of the chemical structure of edible oils using Raman With regard to the use of the TBA test, we believe that while spectroscopy during the oxidation process. The different Raman absolute values provided by this method are not necessarily reliable peaks such as 1,640, 1,658, and 1,747 cm−1, which accounts for due to interferences from other components present in the food, the C=C double bonds of trans, trans-2,4 decadienal, C=C cis double relative values to be used for comparative purposes show trends that bond stretching, and C=O ester-carbonyl stretching, respectively, are trustworthy. Hence, the use of this colorimetric assay provides were identified in oxidized canola oil and the method could be used useful information if care is exercised in the use of the methodol- for the prediction of the level of oxidation. ogy. In addition, since MDA is produced when at least 2 or 3 double bonds are present in the molecules, authors using this method 7.7. Chromatographic techniques for the measurement of lipid for evaluating oxidation of oils when there are no polyunsaturated fatty acids present are cautioned about their misuse of the assay. Fi- oxidation nally, mention should be made that when reporting results from the TBA test, results are always reported as TBA reactive substances Gas-chromatography (GC) is a useful tool to measure volatile (TBARS) and as MDA equivalents and not MDA. MDA can only compounds generated in lipid oxidation. A major volatile com- be reported as such when HPLC determination is carried out. pound in lipid oxidation is hexanal formed from omega-6 fatty acids such as linoleic, gamma-linolenic, and arachidonic acids by the β-scission of alkoxyl radical (RO•), which can be analyzed by 7.5. p-Anisidine and TOTOX value using GC with a number of different detectors.Panseri et al. (2011) utilized headspace solid-phase microextraction (HS-SPME) meth- p-Anisidine value is also one of the classical measurements to test od to isolate volatile compounds from the oxidized butter and the level of oxidation in animal and vegetable oils. The p-anisidine injected to the gas chromatography-mass spectrometry (GC/MS) reagent reacts with aldehydes such as 2-alkenal and 2,4-alkedienal system to analyze hexanal content. CAR/PDMS fibre was used for under acidic medium, forming yellowish complex that is detect- the extraction of hexanal in samples, and the optimum extraction able at 350 nm. In the industry, p-anisidine is usually used to cal- condition was 180 min at 4 °C. This approach displayed a great culate TOTOX value, defined as follow: performance in assessing the lipid oxidation of butter by showing TOTOX value = 2 peroxide a linear increase of hexanal content during the storage of butter value (PV) + p-anisidine value (p-AV) for six months. Iglesias et al. (2009) employed SPME-GC/MS to scan volatile compounds in the oxidized minced fish muscle of sea This method does not have any real meaning as variables with bream and detected 3-hexanone as a major oxidation compound. different units are added. Meanwhile, Wanasundara and Shahidi Chen et al. (2018) investigated the relationship between SPME- (1995) proposed the replacement of p-anisidine value with TBA GC/MS data and the sensory scores of dairy powders fortified with value in the calculation of TOTOX and reported the results ad long-chain polyunsaturated fatty acids and iron. They identified 10 TOTOX-TBA. volatile compounds including 1-penten-3-ol, 2-octanone, 3,5-octadien-2-one, decanoic acid, 2-(2-pentenyl) furan, (Z)-4-heptenal, 7.6. Fourier transform infrared (FTIR) and Fourier transform octanoic acid, (E,Z)-2,6-nonadienal, 3-hexanone, and nonanal and Raman spectroscopy concluded that the fishiness of the oxidized samples in sensory test was strongly related with alcohols, ketones, aldehydes, and furans detected in SPME-GC/MS. Oh et al. (2015) measured the Fourier transform infrared (FTIR) and Fourier transform Raman level of volatile compounds such as pentane, 2-propenal, hexanal, spectroscopy have successfully been used for the determination and trans-2-heptenal during oxidation of linoleic acid in the pres- of lipid oxidation. Cebi et al. (2017) employed the attenuated total ence of deuterium oxide using SPME-GC/MS. In addition, Shahidi reflectance (ATR)-Fourier transform infrared spectroscopy (FTIR) (2001) reported that propanal was the dominant oxidation product to predict the peroxide value of microalgal oil. They monitored the by the breakdown of ω-3 fatty acids such as eicosapentaenoic and shift of band at 990–940 cm−1, which provides information about docosahexaenoic acids. the alteration of C-H bond from the plane deformation vibration of Kim et al. (2013) proposed the ratio of saturated fatty acids trans double bond. The results displayed a high relationship with (SFAs) to unsaturated fatty acids (UFAs) after analysis of GC- PV. Muik et al. (2005) employed Fourier transform Raman spec- flame ionization detector (FID) as an efficient means for the evalu- troscopy to assess the oxidation of six different vegetable oils. This ation of oxidation of oil/fat. Gas-chromatography (GC) with the approach allowed the detection of saturated and α, β-unsaturated different extraction methods and detectors showed an excellent aldehydes formed during lipid oxidation as well as the formation of performance in the detection of oxidation products during lipid conjugated double bonds and the isomerisation of cis to trans dou- oxidation. ble bonds in the C=C stretching region. Each oil showed a distinct High performance liquid chromatography (HPLC) is also used pattern in the spectrum during oxidation, and the results showed a for the determination of lipid oxidation of food and biological good correlation with other classic oxidation parameters such as samples. Jeong et al. (2013) employed 2,4-dinitrophenylhydrazine

Journal of Food Bioactives | www.isnff-jfb.com 31 Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? Shahidi et al.

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Journal of Food Bioactives | www.isnff-jfb.com 35 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Review J. Food Bioact. 2020;9:36–39

Brazil nut: nutritional benefits from a unique combination of antioxidants

Alan Giovanini de Oliveira Sartoria*, Marisa A.B. Regitano-d’Arcea and Leif H. Skibstedb aDepartment of Agri-food Industry, Food and Nutrition, Luiz de Queiroz College of Agriculture, University of São Paulo, Piracicaba, SP, Brazil bDepartment of Food Science, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark *Corresponding author: Alan Giovanini de Oliveira Sartori, Department of Agri-food Industry, Food and Nutrition, Luiz de Queiroz Col- lege of Agriculture, University of São Paulo, Piracicaba, SP, Brazil. E-mail: [email protected] DOI: 10.31665/JFB.2020.9216 Received: March 27, 2020; Revised received & accepted: March 31, 2020 Citation: de Oliveira Sartori, A.G, Regitano-d’Arce, M.A.B., and Skibsted, L.H. (2020). Brazil nut: nutritional benefits from a unique combination of antioxidants. J. Food Bioact. 9: 36–39.

Abstract

Brazil nut is the seed of the Bertholletia excelsa tree, which grows naturally in the rainforests of South America. The production chain is environmentally sustainable as seeds are collected from the hard-shelled indehiscent fruits as they fall on the forest floor, without the need of deforestation. Brazil nut is among the most popular and commercially important tree nuts, and a reduced risk of coronary heart disease among other health benefits have been related to its consumption. A high content of both soluble and bound phenolic antioxidants, especially in the brown skin of the kernel, partly explains this positive health effect together with a high content of oleic acid and linoleic acid. The ω-3/ω-6 is less favorable, but the relatively high content of delta-tocopherol for a tree nut and the highest selenium (Se) level among all foods warrant other health benefits such as antioxidant activity and anticancer effects. Se levels in Brazil nuts vary considerably depending on origin, and recommendations for their safe consumption should be made and added in food labels in order to avoid selenosis.

Keywords: Bertholletia excelsa; Brazil nut; Phenolic compounds; Selenium; Tocopherols; Antioxidant.

Brazil nut is among the most popular and commercially impor- fects related to a regular Brazil nut consumption (Cardoso et al., tant tree nuts, along with pecans, walnuts, pine nuts, pistachios, 2017). Lipid-rich Brazil nuts have a unique composition of plant macadamia nuts, hazelnuts, almonds and cashews (Venkatachalam phenols and tocopherol homologues that are atypically combined and Sathe, 2006). Brazil nut, with the Portuguese name Castanha with seleno-amino acids, for which synergistic antioxidant effects do Pará, named after the Brazilian state of Pará, is available raw are unexplored or at least not understood. Table 1 summarizes the in-shell or shelled and dried. Dried Brazil nuts are increasingly major findings reported in this article concerning the chemical being added to foods such as baked goods and breakfast cereals. composition, especially antioxidants, of Brazil nut and the health Botanically, nuts are fruits with one seed, but Brazil nut as used in effects related to its regular consumption. food, is rather a single seed of a fruit with up to 24 seeds inside an As the other tree nuts, Brazil nut has a low water activity with indehiscent shell. The Food and Drug Administration (FDA) of the lipid and protein content of 60–70% and 15%, respectively (Uni- United States now allows a health claim for tree nuts, including camp, 2006; Venkatachalam and Sathe, 2006; USDA, 2019). The Brazil nuts (FDA, 2003). The recommendation of a daily intake of lipid content is high and almost comparable to macadamia nut with approximately 40 g of tree nuts as part of a diet low in saturated a lipid profile favorable to human health due to the high proportion fats and cholesterol in order to reduce the risk of coronary heart of unsaturated fatty acids, mainly the essential linoleic acid and diseases is based on epidemiological studies (FDA, 2003). There oleic acid. An effect of lowering total and low-density lipoprotein- is also evidence of anti-cancer and more general antioxidant ef- cholesterol (LDL-c) while maintaining or increasing high-density

Table 1. : The chemical composition, especially antioxidants, of Brazil nuts and the health effects related to its regular consumption Subject Key findings References Hydrosoluble Predominately found in the brown skin that covers the kernel, detected so far are John and Shahidi phenolic phenolic acids and flavonoid derivatives like gallic acid, gallocatechin, protocatechuic (2010); Gomes and compounds acid, catechin, vanillic acid, taxifolin, myricetin, ellagic acid, sinapic acid and quercetin Torres (2016) Relevant amounts of ellagic acid derivatives found in aqueous Silva et al. (2019) extracts of bark biomass residues of the Brazil nut tree Tocopherols Brazil nut has remarkable levels of delta-tocopherol, in comparison with other tree nuts Miraliakbari and Shahidi (2008b) Selenium Selenium levels vary significantly depending on the nut origin. Silva Jr. et al. (2017) Therefore, recommendations of adding Se levels information based on the origin of the seeds in retail labelling should be made Effects of Selenium levels decreased up to 73% during water-extraction process Sartori et al. (2020) processing on Alpha-tocopherol losses are higher than gamma-tocopherol Sartori et al. (2018a) nutrient levels losses in Brazil nut oil during storage Health effects No selenosis reported in health populations with diets rich in selenium from Brazil nuts Martens et al. (2015); Lemire et al. (2012) Consumption of Brazil nuts with high Se levels may have Duarte et al. (2019) a pro-inflammatory effect in obese women Anti-cancer and antioxidant effects related to a regular Cardoso et al. (2017) Brazil nut consumption was reported lipoprotein-cholesterol (HDL-c), as seen for the intake of olive oil, during water-extraction to produce water-soluble Brazil nut extract is assigned to oleic acid. (Brazil nut milk) and Brazil nut flour to be 73%. Further studies are Se levels vary from one seed to another (<0.5 to 146.6 µg Se/g), needed in order to identify the effect of every process step, although and a single Brazil nut (ca. 3 g) may overcome the daily dietary Se may be predominantly lost during the flour-drying step, since or- recommended intake for this mineral of 70 µg (Silva Jr. et al., ganoseleno compounds formed by selenomethionine in the presence 2017). Se levels also vary according to the seed origin, mostly due of glucose under heating may volatilize (Tsai et al., 1998). to Se soil levels and soil acidity, and average values of 66.1 µg Along with Se, Brazil nut contains other compounds with an- Se/g (Amazonas), 51.2 µg Se/g (Pará), 10.2 µg Se/g (Roraima), 3.0 tioxidant activity, such as zinc and phenolics, mainly tocopherols, µg Se/g (Acre), and 2.4 µg Se/g (Mato Grosso) were reported ac- which play a role in human health. Antioxidants are substances cording to the Brazilian state where the seeds were collected (Silva that when present at low concentrations compared with those of Jr. et al., 2017). Individuals from some Amazonian populations an oxidizable substrate significantly delay or prevent oxidation with selenium-rich diets, especially due to Brazil nut consump- (Halliwell, 1990). In Brazil nuts, there are oxidizable unsaturated tion, and consequently, Se blood levels above the safe levels, did fatty acids as substrates, and antioxidants are important in nature not show toxicity symptoms (Martens et al., 2015; Lemire et al., for preserving the vitality of the seed. During processing and stor- 2012). However, preliminary studies revealed that consumption of age of Brazil nuts as a food, antioxidants become important for Brazil nuts with high Se levels may have a pro-inflammatory effect protection of the lipids to keep them nutritionally and sensorially in obese women (Duarte et al., 2019). Thus, the consumption of acceptable, as lipid oxidation implies loss of nutritional value and Brazil nuts should be moderate. Furthermore, consumption recom- generation of off-flavors (Miraliakbari and Shahidi, 2008a; Za- mendations from the Government should be considered in order to jdenwerg et al., 2011). prevent selenosis. Since analysis of Se levels in individual nuts is There are several studies concerning the oxidative stability of expensive and labor-demanding, an affordable alternative could be Brazil nuts, Brazil nut oil, and Brazil nut flour during storage, the description of the expected Se level in the labels of Brazil nuts mainly monitoring hydroperoxides, volatiles or the tendency of and products containing Brazil nuts per portion, based on the seeds free radicals formation (Gutierrez, Regitano-d’Arce, and Rau- origin. This labeling initiative could also raise worldwide consum- en-Miguel, 1997; Regitano-d’Arce, 1998; Vieira and Regitano- er awareness on the unique health benefits related to Brazil nuts. d’Arce, 1999; Miraliakbari and Shahidi, 2008a; Zajdenwerg et In Brazil nuts, approximately 85% of Se is covalently bound to al., 2011; Sartori et al., 2018a, Sartori et al., 2018b, Sartori et al., proteins, mainly replacing sulfur in selenomethionine and seleno- 2018c). When compared with pine nut, walnut and hazelnut oils, cysteine (Kannamkumarath et al., 2002; Chunhieng et al., 2004). Brazil nut oil along with pecan oil, showed to be the most stable The biological activity of Se in mammals is normally assigned to oil during a storage test at 60 °C for 12 days, by monitoring the its antioxidant activity when incorporated in glutathione peroxi- formation of headspace hexanal both for extraction with hexane dases (GPx), iodothyronine deiodinases (IDI), thioredoxin reduc- and with a mixture of chloroform and methanol (Miraliakbari and tases (TR) and selenoprotein P (SePP), which are among the 25 Shahidi, 2008a). The resistance against autoxidation was hypoth- selenoenzymes identified so far (Cardoso et al., 2017). The effect esized to be due to the lower degree of unsaturation of lipids and of processing, such as heating or pasteurization, on Se levels in especially the absence of alpha-linolenic acid and to the presence Brazil nuts is still not clear, despite the increasing use of this tree of tocols and phospholipids in oils of pecans and Brazil nuts. The nut in food formulations. Sartori et al. (2020) estimated Se losses antioxidant mechanism of phospholipids in vegetable oils still re-

Journal of Food Bioactives | www.isnff-jfb.com 37 Brazil nut: nutritional benefits de Oliveira Sartori et al. mains unexplained with no studies evaluating antioxidant activ- for the content of phenolics in hazelnuts (Ghirardello et al., 2016). ity of phospholipids in Brazil nut oil. In contrast, tocopherols are Aqueous extracts of bark biomass residues of the Brazil nut tree known to donate hydrogen atoms to lipid peroxyl radicals halting may be a relatively inexpensive source of antioxidants, with high propagation of autoxidation (Kamal-Eldin and Appelqvist, 1996). amounts of ellagic acid derivatives present (5.0 g/kg for the outer In Brazil nut oil, the alpha-tocopherol homologue seems to have bark and 44 g/kg for the inner bark) (Silva et al., 2019). The major the highest ability to act as antioxidant, as observed in a study with derivatives identified by RP-HPLC-DAD-MS/MS and NMR are storage at a high temperature of 80 °C (Zajdenwerg et al., 2011). eschweilenol C, ellagic acid and valoneic acid dilactone (Silva et The chain-breaking mechanism operating was confirmed in anoth- al., 2019). The hard shell of the Brazil nut fruit should also be in- er study, where the contents of alpha- and gamma-tocopherol pre- vestigated as a source of antioxidants. sent in cold-pressed Brazil nut oil were monitored under simulated In summary, Brazil nut is a relevant source of antioxidant com- retail conditions during five months Sartori( et al., 2018a). pounds, which are related to health benefits. These antioxidant Beta-tocopherol and gamma-tocotrienol are present only in compounds include Se, tocopherols, phenolic acids, and various trace amounts and vitamin C and carotenoids with antioxidant flavonoid-derivatives. More investigations are needed to shed activity seems completely absent in Brazil nuts (USDA, 2019). light on the effect of processing and storage on some of the bioac- In contrast, the content of delta-tocopherol is remarkable, as it tives in Brazil nuts and in their co-products. Less utilized parts of amounts to almost 10% of the total concentration of the tocopherol the Bertholletia excelsa tree may also be sources of antioxidants, homologues in marked contrast to other tree nut oils like hazelnuts, which can be extracted in a relatively simple way and in abundant almonds and pecan oil (Miraliakbari and Shahidi, 2008b). Delta- amounts. Brazil nuts have very high amounts of Se and could be tocopherol is the least reducing of the tocopherol homologues, more widely consumed, especially in regions of China and Scan- the least efficient as radical scavenger (Mortensen and Skibsted, dinavia, which have a soil depleted in Se and where Se deficiency 1997), and probably has little effect as antioxidant in the oil. How- is a common problem among their populations. Se level in Brazil ever, delta-tocopherol, along with gamma-tocopherol, delta-tocot- nuts vary from one seed to another and is strongly dependent on rienol and gamma-tocotrienol seems to have cancer-preventing the origin. Hence, in order to assure safe consumption of Brazil effects stronger than alpha-tocopherol, by inhibiting the growth nuts, recommendations of adding Se levels information based on and inducing death of different types of cancer cells, and by being the origin of the seeds in retail labelling should be made. Such la- capable of suppressing cancer development in preclinical cancer belling could also improve the image of Brazil nuts as a functional models (Jiang, 2019; Li et al., 2011). food worldwide. As with peanuts, phenolic compounds other than tocopherols are predominantly present in the brown skin that covers Brazil nut kernels (John and Shahidi, 2010). There are several studies con- References cerning the in vitro antioxidant activity of Brazil nuts (Wu et al., 2004; Kornsteiner et al., 2006; Miraliakbari and Shahidi, 2008c; Abe, L.T., Lajolo, F.M., and Genovese, M.I. (2010). Comparison of phenol Abe et al., 2010; John and Shahidi, 2010; Gomes and Torres, 2016), content and antioxidant capacity of nuts. Ciência e Tecnologia de Ali- which present inconsistent results, likely due to the variation in the mentos. 30: 254–259. presence (and if, in which proportion) or absence of the brown skin Cardoso, B.R., Duarte, G.B.S., Reis, B.Z., and Cozzolino, S.M.F. (2017). Food in the analyzed samples. Only two studies identified the bioactive Res. Int. 100: 9–18. compounds related to the antioxidant activity of Brazil nuts (John Chunhieng, T., Petritis, K., Elfakir, C., Brochier, J., Goli, T., and Montet, D. and Shahidi, 2010; Gomes and Torres, 2016). The brown skin was (2004). Study of selenium distribution in the protein fractions of the found to have more than 1.2 g of gallic acid equivalents (GAE) Brazil nut, Bertholletia excelsa. J. Agric. Food Chem. 52: 4318–4322. Duarte, G.B.S., Reis, B.Z., Rogero, M.M., Vargas-Mendez, E., Barbosa Jr, per 100 g of soluble phenolics and 0.35 g GAE/100 g of bound F., Cercato, C., and Cozzolino, S.M.F. (2019). Consumption of Brazil phenolics, while the whole nut had 0.52 g GAE/ 100 g of soluble nuts with high selenium levels increased inflammation biomarkers phenolics and 18 mg GAE/100 g of bound phenolics (John and in obese women: A randomized controlled trial. Nutrition. 63-64: Shahidi, 2010). Extracts prepared from the brown skin clearly had 162–168. the highest in vitro antioxidant activities, and the major phenolics Food and Drug Administration–FDA. (2003). Qualified Health Claims: Let- identified by HPLC-ESI(-)-MS were phenolic acids and flavonoid ter of Enforcement Discretion–Nuts and Coronary Heart Disease, derivatives like gallic acid, gallocatechin, protocatechuic acid, cat- Docket n° 02P-0505. FDA, Washington, DC. echin, vanillic acid, taxifolin, myricetin, ellagic acid and quercetin Ghirardello, D., Bertolino, M., Belviso, S., Dal Bello, B., Giordano, M., Rolle, (John and Shahidi, 2010). Seventy percent aqueous acetone was L., Gerbi, V., Antonucci, M., Spigolon, N., and Zeppa, G. (2016). Phe- used for extraction, which was the solvent mixture with the highest nolic composition, antioxidant capacity and hexanal content of hazelnuts (Corylus avellana L.) as affected by different storage conditions. yield for total phenolics for both, kernel and brown skin, defat- Postharvest Biol. Technol. 112: 95–104. ted or whole, when compared to methanol and ethanol (John and Gomes, S., and Torres, A.G. (2016). Optimized extraction of polyphenolic Shahidi, 2010). In another study, a higher yield was obtained using antioxidant compounds from Brazil nut (Bertholletia excelsa) cake 40% aqueous ethanol, when compared to acetone and water com- and evaluation of the polyphenol profile by HPLC. J. Sci. Food Agric. binations under optimized conditions for obtaining antioxidant ex- 96: 2805–2814. tracts of the whole defatted nuts. Gallic acid, protocatechuic acid, Gutierrez, E.M.R., Regitano-d’Arce, M.A.B., and Rauen-Miguel, A.M.O. 2,4-dihydroxybenzoic acid, p-hydroxybenzoic acid, p-coumaric (1997). Estabilidade oxidativa do óleo bruto da castanha-do-pará acid, sinapic acid and catechin were identified in the ethanol/water (Bertholletia excelsa). Ciência e Tecnologia de Alimentos. 17: 22–27. extracts by RP-HPLC-DAD (Gomes and Torres, 2016). Phenolic Halliwell, B. (1990). How to characterize a biological antioxidant. Free compounds have been demonstrated to render protective effects Radical Res. Commun. 9: 1–32. Jiang, Q. (2019). Natural forms of vitamin E and metabolites — regula- against coronary heart disease such as by reducing platelet activity tion of cancer cell death and underlying mechanisms. IUBMB Life. and inhibiting LDL oxidation (Lutz et al., 2019). To the best of our 71: 495–506. knowledge, there are no studies concerning stability of these phe- John, J.A., and Shahidi, F. (2010). Phenolic compounds and antioxidant nolics during storage of Brazil nuts. Nevertheless, for comparison, activity of Brazil nut (Bertholletia excelsa). J. Functional Foods 2: no significant changes during 2 years of storage were observed 196–210.

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Kamal-Eldin, A., and Appelqvist, L.Å. (1996). The chemistry and antioxi- Sartori, A.G.O., Sampaio, G.R., Bastos, D.H.M., Regitano d’Arce, M.A.B., dant properties of tocopherols and tocotrienols. Lipids. 31: 671–701. and Skibsted, L.H. (2018a). Volatiles and tendency of radical forma- Kannamkumarath, S.S., Wrobel, K., Vonderheide, A., and Caruso, J.A. tion of cold-pressed Brazil nut oil during ambient storage. J. Am. Oil (2002). HPLC-ICP-MS determination of selenium distribution and spe- Chem. Soc. 95: 721–730. ciation in different types of nut. Anal. Bioanal. Chem. 373: 454–460. Sartori, A.G.O., Sampaio, G.R., Bastos, D.H.M., Spoto, M.H.F., Skibsted, Kornsteiner, M., Wagner, K.H., and Elmadfa, I. (2006). Tocopherols and L.H., and Regitano-d’Arce, M.A.B. (2018c). Tendency of lipid radical total phenolics in 10 different nut types. Food Chem. 98: 381–387. formation and volatiles in lose or vacuum-packed Brazil nuts stored Lemire, M., Philibert, A., Fillion, M., Passos, C.J.S., Guimarães, J.R.D., Bar- at room temperature or under refrigeration. Grasas y Aceites 69: bosa, F., and Mergler, D. (2012). No evidence of selenosis from a se- e283. lenium-rich diet in the Brazilian Amazon. Environment international. Silva, E.C. Jr, Wadt, L.H.O., Silva, K.E., Lima, R.M.B., Batista, K.D., Guedes, 40: 128–136. M.C., Carvalho, G.S., Carvalho, T.S., Reis, A.R., Lopes, G., and Guil- Li, G.X., Lee, M.J., Liu, A.B., Yang, Z., Lin, Y., Shih, W.J., and Yang, C.S. (2011). herme, L.R.G. (2017). Natural variation of selenium in Brazil nuts and δ-Tocopherol is more active than α- or γ-tocopherol in inhibiting lung soils from the Amazon region. Chemosphere 188: 650–658. tumorigenesis in vivo. Cancer Prev. Res. 4: 404–413. Silva, F.M.A., Hanna, A.C.S., Souza, A.A., Silva-Filho, F.A., Canhoto, O.M.F., Lutz, M., Fuentes, E., Ávila, F., Alarcón, M., and Palomo, I. (2019). Roles of Magalhães, A., Benevides, P.J.C., Azevedo, M.B.M., Siani, A.C., Pohlit, Phenolic Compounds in the Reduction of Risk Factors of Cardiovascu- A.M., Souza, A.D.L., and Koolen, H.H.F. (2019). Integrative Analysis lar Diseases. Molecules. 24: 366. Based on HPLC-DAD-MS/MS and NMR of Bertholletia excelsa Bark Martens, I.B.G., Cardoso, B.R., Hare, D.J., Niedzwiecki, M.M., Lajolo, F.M., Biomass Residues: Determination of Ellagic Acid Derivatives. J. Braz. Martens, A.M., and Cozzolino, S.M.F. (2015). Selenium status in pre- Chem. Soc. 30: 830–836. school children receiving a Brazil nut–enriched diet. Nutrition. 31: Tsai, J.H., Hiserodt, R.D., Ho, C.T., Hartman, T.G., and Rosen, R.T. (1998). De- 1339–1343. termination of volatile organic selenium compounds from the Mail- Miraliakbari, H., and Shahidi, F. (2008a). Oxidative stability of tree nut oils. lard Reaction in a selenomethionine-glucose model system. J Agric. J. Agric. Food Chem. 56: 4751–4759. Food Chem. 46: 2541–2545. Miraliakbari, H., and Shahidi, F. (2008b). Lipid class compositions, toco- United States Department of Agriculture–USDA. (2019). National nutrient pherols and sterols of tree nut oils extracted with different solvents. database for standard reference. https://fdc.nal.usda.gov/fdc-app. J. Food Lipids. 15: 81–96. html#/?query=ndbNumber:12078. Accessed 22 Jan 2020. Miraliakbari, H., and Shahidi, F. (2008c). Antioxidant activity of minor com- Universidade Estadual de Campinas –Unicamp. (2006). Tabela Brasileira ponents of tree nut oils. Food Chem. 111: 421–427. de Composição de Alimentos. Unicamp, Campinas. Mortensen, A., and Skibsted, L.H. (1997). Relative stability of carotenoid Venkatachalam, M., and Sathe, S.K. (2006). Chemical composition of se- radical cations and homologue tocopheroxyl radicals. A real time ki- lected edible nut seeds. J. Agric. Food Chem. 54: 4705–4714. netic study of antioxidant hierarchy. FEBS Letters. 417: 261–266. Vieira, T.M., and Regitano-d’Arce, M.A. (1999). Antioxidant concentration Regitano-d’Arce, M.A.B. (1998). Castanha do Pará: óleo e subprodutos sob effect on stability of Brazil nut (Bertholletia excelsa) crude oil. Archi- a ótica da lipidologia. University of São Paulo, Piracicaba, p. 64. vos Latinoamericanos de Nutricion 49: 271–274. Sartori, A.G.O., Alencar, S.M., Bastos, D.H.M., Regitano-d’Arce, M.A.B., and Wu, X., Beecher, G.R., Holden, J.M., Haytowitz, D.B., Gebhardt, S.E., and Skibsted, L.H. (2018b). Effect of water activity on lipid oxidation and Prior, R.L. (2004). Lipophilic and hydrophilic antioxidant capacities of nonenzymatic browning in Brazil nut flour. Eur. Food Res. Technol. common foods in the United States. J. Agric. Food Chem. 52: 4026– 244: 1657–1663. 4037. 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Journal of Food Bioactives | www.isnff-jfb.com 39 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Review J. Food Bioact. 2020;9:40–51

Blackberry polyphenols: review of composition, quantity, and health impacts from in vitro and in vivo studies

Julie A. Robinsona, Julianne E. Bierwirtha, Phillip Greenspanb and Ronald B. Pegga* aDepartment of Food Science & Technology, College of Agricultural and Environmental Sciences, The University of Georgia, 100 Cedar Street, Athens, GA 30602, USA bDepartment of Pharmaceutical & Biomedical Sciences, College of Pharmacy, The University of Georgia, 250 W. Green Street, Athens, GA 30602, USA *Corresponding author: Department of Food Science & Technology, College of Agricultural and Environmental Sciences, The University of Georgia, 100 Cedar Street, Athens, GA 30602, USA. Tel: +1-706-542-1099; Fax: +1-706-542-1050; E-mail: [email protected] DOI: 10.31665/JFB.2020.9217 Received: March 31, 2020; Revised received & accepted: March 31, 2020 Abbreviations: C3G, cyanidin-3-O-glucoside; DoP, degree of polymerization; DAD, diode array detection; DPPH, 2,2′-diphenyl-1-picrylhydrazyl; FW, fresh weight; HHDP, hexahydroxydiphenic acid; MI, myocardial infarction; MS, mass spectrometry; NF-κB, nuclear factor kappa-B; PACs, proanthocyanidins; RP-HPLC, reversed-phase high performance liquid chromatography Citation: Robinson, J.A., Bierwirth, J.E., Greenspan, P., and Pegg, R.B. (2020). Blackberry polyphenols: review of composition, quantity, and health impacts from in vitro and in vivo studies. J. Food Bioact. 9: 40–51.

