Journal of Food Bioactives

Volume 7 September 2019

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

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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)

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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

Viewpoint J. Food Bioact. 2019;7:1–6

Are there serious adverse effects of omega-3 polyunsaturated fatty acid supplements?

Klaus W. Langea*, Yukiko Nakamuraa, Alexander M. Gosslaub,c and Shiming Lic aDepartment of Experimental Psychology, University of Regensburg, Germany bDepartment of Science (Biology), City University of New York, United States cDepartment of Food Science, Rutgers University, New Brunswick NJ, United States *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.2019.7192 Received: September 27, 2019; Revised received & accepted: September 28, 2019 Citation: Lange, K.W., Nakamura, Y., Gosslau, A.M., and Li, S. (2019). Are there serious adverse effects of omega-3 polyunsaturated fatty acid supplements? J. Food Bioact. 7: 1–6.

Abstract

Dietary omega-3 polyunsaturated fatty acids (PUFAs) may reduce the risk of various diseases. Marine oils rich in omega-3 PUFAs have therefore become popular dietary supplements. Adverse effects need to be considered when administering omega-3 PUFAs. While unproblematic short-term adverse events linked to omega-3 PUFAs have been reported, long-term PUFA supplementation may be associated with increased cancer risk, possibly due to PUFAs, their oxidation products or added vitamin E. Large-scale trials have shown increased rates of prostate cancer in men taking α-tocopherol supplementation. Omega-3 PUFAs are highly prone to oxidative degradation to lipid peroxides and secondary oxidation products, which may render them ineffective or harmful. Oil contained in omega-3 supplements may contain a mixture of omega-3 PUFAs, problematic additives, and unspecified levels of potentially toxic oxidation products. The health consequences of oxidized fish oil intake remain unclear. Given the harmful effects of oxidized lipid products demonstrated in animal experiments, caution is needed in the supplementation of PUFAs at high doses over extended periods of time and during vulnerable phases of life, such as prenatal development, childhood, and adolescence. A balanced approach should weigh the overall health benefits of omega-3 PUFAs against potentially harmful effects of their supplementation. Future research should address the development of effective antioxidants without side effects.

Keywords: Omega-3 fatty acids; Fish oil supplement; Oxidation; Vitamin E; Adverse effects; Cancer.

1. Introduction healthy nutrients are administered, adverse effects should also be considered. Omega-3 polyunsaturated fatty acids (PUFAs) have attracted in- A consideration in regard to undesired side effects of omega-3 creasing attention in recent years due to claims of their beneficial PUFAs is the form in which they are administered. Both dietary role in the promotion of health and reduction in the risk of various consumption and supplement intake could be problematic. Sev- diseases, such as cardiovascular diseases, type 2 diabetes, cancer, eral potential risks need to be considered when omega-3 PUFAs cognitive decline, neurodegenerative diseases, and mental dis- are administered as part of the diet. An important natural source orders (Shahidi, 2015; Shahidi and Ambigaipalan, 2018; Lange, of these bioactives is fish and seafood; these may be contaminat- 2019a,b,c). Marine oils, rich in omega-3 PUFAs, have therefore ed with methylmercury, dioxins, and polychlorinated biphenyls, become one of the most popular dietary supplements globally which may increase the risk for some cancers or may harm unborn (Barnes et al., 2008). However, even when seemingly natural and children when consumed by the mother during pregnancy (Mozaf-

Copyright: © 2019 International Society for Nutraceuticals and Functional Foods. 1 All rights reserved. Adverse effects of omega-3 supplements Lange et al. farian and Rimm, 2006). The consumption of fish once or twice a For example, a prospective study from Norway investigating the week has been claimed to be unproblematic in this respect (Mozaf- relationship between diet, as assessed using a semi-quantitative farian and Rimm, 2006). However, endocrine disruptors contained food-frequency questionnaire, and the subsequent risk of cutane- in fish and fish oil, such as bisphenols and small amounts of pesti- ous malignant melanoma in 50,757 individuals attending a health cides, may be associated with hormonal-based cancers, including screening program (mean follow-up time of 6.9 years) found that prostate cancer (Gerber, 2012). Omega-3 fatty acid supplements cod liver oil supplementation and the intake of polyunsaturated fat may also contain contaminants. Commercially available fish-oil were associated with a significantly increased risk of melanoma in capsules should therefore be assessed for pollutants and certified women (Veierød et al., 1997). by health agencies. Inflammation appears to play a role in carcinogenesis, includ- Intervention trials with omega-3 fatty acid supplements have ing in the etiology of prostate cancer (Sfanos and De Marzo, 2012; reported no serious adverse reactions at the doses administered Wang et al. 2009). Omega-3 PUFAs may have anticarcinogenic (Sydenham et al., 2012). The more common adverse effects of effects due, in part, to their anti-inflammatory activity (Larsson fish oil preparations, particularly in higher dosages, include nau- et al. 2004; Chapkin et al. 2009). However, a large prospective sea, fishy belching and loose stools (Mehta, 2004). These effects study examining the relationship between inflammation-associated have caused the blinding of some of the earlier treatment stud- phospholipid fatty acids and prostate cancer risk found no support ies of fish oils to be questioned (Damico et al., 2002). While, in for the claim that omega-3 fatty acids reduce the risk (Brasky et general, omega-3 PUFAs do not seem to have clinically relevant al., 2011). The findings of this study, in fact, suggest that doco- effects on bleeding time (Eritsland et al., 1995), caution is needed sahexaenoic acid (DHA) may increase high-grade prostate cancer when administering high doses, since these have been shown to risk (Brasky et al., 2011). Another large, prospective case-cohort prolong bleeding time (Emsley et al., 2002; Emsley et al., 2006). study confirmed the finding of an elevated prostate cancer risk Since clinical researchers tend to distance themselves from ad- among men with high blood levels of omega-3 PUFAs. Statisti- verse events (Ioannidis, 2009), underreporting of such events is cally significant increases in risk were found for high-grade (by common. 71%) and low-grade (by 44%) disease and for eicosapentaenoic When similar rates of adverse events for omega-3 PUFAs com- acid (EPA), docosapentaenoic acid (DPA) and DHA (Brasky et pared with placebo are reported, this concerns unwanted effects in al., 2013). In a case-control study, an increase in prostate cancer the short term only. A commonly neglected consideration is that risk was observed in the highest compared to the lowest quintile adverse effects of omega-3 PUFAs may occur many years follow- of plasma phospholipid EPA and DPA, by 14% and 16%, respec- ing supplementation and may therefore fail to be detected. Rand- tively (Crowe et al., 2014). Although the above correlations do omized controlled trials are the gold standard for measuring treat- not provide conclusive evidence that the consumption of fish oil ment effects. However, randomized clinical drug trials are often supplements or fish causes prostate cancer, the consistency of the underpowered to detect late occurring and rare adverse effects. In results suggests that omega-3 fatty acids may be a factor in pros- contrast, non-randomized studies can include much larger samples tate tumorigenesis. The associations between prostate cancer and and can follow patients for considerably longer periods in com- exposure to omega-3 fatty acids, as assessed by their blood levels, parison with randomized trials, allowing for detection of rare and cannot distinguish between fish consumption and omega-3 supple- late adverse events. While non-randomized studies are of limited mentation. In addition, omega-3 PUFA supplements contain other use in establishing the benefits of drugs, they appear more reliable compounds, such as oxidation products and vitamin E, which may in establishing harms. be related to carcinogenesis. A variety of observational and epidemiological studies can provide information regarding potential harms of PUFA supplementation. Available studies suggest that long-term administration 3. Oxidation products of omega-3 fatty acids of omega-3 PUFAs could cause adverse effects due to the PUFAs themselves, compounds produced by PUFA oxidation or antioxi- As early as the 1950s, concerns were raised regarding the safety dants added to PUFA supplements, such as vitamin E. of oxidized fish oil (Kaneda and Ishii, 1953; Matsuo, 1954). The toxic effects of some fish and marine animal oils in rats (Sahashi, 1933; Somekawa, 1933) were shown not to be caused by fish oil 2. Omega-3 fatty acids per se but rather by its oxidized form (Kaneda and Ishii, 1953; Matsuo, 1954). On the basis of epidemiological, clinical, and experimental studies, Omega-3 PUFAs are highly prone to oxidation due to the large it is commonly assumed that omega-3 PUFAs reduce the risk of number of double bonds in their chemical structures (Arab-Tehra- various kinds of cancer (for review see Shahidi and Ambigaipalan, ny et al., 2012; Benzie, 1996; Shahidi and Zhong, 2010). Oxidized 2018). These purported effects are supported by several clinical fish oils could have altered biological activity rendering them in- studies. However, some systematic reviews have reported that the effective or even harmful. The oxidation of PUFAs leads to the available evidence is insufficient to suggest a significant relation- formation of free radicals as demonstrated both in vitro and in ship between omega-3 PUFAs and cancer incidence (e.g. MacLean vivo (Spiteller, 2005). Omega-3 PUFAs are oxidized to primary et al., 2006; Gerber, 2012). Factors that may have influenced the lipid hydroperoxides and secondary oxidation products, i.e. alde- inconsistent results of omega-3 PUFAs in relation to cancer in- hydes and ketones (Albert et al., 2013; Arab-Tehrany et al., 2012; clude differences in the amount, source, type, and form (ethyl es- Shahidi and Zhong, 2010). In cascade-like chain reactions, lipid ters or triacylglycerols) of omega-3 PUFAs, the ratio of omega-6 peroxides hasten oxidation of other fatty acids, producing further to omega-3 PUFAs, the proportions of EPA, DHA and docosap- lipid peroxides. The radicals and lipid peroxides formed follow- entaenoic acid in the preparations, and genetic factors (Berquin et ing omega-3 PUFA oxidation are relatively stable compared to al., 2007). other reactive oxygen species (Gutowski and Kowalczyk, 2013). The findings of several studies suggest that the intake of ome- It has been speculated that omega-3 peroxides may lead to lipid ga-3 fatty acids can be associated with an elevated cancer risk. membrane peroxidation, cell damage, and oxidative stress (Albert

2 Journal of Food Bioactives | www.isnff-jfb.com Lange et al. Adverse effects of omega-3 supplements et al., 2013). Evidence of harmful effects of oxidized lipids has effects of chronic, low grade exposure to peroxides, aldehydes, or been provided by animal studies (Esterbauer, 1993). The findings malondialdehyde. Furthermore, the study did not examine other of such studies suggest that lipid peroxidation may contribute to important pathological markers, such as low density lipoprotein, the pathophysiological changes in inflammation-associated dis- specific inflammatory markers or markers of DNA damage. eases, including neurodegenerative diseases (Grimm et al., 2016; Oxidation of trial oils may be responsible, at least partly, for Maruyama et al., 2014; Pamplona et al., 2005; Yakubenko and By- the inconsistent effects of fish oil on health, including the primary zova, 2017). Chronic lipid peroxidation may be involved in the and secondary prevention of cardiovascular disease (Bosch et al., pathogenesis of Alzheimer’s disease (Sayre et al., 1997) and in 2012; Rizos et al., 2012; Wang et al., 2006; Yokoyama et al., 2007). carcinogenesis (Bartsch and Nair, 2006). The intake of marine oil Omega-3 supplementation trials have not reported the oxidative leads to increased plasma (Haglund et al., 1991) and urinary (Piche status of the oils investigated. The influence of oxidation on the et al., 1988) malondialdehyde levels in mice and humans, due to efficacy or potential harmful effects of the supplements therefore absorption of peroxidized oil and in vivo oxidation (Piche et al., remains unknown. However, studies in animals have demonstrated 1988), which can only partially be reduced by added antioxidants that oxidized lipid products can cause harm. The 2010 conclusion (Cho and Choi, 1994; Gonzalez et al., 1992; Piche et al., 1988). of the European Food Standards Authority Panel on Biological Malondialdehyde has been shown to induce DNA mutations (Basu Hazards in regard to fish oil consumed by humans that, “informa- and Marnett, 1983) and to cause thyroid tumors and skin cancer in tion on the level of oxidation of fish oil ……. and related toxico- animals (Marnett, 1999). Malondialdehyde exposure may also be logical effects in humans is lacking” (EFSA Panel on Biological related to an increased risk of breast cancer in women (Wang et Hazards, 2010), remains salient today. al., 1996). In conclusion, it is essential to use formulations that are able to effectively preserve omega-3 PUFA bioactivity. In animal studies, DHA molecules can be protected from oxi- 4. Vitamin E dation by using microencapsulated omega-3 PUFA formulations (Hogan et al., 2003; Kolanowski et al., 2004). Dietary omega-3 supplements in humans should also be prevented from oxidizing. Many epidemiological studies have suggested preventive activ- Even oils stored in the dark at 4 °C could oxidize to unacceptable ity of vitamin E against cancer (Ju et al., 2010). However, several levels within a few weeks of storage (Pak, 2005). Commercially large-scale trials assessing the effects of α-tocopherol, the most available omega-3 PUFA supplements have been shown to be fre- commonly used form of vitamin E, failed to find a cancer preven- quently oxidized (Fantoni et al., 1996, Fierens and Corthout, 2007; tive effect (Gaziano et al., 2009; Lee et al., 2005; Lippman et al., Halvorsen and Blomhoff, 2011; Kolanowski, 2004; Opperman and 2009). Benade, 2013). For example, the majority of fish oil supplements Based on findings suggesting that vitamin E intake is associated on sale in New Zealand were shown to contain PUFAs below the with a reduced prostate cancer risk (Chan et al., 1999; Kirsh et concentrations claimed by labels and also to be oxidized, with al., 2006), the phase III, randomized, placebo-controlled Selenium peroxide (primary oxidation product), anisidine (reflecting sec- and Vitamin E Cancer Prevention Trial (SELECT) of prostate can- ondary oxidation products), and total oxidation values exceeding cer prevention was conducted, with incident prostate cancer as the the recommended thresholds (Albert et al., 2015). Another study primary endpoint (Lippman et al., 2009). The trial was terminated from New Zealand found that 14−28% of fish oil products did not after 7 years of follow-up, since no beneficial effects of selenium, comply with voluntary industry-set maximum limits on peroxide, vitamin E, or a combination of both were found, and a statisti- anisidine, and total oxidation values (Bannenberg et al., 2017). In cally significant benefit was impossible to ascertain with additional North America, 50% of over-the-counter omega-3 PUFA supple- follow-up (Lippman et al., 2009). Furthermore, extended follow- ments have been found to fall below at least one of the voluntary up analysis found an elevated risk, by 17%, of incident prostate safety standards recommended for primary and secondary oxida- cancer in the vitamin E alone group (Klein et al., 2011). tion as well as total oxidation (Jackowski et al., 2015). Several questions have been raised in regard to the association The antioxidant most commonly added to omega-3 supple- of vitamin E and cancer. For example, it has been proposed that, at ments is α-tocopherol. Phenolic compounds appear to be efficient the nutritional level, all forms of vitamin E are cancer preventive in delaying omega-3 PUFA oxidation (Hasiewicz-Derkacz et al., (Yang et al., 2012), since many observations have suggested that 2015). Natural antioxidants such as myricetin, rosmarinic and car- the dietary consumption or plasma levels of α-tocopherol and other nosic acids have recently been demonstrated to be more effective tocopherols correlates inversely with cancer risk (Helzlsouer et al., in preventing oxidation of omega-3 PUFA oils than α-tocopherol 2000; Huang et al., 2003; Mahabir et al., 2008; Ju et al., 2010). and synthetic antioxidants (Guitard et al., 2016). It should be em- However, at the supranutritional level, α-tocopherol administered phasized, however, that the addition of antioxidants to fish oils as a supplement does not appear to be cancer preventive, as dem- decreases but does not prevent oxidation (Zuta et al., 2007). New onstrated in several cancer prevention trials (Gaziano et al., 2009; antioxidants should be investigated in future studies. For example, Klein et al., 2011; Lee et al., 2005; Lippman et al., 2009). This view esters of DHA with the main tea polyphenol epigallocatechin gal- is supported by observations in animal models, which have shown late have been shown to help stabilize DHA and also provided an the cancer preventive activity of γ- and δ-tocopherols as well as of antioxidant preparation (Zhong and Shahidi, 2011). a naturally occurring mixture of tocopherols, while cancer preven- A randomized placebo-controlled study has investigated in hu- tive activity of α-tocopherol was not apparent (for review see Yang mans the effects of oxidized versus non-oxidized oil administered et al., 2012). This suggests that vitamin E, administered in the diet for 7 weeks (Ottestad et al. 2012). No difference was observed or via supplements rich in γ- and δ-tocopherols, is cancer preven- between groups in respect to markers of in vivo lipid peroxida- tive, while high doses of α-tocopherol supplements are not. Both tion, markers of antioxidant activity, C-reactive protein, or liver mechanistic and preclinical animal studies have demonstrated that, function tests (Ottestad et al. 2012). These findings suggest that in comparison with α-tocopherol, other forms of vitamin E, such oxidized marine omega-3 PUFAs may be unrelated to acute oxida- as γ-tocopherol, δ-tocopherol, γ-tocotrienol, and δ-tocotrienol are tive toxicity. However, the duration of this trial study was short, more potent in inhibiting multiple cancer-promoting pathways and and long-term follow-up may be required to identify the health have far superior cancer preventive efficacy (Jiang, 2017). The

Journal of Food Bioactives | www.isnff-jfb.com 3 Adverse effects of omega-3 supplements Lange et al. question of whether there are optimal ratios for these agents in acids should be investigated. regard to cancer prevention needs to be investigated. The measure- In summary, short-term adverse effects of omega-3 PUFAs do ment of baseline blood levels of the different forms of tocopherol not appear to give cause for concern. However, in view of po- is of importance, since cancer preventive effects may be confined tentially increased cancer risks associated with omega-3 supple- to individuals with low blood levels (Yang et al., 2012). mentation, possibly due to PUFAs, oxidation products or added In addition, the findings of recent gene-supplement interaction vitamin E, caution is advised in the recommendation of PUFA sup- studies suggest that the effects of vitamin E on total risk of cancer plementation over extended periods of time. Deleterious effects of (Hall et al. 2019a) and risk of colorectal cancer (Hall et al., 2019b) omega-3 PUFA supplements may be particularly relevant when may be influenced by genetic variation in catechol-O-methyltrans- administered during vulnerable phases of life, such as prenatal ferase (COMT), an enzyme that metabolizes catecholamines. This development, childhood, and adolescence. A balanced approach variation may increase the risk in some people while decreasing should carefully weigh potentially harmful effects of the supple- it in others. Pharmacogenetic analysis of COMT gene and cancer mentation of omega-3 PUFAs against their overall health benefits. prevention in two large randomized trials revealed a statistically significant decline in colorectal cancer by alpha-tocopherol when COMT was inhibited by siRNA (Hall et al., 2019a). 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6 Journal of Food Bioactives | www.isnff-jfb.com Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Perspective J. Food Bioact. 2019;7:7–17

A perspective on phenolic compounds, their potential health benefits, and international regulations: The revised Brazilian normative on food supplements

Adriano Costa de Camargoa* and Renan da Silva Limab aDepartamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Casilla 306- 22, Santiago, Chile bDepartment of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, A1B 3X9, Canada *Corresponding author: Adriano Costa de Camargo, Departamento 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.2019.7193 Received: September 11, 2019; Revised received & accepted: September 29, 2019 Citation: de Camargo, A.C., and Lima, R.S. (2019). A perspective on phenolic compounds, their potential health benefits, and international regulations: The revised Brazilian normative on food supplements. J. Food Bioact. 7: 7–17.

Abstract

Phenolic compounds possess a myriad of health benefits, thus making them potential ingredients for the procurement of food supplements. Anvisa, Brazil’s national regulatory body, has recently revised the regulation on food supplements and only a few phenolic compounds (chlorogenic acid, rutin, proanthocyanidins, and α-tocopherol) have been mentioned. Despite several scientific evidences on the bioactivity of phenolics, especially concerning their antioxidant activity, Anvisa does not authorize any claims for supplements containing these compounds, except for α-tocopherol, which was mentioned as “vitamin E”. The upper limit doses allowed for the phenolics appear to be lower than what the literature suggests as necessary to achieve potential health benefits and might be prohibitive for supplements available in the international market. Moreover, Brazilian sources of phenolic compounds are not listed in the Normative Instruction (NI) as authorized ingredients for supplements. The Brazilian NI on food supplements has moved forward, but it is still limited.

Keywords: Normative Instruction (NI) N° 28; Flavonoids; Tocopherols; Brazil; Phenolic acids.

1. Introduction each authorized ingredient for this type of product. The tool created by Anvisa is a set of interactive tables separat- Brazil’s National Health Surveillance Agency (Agência Nacional ed by ingredient category (e.g., bioactive substances, protein, fibre, de Vigilância Sanitária–Anvisa) is a regulatory body linked to the enzymes, among others). It provides details regarding the lower country’s Ministry of Health. The agency is responsible for the and upper limits for each nutrient/bioactive substance/enzyme supervision and approval of food, cosmetics, tobacco, pharmaceu- according to age group and individual condition (e.g., pregnant ticals, health services, medical devices, among others. In addition, and breastfeeding women). There are also tables specifying the food supplements, which are regulated by the Normative Instruc- authorized ingredients that can be used to provide the target com- tion (NI) N° 28 from July 26th, 2018, fall under the Anvisa’s scope. pounds, as well as their function, specifications, allowed claims The normative establishes the list of components, limits of usage, and requirements to produce them, requisites for complementary claims, and labeling for food supplements (Agência Nacional de labels as well as other relevant information (Agência Nacional de Vigilância Sanitária, 2019a). Recently, Anvisa updated the infor- Vigilância Sanitária, 2019c). The objective is to facilitate the ac- mation contained in NI N° 28 and created a tool (Agência Nacional cess to information about food supplements to agencies connected de Vigilância Sanitária, 2019c) that enables anyone to easily track to the National System of Health Surveillance, manufacturers, and

Copyright: © 2019 International Society for Nutraceuticals and Functional Foods. 7 All rights reserved. Phenolic compounds in food supplements regulation de Camargo et al. final consumers. It is not uncommon to find supplements that are legally com- mercialized in other countries but do not fall under the revised Brazilian regulation. This may be explained by the different approaches regarding food regulation in each country. In the United States, for example, the manufacturer is entirely responsible for the safety of food supplements, their efficacy, and health claims. Regulatory agencies monitor the products in the market, interfering when the product’s safety is not adequate. Food companies that put consumers’ health at risk may suffer severe consequences. In Brazil, the approach is focused on the prevention of risk exposure. Therefore, a mandatory registration under Anvisa’s rules is required before the product reaches the market. Usually, the inci- dents reported are associated with the commercialization of illicit products. If manufacturers intend to use an ingredient that is not specified in the NI, they should submit a petition to Anvisa to get the approval. Currently, depending on its complexity, Anvisa may Observations – – – includes α-Tocopherol As α-tocopherol. the only α-tocopherol RRR-α-tocopherol, and the in food., occurs naturally that form (RRR-, of α-tocopherol 2R-stereoisomers that and RSS-α-tocopherol) RRS-, RSR-, foods and supplements. occur in fortified available Considering the commercially with form (rac-α-tocoferyl) synthetic an activity of 0.67 x RRR-α-tocopherol, E as 1 1 IU of vitamin considering of rac-α-tocoferyl. mg of acetate take about 55 days to reach a decision on a petition (Agência de Vigilância Sanitária, 2019b). Pregnant Pregnant women Not allowed Not allowed Not allowed Min: 2.25 mg Max: 800 mg 2. Phenolics compounds as bioactive substances

Phenolic compounds are secondary metabolites, with one or more hydroxyl groups on the aromatic ring(s). They are widely studied Not allowed Not allowed Min: 2.85 mg Not allowed 800 Max: mg due to their potential beneficial health effects, such as antioxidant, - Breastfeed ing women anti-inflammatory, anti-microbial, and cardioprotective properties (Shahidi et al., 2019). Therefore, as bioactive substances, they can be used in the formulation of food supplements. More than 8,000 phenolic compounds have been reported in the literature (de Camargo et al., 2018). However, as specified in the bioactive substances section (Table 1), Anvisa mentions the use of only some phenolics, namely chlorogenic acid, rutin, proanthocyanidins and Min: Not established Min: Not established Min: 2.25 mg Min: Not established 0.12 mg Max: 0.6 mg Max: 7.5 mg Max: 1,000 mg Max: α-tocopherol, this latter as “vitamin E”. The list of approved ingre- old +19 years dients that can be used as sources of such substances is shown in Table 2. Due to the importance of phenolic compounds for food supplements formulation, one should explore in more detail the aspects concerning the revised NI. Accordingly, the rationale behind Not allowed Not allowed Min: 2.25 mg Not allowed Max: 600 mg the limits established, approved claims, as well as the scientific 9-18 years old support about the potential health benefits associated with them will be discussed. A quick comparison with similar regulations from other countries will be addressed. Not allowed Not allowed Min: 1.05 mg Not allowed Max: 300 mg 4-8 years 4-8 years old 2.1. Chlorogenic acid (CGA)

The term CGA corresponds to a wide range of compounds gener- Not allowed Not allowed Min: 0.9 mg Not allowed Max: 200 mg ated via condensation of the carboxy group of trans-caffeic acid 1-3 years old with the 3-hydroxy group of quinic acid. Figure 1 shows the basic chemical structure of CGA and other phenolic compounds addressed in NI N° 28. CGA exhibits antioxidant activity due to the Not allowed Not allowed Not allowed Not allowed 7-11 months presence of vicinal hydroxyl groups on the aromatic ring, allowing the donation of hydrogen atoms to reduce free radicals. Each chlorogenic acid subgroup possesses several isomers (Celli and de Not allowed Not allowed Not allowed Not allowed Camargo, 2019). In green coffee beans, for example, the predomi- 0-6 months nant isomer (3-(3,4-dihydroxycinnamoyl)quinic acid) accounts for 76-84% of the total CGA content (Liang and Kitts, 2016). Coffee is the primary source of chlorogenic acid in nature, with green coffee beans presenting around 5-12 g CGA/100 g on a dry weight basis (DW) while a cup of 200 mL of standard brewed coffee has 0.07- 0.35 g (Farah et al., 2008). Coffee and coffee-based products are subjects of a fair number Rutin Proanthocyanidins E Vitamin Chlorogenic acid Chlorogenic Phenolic compound Agência Nacional de Vigilância Sanitária (2019c) . Nacional de Vigilância Sanitária Agência from Adapted Source: of studies associating the intake of CGA with health-promoting from July 26th, 2018 (NI N° 28) Instruction Normative Anvisa’s by authorized supplements food for of phenolic compounds 1. List Table

8 Journal of Food Bioactives | www.isnff-jfb.com de Camargo et al. Phenolic compounds in food supplements regulation – – – Other in - formation – – – The warning “ThisThe warning product by should not be consumed and breastfeeding pregnant must and children” women on the label. be written “ThisThe warning product by should not be consumed and breastfeeding pregnant must and children” women on the label. be written No additional requirements. No additional Requirements for Comple - for Requirements Labelling and other mentary “ThisThe warning product by should not be consumed and breastfeeding pregnant must and children” women on the label. be written “ThisThe warning product by should not be consumed and breastfeeding pregnant must and children” women on the label. be written “ThisThe warning product by should not be consumed and breastfeeding pregnant must and children” women on the label. be written No claims 1) Claim: Caffeine enables an elevated state of alert state enables an elevated 1) Claim: Caffeine Requirements: the concentration. and improves supplements the dietary for this claim is exclusive the minimum meets content in which the caffeine IN 28/2018–Appendix III. by established amount and endurance improves 2) Claim: Caffeine activities. physical during endurance performance the dietary to this claim is exclusive Requirements: is 200 content in which the caffeine supplements activity. the physical mg, 1 h prior to consumed 1) Claim: Vitamin E is an antioxidant that helps helps that E is an antioxidant 1) Claim: Vitamin free by caused the damages against protect this claim is exclusive Requirements: radicals. in which the supplements the dietary to the minimum amount meets E content vitamin in IN 28/2018–Appendix III established this E. Requirements: of vitamin 2) Claim: Source in supplements the dietary to claim is exclusive the minimum meets E content which the vitamin in IN 28/2018–Appendix III. established amount Authorized claims and requirements to make them make to requirements and claims Authorized No claims No claims No claims Source of phenolic Source compounds Source of caffeine Source Source of Source E Vitamin Function Source of Source acid chlorogenic of rutin Source of Source proanthocyanidins , 2016 th , 2016 , 2001 nd th These components must must These components the specifications follow and approved evaluated ANVISA. by FCC 6 FCC 10 FCC 10 February ADOPTED: doi: 10.2903/j.efsa.2016.4412 Specifications For this component, the this component, For specifications approved the following belong to C.A.S. S.p.A – manufacturers: Hans Zipperle – Italy; Verona – Italy; AG/S.p.A – Merano – France Indena S.A.S. – Tours the this component, For specifications approved the following belong to C.A.S. S.p.A – manufacturers: Hans Zipperle – Italy; Verona – Italy; AG/S.p.A – Merano – France Indena S.A.S. – Tours the this component, For belong specifications approved manufacturer: the following to – Sagamore – DBS LLC Naturex – USA – Massachusetts N° 3, Instruction Normative January 19 DE from PUBLISHED: March 2 PUBLISHED: March 58-95-7 CAS ) Powdered guarana guarana Powdered cupana ( Paulinia Dextroalphatocopherol acetate/D-alpha- acetate tocopherol Authorized Component Authorized Tomato hydrosoluble hydrosoluble Tomato concentrate ( Lycopersicon ) esculentum hydrosoluble Tomato concentrate ( Lycopersicon ) esculentum cranberry Powdered ( Vaccinium ) macrocarpon extract Propolis Table 2. List of source ingredients of bioactive compounds for food supplements authorized by Anvisa’s Normative Instruction (NI N° 28) from July 26th, 2018 (NI N° 28) Instruction Normative by Anvisa’s authorized food supplements for compounds of bioactive ingredients of source 2. List Table

Journal of Food Bioactives | www.isnff-jfb.com 9 Phenolic compounds in food supplements regulation de Camargo et al. – Other in - formation – - (continued) No additional requirements. No additional Requirements for Comple - for Requirements Labelling and other mentary No additional requirements. No additional 3) Claim: Rich in/High content of vitamin E. of vitamin 3) Claim: Rich in/High content the dietary to this claim is exclusive Requirements: E content in which the vitamin supplements the double of minimum to corresponds in IN 28/2018–Appendix established amount III, since it does not surpass the maximum in the Appendix IV. established amount 1) Claim: Vitamin E is an antioxidant that helps helps that E is an antioxidant 1) Claim: Vitamin free by caused the damages against protect this claim is exclusive Requirements: radicals. in which the supplements the dietary to the minimum amount meets E content vitamin in IN 28/2018–Appendix III established this E. Requirements: of vitamin 2) Claim: Source in supplements the dietary to claim is exclusive the minimum meets E content which the vitamin in IN 28/2018–Appendix III. established amount E. of vitamin 3) Claim: Rich in/High content the dietary to this claim is exclusive Requirements: E content in which the vitamin supplements the double of minimum to corresponds in IN 28/2018–Appendix established amount III, since it does not surpass the maximum in the Appendix IV. established amount Authorized claims and requirements to make them make to requirements and claims Authorized 1) Claim: Vitamin E is an antioxidant that helps helps that E is an antioxidant 1) Claim: Vitamin free by caused the damages against protect this claim is exclusive Requirements: radicals. in which the supplements the dietary to the minimum amount meets E content vitamin in IN 28/2018–Appendix III established this E. Requirements: of vitamin 2) Claim: Source in supplements the dietary to claim is exclusive the minimum meets E content which the vitamin in IN 28/2018–Appendix III. established amount E. of vitamin 3) Claim: Rich in/High content the dietary to this claim is exclusive Requirements: E content in which the vitamin supplements the double of minimum to corresponds in IN 28/2018–Appendix established amount III, since it does not surpass the maximum in the Appendix IV. established amount Source of Source E Vitamin Function Source of Source E Vitamin , 2016 th , 2016 nd JECFA JECFA th JECFA (1986). JECFA th JECFA (1986) and JECFA th ADOPTED: February 10 February ADOPTED: (2000) and published in FNP 52 Add 8 (2000), tentative superseding at prepared specifications the 30 published in FNP 37 (1986) and in FNP 52 (1992). A ADI of 0.15-2 mg/kggroup dl-alpha-tocopherol for bw and d-alpha-tocopherol, singly or in concentrate, was established combination, the 30 at doi: 10.2903/j.efsa.2016.4412 9.0 pharmacopoeia European European pharmacopoeia 9.0 pharmacopoeia European 6 FCC the 55 at Prepared Specifications PUBLISHED: March 2 PUBLISHED: March 7695- 91-2 59-02-9 CAS DL-alpha-tocopherol DL-alpha-tocopherol acetate/rac- alpha-tocopherol acetate/DL-alpha- acetate tocopheryl Dextro-alpha- tocopherol/D- alpha-tocopherol Authorized Component Authorized Table 2. List of source ingredients of bioactive compounds for food supplements authorized by Anvisa’s Normative Instruction (NI N° 28) from July 26th, 2018 (NI N° 28) Instruction Normative by Anvisa’s authorized food supplements for compounds of bioactive ingredients of source 2. List Table

10 Journal of Food Bioactives | www.isnff-jfb.com de Camargo et al. Phenolic compounds in food supplements regulation Other in - formation – – – - (continued) Requirements for Comple - for Requirements Labelling and other mentary No additional requirements. No additional requirements. No additional requirements. No additional Authorized claims and requirements to make them make to requirements and claims Authorized 1) Claim: Vitamin E is an antioxidant that helps helps that E is an antioxidant 1) Claim: Vitamin free by caused the damages against protect this claim is exclusive Requirements: radicals. in which the supplements the dietary to the minimum amount meets E content vitamin in IN 28/2018–Appendix III established this E. Requirements: of vitamin 2) Claim: Source in supplements the dietary to claim is exclusive the minimum meets E content which the vitamin in IN 28/2018–Appendix III. established amount E. of vitamin 3) Claim: Rich in/High content the dietary to this claim is exclusive Requirements: E content in which the vitamin supplements the double of minimum to corresponds in IN 28/2018–Appendix established amount III, since it does not surpass the maximum in the Appendix IV. established amount helps that E is an antioxidant 1) Claim: Vitamin free by caused the damages against protect this claim is exclusive Requirements: radicals. in which the supplements the dietary to the minimum amount meets E content vitamin in IN 28/2018–Appendix III established this E. Requirements: of vitamin 2) Claim: Source in supplements the dietary to claim is exclusive the minimum meets E content which the vitamin in IN 28/2018–Appendix III. established amount E. of vitamin 3) Claim: Rich in/High content the dietary to this claim is exclusive Requirements: E content in which the vitamin supplements the double of minimum to corresponds in IN 28/2018–Appendix established amount III, since it does not surpass the maximum in the Appendix IV. established amount helps that E is an antioxidant 1) Claim: Vitamin free by caused the damages against protect this claim is exclusive Requirements: radicals. in which the supplements the dietary to the minimum amount meets E content vitamin in IN 28/2018–Appendix III established this E. Requirements: of vitamin 2) Claim: Source in supplements the dietary to claim is exclusive the minimum meets E content which the vitamin in IN 28/2018–Appendix III. established amount E. of vitamin 3) Claim: Rich in/High content Function Source of Source E Vitamin of Source E Vitamin of Source E Vitamin JECFA JECFA JECFA th th JECFA (1986). JECFA (1986). JECFA th th JECFA (2003). A JECFA (2003). A JECFA st st (1986), published in FNP 37 (1986) and in FNP 52 and arsenic (1992). Metals at revised specifications the 61 (1986), published in FNP 37 (1986) and in FNP 52 and arsenic (1992). Metals at revised specifications the 61 group ADI of 0.15-2 mg/kggroup dl-alpha-tocopherol for bw and d-alpha-tocopherol, singly or in concentrate, was established combination, the 30 at ADI of 0.15-2 mg/kggroup dl-alpha-tocopherol for bw and d-alpha-tocopherol, singly or in concentrate, was established combination, the 30 at Specifications Prepared at the 30 at Prepared FCC10 the 30 at Prepared European pharmacopoeia 9.0 pharmacopoeia European 10 FCC 9.0 pharmacopoeia European 10 FCC CAS 10191- 41-0 893081 Authorized Component Authorized DL-alpha-tocopherol Mixture of tocopherols Mixture of Acidic succinate D-alpha-tocopheryl Table 2. List of source ingredients of bioactive compounds for food supplements authorized by Anvisa’s Normative Instruction (NI N° 28) from July 26th, 2018 (NI N° 28) Instruction Normative by Anvisa’s authorized food supplements for compounds of bioactive ingredients of source 2. List Table

