There are also a number of chalcones (the precursors of flavonoids and isoflavonoids), such as licochalcone A, kuraridin, kuraridinol, 2,4,20,40-tetrahydroxy-3-(3-methyl-2-butenyl) and morachalcone A, with remarkable tyrosinase inhibitory activity [72]. Additionally, due to the versatile bioactivity and unique structural motif of chalcones, a number of derivatives have

Although a great number of phenolic compounds revealed tyrosinase inhibitory capacity, the necessity to clarify the viability of these inhibitors in terms of their skin-whitening efficiency

The control of some important human diseases includes the use of enzyme inhibitors; however, there is some concern about the use of synthetic inhibitors due to their side effects. Thus, the use of natural enzyme inhibitors, particularly from plant origin, is encouraged nowadays. Indeed, up to date, extensive research has been conducted to study the enzyme inhibitory properties of phenolic compounds. Data revised showed that different phenolic compounds are efficient inhibitors of the activity of a broad number of enzymes linked with important human conditions, such as hypertension, type II diabetes, obesity, Alzheimer's diseases, inflammation, and skin disorders. It is unmistakable that phenolic compounds are multifunctional compounds that provide a wide spectrum of biological actions beneficial for human health. These compounds exert their action by different mechanisms and have a huge potential in the prevention and treatment of several human diseases. Our bibliographic survey also indicates that flavonoids are probably the group of phenolic compounds with great capacity to inhibit the activity of all the human enzymes analyzed. Several structure-activity relationships studies can be found for some flavonoid compounds. In addition to the enzyme inhibitory capacity, several studies demonstrated the strong antioxidant and anti-inflammatory properties of flavonoids which highlights the relevance of these compounds for the preven-

For the future, standardized protocols to search potential inhibitors should be designed in order to minimize the differences among obtained results. Despite the vast number of phenolic compounds studied *in vitro,* few compounds have continued to *in vivo* tests. In addition,

tion and control of diseases involving oxidative stress or inflammation.

further studies should be performed to predict drug-likeness and drug ability.

necessary for tyrosinase inhibitory activity [71].

comparably effective and much more stable than resveratrol [75].

been developed as effective tyrosinase inhibitor candidates.

has become an urgent task.

112 Phenolic Compounds - Biological Activity

**5. Concluding remarks**

S. Gonçalves acknowledges a grant from the Foundation for Science and Technology (FCT), Portugal (SFRH/BPD/84112/2012), financed by POPH-QREN and subsidized by the European Science Foundation.

## **Author details**

Sandra Gonçalves and Anabela Romano\*

\*Address all correspondence to<EMAIL_ADDRESS>
Faculty of Sciences and Technology, MeditBio, University of Algarve, Campus de Gambelas, Faro, Portugal

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Additional information is available at the end of the chapter

Anthony Ananga, James Obuya, Joel

http://dx.doi.org/10.5772/66894

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118 Phenolic Compounds - Biological Activity

Grapes (*Vitis* spp.) are consumed as fresh table fruits, raisins, and processed into wine, juice, jelly and other value-added products. Grapes contain bioactive secondary metabolites (polyphenols), such as proanthocyanins (oliogemeric flavonoids), flavonoids (catechin, epicatechin, and quercetin), and anthocyanins. They have non-flavonoids such as hydroxycinnamic acids (p-coumaric, cinnamic, caffeic, gentisic, ferulic, and vanillic acids), and hydroxybenzoic acids: trihydroxy stilbenes (resveratrol and polydatin). These phytochemicals are of economic importance to pharmaceutical, food and cosmetic industries. Nutraceuticals from grape seeds have potential cardioprotective, anti-cancer, antioxidant, anti-inflammatory, antiviral, neuroprotective, hepatoprotective and antimicrobial properties. Grape seed nutraceuticals have been re-invented in the past few years as a new paradigm in human medicine. In particular, nutraceuticals from grape seeds have been used in stopping wound bleeding, anti-inflammatory agents, pain relief, and anti-diarrhea. In addition, they can be used for the treatment of various human health conditions such as cancer, cholera, smallpox, and nausea as well as eye infections, skin, kidney, liver diseases, etc. Nowadays, consumers are demanding for healthy supplements and personal care products with natural ingredients. Therefore, the present review highlights recent developments and future opportunities of grape seed nutraceuticals for the prevention of human diseases.

**Keywords:** grape seeds, polyphenols, oxidation, nutraceuticals, human diseases

## **1. Introduction**

Grape (*Vitis* spp.) is one of the most economically important fruit crops worldwide [1]. Grapevine has a rich diversity, as reflected by global variations between wines from different continents or adjacent vineyards. These differences can be attributed to geographical locations, diversity in

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

climate conditions, or by human interventions arising from breeding and other vineyard management practices. Grapes are consumed fresh as table fruits or raisins and can be processed into wine, jam, jelly, grape seed oil, vinegar, grape seed extracts (GSE) and other products [1, 2]. The quality of grape products depends on a wide range of factors such as variety, environmental conditions, viticultural practices and, more importantly, chemical properties of their secondary metabolites [1]. Secondary metabolites found in grape seeds include phytochemicals such as flavonoids, which are a group of natural polyphenols derived from phenylpropanoid pathway [2]. The stability of secondary metabolites may be impacted by external factors, such as environmental conditions (pH, light, temperature, etc.), due to the nature of physiological functions in growth and development of grapevines [3, 4]. Grape seeds contain phytochemicals such as alkaloids, terpenes, volatile oils, resins, glycosides, tannins, sterols, saponins, and phenolics. These polyphenols have important applications in pharmaceutical, agrochemical, food, and cosmetic industries [1, 5, 6]. More importantly, phenolic compounds are key determinants of wine quality such as aroma, color, and taste and are collectively referred to as organoleptic characteristics of grape products [7, 8]. For example, the quality of grape products is characterized by polyphenolic compounds such as flavonoids and anthocyanins. These natural molecules are generated through specific biosynthetic pathways in grapes.

Flavonoids are primarily located in the epidermal layer of berry skin and seeds in grapes [9]. They have biological attributes such as cytotoxicity and antioxidants, and their biochemical properties play important functions in the defense against biotic and abiotic stresses in grapevines [10, 11]. These phenolic-rich compounds are potential nutraceuticals with biological properties for treatment and/or prevention of various human diseases, whereby they can be used as antioxidants, anti-inflammatory, and antimicrobial agents. Application of nutraceuticals from grape seeds in human health is not new, because wine has been used for medicinal purposes since the medieval period. However, it has reemerged as an important field of human medicine and human nutrition in the past few years [12]. For instance, ethanol in "alcoholic" beverages has potential application in human health due to its properties in inhibition of platelet aggregation, eicosanoid biosynthesis, and an antioxidant for free radicals [13]. Hence, it is important to analyze and characterize grape seed products for biochemical composition, biological activity, and bioavailability of phytochemicals and investigate their correlation to human health.

## **2. History and establishment of grapes in North America for food and wine industries**

Domesticated crops appeared after the Neolithic period (4000–1000 BC) and included grape varieties that were selected for wine production, because they were easier to propagate and had higher proportion of flesh compared to seeds. Wine production from grape has been part of human culture for nearly 6000 years, with the earliest evidence dating back to between 7000 and 5000 BC. Wine produced from grapes was used in dietary applications, medicine, and socioreligious activities. The earliest known winemaking facility, or at least its development, was considered to be in the Caucasus Mountains (4100–4000 BC), near the village of Areni in Southern Armenia. The region is considered as the origin of domesticated wine grapes (*Vitis vinifera*) [13]. However, the transfer of winemaking knowledge and technology has been somewhat linear, as it moved from Western Asia to Eastern Mediterranean region such as Egypt, Greece, and Southern Italy, before reaching the rest of Europe c.a. 2000 years ago. Today, for instance, wine grape is produced in every continent worldwide, and its chemical composition is profoundly influenced by factors such as improved enological techniques, region-specific grape cultivars, and local climatic conditions [13, 14]. The development of grape production and winemaking methods has been parallel to the overall technological advances, which came after Western civilization. There has been tremendous progress made in grape production, especially with respect to vine cultivation, vinification equipment, and winemaking practices such as fermentation, as compared with those used during the Neolithic era, including contemporary Egypt, ancient Greece, Western Asia, Ancient Israel, or the Roman Empire. These techniques plateaued around 200–400 AD and was followed by a period of between 1200 and 1400 years when the progress of wine technology slowed down and was generally restricted to monastic religious orders of Western Europe [15]. Wine production methods increased during the eighteenth century, and the phenomenon was likely due to positive changes in trade relations in Europe, which led to increased demand for vintage and age-worthy wines associated with higher quality [15].

climate conditions, or by human interventions arising from breeding and other vineyard management practices. Grapes are consumed fresh as table fruits or raisins and can be processed into wine, jam, jelly, grape seed oil, vinegar, grape seed extracts (GSE) and other products [1, 2]. The quality of grape products depends on a wide range of factors such as variety, environmental conditions, viticultural practices and, more importantly, chemical properties of their secondary metabolites [1]. Secondary metabolites found in grape seeds include phytochemicals such as flavonoids, which are a group of natural polyphenols derived from phenylpropanoid pathway [2]. The stability of secondary metabolites may be impacted by external factors, such as environmental conditions (pH, light, temperature, etc.), due to the nature of physiological functions in growth and development of grapevines [3, 4]. Grape seeds contain phytochemicals such as alkaloids, terpenes, volatile oils, resins, glycosides, tannins, sterols, saponins, and phenolics. These polyphenols have important applications in pharmaceutical, agrochemical, food, and cosmetic industries [1, 5, 6]. More importantly, phenolic compounds are key determinants of wine quality such as aroma, color, and taste and are collectively referred to as organoleptic characteristics of grape products [7, 8]. For example, the quality of grape products is characterized by polyphenolic compounds such as flavonoids and anthocyanins. These natural mol-

Flavonoids are primarily located in the epidermal layer of berry skin and seeds in grapes [9]. They have biological attributes such as cytotoxicity and antioxidants, and their biochemical properties play important functions in the defense against biotic and abiotic stresses in grapevines [10, 11]. These phenolic-rich compounds are potential nutraceuticals with biological properties for treatment and/or prevention of various human diseases, whereby they can be used as antioxidants, anti-inflammatory, and antimicrobial agents. Application of nutraceuticals from grape seeds in human health is not new, because wine has been used for medicinal purposes since the medieval period. However, it has reemerged as an important field of human medicine and human nutrition in the past few years [12]. For instance, ethanol in "alcoholic" beverages has potential application in human health due to its properties in inhibition of platelet aggregation, eicosanoid biosynthesis, and an antioxidant for free radicals [13]. Hence, it is important to analyze and characterize grape seed products for biochemical composition, biological activity, and bioavailability of phytochemicals and investigate their

**2. History and establishment of grapes in North America for food and**

Domesticated crops appeared after the Neolithic period (4000–1000 BC) and included grape varieties that were selected for wine production, because they were easier to propagate and had higher proportion of flesh compared to seeds. Wine production from grape has been part of human culture for nearly 6000 years, with the earliest evidence dating back to between 7000 and 5000 BC. Wine produced from grapes was used in dietary applications, medicine, and

ecules are generated through specific biosynthetic pathways in grapes.

correlation to human health.