Abstract

Blackberries possess marked concentrations of antioxidant polyphenols, most notably anthocyanins, ellagic acid, ellagitannins, epi/catechin, and proanthocyanidins. While the quantity and type of polyphenols can vary, cyanidin-3-O-glucoside (C3G) is consistently the most abundant polyphenol found in blackberries. Though blackberry antioxidants have not demonstrated significant bioavailability or bioaccessibility in their native form, emerging research suggests that antioxidant metabolites, such as protocatechuic acid derived from C3G, may account for some of the in vivo benefits. Cell studies conducted with blackberry phenolic extracts have demonstrated anti- inflammatory, chemopreventive, and neuroprotective effects; whereas, animal models given blackberries have exhibited reduced insulin resistance, attenuated weight gain, and a decrease in inflammatory markers. Blackberry consumption can lead to positive health outcomes. In human health cross-sectional and longitudinal studies, fresh berry consumption has been associated with better long-term insulin resistance, cognitive function, bone density, and cardiovascular function. Research studies, combiningin vitro digestion and absorption with targeted cell studies, are being performed to better understand the metabolism and bioactivity of blackberry phenolics. These compounds are not only absorbed by the body, but also can attach to the lining of the digestive tract or be fermented in the colon, contributing to the health outcomes afforded by blackberry consumption.

Keywords: Blackberries; Polyphenols; Antioxidants; Anthocyanins; Ellagitannins; Proanthocyanidins.

1. Introduction disease progression (Olas, 2018). Antioxidant research has demonstrated that increased consumption of polyphenol-rich foods, such Blackberries, Rubus spp., are cultivated worldwide and are of as blackberries, can have anticancer, antimutagenic, antimicrobial, growing commercial relevance. Blackberries are usually con- anti-inflammatory, and neuroprotective efficacy (Nile and Park, sumed fresh, but are also made into jams, juices, pies/cobblers, 2014). Blackberries are a source of non-nutritive compounds, and extracts. Modern consumers have become keenly aware of such as fiber and polyphenols, as well as nutrients including vita- the potential of functional foods for preventing or slowing chronic mins, minerals, and sugars (Kaume et al., 2012; Olas, 2018). The

Figure 1. Chemical structures of selected non-tannin polyphenolic antioxidants reported in blackberries. (a) protocatechuic acid glucoside; (b) ellagic acid; (c) hexahydroxydiphenic acid (HHDP); (d) cyanidin-3-O-glucoside (C3G); and (e) quercetin-3-O-galactoside. phenolics most often found in blackberries include anthocyanins, mendations from the USDA, based on research that demonstrates tannins, hydroxybenzoic acids, and flavan-3-ols. Anthocyanins, increased consumption can protect against disease. Antioxidant particularly cyanidin-3-O-glucoside (C3G), are consistently the compounds, including phenolics, do not have established govern- predominant phenolic antioxidant found in blackberries, and have mental recommendations. Because polyphenols have been shown been shown to have prominent bioactivity (Schulz et al., 2019). to ameliorate diseases that arise from reactive oxygen species, there The composition and content of blackberry phenolics can vary could be a benefit to establishing governmental recommendations due to location, cultivar, and time of harvest. Different varieties for phenolic antioxidants (Olas, 2018). Research on high-dose anti- grown in the same region have been found to contain different oxidant supplementation with vitamin C, vitamin E, and β-carotene amounts of phenolics (Liao et al., 2020; Fan-Chiang and Wrolstad, have failed to demonstrate an increase in health benefits, and in 2005). Cultivars harvested later in the year or at greater degrees some studies have shown harmful effects (National Center for Com- of ripeness can possess an elevated total phenolics content (TPC) plimentary and Integrative Health [NCCIH], 2013). Polyphenols (Liao et al., 2020; Mikulic-Petkovsek et al., 2017; Siriwoharn and can have a pro-oxidant effect under conditions that favor autoxida- Wrolstad, 2004). Not all studies have reported the same finding: tion, but berry extracts have not been found to have the same po- Siriwoharn et al. (2004), for instance, did not find an increase in tentially toxic health effects, which high doses of other antioxidants the TPC based on ripeness or harvest time; so, this could be spe- have demonstrated (Olas 2018). This review focuses on recent work cific to certain growing locations or cultivars. There is a variety characterizing blackberry antioxidants as well as the in vitro and in of different blackberry categories based on how they grow: these vivo studies conducted to determine their physiological effects. include thorny or thornless as well as erect, semi-erect, or trailing. Depending on which category is investigated, different profiles of the blackberry phenolics will be observed (Kolniak-Ostek et al., 2. Phenolic composition of blackberries 2015). The soil composition also can modify the phenolics content in blackberries. For instance when vermicompost was added to Thousands of phenolic antioxidant compounds exist and are classi- soil, the TPCs and anthocyanin contents were greater (Abud-Arch- fied into several categories based on structural similarities. For ex- ila et al., 2018). Bioactive compounds also increased when the soil ample, Craft et al. (2012) separated phenolics into seven categories: was irrigated, and the climate was temperate (Croge et al., 2019). phenolic acids, coumarins, flavonoids, isoflavonoids, stilbenes, lig- Current US antioxidant recommendations have focused on nans, and phenolic polymers (tannins). These phenolic groups differ studies of vitamins and minerals such as vitamin C, vitamin E, from each other in functional group placement, or the addition of a β-carotene (a precursor to vitamin A), selenium, and zinc (National chemical moiety as in glycosylation (Craft et al., 2012). Chemical Center for Complimentary and Integrative Health [NCCIH], 2013). structures of selected non-tannin polyphenolic antioxidants and tan- These vitamins and minerals have established dietary intake recom- nins of blackberries are illustrated in Figures 1 and 2, respectively.

Journal of Food Bioactives | www.isnff-jfb.com 41 Blackberry phenolics Robinson et al.

Figure 2. Chemical structures of selected tannin constituents reported in blackberries. (a) pedunculagin; (b) castalagin; (c) sanguiin H-6; (d) lambertianin C; and (e) procyanidin B-type dimer.

The TPC of blackberries varied between 140 and 960 mg gallic is the phenolic most often reported for comparison against litera- acid equivalents (GAE) per 100 g FW (Table 1). Gallic acid is only ture results (Liao et al., 2020; Pantelidis et al., 2007). In terms of present in small quantities in blackberries, and therefore is not the blackberries, phenolic acids, flavonoids, and tannins are the most desired phenolic acid standard for the Folin-Ciocalteu assay; yet, it prevalent phenolic classes.

Table 1. Range of total phenolics content, total monomeric anthocyanin content, and total ellagitannins content in blackberries TPC (mg GAE/100 TMAC (mg C3G Total Ellagitannins Reference Location g FW) eq./100 g FW) (mg EAE/100 g FW) Liao et al., 2020 Georgia, USA 409–459 140–150 10.4–27.8 Sellappan et al., 2002 Georgia, USA 418–555 111–123 – Siriwoharn & Oregon, USA 822–844 154–225 – Wrolstad, 2004 Siriwoharn et al., 2004 Oregon, USA 903–960 131–221 20.7–25.4 Fan-Chiang & Oregon, Oklahoma, France, – 70.3–201 – Wrolstad, 2005 Macedonia, Mexico and Chile Van de Velde et al., 2016 Argentina 140–162 107–124 8.9–10.3

Abbreviations: TPC, total phenolics content; GAE, gallic acid equivalents; FW, fresh weight; TMAC, total monomeric anthocyanins; C3G eq., cyanidin-3-O-glucoside equivalents; EAE, ellagic acid equivalents; –, not reported or measured.

42 Journal of Food Bioactives | www.isnff-jfb.com Robinson et al. Blackberry phenolics

2.1. Phenolic acids other derivatives, while in other works, they are segregated into free catechin/epicatechin from their derivative forms. The primary phenolic acid in blackberries is ellagic acid; that is, a polyphenol with four resonance stabilized hydroxy groups, clas- 2.2.2. Anthocyanins sified in the hydroxybenzoic acid family of compounds (Fig. 1). Ellagic acid is commonly glycosylated, and is synthesized during Anthocyanins contribute to the dark blue color in blackberries, and fruit ripening as ellagitannins are degraded (Komorsky-Lovrić and are the antioxidant class most associated with red berries and fruits. Novak, 2011). Blackberries are a significant source of ellagitan- An anthocyanin (i.e., a glycoside in which the sugar is generally at- nins as well as their hydrolytic breakdown products, ellagic acid tached at the 3-position of the C-ring) forms a pH dependent 2-phe- and gallic acid. Ellagic acid concentration is highest in the seeds; nylbenzopyrylium (flavylium) cation that expresses the deep red so, juice processing that homogenizes the seed as well as the flesh color of the blackberries in acidic environments. Anthocyanin intake and skin retains roughly the same ellagic acid content as that of the has been associated with a reduction in insulin resistance, a decreased whole fruit (Djurić et al., 2014). There is evidence that ellagic acid risk of myocardial infarction (MI), and moderating the inflammatory metabolites can reduce the risk of cancer and mitigate inflamma- response (Cassidy et al., 2013; Jennings et al., 2012; 2014). tory response in humans (Espín et al., 2013; Larrosa et al., 2010). While the coloration of anthocyanins allows for easy spectro- Ellagic acid absorbs electromagnetic radiation at wavelengths of photometric detection at a wavelength of 520 nm, the diversity of 255 and 320 nm, and is often quantified by hydrolyzing the glyco- anthocyanins and their derivatives makes isolating individual mol- side and then analyzed by reversed-phase high performance liquid ecules extremely challenging. Separation is often achieved by RP- chromatography (RP-HPLC) coupled with diode array detection HPLC and compound identification by mass spectrometry (MS). (DAD). Concentrations have been found to vary greatly among Fan-Chiang and Wrolstad (2005) determined the anthocyanin con- blackberry species and cultivars, ranging from 30.0 to 54.8 mg/100 tents in eighteen different blackberry varieties from five different g FW (Djurić et al., 2014; Jakobek and Seruga, 2012; Komorsky- locations and found that the total monomeric anthocyanins con- Lovrić and Novak, 2011; Sellappan et al., 2002). Phenolic acids tents (TMACs) ranged from 70.3 to 201 mg/100 g FW (Table 1). sometimes present in lesser concentrations may include gallic acid They reported the presence of five dominant anthocyanins, namely (3.4–6.4 mg/100 g FW), caffeic acid (1.38–3.64 mg/100 g FW), C3G averaging 82.9% of the anthocyanins, cyanidin-3-O-rutino- p-coumaric acid (0.40–2.08 mg/100 g FW), and ferulic acid (2.99– side at 10.2%, cyanidin-3-O-xyloside at 2.5%, cyanidin-3-O-diox- 3.51 mg/100 g FW) (da Rosa et al., 2014; Sellappan et al., 2002). ylglucoside at 2.5%, and cyanidin-3-O-malonylglucoside at 1.9%. As listed in Table 2, C3G is the chief anthocyanin, comprising as 2.2. Flavonoids much as 92% of all anthocyanins in blackberries (Fan-Chiang and Wrolstad, 2005; Niculescu et al., 2013; Ştefănuţ et al., 2011; Zhang et al., 2012). Other cyanidin-based anthocyanins have been identified 2.2.1. Flavonols and flavan-3-ols as well, though inconsistently (Table 1). The composition of anthocyanins in blackberries can vary due to location, growing conditions, (+)-Catechin and its isomer (−)-epicatechin are consistently the time of harvest, and differences in cultivars (Fernandes et al., 2014; most abundant flavan-3-ols in blackberries. Catechin, like ellagic Liao et al., 2020). Cyanidin is the primary aglycone, comprising as acid, possesses two aromatic rings with four resonance-stabilized much as 71 mg/100 g FW in one study (Jakobek and Seruga, 2012). hydroxy groups that can act as hydrogen atom or electron donors. The antiradical activity of the anthocyanins in blackberries, as meas- Proanthocyanidins (PACs or condensed tannins) are the polymeric ured by the DPPH radical assay, is almost seven times that of the form, which is present in the developing berry and is degraded combined flavonols and phenolic acids (Jakobek and Seruga, 2012). during ripening to release free (+)-catechin and (−)-epicatechin. The results of these bench-top measures imply that most health ben- Catechins are found most abundantly in the skins of the fruit, as efits of blackberries are due to the bioactivity of anthocyanins. opposed to the flesh or seeds (Nile and Park, 2014). There is strong evidence for the positive impact of consuming flavan-3-ols on cognitive and cardiovascular function (Mastroiacovo et al., 2015; 2.3. Tannins Milenkovic et al., 2014). Catechin and epicatechin are detectable by RP-HPLC-DAD at a UV wavelength of 280 nm. Catechin and Tannins can be classified as either hydrolyzable or PACs. The epicatechin have been reported at concentrations of 378.7 ± 24.5 breakdown of tannins over the course of fruit ripening is respon- µg/100 g and 448.4 ± 7.5 µg/100 g dry matter, respectively, in sible for many of the visual and sensory characteristics of mature blackberries (Schulz et al., 2019). berries. PACs hydrolyze to form (+)-catechin and (−)-epicatechin. Flavan-3-ols are one of the phenolic classes, whose concen- Ellagitannins, an example of a hydrolyzable tannin, can undergo tration is strongly dependent on varietal or growth conditions. As lactonization spontaneously to form hexahydroxydiphenic acid shown in Table 2, catechin was reported in methanolic/HCl extracts (HHDP, see Fig. 1), a precursor to ellagic acid (Lipińska et al., of Georgia-grown blackberries at concentrations ranging from 266 2014), which also contributes to tartness. Tannins that remain in- to 313 mg/100 g FW (Sellappan et al., 2002), but for berries grown tact are capable of binding to proteins. This both gives an astrin- in Ecuador, the catechin levels in an ethyl acetate extract were re- gent mouth-feel, as tannins bind to the salivary proteins, and reported as being undetectable (Jakobek and Seruga, 2012; Vasco duces the bioavailability of digested protein (Nile and Park, 2014). et al., 2009). When examining the concentrations of flavan-ols in literature, as well as flavonoids in general, one must be careful to note if an acid hydrolysis step has been employed prior to HPLC 2.3.1. Proanthocyanidins (PACs) analysis. If so, then sugar moieties would be cleaved thereby releas- ing aglycones and markedly influencing the quantitative findings. PACs, or condensed tannins, are a polymerized form of (+)-cat- In some papers, flavan-3-ol levels are reported as (+)-catechin and echin and (−)-epicatechin. Their biological significance is largely (−) epicatechin with no mention of the presence of glycosides or dependent on their degree of polymerization (DoP); intestinal ab-

Journal of Food Bioactives | www.isnff-jfb.com 43 Blackberry phenolics Robinson et al.

Table 2. Content of dominant phenolic acids, anthocyanins, flavonols, and tannins reported in blackberries in mg/100 g fresh weight Phenolic Acids ellagic acid gallic acid p-coumaric acid Liao et al., 2020 – – 0.55 Fan-Chiang & Wrolstad, 2005 – – – Kolniak-Ostek et al., 2015 0.66–2.71 – nd–0.47 Mertz et al., 2007 * nd–0.24 0.06–0.22 0.50–0.76 Schulz et al., 2019 * – 0.09–0.18 nd–0.03 Sellappan et al., 2002 30.01–33.81 4.12–6.42 0.4–2.08 Siriwoharn et al., 2004 1.64–3.62 – – Van de Velde et al., 2016 – – –

Anthocyanins and Flavonols cyanidin-3-O- cyanidin-3-O- cyanidin-3-O- cyanidin- cyanidin-3-O- quercetin-3-O- glucoside malonylglucoside rutinoside 3-O-xyloside dioxalylglucoside galactoside Liao et al., 2020 99.9 – – – – 7.06 Fan-Chiang & 30.65–191.35 nd–10.3 nd–107.3 nd–22.71 nd–30 – Wrolstad, 2005 Kolniak-Ostek et al., 2015 25.15–122.54 0.96–3.34 nd–4.66 nd–6.37 nd–3.03 0.72–2.56 Mertz et al., 2007 * 45.6–81.6 nd–4.8 nd–75.6 – – – Schulz et al., 2019 * – – – – – – Sellappan et al., 2002 – – – – – nd Siriwoharn et al., 2004 72.6–81.2 1.12–4.72 2.71–24.2 0.18–6.30 1.91–5.07 – Van de Velde et al., 2016 91.4–107.3 5.8–7.1 – 0.8–2.9 – 2.94–4.02

Proanthocyanidin Monomer and Tannins (−)-epicatechin lambertianin C sanguiin H-6 Liao et al., 2020 – 5.47 – Fan-Chiang & Wrolstad, 2005 – – – Kolniak-Ostek et al., 2015 – 2.70–4.56 1.00–4.82 Mertz et al., 2007 * 0.61–0.76 62.4–71.8 50.4–294 Schulz et al., 2019 * 0.03–0.05 – – Sellappan et al., 2002 nd – – Siriwoharn & Wrolstad, 2004 1.43–4.41 – – Van de Velde et al., 2016 – 3.8–4.7 –

Abbreviations: –, not reported or measured; nd, not detectable but looked for; *, results reported on a dry weight have been converted to fresh weight according to the mass balance details. sorption of PAC dimers is less than 10%, and those with a DoP and carcinogenesis (Manach et al., 2005). Amongst six species of greater than 4 units are not absorbed at all (Ou and Gu, 2014). Un- Mexican blackberries, PAC oligomers were found ranging from fortunately, PACs appear not to hydrolyze or degrade significantly 2 to 6 catechin/epicatechin subunits, with measurement of a PAC to form monomers during human digestion (Rios et al., 2002). Ou content averaging 3.32 to 5.37 g catechin equivalents/100 g FW. and Gu (2014) reported that PACs are digested by intestinal mi- (Cuevas-Rodríguez et al., 2010). crobes to form a series of bioavailable aromatic acid metabolites. While this may be the primary health-promoting mechanism, this digestion also decreases with increasing DoP. Despite absorption 2.3.2. Hydrolyzable tannins limitations, studies have posited that PACs may be beneficial to health by protecting the alimentary canal from oxidative stress An ellagitannin (a class of hydrolyzable tannins) is defined by the

44 Journal of Food Bioactives | www.isnff-jfb.com Robinson et al. Blackberry phenolics formation of an ester bond between HHDP and a monosaccharide. homogenization, oral phase, gastric phase, small intestine phase Ellagitannins exist frequently in nature as high-molecular-weight and isolation of the digesta as well as its uptake by cells. The ef- polymers, but they are highly susceptible to changes including fect of colonic metabolism will be discussed in section 3.2.2. As polymerization to form higher DoP oligomers or hydrolysis by explained by Carbonell-Capella (2014), methodologies in studies intestinal microorganisms to form beneficial urolithins (Tomás- can differ at every step in the process, including level of sample Barberán et al., 2014). The study of Tomás-Barberán et al. (2014) homogenization, incubation time for each phase, and isolation found inter-subject variability in the metabolites formed from el- method for the resulting digesta. Varying the enzyme mixture and lagic acid and ellagitannins, suggesting that the microbial diversity concentration can have a profound impact on the results obtained. of the intestines influences their bioactivity. Like ellagic acid, ella- While gastric pepsin has seemingly no effect on the phenolic gitannins exhibit anti-inflammatory and anti-carcinogenic proper- composition, α-amylase may conjugate with anthocyanins and ties (Espín et al., 2013; Larrosa et al., 2010). Blackberry ellagitan- reduce their bioavailability (Wiese et al., 2009). The condition of nins are primarily a mixture of sanguiin H-6/lambertianin A and the sample prior to digestion influences the result as well. For lambertianin C with total ellagitannin contents ranging from 23 to instance, a homogenized sample may show increased bioavail- 343 mg/100 g FW, making them relatively abundant when com- ability, but this does not accurately mimic human mastication pat- pared to other fruits (Hager et al, 2008; 2010). Like ellagic acid, terns like for a study in which the sample is ground (Alminger et Hager et al. (2010) found the majority of ellagitannins in the seeds al., 2014). A more recent digestion study followed in vitro diges- and that traditional juicing can reduce the ellagitannin content by tion with gut microbiota fermentation to mimic the environment 70 to 82%. in which polyphenols and their derivatives undergo in the colon. The results showed improved antidiabetic and antioxidant activities compared to in vitro gastric and duodenal digestion alone 3. Bioavailability and bioaccessibility of blackberry phenolics (Gowd et al., 2018). A relatively simple and inexpensive way to mimic intestinal absorption is via dialysis tubing, in which a selectively permeable Crucial to the understanding of any functional food or phytochemi- membrane separates the theoretically absorbed molecules from cal is the digestion and absorption process, which the nutrients and unabsorbed colon-bound ones (Tavares et al., 2012; 2013). More bioactives undergo. Research often focuses on biomolecules of complex systems use Caco-2 cells, a line of human colon cancer food that are absorbed from the digestive tract to the bloodstream; cells differentiated to express intestinal epithelial properties. While nonetheless, constituents that are not absorbed should not be dis- a Caco-2 system may be more representative of human absorp- counted in terms of providing a physiological function. Examining tion, laboratory conditions, cost, amount of time required, and the and perceiving how polyphenols, especially anthocyanins, break- necessary equipment can make it prohibitive (Carbonell-Capella down during digestion and interact with intestinal lining and gut et al., 2014). microbiota might possibly explain the health benefits afforded by consuming blackberries, even though the measured bioavailability is low (Fernandes et al., 2014). Though they may not interact with 3.1.2. In vitro digestion and absorption of blackberries and other systemic organ systems, these constituents may still provide relevant compounds a benefit to the epithelial lining of the alimentary canal (Manach et al., 2005). Most research investigating the effect of foods and food An in vitro digestion study (Tavares et al., 2013), in which black- components on cultured cells does not factor in the effect of in berries from Portugal underwent digestion and simulated absorp- vitro digestion and absorption. Such studies determine the poten- tion at physiologically relevant concentrations, reported a 83 to tial health effects of phenolics that may never reach the intestinal 95% post-absorption reduction in antioxidant capacity with a 94.5 epithelium or may not be absorbed into the bloodstream. Further, to 99.5% reduction in the TPC. In another study by the same au- they habitually dismiss the role of the metabolism of ingested thors, the pre-absorption reduction in TPC and antioxidant capac- phenolic compounds (Bowen-Forbes et al., 2010; Calvo-Castro ity was found to be 93 and 49.4%, respectively (Tavares et al., et al., 2013). Other studies ignore matrix effects by assessing the 2012). The reduced antioxidant capacity of dietary phenolics be- absorption of purified compounds, often at higher-than-relevant fore absorption can result from oxidation, non-covalent binding concentrations (Serra et al., 2013). These can address the cellular with intestinal proteins, and transformation into glucuronidated, response to a particular phenolic compound, but they should be sulfated, or methylated metabolites. Noteworthy is that anthocya- framed within an understanding of the physiological concentration nins have been found to be moderately resistant to such modifica- at which that chemical would be exposed to similar cells during ditions (Carbonell-Capella et al., 2014; Yang et al., 2011). Tavares gestion in humans. Nonetheless, an understanding of the chemical et al. (2013) also noted that wild blackberry cultivars best retained modifications that take place during digestion as well as intestinal their antioxidant capacity after digestion as compared to commer- absorption kinetics is essential. This review will cover the research cial cultivars. on the digestion and absorption of blackberries, as well as its iso- Tomas et al. (2020) examined how the phenolic profile of black- lated phenolic constituents. berry puree changed depending on the fiber content added using an in vitro digestion and fermentation study. These researchers found 3.1. In vitro digestion and absorption that the interaction with the fiber during digestion lowered the TPC and changed the composition of the free- and bound-phenolics in the sample similarly to the study of Tavares et al. (2013). Further- 3.1.1. In vitro digestion methodology more, they found that a potential benefit of the added fiber was that anthocyanins were not bioavailable during gastric and duodenal Human digestion varies from person to person; thus, there is not digestion, but were found to offer an increased antioxidant poten- yet one accepted standard for the methodology of in vitro diges- tial in the large intestine (Tomas et al., 2020). tion studies (Carbonell-Capella et al., 2014). The majority of In vitro digestion studies are difficult to perform because poly- in vitro digestion trials include the following steps: grinding or phenols will be absorbed differently depending on the food matrix

