Section 3 Health Effects

*Technological Innovation in the Olive Oil Production Chain*

European Journal of Lipid Science and Technology. 2013;**115**(9):1062-1069

Amirante R, Dugo G, Cicero N, Boskou D. Research and innovative approaches to obtain virgin olive oils with a higher level of bioactive constituents. In: Olive and Olive Oil Bioactive Constituents. Urbana, Illinois; 2015. pp. 179-215

[23] Clodoveo ML, Camposeo S,

[24] Clodoveo ML, Moramarco V, Paduano A, Sacchi R, Di Palmo T, Crupi P, et al. Engineering design and prototype development of a full scale ultrasound system for virgin olive oil by means of numerical and experimental analysis. Ultrasonics Sonochemistry.

[25] Amirante R, Distaso E, Tamburrano P, Paduano A, Pettinicchio D, Clodoveo ML. Acoustic cavitation by means ultrasounds in the extra virgin olive oil extraction process. Energy Procedia.

2017;**37**:169-181

2017;**126**:82-90

[15] Roselli L, Clodoveo ML, Corbo F, De Gennaro B. Are health claims a useful tool to segment the category of extra-virgin olive oil? Threats and opportunities for the Italian olive oil supply chain. Trends in Food Science &

[16] Roselli L, Cicia G, Cavallo C, Del Giudice T, Carlucci D, Clodoveo ML, et al. Consumers' willingness to buy innovative traditional food products: The case of extra-virgin olive oil extracted by ultrasound. Food Research

International. 2018;**108**:482-490

[17] Clodoveo ML. New advances in the development of innovative virgin olive oil extraction plants: Looking back to see the future. Food Research International. 2013;**54**(1):726-729

[18] Clodoveo ML. An overview of emerging techniques in virgin olive oil extraction process: Strategies in the development of innovative plants. Journal of Agricultural Engineering.

[19] Clodoveo ML, Dipalmo T, Rizzello CG, Corbo F, Crupi P. Emerging technology to develop novel red winemaking practices: An overview. Innovative Food Science & Emerging

[20] Clodoveo ML, Durante V, La Notte D. Working towards the development of innovative ultrasound equipment for the extraction of virgin olive oil. Ultrasonics Sonochemistry. 2013;**20**(5):1261-1270

[21] Clodoveo ML, Hbaieb RH. Beyond the traditional virgin olive oil extraction

[22] Clodoveo ML, Durante V, La Notte D, Punzi R, Gambacorta G. Ultrasoundassisted extraction of virgin olive oil to improve the process efficiency.

systems: Searching innovative and sustainable plant engineering solutions. Food Research International.

2013;**54**(2):1926-1933

2013;**44**(2s):297-305

Technologies. 2016;**38**:41-56

Technology. 2017;**68**:176-181

**32**

**35**

**Chapter 4**

**Abstract**

**1. Introduction**

*Amany M. Basuny*

well as their contribution to human health.

Antioxidants in Olive Oil

Olive oil contains polyphenols, vitamin E, and other natural antioxidants that are the oil's own natural preservatives. Antioxidants dampen the autogeneration of peroxides, delaying the onset of oxidation and rancidity. As a result, antioxidants increase the oil's shelf life. Among the antioxidants, there are compounds that have been associated with human health benefits. They absorb free radicals and appear to have a positive impact on cardiovascular and cancer ailments, as attributed to the Mediterranean diet. The main objectives of this chapter were to investigate the phytochemical profile such as phenolic compounds and tocopherols, and in vitro, to study the biological potential (antioxidant capacity) of the olive oil. Furthermore, the relationship and correlations between phytochemical and antioxidant capacity have been highlighted. The investigation of these compounds supported by verifiable evidence may explain their role in the quality and authenticity of olive oil as

**Keywords:** antioxidants, polyphenols, tocopherols, olive oil, phytochemical

Olive oil is obtained from the fruits—technically named drupes-of *Olea europea* L., a tree that is best grown between the 30 and the 45 parallel. Accordingly, the Mediterranean countries supply more than 95% of the world olive oil production, 75% of which comes from the European Union (mostly Spain, Italy, and Greece) and the rest from Maghrebian countries. Olive oil contributes 4% of total vegetable oil production: its world production is around 2,000,000 tons/year. Due to the accretion aloft of the Mediterranean diet, during which oil is that the aloft fat element, its assembly is currently accretion to non-traditional producers like the U. S., Canada, Australia, South America, and Japan. reckoning on its actinic backdrop and its aggregate of acidity, oil is classed into actually altered grades [1] that additionally action pointers for the client aural the accession of the admired analytic oil. From this classification, it may be all over that the foremost admired analytic oil is that the added abstinent one, acquired from complete olives that are bound candy and coldpressed. During this approach, activation of cellular lipases and abasement of the triglycerides is decreased. One allotment of the needs of this argument is to adduce the phenoplast fraction responsible for the acumen and acidity of oil and endued with "pharmacological" properties as a further, admired brand of oil quality.

Olive oil contains polyphenols, vitamin E, and accession accustomed antioxidants that are the oil's own accustomed preservatives. Antioxidants bedew the car address of peroxides, dabbling the access of agitation and rancidity. As a result, antioxidants access the oil's time period. A allotment of the antioxidants, there are compounds that are accompanying to beastly bloom advantages. They blot

## **Chapter 4** Antioxidants in Olive Oil

*Amany M. Basuny*

### **Abstract**

Olive oil contains polyphenols, vitamin E, and other natural antioxidants that are the oil's own natural preservatives. Antioxidants dampen the autogeneration of peroxides, delaying the onset of oxidation and rancidity. As a result, antioxidants increase the oil's shelf life. Among the antioxidants, there are compounds that have been associated with human health benefits. They absorb free radicals and appear to have a positive impact on cardiovascular and cancer ailments, as attributed to the Mediterranean diet. The main objectives of this chapter were to investigate the phytochemical profile such as phenolic compounds and tocopherols, and in vitro, to study the biological potential (antioxidant capacity) of the olive oil. Furthermore, the relationship and correlations between phytochemical and antioxidant capacity have been highlighted. The investigation of these compounds supported by verifiable evidence may explain their role in the quality and authenticity of olive oil as well as their contribution to human health.

**Keywords:** antioxidants, polyphenols, tocopherols, olive oil, phytochemical

### **1. Introduction**

Olive oil is obtained from the fruits—technically named drupes-of *Olea europea* L., a tree that is best grown between the 30 and the 45 parallel. Accordingly, the Mediterranean countries supply more than 95% of the world olive oil production, 75% of which comes from the European Union (mostly Spain, Italy, and Greece) and the rest from Maghrebian countries. Olive oil contributes 4% of total vegetable oil production: its world production is around 2,000,000 tons/year. Due to the accretion aloft of the Mediterranean diet, during which oil is that the aloft fat element, its assembly is currently accretion to non-traditional producers like the U. S., Canada, Australia, South America, and Japan. reckoning on its actinic backdrop and its aggregate of acidity, oil is classed into actually altered grades [1] that additionally action pointers for the client aural the accession of the admired analytic oil. From this classification, it may be all over that the foremost admired analytic oil is that the added abstinent one, acquired from complete olives that are bound candy and coldpressed. During this approach, activation of cellular lipases and abasement of the triglycerides is decreased. One allotment of the needs of this argument is to adduce the phenoplast fraction responsible for the acumen and acidity of oil and endued with "pharmacological" properties as a further, admired brand of oil quality.

Olive oil contains polyphenols, vitamin E, and accession accustomed antioxidants that are the oil's own accustomed preservatives. Antioxidants bedew the car address of peroxides, dabbling the access of agitation and rancidity. As a result, antioxidants access the oil's time period. A allotment of the antioxidants, there are compounds that are accompanying to beastly bloom advantages. They blot

chargeless radicals and assume to own an absolute appulse on barge and blight ailments, as attributed to the Mediterranean diet. Polyphenols are a basal class of inhibitor in oil. Over thirty polyphenols are accustomed in olives. Absolute phenol account (or absolute arctic phenol value) is their aggregate live.

#### **2. Classification and allure of phenoplast compounds**

The bulb phenols are ambrosial accessory metabolites that embrace a abounding alter of drugs possessing associate in nursing ambrosial ring address one or a lot of actinic accumulation substituent's. aural the allowance context, this analogue is not actually satisfactory back it accordingly includes compounds like estrogen, the feminine steroid hormone (which is in the capital terpenoid in origin). For this reason, an analogue accurate metabolic abettor is preferred, the bulb phenols getting advised those substances acquired from the shikimate alleyway and phenylpropanoid metabolism phenoplast compounds are accessory bulb metabolites actinic throughout acceptable development or in bellicose situations (**Figure 1**). In abstinent olive oils, the amalgam of those compounds happens already the olive fruits are ashamed throughout the bartering adjustment to get the oil. Thus, the presence of phenolic compounds is directly related to glycosides initially present in the fruit tissue, and the activity of hydrolytic and oxidative enzymes. In terms of chemical structure, they have at least one hydroxyl attached to an aromatic ring [2].

Major arctic phenoplast compounds allowance in abstinent oil are detected and quantified. These phenoplast compounds is as well phenoplast acids, aboveboard phenols like tyrosol and hydroxytyrosol, secoiridoid derivatives of the glycosides oleuropein and ligstroside, lignans, flavonoids and hydroxyl-isochromans [3, 4]. The appellation "polar phenoplast compounds" is alive to differentiate them from accession class of phenols, the tocopherols. Oil arctic phenol fraction, accustomed for several years as "polyphenols," is in fact a chic admixture of compounds with assorted actinic structures acquired from abstinent oil by liquid-liquid allotment with methanol: water.

**37**

acid.

**2.1 Phenolic acids**

**2.2 Phenolic alcohols**

hydroxytyrosol), and homovanillyl alcohol.

**2.3 Derivatives of phenoplast alcohols**

compound of methyl group malate.

*Antioxidants in Olive Oil*

*DOI: http://dx.doi.org/10.5772/intechopen.84540*

Furthermore the types of components mentioned above, other phenolic compounds with different structure (e.g., vanillin) have been identified. Litridou et al., [5] found that the presence of an ester of tyrosol with a dicarboxylic acid. Litridou et al., [5] reported that total polar phenol and ortho-diphenol content recorded higher in the less polar part of the methanol extract. This part contains primarily the dialdehydic and decarboxymethyl pattern of elenolic acerbic abutting to hydroxytyrosol and tyrosol, hydroxytyrosol acetate, lignans and luteolin. Brenes et al., [6] accustomed 4-ethylphenol all told oils declared for clarification and conspicuously

aural the "added action olive oils," acknowledgment to the adhesive storage.

another class of compounds, hydroxy-isochromans, was identified by [7]. According to the authors the formation of such compounds is due to a reaction between hydroxytyrosol and aromatic aldehydes (vanillin, benzaldehyde). The phenol allowance in olive bake-apple abutting as associate in nursing amoebic admixture to the aglycon atom of oleuropein is freed throughout malaxation of the olive lurid by enzymes. This actinic acknowledgment adjustment additionally favors the accumulation of carbonyl compounds and so hydroxy-isochromans are fashioned. Some of the accustomed secoiridoid compounds just like the amoebic admixture blazon of oleuropein accept stereo chemical isomers. The attendance of such isomers was accustomed by coupling aloft liquid action with cavalcade column solid-phase extraction to nuclear resonance spectrometry. The methyl acetals of the aglycons; ligstroside and the β-hydroxytyrosol ester of methyl malate was identified [8], the investigated of oleocanthal by Beauchamp et al., [9], a derivative of tyrosol that has the same pharmacological activity as the anti-inflammatory drug ibuprofen, and some investigation indicating an anti-inflammatory activity, provide important new information for the forms of tyrosol and hydroxytyrosol derivatives present in olive oil and olives, some of which may be antioxidant and/or biologically active. Thus, altitude of some styles of aglycons is as well all-important (in accession to the all-embracing arctic phenols content) for the assay of quality, adherence and biological action worth. The arctic atom can as well accommodate non phenoplast about affiliated compounds like cinammic acerbic and elenolic acid. The a lot of phenoplast and non phenoplast compounds arise to be allowance aural

the arctic atom of oil abstinent oil accord to the consecutive classes:

There are many phenolic acids was found in olive oil such as, Hydroxybenzoic acids, 4-hydroxybenzoic, protocatechuic, gallic acid, vanillic acid, syringic acid hydroxyphenylacetic acids, 4-hydroxyphenylacetic, hydroxycinnamic acids, o-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, and finally sinapic,

Many alcoholic phenols are found in olive oil for example, (p-hydroxyphenyl) ethyl alcohol (p-HPEA, tyrosol), (3,4-dihydroxyphenyl) ethanol (3,4 DHPEA,

Also some components from derivatives of phenoplast alcohols appeared in olive oil for example, 4-(acetoxyethyl)-1,2-dihydroxybenzene, hydroxytyrosol organic

Also glycosides compounds were found in olive oil but only in trace amounts

**Figure 1.** *Metabolic pathways leading to the formation of phenolic compounds.*

#### *Antioxidants in Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84540*

*Technological Innovation in the Olive Oil Production Chain*

chargeless radicals and assume to own an absolute appulse on barge and blight ailments, as attributed to the Mediterranean diet. Polyphenols are a basal class of inhibitor in oil. Over thirty polyphenols are accustomed in olives. Absolute phenol

The bulb phenols are ambrosial accessory metabolites that embrace a abounding alter of drugs possessing associate in nursing ambrosial ring address one or a lot of actinic accumulation substituent's. aural the allowance context, this analogue is not actually satisfactory back it accordingly includes compounds like estrogen, the feminine steroid hormone (which is in the capital terpenoid in origin). For this reason, an analogue accurate metabolic abettor is preferred, the bulb phenols getting advised those substances acquired from the shikimate alleyway and phenylpropanoid metabolism phenoplast compounds are accessory bulb metabolites actinic throughout acceptable development or in bellicose situations (**Figure 1**). In abstinent olive oils, the amalgam of those compounds happens already the olive fruits are ashamed throughout the bartering adjustment to get the oil. Thus, the presence of phenolic compounds is directly related to glycosides initially present in the fruit tissue, and the activity of hydrolytic and oxidative enzymes. In terms of chemical structure, they have at least one hydroxyl attached to an aromatic ring [2]. Major arctic phenoplast compounds allowance in abstinent oil are detected and quantified. These phenoplast compounds is as well phenoplast acids, aboveboard phenols like tyrosol and hydroxytyrosol, secoiridoid derivatives of the glycosides oleuropein and ligstroside, lignans, flavonoids and hydroxyl-isochromans [3, 4]. The appellation "polar phenoplast compounds" is alive to differentiate them from accession class of phenols, the tocopherols. Oil arctic phenol fraction, accustomed for several years as "polyphenols," is in fact a chic admixture of compounds with assorted actinic structures acquired from abstinent oil by liquid-liquid allotment

account (or absolute arctic phenol value) is their aggregate live.

**2. Classification and allure of phenoplast compounds**

**36**

**Figure 1.**

*Metabolic pathways leading to the formation of phenolic compounds.*

with methanol: water.

Furthermore the types of components mentioned above, other phenolic compounds with different structure (e.g., vanillin) have been identified. Litridou et al., [5] found that the presence of an ester of tyrosol with a dicarboxylic acid. Litridou et al., [5] reported that total polar phenol and ortho-diphenol content recorded higher in the less polar part of the methanol extract. This part contains primarily the dialdehydic and decarboxymethyl pattern of elenolic acerbic abutting to hydroxytyrosol and tyrosol, hydroxytyrosol acetate, lignans and luteolin. Brenes et al., [6] accustomed 4-ethylphenol all told oils declared for clarification and conspicuously aural the "added action olive oils," acknowledgment to the adhesive storage.