120 Phenolic Compounds - Biological Activity

**wine industries**

Wine production began to expand during the seventeenth century, whereby North Americans became latecomers to join the viticulture industry after the arrival of the first European settlers. This was catapulted in part by the quest of the British government to produce its own wine in North America instead of France. The Franciscan missionaries planted the first largescale vineyards in California c.a. 200 years ago and reestablished them shortly after the repeal of prohibition [16]. However, the "Great French Wine Blight" that originated from California vineyards in the 1850s almost wiped out the European grape production. Grape phylloxera, *Phylloxera vitifoliae*, was transported to Europe on infected California rootstocks, and the pandemic almost wiped out c.a. 2.5 million acres of vineyards in France alone. The tide turned around when vineyards in Europe were replanted with *V. vinifera* grafted on Phylloxeraresistant rootstocks, *V. labrusca*, a fox grape species native to the eastern North America [13].

With the rapid advancement of science, it was only natural that research on grapes with focus on value addition became such a huge subject worldwide. This led to increased demand for improved quality attributes such as wine color, flavor, and chemical composition of phenolic compounds as well as the growth of wine and grape associated-food industries. High demand for vintage and/or age-worthy wines led to increased research in wine and other grape products. The high demand for quality was driven by the fact that wine became an integral part of human lifestyle from various cultural backgrounds worldwide. For example, wine- and grape-related products were used during social events in many countries, whereas in a few other places, these products were used for either religious practices or medicinal purposes.

## **3. Grape seed polyphenols and their benefits to human health**

Grape seeds contain polyphenol-rich phytochemicals such as proanthocyanidins (oligomeric flavonoids), flavonoids (catechin, epicatechin, and quercetin), and anthocyanins. They also contain non-flavonoids such as hydroxycinnamic acids (p-coumaric, cinnamic, caffeic, gentisic, ferulic, and vanillic acids) and hydroxybenzoic acids: trihydroxy stilbenes (resveratrol and polydatin) as well as vitamin E [12, 13, 17–19]. These secondary metabolites are responsible for blue, purple, and red colors in many plant tissues. Also, grape seeds are rich in proanthocyanidins, which are oligomeric anthocyanins [20, 21], and a few monomeric flavonoids. Previous studies indicated that these extracts could be used as supplements [22]. The polyphenols from grape seeds are potent antioxidants (**Figure 1**) that can protect the body from wide range of health conditions such as premature aging, numerous diseases, and decay [23]. During winemaking process, polyphenols from grape seeds can also infiltrate into wine products. Therefore, moderate wine consumption has been associated with reduced mortality from coronary heart disease, because it increases high-density lipoprotein (HDL) cholesterol content and, as a result, inhibits platelet aggregation [13]. The antioxidant effects of red wine and its major polyphenols have been demonstrated in many experimental systems spanning from *in vitro* studies (human low-density lipoprotein, liposomes, macrophages, cultured cells) to *in vivo* involving animal models as well as healthy human subjects [13].

 **Figure 1.** Muscadine grape seeds and their beneficial effects to human health.

## **4. Chemical composition**

**3. Grape seed polyphenols and their benefits to human health**

122 Phenolic Compounds - Biological Activity

*in vivo* involving animal models as well as healthy human subjects [13].

 **Figure 1.** Muscadine grape seeds and their beneficial effects to human health.

Grape seeds contain polyphenol-rich phytochemicals such as proanthocyanidins (oligomeric flavonoids), flavonoids (catechin, epicatechin, and quercetin), and anthocyanins. They also contain non-flavonoids such as hydroxycinnamic acids (p-coumaric, cinnamic, caffeic, gentisic, ferulic, and vanillic acids) and hydroxybenzoic acids: trihydroxy stilbenes (resveratrol and polydatin) as well as vitamin E [12, 13, 17–19]. These secondary metabolites are responsible for blue, purple, and red colors in many plant tissues. Also, grape seeds are rich in proanthocyanidins, which are oligomeric anthocyanins [20, 21], and a few monomeric flavonoids. Previous studies indicated that these extracts could be used as supplements [22]. The polyphenols from grape seeds are potent antioxidants (**Figure 1**) that can protect the body from wide range of health conditions such as premature aging, numerous diseases, and decay [23]. During winemaking process, polyphenols from grape seeds can also infiltrate into wine products. Therefore, moderate wine consumption has been associated with reduced mortality from coronary heart disease, because it increases high-density lipoprotein (HDL) cholesterol content and, as a result, inhibits platelet aggregation [13]. The antioxidant effects of red wine and its major polyphenols have been demonstrated in many experimental systems spanning from *in vitro* studies (human low-density lipoprotein, liposomes, macrophages, cultured cells) to

Research in the chemical composition of grape and wine has advanced greatly in the past 30–40 years. Currently, more than 500 compounds have been identified from wine, out of which 160 of these phytochemicals are esters. Each individual compound may be either insignificant or has no role in human organoleptic (taste) perception, but collectively contribute to wine taste [13]. In grape seeds, the primary compounds such as water, ethanol, organic acids, sugars, and glycerol are present at high concentrations (>100 mg/L), and the rest are polyphenols. These polyphenols are particularly large and complex group of compounds, which are key determinants of quality of red wines. The identification of phenolic compounds in grapes and wine began in the late nineteenth century [15]. Previous studies indicated that grapes contain phytochemicals with antioxidant properties [16, 23].

Chemically, polyphenols are cyclic benzene compounds that contain one or more hydroxyl groups associated directly with the hydroxy-substituted benzene ring structure such as catechins, resveratrol, cyanidin (**Figure 2**), and proanthocyanidins, which are oligomers of resveratrol, catechin, and epicatechin.

 **Figure 2.** Structures of common healthy compounds found in grape seeds: (A) catechins, (B) resveratrol, and (C) cyanidin.

The two primary groups of polyphenols that occur in grape seeds and wine are flavonoids and non-flavonoids. Flavonoids have been characterized as molecules possessing two phenols, which are joined by a pyran (oxygen containing) carbon ring structure. They have a distinct C6 -C3 -C3 three-ring structure. The most common flavonoids in wine are flavanols (flavan-3-ols), which are the building block of grape tannins; whereas anthocyanins are predominantly present in red wines. Flavan-3-ol monomers (catechin) are responsible for the bitterness in wine and are associated with astringency in wine. The major flavan-3-ol monomers found in grapes and wine include (+)-catechin, (−)-epicatechin, and (−)-epicatechin-3-*O*-gallate [24]. Flavonoids are primarily from the skins, seeds, and stems of the fruit. Grape tannins, which are polymers of flavanols, are also known as condensed tannins or proanthocyanidins. Proanthocyanidins contribute to the complexity of wine taste and mouthfeel. Flavanol monomers and oligomers (links of two to four monomers) contribute to the bitterness, and their polymers contribute to astringency in wine. There are five anthocyanidins (cyanidin, peonidin, delphinidin, petunidin, and malvidin) in grapes. Anthocyanin with sugar bound to the anthocyanidin moiety may be acylated (acid linked to the sixth position of the sugar) such as acetic, coumaric, and caffeic acids. Flavonols (kaempferol, quercetin, and myricetin) are present in grape seeds and wine and are esterified to sugars to form glycosides. Flavonols are important cofactors for color enhancement. They also act as a natural sunscreen in the skin of grape berries.

Non-flavonoids have either C<sup>1</sup> -C6 or C3 -C6 structures, meaning that either one carbon or three carbons are attached to the primary benzene ring (six carbons). The majority of the non-flavonoids found in grapes are phenolic acids: hydroxycinnamic acids or hydroxycinnamates (esterified with tartaric acid: caftaric acid, coutaric acid, and fertaric acid), hydroxybenzoic acids (gallic acid, a hydrolysis product from grape or oak tannins), and stilbenes (resveratrol and piceid). They are predominantly present in pulp of grapes and are the major phenolic compounds in white wine [15]. Previous studies indicated that non-flavonoids are produced in the grape berry before veràison [25]. Polymerization of polyhydroxy flavan-3-ol units, (+)-catechin and (−)-epicatechin, and their gallate esters produces oligomers and polymers called proanthocyanidins. Resveratrol is mainly found in the grape skin, whereas proanthocyanidins are found in the seeds [26]. Previous studies indicated that these compounds are high in seed material [27] and are produced before veràison and change during fruit ripening [15]. Tannins on grapes protect wine against oxidation, stabilize wine color, and enhance mouthfeel [13]. Proanthocyanidins are members of a class of compounds described as anthocyanogens, leucoanthocyanidins, flavan-3,4-diols, condensed tannins, and tannins [15]. Proanthocyanidins are polymers of flavan-3-ol subunits, meaning that they have a wide range of molecular weight. These phenolic compounds mainly impact the astringency in red wines, and they have been extracted from the skin, seeds, and stems of the grape berry. Recent studies demonstrated that proanthocyanidins (flavonoids) are some of the major compounds present in grapes and wines.

Flavonoids are a class of secondary plant phenolics with significant antioxidant and chelating properties. For example, their cardioprotective characteristics stem from their inhibition of lipid peroxidation, chelation of redox-active metals, and attenuation of reactive oxygen species [28]. Primarily flavonoids occur in food polymers that are degraded to variable extents in the digestive tract. Although metabolism of these compounds has remained elusive, it has been established that their enteric absorption is correlated to reduction in reactive oxidative species in blood plasma [28]. The propensity of flavonoids to inhibit free radical activities is mediated by their chemical structure. However, their physical properties such as flavan nucleus, number, positions, and types of substitutions are considered to play a major role in their radical scavenging and chelating activity [28].

Structures of common anthocyanins were identified from *V. vinifera* and were determined wine in 1959. It was noted that malvidin-3-*O*-glucoside was a major grape anthocyanin and was present along with its acylated forms [15]. Similarly, it was noted that anthocyanins from *V. vinifera* showed different structural compositions to those isolated from non-*vinifera* species, because they were exclusively monoglucosides and those in non-*vinifera* species were present as 3,5-diglucosides [15]. Because of the unique hue manifestation of both grape and wine phenolics, several studies have been performed on anthocyanins as compared to other compounds [15]. These studies have focused on understanding the changes in anthocyanidins in respect to berry development [29], potential impact of cultural practices on production [30], and techniques for their extraction from wine and/or grape seed extracts [31].