Journal of Food Bioactives | www.isnff-jfb.com 45 Blackberry phenolics Robinson et al. in question, and can bind to the lining of the gastrointestinal tract. 2014). Similarly, PACs with high DoP were not shown to degrade Surveys on the bioavailability of individual polyphenolic classes significantly during gastrointestinal digestion in rats, but were find that most abundant dietary components are not necessarily the metabolized by colonic bacteria to form phenolic acids and other best absorbed. As discussed earlier, molecular weight/size often metabolites; this may account for their reported bioactivity (Ou plays a critical role in absorption. In general, small molecules such and Gu, 2014). These authors reported absorption rates of 10% as phenolic acids and catechins are all easily absorbed by the epi- and less for PAC dimers, trimers, and tetramers with decreasing thelium, whereas large and highly polymerized PACs and ellagi- absorption as the DoP increases. To further emphasize the role of tannins are not (Carbonell-Capella et al., 2014). microbiota-associated digestion, a study comparing C3G metabo- C3G and other anthocyanins are often the molecules of interest lism in rats with translocated human intestinal bacteria versus their in studies on the health effects of berry phenolics. Research find- ‘clean’ equivalents found over twice the recovery of C3G and its ings have repeatedly shown poor absorption kinetics for C3G and byproducts in the urine of the bacteria-rich group (Hanske et al., associated anthocyanins, but yet increased intake is still associated 2013). Digestion and absorption research increasingly points to with improved health outcomes (Cassidy et al., 2013; Jennings et intestinal microorganisms and their key role in metabolizing unab- al., 2014). Degradation products that occur in the small intestine, sorbed polyphenols into biologically active substrates. particularly the formation of protocatechuic acid, phloroglucinal- A 13C tracer can be used to help elucidate the metabolic path- dehyde and metabolites thereof, appear to be the absorbed bioac- way of a specific bioactive. Five hundred milligrams of 13C-la- tives of interest (Carbonell-Capella et al., 2014; Kay et al., 2009) beled C3G (with three 13C atoms on the A-ring and two 13C atoms on the B-ring) were fed to eight male participants with urine, 3.2. In vivo digestion and absorption blood, breath, and feces collected over 48 h (Czank et al., 2013). Urinary and blood concentrations peaked within an hour of consumption. Maximum elimination occurred from the breath at 6 h 3.2.1. In vivo digestion of blackberries and in feces between 6 and 24 h. At its height, fecal elimination was more than four times that of urinary elimination. Altogether, In a simple bioavailability study, eight healthy subjects consumed twenty-four metabolites of C3G were identified by HPLC-MS. 180 g of blackberries and were subsequently analyzed for plasma The primary metabolites at each stage were phenylacetic and and urine anthocyanin concentrations. C3G was metabolized pri- phenylpropenoic acids. Other metabolite classes included phase marily to peonidin-O-glucuronide. The concentrations of C3G and II conjugates possessing methylated and glucuronidated moie- its metabolites in the plasma (5 to 20 nmol/L) and the urine (0.08% ties, degradants such as protocatechuic acid and phloroglucinal- of ingested mass) were low compared to the amount consumed dehyde, conjugates of protocatechuic acid including vanillic acid (AlGamdi, 2013). A subsequent study was performed on five il- and 2-hydroxy-4-methoxybenzoic acid, and hippuric acid. The eostomy subjects to determine the influence of digestion and ab- most prevalent C3G metabolites in serum were phase II conju- sorption via the small intestine: unaltered blackberry anthocyanins gates of protocatechuic acid. The minimum bioavailability of were recovered at 5.1% of the initial dose (AlGamdi, 2013). In an C3G was 12.38% in this study (Czank et al., 2013). These results experiment to determine the distribution of anthocyanins and their further confirm the degradation and metabolism of C3G, account- derivatives in target organs, sixteen rats were fed a blackberry-en- ing for the low recovery of the parent compound in urine and its riched diet for twelve days. Urine, plasma, bladder, prostate, heart, low concentrations in serum. testes, and adipose tissue were analyzed for C3G and total antho- While only a small percentage of polyphenols are absorbed into cyanins concentration. The highest levels of C3G and anthocya- the blood stream, polyphenols and their metabolites could also po- nins were found in the bladder (1.37 nmol C3G/g tissue and 2.37 tentially attach to the intestinal epithelium and be important for the nmol C3G equivalents/g tissue, respectively). Very low concen- health of beneficial bacteria in the colon. The effect of polyphenols trations were recovered from blood serum and urine. The second on the gut microbiome has just recently begun to be investigated highest concentration of anthocyanins was found in the prostate in detail. For example, Ma and Chen (2020) determined that poly- (Felgines et al., 2009). Because studies have shown health benefits phenolic supplementation changed the types of bacteria found in from blackberry consumption, these rather low concentrations of the large intestine without significantly increasing or decreasing phenolics have either significant bioactivity or there are other ways total bacterial counts. Beneficial bacteria such as Lactobacillus in which blackberry components impart health benefits. spp. and Bifidobacterium spp. increased with polyphenolic supplementation by 220 and 56%, respectively. Other bacterial species such as Eubacterium, Bacteroides, Prevotella, Enterococcus, and 3.2.2. In vivo digestion of isolated phenolics of interest Enterobacteria were not consistently changed with polyphenolic supplementation (Ma and Chen, 2020). The following paragraphs summarize research on the bioavailability and bioabsorbability of isolated phenolic molecules commonly found in blackberries, which include epi/catechin, ellagic acid, 4. In Vitro cell studies ellagitannins, PACs, and anthocyanins. Epi/catechins and other flavan-3-ols are partially absorbed in the small intestines, but the Berry extracts have been employed in cultured in vitro cell studies majority travel to the large intestine where they are broken down in an effort to understand their potential for preventing or slowing into absorbable phenolic acids by colonic bacteria (Calani et al., the progression of cancer, diabetes, neurodegeneration, inflam- 2012). Free ellagic acid can be absorbed beginning in the gastric mation, and oxidative damage. It is important to note that, unless compartment and continuing in the small intestine. Ellagitannins, otherwise stated, the extracts investigated were undigested and on the other hand, must first be hydrolyzed in the small intestine unabsorbed, and therefore represent an incomplete estimation of to form ellagic acid and then transformed by intestinal microbiota the influence of orally consumed blackberries on target cells. Fur- to dibenzopyranone metabolites—urolithin A and B—which are thermore, different types of cell lines have been used in assays and absorbed in the colon and undergo glucuronidation (Lipińska et al., include Caco-2, HepG2, and BV-2 microglia cells, depending on

46 Journal of Food Bioactives | www.isnff-jfb.com Robinson et al. Blackberry phenolics whether the study was focused on absorption or neuroprotective complications by having the ingested carbohydrates remain intact, properties (Kellett et al., 2018; Ma et al., 2018). which Spínola et al. (2019) was able to demonstrate in vitro. When exposed to blackberry extracts (especially those rich in C3G), glucosidases, but not amylases, were significantly inhibited. Pro- 4.1. Chemoprevention tection from oxidative stress was also improved by a blackberry extract (Spínola et al., 2019). Research investigations on the effects of blackberry extracts on cancer progression are numerous. In a study of Jamaican- and Michigan-grown blackberry as well as raspberry fruits, Jamaican 4.5. Considerations blackberry extracts at a concentration of 250 μg/mL were found to significantly reduce the proliferation of breast, lung, colon and The duration of sample exposure presents a challenge to all cell gastric cancer cells by 24, 54, 50, and 57%, respectively (Bowen- studies. For example, cancer cells are exposed to high concen- Forbes et al., 2010). Promising findings have been documented for trations of anthocyanins for upwards of 24 h after one hour of leukemia; that is, over 24% of cells exposed to blackberry extracts consumption (Bowen-Forbes et al., 2010; Czank et al., 2013). at 100 μg/mL were converted to the G2/M phase of the cell cy- This would imply that chronic and sustained consumption of ber- cle (Zunino et al., 2010). Similarly, cells incubated with a Costa ries, or at least consumption concurrent with events like initia- Rica blackberry juice extract initiated apoptosis of UVB-damaged tion of inflammatory response, is necessary in order to achieve keratinocytes to a greater extent than that of control cells (Calvo- the desired benefit. Of further note, while in vitro cell studies Castro et al., 2013). Suppression of the inflammatory macrophage test the sample extracts for cytotoxicity, the lack of deleterious cytokine tumor necrosis factor-alpha, which can exacerbate cell effects from phenolics does not imply that physiological dosage proliferation and metastasis in cancers with transcriptional dysreg- would be the same. One study cites 4 μM as being the highest ulation by nuclear factor kappa-B (NF-κB), and AP-1 transcription physiological dose of serum polyphenols; yet, many studies have factors, is a postulated mechanism for these effects (Ding et al., used as much as 100 μM (Manach et al., 2005; Tavares et al., 2006; Milenkovic et al., 2014). 2012). The employment of higher than normal concentrations with longer than typical exposure times may mimic repeated long-term consumption effects, or it may simply create enough 4.2. Inflammation statistical power to achieve significant findings in, say, a model cell experimental system. Ideally, future cell studies will take Fermented blueberry- and blackberry-mixed juices at 100 μM C3G into account the in vitro digestive process and the bioactivity of equivalents concentration were able to drastically decrease inflam- the absorbed metabolites. mation by as much as 80% in murine RAW 264.7 macrophage cells, as measured by cyclooxygenase-2 and NF-κB inhibition (Johnson et al., 2013). C3G has shown even greater anti-inflam- 5. Animal studies matory activity over some anti-inflammatory drugs. Noteworthy is that C3G reduced cytokine-induced inflammation in human intes- Next to human clinical trials, animal studies are the best way in tinal HT-29 cells at a lower concentration than that of 5-aminosali- which to explore the bioactivity of blackberry extracts. Blackberry cylic acid (Serra et al., 2013). Ellagitannins from blackberries have juices and extracts administered to mice and rats have had many been reported to reduce inflammation via inhibition of the NF-κB effects including reductions in insulin resistance, inflammation, pathway in gastric cells (Sangiovanni et al., 2013). oxidative stress, and weight gain. In an assessment of the anti- hyperglycemic effect of berries, blackberry phenolic extracts were 4.3. Neurodegeneration administered to diabetic rats over the course of five weeks; the average serum glucose concentrations decreased significantly from The influence of digestion on thein vitro cellular response to black- 360 to 270 mg/dL (Ştefănuţ et al., 2013). berry extracts has been studied when neuroblastoma cells were ex- In a study on gastric inflammation, rats treated orally for ten days with blackberry ellagitannins showed reduced peptic ulcers, posed to H2O2 to mimic age-related neurodegeneration and then treated with either raw or digested, as well as dialyzed, blackberry protection from ethanol-induced stress, and inhibition of the extracts at physiological concentrations. The results showed not NF-κB pathway (Sangiovanni et al., 2013). A comparison of the only a protective effect at the lowest dose of the digested extract antioxidant potential of ellagitannins, anthocyanins, and whole (1.5 µM), but also no protective effect from the undigested frac- blackberry aqueous extracts was performed by feeding healthy tion, even at the highest concentration employed (Tavares et al., rats for 35 days. Antioxidation markers such as catalase, glu- 2012). The enhanced response of cells to the digested fraction sug- tathione, and superoxide dismutase levels were determined in the gests an adaptive response by mammalian cells to favor biologi- serum, erythrocytes, liver, kidney, spleen, and brain. The find- cally available polyphenols. This finding matches later research by ings indicated an antioxidant response to blackberry consump- the same investigators, who found an enhanced effect for digested tion (Hassimotto and Lajolo, 2011): this result is supported by wild blackberry species in preventing neuroblastoma cells from similar research in oxidatively stressed mice (Cho et al., 2011; oxidative neurodegeneration (Tavares et al., 2013). Hassan and Abdel-Aziz, 2010). Other effects of blackberry consumption on rats and mice include a shift in the microflora composition of mice fed blackberry powder (Park et al., 2013) and 4.4. Diabetes and obesity attenuated weight gain as well as inflammation in post-ovariectomized female rats consuming whole blackberries (Kaume et al., When carbohydrates are ingested they are broken down by en- 2012). When rats were given a blackberry extract before being zymes, including glucosidases and amylases, into monosaccha- induced into a manic state with ketamine, brain interleukin pro- rides. A type-2 diabetic could possibly prevent hyperglycemic teins, markers of the immune response, were reduced (Chaves

Journal of Food Bioactives | www.isnff-jfb.com 47 Blackberry phenolics Robinson et al. et al., 2020). These studies indicate that at physiological doses, encompassing 3,160 participants, total flavonoid and sub-categori- blackberries and their extracts can have a multitude of positive cal consumption acquired from food-frequency questionnaires was influences on a living organism. analyzed along with bone density at the spine and hip. Total flavonoid intake was deemed to be positively associated with higher bone mineral density. The strongest association among the sub-cat- 6. Human studies egories of flavonoids was with anthocyanins, which demonstrated a 3.4 and 3.1% greater mineral density at the spine and hip, respec- There exist human studies on blackberry consumption; however, tively. The second greatest association was found among flavones, most are not clinical trials, rather they are cross-sectional and lon- while all other subcategories had non-significant results (Welch et gitudinal studies of dietary patterns along with a number of habits al., 2012). and risk factors. This section describes research specific to blackberries, but also research on overall berry consumption, anthocya- 6.4. Heart health nin intake, and cyanidin supplementation. The Nurses’ Health Study population was used once again, this 6.1. Anti-hypoglycemic effects time to demonstrate the correlation between flavonoid intake and risk of MI. Out of 93,600 healthy participants aged 25 to 42 years, Diabetes complications can be exacerbated by oxidative stress. 405 cases of MI were documented over the 18-year follow up. Cor- Antioxidant consumption has been reported to help prevent such relation with food frequency questionnaires found that increased complications from worsening (Solverson et al., 2018). These au- intake of anthocyanin-rich foodstuffs significantly reduced the risk thors tested seventeen overweight or obese men by feeding them of MI, with high anthocyanin consumers approximately 32% less either 600 g of blackberries, or carbohydrate-matched gelatin of likely to suffer MI (Cassidy et al., 2013). Another study found that the same caloric content. The results showed that fat oxidation measures of arterial stiffness and blood pressure were improved and insulin sensitivity increased for the men fed blackberries, with among high anthocyanin and flavone consumers in a cross sec- younger subjects demonstrating the best improvement (Solverson tional study of 1,898 twins (Jennings et al., 2012). et al., 2018). A cross sectional study of 1,997 women investigated flavonoid consumption broken down by class against diabetes- related markers such as fasting glucose, insulin, and C-reactive 7. Conclusions protein. Of all classes of flavonoids, only anthocyanins and flavones imparted beneficial effects; that is, increased anthocyanin Blackberries possess a rich array of potentially bioactive constitu- consumption correlated with reduced peripheral insulin resistance, ents outside those with current dietary recommendations. C3G is reduced C-reactive protein, reduced insulin levels, and reduced in- the most studied and most understood of these phytochemicals, flammation (Jennings et al., 2014). A 2012 analysis of participants and may exhibit a greater influence on the health of the consumer in the Nurses Health Studies (NHS and NHS II) combined with the than any other individual molecule found in the fruit. Despite the Health Professionals Follow-Up Study confirmed this correlation. low reported absorption of many key bioactives in their native With a cohort of > 200,000 healthy individuals, 12,611 cases of form, modifications that occur during digestion may, in fact, im- type-2 diabetes were documented over the duration of the respec- prove intestinal absorption and effectiveness. Studies that do not tive studies. Of all of the flavonoid classes and subclasses, only consider digestion and absorption of phenolic constituents may anthocyanins were associated with a reduced risk in developing actually see a diminished effect in the raw, undigested samples as type-2 diabetes (Wedick et al., 2012). compared to the breakdown products and metabolites. In vitro cell studies, as well as animal studies, have shown sig- 6.2. Cognitive function nificant benefits of blackberry phenolics, including apoptosis of carcinogenic cells, reduced inflammation, lowered blood sugar, Research on the relationship between blackberry consumption and and decreased weight gain. Anthocyanins, as a class, are consist- cognitive function in humans is extremely limited. In a study of ently the most efficacious of blackberry phenolics in human cross- the long-term cognitive function of > 16,000 aged 70+ NHS par- sectional studies, longitudinal studies, and controlled clinical ticipants, dietary analysis identified that greater berry intake was trials. Human studies have correlated anthocyanin consumption associated with slower loss of cognition due to age. Participants also with improved bone density, improved cardiovascular func- who consumed large amounts of berries delayed that cognition loss tioning, improved memory, and reduced insulin resistance. Further by roughly 2.5 years over the course of the 6-year study (Devore research to understand the influence of blackberry phenolics on et al., 2012). cultured cell systems should attempt to mimic the modifications that occur in the alimentary canal employing relevant physiological concentrations. 6.3. Bone health Understanding of the function of blackberries in human health would be greatly augmented by additional randomized controlled Bone loss occurs naturally over time, but can be exacerbated by clinical trials. Ideally such studies would supplement the diet with lifestyle choices. In a clinical trial of female post-menopausal isolated blackberry phenolics, or else control for calories and other smokers, the women were assigned to different levels and sources nutrients found in blackberries, in order to determine the influ- of berry consumption. Participants were analyzed at 0, 3, 6, and 9 ence of the phenolic antioxidants alone. Despite gaps in current months for oxidative stress, bone density measurements, and in- research, the existing data demonstrates the potential for black- flammatory markers. Of the berries consumed in the study, blackberries as a healthy dietary constituent with multi-functionality in berries were the only type that exhibited low-level prevention of its influence on chronic disease progression (Van de Velde et al., smoking-induced bone loss (Kaume et al., 2014). In a twin-study 2016).

48 Journal of Food Bioactives | www.isnff-jfb.com Robinson et al. Blackberry phenolics

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Journal of Food Bioactives | www.isnff-jfb.com 51 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Original Research J. Food Bioact. 2020;9:52–57

Phenethylamine in hot water extract of Chlorella pyrenoidosa expands lifespan of SOD1 mutant adults of Drosophila melanogaster at very low dose

Yifeng Zhenga, Yoshihiro H. Inoueb, Nagi Kohnob, Masaki Fujishimac, Eri Okumurac and Kenji Satoa* aDivision of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Kyoto 606-8502, Japan bDepartment of Insect Biomedical Research, Center for Advanced Insect Research Promotion, Kyoto Institute of Technology, Matsuga- saki, Kyoto, 606-0962, Japan cResearch & Development Group, Sun Chlorella Co., Ltd., Osaka-cho, Kyoto, 600-8177, Japan *Corresponding author: Kenji Sato, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa- oiwake-cho, Kyoto 606-8502, Japan. Tel: +81 75 753 6444; Fax: +81 753 6400; E-mail: [email protected] DOI: 10.31665/JFB.2020.9218 Received: March 17, 2020; Revised received & accepted: March 31, 2020 Citation: Zheng, Y., Inoue, Y.H., Kohno, N., Fujishima, M., Okumura, E., and Sato, K. (2020). Phenethylamine in hot water extract of Chlorella pyrenoidosa expands lifespan of SOD1 mutant adults of Drosophila melanogaster at very low dose. J. Food Bioact. 9: 52–57.

Abstract

Hot water extract of chlorella (WEC) increased the lifespan of superoxide dismutase (SOD1)mutant adults of Dros- ophila melanogaster in a dose dependent manner (200–800 µg/mL). Compounds in WEC were successively fractionated by solid phase extraction using a Sep-Pak C18 cartridge and size exclusion chromatography (SEC). Amino compounds in SEC fractions were derivatized with 6-aminoquinolyl-N-hydroxylsuccinimidyl carbamate and analyzed by reversed phased-liquid chromatography-tandem mass spectrometry. Phenylalanine, phenethylamine, isopentylamine, and 2-methylbutylamine were identified in the SEC fraction, which increased the lifespan of the D. melanogaster mutant adults. Phenethylamine, at very low doses (6–60 ng/g of diet) that roughly corresponded to those of phenethylamine in WEC (200–800 μg/mL), increased the lifespan of the D. melanogaster adults, while isopentylamine did not exert the lifespan elongation activity. Since phenethylamine did not show SOD-like activity, it did not increase lifespan by direct antioxidant activity.

Keywords: Chlorella; Lifespan; Superoxide dismutase-1; Drosophila melanogaster; Phenethylamine.

1. Introduction In addition to the dried form of C. pyrenoidosa, hot water extract of C. pyrenoidosa (WEC) was prepared to concentrate potential bioactive compounds, which has been referred as chlorella growth Chlorella is a fresh water unicellular green alga. It is rich in vita- factor (CGF) in some articles (Merchant and Andre, 2001). WEC mins, minerals, dietary fiber, nucleic acids, amino acids, and other was demonstrated to suppress ovariectomy-induced body weight bioactive substances (Merchant and Andre, 2001). Dried chlo- gain and dyslipidemia in rats (Hidaka et al., 2004). WEC extended rella, Chlorella pyrenoidosa and Chlorella vulgaris, has a long the lifespan of tumor-bearing mice possibly by exhibiting immu- history of being used as a food supplement. Beneficial effects of nomodulatory activity (Miyazawa et al., 1988). These facts sug- ingestion of dried C. pyrenoidosa have been demonstrated. C. gested that some water-soluble components of C. pyrenoidosa pyrenoidosa ameliorated high fat diet-induced dyslipidemia in were responsible for these beneficial effects. In vitro experiments rats (Cherng and Shih, 2005), and reduced the risk of anemia, also demonstrated that WEC stimulated cytokine production in proteinuria, and edema in pregnant women (Nakano et al., 2010). human peripheral blood mononuclear cells (Ewart et al., 2007).

These beneficial activities of WEC could not solely be due to its compounds in WEC were re-fractionated. WEC (1% water solu- conventional nutrients, even though it contained relatively high tion, 100 mL) was loaded onto the Sep-Pak C18 35 cc Vac Car- contents of vitamins, amino acids, and other nutrients. There is tridge. After the UA fraction was eluted, the cartridge was washed limited information on other active compounds, beyond conven- with distilled water (100 mL). The compounds absorbed by the tional nutrients in WEC. column were eluted by 30% (v/v) acetonitrile (100 mL). The ef- The objective of the present study was to identify active com- fluent was freeze-dried and dissolved in distilled water to obtain a pounds responsible for the beneficial effects of WEC. However, 1% (w/v) solution. The solution was filtered through a Cosmonice it was difficult to detect active compounds in chlorella using in filter W (0.45 μm, Nacalai Tesque). Aliquot (200 μL) of the filtrate vitro assay, because the mechanism for the reported beneficial was loaded onto the size exclusion chromatography (SEC) column effects of chlorella was unknown. Animal experiments using ro- (Superdex Peptide, 10/300, GE Healthcare, Buckinghamshire, dents required a relatively large sample of fraction for evaluation UK), which was equilibrated with 30% acetonitrile containing of activity. Therefore, it was also difficult to identify active com- 0.1% formic acid. Elution was performed at 0.5 mL/min. The frac- pounds by animal experiments. On the contrary, the superoxide tions were collected every minute, and these steps were repeated dismutase (SOD1) mutant adults of Drosophilia melanogaster 10 times. showed markedly short lifespan (10–20 days) (Reveillaud et al., 1994; Oka et al., 2015; Le et al., 2019). It was demonstrat- 2.3. Liquid chromatography-tandem mass spectrometry (LC- ed that some food compounds extended the lifespan of the D. MS/MS) analyses melanogaster adults, and this lifespan-elongation activity was associated with the health promoting activity of these food compounds (Jafari, 2011; Zuo et al., 2013; Lee and Min, 2015; Le Compounds in the SEC fraction were resolved by reversed et al., 2019). This assay compared to the one using rodents, was phase-high performance liquid chromatography (RP-HPLC) and relatively shorter and required a smaller fraction. In the present detected by electrospray ionization-mass spectrometry (ESI- study, a compound, at a very low dose, with lifespan-elongation MS) in positive and negative modes. However, only a few peaks activity towards Sod1 mutant adults of D. melanogaster was of nucleoside and amino acids were detected (data not shown). identified in WEC. To improve resolution and detection of compounds with primary and secondary amines, derivatization with AccQ, followed by LC-MS/MS, was performed (Ejima et al., 2018). Aliquots (200 2. Materials and methods μL) of SEC fractions were dried under vacuum. The residue was dissolved in distilled water (20 μL), and 0.3% AccQ acetonitrile solution (20 μL) and 50 mM sodium borate buffer (60 μL) (pH 2.1. Materials 8.8) were added. The resultant solution was kept at 50 °C for 10 min. The reactant was clarified by passing it through a Cos- Dried C. pyrenoidosa, and WEC were prepared and supplied monice filter W. The filtrate (20 μL) was analyzed by LC-MS at by Sun Chlorella (Kyoto, Japan). Isopentylamine, pentylamine, precursor ion scan mode with an LCMS 8040 (Shimadzu, Kyoto, 2-methylbutylamine, and phenethylamine were purchased from Japan) equipped with RP-HPLC column (Cosmosil 5C18 MS-II, Nacalai Tesque (Kyoto, Japan) and Tokyo Chemical Industry (To- Nacalai Tesque), targeting the AccQ-derived product ion (m/z kyo, Japan), and 6-aminoquinolyl-N-hydroxysuccinimidyl carba- 171.1) at collision energy −35 eV. A binary linear gradient, with mate (AccQ) was purchased from Toronto Research Chemicals 0.1% formic acid (solvent A) and 0.1% formic acid containing (Toronto, ON, Canada). Acetonitrile (HPLC grade) was purchased 80% acetonitrile (solvent B), was used at a flow rate of 0.2 mL/ from Nacalai Tesque. WST-1 SOD assay kit was purchased from min. The gradient program was as follows: 0–15 min, 0–50% Dojindo laboratory (Kumamoto, Japan). B; 15–20 min, 50–100% B; 20–25 min, 100% B; 25–25.1 min, 100–0% B; 25.1–35 min, 0% B. The column was maintained at 2.2. Chromatographic fractionation 40 °C.

Sep-Pak C18 35 cc Vac Cartridge (Waters, Miliford, MA) was 2.4. Determination of monoamines in WEC and crude chlorella successively pretreated with acetonitrile (50 mL) and water (50 mL). WEC was dissolved in distilled water to obtain 5% Monoamines were dissolved in distilled water or ethanol to obtain (w/v) solution. After centrifugation of the solution at 3,000 g 10 μM solutions, which were used as standard solutions. Crude for 10 min, the supernatant was collected. The supernatant (300 chlorella and WEC powders were suspended in distilled water to mL) was loaded onto the Sep-Pak C18 35 cc Vac Cartridge, obtain 1% (w/v) solutions, which were vigorously stirred and then and the unabsorbed fraction was collected. The cartridge was centrifuged at 15,000 g for 10 min. The supernatants were collect- then washed with distilled water (50 mL). The compounds ab- ed. Monoamines in standard and sample solutions were derivatized sorbed by the column were successively eluted using 10, 30, with AccQ, as described above. The AccQ-derivatives were de- 100% (v/v) of acetonitrile, acetone, and hexan (50 mL). The termined by LC-MS/MS at multiple-reaction monitoring (MRM) unabsorbed fraction and fractions eluted with 10% and 30% mode. The elution conditions were the same as described above. acetonitrile were referred as the UA, 10ACN, and 30ACN frac- MRM conditions for the AccQ-derivatives of standard monoam- tions, respectively. These fractions were freeze-dried. Effluents ines were optimized using LabSolutions Version 5.65 (Shimadzu, with acetonitrile, acetone, and hexan were combined and dried Kyoto, Japan). in a rotary evaporator, and the resultant fraction was referred as the AAH fraction. These fractions were then evaluated for lifespan-elongation activity towards Sod1n1 mutant adults of D. 2.5. Survival assay using Sod1 mutant D. melanogaster flies melanogaster. Based on the results of the first Sep-Pak C18 fractionation, The Sod1n1 mutant flies were prepared by a method as described

Journal of Food Bioactives | www.isnff-jfb.com 53 Lifespan-elongating compound in chlorella extract Zheng et al.