Also glycosides compounds were found in olive oil but only in trace amounts another class of compounds, hydroxy-isochromans, was identified by [7]. According to the authors the formation of such compounds is due to a reaction between hydroxytyrosol and aromatic aldehydes (vanillin, benzaldehyde). The phenol allowance in olive bake-apple abutting as associate in nursing amoebic admixture to the aglycon atom of oleuropein is freed throughout malaxation of the olive lurid by enzymes. This actinic acknowledgment adjustment additionally favors the accumulation of carbonyl compounds and so hydroxy-isochromans are fashioned.

Some of the accustomed secoiridoid compounds just like the amoebic admixture blazon of oleuropein accept stereo chemical isomers. The attendance of such isomers was accustomed by coupling aloft liquid action with cavalcade column solid-phase extraction to nuclear resonance spectrometry. The methyl acetals of the aglycons; ligstroside and the β-hydroxytyrosol ester of methyl malate was identified [8], the investigated of oleocanthal by Beauchamp et al., [9], a derivative of tyrosol that has the same pharmacological activity as the anti-inflammatory drug ibuprofen, and some investigation indicating an anti-inflammatory activity, provide important new information for the forms of tyrosol and hydroxytyrosol derivatives present in olive oil and olives, some of which may be antioxidant and/or biologically active. Thus, altitude of some styles of aglycons is as well all-important (in accession to the all-embracing arctic phenols content) for the assay of quality, adherence and biological action worth. The arctic atom can as well accommodate non phenoplast about affiliated compounds like cinammic acerbic and elenolic acid. The a lot of phenoplast and non phenoplast compounds arise to be allowance aural the arctic atom of oil abstinent oil accord to the consecutive classes:

#### **2.1 Phenolic acids**

There are many phenolic acids was found in olive oil such as, Hydroxybenzoic acids, 4-hydroxybenzoic, protocatechuic, gallic acid, vanillic acid, syringic acid hydroxyphenylacetic acids, 4-hydroxyphenylacetic, hydroxycinnamic acids, o-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, and finally sinapic, acid.

#### **2.2 Phenolic alcohols**

Many alcoholic phenols are found in olive oil for example, (p-hydroxyphenyl) ethyl alcohol (p-HPEA, tyrosol), (3,4-dihydroxyphenyl) ethanol (3,4 DHPEA, hydroxytyrosol), and homovanillyl alcohol.

#### **2.3 Derivatives of phenoplast alcohols**

Also some components from derivatives of phenoplast alcohols appeared in olive oil for example, 4-(acetoxyethyl)-1,2-dihydroxybenzene, hydroxytyrosol organic compound of methyl group malate.

#### **2.4 Glycosides**

Oleuropein it is the most important compound in olive oil where it consists of (an organic compound of hydroxytyrosol with β-glucosylated elenolic acid).

#### **2.5 Ligstroside**

Ligstroside are derivatives for aglyconic of oleuropein and ligstroside dialdehydic type of elenolic acid joined to three, 4-DHPEA(3,4-DHPEA-EDA). Dialdeyhydic type of elenolic acid joined to p-HPEA(p-HPEA-EDA). Dialdehydic type of decarboxymethyl elenolic acid joined to 3,4-DHPEA. Dialdehydic type of decarboxymethyl elenolic acid joined to p-DHPEA.

#### **2.6 Lignans**

Lignans for example (+)-1-acetoxypinoresinol, (+)-pinoresinol, (+)-1-hydroxypinoresinol, syringaresinol.

#### **2.7 Flavonoids**

Flavonoids such as apigenin, luteolin, taxifolin, hydroxy-isochromans, 1-phenyl-6,7-dihydroxy-isochroman 1-(3′-methoxy-4′hydroxy) pheny l-6,7-dihydroxy-isochroman.

#### **2.8 Other phenols**

Vanillin compound (4-hydroxy-3-methoxybenzaldeyde). 4-ethylphenol cmpound (not found in virgin olive oils however in oils of "second centrifugation," supposed for refining).

**39**

*Antioxidants in Olive Oil*

(**Figure 2**).

*DOI: http://dx.doi.org/10.5772/intechopen.84540*

**3. Antioxidant activity of phenolic compounds**

advised to own inhibitor and atom scavenging capabilities.

Cinnamic acid, elenolic acid, elenolic acid organic compound, 11-methyl oleosid

The accretion absorption for the inhibitor backdrop of accustomed compounds and aliment locations is acknowledgment to their adeptness to absorber fats allowance in foods and as well the antecedent that they apprehend the after-effects of acknowledging breed on the concrete structure. Phenols allowance in oil are advised a lot of and a lot of actively, as abstracts accumulated indicates a butt of biological activities suggesting that these compounds could accept an absolute aftereffect on bloom and oil is an allotment of those accustomed agents that accept advanced been

As a aftereffect of their basal actinic properties, the phenolics arrest lipid peroxidation and display abounding physiological activities. The inhibitor backdrop of the phenolics is accustomed and still allure advanced attempt. Thus, plants like the assemble rosemary are acutely acclaimed for his or her inhibitor properties, that accept mostly been attributed to the phenoplast compounds carnosol, rosmanol and rosmadial. Similarly, the phenolics in olives accept admiring absorption as antioxidants. Absolute aqueous phenols and as well the oleosidic styles of 3,4-dihydroxyphenylethanol (hydroxytyrosol) were accompanying with the aerophilic adherence of abstinent olive oil admitting tocopherols showed low correlation. a lot of specifically, inhibitor action in esthetic oil aside aural the alternation hydroxytyrosol, caffeic acerbic > butylated hydroxytoluene (BHT) > protocatechuic acid, syringic acid. Tyrosol, p-hydroxyphenylacetic acid, o-coumaric acid, p-coumaric acid, p-hydroxybenzoic acerbic and vanillic acerbic had little or no inhibitor activity, and their accession to the acumen of the oil was negligible. a advance of strategies are wont to appraise the inhibitor action of awkward olive extracts and esthetic phenolics. One access involves barometer of the inhibition of aerophilic abasement of Associate in Nursing oil or archetypal substance, like methyl accumulation linoleate. This can be calmly performed aural the Rancimat equipment, that has been wont to demonstrate that the action of tyrosol (in esthetic tallow) was beneath than that of the bogus BHT admitting oleuropein showed a stronger action admitting the a lot of regulative attention aftereffect was acquired with acerbic esters and hydroxytyrosol. Care should be acclimatized aural the estimation of adeptness about inhibitor action because the substrate and additionally the analytic address influences the results. The after effect of substrate may be attributed to the athletic access of the unsaturation affectionate and aggregate of the lipid arrangement on the dynamics and apparatus of the antioxidative action of the phenols. For instance, already yield a attending acted in accession accelerated kitchen apparatus assay on esthetic vegetable oil angular films, the action of hydroxytyrosol was beneath than that of acerbic esters. Similarly, the trends in inhibitor action of phenolics differed in band with whether or not hydroperoxide accumulation (peroxide value) or atomization (hexanal and volatiles) was abstinent in accelerated adherence tests on oil. These after-effects emphasize the claim to reside a minimum of 2 agitation ambit to college appraise antioxidants and as well the aerophilic adherence of olive oils. on esthetic vegetable oil angular films, the action of hydroxytyrosol was beneath than that of acerbic esters. Similarly, the trends in inhibitor action of phenolics differed in band with whether or not hydroperoxide accumulation (peroxide value) or atomization (hexanal and volatiles) was abstinent in accelerated adherence tests

**2.9 Non phenoplast compounds**

**Figure 2.** *Chemical structures of predominant olive plant polyphenols.*

#### **2.9 Non phenoplast compounds**

*Technological Innovation in the Olive Oil Production Chain*

methyl elenolic acid joined to p-DHPEA.

Oleuropein it is the most important compound in olive oil where it consists of

Ligstroside are derivatives for aglyconic of oleuropein and ligstroside dialdehydic type of elenolic acid joined to three, 4-DHPEA(3,4-DHPEA-EDA). Dialdeyhydic type of elenolic acid joined to p-HPEA(p-HPEA-EDA). Dialdehydic type of decarboxymethyl elenolic acid joined to 3,4-DHPEA. Dialdehydic type of decarboxy-

Lignans for example (+)-1-acetoxypinoresinol, (+)-pinoresinol, (+)-1-hydroxy-

Flavonoids such as apigenin, luteolin, taxifolin, hydroxy-isochromans, 1-phenyl-

Vanillin compound (4-hydroxy-3-methoxybenzaldeyde). 4-ethylphenol cmpound (not found in virgin olive oils however in oils of "second centrifugation,"

6,7-dihydroxy-isochroman 1-(3′-methoxy-4′hydroxy) pheny

(an organic compound of hydroxytyrosol with β-glucosylated elenolic acid).

**2.4 Glycosides**

**2.5 Ligstroside**

**2.6 Lignans**

**2.7 Flavonoids**

**2.8 Other phenols**

supposed for refining).

pinoresinol, syringaresinol.

l-6,7-dihydroxy-isochroman.

**38**

**Figure 2.**

*Chemical structures of predominant olive plant polyphenols.*

Cinnamic acid, elenolic acid, elenolic acid organic compound, 11-methyl oleosid (**Figure 2**).

#### **3. Antioxidant activity of phenolic compounds**

The accretion absorption for the inhibitor backdrop of accustomed compounds and aliment locations is acknowledgment to their adeptness to absorber fats allowance in foods and as well the antecedent that they apprehend the after-effects of acknowledging breed on the concrete structure. Phenols allowance in oil are advised a lot of and a lot of actively, as abstracts accumulated indicates a butt of biological activities suggesting that these compounds could accept an absolute aftereffect on bloom and oil is an allotment of those accustomed agents that accept advanced been advised to own inhibitor and atom scavenging capabilities.

As a aftereffect of their basal actinic properties, the phenolics arrest lipid peroxidation and display abounding physiological activities. The inhibitor backdrop of the phenolics is accustomed and still allure advanced attempt. Thus, plants like the assemble rosemary are acutely acclaimed for his or her inhibitor properties, that accept mostly been attributed to the phenoplast compounds carnosol, rosmanol and rosmadial. Similarly, the phenolics in olives accept admiring absorption as antioxidants. Absolute aqueous phenols and as well the oleosidic styles of 3,4-dihydroxyphenylethanol (hydroxytyrosol) were accompanying with the aerophilic adherence of abstinent olive oil admitting tocopherols showed low correlation. a lot of specifically, inhibitor action in esthetic oil aside aural the alternation hydroxytyrosol, caffeic acerbic > butylated hydroxytoluene (BHT) > protocatechuic acid, syringic acid. Tyrosol, p-hydroxyphenylacetic acid, o-coumaric acid, p-coumaric acid, p-hydroxybenzoic acerbic and vanillic acerbic had little or no inhibitor activity, and their accession to the acumen of the oil was negligible. a advance of strategies are wont to appraise the inhibitor action of awkward olive extracts and esthetic phenolics. One access involves barometer of the inhibition of aerophilic abasement of Associate in Nursing oil or archetypal substance, like methyl accumulation linoleate. This can be calmly performed aural the Rancimat equipment, that has been wont to demonstrate that the action of tyrosol (in esthetic tallow) was beneath than that of the bogus BHT admitting oleuropein showed a stronger action admitting the a lot of regulative attention aftereffect was acquired with acerbic esters and hydroxytyrosol. Care should be acclimatized aural the estimation of adeptness about inhibitor action because the substrate and additionally the analytic address influences the results. The after effect of substrate may be attributed to the athletic access of the unsaturation affectionate and aggregate of the lipid arrangement on the dynamics and apparatus of the antioxidative action of the phenols. For instance, already yield a attending acted in accession accelerated kitchen apparatus assay on esthetic vegetable oil angular films, the action of hydroxytyrosol was beneath than that of acerbic esters.

Similarly, the trends in inhibitor action of phenolics differed in band with whether or not hydroperoxide accumulation (peroxide value) or atomization (hexanal and volatiles) was abstinent in accelerated adherence tests on oil. These after-effects emphasize the claim to reside a minimum of 2 agitation ambit to college appraise antioxidants and as well the aerophilic adherence of olive oils. on esthetic vegetable oil angular films, the action of hydroxytyrosol was beneath than that of acerbic esters. Similarly, the trends in inhibitor action of phenolics differed in band with whether or not hydroperoxide accumulation (peroxide value) or atomization (hexanal and volatiles) was abstinent in accelerated adherence tests

on oil. These after-effects emphasize the claim to reside a minimum of 2 agitation ambit to college appraise antioxidants and as well the aerophilic adherence of olive oils. In oil, the phenoplast agreeable is a basal qualitative constant acknowledgment to its alternation with the achromatize range, chargeless blubbery acidity, and acoustic quality. Chargeless blubbery acids (FFA) accord associate in nursing basis of the aggregate of agitator action and already allowance at top concentrations, about-face out abominable aromas aural the oil.65 as a aftereffect of phenolics accomplish as inhibitor capacity of oil, a top FFA agreeable consistently indicates a top aggregate of agitator action and accordingly a bargain inhibitor content. Similarly, achromatize range, or achromatize account (PV) monitors the antecedent artifact of oxidation; that is, the hydroperoxides (**Figure 3**). The PV so offers one allotment of the foremost absolute measures of lipid peroxidation. The aggregate of peroxides that has got to be ancient to accommodate apparent rancidity depends aloft the agreement of the oil and, particularly, the aggregate of unsaturation and as well the attendance of antioxidants, notably, the phenolics.

Evaluation of inhibitor action of the all-embracing arctic phenol atom or alone phenols are about accurate determinations of the shelf-life of the oil or accelerated tests like Rancimat assay at 120°C. Methods are developed to reside the inhibitor action anon aural the oil additionally to strategies for the aftereffect of phenoplast extracts, authentic phenols or fractions acquired by basic HPLC. Papadopoulos and Boskou [10] compared the inhibitor aftereffect of phenoplast acids and simple phenols on esthetic oil. Hydroxytyrosol and caffeic acerbic were begin to be a lot of able antioxidants in advertence to BHT, already the acumen and keep ability of the oil containing these additives were examined. Baldioli et al., [11] acclimated Rancimat to assay the aftereffect of assorted phenols and secoiridoid derivatives on esthetic oil stability. The absorption of hydroxytyrosol, the dialdehydic blazon of elenolic acerbic abutting to hydroxytyrosol Associate in Nursingd and actinic admixture of oleuropein aglycon were begin to associate able-bodied to the aerophilic adherence of esthetic oil.

Fogliano et al., [12] acquired by semi basic HPLC fractions absolute alone phenols and evaluated the about inhibitor authority in advertence to BHT by ascertainment the peroxidation at 240 nm abusage the ABAP (2,2-azo-bis-2-amidinopropane hydrochloride) actinic agent. Gas abolitionist absorbance adequacy (ORAC) of oil was advised by Ninfali et al., [13] employing a spectrofluorometric address that measures the aegis of the phenoplast substances of the oil on the b-phycoerythrin ablaze adulteration as compared with Trolox. This value, that indicates the adequacy to attract peroxyl radicals, was projected as a backup constant to appraise the accepted and adherence adjoin agitation of added abstinent oil.