## **5. Biological activities**

to the anthocyanidin moiety may be acylated (acid linked to the sixth position of the sugar) such as acetic, coumaric, and caffeic acids. Flavonols (kaempferol, quercetin, and myricetin) are present in grape seeds and wine and are esterified to sugars to form glycosides. Flavonols are important cofactors for color enhancement. They also act as a natural sunscreen in the skin

carbons are attached to the primary benzene ring (six carbons). The majority of the non-flavonoids found in grapes are phenolic acids: hydroxycinnamic acids or hydroxycinnamates (esterified with tartaric acid: caftaric acid, coutaric acid, and fertaric acid), hydroxybenzoic acids (gallic acid, a hydrolysis product from grape or oak tannins), and stilbenes (resveratrol and piceid). They are predominantly present in pulp of grapes and are the major phenolic compounds in white wine [15]. Previous studies indicated that non-flavonoids are produced in the grape berry before veràison [25]. Polymerization of polyhydroxy flavan-3-ol units, (+)-catechin and (−)-epicatechin, and their gallate esters produces oligomers and polymers called proanthocyanidins. Resveratrol is mainly found in the grape skin, whereas proanthocyanidins are found in the seeds [26]. Previous studies indicated that these compounds are high in seed material [27] and are produced before veràison and change during fruit ripening [15]. Tannins on grapes protect wine against oxidation, stabilize wine color, and enhance mouthfeel [13]. Proanthocyanidins are members of a class of compounds described as anthocyanogens, leucoanthocyanidins, flavan-3,4-diols, condensed tannins, and tannins [15]. Proanthocyanidins are polymers of flavan-3-ol subunits, meaning that they have a wide range of molecular weight. These phenolic compounds mainly impact the astringency in red wines, and they have been extracted from the skin, seeds, and stems of the grape berry. Recent studies demonstrated that proanthocyanidins (flavonoids) are some of the

Flavonoids are a class of secondary plant phenolics with significant antioxidant and chelating properties. For example, their cardioprotective characteristics stem from their inhibition of lipid peroxidation, chelation of redox-active metals, and attenuation of reactive oxygen species [28]. Primarily flavonoids occur in food polymers that are degraded to variable extents in the digestive tract. Although metabolism of these compounds has remained elusive, it has been established that their enteric absorption is correlated to reduction in reactive oxidative species in blood plasma [28]. The propensity of flavonoids to inhibit free radical activities is mediated by their chemical structure. However, their physical properties such as flavan nucleus, number, positions, and types of substitutions are considered to play a major role in

Structures of common anthocyanins were identified from *V. vinifera* and were determined wine in 1959. It was noted that malvidin-3-*O*-glucoside was a major grape anthocyanin and was present along with its acylated forms [15]. Similarly, it was noted that anthocyanins from *V. vinifera* showed different structural compositions to those isolated from non-*vinifera* species, because they were exclusively monoglucosides and those in non-*vinifera* species were present as 3,5-diglucosides [15]. Because of the unique hue manifestation of both grape and wine phenolics, several studies have been performed on anthocyanins as compared to other

structures, meaning that either one carbon or three

of grape berries.

Non-flavonoids have either C<sup>1</sup>

124 Phenolic Compounds - Biological Activity

major compounds present in grapes and wines.

their radical scavenging and chelating activity [28].

Grape seed products are nutraceutical agents commonly consumed as a health/dietary supplement or are sold as nutrition supplements (100–500 mg). Grape seeds are rich sources of catechins and procyanidins, which are present in red wine and grape juice. Previous studies indicated that these compounds have shown potent antioxidant activities [32–34] and scavenging activity against free radicals [35–38]. The antioxidant capacity of grape seed extracts is considered to perform better compared with vitamins C and E [39]. It was reported that procyanidins inhibited platelet aggregation [40] and had also successfully inhibited the oxidation of low-density lipoprotein (LDL) as well as contributed to reduction in risks of heart disease associated with atherosclerosis [41]. In addition, procyanidins performed very well as anti-inflammatory [42, 43], antimutagens [44], anticancer [45–47], and antiviral [48] agents.

Grape seed extracts are potential antimicrobial for disease control [49] and can be used in food preservatives to ensure food safety [50]. Currently, there has been growing interest on the use of natural antibacterial compounds, such as herbal-based extracts for the preservation of foods, because they possess a unique characteristic flavor and have shown antioxidant and antimicrobial activities [51]. In general, grape seed extracts can be used as antibiotics, antidiarrhea, antiulcer, and anti-inflammatory agents [52, 53]. For example, the mechanism behind their antiulcer is considered to be due to their ability to protect the stomach wall from injuries caused by free radicals as well as the ability of procyanidins to bind other protein targets [53]. Flavonoids have shown success as antiulcer agents, because their suppressive effect depends on the presence of procyanidin oligomers. Previous studies indicated that procyanidins such as catechin oligomers significantly reduced gastric mucosal damage [53]. Furthermore, the binding ability of procyanidin oligomers to bind bovine serum albumin got strengthened in acidified solution. Thus, understanding the biosynthesis of these phenolic compounds may be important to efficiently manage their production as well as insure their bioavailability after wine production. However, it is important to further explore the *in vivo* potential activities of these secondary metabolites from grape seed extract, to determine their potential pharmacological applications in human medicine.

## **6. Health benefits**

Grape seed extracts have potential to be used as nutraceuticals [46, 54]. For instance, red wine constitutes a reliable and rich source of biologically active phytochemicals, such as phenolic acids and polyphenols, whose individual and summated actions are believed to confer health benefits in humans. Epidemiologic studies revealed that individuals with the habit of daily moderate wine consumption can experience significant reductions in ill-health conditions, including those leading to cardiovascular mortality as compared with individuals who either abstain from drinking or consume excess alcohol [55]. Moderate consumption of wine, for example, was implicated in reduced atherosclerosis cardiovascular heart disease in the French population [56]. Heart disease is lower among the French (who have a relatively high red wine intake) as compared with other Europeans, despite their propensity to consume foods known to be rich in cholesterol, which was referred to as the "French paradox" due to their low incidence of coronary heart disease. Previous studies determined that drinking one to two glasses of wine a day can help protect against heart disease. In addition, phenolics in grapes and red wines have been demonstrated to inhibit oxidation of human low-density lipoproteins (LDL) *in vitro* [57]. Hence, researchers are working toward understanding both molecular and nutritional basis for these anecdotal observations. Although the benefits of polyphenols from fruits and vegetables is gaining ground on how they are making significant contribution to human health, but consensus toward increasing the rate of wine consumption is developing quite slowly [55]. This could be due to the negative perception that a huge segment of the society has toward alcohol consumption.

Recently, plant polyphenols have generated increased attention due to their potent antioxidant properties and ability to prevent various diseases associated with oxidative stress [58]. This led to identification and development of phenolic compounds or extracts from different plants with health and related medical applications in the past few years. Medicinal and nutritional values of grapes have been known to exist for thousands of years. For example, several ancient Greek philosophers exalted the healing power of wine from grapes, and in places, such as Ancient Egypt, wine was mythologically considered as "The Gift of Osiris." They also used wine as a solvent for other medicinal products and in combination with other medicines ("polypharmacy"), and wine has been used in prescriptions dating back to between 3400 and 2550 BC [59]. More importantly, wine was at the core of Mediterranean civilization and was the basis of vast seaborne trade that contributed to the spread of Greek civilization beginning the sixth century, including western Asia Minor, southern Italy, Sicily, North Africa, southern France, and Spain [59]. Wine was held in high esteem by the Persians because of its fame as a cure, which was epitomized by the oldest desert proverb: "He that has health has hope; and he that has hope, has everything." Through the medieval period, the conversion of grapes into wine was considered as a gift from the gods, and the best wines were mainly preserved for ruling elite of the society, although the latter may have been associated with the high cost of wine. In addition, wine was used for medicinal purposes during the medieval period in areas such as ancient India in 2000 BC, ancient China in the past 5000 years, and ancient Rome after 146 BC as well as during the era of Biblical Jewish and Arabic period [59]. The rich history of wine provides an insight of its benefit to human health for several years. It is, therefore, the reason why modern science continues to explore benefits of grape seeds as nutraceuticals with the potential to revolutionize modern human medicine.

To date, various parts of grape are known to confer therapeutic benefits in humans. For example, grape leaves can be used to alleviate wound bleeding, inflammation, muscle pain, and diarrhea; whereas unripe grapefruits are recommended for the treatment of sore throat. Raisins (dried grape fruits) can be used to provide relief against constipation and are recommended for treatment of human diseases, including anemia in pregnant women. In addition, ripe and sweet grapes can be used for treatment of diseases such as cancer, cholera, smallpox, nausea, and eye infections as well as can provide relief against skin, kidney, and liver disorders [23]. Grape extracts are rich in bioflavonoids (procyanidins) and are some of the most commonly consumed dietary supplements in the United States due to their potential health benefits. Previous studies indicated that grape extracts are beneficial to human health, because they have showed some efficacy against several diseases such as prostate carcinoma, which causes prostate cancer in men; cardiovascular-related conditions such as hypertension; vascular fragility; and other health conditions such as allergies, hypercholesterolemia, LDL-cholesterol oxidation, and platelet aggregation disorder [60–63]. Other studies have showed that grape extracts can be used as therapeutic agents against diabetic cardiomyopathy [64].

Phenolic compounds such as catechin, quercetin, and resveratrol been known to promote the production of nitric oxide by vascular endothelium cells and, as a result, inhibit biosynthesis of thromboxane in platelets and leukotriene in neutrophils demonstrated to modulate biosynthesis and secretion of lipoproteins in animal models and human cell lines, and arrest tumorigenesis and subsequent carcinogenesis [13]. However, lack of statistical correlation between wine consumption and lower rates of atherosclerosis has made it difficult to resolve the key question of whether moderate consumption may likely lead to decreased atherosclerotic mortality [55]. However, some studies have suggested that there is a relationship between wine consumption and atherosclerotic mortality, which may likely be associated with its direct effect on lipoprotein metabolism.

Moderate ethanol intake is not generally contraindicated in diabetes, although diabetic patients have showed high risk for atherosclerotic cardiovascular disease [65], initiated from oxidative damage of glycosylated proteins and lipids [66–68]. Thus, diabetics as well as nondiabetics are likely to benefit from moderate ethanol intake, because it can help ameliorate risk factors associated with atherosclerotic heart disease that arise from lipoprotein metabolism. Although little information is available on the effects of polyphenols in wine and related grape seed products on diabetics, there may be confounding lifestyle variables associated with spontaneous wine consumption, such as reduced smoking, increased exercise frequency, and better dietary habits that mimic general recommendations given to diabetic patients [55].

## **7. Bioavailability**

moderate wine consumption can experience significant reductions in ill-health conditions, including those leading to cardiovascular mortality as compared with individuals who either abstain from drinking or consume excess alcohol [55]. Moderate consumption of wine, for example, was implicated in reduced atherosclerosis cardiovascular heart disease in the French population [56]. Heart disease is lower among the French (who have a relatively high red wine intake) as compared with other Europeans, despite their propensity to consume foods known to be rich in cholesterol, which was referred to as the "French paradox" due to their low incidence of coronary heart disease. Previous studies determined that drinking one to two glasses of wine a day can help protect against heart disease. In addition, phenolics in grapes and red wines have been demonstrated to inhibit oxidation of human low-density lipoproteins (LDL) *in vitro* [57]. Hence, researchers are working toward understanding both molecular and nutritional basis for these anecdotal observations. Although the benefits of polyphenols from fruits and vegetables is gaining ground on how they are making significant contribution to human health, but consensus toward increasing the rate of wine consumption is developing quite slowly [55]. This could be due to the negative perception that a huge seg-

Recently, plant polyphenols have generated increased attention due to their potent antioxidant properties and ability to prevent various diseases associated with oxidative stress [58]. This led to identification and development of phenolic compounds or extracts from different plants with health and related medical applications in the past few years. Medicinal and nutritional values of grapes have been known to exist for thousands of years. For example, several ancient Greek philosophers exalted the healing power of wine from grapes, and in places, such as Ancient Egypt, wine was mythologically considered as "The Gift of Osiris." They also used wine as a solvent for other medicinal products and in combination with other medicines ("polypharmacy"), and wine has been used in prescriptions dating back to between 3400 and 2550 BC [59]. More importantly, wine was at the core of Mediterranean civilization and was the basis of vast seaborne trade that contributed to the spread of Greek civilization beginning the sixth century, including western Asia Minor, southern Italy, Sicily, North Africa, southern France, and Spain [59]. Wine was held in high esteem by the Persians because of its fame as a cure, which was epitomized by the oldest desert proverb: "He that has health has hope; and he that has hope, has everything." Through the medieval period, the conversion of grapes into wine was considered as a gift from the gods, and the best wines were mainly preserved for ruling elite of the society, although the latter may have been associated with the high cost of wine. In addition, wine was used for medicinal purposes during the medieval period in areas such as ancient India in 2000 BC, ancient China in the past 5000 years, and ancient Rome after 146 BC as well as during the era of Biblical Jewish and Arabic period [59]. The rich history of wine provides an insight of its benefit to human health for several years. It is, therefore, the reason why modern science continues to explore benefits of grape seeds as nutraceuticals

To date, various parts of grape are known to confer therapeutic benefits in humans. For example, grape leaves can be used to alleviate wound bleeding, inflammation, muscle pain, and diarrhea; whereas unripe grapefruits are recommended for the treatment of sore throat.

ment of the society has toward alcohol consumption.