Figure 1. Effect of different concentration of WEC (a) and its Sep-Pak C18 fractions (200 μg/g) (b) on survival rate ofSod1 mutant flies. previously (Le et al., 2019). This mutant showed a considerably 3. Results declined SOD1 activity due to an amino acid substitution without the reduced mRNA level (Oka et al., 2015). The young adult 3.1.. Lifespan-elongation activity of WEC mutant males, homozygous for Sod1n1 were collected within 24 hrs after eclosion and 20 flies were added into the tube (100 mm in height and 15 mm in diameter) with medium containing sam- As shown in Figure 1a, WEC extended the survival period of the ple at suitable contents and incubated at 25 °C and 50–55% hu- Sod1 mutant flies in a dose dependent manner to 800 μg/mL in midity. The assay was repeated three to five times (using 60–100 diet. WEC (3,200 μg/mL), however, showed a survival period Sod1 mutant flies). Dead adults in each vial were counted every similar to that of WEC (200 µg/mL) and shorter than that of WEC 12 h for the Sod1 mutants. Food vials were changed every three (800 μg/mL). days. 3.2. Identification of compounds with life span elongation activ- 2.6. SOD-like activity assay ity

Phenethylamine was dissolved in water to obtain the resultant WEC was first fractionated by solid phase extraction using a Sep- solution (0.6–600 ng/mL). SOD-like activity was examined by Pak C18. As shown in Table 1, the UA fraction accounted for ap- using a WST SOD assay kit. In addition, wild D. melanogaster proximately 70% (w/w) of the initial materials. The 10ACN and was homogenized with phosphate buffer saline (PBS) (10 μL/ 30ACN fractions approximately accounted for 4% of the initial individual). The homogenates were mixed with the same volume materials, respectively, while only small amounts of compounds of water or phenethylamine above and incubated at 25 °C for 30 were recovered into the AAH fraction. As shown in Figure 1b, un- min. The mixture was then centrifuged at 15,000 g for 10 min. til day 9, survival rates of all groups treated with Sep-Pak C18 The supernatants were collected, and SOD-like activity was ex- fractions were higher than that of the control group. Thereafter, amined. survival rate of the AAH group drastically decreased. After day 10, survival rate of the UA group also decreased. On the contrary, the 10ACN and 30ACN groups showed survival curves similar to that 2.7. Statistics of crude WEC. Based on these results, active compounds in WEC were eluted with 30% acetonitrile from the solid phase column A survival curve was calculated based on Kaplan-Meier survival after washing the cartilage with water. The active fraction was fur- estimation and analyzed by log-rank test between the drug-treated ther fractionated using SEC. As shown in Figure 2a, fractions 16– and control groups. For comparison of the two groups, we used 35, 36–40, 41–44, 45–50, and 51–70 were collected and referred the Student’s t-test. One-way ANOVA with post-hoc Tukey’s test as SEC Fr. I, II, III, IV, and V, respectively. Lifespan-elongation was applied to assess the differences among the groups. Data were activity of these fractions was evaluated. As shown in Figure 2b, considered significant at p-values < 0.05. SEC Fr. III and V (200 μg/g)-administered fractions, compared to

Table 1. Recovery of solid phase extraction fractions using Sep-Pac C18 cartilage Fraction Dry weight (g) Recovery to WEC (%) unabsorbed 10.3 68.67 10% acetonitrile 0.6 4 30% acetonitrile 0.59 3.9 AAH 0.06 0.404

54 Journal of Food Bioactives | www.isnff-jfb.com Zheng et al. Lifespan-elongating compound in chlorella extract

Figure 2. Size exclusion chromatography (SEC) elution pattern of compounds in Sep-Pak C18 30% acetonitrile fraction (a) and effect of SEC fractions (I–V) (200 μg/g) on survival rate of Sod1 mutant flies (b). the control, showed a decreased survival rate after day 7. SEC Fr. ed isoleucine (2-methylbutylamine) and leucine (isopentylamine). II-administered group, compared to the control, showed a reduced Retention times of peaks 1–4 coincided with those of AccQ de- survival rate between days 2 and 7. Before day 8, SEC Fr. I and rivatives of authentic phenylalanine, 2-methylbutylamine, isopen- IV-administered fractions showed survival curves similar to each tylamine, and phenethylamine. Product ion patterns of these peaks other and survival rates higher than that of the control. However, by LC-MS/MS also coincided with those of the authentic amines, after day 8, survival of D. melanogaster of the SEC Fr. I group de- respectively. Therefore, peaks 1–4 were identified as phenylala- creased. SEC Fr. IV-administered group, compared to the control, nine, 2-methylbutylamine, isopentylamine, and phenethylamine. showed an increased survival rate and lifespan of D. melanogaster Contents of 2-methylbutylamine, isopentylamine, and phenethyl- throughout the experiment (P < 0.001). Thus, SEC Fr. IV was used amine in WEC were 2.5 ± 0.2, 10.7 ± 0.2, and 11.7 ± 0.4 μg/g, as the active fraction for the following experiments. respectively, and those in dry chlorella powder were 0.0 ± 0.00, Amino compounds in SEC Fr. III and IV were detected by de- 0.5 ± 0.02, and 0.8 ± 0.04 μg/g, respectively. As shown in Fig- rivatization with AccQ, followed by LC-MS/MS at precursor scan ure 1a, WEC (800 μg/mL), which contained isopentylamine and mode targeting product ions from AccQ (m/z = 171.1). As shown phenethylamine at 8.6 ng/mL and 10 ng/mL, respectively, showed in Figure 3, some peaks marked with asterisk (*) appeared in both the best lifespan-elongation activity towards the Sod1 mutant flies. SEC Fr. III (inactive fraction) and SEC Fr. IV (active fraction). On As shown in Figure 4b, administration of isopentylamine (8.6 ng/ the other hand, four peaks indicated as 1–4 that appeared in SEC mL) did not significantly affect the survival rate. A higher dose Fr. IV were not present in SEC Fr. III. The mass to charge ratio (86 ng/mL) of isopentylamine showed survival rates higher than (m/z) of compounds in peaks 1–4 were 336, 258, 258, and 292, re- that of the control before day 5. However, after day 6, the survival spectively. The m/z 336 and 292 corresponded to protonated AccQ rate drastically decreased, and maximum lifespan was shorter than derivatives of phenylalanine and its decarboxylated form, pheneth- that of the control group. Phenethylamine (60 ng/mL) showed a ylamine, respectively. The m/z 258 corresponded to decarboxylat- survival rate significantly higher (P < 0.001) than that of the con-

Figure 3. Precursor ion scan of AccQ derivatives in SEC fraction III and IV. *Shows reagent peak.

Journal of Food Bioactives | www.isnff-jfb.com 55 Lifespan-elongating compound in chlorella extract Zheng et al.

Figure 4. Effect of different concentration of phenethylamine (a), isopentylamine (b) and pentylamine (c) on survival rate of Sod1 mutant flies. trol group and a maximum survival period approximately 150% and apple polyphenols (10 mg/g) in the diet extended the lifespan of that of the control group. However, phenethylamine (600 ng/ of D. melanogaster, respectively (Bahadorani et al., 2008; Zhang mL) showed a survival rate lower than that of the control group. et al., 2014; Peng et al., 2011). Vitamin E (16.7 mg/g of diet) also On the other hand, pentylamine, a non-natural monoamine, exerted extended the lifespan of the Sod1 mutant flies (Bahadorani et al., lifespan-elongation activity at doses of 20 and 200 ng/mL (p < 2008). Coenzyme Q10 (50 μg/g of diet) elongated the lifespan of C. 0.001 and 0.0001 respectively). elegans (Ishii et al., 2004). A strong superoxide dismutase/catalase mimetic, EUK-134, at a very low dose of 50 μM (ca.20 μg/g) in the diet, extended the lifespan of C. elegans (Melov et al., 2000). The 4. Discussion present study demonstrated that phenethylamine (6–60 ng/g of diet) expanded the lifespan of the Sod1 mutant flies, and these doses were Phenethylamine is an endogenous trace amine, and is contained in far less than those reported in the previous studies. On the contrary, some food items such as cheeses (0–3 μg/g) (Novella-Rodriguez isopentylamine did not expand the lifespan of the Sod1 mutant flies; et al., 2003), a chocolate (2.6 μg/g) (Mayr and Schieberle, 2012), however, pentylamine, a linear chain monoamine, did, suggesting and wines (approximately 1 μg/mL) (Landete et al., 2005). The that all hydrophobic monoamines did not always exhibit lifespan- present study demonstrated that WEC contained phenethylamine elongation activity and structure of the hydrophobic moiety contrib- (10 μg/g of dry matter). It has been reported that oral supplementa- uted to the elongation activity. To our best knowledge, there is no tion of phenethylamine (10–60 mg/day) with selegiline (monoam- food compound, except some trace metals such as selenium, which ine oxidase-B inhibitor, 10 mg/day) relieved depression (Sabelli et exerts beneficial activity in such low doses (Diplock, 1987). al., 1996). On the other hand, high dose of phenethylamine (25–75 Inhibition of SOD1 resulted in the accumulation of superox- − − mg/kg body weight of mice) induced psychomotor dysfunction ide radicals (O2 ) (Huang, 2000). Over production of O2 caused and decrease in striatal biogenic amines (Sengputa and Mohanaku- oxidation of some biomolecules, such as DNA, protein, and lipids mar, 2010). However, the amount of phenethylamine (10–100 μg) (Ames et al., 1993), damaging cells. However, phenethylamine obtained from consumption of a few grams of WEC was similar or (6–6,000 ng/mL) did not show significant SOD-like activity. In less than that obtained from one serving of cheese, chocolate, or addition, phenethylamine, which was treated with a homogenate wine. Therefore, such low doses of phenethylamine did not exert of D. melanogaster, did not show significant SOD-like activity at any adverse effects on human health. the same dose. As mentioned above, the dose of phenethylamine It has been demonstrated that some antioxidant vitamins and for elongation of lifespan of the Sod1 mutant flies was far less food compounds extended the lifespan of D. melanogaster and than that of vitamins E and C, which are strong antioxidants. It was, Caenorhabditis elegans. Vitamin C (20 mM), lutein (0.1 mg/g), therefore, unlikely that antioxidant activities of phenethylamine

56 Journal of Food Bioactives | www.isnff-jfb.com Zheng et al. Lifespan-elongating compound in chlorella extract and its metabolites contributed to the lifespan extension of the or iconoclast? Pharmacol. Therapeut. 116: 355–390. Sod1 mutant flies. Although the mechanism of lifespan extension Hidaka, S., Okamoto, Y., and Arita, M. (2004). A Hot Water Extract of Chlo- by phenethylamine remains unknown, it is possible that phenethyl- rella pyrenoidosa Reduces Body Weight and Serum Lipids in Ovariec- amine exerted this activity by binding to some cell surface recep- tomized Rats. Phytother. Res. 18: 164–168. tors, intracellular transcription factors, or their regulators. It was Huang, P., Feng, L., Oldham, E.A., Keating, M.J., and Plunkett, W. (2000). Superoxide dismutase as a target for the selective killing of cancer demonstrated that some trace amines, such as tyramine and octopa- cells. Nature 407: 390–395. mine, exerted significant bioactivities via G protein-coupled recep- Ishii, N., Senoo-Matsuda, N., Miyake, K., Yasuda, K., Ishii, T., Hartman, P.S., tors (Grandy, 2007). However, the specific receptor for phenethyl- and Furukawa, S. (2004). Coenzyme Q10 can prolong C. elegans lifes- amine was not identified. Now, further studies on target proteins of pan by lowering oxidative stress. Mech. Ageing Dev. 125: 41–46. phenethylamine in D. melanogaster are being conducted. Jafari, M. (2011). Drosophila melanogaster as a model system for the eval- Effects of oral administration of such low doses of trace amines, uation of anti-aging compounds. Fly (Austin) 4: 253–257. such as phenethylamine, on animal and human health have not Landete, J.M., Ferrer, S., Polo, L., and Pardo, I. (2005). Biogenic amines been examined. Now, effects of low dose phenethylamine on high in wines from three Spanish regions. J. Agric. 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Development of Stable Isotope Di- onstrated to exhibit lifespan-elongation activity. Phenethylamine, lution Assays for the Simultaneous Quantitation of Biogenic Amines compared to compounds including antioxidant vitamins and phyto- and Polyamines in Foods by LC-MS/ MS. J. Agric. Food Chem. 60(12): chemicals, increased life span at an extensively lower dose (6–60 3026–3032. ng/g of diet). Since phenethylamine did not show SOD-like activity, Melov, S., Ravenscroft, J., Malik, S., Gill, M.S., Walker, D.W., Clayton, P.E., it did not exert lifespan elongation activity towards the Sod1 mutant Wallace, D.C., Malfroy, B., Doctrow, S.R., and Lithgow, G.J. (2000). Extension of life-span with superoxide dismutase/catalase mimetics. flies by only direct antioxidant activity. While there is no report Science 289: 1567–1569. on the beneficial effects of ingestion of trace amounts of monoam- Merchant, R.E., and Andre, C.A. (2001). A review of recent clinical trials of ine, phenethylamine obtained from ingestion of chlorella and other the nutritional supplement Chlorella pyrenoidosa in the treatment foods might show some beneficial effects on human health. To ex- of fibromyalgia, hypertension, and ulcerative colitis. Altern. Ther. amine the effects of trace amounts of phenethylamine, an animal Health M. 7(3): 79–91. experiment using NAFLD mouse model is being conducted. Miyazawa, Y., Murayama, T., Ooya, N., Wang, L.F., Tung, Y.C., and Yama- guchi, N. (1988). Immunomodulation by a unicellular green algae (Chlorella Pyrenoidosa) in tumor bearing mice. J. Ethnopharmacol. Acknowledgments 24: 135–146. Nakano, S., Takekoshi, H., and Nakano, M. (2010). Chlorella pyrenoidosa Supplementation Reduces the Risk of Anemia, Proteinuria and Ede- We would like to thank Editage (www.editage.jp) for English ma in Pregnant Women. Plant Foods Hum. Nutr. 65: 25–30. language editing. 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Journal of Food Bioactives | www.isnff-jfb.com 57 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Original Research J. Food Bioact. 2020;9:58–69

Low frequency, high power ultrasound: a non-thermal green technique improves phenolic fractions (free, conjugated glycoside, conjugated esters and bound) in fermented seabuckthorn beverage

Kelly Dornana, Aynur Gunenca, Azza Ferichichib and Farah Hosseiniana,c* aFood Science, Chemistry Department, Carleton University, Ottawa, ON, Canada bUniversity of Tunis El Manar, Tunis, Tunisia cInstitute of Biochemistry, Carleton University, Ottawa, ON, Canada *Corresponding author: Farah Hosseinian, Food Science, Chemistry Department, Carleton University, Ottawa, ON, Canada. Tel: +1 613 520-2600, ext. 2048; Fax: +1 613 520-3729; E-mail: [email protected] DOI: 10.31665/JFB.2020.9219 Received: March 04, 2020; Revised received & accepted: March 31, 2020 Citation: Dornan, K., Gunenc, A., Ferichichi, A., and Hosseinian, F. (2020). Ultrasound, a powerful non-thermal and green technique on phenolic fractional profiles (free, conjugated glycoside, conjugate esters and bound) in fermented seabuckthorn beverage. J. Food Bioact. 9: 58–69.

Abstract

Phenolic compounds were characterized after traditional extraction method (TEM) and ultrasound-assisted- ex tractions (UAEs) for 10 min (US10) and 15 min (US15). Four fractions (free, bound, conjugated esters, and conjugated glycosides) were obtained and characterized using RP-HPLC-PDA. The US10 extracted 22.1% (454.3 mg/kg) phenolic compounds while US15 extracted 66.6% (1,369.7 mg/kg) phenolic compounds compared to traditional extraction method (TEM) after 1 hr of extraction. US10 and TEM extracted a similar amount of aglycones (112.3 ± 4.0 and 109.0 ± 12.9 mg/kg, respectively) while US15 showed a significant decrease in aglycone extraction (58.9 ± 4.1 mg/kg) (P < 0.05). In seabuckthorn kombucha, US decreased initial microbial load by 2.6 log CFU/mL, increased ORAC value by 3% and increased water solubility index (WSI) by 40% (from 6.64 to 9.29 g/g) without syneresis. Results from this study suggest that application of US can enhance phenolic functionality during fermentation and is capable of decreasing syneresis, increasing oil yield, decreasing microbial load, and increasing ORAC with minimal loss of nutritional quality.

Keywords: Kombucha; Phenolics; Rancimat; SCOBY; Seabuckthorn; Ultrasound.

1. Introduction more bioavailable due to its low molecular weight and lipophilicity making it ready for passive diffusion across the intestinal mem- Seabuckthorn berries (SB) are small-sized berries that grow on de- brane (Bilal Hussain et al., 2018). ciduous shrubs, with some varieties capable of surviving Canadian SB have been shown to increase probiotic viability when added winters (Schroeder et al., 2014). The health promoting property of to yogurt (Gunenc et al., 2016). Its functional phenolics and high SB is attributed to their high antioxidant content, including ascor- antioxidant content make SB an ideal starting ingredient for pro- bic acid, tocopherols, carotenoids, anthocyanins and other phenol- duction of a health promoting kombucha-like beverage. Kombu- ic compounds (Chauhan et al., 2001). Phenolic compounds, sec- cha (K) is a fermented beverage traditionally made with sweet- ondary metabolites in plants, exists in free, bound or conjugated ened tea and a symbiotic culture of bacteria and yeast (SCOBY) (glycoside or ester) form. The free, or aglycone, form is typically (Chakravorty et al., 2015). There are products on the market using

SB juice and leaves and SCOBY to create a semi-clear sparkling K method (Kostecka-Gugala et al., 2015; Vitas et al., 2018) by mix- beverage. The oil from the seed and pulp of SB are commercially ing 0.1 mL of samples with 7.9 mL of distilled water, 0.5 mL of available and highly sought after for their, high unsaturated fatty Folin-Ciocalteu reagent and 1.5 mL of sodium carbonate (20%, acids, phytosterols, and fat soluble vitamins A and E content (Olas, w/w). Absorbance was measured at 750 nm after a 1 hr incubation 2018). In this study, we investigated the use of whole SB, includ- at room temperature. Blank consisted of the same reaction mixing pulp and seeds, in the fermentation process. ture with distilled water replacing sample. A chlorogenic stand- Application of ultrasound (US) to a liquid acoustic cavitation ard curve was prepared. Results are expressed as chlorogenic acid generates micro-bubbles that grow rapidly and collapse, producing equivalents per mL of sample (mg CAE/mL). an area of high temperature and high pressure that leads to production of a shockwave capable of disrupting the biological cell wall 2.3. Fractionation and characterization of phenolic compounds (Cruz-Cansino et al., 2016). Through this same phenomenon, US has been shown to be a promising and versatile nonthermal technol- in SB ogy for increase extraction of phenolics; as an emerging preservation technique to decrease spoilage and pathogenic microorganisms in 2.3.1. Extraction of crude phenolics fruit juice with minimal nutritional loss; and can also be used as an emulsifier by promoting of small and even sized crystals which was Whole SB (H. rhamnoides cv. Sunny) kindly provided by Omega of particular interest in this study due to the high oil content of SB. Fruit (Magog, QC, Canada), were freeze-dried and powdered us- Our aim was to investigate use of US for phenolic extraction and characterization of whole SB and for fermentation of diluted whole ing a grinder. Free, conjugated and bound phenolics were extracted SB and its effects on physicochemical properties. Moisture content, according to a method used by Gunenc et al. (2015) and Arimboor juice yield, pH, acidity, and total phenolics content (TPC) of SB were et al. (2008). Two grams of powdered whole berries were defatted measured before and after US. Moisture, protein, ash and fat content by mixing with 10 mL of hexane for 1 hr at room temperature. The were determined for dilute SB puree without (P) and with US (P+US). mixture was filtered, and the extraction was repeated. The resi- Total microbial count, oxidative stability, oxygen radical absorbance due was dried in an oven at 40 °C and then extracted with 70% capacity (ORAC), vitamin C concentration, water solubility index methanol (1:10 w/v) traditionally for 1 hr at room temperature; (WSI), and water absorption index (WAI), were also determined for with direct high power, high intensity, low frenquency ultrasound P and P+US, as well as for fermented diluted seabuckthorn purée (K), treatment (90 W, 20 kHz) using a UIP500hd ultrasonic processor fermented diluted seabuckthorn purée with US (K+US), fermented (Hielscher, Germany) for 10 min and 15 min, respectively. The diluted seabuckthorn purée with sugar (K+S), and fermented diluted mixture was filtered, and the extraction was repeated one more seabuckthorn purée with sugar and US (K+S+US). time. The filtrates were combined, dried under vacuum at <50 °C and referred to as crude phenolic extracts.

2. Materials and methods 2.3.2. Fractionation of phenolic compounds

2.1. Chemicals and reagents The residue from the previous extraction was dried in an oven at 40 °C, re-dissolved in 40 mL 2 M NaOH and mixed for 4 hr at room Sodium chloride, sodium hydroxide, phenolphthalein, agar, and temperature. The mixture was then acidified to pH 2 using 6 N HCl ascorbic acid were from Bioshop Canada Inc. (Burlington, ON, and extracted with ethyl ether (3 × 40 mL) at room temperature. The Canada). Nutrition broth with 1% peptone was from HeMedia ethyl ether extracts were combined and evaporated under vacuum Laboratories (West Chester, PA, USA). Trolox, fluorescein, rutin, at <40 °C and referred as the bound phenolics fraction. For extrac- AAPH, potassium iodide, potassium iodate, N-hexane (99%), Fo- tion of free and conjugated phenolics, crude phenolic extracts were lin-Ciocalteu reagent, sodium carbonate, and chlorogenic acid were dissolved in 10 mL of water, acidified with 6 N HCl to pH 2, and from Sigma (Canada Ltd., Oakville, ON, Canada). Sulfuric acid, extracted with ethyl ether (3 × 10 mL) at room temperature. The methanol, acetonitrile were from VWR International (Radnor, PA, ethyl ether extracts were combined and evaporated under vacuum USA). Hydrochloric and formic acid were from Anachemia (Mon- at <40 °C and referred to as the soluble free phenolics fraction. The treal, QC, Canada) Ethyl ether was from Fisher Chemical (Ottawa, water phase was neutralized using 2 M NaOH and dried to near dry- ON, Canada). Phenolic acid standards (over 98% pure): gallic, pro- ness under vacuum at <40 °C, dissolved in 10 mL of 2 M NaOH and to-catechuic, chlorogenic, caffeic, vanillic, syringic, p-coumaric, mixed for 4 hr at room temperature. The solution was then acidi- ferulic, o-coumaric, trans-cinnamic; and flavonoid standards: cat- fied to pH 2 and extracted with ethyl ether (3 × 10 mL). The ether echin, rutin, myricetin, quercetin, apigenin and kaempferol, were phase was dried as before and analyzed as phenolics liberated from purchased from Sigma-Aldrich (St. Louis, MO, USA). esters. The water phase was neutralized, dried as before and then hydrolyzed with 50 mL 2 M HCl for 30 min at 95 °C, cooled and extracted with ethyl ether (3 × 50 mL). The water phase was discarded 2.2. Chemical properties: pH, % acidity, total soluble solids, while the ether phase was combined, dried as before and analyzed total phenols content as phenolics liberated from glycosides. Samples were re-dissolved in methanol and filtered through 0.45 μm membrane filters (Celltreat The pH of pureed SB before and after US was measured using a scientific product, China) for HPLC analysis. bench top pH-meter (Thermo Scientific, United States). Titratable acidity was determined using a sodium hydroxide standard solution and phenolphthalein indicator and results are expressed as % 2.3.3. Chromatographic conditions acidity (Vitas et al., 2018). Total soluble solids (TSS), expressed as °Brix, were measured using a refractometer (Erma, Japan). Total The Alliance® HPLC system e2695, Separation Module with the phenolic content (TPC) was determined using the Folin-Ciocalteu 2998 Photodiode Array Detector (PDA) from Waters (Milford,

Journal of Food Bioactives | www.isnff-jfb.com 59 Ultrasound, a powerful non-thermal and green technique Dornan et al.

MA, USA) and Empower 3 software was used. A reverse phase The oil samples were weighed, and fat content was calculated and (RP) column (Atlantis R T3, 150 × 4.6 mm, 5 μm) was used as the expressed as % fat. stationary phase with the column temperature maintained at 30 °C. A gradient elution was used for the mobile phase with 0.5% (v/v) 2.4.5. Ash content formic acid in water (Solvent A) and 100% acetonitrile (Solvent B) at flow rate rate of 1 mL/min. The gradient program for solvent A was as follows: 0 min, 95% A; 0–35 min, 50% A; 35–40 min, 90% Dry ash method was used based on the AOAC official method A; 40–50 min, 95% A. Chromatograms were recorded at 280 and 930.05 (AOAC, 1990). Freeze-dried samples of P and P+US were 320 nm for phenolic acids and flavonoids, respectively. used to determine ash content. Samples were weighed and pre-ig- nited using a muffle furnace at 550 °C and left overnight. Remain- ing ash was weighed, and ash percentage was calculated. 2.4. Application of US for SB kombucha beverage

2.4.6. Fermentation protocol for SB kombucha 2.4.1. Sample preparation

Four samples of kombucha (K) were made: K, K+US, K+S, and Fresh whole SB (H. rhamnoides cv. Sunny) were puréed using a K+S+US. All samples were prepared using 200 mL of P or P+US Vitamix blender for 2 min (Vitamix®, USA). A volume of dH O 2 and 12.5 g of SCOBY. K consisted of P and SCOBY. K+US con- equal to 30% of the original purée volume was added and blend- sisted of P+US and SCOBY. K+S consisted of P, 15.0 g of sucrose ed. US (90 W, 20 kHz, 10 min) was applied to 200 mL of the and SCOBY. K+S+US consisted of P+US, 15.0 g of sucrose and diluted purée using a UIP500hd ultrasonic processor (Hielscher, Germany). Treatment time was chosen to limit degradation of nu- SCOBY. All samples were left to ferment in a dark place at room trients and keep the sample fresh-like based on our preliminary temperature for five days. A second US treatment (90 W, 20 kHz,10 optimization of ultrasound treatment. The diluted SB purée (P) min) was applied to K+US and K+S+US to halt fermentation on and the diluted SB purée with ultrasonic treatment (P+US) were day 5. All samples were then stored at 4 °C. Part of the samples then stored at 4 °C. Some sample was also freeze-dried for analy- were freeze-dried, at −50 °C for 3 days, on day 5 of fermentation sis. Freeze drying was done using a Labconco freeze dryer/lyo- and stored at −20 °C for further analysis (WSI and WAI). philizer (Kansas CIty, MO, USA). The samples were first frozen at −80 °C and then freeze dried at −50 °C for 3 days using vacuum 2.4.7. Total microbial count pressure and sublimation to remove unbound water without the use of heat. The total microbial count, based on the ISO method 4833 (1991), was used to determine the log colony forming units (log CFU/ 2.4.2. Moisture content mL). Samples were diluted in saline solution and plated on nutrient broth agar. Plates were incubated at 30 °C for 48 hr before Moisture content was determined by lyophilization and calculated enumeration. Total count was determined on day 0, 2, 5, 14 and according to the AOAC official method 930.04 (AOAC, 1998a). 21 for all samples. After preparation, P and P+US were weighed and freeze dried at −50 °C for 3 days. After freeze-drying the samples were weighed 2.4.8. Oxidative stability again and water content was calculated and expressed as % moisture. Oxidative stability was determined, according to the AOAC official method Cd12b-92 (AOCS, 2017b), on day 0 for P and P+US, 2.4.3. Protein content and on day 5 of fermentation for K, K+US, K+S, K+S+US using Rancimat (Metrohm, Switzerland). For this, 3 g of sample was Dumas method was used to determine protein content for P and used and Rancimat was set at 120 °C with air flow at 20 L/h. P+US. The protocol was based on the AOCS official method Ba 4e-93 (AOCS, 2017a). After freeze-drying, 1 mg of sample was 2.4.9. Oxygen radical absorbance capacity (ORAC) added to tin weigh boats. Apple leaves were used as the standard. Samples were combusted in an oxygen-enriched atmosphere at a high temperature using N-cube. Vario Micro software was used to ORAC was used to determine the antioxidant activity on day 0, measure % N in the sample. Assuming that nearly all nitrogen in 7, and 21 for P and P+US and on day 2, 5, and 21 for kombucha the sample is present as amino acids in proteins, a default Jones samples (K, K+US, K+S, K+S+US) based on protocol adapted factor of 5.6, as proposed by Mariotti et al. (2008), was used to from Huang et al. (2002). Antioxidant standard was trolox, per- calculate protein content (% protein) on a dry basis. oxyl radical generator was AAPH, positive control was rutin, and probe was fluorescein. Samples were centrifuged at 4,000 RPM for 20 min and the supernatant was used to measure ORAC. The 2.4.4. Fat content standards, samples and rutin were placed in a 96-well fluoromet- ric microplate. Fluorescein was added to each well. The plate was Seeds were separated from pulp and crushed using a mortar. Li- incubated at 37 °C for 20 min before adding AAPH. Absorbance pids were extracted according to the method of Ferreira-Dias et al. was read using the FLx800TM Multi-Detection Microplate Reader (2003). Lipid extracts were obtained in a Soxhlet apparatus for 5 with Gen5TM software (excitation wavelength=485 nm; emission h at the boiling point (67–69 °C) of n-hexane. A ratio of crushed wavelength= 525 nm) for 60 min. Final ORAC values were calcu- sample to solvent of 1:6 (m/v) was used. The solvent was evapo- lated using a Trolox standard curve and the measured net AUC for rated using a rotary evaporator at 40 °C (Kozlowska et al., 2016). each sample. Values are expressed as µmol Trolox equivalent per