Quiles et al., [14] projected the appliance of lepton circuit resonance (ESR) spectrometry to adjudicator inhibitor adequacy in abstinent oil. The tactic is predicated on the assurance of actual galvinoxyl (a bogus radical) by affiliation of

**41**

*Antioxidants in Olive Oil*

*DOI: http://dx.doi.org/10.5772/intechopen.84540*

bly access the atypical concentration in olive oils.

during which astringent agitation altitude are used.

as associate in nursing inhibitor.

the ESR spectrum already accession of associate in nursing ethyl booze acknowledgment of the oil. Lepton allurement resonance was additionally activated by Ottaviani et al., [15] United Nations bureau accustomed and quantified chargeless radicals by suggests that of the spin-trapping address abusage alpha-phenylnutylnitrone (PBN) as circuit entice. From their absorption the authors all over that EPR may be activated to appraise accumulator and administration altitude that apprecia-

In accepted the inhibitor action of phenols is college in ortho-diphenols or phenols with o-methoxy teams. The action of aboveboard phenols, secoiridoids and lignans as antioxidants was afresh advised by Carrasco-Pancorbo et al., [16] by the DPPH abolitionist yield a attending at and barometer of agitation stability. The absorption accustomed antecedent allegation advertence that the attendance of added accumulation at ortho-position enhances significantly the adaptability to act

A abstract access to the atypical scavenging abeyant of phenoplast compounds encountered in olives and oil and olive leaves was arise by Nenadis et al., [17]. This access is predicated on breakthrough actinic calculations of band break calefaction agreeable (BDE) of phenoplast abolitionist teams and as well the ionization abeyant (P) ethics and aims at admiration the H-donating and electron-donating talents. Catechols were begin to own best low BDE values. Lignans and monophenols had abounding college BDE ethics (a lower abeyant for abolitionist scavenging). In absolute systems, however, action could alter do to variations in lipophility.

Roche et al., [18] characterized oil phenols by the aggregate of radicals cornered

The after effect of hydrogen ion absorption and brownish aspect anions on the inhibitor action of oil polyphenols in oil-in-water emulsions was advised by Paiva-Martins and Gordon [20]. inhibitor behavior is a lot of complicated in emulsions than in aggregate oil as there are a lot of variables anxious in lipid oxidization, (pH, emulsifiers). Four oil phenols were examined, oleuropein, hydroxytyrosol, 3,4-dihydroxyplenylethanol-elenolic acerbic and three,4-dihydroxyphenylethanolelenolic acerbic dialdehyde. The aftereffect of every inhibitor on DPPH abolitionist absorption (and additionally the) ferric-reducing inhibitor abeyant (FRAP) were as well determined. The plan has apparent that phenoplast compounds of oil accept a top inhibitor adequacy at hydrogen ion absorption alter three. 5–7.4, about their

action is as well bargain aural the attendance of brownish aspect anions.

In vitro and animal studies showed that polyphenols from olives have potent antioxidant activities; 50% of the phenolic compounds contained in olives and virgin olive oil are hydroxytyrosol and derivatives. These compounds seem to have the highest antioxidant potency compared to the other olive polyphenols. The radical

per inhibitor atom and by the acceleration constants K1for the primary H-atom absorption by the atypical DPPH. Oleuropein, hydroxytyrosol and caffeic acerbic accept the a lot of important K.1 ethics admitting dihydrocaffeic acid, associate in nursing centralized agency bulk of caffeic acid, was begin to be the simplest inhibitor in agreement of arrangement (number of radicals cornered per molecule). The absorption adumbrated that overall olive phenols are economical scavengers of aqueous peroxyl radicals with an continued abiding inhibitor aftereffect. The closing is acknowledgment to the balance action of their agitation product. An audible access for assay of the inhibitor adeptness of oil was projected by [19]. The tactic is predicated on a FIA arrangement with associate in nursing amperometric detector in band with the authors the strategy is acute and offers an alternating to the Rancimat adjustment for absolute and reliable ascertainment of the all-embracing inhibitor adeptness of oil. The strategy is additionally college accompanying to the \$64,000 accumulate adeptness than the Rancimat method,

**Figure 3.** *Mechanism of the antioxidant activity of olive phenols.*

#### *Antioxidants in Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84540*

*Technological Innovation in the Olive Oil Production Chain*

well the attendance of antioxidants, notably, the phenolics.

accepted and adherence adjoin agitation of added abstinent oil.

Quiles et al., [14] projected the appliance of lepton circuit resonance (ESR) spectrometry to adjudicator inhibitor adequacy in abstinent oil. The tactic is predicated on the assurance of actual galvinoxyl (a bogus radical) by affiliation of

on oil. These after-effects emphasize the claim to reside a minimum of 2 agitation ambit to college appraise antioxidants and as well the aerophilic adherence of olive oils. In oil, the phenoplast agreeable is a basal qualitative constant acknowledgment to its alternation with the achromatize range, chargeless blubbery acidity, and acoustic quality. Chargeless blubbery acids (FFA) accord associate in nursing basis of the aggregate of agitator action and already allowance at top concentrations, about-face out abominable aromas aural the oil.65 as a aftereffect of phenolics accomplish as inhibitor capacity of oil, a top FFA agreeable consistently indicates a top aggregate of agitator action and accordingly a bargain inhibitor content. Similarly, achromatize range, or achromatize account (PV) monitors the antecedent artifact of oxidation; that is, the hydroperoxides (**Figure 3**). The PV so offers one allotment of the foremost absolute measures of lipid peroxidation. The aggregate of peroxides that has got to be ancient to accommodate apparent rancidity depends aloft the agreement of the oil and, particularly, the aggregate of unsaturation and as

Evaluation of inhibitor action of the all-embracing arctic phenol atom or alone phenols are about accurate determinations of the shelf-life of the oil or accelerated tests like Rancimat assay at 120°C. Methods are developed to reside the inhibitor action anon aural the oil additionally to strategies for the aftereffect of phenoplast extracts, authentic phenols or fractions acquired by basic HPLC. Papadopoulos and Boskou [10] compared the inhibitor aftereffect of phenoplast acids and simple phenols on esthetic oil. Hydroxytyrosol and caffeic acerbic were begin to be a lot of able antioxidants in advertence to BHT, already the acumen and keep ability of the oil containing these additives were examined. Baldioli et al., [11] acclimated Rancimat to assay the aftereffect of assorted phenols and secoiridoid derivatives on esthetic oil stability. The absorption of hydroxytyrosol, the dialdehydic blazon of elenolic acerbic abutting to hydroxytyrosol Associate in Nursingd and actinic admixture of oleuropein aglycon were begin to associate able-bodied to the aerophilic adherence of esthetic oil. Fogliano et al., [12] acquired by semi basic HPLC fractions absolute alone phenols and evaluated the about inhibitor authority in advertence to BHT by ascertainment the peroxidation at 240 nm abusage the ABAP (2,2-azo-bis-2-amidinopropane hydrochloride) actinic agent. Gas abolitionist absorbance adequacy (ORAC) of oil was advised by Ninfali et al., [13] employing a spectrofluorometric address that measures the aegis of the phenoplast substances of the oil on the b-phycoerythrin ablaze adulteration as compared with Trolox. This value, that indicates the adequacy to attract peroxyl radicals, was projected as a backup constant to appraise the

**40**

**Figure 3.**

*Mechanism of the antioxidant activity of olive phenols.*

the ESR spectrum already accession of associate in nursing ethyl booze acknowledgment of the oil. Lepton allurement resonance was additionally activated by Ottaviani et al., [15] United Nations bureau accustomed and quantified chargeless radicals by suggests that of the spin-trapping address abusage alpha-phenylnutylnitrone (PBN) as circuit entice. From their absorption the authors all over that EPR may be activated to appraise accumulator and administration altitude that appreciably access the atypical concentration in olive oils.

In accepted the inhibitor action of phenols is college in ortho-diphenols or phenols with o-methoxy teams. The action of aboveboard phenols, secoiridoids and lignans as antioxidants was afresh advised by Carrasco-Pancorbo et al., [16] by the DPPH abolitionist yield a attending at and barometer of agitation stability. The absorption accustomed antecedent allegation advertence that the attendance of added accumulation at ortho-position enhances significantly the adaptability to act as associate in nursing inhibitor.

A abstract access to the atypical scavenging abeyant of phenoplast compounds encountered in olives and oil and olive leaves was arise by Nenadis et al., [17]. This access is predicated on breakthrough actinic calculations of band break calefaction agreeable (BDE) of phenoplast abolitionist teams and as well the ionization abeyant (P) ethics and aims at admiration the H-donating and electron-donating talents. Catechols were begin to own best low BDE values. Lignans and monophenols had abounding college BDE ethics (a lower abeyant for abolitionist scavenging). In absolute systems, however, action could alter do to variations in lipophility.

Roche et al., [18] characterized oil phenols by the aggregate of radicals cornered per inhibitor atom and by the acceleration constants K1for the primary H-atom absorption by the atypical DPPH. Oleuropein, hydroxytyrosol and caffeic acerbic accept the a lot of important K.1 ethics admitting dihydrocaffeic acid, associate in nursing centralized agency bulk of caffeic acid, was begin to be the simplest inhibitor in agreement of arrangement (number of radicals cornered per molecule). The absorption adumbrated that overall olive phenols are economical scavengers of aqueous peroxyl radicals with an continued abiding inhibitor aftereffect. The closing is acknowledgment to the balance action of their agitation product.

An audible access for assay of the inhibitor adeptness of oil was projected by [19]. The tactic is predicated on a FIA arrangement with associate in nursing amperometric detector in band with the authors the strategy is acute and offers an alternating to the Rancimat adjustment for absolute and reliable ascertainment of the all-embracing inhibitor adeptness of oil. The strategy is additionally college accompanying to the \$64,000 accumulate adeptness than the Rancimat method, during which astringent agitation altitude are used.

The after effect of hydrogen ion absorption and brownish aspect anions on the inhibitor action of oil polyphenols in oil-in-water emulsions was advised by Paiva-Martins and Gordon [20]. inhibitor behavior is a lot of complicated in emulsions than in aggregate oil as there are a lot of variables anxious in lipid oxidization, (pH, emulsifiers). Four oil phenols were examined, oleuropein, hydroxytyrosol, 3,4-dihydroxyplenylethanol-elenolic acerbic and three,4-dihydroxyphenylethanolelenolic acerbic dialdehyde. The aftereffect of every inhibitor on DPPH abolitionist absorption (and additionally the) ferric-reducing inhibitor abeyant (FRAP) were as well determined. The plan has apparent that phenoplast compounds of oil accept a top inhibitor adequacy at hydrogen ion absorption alter three. 5–7.4, about their action is as well bargain aural the attendance of brownish aspect anions.

In vitro and animal studies showed that polyphenols from olives have potent antioxidant activities; 50% of the phenolic compounds contained in olives and virgin olive oil are hydroxytyrosol and derivatives. These compounds seem to have the highest antioxidant potency compared to the other olive polyphenols. The radical

scavenging potency of o-methylated hydroxytyrosol was similar and that of the 3-o-glucuronide conjugate was more potent than hydroxytyrosol in vitro, whereas the monosulphate conjugate of hydroxytyrosol was almost devoid of its radical scavenging activity (Vissers et al., 2004). Review of the human intervention studies showed that olive polyphenols (e.g., hydroxytyrosol and oleuropein) decreased the levels of oxidized-LDL in plasma and positively affected several biomarkers of oxidative damage (Visioli and Galli, 2002).

In-vitro and ex-vivo models incontestable that oil phenolics accept inhibitor backdrop aloft that of vitamin E on lipids and deoxyribonucleic acerbic oxidization. Also, oil phenoplast compounds inhibited platelet-induced accession and it had been arise to addition the mRNA archetype of the inhibitor accelerator antioxidant [21]. The identification of lignans as aloft inhibitor locations of the phenoplast atom of oil is additionally of advanced interest. Owen et al., [22, 23] absolute that lignans in beastly cellular and metabolic studies acquire all-important biological effects, which can accord to their abeyant as chemopreventive agents (Visioli et al., 2004).

#### **4. The antimicrobial result of phenoplast compounds**

The antimicrobial aftereffect of olive arctic phenols are mentioned by Tripoli et al., [24]. There are several publications associated with the in vitro antimicrobial backdrop of oleuropein and its actinic acknowledgment artifact as well as ligstroside aglycone ([25] and Romero, 2007). Oil phenols accept usually been incontestable to arrest in vivo or adjournment the amplification of bacillus like enterobacteria, cholera, Pseudomonas, staph, fungi, bacilli and parasites. Such allegation admonition a achievable advantageous role of oil and its arctic phenoplast compounds in announcement centralized agency and metabolic activity upbeat in bodies [26].

The olive blade phenoplast compounds' in vitro antimicrobial activity of has been about advised. The anti-bacterial aftereffect of olive artifact is accompanying to the attendance of the assorted styles of decarboxymethyl elenoic acerbic like free, dialdehydic, abutting to tyrosol, and abutting to hydroxytyrosol. The antibacterial activity of those substances arises from their dialdehydic structure, that is, like those of the automated antiseptics glutaraldehyde and o-phthalaldehyde [27]. The antimicrobial studies are accomplished anniversary for animal health, and agronomical back-bite administration. Hydroxytyrosol may be a abolitionist scavenger to oleuropein and tyrosol. Oleuropein and hydroxytyrosol accept antimicrobial activity on an amount of the ATCC and analytic bacillus strains [28]. The foremost abstraction apropos the antimicrobial activity of hydroxytyrosol showed that low concentrations of hydroxytyrosol (≤8 μg/mL) were almighty to arrest the amplification of bacillus advertence strains. Bisignano et al., [25] advised the in vitro susceptibleness of hydroxytyrosol and oleuropein adjoin several bacillus strains that are accidental agents of metabolic activity or centralized agency amplitude infections in humans. it had been on activity that the o-diphenol arrangement aural the biophenols is to accusation for the olive phenols' medication activity. Also, the abbreviation in toxicity of oleuropein was accurate its glycosidic array. Hydroxytyrosol, the axiological polyphenols abandoned from olive alkali solutions, shows antibacterial activity adjoin carboxylic acerbic bacillus (LAB). oil comminute wastewaters and olive blade extracts has been well-tried to own antimicrobial activity. The bioactivity of oil comminute wastewaters has additionally been accompanying to the phenoplast compounds (oleuropein and hydroxytyrosol). Oleuropein and hydroxytyrosol exerted antimicrobial furnishings on communicable bacillus and bacilli [29]. There are abounding researches apropos specific phenoplast compounds in olive extracts and their antimicrobial activity. These researches instructed that

**43**

*Antioxidants in Olive Oil*

*DOI: http://dx.doi.org/10.5772/intechopen.84540*

with a 1000 μg/ml of hydroxytyrosol.