Journal of Food Bioactives | www.isnff-jfb.com 11 Phenolic compounds in food supplements regulation de Camargo et al. – – Other in - formation - (continued) No additional requirements. No additional requirements. No additional Requirements for Comple - for Requirements Labelling and other mentary Requirements: this claim is exclusive to the dietary the dietary to this claim is exclusive Requirements: E content in which the vitamin supplements the double of minimum to corresponds in IN 28/2018–Appendix established amount III, since it does not surpass the maximum in the Appendix IV. established amount 1) Claim: Vitamin E is an antioxidant that helps helps that E is an antioxidant 1) Claim: Vitamin free by caused the damages against protect this claim is exclusive Requirements: radicals. in which the supplements the dietary to the minimum amount meets E content vitamin in IN 28/2018–Appendix III established this E. Requirements: of vitamin 2) Claim: Source in supplements the dietary to claim is exclusive the minimum meets E content which the vitamin in IN 28/2018–Appendix III. established amount E. of vitamin 3) Claim: Rich in/High content the dietary to this claim is exclusive Requirements: E content in which the vitamin supplements the double of minimum to corresponds in IN 28/2018–Appendix established amount III, since it does not surpass the maximum in the Appendix IV. established amount helps that E is an antioxidant 1) Claim: Vitamin free by caused the damages against protect this claim is exclusive Requirements: radicals. in which the supplements the dietary to the minimum amount meets E content vitamin in IN 28/2018–Appendix III established this E. Requirements: of vitamin 2) Claim: Source in supplements the dietary to claim is exclusive the minimum meets E content which the vitamin in IN 28/2018–Appendix III. established amount E. of vitamin 3) Claim: Rich in/High content the dietary to this claim is exclusive Requirements: E content in which the vitamin supplements the double of minimum to corresponds in IN 28/2018–Appendix established amount III, since it does not surpass the maximum in the Appendix IV. established amount Authorized claims and requirements to make them make to requirements and claims Authorized Source of Source E Vitamin of Source E Vitamin Function European pharmacopoeia 9.0 pharmacopoeia European USP NF 34 Specifications 17407- 37-3 CAS Acidic succinate of Acidic succinate DL-alpha-tocopheryl Acidic succinate of Acidic succinate D-alpha-tocopheryl- polyethylene glycol-1000 Authorized Component Authorized Table 2. List of source ingredients of bioactive compounds for food supplements authorized by Anvisa’s Normative Instruction (NI N° 28) from July 26th, 2018 (NI N° 28) Instruction Normative by Anvisa’s authorized food supplements for compounds of bioactive ingredients of source 2. List Table (2019c) . Nacional de Vigilância Sanitária Agência from Adapted Source:

12 Journal of Food Bioactives | www.isnff-jfb.com de Camargo et al. Phenolic compounds in food supplements regulation

Figure 1. Chemical structures of phenolic compounds mentioned in the Brazilian Normative Instruction N° 28. (a) chlorogenic acid, (b) rutin, (c) procyanidin B2, and (d) α-tocopherol. effects (Naveed et al., 2018). Johnston et al. (2003) observed anti- due to its potential as a source of CGA and economic importance, obesity and anti-diabetic properties promoted by CGA when ad- coffee would be the most reasonable choice as a source ingredient ministering coffee (caffeinated and decaffeinated, 0.88 g CGA/L) in the development of CGA-based supplements. to nine healthy volunteers. According to these authors, this was Furthermore, the outcome of studies (Johnston et al., 2003; due to the fact that CGA inhibits the activity of hepatic glucose- Zhao et al., 2008) investigating the health benefits of CGA suggest 6-phosphatase, reducing glucose release, as well as diminishing that in order to efficiently act as an antioxidant, anti-inflammatory, the glucose absorption in the small intestine via inhibition of glu- anti-obesity, and anti-diabetic factor, the dosage of CGA needs cose-6-phosphate translocase 1. The reduced amount of glucose in to be around 700-880 mg/L of coffee/coffee extract, much higher the blood circulation decreased insulin activity, which led to burn- than the upper limit established by the regulation (0.12 mg). This ing excessive adipose tissue. raises the question if the approved maximum CGA dosage (Table The antioxidant and anti-inflammatory properties of CGA have 1) would be sufficient to promote the full range of health benefits. also been investigated. A study by Zhao et al. (2008) using Caco- In comparison with regulations from other countries, the Bra- 2 cell line model verified that this phenolic acid was capable of zilian one is stricter regarding the limit of usage, and it lacks de- inhibiting oxidative stress-induced secretion of interleukin-8 (IL- tailed information. The European Food Safety Authority (EFSA) 8), a chemokine associated with the initiation of pro-inflammatory released in 2011 a scientific opinion on the use of coffee and cof- responses, in human intestinal epithelial cells in vitro. A dose-de- fee extracts as a source ingredient in CGA-based supplements. It pendent response was observed, and the highest bioefficacy was was suggested that coffee extract should be limited to a maximum found in cells exposed to 0.7 g/L. Kozuma et al. (2005) also veri- of 400 mg/day, containing 45% CGA (180 mg/day). According to fied a dose-dependent relationship between the administration of EFSA (2011a), the proposed claims “chlorogenic acids from coffee CGA-rich green coffee bean extracts and antihypertensive effects. extract contributes to keeping normal blood glucose levels” and In a human trial involving 117 male volunteers, the administration “chlorogenic acids from coffee extract have a beneficial effect on of 93 and 185 mg of extract (50.22 and 100 mg of CGA, respec- glucose/insulin metabolism” are allowed. tively) was capable of reducing both systolic and diastolic blood Coffee extract (as a source of CGA) is also included in the U.S. pressures. Furthermore, adverse effects on hematological param- Department of Health and Human Services list as an approved in- eters and/or blood chemistry were not observed. gredient for the manufacture of food supplements aiming weight Judging by the studies presented, chlorogenic acid seems to be loss and/or weight management. However, the agency states that responsible for a myriad of health benefits. Therefore, it is indeed clinical trials on the efficacy of CGA for weight reduction are lim- an unusual choice that the revised regulation does not mention any ited and of poor methodological quality. Additionally, some safety claims associated with the intake of chlorogenic acid (Table 2), not concerns have been reported in a few studies when the coffee ex- even its well-reported antioxidant properties (Celli and de Cama- tract dosage was above 200 mg/day (U.S. Department of Health rgo, 2019; Kamiyama et al., 2015; Yao et al., 2019). In addition, and Human Services, 2019). Table 2 only suggests tomato hydrosoluble concentrate as an approved source ingredient for the manufacture of CGA-based supplements. Tomato is indeed a rich source of CGA, showing con- 2.2. Rutin centrations ranging from 14.31 to 32.84 mg/kg on a fresh weight (FW) basis (Martínez-Valverde et al., 2002). However, coffee re- Rutin is a glycoside form of quercetin (Ganeshpurkar and Salu- mains the major source of this phenolic compound. Furthermore, ja, 2017). Its chemical structure is shown in Figure 1. Some is- Brazil is among the most important suppliers of coffee. Therefore, sues concerning other phenolic compounds, such as rutin, are

Journal of Food Bioactives | www.isnff-jfb.com 13 Phenolic compounds in food supplements regulation de Camargo et al. also noticeable when one analyzes the revised Anvisa regulation. g, respectively), esterified (7.22–17.1 mg CE/g and 25–45.6 mg Some of the primary sources of this phenolic include capers (332 CE/g, respectively), and insoluble-bound (91.3–142 mg CE/g and mg/100 g FW), black olive (45.36 mg/100 g FW), and buckwheat 85.7–145 mg CE/g, respectively), depending on the grape variety. (36.14 mg/100 g FW) (Inocencio et al., 2000; Romani et al., 1999; Furthermore, PAC-rich extracts (also composed of other pheno- Vlahov, 1992; Oomah and Mazza, 1996; Holasova et al., 2002; lics) were able to inhibit copper-induced LDL-cholesterol (LDL- Steadman et al., 2001; Dietrych-Szostak and Oleszek, 1999). Nev- c) oxidation in vitro (de Camargo et al., 2014). The presence of ertheless, neither of these sources are listed by Anvisa amongst oxidized LDL-c has been accepted as a biomarker that indicates the approved ingredients (as rutin sources) for the development a risk factor for the development of cardiovascular diseases (Am- of rutin-based supplements. Furthermore, rutin has been found in arowicz, 2016), while DNA-damage signaling and repair are cru- fruits, including orange peels (Gosslau et al., 2019; Omoba et al., cial pathways to the etiology of most, if not all, human cancers 2015). Therefore, as an important orange producer, the Brazilian (Khanna and Jackson, 2001). The same study (de Camargo et al., industry would benefit if orange peel extract could be included as 2014), reported the protection of DNA against oxidation induced a natural source of rutin. by peroxyl radicals. Both in vitro biological assays correlated with Similar to what was addressed for chlorogenic acid, tomato total phenolics and proanthocyanidin contents of the test sample, hydrosoluble concentrate appears as the only authorized source which suggests a dose-dependent relationship between PACs and ingredient for rutin (Table 2). This phenolic compound has been specific health-promoting effects. shown to possess in vivo antioxidant activity in a 6-week clinical PACs were also found in high amounts in peanut skin (free trial (Boyle et al., 2000) with 18 female volunteers. The admin- soluble = 20.33–36.25 mg CE/g, esterified = 0.91–4.34 mg CE/g, istration of a supplement containing 500 mg of rutin was able to and insoluble-bound = 1.33–3.03 mg CE/g) (de Camargo, et al., elevate plasma flavonoids and decrease endogenous oxidation of 2015) and guarana seeds extract (free soluble = 13.1-60.5 mg/g, pyrimidines. Rutin has also been associated with antidiabetic and depending on the extraction solvent) (Majhenič et al., 2007). Pow- antihypertensive effects. In another clinical trial (Sattanathan et al., dered guarana has been approved by Anvisa as a source of caffeine. 2011), 40 patients with type 2 diabetes mellitus (40-60 years old) However, guarana possesses many other bioactive compounds, in- received tablets with 500 mg of rutin daily over 120 days. By the cluding PACs, that exhibit in vitro and in vivo health-promoting end of the trial, the subjects presented lower blood sugar levels and effects. In fact, their potential anticancer, antioxidant, antihyper- reduced systolic and diastolic blood pressures. glycemic and anti-obesity effects have recently been reported (da Once again, the maximum dose established by Anvisa appears Silva et al., 2019). to be an issue that needs to be addressed. The regulation limits Like coffee, which has not been mentioned as a source of CGA, rutin to 0.6 mg, much lower than the dose administered in the and despite of their well-documented potential as sources of PACs, clinical trials that presented favorable outcomes. A scientific opin- grapes, peanuts, cocoa, guarana, and their processing by-products ion released by EFSA in 2010, recommends the use of rutin as are not mentioned in the new version of the Brazilian regulation an antioxidant compound, allowing a daily dosage of up to 300 (Agência Nacional de Vigilância Sanitária, 2019c) as approved mg, which is 50 times higher than the value indicated by Anvisa. sources of PACs. Instead, cranberry powder is the mentioned in- The same document also acknowledges that, given the available gredient for this purpose (Table 2). Cranberries are indeed rich scientific data, rutin may have beneficial physiological effects by sources of PACs, with 4.18 mg CE/g of fruit DW (Kruger et al., counteracting protein-induced, lipid-induced, and DNA-induced 2014). Therefore, establishing them as the source ingredient for oxidative damage. The agency allows the claims “a strong anti- PACs is coherent in countries (e.g., USA, Canada, and Chile) with oxidant that protects the body’s cells from the harmful effects of a considerable production of this feedstock (FAOSTAT, 2017). free radicals” and “improves the immune system” to be used in the However, Brazil is not known as a producer of cranberries. There- label of rutin-based supplements (European Food Safety Author- fore, indicating national sources of PACs would be expected. ity, 2010a). In contrast, the revised version of Anvisa’s regulation EFSA also recognizes grape seeds extract as a source of PACs. does not cite any approved claim for this substance. A scientific opinion from 2010 recommends a dose of 25–50 mg PAC/day and proposes a variety of claims, such as “helps improve 2.3. Proanthocyanidins (PACs) the general aging process, protecting skin from environmental stress” and “scientifically proven mix of naturally occurring antioxidants that work in synergy helping protect cells from free PACs are also present in the revised regulation. This phenolic class radical damage” (European Food Safety Authority, 2010b). The of compounds, also known as condensed tannins, includes dimeric, Brazilian regulation does not bring any authorized claim for PACs- polymeric or oligomeric flavonoids. Procyanidins are composed of based supplements, as it is the case for other phenolic compounds catechin and epicatechin, while prodelphinidins contain (epi) gal- (Table 2). locatechin in their structures (de Camargo et al., 2015). Cocoa is a rich source of proanthocyanidins (119.78 mg catequin eviquiva- lent (CE)/100 g of cocoa powder), with procyanidin B2 (Figure 2.4. Tocopherols and tocotrienols 1) as the major compound (Tomas-Barberán et al., 2007). Peanuts are sources of proanthocyanidin A while grape is a rich source of The only phenolic class addressed in the revised version of the proanthocyanidin B (de Camargo et al., 2017; Ma et al., 2014; de regulation comprises tocopherols, generically called vitamin E in Camargo et al., 2014; de Camargo et al., 2015; de Camargo et al., the normative. This group consists of eight fat-soluble monophe- 2016). Therefore, PACs type B are amongst the major polyphenols nols. They are composed of a chromanol ring carrying a hydroxyl in red wine, grape juice, and other grape-based products and by- group. The chromanol ring is bound to a hydrophobic side chain, products (de Oliveira et al., 2017; Toscano et al., 2017; da Silva et responsible for differentiating between tocopherols and tocot- al., 2015). rienols (Figure 1). In tocopherols, the side chain is completely According to the literature (de Camargo et al., 2014), grape by- saturated, whereas, in tocotrienols, there are three double bonds products from juice and winemaking are sources of different forms at carbons 3′, 7′, and 11′. Tocopherols and tocotrienols each have of PACs, namely free (26.6–68.1 mg CE)/g and 20.2–23 mg CE four possible homologs (α, β, γ, δ), determined by the number and

14 Journal of Food Bioactives | www.isnff-jfb.com de Camargo et al. Phenolic compounds in food supplements regulation

Table 3. α-Tocopherol content of common and specialty oils of health-promoting effects. A supplementation with α-tocopherol at doses ranging from 400 to 800 IU/day showed a 20% decrease Oil Concentration (mg/100 g of oil) of fatal myocardial infarction, as evidenced by a meta-analysis Almonds nd–34.9 that evaluated randomized clinical trials (Loffredo et al., 2015). γ-Tocopherol (300 mg) also had a positive impact on cardiovas- Barley 14.2–20.1 cular diseases (Christen et al., 1997). Tocotrienol homologs have Brazil nuts nd–2.2 displayed anticarcinogenic effects in breast cancer cell lines, while Camelina 2.81–3.80 anti-diabetes and anti-obesity effects have also been widely observed both for tocopherol and tocotrienol homologs (Shahidi and Cashew nd–7.84 De Camargo, 2016). However, tocotrienols are not mentioned Coconut 0.20–1.82 in the revised version of the normative instruction, even though γ-tocotrienol is abundant in soybean (61.0-69.0 mg/100 g of oil) Corn 18.0–25.7 (de Camargo et al., 2019), one of the major commodities produced Cottonseed 30.5–57.3 in Brazil. EFSA acknowledges the scientific evidence on these benefits Grape 11.8–18.8 and proposes an array of claims that range from “helps memory Hazelnut 15.7–42.1 and perception retention, especially in the elderly” to “due to vitamin E favorable effects on free radicals it could support reduc- Linseed 0.54–1.20 ing the age-related cognitive decline.” These claims, however, can Macadamia 0.08–0.11 never suggest that such supplements are able to prevent or cure diseases (European Food Safety Authority, 2010b). A similar ap- Olive 11.0–17.0 proach is taken by Health Canada, which states that selling vitamin Palm 6.05–42.0 E with the purpose of preventing diseases, including cardiovascu- Peanut 8.86–30.4 lar ailments and cancer, is not permitted. The agency considers 40 IU a normal dose of vitamin E while 400 IU is considered a “high Pecan nd–1.82 dose” or “megadose” and explains that 15 mg of α-tocopherol/day Pine 2.2–16.6 corresponds to 22 IU (from natural sources) or 33 IU (from synthetic sources). Canadian regulation establishes a maximum daily Pistachios nd–32.8 intake of 1,000 mg for any form of vitamin E (Health Canada, Rapeseed 18.9–24.0 2019). This is also the upper limit for individuals over 19 years old in Brazilian law (Table 1). However, no claims related to health Rice bran 0.73–15.9 benefits, other than antioxidant activity, are allowed (Table 2). Safflower 36.7–47.7 In other countries in South America, such as Chile and Argen- tina, vitamin E is included in the list of approved compounds for Sesame 0.24–36.0 food supplements. The Chilean regulation does not specify what Soybean 9.53–12.0 form of vitamin E should be used and limits its dose to a maximum of 500 mg (Ministerio de salud, 2002). On the other hand, the Ar- Sunflower 32.7–59.0 gentinian regulation allows up to 1,000 mg of vitamin E in food Walnut nd–3.80 supplements, expressed as α-tocopherol equivalent (Conal, 2014). However, neither of them mentions other phenolics compounds Wheat germ 151–192 nor brings a list of authorized health claims (Ministerio de salud, Source: Adapted from Shahidi and de Camargo (2016) and de Camargo et al. (2019). 2002; Conal 2014). nd, non-detected. The knowledge about bioactive substances, especially phenolic compounds, has been continuously evolving. Some aspects regard- position of methyl groups on the chromanol ring (Shahidi and de ing the bioavailability and bioaccessibility of many phenolics still Camargo, 2016). This phenolic class is well-known for its capacity are not completely elucidated, and that makes it hard to reach a to donate a hydrogen atom to stabilize free radicals (Kamal-Eldin consensus in order to establish lower and upper dose limits (Sha- and Appelqvist, 1996). Thus, they are widely used to protect oils hidi and Peng, 2018). The approach required for Anvisa to author- and lipid-rich foods against oxidation (Shahidi and de Camargo, ize an ingredient as part of food supplements requires that industry 2016; Kamal-Eldin and Appelqvist, 1996). The homologs have be capable of gathering as much scientific evidence as possible to different levels of vitamin E activity. α-Tocopherol has the high- petition for approval. However, changing the regulation in rela- est vitamin E activity due to the presence of α-tocopherol transfer tion to phenolic compounds should be a collective effort involv- protein (α-TTP) in the liver, which facilitates the absorption of this ing food legislators, academia, and manufacturers of supplement specific form (Kamal-Eldin and Appelqvist, 1996). ingredients. Tocopherols and tocotrienols (tocols) are mainly found in vegetable oils (Table 3). Wheat germ oil is the most abundant source of α-tocopherol (151–192 mg/100 g of oil) and β-tocopherol (31.2– 3. Conclusion 65 mg/100 g of oil). Sunflower oil is also rich in α-tocopherol (32.7–59 mg/100 g of oil), while palm and soybean oil, the most In summary, the inclusion of phenolic compounds in the revised produced vegetable oils worldwide, have significant amounts of version of the normative NI N° 28 demonstrates a natural evolu- γ-tocotrienol (11.3–36 mg/100 g of oil) and γ-tocopherol (61–69.9 tion. However, the number of approved compounds is still limited, mg/100 g of oil), respectively (Shahidi and de Camargo, 2016). and for the majority of them, the established upper limit doses may The different forms of vitamin E are associated with a variety be prohibitive for products available in the international market.

Journal of Food Bioactives | www.isnff-jfb.com 15 Phenolic compounds in food supplements regulation de Camargo et al.

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Journal of Food Bioactives | www.isnff-jfb.com 17 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Review J. Food Bioact. 2019;7:18–26

Anticancer and antiproliferative properties of food-derived protein hydrolysates and peptides

Ifeanyi D. Nwachukwua,b and Rotimi E. Alukoa,c* aDepartment of Food and Human Nutritional Sciences, 190 Dysart Road, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada bCenter for Nutrition and Healthy Lifestyles, School of Public Health, 24951 North Circle Drive, Loma Linda University, Loma Linda, CA 92350, USA cRichardson Centre for Functional Foods and Nutraceuticals, 196 Innovation Drive, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada *Corresponding author: Rotimi E. Aluko, Department of Food and Human Nutritional Sciences, 190 Dysart Road, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada. Tel: +1-204-474-9555; Fax: +1-204-474-7593; E-mail: [email protected] DOI: 10.31665/JFB.2019.7194 Received: September 12, 2019; Revised received & accepted: September 29, 2019 Citation: Nwachukwu, I.D., and Aluko, R.E. (2019). Anticancer and antiproliferative properties of food-derived protein hydrolysates and peptides. J. Food Bioact. 7: 18–26.

Abstract

Cancers of all types are among the four main non-communicable diseases, a category of diseases responsible for 38 million yearly deaths worldwide. Although various medical procedures including surgery, immunotherapy, radiation therapy, hormone therapy, stem cell transplant and chemotherapy have been used for decades in the treatment and control of cancer, current survival rates suggest that more definitive and effective treatment strategies are warranted. This work provides a succinct summary of the various methods used for producing anticancer peptides and protein hydrolysates from food sources, their modes of action, as well as descriptions of their antitumour properties in cellular and animal models. Although the mechanisms by which protein hydrolysates and peptides exert antitumor and antiproliferative effects are not entirely elucidated, there is evidence pointing to antioxidative function as an important predictor of their anticancer property.

Keywords: Antitumor; Lunasin; Cancer; Apoptosis; Bioactive peptides; Antiproliferative.

1. Introduction surgery and certain types of chemotherapy have been shown to adversely affect fertility and reproductive organs (Barton et al., 2013; According to data on the global cancer incidence and mortality Wasilewski-Masker et al., 2014). Furthermore, irritability, loss of burden released in 2014 by the WHO, about 8.2 million cancer- libido, hot flashes and night sweats have been reported in patients related fatalities and 14.1 million new cases of cancer (excluding undergoing hormone therapy, with certain types of hormone thera- non-melanoma skin cancer) were diagnosed worldwide in 2012 py increasing the potential for subsequent diagnosis with diabetes, (WHO, 2014). In spite of the promising advances made over the osteoporosis and obesity (Keating et al., 2010; Saylor and Smith, years in medical sciences in general, and in cancer treatment in 2013; Wadhwa et al., 2009). Studies have also shown that cancer particular, current strategies for the treatment of cancer remain survivors who underwent stem cell transplantation are prone to inadequate and unsatisfying in terms of treatment outcomes, as subsequently face the challenge of chronic anemia and recurrent well as short and long term health effects (Miller et al., 2016). For infections (Miller et al., 2016). Given cancer’s standing as a major instance, apart from aesthetic concerns and post-surgery negative cause of morbidity and mortality, which affects people in every body image, it has been reported that about 25–60% of women country and region as well as the expectation that the global cancer develop chronic pain following mastectomy (Miller et al., 2016; burden will surpass 20 million new cancer cases by 2025 (WHO, Vilholm et al., 2008). In both men and women, radiation therapy, 2014), the development of alternative and/or supplementary strate-

18 Copyright: © 2019 International Society for Nutraceuticals and Functional Foods. All rights reserved. Nwachukwu et al. Anticancer and antiproliferative properties gies for reducing the risk of cancer is critically needed. cycle arrest, inhibition of intracellular signaling systems, regulation The idea that cancer incidence and/or progression can be pre- of immune system, protease inhibition, and nucleic acid impairment vented, delayed, suppressed or reversed by lifestyle modification (Ortiz-Martinez et al., 2014; Rajendran et al., 2017). Apoptosis, the such as the administration or consumption of natural or biologi- carefully controlled and programmed death of cells is widely rec- cal (including dietary) substances with the capacity to enhance the ognized as one of the most effective means through which the body host organism’s defense mechanisms or limit exposure to and/or regulates cell death and division, with homeostatic maintenance of interaction with carcinogens is not new (Munjal et al., 2012; Stew- the appropriate number of cells (Indran et al., 2011). Anticancer ard and Brown, 2013). Studies have reported increased cancer risk protein hydrolysates and peptides are known to induce apoptosis in in subjects with lower intakes of fruits, vegetables, whole grains cancer cells by upregulating apoptotic gene expression as was ob- and red meat (Hoang et al., 2018; Levi et al., 1999; McCullough served with the enhancement of caspase-3, p21 and p53 expressions et al., 2003). Improved diet and other lifestyle changes like weight in MCF-7 human breast cancer cells by soybean-derived peptides management, increased physical activity and minimal or zero alco- (Park et al., 2009), as well as downregulating tumorigenic genes hol consumption have all been shown to lower the disease risk for as seen with the suppression of PTTG1 and TOP2A gene expres- cancer (McCullough et al., 2011). However, a considerable pro- sions by soybean-derived peptide fractions in HeLa cells (Robles- portion (37–87%) of cancer patients are known to take advantage Ramírez et al., 2012). Bioactive peptides and protein hydrolysates of alternative cancer therapies due to a number of reasons as pre- are also known to exert their anticancer effects by orchestrating viously summarized (Rajendran et al., 2017), including increased cellular DNA damage as was the case with common bean-derived patient awareness and access to information, the belief that “natu- peptide GLTSK whose impairment of DNA function was observed ral is safer”, the side effects of conventional drugs, and increased as the overexpression of the histone γH2AX in HCT116 human patient autonomy. Furthermore, apart from the adoption of specific colorectal cancer cells (Luna-Vital et al., 2016). The histone variant dietary habits and the increased consumption of certain classic nu- H2AX is known to be rapidly phosphorylated at the Ser-139 residue trients as a strategy for reducing cancer risk (Milner, 2002), there as a result of DNA double-strand breakage yielding γH2AX, whose is a growing body of evidence (Chatterjee et al., 2018; Daliri et al., formation is routinely used as a highly specific and sensitive indica- 2017; Hsu et al., 2011; Sheih et al., 2010; Suarez-Jimenez et al., tor of DNA damage (Luna-Vital et al., 2016; Mah et al., 2010). Ad- 2012) suggesting that food-derived protein hydrolysates and pep- ditionally, the polypeptide lunasin was reported to reduce KM12L4 tides could be important for reducing cancer risk thus explaining human colon carcinoma cells adhesion by disrupting α5β1 integrin the significant attention this area of research currently enjoys and and extracellular matrix interaction (Dia and González de Mejia, justifying the need for this work. 2011; Rajendran et al., 2017) and thus impair tumor progression. This is because integrin adhesion to extracellular matrix is critical for intra-tumor endothelial cell proliferation and migration (Con- 2. Food sources of anticancer peptides and potential- mecha coni et al., 2010). In an earlier study in which lunasin induced apop- nisms of action tosis in HT-29 human colon cancer cells by means of mitochondrial pathway activation and induction of nuclear clusterin expression, Dia and González de Mejia (2010) credited the anticancer property Food-derived bioactive peptides and protein hydrolysates with an- of the polypeptide to its RGD motif, which promotes adhesion and ticancer properties have been produced from a variety of animal internalization of lunasin into the cell through interaction with the and plant sources including soybean (Mora-Escobedo et al., 2009), extracellular matrix (Rajendran et al., 2017). Also, using molecular half-fin anchovy (Song et al., 2014), chickpea (Xue et al., 2012), docking studies, Wang et al. (2008) revealed the potential of three common bean (Luna-Vital et al., 2016), sea cucumber (Pérez-Vega soy bean-derived peptides (FEITPEKNPQ, IETWNPNNKP, and et al., 2013), tuna cooking juice (Hung et al., 2014), blood clam VFDGEL) to inhibit topoisomerase II. Other described mecha- muscle (Chi et al., 2015), walnut (Jahanbani et al., 2016), maize nisms of action include histone deacetylation, protease inhibition, (Ortiz-Martinez et al., 2017), mung bean (Gupta et al., 2018), membrane permeation and disruption, and calcium modulation as loach (You et al., 2011), fish protein (Picot et al., 2006), tunicate reviewed by Rajendran et al. (2017) and Sah et al. (2015). (Kim, 2011), as well as whey and casein (Sah et al., 2018). Table 1 contains additional sources of protein hydrolysates and peptides with anticancer properties. In general, these products are gener- 2.2. Food protein anticancer properties—the antioxidant con- ated from the enzymatic hydrolysis of food proteins to produce a nection complex matrix of peptides called protein hydrolysate. The hydrolysates are usually composed of active and non-active peptides; Reactive oxygen species (ROS) such as hydroxyl radicals, hydro- therefore, further processing to obtain fractions that are enriched gen peroxide, singlet oxygen and superoxide anions are typically with active fractions may be carried out (Doyen et al., 2011; Pere- produced as a result of endogenous and exogenous stimuli, and go et al., 2011). In some cases, the active peptide fractions are sub- are often routinely neutralized by living organisms using well- jected to additional separation and purification protocols that yield established endogenous antioxidant defense systems (Nwachukwu homogenous peptides suitable for amino acid sequence analysis and Aluko, 2019). When produced in excess, ROS can overwhelm (Hung et al. 2014; Ma et al. 2015; Pan et al., 2016; Wang and natural defense systems resulting in a state of oxidative stress. Sus- Zhang, 2017; Wang et al., 2013; You et al., 2011). tained and cumulative oxidative stress has the potential to cause deleterious oxidative damage to cellular macromolecules such as proteins, lipids and nucleic acids resulting in irreversible altera- 2.1. Anticancer peptides and hydrolysates: mechanisms of tion of cellular functions (Fuchs-Tarlovsky, 2013; Nwachukwu action and Aluko, 2019). In the case of oxidative damage to DNA for instance, ROS can react with cellular components such as phos- Food-derived protein hydrolysates and peptides are believed to pholipids and proteins to form secondary reactive intermediates, modulate their anticancer functions through a number of well delin- which can irreversibly bind to DNA bases to form DNA adducts eated mechanisms of action including apoptosis induction and cell (Marnett, 2000). Since DNA adducts can promote miscoding and

Journal of Food Bioactives | www.isnff-jfb.com 19 Anticancer and antiproliferative properties Nwachukwu et al. Park et et Park al., 2009 Kim et al., Kim et 2000 Dia and Mejia, 2010 González González de Mejia et al, 2010 Robles- et Ramírez al., 2012 Ortiz- et Martinez al., 2017 Xue et al., et Xue 2012 Gupta et et Gupta al., 2018 Wang and Wang Zhang, 2017 Hung et Hung et al., 2014 References Apoptosis via p21, p53, and Apoptosis and expression, caspase-3 and HSP90 inhibition of VEGF Not investigated Activation of mitochondrial of mitochondrial Activation and increased pathways induction of pro-apoptotic expression nuclear clusterin Activation of Caspase-3 Activation and dose-dependent arrest G2 cell cycle Apoptosis induction Apoptosis regulation and down and TOP2A of PTTG1 genes tumorigenic Apoptosis induction, Apoptosis inhibition cell growth Tumour suppression Tumour Tumour suppression Tumour Tumour suppression Tumour Apoptosis induction and Apoptosis tumour suppression Mechanism value 50 Not stated 0.16 mg/mL 61.7 µM 14 µM 1.83 mg/mL 1.5–150 µg/ peptide mL for and fractions 0.001–1.5 mM derived for peptides. Not mentioned 0.32, 0.73 and 0.45 mg/mL for MCF-7; 0.26, 0.48 and 0.54 mg/ml MDA- for MB-231 cells 99.88 µg/mL 1.39 mg/mL IC MCF-7 P388D1 HT-29 colon colon HT-29 cells cancer L1210 leukemia cells L1210 leukemia HeLa cells Hepatocarcinoma Hepatocarcinoma human HepG2 cells Tumour-bearing mice Tumour-bearing MCF-7 and MDA- MB-231 breast cells cancer HT-29 cancer cells cancer HT-29 Human breast cancer cancer Human breast MCF-7 cells and MCF-10A mammary epithelial cells Cellular and/or Cellular and/or animal model Sequential hydrolysis hydrolysis Sequential with endopeptidase, and exopeptidase order in that amylase Thermoase and extraction ethanol Lunasin obtained by by Lunasin obtained from aqueous extraction by followed soybean and size ion exchange chromatography, exclusion and further purified separation HPLC by Simulated GIT digestion GIT digestion Simulated and pancreatin with pepsin Extraction, precipitation, precipitation, Extraction, and hydrolysis enzymatic fractionation membrane Alcalase hydrolysis hydrolysis Alcalase ion exchange by followed chromatography column fractionation Alcalase hydrolysis Alcalase Ammonium sulfate Ammonium sulfate of vicilin fractionation separate by followed with hydrolyses and trypsin alcalase Enzymatic hydrolysis hydrolysis Enzymatic and fractionation Enzymatic hydrolysis hydrolysis Enzymatic XXIII with Protease and ultrafiltration Production Method Production Isoflavone-derived soy Isoflavone-derived XMLPSYSPY Lunasin Lunasin Peptide fractions Peptide Peptides Protein hydrolysates Protein Protein hydrolysates and hydrolysates Protein vicilin seed extracts HVLSRAPR KPEGMDPPLSEPEDRRDGAAGPK, KPEGMDPPLSEPEDRRDGAAGPK, KLPPLLLAKLLMSGKLLAEPCTGR Protein hydrolysates/Peptide Protein Soybean Soybean Soybean Soybean Soybean Soybean (germinated) Maize Chickpea Mung bean vicilin Peptides from from Peptides Spirulina platensis Tuna cooking cooking Tuna juice Source Table 1. Sources, methods of production and mechanisms of action of anticancer food-derived protein hydrolysates and peptides* and hydrolysates protein food-derived anticancer and mechanisms of action of production methods 1. Sources, Table

20 Journal of Food Bioactives | www.isnff-jfb.com Nwachukwu et al. Anticancer and antiproliferative properties Pan et al, et Pan 2016 Song et Song et al., 2011 Hsu et al., Hsu et 2011 You et al., et You 2011 Ding et Ding et al, 2011 Lin et al., Lin et 2009 Chang et Chang et al, 2011 Ma et al., Ma et 2015 Barrio and Añón, 2010 Xue et al, et Xue 2010 Wang et et Wang al., 2008 Kobayashi Kobayashi al, 2004 et References Apoptosis and tumour Apoptosis suppression Cell proliferation inhibition Cell proliferation Inhibition of cell viability and proliferation Inhibition of cell proliferation Inhibition of cell viability and growth Inhibition of cell growth Inhibition of cell growth and cell lysis Downregulation of Downregulation (IL)-6, IL-8, IL-12, interleukin Bcl-2 IL-15, interferon-γ, as as well and caspase-7, of IL-2 and IL-8 upregulation Induction of apoptosis Induction of apoptosis and autophagy Inhibition of cell adhesion, and apoptosis necrosis DNA structural damage damage DNA structural induction and apoptosis Human topoisomerase Human topoisomerase II inhibition Inhibition of the upregulation Inhibition of the upregulation of urokinase-type uPA, activator, plasminogen uPAR receptor, specific its and Mechanism - (continued) value 50 4.81 mg/mL 13.67, 25.17 and 40.28 mg/ mL respectively 8.1 µM and 8.8 µM respectively Not stated Variable Not stated Not stated 0.60 and 0.65 mg/mL respectively 1.0, 1.5 and 2.5 and 2.5 mg/mL respectively Not stated 7.9, 4.0 and 2.4 mM in order that 2–3 µM IC HeLa DU-145 prostate, DU-145 prostate, 1299 lung, and 109 esophagus cells cancer MCF-7 HepG2, MCF-7 cells and Caco-2 DU-145 prostate DU-145 prostate cells cancer A549, HepG2, HT1080, U937, NIH3T3, RAW264.7 cells cancer HeLa, HepG2, HT1080 Human HeLa cervical and MCF-7 cancer cells cancer breast UMR106, Caco-2, UMR106, Caco-2, MC3T3E1 TC7, HeLa L1210 HRA human ovarian HRA human ovarian cells cancer Cellular and/or Cellular and/or animal model Enzymatic hydrolysis hydrolysis Enzymatic trypsin with pepsin, and chymotrypsin Peptic hydrolysates heated heated hydrolysates Peptic 95 °C 10 min at for Papain and protease XXIII and protease Papain Loach tissue followed homogenization papain hydrolysis by Trypsin Chemically synthesized Chemically Chemically synthesized Chemically Alkaline extraction, extraction, Alkaline by precipitation and centrifugation with papain hydrolysis Alcalase Alcalase and flavourzyme Alcalase Pepsin and pancreatin Pepsin Production Method Production FIMGPY Heated products from peptic peptic from products Heated of marine hydrolysates anchovy) fish (half-fin LPHVLTPEAGAT PTAEGGVYMVT LPHVLTPEAGAT Loach peptide fractions Loach peptide An oligopeptide, N Gln-Pro-Lys An oligopeptide, Epinecidin-1 Tilapia hepcidin (TH) 1–5 Cys-Thr-Leu-Glu-Trp Mantegazzianus peptide isolate peptide Mantegazzianus Rapeseed peptide VFDGEL, IETWNPNNKP and FEITPEKNPQ Kunitz trypsin inhibitor Kunitz Protein hydrolysates/Peptide Protein )

) ) Skate ( Raja Skate porosa protein cartilage hydrolysate Loach ( Misgurnus anguillicaudatus Setipinna taty Setipinna Tuna dark muscle Tuna byproduct Cuttlefish ink Cuttlefish ) ( Sepia esculenta Estuary cod cod Estuary ( Epinephelus coioides Tilapia Walnut (seed) Amaranthus Amaranthus mantegazzianus Rapeseed Soybean Soybean Source Table 1. Sources, methods of production and mechanisms of action of anticancer food-derived protein hydrolysates and peptides* and hydrolysates protein food-derived anticancer and mechanisms of action of production methods 1. Sources, Table