126 Phenolic Compounds - Biological Activity

with the potential to revolutionize modern human medicine.

Some flavonoids found in wine, such as tannins, are in the form of polymers that may not readily break down under physiological conditions and may not be expected to be available for absorption [69]. Conversely, for non-tannin flavonoids in red wine, about half are present as glycosides, and the rest are present as aglycones (glycosyl group replaced by hydrogen). It has been shown that flavan-3-ols, flavonols, anthocyanins, and non-flavonoid stilbenes, which are present in red wine and related grape products, can be absorbed in the gastrointestinal tract [55]. Previous studies reported that anthocyanin pigments, which are responsible for the red color of wine, were found in human urine [70] and human plasma [71] after wine consumption. Most glycosides that pass into the large intestine generally end up being hydrolyzed by fecal microflora, rendering them as free aglycones, which leads to their poor absorption in the small intestines. Resveratrol, a non-flavonoid trihydroxystilbene, which is a relatively minor component of red wine [72], is present at insignificant low levels in white wines [55, 73]. However, long-term wine consumption can increase tissue-specific resveratrol concentrations in the body [74].

Flavonoids can travel in the body while bound to plasma proteins [75, 76]. The metabolism of flavonoid glycosides to aglycones and specific glucuronides occurs in the intestinal tissue, which depends on the structure of the flavonoid [77]. The metabolism of esterified flavonoids to aglycones leads to increased lack of absorption in the small intestine. However, conjugated derivatives of quercetin have retained partial antioxidant activity, and it has been demonstrated that different combinations of these conjugates have potential additive effects [78]. Phenolic acids and polyphenols possess multiple hydroxyl groups and are subject to further metabolism by enzymes in the intestine, liver, and kidney [79, 80]. However, the main drawback is that other important nutraceuticals may not be fully absorbed in the human body. Besides, bioavailability under in vivo conditions needs to be resolved as well as their absorption in the gastrointestinal tract in order to keep them at sustainable pharmacokinetic levels in the blood system.

## **8. Safety concerns for application of grape seed products from American native grapes in foods and cosmetics**

Today, consumer demand for health supplements as well as personal care products with natural and/or organic ingredients is promising. Consumption of a large amount of grapes and related products, such as wine, has contributed to the low risk of chronic diseases, such as coronary heart disease and certain cancers [81]. Grape seed extract, which is primarily a mixture of proanthocyanidins, has been shown to modulate a wide range of biological, pharmacological, and toxicological effects as well as cytoprotective functions. Previous studies investigated the ability of IH636 from grape seed proanthocyanidin extract (GSPE) for the prevention of acetaminophen (AAP)-induced nephrotoxicity, amiodarone (AMI)-induced lung toxicity, and doxorubicin (DOX)-induced cardiotoxicity in mice [82]. However, there are safety concerns on the use of these natural products in humans.

Probably one of the best-known properties of polyphenolic compounds, which may also be of safety concern, is their binding and precipitation of protein targets [52]. Most polyphenols have the capacity to bind proteins because of their high affinity for hydroxylation, that is, introduction of hydroxyl groups on other essential biological compounds. Conversely, low-molecular-weight phenols lack the ability to precipitate proteins, unless their oligomeric composition has at least three flavonol subunits, which may mainly be found on highly polymerized tannin molecules. Tannin-protein complexes arise from interactions between hydrogen molecules and hydrophobic moieties without the participation of their respective covalent or ionic bonds [52]. The safety of plant products may vary due to several factors such as geographical origin, growth conditions, and other production processes [83]. It is therefore important to conduct studies to characterize these phytochemicals from plant-based products, in order to investigate whether they are safe for use by humans.

To date, there are no conclusive studies on contraindication of grape products for either treatment or prevention of human diseases as well as their use in cosmetics. However, recent studies suggest that grape seed products may be safe for treatment of important human diseases. For example, in one particular study which involved the administration of grape seed extract (GSE) as a dietary admixture at levels of 0.02, 0.2, and 2% (w/w) to rats for 90 days, it was demonstrated that GSE showed no toxicity. Results indicated lack of toxicity, which supported the use of proanthocyanidin-rich grape seed extracts [22]. Similarly, in a different study that the administration of GSE IH636 to male and female Sprague-Dawley rats in the feed at levels of 0.5, 1.0, or 2.0% for 90 days, it was reported there were no significant toxicological effects [84]. Besides, Wren and colleagues found no significant changes in clinical signs, hematological parameters, organ weights, ophthalmology evaluations, or histopathology, and identical results were reported from a different study that was conducted on IGS BR rats, which were fed with dietary supplements of GSE at concentrations of 0, 0.63, 1.25, or 2.5% (w/w), which found lack of adverse effects on mouse models [85].

These studies pointed to lack of toxicity and supported the use of proanthocyanidinrich extracts from grape seeds, except for personal care products, in which there has been some evidence for minimal side effects on the skin surface such as irritation, sensitization, phototoxicity, and immediate-type allergy [86].

## **9. Conclusions and future remarks**

present in red wine and related grape products, can be absorbed in the gastrointestinal tract [55]. Previous studies reported that anthocyanin pigments, which are responsible for the red color of wine, were found in human urine [70] and human plasma [71] after wine consumption. Most glycosides that pass into the large intestine generally end up being hydrolyzed by fecal microflora, rendering them as free aglycones, which leads to their poor absorption in the small intestines. Resveratrol, a non-flavonoid trihydroxystilbene, which is a relatively minor component of red wine [72], is present at insignificant low levels in white wines [55, 73]. However, long-term wine consumption can increase tissue-specific resveratrol concentrations

Flavonoids can travel in the body while bound to plasma proteins [75, 76]. The metabolism of flavonoid glycosides to aglycones and specific glucuronides occurs in the intestinal tissue, which depends on the structure of the flavonoid [77]. The metabolism of esterified flavonoids to aglycones leads to increased lack of absorption in the small intestine. However, conjugated derivatives of quercetin have retained partial antioxidant activity, and it has been demonstrated that different combinations of these conjugates have potential additive effects [78]. Phenolic acids and polyphenols possess multiple hydroxyl groups and are subject to further metabolism by enzymes in the intestine, liver, and kidney [79, 80]. However, the main drawback is that other important nutraceuticals may not be fully absorbed in the human body. Besides, bioavailability under in vivo conditions needs to be resolved as well as their absorption in the gastrointestinal tract in order to keep them at sustainable pharmacokinetic levels

**8. Safety concerns for application of grape seed products from American**

Today, consumer demand for health supplements as well as personal care products with natural and/or organic ingredients is promising. Consumption of a large amount of grapes and related products, such as wine, has contributed to the low risk of chronic diseases, such as coronary heart disease and certain cancers [81]. Grape seed extract, which is primarily a mixture of proanthocyanidins, has been shown to modulate a wide range of biological, pharmacological, and toxicological effects as well as cytoprotective functions. Previous studies investigated the ability of IH636 from grape seed proanthocyanidin extract (GSPE) for the prevention of acetaminophen (AAP)-induced nephrotoxicity, amiodarone (AMI)-induced lung toxicity, and doxorubicin (DOX)-induced cardiotoxicity in mice [82]. However, there are

Probably one of the best-known properties of polyphenolic compounds, which may also be of safety concern, is their binding and precipitation of protein targets [52]. Most polyphenols have the capacity to bind proteins because of their high affinity for hydroxylation, that is, introduction of hydroxyl groups on other essential biological compounds. Conversely, low-molecular-weight phenols lack the ability to precipitate proteins, unless their oligomeric composition has at least three flavonol subunits, which may mainly be found on highly

in the body [74].

128 Phenolic Compounds - Biological Activity

in the blood system.

**native grapes in foods and cosmetics**

safety concerns on the use of these natural products in humans.

Phenolic compounds found in grape seeds have desirable biological activities, which are related to their antioxidant properties. These compounds have the ability to scavange for free radicals and inhibit non-desirable enzymatic activities, including the modulation of essential biosynthetic pathways for the metabolism of cellular and extracellular products such as membrane proteins and lipids. Moderate consumption of red wine, for example, can lead to increased plasma concentrations of HDL cholesterol and decreased adhesion of platelets, which may be beneficial to treating platelet aggregration in humans. Biosynthesis of pro-aggregatory eicosanoids such as thromboxane A<sup>2</sup> and synthesis of leukocytes by proinflammatory leukotrienes to inhibit the formation of atherosclerosis can reduce the risk of coronary arterial disease by promoting the relaxation of vascular smooth muscle [16, 87]. In addition, some polyphenols are capable of promoting the synthesis of prostacyclin (prostaglandin member of eicosanoids) and nitric oxide, which can lead to optimized blood flow through the arterial system [13].

Use of nutraceuticals from grape seeds for the mitigation of grape diseases for which biomarkers are known have not been validated. Although nutraceuticals have shown a great promise in the treatment and/or prevention of human diseases, consensus on their wide acceptance as alternative therapeutic agents is developing very slowly. Previous studies demonstrated that nutraceuticals found in grape seeds likely have the ability to modulate cellular metabolism and signaling, which is consistent with reduced coronary arterial disease [16, 87]. However, additional research is needed to address crucial important issues such as the mechanisms by which phytochemicals in grape seeds can be used as nutraceuticals, develop biomarkers for their role in disease prevention, and determine the appropriate dosage for their application in human health medicine.

There are still several gaps that need to be addressed. First, there should be a broad framework for research to understand the mechanism by which polyphenols from grape seed products confer dietary health benefits to humans. Second, additional clinical studies are needed to determine their pharmacokinetic properties in human disease prevention. Third, appropriate biomarkers or mechanistic end points should be developed to determine the interactions between diseases and polyphenols at cellular and subcellular levels. Lack of adequate clinical data on nutraceuticals from grape seed products precludes their use in human health. First, it is not known whether the benefit of red wine originates from the tendency to consume them during meal time, or their consumption together with other macronutrients improves their absorption in the gastrointestinal tract. Second, it is not known whether the ability of wine to scavenge for free radicals originates from its chemical properties as a biofuel, through its ability to cause oxidative stress, or using both mechanisms, which may suggest that it has multiple effects at the cellular or subcellular level.

The success of human genome project and recent developments in molecular biology has led to major revolution in biological sciences. Thus, the application nutraceuticals from grape seeds to human health need to be prioritized as one of the areas of current research. Additional research should focus on understanding potential effects of confounding factors that may be associated with the use of nutraceuticals from grape seeds. This is because preliminary data suggest that nutraceuticals from grape seeds have valuable applications in the prevention and/or treatment of several human diseases. Hence, there are unlimited opportunities presented by the application of polyphenols from grape seeds as nutraceuticals. Therefore, it is important to investigate not only their bioavailability and chemical composition but also to characterize their biological properties to determine their mechanisms as well as their synergistic properties and other components of human diet.