60 Journal of Food Bioactives | www.isnff-jfb.com Dornan et al. Ultrasound, a powerful non-thermal and green technique

L of sample (µmol TE/L). Table 1. Chemical and nutritional analysis of seabuckthorn berries Fruit No-US After US 2.4.10. Vitamin C Moisture% 80.00 78.80

Vitamin C concentration was determined on day 0 for P and P+US, Juice (Yield %) 66.10 65.90 and on day 5 of fermentation for K, K+US, K+S, K+S+US using pH 2.60–2.80 2.60–3.20 protocol adapted from Sapei and Hwa (2014). Direct titration was used with a vitamin C standard solution, a 3% starch solution as Acidity% 2.80–2.90 2.70–2.80 indicator, and an iodine solution as titrant. °Brix (TSS) 8.20 8.30 TPC (mg CAE/100g) 550 600 2.4.11. Water solubility index (WSI) and water absorption index Synersis After 2 hr After 4 weeks (WAI) CAE: Chlorogenic acid equivalent US: Ultrasound. WSI and WAI was determined using protocol adapted from Kyri- akopoulou et al. (2013) on day 0 for P and P+US, and day 5 of acoustic cavitation, and release of phenolic compounds into the fermentation for K, K+US, K+S, K+S+US by dissolving 1.00 g of solution (Golmohamadi et al., 2013). freeze dried sample in 25.00 mL of ddH2O, and then centrifuging at 4,000 RPM for 20 min. Supernatant was separated and freeze 3.2. Fractional extraction of phenolic compounds from SB dried at −50 °C for 3 days. Remaining hydrogel was weighted, and the WAI was calculated as: UAE can be used to decrease the time required for extraction of mass hydrogel (g) phenolics compounds compared to traditional methods. The phe- WAI = (1) mass sample (g) nomenon of acoustic cavitation, as a result of repeated compression and expansion of bubbles when acoustic waves propagate WSI was determined from the weight of dry solids remaining after through an aqueous medium, increases extraction power. The rar- freeze-drying the supernatant, using the following equation: efaction of sound waves in an aqueous medium produce bubbles that compress and expand creating an area of high temperature and mass dried SN (g) WSI = ×100 (2) pressure leading to implosion of the bubble. This implosion cre- mass sample (g) ates microstreams within the solvent capable of penetrating and disrupting cell walls ultimately increasing solvent penetration and release of solutes within cells (Ashokkumar, 2015). In this study 2.5. Statistical analysis we used high power (90 W) low frequency (20,000 kHz) direct US with two different treatment times (10 min & 15 min) to com- All trials were done in triplicates and results presented as means ± pare and characterize different fractions (bound, conjugated esters, standard deviations. One-way ANOVA was performed for each set conjugated glycosides, and free) of phenolic compounds found in of results using Statistical Analysis System (SAS) software (SAS defatted whole SB with a traditional extraction method (1 hr). The Institute Inc., Cary, NC, USA). Statistical significance was set at results are presented in Figure 1 and Table 2, and are expressed as P < 0.05. mg/kg of dry sample. The total amount of extracted and characterized phenolic compounds was 2,054.4 mg/kg for the TEM, 454.3 mg/kg for US10, 3. Results and discussion and 1,369.7 mg/kg for US15. Although neither UAE gave as high of a result as the TEM, a 201.5% increase was seen with the US15 3.1. Chemical properties compared to US10. Compared to the 1 hr TEM, the 15 min UAE extracted 66.7% of the phenolic compounds in a quarter of the time. This shows that US is an efficient method to extract phenolic Chemical properties for SB with and without US are presented compounds in less time. in Table 1. Moisture content of SB was 80% and juice yield was For all extractions, Figure 1 shows the predominant fraction ex- 66.10%. SB are acidic with a pH between 2.60 and 2.80 and acid- tracted was conjugated glycosides representing 80% of phenolic ity between 2.80 and 2.90% measured in this study. TSS in fresh compounds extracted with TEM, 56.6% for US10, and 86.7% for SB was measured as 8.20 °Brix. These results are similar to those US15. Conjugated esters represented 4.9% of phenolic compounds published by Beveridge et al. (1999). Sonication of SB had no ef- for TEM, 7% for US10, and 5% for US15. Only phenolic acids fect on moisture content, juice yield, pH, % acidity, and/or TSS were found in bound form with this fraction representing 9.8% with only slight variations observed, as seen in Table 1. TPC of of phenolic compounds for TEM, 11.6% for US10, and 4.0% for SB measured in this study was 550 mg chlorogenic acid equiva- US15. Free phenolic compounds represented 5.3% of phenolic lent/100 g, higher than 175.25 mg gallic acid equivalent/100 g re- compounds extracted for TEM, 24% for US10, and 4.3% for US15. ported by Araya-Faria et al. (2011) but lower than 964 to 10 704 Gallic acid was the main phenolic identified in the glycoside mg gallic acid equivalent/100 g reported by Korekar et al. (2014). conjugated fraction (1,492.7 ± 184.5 mg/kg for TEM, 212.1 ± 2.9 Phenolics are secondary metabolites produced by plants that de- mg/kg for US10, 1,080.1 ± 48.5 mg/kg for US15). Other phenolic fend against oxidative stress, considered antioxidants capable of acids identified in the conjugated glycoside fraction were vanillic scavenging free radicals (Vitas et al., 2018). US led to a 9% in- acid (52.5 ± 27.5 mg/kg) and P-coumaric acid (3.4 ± 0.9 mg/kg) crease in TPC (600 mg CAE/100 g) compared to SB without US for TEM, protocatechuic acid (25.3 ± 0.1 mg/kg) and vanillic acid (550 mg CAE/100 g) possibly through disruption of cell wall, by (19.4 ± 5.8 mg/kg) for US10. Only gallic acid was identified in this

Journal of Food Bioactives | www.isnff-jfb.com 61 Ultrasound, a powerful non-thermal and green technique Dornan et al.

Figure 1. Phenolic fractional profile (free, bound, glycoside and ester conjugated (a) and free vs bound, glycoside and ester conjugated (b) of defatted whole SB using traditional (TEM), 10 min US (US10), and 15 min US (US15) assisted extractions. fraction for US15. Identified glycoside conjugated flavonoids were Only phenolic acids were identified in bound form. All phenolic kaempferol (68.6 ± 9.0 and 85.4 ± 9.2 mg/kg) and quercetin (26.5 acids tested were identified: protocatechuic acid (139.6 ± 5.3, 13.6 ± 3.7 and 21.4 ± 2.9 mg/kg) for TEM and US15, respectively. No ± 2.9 and 13.6 ± 3.0 mg/kg), p-coumaric (39.3 ± 2.4, 26.7 ± 6.5 and flavonoids were found in this fraction for US10. 28.1 ± 4.1 mg/kg), ferulic acid (9.6 ± 1.8, 6.5 ± 0.4, and 5.4 ± 0.2), Ester conjugated phenolic acids extracted and identified were gallic acid (7.7 ± 1.0, 4.5 ± 1.1 and 6.6 ± 3.0 mg/kg), and vanillic ferulic acid (33.9 ± 7.0, 11.9 ± 2.7 and 31.6 ± 0.1 mg/kg), proto- acid (5.4 ± 0.8, 1.6 ± 0.2, and 1.6 ± 0.3 mg/kg) for TEM, US10 and catechuic acid (24.8 ± 5.0, 6.9 ± 1.2 and 11.7 ± 2.8 mg/kg), p-cou- US15, respectively. maric acid (7.2 ± 3.2, 2.7 ± 0.8 and 7.8 ± 0.4 mg/kg) and vanillic Protocatechuic acid was the only phenolic acid identified in the acid (2.0 ± 0.8, 0.7 ± 0.1 and 0.7 ± 0.4 mg/kg) for TEM, US10 and free fractions for all samples (20.7 ± 2.3 mg/kg for TEM, 3.7 ± US15, respectively. Rutin was the only ester conjugated flavonoid 0.5 mg/kg for US10, and 8.7 ± 0.9 mg/kg for US15). Flavonoids extracted and identified in all extraction methods, 32.5 ± 5.5 mg/ represented the bulk of this fraction, specifically rutin (39.0 ± 7.8, kg for TEM, 10.0 ± 1.1 mg/kg for US10, and 16.2 ± 0.6 mg/kg for 23.4 ± 3.5, and 15.2 ± 0.7 mg/kg), quercetin (23.2 ± 5.4, 47.6 ± 0.1, US15. and 19.4 ± 1.9 mg/kg), and kaempferol (18.9 ± 6.5, 37.7 ± 0.1, and

62 Journal of Food Bioactives | www.isnff-jfb.com Dornan et al. Ultrasound, a powerful non-thermal and green technique a b de cd de de c e e e e e 256.8 ± 5.0 2,054.5 454.3 1,369.7 Total 201.5 ± 5.8 52.9 ± 9.0 55.3 ± 9.8 109.0 ± 12.9 1,643.7 ± 176.1 112.3 ± 4.0 58.9 ± 4.1 1,186.9 ± 40.4 100.3 ± 6.7 32.3 ± 1.8 68.5 ± 3.0 d b c d a Kaempferol ND 94.6 37.7 100.9 ND ND ND 18.9 ± 6.5 68.6 ± 9.0 37.7 ± 0.1 15.6 ± 2.0 85.4 ± 9.2 ND ND ND bc b a c bc Flavonoids Quercetin ND 49.7 47.6 40.8 26.5 ± 3.7 21.4 ± 2.9 ND ND ND 23.2 ± 5.4 ND 47.6 ± 0.1 ND 19.4 ± 1.9 ND a a b c c bc Rutin 39.0 ± 7.8 23.4 ± 3.5 ND 15.2 ± 0.7 71.5 33.4 31.4 ND ND ND 32.5 ± 5.5 ND 10.0 ± 1.1 ND 16.2 ± 0.6 a a b bc bc c Ferulic acid Ferulic ND ND ND ND 43.5 18.4 37.0 9.6 ± 1.8 33.9 ± 7.0 ND 6.5 ± 0.4 11.9 ± 2.7 5.4 ± 0.2 31.6 ± 0. 1 ND a b b c c c c -coumaric acid P -coumaric ND 39.3 ± 2.4 7.2 ± 3.2 3.4 ± 0.9 ND 26.7 ± 6.5 2.7 ± 0.8 ND ND 28.1 ± 4.1 7.8 ± 0.4 49.8 29.5 36.0 ND a b b b b b b b Vanillic acid Vanillic ND 5.4 ± 0.8 2.0 ± 0.8 ND 1.6 ± 0.2 0.7 ± 0.1 19.4 ± 5.8 ND 1.6 ± 0.3 0.7 ± 0.4 59.9 21.7 2.2 52.5 ± 27.5 ND Phenolic acids a b b c b c cd e de cde 20.7 ± 2.3 3.7 ± 0.5 8.7 ± 0.9 Proto catechuic acid catechuic Proto 24.8 ± 5.0 185.1 6.9 ± 1.2 49.6 34.0 139.6 ± 5.3 ND 13.6 ± 2.9 25.3 ± 0.1 13.6 ± 3.0 11.7 ± 2.8 ND a b c d d d d ND ND ND Gallic acid 7.7 ± 1.0 ND 184.5 ± 1,492.7 1,500.4 4.5 ± 1.1 ND 212.1 ± 2.9 216.5 6.6 ± 3.0 0.6 ± 0.1 1,080.1 ± 48.5 1,087.3 Traditional Free US-10 Free US-15 Free Bound Conjugated Esters Glycosides Total Bound Conjugated Esters Glycosides Total Bound Conjugated Esters Glycosides Total Table 2. Free, bound, and conjugated phenolic acids and flavonoids in SB berries (mg/kg phenolic acids and flavonoids of dry matter) bound, and conjugated 2. Free, Table determined. P < 0.05). ND: not ANOVA (significance: using one-way difference no significant column indicate in a The same letters deviation. ± standard means of triplicates are Values

Journal of Food Bioactives | www.isnff-jfb.com 63 Ultrasound, a powerful non-thermal and green technique Dornan et al.

15.6 ± 2.0 mg/kg) for TEM, US10 and US15, respectively. The importance of solubility and biotransformation of plant phenolics and food matrix have not been examined in much detail, as they ultimately effect phenolics bioavailability. Although phenolic/polyphenols are very abundant in our diet, majority of phenolics (e.g. proanthocyanidins/tannins, anthocyanins) are either very poorly absorbed or not absorbed at all. It has been reported only 5 to 20% of phenolics are ready for absorption and research evidences suggested that solubility of plant phenolics might be the primary reason that limit phenolic bioavailability. Transformation of lipophilic compounds into hydrophilic compounds is called het- erologous compounds, in which compounds are easily absorbed and excreted in the human body (Karakaya, 2004; Kawabata et al., 2015; Shahidi et al., 2019). This means the plant phenolics in aglycone forms (more lipophilic) have higher absorption, bioavailability, and biotransformation than their bound, ester, and glycosidic forms. It is interesting to note that with US10 was able to extract a similar amount of aglycones (112.3 ± 4.0 mg/kg) compared with TEM (109.0 ± 12.9 mg/kg), both of which had a significantly higher free phenolics fraction compared to US15, see Figure 1. Further- more, as seen in Table 2, no quercetin or kaempferol glycosides were observed for US10, as opposed to TEM and US15, while a significant increase in the aglycone form of these two flavonoids was observed for US10 compared to TEM and US15. This significant increase could be due to hydrolysis of the glycoside bond facilitated by US when comparing US10 to TEM. But when comparing US10 to US15, an extra 5 min of US led to a decrease in the free form of quercetin and kaempferol and a significant increase in their glycoside derivative. In this case the extra 5 min of US has a negative effect on the free form nature of the flavonoids either through degradation by radicals produced by US or reversion back to their glycosidic derivative perhaps facilitated by prolonged US. Although the significant increase of glycosidic derivatives in US15 compared to US10 is likely due to further extraction of these compounds as there exists no evidence that US may lead to glycosidic bond formation. US10 was chosen as having the optimal US treatment time to prevent degradation and get the higher amount of Figure 2. Seabuckthorn puree (P) without and with ultrasound treatment bioavailable aglycones. (P+US) on day 0 and 21.

3.3. Application of US for SB kombucha physical characteristics.

3.3.1. Physical observations 3.3.2. Nutritional properties

Images were taken on day 0 and 21 for P and P+US, shown in Moisture, ash, protein and fat content of P and P+US are presented Figure 2, and on day 0, 5 and 21 for kombucha samples, shown in Table 3. US significantly (P < 0.05) increased by 10% extraction in Figure 3. On day 0, all sonicated samples (P+US, K+S+US, yield from pulp (from 19.04 ± 0.08 to 20.97 ± 0.29%) and by 7% and K+US) were homogenous while syneresis can be seen in all for seed (from 14.81 ± 0.08 to 15.83 ± 0.28%). This increase in non-sonicated samples (P, K+S, and K) approximately 2 h after oil yield highlights the functionality of US as a green technology sample preparation. By day 5 of fermentation, no phase separation to maximize raw material value by increasing oil yield while reis seen in all kombucha samples. The disappearance of syneresis ducing processing time, power consumption, and use of hazardous in K and K+S by day 5 shows the emulsifying effect of fermen- solvents (Hernandez-Santos et al., 2016). It has been shown that tation, possibly explainable through hydrolysis of insoluble high sonication can lead to higher extraction yields through disruption molecular weight polysaccharides by SCOBY into soluble low of the cell wall leading to increased solvent penetration and surface molecular weight sugars (Septembre-Malaterre et al., 2018). By area between solvent and oil through agitation (Zhang et al., 2008). day 21 of storage, P+US remained homogenous. This shows that US alone is an effective emulsification technology for this product, 3.3.3. Total microbial count with no syneresis observed in P+US for the whole study (21 days). All kombucha samples also remained stable for the whole study with no mold observed showing the preservative effects of fermen- To investigate US as a co-sterilizer, total microbial count (log tation. Based on visual observations, sonicated samples were also CFU/mL) for each sample was measured and results are presented thicker in texture compared to non-sonicated samples, however, in Figure 4. The US treatment applied to P+US on day 0 resulted rheological study would be required to determine effect of US on in a 2.5-log reduction in CFU/mL compared to P. By day 21, total

64 Journal of Food Bioactives | www.isnff-jfb.com Dornan et al. Ultrasound, a powerful non-thermal and green technique

Figure 3. Kombucha (K) samples without and with sugar (S) and without and with ultrasound treatment (US) on day 0, 5 and 21. microbial count was significantly lower (P < 0.05) in P+US com- by the decrease in temperature on day 5 (stored at 4 °C) and by pared to P with a 2.8-log difference in CFU/mL. This shows the similar results seen by Ayed et al. (2017) who reported an initial efficacy US technology for reduction of microbial load. Also, on increase in microbial count during fermentation of red grape juice day 0, log CFU/mL of kombucha samples with US (K+US and and a decrease after day 6 attributed to a decrease in oxygen, creat- K+S+US) was slightly lower than non-US samples (K and K+S). ing an anaerobic and starved environment for the SCOBY (Chen Also seen on day 0, sonicated kombucha samples had a slightly and Liu, 2000). higher log CFU/mL compare to P+US due to microbial repopula- US did not result in a complete elimination of CFUs in samples. tion by SCOBY added after US. On day 2, there was an increase in Khandpur et al. (2015) reported 5 log reductions with 100 W ultra- log CFU/mL for all kombucha samples with no significant differ- sound treatment at 20 kHz for 15 min at less than 30 °C in combi- ence between them. A second US treatment was applied to K+US nation with solvent extracts of orange peel oil in fruit and vegeta- and K+S+US on day 5 which led to a 2.4 and 2-log decrease in ble juice, which satisfied safety limits in refrigerated conditions for total microbial count compared to K and K+S, respectively. A 20 days. A 5-log pathogen reduction in fruit juice is required by the 2-log decrease was then observed for non-sonicated samples (K FDA. In this study pathogen count was not determined but rather and K+S) on day 14 compared to day 5. This could be explained total microbial count. Also, seeing as kombucha is fermented with

Table 3. Moisture, ash, protein and fat contents as percentage of P and P+US Sample Moisture Ash Protein Fat content pulp (%) Fat content seed (%) P 87.5 ± 0.2a 1.52 ± 0.00a 9.80 ± 0.46a 19.04 ± 0.08a 14.81 ± 0.08a P+US 88.7 ± 1.8a 1.51 ± 0.00a 9.84 ± 0.85a 20.97 ± 0.29b 15.83 ± 0.28b

P: purée with 30% water; US: ultrasonic treatment. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one- way ANOVA (significance: P < 0.05).

Journal of Food Bioactives | www.isnff-jfb.com 65 Ultrasound, a powerful non-thermal and green technique Dornan et al.

Figure 4. Total microbial count (log CFU/mL) of samples with and without US on day 0, 2, 5, 14 and 21. probiotics, a complete reduction in microbial count may not be fa- nolics or volatile short chain fatty acids, which are byproducts in vourable. In Canada, in order to be able to make a probiotic health fermentation of dietary fibre (den Besten et al., 2013). No previous claim, a serving of stated size of a product must contain a mini- research exists in the literature investigating oxidative stability of mum level of 1.0 × 109 CFU of specified Bifidobacterium and/or kombucha or seabuckthorn puree using Rancimat but a study as- Lactobacillus strains (CFIA, 2019). Future studies are required to sessing the oxidative stability of flaxseed-enriched lasagna using isolate, characterize and count the microbial strains present in the the Rancimat method, by Mercier et al. (2015), reported an initial beverage to determine its eligibility as a probiotic product. increase in conductivity, presumably explained by the presence of Temperature, time, power and frequency of US are main factors volatile short chain fatty acids. It would be interesting to investi- affecting the overall reduction of microbial count (Ashokkumar, gate the presence of volatile short chain fatty acids and volatile 2015). Our study had no temperature control during application phenols in kombucha in future studies using gas chromatographic of US. Sample temperature reached 60 °C after 4 min and a maxi- headspace analysis. mum of 88 °C after 10 min, thereby exposing the samples to temperatures similar to conventional thermal treatment of juice (75–80 °C) for 6 min. Despite this, no significant degradation of nutrients, 3.3.5. Oxygen radical absorbance capacity (ORAC) antioxidant activity or vitamin C content was seen in this study. The parameters used represent a high power low frequency sys- ORAC results are shown in Table 5. US led to 3% increase in an- tem capable of generating acoustic cavitation leading to rupturing tioxidant capacity in P+US (315.42 ± 1.76 µmol TE/L) compared of microbial cell walls with minimal generation of free radicals to P (305.96 ± 0.29 µmol TE/L) on day 0. This increase is less than (Ashokkumar, 2015). The combined acoustic cavitation, heat gen- that observed by Golmohamadi et al. (2013) who found that soni- erated from US, and the production of ethanol and organic acids cation of raspberry purée for 10 min at 20 kHz resulted in a 17.3% during fermentation may act in synergy to prevent the growth of increase in total antioxidant activity, measured by the photochemi- pathogenic organisms in kombucha without decreasing nutritive luminescence method, attributed to disruption of the plant cell wall qualities. by US and release of bioactive compounds. The explanation for differences observed between studies may be attributed to methods 3.3.4. Oxidative stability Table 4. Rancimat results (Induction time, h) as a measure of oxidative stability for samples with and without US Due to the high oil and unsaturated fatty acid content of SB, oxidative stability of the samples was determined using Rancimat to Sample Induction time measure induction time (h), presented in Table 4. US had no effect P 2.66 ± 0.35a on oxidative stability, with no significant differences observed be- b tween P (2.66 ± 0.35 h) and P+US (2.91 ± 1.46 h). Fermentation K 0.10 ± 0.01 decreased induction time for K (0.10 ± 0.01 h), K+US (0.22 ± 0.10 K+S 0.13 ± 0.02b h), K+S (0.13 ± 0.02 h) and K+S+US (0.40 ± 0.28 h) with no a significant difference between kombucha samples. These results P + US 2.91 ± 1.46 were not expected since fermentation should lead to an increase K+US 0.22 ± 0.10b in antioxidant activity and thus an increase in oxidative stability, b therefore an increase in induction time, measured by Rancimat, K+S+US 0.40 ± 0.28 would be expected. However, the decrease in induction time ob- P: purée with 30% water; US: ultrasonic treatment; K: kombucha; S: sugar. Values served in this study may be explained by the production of volatile are means of triplicates ± standard deviation. The same letters indicate no significant by products from fermentation, such as low molecular weight phe- difference using one-way ANOVA (significance: P < 0.05).

66 Journal of Food Bioactives | www.isnff-jfb.com Dornan et al. Ultrasound, a powerful non-thermal and green technique

Table 5. ORAC (µmol TE/L) results for samples with and without US on day 0,2,5,7 and 21 Day ORAC 0 2 5 7 21 P 305.96 ± 0.29b N/A N/A 300.83 ± 3.89b 298.30 ± 2.35ab K N/A 301.18 ± 3.24a 299.51 ± 8.07a N/A 280.01 ± 7.18c K+S N/A 302.23 ± 1.33a 286.01 ± 4.94b N/A 283.39 ± 14.06c P+US 315.42 ± 1.76a N/A N/A 309.99 ± 0.85a 300.40 ± 1.58ab K+US N/A 311.65 ± 15.12a 305.87 ± 9.95a N/A 308.98 ± 8.15a K+S+US N/A 298.12 ± 4.66a 282.66 ± 11.22b N/A 289.09 ± 5.97ab

N/A: not applicable; P: purée with 30% water; K: kombucha; S: sugar; US: ultrasonic treatment. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05). used to measure antioxidant activity. 0.12 g/L). Vitamin C measurement is useful to predict nutrient loss The ORAC values of kombucha shows that the addition of su- during food processing and storage due to its easily deteriorative crose increased the rate of antioxidant depletion, as ORAC for nature. If vitamin C is retained, other nutrients are most likely to K+S (286.01 ± 4.94 µmol TE/L) on day 5 was significantly (P also be retained (Araya-Farias et al., 2011). Vitamin C content for < 0.05) lower than P (305.96 ± 0.29 µmol TE/L) on day 0 while K, K+US, K+S and K+S+US were 0.20 ± 0.02, 0.10 ± 0.03, 0.15 K (299.51 ± 8.07 µmol TE/L) was not. The same is observed ± 0.00 and 0.19 ± 0.10 g/L, respectively, with no significant dif- when comparing P+US (315.42 ± 1.76 µmol TE/L) on day 0 to ference between treatments. Vitamin C for the kombucha samples K+S+US (282.66 ± 11.22 µmol TE/L) and K+US (305.87 ± 0.85 were significantly (P < 0.05) lower than P, P+US with a 11-to µmol TE/L) on day 5. Shalaby et al. (2016) reported a decrease 16-fold reduction. Aerobic fermentation at room temperature for 5 in antioxidant activity in green tea with addition of sucrose poten- days likely contributed to this decrease. Fortification with ascorbic tially attributable to the condensation reaction between hydroxyl acid, often done with juices from fruit naturally low in vitamin C, groups of phenolics and sucrose molecules to form glycosides. No may serve as a solution to overcome the observed loss of vitamin significant difference was observed in ORAC between P (298.30 C during fermentation and should be investigated to determine its ± 2.35 µmol TE/L), P+US (300.40 ± 1.58 µmol TE/L), K+US effects since ascorbic acid may have negative depending on food (308.98 ± 8.15 µmol TE/L) and K+S+US (289.09 ± 5.97 µmol matrix and composition (Pacheco-palencia et al., 2007). TE/L) on day 21 while K (280.01 ± 7.18 µmol TE/L) and K+S (283.39 ± 14.06 µmol TE/L) had significantly lower ORAC val- 3.3.7. WSI and WAI of freeze-dried powder ues. This suggests that combining US and fermentation may protect against depletion of antioxidants during shelf life compared to fermentation alone perhaps through the breakdown of plant WSI and WAI for all samples are presented in Table 7. US signifi- cell walls by sonication and release of bioactive compounds into cantly increased WAI by 40% for P+US (6.64 ± 0.70 g/g) com- the solution (Golmohamadi et al., 2013), combined with release pared to P (9.29 ± 1.56 g/g) but had no effect on WSI when com- of polysaccharide conjugated antioxidants through fermentation paring P+US (44.2 ± 1.93%) and P (46.81 ± 3.03%). The increase (Wang et al., 2016). in WAI by US may be explained by an increase in pore space and/ or damage to the plant cell wall by sonication (Golmohamadi et al., 2013). When investigating the effects of different drying tech- 3.3.6. Vitamin C niques on seabuckthorn berries, Kyriakopoulou et al. (2013) found that freeze drying resulted in an increase in WAI compared to air The concentration of vitamin C, for all samples, is presented in drying, and accelerated solar drying, attributed to an increase in Table 6. US had no effect on vitamin C content with no signifi- pore space. In this study both freeze-drying and US were used cant difference between P (2.31 ± 0.83 g/L) and P+US (1.65 ± Table 7. Water Solubility Index (%) and Water Absorption Index (g/g) of Table 6. Vitamin C concentration (g/L) for samples with and without US freeze dried samples without and without US Sample Vitamin C Sample Water Solubility Index Water Absorption Index P 2.31 ± 0.83a P 46.81 ± 3.03d 6.64 ± 0.70b K 0.20 ± 0.02b K 61.60 ± 1.65c 3.97 ± 0.75c K+S 0.15 ± 0.00b K+S 83.31 ± 1.34a 1.19 ± 0.02d P + US 1.65 ± 0.12a P + US 44.62 ± 1.93d 9.29 ± 1.56a K+US 0.10 ± 0.03b K+US 59.25 ± 0.49c 4.62 ± 2.58bc K+S+US 0.19 ± 0.10b K+S+US 89.93 ± 5.42a 1.39 ± 0.18d

P: purée with 30% water; US: ultrasonic treatment; K: kombucha; S: sugar. Values are P: purée with 30% water; K: kombucha; US: ultrasonic treatment; S: sugar. Values are means of triplicates ± standard deviation. The same letters in a column indicate no means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05). significant difference using one-way ANOVA (significance: P < 0.05).