Journal of Food Bioactives | www.isnff-jfb.com 21 Anticancer and antiproliferative properties Nwachukwu et al. Wang et et Wang al., 2013 Castro et et Castro al, 2009 Perego et et Perego al, 2011 Mader et Mader et al, 2006 Jang et Jang et al., 2008 Naqash and Nazeer, 2012 Kim, 2011 Alemán et Alemán et al., 2011 Doyen et et Doyen al., 2011 Kannan et et Kannan al., 2010 Picot et et Picot al., 2006 References Bcl-2 downregulation, Bcl-2 downregulation, and inhibition apoptosis of cell proliferation Apoptosis (increased (increased Apoptosis expression) caspase-3 and necrosis Apoptosis Inhibition of angiogenesis Inhibition of cell viability Inhibition of cell viability Not mentioned Not stated Not mentioned Not mentioned Inhibition of cell growth Inhibition of cell growth and proliferation Mechanism - (continued) value 50 Not stated 0.19–156.9 mg/mL Not mentioned Not stated Not mentioned 61.1 µg/mL 577.1–1,240.0 µg/mL 130 and 100 µg/mL for MCF-7 and U87 respectively Not mentioned Not mentioned Not stated IC SKOV-3 B1F610 HT-29 intestinal intestinal HT-29 tumour cells Human umbilical Human umbilical endothelial cells vein MCF-7, AGS, A549 MCF-7, AGS, HepG2 liver HepG2 liver cells cancer AGS, DLD-1, HeLa AGS, MCF-7, U87 A549, HCT15, PC3 BT549, Caco-2, HepG2, Caco-2, MCF-7, HCT-116, MDA-MB-231 MCF-7/6, MDA- MB-231 and breast cells cancer Cellular and/or Cellular and/or animal model Membrane ultrafiltration ultrafiltration Membrane fractionation and RP-HPLC Chemically synthesized Chemically Synthetic Extraction with phosphate with phosphate Extraction by followed buffer with thermolysin hydrolysis A, and proteinase K, proteinase trypsin, papain, pepsin, tyrosinase, (sequentially) protease 4 h hydrolysis with 4 h hydrolysis 37 °C at trypsin Alcalase, Thermoase Alcalase, pepsin, PC10F, Protamex, trypsin, trypsin, Protamex, Savinase, Neutrase, NS37005, Esperase, Alcalase Enzymatic hydrolysis with hydrolysis Enzymatic electrodialysis, Protamex, ultrafiltration Gastrointestinal juice Gastrointestinal pH-shift extraction extraction pH-shift Protamex by followed hydrolysis and alcalase Production Method Production )

PGPIPN immunomodulatory PGPIPN immunomodulatory peptide Whey protein isolate and isolate protein Whey hydrolysate collagen Casein phosphopeptide LfcinB FKCRRWQWRMK FKCRRWQWRMK LfcinB KLGAPSITCVRRAF Gly-Phe-His-Ile, Asp-Phe-His- Gly-Phe-His-Ile, Phe-His-Asp, and Ile-Asn-Gly, Gly-Leu-Ser-Asp-Gly-Glu-Trp-Gln Nemipterus japonicus Nemipterus tryptic hydrolysates tryptic Solitary tunicate ( Styela clava tunicate Solitary hydrolysates enzymatic Squid gelatin protein protein Squid gelatin hydrolysates KCl2 cationic peptide cationic KCl2 Alacalase hydrolysis followed followed hydrolysis Alacalase GIT digestion simulated by Fish protein hydrolysates Fish protein Protein hydrolysates/Peptide Protein Bovine milk Bovine Bovine milk Bovine Milk Bovine Bovine lactoferrin Beef Beef sarcoplasmic protein hydrolysates Japanese bream threadfin Rough squirt/ sea Rough tunicate solitary Squid gelatin Snow crab crab Snow (by-products) Rice bran Blue whiting Plaice, cod, Salmon Atlantic Source Table 1. Sources, methods of production and mechanisms of action of anticancer food-derived protein hydrolysates and peptides* and hydrolysates protein food-derived anticancer and mechanisms of action of production methods 1. Sources, Table U87-glioma; HT1080–fibrosarcoma; fibroblast; NIH3T3–mouse cancer; DU-109–esophageal cancer; DLD-1–colon TC7, HCT-116, Caco-2, cancer; HeLa–cervical cancer; BT549–breast MDA-MB-231, MCF-7, MCF-6, Key: lines *Cell L1210- lymphocytic HepG-2–liver leukemia; cancer; A549, DU-1299–lung HT-29,cancer; P388D1-mouse U937–histiocytic monocyte lymphoma; macrophage cell RAW264.7 line; (mouse)–macrophage; MC3T3E1–osteoblastic B1F610-melanoma cells; SKVO-3–ovarian C33–epidermoid cervical carcinoma; cancer; HRA–human ovarian adenocarcinoma; cells; AGS–stomach osteosarcoma-derived UMR106–rat cells; DU-145–prostate; calvaria-derived endothelial cells. vein umbilical cell; HUVEC–Human cancer

22 Journal of Food Bioactives | www.isnff-jfb.com Nwachukwu et al. Anticancer and antiproliferative properties even mutations if they evade cellular repair mechanisms, their for- mon bean (Phaseolus vulgaris L.) showed that the two most potent mation is important in the carcinogenic process (Fuchs-Tarlovsky, peptides (GLTSK and GEGSGA) selectively inhibited the prolif- 2013). Studies have linked oxidative stress with the pathogenesis eration of HCT116 colon cancer cells but not that of CCD-33Co of inflammation-related cancers, and agents with the capacity to normal colon cells (Luna-Vital et al., 2016). While the inhibition protect cells against ROS attack by quenching free radicals, are of HCT116 cells by GLTSK was thought to be through loss of thought to be potent chemopreventive candidates (Chi et al., 2015; mitochondrial membrane potential, GEGSGA peptide suppressed Sheih et al., 2010). In fact, the correlation of antioxidative func- the proliferation of the cancer cells via DNA damage. Of particu- tion with anticancer property has traditionally been reported in a lar importance was the finding that when combined, either peptide range of biological and chemical agents, including dietary kelp induced apoptosis and synergistically enhanced the effect of the and plant phenolics (Cai et al., 2004; Dai and Mumper, 2010; chemotherapy drug oxaliplatin on HCT116 cells—a result that Maruyama et al., 1991). Against this backdrop, peptides with could have wide implications for peptide use in cancer combina- antioxidative functions have been shown to also possess antican- tion therapy. Confocal microscopy data also revealed that when cer properties. For example, an antioxidant peptide fraction from combined with oxaliplatin, GEGSGA promoted PARP cleavage, algae protein waste was found to induce cell cycle arrest in and decreased the levels of antiapoptotic Bcl-2 and caused the activa- dose-dependently inhibit the growth of AGS human gastric can- tion and nuclear translocation of p53 protein (Luna-Vital et al., cer cells (Sheih et al., 2010). Additionally, the anticancer activity 2016). The antioxidant peptide, WPP, from blood clam protein of protein hydrolysates and peptides with antioxidative properties hydrolysates was shown to selectively inhibit the proliferation of such as the oyster-derived LANAK (Umayaparvathi et al., 2014), PC-3 and DU-145 human prostate cancer cells as well as HeLa and a fresh water fish-derived peptide fraction, LPH-IV (You et al., H1299 non–small-cell lung cancer cell lines while showing hardly 2011), and H3 (a polypeptide with 117 amino acid residues) from any cytotoxicity to normal NIH3T3 mouse fibroblast cells (Chi et the marine invertebrate, Arca subcrenata (Chen et al., 2013) have al., 2015). WPP also induced apoptosis in PC-3 cells with classical also been demonstrated. Due to the proven capacity of antioxidants apoptotic changes in morphology such as chromatin condensation, to protect healthy cells from oxygen-based radicals during cancer cytoplasmic blebbing and nuclear fragmentation being observed in therapy and the use of antioxidant compounds in combination with the cells (Chi et al., 2015). anticancer drugs such as doxorubicin and anthracycline (Fuchs- Also, using an MTT-based spectrophotometric assay, a recent Tarlovsky, 2013), antioxidant peptides hold great promise for simi- study found that alcalase and trypsin protein hydrolysates from lar chemotherapeutic applications. mung bean vicilin inhibited the proliferation of human breast cancer cell lines MCF-7 and MDA-MB-231 (Gupta et al., 2018). Addi- tional studies have also demonstrated the anticancer effects of food- 3. Ex-vivo anticancer protein hydrolysates and peptides derived protein hydrolysates and peptides in cellular models such as the cytotoxic effects of walnut protein hydrolysates on human The antitumor and/or antiproliferative capacity of food-derived breast (MDA-MB231) and colon (HT-29) cancer cells (Jahanbani et protein hydrolysates and peptides have been amply demonstrated al., 2016), sea cucumber hydrolysates and peptide fractions on HT- in various cancer cell models, using hydrolysates and peptides 29 colorectal cancer cells (Pérez-Vega et al., 2013), and tuna dark that vary in both their sources and chemical structures. Lunasin, muscle hydrolysates and peptides (LPHVLTPEAGAT and PTAE- the naturally-occurring 43-amino acid peptide first identified in GGVYMVT) on MCF-7 human breast cancer cell line (Hsu et al., soybean was found to induce apoptosis and cause cytotoxicity 2011). Other studies include algae protein-derived peptide (VE- to HT-29 human colon cancer cells (Dia and González de Mejia, CYGPNRPQF) on AGS human gastric adenocarcinoma cell model 2010). In a recent study, Ortiz-Martinez et al. (2017) reported the (Sheih et al., 2010), germinated soybean protein hydrolysates on antiproliferative effect of peptide fractions from albumin alcalase HeLa and C-33 cervical cancer cells (Mora-Escobedo et al., 2009), hydrolysates of a white hybrid maize (Asgrow-773) and a quality walnut protein hydrolysates on UACC-62 melanoma cells (Carrillo protein (CML-502) maize as well as that of their derived peptides et al., 2017), and rice bran protein hydrolysates on human colon on HepG2 cells, an in vitro model of human liver cancer. The pep- (Caco-2) and liver (HepG2) cancer cells (Kannan et al., 2008). The tide fractions from both maize samples were found to inhibit the effect of processing on the anticancer property of food-derived pro- growth of HepG2 cells in a dose-dependent manner by up to 94% tein hydrolysates has also been studied. Song et al. (2011) showed and to increase the rate of apoptosis induction in HepG2 cells by a that the heated products of half-fin anchovy peptic hydrolysates had staggering 400% on the average. Unlike peptide fractions obtained stronger antiproliferative effects on DU-145, H1299 and ECA-109 following the alcalase hydrolysis of corn gluten meal, which were human esophageal cancer cells than the unheated samples. Further shown to block HepG2 DNA replication in the S phase of the cell studies with peptide fractions and a purified peptide (YALPAH) cycle in a different study (Li et al., 2013), the maize peptide frac- from the heated (121 °C for 30 min) protein hydrolysate products tions in this study had no modulatory effect on HepG2 cell cycle showed potent antiproliferative effects on PC-3 cancer cells and the (Ortiz-Martinez et al., 2017). The study by Li et al. (2013) also induction of apoptosis by YALPAH (Song et al., 2014). found that corn peptides induced apoptosis in HepG2 cells in a dose-dependent manner by significantly inhibiting the expression of antiapoptotic Bcl-2 protein and upregulating the expression of 4. Animal studies p53 and cleaved caspase-3. It has also been reported that apoptosis induction via the downregulation of Bcl-2, PARP and caspase-9 The anticancer properties of food-derived peptides and protein levels as well as the upregulation of p53, Bax and cleaved cas- hydrolysates have been evaluated in animal models and reported pase-3 expressions was central to the antiproliferative effects of by various researchers. In one study where the hepatic carcinoma tuna cooking juice protein hydrolysates and ultrafiltration peptide cell line H-22 was subcutaneously injected into Chinese Kun Ming fractions on MCF-7 human breast cancer cells (Hung et al., 2014). (KM) mice, the administration of chickpea albumin hydrolysates In addition, a study which evaluated the effect of five pure pep- to the mice by oral gavage was found to significantly increase tide sequences derived from the non-digestible fraction of com- tumor inhibition rate and decrease tumor volume after a 12-day

Journal of Food Bioactives | www.isnff-jfb.com 23 Anticancer and antiproliferative properties Nwachukwu et al. treatment (Xue et al., 2012). In addition, chickpea albumin hydro- enzyme (ACE)-inhibitory peptides were found in plasma collected lysate also significantly increased the proliferation of spleen lym- from human subjects who had consumed a peptide-enriched drink phocytes and enlargement of splenic volume thus suggesting that (van Platerink et al., 2006). In another study, MALDI-MS imag- administration of the hydrolysate enhanced the immune system ing analysis revealed that Trp-His, an anti-atherosclerotic dipep- and increased the weight of the immune organ. This is particularly tide administered by oral gavage to Sprague-Dawley rats, was noteworthy given the historical dilemma of administering anti-tu- absorbed intact into the systemic circulation due to its selective mor drugs and contending with the concomitant impairment of the transport across the rat intestinal epithelium by the peptide trans- immune system (Xue et al., 2012). In H-22 tumor-bearing BALB/c porter, PepT1 (Tanaka et al., 2015). Recently, our work also report- mice, corn peptide fractions dose-dependently suppressed tumor ed the detection in blood plasma of certain calmodulin-dependent growth and enhanced spleen volume while upregulating the level phosphodiesterase (CaMPDE)-inhibitory peptides administered of serum cytokines IL-2, IFN-γ and TNF-α in a concentration-de- by oral gavage to adult Wistar rats and absorbed intact across the pendent manner (Li et al., 2013). Furthermore, the cationic and an- intestinal epithelium (Nwachukwu et al., 2019). timicrobial peptide lactoferricin was found to extend the survival of immune-deficient SCID/beige mice inoculated with Ramos human B-lymphoma cells (Furlong et al., 2010). After B-lymphoma 5. Conclusions cell inoculation via the tail vein and subsequent intraperitoneal injection of bovine lactoferricin, adult mice were weighed and The common practice of grouping cancer types according to the examined daily for signs of distress while hind limb paralysis as a result of B-lymphoma cell dissemination to the central nervous major anatomical sites affected in various reports of global mortal- system was taken as a measure of survival (Furlong et al., 2010). ity tends to downplay cancer’s standing as a leading cause of death Bovine lactoferricin is an amphipathic, 25-amino acid polypeptide (WHO, 2014). It is arguably the top and most widespread cause of obtained following peptic digestion of the iron-binding protein, death as it affects populations in all countries and all regions of the lactoferrin in cow milk. It is thought that bovine lactoferricin binds world. The studies reviewed in this work represent ample proof of to negatively charged structures on cancer cells and then disrupts the promising potential of food-derived protein hydrolysates and the membrane of those cells by inserting its bulky hydrophobic peptides to function as anticancer agents. Given that these bioac- amino acid residues into the phospholipid bilayer of the cell mem- tive agents are not drugs and are therefore, not designed for use in branes (Furlong et al., 2010; Hoskin and Ramamoorthy, 2008). In acute conditions, their consumption as part of the daily diet or as an earlier study, the antitumour effect of bovine lactoferricin on components of nutraceuticals and/or functional foods should be mice neuroblastoma xenograft tumors was shown (Eliassen et al., geared towards health promotion and prevention of cancer. Since 2006). The results indicated that tumors from rats treated with the over 60% of global cancer cases and 70% of cancer deaths occur polypeptide and weighed at autopsy had significant reductions in in the low-income countries of Africa, Asia, and Central and South weight compared with those from control subjects. Furthermore, in America (WHO, 2014), which typically have poorly developed a different study Eliassen et al. (2006) studied the effects of P2, a healthcare systems, the gains of an early and habitual adoption of polypeptide fraction from Arca subcrenata, on KM mice subcuta- bioactive peptides and functional protein hydrolysates as part of a neously inoculated with S-180 sarcoma or H-22 hepatoma tumors regular diet cannot be overemphasized. In addition, given the use and reported significant reductions in tumor weights. P2 reduced of antioxidants in cancer treatment, future studies should focus on tumor weight by up to 60% in S-180 tumor-bearing mice and 46% the prospect of utilizing antioxidant bioactive peptides in cancer in H-22 tumor-bearing mice at a dosage of 63 mg/kg/day. Also, combination therapy. using C57BL/6J mice subcutaneously inoculated with B16F10 melanoma cells, the cationic peptide INKKI isolated from bovine β-casein was tested for antitumor activity (Eliassen et al., 2006). Acknowledgments Results showed that following peritumoral injection of the peptide, INKKI-treated tumor-bearing mice had significantly reduced tu- The authors acknowledge support of the Natural Sciences and En- mor volume (up to 72%) and decreased metastasis loci in compari- gineering Council of Canada (NSERC), funding reference number son with the untreated control. Furthermore, the peptide treatment RGPIN-2018-06019. Cette recherche a été financée par le Con- led to a significant delay in tumor growth and doubling time (Elias- seil de recherches en sciences naturelles et en génie du Canada sen et al., 2006). Lastly, it was reported that female Sprague-Daw- (CRSNG), numéro de référence RGPIN-2018-06019. ley rats with 7,12-dimethylbenz[α]anthracene (DMBA)-induced mammary tumorigenesis, which received 3.3 g soy peptide daily as part of their diet, had significantly reduced (up to 50%) incidence Conflict of interest of ductal carcinomas (Eliassen et al., 2006). Additionally, the soy peptide (XMLPSYSPY) also induced apoptosis and the expression of p21, p53, and caspase-3 proteins, significantly reduced the num- The authors declare no conflict of interest. ber of tumors per mice, the weight of ductal carcinomas, and also extended the latency period of tumor development when compared References to the control (Eliassen et al., 2006). Importantly, the soy peptide used in this study was purified to be isoflavone-free. Questions surrounding the continued efficacy of orally-ingested bioactive Alemán, A., Pérez-Santín, E., Bordenave-Juchereau, S., Arnaudin, I., peptides in the face of the degradative action of digestive enzymes Gómez-Guillén, M.C., and Montero, P. (2011). Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity. as well as their absorbability and bioavailability have been duly Food Res. Int. 44(4): 1044–1051. answered by studies showing the detection of intact peptides in Barrio, D., and Añón, M. (2010). Potential antitumor properties of a pro- blood circulation following oral gavage (van Platerink et al., 2006; tein isolate obtained from the seeds of Amaranthus mantegazzianus. Foltz et al., 2007; Tanaka et al., 2015; Matsui, 2018; Nwachukwu Eur. J. Nutr. 49(2): 73–82. et al., 2019). In one study, up to 17 small angiotensin converting Barton, S.E., Najita, J.S., Ginsburg, E.S., Leisenring, W.M., Stovall, M.,

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

Review J. Food Bioact. 2019;7:27–35

UF-LC-DAD-MSn for discovering enzyme inhibitors for nutraceuticals and functional foods

Li Lia and Rong Tsaob* aThe College of Chemistry, Changchun Normal University, Changchun 130032, China bGuelph Research and Development Center, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, N1G 5C9 Canada *Corresponding author: Rong Tsao, Guelph Research and Development Center, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, N1G 5C9 Canada. Tel: +1 226-217-8108; Fax: +1 226-217-8183; E-mail: [email protected] DOI: 10.31665/JFB.2019.7195 Received: September 29, 2019; Revised received & accepted: September 30, 2019 Citation: Li, L., and Tsao, R. (2019). UF-LC-DAD-MSn for discovering enzyme inhibitors for nutraceuticals and functional foods. J. Food Bioact. 7: 27–35.

Abstract

The technique of ultrafiltration liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UF-LC-MSn) in screening enzyme inhibitors has emerged as an efficient tool for high-throughput screening of bioactives. The mini-review discusses about the principles of the UF-LC-MSn method and its applications in the discovery of bioactives in herbs and foods that are specific inhibitors of important proteins/enzymes related to human health. The advantageous and disadvantages of the method is critically assessed, and its potential use in functional food and nutraceutical research is addressed.

Keywords: Ultrafiltration; LC-MS; High-throughput screening; Enzyme inhibitors; Herb extracts; Food bioactives.

1. Introduction function, arthritis and fever (Chandrasekharan and Simmons, 2004; Yokoyama and Tanabe, 1989). It is also a key enzyme responsible for the production of inflammatory mediators prostaglandins The pharmacological activity of many drugs or food bioactives (PG) and their metabolites, and has been associated with pro-in- comes from the interactions between small bioactive molecules and flammatory activities that are related to several acute and chronic biological targets e.g. macromolecules like proteins in vivo. Exist- diseases. α-Glucosidase is closely related to type 2 diabetes (van de ing studies have provided convincing evidence that enzymes are Laar et al., 2005), which has become a worldwide health problem. major biomarkers in many pathological processes, which are attrac- α-Glucosidase inhibitors are widely used in the treatment of type 2 tive targets for new drug discovery and for understanding the rela- diabetes, and many dietary components including polyphenols have tionship between diet and health. Recent surveys found that nearly been found to exhibit excellent α-glucosidase inhibitory activities, half of all the marketed small-molecule drugs are enzyme inhibitors thus play significant role in dietary intervention for metabolic syn- (Jin et al., 2016; Newman and Cragg, 2007; Wang et al., 2011). dromes like type 2 diabetes(Wang et al., 2014; Zhang et al., 2015; Many enzymes play critical roles in non-communicable chronic Tang et al., 2016). Xanthine oxidase (XOD) can generate reactive diseases. For example, matrix metalloprotease-2 (MMP-2) is key oxygen species and catalyze the metabolism of hypoxanthine and enzyme involved in cancer progression, tissue remodeling, bone xanthine to produce uric acid, the over production of which could remodeling, wound healing and several aspects of immunity (Sza- cause hyperuricemia, a risk factor for gout (Lin et al., 2000; Liu boa et al., 2004). 5-Lipoxygenase (5-LOX) is an important enzyme et al., 2012a; Wang et al., 2014). The angiotensin-converting en- in leukotriene biosynthesis and catalysis, and is involved in sev- zyme (ACE) converts the hormone angiotensin I to angiotensin II eral pathological processes related to pancreatic cancer, rheumatoid which is the active vasoconstrictor controlling the blood pressure arthritis and inflammatory reactions (Ding et al., 2011; Schneider via regulating the volume of fluids in the body. As a result, ACE and Bucar, 2005; Steinhilber, 1994). Cyclooxygenase-2 (COX-2) indirectly increases blood pressure. ACE inhibitors are widely used is essential for the maintenance of the gastrointestinal tract, renal as pharmaceutical drugs for treatment of cardiovascular diseases,

Figure 1. Typical procedures of the ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS) method. and many food-originated inhibitors of ACE have been found to tration with different molecular cut-offs has been used for isolation exhibit therapeutic value in the prevention of hypertension (Inoue et and purification of macromolecules (He et al, 2013). However in al., 2011). Acetylcholinesterase (AChE) is an acetylhydrolase that the last few years, this technology has been coupled with liquid catalyzes the breakdown of acetylcholine, one of the most neuro- chromatography-diode array detector-mass spectrometry (LC- transmitters. AChE inhibitors prevent the hydrolysis of acetylcho- DAD-MS) for high-throughput screening and discovery of enzyme line into choline and acetate, thus increase both the level and dura- inhibitors from herb extracts and plant foods (Cao et al., 2010; Choi tion of action of the neurotransmitter acetylcholine in the central et al., 2011; Li et al., 2014; Li et al., 2015; Li et al., 2009; Liu et al., nervous system, which is considered beneficial to alleviating Alz- 2013; Liu et al., 2013; Liu et al., 2012a, b; Liu et al., 2011; Shi et heimer’s disease. Phenolics such as cinnamic acids and flavonoids, al., 2013; van Breemen et al., 2011; Wang et al., 2014; Wang et al., and other phytochemicals such as ginsenosides have been found to 2014; Yang et al., 2012; Zhao et al., 2016; Zhang et al., 2012; Shi et inhibit AChE activity and (Zhang et al., 2018; Yang et al., 2019). al., 2010; Zhou et al., 2012 ). In ultrafiltration LC-DAD-MS (UF- Other enzymes have been known for their role in tumour formation LC-MS) experiments, when a mixture of compounds (ligands) is and cancer. Quinone reductase-2 (QR-2) is a cytosolic enzyme in- in contact with the target enzyme (a macromolecular receptor), the volved in several possible mechanisms of action including antima- ultrafiltration membrane retains the macromolecules and the ligand- larial and antitumor activities (Fu et al., 2005; Kwiek et al., 2004), receptor complexes, but allows unbound, low molecular weight and aromatase represents an important target for the treatment of compounds to pass through. Bound ligands are then dissociated by hormone dependent breast cancer (Kendall and Dowsett, 2006). disrupting the ligand-receptor complex with an organic solvent or Another target of antiviral agents is neuraminidase (NA) which is changing pH. The released ligands in the organic solvent are then a glycoside hydrolase enzyme that breaks the glycosidic linkages analyzed and identified on-line using LC-DAD-MSn (Johnson et of neuraminic acids. Inhibitors of neuraminidase are particularly al., 2002) (Figure 1). In UF-LC-MSn, the tandem mass spectromet- important drugs against influenza infection (Meanwell and Krys- ric fragmentation pattern of compounds has been studied and used tal, 1996). Discovering new inhibitors of these important enzymes extensively in the characterization of unknown compounds. This is from medicinal and functional foods is a good practice although especially useful when standard references are not available (Cuy- there are challenges. Synthetic enzyme inhibitors are mostly phar- ckens and Claeys, 2004; 2005). UF-LC-MSn is a high throughput maceutical drugs that can ameliorate symptoms of various diseases, screening and identification technique for active compounds, and is however, they can also cause severe side effects as have been found advantageous in low sample requirement and reuse of enzymes, and in clinical applications in recent years (Borges et al., 2002; Gnant, it does not involve any enzyme immobilization process (Zhou et 2006; Kitching et al., 2009; Nelson et al., 2000; van de Laar et al., al., 2012) (Figure 1). More importantly, UF-LC-MSn can effectively 2005). For this reason, increasing attention has been given to resolve one of the mostchallenging problems in functional foods and searches targeting on naturally occurring enzyme inhibitors such as nutraceuticals research, i.e. thecompound with the highest concen- those derived from herb or food extracts. However, screening and tration in a particular food or its extract is often assumed as the identifying active constituent(s) by conventional methods are time most active component, yet in many cases, minor components of consuming and inefficient (Wen et al., 2007; Shi et al., 2013). Rapid a mixture may be more bioactive when compounds are purified. and effective screening strategies must therefore be developed for There lacks information on the bioactivity of individual compounds identifying inhibitors from complex mixtures. Membrane ultrafil- of mixtures.

28 Journal of Food Bioactives | www.isnff-jfb.com Li et al. UF-LC-DAD-MSn for discovering enzyme inhibitors

Table 1. Major MMP-2/5-LOX inhibitors identified in plant extracts by UF-LC-MS Relative binding to enzymeb IC d (µM) Compounds MWa 50 Source Referencese MMP-2c 5-LOXc MMP-2 5-LOX Hydroxyl safflower yellow A 612 2.01 5.76 137.3 3.72 C. tinctorius Li et al. 2014; Zhao et al. 2016 Anhydrosafflor yellow B 1,062 16.29 22.93 2.82 3.45 Astilbin 450 14.44 40.78 107.8 8.45 S. glabra Li et al. 2015; Zhao et al. 2016 Isoastilbin 450 - 4.30 - 46.6 Engelitin 434 21.14 5.07 106.9 21.33 Isoengelitin 434 - 1.54 - 191.31 Resveratrol 228 41.59 44.26 53.1 4.38 prim-O-Glucosylcimifugin 468 4.40 9.22 108.87 9.76 S. divaricata 4′-O-β-D-Glucosyl-5-O- 452 12.42 32.55 15.6 15.53 methylvisamminol Cimifugin 307 0.80 1.35 313.25 93.66 sec-O-Glucosylhamaudol 438 0.63 35.96 344.4 7.45 Puerarin 416 - 25.8 P. lobata Zhao et al. 2016 Daidzin 416 40.41 4.32 3′-Methoxy-puerarin 432 - - 3′-Hydroxy-puerarin 446 18.28 7.36 Daidzein 254 19.68 6.65 aMW: molecular weight; bRelative binding to enzyme = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; cConcentration of MMP-2 d e and 5-LOX were 20 and 0.1ng/μL, respectively. IC50: Concentration required for 50% inhibition of the enzyme activity under the assay conditions. Refer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

While the UF-LC-MSn technique has been used in discovering ed by LC-DAD-MSn after affinity based binding and ultrafiltration bioactives from medicinal herbs, its application in food bioactives purification, and the selected results are shown inTable 1 and Figure has not been widely adopted. This paper is therefore intended to re- 2. Resveratrol had the highest binding ability to 5-LOX, followed view the most recent studies reported for the utilization of UF-LC- by astilbin, daidzin, sec-O-glucosylhamaudol, 4′-O-β-D-glucosyl-5- MSn for high-throughput screening and identification of food and O-methylvisamminol, anhydrosafflor yellow B. This corresponded herb originated enzyme inhibitors, and to discuss its possibility in exactly to the results found in vitro enzyme assays (Table 1). Results functional foods and nutraceuticals, and health research. from the ultrafiltration LC-DAD-MS assay proved that the relative quantity of the compounds in an extract mixture may not automati- cally guarantee the same in bioactivity, and minor components such 2. Ultrafiltration LC-MS screening of enzyme inhibitors as anhydrosafflor yellow B, sec-O-glucosylhamaudol, resveratrol and daidzin in extracts may play more important role than major components in bioactivity, in this case 5-LOX inhibition. This can 2.1. Screening for 5-LOX, MMP-2 and COX-2 inhibitors be exemplified in the study of kudzu, a widely accepted functional food in China. The results also suggest a greater number of binding 5-LOX, MMP-2 and COX-2 are three key enzymes that are closely can be achieved by increasing receptor concentrations, and revealed related to immunity and inflammation, and are involved in sev- that enzyme inhibition can also be a competitive process among eral pathological processes, such as cancer, fever, wound healing, compounds coexisted in herb extracts (Figure 2). HPLC-UV chro- rheumatoid arthritis and inflammatory reactions (Brown, 1997; matogram of the extract shows that not all detected peaks bound to Ding et al., 2011; Ra and Parks, 2007; Schneider and Bucar, 2005; the enzyme. The largest peak (peak 1, puerarin) detected by DAD Steinhilber, 1994; Yokoyama and Tanabe, 1989). Finding selec- did not have any affinity. Binding capacity or affinity also depended tive inhibitors of MMP-2, 5-LOX or COX-2 from herb extracts on the concentration of enzyme (Figure 2). has become increasingly a hot research topic in recent years. UF- Similar ultrafiltration LC-MS screening has been conducted for LC-MSn has been proven useful for high-throughput screening and MMP-2 inhibitors from C. tinctorius, S. glabra, Smilax china L. and identification of MMP-2, 5-LOX or COX-2 inhibitors from some S. divaricate (Li et al., 2014; Li et al., 2015). S. glabra and S. china plant extracts (Li et al., 2014; Li et al., 2015; Zhao et al., 2016). are widely consumed by Chinese as functional/medicinal foods for UF-LC-MSn was successfully used to screen and identify a high- improving immunity and general health. Among the compounds affinity inhibitor of 5-LOX from four Chinese medicinal herbs/ identified, resveratrol showed the strongest binding to MMP-2, fol- foods, Carthamus tinctorius L. Smilax glabra Roxb., Saposhnikovia lowed by engelitin, anhydrosafflor yellow B, astilbin and 4′-O-O- divaricata (Turcz.) Schischk and Pueraria lobata (Zhao et al., 2016). D-glucosyl-5-O-methylvisamminol (Table 1). These studies proved Only compounds with certain binding ability to 5-LOX were detect- that the relative quantity of the compounds in an extract may not

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Figure 2. Ultrafiltration LC-DAD chromatograms of potential 5-LOX inhibitors in kudzu (Pueraria lobate) as compared to that of the crude extract without affinity purification. 5-LOX concentration: a, 0 ng/µL; b, 0.1 ng/µL; c, 0.2 ng/µL; d, 0.3 ng/µL; e, crude extract of P. lobata. Peaks 1, Puerarin; 2, Daidzin; 3, 3′-Methoxy-puerarin; 4, 3′-Hydroxy-puerarin; 5, Daidzein. Figure adopted from Zhao et al., 2016 with permission. follow the relative strength in bioactivity. It was also found that the 10-gingerol, 8-shogaol and 10-shogaol showed specific binding same compound may have binding ability to different enzymes. to the active site of COX-2 with IC50’s of 32, 17.5 and 7.5 µM, Compounds such as resveratrol and daidzin showed strong inhibition respectively (Table 2). UF-LC-MSn has also been used to iden- against both 5-LOX and MMP-2, suggesting a wider significance tify COX-2 inhibitors from traditional Chinese herbs; some of the as these are phytochemicals found in many commonly consumed COX-2 inhibitors identified such as phenyl ferulate are also known foods, such as grapes, mulberries, peanuts, grapes, red wine, soy- for common foods (Cao et al., 2010). bean and soy-based foods (Tsao, 2010; Lastra and Villegas, 2005). Ginger root (Zingiber officinale) has been used for both culinary and medicinal purposes, and UF-LC-MSn studies have shown that 2.2. Screening for α-glucosidase inhibitors gingerol-related compounds from the roots of Z. officinale were inhibitors of COX-2 (van Breemen et al., 2011). Among them, α-Glucosidase is an enzyme present in the brush border of the small

Table 2. Major COX-2 inhibitors identified in plant extracts by UF-LC- MS a b c d Compounds MW Relative binding to COX-2 IC50 (μM) Source References 10-Gingerol 350 0.17 32.0 Z. officinale Van et al. 2011 8-Shogaol 304 0.27 17.5 10-Shogaol 332 0.35 7.5 Senkyunolide O 380 52 5 L. chuanxiong Cao et al. 2010 Phenethyl-trans-ferulate 297 25 31 N. incisum Cryptoanshinone 296 31 22 S. miltiorrhiza Roburic acid 440 45 9 G. macrophylla a b c MW: molecular weight; Relative binding to COX-2 = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; IC50: Concentration required for 50% inhibition of the enzyme activity under the assay conditions; dRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

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Table 3. α-Glucosidase inhibitors identified by UF-LC-MS Compounds MWa Source Referencesb Quercetin 448 G. biloba; A. senticosus Liu et al. 2013; Zhou et al. 2012 Apigenin 270 G. biloba Liu et al. 2013 Kaempferol 286 Isorhamnetin 316 Rutin 610 A. senticosus Zhou et al. 2012 Hyperin 464 Isoquercitrin 464 1,5-Dicaffeoylquinic acid 516 1,4-Dicaffeoylquinic acid 516 4,5-Dicaffeoylquinic acid 516 5,7,3,2′,6′-Pentahydroxy flavanone 304 S. baicalensis Wang et al. 2014 Chrysin-6-C-arabinosyl-8-C-glucoside 548 Chrysin-6-C-glucosyl-8-C-arabinoside 548 5,7,2′,5′-Tetrahydroxy-8,6′-dimethoxy flavonne 346 Baicalin 446 Oroxylin A-7-O- glucuronide 460 Wogonoside 460 Baicalein 270 Skulllcapflavon II 374 Wogonin 284 Oroxylin A 284 Quercetin-3-O-rha- (1–4)-glcosyk-rhamnoside 756 C. oxyacantha Li et al. 2009 Vitexin-2″-O-glucoside 594 Vitexin-2″-O-rhamnoside 578 Vitexin 432 aMW: molecular weight; bRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry. intestine, whose inhibitors act by a reversible inhibition, could de- nols are commonly found in fruits, vegetables, cereals and pulse lay the absorption of carbohydrates from the small intestine, and grains, thus consumption of these foods are beneficial to blood suppress the postprandial blood glucose and insulin levels (Laar et glucose management Ramdath et al., 2014; Tang et al., 2016; al., 2005). Recent studies in the discovery of food and other natural Zhou et al., 2012; Zhang et al., 2015). UF-LC–MSn was used to sources of α-glucosidase inhibitors have attracted considerable in- rapidly and selectively screen and identify major α-glucosidase terest as α-glucosidase is an important biomarker for treating type ligands or inhibitors in Radix Astragali and polyphenols particu- 2 diabetes mellitus (Li et al., 2009; Liu et al., 2013; Ramdath et larly calycosin, formononetin and other isoflavones were found to al., 2014; Tang et al., 2016; Wang et al., 2014; Zhou et al., 2012; be strong α-glucosidase inhibitors. The activity of eight isoflavone Zhang et al., 2015). aglycones was evaluated and confirmed using in vitro enzymatic The fruit of hawthorn (Crataegus oxyacantha L.) has been assay (Zhao et al., 2015). part of foods or confectionaries in different cultures, however hawthorn tree leaves also contained flavonoids that were 2.3. Screening for xanthine oxidase inhibitors α-glucosidase inhibitors in a UF-LC-MSn screening study (Li et al., 2009). Similar results were found in other studies. Polyphe- nols including flavonoids and phenolic acids of Siberian ginseng Xanthine oxidase (XOD) is an important enzyme involved in Acanthopanax senticosus Harms, Ginkgo biloba leaves and other chronic gout, cardiovascular disease and many other diseases re- medicinal foods, especially rutin, hyperin, isoquercitrin, quer- lated to hyperuricemia (Dawson et al., 2007; Lin et al., 2000). In citrin, 1,5-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid and purine metabolism, xanthine and hypoxanthine are oxidized to uric 4,5-dicaffeoylquinic acid were found to be strong inhibitors of acid by XOD, which is highly expressed in the liver and intestine α-glucosidase due to their higher affinity (Zhou et al., 2012) (Table (Borges et al., 2002). UF-LC-MS has been used for the discovery 3). ( Liu et al., 2013; Wang et al., 2014) (Table 3). These polyphe- of XOD inhibitors (Table 4) (Liu et al., 2012a; Liu et al., 2013;

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Table 4. XOD inhibitors identified by UF-LC-MS Compounds MWa Source Referencesb Amentoflavone 538 S. tamariscina Wang et al. 2014 Robustaflavone 538 Tanshinone II B 310 S. miltiorrhizae Liu et al. 2013 Tanshindiol B 312 Tanshindiol A 312 15,16-Dihydrotanshinone I 278 1,2-Dihydrotanshinone I 278 Danshenxinkun B 280 Cryptotanshinone 296 Tanshinone I 276 3-Hydroxy methylenetanshinquinone 294 Mmethylene tanshinquinone 278 Tanshinone II A 294 aMW: molecular weight; bRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

Wang et al., 2014). were important for the affinity, and inhibited XOD activity signifi- An ultra high performance liquid chromatography and triple cantly (Liu et al., 2013). Application of UF-LC-MSn in screening quadrupole mass spectrometry (UHPLC-TQ-MS) method was food bioactives needs to be further explored considering the im- established to simultaneously screen and identify inhibitors of portance of the two marker enzymes in inflammation and chronic both superoxide anion scavengers and XOD in a single analysis diseases. by adding WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium sodium salt) to the enzymatic re- 2.4. Screening for neuraminidase inhibitors action (Liu et al., 2012a). This method not only distinguishes the specific inhibitory effects of some of the commonly found polyphenols in foods, but more interestingly, because of the introduc- Neuraminidase (NA) is an enzyme present in the surface of the tion of LC-MS which can directly measure substrates and/or prod- influenza virus. This enzyme is recognized as an attractive target ucts of the enzyme reaction, it can also effectively eliminate the for developing agents against influenza infection and its inhibi- false positive and negative results. Some food flavonoids such as tors have been widely used in the treatment (Meanwell and Krys- quercetin and apigenin were found to be strong inhibitors of both tal, 1996; von Itzstein et al., 1993; Palese et al., 1974). Although superoxide anion radical and XOD, and others such as catechin synthetic NA inhibitors exemplified by oseltamivir and zanamivir was a strong radical scavenger but lacked XOD inhibitory activity, have been designed to halt the spread of the virus in the body, or in the case of the isoflavone genistein, a strong XOD inhibitor adverse side effects such as nausea, vomiting, diarrhea, abdominal but not a radical scavenger. Although ultrafiltration was not part pain have been observed (Kitching et al., 2009). Hence, naturally of this unique method, it proved the usefulness of LC-MS in ki- occurring NA inhibitors have been intensely studied as safer al- netic studies of enzyme inhibition by food bioactives. UF-LC-MSn ternatives. Liu et al. (2011) reported an UF-LC-MSn method for has been used to screen and identify XOD inhibitors including the screening NA inhibitors from Radix scutellaria extract. As a result, unique flavonoids from medicinal foods Selaginella tamariscina some unique flavonoids have been identified NA inhibitors (Table (Beauv) Spring and Radix Salviae Miltiorrhizae extracts (Liu et 5). However, not all flavonoids are NA inhibitors. Using the same al., 2013, Wang et al., 2014). Structural features of these bioactives screening method, Liu et al. (2012b) found no compounds in the

Table 5. Screening the NA inhibitors from R. scutellaria extract by ultrafiltration LC-MS a b c d Compounds MW Relative binding to NA IC50 (μM) Source References Baicalin 446 20.32 49.34 R. scutellaria Liu et al. 2011 oroxylin A-7-O-gluacid 460 18.05 wogonin 460 20.01 29.83 Baicalein 270 25.16 30.52 wogonoside 284 26.08 oroxylin A 284 28.23 a b c MW: molecular weight; Relative binding to Neuraminidase = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; IC50: Concentration required for 50% inhibition of the enzyme activity under the assay conditions; dRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry.