## **Acknowledgements**

The authors are grateful to the Florida A&M University, College of Agriculture and Food Science. The research work has been done with the financial support of USDA/NIFA/AFRI Plant Biochemistry program grant # 2015-12398.

## **Author details**

Use of nutraceuticals from grape seeds for the mitigation of grape diseases for which biomarkers are known have not been validated. Although nutraceuticals have shown a great promise in the treatment and/or prevention of human diseases, consensus on their wide acceptance as alternative therapeutic agents is developing very slowly. Previous studies demonstrated that nutraceuticals found in grape seeds likely have the ability to modulate cellular metabolism and signaling, which is consistent with reduced coronary arterial disease [16, 87]. However, additional research is needed to address crucial important issues such as the mechanisms by which phytochemicals in grape seeds can be used as nutraceuticals, develop biomarkers for their role in disease prevention, and determine the appropriate

There are still several gaps that need to be addressed. First, there should be a broad framework for research to understand the mechanism by which polyphenols from grape seed products confer dietary health benefits to humans. Second, additional clinical studies are needed to determine their pharmacokinetic properties in human disease prevention. Third, appropriate biomarkers or mechanistic end points should be developed to determine the interactions between diseases and polyphenols at cellular and subcellular levels. Lack of adequate clinical data on nutraceuticals from grape seed products precludes their use in human health. First, it is not known whether the benefit of red wine originates from the tendency to consume them during meal time, or their consumption together with other macronutrients improves their absorption in the gastrointestinal tract. Second, it is not known whether the ability of wine to scavenge for free radicals originates from its chemical properties as a biofuel, through its ability to cause oxidative stress, or using both mechanisms, which may suggest that it has

The success of human genome project and recent developments in molecular biology has led to major revolution in biological sciences. Thus, the application nutraceuticals from grape seeds to human health need to be prioritized as one of the areas of current research. Additional research should focus on understanding potential effects of confounding factors that may be associated with the use of nutraceuticals from grape seeds. This is because preliminary data suggest that nutraceuticals from grape seeds have valuable applications in the prevention and/or treatment of several human diseases. Hence, there are unlimited opportunities presented by the application of polyphenols from grape seeds as nutraceuticals. Therefore, it is important to investigate not only their bioavailability and chemical composition but also to characterize their biological properties to determine their mechanisms as well as their syner-

The authors are grateful to the Florida A&M University, College of Agriculture and Food Science. The research work has been done with the financial support of USDA/NIFA/AFRI

dosage for their application in human health medicine.

130 Phenolic Compounds - Biological Activity

multiple effects at the cellular or subcellular level.

gistic properties and other components of human diet.

Plant Biochemistry program grant # 2015-12398.

**Acknowledgements**

Anthony Ananga1, 2\*, James Obuya<sup>1</sup> , Joel Ochieng<sup>3</sup> and Violeta Tsolova1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Center for Viticulture and Small Fruit Research, College of Agriculture and Food Science, Florida A&M University, Tallahassee, FL, USA

2 Food Science Program, College of Agriculture and Food Sciences, Florida A&M University, Tallahassee, FL, USA

3 Faculties of Agriculture and Veterinary Medicine, University of Nairobi, Nairobi, Kenya

## **References**

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## **Phenolic Compounds with Anti-virulence Properties Phenolic Compounds with Anti-virulence Properties**

Naybi Muñoz-Cazares, Rodolfo García-Contreras, Contreras, Macrina Pérez-López and Israel

Macrina Pérez-López and Israel Castillo-Juárez Castillo-Juárez

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Naybi Muñoz-Cazares, Rodolfo García-

http://dx.doi.org/10.5772/66367

#### **Abstract**

Natural products represent the major source of approved drugs and still play an important role in supplying chemical diversity as well as new structures for designing more efficient antimicrobials. They are also the basis for the discovery of new mechanisms of antibacterial action. In this regard, a large number of substances, mainly extracts from natural sources, have been obtained in order to identify their anti-virulence activity. In recent years, there is an increase in the study of anti-virulence natural product derivatives. Different targets have been proposed as a solution to the serious problem of bacterial antibiotic resistance. Inhibition of bacterial quorum-sensing systems has been one of the most studied; however, there are other mechanisms involved in virulence regulation, damage to the host and bacterial survival, which suggests that there are another good targets such as bacterial secretion systems, biofilm formation, two-component systems, flagellum, fimbriae, toxins and key enzymes. Within the natural products, the main anti-virulence compounds are phenolic in nature, so that the next chapter describes and analyzes the relationship between chemical structure and activity of the main phenolic compounds reported.

**Keywords:** anti-virulence, quorum sensing, antibiotic resistance, phytochemicals, antibiofilms

## **1. Introduction**

Since their introduction in the middle forties, antibiotics had been extensively used for the treatment of infectious diseases, producing remarkable results and saving millions of lives worldwide [1]; nevertheless, bacteria are very dynamic organisms able to interchange genes by several mechanisms including conjugation, transformation and transfection via

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

bacteriophages [1]. In addition, they usually replicate at high rates and hence have the ability to evolve quickly and adapt to strong selective pressures; this combined with the selfprescription, inadequate prescription by some physicians (e.g., to treat viral diseases) and their improper use by patients who do not complete the recommended treatment scheme has derived in an alarming situation since to date antibiotic resistance (including multiresistance and panresistance) is a common trend in most of hospital-acquired infections and is becoming more common in community-acquired ones [2, 3]. In fact, the situation is so delicate that recently, the OMS warned that if the current trends are still observed, then by the year 2050 we will enter the post-antibiotic era and previously treatable infectious diseases will cause more deaths than other important diseases such as cancer [4].

Hence, the discovery of new antibiotics as well as the development of alternative approaches to combat bacterial infections is urgently needed [5]; among such new approaches are the inhibition of bacterial antibiotic resistance mechanisms, the utilization of non-antibiotic bactericide agents such as bacteriophages, the repurposing of clinically approved drugs, and the inhibition of bacterial virulence [5]. For the first approach, already successful examples can be found in the clinic; by instance, the co-utilization of clavulanic acid (an inhibitor of β-lactamases) and amoxicillin is commonly administrated [6]; and current research is focused on the utilization of broad spectrum anti-resistance compounds such as those inhibiting multidrug efflux pumps [7]. Regarding the second approach, it was recently demonstrated that some anticancer drugs such as 5-fluorouracil [8], mitomycin C [9] and cisplatin [10] have remarkable antibacterial properties, while bacteriophages had been used in east European countries for the treatment of diverse bacterial infections, and currently, its utilization in the occidental medicine is being proposed [11, 12]. Finally, targeting bacterial virulence instead of their viability is a concept that had derived in several publications, mostly centered in the inhibition of master virulence regulators such as quorum-sensing (QS) systems, which allow several Gram-negative and Gram-positive bacteria to coordinate the production of several virulence factors, once a high population density is reached (**Figure 1A**). Indeed, initially, it was claimed that this approach will be impervious to the generation of resistance since *in vitro* in rich media QS does not control metabolic processes linked to growth; nevertheless, in some conditions, QS inhibition can promote resistance [13–15] and not all clinical strains are sensitive toward current QS inhibitors [16]. However, since QS also regulates the stress response, it has been shown that QS-inhibited bacteria are more susceptible to the action of disinfectants, antibiotics and the immune system [17, 18], and hence, QS inhibition may be a valuable adjuvant therapy for recalcitrant bacterial infections [15].

As mentioned previously, QS is a master regulator of the production of several bacterial virulence factors, such as: exoproteases that degrade connective tissue such as elastase and alkaline protease (collagenase), phenazines that promote the generation of reactive oxygen species, siderophores that facilitate iron uptake, toxins that disrupt cellular processes and exopolysaccharides that form phagocytosis-resistant capsules and participate in the generation of the biofilm matrix [19] (**Figure 1C**).

Another key factor for the development of chronic infections and colonization of surfaces is the formation of biofilms, which is the main way the bacteria are found in nature [20].

bacteriophages [1]. In addition, they usually replicate at high rates and hence have the ability to evolve quickly and adapt to strong selective pressures; this combined with the selfprescription, inadequate prescription by some physicians (e.g., to treat viral diseases) and their improper use by patients who do not complete the recommended treatment scheme has derived in an alarming situation since to date antibiotic resistance (including multiresistance and panresistance) is a common trend in most of hospital-acquired infections and is becoming more common in community-acquired ones [2, 3]. In fact, the situation is so delicate that recently, the OMS warned that if the current trends are still observed, then by the year 2050 we will enter the post-antibiotic era and previously treatable infectious diseases will cause

Hence, the discovery of new antibiotics as well as the development of alternative approaches to combat bacterial infections is urgently needed [5]; among such new approaches are the inhibition of bacterial antibiotic resistance mechanisms, the utilization of non-antibiotic bactericide agents such as bacteriophages, the repurposing of clinically approved drugs, and the inhibition of bacterial virulence [5]. For the first approach, already successful examples can be found in the clinic; by instance, the co-utilization of clavulanic acid (an inhibitor of β-lactamases) and amoxicillin is commonly administrated [6]; and current research is focused on the utilization of broad spectrum anti-resistance compounds such as those inhibiting multidrug efflux pumps [7]. Regarding the second approach, it was recently demonstrated that some anticancer drugs such as 5-fluorouracil [8], mitomycin C [9] and cisplatin [10] have remarkable antibacterial properties, while bacteriophages had been used in east European countries for the treatment of diverse bacterial infections, and currently, its utilization in the occidental medicine is being proposed [11, 12]. Finally, targeting bacterial virulence instead of their viability is a concept that had derived in several publications, mostly centered in the inhibition of master virulence regulators such as quorum-sensing (QS) systems, which allow several Gram-negative and Gram-positive bacteria to coordinate the production of several virulence factors, once a high population density is reached (**Figure 1A**). Indeed, initially, it was claimed that this approach will be impervious to the generation of resistance since *in vitro* in rich media QS does not control metabolic processes linked to growth; nevertheless, in some conditions, QS inhibition can promote resistance [13–15] and not all clinical strains are sensitive toward current QS inhibitors [16]. However, since QS also regulates the stress response, it has been shown that QS-inhibited bacteria are more susceptible to the action of disinfectants, antibiotics and the immune system [17, 18], and hence, QS inhibition may be a valuable adjuvant therapy for recalcitrant bacterial

As mentioned previously, QS is a master regulator of the production of several bacterial virulence factors, such as: exoproteases that degrade connective tissue such as elastase and alkaline protease (collagenase), phenazines that promote the generation of reactive oxygen species, siderophores that facilitate iron uptake, toxins that disrupt cellular processes and exopolysaccharides that form phagocytosis-resistant capsules and participate in the genera-

Another key factor for the development of chronic infections and colonization of surfaces is the formation of biofilms, which is the main way the bacteria are found in nature [20].

more deaths than other important diseases such as cancer [4].

140 Phenolic Compounds - Biological Activity

infections [15].

tion of the biofilm matrix [19] (**Figure 1C**).