Journal of Food Bioactives | www.isnff-jfb.com 67 Ultrasound, a powerful non-thermal and green technique Dornan et al. which may have led to a synergistic effect on pore space through sonch.2014.08.012. damage to the cell wall, leading to an increase in water absorption. Ayed, L., Abid, S.B., and Hamdi, M. (2017). Development of a beverage Fermentation alone led a 32% increase in WSI when comparing K from red grape juice fermented with the Kombucha consortium. (61.50 ± 1.65%) to P (46.81 ± 3.03%). This may be attributed to Ann. Microbiol. 67(1): 111–121. doi:10.1007/s13213-016-1242-2. the breakdown of polysaccharides to lower molecular weight sug- Beveridge, T., Li, T.S.C., Oomah, B.D., and Smith, A. (1999). Sea Buckthorn Products: Manufacture and Composition†. J. Agric. Food Chem. ars that are more water-soluble (Septembre-Malaterre et al., 2018). 47(9): 3480–3488. doi:10.1021/jf981331m. The addition of sucrose increased WSI by 35% when comparing Bilal Hussain, M., Hassan, S., Waheed, M., Javed, A., Adil Farooq, M., and K+S and K and by 52% when comparing K+S+US and K+S, as Tahir, A. (2018). Bioavailability and Metabolic Pathway of Phenolic expected with water-solubility of sucrose. Compounds. Plant Physiol. Aspects Phenolic Compd.. IntechOpen, Fermentation alone led to a 40% decrease in WAI when com- Available from: https://www.intechopen.com/books/plant-physio- paring K (3.97 ± 0.75 g/g) to P (6.64 ± 0.70 g/g). This is may be logical-aspects-of-phenolic-compounds/bioavailability-and-meta- attributable to the breakdown of pectin networks that are normally bolic-pathway-of-phenolic-compounds. capable of trapping water (Sila et al., 2009). US also increased CFIA. (2019). Probiotic Claims. Retrieved from http://inspection.gc.ca/ food/requirements-and-guidance/labelling-standards-of-identity- the WAI of kombucha samples, with exception of samples with and-grades/for-industry/health-claims/eng/1392834838383/13928 added sucrose. The addition of sucrose led to a significant (P < 34887794?chap=10. 0.05) decrease in WAI when comparing K+S and K+S+US, to K Chakravorty, S., Bhattacharya, S., Chatzinotas, A., Chakraborty, W., Bhat- and K+US, respectively. tacharya, D., and Gachui, R. (2016). Kombucha tea fermentation: Mi- crobial and biochemical dynamics. Int. J. Food Microbiol. 220(Com- plete): 63–72. 4. Conclusion Chauhan, A.S., Rekha, M.N., Ramteke, R.S., and Eipeson, W.E. (2001). Preparation and quality evaluation of processed products from Seabuckthorn (Hippophae rhamnoides Lin.) berries. Beverage and Ultrasound extracted a similar amount of phenolics in aglycone/ Food World 28(3): 31–34. free form in 10 min compared to the 1 hr traditional extraction Chauhan, A.S., Rekha, M.N., Ramteke, R.S., and Eipeson, W.E. (2001). method, while the 15 min UAE showed a significant decrease in Preparation and quality evaluation of processed products from aglycones. For this reason, a 10 min US treatment was chosen for Seabuckthorn (Hippophae rhamnoides Lin.) berries. Beverage and the rest of the study. SB was an effective substrate for fermentation Food World 28(3): 31–34. Chen, C., and Liu, B.Y. (2000). Changes in major components of tea in production of kombucha. US resulted prevented phase separa- fungus metabolites during prolonged fermentation - Chen - 2000 tion in P+US for the whole study (21 days). Fermentation was also - Journal of Applied Microbiology - Wiley Online Library. Journal an effective emulsifier with no syneresis observed in all kombucha of Applied Microbiology 89(5): 834–839. doi:10.1046/j.1365- samples for the whole study. US increased oil content extracted 2672.2000.01188.x. from pulp by 10% and from seed by 7% and had no effect on mois- Cruz-Cansino, N.d.S., Reyes-Hernandez, I., Delgado-Olivares, L., Jaramillo- ture, protein or ash content of P+US compared to P. US application Bustos, D.P., Ariza-Ortega, J.A., and Ramirez-Moreno, E. (2016). Ef- decreased microbial load by 2 or 3 log CFU/mL, had no effect fect of ultrasound on survival and growth of Escherichia coli in cactus on oxidative stability, and increased ORAC value by 3% in P+US pear juice during storage. Braz. J. Microbiol. 47(2): 431–437. compared to P on day 0. US had no effect on vitamin C content den Besten, G., van Eunen, K., Groen, A.K., Venema, K., Reijngoud, D.-J., and Bakker, B.M. (2013). 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Original Research J. Food Bioact. 2020;9:70–78

Physicochemical characteristics, phenolic profile, and antioxidant capacity of Syrah tropical wines: effects of vineyard management practices

Erika Samantha Santos de Carvalhoa, Aline Camarão Telles Biasotob*, Rita de Cássia Mirela Resende Nassurc, Ana Paula André Barrosd, Patrícia Coelho Souza Leãob, Renan da Silva Limae, Adriano Costa de Camargof* and Maria Eugênia de Oliveira Mamedea aUniversidade Federal da Bahia–UFBA, Departamento de Análises Bromatológica Faculdade de Farmácia–Salvador–BA, Brasil bEmpresa Brasileira de Pesquisa Agropecuária–Embrapa Semiárido, Rodovia BR 428, Km 152, Caixa Postal 23. CEP. 56.302-970 Petro- lina–PE, Brasil cUniversidade do Estado da Bahia–UNEB, Av. Edgard Chastinet, s/n–Campus Juazeiro, Juazeiro–BA, Brasil dInstituto Federal de Educação, Ciência e Tecnologia do Sertão Pernambucano–Campus Petrolina Zona Rural–Petrolina–PE, Brasil eDepartment of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, A1B 3X9, Canada fDepartamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad, Católica de Chile, Casilla 306- 22, Santiago, Chile *Corresponding author: Aline Camarão Telles Biasoto, Empresa Brasileira de Pesquisa Agropecuária–Embrapa Semiárido, Rodovia BR 428, Km 152, Caixa Postal 23. CEP. 56.302-970 Petrolina–PE, Brasil. E-mail: [email protected]; Adriano Costa de Camargo, De- partamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad, Católica de Chile, Casilla 306-22, Santiago, Chile. E-mail: [email protected] DOI: 10.31665/JFB.2020.9220 Received: March 17, 2020; Revised received & accepted: March 31, 2020 Citation: de Carvalho, E.S.S, Biasoto, A.C.T, de Cássia Mirela Resende Nassur, R., Barros, A.P.A., Leão, P.C.S, Lima, R.S., de Camargo, A.C., and de Oliveira Mamede, M.E. (2020). Physicochemical characteristics, phenolic profile, and antioxidant capacity, of Syrah tropical wines: effects of vineyard management practices. J. Food Bioact. 9: 70–78.

Abstract

The present study evaluated the influence of training systems and rootstocks on the quality of Syrah tropical wines, produced at São Francisco Valley, Brazil. For this purpose, physicochemical characteristics, phenolic composition, and antioxidant activity were assessed in wines produced with grapes grown under divided trellis system (lyre) and esparlier or vertical shoot position (VSP) training systems, grafted on IAC 572, IAC 766 and Paulsen 1103 rootsotcks and harvested at two different periods. Harvest season had the strongest influence on wine quality, followed by the rootstock. Regardless of the training system and climatic variability between the harvests, the use of the IAC 766 rootstock led to wines with higher alcohol, anthocyanins contents and color intensity. The interaction between the espalier training system and the rootstock IAC 766 resulted in higher flavonol content, phenolic acids, and malvidin-3-O-glucoside, which was detected as the major phenolic as quantified by HPLC. This wine also presented significant levels of procyanidins A2 and B2, which showed a positive correlation with the antioxidant activity.

Keywords: São Francisco Valley; Vitis vinifera L; Phenolic compounds; Antioxidant activity; Physicochemical composition.

1. Introduction systems, such as espalier and lyre, promote bunches exposure and aeration between the lines, making the microclimate warmer and Wine consumption has long been related to a myriad of health- less humid, which favors grape maturation and reduces rot occur- promoting effects, such as lower incidence of some types of cancer rence (Regina et al., 1998). In addition, the divided trellis system and cardiovascular ailments (Forester and Waterhouse, 2009). The (e.g., lyra) almost doubles grape yield when compared to the VSP impact of wine components on health continue to be assessed by system (Carbonnea et al., 2007). Nevertheless, the rootstock influ- many studies, and it seems to be associated with the presence of ences the plant vegetative growth, yield, and grape quality (Li et al., 2019), with significant soil-climatic interference, responding antioxidant substances, namely phenolic compounds (Cueva et al., differently according to the grafted canopy variety (Nassur et al., 2016; Fernandes et al. 2017). Phenolics are plant secondary me- 2014). tabolites capable of mitigating oxidative stress mainly through free With that being said, the study of more suitable techniques radical scavenging, reducing properties as well as metal chelation for the agronomic management of Syrah vines cultivated under (Shahidi and Ambigaipalan, 2015). semi-arid tropical climate conditions may play a role in improving Wine possesses a great variety of phenolic groups, namely phe- the quality of red wine produced in São Francisco Valley, Brazil. nolic acids and flavonoids, including anthocyanins and proantho- Therefore, the objective of this study was to evaluate the effect cyanidins (de Oliveira et al., 2017). Additionally, phenolic-rich of different rootstocks and training systems on the physicochemi- extracts from winemaking by-products have displayed in vitro cal parameters, phenolic composition, and antioxidant activity of inhibition of copper-induced human LDL-cholesterol oxidation, Syrah red wines from the São Francisco Valley. as well as prevention of peroxyl radical-induced DNA strand scission, which is believed to be related to their antioxidant capacity (de Camargo et al., 2014). 2. Material and methods Grape cultivation practices play an important role in wine’s phenolic composition, and, consequently, the health-promoting benefits associated with these compounds (Sartor et al., 2017). 2.1. Chemicals The recently established wine production in São Francisco Valley, Brazil, has been gaining attention due to the product’s unique char- Phenolic standards ferulic, syringic, chlorogenic, caffeic acids, acteristics. The region is located in an area of a semiarid tropical and quercetin were obtained from Chem Service (WestChester, climate, in the Brazilian Northeast (latitude of 8° South, longitude USA) and Sigma-Aldrich (St Louis, MO, USA), while the other of 40° West, and altitude of 350 m). São Francisco Valley has an standards were purchased from Extrasynthese (Genay, France). annual average temperature of 26 °C, insolation of 3,000 h/year, Orthophosphoric acid was obtained from Fluka (Switzerland), and 3 and maximum rainfall of 500 mm /year (Padilha et al., 2016). Due HPLC grade acetonitrile was purchased from JT Baker (Phillips- to the absence of low temperatures during the autumn and winter burg, NJ, USA). seasons and the availability of water for irrigation, year-round production can be carried out, with two annual harvests. Currently, the wine production in São Francisco Valley accounts for about 4 2.2. Grape cultivation million liters per year, being majorly destined to sparkling wines manufacture (70%) (Pereira et al., 2016). Unlike other Brazilian Grapes of the Syrah variety were harvested from ‘Bebedouro’ ex- wine regions, São Francisco Valley wines, which are classified as perimental field–Embrapa Semiarid (09° 09′S, 40° 22′W, 365.5 m, tropical wines, are mainly produced from Vitis vinifera L. grapes, Petrolina-PE, Brazil) in June 2014 (harvest I) and December 2014 being Syrah the main variety. (harvest II), corresponding to the 5th and 6th production cycles, re- However, as grape production for winemaking in São Francisco spectively. The treatments in the field were arranged in subdivided is still recent, all the optimum agricultural practices for the grape plots, where two training systems represented the main treatments: varieties grown in this area have not been totally established thus divided trellis (lyre) and vertical shoot position (VSP), and the far. In some cases, the grapes used for winemaking are cultivated secondary treatments by three rootstocks (Paulsen 1103, IAC 572, using the same practices used to produce table grapes. Techniques and IAC 766). The experimental design was a randomized block applied to vines grown in temperate climate conditions have also with four replicates. During the crop year, the climatic conditions been applied. However, neither approach is consistent with the presented the following means and deviations: temperature (26.2 correct cultivation practices for tropical winemaking grapes. The ± 0.9 °C), relative humidity (64.4 ± 5.5%), precipitation (549.8 production of quality wines is directly related to the climatic con- ± 181.8 mm), solar brightness (7.5 ± 1.1 hours), solar radiation ditions of the vineyard and the agronomic techniques adopted, in- (442.3 ± 32.3 W m2), and air velocity (190.4 ± 27.2 km day−1). cluding the choice of vine rootstock and training system. Some studies have highlighted the relevance of climatic conditions, geo- graphic origin, and crop management practices for wine quality 2.3. Winemaking (Alañón et al., 2015; Cassino et al., 2017; Moreno et al., 2017). A suitable training system favors microclimatic conditions, After manual harvesting, 40 kg of grapes (four replicates of each such as appropriate balance for the vegetative growth of grape- treatment in the experimental area) were homogenized and experi- vine, increased production, and maximized oenological potential mentally vinified at the Embrapa Semi-Arid Oenology - Labora of the grapes (Santos et al., 2006). However, the effect of grape- tory, Petrolina-PE, Brazil. Vinification was carried out in duplicate vine grafting on wine composition remains controversial, since based on the traditional method for young red wines (Peynaud, other variables such as cultivar and environment may affect the 1997). After destemming and crushing the grapes, the wort must resulting performance (Nassur et al., 2017). Among the adopted placed in 20 L glass bottles, capped with a glass airlock valve, management systems in traditional wine-producing areas, the ver- and sulfided with the addition of potassium metabisulphite (100 tical shoot position system is considered the most suitable for Vitis mg kg−1). The alcoholic fermentation was carried out in an air- vinifera grapes (Miele and Mandelli, 2014). Vertical and oblique conditioned room (25 ± 1 °C), followed by 7-day maceration after

Journal of Food Bioactives | www.isnff-jfb.com 71 Physicochemical characteristics, phenolic profile, and antioxidant capacity de Carvalho et al. addition of yeast Saccharomyces cerevisiae Maurivin PDM® (200 ANOVA and means compared by Tukey’s test (p ≤ 0.05). Princi- mg Kg−1), ammonium phosphate activator Gesferm Plus® (200 pal Component Analysis (PCA) and Pearson’s correlation analyses mg Kg−1), and Everum Thermp pectinolytic enzyme (0.008 mL were also performed. Kg−1). Then, spontaneous malolactic fermentation (18 ± 1 °C), cold tartaric stabilization (0 °C), and the addition of a stabilizer composed of Arabic gum and metatartaric acid–Stabigum® (400 3. Results and discussion mg L−1) were performed. Before bottling, the free sulfur dioxide −1 (SO2) content of the wines was corrected to 50 mg L , and the 3.1. Physicochemical characteristics wines were stored in an air-conditioned wine cellar at 18 ± 1 °C one month before the analyses were carried out. The physicochemical characteristics of the wines are presented in Table 1. The pH value of the wines varied slightly between the 2.4. Physicochemical analyses evaluated harvests, from 3.77 to 3.96 for the harvest I (June) and from 3.70 to 3.90 for the harvest II (December). According to Jack- The physicochemical parameters were determined following the son (2008), the ideal pH for red wines should be between 3.3 and OIV (2015) procedures. The pH of each sample was measured us- 3.6. However, due to the intense solar radiation in the São Fran- ing a pH meter Tec-3MP (TECNAL, Piracicaba, SP, Brazil). After cisco Valley and the high levels of potassium in the soil, the red the simple distillation of the samples in SuperDee automatic dis- wines of this region usually have higher pHs, reaching values as tiller (Gibertini, Italy), the alcohol content and the dry extract con- high as 4.0. Padilha et al. (2016) evaluated commercial red wines tent in Super Alcomat electronic hydrostatic balance (Gibertini, and observed pH values between 3.60 and 4.09. The influence of Italy) were determined at 20 °C. The total acidity was quantified the training system on pH was significant (p ≤ 0.05) only for sam- by titration of the samples with 0.1 N NaOH to pH 8.2. The volatile ples from grapes grown under the IAC 766 rootstock from harvest acidity was determined after the steam distillation of the samples II (lower pH value). Reduced pH in wines can be associated with in the SuperDee distiller and titration with 0.1 N NaOH. The free higher color intensity and phenolic compounds concentration (Pic- and total sulfur dioxide contents were determined by iodometry cardo et al., 2019). Grape pulp usually ripens faster than skins, re- according to the Ripper method, which uses a 0.02 N iodine solu- ducing pH and sugar content in the must and results in high pH and tion as titrant and starch as an indicator. The content of total reduc- sugar concentrations in the must (Llaudy et al., 2008) and wine. ing sugars was determined by the Lane-Eynon method, based on The period of the year and the rootstock can influence ripening and the procedures described by Ribéreau-Gayon et al. (2006). quality attributes in wines. The total titratable acidity (TTA) ranged from 5.20 to 6.55 g L−1 in wines from harvest I and 4.03 and 4.63 g L−1 in wines from 2.5. Phenolic composition, color intensity, and antioxidant harvest II (Table 1). Different TTA values between harvests could capacity be due to the higher degree of grape maturation during the harvest II period. The Brazilian legislation (Brasil, 2010) establishes The total phenolic content (TPC) was determined by the Folin- that the TTA content of red wines should be between 55 and 130 Ciocalteu method (Singleton and Rossi, 1965). Absorbance read- mEq L−1 (4.12 to 9.75 g L−1) and, in general, the evaluated wines ing was performed on a Genesys™ 10S UV-VIS spectrophotome- followed this specification, except for harvest II wines. For both ter (Thermo Fisher Scientific, Waltham, MA, USA) and a standard harvests, the training system did not promote a significant differ- curve was prepared using gallic acid. The concentration of total ence in TTA. The Paulsen 1103 rootstock resulted in wines with monomeric anthocyanins was determined spectrophotometrically the highest TTA, considering the ones produced from harvest I. by the method described by Lee et al. (2005). Monomeric anthocy- The same was observed for rootstock IAC 572 in harvest II. TTA anin contents were expressed as malvidin-3-O-glucoside equiva- values reported by Andrade et al. (2008) in red wines from the lent (MGE). The color intensity (CI) was determined from the sum São Francisco Valley ranged from 3.6 to 6.3 g L−1, similar to what of the absorbance readings at 420 nm, 520 nm and 620 nm (Ough was found in the present study, demonstrating that lower TTAs is and Amerine, 1988), and the antioxidant capacity was evaluated a characteristic of wines produced in this location. According to by the DPPH method (2,2-diphenyl-1-picryl-hydrazyl) according Ordunã (2010), lower TTAs are correlated to higher pH in grapes, to the procedure described by Brand-Williams et al. (1995), us- which is influenced by the potassium concentration. Higher tem- ing Trolox (6-hydroxy-2,5,7,8-tetramethylchromate-2-carboxylic peratures, such as the ones recorded in the São Francisco Valley acid) to prepare the standard curve. during fruit maturation, can significantly increase the potassium level in the fruit. Lima et al. (2015) also stated that the high temperatures and luminosity in this area could lead to an acidity de- 2.6. Identification and quantification of phenolic compounds crease, with higher pH values. The alcohol content of the wines ranged from 10.50 to 11.14% Phenolic compounds were identified and quantified by high-per- (v/v) for the harvest I and 12.11 to 13.40% (v/v) for the harvest II. formance liquid chromatography (HPLC) using a Waters 2695 The increased alcohol content in harvest II may be the result of a Alliance system (Milford, MA, USA) equipped with DAD-array higher degree of grape maturation, which, consequently generates and FD-fluorescence detectors working simultaneously using a a higher content of total soluble solids, mostly sugars. Regardless validated method described elsewhere (Natividade et al., 2013). of the training system, the IAC 766 rootstock resulted in wines with the highest alcoholic content for the harvest I, with no significant differences concerning the rootstock in harvest II for the 2.7. Statistical analysis majority of samples. Miele and Rizzon (2019) suggested that alcohol content is one of the variables most affected by the rootstocks The Statistical analysis was carried out using the SAS software system in Cabernet Sauvignon wines. (SAS® Institute Inc, Cary, NC, 2015). Results were submitted to The dry extract concentration of the wines ranged from 24.70 g

72 Journal of Food Bioactives | www.isnff-jfb.com de Carvalho et al. Physicochemical characteristics, phenolic profile, and antioxidant capacity

Table 1. Physicochemical characteristics, total phenolic content, total monomeric anthocyanin content, and antioxidant activity of Syrah tropical wines from grapes with different training systems and rootstocks Rootstock2 Parameter Training systems1 Paulsen 1103 IAC-766 IAC-572 Paulsen 1103 IAC-766 IAC-572 June, 2014 (Harvest I) December, 2014 (Harvest II) pH Lyre 3.77 Ac 3.93 Aa 3.85 Ab 3.83 Aa 3.79 Ab 3.80 Aa VSP 3.79 Ab 3.96 Aa 3.94 Aa 3.83 Aa 3.70 Bb 3.90 Aa Total acidity (g L−1) Lyre 5.90 Aa 5.30 Ab 5.55 Ab 4.40 Ab 4.40 Ab 4.63 Aa VSP 6.55 Aa 5.45 Ab 5.20 Ab 4.03 Ab 4.10 Aa 4.10 Aa Alcohol (% v/v) Lyre 10.89 Ab 11.02 Aa 10.79 Bc 12.29 Aa 12.24 Aa 12.55 Aa VSP 10.5 Bb 11.14 Aa 11.01 Aa 13.40 Aa 12.11 Ab 13.29 Aa Dry extract (g L−1) Lyre 26.90 Ac 28.60 Bb 29.50 Aa 27.60 Ab 30.29 Aa 26.83 Ab VSP 27.30 Ac 30.75 Aa 29.65 Ab 28.36 Aa 24.70 Bb 29.30 Aa −1 a a a b a b Free SO2 (mg L ) Lyre 30.55 A 30.72 A 30.38 A 45.91 A 49.66 A 42.32 A VSP 26.28 Bb 30.21 Aa 25.77 Bb 39.70 Bc 50.00 Aa 46.50 Ab −1 c a b b a b Total SO2 (mg L ) Lyre 108.88 A 154.62 A 13.00 B 68.4 B 77.6 A 70.3 B VSP 91.39 Bc 106.49 Bb 153.77 Aa 78.0 Aa 73.5 Ab 75.4 Aa Volatile acidity (g L−1) Lyre 0.62 Aa 0.59 Aa 0.62 Aa 0.98 Aa 0.99 Aa 0.55 Ab VSP 0.63 Aa 0.62 Aa 0.48 Bb 0.41 Bb 0.72 Aa 0.39 Bb Reduced sugars (g L−1) Lyre 3.33 Aa 2.27 Ac 2.64 Bb ND 1.33 Aa 1.39 Aa VSP 3.35 Aa ND 2.79 Ab ND ND 1.66 Aa Total phenolics (mg GAE L−1) Lyre 1,900 Ab 2,290 Aa 1,880 Ab 2,270 Ab 2,500 Aa 2,040 Ab VSP 1,780 Aa 1,520 Ab 1,500 Bb 2,180 Aa 1,680 Bb 2,400 Aa Monomeric anthocyanins Lyre 169.07 Ab 183.06 Aa 178.67 Ab 166.99 Ab 191.20 Aa 185.77 Ab (mg MGE L−1) VSP 143.40 Bb 253.61 Aa 203.10 Ac 162.41 Aa 177.84 Aa 162.41 Aa IC (420nm+520nm+620nm) Lyre 6.91 Ab 6.24 Bc 7.64 Aa 4.33 Bc 5.09 Bb 9.17 Aa VSP 7.04 Ac 9.75 Aa 8.03 Ab 6.58 Ac 9.47 Aa 8.83 Bb DPPH (µmol TE mL−1) Lyre 9.23 Aa 8.25 Bb 9.24 Aa 8.33 Aa 7.60 Bc 7.97 Ab VSP 8.95 Aa 9.23 Aa 9.42 Aa 7.97 Ab 9.54 Aa 9.26 Aa

L−1 to 30.75 g L−1 (Table 1). The values for dry extract are lower ards, which recommends volatile acidity up to 20 mEq L−1 (Brazil, than those reported by Oliveira et al. (2011) in studies with com- 2010). The importance of maintaining wines with lower contents mercial red wines (V. vinifera L) from the São Francisco Valley, of volatile acidity relies on the fact that this parameter may nega- ranging from 32.90 to 38.37 g L−1. The present values are above tively influence wine nutritional quality and sensory characteris- the minimum dry extract limit established by the Brazilian leg- tics. Miele and Rizzon (2019) observed no statistical differences in islation, which is 13.0 g L−1 (Brazil, 2010). The training system volatile acidity (ranging from 12–14 meq L−1) for Cabernet Sauvi- influenced the wines’ dry extract content when the grapes were gnon wines grafted under different rootstocks. In the present study, grafted under the IAC 766 rootstock. According to Miele and Riz- rootstocks IAC-572 (harvest I and II) and Paulsen 1103 (harvest II) zon (2019), the main components of the dry extract are organic rendered a decreased volatile acidity. acids and glycerol, being this an important parameter to regulate the wine body. 3.2. Phenolic composition and antioxidant activity In addition, the total SO2 content of the wines ranged from 70.30 to 154.62 mg L−1 (Table 1), below the maximum limit allowed by the Brazilian legislation, which is 350 mg L−1 (Brazil, The total phenolic content (TPC) of wines produced from grapes 2010). The values for volatile acidity varied between 0.39 and with different training systems and rootstocks ranged from 1,500 0.99 g L−1, which is equivalent to 6.50 mEq L−1 and 16.50 mEq to 2,290 mg gallic acid equivalent (GAE) L−1 for wines from the L−1, respectively, and are also within Brazilian legislation stand- harvest I and from 1,680 to 2,500 mg GAE L−1 for the second