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Table 6. Screening the QR-2 inhibitors from extracts of marine sediment bacterial cultures by ultrafiltration LC-MS a b c d Compounds MW Relative binding To QR-2 IC50 (μM) source References xanthohumol 354 196 H.lupulus Choi et al. 2011 xanthohumol D 370 110 tetrangulol methyl ether 318 0.16 Actinomycessp a b c MW: molecular weight; Relative binding to Quinone reductase-2 = (amount of compound specifically bound)/(total amount of compound in incubation) × 100%; IC50: Concen- tration required for 50% inhibition of the enzyme activity under the assay conditions; dRefer to main text. UF-LC-MS: Ultrafiltration liquid chromatography-mass spectrometry. n-butanol extract of Indigowoad Root Granule had NA inhibitory rally occurring aromatase inhibitors of food origin can be used for activity. chemoprevention or even the treatment of breast cancer in women of high risk (Bulun et al., 2005; Santen, 2003; Brueggemeier, 2006; Brueggemeier et al., 2005; Evans et al., 1986). Studies on use of 2.5. Screening for certain QR-2 inhibitors UF-LC-MS-MS toward aromatase screening are relatively scarce, but it has been used for screening aromatase inhibitors from a well Quinone reductase-2 (QR-2) is a cytosolic enzyme that is a tar- known medicinal herb Corydalis yanhusuo (Shi et al., 2010), which get for antimalarial activity and antitumor activities due to several successfully identified several quaternary protoberberine alkaloids possible mechanisms of action. QR-2 inhibitors act to antimalarial including berberine, which is a common bioactive found in barber- activity and antitumor activities or can function as chemopreven- ry and Oregon grape and other edible wild fruits. Considering the tion agents by prevent the metabolic activation of toxic quinones prevalence of the breast cancer, further studies using UF-LC-MSn (Table 6). A well known polyphenol resveratrol has been shown to for identifying aromatase and other important enzyme inhibitors in be a good QR-2 inhibitor and recognized as a cancer chemopre- food plants should be carried out. UF-LC-MSn has also been used ventive agent (Knox et al., 2000). Discovery of natural QR-2 in- to screen for synthetic antimicrobial agents that inhibit shikimate hibitors like resveratrol has instigated further studies in this direc- kinase (Mulabagal and Calderón, 2010). Yang et al. found that cin- tion using effective high-throughput screening methods.Choi et al. namic acids such as chlorogenic acid and other caffeic acid deriva- (2011) developed an UF-LC-MS method to screen ligands of QR-2 tives, and flavonol glycosides such as rutin, isoquercitrin and other from a hop (Humulus lupulus L.), and identified two prenylated quercetin and kaempferol glycosides, were inhibitors of tyrosinase, chalcones (open chain flavonoids) xanthohumol and xanthohumol a key enzyme involved in browning of fruits and vegetables, and in D as strong QR-2 inhibitors. Hops are widely used in the brew- melanin synthesis. Inhibitors of tyrosinase are widely used in the ery of beers, and xanthohumols are known as the bittering agents. agri-food industry and in the cosmeceutical industry for treating Other research has also led to the discovery of novel prenylated hyperpigmentation in human skin (Yang et al., 2012). bichalcone and chalcone compounds from hops as -2 inhibitors (Yu et al., 2014). 3. Pros and cons of ultrafiltration LC-MSn as a method of screen- 2.6. Screening for ACE and AChE inhibitors ing enzyme inhibitors

n Some natural food colourants and food and beverage formulations Ultrafiltration LC-MS as a screening method has been proven with different food matrices, including infant formula, soy paste, to be efficient, and can be used to find specific enzyme inhibitors ketchup, mayonnaise, wheat flour, orange juice, supplement drink, from a mixture of natural compounds. While enzyme inhibition is tea and coffee were found to contain strong angiotensin-converting a complexed process, and many factors, including pH, tempera- enzyme (ACE) inhibitory activities, using high-throughput LC/ ture, substrate concentration and the amount of enzyme, can all SID-MS/MS. ACE is a target enzyme that regulates blood pres- affect the outcome. There are different types of enzyme- inhibi sure, and inhibitors of ACE therefore have therapeutic value in the tion, but most food bioactives are reversible inhibitors. Natural prevention of hypertension (Inoue et al., 2011). inhibitors such as flavonoids and phenolic acids bind with target UF-LC-MS screening has led to the discovery of several cin- enzymes by non-covalent bonding, i.e. via hydrogen bonds, hy- namic acids and flavonoids as acetylcholinesterase (AChE) inhibi- drophobic interactions, and ionic bonds. For this reason, binding tors that might be potentially used for the treatment and preven- of food bioactives in a mixture to a specific enzyme can be com- tion of Alzheimer’s disease (Zhang et al., 2018). Most recently, the plicated. Different compounds in a extract or a particular food are same researchers also used this approach to screen AChE inhibitors likely to be competitive inhibitors of an enzyme. These potential from stem-leaf and found saponins, particularly the ginsenosides inhibitors compete with each other to bind to the enzyme, and may have similar affinity for the active site. Increasing concentration F1 , Rd, Rk3 , 20(S)-Rg3 , F2 and Rb2 that are also main bioactives of ginseng root, were strong AChE inhibitor (Yang et al., 2019). of these compounds or enzyme can normally separate the strong Similarly were some alkaloids (Zhao et al., 2016). inhibitors from weaker ones. Binding of a compound to a target enzyme may not necessarily suggest inhibition of the enzyme activity, as study on the binding 2.7. Screening for other inhibitors to a specific site of the enzyme, i.e. it may be caused by simple adsorption of a compound to the enzyme. Such false positive iden- Inhibitors of other enzymes such as aromatase have also been stud- tification can be easily avoided by using denatured enzyme fol- ied using UF-LC-MSn. Aromatase is the enzyme that catalyzes a lowing the same incubation protocol. Merchanism of binding of a key aromatization step in the synthesis of estrogen from androgens bioactive compound to a specific target protein/enzyme can also be which stimulates growth of breast and ovarian cancers, thus natu- assessed using molecular docking approach (Zhang et al., 2017).

Journal of Food Bioactives | www.isnff-jfb.com 33 UF-LC-DAD-MSn for discovering enzyme inhibitors Li et al.

4. Conclusion C.R. (1986). Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P450 mRNA. Proc. Natl. Acad. Sci. USA 83: 6387–6391. This mini-review confirms the increasing role of UF-LC-MS in Fu, Y., Buryanovskyy, L., and Zhang, Z. (2005). Crystal structure of quinone the searching for enzyme inhibitor from different herb extracts. reductase 2 in complex with cancer prodrug CB1954. Biochem. Bio- In summary, we found that only certain components of the herb phys. Research Commun. 336(1): 332–338. extracts actively and competitively bound to the enzyme using Gnant, M. (2006). Management of bone loss induced by aromatase inhibi- UF-LC-MS, suggesting these phytochemicals can play a potential tors. Cancer Invest. 24: 328–330. role in preventing and maintaining good health. Moreover, sev- He, R., Girgih, A.T., Malomo, S.A., Ju, X.R., and Aluko, R.E. (2013). Antioxi- eral 5-LOX or MMP-2 inhibitors such as resveratrol, daidzin, and dant activities of enzymatic rapeseed protein hydrolysates and the membrane ultrafiltration fractions. J. Funct. Foods 5(1): 219–227. α-glucosidase inhibitors, i.e. rutin, hyperin, isoquercitrin, quer- Inoue, K., Kitade, M., Hino, T., and Oka, H. (2011). Screening assay of angi- citrin are also major components of foods. Z. officinale and C. otensin-converting enzyme inhibitory activity from complex natural oxyacantha screened for COX-2 and α-glucosidase inhibitors are colourants and foods using high-throughput LC-MS/MS. Food Chem. consumed as foods all around the world. The usefulness of the UF- 126: 1909–1915. LC-MSn assay therefore can be a valid and effective screening and Jin, Y., Cheng, X., Jiang, F., Guo, Z., Xie, J., and Fu, L. (2016). 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Journal of Food Bioactives | www.isnff-jfb.com 35 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Original Research J. Food Bioact. 2019;7:36–42

Revisiting DPPH (2,2-diphenyl-1-picrylhydrazyl) assay as a useful tool in antioxidant evaluation: a new IC100 concept to address its limitations

JuDong Yeo and Fereidoon Shahidi*

Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9 *Corresponding author: Fereidoon Shahidi, 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.2019.7196 Received: September 06, 2019; Revised received & accepted: September 28, 2019 Citation: Yeo, J.D., and Shahidi, F. (2019). Revisiting DPPH (2,2-diphenyl-1-picrylhydrazyl) assay as a useful tool in antioxidant evaluation: a new IC100 concept to address its limitations. J. Food Bioact. 7: 36–42.

Abstract

The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay has been widely used in antioxidant evaluation. However, it suffers from certain limitations that are addressed in this contribution. The limitations discussed in this work were the ratio of DPPH radicals to antioxidants and the presence of pigments in the reaction medium which interferes with absorbance readings. To do so, we used eight different concentrations of DPPH solution. The modified DPPH assay proposes a new concept, IC100, that is defined as the amount of DPPH radical required to oxidize all antioxidants present in the reaction medium. The modified DPPH assay does not suffer from an underestimation of antioxidant activity found in the original DPPH procedure due to the decrease in the ratio of DPPH radicals to antioxidants. Moreover, the modified method was not influenced by interference from coexisting pigments in the measurement of radical scavenging potential of extracts. To the best of our knowledge, this is the first attempt to effectively resolve the above-mentioned limitations of the DPPH assay.

Keywords: IC100; DPPH; Radical scavenging ability; Stoichiometric factor; Antioxidant capacity.

1. Introduction as flavonoids including quercetin, kaempferol, and catechins that are found in seeds, cereals, legumes, fruits, and vegetables (Shahidi Free radicals with one or more unpaired electron are naturally oc- and Yeo, 2016; Shahidi and Peng, 2018). Natural antioxidants also curring in biological and food systems; these include hydroxyl exhibit a potent free radical scavenging potential by displaying ex- (HO•), lipid alkyl (L•), alkoxyl (LO•), and lipid peroxyl (LOO•) cellent electron or hydrogen atom donating ability to free radicals radicals (Pryor, 1986). As evidenced in their short lifetime of 10-9 (Litwinienko and Ingold, 2003; Foti and Ruberto, 2000). In this con- to 10 seconds, they are highly reactive and unstable entities. This tribution, the traditional 2,2-diphenyl-1-picrylhydrazyl (DPPH) as- aggressive property of free radicals causes damage to the cells, say that is popularly used in the evaluation of free radical scaveng- leading to many chronic diseases such as atherosclerosis, ageing, ing potential of antioxidants is revisited to address its shortcomings. cancer and several other ailments (Aruoma, 1998). In 1958, Blois reported the oxidation of antioxidants such as Recently, a large body of research has been conducted in order cysteine, glutathione, ascorbic acid, tocopherol, as well as polyhy- to explore natural radical scavengers which can suppress the ag- droxy and aromatic compounds by DPPH radical. Since then DPPH gressive tendency of free radicals. In this connection, plant-based radical has been widely used as a popular reagent for the determina- foods provide excellent sources of free radical scavengers such as tion of electron or hydrogen atom donating potential of antioxidants phenolic compounds. These include phenolic acids such as gallic in food and in polymer chemistry research (Blois, 1958; Fargere et acid, protocatechuic acid, p-coumaric acid, and ferulic acid as well al., 1995). In the UV-visible spectrum, DPPH radical has three main

Figure 1. The changes of chemical structure (a), color (a), and the absorption spectrum of DPPH radical after reduction by an antioxidant. peaks in which two peaks below 400 nm are derived mainly from toluene (BHT), α-tocopherol, ascorbyl palmitate, and catechin the DPPH. Another peak around 517 nm is due to the resonance of were purchased from Sigma– Aldrich Canada Ltd. (Oakville, ON, the radical, that changes upon the reduction of DPPH radical by an- Canada). Methanol and ethanol were bought from Fisher Scientific tioxidants. However, peaks at 220 and 324 nm are not affected to any Co. (Nepean, ON, Canada). Blackberry, raspberry, bell pepper and great extent and with only a slight blue shift of the 324 nm peak due beet were bought from a local market in St. John’s, NL, Canada. to the stabilization of the DPPH molecule after reduction (Figure 1). All other chemicals and reagents were procured from commercial Thus, the absorbance at 517 nm is monitored in the DPPH assay and sources and were used without any further purification. the reason for routinely using DPPH assay for the determination of hydrogen atom or electron donating capacity of bioactive molecules. Meanwhile, there are certain shortcomings in the DPPH assay 2.2. The original DPPH radical scavenging assay which may adversely affect the accuracy of the results; firstly, the ratio of DPPH radical to antioxidant in the reaction medium af- The original DPPH radical scavenging assay of representative an- fects the electron transfer or hydrogen atom donating potential of tioxidants and food-extracts was performed according to the pro- antioxidants. The lack of standardization in the ratio of DPPH· to cedure described by Madhujith and Shahidi (2006) with minor antioxidant makes it hard to compare the results with those in the modifications. The DPPH solution (0.2 mM, 1.9 mL) in methanol existing literature. This limitation has already been noted in the was allowed to react with 0.1 mL of each sample (1 g of dried published work. Deng et al. (2011) argued that “IC50 value changes weight/30mL) and kept in the dark for 30 min. DPPH radical scav- depending on the final concentration of antioxidant in the DPPH enging activity was measured by reading the absorbance at 517 nm. solution”. For example, IC50 of 10 antioxidants varied about 2–3 times when three different DPPH concentrations were used. Other studies have also pointed out to similar limitations of the DPPH 2.3. Modified DPPH assay assay (Scherer and Godoy, 2009; Sun and Ho, 2005; van den Berg et al., 1999). Furthermore, the pigments which may coexist in the The modified DPPH method was employed by using a wide range extracts could interfere with the determinations by absorbing in the of DPPH concentrations as graphically shown in Figure 2. Briefly, same wavelength range of DPPH radicals (around 517 nm), lead- 0.1 mL of antioxidant (0.25, 0.50, and 1.00 mM) or extract (25, 50, ing to underestimation of the final results. Thus, this study was de- and 100 mg/mL) was mixed with 1.9 mL of different concentra- signed to propose a possible solution to address concerns about the tions of DPPH solution (0.1–0.8 mM), followed by 30 min reac- accuracy and reproducibility of the original DPPH method which has been widely used to measure antioxidant capacity of molecules tion time in the dark. The absorbance was read using a diode array since it was first considered by Blois (1958). In a recent publica- spectrophotometer (Agilent, Palo Alto, CA, USA) at 517 nm. The tion, Yeo and Shahidi (2019) suggested the use of electron para- intensity of the remaining purple-colored DPPH radicals increased magnetic resonance (EPR) for evaluation of the DPPH scavenging gradually depending on the concentration of the DPPH solution. of colored extracts to eliminate interference The core hypothesis of This allowed determination of the specific point that the absorb- the present study was to adopt a wide range of DPPH concentra- ance began to increase, which we refer to as the “Inhibitory Con- tions to circumvent the ratio of DPPH· to antioxidant and pigment centration 100 or (IC100)”. The IC100 indicates “the concentration interference in the original DPPH assay and to device a new index of DPPH radical needed to abstract 100% of the available hydro- which can express the results so obtained in a precise manner. gen atoms of the antioxidant present in the reaction medium”. This new concept was used again to calculate the stoichiometry factor of the pure antioxidant compound or extract. 2. Materials and methods

2.4. Determination of the IC100, the stoichiometry factor, and a 2.1. Materials new unit of scavenged DPPH radical in mg/g of sample

The 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxy- The determination of IC100 was carried out as summarized in Fig-

Journal of Food Bioactives | www.isnff-jfb.com 37 DPPH assay revisited Yeo et al.

Figure 2. Procedure of the modified DPPH procedure. ure 3. The IC100 indicates the specific concentration of the DPPH Scavenged DPPH mg/g of DW solution at which absorbance begins to increase after reaction with IC100 (mM)× 394 (MW of DPPH) an antioxidant, defined as the IC100. For the determination of IC100, = two equations are proposed using the pattern in the graph before Concentration of extract (mg/Ml) and after the IC100, followed by calculating the exact IC100, as explained in Figure 3. The IC100 was further used to determine the stoichiometry factors of the pure representative antioxidants. Stoi- 2.5. Extraction of blackberry, raspberry, bell pepper, and beet chiometry factor was calculated by dividing IC100 by the concentration of antioxidant used to obtain the number of hydrogen atoms The extraction was carried out as described by Yeo and Shahidi or electrons to scavenge one molecule of DPPH radical. The IC100 (2019) with slight modification. The fresh blackberry, raspberry, so obtained was also converted into the scavenged DPPH radical in bell pepper, and beet were finely ground using a blender, followed mg/g of dry weight (DW) of defatted samples for the extracts using by lyophilization. One gram of dried sample was subsequently the following equation. mixed with 10 mL of 70% methanol for 20 min at ambient tem-

Figure 3. Determination of IC100 by using two equations having a different pattern in the measurement of DPPH radical scavenging ability.

38 Journal of Food Bioactives | www.isnff-jfb.com Yeo et al. DPPH assay revisited

Table 1. DPPH radical scavenging ability of representative antioxidants using the original DPPH assay Concentration Ratio Scavenged DPPH Stoichiometric factor (mM) (DPPH/Antioxidant) (%) (n, DPPH/Antioxidant) α-Tocopherol 1.000 2/1 65.7 ± 0.5 1.3 0.500 4/1 51.8 ± 1.1 2.1 0.250 8/1 27.5 ± 1.3 2.2 0.125 16/1 17.1 ± 0.8 2.7 0.063 32/1 10.1 ± 0.5 3.2 Ascorbyl-6-palmitate 1.000 2/1 31.1 ± 2.1 0.6 0.500 4/1 17.5 ± 0.6 0.7 0.250 8/1 10.4 ± 1.5 0.8 0.125 16/1 6.7 ± 0.8 1.1 0.063 32/1 4.1 ± 0.1 1.3 BHT 1.000 2/1 62.7 ± 0.6 1.3 0.500 4/1 46.9 ± 1.4 1.9 0.250 8/1 30.1 ± 0.3 2.4 0.125 16/1 17.7 ± 0.1 2.8 0.063 32/1 11.4 ± 0.3 3.6 Catechin 1.000 2/1 67.9 ± 2.1 1.4 0.500 4/1 68.1 ± 1.7 2.7 0.250 8/1 61.2 ± 1.5 4.9 0.125 16/1 41.6 ± 1.0 6.7 0.063 32/1 26.9 ± 0.3 8.6 perature, and this step was repeated two more times. The extracts a large variation existed among the reports in the determination of were then filtered through a Whatman No. 1 filter paper in order to IC50 of ascorbic acid, being 5.8, 50.0, 62.4, and 110.7 µg/mL (Kan- remove any remaining solid particles in the samples. The extracts imozhi and Prasad, 2009; Charrier et al., 2006; Shirwaikar et al., were then kept at −40 C for further analysis. 2006; Ricci et al., 2005). This observation was also made by Deng et al. (2011), Scherer and Godoy (2009), Sun and Ho (2005) as well as van den Berg et al. (1999). The reason for the discrepancy 3. Results and discussion might be due to the reversibility of the reaction. According to Bon- det et al. (1997) and Huang et al. (2005), some of DPPH-H which is formed after receiving a hydrogen atom from the antioxidants 3.1. Limitation of the DPPH assay as reflected in the ratio of may be converted back to DPPH radical due to the chemical equi- DPPH radicals to antioxidants librium of the reaction medium. Therefore, at a low concentration of antioxidant in the reaction medium, a low amount of DPPH- The results in the DPPH assay are affected by the ratio of DPPH H will be produced, followed by a low amount of re-conversion radical to the antioxidant in the reaction medium as summarized in of DPPH-H to DPPH radical. Conversely, at a high concentration Table 1. The ratios of DPPH· to antioxidant selected were 2/1, 4/1, of antioxidant a high amount of DPPH-H is re-converted to the 8/1, 16/1, and 32/1. For comparison of DPPH radical scavenging DPPH radical, hence this may lead to the more underestimation activity (DRSA), the results were expressed as the stoichiometry of the DPPH radical scavenging potential of antioxidant. Thus, to factor which is defined as the number of hydrogen atoms or elec- address this limitation of the DPPH assay a viable solution that trons that the antioxidant molecule may donate to the DPPH radi- could circumvent the underestimation of DPPH radical scavenging cal. As expected, the stoichiometry factors of antioxidants were ability of antioxidants is deemed necessary. greatly affected by the ratios of DPPH· to antioxidant, namely the stoichiometry factors of α-tocopherol, ascorbyl-6-palmitate, buty- 3.2. Applying the modified DPPH assay to representative lated hydroxytoluene (BHT) and catechin were significantly increased approximately 2-6 times with the increase of the ratio from antioxidants 2/1 to 32/1. This indicates that the number of hydrogen atoms or electrons that the antioxidants can donate varies depending on the As mentioned earlier, one needs to overcome the reversibility of ratio of DPPH radical to antioxidant in the reaction medium. Sev- DPPH-H back to the DPPH radical in the reaction medium to ad- eral studies have shown the discrepancy of DRSA of antioxidants dress the ratio issue. For that, we employed a wide range of DPPH at the different ratios of DPPH radical to antioxidant. For example, concentrations ranging from 0.1 to 0.8 mM to avoid the reversibil-

Journal of Food Bioactives | www.isnff-jfb.com 39 DPPH assay revisited Yeo et al.

Table 2. The IC100 and stoichiometric factors of representative antioxidants using the modified DPPH method Stoichiometric factor Concentration (mM) IC (mM) 100 (n, DPPH/Antioxidant) α-Tocopherol 1.00 0.124 2.488 0.50 0.060 2.389 0.25 0.025 1.985 Ascorbyl 6-palmitate 1.00 0.133 2.655 0.50 0.060 2.396 0.25 ND ND BHT 1.00 0.011 0.221 0.50 0.007 0.265 0.25 ND ND Catechin 1.00 0.198 3.963 0.50 0.103 4.106 0.25 0.045 3.637 ity. Four representative antioxidants, α-tocopherol, ascorbyl-6-pal- lower stoichiometry factors than it is expected by displaying 1.4 mitate, BHT, and catechin, were tested to evaluate the suitability and 0.7, respectively. Thus, a discrepancy between stoichiometry of the modified DPPH assay, and the following results were cal- factor and number of hydroxyl groups was found in the experimen- culated into IC100 and the stoichiometry factor. The stoichiometry tal data depending on the reaction environment. factor is a unique property of individual antioxidants that should In the present study, the correlation coefficient between stoi- provide the same value, regardless of the different ratios of the chiometry factor calculated by the modified method and number DPPH radical to the antioxidant; thus, the stoichiometry factor of hydroxyl groups was 0.98, showing a high value with the ex- can serve as an appropriate parameter to test the suitability of the ception of α-tocopherol. Once α-tocopherol is included, the cor- modified method (Table 2). Unlike the original DPPH assay given relation coefficient dropped to 0.70, which means that the number in Table 1, the stoichiometry factor by the modified method did of hydroxyl groups of α-tocopherol (only 1) does not match with not show any marked increasing or decreasing trend due to the the number of hydrogen atom or electron it can donate; in other changes in the ratio of DPPH radical to the antioxidant. Thus, the words, α-tocopherol donates one more hydrogen atom or electron ratio of DPPH· to antioxidant is no longer an issue in the modified to the DPPH radical. The difference between the number of hy- method, possibly due to the minimization of the reversibility of droxyl groups of α-tocopherol and the number of hydrogen atoms DPPH-H back to the DPPH radical by adopting a wide range of or electrons it can donate is related to the hydrogen atom or elec- DPPH concentrations. tron donating mechanism; in this α-tocopherol becomes phenoxyl Meanwhile, the modified method is considered as a more ap- radical after donating a hydrogen atom, subsequently it can do- propriate way to yield stoichiometry factor of antioxidants com- nate one more electron, leading to the formation of α-tocopheryl pared to the original DPPH assay. According to the literature, IC50 quinone (Decker, 2002). Thus, α-tocopherol can donate one more obtained by the original method suffers from serious errors itself; electron or hydrogen atom in the process, which may be the rea- that is, the IC50 showed marked variations depending on the ratio son for the observed disagreement. Therefore, determination of the of DPPH radical to antioxidants adopted, leading to the errone- stoichiometry factor based on the modified DPPH assay not only ous stoichiometry factor (Scherer and Godoy, 2009; Sun and Ho, can improve the accuracy in the evaluation of hydrogen atom or 2005; van den Berg et al., 1999). However, the stoichiometry fac- electron donating ability of antioxidant, but also allows a critical tor yielded by the modified method considering the IC100 displayed discussion about the possible hydrogen atom or electron donating a constant value of stoichiometry factor at different ratios of DPPH mechanism of antioxidant by comparing the stoichiometry factor radical to the antioxidant. and the number of hydroxyl groups present. In addition, the stoichiometry factor provides a valuable op- Up to the present day, many attempts for the expression of portunity to explore the mechanism of hydrogen atom or electron DPPH radical scavenging potential of antioxidants have been donating potential of antioxidants. For example, stoichiometry made, namely IC50, EC50, and the inhibition of the DPPH radical factors of α-tocopherol, ascorbyl-6-palmitate, BHT, and catechin, in percent (I%) (Ani et al., 2006; Elzaawely et al., 2007; Guerrero determined by modified method, were 2.3, 2.5, 0.2, and 3.9, re- et al., 2006). Moreover, Scherer and Godoy (2009) proposed a new spectively. Given the corresponding number of hydroxyl groups formula referred to as antioxidant activity index (AAI) and Deng et in their chemical structures as 1, 3, 1, and 5, respectively, a high al. (2011) suggested a novel index such as antioxidant activity unit dependency between stoichiometry factors by the modified meth- (AAU) by considering the volume ratio of the sample to DPPH od and the number of hydroxyl groups is expected. Shi and Niki solution and the molecular weight of antioxidants as well. All such (1998) reported that kaempferol showed the experimental stoichio- approaches for the expression of yielded results have successfully metric numbers of 1.9, which is lower than the number of hydroxyl been adopted. However, since they used only one concentration groups (4) of kaempferol. Cheng et al. (1998) noted that butein of DPPH solution (one ratio of DPPH radical/antioxidants) in the and ascorbic acid, which have four hydroxyl groups, also showed measurement, the expressed values cannot be free from the ratio

40 Journal of Food Bioactives | www.isnff-jfb.com Yeo et al. DPPH assay revisited

Table 3. The calculation of IC100 and scavenged DPPH radical by different food extracts

Concentration(mg/mL) IC100 (mM) Scavenged DPPH mg/g of DW Bell pepper 100 0.296 1.164 50 0.156 1.229 25 0.072 1.134 Correlation coefficient 0.998 Blackberry 100 0.521 2.052 50 0.242 1.908 25 0.132 2.081 Correlation coefficient 0.997 Raspberry 100 0.345 1.360 50 0.170 1.338 25 0.084 1.324 Correlation coefficient 1.000 Beet 200 0.072 0.141 100 0.034 0.133 50 0.021 0.162 Correlation coefficient 0.993

issue. Thus, the IC100, which considers a wide range of DPPH so- ing a standard deviation of only 0.048 or 4.13% difference among lution concentrations, is expected to offer a promising alternative the three tests. This indicates that the modified DPPH method that avoids the ratio issue in the DPPH assay. effectively reduces variations caused by the presence of interfering pigments. The efficiency of the modified DPPH method for 3.3. A solution for the presence of interfering pigments in the addressing the pigment issue was also proven for other extracts such as blackberry, raspberry, and beet, which showed standard de- DPPH assay using a modified method viations of 0.092, 0.018, and 0.014, respectively. This observation confirms the suitability and feasibility of the modified DPPH assay As mentioned earlier, the co-existing pigments in extracts with ab- in addressing the interference due to the co-existing pigments in sorption in the same wavelength range of DPPH radicals (around the original DPPH assay. Moreover, the new unit may provide an 517 nm) interfere with the absorbance readings. For example, the effective way to express the antioxidant capacity of samples, as it underestimation of DPPH radical scavenging potential of extract does not require any standard curve to obtain standard compound occurs for samples containing a high concentration of pigments equivalents. This reduces the required time for such determina- that can absorb at 517 nm. This is bacause the pigments present are tions. considered as unreacted DPPH radical in the UV-vis range (data In summary, the modified DPPH assay proposed here efficiently not shown). To resolve this issue, the modified DPPH assay was addresses the interference by co-existing pigments in the medium employed, and its dependability and accuracy were tested by us- which absorb in the same wavelength range. The core principle ing pigment-containing extracts such as bell pepper, blackberry, behind this achievement is that IC100, which is the required DPPH raspberry, and beet (Table 3). The modified method removed the concentration to be reduced by all antioxidants in the extract, re- limitation of the original DPPH assay; that is, the IC100 was not influenced by the presence of pigments in the extracts, which is not possible in the original DPPH assay. The effectiveness of the modified method is easily demonstrated as the correlation coefficient between the concentrations of extracts and IC100; were 0.998, 0.997, 1.000, and 0.993 for bell pepper, blackberry, raspberry, and beet, respectively. Moreover, the IC100 may be calculated into a new unit in which the scavenged DPPH radical is given in mg (DPPH radical)/g of DW of the sample. The new unit was calculated based on the IC100 proposed here as a unit for the extracts of natural sources, which are difficult to define their molecular weight and following stoichiometry factor. The DPPH radical scavenging ability of the coloured extracts showed a nearly constant value for different concentrations of the extracts. For example, bell pepper gave values of 1.134, 1.229, and 1.164 DPPH radical in mg/g of DW at 25, Figure 4. The IC100 of new DPPH method in the presence or absence of 50, and 100 mg/mL concentrations of extract, respectively, show- pigments.

Journal of Food Bioactives | www.isnff-jfb.com 41 DPPH assay revisited Yeo et al. mains unchanged regardless of the presence or absence of any dant properties of butein isolated from Dalbergia odorifera. Bioch et pigments, as depicted in Figure 4. Therefore, the modified DPPH Bioph. Acta 1392: 291–299. assay also overcomes the issue associated with the colour interfer- Decker, E.A. (2002). Antioxidant mechanisms. In: Akoh, C.C., and Min, D.B. ence caused by commonly used original DPPH assay. As already (Ed.). Food lipids. Marcel Dekker, Inc., New York, pp. 520–525. noted in the previous study we proposed the use of EPR spectros- Deng, J., Cheng, W., and Yang, G. (2011). A novel antioxidant activity index (AAU) for natural products using the DPPH assay. Food Chem. 125: copy to effectively address the concern about interference from 1430–1435. colored material in the same absorption range (Yeo and Shahidi, Elzaawely, A.A., Xuan, T.D., and Tawata, S. (2007). Essential oils, kava py- 2019). Thus, this study will be a useful alternative for the research- rones and phenolic compounds from leaves and rhizomes of Alpinia ers who have no access to EPR machine by employing UV-visible zerumbet (Pers.) B.L. Burtt. and R.M. Sm. and their antioxidant activ- spectroscopy. ity. Food Chem. 103: 486–494. Fargere, T., Abdennadher, M., Delmas, M., and Boutevin, B. (1995). Deter- mination of peroxides and hydroperoxides with 2,2-diphenyl-1-pic- 4. Conclusion rylhydrazyl (DPPH). Application to ozonized ethylene vinyl acetate copolymers (EVA). Eur. Polym. J. 31: 489–497. Foti, M.C., and Ruberto, G. (2000). Kinetic solvent effects on phenolic anti- The ratio of DPPH radical to antioxidants which influences the oxidants determined by spectrophotometric measurements. J. Agric. results in the original DPPH assay was resolved by employing a Food Chem. 49: 342–348. Guerrero, J.L.G., Guirado, C.M., Fuentes, M.M.R., and Pérez, A.C. (2006). modified DPPH method. The revisited method introduced100 IC which successfully eliminated/reduced the effects caused by Nutrient composition and antioxidant activity of 10 pepper (Capsi- changes in the ratio of DPPH·/antioxidant. In addition, the stoichi- cum annuun) varieties. Eur. Food Res. Technol. 224: 1–9. ometry factor calculated from IC allowed to critically discuss Huang, D., Ou, B., and Prior, R.L. (2005). The chemistry behind antioxidant 100 capacity assays. J Agric. Food Chem. 53: 1841–1856. the possible hydrogen atom or electron donating mechanism of Kanimozhi, P., and Prasad, N.R. (2009). Antioxidant potential of sesamol antioxidants by comparing the stoichiometry factor and the num- and its role on radiation-induced DNA damage in whole body irradi- ber of hydroxyl groups in the structure of antioxidants. Moreover, ated Swiss albino mice. Env. Toxicol. Pharmacol. 28: 192–197. the pigment interference issue of the original DPPH assay was re- Litwinienko, G., and Ingold, K.U. (2003). Abnormal effects on hydrogen solved by applying the modified DPPH assay. The modified DPPH atom abstractions. The reactions of phenols with 2,2-diphenyl-1-pic- assay might also lend itself to the exploitation of other methods rylhydrazyl (DPPH•) in alcohols. J. Org. Chem. 68: 3433–3438. when the issue of colour interference is encountered. Madhujith, T., and Shahidi, F. (2006). Optimization of the extraction of antioxidative constituents of six barley cultivars and their antioxidant properties. J. Agric. Food Chem. 54: 8048–8057. Pryor, W.A. (1986). Oxy-radicals and related species: their formation, life- Acknowledgments times and reactions. Annu. Rev. Physiol. 48: 657–667. Ricci, D., Fraternale, D., Giamperi, L., Bucchini, A., Epifano, F., Burini, G., We are grateful to the Natural Science and Engineering Research and Curini, M. (2005). 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42 Journal of Food Bioactives | www.isnff-jfb.com Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Original Research J. Food Bioact. 2019;7:43–47

Antioxidant activity of soybean peptides on human hepatic HepG2 cells

Carmen Lammi*, Carlotta Bollati and Anna Arnoldi

Department of Pharmaceutical Sciences, University of Milan, 20133 Milan, Italy *Corresponding author: Carmen Lammi, Department of Pharmaceutical Sciences, University of Milan, via Mangiagalli 25, 20133 Milan, Italy. Tel: +39 02-50319372; E-mail: [email protected] DOI: 10.31665/JFB.2019.7197 Received: September 28, 2019; Revised received & accepted: September 28, 2019 Abbreviations: CVD, Cardiovascular disease; DPP-IV, dipeptidyl peptidase-IV; DPPH, 1,1-Diphenyl-2-picrylhydrazyl radical; HMG- CoAR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; NO, nitric oxide; RNS, reactive nitrogen species; ROS, reactive oxygen species Citation: Lammi, C., Bollati, C., and Arnoldi, A. (2019). Antioxidant activity of soybean peptides on human hepatic HepG2 cells. J. Food Bioact. 7: 43–47.