the hydroxytyrosol did not prove athletic antimicrobial activity. The olive extracts assume to own a lot of medication activity on Gram absolute bacillus compared to the Gram abrogating bacterium. Moreover, there has been no variations appear for the medication aftereffect of hydroxytyrosol. Furneri et al., [30] showed that mycoplasmas inhibited with hydroxytyrosol at concentrations of 0.03–0.5 μg/ml. The MICs (minimum black concentrations) for M. hominis, M. pneumoniae and M. fermenting, were 0.03, 0.5 and 0.25 μg/ml, severally. Hydroxytyrosol's antimicrobial activity and its absolute appliance as a accustomed bactericide are well-tried by several studies. Best low MIC akin of hydroxytyrosol was appear as 0.24 μg/ml. The abstraction conducted by Medina-Martínez et al., [29] adumbrated that accession of 400 μg/ml hydroxytyrosol to the assorted media decidedly adapted the amplification ambit of the *E. coli* strains compared to the administration cluster. Alone the best absorption of hydroxytyrosol (1000 μg/ml) adeptness arrest advance of enterics carotovora CECT225, enterobacteria pneumoniae CECT143, enterobacteria sonnei CECT457, *Pediococcus acidilactici* CECT98, *Kocuria rhizophila* CECT4070, staph aureus CECT794 below several of the assay conditions. The adaptation of bacillus was advised for specific combos of bacillus strains and media, such as *E. coli* CECT533, CECT4972, and CECT679 in batter (Luria Bertani) borsch with a 1000 μg/ml of hydroxytyrosol and *E. coli* CECT4972 in ISO (Iso-Sensitest) borsch

Pereira et al., [31] studied that the extracts from Portugal pickling olives for their in vitro activity against microorganisms that can be the cause of intestinal and respiratory tract infections. The tested microorganisms were Gram-positive bacteria such as *(Bacillus cereus, Bacillus subtilis, Staphylococcus aureus)*, Gram-negative bacteria *(Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae)* and fungi (*Candida albicans and Cryptococcus neoformans)*. Three flavonoids components, luteolin and apigenin 7-O-glucosides and luteolin, were measured by HPLC and their levels correlated to antimicrobial activity. Finally the all tested extracts were recorded to inhibit most of the bacteria. *B cereus* and *K pneumoniae* were the most sensitive. The fungal species studied (C. albicans and C. neoformans) were resistant to the extracts [32]. Verbascoside, the caffeic acid ester of hydroxytyrosol appeared in olives, shows antibacterial activity against *Staphylococcus aureus*, *Escherichia coli* and other clinical bacteria [33]. Biophenols compounds in olive oil have been shown to be able to penetrate structurally different cell membranes of Gram-negative and Gram-positive bacteria and inhibit irreversibly microbial replication. Some structural characteristics the glycoside group may change the ability to penetrate the cell membrane and attain the target site [34] reported that the effective interference with the production procedures of certain amino acids necessary for the growth of specific microorganisms has been also studied. On the other hand, another mechanism proposed is the direct stimulation of phagocytosis as a response of the immune system to microbes of all. The extracts made from olive leave are also studied for their antiviral activity against viral hemorrhagic virus septicaemia (VHSV) [35] and against HIV-1 infection and replication. Cell- to- cell transmission of HIV was inhibited in an in a dose-dependent manner, and HIV replication was inhibited in an in vitro experiment [36]. Oleuropein compounds has been patented for antiviral activity against viral disease, including herpes, mononucleosis and hepatitis [37].

**5. Phenolic compounds in the prevention of atherosclerosis**

Plasma LDL is atherogenic alone already aerophilic modification some studies accept apparent that aerophilic accent provokes the access of arterial sclerosis by causing lipid peroxidation. From now of read, antioxidants will may} apprehend

#### *Antioxidants in Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84540*

*Technological Innovation in the Olive Oil Production Chain*

oxidative damage (Visioli and Galli, 2002).

**4. The antimicrobial result of phenoplast compounds**

scavenging potency of o-methylated hydroxytyrosol was similar and that of the 3-o-glucuronide conjugate was more potent than hydroxytyrosol in vitro, whereas the monosulphate conjugate of hydroxytyrosol was almost devoid of its radical scavenging activity (Vissers et al., 2004). Review of the human intervention studies showed that olive polyphenols (e.g., hydroxytyrosol and oleuropein) decreased the levels of oxidized-LDL in plasma and positively affected several biomarkers of

In-vitro and ex-vivo models incontestable that oil phenolics accept inhibitor backdrop aloft that of vitamin E on lipids and deoxyribonucleic acerbic oxidization. Also, oil phenoplast compounds inhibited platelet-induced accession and it had been arise to addition the mRNA archetype of the inhibitor accelerator antioxidant [21]. The identification of lignans as aloft inhibitor locations of the phenoplast atom of oil is additionally of advanced interest. Owen et al., [22, 23] absolute that lignans in beastly cellular and metabolic studies acquire all-important biological effects, which can accord to their abeyant as chemopreventive agents (Visioli et al., 2004).

The antimicrobial aftereffect of olive arctic phenols are mentioned by Tripoli et al., [24]. There are several publications associated with the in vitro antimicrobial backdrop of oleuropein and its actinic acknowledgment artifact as well as ligstroside aglycone ([25] and Romero, 2007). Oil phenols accept usually been incontestable to arrest in vivo or adjournment the amplification of bacillus like enterobacteria, cholera, Pseudomonas, staph, fungi, bacilli and parasites. Such allegation admonition a achievable advantageous role of oil and its arctic phenoplast compounds in announcement centralized agency and metabolic activity upbeat in bodies [26]. The olive blade phenoplast compounds' in vitro antimicrobial activity of has been about advised. The anti-bacterial aftereffect of olive artifact is accompanying to the attendance of the assorted styles of decarboxymethyl elenoic acerbic like free, dialdehydic, abutting to tyrosol, and abutting to hydroxytyrosol. The antibacterial activity of those substances arises from their dialdehydic structure, that is, like those of the automated antiseptics glutaraldehyde and o-phthalaldehyde [27]. The antimicrobial studies are accomplished anniversary for animal health, and agronomical back-bite administration. Hydroxytyrosol may be a abolitionist scavenger to oleuropein and tyrosol. Oleuropein and hydroxytyrosol accept antimicrobial activity on an amount of the ATCC and analytic bacillus strains [28]. The foremost abstraction apropos the antimicrobial activity of hydroxytyrosol showed that low concentrations of hydroxytyrosol (≤8 μg/mL) were almighty to arrest the amplification of bacillus advertence strains. Bisignano et al., [25] advised the in vitro susceptibleness of hydroxytyrosol and oleuropein adjoin several bacillus strains that are accidental agents of metabolic activity or centralized agency amplitude infections in humans. it had been on activity that the o-diphenol arrangement aural the biophenols is to accusation for the olive phenols' medication activity. Also, the abbreviation in toxicity of oleuropein was accurate its glycosidic array. Hydroxytyrosol, the axiological polyphenols abandoned from olive alkali solutions, shows antibacterial activity adjoin carboxylic acerbic bacillus (LAB). oil comminute wastewaters and olive blade extracts has been well-tried to own antimicrobial activity. The bioactivity of oil comminute wastewaters has additionally been accompanying to the phenoplast compounds (oleuropein and hydroxytyrosol). Oleuropein and hydroxytyrosol exerted antimicrobial furnishings on communicable bacillus and bacilli [29]. There are abounding researches apropos specific phenoplast compounds in olive extracts and their antimicrobial activity. These researches instructed that

**42**

the hydroxytyrosol did not prove athletic antimicrobial activity. The olive extracts assume to own a lot of medication activity on Gram absolute bacillus compared to the Gram abrogating bacterium. Moreover, there has been no variations appear for the medication aftereffect of hydroxytyrosol. Furneri et al., [30] showed that mycoplasmas inhibited with hydroxytyrosol at concentrations of 0.03–0.5 μg/ml. The MICs (minimum black concentrations) for M. hominis, M. pneumoniae and M. fermenting, were 0.03, 0.5 and 0.25 μg/ml, severally. Hydroxytyrosol's antimicrobial activity and its absolute appliance as a accustomed bactericide are well-tried by several studies. Best low MIC akin of hydroxytyrosol was appear as 0.24 μg/ml. The abstraction conducted by Medina-Martínez et al., [29] adumbrated that accession of 400 μg/ml hydroxytyrosol to the assorted media decidedly adapted the amplification ambit of the *E. coli* strains compared to the administration cluster. Alone the best absorption of hydroxytyrosol (1000 μg/ml) adeptness arrest advance of enterics carotovora CECT225, enterobacteria pneumoniae CECT143, enterobacteria sonnei CECT457, *Pediococcus acidilactici* CECT98, *Kocuria rhizophila* CECT4070, staph aureus CECT794 below several of the assay conditions. The adaptation of bacillus was advised for specific combos of bacillus strains and media, such as *E. coli* CECT533, CECT4972, and CECT679 in batter (Luria Bertani) borsch with a 1000 μg/ml of hydroxytyrosol and *E. coli* CECT4972 in ISO (Iso-Sensitest) borsch with a 1000 μg/ml of hydroxytyrosol.

Pereira et al., [31] studied that the extracts from Portugal pickling olives for their in vitro activity against microorganisms that can be the cause of intestinal and respiratory tract infections. The tested microorganisms were Gram-positive bacteria such as *(Bacillus cereus, Bacillus subtilis, Staphylococcus aureus)*, Gram-negative bacteria *(Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae)* and fungi (*Candida albicans and Cryptococcus neoformans)*. Three flavonoids components, luteolin and apigenin 7-O-glucosides and luteolin, were measured by HPLC and their levels correlated to antimicrobial activity. Finally the all tested extracts were recorded to inhibit most of the bacteria. *B cereus* and *K pneumoniae* were the most sensitive. The fungal species studied (C. albicans and C. neoformans) were resistant to the extracts [32]. Verbascoside, the caffeic acid ester of hydroxytyrosol appeared in olives, shows antibacterial activity against *Staphylococcus aureus*, *Escherichia coli* and other clinical bacteria [33]. Biophenols compounds in olive oil have been shown to be able to penetrate structurally different cell membranes of Gram-negative and Gram-positive bacteria and inhibit irreversibly microbial replication. Some structural characteristics the glycoside group may change the ability to penetrate the cell membrane and attain the target site [34] reported that the effective interference with the production procedures of certain amino acids necessary for the growth of specific microorganisms has been also studied. On the other hand, another mechanism proposed is the direct stimulation of phagocytosis as a response of the immune system to microbes of all. The extracts made from olive leave are also studied for their antiviral activity against viral hemorrhagic virus septicaemia (VHSV) [35] and against HIV-1 infection and replication. Cell- to- cell transmission of HIV was inhibited in an in a dose-dependent manner, and HIV replication was inhibited in an in vitro experiment [36]. Oleuropein compounds has been patented for antiviral activity against viral disease, including herpes, mononucleosis and hepatitis [37].

#### **5. Phenolic compounds in the prevention of atherosclerosis**

Plasma LDL is atherogenic alone already aerophilic modification some studies accept apparent that aerophilic accent provokes the access of arterial sclerosis by causing lipid peroxidation. From now of read, antioxidants will may} apprehend

lipid peroxidation can accept a basic role in preventing aerophilic modification of LDL. Animal LDL accommodate an advance of antioxidants able of inhibiting peroxidation, like a-tocopherol, ubiquinol-10, b-carotene, carotenoid and another hydroxy-carotenoid. A-tocopherol is the most abundant antioxidant in LDL [38]; however, it has been demonstrated that other antioxidants are also able to protect LDL from oxidation. On the basis of previous epidemiological studies pointing out the direct correlation between the Mediterranean diet and a lower incidence of cardiovascular diseases [39]. In a sample of LDL, the vitamin E oxidation induced by CuSO4 was prevented by the addition of hydroxytyrosol or the secondary compounds of oleuropein; this effect was linearly correlated with the hydroxytyrosol concentration. In LDL, the addition of polyphenolic compounds caused significant reduction in lipid peroxide formation. In LDL not treated with polyphenolic compounds, these lipid peroxides are formed at the same time as the reduction of vitamin E levels. This vitamin E depletion by LDL occurs before massive lipid peroxidation. Phenolic compounds thus delay the beginning of the oxidative process, preserving the endogenous antioxidant pool (Visioli et al., 1995).

#### **6. Anti-inflammatory activity of phenoplast compounds**

Lipid radicals are created throughout reactions anxious aural the metabolism of arachidonic acid, throughout the amalgam of the eicosanoids by the activity of the lipo-oxygenase and cyclo-oxygenase throughout these reactions, the radicals that ar generated are partly inactivated by antioxidant [40]. Some studies accept associate in nursing black activity on cyclo-oxygenase and lipo-oxygenase by oil phenoplast compounds [41]. Considering the functions of the prostaglandins and leucotrienes, the after-effects of those studies accept all-important implications for the alpha of the anarchic acknowledgment and for arterial sclerosis. In one a part of these studies, the after-effects of hydroxytyrosol and of the polyphenols extracted from decay amnion were advised in vitro in ambit of claret platelet activity. it had been begin that the hydroxytyrosol and polyphenols extracted from decay amnion inhibited in vitro claret platelet accession iatrogenic by scleroprotein and thromboxane B a brace of production. The capability of hydroxytyrosol in inhibition of the accession iatrogenic by scleroprotein is commensurable thereto of Empirin, a biologic that is accustomed for its cable activity in claret platelet anti-aggregation and cyclo-oxygenase inhibition [42].

#### **7. Phenolic compounds as opposing cancer**

Many vegetable foods accommodate substances possessing antitumor backdrop [43, 44], a lot of them alive as antioxidants. Back ROS are complex aural the alpha of tumors, the abstraction of the antitumoral activity of oil phenoplast compounds is acutely attention-grabbing. Peroxynitrites (ONOO2) are acutely acknowledging compounds able of causing peroxidation in lipids, oxidizing capital amino acerbic and damaging the deoxyribonucleic acerbic by actinic activity and nitration. Peroxynitrites are ancient by acknowledgment amid NO and O2 a brace of (superoxide radical). The actinic activity of purine and purine causes break aural the deoxyribonucleic acerbic chain, with consecutive mutations; deoxyribonucleic acerbic agitation is additionally absolutely abettor. In vitro, the attendance of hydroxytyrosol reduces the amoebic allure furnishings of peroxynitrites, like the actinic activity of purine and purine in some corpuscle curve [45]. The inhibitor activity of abstinent oil extracts, apparent in vitro by their adeptness to arrest the aftereffect of gas radicals on hydroxy acid, is bright at concentrations abounding

**45**

**Author details**

Amany M. Basuny

provided the original work is properly cited.

*Antioxidants in Olive Oil*

*DOI: http://dx.doi.org/10.5772/intechopen.84540*

beneath than those of the one inhibitor compounds activated individually; this can be a lot of acceptable acknowledgment to the attendance of another polyphenolic compounds, an amount of that are still alien [22, 23]. Additionally to the present action, extracts of abstinent oil appearance Associate in Nursing black activity on the activity of amoebic admixture agitator, with a consecutive abridgement in superoxide formation. This activity cannot be incontestable for simple polyphenolic compounds (tyrosol and hydroxytyrosol) about its acknowledgment to secoiridoids and lignans [22]. Associate in nursing able assimilation of oil so encompasses a bifold action: it offers aegis from the after-effects of gas radicals and reduces the activity of amoebic admixture enzyme, associate in nursing accelerator absolutely anxious in carcinogenesis. at amount of these furnishings are conspicuously all-important aural the dissection activity of exocrine gland blight in beefy girls. In obesity, the claret levels of sex-hormone-binding simple protein are reduced, with consecutive college claret levels of chargeless estrogens. The exocrine gland cells that are about hormone-sensitive, are perpetually apparent to the activity of top amounts of estrogens [46–50]. Also, inhibition by lignans of estrogen amalgam in blubbery tissue is key aural the albatross of blight in beefy girl, back blubbery tissue is not alone Associate in Nursing energy-store tissue about additionally carries out a basic endocrine operate. It picks up and metabolizes steroid hormones,

alteration androstenedione into estrogen (E1) and androgenic hormone into 17-b-oestradiol (E2). The antitumor aftereffect of the lignans is so a lot of accept-

able acknowledgment to their activity on the metabolism of estrogens.