Journal of Food Bioactives | www.isnff-jfb.com 73 Physicochemical characteristics, phenolic profile, and antioxidant capacity de Carvalho et al. harvest of the 2014 year, being higher for this period, when grapes 0.01) and B2 (r = 0.57, p = 0.05). The wine containing the highest were harvested at a more advanced maturation stage (Table 1). levels of procyanidin A2, B2, and total flavan-3-ols was obtained Biasoto et al. (2014) reported that TPC in red wines usually vary from grapes grown in a espalier system and grafted on IAC 766 from 1,900 to 3,800 mg GAE L−1. In the present study, most of rootstock. the evaluated wines presented phenolic content within the range A higher concentration of flavonols was observed for the wines suggested by these authors. The combination of lyre training sys- produced in December, when high temperatures are recorded in tem with rootstock IAC 766 yielded the highest concentrations Brazil. Similar findings were obtained by Pereira et al. (2006) that of TPC both for harvest I (2,290 mg L−1) and II (2500 mg L−1). investigated the climate influence on Merlot wine metabolic pro- These differences indicate that grape growing practices have a sig- files with or without the exposition to higher solar radiation. In the nificant influence on wine phenolic composition, as suggested by present study, an increased amount of flavonols was observed in Gutiérrez-Gamboa et al. (2019). Other factors that can affect this grapes and wines (harvest II) due to higher solar exposition and parameter include climatic and processing conditions. temperature. The content of total monomeric anthocyanins (TMA) in wines Flavonols are also considered fundamental for the intensity and ranged from 143.40 to 253.61 mg MGE L−1 for the harvest I and stability of red wine color, and also play an important role for their from 162.41 to 191.20 mg MGE L−1 for those from the harvest II. astringency and bitterness (Niculcea et al., 2015). Regardless of The concentrations of total monomeric anthocyanins obtained in the training system, IAC 766 rootstock resulted in higher flavonol red wines are in accordance with the results obtained by Padilha contents in grapes from harvest I. On the other hand, IAC 572 et al. (2016), who analyzed commercial red wines of São Fran- rootstock originated wines with higher contents of flavonols in the cisco Valley and found TMAs between 36.2 and 351.3 mg L−1. In harvest II. Thus, the choice of rootstock possibly influenced the general, wines from grapes grown under the IAC 766 rootstock synthesis of flavonols in grapes as a function of the period of the presented greater TMA, regardless of the harvest and the evaluated year. Isoquercetin and isorhanmetin-3-O-glucoside were the main training system (Table 1). Similarly, other authors have shown the flavonols present in the tested samples, ranging from 9.35 to 41.25 influence of the rootstock on grape and wine anthocyanins content mgL−1 for isoquercetin, and 8.06 to 26.45 mg L−1 for isorhanme- (Walker et al., 2000; Satisha et al., 2010; Chou and Li 2014; Li et tine-3-O-glucoside. The wine produced with grapes obtained from al., 2019; Gutiérrez-Gomboa et al., 2019). harvest II and grown under lyre system and IAC 572 rootstock The higher ability of phenolic compounds in scavenging DPPH showed to be particularly rich in these two compounds. Evaluating may be linked to higher biological activities as recently demon- grape juices produced in the São Francisco Valley, Nassur et al. strated in cell model systems (Falcão et al., 2019). The antioxidant (2014) observed that IAC 572 rootstock resulted in significantly activity values evaluated by the DPPH method ranged from 7.60 high phenolic compounds and anthocyanins levels. to 9.54 μmol Trolox equivalent (TE) mL−1 (Table 1). The antioxi- The levels of trans-resveratrol in the wines ranged from 0.20 dant activity of the wines did not vary according to the harvest to 0.50 mg L−1, exhibiting the lowest contribution amongst the season. The training system influenced the antioxidant activity of phenolic compounds. However, trans-resveratrol content was not the wine, only when VSP training system was used in combination influenced by the training system and rootstocks. In contrast, the with IAC-766 rootstock, yielding slightly lower values. In general, grapes from harvest II resulted in wines containing higher contents the majority of the wines displayed satisfactory scavenging activ- of trans-resveratrol. Similar results were observed by Lucena et ity towards the DPPH radical, which is consistent with the pres- al. (2010) for wines from the São Francisco Valley, with contents ence of phenolic antioxidants in abundance. between 0.04 and 1.26 mg L−1. However, the authors reported that resveratrol in the cis form is found in higher levels than the trans form in wines from this location. Consumption of resvera- 3.3. Identification and quantification of phenolic compounds trol is associated with the prevention of a wide range of chronic- degenerative diseases (Gresele et al., 2011). According to Urvieta With the use of HPLC-DAD-FD analysis, it was possible to iden- et al. (2018), fruits that receive higher UV-B incidence may result tify and quantify 24 phenolic compounds in the wines (Table 2). in wines with higher resveratrol content, which lends support to Among them, phenolics from different classes, namely flavan- the differences observed in the present study. In fact, the wines 3-ols (n = 7), flavonols (n = 6), stilbenes (n = 1), phenolic acids produced from grapes from harvest I, that received a lower UV-B (n = 5), and anthocyanins (n = 5) were positively identified. The incidence, also had lower levels of trans-resveratrol, while the op- wines produced with the grapes cultivated in the VSP system and posite was found for the wines produced from the second harvest, IAC 766 rootstock obtained the highest total flavan-3-ol content in which may be explained by the higher UV-B incidence. both harvests (I = 73.26 mg L−1 , II = 37.50 mg L−1). The training system and rootstock also influenced the content of Generally, the wines showed significant amounts of (+) catechin phenolic acids of the wines (Table 2). Similar to flavonoids, phe- (5.30 to 27.00 mg L−1), procyanidin B1 (5.50–14.96 mg L−1), and nolic acids are of great importance due to their antioxidant activity procyanidin B2 (3.83–14.00 mg L−1). Padilha et al. (2016) found and can influence the wine quality as well as its stability (Rodtjer (+)-catechin to be the major component of red wine, with amounts et al., 2006). The climatic variability between harvests during the ranging from 2.20 to 40.40 mg L−1. The presence of oligomeric crop year did not play a strong influence on the content of phenolic flavan-3-ols (e.g., proanthocyanidins) leads to perceived astrin- acids. Gallic (10.50–22.30 mg L−1) and caffeic (7.00–20.76 mg gency, rich body, and improved aging properties (Waterhouse et L−1) acids were found to be the primary ones in the wine. Regard- al., 2002). The phenolic-driven sensory changes in several feed- less of the harvest, the wine obtained from the grapes grown under stocks has been recently discussed by de Camargo and Schwember a VSP system and grafted on IAC 766 rootstock presented higher (2019). total phenolic acids, gallic, and caffeic acids contents. The concen- The Pearson’s correlation analysis (p ≤ 0.05) aimed to verify trations of gallic acid in the wines evaluated are similar to those the correlation between the antioxidant activity and the quantified found by Padilha et al. (2016) that studied commercial wines from phenolic compounds. A positive and significant correlation existed the São Francisco Valley region. Caraguso and Nardini (2015) between the antioxidant activity and the total content of flavan- evaluated commercial red wines from Italy. The contents of caf- 3-ols (r = 0.56; p = 0.05), as well as procyanidins A2 (r = 0.74, p = feic, syringic, and ferulic acids found in the present study are also

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Table 2. Phenolic compounds identified and quantified in Syrah wines from grapes with different rootstock and training systems Rootstock2 Phenolic compounds (mg L−1) Training system1 Paulsen 1103 IAC-766 IAC-572 Paulsen 1103 IAC-766 IAC-572 June, 2014 (Harvest I) December, 2014 (Harvest II) (+)-Catechin VSP 16.30 Ac 27.00 Aa 22.70 Ab 7.30 Ab 9.40 Aa 6.25 Bc Lyre 13.86 Ab 16.73 Ab 17.40 Aa 6.90 Ab 5.30 Bc 8.20 Aa (−)-Epicatechin VSP 7.50 Ab 10.33 Aa 7.00 Ac 3.40 Ab 4.85 Ab 6.25 Aa Lyre 6.00 Bc 6.16 Ab 6.90 Aa 3.23 Bb 5.30 Bc 8.20 Aa (−)-Epicatechin Gallate VSP 1.40 Ab 2.30 Aa 1.43 Ab 0.90 Ac 1.30 Aa 1.10 Ab Lyre 1.40 Aa 1.36 Ab 1.43 Aa 1.16 Aa 1.23 Aa 1.10 Aa (−)-Epigallocatechin VSP 3.83 Aa 3.36 Aa 2.83 Ab 2.35 Ab 4.80 Aa 2.35 Ab Lyre 3.30 Aa 2.60 Bc 2.83 Ab 2.73 Aa 2.53 Bb 2.45 Ab Procyanidin A2 VSP 1.10 Ab 1.40 Aa 1.00 Bc 0.60 Ab 0.90 Aa 0.80 Aa Lyre 1.00 Bb 0.90 Bc 1.10 Aa 0.60 Aa 0.50 Bb 0.60 Aa Procyanidin B1 VSP 13.50 Ab 14.96 Aa 11.13 Ac 7.52 Aa 6.00 Ac 7.15 Bb Lyre 13.93 Aa 9.13 Bc 10.63 Bb 7.10 Bb 5.50 Bc 7.30 Aa Procyanidin B2 VSP 8.53 Ac 14.00 Aa 10.03 Ab 5.55 Ab 10.25 Aa 7.15 Bb Lyre 6.50 Bc 9.80 Aa 9.03 Bb 5.06 Ab 3.83 Bc 7.30 Aa Ʃ Flavan-3-ols VSP 52.16 Ac 73.26 Aa 56.13 Ab 27.62 Ab 37.50 Aa 24.20 Bc Lyre 46.00 Bc 46.70 Bb 49.33 Aa 26.78 Ab 24.19 Ab 35.15 Aa Kaempferol-3-O-glucoside VSP 0.66 Ab 1.20 Aa 0.56 Ab 5.70 Bb 6.35 Aa 6.10 Ab Lyre 0.66 Aa 0.46 Ab 0.43 Ab 6.36 Ab 5.70 Bc 7.15 Aa Isorhamnetin-3-O-glucoside VSP 9.06 Ab 13.53 Aa 8.66 Ac 11.95 Bb 11.70 Bc 17.35 Aa Lyre 8.06 Bb 8.10 Bb 8.53 Aa 22.83 Ab 19.50 Ab 26.45 Aa Rutin VSP 0.90 Ac 1.30 Aa 1.00 Ab 0.45 Bb 0.50 Bb 1.30 Aa Lyre 0.90 Aa 0.90 Aa 0.83 Ab 1.40 Ab 1.40 Ab 1.75 Aa Quercetin VSP 0.20 NS 0.20 NS 0.20 NS 0.30 Bc 0.70 Aa 0.40 Bb Lyre 0.20 NS 0.20 NS 0.20 NS 0.60 Ab 0.60 Bb 0.90 Aa Myricetin VSP 0.86 Ab 1.33 Aa 0.96 Ab 0.60 Ab 1.15 Aa 0.60 Bb Lyre 0.76 Ac 1.10 Ab 1.00 Aa 1.00 Aa 0.70 Bb 1.25 Aa Isoquercetin VSP 13.50 Ac 18.33 Aa 14.0 Ab 15.90 Bb 9.35 Bc 29.45 Aa Lyre 12.90 Aa 12.80 Ab 12.26 Ab 30.75 Ab 29.16 Ab 41.25 Aa Ʃ Flavonols VSP 25.20 Ab 35.90 Aa 25.40 Ab 34.90 Bb 29.75 Bc 55.20 Aa Lyre 23.50 Ab 24.00 Aa 23.25 Ab 62.96 Ab 57. 06 Ac 78.75 Aa Trans-resveratrol (Stilbene) VSP 0.20 NS 0.20 NS 0.20 NS 0.50 NS 0.50 NS 0.50 NS Lyre 0.20 NS 0.20 NS 0.20 NS 0.46 NS 0.46 NS 0.50 NS Gallic acid VSP 13.83 Ac 20.73 Aa 14.56 Ab 16.85 Ab 22.30 Aa 12.20 Bc Lyre 13.16 Aa 12.00 Bb 13.03 Ab 16.16 Aa 10.50 Bc 15.25 Ab Caffeic acid VSP 7.00 Bc 20.76 Aa 8.40 Bb 12.20 Ab 20.40 Aa 10.90 Bc Lyre 15.60 Aa 12.46 Bb 8.96 Ac 10.36 Bb 9.80 Bc 13.80 Aa Chlorogenic acid VSP 5.20 Aa 0.93 Bc 4.30 Bb 0.40 NS 0.40 NS ND Lyre 2.30 Bc 2.90 Ab 5.13 Aa 0.40 NS 0.40 NS ND

Journal of Food Bioactives | www.isnff-jfb.com 75 Physicochemical characteristics, phenolic profile, and antioxidant capacity de Carvalho et al.

Table 2. Phenolic compounds identified and quantified in Syrah wines from grapes with different rootstock and training systems - (continued) Rootstock2 Phenolic compounds (mg L−1) Training system1 Paulsen 1103 IAC-766 IAC-572 Paulsen 1103 IAC-766 IAC-572 June, 2014 (Harvest I) December, 2014 (Harvest II) Syringic acid VSP 3.63 Ab 4.56 Aa 2.90 Ac 1.30 Ab 1.50 Aa 1.10 Bc Lyre 3.23 Ab 3.06 Bb 4.20 Aa 1.20 Bb 0.93 Bc 1.50 Aa Ferulic acid VSP 0.30 Ab 0.56 Aa 0.33 Ab 0.95 Ac 1.20 Aa 1.05 Ab Lyre 0.43 Aa 0.36 Aa 0.36 Aa 0.96 Ab 0.80 Bc 1.10 Aa Ʃ Phenolic acids VSP 29.96 Ab 47.56 Aa 30.50 Ab 31.70 Ab 45.80 Aa 25.25 Bc Lyre 34.73 Aa 30.80 Bb 31.70 Aa 29.10 Bb 22.43 Bc 31.65 Aa Malvidin 3-O-glucoside VSP 63.30 Ac 105.13 Aa 86.16 Ab 55.05 Bc 83.20 Aa 63.95 Bb Lyre 53.53 Bc 84.50 Aa 73.10 Ab 60.83 Ab 52.90 Bc 70.20 Aa Delphinidin 3-O-glucoside VSP 1.10 Ac 1.80 Aa 1.50 Ab 1.60 Ab 1.50 Ac 2.95 Aa Lyre 0.90 Bc 1.10 Bb 1.43 Aa 1.80 Bb 1.10 Bc 2.05 Aa Peonidin 3-O-glucoside VSP 1.90 Ac 2.53 Aa 2.03 Ab 2.85 Bb 1.90 Bc 4.05 Aa Lyre 1.63 Bc 1.80 Bb 1.90 Aa 3.20 Aa 2.00 Ac 3.10 Bb Pelargonidin 3-O-glucoside VSP 4.86 Ac 9.03 Aa 7.33 Ab 6.40 Bc 7.15 Ab 9.65 Aa Lyre 4.20 Bc 5.93 Ab 6.76 Aa 6.76 Ab 4.93 Bc 8.20 Aa Petunidin 3-O-glucoside VSP 0.50 Ab 0.60 Aa 0.50 Ab 0.60 Aa 0.55 Aa 0.60 Aa Lyre 0.66 Aa 0.50 Bb 0.43 Ab 0.60 Aa 0.50 Bb 0.60 Aa Ʃ Anthocyanins VSP 71.66 Ac 119.10 Aa 47.53 Ab 66.45 Bc 94.35 Aa 81.20 Bb Lyre 60.93 Bc 93.83 Aa 83.63 Bb 73.20 Ab 61.43 Ab 84.15 Aa

1Results followed by the same uppercase letter in the column (training system) do not differ significantly, according to Tukey’s Test (p ≤ 0.05). 2 Results followed by the same low- ercase letter in the row (rootstock) do not differ significantly, according to Tukey’s test (p ≤ 0.05). ND: Not detected; NS: not significant; Lyre: divided trellis system; VSP: vertical shoot positioning, sparlier system. close to those reported by these authors. tem. The wines from harvest I are located on the positive side of Irrespective of the harvesting season and training system, in- CP1, which explains 48.26% of the variability among the samples, creased anthocyanin levels were found in wines from grapes graft- while the wines from the harvest II were located on the negative ed under the IAC 766 rootstock. Wines from grapes of the harvest I side of this component. The wines of the harvest II were highlight- cultivated in a VSP system with the IAC 766 rootstock presented a ed in the anthocyanins pelargonidin-3-O-glucoside, petunidin- total anthocyanin content of 119.10 mg L−1, while, under the same 3-O-glucoside, delfinidin-3-O-glucoside, peonidin-3-O-glucoside, training system and rootstock, the wines elaborated from grapes of trans-resveratrol, ferulic acid, and of most flavonols, except in the harvest II resulted in 94.35 mg L−1 (Table 2). The total antho- myricetin. In contrast, the wines from harvest I were closer to the cyanin content of the evaluated wines is within the range found by vectors that represent all the other phenolic compounds that were Padilha et al. (2016), who analyzed VSF commercial red wines of quantified in the samples. differentVitis vinifera L. varieties, and found concentrations ranging between 6.5 and 141.7 mg L−1. Through co-pigmentation and formation of polymeric pigments, anthocyanins participate in the 4. Conclusions main reactions resulting in wine color, being responsible for the red, purple, and blue tones (Lee et al., 2008). Regardless of har- The influence of training systems and rootstocks on the quality of vest season it can be observed (Table 1) that the combination of the Syrah tropical wines from São Francisco Valley, Brazil, was IAC 766 and the VSP system resulted in wines with higher color addressed in this study. Both affected the wine quality in different intensity. Malvidin-3-O-glucoside rendered the highest contribu- ways. Among the variables (e.g. harvests, training systems, and tion to the phenolic profile in all wines analyzed. In the samples rootstocks), the PCA analysis showed that the climatic variability obtained from the combination of VSP and IAC 766, the values of between the two harvests of the year had the most prominent in- this anthocyanin for harvests I and II were 105.13 and 83.20 mg fluence. In general, the use of espalier training system combined L−1, respectively. with rootstock IAC-766 rendered wines with increased contents Figure 1 shows about the Principal Component Analysis (PCA). of flavan-3-ols, flavonols, phenolic acids, and anthocyanins. How- The results demonstrate that the climatic variability between the ever, in order to recommend an optimum combination of training harvesting seasons influenced the profile of phenolic compounds system and rootstock, further studies under field conditions and the of the wines more than the evaluated rootstock or training sys- evaluation of a larger number of harvests are required.

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Figure 1. Principal Component Analysis (PCA) of the phenolics quantified (n = 24) by HPLC-DAD-FD in Syrah tropical wines from grapes grown under lyre and VSP training systems using the IAC-766, IAC-572, and Paulsen 1103 rootsotcks, harvested during two periods of the year in 2014 (Harvest I and II). Esp = VSP, SI = harvest I, SII = harvest II, P1103 = Paulsen 1103, Ac. = acid.

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Original Research J. Food Bioact. 2020;9:79–87

Promoting effect of Se-allylselenocysteine on 7,12-dimethylbenz[a] anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)- induced skin tumorigenesis

An-Chin Chenga#, Wan-Ru Jiangb#, Yu-Hsuan Hsiaob, Vladimir Badmaevc, Chi-Tang Hod, Roch-Chui Yu and Min-Hsiung Panb,e,f* aDepartment of Nutrition and Health Sciences, Chun Jung Christian University, Tainan, Taiwan bInstitute of Food Science and Technology, National Taiwan University, Taipei 10617, Taiwan cAmerican Medical Holdings Incorporated, New York, New York dDepartment of Food Science, Rutgers University, New Brunswick, NJ 08901, USA eDepartment of Medical Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan fDepartment of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan #These authors contributed equally to this work. *Corresponding author: Min-Hsiung Pan, Institute of Food Science and Technology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan. Tel: +886 2 33664133; Fax: +886-2-33661771; E-mail: [email protected] DOI: 10.31665/JFB.2020.9221 Received: February 26, 2020; Revised received & accepted: March 31, 2020 Citation: Cheng, A.-C., Jiang, W.-R., Hsiao, Y.-H., Badmaev, V., Ho, C.-T., Yu, R.-C., and Pan, M.-H. (2020). Promoting effect of Se- allylselenocysteine on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumorigenesis. J. Food Bioact. 9: 79–87.

Abstract

Se-allylselenocysteine (ASC), an analogue of garlic bioactive compound, has been shown to inhibit mammary carcinogenesis in vivo and cell growth in vitro. However, the function of ASC on anti-inflammatory effects remains largely unknown. Therefore, we investigated whether ASC has an anti-inflammatory effect on lipopolysaccharide (LPS)-induced inflammation or an anti-tumor effect promoting on DMBA/TPA-induced skin tumorigenesis and tried to elucidate the mechanisms involved. Herein, the results showed that ASC inhibited LPS-induced production of nitric oxide (NO) with a decreased protein level of inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. However, ASC enhanced LPS-induced cyclooxygenase-2 (COX-2) protein levels and mRNA expression. In- terestingly, we found for the first time that topical application of ASC on the dorsal skin of DMBA-initiated and TPA-promoted mice significantly accelerated skin tumorigenesis and raised tumor multiplicity as compared to the positive control group (DMBA/TPA). The number of tumours that were 1–3, 3–5, and >5 mm in size per mouse increased in a dose-dependent manner in the ASC pre-treated groups. Pre-treatment with ASC showed a significant increase in the expression of COX-2 compared with the positive control group. Thus, ASC may modulate the COX-2 protein expression and promote DMBA/TPA-induced skin cancer in mice.

Keywords: Anti-inflammation; Se-allylselenocysteine (ASC); Nitric oxide synthase (iNOS); Cyclooxygenase-2 (COX-2); 7,12-dimethylbenz [a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumorigenesis.

1. Introduction spectively, are considered the most prominent molecular mechanisms during inflammatory responses (Florentino et al., 2017; Kim Production of NO and prostaglandins by iNOS and COX-2, re- and Kang, 2016) and are also involved in multistage carcinogen-

Copyright: © 2020 International Society for Nutraceuticals and Functional Foods. 79 All rights reserved. Promoting effect of Se-allylselenocysteine on DMBA/TPA-induced skin tumorigenesis Cheng et al. esis, especially the promotion stage (Pan and Ho, 2008). Exces- 2. Materials and methods sive and prolonged NO generation caused by the overexpression of iNOS has also been implicated in inflammation tumorigenesis, 2.1. Materials while COX-2-mediated prostaglandin production stimulates cell proliferation, invasion, and angiogenesis in cancer development (Qiu et al., 2017). Lipopolysaccharide (LPS) (Escherichia coli 0127:E8), Phorobol Garlic extract contains diverse array of organic polysulfide com- 12-myristate 13-acetate (TPA), and 7,12-dimethylbenz[a]anthra- pounds such as diallyl sulfide (DAS), diallyl disulfide (DADS), cene (DMBA) were purchased from Sigma Chemical (St. Louis, and S-allylcysteine (SAC) which have potent anti-inflammation MO). Se-allylselenocysteine was provided by American Medical and anti-cancer properties. LPS activates NF-κB, a transcription Holding, Inc. (New York, NY, USA). Reverse transcription poly- factor that is involved in inflammatory response. Previous studies merase chain reaction (RT-PCR) reagents were purchased from have indicated that aged red garlic extract and garlic oil deriva- TaKaRa (Mountain View, CA, USA). tives such as DAS, DADS, and allyl methyl sulfide (AMS) could exert anti-inflammatory effects in LPS-stimulated RAW 264.7 2.2. Animals macrophages through inhibition of iNOS expression, NO production, prostaglandin E2 (PGE2), and NF-κB (Liu et al., 2006; Shin et al., 2013; Lee et al., 2015; Ryu et al., 2015). ASC, an analogue Female Institute of Cancer Research mice at 5–6 weeks age were of garlic compound, has been shown to exert an inhibitory effect obtained from the BioLASCO Experimental Animal Center (Bio- on the growth of TM12 cells through modulating the expression LASCO Taiwan Co., Ltd, Taipei, Taiwan). All animals were housed of cell cycle regulatory proteins, inducing the loss of DNA integ- in a controlled atmosphere (25 ± 1 °C at 50% relative humidity) rity, and increasing the rate of apoptosis (Zhu et al., 2000a; Zhu and with a 12 hr light-12 hr dark cycle. All experimental protocols et al., 2000b; Jiang et al., 2001). ASC was also found to inhib- used in these animal experiments were approved by the Institu- it mammary carcinogenesis in vivo and cell growth in vitro (Ip tional Animal Care and Use Committee (IACUC B201700174) of et al., 1999). ASC did not induce the expression of cytochrome National Taiwan University. P450s (CYP) at a concentration of 100 μM. CYP, which is an im- The dorsal skin of each mouse was shaved with surgical clip- portant factor result in a risk at bioactivation of pro-carcinogens. pers before the application of the tested compound. ASC and TPA ASC also elevates glutathione-S-transferase (GST) mRNA level, were dissolved in 200 μL of acetone and applied topically to the the induction of GST normally enhances the detoxification ability shaved area of each mouse. (AC’t Hoen et al., 2002). Our previous study found that ASC could induce autophagy and epigenetic regulation of protocadherin 17 2.3. Cell culture (PCDH17) in human colorectal adenocarcinoma cells (Wu et al., 2015). However, the function of ASC on anti-inflammatory effects RAW 264.7 murine macrophage cells obtained from the American remains unknown. Type Culture Collection (Rockville, MD, USA) were cultured in The two-stage skin carcinogenesis model is a well-char- Dulbecco’s Modified Eagle Medium (DMEM), supplemented with acterized model of multistep carcinogenesis. A single dose of 10% endotoxin-free, heat-inactivated fetal bovine serum, 10,000 7,12-dimethylbenz[a]anthracene (DMBA) was applied on mouse units/mL penicillin, and 10,000 μg/mL streptomycin (GIBCO, skin to mutate the Ha-ras gene at codon 61 (A to T) (Nelson et Grand Island, NY, USA) and kept at 37 °C in a humidified atmos- al., 1992), which resulted in the transformation of the original phere with 5% CO2 in air, according to ATCC recommendations. normal cells to cancer cells (Shen et al., 2014). Repeated treat- When the cells reached a density of 2–3 × 106 cells/mL, they were ment with TPA could support the growth of transformed cells activated by incubation in medium containing LPS (100 ng/mL). by activating inflammation-related cytokines (Chun et al., 2004; Various concentrations of test compounds dissolved in ddH2O Passos et al., 2013), such as epidermal growth factor receptor were added to the LPS. (EGFR) (Casanova et al., 2002) and extracellular signal-regulated kinase (ERK) (Bourcier et al., 2006).The development of skin tumors in female ICR mice in response to treatment with DMBA 2.4. Cell viability assay and phorobol 12-myristate 13-acetate (TPA) can explain the multistep process of carcinogenesis including initiation, promotion, The RAW 264.7 cells were cultivated at a density of 1 × 106 cells and progression (Balmain et al., 1984; Arora et al., 2013; Ma et in a 24-well plate. The ASC studied was added to the medium 12 al., 2013). hr after inoculation. The cells were harvested after 24 hr. Viability In this study, we examined the effects of ASC on LPS-induced was determined by trypan blue. inflammatory responses of RAW 264.7 murine macrophages. The association between the suppression of NF-κB and the inhibition of NO production was also assessed. We also investigated its an- 2.5. Nitrite assay ticancer potential against DMBA and TPA-induced skin cancer. The results indicated that ASC suppressed the production of NF- The nitrite concentration in the culture medium was measured as κB, thereby inhibiting the expression of iNOS in activated mac- an indicator of NO production, according to the Griess reaction. rophages. However, this study evidenced that ASC had tumor The cells were treated with LPS (100 ng/mL) for 24 h in the pres- induction effects on a DMBA/TPA skin carcinogenesis protocol. ence of ASC or vehicle solution. The conditional medium (100 μL) Data showed that ASC could significantly accelerate mouse skin was taken and mixed with an equal volume of the Griess reagent tumorigenesis and raise tumor multiplicity more than the positive (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyleth- control group. However, future investigations are warranted to ylenediamine dihydrochloride in water). Nitrite production was determine the mechanisms of tumorigenesis effects of ASC on determined by reading the absorbance at 550 nm. A standard curve DMBA/TPA induced skin cancer. was generated with NaNO2.