Abstract

Soybean is an interesting source of bioactive peptides useful for the development of functional foods and nutraceuticals. In this study, the antioxidant activity of peptic (P) and tryptic (T) soybean hydrolysates was characterized. Results suggest that both hydrolysates are able to scavenge DPPH radical. Moreover, after induction of oxidative stress by using H2O2, either Soybean P or T pre-treatment reduces the ROS, lipid peroxidation, and NO levels in human hepatic HepG2 cells. HepG2 cells, exposed to 2H O2 alone, produce a significant augmentation of intracellular reactive oxygen species (ROS) levels by 29.5%, with the consequence of an augmentation of cellular lipid peroxidation levels up to 112.4 ± 0.5%. The pre-treatment with soybean hydrolysates restores the basal level of ROS and induces a reduction of cellular lipid peroxidation. The antioxidant ability of Soybean P and T are also confirmed by their ability to reduce the 2H O2-induced NO levels in HepG2 cells.

Keywords: Antioxidant; Bioactive peptides; Food bioactive peptide; Hydrolysates; Ros; Soybean.

1. Introduction of ROS. However, in pathological conditions, the antioxidant systems can be destroyed and the use of food-derived antioxidant Cardiovascular disease (CVD) is a leading cause of death world- agents could be a good strategy to impair the progression of dis- wide. Many risk factors are responsible for the development of this ease related to oxidative stress (Lorenzo et al., 2018). For instance, multifactorial disease, with a prevalence of those related to ath- egg, milk, meat, and fish have been identified as a good sources erosclerosis, which is strictly connected with oxidative stress and of peptides with interesting antioxidant activity (Ibrahim, Isono, inflammatory processes Wu,( Xia, Kalionis, Wan, & Sun, 2014). & Miyata, 2018; R. Liu, Xing, Fu, Zhou, & Zhang, 2016; Nazeer, Although reactive oxygen species (ROS) are produced by liv- Kumar, & Jai Ganesh, 2012; Zambrowicz et al., 2015). ing organisms as a result of normal cellular metabolism, at high Digestion or suitable technological treatments can deliver from concentrations they produce adverse effects on cell components, food proteins bioactive peptides, some of which showing a multi- such as lipids, proteins, and DNA. Oxidative stress, which refers functional behaviour (Lammi, Aiello, Boschin, & Arnoldi, 2019). to the shift in balance between oxidants/antioxidants in favour of In particular, some anti-diabetic, hypotensive, and hypocholes- oxidants, contributes to many pathological conditions (Dhalla, terolemic peptides display also antioxidant activity (Girgih et al., Temsah, & Netticadan, 2000). Aerobic organisms have integrated 2014; Iqbal & Hussain, 2009; Siow & Gan, 2013; Zambrowicz et antioxidant systems, which include enzymatic and nonenzymatic al., 2015). antioxidants, that are usually effective in blocking harmful effects Numerous clinical studies have associated soy food consump-

Copyright: © 2019 International Society for Nutraceuticals and Functional Foods. 43 All rights reserved. Antioxidant activity of soybean peptides on human hepatic HepG2 cells Lammi et al. tion with a reduced risk of developing some chronic diseases, 2.4. Cell culture such as obesity, hypercholesterolemia, and insulin-resistance/type II diabetes (Velasquez & Bhathena, 2007). As for the active sub- HepG2 cell line was bought from ATCC (HB-8065, ATCC from stance in soy foods, the protein plays a role in cholesterol reduc- LGC Standards, Milan, Italy) and was cultured following the con- tion (Fukui et al., 2002; Liu et al., 2014) and some hypocholes- ditions previously described (Lammi et al., 2015b). terolemic and anti-diabetic peptides have been already identified in the sequences of glycinin and β-conglycinin (Lammi, Zanoni, & Arnoldi, 2015a; Lammi, Zanoni, & Arnoldi, 2015b). Soybean 2.5. Nitrite/nitrate assay represents thus a promising source of protein hydrolysates with a multifunctional behaviour that has been recently investigated. In A total of 3 × 104 HepG2 cells/well were seeded on a 96-well particular, it has been demonstrated that peptic (P) and tryptic (T) plate. The next day, cells were treated with Soybean P and Soy- hydrolysates from soybean protein show an in vitro hypocholes- bean T at different concentrations (0.5 and 1.0 mg/mL) overnight terolemic activity targeting 3-hydroxy-3-methylglutaryl coenzyme and then 0.5 mM H2O2 was added in each well for 30 min at 37 A reductase (HMGCoAR). Through the inhibition of this enzyme, °C. After incubation, cells were centrifuged at 1,000 g for 15 min both hydrolysates lead to an augmentation of the low-density li- to remove insoluble material. The supernatant was transferred in poprotein (LDL) receptor (LDLR) protein levels producing an a 96-well plate, then 10 µL of nitrate reductase solution and 10 increased ability of hepatic HepG2 cells to clear LDL from the µL of the enzyme co-factors solution were added to the samples extracellular space(Lammi, Arnoldi, & Aiello, 2019). Moreover, and the plate was incubated at 25 °C for 2 h. Afterward, 50 µL the same hydrolysates are able of inhibiting dipeptidyl peptidase- of Griess Reagent A was added to each well and, after 5 min, 50 IV (DPP-IV) in vitro on the human recombinant enzyme as well as µL of Griess Reagent B was added for 10 min. For the detection in human intestinal Caco-2 cells expressing DPP-IV, suggesting a step, the absorbance at 540 nm was measured using a Synergy H1 potential anti-diabetic effect. microplate reader. Considering that both diabetes and hypercholesterolemia are correlated to oxidative stress, this study was aimed at characterizing the antioxidant properties of the same soybean hydrolysates. 2.6. Fluorometric intracellular ROS assay This was done, initially, by evaluating in vitro the antioxidant activity employing the 1,1-diphenyl-2-picrylhydrazyl radical For cells preparation, 3 × 104 HepG2 cells/well were seeded on (DPPH) reagent, then by pre-treating HepG2 cells with the hy- a 96-well plate overnight in growth medium. The day after, the drolysates after the induction of oxidative stress using H2O2 and medium was removed, 50 μL/well of Master Reaction Mix was assessing their ability to reduce the ROS, lipid peroxidation, and added and the cells were incubated at 5% CO2, 37 °C for 1 h in nitric oxide (NO). the dark. Then, cells were treated with 5 μL of 12× Soybean P and Soybean T to reach the final concentrations of 0.5 mg/mL and 1.0 mg/mL and incubated at 37 °C for 1 h in the dark. To induce 2. Material and methods ROS, cells were treated with H2O2 at a final concentration of 0.5 mM for 30 min a 37 °C in the dark and fluorescence signals (ex./ 2.1. Materials and cell cultures em. 490/525 nm) were recorded using a Synergy H1 microplate reader.

All chemicals and reagents were of analytical grade. DPPH, ROS, lipid peroxidation and Nitrite/Nitrate assays were from Sigma- 2.7. Lipid peroxidation (MDA) assay Aldrich (St. Louis, MO, USA). The HepG2 cell line was bought from ATCC (HB-8065, ATCC from LGC Standards, Milan, Italy) HepG2 cells (5 × 105 cells/well ) were seeded in a 6 well plate and, and was cultured following the conditions previously described the following day, they were treated with 0.5–1.0 mg/mL of Soy- (Lammi et al., 2015b). bean P and T for 24 h at 37 °C under 5% CO2 atmosphere. The day after, cells were incubated with H2O2 1mM or vehicle (H20) for 30 min, than collected and homogenized in 300 µL ice-cold MDA 2.2. Production of Soybean P and T hydrolysates lysis buffer containing 3 µL of BHT (100×). Samples were centrifuged at 13,000 × g for 10 min, then they were filtered through a Soybean P and Soybean T hydrolysates were obtained by extract- 0.2 µm filter to remove insoluble material. To form the MDA-TBA ing the proteins from 2 g of defatted soybean flour and by hydro- adduct, 300 µL of the TBA solution were added into each vial con- lysing them with pepsin or trypsin. Their production and analysis taining samples and incubated at 95 °C for 60 min, then cooled to of these materials are described elsewhere (Lammi et al., 2019). RT for 10 min in an ice bath. For analysis, 100 µL of each reaction mixture were pipetted into a 96 well plate and the absorbance was 2.3. DPPH assay measured at 532 nm using the Synergy H1 fluorescent plate reader from Biotek. To normalize the data, total proteins for each sample were quantified by Bradford method. The DPPH assay to determine the antioxidant activity in vitro was performed by a standard method with slight modifications. The DPPH solution (0.0125 mM in methanol, 45 μL) was added 2.8. Statistically analysis to 15 μL of the Soybean P and Soybean T hydrolysates at different concentrations (0.5–5.0 mg/mL). The reaction for scavenging Statistical analyses were carried out by One-way ANOVA (Graph- DPPH radicals was performed in the dark at room temperature pad Prism 8) followed by Brown-Forsythe’s test. Values were and the absorbance was measured at 520 nm after 30 min incuba- expressed as means ± sd; P-values < 0.05 were considered to be tion. significant.

44 Journal of Food Bioactives | www.isnff-jfb.com Lammi et al. Antioxidant activity of soybean peptides on human hepatic HepG2 cells

Figure 1. In vitro evaluation of the DPPH radical scavenger activity of Soybean P (a) and Soybean T (b) hydrolysates. The data points represent the averages ± sd of four independent experiments in duplicate. (*) p < 0.05. C: control sample.

3. Results and discussion to be more active that a hempseed protein hydrolysate, obtained by co-digesting the proteins with pepsin and pancreatin, which has shown a poor scavenger of DPPH activity, i.e. about 4% (Gir- 3.1. In vitro radical scavenging activity of Soybean P and T hy- gih, Udenigwe, & Aluko, 2011). Instead, rice bran protein hydro- drolysates lysates, obtained after the hydrolysis of the proteins with Alcalase, displayed a DPPH free radical scavenging activity of about 32% In order to evaluate the in vitro radical scavenging activity of Soy- at 20 mg/mL (Wattanasiritham, 2015). Finally, fish and chicken bean P and T hydrolysates, the DPPH assay was employed. The bones hydrolysates, obtained using trypsin, have shown an anti- hydrolysates were tested in the range from 0.5 to 5.0 mg/mL. The oxidant activity of about 15% and 10%, respectively, at 5.0 mg/mL results clearly suggested that both hydrolysates have a modest abil- (Centenaro, Mellado, & Prentice-Hernandez, 2011). ity to scavenge DPPH radical (Figure 1a–b). Soybean P reduces the DPPH radicals by 13.7%, 15.5, and 22.1% at 0.5, 1.0, and 5.0 3.2. Soybean P and T hydrolysates modulate the H O -induced mg/mL, respectively (Figure 1a), whereas Soybean T diminishes 2 2 the DPPH radicals by 5.1%, 11.3%, and 18.4%, respectively (Fig- oxidative stress in human hepatic HepG2 cells ure 1b), indicating that the former hydrolysate has a better radical scavenging activity than the latter. This different behaviour may be Excessive production of intracellular ROS leads to severe cellu- explained considering the different physiochemical properties of lar damage, which may affect proteins, DNA and lipid stability. these hydrolysates. Thus, Soybean P is predominantly character- (Dhalla et al., 2000) For this reason, in order to evaluate whether ized by peptides ranging from 1 to 1.2 kDa, whereas Soybean T Soybean P and T hydrolysates modulate the H2O2-induced ROS contains mostly large amounts of medium and long peptides with a production, HepG2 cells were pre-treated with both hydrolysates molecular weight larger than 2 kDa. Moreover, the average hydro- (0.5 and 1.0 mg/mL) O/N at 37 °C. The following day, the same phobicity of pepsin peptides is larger than that of trypsin peptides cells were treated with 0.5 mM H2O2 for 30 min at 37 °C. Results (Lammi et al., 2019). Instead, Soybean P contains 22.2% peptides (Figure 2) clearly highlight that HepG2 cells, exposed to H2O2 with lengths ranging from 8 to10 amino acid residues and an av- alone, produce a significant augmentation of intracellular ROS lev- erage hydrophobicity of 48.1 kcal mol−1, 73.6% peptides with a els by 29.5% vs control cells (p < 0.01), which was attenuated by length of 11–20 amino acid residues and an average hydrophobic- the pre-treatment with Soybean P and T hydrolysates: Soybean P −1 ity of 44.5 kcal mol , and 4.2% of peptides with a length of 20–21 reduced the H2O2-induced intracellular ROS by 19.5% and 14.2% amino acids and an average hydrophobicity of 50.7 kcal mol−1. On at 0.5 and 1.0 mg/mL, respectively, whereas Soybean T by 17.3% the contrary, Soybean T contains 6.2% peptides with a length of and 13.3% at 0.5 and 1.0 mg/mL, respectively (p < 0.05). These 9–10 amino acid residues and an average hydrophobicity of 32.2 findings underline a dramatic increase of intracellular ROS, but kcal mol−1, 67.2% peptides with a length of 11–20 amino acid resi- the pre-treatment with Soybean P and T hydrolysates significantly dues and an average hydrophobicity of 39.2 kcal mol−1, and 26.6% protects the HepG2 cells restoring the ROS level to basal condition peptides with a length of 20–27 amino acids and an average hydro- and confirming their good ability to act as natural antioxidant. As phobicity of 40.4 kcal mol−1. already underlined, high oxidative stress results in significant dam- Even though, the radical scavenging activity of food protein age to human cells by altering proteins, lipids and DNA, leading hydrolysates is influenced by many factors (i.e. the proteases to several simultaneous processes, which may culminate in patho- used for the generation of the hydrolysates, size and amino acid logical conditions involved in CVD progression. composition of the peptides, and the DPPH assay conditions), our Lipid of cellular membranes are susceptible to an oxidative findings are in line with previous studies (Aluko & Monu, 2003; attack, typically by ROS, resulting in a well-defined chain reac- Li, Jiang, Zhang, Mu, & Liu, 2008; Udenigwe, Lu, Han, Hou, & tion with the production of end products such as malondialdehyde Aluko, 2009). In particular, soybean P and T hydrolysates resulted (MDA). Based on these considerations, the capacity of Soybean P

Journal of Food Bioactives | www.isnff-jfb.com 45 Antioxidant activity of soybean peptides on human hepatic HepG2 cells Lammi et al.

Figure 2. Evaluation of the effects of Soybean P and T hydrolysates on the H2O2-induced ROS production levels at human hepatic HepG2 cells. The Figure 4. Investigation of the ability of Soybean P and T hydrolysates data points represent the averages ± sd of six independent experiments in to modulate the H2O2-induced NO level production at human hepatic duplicate. (*) p < 0.05, (**) p < 0.01. C: control cells. HepG2 cells. The data points represent the averages ± sd of six independent experiments in duplicate. (*) p < 0.05, (**) p < 0.01. C: control cells. and T hydrolysates to modulate the H2O2-induced lipid peroxidation in human hepatic HepG2 cells was assessed measuring the 0.5% was observed (p < 0.001). In addition, the pre-treatment of reaction of MDA with thiobarbituric acid (TBA) to form fluoro- HepG2 cells with both Soybean P and T hydrolysates determine a metric (λex = 532/λem = 553 nm) product, proportional to the significant reduction of lipid peroxidation even under basal con- MDA present. Figure 2 clearly suggests that, in agreement with the ditions (p < 0.01). As illustrated in the Figure 3, Soybean P de- observed increase of ROS after the H2O2 treatment, a significant creases the lipid peroxidation up to 108.1 ± 2.2% and 95.8 ± 0.2% increase of the lipid peroxidation at cellular level up to 112.4 ± at 0.5 and 1.0 mg/mL, respectively, whereas Soybean T up to 93.1 ± 5.3% and 93.3 ± 1.0% at 0.5 and 1.0 mg/mL, respectively. Since the lipid peroxidation is a validated marker of oxidative stress, these findings confirm the good antioxidant property of soybean hydrolysates and that the tryptic hydrolysate is more active than the peptic one.

3.3. Soybean P and T hydrolysates modulate the H2O2-induced NO production in human hepatic HepG2 cells

The ROS act either as a signalling molecule or a mediator of inflammation (Mittal, Siddiqui, Tran, Reddy, & Malik, 2014). Su- peroxide can rapidly combine with NO to form reactive nitrogen species (RNS), such as peroxynitrite, with a reaction rate that is faster than the dismutation of superoxide by superoxide dismutase (Beckman, 1996). In addition, the RNS lead to a nitrosative stress, which parallels the pro-inflammatory activity of ROS (Sunil, Shen, Patel-Vayas, Gow, Laskin, & Laskin, 2012). More and more emerging evidences clearly underline the intricate relation between oxidative stress and inflammation Mittal( et al., 2014). Based on these considerations, the Soybean P and T hydrolysates effects on the NO level production were evaluated at human hepatic HepG2 cells, after oxidative stress induction. A H2O2 treatment was used to induce the oxidative stress and the NO levels, produced at intracellular levels, were measured. Figure 4 clearly indicates that the H2O2 treatment dramatic increases the NO levels Figure 3. Evaluation of the effects of Soybean P and T hydrolysates on the up to 383.6 ± 94.1% (p < 0.05) and that the pre-treatment with soybean peptides reduces the H O -induced NO levels leading their H2O2-induced lipid peroxidation levels at human hepatic HepG2 cells. 2 2 The data points represent the averages ± sd of six independent experi- values closer to the basal levels (p < 0.01). In particular, Soybean P ments in duplicate. (***) p < 0.001. C: control cells. (0.5 and 1.0 mg/mL) decreases the NO level up to 139.1 ± 34.7%

46 Journal of Food Bioactives | www.isnff-jfb.com Lammi et al. Antioxidant activity of soybean peptides on human hepatic HepG2 cells and 125.9 ± 14.7%, whereas Soybean T (0.5 and 1.0 mg/mL) up duces oxidative stress factors in spontaneously hypertensive rats. to 125.8 ± 22.6% and 125.6 ± 35.8%, respectively. Interestingly, Nutrients 6(12): 5652–5666. these findings confirm the same trend that was observed assessing Girgih, A.T., Udenigwe, C.C., and Aluko, R.E. (2011). In vitro antioxidant properties of hemp seed (Cannabis sativa L.) protein hydrolysate the effect of soybean peptides on the modulation of H2O2-induced cellular lipid peroxidation. In particular, Soybean T hydrolysate fractions. J Am Oil Chem Soc 88(3): 381–389. Ibrahim, H.R., Isono, H., and Miyata, T. (2018). Potential antioxidant bio- seems to be also in this case slightly more active to restore the active peptides from camel milk proteins. Anim Nutr 4(3): 273–280. basal intracellular NO levels. Iqbal, J., and Hussain, M.M. (2009). Intestinal lipid absorption. Am J Physi- ol Endocrinol Metab 296(6): E1183–1194. Lammi, C., Aiello, G., Boschin, G., and Arnoldi, A. (2019). Multifunctional 4. Conclusion peptides for the prevention of cardiovascular disease: A new concept in the area of bioactive food-derived peptides. J Funct Foods Soybean is an interesting source of bioactive peptides useful for 55: 135–145. Lammi, C., Arnoldi, A., and Aiello, G. (2019). Soybean Peptides Exert Mul- the development of functional foods and nutraceuticals. Many tifunctional Bioactivity Modulating 3-Hydroxy-3-Methylglutaryl-CoA evidences clearly suggest that soybean peptides mediate hypocho- Reductase and Dipeptidyl Peptidase-IV Targets in Vitro. J Agric Food lesterolemic, hypotensive and hypoglycaemic activities which are Chem 67(17): 4824–4830. strictly related to oxidative stress. Our results indicate that soybean Lammi, C., Zanoni, C., and Arnoldi, A. (2015a). IAVPGEVA, IAVPTGVA, and peptides could also contribute to an antioxidant activity which is LPYP, three peptides from soy glycinin, modulate cholesterol- me linked to the modulation of intracellular ROS and NO levels lead- tabolism in HepG2 cells through the activation of the LDLR-SREBP2 ing to a reduction of lipid degradation. pathway. J Funct Foods 14: 469–478. Lammi, C., Zanoni, C., and Arnoldi, A. (2015b). Three peptides from soy glycinin modulate glucose metabolism in human hepatic HepG2 Acknowledgments cells. Int J Mol Sci 16(11): 27362–27370. Li, Y., Jiang, B., Zhang, T., Mu, W., and Liu, J. (2008). Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH). We acknowledge Carlo Sirtori Foundation (Milan, Italy) for hav- Food Chem 106(2): 444–450. ing provided part of equipment used in this experimentation and Liu, R., Xing, L., Fu, Q., Zhou, G.H., and Zhang, W.G. (2016). A Review of the Fondazione Cariplo, project “SUPER-HEMP: Sustainable Antioxidant Peptides Derived from Meat Muscle and By-Products. Process for Enhanced Recovery of Hempseed Oil” number: 2017- Antioxidants (Basel) 5(3): 32. 1005. Moreover, we thank the project “DISCOVERY—Disaggre- Liu, Z.-M., Ho, S.C., Chen, Y.-M., Ho, S., To, K., Tomlinson, B., and Woo, J. (2014). Whole soy, but not purified daidzein, had a favorable ef- gation of conventional vegetable press cakes by novel techniques fect on improvement of cardiovascular risks: A 6-month randomized, to receive new products and to increase the yield”, bando ERA- double-blind, and placebo-controlled trial in equol-producing post- NET SUSFOOD2 menopausal women. Mol. Nutr. Food Res. 58(4): 709–717. Lorenzo, J.M., Munekata, P.E.S., Gómez, B., Barba, F.J., and Toldrá, F. (2018). Bioactive peptides as natural antioxidants in food products– Author contributions A review. Trends Food Sci Tech. 79: 136–147. Mittal, M., Siddiqui, M.R., Tran, K., Reddy, S.P., and Malik, A.B. (2014). Reactive oxygen species in inflammation and tissue injury. Antioxid Experiment ideation and design, CL.; Biological experiments, Redox Signal 20(7): 1126–1167. C.L. C.B. Data analysis, C.L. and C.B.; Discussion of the results, Nazeer, R.A., Kumar, N.S., and Jai Ganesh, R. (2012). In vitro and in vivo C.L., Manuscript writing, C.L. and A.A. studies on the antioxidant activity of fish peptide isolated from the croaker (Otolithes ruber) muscle protein hydrolysate. Peptides 35(2): 261–268. Conflict of interest Siow, H.L., and Gan, C.Y. (2013). Extraction of antioxidative and antihypertensive bioactive peptides from Parkia speciosa seeds. Food Chem 141(4): 3435–3442. The authors declare they have no conflicts of interest. Sunil, V.R., Shen, J., Patel-Vayas, K., Gow, A.J., Laskin, J.D., and Laskin, D.L. (2012). Role of reactive nitrogen species generated via inducible nitric oxide synthase in vesicant-induced lung injury, inflammation and References altered lung functioning. Toxicol Appl Pharmacol 261(1): 22–30. Udenigwe, C.C., Lu, Y.L., Han, C.H., Hou, W.C., and Aluko, R.E. (2009). Flax- Aluko, R.E., and Monu, E. (2003). Functional and bioactive properties of seed protein-derived peptide fractions: Antioxidant properties and quinoa seed protein hydrolysates. J Food Sci 68(4): 1254–1258. inhibition of lipopolysaccharide-induced nitric oxide production in Beckman, J.S. (1996). Oxidative damage and tyrosine nitration from perox- murine macrophages. Food Chem 116(1): 277–284. ynitrite. Chem Res Toxicol 9(5): 836–844. Velasquez, M.T., and Bhathena, S.J. (2007). Role of dietary soy protein in Centenaro, G.S., Mellado, F.S., and Prentice-Hernandez, C. (2011). Antioxi- obesity. Intern J Med Sci 4(2): 72–82. dant activity of protein hydrolysates of fish and chicken bones. Adv J Wattanasiritham, L.K., and S Theerakulkait, C. (2015). Antioxidant activity Food Sci Technol. 3: 280–288. of rice bran protein extract, its enzymatic hydrolysates and its com- Dhalla, N.S., Temsah, R.M., and Netticadan, T. (2000). Role of oxidative bination with commercial antioxidants. Pakistan J Nut. 14: 647–652. stress in cardiovascular diseases. J Hypertens 18(6): 655–673. Wu, J., Xia, S., Kalionis, B., Wan, W., and Sun, T. (2014). The role of oxida- Fukui, K., Tachibana, N., Wanezaki, S., Tsuzaki, S., Takamatsu, K., Yamamo- tive stress and inflammation in cardiovascular aging. Biomed Res Int. to, T., and Shimoda, T. (2002). Isoflavone-free soy protein prepared 2014: 615312. by column chromatography reduces plasma cholesterol in rats. J Ag- Zambrowicz, A., Pokora, M., Setner, B., Dąbrowska, A., Szołtysik, M., Ba- ric Food Chem 50(20): 5717–5721. bij, K., and Chrzanowska, J. (2015). Multifunctional peptides derived Girgih, A.T., Alashi, A.M., He, R., Malomo, S.A., Raj, P., Netticadan, T., and from an egg yolk protein hydrolysate: isolation and characterization. Aluko, R.E. (2014). A novel hemp seed meal protein hydrolysate re- Amino Acids 47(2): 369–380.

Journal of Food Bioactives | www.isnff-jfb.com 47 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Original Research J. Food Bioact. 2019;7:48–55

Intestinal permeability and transport of apigenin across Caco-2 cell monolayers

Marina Zulkiflia*, Amin Ismailb,c*, Loh Su Pengb,c, Fadhilah Jailanid and Nur Kartinee Kassime aAlliance of Research and Innovation for Food (ARIF), Faculty of Applied Sciences, Universiti Teknologi MARA Negeri Sembilan, Kuala Pilah Campus, 72000 Kuala Pilah, Negeri Sembilan, Malaysia bDepartment of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia cResearch Centre of Excellence, Nutrition and Non-communicable Diseases, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia dDepartment of Food Science and Technology, Faculty of Applied Sciences, Universiti Teknologi MARA Shah Alam, 40450 Shah Alam, Selangor, Malaysia eDepartment of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia *Corresponding author: Marina Zulkifli, Alliance of Research and Innovation for Food (ARIF), Faculty of Applied Sciences, Universiti Teknologi MARA Negeri Sembilan, Kuala Pilah Campus, 72000 Kuala Pilah, Negeri Sembilan, Malaysia. E-mail: [email protected]. Amin Ismailand, Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. E-mail: [email protected] DOI: 10.31665/JFB.2019.7198 Received: September 22, 2019; Revised received & accepted: September 29, 2019

Abbreviations: CO2, carbon dioxide; DAD, diode array detector; DMEM, dulbecco’s modified eagle’s medium; ESI, electrospray ionization; ESI-QTOF-MS, electrospray ionization-quadrupole time of flight-tandem mass spectrometry; FBS, fetal bovine serum; HPLC-DAD, high performance liquid chromatography diode array detector; HBSS, hank’s balance salt solution; LCMS-QTOF-MS, liquid chromatography mass spectrometry- quadrupole time of flight-tandem mass spectrometry; mm, millimetre; min, minute; MRPs, Multidrug resistance- related proteins; MS, Mass spectrometry; °C, Degree celcius; PBS, Phosphate buffer solution; µl, Microlitre; MW, Molecular weight; P-gp, P-glycoprotein; RPM, Route per minute; secs, Seconds; SD, Standard deviation; SPSS, Statistical package for the social sciences Citation: Zulkifli, M., Ismail, A., Peng, L.S, Jailani, F., and Kassim, N.K. (2019). Intestinal permeability and transport of apigenin across Caco-2 cell monolayers. J. Food Bioact. 7: 48–55.

Abstract

Investigation on bioavailability was carried out by determining the absorption and transport of bioaccessible apigenin from fruit of mango (Mangifera indica var. Water Lily) into Caco-2 human intestinal cell using a reliable and sensitive analytical method of LC-MS/MS. Results revealed that the concentration of glucuronidated apigenin was lower than apigenin. The apigenin was metabolised inside the cells through glucuronidation process, and cross the monolayer to reach the basolateral sides or effluxed back to the apical side. The permeability coefficient of apigenin from apical to basolateral sides and basolateral to apical sides showed a medium permeability of less than 20 ×10−6 cm·sec−1. Since the value of efflux ratio was 1.5, it suggested that apigenin was absorbed and transported through a simple diffusion mechanism.

Keywords: Bioavailability; Permeability coefficient; Apigenin; LC-MS/MS.

1. Introduction the target sites in the body. The fraction must be available to achieve any effect in a specific tissue, in which it is able tobe Bioavailability is the fraction of a nutrient that is digested, ab- extracted from the food matrix and subsequently be absorbed from sorbed, metabolized through the systemic circulation and reached the gut through the intestinal cells by passive diffusion or by active

48 Copyright: © 2019 International Society for Nutraceuticals and Functional Foods. All rights reserved. Zulkifli et al. Intestinal permeability and transport of apigenin across Caco-2 cell monolayers transport (Soler et al., 2010; Stahl et al., 2005). The bioavailability 2.2. Maintenance of Caco-2 cell lines of polyphenol varies for each compound and the highest concentration in the food is not necessarily high bioavailability (Manach The human colorectal adenocarcinoma cell line (Caco-2) cells were et al., 2005). The polyphenol bioavailability is reported to be low purchased from the American Type Culture Collection (Manassas, due to the sulfation and glucuronidation of free hydroxyl groups in VA, USA). Frozen Caco-2 cells were defrosted and routinely cul- its structure (Tang et al., 2012). In spite of its low bioavailability, tured in 25 cm2 flasks before transferred to 75 cm2 flasks in Dul- the polyphenol may be retain in the gastrointestinal lumen with becco’s Modified Eagle’s Medium (DMEM) consisting of 10% fe- greater amount in which they play an important function in pre- tal bovine serum (FBS) and 1% penicillin/streptomycin. The cells serving the oxidative damage and in preventing the stomach, colon were grown and differentiated in a humidified atmosphere of 5% and rectal cancer (Halliwell et al., 2000). CO2 and 95% air at 37 °C (Esco, Singapore). Media were changed Human colorectal adenocarcinoma Caco-2 cells have been three times per week by discarding the culture medium and rinsed widely used in in vitro studies on transport of drug across the in- with phosphate buffer solution (PBS). The cells were subcultured testinal epithelial cells for more than 30 years. The Caco-2 cell when it reached 80% confluency by treatment with a solution con- line is a heterogeneous population due to different transport rates, taining 1.5 ml trypsin/ethylenediamine tetraacetic acid (0.05%, 1 brush border assembly, and other differentiation characteristics of mL) for 7 min. When the cells detached, the reaction was stopped the population (Gonzales et al., 2015). Currently, Caco-2 cells are by adding 3 mL of complete supplemented media. About 2 mL of commonly used in the development and discovery of drug. During cells were transferred into 75 cm2 flask for at least three passages the growth phase, the Caco-2 cells form semi-spheroid clusters, before being cultured in a 6 well Transwell® plate for transport with the formation of tight junction between adjacent cells. When experiment. The remaining cell suspensions were centrifuged (Tut- it reached confluence and differentiation, brush border microvilli tlingen, Germany) at 210 × g for 5 min at 4 °C for cell preservation. will cover the apical membrane, leading to the morphological po- larization of the monolayer. Caco-2 cells also participate in mechanism of apical efflux, which is responsible in restricting oral drug 2.3. Culturing Caco-2 cell monolayer absorption. There are various morphological and functional properties of Caco-2 cells were seeded at 6 × 104 cells/cm2 and subsequently small bowel enterocytes expressed by Caco-2 cell such as for- cultured on a 6 well Transwell® plate (polycarbonate membrane, mation of tight junctions and microvilli, brush border enzymes, pore size of 0.4 µm, membrane diameter of 24.5 mm, surface area phase II conjugation enzymes as well as membrane transport of 4.7 cm2) then incubated in humidified atmosphere (37 °C, 5% proteins. Two main carrier superfamilies are associated in drug CO2, 95% air). Cells were allowed to grow and differentiate to absorption are ATP-binding cassettes (ABC) and solute carri- confluent monolayers for 21–27 days post seeding. Trypan blue ers (SLCs). The ABC proteins like P-glycoprotein (P-gp) and and haemocytometer were used during cell seeding for determina- Multidrug Resistance Protein (MRP) are involved in the intes- tion of total cell counts and viable cell number. The medium was tinal transport and absorption mechanism of several compounds changed three times per week. The passage number used for the (Crowe and Wright, 2012). Hence, the cell has been widely used experiment was between 35–45. to study the characteristics of polyphenols metabolism in the small intestine. 2.4. Evaluation of compound stability in cell culture transport To date, there is a lack of knowledge concerning the transport of apigenin particularly from mango fruit through the human intes- media tinal cell monolayers. Hence, this study focused on investigating the absorption and transport of bioaccessible fraction of apigenin The stability of apigenin was evaluated based on modified method from Water Lily mango obtained during simulation gastrointesti- by Fadhilah and Williamson (2014). About 1 mL of digested sam- nal in epithelial cell monolayers using Caco-2 cell culture model ple was mixed with 1 mL of Hank’s Balance Salt Solution (HBSS). and paid particular attention to the production of conjugates within The pH was adjusted to 7.4 with 1M sodium hydroxide or 1M the cells and its efflux from the cells. hydrochloric acid. Next, the solution was kept in an incubator of 5% CO2 and 95% air at 37 °C. The sample was collected at 0 min and after 30, 60, 90, 120 and 180 min incubation. Then, 100 µL of 2. Material and methods sample were mixed with 100 µL ethanol containing genistein as an internal standard. The sample was vortexed, mixed and centrifuged at 17,000 × g for 10 min before being analysed using a high perfor- 2.1. Preparation of sample mance liquid chromatography-diode array detector (HPLC-DAD).