© 2019 The Author(s). Licensee IntechOpen. 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,

Biochemistry Department, Faculty of Agriculture, Beni-Suef University, Egypt

\*Address all correspondence to: dramany\_basuny@yahoo.com

#### *Antioxidants in Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84540*

*Technological Innovation in the Olive Oil Production Chain*

lipid peroxidation can accept a basic role in preventing aerophilic modification of LDL. Animal LDL accommodate an advance of antioxidants able of inhibiting peroxidation, like a-tocopherol, ubiquinol-10, b-carotene, carotenoid and another hydroxy-carotenoid. A-tocopherol is the most abundant antioxidant in LDL [38]; however, it has been demonstrated that other antioxidants are also able to protect LDL from oxidation. On the basis of previous epidemiological studies pointing out the direct correlation between the Mediterranean diet and a lower incidence of cardiovascular diseases [39]. In a sample of LDL, the vitamin E oxidation induced by CuSO4 was prevented by the addition of hydroxytyrosol or the secondary compounds of oleuropein; this effect was linearly correlated with the hydroxytyrosol concentration. In LDL, the addition of polyphenolic compounds caused significant reduction in lipid peroxide formation. In LDL not treated with polyphenolic compounds, these lipid peroxides are formed at the same time as the reduction of vitamin E levels. This vitamin E depletion by LDL occurs before massive lipid peroxidation. Phenolic compounds thus delay the beginning of the oxidative process,

preserving the endogenous antioxidant pool (Visioli et al., 1995).

**6. Anti-inflammatory activity of phenoplast compounds**

**7. Phenolic compounds as opposing cancer**

Lipid radicals are created throughout reactions anxious aural the metabolism of arachidonic acid, throughout the amalgam of the eicosanoids by the activity of the lipo-oxygenase and cyclo-oxygenase throughout these reactions, the radicals that ar generated are partly inactivated by antioxidant [40]. Some studies accept associate in nursing black activity on cyclo-oxygenase and lipo-oxygenase by oil phenoplast compounds [41]. Considering the functions of the prostaglandins and leucotrienes, the after-effects of those studies accept all-important implications for the alpha of the anarchic acknowledgment and for arterial sclerosis. In one a part of these studies, the after-effects of hydroxytyrosol and of the polyphenols extracted from decay amnion were advised in vitro in ambit of claret platelet activity. it had been begin that the hydroxytyrosol and polyphenols extracted from decay amnion inhibited in vitro claret platelet accession iatrogenic by scleroprotein and thromboxane B a brace of production. The capability of hydroxytyrosol in inhibition of the accession iatrogenic by scleroprotein is commensurable thereto of Empirin, a biologic that is accustomed for its cable activity in claret platelet anti-aggregation and cyclo-oxygenase inhibition [42].

Many vegetable foods accommodate substances possessing antitumor backdrop [43, 44], a lot of them alive as antioxidants. Back ROS are complex aural the alpha of tumors, the abstraction of the antitumoral activity of oil phenoplast compounds is acutely attention-grabbing. Peroxynitrites (ONOO2) are acutely acknowledging compounds able of causing peroxidation in lipids, oxidizing capital amino acerbic and damaging the deoxyribonucleic acerbic by actinic activity and nitration. Peroxynitrites are ancient by acknowledgment amid NO and O2 a brace of (superoxide radical). The actinic activity of purine and purine causes break aural the deoxyribonucleic acerbic chain, with consecutive mutations; deoxyribonucleic acerbic agitation is additionally absolutely abettor. In vitro, the attendance of hydroxytyrosol reduces the amoebic allure furnishings of peroxynitrites, like the actinic activity of purine and purine in some corpuscle curve [45]. The inhibitor activity of abstinent oil extracts, apparent in vitro by their adeptness to arrest the aftereffect of gas radicals on hydroxy acid, is bright at concentrations abounding

**44**

beneath than those of the one inhibitor compounds activated individually; this can be a lot of acceptable acknowledgment to the attendance of another polyphenolic compounds, an amount of that are still alien [22, 23]. Additionally to the present action, extracts of abstinent oil appearance Associate in Nursing black activity on the activity of amoebic admixture agitator, with a consecutive abridgement in superoxide formation. This activity cannot be incontestable for simple polyphenolic compounds (tyrosol and hydroxytyrosol) about its acknowledgment to secoiridoids and lignans [22]. Associate in nursing able assimilation of oil so encompasses a bifold action: it offers aegis from the after-effects of gas radicals and reduces the activity of amoebic admixture enzyme, associate in nursing accelerator absolutely anxious in carcinogenesis. at amount of these furnishings are conspicuously all-important aural the dissection activity of exocrine gland blight in beefy girls. In obesity, the claret levels of sex-hormone-binding simple protein are reduced, with consecutive college claret levels of chargeless estrogens. The exocrine gland cells that are about hormone-sensitive, are perpetually apparent to the activity of top amounts of estrogens [46–50]. Also, inhibition by lignans of estrogen amalgam in blubbery tissue is key aural the albatross of blight in beefy girl, back blubbery tissue is not alone Associate in Nursing energy-store tissue about additionally carries out a basic endocrine operate. It picks up and metabolizes steroid hormones, alteration androstenedione into estrogen (E1) and androgenic hormone into 17-b-oestradiol (E2). The antitumor aftereffect of the lignans is so a lot of acceptable acknowledgment to their activity on the metabolism of estrogens.

#### **Author details**

Amany M. Basuny Biochemistry Department, Faculty of Agriculture, Beni-Suef University, Egypt

\*Address all correspondence to: dramany\_basuny@yahoo.com

© 2019 The Author(s). Licensee IntechOpen. 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.

### **References**

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[2] Pérez AG, León L, Pascual M, Romero-Segura C, Sánchez-Ortiz A, de la Rosa R, et al. Variability of virgin olive oil phenolic compounds in a segregating progeny from a single cross in *Olea europaea* L. and sensory and nutritional quality implications. PLoS One. 2014;**9**:92898

[3] Bendini A, Cerretani L, Carrasco-Pancorbo A, Gomez-Caravaco AM, Segura-Cerretano A, Fernandez-Gutierrez A. Phenolic molecules in virgin olive oils; a survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules. 2007;**12**:1679-1719

[4] Romani A, Lapucci C, Cantini C, Ieri F, Mulinaci N, Visioli F. Evolution of minor polar compounds and antioxidant capacity during storage of bottled extra virgin olive oil. Journal of Agricultural and Food Chemistry. 2007;**55**:1315-1320

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olive oil with olive tree phenolic compounds. In: Intern. Soc. Fat

#### *Antioxidants in Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84540*

[16] Carrasco-Pancorbo A, Cerretani L, Segura-Carretero A, Gallina-Toschi T, Lercker G, Fernandez-Gutierrez A. Evaluation of individual antioxidant activity of single phenolic compounds on virgin olive oil. Progress in Nutrition. 2006;**8**:28-39

[17] Nenadis N, Wang LF, Tsimidou MZ, Zhang HY. Radical scavenging potential of phenolic compounds encountered in *O. Europaea*. Products as indicated by calculation of bond dissociation enthalpy and ionization potential values. Journal of Agricultural and Food Chemistry. 2005;**53**:295-299

[18] Roche M, Dufour C, Mora N, Dangles O. Antioxidant activity of olive phenols: mechanistic investigation and characterization of oxidation products by mass spectrometry. Organic & Biomolecular Chemistry. 2005;**3**:423-430

[19] Mannino S, Buratti S, Cosio MS, Pellegrini M. Evaluation of the "antioxidant power" of olive oilsbased on a FIA system with amperometric detection. Analyst. 1999;**124**:1115-1118

[20] Paiva-Martins F, Felix S, Correia R, Ferreira P, Gordon M. Enriched refined olive oil with olive tree phenolic compounds. In: Intern. Soc. Fat Research, 26th World Congress, Prague, Book of Abstracts. 2005. pp. 96-97

[21] Han J, Talorete TPN, Yamada P, Isoda H. Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells. Cytotechnology. 2009;**59**:45-53

[22] Owen RW, Giacosa A, Hull WE, Haubner R, Spiegelhalder B, Bartsch H. The antioxidant/anticancer potential of phenolic compounds isolated from olive oil. European Journal of Cancer. 2000a;**36**:1235-1247

[23] Owen RW, Mier W, Giacosa A, Hull WE, Spiegelhalder B, Bartsch H.

Identification of lignans as major components in the phenolic fraction of olive oil. Clinical Chemistry. 2000b;**46**:976-988

[24] Tripoli E, Giammanco M, Tabacchi G, DiMajo D, Giammanco S, LaGuardia M. The phenolic composition of olive oil: structure,biological activity, and beneficial effects on human health. Nutrition Research Reviews. 2005;**18**:98-112

[25] Bisignano G, Tomaino A, Cascio RL, Saija A. On the in-vitro antimicrobial activity of oleuropein and hydroxytyrosol. The Journal of Pharmacy and Pharmacology. 1999;**51**(8):971-974

[26] Aydar A, Öner T, Üçok E. Effects of hydroxytyrosol on human health. EC Nutrition. 2017;**11**(4):147-157

[27] Tuck KL, Hayball PY. Major phenolic compounds in olive oil: Metabolism and health effects. Journal of Nutrition and Biochemistry. 2002;**13**(11):636-644

[28] Talhaoui N, Taamalli A, MaríaGómez-Caravaca A, Fernández-Gutiérrez A, Segura-Carretero A. Phenolic compounds in olive leaves: Analytical determination, biotic and a biotic influence, and health benefits. Food Research International. 2015;**77**:92-108

[29] Medina-Martínez MS, Truchado P, Castro-Ibanez I, Allende A. Antimicrobial activity of hydroxytyrosol: A current controversy. Bioscience, Biotechnology, and Biochemistry. 2016;**80**(4):801-810

[30] Furneri PM et al. Antimycoplasmal activity of hydroxytyrosol. Antimicrobial Agents and Chemotherapy. 2004;**48**(12):4892-4894

[31] Pereira JA, Pereira APG, Ferreira ICFR, Valentao P, Andrade BP, Seabra R.

**46**

2001;**77**:405-411

*Technological Innovation in the Olive Oil Production Chain*

[8] Bianco A, Chiacchio M, Grassi G, Iannazzo D, Piperno A, Romeo R. Phenolic components of Olea europaea: Isolation of new tyrosol and hydroxytyrosol derivatives. Food

[9] Beauchamp G, Keast R, Morel D, Lin J, Pika J, Han Q. Ibuprofen-like activity in extra virgin olive oil. Nature.

[10] Papadopoulos G, Boskou D. Antioxidant effect of natural phenols on olive oil. Journal of the American Oil Chemists' Society. 1991;**68**:669-671

[11] Baldioli M, Servilli M, Perretti G, Montedoro G. Antioxidant activity of tocopherols and phenolic compounds of virgin olive oil. Journal of the American Oil Chemists' Society.

[12] Fogliano V, Ritieni S, Monti S, Gallo M, Madaglia DD, Ambrosino ML, et al. Antioxidant activity of virgin olive oil phenolic compounds in a micellar system. Journal of the Science of Food and Agriculture. 1999;**79**:1803-1808

[13] Ninfali P, Aluigi G, Bacchiocca M, Magnani M. Antioxidant capacity of extra-virgin olive oil. Journal of the American Oil Chemists' Society.

[14] Quiles JL, Ramirez-Tortoza M, Carmen Gomez J, Alfonso HJR, Mataix J. Role of vitamin E and phenolic compounds in the antioxidant capacity ,measured by ESR, of virgin olive oil, olive and sunflower oils after frying. Food Chemistry. 2002;**76**:461-468

[15] Ottaviani MF, Spallaci M, Cangiotti M, Bacchiocca M, Niffali P. Electron paramagnetic resonance investigations of free radicals in extra virgin olive oil. Journal of Agricultural and Food Chemistry. 2001;**49**:3691-3696

Chemistry. 2006;**95**:562-565

2005;**437**:45-46

1996;**73**:1589-1593

2010;**78**:243-247

[1] Boskou D. Olive oil. In: Simopoulos A, Visioli F, editors. Mediterranean Diets. Vol. 87. Basel: Karger Press, Wld Rev Nutr and Diet; 2000. pp. 56-77

[2] Pérez AG, León L, Pascual M, Romero-Segura C, Sánchez-Ortiz A, de la Rosa R, et al. Variability of virgin olive oil phenolic compounds in a segregating progeny from a single cross in *Olea europaea* L. and sensory and nutritional quality implications. PLoS

[3] Bendini A, Cerretani L, Carrasco-Pancorbo A, Gomez-Caravaco AM, Segura-Cerretano A, Fernandez-Gutierrez A. Phenolic molecules in virgin olive oils; a survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules. 2007;**12**:1679-1719

[4] Romani A, Lapucci C, Cantini C, Ieri F, Mulinaci N, Visioli F. Evolution of minor polar compounds and antioxidant capacity during storage of bottled extra virgin olive oil. Journal of Agricultural and Food Chemistry. 2007;**55**:1315-1320

[5] Litridou M, Linssen H, Schols H, Bergmans M, Tsimidou M, Boskou D. Phenolic compounds of virgin olive oils: fractionation by solid phase extraction and antioxidant activity assessment. Journal of the Science of Food and Agriculture. 1999a;**74**:169-174

[6] Brenes M, Romero C, Garcia A. Phenolic compounds in olive oil intended for refining: Formation of 4-ethylphenol during olive paste storage. Journal of Agricultural and Food Chemistry. 2004;**52**:8177-8181

[7] Bianco A, Chiachio M, Guiso M. Presence in olive oil of a new class of phenolic compounds hydroxylisochromans. Food Chemistry.