**Figure 1.** Main targets of anti-virulence of phenolic compounds. A: Quorum-sensing system, B: biofilm formation, C: toxins, D: two-component systems, E: curli fibers, F: bacterial type III secretion systems, G: flagellum, H: fimbriae, I: sortase enzymes.

These structures consist of multicellular communities enclosed in a matrix which makes them extremely resistant to antibacterial agents (**Figure 1B**) [21]. They also provide robust niches that allow the bacteria to protect themselves from environmental fluctuations and against the immune system, which drastically reduces the effectiveness of antimicrobial therapy [20].

Since for many pathogenic bacteria QS is the main regulator of expression of bacterial virulence factors [19], its disruption has been the main anti-virulence strategy investigated to date [19]. However, another alternative that has also been reported is the *direct inhibition of* individual *virulence factors*, such as toxins, response regulators (two-component regulatory systems (TCS) and processes involved in the formation and maturation of structures such as the curli, the bacterial type III secretion system (T3SS), fimbriae and flagellum.

TCS are response regulators which are formed by a protein localized in the cytoplasmic membrane called histidine kinase sensory protein (HKSP), which acts as an environmental sensor that is activated in ATP-dependent way (**Figure 1D**) [22]. HKSP then activates a response regulator protein (RRP) found in the cytoplasm which is responsible for recognizing DNA sequences that modulate the expression of genes involved in various functions such as chemotaxis, porin expression and expression of virulence factors among others (**Figure 1D**) [22]. An important feature is that TCRs have not detected in mammalian cells, so there are a suitable specific target to treat bacterial infections [23].

The curli (**Figure 1E**) is the major protein component of the extracellular matrix and is mainly produced by enterobacteria to aid in the formation of three-dimensional structures such as biofilms [24]. Curli fibers belong to a growing class of fibers known as amyloid fibers, which are also involved in host cell adhesion and invasion, and are also strong inducers of host inflammatory response [24]. The structure and biogenesis of curli are unique among bacterial fibers and represent an excellent anti-virulence target [25].

The type III secretion system (T3SS) also known as the injectisome is a multiprotein apparatus that facilitates the secretion and translocation of toxins or effector proteins from the bacterial cytoplasm directly to eukaryotic cells (**Figure 1F**) [26, 27]. It is highly conserved in most Gram-negative pathogens, but its presence is not a necessary condition for bacterial survival *in vitro* [27].

Motility and recognition surfaces are key factors for the dispersal and colonization of new niches by bacteria [28]. For that, the flagellum and the fimbriae are target structures suitable for anti-virulence molecules [28, 29]. The flagella (**Figure 1G**) are multiprotein complexes based on flagellin, which rotate allowing bacterial displacement in aqueous media [29], while fimbriae (**Figure 1H**) are extracellular protein structures mainly constituted by pilin, which start in the plasma membrane, cross the cell wall and extend around the cell. These structures allow the adhesion of bacteria mainly to epithelial cells [30].

Another important virulence factors are the sortase enzymes (cysteine transpeptidases) (**Figure 1I**), which are used by Gram-positive bacteria to display proteins in cell surface, such as glycoproteins [30], and they can also attach to proteins in the cross-bridge peptide of the cell wall or link other proteins together to form pilin [31]. The phenomenon of protein deployment is essential for the development of virulence factors and promotes nutrient acquisition, adhesion and immune system evasion [30]. Because surface proteins play a fundamental role in microbial physiology and are frequently virulence factors, sortase enzymes are a very important target [31].

Reports related to the study of natural products as anti-virulence molecules had increased in the last decade. Their powerful attack against bacterial infections without promoting resistance and the elimination of antibiotic-resistant strains are the most attractive features of this kind of compounds. Among natural products with anti-virulence activity, those derived from plants with anti-QS and antibiofilm activity are the most common [32]. Phenolic compounds are secondary metabolites present in plants, which are crucial in many aspects of their lives, especially during the interactions with the environment, since they are used in the defense of plants against bacterial pathogens. Similarly, compounds of phenolic type are the major metabolites with anti-virulence properties described so far, and specifically, the flavonoids are the main representatives [33].

Most of the biologically active reported phenolic compounds have chemical structures with previously identified antimicrobial, antioxidant and anticancer activity. Similarly, for some of them their participation in the regulation of various physiological functions in plants and animals is well known. In recent years, the anti-virulence properties of phenolic compounds are being unravel, and most of the cases depend on the compound concentration and the bacterial system in which the phenolic compounds can exhibit bactericidal or anti-virulence effects. In the next chapter, we discuss studies of phenolic compounds derived mainly from plant species, starting with those that are better characterized and that have more anti-virulence reported properties. We focus on the relationship between their structures and their activity.

## **2. Phenolic compounds anti-virulence**

The curli (**Figure 1E**) is the major protein component of the extracellular matrix and is mainly produced by enterobacteria to aid in the formation of three-dimensional structures such as biofilms [24]. Curli fibers belong to a growing class of fibers known as amyloid fibers, which are also involved in host cell adhesion and invasion, and are also strong inducers of host inflammatory response [24]. The structure and biogenesis of curli are unique among bacterial

The type III secretion system (T3SS) also known as the injectisome is a multiprotein apparatus that facilitates the secretion and translocation of toxins or effector proteins from the bacterial cytoplasm directly to eukaryotic cells (**Figure 1F**) [26, 27]. It is highly conserved in most Gram-negative pathogens, but its presence is not a necessary condition for bacterial survival

Motility and recognition surfaces are key factors for the dispersal and colonization of new niches by bacteria [28]. For that, the flagellum and the fimbriae are target structures suitable for anti-virulence molecules [28, 29]. The flagella (**Figure 1G**) are multiprotein complexes based on flagellin, which rotate allowing bacterial displacement in aqueous media [29], while fimbriae (**Figure 1H**) are extracellular protein structures mainly constituted by pilin, which start in the plasma membrane, cross the cell wall and extend around the cell. These structures

Another important virulence factors are the sortase enzymes (cysteine transpeptidases) (**Figure 1I**), which are used by Gram-positive bacteria to display proteins in cell surface, such as glycoproteins [30], and they can also attach to proteins in the cross-bridge peptide of the cell wall or link other proteins together to form pilin [31]. The phenomenon of protein deployment is essential for the development of virulence factors and promotes nutrient acquisition, adhesion and immune system evasion [30]. Because surface proteins play a fundamental role in microbial physiology and are frequently virulence factors, sortase enzymes are a very

Reports related to the study of natural products as anti-virulence molecules had increased in the last decade. Their powerful attack against bacterial infections without promoting resistance and the elimination of antibiotic-resistant strains are the most attractive features of this kind of compounds. Among natural products with anti-virulence activity, those derived from plants with anti-QS and antibiofilm activity are the most common [32]. Phenolic compounds are secondary metabolites present in plants, which are crucial in many aspects of their lives, especially during the interactions with the environment, since they are used in the defense of plants against bacterial pathogens. Similarly, compounds of phenolic type are the major metabolites with anti-virulence properties described so far, and specifically, the flavonoids

Most of the biologically active reported phenolic compounds have chemical structures with previously identified antimicrobial, antioxidant and anticancer activity. Similarly, for some of them their participation in the regulation of various physiological functions in plants and animals is well known. In recent years, the anti-virulence properties of phenolic compounds are being unravel, and most of the cases depend on the compound concentration and the bacterial

fibers and represent an excellent anti-virulence target [25].

allow the adhesion of bacteria mainly to epithelial cells [30].

*in vitro* [27].

142 Phenolic Compounds - Biological Activity

important target [31].

are the main representatives [33].

#### **2.1. Epigallocatechin gallate and related compounds**

It is well documented that this kind of compounds has antimicrobial, antioxidant, anti-inflammatory, hypocholesterolemic and cancer-preventive properties [34, 35]. The *epigallocatechin gallate* (EGCG) (**Figure 2A**) is one of the flavonoids with the largest number of reports related to its antibiofilm activity; remarkably high compound doses can inhibit bacterial growth, but sublethal concentrations exhibit anti-virulence properties.

At the same concentration, *catechin* (**Figure 2B**) and EGCG inhibit the formation of biofilms of *P. aeruginosa;* however, only catechin do not affect the growth [36], so the presence of galloyl

**Figure 2.** Epigallocatechin gallate and related compounds with anti-virulence properties. **A**: Epigallocatechin gallate, **B**: catechin, **C**: catechin-gallate, **D**: catechin-gallate, **E**: (−) epicatechin gallate.

group in EGCG seems to favor the bactericidal effect. In this regard, it is suggested that EGCG affect the viability because it binds to peptidoglycan, hence directly disrupting the integrity of the bacterial cell wall. Similarly, EGCG at concentrations that affect bacterial viability inhibit the biofilm of *Enterococcus faecalis*, an opportunistic pathogen implicated in urinary tract infections, endocarditis and root canal infections [37]. In this case, biofilm inhibition is attributed to a bactericidal effect, where the EGCG induces hydroxyl radicals that can damage DNA, proteins and lipids [37].

However, using sublethal concentrations, it has been found that EGCG significantly decreased the expression of virulence genes that regulate the expression of cytolysins, gelatinase and serine protease in *E. faecalis* [37]. It also inhibits biofilm formation of Staphylococcal isolates by interfering directly with polysaccharides of the glycocalyx [38]. Similarly, it inhibits swarming and biofilm formation of *Burkholderia cepacia* without affecting the growth, likely through QS inhibition [39].

EGCG and *catechin gallate* (**Figure 2C**) directly inhibit the anthrax lethal factor (LF) produced by *Bacillus anthracis*, which has a key role in the development of anthrax [40]. LF is a zinc metalloprotease that directly affects MAPK-signaling kinases, which are essential for transmitting signals in eukaryotes. EGCG and *catechin gallate* block the activity of LF, preventing MAPK-kinases cleavage and macrophages death [40]. In the case of EGCG, it also delays the death of mice exposed to the anthrax toxin [40]. It is noteworthy that although other catechins were evaluated, the presence of a galloyl group in the structure seems to be essential for this anti-virulence activity.

For the case of *catechin* (**Figure 2B**), it has also been reported that it inhibits the production of virulence factors regulated by QS in *P. aeruginosa*, such as pyocyanin and elastase [41]. Also, it was found to have a negative impact on the transcription of several genes involved in QS, such as those codifying proteins involved in the synthesis of autoinducer molecules [41].

Dental plaque is a complex biofilm that allows the survival and development of *Streptococcus mutans*. It has been reported that EGCG shows bactericidal activity against *S. mutans;* in addition, its antibiofilm activity is due to reducing the adherence of bacteria to surfaces by direct inhibition of glucosyltransferases [42], which are enzymes that synthesize polysaccharides [43, 44]. However, at sublethal concentrations, EGCG reduces biofilm by interfering with gene regulation, specifically by inhibiting the expression of the *gtf* genes (encoding glucosyltransferases), which are associated with adhesion and formation of biofilms [45]. Moreover, it represses genes encoding virulence factors associated with acidogenicity and acidurity, such as *ldh*, *eno*, dATP, Agud and the activity of the F₁F₀-ATPase and lactate dehydrogenase [42].

EGCG at sublethal concentrations also inhibits motility and biofilm formation of *Campylobacter jejuni*, a foodborne pathogen which is one of the main causes of gastrointestinal infections worldwide [46]. In this case, the mechanism involved in biofilm inhibition is related to QS inhibition [46].