80 Journal of Food Bioactives | www.isnff-jfb.com Cheng et al. Promoting effect of Se-allylselenocysteine on DMBA/TPA-induced skin tumorigenesis

2.6. Total protein extraction standard initiation-promotion with DMBA and TPA, as reported previously (DiGiovanni, 1992). One group was composed of 12 Total protein extracts (for iNOS, COX-2, β-actin, p-IκBα, and IκBα) female ICR mice. These mice were given commercial rodent pel- were prepared in a gold lysis buffer (10% Glycerol, 1% Triton X-100, lets and fresh tap water ad libitum, both of which were changed 137 mM NaCl, 10 mM NaF, 5 mM ethylenediaminetetraacetic acid twice a week. The dorsal region of each mouse was shaved with (EDTA), 1 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid an electric clipper 2 days before initiation. Mice at 6 weeks old were started on 200 nmol DMBA in 200 μL acetone; control mice (EGTA), 20 mM Tris pH 7.9, 1 mM Na3VO4, 100 mM-glycerol phosphate, and 1 mM sodium pyrophosphate) for 30 min at 4 °C. The received 200 μL acetone only. One week after initiation, the mice supernatants containing total proteins were obtained by centrifuga- were treated topically with 200 μL acetone or promoted with TPA tion at 10,000 × g for 30 min and stored at −20 °C until tested. (5 nmol in 200 μL acetone) twice a week for 20 weeks. In the other two groups, the mice were treated with ASC (1 and 5 mol in 200 μL acetone) 30 min before each TPA treatment. Tumors of at least 2.7. Western blotting 1 mm in diameter were counted and recorded every week. The results were expressed as the average number of tumors per mouse, Whole protein extracts were obtained by homogenizing skin sam- percentage of tumor-bearing mice, and average tumor weight per ples and cells, flash-frozen in liquid nitrogen, in a whole cell lysis mouse. buffer. Proteins from (50 μg) whole-cell lysates were resolved by 10% SDS-PAGE, transferred onto polyvinylidene difluoride mem- 2.11. Statistical analysis branes (Immobilon P, Millipore, Bedford, MA, USA), and then probed with a primary antibody followed by a secondary antibody conjugated with horseradish peroxidase. The immunocomplexes Quantitative data represent mean values with the respective stand- were visualized with Western Chemiluminescent HRP Substrate ard error of the mean (SE) corresponding to three or more repli- (ECL) (Millipore, Amersham, UK). cates. A One-Way Student’s t-test was used to assess the statistical significance between the LPS and ASC plus LPS-treated cells. Data were considered statistically significant atp < 0.05. 2.8. Reverse transcription-polymerase chain reaction (RT-PCR)

The level of COX-2 mRNA expression was measured by RT-PCR. 3. Results Total RNA was isolated using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) as recommended by the manufacturer’s instruc- 3.1. ASC suppresses LPS-induced NO production in RAW 264.7 tions. Briefly, total RNA (1 μg) was performed by PCR in a final macrophages volume of 50 μL containing 25 L 2 × 1 step buffer, 2 L PrimeScript 1 step enzyme mix, 1 L upstream primer (20 mer), 1 L downstream We first tested the effect of ASC on cell viability to exam the cy- reverse primer (20 mer), and RNase free dH2O. The specific PCR primers used in this experiment are listed as follow. COX-2: prim- totoxic effects of ASC on RAW 264.7 macrophages. RAW 264.7 er sequence, sense: 5′-GGAGAGACTATCAAGATAGTGATC-3′, macrophages were treated with different concentrations of ASC as antisense: 5′-ATGGTCAGTAGACTTTTACAGCTC-3′; β-actin, indicated. After 24 hr of treatment, the viability of cells was de- sense: 5′-AAGAGAGGCATCCTCACCCT-3′, antisense: 5′-TA- termined by trypan blue exclusion assay. As shown in Figure 1, CATGGCTGGGGTGTTGAA-3′. ASC did not show cytotoxic effects on RAW 264.7 macrophages The PCR conditions were as follows. After an initial denaturation at 5–50 μM of treatment. To evaluate the inhibitory effects of ASC for 2 min at 94 °C, 30 cycles of amplification (denaturation at 94 °C on LPS-stimulated NO production in RAW 264.7 cells, the cells for 30 s, primer annealing at 50 °C for 30 s and extension at 72 °C were treated with LPS (100 ng/mL) only or with different concen- for 1 min) were performed and samples were kept at 4 °C following trations of ASC for 24 hr. At the end of incubation time, 100 μL PCR. A 15 μL sample of each PCR product was electrophoresed on of the culture medium was collected for nitrite assay. As shown in a 2% agarose gel and visualized by ethidium bromide staining. Each Figure 2, the nitrite in LPS-stimulated cells is about 10 times the value was normalized to the expression of β-actin. The values pre- concentration of the negative control group, and the LPS-induced sented are the mean ± SE of at least triplicate measurements. nitrite production was reduced significantly in 50 μM of ASC treated RAW 264.7 macrophages. 2.9. Transient transfection and luciferase assay 3.2. Effects of ASC on the LPS-induced expressions of iNOS and Cox-2 proteins and mRNA NF-κB luciferase-stable RAW 264.7 cells were seeded and cultivated at a density of 1 × 106 cells in a 24-well plate. After 12 h, the cells were co-incubated with 100 ng/mL LPS with or without ASC In unstimulated RAW 264.7 cells, iNOS and COX-2 proteins were for an additional 24 h. The cells were collected into an Eppendorf undetectable. Following stimulation with LPS, elevated levels of tube, centrifuged (1,000 × g, 4 °C, 10 min) and then we removed iNOS and COX-2 proteins were detected in RAW 264.7 cells. Ac- the supernatant. Luciferase activity was assayed by means of the cording to previous data, nitrite production was reduced signifi- reporter gene assay system (Perkin Elmer, Waltham, MA, USA), cantly in RAW 264.7 cells treated with 50 μM of ASC. Likewise, with 100 μL of cell lysate used in each assay. iNOS expression was inhibited markedly by ASC at 50 μM in a similar manner (Fig. 3). These results indicate that the reduced expressions of iNOS by ASC were responsible for the inhibition of 2.10. Two-stage tumorigenesis in mouse skin LPS-induced NO production. However, ASC could significantly enhance the levels of COX-2 protein (Fig. 3). To detect the effects The anti-tumor promoting activity of ASC was examined by a of ASC on LPS-induced COX-2 mRNA expression by RT-PCR,

Journal of Food Bioactives | www.isnff-jfb.com 81 Promoting effect of Se-allylselenocysteine on DMBA/TPA-induced skin tumorigenesis Cheng et al.

Figure 3. Effects of ASC on LPS-induced iNOS and COX-2 protein level in RAW 264.7 cells. The cells were treated with different concentrations of ASC and LPS (100 ng/mL) for 24 hr. An equal amount of total proteins (50 μg) were subjected to 8% SDS-PAGE. The expression of iNOS, COX-2 and β-actin was detected by western blot using specific antibodies. These- ex periments were repeated three times with similar results. Relative protein levels were quantified using Image J.

3.3. Effects of ASC on the LPS-induced IκB phosphorylations and NF-κB activation

In this study, we investigated whether ASC inhibited the LPS-stimulated degradation of IκB in RAW 264.7 macrophages by Western Figure 1. Effects of ASC on cell viability. RAW 264.7 cells were treated blotting. Figure 5a shows that LPS-induced IκB-α degradation was with 5, 10, 25, 50 μM of ASC for 24 hr. The viability of cells was deter- significantly blocked by ASC. Transient transfection with pNF- mined by trypan blue assay. The values are expressed as means S.E. of κB-Luc reporter plasmid was applied to confirm whether ASC in- triplicate tests. *P < 0.05, **P < 0.01, ***P < 0.001. hibited NF-κB activity in LPS-activated macrophages. As shown in Figure 5b, LPS-induced NF-κB activity was elevated fivefold in analysis showed that the expression of COX-2 mRNA was cor- these transfected cells, but the effects were reduced by ASC treat- related with the levels of COX-2 protein (Fig. 4). ment in a dose-dependent manner.

Figure 2. Inhibitory effects of ASC on NO production in LPS-stimulated RAW 264.7 cells. The cells were treated with LPS (100 ng/mL) only or with different concentrations of ASC for 24 hr. At the end of incubation time, 100 μL of the culture medium was collected for nitrite assay. The values are expressed as means S.E. of triplicate tests. *P < 0.05, **P < 0.01, ***P < 0.001 (control versus LPS alone; LPS alone versus ASC 5, 10, 25 and 50 μM).

82 Journal of Food Bioactives | www.isnff-jfb.com Cheng et al. Promoting effect of Se-allylselenocysteine on DMBA/TPA-induced skin tumorigenesis

groups (Fig. 6e). As shown in Figure 6f, characteristic squamous pearls were clearly observed on the uncovered dorsal skin of mice with topical applications of DMBA/TPA. ASC mice combined could induce more papillomas per mouse in total and larger size tumours per mouse than the positive control group in a dose-dependent manner.

3.5. Pro-inflammation effect of ASC on DMBA/TPA-induced Figure 4. RT-PCR analysis of the effects of ASC on LPS-induced COX-2 mouse skin tumorigenesis mRNA expression. Cells were treated with LPS (100 ng/mL) and ASC (5, 10, 25, 50 μM) for 6 hr, and total RNA was subjected to RT-PCR with the primers COX-2 with β-actin as internal control. The PCR product was re- COX-2 is often undetectable in normal tissue, whereas its ex- solved in 2% agarose gel. These experiments were repeated three times pression is observably higher in tumour tissue (Cao and Prescott, with similar results. Quantification of COX-2 expression was normalized to 2002). The pro-inflammatory activity of ASC can be demonstrated β-actin using Image J. by its effect on COX-2 expression in TPA-induced mouse skin. The results showed that topical application of TPA to mouse dorsal 3.4. Tumour-promoting effect of ASC on DMBA/TPA-induced skin could upregulate the expression of COX-2 compared to the mouse skin tumorigenesis acetone treated control group, whereas pre-treatment with ASC caused a marked increase in COX-2 expression in a dose-depend- For testing the effect of ASC on DMBA/TPA-induced mouse skin ent manner compared to the DMBA/TPA control. (Fig. 7). tumourigenesis, we examined the body weight of the mice for 20 weeks and found no difference in weight between the ASC treated 4. Discussion mice and those without treatment (Fig. 6a). Subsequently, tumours were analyzed histologically. As shown in Figure 6b and c, ASC significantly accelerated the frequency and progression of chemi- First, we tested the effect of ASC on cell viability to exclude the cally-induced papillomas of the skin and raised an average number possibility that the decreased nitrite production in ASC-treated of tumours in the mice. 7 weeks after the ASC administration, the cells was due to growth inhibition. As shown in Figure 1, ASC did first skin tumours (≥1 mm diameter) occurred in mice treated with not show cytotoxic effects on RAW 264.7 macrophages at 5–50 1 μmol/200 μL and 5 μmol/200 μL. Over the subsequent 11 weeks μM treatment. According to Figure 2, nitrite production was re- of tumour promotion, the number of tumours per mouse increased duced significantly in RAW 264.7 cells treated with 50 μM of ASC. much faster in ASC 1 μmol /200 μL treated and the positive control We next tested the level of iNOS expression, in unstimulated RAW mice. Moreover, 100% of the ASC 5 μmol/200 μL treated mice had 264.7 cells, iNOS proteins were undetectable. Following stimu- developed numerous tumours at week 9. lation with LPS, elevated levels of iNOS proteins were detected Counting of tumours with an area of ≥1 mm2 revealed that ASC in RAW 264.7 cells. As shown in Figure 3, iNOS expression was 1 μmol/200 μL treated mice developed on average 31 and ASC 5 also markedly inhibited by ASC at 50 μM. These results indicate μmol/200 μL treated mice displayed 35 papillomas of this size, that ASC could decrease inflammation by inhibiting LPS-induced both of which were higher than the positive control. The number of iNOS expression and NO production. tumours that were 1–3, 3–5, 5–7, and >7 mm in size per mouse in- NF-κB is a key regulator of the various genes involved in increased in a dose-dependent manner in the ASC pre-treated groups flammatory responses (Xie et al., 1994). In unstimulated cells, NF- (Fig. 6d). Tumour weight was also increased in ASC pre-treated κB is sequestered in the cytoplasm by its inhibitor, IκB. Under LPS

Figure 5. Effects of ASC on LPS-induced phosphorylation and degradation of IκB and NF-κB-luciferase (luc) activation. (a) RAW 264.7 cells were treated with LPS (100 ng/mL) and ASC, and total cellular lysates were prepared and analysed for the content of IκBα, p-IκBα, and β-actin by western blot. Quantifi- cation of IκB and p-IκBα protein level were normalized to β-actin using Image J. (b) The cells were treated with LPS (100 ng/mL) with or without ASC for 24 hr. Then, cells were collected for measuring luciferase activity assay. *P < 0.05, **P < 0.01, ***P < 0.001 (control versus LPS alone; LPS alone versus ASC 5, 10, 25, and 50 μM)

Journal of Food Bioactives | www.isnff-jfb.com 83 Promoting effect of Se-allylselenocysteine on DMBA/TPA-induced skin tumorigenesis Cheng et al.

Figure 6. The promoting effect of ASC on DMBA/TPA-induced skin tumorigenesis in ICR mice. Female ICR mice received vehicle (acetone) or ASC (1 or 5 μmol) 30 min prior to each topical application of TPA (5 nmol) twice weekly for 20 weeks following DMBA-initiation, as described in materials and methods. Control animals received vehicle alone and did not produced papillomas. (a) The body weight of mice during skin tumour promotion. (b) Average number of tumours per mouse (tumour multiplicity). (c) Percentage of mice with papillomas (tumour incidence). (d) Size distribution of papillomas. (e) Tumour weight per mouse on control groups and ASC-treated mice. (f) Representative photographs of each group are shown at the end of week 20. *Significantly different from the corresponding TPA value at *P<0.05, **P<0.01, and ***P<0.001.

84 Journal of Food Bioactives | www.isnff-jfb.com Cheng et al. Promoting effect of Se-allylselenocysteine on DMBA/TPA-induced skin tumorigenesis

and those without treatment (Fig. 6a). In our previous study, ASC showed an anti-proliferative activity in colorectal cancer cell lines including HT-29 and COLO 205 cells at the concentration of ASC in 75 and 100 μM (Wu et al., 2015). These results indicated that the topical application of ASC did not result in systemic toxicity under a range of dosage condition. However, high dosage of Se could cause toxicity in plants. In general, the content of Se in plant tissues caused toxicity is around 5 mg kg−1 dry weight. The Se tolerance range in crop plants are quietly different, for instance, in rice 2 mg kg−1 dry weight is the maximum levels of Se, in wheat is 4.9 mg kg−1 dry weight, while Dutch clover can tolerant 330 mg kg−1 dry weight (Kolbert et al., 2018). Based on these results, we Figure 7. ASC up-regulate COX-2 protein level on DMBA/TPA-induced suggested that a relatively higher dosage of Se-compounds may skin tumorigenesis. Shaven backs of female ICR mice were treated with cause cell toxicity and induce inflammation by increasing COX- ASC (1 or 5 μmol) 30 min prior to TPA (5 nmol) twice weekly for 20 weeks 2 expression. On the other hand, topical application of ASC on following DMBA-initiation, and control animals were treated with acetone the dorsal skin of DMBA-initiated and TPA-promoted mice did only for 20 weeks following DMBA-initiation. Mice were sacrificed after 20 not suppress but significantly accelerated skin tumorigenesis and weeks and skin fractions were prepared from the skin/tumours of each raised tumor multiplicity (Fig. 6b, c, d). Because our study is based group. The protein expression was determined by western blot analysis as described in the text. Quantification of COX-2 protein levels were normal- on the model of applying the drug to mouse skin, it is a reasonable ized to β-actin using Image J. The data is representative of three different inference that ASC needs to metabolize to other derivatives in vivo sets of animals giving a similar trend. to exert its inhibitory effect on cancer. Capsaicin, a pungent ingredient of chili peppers, has widely stimulation, IκB is phosphorylated and degraded by IκB kinase, reported anticancer activities (Lin et al., 2016; Qian et al., 2016) ubiquitinated, and rapidly breaks away from NF-κB. NF-κB then and was suggested to have a clinical significance in tumor therapy translocate to the nucleus, where it binds to DNA and activates (Dasgupta et al., 1998; Sharma et al., 2013). However, topical ap- the transcription of iNOS and COX-2 (Rice and Ernst, 1993). As plication of capsaicin on the dorsal skin of DMBA/TPA-induced shown in Figure 5a and b, the LPS-induced IκB-α degradation was skin tumorigenesis could significantly promote tumor formation significantly blocked by ASC, and LPS-induced NF-κB activity and induce more numerous and larger skin tumors by modulating was reduced by ASC treatment in a dose-dependent manner. The inflammation (Liu et al., 2015). The similarity between our find- above findings show that ASC suppressed iNOS expression at least ings and Liu’s report suggests that the tumour promoting effects in part via the NF-κB-dependent mechanism. by ASC may occur via inflammation regulating (Liu et al., 2015). COX-2 is a rate-limiting enzyme produced during the produc- Previous studies have indicated COX-2 overproduction following tion of prostaglandins, and prostaglandins play an important role DMBA/TPA treatment in a variety of cell types, leading to the sug- in inflammation and tumour progression (Seibert and Masferrer, gestion that COX-2 production in microenvironment cells modu- 1994; Wang et al., 2007). COX-2 is often undetectable in normal lates tumor progression (Müller-Decker et al., 2002). Jiao et al. tissue, whereas its expression is observably higher in many epi- (2014) also indicated that COX-2 elevation in DMBA/TPA-treated thelial cancers (Cao and Prescott, 2002; Wang and Dubois, 2006). epithelial cells is required for DMBA/TPA-driven papilloma ap- Epidemiological and clinical study has shown as higher as COX- pearance and progression. The effect of ASC on DMBA/TPA-in- 2 is expressed, a poorer of prognosis is associated (Sobolewski duced mouse skin tumourigenesis showed that pre-treatment with et al., 2010). These reports showed that COX-2 has a multi-step ASC causes a marked increase in COX-2 expression in a dose- role in tumorigenesis, whether in tumour promotion at early stage dependent manner compared to the DMBA/TPA control (Fig. 7). or at late development of chemoresistance and metastatic forma- These results demonstrated that the COX-2-promoting effect of tion (Sarkar et al., 2007). According to Figure 3, ASC significantly ASC was responsible for the pro-inflammatory and papilloma pro- enhanced the levels of COX-2 protein. RT-PCR analysis of the ef- gression activity in DMBA/TPA induced mouse skin. fects of ASC on LPS-induced COX-2 mRNA expression showed Several studies have indicated that angiogenic activators play that pre-treatment with ASC caused a marked increase in COX-2 an important part in the growth and spread of tumors. Disturbance mRNA expression in a dose-dependent manner (Fig. 4). These re- of the balance between endogenous activators such as vascular sults demonstrated that the COX-2-promoting effect of ASC was endothelial growth factor (VEGF) and inhibitors of angiogenesis responsible for the pro-inflammatory effects in LPS-stimulated in the tumor microenvironment strongly induce tumor angiogen- RAW 264.7 cells. esis (Yamamizu et al., 2015). Angiogenesis is a normal and vital Selenium is one of the essential minerals for life but it detected process in growth and development as well as in wound healing. to be toxic at low concentration and has been studied many years Previous study substantiated that aged garlic extract exerts benefi- (Arnault and Auger, 2006). On the other hand, evidence showed cial effects on wound healing (Ejaz et al., 2009), and garlic likely that selenium could alter the metabolism of carcinogens in liver prevents the formation of peritoneal adhesions in a rat model (Sah- by played role in the mixed function oxidase system (Steinmetz baz et al., 2014). Therefore, we presume that the tumor-promoting and Potter, 1996). There are many studies demonstrating that ASC effect of ASC on DMBA-initiated and TPA-promoted skin tumo- exhibits anti-carcinogenesis effects in vivo and in vitro (Zhu et al., rigenesis may be in part due to its effects on wound healing via 2000a; Zhu et al., 2000b; Jiang et al., 2001). In order to study the angiogenesis. effect of ASC on the tumourigenesis, we employed a well-estab- In summary, the present results speculate that ASC may modu- lished two-stage model of chemical skin carcinogenesis in female late inflammatory processes via different signal-generating path- ICR mice based on DMBA/TPA treatments (DiGiovanni, 1992) as ways in complex microenvironments. COX-2 plays a crucial role papilloma formation can be induced by the two-stage carcinogen- in the tumour-promoting and papilloma progression effects of ASC esis protocol (Kemp, 2005). Throughout the experiment, there was on DMBA/TPA-induced skin cancer in mice. However, future in- no noticeable difference in weight between the ASC-treated mice vestigations need to clarify the underlying molecular mechanisms

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Journal of Food Bioactives | www.isnff-jfb.com 87 Journal of Food Bioactives

The Journal of Food Bioactives (JFB), a publication of the International Society for Nutraceuticals and Functional Foods (ISNFF), aims to bring together the results of fundamental and applied research on food bioactives, functional food ingredients, nutraceuticals and natural health products that are known to possess or perceived to have health-promoting properties.

Meeting Report

1 Does COVID-19 affect food safety and security? Fereidoon Shahidi

The goal of this contribution is to provide a summary report on the Extraordinary Scientific Roundtable on COVID-19 and Food Safety co- organized by the International Union of Food Science and Technology (IUFoST) and the Chinese Institute of Food Science and Technology (CIFST).

Viewpoint

4 Is there a role of gut microbiota in mental health? Klaus W. Lange, Katharina M. Lange, Yukiko Nakamura, Shigehiko Kanaya

Research on the interaction between gut microbiota and the brain may have implications for our understanding of brain function, cognition, behavior and mental health.

Reviews

10 Pomegranate as a natural source of phenolic antioxidants: a review Fellipe Lopes de Oliveira, Thaise Yanka Portes Arruda, Renan da Silva Lima, Sabrina Neves Casarotti, Maressa Caldeira Morzelle

Pomegranate, a recognized source of phenolic compounds, has been associated with health-promoting benefits, mostly due to its antioxidant activity.

88 23 Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? A critical review focusing on bioactive molecule screening assays in in vitro and in vivo models Fereidoon Shahidi, JuDong Yeo

Increasing evidence has proven the potent antioxidative effectiveness of bioactives in natural products for preventing/suppressing chronic diseases.

36 Brazil nut: nutritional benefits from a unique combination of antioxidants Alan Giovanini de Oliveira Sartori, Marisa A.B. Regitano-d’Arce, Leif H. Skibsted

Brazil nut is the seed of the Bertholletia excelsa tree, which grows naturally in the rainforests of South America.

40 Blackberry polyphenols: review of composition, quantity, and health impacts from in vitro and in vivo studies Julie A. Robinson, Julianne E. Bierwirth, Phillip Greenspan, Ronald B. Pegg

Blackberries possess marked concentrations of antioxidant polyphenols, most notably anthocyanins, ellagic acid, ellagitannins, epi/catechin, and proanthocyanidins.

Original Research

52 Phenethylamine in hot water extract of Chlorella pyrenoidosa expands lifespan of SOD1 mutant adults of Drosophila melanogaster at very low dose Yifeng Zheng, Yoshihiro H. Inoue, Nagi Kohno, Masaki Fujishima, Eri Okumura, Kenji Sato

89 Hot water extract of chlorella (WEC) increased the lifespan of superoxide dismutase (SOD1) mutant adults of Drosophila melanogaster in a dose dependent manner (200–800 μg/mL).

58 Low frequency, high power ultrasound: a non-thermal green technique improves phenolic fractions (free, conjugated glycoside, conjugated esters and bound) in fermented seabuckthorn beverage Kelly Dornan, Aynur Gunenc, Azza Ferichichi, Farah Hosseinian

Phenolic compounds were characterized after traditional extraction method (TEM) and ultrasound-assisted extractions (UAEs) for 10 min (US10) and 15 min (US15).

70 Physicochemical characteristics, phenolic profile, and antioxidant capacity of Syrah tropical wines: effects of vineyard management practices Erika Samantha Santos de Carvalho, Aline Camarão Telles Biasoto, Rita de Cássia Mirela Resende Nassur, Ana Paula André Barros, Patrícia Coelho Souza Leão, Renan da Silva Lima, Adriano Costa de Camargo, Maria Eugênia de Oliveira Mamede

The present study evaluated the influence of training systems and root-

90 stocks on the quality of Syrah tropical wines, produced at São Francisco Valley, Brazil.

79 Promoting effect of Se-allylselenocysteine on 7,12-dimethylbenz[a] anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)- induced skin tumorigenesis An-Chin Cheng, Wan-Ru Jiang, Yu-Hsuan Hsiao, Vladimir Badmaev, Chi-Tang Ho, Roch-Chui Yu, Min-Hsiung Pan

Se-allylselenocysteine (ASC), an analogue of garlic bioactive compound, has been shown to inhibit mammary carcinogenesis in vivo and cell growth in vitro.

91 Journal of Food Bioactives

Table of Contents

Meeting Report Does COVID-19 affect food safety and security? Fereidoon Shahidi...... 1

Viewpoint Is there a role of gut microbiota in mental health? Klaus W . Lange, Katharina M . Lange, Yukiko Nakamura, Shigehiko Kanaya ...... 4

Reviews Pomegranate as a natural source of phenolic antioxidants: a review Fellipe Lopes de Oliveira, Thaise Yanka Portes Arruda, Renan da Silva Lima, Sabrina Neves Casarotti, Maressa Caldeira Morzelle. . . . .10

Should the in vitro colorimetric assays in antioxidant and lipid oxidation evaluation be abandoned? A critical review focusing on bioactive molecule screening assays in in vitro and in vivo models Fereidoon Shahidi, JuDong Yeo ...... 23

Brazil nut: nutritional benefits from a unique combination of antioxidants Alan Giovanini de Oliveira Sartori, Marisa A .B . Regitano-d’Arce, Leif H . Skibsted...... 36 .

Blackberry polyphenols: review of composition, quantity, and health impacts from in vitro and in vivo studies Julie A . Robinson, Julianne E . Bierwirth, Phillip Greenspan, Ronald B . Pegg...... 40

Original Research Phenethylamine in hot water extract of Chlorella pyrenoidosa expands lifespan of SOD1 mutant adults of Drosophila melanogaster at very low dose Yifeng Zheng, Yoshihiro H . Inoue, Nagi Kohno, Masaki Fujishima, Eri Okumura, Kenji Sato ...... 52

Low frequency, high power ultrasound: a non-thermal green technique improves phenolic fractions (free, conjugated glycoside, conjugated esters and bound) in fermented seabuckthorn beverage Kelly Dornan, Aynur Gunenc, Azza Ferichichi, Farah Hosseinian ...... 58

Physicochemical characteristics, phenolic profile, and antioxidant capacity of Syrah tropical wines: effects of vineyard management practices Erika Samantha Santos de Carvalho, Aline Camarão Telles Biasoto, Rita de Cássia Mirela Resende Nassur, Ana Paula André Barros, Patrícia Coelho Souza Leão, Renan da Silva Lima, Adriano Costa de Camargo, Maria Eugênia de Oliveira Mamede...... 70

Promoting effect of Se-allylselenocysteine on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumorigenesis An-Chin Cheng, Wan-Ru Jiang, Yu-Hsuan Hsiao, Vladimir Badmaev, Chi-Tang Ho, Roch-Chui Yu, Min-Hsiung Pan ...... 79