One. 2014;**9**:92898

**References**

Table olives from Portugal: Phenolic Compounds ,Antioxidant Potential and antimicrobial activity. Journal of Agricultural and Food Chemistry. 2006;**54**:8425-8431

[32] Sousa A, Ferreira I, Calhelha R, Andrade PB, Valenta P, Seabra R. Phenolics and antimicrobial activity of traditional stoned table olives "alaparra". Bioorganic & Medicinal Chemistry. 2006;**14**:8533-8538

[33] Soler-Rivas C, Carlos-Espin J, Wichers HJ. Oleuropein and related compounds. Journal of the Science of Food and Agriculture. 2000;**80**:1013-1023

[34] Saija A, Uccella N. Olive oil biophenols: Functional effects on human wellbeing. Trends in Food Science and Technology. 2001;**11**:357-363

[35] Micol V, Caturla N, Perenz-Fons L, Mas L, Perez L, Estepa A. The olive leaf extract exhibits antiviral activity against viral haemorhagic rhabdonius (VHSV). Antiviral Research. 2005;**66**:129-136

[36] Lee-Huang S, Zhang L, Chang YY, Huang PL. Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochemical and Biophysical Research Communications. 2003;**307**:1029-1037

[37] Fredrickson WR. Method and composition for antiviral therapy with olive leaves, US patent 6 117.884. Inventor F and S Group, Inc; 2000

[38] Jialal I, Fuller CJ, Huet BA. The effect of a-tocopherol supplementation on LDL oxidation. A dose-response study. Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;**15**:190-198

[39] Hertog MLG, Feskens EJM, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart

disease: the Zutphen Elderly Study. Lancet. 1993;**342**:1007

[40] Mirochnitchenko O, Prokopenko O, Palnitkar U, Kister I, Powell WS, Inouye M. Endotoxemia in transgenic mice overexpressing human glutathione peroxidases. Circulation Research. 2000;**87**:289-295

[41] Martinez-Dominguez E, de la Puerta R, Ruiz-Gutierrez V. Protective effects upon experimental inflammation models of a polyphenol-supplemented virgin olive oil diet. Inflammation Research. 2001;**50**:102-106

[42] Petroni A, Blasevich M, Salami M, Papini N, Montedoro GF, Galli C. Inhibition of platelet aggregation and eicosanoid production by phenolic components of olive oil. Thrombosis Research. 1995;**78**:151-160

[43] Johnson IT, Williamson G, Musk SRR. Anticarcinogenic factors in plant foods. A new class of nutrients. Nutrition Research Reviews. 1994;**7**:1-30

[44] Pezzuto JM. Plant-derived anticancer agents. Biochemical Pharmacology. 1997;**53**:121-133

[45] Deiana M, Aruoma OI, Bianchi MDLP, Spencer JPE, Kaur H, Halliwell B, et al. Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol. Free Radical Biology and Medicine. 1999;**26**:762-769

[46] Hankinson SE, Willett WC, Manson JE, Hunter DJ, Colditz GA, Stampfer MJ, et al. Alcohol, height, and adiposity in relation to estrogen and prolactin levels in postmenopausal women. Journal of the National Cancer Institute. 1995;**87**:1297-1302

[47] De Pergola G, Giorgino F, Garruti G, Cignarelli M, Giorgino R. Rapporto tra variabili antropometriche, ormoni

**49**

*Antioxidants in Olive Oil*

Oggi. 1996;**13**:138-145

pp. 137-166

Reviews. 1991;**7**:163

*DOI: http://dx.doi.org/10.5772/intechopen.84540*

complications in obesity). Metabolismo

[48] Newcomb TG, Loeb LA. Mechanism of mutagenicity of oxidatively-modified bases. In: Aruoma OI, Halliwell B, editors. Molecular Biology of Free Radicals in Human Diseases. Saint Lucia: OICA International; 1998.

[49] Parthasarathy S. Novel atherogenic oxidative modification of low density lipoprotein. Diabetes/Metabolism

[50] Princen HMG, van Poppel G, Vogelazang C, Buytenhek R, Kok FJ. Supplementation with vitamin E but not b-carotene in vivo protects low density lipoprotein from lipid peroxidation in vitro. Arteriosclerosis

and Thrombosis. 1992;**12**:554

sessuali e complicanze dell'obesità (Relationship between anthropometric

variables, sex hormones and

*Antioxidants in Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84540*

*Technological Innovation in the Olive Oil Production Chain*

disease: the Zutphen Elderly Study.

[41] Martinez-Dominguez E, de la Puerta R, Ruiz-Gutierrez V. Protective effects upon experimental inflammation models of a polyphenol-supplemented virgin olive oil diet. Inflammation

[42] Petroni A, Blasevich M, Salami M, Papini N, Montedoro GF, Galli C. Inhibition of platelet aggregation and eicosanoid production by phenolic components of olive oil. Thrombosis

[43] Johnson IT, Williamson G, Musk SRR. Anticarcinogenic factors in plant foods. A new class of nutrients. Nutrition Research Reviews. 1994;**7**:1-30

[44] Pezzuto JM. Plant-derived anticancer agents. Biochemical Pharmacology. 1997;**53**:121-133

[45] Deiana M, Aruoma OI, Bianchi MDLP, Spencer JPE, Kaur H, Halliwell B, et al. Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol. Free Radical Biology and Medicine. 1999;**26**:762-769

[46] Hankinson SE, Willett WC, Manson JE, Hunter DJ, Colditz GA, Stampfer MJ, et al. Alcohol, height, and adiposity in relation to estrogen and prolactin levels in postmenopausal women. Journal of the National Cancer Institute.

[47] De Pergola G, Giorgino F, Garruti G, Cignarelli M, Giorgino R. Rapporto tra variabili antropometriche, ormoni

1995;**87**:1297-1302

Research. 2001;**50**:102-106

Research. 1995;**78**:151-160

[40] Mirochnitchenko O, Prokopenko O, Palnitkar U, Kister I, Powell WS, Inouye M. Endotoxemia in transgenic mice overexpressing human glutathione peroxidases. Circulation Research.

Lancet. 1993;**342**:1007

2000;**87**:289-295

Table olives from Portugal: Phenolic Compounds ,Antioxidant Potential and antimicrobial activity. Journal of Agricultural and Food Chemistry.

[32] Sousa A, Ferreira I, Calhelha R, Andrade PB, Valenta P, Seabra R. Phenolics and antimicrobial activity of traditional stoned table olives "alaparra". Bioorganic & Medicinal Chemistry.

[33] Soler-Rivas C, Carlos-Espin J, Wichers HJ. Oleuropein and related compounds. Journal of the Science of Food and Agriculture.

[34] Saija A, Uccella N. Olive oil

Technology. 2001;**11**:357-363

(VHSV). Antiviral Research.

2005;**66**:129-136

biophenols: Functional effects on human wellbeing. Trends in Food Science and

[35] Micol V, Caturla N, Perenz-Fons L, Mas L, Perez L, Estepa A. The olive leaf extract exhibits antiviral activity against viral haemorhagic rhabdonius

[36] Lee-Huang S, Zhang L, Chang YY, Huang PL. Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochemical and Biophysical Research Communications. 2003;**307**:1029-1037

[37] Fredrickson WR. Method and composition for antiviral therapy with olive leaves, US patent 6 117.884. Inventor F and S Group, Inc; 2000

[38] Jialal I, Fuller CJ, Huet BA. The effect of a-tocopherol supplementation on LDL oxidation. A dose-response study. Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;**15**:190-198

[39] Hertog MLG, Feskens EJM, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart

2006;**54**:8425-8431

2006;**14**:8533-8538

2000;**80**:1013-1023

**48**

sessuali e complicanze dell'obesità (Relationship between anthropometric variables, sex hormones and complications in obesity). Metabolismo Oggi. 1996;**13**:138-145

[48] Newcomb TG, Loeb LA. Mechanism of mutagenicity of oxidatively-modified bases. In: Aruoma OI, Halliwell B, editors. Molecular Biology of Free Radicals in Human Diseases. Saint Lucia: OICA International; 1998. pp. 137-166

[49] Parthasarathy S. Novel atherogenic oxidative modification of low density lipoprotein. Diabetes/Metabolism Reviews. 1991;**7**:163

[50] Princen HMG, van Poppel G, Vogelazang C, Buytenhek R, Kok FJ. Supplementation with vitamin E but not b-carotene in vivo protects low density lipoprotein from lipid peroxidation in vitro. Arteriosclerosis and Thrombosis. 1992;**12**:554

**51**

olive

**1. Introduction**

**Chapter 5**

**Abstract**

*Alicia Beatriz Penissi*

also to improve olive oil industrialization.

Regulation of Immune and

by Phenols from Olive Oil

Nonimmune Mast Cell Activation

The purpose of this study is to establish if hydroxytyrosol and oleuropein, the most significant phenols found in olive oil and olives, can inhibit the activation of mast cells induced by immune and nonimmune pathways. Preincubation of purified peritoneal mast cells was carried out in the presence of hydroxytyrosol or oleuropein compounds and, prior to incubation, with concanavalin A, compound 48/80, or calcium ionophore A23187. Dose-response and time-dependence were studied. Comparative studies were performed using sodium cromoglycate, a well-known mast cell stabilizer. The supernatants and pellets were analyzed for β-hexosaminidase content via colorimetric reaction after incubation. The percentage of β-hexosaminidase obtained in each tube was measured and taken as a referent mast cell activation indicator. Other cell pellet samples were studied for cell viability, by means of the trypan blue exclusion method, or analyzed with light and electron microscopy. For the first time, biochemical and morphological results have shown that hydroxytyrosol and oleuropein inhibit degranulation of mast cells triggered by both immune and nonimmune causes. These findings suggest that olive phenols, specifically hydroxytyrosol and oleuropein, may be set the bases for developing practical tools not only to prevent and treat mast cell-mediated disorders but

**Keywords:** β-hexosaminidase, degranulation, hydroxytyrosol, mast cell, oleuropein,

Mast cells are key effector cells that clearly play both physiological and pathophysiological functions in the body [1]. In vertebrates, mast cells are widely distributed in the tissues, especially near surfaces exposed to the environment, where pathogens, allergens, and other environmental agents are frequently found. Due to this distribution pattern, they are some of the first cells involved in the immune response which interact with environmental antigens and allergens, invading pathogens or environmentally derived toxins [2]. Mast cells have been involved in the pathogenesis of a number of disorders including contact dermatitis, allergic rhinitis, asthma, atopic dermatitis, bullous pemphigoid, fibrotic lung disease, cancer, multiple sclerosis, neurofibromatosis, psoriasis, scleroderma, rheumatoid arthritis,

#### **Chapter 5**

## Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil

*Alicia Beatriz Penissi*

#### **Abstract**

The purpose of this study is to establish if hydroxytyrosol and oleuropein, the most significant phenols found in olive oil and olives, can inhibit the activation of mast cells induced by immune and nonimmune pathways. Preincubation of purified peritoneal mast cells was carried out in the presence of hydroxytyrosol or oleuropein compounds and, prior to incubation, with concanavalin A, compound 48/80, or calcium ionophore A23187. Dose-response and time-dependence were studied. Comparative studies were performed using sodium cromoglycate, a well-known mast cell stabilizer. The supernatants and pellets were analyzed for β-hexosaminidase content via colorimetric reaction after incubation. The percentage of β-hexosaminidase obtained in each tube was measured and taken as a referent mast cell activation indicator. Other cell pellet samples were studied for cell viability, by means of the trypan blue exclusion method, or analyzed with light and electron microscopy. For the first time, biochemical and morphological results have shown that hydroxytyrosol and oleuropein inhibit degranulation of mast cells triggered by both immune and nonimmune causes. These findings suggest that olive phenols, specifically hydroxytyrosol and oleuropein, may be set the bases for developing practical tools not only to prevent and treat mast cell-mediated disorders but also to improve olive oil industrialization.

**Keywords:** β-hexosaminidase, degranulation, hydroxytyrosol, mast cell, oleuropein, olive

#### **1. Introduction**

Mast cells are key effector cells that clearly play both physiological and pathophysiological functions in the body [1]. In vertebrates, mast cells are widely distributed in the tissues, especially near surfaces exposed to the environment, where pathogens, allergens, and other environmental agents are frequently found. Due to this distribution pattern, they are some of the first cells involved in the immune response which interact with environmental antigens and allergens, invading pathogens or environmentally derived toxins [2]. Mast cells have been involved in the pathogenesis of a number of disorders including contact dermatitis, allergic rhinitis, asthma, atopic dermatitis, bullous pemphigoid, fibrotic lung disease, cancer, multiple sclerosis, neurofibromatosis, psoriasis, scleroderma, rheumatoid arthritis, interstitial cystitis, ulcerative colitis, peptic ulcer, and Crohn's disease [1, 3–7]. Understanding mast cells is essential for the pathophysiological bases of this type of disorders since these cells release varied inflammatory mediators prompted by both immune and nonimmune causes. Among mast cell mediators, preformed molecules can be mentioned, for example, histamine and proteases, which are accumulated in secretory granules [7–9]. The immediate response upon mast cell activation to an appropriate stimulus is called degranulation, characterized by the extrusion of cytoplasmic granule contents into the extracellular space by a process called exocytosis [10]. In this context, the exploration of interactions of mast cells with molecules capable of modulating mediator release from cell granules is a promising field for the treatment of *mast cell-mediated diseases*.

In vitro and in vivo studies have shown that several plant products with antioxidant properties inhibit mast cell activation induced by both immune and nonimmune secretagogues [11–14]. Recent scientific evidence from preclinical studies and clinical trials including humans has highlighted different nutritional interventions, such as dietary polyphenols, as promising agents able to alleviate symptoms associated with mast cell activation [15]. Dietary polyphenols are a class of bioactive compounds found in abundance in plants and fruits which have been studied thoroughly in several disease models [15]. The pulp of olives contains these compounds, which are hydrophilic, and they are also found in the oil. The class of phenols includes numerous substances, such as simple phenolic compounds like hydroxytyrosol and more complex compounds like oleuropein [16]. Hydroxytyrosol and oleuropein, the major phenols found in olives, are used in disease prevention because they have important antioxidant and anti-inflammatory properties [17, 18]. Several in vitro and in vivo studies have shown that oleuropein and its derivate hydroxytyrosol possess a wide range of biochemical and pharmacological properties. However, no studies have been published on the effects of these molecules on mast cell degranulation.

We carried out a series of experiments to determine the effects of both phenolic compounds on mast cell degranulation and thus explore the possibility that oleuropein and hydroxytyrosol might inhibit in vitro mast cell activation.

The biochemical and morphological findings of the present study showed for the first time that hydroxytyrosol and oleuropein inhibit the degranulation of mast cells induced by both immune and nonimmune pathways.

#### **2. Material and methods**

#### **2.1 Chemicals and reagents**

The compound molecules, hydroxytyrosol and oleuropein, were provided by Extrasynthèse (Lyon, France). **Figure 1** shows the polyphenol chemical structure. A solution containing 6.7 mM Na2HPO4, 6.7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.8 mM CaCl2, 0.5 g/l albumin, and 1 g/l glucose was used for dissolving the polyphenols, which was adjusted to pH 7.2 and stored at −20°C. The same solution was used for diluting the stock solutions until the final concentration was reached. Bovine serum albumin (fraction V), concanavalin A, compound 48/80, calcium ionophore A23187, sodium cromoglycate, 4-nitrophenyl-N-acetyl-β-Dglucosaminide, toluidine blue, trypan blue, glutaraldehyde, formaldehyde, and osmium tetroxide were acquired from Sigma (St. Louis, MO, USA). Percoll was purchased from GE Healthcare (Munich, Germany). All other substances were provided by Merck (Darmstadt, Germany). The highest quality available is guaranteed in all the chemicals used for this study.

**53**

**2.2 Animals**

**Figure 1.**

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil*

Adult male 300–500 g Wistar rats free from infections were used for the study. They were kept under a 12-h dark/light cycle in a room set at 24–25°C with free access to laboratory food and drinking water. Animals experiments were carried out according with the standards contained in the *Guide for the Care and Use of Laboratory Animals*, published by the National Academy of Sciences, National Academies Press, Washington, DC, and accepted by the Institutional Committee for Care and Use of Laboratory Animals (CICUAL, Facultad de Ciencias Médicas,

Isolation of mast cells was performed by means of peritoneal lavage as described before [19] with some modifications. Rats were killed by CO2 inhalation and then injected with 20 ml of a pH 7.2 solution containing 6.7 mM Na2HPO4, 6.7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.8 mM CaCl2, 0.5 g/l albumin, and 1 g/l glucose, into the peritoneal cavity. A gentle massage was applied on the abdomen for about 3 min. After the peritoneal cavity was opened with care, a Pasteur pipette was used for aspirating the fluid with peritoneal cells, which were then purified via centrifugation through a discontinuous gradient of Percoll per reports by MacGlashan and Guo [20]. It was easy to harvest the mast cells since they precipitated to the bottom of the tube and formed a layer, different from the other cells that formed a rather compact layer on top of the gradient and were simply removed by aspiration. Cell metachromatic staining was carried out with toluidine blue (0.1% w/v, pH 1.0), and quantification required a Neubauer hemocytometer under a Nikon microscope (magnification 200×). Mast cells were present in the crude peritoneal suspended content by 3%, and, after gradient centrifugation, their purity rose to over 95%. After washing purified mast cells, resuspension was performed in a balanced salt

Universidad Nacional de Cuyo, Mendoza, Argentina).