The fruit pulp of mango (Mangifera indica var. Water Lily) was cut into small pieces then freeze-dried at −40 °C for 28 hr. Then, 2.5. Evaluation of monolayer integrity 3 g of freeze-dried sample were homogenized in 20 mL of 80% methanol using a homogenizer. The mixture was sonicated for 30 The monolayer integrity in transwells was determined by phenol min at 30 °C in ultrasonic bath, followed by the centrifugation at red method as described by Soler et al. (2010) with slight modi- 12,000 × g for 15 min at 5 °C, and filtered through a Whatman fications. The culture medium from apical and basolateral com- filter paper No. 1. The extract was concentrated to dryness in a partments were removed and washed three times with phosphate rotary evaporator. Then 100 mg of extract were mixed with 10 mL buffer solution (PBS). About 0.6 mL of DMEM containing phe- of 0.9% saline. All solutions were prepared freshly and subjected nol red was filled to the apical compartment whereas 1.5 mL of to in vitro digestion immediately after preparation. The digested PBS was loaded to the basolateral compartment. Then cells were extract was stored at −20 °C in a tightly sealed container for trans- placed in the incubator at 37 °C (5% CO2 and 95% O2) and diffu- port experiment. sion of phenol red across the monolayer from apical to basolateral

Journal of Food Bioactives | www.isnff-jfb.com 49 Intestinal permeability and transport of apigenin across Caco-2 cell monolayers Zulkifli et al. was allowed for 1 h. Aliquots of 100 μL were removed from both min). Identification of compounds was done by comparing the compartments and absorbance were measured at 570 nm. Well that spectra and retention time of pure standards. The concentration of supported less than 1% of phenol red transport was considered ac- apigenin aglycone and metabolite were calculated for both apical ceptable for transport experiment. The integrity of monolayer was and basolateral compartments by constructing the standard curve performed before and after the cell treatment. The percentage of ranging from 20–100 µg/mL. phenol red diffusion was calculated as follows: Basolateral× 100 2.9. Confirmation by LCMS-QTOF-MS Percentage of diffusion = abs Apicalabs Mass spectrometry analysis of apigenin aglycone and its metabolite after incubation of bioaccessible fraction were conducted by LC- 2.6. Determination of transepithelial transport across Caco-2 QTOF-MS (Agilent Technologies Inc., Palo Alto, CA) equipped cell monolayer with electrospray ionization source. Ionisation was achieved using an electrospray (ESI) interface operating in the negative mode − Transport experiment was performed according to the method of [M-H] . Spectral data in the range of m/z 50 to 1,100 were applied Fadhilah and Williamson (2014) with some modifications. The with negative ionization mode. High purity nitrogen was used as transport buffer solution containing Hank’s Balance Salt Solution the dry gas at a flow rate of 10 L/min, and at a pressure of 30 psi. (HBSS) supplemented with 1.8 mM calcium chloride (to preserve Gas and vapourizer temperature were set at 325 °C, and the scan the integrity of the tight junction) and 100 µM ascorbic acid (to source parameters were capillary voltage and fragmentor with 4 reduce the degradation of apigenin) was prepared freshly. Medium kV and 175, respectively. Data were acquired in MS and MS/MS was aspirated and rinsed three times with transport buffer (modi- scanning modes. The intestinal apparent permeability coefficient fied HBSS, pH 7.4). Both apical and basolateral compartments (Papp) was calculated for bidirectional transport experiments us- were filled with 2 mL of modified HBSS and incubated (37 °C, ing the following equation: 5% CO /95% O ) in order to equilibrate the pH of cell monolay- V dC 2 2 Papp (cm/sec) = × ers to pH 7.4. After 30 min, the HBSS was aspirated to waste. The CoA dt digested sample solution (1 mL) containing apigenin in modified HBSS (40 µM) was added to the apical compartment whereas the where: V = volume of the solution in the receiving compartment, basolateral compartment was filled with 2 mL of modified HBSS A = membrane surface area, Co = initial concentration in donor and equivalent amount of DMSO. For blank transport control, only compartment, dC/dt = change in apigenin concentration in the re- modified HBSS was loaded to both apical and basolateral com- ceiver over time. partments. After 2 h incubation in humidified atmosphere, sample from two compartments were collected, deproteinated and imme- 2.10. Statistical analysis diately frozen at −80 °C prior to analysis. The monolayer integrity was performed before and after the experiment by phenol red method. The same protocol was repeated from basolateral to apical Data were average of three independent experiments. Statistical compartment for efflux study. analysis was conducted using a Analysis of Variance (ANOVA) by SPSS statistical package for Windows (21.0; SPSS Inc., Chicago, IL, USA). The level of significance was set at p < 0.05. 2.7. Sample preparation

Samples from transport study were defrosted at room temperature 3. Results and discussion and 300 µl solutions were mixed with 300 µl ethanol consisting of genistein (2 µM) as an internal standard. The mixture was then 3.1. Stability of apigenin vortexed and centrifuged at 17,000 × g for 10 min. The supernatant was transferred to an Eppendorf tube and evaporated to dryness under nitrogen gas. Next, dried samples were reconstituted in 600 The stability of apigenin in transport buffer solution was - per µL of 50% ethanol in water (50:50, v/v) containing ferulic acid formed to ensure that the compound withstand the experimental (internal standard, 2 µM), vortexed and subsequently centrifuged conditions throughout the transport study. Figure 1 indicates the for 10 min at 17,000 × g. Samples were analysed using a HPLC- changes in concentration of apigenin at time intervals up to 180 DAD and confirmed by electrospray ionization-quadrupole time of min incubation at 37 °C in HBSS transport buffer solution (pH flight-tandem mass spectrometry (ESI-QTOF-MS) 7.4). At 0 min, there was 98.82% of apigenin initial concentration and left about 88.68% concentration after 180 min incubation. The apigenin observed to be stable in HBSS buffer solution as only 2.8. Quantification by HPLC-DAD slight changes noticed. Even though no data on the stability of apigenin reported, but HPLC separation and quantification of apigenin aglycone and its these results are in accordance with other studies by Kosinka et al. metabolite produced by Caco-2 permeates were carried out using (2012) indicating that the catechin, epicatechin and procyanidin an Agilent series 1100 system (Agilent, Germany) equipped with in cocoa extract were stabled during incubation at 37 °C for 120 diode array detector (DAD). The analytical column used was 250 min. Stability of pelargonidin extracted from strawberry was also × 4.0 mm i.d, 5 µm Lichrosper C18, kept at 25 °C. The mobile constant in HBSS buffer at pH 7.4 for 120 min. phase applied was 0.5% (v/v) acetic acid in water (eluent A) and HBSS is widely used as a buffer for transport study in cell cul- 100% methanol (eluent B) with a total run time of 30 min at a ture models (Hubatsch et al., 2007). The buffer was supplemented flow rate of 0.6 ml/min. Gradient elution was conducted as fol- with 1.8 mM CaCl2 with the purpose to maintain the integrity of lows; 0–90% B (0–20 min); 90% B (20–25 min); 90–0% B (25–30 the tight junction and addition of 100 µM ascorbic acid to prevent

50 Journal of Food Bioactives | www.isnff-jfb.com Zulkifli et al. Intestinal permeability and transport of apigenin across Caco-2 cell monolayers

Figure 1. Stability of apigenin in transport buffer solution at pH 7.4 incubated over 180 min at 37 °C. Stock solution of apigenin was diluted to a final concentration of DMSO (0.4%) in transport buffer solution containing HBSS supplemented with 1.8 mM CaCl2 and 100 µM ascorbic acid. the compound degradation. A study by Halliwell (2003) showed transport experiments. that most polyphenol compounds were readily oxidized in aqueous media with pH higher than 7, resulted in the formation of poly- 3.2. Monolayer integrity phenolic oxidation products and H2O2. The information on compound stability in transport buffer solution is necessary to ensure the amount present in apical and basolateral compartments during Due to the absence of transepithelial electrical resistance (TEER)

Figure 2. Passage of phenol red across Caco-2 cell monolayers after 1 h incubation. DMEM containing phenol red was applied to the apical compartment whereas PBS was loaded to the basolateral compartment, then incubated for 1 h at 37 °C.

Journal of Food Bioactives | www.isnff-jfb.com 51 Intestinal permeability and transport of apigenin across Caco-2 cell monolayers Zulkifli et al.

Table 1. LC-MS/MS of apigenin and its metabolite formed after incubation using Caco-2 cells monolayer as a model of the human intestinal epithelium Peak Compound Retention time (min) Theoretical [M-H]− (m/z) Experimental [M-H]− (m/z) Fragment ion [M-H]− (m/z) 1 Apigenin 17.29 269.0467 269.0498 176.9943 112.9858 68.9968 2 Apigenin 14.86 445.0849 445.0767 269.0453 glucuronide 217.0033 141.0168 113.0241 with epithelial voltohmeter instrument, the integrity of Caco-2 cell apical compartment after incubation of bioaccessible apigenin pre- monolayer was measured using the phenol red permeability meth- pared in modified HBSS. Both figures show similar fragmentation od. The stability of Caco-2 cells monolayer integrity in the pres- patterns of ions for apigenin and its metabolite but only differ on ence of solution treatments were determined by changes in percent the bidirectional transport from apical to basolateral (a→b) direc- diffusion values as illustrated in Figure 2. There is a clear trend of tion and from basolateral to apical (b→a) direction. decreasing percentage diffusion of phenol red from upper to lower Using the Caco-2 cell monolayer, the bidirectional transport and compartments both for the control (without cell) and Caco-2 cells. metabolism of apigenin and glucuronidated metabolite in bioacces- The values decreased gradually for Caco-2 cells until reached the sible fraction were investigated from a to b direction and from b to confluency at day 21. After the transport experiment, the mon- a direction. Table 2 demonstrates the concentration of apigenin and olayer integrity of Caco-2 cell was 1.2% but still considered as glucuronidated apigenin produced in the apical and basolateral com- acceptable value. Statistically, there was no significant difference partments after 2 h incubation. The concentration of apigenin agly- in percentage of phenol red diffusion after cell treatment, probably cone absorbed into the basolateral compartment from a to b direction that the integrity of cell monolayer maintained and no disruption was lower than the concentration effluxed to the apical compartment of the monolayer or cell lysis observed. from b to a direction with the values of 27.99 pmol/cm2·min and It was suggested that cells formed confluent monolayer at day 37.29 pmol/cm2·min, respectively. Meanwhile, the glucuronidated 21 as the percentage diffusion of phenol red was 0.1%. According apigenin showed preferential apical efflux (6.55 pmol/cm2·min) to Fale et al. (2010), well with the value less than 1% of phenol compared to the basolateral absorption (5.71 pmol/cm2·min). red transport under this condition was considered suitable for use Previous study reported that during transport through the in the transport study. The Caco-2 cell monolayers were believed Caco-2 cell monolayer, apigenin was metabolized into a number to form a brush border microvilli that resembles the luminal mem- of Phase II conjugates, most notably glucuronides and sulphates brane of the intestinal epithelium. The structural and functional (Gradolatto et al., 2008). The main reason for its poor bioavail- differentiation of the brush border microvilli is associated with po- ability could be due to the extensive first-pass metabolism (Tang et larization of the epithelial monolayers with the presence of tight al., 2012). In the current study, it demonstrated that the major form junctions. The present study exhibited that Caco-2 cultured on of compound transported across the cells was aglycone. Glucuro- Transwell insert can differentiate spontaneously as well as express nidation of apigenin also occurred at the highest concentration in numerous morphological and functional properties of character- apical efflux metabolized by UDP-glucuronosyltransferase (UGT). istics of mature enterocytes. The main advantage of this cellular The enzyme that contributes to glucuronidation of flavonoids in line was associated with its stability when grown as monolayers on humans is also present in Caco-2 cells (Galijatovic et al., 2001). Transwell inserts that resemble the parental Caco-2 parent cell line Contrary to expectations, no sulfated apigenin detected in media (Soler et al., 2010). solutions, and possible explanation for this could be related to the structure of apigenin with only one substituted hydroxyl group on the b ring, which is less susceptible to sulphation. Lower conjuga- 3.3. Identification of apigenin and its metabolite tion activity of the Caco-2 strain used in the study might also be a possible reason for the present result as reported by Fadhilah and Identification of apigenin and its metabolite in the basolateral com- Williamson (2014). partment after incubation for 2 h in humidified atmosphere is tabu- Since Caco-2 cell is an in vitro model for the intestinal absorp- lated in Table 1. The compounds produced by the intestinal cells tion, it is important to know the directional movement of parent were identified and detected by comparing the ESI-MS spectra compound in Caco-2 cells from apical to basolateral and from in full scan mode and its retention time with those the standards. basolateral to apical directions. The intestinal apparent perme- Results of LC-MS/MS analysis revealed that apigenin was identi- ability coefficient (Papp) of apigenin aglycone was calculated to fied on the basis that the peak of parent ion (M+) at m/z 269.0498 facilitate the estimation of absorption and transport through the and fragment ions were at m/z 176.9943, 112.9858, and 68.9968 Caco-2 cell monolayer. As shown in Figure 5, the apigenin agly- at 17.29 min. The ESI-MS spectrum of the bioaccessible apigenin cone demonstrates higher permeability value (Papp) in b to a di- eluted at 14.86 min produced a parent ion (M+) at m/z 445.0767, rection (17.89 × 10−6 cm·sec−1) than its permeability in opposite with formation of four fragment ions at m/z 269.0453, 217.0033, direction (12.44 × 10−6 cm·sec−1), suggesting that the permeabil- 141.0168 and 113.0241. Therefore, it was confirmed the identifica- ity value is dependent on direction. This observation supports the tion of metabolite as apigenin glucuronide. idea that the secretion rate of apigenin transport (Papp from b to Meanwhile, Figure 3 demonstrates the ESI-MS/MS spectra of a) direction was greater than the absorptive rate of transport (Papp apigenin (a) and apigenin glucuronide (b) detected in the basolat- from b to a) direction, and considered as moderate permeability eral compartment while the Figure 4 shows the ESI-MS/MS spec- based on the Papp values. Nevertheless, there were no changes tra of apigenin (a) and apigenin glucuronide (b) detected in the in permeability value of apigenin glucuronide in both directions,

52 Journal of Food Bioactives | www.isnff-jfb.com Zulkifli et al. Intestinal permeability and transport of apigenin across Caco-2 cell monolayers

Figure 3. LC-ESI-MS/MS spectra of apigenin (a) and generated metabolite (b) detected in the basolateral compartment after incubation of bioaccessible apigenin prepared in modified HBSS on the apical compartment.

Figure 4. LC-ESI-MS/MS spectra of apigenin (a) and generated metabolite (b) detected in the apical compartment after incubation of bioaccessible apigenin prepared in modified HBSS on the basolateral compartment.

Journal of Food Bioactives | www.isnff-jfb.com 53 Intestinal permeability and transport of apigenin across Caco-2 cell monolayers Zulkifli et al.

Table 2. Metabolism of apigenin by Caco-2 cells Metabolite production (pmol/cm2·min) Metabolite Transport Apical Basolateral Apigenin a→b 51.33 ± 2.08 27.99 ± 1.40 b→a 37.29 ± 1.94 43.65 ± 2.22 Apigenin glucuronide a→b 4.61 ± 0.56 5.71 ± 0.48 b→a 6.55 ± 0.87 5.31 ± 0.52

The culture incubated for 2 h in the presence of bioaccessible apigenin. a→b indicates the transport of compounds from apical to basolateral, b→a indicates the transport of compounds from basolateral to apical. a: Apical; b: Basolateral which is also suggested as moderate permeability since the Papp in decreasing its bioavailability. These carrier proteins were abun- value were less than 20 × 10−6 cm·sec−1 (Breemen and Li, 2005). dant in apical membranes of pharmacologically important epithe- Indication of transport mechanism can be determined based on lial barriers such as the intestinal epithelium (Ueda et al., 1999). the efflux ratio of apparent permeability values (Pappba/Pappab). In In Caco-2 cells, P-gp has shown to be highly expressed, make it general, efflux ratio more than 2 is an indicative of active efflux in suitable as cellular model for studying the intestinal permeability which the compound is actively pumped back into the intestinal of flavonoids. lumen, resulting in reducing its intestinal absorption. The values lower than 0.5 indicated an active influx, while a value equal to 1 was a predictive of passive diffusion (Hubatsch et al., 2007). It is 4. Conclusion noticeable that the efflux ratio of apigenin aglycone was 1.5, suggesting a passive diffusion transport mechanism. The results were Data obtained could support the use of in vitro Caco-2 cell model consistent with previous study by Tang et al. (2017), where the as a standard tool for prediction of in vivo studies in determining efflux ratio of apigenin was reported as 1, which also indicated the the intestinal absorption of apigenin. The present study has shown same transport mechanism. that apigenin from Water Lily mango pulp was taken up and me- In line with previous observation, it was observed that glucu- tabolized into glucuronide conjugate possibly by a phase II meta- ronidation of apigenin occurred inside the Caco-2 cells, and trans- bolic glucuronyltransferase when incubated in Caco-2 cell mon- ported back to the culture medium. According to Hu et al. (2003), olayer. However, no sulfate conjugate present, which is in contrast when the apigenin permeates the Caco-2 cell monolayer, the glu- to the expectation from the in vivo study. It was noticeable that curonidated apigenin was biotransformed in the cells and subse- the apigenin glucuronide was preferentially effluxed to the apical quently expelled back to apical side mainly by P-glycoprotein compartment possibly by the action of P-glycoprotein and multi- (P-gp) and multidrug resistance-related proteins (MRPs). Thus, drug resistance proteins. Apigenin transport demonstrated a medi- the intracellular concentration of apigenin has reduced, resulting um permeability for both directions from apical to basolateral and

Figure 5. Transport of apigenin and its metabolite across Caco-2 cell monolayers from apical to basolateral (a→b) and basolateral to apical (b→a) directions.

54 Journal of Food Bioactives | www.isnff-jfb.com Zulkifli et al. Intestinal permeability and transport of apigenin across Caco-2 cell monolayers basolateral to apical directions based on the apparent permeability CaCo-2 cells–Potential role in carginogen bioactivation. Pharmaceu- values, which was less than 20 × 10−6 cm·sec−1. Results on efflux tical Research 18: 374–379. ratio indicated that both compounds were transported through the Gonzales, G.B., Camp, J.V., Vissenaekens, H., Raes, K., Smagghe, G., and simple diffusion mechanism as the value of efflux ratio forapi- Grootaert, C. (2015). Review on the use of cell cultures, to study me- genin and apigenin glucuronide reported as 1.5 and 1, respectively. tabolism, transport and accumulation of flavonoids: From mono-cultures to co-culture systems. Comprehensive Reviews in Food Science and Food Safety 14: 741–754. Gradolatto, A., Canivence-Lavier, M.C., Basly, J.P., Siess, M.H., and Teys- Availability of data and material sier, C. (2008). Metabolism of apigenin by rat liver phase I and phase II enzymes and by isolated perfused rat liver. Drug metabolism and All data are presented in the main paper. Disposition 32: 58–65. Halliwell, B. (2003). Oxidative stress in cell culture: an under-appreciated problem. Federation of European Biochemical Societies Letters 540: 3–6. Acknowledgments Halliwell, B., Zhao, K., and Whiteman, M. (2000). The gastrointestinal tract: a major site of antioxidant action. Free Radical Research 33: The authors would like to thank the Ministry of Higher Education, 819–830. Universiti Teknologi MARA and Universiti Putra Malaysia for the Hu, F. (2003). Plant-based foods and prevention of cardiovascular disease: an overview. American Journal of Clinical Nutrition 78: 544S. technical support throughout the study. We also thank to Mr. Mohd Hubatsch, I., Ragnarsson, E.G.E., and Artursson, P. (2007). Determination Izwan Mohamad Yusof from iPROMISE UiTM for providing of drug permeability and prediction of drug absorption in Caco-2 service, advice and shared knowledge on LCMS-QTOF-MS. Our monolayers. Nature Protocols 2: 2111–2119. appreciation goes to all the highly dedicated, helpful, knowledge- Kosinka, A., Xie, Y., Diering, S., Heritier, J., and Andlauer, W. (2012). Stabil- able and very responsible technical staffs of Nutrition and Dietet- ity of phenolic compounds isolated from cocoa, green tea and straw- ics Department, Faculty of Medicine and Health Sciences, UPM berries in Hank’s balanced salt solution under cell culture conditions. especially to Mr. Syed Hasbullah, Mr. Eddie, Mrs. Maznah Ahmad Food Chemistry 62: 91–96. and Mrs. Suryani. Manach, C., Williamson, G., Morand, C., Scalbert, A., and Rémésy, C. (2005). Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. America Journal of Clinical Nutri- tion 81: 230S–242S. Conflict of interest Soler, A., Romero, M.P., Macia, S., Furniss, C.S.M., Kroon, P.A., and Motilva, M.J. (2010). Digestion stability and evaluation of the metabolism and The authors declare no competing interests. transport of olive oil phenols in the human small intestinal epithelial Caco-2/TC7 cell line. Food Chemistry 119: 703–714. Stahl, W., Berg, H.V.D., Arthur, J., Bast, A., Dainty, J., Richard, M.F., Gartner, References C., Haenen, G., Hollman, P., Holst, B., Frank, J.K., Polidaro, C., Rice- Evans, C., Southon, S., Vliet, T.V., Vina-Rebes, J., Williamson, G., and Astley, S.B. (2005). Bioavailability and metabolism. Molecular As- Breemen, R.B.V., and Li, Y. (2005). Caco-2 cell permeability assays to meas- pects of Medicine. 23: 39–100. ure drug absorption. Expert Opinion Drug Metabolism Toxicology Tang, D., Chen, K., Huang, L., and Li, J. (2017). Pharmacokinetics properties 1(2): 175–185. and drug interactions of apigenin, a natural flavone. Expert Opinion Crowe, A., and Wright, C. (2012). The impact of P-glycoprotein mediated on Drug Metabolism and Toxicology 13: 323–330. efflux on absorption of 11 sedating and less-sedating antihistamines Tang, L., Zhou, J., Yang, C.H., Xia, B.J., Hu, M., and Liu, Z.Q. (2012). Sys- using Caco-2 monolayers. Xenobiotica 42(6): 538–549. tematic studies of sulfation and glucuronidation of 12 flavonoids in Fadhilah, J., and Williamson, G. (2014). Effect of edible oils on quercetin, the mouse liver S9 fraction reveal both unique and shared positional kaempferol and galangin transport and conjugation in the intestinal preferences. Journal of Agricultural and Food Chemistry 60(12): Caco-2/HT29-MTX co-cultured model. Food and Function 5: 653–662. 3223–3233. Galijatovic, A., Otake, Y., Walle, U.K., and Walle, T. (2001). Induction of Ueda, K., Yoshida, A., and Amachi, T. (1999). Recent progress in P-glycopro- UPD-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in tein research. Anticancer Drug Design 14(2): 115–121.

Journal of Food Bioactives | www.isnff-jfb.com 55 Journal of International Society for Food Bioactives Nutraceuticals and Functional Foods

Original Research J. Food Bioact. 2019;7:56–62

Modulatory effect of selected phenolic acids on enzyme activities of purinergic signaling

Adedayo O. Ademiluyia*, Taiwo A. Adeyeyea, Opeyemi B. Ogunsuyia,b, Damilola M. Olatundea and Ganiyu Oboha aDepartment of Biochemistry, Federal University of Technology, P.M.B. 704, Akure. Nigeria bDepartment of Biomedical Technology, Federal University of Technology, P.M.B. 704, Akure. Nigeria *Corresponding author: Adedayo O. Ademiluyi, Department of Biochemistry, Federal University of Technology, P.M.B. 704, Akure. Ni- geria. Tel: +234(0)8038044248; E-mail: [email protected], [email protected] DOI: 10.31665/JFB.2019.7199 Received: May 28, 2019; Revised received & accepted: September 29, 2019 Citation: Ademiluyi, A.O., Adeyeye, T.A., Ogunsuyi, O.B., Olatunde, D.M., and Oboh, G. (2019). Modulatory effect of selected phenolic acids on enzyme activities of purinergic signaling. J. Food Bioact. 7: 56–62.

Abstract

The antioxidant activities and effects of selected phenolic acids (ferulic, caffeic,p -coumaric and chlorogenic acids) on enzymes (ATPdase, ecto-5′ nucleotidase, phosphodiesterase-5′ and Na+/K+ ATPase) of the purinergic signalling in isolated rat brain were assessed. The antioxidant properties as typified by the DPPH and ABTS radical scavenging ability as well as inhibition of thiobarbituric acid reactive substances (TBARS) production in isolated rat brain revealed that chlorogenic acid had the highest antioxidant effect. Furthermore, ferulic acid had the highest +Na / + K -ATPase (IC50 = 3.48 μM) stimulatory activity, as well as the highest ecto-5′ nucleotidase (IC50 = 6.22 μM) inhibitory activity. In addition, chlorogenic acid had the highest phosphodiesterase-5′ (IC50 = 21.03 μM) inhibitory activity while p-coumaric acid caused the highest inhibition of ATPdase (IC50 = 17.56 μM) activity. This study revealed that the phenolic acids altered critical enzymes of the purinergic signaling in addition to their antioxidant properties thus, suggesting their possible neuromodulatory effects.

Keywords: Purinergic system; Phenolic acids; Neuromodulation; Oxidative stress; Antioxidants.

1. Introduction cleoside triphosphate diphosphohydrolases, ecto-5′ nucleotidase and Na+/K+ ATPase which have been implicated in neurodegenerative diseases. Ecto-5′ nucleotidase is a magnesium-dependent The role of purinergic system in neuronal function has been well glycoprotein which functions in the hydrolysis of adenosine established and studied (Ademiluyi et al., 2016). Consequently, monophosphate to the nucleoside adenosine that consequently interest has been directed towards characterization of purinergic activate the inorganic phosphate (Pi) adenosine receptors (Sinha signalling in different regions of the brain and spinal cord. Vari- et al., 2016). Na+/K+-ATPase is a crucial enzyme responsible for ous purinergic-receptor subtypes have been shown to be widely maintaining the ionic gradient necessary for neuronal excitabil- distributed throughout the CNS, being present in neurons and ity. It is present at high concentrations in brain cellular mem- glia (Burnstock, 2007). It is now well established that ATP both branes, consuming about 40–50% of the ATP generated in this acts as a fast excitatory neurotransmitter and has potent long-term tissue, Na+/K+-ATPase activity is decreased in various chronic (trophic) role in cell proliferation, growth and development, and in neurodegenerative disorders. disease and cytotoxicity (Zimmermann et al., 2006). Nucleoside triphosphate diphosphohydrolases (EC 3.6.1.5, The purinergic signalling system is a ubiquitous extracellular NTPDases) are well characterized ectoenzymes in the central communication channel mediated by ATP and its extracellular nervous system. The phosphodiesterases (PDEs) are a super breakdown product adenosine as well as enzymes such as nu- family of enzymes that catalyze the hydrolysis of the nucleotide

56 Copyright: © 2019 International Society for Nutraceuticals and Functional Foods. All rights reserved. Ademiluyi et al. Modulatory effect of selected phenolic acids on enzyme activities monophosphates, cyclic adenosine monophosphate (cAMP), and 2.3. DPPH radical scavenging ability cyclic guanosine monophosphate (cGMP) to the corresponding nucleoside monophosphates. Several of the PDEs have already The scavenging ability of the phenolic acids against DPPH (1,1-di- been identified and characterized, including PDE-5, PDE-6, phenyl-2 picrylhydrazyl) radical was evaluated as described by PDE-8, PDE-9, PDE-10, and PDE-11, to name a few (Soderling Gyamfi et al. (1999). Briefly, appropriate dilution (50–1,000 µM) et al., 1999; Yuasa et al., 2000). Moreover, the NTPDase family, of the phenolic acid (1 mL) was mixed with 1 mL, 0.4 mM DPPH composed of the eight members NTPDase 1 to 8, is involved in in methanolic solution, the mixture was left in the dark for 30 min controlling nucleotide and nucleoside concentrations, acting to and the absorbance was taken at 516 nm. The DPPH free radical regulate purinergic neurotransmission (Rico et al., 2008) of which scavenging ability was subsequently calculated as percentage of E-NTPDase 1, 2, 3 and 8 are extracellular. NTPDase 1 hydro- control. lyses ATP directly to AMP, and UTP to UDP, whereas NTPDase 2 converts ATP to ADP. Immunohistochemical studies revealed that this enzyme is strongly associated with axon-like neuronal 2.4. ABTS radical scavenging ability structures in the brain, where it may act as a main regulator of extracellular ATP levels. The ability of the phenolic acids to scavenge 2,2′-azino-bis(3- Phenolic compounds are a large class of plant secondary me- ethylbenzthiazoline-6-sulphonic acid (ABTS) radical cation was tabolites, showing a diversity of structures, from rather simple determined according to the method of Re et al. (1999). The structures, e.g. phenolic acids, through polyphenols such as fla- ABTS.+ was generated by reacting an aqueous solution of ABTS vonoids, that comprise several groups, to polymeric compounds (7 mmol/L) with K2S2O8 (2.45 mmol/L, final concentration) in the based on these different classes. Phenolic compounds are known dark for 16 h and adjusting the absorbance at 734 nm to 0.700 to be responsible for the free radical scavenging and antioxidant with ethanol. Thereafter, 0.2 mL of appropriate dilution (0.05–13 activities of plants; they possess many biological effects, mainly mM) of the phenolic acid was added to 2.0 mL of ABTS.+ solution attributed to their antioxidant activities in scavenging free radi- and the absorbance was read at 734 nm after 15 min. The Trolox cals, inhibiting peroxides and chelating transition metals and can equivalent antioxidant capacity was subsequently calculated using protect against neurodegenerative diseases, (Adefegha et al., 2018; Trolox as the standard. Shahidi and Yeo 2018). Phenolic compounds have the potential to act against oxidative damage; therefore play a protective role in living organism. Because of their antioxidant activities, they 2.5. Lipid peroxidation and thiobarbibutric acid reactions are widely used in processed foods as a natural antioxidant. This study investigated the effects of some phenolic acids such as; caf- The lipid peroxidation assay was carried out using the modified feic, ferulic, p-coumaric and chlorogenic acid on some enzymes method of Ohkawa et al. (1979). Briefly, 100 μL of rat brain ho- of the purinergic system in isolated rat brain homogenate in vitro. mogenate fraction was mixed with a reaction mixture containing These phenolic acids are ubiquitous in plant foods such as caffeic 30 μL of Tris-HCl buffer (0.1 M, pH 7.4), phenolic acid solutions and chlorogenic acid from coffee (Oboh et al., 2013; Celli and de (50 μL) and 30 μL of 250 μM of freshly prepared FeSO4. The vol- Camargo 2019), ferrulic acid from millet and whole wheat (Gaw- ume was made up to 300 μL with distilled water before incuba- lik-Dziki et al., 2017; Kumari et al., 2019) and p-coumaric acid tion at 37 °C for 1 h. Thereafter, 300 μL of 8.1% sodium dodecyl from grains (wheat, rice, and corn) and fruits (apple, pears and sulfate (SDS) was added to the reaction mixture and was followed grapes (Pei et al., 2016; Shahidi et al., 2019). by addition of 600 μL of acetic acid/HCl (pH 3.4) mixture and 600 μL of 0.8% TBA (thiobarbituric acid). This mixture was incubated at 100 °C for 1 h. TBARS (thiobarbituric acid reactive substances) 2. Materials and methods produced were measured at 532 nm and expressed as percentage of control. 2.1. Sample preparation 2.6. Na+/K+-ATPase assay Based on the physiological plasma peak concentration of phenolic acids as described by Ishisaka et al. (2011), the concentrations of + + the phenolic acids (caffeic acid, ferulic acid, chlorogenic acid, The Na /K -ATPase activity was measured in whole rat brain ho- p-coumaric acid) used in this study were between 50–1,000 µM mogenate and incubated with the selected phenolic acids as de- (aqueous solutions). scribed by Wyse et al. (2000). The assay mixture consisted of 50 μL of Na+/K+-ATPase substrate buffer (pH 7.4) (containing in mM, 30 Tris-HCl, 0.1 EDTA, 50 NaCl, 5 KCl, and 6 MgCl2), 50 μL of 2.2. Chemicals and reagents each of the selected phenolics (0.05–13 mM), 50 μL of supernatant (50 μg of protein) in the presence or absence of 50 μL of ouabain Chemicals such as caffeic acid, ferulic acid, chlorogenic acid, (1 mM), in a final volume of 200 μL. The reaction was initiated p-coumaric acid, adenosine triphosphate (ATP), adenosine by the addition of 50 μL adenosine triphosphate (ATP) to a final monophosphate (AMP), ouabain, ammonium molybdate, thio- concentration of 3 mM. After incubating for 30 min at 37 °C, the barbituric acid, (TBA), phosphate buffer of different molarity, reaction was terminated by the addition of 70 μL of 50% (w/v) sodium hydroxide, benzene, and 5,5′-dithio-bis(2-nitrobenzoic trichloroacetic acid (TCA). The amount of inorganic phosphate acid) were purchased from Sigma-Aldrich, ChemieGmH (Stein- (Pi) released was quantified as described by Fiske and Subbarow heim, Germany). Acetic acid was procured from BDH Chemical (1925) using a reaction mixture that contained 100 μL of ammo- Ltd., (Poole, England). Except otherwise stated, all other chemi- nium molybdate (50 mM), 40 μL of reaction mixture from first cals and reagents are of analytical grade while the water was glass grid and 10 μL of ascorbic acid (8%). The enzyme activity was distilled. expressed in nmol. of Pi/mg of protein/min.

Journal of Food Bioactives | www.isnff-jfb.com 57 Modulatory effect of selected phenolic acids on enzyme activities Ademiluyi et al.

2.7. ATPDase assay

The effect of the phenolic acids on ATPDase activity was determined as described by Schetinger et al. (2000), with slight modification. The assay mixture consisted of 5 mM CaCl2, 5 mM KCl, 0.1 mM EDTA, and 50 mM Tris-HCl buffer (pH 7.4) in a final reaction volume of 200 μL. Twenty microliters of the supernatant (enzyme preparation) with protein content of 8–12 μg were added to the reaction mixture and pre-incubated at 37 °C for 10 min. The reaction was initiated by the addition of ATP as substrate to obtain a final concentration of 1.0 mM, followed by incubation for 20 min. The reaction was stopped by the addition of 200 µL of 10% trichloroacetic acid (TCA) to obtain a final concentration of Figure 1. Effect of the phenolic acids (caffeic, ferulic, chlorogenic and + + 5% and the tubes were chilled on ice for 10 min. The released p-coumaric acid) on Na /K -ATPase Activity in isolated rat brain. Values inorganic phosphate (Pi) was assayed by the method of Fiske and represent mean ± SD of triplicate experimental readings. Subbarow (1925) using ascorbic acid as colorimetric reagent and KH2PO4 as standard. Enzyme activity was expressed as expressed KH2PO4 as standard. Enzyme activity was expressed as expressed in nmol. of Pi/mg of protein/min. in nmol. of Pi/mg of protein/min.

2.8. Phosphodiesterase type 5 (PDE-5) assay 2.10. IC50

The effect of the phenolic acids on phosphodiesterase-5′ activity The concentration of the phenolic acids causing 50% inhibition of was tested using the method of Kelly and Butler (1977) with slight ATPDase, phosphodiesterase-5′ and ecto-5′ nucleotidase activities as well as stimulation of Na+/K+-ATPase activity (IC ) was calcu- modification. The substrate, p-nitrophenylphenylphosphonate, 50 lated by nonlinear regression analysis. (55.84 mg) was dissolved in 20 mL of Tris buffer pH 7.4 to produce 5 mM working solution. The substrate, enzyme, and the phenolic solution were incubated separately in a water bath at 37 °C 2.11. Data analysis for 10 min to equilibrate. Different concentration of the phenolic acids was mixed with 100 μL of the enzyme and incubated in water The results of triplicate experimental readings were pooled and bath maintained at 37 °C. Thereafter, 1 mL of the substrate was expressed as mean ± standard deviation (S.D.). Student t-test, subsequently added to the mixture and was left to stand for 30 one-way analysis of variance (ANOVA) and least significance dif- seconds. Change in absorbance after 5 min at 400 nm was taken as ference (LSD) were carried out. Significance was accepted at p ≤ a function of the enzyme activity. 0.05.

2.9. Ecto-5′ nucleotidase assay 3. Results and discussion Ecto-5′ nucleotidase activity was determined in whole rat brain + + homogenate as described by Heymann et al. (1984). The assay 3.1. Effects of phenolic acids on Na /K -ATPase activity in rat mixture consisted of appropriate dilutions of the phenolic acids, brain homogenate 10 mM MgSO4 and 100 mMTris-HCl buffer (pH 7.5), in a final volume of 200 µL. Twenty microliters of tissue (8–12 µg of pro- Results from the assessment of the effects of phenolic acids on tein) were added to the reaction mixture and pre-incubated at 37 Na+/K+-ATPase activity revealed that the phenolic acids stimulate °C for 10 min. The reaction was initiated by the addition of AMP Na+/K+-ATPase activity in rat brain homogenate in a concentra- to a final concentration of 2.0 mM and proceeded for 20 min. In tion dependent manner (Figure 1 and Table 1). The sodium-potas- all cases, reaction was stopped by the addition of 200 µL of 10% sium-activated adenosine triphosphatase (Na+/K+-ATPase; sodium trichloroacetic acid (TCA) to obtain a final concentration of 5% pump; EC 3.6.1.37) is a plasma membrane-associated protein and the tubes were chilled on ice for 10 min. The released in- complex that directly or indirectly controls essential cellular func- organic phosphate (Pi) was assayed by the method of Fiske and tions which include but not limited to free calcium concentration, Subbarow (1925) using ascorbic acid as colorimetric reagent and and membrane potential. By coupling the energy released in the

+ + Table 1. IC50 values (µM) for the inhibitory effect of selected phenolic acids (caffeic, ferulic, chlorogenic and p-coumaric acids) on Na /K -ATPase, ATP- dase, Ecto-5′nucleotidase and phosphodiesterase-5′activities in isolated rat brain homogenates Enzyme activity Caffeic Acid Ferulic Acid Chlorogenic Acid p-coumaric Acid Na+/K+-ATPase 75.57 ± 0.22c 3.48 ± 0.09a 253.9 ± 0.14d 48.59 ± 0.15b ATPDase 346.9 ± 0.03d 27.48 ± 0.02c 18.37 ± 0.07b 17.56 ± 0.04a Ecto-5′ nucleotidase 8.25 ± 4.13a 6.22 ± 1.76a,b 12.48 ± 3.26b 35.31 ± 1.59c Phosphodiesterase-5′ 35.73 ± 0.11b 400.3 ± 0.05d 21.03 ± 0.21a 128.4 ± 0.08c

Values represent means ± standard deviation (n = 3). a–dMean values with the same superscript letter on the same row are not significantly different (p > 0.05).