It is worth noting that to date there are no studies to investigate its structure-activity relationship, so it is not yet known which parts of the structure are critical to their anti-virulence effects. However, for the (−) *epicatechin* (**Figure 2D**) which also possesses anti-QS activity against *Chromobacterium violaceum*, a Gram-negative bacteria with AHLs mediated QS [47]. The (−) *epicatechin gallate* (**Figure 2E**) at sublethal concentrations inhibits two of the major determinants of virulence in *S. aureus*, the α-toxin and the coagulase [48]. Furthermore, it has been shown that in combination with β-lactams, it is efficient to eliminate multiresistant strains of *S. aureus*. Although it has been observed that some synthetic analogs have better pharmacokinetic properties than the native (−) epicatechin gallate [49, 50].

#### **2.2. Cinnamaldehyde and related compounds**

group in EGCG seems to favor the bactericidal effect. In this regard, it is suggested that EGCG affect the viability because it binds to peptidoglycan, hence directly disrupting the integrity of the bacterial cell wall. Similarly, EGCG at concentrations that affect bacterial viability inhibit the biofilm of *Enterococcus faecalis*, an opportunistic pathogen implicated in urinary tract infections, endocarditis and root canal infections [37]. In this case, biofilm inhibition is attributed to a bactericidal effect, where the EGCG induces hydroxyl radicals that can damage DNA,

However, using sublethal concentrations, it has been found that EGCG significantly decreased the expression of virulence genes that regulate the expression of cytolysins, gelatinase and serine protease in *E. faecalis* [37]. It also inhibits biofilm formation of Staphylococcal isolates by interfering directly with polysaccharides of the glycocalyx [38]. Similarly, it inhibits swarming and biofilm formation of *Burkholderia cepacia* without affecting the growth, likely through

EGCG and *catechin gallate* (**Figure 2C**) directly inhibit the anthrax lethal factor (LF) produced by *Bacillus anthracis*, which has a key role in the development of anthrax [40]. LF is a zinc metalloprotease that directly affects MAPK-signaling kinases, which are essential for transmitting signals in eukaryotes. EGCG and *catechin gallate* block the activity of LF, preventing MAPK-kinases cleavage and macrophages death [40]. In the case of EGCG, it also delays the death of mice exposed to the anthrax toxin [40]. It is noteworthy that although other catechins were evaluated, the presence of a galloyl group in the structure seems to be essential for this

For the case of *catechin* (**Figure 2B**), it has also been reported that it inhibits the production of virulence factors regulated by QS in *P. aeruginosa*, such as pyocyanin and elastase [41]. Also, it was found to have a negative impact on the transcription of several genes involved in QS, such as those codifying proteins involved in the synthesis of autoinducer molecules [41].

Dental plaque is a complex biofilm that allows the survival and development of *Streptococcus mutans*. It has been reported that EGCG shows bactericidal activity against *S. mutans;* in addition, its antibiofilm activity is due to reducing the adherence of bacteria to surfaces by direct inhibition of glucosyltransferases [42], which are enzymes that synthesize polysaccharides [43, 44]. However, at sublethal concentrations, EGCG reduces biofilm by interfering with gene regulation, specifically by inhibiting the expression of the *gtf* genes (encoding glucosyltransferases), which are associated with adhesion and formation of biofilms [45]. Moreover, it represses genes encoding virulence factors associated with acidogenicity and acidurity, such as *ldh*, *eno*, dATP, Agud and the activity of the F₁F₀-ATPase and lactate dehydrogenase [42].

EGCG at sublethal concentrations also inhibits motility and biofilm formation of *Campylobacter jejuni*, a foodborne pathogen which is one of the main causes of gastrointestinal infections worldwide [46]. In this case, the mechanism involved in biofilm inhibition is related to QS

It is worth noting that to date there are no studies to investigate its structure-activity relationship, so it is not yet known which parts of the structure are critical to their anti-virulence effects. However, for the (−) *epicatechin* (**Figure 2D**) which also possesses anti-QS activity

proteins and lipids [37].

144 Phenolic Compounds - Biological Activity

QS inhibition [39].

anti-virulence activity.

inhibition [46].

*Cinnamaldehyde* (CN) (**Figure 3A**) is a major constituent of cinnamon essential oils and occurs naturally in the bark and leaves of cinnamon trees of the genus *Cinnamomum* [51]. The antimicrobial activity of this compound has been proven [52, 53], but new studies have explored their anti-virulence properties, and in contrast to another compounds, it is considered a nontoxic substance widely used in food and in the cosmetic industry and their use is generally recognized as safe [54].

In *P. aeruginosa*, the acylated homoserine lactones (AHLs) are their main autoinducer molecules (**Figure 1A**) and the CN can inhibit their synthesis as well as the production of the phen-

**Figure 3.** Cinnamaldehyde and related compounds with anti-virulence properties. **A**: Cinnamaldehyde, **B**: 2-nitrocinnamaldehyde, **C**: 4-methoxy-cinnamaldehyde, **D**: 3,4-dichloro-cinnamaldehyde, **E**: (*E*)-4-phenyl-3-buten-2-one, **F**: (*E*)-3-decen-2-one, **G**: 4N-4-nitrocinnamaldehyde, **H**: 4D-4-dimethylaminocinnamaldehyde, **I***:* caffeic acid, **J**: ferulic acid, **K**: *p-*coumaric acid, **L**: TS027, **M**: TS110, **N**: 4-methoxy-cinnamic acid, **O**: *trans-2-methoxy-cinnamic acid*.

azine, pyocyanin and swarming motility [55]. Remarkably, CN also has antitoxin production and anti-hemolytic activities [56]. Similarly, in *C. violaceum*, *Yersinia entrerolitica* and *Erwinia carotovora*, the concentration of AHLs was also reduced by CN and the mechanism proposed was the inhibition of synthesis or degradation transformation of the autoinducer [57].

The antibiofilm properties of CN have been widely documented; for example, in *P. aeruginosa* and in enterohemorrhagic *Escherichia coli*, this compound markedly abolished the biofilm formation in a dose-dependent manner by reducing the swarming motility and fimbriae production, respectively. In a previous report, it was shown that for the uropathogenic *E. coli*, CN prevented biofilm formation on plates and catheters, furthermore effectively inactivated preformed biofilms [54]. The mechanism proposed for the biofilm inhibition was related to the hydrophobicity of this compound, which helps to target lipids located in the bacterial cell membrane and mitochondria, increasing the membrane permeability, leading to the leakage of ions and other cell contents [54, 58]. The foodborne pathogen *Listeria monocytogenes* forms biofilm for persistence and survives in which CN has inhibitory effect on formation and inactivating mature biofilm by means of the down-regulated critical genes for biofilm formation in this bacteria [59].

In *Vibrio harveyi*, the autoinducer-2 (A2) is also blocked by CN in a concentration-dependent way by decreasing the binding ability of the autoinducer to its response regulator protein. Between cinnamaldehyde derivatives, the *2-nitro-cinnamaldehyde* (**Figure 3B**) was the most active compound yielding an inhibition of A2 similar to CN [60]. Similarly, the 2-nitro-cinnamaldehyde and *4-methoxy-cinnamaldehyde* (**Figure 3C**) inhibit pigment production and protease activity in *Vibrio anguillarum* [60]. The CN is an aromatic carboxylic acid, and its inhibitory was highly dependent on the substitution pattern of the aromatic ring. Replacement of the dimethylamine (Me<sup>2</sup> N) substituent with a methoxy (MeO) or a nitro (NO<sup>2</sup> ) group enhanced the activity [60].

Various cinnamaldehyde analogs were also evaluated against *Vibrio* spp. The most active compounds were *2-nitro-cinnamaldehyde*, *3, 4-dichloro-cinnamaldehyde* (**Figure 3D**), *(E)-4-phenyl-3 buten-2-one* (**Figure 3E**) and *(E)-3-decen-2-one* (**Figure 3F**), which show inhibitory activity in A2, bioluminescence, pigment and protease production [61]. In this case, also the inhibitory effect of cinnamaldehyde analogs was dependent on the structure, and analogs in which the aromatic ring was replaced by an alkyl moiety, but which still contain the acrolein group, proved also to be active inhibitors [61]. In general, the inhibitory effect of cinnamaldehyde analogs is highly dependent on the nature and degree of substitution of the aromatic ring, and the substituents with electron-withdrawing properties increase its activity. The CN and their analogs furthermore proved to be active blockers of virulence in vivo in different models, suggesting that they may have potential for therapeutic applications in humans and animals [61].

The CN also has inhibitory activity on biofilm formation in a methicillin-resistant *Staphylococcus aureus* at dose-dependent manner and represses the expression of *sarA*, a gene implicated in the regulation of its biofilm [51]. In *Streptococcus pyogenes*, when the biofilm was treated with CN and their derivatives the *2-nitro-cinnamaldehyde* **(Figure 3B)**, *4N-4-nitrocinnamaldehyde* (**Figure 3G**) and *4D-4-dimethylaminocinnamaldehyde* (**Figure 3H**), the biomass, average thickness and colony size at substratum were decreased and the molecular docking shows sequence and structure similarity with the active site for QS inhibition [62].

Among the cinnamaldehyde-related molecules, the *caffeic acid* (CA) (**Figure 3I**) and *ferulic acid* (FA) (**Figure 3J**) have shown antibiofilm properties. CA is the first phenolic acid compound that has been reported to have inhibitory activity on biofilm formation in *Staphylococcus epidermis* by a mechanism that did not involve bacterial death [63]. The potential of FA to control biofilm formation has been demonstrated by the reduction in mass and metabolic activity in *Escherichia coli* and *Listeria monocytogenes* biofilms, and also this compound caused the total inhibition of motility in both bacteria and the colony spreading in *S. aureus;* a form of passive bacterial movement was also inhibited [64].

The QS inhibitory activity of CA and FA also was evaluated in *C. violaceum*, and the results revealed that the activity was mediated by their ability to modulate AHL activity and synthesis [47]. Other related compound the *p-coumaric acid* (**Figure 3K**) showed QS inhibition in reporter strains like *C. violaceum*, *Agrobacterium tumefaciens* and *Pseudomonas chlororaphis* [65]. In addition, it represses the expression of regulatory genes of the T3SS of the phytopathogenic bacteria *Dickeya dadantti*, and for this activity, its hydroxyl group on the phenyl ring and the double bond are important [66]. Some of their derivatives such as TS027 (**Figure 3L**) and TS110 (**Figure 3M**) also repress the expression of T3SS regulatory genes and inhibit T3 effector protein in *P. aeruginosa* without affecting its growth [67]. While the cinnamic acid and *4-methoxy-cinnamic acid* (**Figure 3N**) suppress the expression of T3SS in *Erwinia amylovora* [68], the *o-coumaric acid* (isomer of 3M) and *trans-2-methoxy-cinnamic acid* (**Figure 3O**) suppress translocation of two effector proteins of T3SS in *Xanthomonas oryzae* [69].