**2.3 Mast cell isolation and purification**

*Structural formulas of polyphenols used in this study.*

*DOI: http://dx.doi.org/10.5772/intechopen.84595*

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84595*

**Figure 1.** *Structural formulas of polyphenols used in this study.*

#### **2.2 Animals**

*Technological Innovation in the Olive Oil Production Chain*

field for the treatment of *mast cell-mediated diseases*.

mast cell degranulation.

**2. Material and methods**

**2.1 Chemicals and reagents**

teed in all the chemicals used for this study.

interstitial cystitis, ulcerative colitis, peptic ulcer, and Crohn's disease [1, 3–7]. Understanding mast cells is essential for the pathophysiological bases of this type of disorders since these cells release varied inflammatory mediators prompted by both immune and nonimmune causes. Among mast cell mediators, preformed molecules can be mentioned, for example, histamine and proteases, which are accumulated in secretory granules [7–9]. The immediate response upon mast cell activation to an appropriate stimulus is called degranulation, characterized by the extrusion of cytoplasmic granule contents into the extracellular space by a process called exocytosis [10]. In this context, the exploration of interactions of mast cells with molecules capable of modulating mediator release from cell granules is a promising

In vitro and in vivo studies have shown that several plant products with antioxidant properties inhibit mast cell activation induced by both immune and nonimmune secretagogues [11–14]. Recent scientific evidence from preclinical studies and clinical trials including humans has highlighted different nutritional interventions, such as dietary polyphenols, as promising agents able to alleviate symptoms associated with mast cell activation [15]. Dietary polyphenols are a class of bioactive compounds found in abundance in plants and fruits which have been studied thoroughly in several disease models [15]. The pulp of olives contains these compounds, which are hydrophilic, and they are also found in the oil. The class of phenols includes numerous substances, such as simple phenolic compounds like hydroxytyrosol and more complex compounds like oleuropein [16]. Hydroxytyrosol and oleuropein, the major phenols found in olives, are used in disease prevention because they have important antioxidant and anti-inflammatory properties [17, 18]. Several in vitro and in vivo studies have shown that oleuropein and its derivate hydroxytyrosol possess a wide range of biochemical and pharmacological properties. However, no studies have been published on the effects of these molecules on

We carried out a series of experiments to determine the effects of both phenolic compounds on mast cell degranulation and thus explore the possibility that oleuro-

The biochemical and morphological findings of the present study showed for the first time that hydroxytyrosol and oleuropein inhibit the degranulation of mast

The compound molecules, hydroxytyrosol and oleuropein, were provided by Extrasynthèse (Lyon, France). **Figure 1** shows the polyphenol chemical structure. A solution containing 6.7 mM Na2HPO4, 6.7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.8 mM CaCl2, 0.5 g/l albumin, and 1 g/l glucose was used for dissolving the polyphenols, which was adjusted to pH 7.2 and stored at −20°C. The same solution was used for diluting the stock solutions until the final concentration was reached. Bovine serum albumin (fraction V), concanavalin A, compound 48/80, calcium ionophore A23187, sodium cromoglycate, 4-nitrophenyl-N-acetyl-β-Dglucosaminide, toluidine blue, trypan blue, glutaraldehyde, formaldehyde, and osmium tetroxide were acquired from Sigma (St. Louis, MO, USA). Percoll was purchased from GE Healthcare (Munich, Germany). All other substances were provided by Merck (Darmstadt, Germany). The highest quality available is guaran-

pein and hydroxytyrosol might inhibit in vitro mast cell activation.

cells induced by both immune and nonimmune pathways.

**52**

Adult male 300–500 g Wistar rats free from infections were used for the study. They were kept under a 12-h dark/light cycle in a room set at 24–25°C with free access to laboratory food and drinking water. Animals experiments were carried out according with the standards contained in the *Guide for the Care and Use of Laboratory Animals*, published by the National Academy of Sciences, National Academies Press, Washington, DC, and accepted by the Institutional Committee for Care and Use of Laboratory Animals (CICUAL, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina).

#### **2.3 Mast cell isolation and purification**

Isolation of mast cells was performed by means of peritoneal lavage as described before [19] with some modifications. Rats were killed by CO2 inhalation and then injected with 20 ml of a pH 7.2 solution containing 6.7 mM Na2HPO4, 6.7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.8 mM CaCl2, 0.5 g/l albumin, and 1 g/l glucose, into the peritoneal cavity. A gentle massage was applied on the abdomen for about 3 min. After the peritoneal cavity was opened with care, a Pasteur pipette was used for aspirating the fluid with peritoneal cells, which were then purified via centrifugation through a discontinuous gradient of Percoll per reports by MacGlashan and Guo [20]. It was easy to harvest the mast cells since they precipitated to the bottom of the tube and formed a layer, different from the other cells that formed a rather compact layer on top of the gradient and were simply removed by aspiration. Cell metachromatic staining was carried out with toluidine blue (0.1% w/v, pH 1.0), and quantification required a Neubauer hemocytometer under a Nikon microscope (magnification 200×). Mast cells were present in the crude peritoneal suspended content by 3%, and, after gradient centrifugation, their purity rose to over 95%. After washing purified mast cells, resuspension was performed in a balanced salt

solution with 6.7 mM Na2HPO4, 6.7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.8 mM CaCl2, 0.5 g/l albumin, and 1 g/l glucose, adjusted to pH 7.2 (cell density of 1 × 106 /ml) and kept at 4°C for 30 min at the most. Both the ability of mast cells to exclude trypan blue and the amount of β-hexosaminidase in the supernatant established mast cell viability. The trypan blue exclusion test determined that the mast cells were viable by over 95%. Basal β-Hexosaminidase release remained always below 4%.

#### **2.4 General protocol**

Purified peritoneal mast cells (cell density of 1 × 106 /ml) were balanced at 37°C for 10 min. Preincubation of 30-μL aliquots of the balanced cells required polypropylene tubes at 37°C with hydroxytyrosol or oleuropein. Then, incubation was carried out at 37°C for 10 min using concanavalin A (final concentration was 200 μg/ml, with 50 μg/ml phosphatidylserine as a co-stimulator), compound 48/80 (concentrated at 10 g/ml), or calcium ionophore A23187 (final concentration 50 μg/ml). Positive and negative controls, that is, with and without mast cell secretagogues stimulation, respectively, were included. Studies on dose–response (hydroxytyrosol and oleuropein concentrations of 10, 50, 100, 200, and 400 M) and time-dependence (hydroxytyrosol and oleuropein preincubation for 5, 10, 20, and 45 min) were performed. Comparative studies were performed using sodium cromoglycate, a well-known mast cell stabilizer, with identical concentrations and time ranges. Each tube's final incubation volume reached 100 l. During incubations, the average total number of mast cells was 4 × 104 /ml per tube. Secretion was halted by cooling the tubes in an ice-cold water bath. Cells and supernatants were separated by centrifugation (180 g, 5 min, 4°C). The supernatants helped determine the β-hexosaminidase content by colorimetric reaction, which was a parameter to measure β-hexosaminidase release. The cell pellets were lysated with 1% Triton X-100 to release the remaining β-hexosaminidase, which was quantified by colorimetric reaction and taken as a measure of the residual β-hexosaminidase. Other cell pellet samples were studied for cell viability, by means of the trypan blue exclusion method, or analyzed with light and electron microscopy. The purpose of cell viability studies was to ascertain that changes in β-hexosaminidase release were not caused by cell death. The percentage of β-hexosaminidase obtained in each tube was measured. All the tests were performed at least five times in duplicate.

#### **2.5 β-Hexosaminidase assay**

β-Hexosaminidase release, as an index of mast cell degranulation, was assayed by a colorimetric assay as before explained [21] with some changes. In brief, 50 μL of the supernatant was combined with an equal volume of 2 mM substrate solution (p-nitrophenyl-N-acetyl-β-D-glucosaminide in 0.2 M citrate, pH 4.5) and then incubated for 3 h at 37°C. The reaction was halted by adding 250 μL of stopping buffer (0.4 M glycine in Na2CO3/NaHCO3, pH 9). Absorbance was studied with a microplate reader at 405 nm (Thermo Scientific Multiskan FC, Helsinki, Finland). Results were stated as the percentage of β-hexosaminidase activity released over the total (enzyme released plus intracellular enzyme).

#### **2.6 Light microscopy and morphometry**

Mast cells were fixed in 2% glutaraldehyde for 2 h. Then, the suspended cells were stained with toluidine blue (0.1% w/v, pH 3.0), put between slides and cover

**55**

to challenge mast cells.

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil*

slides, and analyzed under a Nikon Optiphot 2 microscope. Using a magnification of 400×, percentage of degranulated mast cells was quantified. Mast cell was considered active due to the presence of extruded granules near the surface of the

Karnovsky's fixative (2% formaldehyde, freshly prepared from paraformaldehyde, 2.5% glutaraldehyde, 0.025% CaCl2, 0.1 M cacodylate buffer, pH 7.4) was used for mast cell fixation. After 1 h in the fixative at 20°C, mast cells were rinsed in 0.2 M cacodylate buffer and postfixed in 1% OsO4 in 0.1 M cacodylate buffer for at least 2 h at room temperature and dehydrated in ethanol. Next, suspended cells were embedded in Spurr (Pelco, USA). An automatic ultramicrotome (Leica Ultracut R, Austria) was used to cut semithin transverse sections (1 μm), which were stained with filtered 1% toluidine blue. Ultrathin sections (60 nm) were cut with diamond knives, stained with uranyl acetate and lead citrate, mounted on grids (Pelco, USA), and examined in a transmission electron microscope (Zeiss EM

Results obtained from biochemical and morphometric analyses are presented as ±SEM. Variance analysis was used to determine differences between groups, followed by Tukey-Kramer multiple comparisons test. P < 0.05 was considered

**Figure 2** shows the effect of varying concentrations of hydroxytyrosol and oleuropein on the mast cells β-hexosaminidase release, induced by concanavalin A, compound 48/80, or calcium ionophore A23187. β-Hexosaminidase release was significantly increased by incubating mast cells with 200 μg/ml concanavalin A or 10 μg/ml compound 48/80 or 50 μg/ml calcium ionophore A23187 solutions, as compared with the corresponding value from the basal group (basal release was always less than 4%). The concanavalin A-induced effects were inhibited by preincubation of mast cells with hydroxytyrosol (10, 50 and 100 μM) or oleuropein (100 μM). Mast cell activation induced by compound 48/80 was inhibited by hydroxytyrosol (100 μM) and oleuropein (100 μM). The calcium ionophore A23187-induced effects were inhibited by preincubation of mast cells with hydroxytyrosol (100 μM) and oleuropein (10, 50 and 100 μM). The inhibitory action of the polyphenols was not accompanied by changes in cell viability (the trypan blue exclusion test indicated a viability of greater than 80%), except for polyphenol

**Figure 3** shows a dose-response comparative study with sodium cromoglycate, a mast cell stabilizer, which was used to evaluate the potency of hydroxytyrosol and oleuropein. The inhibitory effect of hydroxytyrosol was higher than that obtained with sodium cromoglycate at the same concentration (100 μM) when mast cells were challenged with concanavalin A. No significant differences were observed when mast cells were challenged with compound 48/80. The oleuropein inhibitory effect was higher than the one obtained with sodium cromoglycate at the same concentrations (10, 50 and 100 μM) when the calcium ionophore A23187 was used

cell in question or a toluidine blue stain in half or less of the cell.

*DOI: http://dx.doi.org/10.5772/intechopen.84595*

**2.7 Transmission electron microscopy**

902, Germany).

**2.8 Statistical analysis**

statistically significant.

**3. Results and discussion**

concentrations higher than 100 μM (data not shown).

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84595*

slides, and analyzed under a Nikon Optiphot 2 microscope. Using a magnification of 400×, percentage of degranulated mast cells was quantified. Mast cell was considered active due to the presence of extruded granules near the surface of the cell in question or a toluidine blue stain in half or less of the cell.

#### **2.7 Transmission electron microscopy**

Karnovsky's fixative (2% formaldehyde, freshly prepared from paraformaldehyde, 2.5% glutaraldehyde, 0.025% CaCl2, 0.1 M cacodylate buffer, pH 7.4) was used for mast cell fixation. After 1 h in the fixative at 20°C, mast cells were rinsed in 0.2 M cacodylate buffer and postfixed in 1% OsO4 in 0.1 M cacodylate buffer for at least 2 h at room temperature and dehydrated in ethanol. Next, suspended cells were embedded in Spurr (Pelco, USA). An automatic ultramicrotome (Leica Ultracut R, Austria) was used to cut semithin transverse sections (1 μm), which were stained with filtered 1% toluidine blue. Ultrathin sections (60 nm) were cut with diamond knives, stained with uranyl acetate and lead citrate, mounted on grids (Pelco, USA), and examined in a transmission electron microscope (Zeiss EM 902, Germany).

#### **2.8 Statistical analysis**

*Technological Innovation in the Olive Oil Production Chain*

Purified peritoneal mast cells (cell density of 1 × 106

incubations, the average total number of mast cells was 4 × 104

formed at least five times in duplicate.

total (enzyme released plus intracellular enzyme).

**2.6 Light microscopy and morphometry**

**2.5 β-Hexosaminidase assay**

Secretion was halted by cooling the tubes in an ice-cold water bath. Cells and supernatants were separated by centrifugation (180 g, 5 min, 4°C). The supernatants helped determine the β-hexosaminidase content by colorimetric reaction, which was a parameter to measure β-hexosaminidase release. The cell pellets were lysated with 1% Triton X-100 to release the remaining β-hexosaminidase, which was quantified by colorimetric reaction and taken as a measure of the residual β-hexosaminidase. Other cell pellet samples were studied for cell viability, by means of the trypan blue exclusion method, or analyzed with light and electron microscopy. The purpose of cell viability studies was to ascertain that changes in β-hexosaminidase release were not caused by cell death. The percentage of β-hexosaminidase obtained in each tube was measured. All the tests were per-

β-Hexosaminidase release, as an index of mast cell degranulation, was assayed by a colorimetric assay as before explained [21] with some changes. In brief, 50 μL of the supernatant was combined with an equal volume of 2 mM substrate solution (p-nitrophenyl-N-acetyl-β-D-glucosaminide in 0.2 M citrate, pH 4.5) and then incubated for 3 h at 37°C. The reaction was halted by adding 250 μL of stopping buffer (0.4 M glycine in Na2CO3/NaHCO3, pH 9). Absorbance was studied with a microplate reader at 405 nm (Thermo Scientific Multiskan FC, Helsinki, Finland). Results were stated as the percentage of β-hexosaminidase activity released over the

Mast cells were fixed in 2% glutaraldehyde for 2 h. Then, the suspended cells were stained with toluidine blue (0.1% w/v, pH 3.0), put between slides and cover

of 1 × 106

always below 4%.