58 Journal of Food Bioactives | www.isnff-jfb.com Ademiluyi et al. Modulatory effect of selected phenolic acids on enzyme activities

NTPDases (nucleosidetriphosphate diphosphohydrolases), ecto- NPPs (nucleotide pyrophosphohydrolases/phosphodiesterases), ecto-alkaline phosphatases, and ecto-5′ nucleotidase. The ecto- nucleosidase are involved in the breakdown of released ATP into ADP, AMP, adenosine, inosine and hypoxanthine (Zimmermann, 2006). Disturbances in the elements of purinergic pathway within the CNS underline the induction and amplification of several neu- rological pathologies. As observed in the study, an inhibitory effect of the ATPDase was observed in the presence of the phenolic acids with p-coumaric acid having the highest inhibitory activity. This suggests modulatory effect of this enzyme by the phenolic acids. Previous study Figure 2. Effect of the phenolic acids (caffeic, ferulic, chlorogenic and p- revealed that this enzyme is strongly associated with axon-like coumaric acid) on ATPDase activity in isolated rat brain. Values represent neuronal structures in the brain, where it may act as a main regula- mean ± SD of triplicate experimental readings. tor of extracellular ATP levels (Sebastián-Serrano et al., 2018). In the nervous system, purines are important neuromodulators, acting intracellular hydrolysis of ATP to the export of three intracellu- at pre- and post-synaptic sites. Consequently, ATPDase may play lar Na+ ions and the import of two extracellular K+ ions, Na+/K+ an indirect role in the modulation of nucleotide- and nucleoside- electrochemical gradient which is essential for the regulation of mediated processes (Sebastián-Serrano et al., 2018). The inhibi- cell volume action potential of muscle and nerve is maintained (de tion of ATPDase-like activity can impair the hydrolysis of the Lores Arnaiz and Ordieres 2014). Hence, proper and coordinated neurotransmitter ATP and, consequently, higher concetrations of regulation of this enzyme is crucial to prevention of diseases. The ATP and other purine nucleotides accumulates in the extracellular phenolic acid thus binds to and enhance the activity of this enzyme space and is afforded more time of exposure and interaction with in the brain. the purinoreceptors, this could promote an excessive activation of The IC50 (Table 1) revealed that these phenolic acids exerted all P2 purinoreceptors, consequently altering purinergic neuro- various degrees of stimulatory effects with respect to their type transmission. The high extracellular ATP concentration activating and concentration. Ferulic acid caused the highest stimulatory ef- specific receptors ensures continuous twitching of the nerve fibres fect while chlorogenic acid caused the least. Na+/K+-ATPase is a and the propagation of inflammatory processes; a process which crucial enzyme responsible for maintaining the ionic gradient nec- is involved in a variety of CNS morbidities which includes brain essary for neuronal excitability. It is present at high concentrations injury and ischemia, neurodegenerative diseases involving neuro- in brain cellular membranes, consuming about 40–50% of the ATP immune and neuro-inflammatory reactions, as well as neuropathic generated in the tissue (de Lores Arnaiz and Ordieres 2014). With pain and migraine (Burnstock, 2007). regard to the importance of this enzyme for the proper function- It has been acknowledged that the size and the chemical struc- ing of cells and tissues and especially in nerve cells, this enzyme ture and composition of compounds are usually some of the de- regulates the entry of K+ with the exit of Na+ from cells, being termining factors for their inhibitory effects (Berlin et al., 2006). responsible for Na+/K+ equilibrium maintenance through neuronal Phenolics are a class of polyphenolic compounds which have been membranes. Hence, the observed stimulatory effect of the phenolic suggested to modulate several critical enzyme systems and cas- acids on Na+/K+-ATPase activity could be beneficial to the proper cades that are directly involved in several biological processes functioning of this critical membrane bond enzymes system and (Crozier et al., 2009). Some studies have demonstrated the mod- for the maintenance of cellular integrity (Jorgensen and Pedersen, ulatory effects of some polyphenols on the purinergic system as 2001). Studies have shown that Na+/K+ activity in aged rat brains a function of their health promoting effect (Crozier et al., 2009). is significantly lower than that at other stages of brain develop- Phenolic carbonyl compounds tend to interact strongly with pro- ment. This may be related to the depression of neuronal excitabili- teins, nucleic acids, or related biological molecules, which signifi- ty with impairment of cognitive functions being advanced (Koçak- cantly inhibited the protein functions, DNA duplication, or even Toker et al., 2002). Thus, foods rich in phenolic acids with Na+/ loss of cellular activity (Cilliers and Singleton, 1990). Berlin et K+-ATPase stimulatory property could help counteract the effect al. (2006) also showed that the smallest phenolics often possesses of age-induced depression of neuronal excitability. the ability to assess the active sites of enzyme domains and more easily serve as more potent inhibitors of ATPDase when compared to the large polymerized phenolics with high molecular weight. 3.2. Effects of phenolic acids on nucleoside triphosphate diphos- Hence, p-coumaric acid with its higher inhibitory activity com- phohydrolases (ATPDase) activity in rat brain homogenate pared to other phenolics is a function of its small size and chemical composition. The ATPDase inhibitory ability of the phenolic acids in rat brain homogenate revealed that p-coumaric acid (IC = 17.56 μM) had the 50 3.3. Effects of phenolic acids on ecto-5′ nucleotidase activity in highest inhibitory activity with caffeic acid having the least (Figure 2 and Table 1). ATPDase enzyme belongs to a class of Nucleoside rat brain homogenate triphosphate diphosphohydrolases (EC 3.6.1.5, NTPDases) which hydrolyses ATP to ADP and consequently to AMP. Extracellular Assessment of the inhibitory effects of phenolic acids on the ecto- nucleotides and nucleosides are signaling molecules acting in all 5′ nucleotidase activity revealed that phenolic acid inhibited the tissues and organs, including the central nervous system (CNS). enzyme activity in a concentration dependent manner (0.05–13 A wide variety of effects, exerted by ecto-purines, requires that mM). Ecto-5′ nucleotidase (e5NT) belongs to the family of metal- their levels and ATP in particular, must be precisely controlled. lophosphoesterases, which hydrolyses AMP to adenosine, and is a Under physiological conditions, concentration of ecto-purines is regulator of the adenosine signaling pathway (Zimmermann et al., regulated by a complex cascade of ecto-enzymes, including ecto- 2006). One of the more obvious roles of the e5NT is the puriner-

Journal of Food Bioactives | www.isnff-jfb.com 59 Modulatory effect of selected phenolic acids on enzyme activities Ademiluyi et al.

Figure 5. Inhibition of Fe2+ induced TBARS Production in rat’s brain by Figure 3. Effect of the phenolic acids (caffeic, ferulic, chlorogenic and p- phenolic acids (caffeic, ferulic, chlorogenic and p-coumaric acid). Values Val- coumaric acid) on ecto-5′ nucleotidase activity in isolated rat brain. represent mean ± SD of triplicate experimental reading. ues represent mean ± SD of triplicate experimental readings. sine monophosphate (cGMP) to the corresponding nucleoside gic recycling. This signaling involves the conversion of AMP into monophosphates. . PDE-5 is expressed in various tissues of the adenosine and inorganic phosphate (Pi), which allows nucleosides body including the brain where its major function has been spe- produced extracellular to be transported into cells; this could be re- cycled into AMP and subsequently converted into ATP which can cific in catalysing the hydrolysis of cGMP (Reffelman et al., 2003; later be released into the synaptic cleft (Goding, 2000). Senzaki et al., 2001). Studies have hypothesized that blocking the The observed inhibitory effect of the phenolic acids on rat brain action of PDE-5 might result in increased intracellular levels and e5NT activity is a measure of their possible modulatory effect. The prolonged action of cGMP, which is a nitric oxide (NO) donor and inhibition of 5′-nucleotidase could influence adenosine levels in potent vasodilator. Previous studies have reported that inhibition the extracellular space favouring increased adenosine pool and an of PDE-5 improved memory (Prickaerts et al., 2002; Rutten et increased purine salvaging ability and perhaps a down-regulation al., 2005) and counteracts spatial learning impairment induced by of P1 purinoreceptors. Adenosine is a neuromodulator (normally NOS inhibition (Devan et al., 2006). PDE inhibitors have been inhibitory) in the central nervous system and participates in many proposed to be employed as memory enhancers (Blokland et al., metabolic and homeostatic cellular controls (Guieu et al., 1998). 2006; Rutten et al., 2007). Hence, these phenolic acids could be All phenolics inhibited this enzyme in a concentration dependent important dietary components with health promoting effect. Thus, inhibitors of PDE-5 such as the phenolic acids might be an alterna- manner with ferulic acid (IC50 = 6.22 μM) having the significant (p < 0.05) highest inhibition of ecto-5′ nucleotidase activity, while tive and perhaps more suitable for developing drugs targeted to CNS and memory (Puzzo et al., 2009). p-coumaric acid (IC50 = 35.31 μM) had the least (Figure 3 and Table 1). 3.5. Effects of phenolic acids on antioxidant status in rat brain 3.4. Effects of phenolic acids on phosphodiesterase-5′ activity in homogenate rat brain homogenate The incubation of rat’s brain homogenates in the presence of Fe2+ The effect of phenolic acids on phosphodiesterase-5′ activity re- caused a significant increase (p < 0.05) in the TBARS content (96.15%). The phenolic acids inhibited TBARS production in a vealed that chlorogenic acid (IC50 = 21.03 μM) had the highest inhibitory activity than caffeic acid (IC = 35.73 μM), p-coumaric concentration-dependent manner (Figure 5 and Table 2). However, 50 chlorogenic acid has the highest inhibitory ability (IC = 7.54 μM) acid (IC50 = 128.4 μM) with ferulic acid (IC50 = 400.3 μM) having 50 the least inhibition of phosphodiesterase-5′ activity in vitro (Figure and ferulic acid had the least (IC50 = 81.38 μM). Similarly, chlo- 4 and Table 1). rogenic acid (IC50 = 10.33 μM) had significantly higher (p < 0.05) The phosphodiesterases (PDEs) are a super family of enzymes DPPH scavenging ability and caffeic acid (IC50 = 91.73 μM) had that catalyze the hydrolysis of the nucleotide monophosphates, the least. Furthermore, chlorogenic acid (IC50 = 7.21 μM) showed cyclic adenosine monophosphate (cAMP), and cyclic guano- significantly higher (p < 0.05) scavenging ability towards ABTS radical cation while p-coumaric acid (IC50 = 63.07 μM) showed the lowest scavenging activity (Table 2). Oxidative stress plays a major role in aging, and is associated with several diseases including neuropathological conditions (Singh et al., 2019). Oxidative stress occurs when the production of reactive oxygen species overwhelms the antioxidant defence mechanisms leading to cellular damage (Singh et al., 2019). There is an emerging interest in the use of naturally occurring antioxidants for their therapeutic properties. Particularly, phenolics are considered as potential therapeutic agents against a wide range of ailments including neurodegenerative diseases, cancer, diabetes, cardiovascular dysfunction, inflammatory diseases and in ageing Figure 4. Effect of the phenolic acids (caffeic, ferulic, chlorogenic and p- (Shahidi and Yeo, 2018). In recent years, the importance of antiox- coumaric acid) on phosphodiesterase-5-activity in isolated rat brain. Val- idant activities of phenolic compounds and their potential usage as ues represent mean ± SD of triplicate experimental reading. functional foods and nutraceuticals are being promoted and some

60 Journal of Food Bioactives | www.isnff-jfb.com Ademiluyi et al. Modulatory effect of selected phenolic acids on enzyme activities

2+ Table 2. IC50 values (µM) on the inhibitory effect of selected phenolic acids (caffeic, ferulic, chlorogenic and p-coumaric acids) on Fe -induced TBARS production in isolated rat brain homogenates as well as their radical scavenging ability against DPPH and ABTS radicals Caffeic Acid Ferulic Acid Chlorogenic Acid p-coumaric Acid TBAS Production 14.85 ± 0.12b 81.38 ± 0.04d 7.54 ± 6.42a 32.61 ± 2.87c DPPH scavenging ability 91.73 ± 1.70d 79.42 ± 1.59c 10.33 ± 8.89a 20.30 ± 3.01b ABTS scavenging ability* 8.72 ± 0.07b 13.88 ± 0.06c 7.211 ± 0.06a 63.07 ± 0.06d

Values represent means ± standard deviation (n = 3). a–dMean values with the same superscript letter on the same row are not significantly different (p > 0.05). *µmol. Trolox Antioxidant Equivalent. evidence suggests that the biological actions of these compounds this group of phytochemicals an interesting source of compounds are related to their antioxidant activity (Adefegha, 2018). Phenolic for the development of drugs which protects and improves brain acids behave as antioxidants, due to the reactivity of the hydroxyl functions. substituent on the aromatic ring. Although there are several mechanisms, the predominant mode of antioxidant activity for these compounds is believed to be via radical scavenging. Also, their an- References tioxidant activity seems to be related to their molecular structure, more precisely to the presence and number of hydroxyl groups, Adefegha, S.A. (2018). 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Original Research J. Food Bioact. 2019;7:63–72

Optimization of extraction of antioxidants from aromatic herbs and their synergistic effects in a lipid model system

Thais Maria Ferreira de Souza Vieiraa, Marilis Yoshie Hayashi Shimanoa, Renan da Silva Limab and Adriano Costa de Camargoa,b,c* aDepartment of Agri-Food Industry, Food & Nutrition, “Luiz de Queiroz” College of Agriculture (ESALQ-USP), University of São Paulo, Av. Pádua Dias, 11, P.O Box 9, CEP 13418-900, Piracicaba, Brazil bDepartment of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, A1B 3X9, Canada cDepartamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Casilla 306- 22, Santiago, Chile *Corresponding author: Adriano Costa de Camargo, Department of Agri-Food Industry, Food & Nutrition, “Luiz de Queiroz” College of Agriculture (ESALQ-USP), University of São Paulo, Av. Pádua Dias, 11, P.O Box 9, CEP 13418-900, Piracicaba, Brazil. E-mail: [email protected] DOI: 10.31665/JFB.2019.7200 Received: September 24, 2019; Revised received & accepted: September 30, 2019 Citation: Vieira, T.M.F.S., Shimano, M.Y.H., Lima, R.S., and de Camargo, A.C. (2019). Optimization of extraction of antioxidants from aromatic herbs and their synergistic effects in a lipid model system. J. Food Bioact. 7: 63–72.

Abstract

Response surface methodology was applied to improve the polyphenol extraction process of rosemary, oregano, sage, and thyme. Aqueous ethanol (EtOH 50%) rendered the highest polyphenol extraction yield for all tested samples. Based on their total phenolic contents, rosemary, oregano, and thyme were selected for evaluation of their scavenging activities towards DPPH radical and ABTS radical cation and application in an oil model system. All extracts decreased the production of primary oxidation compounds during Schaal oven test storage. The induction period, as evaluated by the Rancimat test, was also reduced. There was an agreement between both oil model system assays, and rosemary extract showed the highest antioxidant capacity, followed by thyme and oregano. A centroid simplex design was used to evaluate the synergistic effect among the samples. Rosemary was able to play a synergistic effect when combined with thyme and oregano, or when used in binary mixtures.

Keywords: Rosemary; Oregano; Thyme; Sage; Phenolic antioxidants.

1. Introduction the inhibition of alpha-glucosidase and lipase, which participate in the breakdown of carbohydrates and triacylglycerols, has been The consumption of natural products has become a worldwide reported (Bautista-Expósito et al., 2018). Therefore, phenolic com- trend, with the food industry making efforts to respond to the pounds may be helpful in the management and/or prevention of needs. The current demand for new sources of bioactive com- type 2 diabetes and obesity. pounds has been reported in several studies (Carmo et al., 2019; Antioxidants delay and/or prevent the oxidative processes of Gremski et al., 2019). Aromatic herbs, which are used as flavoring lipid-rich as well as other foods, such as low moisture breakfast agents in a variety of foods, also serve as rich sources of phenolic cereals, among others. They can be either from synthetic origin antioxidants. Phenolic-driven sensory changes in functional foods [e.g., butylated hydroxyanisole (BHA), butylated hydroxytoluene have recently been discussed (de Camargo and Schwember, 2019). (BHT), and tert-butylhydroquinone (TBHQ)] or extracted from Additionally, phenolic compounds may render antimicrobial, anti- natural sources. Natural antioxidants have been investigated in or- inflammatory, anticarcinogenic, and other properties (Opara and der to replace the synthetic compounds due to the concerns related Chohan, 2014; Falcão et al., 2019; Shahidi et al., 2019). Likewise, to their potential carcinogenic and other adverse health effects

(Taghvaei and Jafari, 2015). Since the eighties (Witschi, 1986; 2.2. Total phenolic content (TPC) Verhagen et al., 1991), some authors have raised the potentially detrimental effects of synthetic antioxidants on health. In fact, TPC was determined spectrophotometrically, according to Swain there is sufficient evidence to support the carcinogenicity of BHA and Hillis (1959). Appropriate dilutions of the extracts (0.5 mL) (Verhagen et al., 1991). Cytotoxic effects of BHA in synergistic were transferred to screw-cap tubes and mixed with 1:10 (v/v) combination with Δ9-tetrahydrocannabinol (THC) were reported aqueous solutions of Folin-Ciocalteau reagent (2.5 mL). The tubes by Sarafian et al. (2002). Likewise, Witschi (1986) reported the were shaken, and after 5 min, a 4% sodium carbonate solution carcinogenic effect of BHT in animal models. Additionally,Imhoff (w/v) was added (2.0 mL). The tubes were kept in the dark for 2 and Hansen (2010) emphasized that the toxicity and carcinogenic h, and the absorbance was then read at 740 nm using a UV-1203 potential of compounds such as TBHQ should not be neglected. spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The re- However, mention should be made that the negative aspects high- sults were expressed as gallic acid equivalents (GAE) per gram of lighted must not overshadow their benefits as often experimental dry sample. animals are over-loaded with such compounds, and the adverse effects may have been exaggerated. Lipid oxidation is a significant problem for the food industry, 2.3. Radical scavenging activity (RSA) leading to unpleasant sensory changes, which vary in intensity depending on the product (da Silva et al., 2014). Many reports re- The extracts that rendered the highest TPC values according to veal the protective action of herb extracts against lipid oxidation the RSM were evaluated for their RSA towards DPPH radical and in food systems (Almeida-Doria and Regitano-d’Arce, 2000; Shan ABTS radical cation. et al., 2011; Racanicci et al., 2004). Their antioxidant properties are mainly correlated to their phenolic compounds (e.g., caffeic acid, rosmarinic acid, carnosic acid, carnosol, rosmanol, thymol, 2.3.1. DPPH radical scavenging activity carvacrol, catechin, kaempferol) (del Bano et al., 2003; Shan et al., 2005; Zheng and Wang, 2001). The antioxidant properties of The antioxidant activity was evaluated using the DPPH (2,2-diphe- each herb depend on its phenolic profile and mechanism of ac- nyl-1-picrylhydrazyl) radical assay, proposed by Brand-Williams tion (Embuscado, 2019). Furthermore, synergistic, additive, and/or et al. (1995) and described by Augusto et al. (2014). Phenolic ex- antagonistic relationships between different phenolic compounds tracts (0.5 mL) were mixed with a 60 μM DPPH ethanolic solution must be considered. (3.0 mL). The absorbance was read at 517 nm using a UV-1203 Spices are often used in combination not only in the food in- spectrophotometer (Shimadzu Corporation), after 45 min standing dustry but also in homemade meals. However, information on the in the dark. The DPPH radical scavenging activity was calculated synergistic antioxidant effect of herb mixtures remains scarce. Ac- using the equation below, and the results were expressed as Trolox cordingly, the present study evaluated the best extraction condi- equivalents (TE) per gram of dry sample (μmol TE/g dry weight). tions to recover phenolic antioxidant compounds from four herbs using response surface methodology (RSM) central composite de- DPPH radical scavenging activity (%) = (1) sign. Furthermore, the radical scavenging activity was investigated [(Abs − Abs )/(Abs )] × 100 towards DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and ABTS control sample control (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) radical where Abscontrol is the absorbance of DPPH radical + ethanol; Ab- cation. The synergistic antioxidant capacity was investigated in ssample is the absorbance of DPPH radical + phenolic extract or two oil model systems using RSM centroid simplex experimental trolox. design.

2.3.2. ABTS radical cation scavenging activity 2. Material and methods The ABTS radical cation scavenging activity was evaluated ac- 2.1. Extraction process cording to Re et al. (1999), as previously described (Augusto et al., 2014). The ABTS radical cation (2,2′-azino-bis(3-ethylbenzo- Dried herbs: rosemary (Rosmarinus officinalis L.), oregano (Ori- thiazoline-6-sulfonic acid), which is generated by oxidation with ganum vulgare L.), thyme (Thymus vulgaris L.), and sage (Salvia potassium persulfate, was used in ethanolic extracts. A 7.00 mM officinalis L.) were obtained from a local market in São Paulo, stock solution of ABTS was prepared one day before the analy- Brazil. The samples were ground and stored in a refrigerator at sis, and the working solution was prepared by diluting the stock 7 °C until further analyses. Refined-bleached-deodorized (RBD) solution until it reached 0.70 ± 0.02 at an absorbance wavelength soybean oil free from synthetic antioxidants was kindly donated by of 734 nm. The diluted extracts (20.00 μL) were mixed with the Cargill and kept frozen (−18 °C) until further analyses. ABTS radical cation work solution (2.00 mL), and the absorbance The extraction of phenolic compounds was performed by mix- was spectrophotometrically (UV-1203, Shimadzu Corporation) ing dried spices (3.0 g) with ethanol/water (30 mL) in a water read at 734 nm after 6 min of reaction. The ABTS radical cation bath, according to Lugemwa (2012). Different concentrations of scavenging activity was calculated using the equation below, and ethanol/water (0, 15, 50, 85, and 99%) and temperatures (30, 35, the results were expressed as μmol TE/g dry weight. 45, 55, and 60 °C) were used as established by the experimental ABTS radical cation scavenging activity (%) = design. All extractions were carried out for 50 min, and the extracts (2) were centrifuged at 4,000 g for 20 min. The supernatant was col- [(Abscontrol − Abssample)/(Abscontrol)] × 100 lected and filtered on a filter paper Whatman n° 2. The extracts where Abscontrol is the absorbance of ABTS radical cation + etha- were stored at 7 °C in amber vials (within 5 days) until the moment nol; Abssample is the absorbance of ABTS radical + phenolic extract of analysis. or trolox.

64 Journal of Food Bioactives | www.isnff-jfb.com Vieira et al. Synergistic antioxidants from aromatic herbs

2.4. Antioxidant capacity in an oil model system mination (R2) and F tests. The fitted model formula is represented by the equation below:

2.4.1. Hydroperoxide content under Schaal oven test condition 2 2 Y = b0 + b1x1 + b2x2 + b11x1 + b22x2 + b12x1x2 (3) where Y is the predicted response, x and x represent the coded The Schaal oven test was carried out as previously reported (Vieira 1 2 levels of independent variables, b0 is the intercept coefficient, and and Regitano-d’Arce, 1999) with slight modifications. Vials with b’s are the coefficients estimated by the least square method. RBD soybean oil (1.5 mL) were mixed with an individual, binary, To study the correlation between TPC and the antioxidant ca- or ternary herb extracts combination and subjected to the Schaal pacity as evaluated by DPPH and ABTS assays, Pearson’s correla- oven test at 60 °C for eight days. The hydroperoxide content was tion analyses (p < 0.01) and (p < 0.05) were carried out using the determined by measuring the formation of ferric ion complex us- ASSISTAT 7.6 software. ing spectrophotometry, according to Shantha and Decker (1994), The second design was planned to investigate the potential syn- with modifications described by de Camargo et al. (2016). For ergistic effect of the three herbs selected according to their TPC better comprehension, all solutions are explained first. Solution 1: +2 yield obtained in the first design. A simplex centroid design was BaCl2 (0.132M) was prepared in HCl (0.4 M). Solution 2: Fe used. The data were analyzed by one-way analysis of variance (0.072 M) was obtained from the mixture (1:1, v/v) of FeSO4 2+ (ANOVA), considering a level of 5% significance. Based on the (0.144) and BaCl2 (0.132M). Solution 3: NH4SCN:Fe (1:1, v/v) +2 experimental design, a cubic model was fitted with the equation was obtained by mixture of NH4SCN (3.94 M) and Fe (0.072 below: M). For analysis, RBD soybean oils were weighed (0.3 g) and mixed with 1.5 mL iso-octane/2-propanol solution (3:1, v/v). The Y = b1x1 + b2x2 + b3x3 + b12x1x2 + mixture was vortexed, and 200 µL of the solution obtained was (4) b13x1x3 + b23x2x3 + b123x1x2x3 mixed with 2.8 mL methanol/n-butanol (2:1, v/v). Finally, 30 µL 2+ where Y = response (dependent variable), x1, x2, x3 = coded levels of NH4SCN:Fe (1:1, v/v) was added. The tubes were incubated at room temperature for 20 min. The absorbances were read at 510 of components proportion, b’s = estimated coefficients by the least nm using a UV-1203 spectrophotometer (Shimadzu Corporation). square method. The hydroperoxide content (mmol LOOH/L oil) was calculated Statistical analyses were conducted using the Statistica 11 soft- using a standard curve of cumene hydroperoxide. Likewise, the ware. UV absorption was evaluated to support the generation of primary oxidation products, such as conjugated dienes. The absorbance at 232 was determined using the same equipment following the Ch 3. Results and discussion 5–91 method from AOCS (2003). 3.1. Extraction process and total phenolic content (TPC) 2.4.2. Oil stability index Different solvents (e.g., acetone, methanol, ethanol, diethyl ether, water, and ethyl acetate) are commonly employed in the extrac- The induction period was evaluated using a Rancimat 743 apparatus (Metrohm Corporation, Herisau, Switzerland), as previously tion of polyphenols. From a toxicological standpoint, ethanol and explained. The oil samples (5.0 g) in the presence of single, bi- water are safer than other organic solvents; thus, more acceptable nary, or ternary extract mixtures were transferred into a vessel and for food applications. Indeed, ethanol and water, individually or in heated to 110 °C and dry airflow of 9 L/h. The induction period combination, have effectively been used in the extraction of phe- was expressed in hours (h). nolic compounds (Almeida-Doria and Regitano-d’Arce, 2000). In the present study, an ethanol/water mixture was chosen as the solvent for the extraction process. Phenolics present in the test 2.5. Experimental design and statistical analysis samples have already been reported (Shimano, 2019). Caffeic and dihydrocaffeic acids were identified in all herbs, while vanillic In the first design, the goal was to optimize the extraction of phe- acid methyl ester of p-hydroxybenzoic acid was detected only in nolic compounds after which three of them were further investigat- rosemary. The presence and identities of other phenolics in these ed for their synergistic effect. The central composite design of two spices are already available in the existing literature (Shahidi and factors/five levels was applied to evaluate the yields of the extrac- Ambigaipalan, 2015; Shahidi and Hossain, 2018). tion process as a function of TPC. Runs 9–12, which represented It is well accepted that several factors such as ethanol percent- the central point, allowed to estimate the variation in the responses age, temperature, extraction time, particle size, and the solvent/ at the central point and provided a basis for the lack-of-fit test solid ratio may affect the extraction process, independently, or via (Rheem and Oh, 2019). The independent variables were ethanol interactions. In the present study, the temperature was chosen as concentration (% EtOH, v/v) and temperature (°C). The range and the second parameter to be investigated. levels of the explanatory variables were, as follows: coded value RSM determined the optimal conditions for the extraction of −1.41 (0% ethanol, 30 °C), −1 (15% ethanol, 35 °C), 0 (50% etha- phenolic compounds from herbs. Phenolics from different plant nol, 45 °C), 1 (85% ethanol, 55 °C), 1.41 (99% ethanol, 60 °C). sources have been reported to display antioxidant action against Experimental data were analyzed with the software Statistica 11. reactive oxygen species (Chandrasekara and Shahidi, 2011; Melo The study of the effect of the extraction conditions was performed et al., 2015). Based on the highest TPC observed in the first de- by response surface methodology and multiple regression analysis. sign (Table 1), three herbs were selected for in vitro antioxidant Second-order mathematical models, including linear and quadratic investigations (ABTS and DPPH methods) and further application terms, and interactions between independent variables were fitted in lipid model systems (Schaal oven test and oil stability index). to define the optimum conditions for extraction of TPC from each Polyphenols from selected herbs were studied for their antioxidant herb. The models were evaluated based on the coefficient of deter- capacity as well as their synergistic effect as binaryor ternary mix-

Journal of Food Bioactives | www.isnff-jfb.com 65 Synergistic antioxidants from aromatic herbs Vieira et al.

Table 1. Total phenolic content of extracts of selected herbs Independent variables TPC (mg GAE/g)* Run % EtOH T (°C) Rosemary Oregano Sage Thyme 1 15 35 43.65 80.58 28.09 46.53 2 85 35 33.77 64.88 13.83 33.77 3 15 55 42.42 94.28 33.76 54.95 4 85 55 30.83 62.09 27.09 35.94 5 0 45 40.15 64.36 27.52 42.27 6 99 45 13.31 21.49 12.69 20.07 7 50 30 36.98 90.73 35.11 45.50 8 50 60 52.25 93.28 53.91 64.55 9 50 45 43.60 91.65 46.20 66.59 10 50 45 56.18 92.36 41.66 59.26 11 50 45 57.93 98.82 46.39 63.32 12 50 45 58.92 94.77 41.80 67.11

*Values expressed as means of triplicate determinations. GAE: gallic acid equivalents, TPC: total phenolic content. Runs 9–12, the central point (n = 4), allows to estimate the variation in the responses at the central point and provides a basis for the lack-of-fit test. tures. factors such as cultivar, geographic origin, harvest time, among In the first design, a central composite design was used to in- others (Shahidi and Ambigaipalan, 2015; Shahidi and Hossain, vestigate the effects of two independent variables—ethanol con- 2018). centration (% EtOH) and temperature (T)—on TPC (Table 1). The ANOVA of the regression models for rosemary, oregano, TPCs ranged from 13.31 to 58.92 (rosemary), from 21.49 to 98.82 sage, and thyme extracts showed that the models were significant (oregano), from 12.69 to 53.91 (sage), and from 20.07 to 67.11 (p < 0.05), with F-values of 22.8, 88.3, 31.6, and 53.9, respec- (thyme), with respective mean values of 42.50, 79.11, 34.00, and tively (Table 2), and they were all three-fold higher than F tabu- 49.99 mg GAE/g dry weight. However, the optimization of the lated. The R2 values were also higher than 0.92. Furthermore, extraction procedure is equally important, as demonstrated in the there was no significance in the lack of fit (p > 0.05). These in- present study, where TPC was increased up to 4.6-fold under op- dicated the predictive capacity of the models. An ideal model timal conditions. should have good significance (p > 0.05), high reliability (data 2 Kim et al. (2011) reported the TPC of 13 spices commonly used within the range of 95%) and low variability (R > 70%). The in meat processing plants. According to these authors, TPCs were models were fit considering only the significant effects for rose- as follows: clove > thyme > savory > rosemary > oregano, basil, mary (Equation 3), oregano, (Equation 4), sage (Equation 5), and and marjoram > caraway, cumin, fennel, coriander, turmeric, and thyme (Equation 6). mace. In this study, oregano presented the highest TPC, followed TPC (mg GAE/g) = 57.67 − 7.44 × (%EtOH) (5) by thyme, rosemary, and sage, respectively. Taking into account − 15.03 × (%EtOH)2 + 2.18 × (T) −6.03 × (T)2 oregano, thyme, and rosemary, the data from the present study is in agreement with the findings of Kim et al. (2011) for thyme and TPC (mg GAE/g) = 94.91 − 13.05 × rosemary, which is not the case for oregano. Nevertheless, this dis- 2 (6) crepancy is not uncommon, since TPC can be affected by several (%EtOH) − 23.40 × (%EtOH)

Table 2. Variance analysis of regression models fitted to experimental designs of TPC of selected herbal extracts Responses Source of variation (sum of squares) Rosemary Oregano Sage Thyme Regression 1,947.536 4,973.575 1,664.749 2,555.898 Residual 131.066 253.406 140.428 82.775 Pure error 3.850 31.397 20.867 39.291 Total 2,078.602 5,226.981 1,805.77 2,638.673

Fcalc* 22.289 88.322 31.612 53.953

Ftab 4.53 4.26 4.07 4.12 R2 0.9307 0.9515 0.9222 0.9671

*If F calculated is three-fold higher than F tabulated, the model may be used for prediction purposes.

66 Journal of Food Bioactives | www.isnff-jfb.com Vieira et al. Synergistic antioxidants from aromatic herbs

Figure 1. Effect of ethanol percentage and temperature on total phenolic content in (a) rosemary; (b) thyme; (c) sage; (d) oregano extracts. TPC, total phenolic contents; GAE, gallic acid equivalent.

TPC (mg GAE/g) = 42.91 − 5.24 × (%EtOH) For oregano, only the linear and quadratic effects of ethanol per- (7) − 13.40 × (%EtOH)2 + 5.70 × (T) centage affected the TPC. Figure 1 shows the response surfaces of the extraction process. An increase from 30 to 45 °C produced extracts from rosemary and TPC (mg GAE/g) = 64.07 − 7.91 × (%EtOH) − 2 2 (8) thyme with higher TPC. Sage showed increases in the TPC, with 16.59 × (%EtOH) + 4.70 × (T) − 4.59 × (T) the temperature rising from 30 to 60 °C. It has previously been The extraction process was significantly affected by ethanol reported that changes in the temperature from 20 to 60 °C causes concentration and temperature. However, interactions of ethanol increases in the TPC of oregano and thyme (Rababah et al., 2010). percentage and temperature were not significant for any of the Based on the response surfaces obtained, the conditions for tested samples. The quadratic effect of ethanol percentage showed efficient extraction of the phenolic compounds were: EtOH 50% the highest absolute value, i.e., with the most significant impact at 45–50 °C (rosemary and thyme), EtOH 50% and temperature on the TPC of all spices, followed by its linear effect. The linear from 57.5 to 60 °C (sage), and EtOH 40–50% at any temperature effect of ethanol percentage in all extracts was negative, meaning ranging from 30 to 60 °C (oregano). Based on the TPC, oregano, that an increase in this factor caused a decrease of TPC. Tempera- thyme, and rosemary with their respective best extraction condi- ture showed a positive linear effect, indicating that increases in the tions were selected for further analysis. temperature also led to increases in the response. However, TPC from oregano extract was not affected by changes in the temperature. For all extracts, the mathematical models presented negative 3.2. Radical scavenging activity (RSA) quadratic parameters, indicating that surfaces have passed by the maximum TPC value. The RSA towards DPPH radical ranged from 295.6 to 178.0 μmol For rosemary and thyme extracts, the linear and quadratic ef- TE/g (Table 3), while the RSA towards ABTS radical cation varied fects of ethanol percentage, as well as the quadratic effect of the from 520.8 to 239.0 μmol TE/g. Regardless of the method em- temperature were negative; the linear effect of the temperature ployed, oregano showed the highest RSA, followed by thyme and was positive. The linear and quadratic effects of explanatory vari- rosemary. Furthermore, a high positive significant correlation (p < ables in sage extracts were similar to those of rosemary and thyme. 0.01) was found between TPC and both RSA assays, which dem- However, the quadratic effect of temperature was not significant. onstrates that the antioxidant activity of the samples is dependent

Journal of Food Bioactives | www.isnff-jfb.com 67 Synergistic antioxidants from aromatic herbs Vieira et al.