#### **2.3. Coumarin and related compounds**

azine, pyocyanin and swarming motility [55]. Remarkably, CN also has antitoxin production and anti-hemolytic activities [56]. Similarly, in *C. violaceum*, *Yersinia entrerolitica* and *Erwinia carotovora*, the concentration of AHLs was also reduced by CN and the mechanism proposed

The antibiofilm properties of CN have been widely documented; for example, in *P. aeruginosa* and in enterohemorrhagic *Escherichia coli*, this compound markedly abolished the biofilm formation in a dose-dependent manner by reducing the swarming motility and fimbriae production, respectively. In a previous report, it was shown that for the uropathogenic *E. coli*, CN prevented biofilm formation on plates and catheters, furthermore effectively inactivated preformed biofilms [54]. The mechanism proposed for the biofilm inhibition was related to the hydrophobicity of this compound, which helps to target lipids located in the bacterial cell membrane and mitochondria, increasing the membrane permeability, leading to the leakage of ions and other cell contents [54, 58]. The foodborne pathogen *Listeria monocytogenes* forms biofilm for persistence and survives in which CN has inhibitory effect on formation and inactivating mature biofilm by means of the down-regulated critical genes for biofilm formation

In *Vibrio harveyi*, the autoinducer-2 (A2) is also blocked by CN in a concentration-dependent way by decreasing the binding ability of the autoinducer to its response regulator protein. Between cinnamaldehyde derivatives, the *2-nitro-cinnamaldehyde* (**Figure 3B**) was the most active compound yielding an inhibition of A2 similar to CN [60]. Similarly, the 2-nitro-cinnamaldehyde and *4-methoxy-cinnamaldehyde* (**Figure 3C**) inhibit pigment production and protease activity in *Vibrio anguillarum* [60]. The CN is an aromatic carboxylic acid, and its inhibitory was highly dependent on the substitution pattern of the aromatic ring. Replacement of the

N) substituent with a methoxy (MeO) or a nitro (NO<sup>2</sup>

Various cinnamaldehyde analogs were also evaluated against *Vibrio* spp. The most active compounds were *2-nitro-cinnamaldehyde*, *3, 4-dichloro-cinnamaldehyde* (**Figure 3D**), *(E)-4-phenyl-3 buten-2-one* (**Figure 3E**) and *(E)-3-decen-2-one* (**Figure 3F**), which show inhibitory activity in A2, bioluminescence, pigment and protease production [61]. In this case, also the inhibitory effect of cinnamaldehyde analogs was dependent on the structure, and analogs in which the aromatic ring was replaced by an alkyl moiety, but which still contain the acrolein group, proved also to be active inhibitors [61]. In general, the inhibitory effect of cinnamaldehyde analogs is highly dependent on the nature and degree of substitution of the aromatic ring, and the substituents with electron-withdrawing properties increase its activity. The CN and their analogs furthermore proved to be active blockers of virulence in vivo in different models, suggesting that they may have potential for therapeutic applications in humans and animals [61]. The CN also has inhibitory activity on biofilm formation in a methicillin-resistant *Staphylococcus aureus* at dose-dependent manner and represses the expression of *sarA*, a gene implicated in the regulation of its biofilm [51]. In *Streptococcus pyogenes*, when the biofilm was treated with CN and their derivatives the *2-nitro-cinnamaldehyde* **(Figure 3B)**, *4N-4-nitrocinnamaldehyde* (**Figure 3G**) and *4D-4-dimethylaminocinnamaldehyde* (**Figure 3H**), the biomass, average thickness and

) group enhanced

was the inhibition of synthesis or degradation transformation of the autoinducer [57].

in this bacteria [59].

146 Phenolic Compounds - Biological Activity

dimethylamine (Me<sup>2</sup>

the activity [60].

The *coumarins* are compounds that have caused great interest for their pharmacological properties such as anti-inflammatory, antitumor, antioxidant and bactericidal activity [70]. Moreover, recently it has also documented that they possess anti-virulence properties. The *coumarin* (**Figure 4A**) and *umbelliferone* (**Figure 4B**) inhibit biofilm formation of *E. coli*, without affecting its growth. By a transcriptional analysis, it was identified that these phenols act by repressing genes related to curli production and motility, which causes a decrease in the production of fimbriae and swarming [71]. For these molecules, the hydroxylation of coumarin is an important determinant for their antibiofilm activity, since the position of hydroxyl groups as well as their number affects the antibiofilm compound activity [71].

Similarly, the presence of characteristic functional groups promotes the effective inhibition of virulence factors, as in the case of the furocoumarins [72], *dihydroxybergamottin* (**Figure 4C**) and *bergamottin* (**Figure 4D**), which exhibit anti-quorum-sensing effect on the AI-1 and AI-2 systems in *Vibrio harveyi*. Similarly, these furocoumarins inhibit biofilm formation of *E. coli*, *V. harveyi*, *Salmonella typhimurium* and *P. aeruginosa* without affecting bacterial growth. Although their mechanism of action is unknown, it is suggested that the presence of a furan residue could be acting as a competitive inhibitor for binding with the receptor protein of natural bacterial autoinducers [72].

**Figure 4.** Coumarin and related compounds with anti-virulence properties. **A:** Coumarin, **B:** umbelliferone, **C:** dihydroxybergamottin, **D:** bergamottin.

#### **2.4. Curcumin and related compounds**

The major constituent of turmeric (*Curcuma longa* L.) roots/rhizomes is the *curcumin* (CUR) (**Figure 5A**), which is an active compound that showed an important antimicrobial activity [73, 74], but several studies also corroborate their inhibitory activity against virulence factors in pathogenic bacteria.

**Figure 5.** Curcumin and related compounds with anti-virulence properties. **A:** Curcumin, **B:** demethoxycurcumin, **C:** bisdemethoxycurcumin.

The secretion of sortase A (SrtA) a surface protein in *S. aureus* involved in bacterial adhesion for pathogenesis was inhibited by CUR, and also on *in vivo* assays, this compound reduces the capacity of bacteria to adhere to surfaces in a dose-dependent manner [75]. The other derivatives present in turmeric extract are *demethoxycurcumin* (**Figure 5B**) and *bisdemethoxycurcumin* (**Figure 5C**), which show inhibitory activity of SrtA [75]. Similarly, in *Streptococcus mutans* CUR inhibited the activity of SrtA and other proteins implicated in bacterial adhesion reducing the biofilm formation in this bacteria [76, 77]. The diverse biological properties of CUR and its derivatives are attributed to the hydroxyl and phenol groups in the molecule [78], and structure-activity relationship studies suggest that a hydroxy group at the para-position is most critical for the expression of biological activity in these compounds [79].

The antibiofilm activity of CUR against uropathogens such as *E. coli*, *Proteus mirabilis* and *Serratia marcescens* was evaluated, and the results showed that their biofilm maturation was disturbed by a biomass reduction and by the interruption of swimming motility [80]. In clinical isolates of *Klebsiella pneumoniae*, the treatment with CUR was also effective for biofilm inhibition [81] as well in enterohemorrhagic *E. coli* [82]. In the same way, in *Vibrio* spp. the inhibitory effect on biofilm formation with the CUR treatment depends on the disruption of the maturation of biofilms and in the reduction of swimming and swarming motility. Further, this compound significantly represses other virulence factors like alginate and exopolysaccharide production and also inhibits bioluminescence. These inhibitory effects were also demonstrated on *in vivo* models in which CUR enhanced the survival rate of *Artemia nauplii* against *Vibrio harveyi* [83].

Diverse virulence factors in *P. aeruginosa* were inhibited by CUR, specifically the elastase, protease and pyocyanin production without affecting bacterial growth in a dose-dependent manner. The biofilm inhibition effect was demonstrated *in vivo* using *Arabidopsis thaliana*, where the treatment with CUR caused a reduction in the plant mortality by suppressing biofilm formation [84]. In the pathogenicity model using *Caenorhabditis elegans*, CUR demonstrate their anti-infective properties by reducing the nematode mortality [84]. Additionally, in *P. aeruginosa* and *C. violaceum*, CUR showed an anti-quorum sensing activity by inhibiting the production of acyl homoserine lactones [84].

#### **2.5. Eugenol and related compounds**

**2.4. Curcumin and related compounds**

dihydroxybergamottin, **D:** bergamottin.

148 Phenolic Compounds - Biological Activity

in pathogenic bacteria.

bisdemethoxycurcumin.

The major constituent of turmeric (*Curcuma longa* L.) roots/rhizomes is the *curcumin* (CUR) (**Figure 5A**), which is an active compound that showed an important antimicrobial activity [73, 74], but several studies also corroborate their inhibitory activity against virulence factors

**Figure 5.** Curcumin and related compounds with anti-virulence properties. **A:** Curcumin, **B:** demethoxycurcumin, **C:**

**Figure 4.** Coumarin and related compounds with anti-virulence properties. **A:** Coumarin, **B:** umbelliferone, **C:**

*Eugenol* (EG) (**Figure 6A**) is a major component of clove oil that possesses various biological properties [85], and their anti-virulence activity also has been evaluated. In pathogenic bacteria that secreted a broad spectrum of virulence factors that contribute to their pathogenicity,

**Figure 6.** Eugenol and related compounds with anti-virulence properties. **A:** Eugenol, **B:** eugenyl acetate, **C:** isoeugenol, **D:** methyl eugenol.

EG showed inhibitory activity. For example, in the nosocomial pathogen *S. aureus*, the hemolysin, staphyloxanthin, toxic shock syndrome toxin 1 (TSST-1) and enterotoxins are the most important virulence factors that were remarkably affected by EG [85]. The expression of virulence-related genes (*sea*, *seb*, *tst* and *hla*) was also decreased after the treatment with this compound [85].

In a methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) S. aureus at subinhibitory concentration, EG eradicates pre-established biofilms and inhibited the colonization of this bacteria in a rat middle ear model, decreasing biofilm in biomass, cell viability and the expression of biofilm-related genes (icaD, sarA and seA), resulting in a low accumulation of polysaccharides and poorly adhesion of cells within biofilms [86]. The biofilm eradication effect of EG was mediated by two mechanisms: bacterial lysis within biofilms and by the disruption of cell-to-cell connections, hence dismantling the biofilm organization, which can be attributed to the hydrophobic and the lipophilic nature of their chemical structure [86].

The biofilm formation and biofilm-related genes in *L. monocytogenes* and *E. coli* also were inhibited by EG at dose-dependent manner [56, 59]. In *P. aeruginosa*, although EG was unable to inhibit biofilm formation, it markedly reduced the production of pyocyanin, fimbriae production, hemolytic activity and other QS-controlled virulence factors in this bacterium such as the pseudomonas quinolone signal (PQS) [56]. Other study showed that EG at subinhibitory concentrations has QS inhibitory activity in *P. aeruginosa* and *C. violaceum* [87].

Moreover, derivatives of EG *eugenyl acetate* (EA) (**Figure 6B**), *isoeugenol* (IE) (**Figure 6C**) and *methyl eugenol* (ME) (**Figure 6D**) showed anti-virulence properties against pathogenic bacteria. In *S. aureus*, EA inhibited the production of virulence factors like hemolysin and staphyloxanthin. Similarly, in *P. aeruginosa* the pyocyanin, pyoverdin and exoprotease production were significantly reduced after the treatment with EA, and it also exhibited QS inhibitory potential in *C. violaceum* [88]. The other derivatives, IE and ME, also presented QS inhibitory against *P. aeruginosa* and *C. violaceum* [89, 90], and in the case of *V. harveyi*, ME have anti-bioluminescence activity [90]. These anti-virulence properties can be attributable to the presence of numerous substituted aromatic molecules like in the case of other phenols [85].

#### **2.6. Long-chain phenols**

Long-chain phenols are a group of metabolites which have extensively studied antitumor, antimicrobial and antioxidant activities; they are also of great interest to the industry because they are used to manufacture different chemicals [91]. Also, different long-chain phenols reported have different anti-virulence properties.