Table 3. Antiradical activities and corresponding Pearson’s correlation with total phenolic contents (TPC) of selected extracts of herbs Antiradical activity (μmol TE/g dry weight) Pearson’s correlation Sample DPPH ABTS TPC × DPPH TPC × ABTS Oregano 295.6 ± 16.4a2 520.8 ± 7.86a 0.9962** 0.9957** Thyme 181.9 ± 2.48b 263.4 ± 3.39b 0.9861** 0.9683** Rosemary 178.0 ± 7.90b 239.0 ± 2.57c 0.9984** 0.9955**

1Data represent the means of triplicate analysis for each sample ± standard deviations; TE is trolox equivalent. 2means with the same letters within a column are not significantly different according to Tukey’s multiple test (p > 0.05); **significant for Pearson’s correlation (p < 0.01). on their phenolic content. a refined-bleached-deodorized (RBD) soybean oil free from syn- The antioxidant activities of phytochemicals depend not only on thetic antioxidant was used as the oxidizable feedstock. RBD soy- the presence of polyphenols but also on their chemical structure. bean oil was chosen due to its relatively high unsaturated fatty acid Thus, the high correlation between RSA assays with TPC is not al- content. Schaal oven test was applied to evaluate the antioxidant ways true. The structure-activity relationship has already been dis- capacity using a mild temperature (60 °C). Every two days, the cussed by Rice-Evans et al. (1996). The high correlation found in oxidation status was assessed using the hydroperoxide content and the present study agrees with the work of Kim et al. (2011), who UV absorption (232 nm) results, which represent the primary oxi- reported a high correlation between TPC and DPPH for hot water dation products. extracts of 13 spices. Their work also supports the information that The ability of the extracts in inhibiting oxidation under extreme the strength of correlation depends on the polyphenols present. Ac- conditions, combining high temperature and oxygen-induced oxi- cording to these authors, the TPC showed a high positive correlation dation, was also evaluated using oil stability index (OSI) method (r = 0.9158, p < 0.001) with DPPH whereas in the same assay the using a Rancimat. This is an automated, efficient, and low-cost flavonoid content exhibited a moderated correlation (r = 0.5430,p < method widely used by the industry. Thus, the results achieved 0.05). It is noteworthy that the correlation depends on the antioxidant may be helpful for future comparison and easy reproduction. method employed. The same authors found no correlation between Based on TPC values, oil samples were prepared with each ex- TPC and hydroxyl radical scavenging activity or superoxide radical tract (100 mg GAE/kg oil) and their binary (50 mg GAE/kg oil for anion scavenging activity. However, the high correlation with DPPH each extract) and ternary (33.3 mg GAE/kg oil for each herb) mix- is useful to anticipate at some point if one feedstock may exhibit tures. The extraction conditions were based on preliminary studies antioxidant capacity in bulk oils (Arranz et al., 2008). Correlations (Table 1). The ability of individual and combined herb extracts between TPC and ABTS were also reported as classified by botani- in preventing oxidation in the oil model system is summarized in cal families in the study of Wojdylo et al. (2007), where the botani- Table 4. cal family had a significant influence on the correlation. The authors The correlation between hydroperoxide concentration and con- found positive correlations only for herbs belonging to Labiatae and jugated dienes increased throughout the experiment and became Compositae family groups when compared to several others. significantly correlated (p < 0.01) on the sixth (r = 0.9483) and eighth (r = 0.9981) days. The highest correlation was found on the eighth day. Thus, such data were chosen to fit the models (Fig. 2a, 3.3. Antioxidant capacity in an oil model system b). The antioxidant capacity of rosemary and oregano ethanolic extracts in soybean oil during thermal oxidation has already been To evaluate the actual antioxidant capacity of the herb extracts, reported by Almeida-Doria and Regitano-d’Arce (2000). The lit-

Table 4. Hydroperoxide content (mmol LOOH/L oil)1, absorbance at 232 nm, and induction period of soybean oil with added herbal extracts subjected to thermal oxidation under Schaal oven test condition (60 °C) and Rancimat testing at 110 °C

Hydroperoxide content 3 4 Relative 3 Absorbance at 232 nm Induction Period Run Mixture2 (mmol LOOH/L oil) concentration Time (days) Time (days) (h)

(x1, x2, x3) 0 4 6 8 0 4 6 8

1 Thyme (x1) (1,0,0) 0.76 2.44 6.59 17.5 5.40 5.52 6.08 8.26 8.26

2 Oregano (x2) (0,1,0) 1.01 4.70 19.8 29.0 5.40 5.76 7.20 10.2 8.03

3 Rosemary (x3) (0,0,1) 0.87 2.43 3.93 5.12 5.34 5.49 5.71 6.21 10.8

4 x1 + x2 (0.5, 0.5, 0) 0.55 2.70 14.2 20.7 5.37 5.64 7.35 8.77 8.37

5 x1 + x3 (0.5, 0, 0.5) 0.37 2.92 4.92 6.37 5.40 5.69 5.86 6.48 9.27

6 x2 + x3 (0, 0.5, 0.5) 0.92 2.97 5.06 6.38 5.41 6.15 5.95 6.61 9.03

7 x1 + x2 + x3 (0.33, 0.33, 0.33) 0.40 2.88 4.56 5.69 5.21 5.51 5.59 6.07 8.94 8 Control (0,0,0) 0.45 2.41 21.1 32.6 5.42 5.78 7.33 10.8 7.82

1values are expressed as means of triplicate determinations;2 Total concentration of phenolics for each run was 100 mg gallic acid equivalent/kg oil and the control was free from any herbal extracts or synthetic antioxidants. 3Results of oven test. 4Results of Rancimat test.

68 Journal of Food Bioactives | www.isnff-jfb.com Vieira et al. Synergistic antioxidants from aromatic herbs erature also provides information on the synergistic effect of individual phenolics using centroid simplex design (Capitani et al., 2009). However, a literature search reveals that there are knowledge gaps regarding common sources of natural antioxidants such as spices. Therefore, the present work may be helpful for future studies and applications of new herb combinations or other sources of phytochemicals presenting a synergistic effect. The F calculated by analysis of variance (ANOVA) was higher than the F tabulated (Table 5). If F calculated is three-fold higher than F tabulated, the model can be used for prediction purposes. In fact, the F-values of 26.2 (hydroperoxide content), 24.13 (absorbance at 232 nm), and 151.17 (induction period) were up to 39 times higher than the F tabulated. Thus, the fit models were valid. The R2 coefficients were 0.9181, 0.9118, and 0.9923 for hydroperoxide content, UV absorbance, and induction period, respectively. Mixtures with three components follow the equation x1 + x2 + x3 = 1, which is equivalent to an equilateral triangle in a cube. The vertices correspond to pure extracts, the edges are binary blends, and the points inside the triangle represent the possible ternary blends. A simplex-centroid design consists of 2q – 1 points, where q corresponds to the components (Myers et al., 2009). The antioxidant capacity of the extracts in RBD soybean oil, as shown by the hydroperoxide content, was effective when ternary or binary mixtures had rosemary in their combination. The antioxidant capacity evaluated by UV absorption is in good agreement with the results evaluated by hydroperoxide content. Among individual extracts, oregano showed the most significant antioxidant capacity as assessed by hydroperoxide content and UV absorption, which was followed by thyme and rosemary, respectively. Taking into account the hydroperoxide contents, UV absorption, and TPC, it is possible to infer that the polyphenols extracted from rosemary exhibited more oxidative protection in RBD soybean oil model system and can be used to induce synergistic protection when combined with thyme and oregano or only with thyme or oregano individually. The Rancimat is an apparatus developed and dedicated to the determination of oil stability index (OSI), which is expressed as an induction period in hours. This test is based on inducing the oil oxidation through continuous aeration at high temperatures. Volatile compounds, formed by hydroperoxide decomposition in secondary oxidation stages, travel to the water, altering its conductivity, which is recorded by the equipment (Velasco et al., 2009). The increase in water conductivity is mainly due to the formation of acetic and formic acids, which are produced by the breakdown of aldehydes, the major secondary oxidation products. The time until such products are detected is called induction period. Therefore, the higher the induction period, the more effective is the antioxidant (Loury, 1972). The induction period of the RBD soybean oil in the presence of herb extracts is shown in Table 4. In this method, the oil was exposed to higher temperatures (110 °C) when compared to the Schaal oven test (60 °C). Phenolic compounds may be less stable at higher temperatures due to extreme conditions of accelerated oxidation. Thus, some loss in their antioxidant activity may occur as a consequence of the degradation of their phenolics into oxidized compounds. However, a significant negative correlation (p < 0.05) existed between hydroperoxide content (r = −0.7978) and Rancimat test or UV absorption (r = −0.7925) and Rancimat test, which demonstrates the same trend among different assays. Figure 2. (a) Hydroperoxide content (HC); (b) UV absorbance at 232 nm of RBD soybean oil in oven test (60 °C) on day 8 and C: Induction pe- Fig. 3a–c shows the percentage of inhibition of hydroperoxides, riod (IP) of RBD soybean oil in Rancimat test. The hydroperoxide con- conjugated dienes, and oxidation in the Rancimat test. Accord- tent (mmol LOOH/L oil) was calculated using a standard curve of cumene ing to the surface response, the worst performance was obtained hydroperoxide. The UV absorption at 232 nm indicates the generation of when oregano extract alone was added to RBD soybean oil, which primary oxidation products (e.g. conjugated dienes). The induction period agrees with the data from the Schaal oven test. The regression was was expressed in hours.

Journal of Food Bioactives | www.isnff-jfb.com 69 Synergistic antioxidants from aromatic herbs Vieira et al.

Table 5. Variance analysis of regression models fitted to experimental designs of hydroperoxide content and absorbance at 232 nm Responses Source of variation (sum of squares) Hydroperoxide content Absorbance (232 nm) Rancimat test Regression 1,617.548 45.59242 9.98347 Residual 144.284 4.40783 0.07705 Total 1,761.32 50.00025 10.06052

Fcalc 26.15872 24.13484 151.1666

Ftab 2.85 2.85 3.87 R2 0.9181 0.9118 0.9923

*If F calculated is three-fold higher than F tabulated, the model may be used for prediction purposes.

2 statistically significant (Fcalculated > Ftabulated), and the R coefficient An apparent synergistic effect can be noted when rosemary is was elevated. Thus, it was possible to fit a model to explain the taken into account. Phenolics from thyme and oregano inhibited variations (Fig. 2c and Table 6). the hydroperoxide generation by 46 and 11%, respectively. How- ever, when combined with those from rosemary, the inhibition increased to 80% in both cases (thyme + rosemary or oregano + rosemary). The same behavior was observed for the inhibition of the formation of conjugated dienes (primary oxidation compounds). In fact, phenolics from oregano enhanced inhibition of formation of conjugated dienes from 2 to 19% when in combination with rosemary (Figure 3). Phenolics from thyme combined with those extracted from rosemary also showed a higher antioxidative effect against conjugated dienes generation when compared with thyme alone. Finally, when combined, phenolics from thyme and rosemary promoted inhibition by 18% in the Rancimat test. In contrast, a lower inhibition (6%) was observed when phenolics from thyme were used alone. The oxidation inhibition of oregano increased from 2.7% (oregano) to 15% (oregano + rosemary). Therefore, the synergistic effect of rosemary was demonstrated in all model systems. Some other studies have employed RSM to improve the extraction of phenolics from other plant materials (Jovanović et al., 2017; Li et al., 2012; Majeed et al., 2016). However, none of them have used this tool to screen the synergistic effect of the obtained extracts in a lipid model system. Finally, predicted values supported those found during our experiments (data not shown). Therefore, this contribution extends the current knowledge and/or application of RSM.

4. Conclusions

The aqueous ethanol (EtOH 50%) yielded the highest total phenolic content (TPC) for all herbs, while the temperature exhibited a different effect for each one of them. A high positive correlation was found between TPC and the antiradical activity towards DPPH radical and ABTS radical cation. DPPH and ABTS assays may be used for initial screening. However, results obtained from practi- cal applications are more relevant. Polyphenols from rosemary had higher antioxidant capacity than the other extracts tested against the production of primary and secondary oxidation compounds in the oil model system subjected to accelerated thermal oxidation. The antioxidant capacity in a refined-bleached-deodorized soybean oil model system can be ranked as rosemary > thyme > oregano. Furthermore, rosemary extract showed a synergistic effect when combined in equal concentration with thyme and oregano (ternary Figure 3. Inhibition percentage of phenolics from herb extracts against mixture, 33.3 mg GAE/kg oil for each herb) or in a binary mixture (a) hydroperoxide and (b) conjugated diene accumulation and (c) oxida- with thyme or oregano (50 mg GAE/kg oil for each herb). This tion in Rancimat test. R is rosemary, T is Thyme, and O is oregano. study was an initial step in the production of polyphenol-rich herb

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Table 6. Special cubic polynomial models of the synergistic effects of mixtures containing phenolic extracts of thyme, oregano and rosemary Hydroperoxide content = 17.48 × Thyme + 29.02 × Oregano + 5.12 × Rosemary − 10.30 × Thyme × Oregano − 19.70 × Thyme × Rosemary − 42.76 × Oregano × Rosemary − 92.54 × Thyme × Oregano × Rosemary Absorbance at 232 nm = 8.26 × Thyme + 10.23 × Oregano + 6.20 × Rosemary − 1.89 × Thyme × Oregano − 3.01 × Oregano × Rosemary − 6.45 × Oregano × Rosemary − 24.16 × Thyme × Oregano × Rosemary Induction Period = 8.26 × Thyme + 8.03 × Oregano + 10.75 × Rosemary + 0.89 × Thyme × Orenago − 0.94 × Thyme × Rosemary − 1.44 × Oregano × Rosemary + 2.46 × Thyme × Oregano × Rosemary extracts aimed to be used as natural antioxidants in food formula- leaves, flowers, stems, and roots of Rosmarinus officinalis. antioxi- tions from vegetable and/or animal origin. dant activity. J. Agric. Food Chem. 51: 4247–4253. Embuscado, M.E. (2019). 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Original Research J. Food Bioact. 2019;7:73–77

Lipophilized resveratrol affects the generation of reactive nitrogen species in murine macrophages and cell viability of human cancer cell lines

Won Young Oha, Yi-Shiou Chioub, Pei-Sheng Leeb, Min-Hsiung Panb* and Fereidoon Shahidia* aDepartment of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9 bInstitute of Food Science & Technology, National Taiwan University, Taipei, Taiwan 10617 *Corresponding author: Min-Hsiung, Institute of Food Science & Technology, National Taiwan University, Taipei, Taiwan 10617. Tel: +886-2-3366-4133 E-mail: [email protected]; Fereidoon Shahidi, 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.2019.7201 Received: September 06, 2019; Revised received & accepted: September 28, 2019 Citation: Oh, W.Y., Chiou, Y.-S., Lee, P.S., Pan, M.-H., and Shahidi, F. (2019). Lipophilized resveratrol affects the generation of reactive nitrogen species in murine macrophages and cell viability of human cancer cell lines. J. Food Bioact. 7: 73–77.

Abstract

Resveratrol was esterified with selected fatty acids to improve its lipophilicity and potential application in food and biological systems. In this study, resveratrol and monoesters of resveratryl propionate (RC3:0) and resveratryl docosahexaenate (RDHA) were examined for their effects on anti-inflammatory and anti-proliferative activity in vitro. All test compounds showed a decreased nitrite production in murine RAW 264.7 cells in a concentration dependent manner. Resveratrol, RC3:0, and RDHA were evaluated for their effects on cell viability using liver cancer (HepG2), colon cancer (HT-29, A431), breast cancer (MCF7), and gastric cancer (AGS) cell lines. All test compounds showed decreased cell viability of HepG2, A431, MCF7, HT-29, and AGS in a concentration- dependent manner. The results suggest that resveratrol esters may serve as potential anti-inflammatory and anti-proliferative agents.

Keywords: Resveratrol ester; Antioxidant activity; Anti-inflammatory activity; Anti-proliferative effect.

1. Introduction biological responses such as gene expression and cell proliferation. However, at high levels, they can also cause harmful effects, in- Inflammation, such as redness, warmth, and swelling, is a normal cluding inflammation, cardiovascular disease, and cancer (Fransen reaction to injuries or infections. However, prolonged inflammation et al., 2012; Zhong et al., 2012). Antioxidative defense systems in can progress further to various chronic diseases such as cardiovas- the body are frequently insufficient to keep ROS/RNS level in bal- cular disease and cancer (Zhong et al., 2012). Reactive oxygen spe- ance, therefore external sources of antioxidants are needed for this cies (ROS) and reactive nitrogen species (RNS) may explain the purpose (Yu, 1994; Loft and Poulsen, 1996). link between inflammation and other chronic diseases (Wiseman Resveratrol is a powerful antioxidant and has been found in and Halliwell, 1996). It is well known that ROS/RNS are continu- more than 70 plant species, especially grape skins. Red wine also ously generated in vivo as a by-product of biological reactions (Hal- contains resveratrol that originates from grapes (Dercks and Creasy, − liwell, 1996, Fransen et al., 2012). Superoxide (O2• ), hydrogen 1989). Resveratrol has received increasing attention since red wine peroxide (H2O2), and nitric oxide (ON•) are the primary ROS/RNS was first shown to display cardioprotective effects (Baur and Sin- in a cell and they can readily form other ROS/RNS (Fransen et al., clair, 2006). In addition, resveratrol prevents oxidation, cancer, 2012). At a low level, ROS/RNS can serve as mediators for various coronary heart disease and inflammation (Jang et al., 1997; Wang et

Copyright: © 2019 International Society for Nutraceuticals and Functional Foods. 73 All rights reserved. Lipophilized resveratrol affects the generation of reactive nitrogen species Oh et al. al., 2002; Donnelly et al., 2004). Although it has beneficial health properties, there are limitations to its bioactivity due to its hydro- philic nature and fast metabolism in the body (Zhong and Shahidi, 2011; Walle et al., 2004). Therefore, structural modification may provide an opportunity to address this concern (Torres et al., 2010; Baur and Sinclair, 2006). Several studies on lipophilic derivatives of phenolic compounds, including resveratrol, have demonstrated their better performance in biological model systems. Acetylated resveratrol showed better inhibition of platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)-induced washed rabbit platelet aggregation and DU-145 human prostate cancer cell-growth than resveratrol itself (Fragopoulou et al., 2007; Cardile et al., 2005). In addition, Cichocki et al. (2008) reported that pterostilbene (dimethylated resveratrol) acted as an inhibitor of tumour promotion biomarker. In addition, Pan et al. (2007) reported that pterostilbene was able to inhibit cell proliferation and induce apoptosis in human gastric carcinoma cells. Moreover, esterifica- tion of resveratrol with polyunsaturated fatty acids (PUFAs) may provide a synergistic effect due to the individual bioactivities of its components. In addition, our preliminary data showed that some of the resveratrol esters had an effect on RNS generation in murine macrophages. For this reason, resveratrol was esterified with propionyl chloride (C3:0) and docosahexaenoyl chloride (DHA, C22:6), as described elsewhere (Oh and Shahidi, 2017). In this study, the mixture of R-3-O- and R-4′-O-monoesters (RC3:0 and RDHA) were evaluated for inhibition against NO production as well as antiproliferative effect in various cancer cell lines.

2. Materials and methods Figure 1. Structures of RC3:0 and RDHA monoesters.

2.1. Materials cultured in Dulbecco’s minimal essential medium (DMEM) containing 10% endotoxin-free, heat-inactivated fetal calf serum, 100 Resveratrol and DHA were acquired from DSM Nutritional Products units/mL penicillin, and 100 μg/mL streptomycin. The AGS cell (Columbia, MD, USA). The RAW 264.7 cells, derived from murine lines in DMEM-F12 were grown at 37 °C in 5% CO2 atmosphere. macrophages, were procured from the American Type Culture Col- HepG2, A431, and MCF7 cell lines were grown in DMEM sup- lection (Rockville, MD, USA). Heat-inactivated fetal calf serum was plemented with 10% heat-inactivated fetal calf serum, 100 units/ purchased from GIBCO (Grand Island, NY, USA). Lipopolysaccha- mL of penicillin, and 100 μg/mL of streptomycin. Those cell lines ride (LPS; Escherichia coli O127:E8, molecular weight, 60 kDa) were kept at 37°C in a humidified atmosphere of 5% CO2. was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Human hepatocellular carcinoma (HepG2) cells (BCRC 60025) 2.4. Nitrite assay were procured from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The human A431, AGS, and MCF7 cell lines were isolated from epidermoid carcinoma (ATCC CRL-1555), The cells were seeded into 24 well plates, after reaching a density gastric adenocarcinoma (ATCC CRL-1739) and breast adenocarci- of 1×106 cells/mL and were activated by changing their medium noma (ATCC HTB-22), respectively. Fetal bovine serum (FBS) was to serum-free DMEM without phenol red containing LPS (Zhong bought from Biological Industries (Cromwell, CT, USA). et al., 2012). The RAW 264.7 cells were treated with different concentrations of each compound and LPS or LPS alone for 24 h. The supernatants (100 μL) were mixed with the Griess reagent 2.2. Preparation of resveratrol esters (100 μL, 1% sulphanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water) in duplicate Preparation of resveratrol esters was the same as that previously in 96-well plates. The absorbance was then read at 570 nm with an described (Oh and Shahidi, 2017). Resveratrol was lipophilized ELISA reader. Sodium nitrite (NaNO2) was employed as a stand- with propionyl chloride and docosahexaenoyl chloride. The mo- ard. noesters were identified by HPLC-MS and several types of NMR (1H NMR, 13C NMR, COSY, HSQC, NOESY, and HMBC). Res- 2.5. Cell viability of HepG2, A431, MCF7, HT-29, and AGS veratrol and its esters (Figure 1) were subjected to NO production and cell viability assay evaluations. Resveratrol and its esters were dissolved in dimethyl sulfoxide (DMSO). Cell viability of various cancer cell lines was assessed 2.3. Cell culture by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium- bromide (MTT) assay (Mosmann, 1983). The cells were plated at The RAW 264.7 cells, derived from murine macrophages, were a density of 2×105 cells/mL into 96 well plates and incubated for

74 Journal of Food Bioactives | www.isnff-jfb.com Oh et al. Lipophilized resveratrol affects the generation of reactive nitrogen species

effective as resveratrol itself at the same concentration. Tsai et al. (1999) reported that resveratrol inhibited the generation of NO and reduced the level of iNOS protein. They also found almost complete suppression of iNOS mRNA by resveratrol, suggesting that the inhibition of iNOS generation might be due to suppression of iNOS mRNA. Bi et al. (2005) reported that resveratrol inhibited the release of NO as well as TNF-α induced by LPS. Zhong et al. (1999) stated that resveratrol showed inhibition of IL-6 release. TNF-α and IL-6 are involved in pro- inflammatory responses. Resveratrol esters have not been studied on the iNOS protein generation, iNOS gene expression, and the release of IL-6 and TNF-α, however they are expected to exert an effect on pro-inflammatory agents due to possessing inhibi- Figure 2. Effect of resveratrol and its monoesters on NO production in tory activity on NO production. This implies that all tested com- LPS-induced RAW 264.7 macrophages. RAW264.7 cells were treated with pounds may render an inhibitory effect on NO production and resveratrol, RDHA and RC3:0 different concentrations and LPS (100 ng/mL) possibly serve as anti-inflammatory agents, but this needs to be for 24 h. Results were statistically analysed with one-way ANOVA followed further investigated. by Tukey’s post hoc test. Statistical significance, *P < 0.05, **P < 0.01 and ***P < 0.001, compared with the LPS-treated group. 3.2. Effect of resveratrol and its esters on the viability of HepG2, 24 h. The cells were then pretreated with test compounds at dif- A431, MCF7, HT-29, and AGS cells ferent concentration for 24 h. The final concentration of DMSO in the culture medium was < 0.05% (v/v). MTT (100 μL) was added, The effect of resveratrol and its esters at various concentrations on and the cells were incubated for a further 1 h. Then the medium the viability of HepG2, A431, MCF7, HT-29, and AGS cells was was aspirated and 100 μL of DMSO were added to dissolve the determined by employing the MTT assay. As shown in Figure 3, MTT-formazan crystals formed by metabolically viable cells. Cell resveratrol and its esters decreased the growth in cultured human viability was scanned with an enzyme-linked immunosorbent as- cancer cells. Resveratrol at 5 μg/mL was able to decrease cell vi- say reader with a 570 nm filter. ability in all tested cancer cell lines except for A431 cells, whereas 5 μg/mL of RC3:0 showed cytotoxicity in all cancer cells. Con- centration at 50-75 μg/mL, resveratrol decreased the cell viability 3. Results and discussions less than 40% in HepG2, MCF7, HT29, and AGS, whereas RC3:0 showed less than 40% cell viability in HepG2, A431, MCF7, and 3.1. Nitric oxide (NO) production AGS. RDHA at all concentrations showed the lowest cytotoxicity in HePG2, A431, HT29, and AGS cells compared to resveratrol and RC3:0. However, 50 and 75 μg/mL of RDHA in MCF7 cells NO is an important molecule with known beneficial activities such showed no cell viability. as vascular relaxation and neurotransmission in mammals (Cole- Several studies have demonstrated the anticancer activity of man, 2001). The solubility and diffusion properties of NO are resveratrol and its derivatives. Jang et al. (1997) studied the an- similar to those of oxygen and it can freely cross cell membranes ticancer activity of resveratrol in three major stages of carcino- and has a long half-life (Coleman, 2001). It is a relatively stable genesis and found its chemopreventive effect due to acting as an − radical, however it can form peroxynitrite anion (ONOO ) which antioxidant, antimutagen, anti-inflammatory, antipromotion, and is a more reactive form of RNS (Kruidenier and Verspaget, 2002). antiprogression agent. In addition, resveratrol inhibited preneo- Moreover, presence of an excessive amount of NO in activated plastic lesions in a mouse mammary glands culture model of car- immune cells leads to tissue damage during inflammation (Zhong cinogenesis and inhibited tumorigenesis in a mouse skin cancer et al., 2012). NO is generally synthesized from L-arginine by three model. Pan et al. (2007) demonstrated that pterostilbene also pos- different types of NO synthase (NOS), namely neural NO synthase sessed effects on cancer cells. They studied the inhibition of cell (nNOS), endothelium NO synthase (eNOS), and inducible NO proliferation in pterostilbene treated human cancer cells such as synthase (iNOS). Unlike nNOS and eNOS, iNOS is continuously COLO 205, AGS, HL-60, HepG2, and HT-29. They reported that active once expressed and produces NO at a sustained high level. pterostilbene (60 μM) showed the largest sensitive inhibitory ef- Expression of iNOS can be induced by LPS or cytokines such as fect on AGS cell-growth compared to the other cell tested. Patel tumour necrosis factor (TNF)-α and interleukin (IL)-1 (Coleman, et al. (2010) revealed the concentration of resveratrol and its me- 2001). tabolites in the colorectal tissue of cancer patients and the poten- In this study, inhibition of LPS-induced NO production in mu- tial benefit of resveratrol as a chemopreventive agent in colorectal rine RAW 264.7 was examined for two different resveratrol esters cancer. They found that daily doses of 0.5 or 1.0 g resveratrol (Figure 1) and compared with resveratrol. The nitrite concentra- could reduce cancer cell proliferation by 5%. However, some tion in the culture medium was considered as NO production. As studies have failed to show such anticancer activity. For example, shown in Figure 2, the nitrite level of LPS treatment alone (LPS) Bove et al. (2002) found that resveratrol showed inhibitory effect increased greatly compared to that of the control (p < 0.005). All on the growth of 4T1 breast cancer cell line (in vitro) in a dose- samples were able to attenuate NO production in murine RAW and time-dependent manner, however it showed no effect on the 264.7 cells. Resveratrol and RC3:0 showed decrease of NO pro- growth of 4T1 breast cancer in mice (in vivo). Baur and Sinclair duction at both 5 and 25 μg/mL. However, RDHA at 5 μg/mL was (2006) suggested that this failure could be due to an inadequate able to attenuate the NO production slightly but an insignificant dose of resveratrol, delivery method, and tumor origin. In phar- difference was observed as compared to LPS; RDHA at 25 μg/mL macokinetic perspective, resveratrol has extremely low bioavail- was able to inhibit overproduction of NO effectively and it was as ability, and increasing the dose of resveratrol to overcome its low

Journal of Food Bioactives | www.isnff-jfb.com 75 Lipophilized resveratrol affects the generation of reactive nitrogen species Oh et al.

Figure 3. Effect of (a) resveratrol, (b) RC3:0, and (c) RDHA on cell viability of various human cancer cells. Cancer cell lines were treated with different concentrations of resveratrol, RDHA and RC3:0 for 24 h and then analysed by MTT assay. Results were statistically analysed with one-way ANOVA followed by Tukey’s post hoc test. Statistical significance,P * < 0.05, **P < 0.01 and ***P < 0.001, compared with the control group.

76 Journal of Food Bioactives | www.isnff-jfb.com Oh et al. Lipophilized resveratrol affects the generation of reactive nitrogen species bioavailability might not be possible due to toxicity. Therefore, Fragopoulou, E., Nomikos, T., Karantonis, H.C., Apostolakis, C., Pliakis, E., lipophilised resveratrols need to be evaluated further in vivo for Samiotaki, M., Panayotou, G., and Antonopoulou, S. (2007). Biologi- their anti-proliferative effect, which may ultimately lead to anti- cal activity of acetylated phenolic compounds. J. Agric. Food Chem. cancer potential. 55: 80–89. Fransen, M., Nordgren, M., Wang, B., and Apanasets, O. (2012). Role of peroxisomes in ROS/RNS-metabolism: Implications for human disease. Biochim. Biophys. Acta. 1822: 1363–1373. 4. Summary Halliwell, B. (1996). Free radicals, proteins and DNA: Oxidative damage versus redox regulation. Biochem. Soc. Trans. 24: 1023–1027. Two resveratrol esters demonstrated anti-inflammatory and anti- Jang, M., Cai, L., Udeani, G.O., Slowing, K.V., Thomas, C.F., Beecher, C.W.W., proliferative activities in this study. The test compounds were able Fong, H.H.S., Farnsworth, N.R., Kinghorn, A.D., Mehta, R.G., Moon, to inhibit NO production in murine RAW 264.7 cells, suggesting R.C., and Pezzuto, J.M. (1997). Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275: their potential anti-inflammatory effect, which needs to be further 218–220. investigated on the generation of iNOS protein, iNOS gene ex- Kruidenier, L., and Verspaget, H.W. (2002). Review article: Oxidative stress pression, the release of IL-6 and TNF-α. In addition, all test com- as a pathogenic factor in inflammatory bowel disease - radicals or pounds in this study showed decreased cell viability of HepG2, ridiculous? Aliment. Pharmacol. Ther. 16: 1997–2015. A431, MCF7, HT-29, and AGS. The results suggest that resvera- Loft, S., and Poulsen, H.E. (1996). Cancer risk and oxidative DNA damage in trol esters need to be further investigated for their anti-inflamma- man. J. Mol. Med. 74: 297–312. tory and anti-proliferative potential. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. lmmunol. Methods. 65: 55–63. Oh, W.Y., and Shahidi, F. (2017). Lipophilization of resveratrol and effects Acknowledgments on antioxidant activities. J. Agric. Food Chem. 65: 8617–8625. Pan, M.-H., Chang, Y.-H., Badmaev, V., Nagabhushanam, K., and Ho, C.-T. One of us (FS) thanks the Natural Science and Engineering Re- (2007). Pterostilbene induces apoptosis and cell cycle arrest in hu- search Council (NSERC) of Canada for partial financial support. man gastric carcinoma cells. J. Agric. Food Chem. 55: 7777–7785. Patel, K.R., Brown, V.A., Jones, D.J.L., Britton, R.G., Hemingway, D., Miller, A.S., West, K.P., Booth, T.D., Perloff, M., Crowell, J.A., Brenner, D.E., References Steward, W.P., Gescher, A.J., and Brown, K. (2010). Clinical pharma- cology of resveratrol and its metabolites in colorectal cancer patients. Cancer Res. 70: 7392–7399. Baur, J.A., and Sinclair, D.A. (2006). Therapeutic potential of resveratrol: Torres, P., Poveda, A., Jimenez-Barbero, J., Ballesteros, A., and Plou, F.J. The in vivo evidence. Nat. Rev. Drug Discov. 5: 493–506. (2010). Regioselective lipase-catalyzed synthesis of 3-O-acyl deriva- Bi, X.L., Yang, J.Y., Dong, Y.X., Wang, J.M., Cui, Y.H., Ikeshima, T., Zhao, Y.Q., tives of resveratrol and study of their antioxidant properties. J. Agric. and Wu, C.F. (2005). Resveratrol inhibits nitric oxide and TNF-α pro- Food Chem. 58: 807–813. duction by lipopolysaccharide-activated microglia. Int. Immunophar- Tsai, S.-H., Lin-Shiau, S.-Y., and Lin, J.-K. (1999). 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Journal of Food Bioactives | www.isnff-jfb.com 77 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.

Viewpoint

1 Are there serious adverse effects of omega-3 polyunsaturated fatty acid supplements? Klaus W. Lange, Yukiko Nakamura, Alexander M. Gosslau, Shiming Li

Dietary omega-3 polyunsaturated fatty acids (PUFAs) may reduce the risk of various diseases.

Perspective

7 A perspective on phenolic compounds, their potential health benefits, and international regulations: The revised Brazilian normative on food supplements Adriano Costa de Camargo, Renan da Silva Lima

Phenolic compounds possess a myriad of health benefits, thus making them potential ingredients for the procurement of food supplements.

Reviews

18 Anticancer and antiproliferative properties of food-derived protein hydrolysates and peptides Ifeanyi D. Nwachukwu, Rotimi E. Aluko

Cancers of all types are among the four main non-communicable diseases, a category of diseases responsible for 38 million yearly deaths worldwide.

78 27 UF-LC-DAD-MSn for discovering enzyme inhibitors for nutraceuticals and functional foods Li Li, Rong Tsao

The technique of ultrafiltration liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UF-LC-MSn) in screening enzyme inhibitors has emerged as an efficient tool for high-throughput screening of bioactives.

Original Research

36 Revisiting DPPH (2,2-diphenyl-1-picrylhydrazyl) assay as a useful tool in antioxidant evaluation: a new IC100 concept to address its limitations JuDong Yeo, Fereidoon Shahidi

The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay has been widely used in antioxidant evaluation.

43 Antioxidant activity of soybean peptides on human hepatic HepG2 cells Carmen Lammi, Carlotta Bollati, Anna Arnoldi

Soybean is an interesting source of bioactive peptides useful for the development of functional foods and nutraceuticals.

48 Intestinal permeability and transport of apigenin across Caco-2 cell monolayers Marina Zulkifli, Amin Ismail, Loh Su Peng, Fadhilah Jailani, Nur Kartinee Kassim

79 Investigation on bioavailability was carried out by determining the absorption and transport of bioaccessible apigenin from fruit of mango (Mangifera indica var. Water Lily) into Caco-2 human intestinal cell using a reliable and sensitive analytical method of LC-MS/MS.

56 Modulatory effect of selected phenolic acids on enzyme activities of purinergic signaling Adedayo O. Ademiluyi, Taiwo A. Adeyeye, Opeyemi B. Ogunsuyi, Damilola M. Olatunde, Ganiyu Oboh

The antioxidant activities and effects of selected phenolic acids (ferulic, caffeic, p-coumaric and chlorogenic acids) on enzymes (ATPdase, ecto-5′ nucleotidase, phosphodiesterase-5′ and Na+/K+ ATPase) of the purinergic signalling in isolated rat brain were assessed.

63 Optimization of extraction of antioxidants from aromatic herbs and their synergistic effects in a lipid model system Thais Maria Ferreira de Souza Vieira, Marilis Yoshie Hayashi Shimano, Renan da Silva Lima, Adriano Costa de Camargo

Response surface methodology was applied to improve the polyphenol extraction process of rosemary, oregano, sage, and thyme.

73 Lipophilized resveratrol affects the generation of reactive nitrogen species in murine macrophages and cell viability of human cancer cell lines Won Young Oh, Yi-Shiou Chiou, Pei-Sheng Lee, Min-Hsiung Pan, Fereidoon Shahidi

Resveratrol was esterified with selected fatty acids to improve its lipophilicity and potential application in food and biological systems.

Journal of Food Bioactives

Table of Contents

Viewpoint Are there serious adverse effects of omega-3 polyunsaturated fatty acid supplements? Klaus W. Lange, Yukiko Nakamura, Alexander M. Gosslau, Shiming Li...... 1

Perspective A perspective on phenolic compounds, their potential health benefits, and international regulations: The revised Brazilian normative on food supplements Adriano Costa de Camargo, Renan da Silva Lima...... 7

Reviews Anticancer and antiproliferative properties of food-derived protein hydrolysates and peptides Ifeanyi D. Nwachukwu, Rotimi E. Aluko . 18

UF-LC-DAD-MSn for discovering enzyme inhibitors for nutraceuticals and functional foods Li Li, Rong Tsao . 27

Original Research Revisiting DPPH (2,2-diphenyl-1-picrylhydrazyl) assay as a useful tool in antioxidant evaluation: a new IC100 concept to address its limitations JuDong Yeo, Fereidoon Shahidi ...... 36

Antioxidant activity of soybean peptides on human hepatic HepG2 cells Carmen Lammi, Carlotta Bollati, Anna Arnoldi...... 43

Intestinal permeability and transport of apigenin across Caco-2 cell monolayers Marina Zulkifli, Amin Ismail, Loh Su Peng, Fadhilah Jailani, Nur Kartinee Kassim...... 48

Modulatory effect of selected phenolic acids on enzyme activities of purinergic signaling Adedayo O. Ademiluyi, Taiwo A. Adeyeye, Opeyemi B. Ogunsuyi, Damilola M. Olatunde, Ganiyu Oboh...... 56

Optimization of extraction of antioxidants from aromatic herbs and their synergistic effects in a lipid model system Thais Maria Ferreira de Souza Vieira, Marilis Yoshie Hayashi Shimano, Renan da Silva Lima, Adriano Costa de Camargo...... 63

Lipophilized resveratrol affects the generation of reactive nitrogen species in murine macrophages and cell viability of human cancer cell lines Won Young Oh, Yi-Shiou Chiou, Pei-Sheng Lee, Min-Hsiung Pan, Fereidoon Shahidi ...... 73