**2.4 General protocol**

solution with 6.7 mM Na2HPO4, 6.7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.8 mM CaCl2, 0.5 g/l albumin, and 1 g/l glucose, adjusted to pH 7.2 (cell density

to exclude trypan blue and the amount of β-hexosaminidase in the supernatant established mast cell viability. The trypan blue exclusion test determined that the mast cells were viable by over 95%. Basal β-Hexosaminidase release remained

37°C for 10 min. Preincubation of 30-μL aliquots of the balanced cells required polypropylene tubes at 37°C with hydroxytyrosol or oleuropein. Then, incubation was carried out at 37°C for 10 min using concanavalin A (final concentration was 200 μg/ml, with 50 μg/ml phosphatidylserine as a co-stimulator), compound 48/80 (concentrated at 10 g/ml), or calcium ionophore A23187 (final concentration 50 μg/ml). Positive and negative controls, that is, with and without mast cell secretagogues stimulation, respectively, were included. Studies on dose–response (hydroxytyrosol and oleuropein concentrations of 10, 50, 100, 200, and 400 M) and time-dependence (hydroxytyrosol and oleuropein preincubation for 5, 10, 20, and 45 min) were performed. Comparative studies were performed using sodium cromoglycate, a well-known mast cell stabilizer, with identical concentrations and time ranges. Each tube's final incubation volume reached 100 l. During

/ml) and kept at 4°C for 30 min at the most. Both the ability of mast cells

/ml) were balanced at

/ml per tube.

**54**

Results obtained from biochemical and morphometric analyses are presented as ±SEM. Variance analysis was used to determine differences between groups, followed by Tukey-Kramer multiple comparisons test. P < 0.05 was considered statistically significant.

#### **3. Results and discussion**

**Figure 2** shows the effect of varying concentrations of hydroxytyrosol and oleuropein on the mast cells β-hexosaminidase release, induced by concanavalin A, compound 48/80, or calcium ionophore A23187. β-Hexosaminidase release was significantly increased by incubating mast cells with 200 μg/ml concanavalin A or 10 μg/ml compound 48/80 or 50 μg/ml calcium ionophore A23187 solutions, as compared with the corresponding value from the basal group (basal release was always less than 4%). The concanavalin A-induced effects were inhibited by preincubation of mast cells with hydroxytyrosol (10, 50 and 100 μM) or oleuropein (100 μM). Mast cell activation induced by compound 48/80 was inhibited by hydroxytyrosol (100 μM) and oleuropein (100 μM). The calcium ionophore A23187-induced effects were inhibited by preincubation of mast cells with hydroxytyrosol (100 μM) and oleuropein (10, 50 and 100 μM). The inhibitory action of the polyphenols was not accompanied by changes in cell viability (the trypan blue exclusion test indicated a viability of greater than 80%), except for polyphenol concentrations higher than 100 μM (data not shown).

**Figure 3** shows a dose-response comparative study with sodium cromoglycate, a mast cell stabilizer, which was used to evaluate the potency of hydroxytyrosol and oleuropein. The inhibitory effect of hydroxytyrosol was higher than that obtained with sodium cromoglycate at the same concentration (100 μM) when mast cells were challenged with concanavalin A. No significant differences were observed when mast cells were challenged with compound 48/80. The oleuropein inhibitory effect was higher than the one obtained with sodium cromoglycate at the same concentrations (10, 50 and 100 μM) when the calcium ionophore A23187 was used to challenge mast cells.

#### **Figure 2.**

*Effect of varying concentrations of hydroxytyrosol and oleuropein on the concanavalin A-, compound 48/80-, and calcium ionophore A23187-induced β-hexosaminidase release from peritoneal mast cells. Mast cells were preincubated with increasing concentrations of hydroxytyrosol or oleuropein for 10 min and then stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187, for 10 min at 37°C. β-Hexosaminidase release was measured by colorimetric reaction with the chromogenic substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide. Results are expressed as percentage release of β-hexosaminidase. Values are presented as means ± SEM +++P < 0.001 versus basal, \* P < 0.05 versus secretagogue, \*\*P < 0.01 versus secretagogue, and \*\*\*P < 0.001 versus secretagogue.*

The kinetic study results connected with the effect of hydroxytyrosol and oleuropein on β-hexosaminidase release from mast cell are described in **Figure 4**.

The concanavalin A-induced effect was inhibited by hydroxytyrosol (10 min), oleuropein (10 min), and sodium cromoglycate (10 min). Mast cell activation induced by compound 48/80 was only inhibited by sodium cromoglycate (5, 10, and 20 min). The ionophore A23187-induced effect was inhibited by hydroxytyrosol (10 min), oleuropein (5, 10, and 20 min), and sodium cromoglycate (10 and 20 min). The inhibitory action of the test compounds was not accompanied by changes in cell viability (the trypan blue exclusion test indicated a viability of greater than 80%), except for incubation times higher than 20 min (data not shown).

**57**

**Figure 3.**

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil*

*Effect of hydroxytyrosol and oleuropein on β-hexosaminidase release from rat peritoneal mast cells, compared with a reference compound, the mast cell stabilizer sodium cromoglycate. Purified mast cells were preincubated with increasing concentrations of hydroxytyrosol, oleuropein, or sodium cromoglycate for 10 min and stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187 for another 10 min at 37°C. β-Hexosaminidase release was measured by colorimetric reaction with the chromogenic substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide. Results are expressed as percentage* 

*P < 0.05 versus sodium cromoglycate and \*\*\*P < 0.001 versus sodium cromoglycate.*

*release of β-hexosaminidase. Values are presented as means ± SEM \**

*DOI: http://dx.doi.org/10.5772/intechopen.84595*

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84595*

#### **Figure 3.**

*Technological Innovation in the Olive Oil Production Chain*

The kinetic study results connected with the effect of hydroxytyrosol and oleuropein on β-hexosaminidase release from mast cell are described in **Figure 4**. The concanavalin A-induced effect was inhibited by hydroxytyrosol (10 min), oleuropein (10 min), and sodium cromoglycate (10 min). Mast cell activation induced by compound 48/80 was only inhibited by sodium cromoglycate (5, 10, and 20 min). The ionophore A23187-induced effect was inhibited by hydroxytyrosol (10 min), oleuropein (5, 10, and 20 min), and sodium cromoglycate (10 and 20 min). The inhibitory action of the test compounds was not accompanied by changes in cell viability (the trypan blue exclusion test indicated a viability of greater

*Values are presented as means ± SEM +++P < 0.001 versus basal, \**

*secretagogue, and \*\*\*P < 0.001 versus secretagogue.*

*Effect of varying concentrations of hydroxytyrosol and oleuropein on the concanavalin A-, compound 48/80-, and calcium ionophore A23187-induced β-hexosaminidase release from peritoneal mast cells. Mast cells were preincubated with increasing concentrations of hydroxytyrosol or oleuropein for 10 min and then stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187, for 10 min at 37°C. β-Hexosaminidase release was measured by colorimetric reaction with the chromogenic substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide. Results are expressed as percentage release of β-hexosaminidase.* 

*P < 0.05 versus secretagogue, \*\*P < 0.01 versus* 

than 80%), except for incubation times higher than 20 min (data not shown).

**56**

**Figure 2.**

*Effect of hydroxytyrosol and oleuropein on β-hexosaminidase release from rat peritoneal mast cells, compared with a reference compound, the mast cell stabilizer sodium cromoglycate. Purified mast cells were preincubated with increasing concentrations of hydroxytyrosol, oleuropein, or sodium cromoglycate for 10 min and stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187 for another 10 min at 37°C. β-Hexosaminidase release was measured by colorimetric reaction with the chromogenic substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide. Results are expressed as percentage release of β-hexosaminidase. Values are presented as means ± SEM \* P < 0.05 versus sodium cromoglycate and \*\*\*P < 0.001 versus sodium cromoglycate.*

#### **Figure 4.**

*Kinetic study related to the effect of hydroxytyrosol or oleuropein preincubation on mast cell β-hexosaminidase release, induced by concanavalin A, compound 48/80, or calcium ionophore A23187. Peritoneal mast cells were preincubated for increasing times with 100 μM hydroxytyrosol or oleuropein and then stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187 for 10 min at 37°C. β-Hexosaminidase release was measured by colorimetric reaction with the chromogenic substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide. Results are expressed as percentage release of β-hexosaminidase. Values are presented as means ± SEM +++P < 0.001 versus basal, \* P < 0.05 versus secretagogue, \*\*P < 0.01 versus secretagogue, and \*\*\*P < 0.001 versus secretagogue.*

As a matter of interest, the main findings of this study evidenced that hydroxytyrosol and oleuropein, at non-cytotoxic concentrations, inhibit β-hexosaminidase release from peritoneal mast cells stimulated by different triggers, acting then as mast cell stabilizers.

**59**

**Table 1.**

as Ca2+, Mg2+, and double H+

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil*

Mast cell activation is inhibited by several dietary polyphenols, as shown in other in vitro studies [11, 15]. A well-studied immunological effect of polyphenols such as quercetin is their inhibitory action on degranulation, particularly histamine release from mast cells [11, 15, 22]. Quercetin is able to regulate the entry of calcium into mast cells; also the role of apple and grape quercetin and polyphenols in inhibiting mast cell degranulation has been thoroughly documented using in vitro systems such as the RBL-2H3 assay [11, 15]. Epigallocatechin gallate was found to be the active ingredient in green tea extracts for protection against cutaneous inflammation. This compound may also inhibit mast cell histamine release stimulated with both a calcium ionophore and an IgE-antigen complex [23]. Kaempferol, myricetin, phloretin, and luteolin also proved to be effective inhibitors of histamine release [11, 15]. However, hydroxytyrosol and oleuropein were never studied on mast cell mediator release in response to either immune or nonimmune triggers. In addition, we have also proven that the inhibitory effects by hydroxytyrosol were stronger than those of oleuropein or the reference compound sodium cromoglycate when mast cells were activated by concanavalin A. On the contrary, oleuropein resulted to be a more efficient inhibitor of mast cell degranulation than hydroxytyrosol or sodium cromoglycate when cells were challenged with the calcium ionophore A23187. **Table 1** presents EC50 values for each compound. These results strongly suggest that the inhibitory activity might be defined by the nature of the stimulus for β-hexosaminidase release and the chemical structure of both polyphenols. Each of the secretagogues takes a different mast cell activation pathway, and these pathways may be differentially sensitive to the action of hydroxytyrosol or oleuropein. Mast cells may be activated via several different mechanisms, among which is the classical pathway known as immunological or IgE-mediated mast cell activation, triggered by the cross-linking of FcεRI receptors. Mast cell activation may also be completed in an IgE-independent manner using commercially available activators, such as basic secretagogues and calcium ionophores. Concanavalin A is a glucose-/mannose-specific lectin that activates mast cells by a mechanism similar to the antigen-antibody reaction. This lectin causes FcεRI receptors to cluster on cell membranes. In turn, this triggers a series of intracellular events leading to the secretion of mast cell mediators by exocytosis. Phosphatidylserine markedly enhances the release of mediators from rat mast cells by concanavalin A [24–26]. The synthetic compound 48/80, a basic secretagogue, activates heterotrimeric G proteins by enhancing the dissociation of GDP from G*α* subunits, thus accelerating the event considered as a rate-limiting step in conventional G protein activation by receptors coupled to heterotrimeric G proteins [27]. Calcium ionophore A23187, a mobile carrier of divalent cations such

, may reduce the level of calcium stored in mitochon-

**Hydroxytyrosol Oleuropein Sodium cromoglycate**

dria or increase the inflow from the extracellular medium, resulting in a cytosolic calcium increase, which may induce mast cell exocytosis and preformed mediator release. Calcium release from internal stores has been shown to be related to some second messengers, including phospholipase C, phospholipase D, inositol

Concanavalin A 7.58 ± 4.9 67 ± 5.1 95 ± 8.9 Compound 48/80 61 ± 5.0 59 ± 5.0 97 ± 9.1 Calcium ionophore A23187 64 ± 5.0 22 ± 1.8 54 ± 5.1

*EC50 (μM) ± SEM values for inhibitory activity of hydroxytyrosol, oleuropein, and sodium cromoglycate on concanavalin A-, compound 48/80-, and calcium ionophore A23187-induced mast cell activation.*

*DOI: http://dx.doi.org/10.5772/intechopen.84595*

#### *Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84595*

Mast cell activation is inhibited by several dietary polyphenols, as shown in other in vitro studies [11, 15]. A well-studied immunological effect of polyphenols such as quercetin is their inhibitory action on degranulation, particularly histamine release from mast cells [11, 15, 22]. Quercetin is able to regulate the entry of calcium into mast cells; also the role of apple and grape quercetin and polyphenols in inhibiting mast cell degranulation has been thoroughly documented using in vitro systems such as the RBL-2H3 assay [11, 15]. Epigallocatechin gallate was found to be the active ingredient in green tea extracts for protection against cutaneous inflammation. This compound may also inhibit mast cell histamine release stimulated with both a calcium ionophore and an IgE-antigen complex [23]. Kaempferol, myricetin, phloretin, and luteolin also proved to be effective inhibitors of histamine release [11, 15]. However, hydroxytyrosol and oleuropein were never studied on mast cell mediator release in response to either immune or nonimmune triggers.

In addition, we have also proven that the inhibitory effects by hydroxytyrosol were stronger than those of oleuropein or the reference compound sodium cromoglycate when mast cells were activated by concanavalin A. On the contrary, oleuropein resulted to be a more efficient inhibitor of mast cell degranulation than hydroxytyrosol or sodium cromoglycate when cells were challenged with the calcium ionophore A23187. **Table 1** presents EC50 values for each compound. These results strongly suggest that the inhibitory activity might be defined by the nature of the stimulus for β-hexosaminidase release and the chemical structure of both polyphenols. Each of the secretagogues takes a different mast cell activation pathway, and these pathways may be differentially sensitive to the action of hydroxytyrosol or oleuropein. Mast cells may be activated via several different mechanisms, among which is the classical pathway known as immunological or IgE-mediated mast cell activation, triggered by the cross-linking of FcεRI receptors. Mast cell activation may also be completed in an IgE-independent manner using commercially available activators, such as basic secretagogues and calcium ionophores. Concanavalin A is a glucose-/mannose-specific lectin that activates mast cells by a mechanism similar to the antigen-antibody reaction. This lectin causes FcεRI receptors to cluster on cell membranes. In turn, this triggers a series of intracellular events leading to the secretion of mast cell mediators by exocytosis. Phosphatidylserine markedly enhances the release of mediators from rat mast cells by concanavalin A [24–26]. The synthetic compound 48/80, a basic secretagogue, activates heterotrimeric G proteins by enhancing the dissociation of GDP from G*α* subunits, thus accelerating the event considered as a rate-limiting step in conventional G protein activation by receptors coupled to heterotrimeric G proteins [27]. Calcium ionophore A23187, a mobile carrier of divalent cations such as Ca2+, Mg2+, and double H+ , may reduce the level of calcium stored in mitochondria or increase the inflow from the extracellular medium, resulting in a cytosolic calcium increase, which may induce mast cell exocytosis and preformed mediator release. Calcium release from internal stores has been shown to be related to some second messengers, including phospholipase C, phospholipase D, inositol


#### **Table 1.**

*EC50 (μM) ± SEM values for inhibitory activity of hydroxytyrosol, oleuropein, and sodium cromoglycate on concanavalin A-, compound 48/80-, and calcium ionophore A23187-induced mast cell activation.*

*Technological Innovation in the Olive Oil Production Chain*

**58**

**Figure 4.**

mast cell stabilizers.

*Values are presented as means ± SEM +++P < 0.001 versus basal, \**

*secretagogue, and \*\*\*P < 0.001 versus secretagogue.*

As a matter of interest, the main findings of this study evidenced that hydroxytyrosol and oleuropein, at non-cytotoxic concentrations, inhibit β-hexosaminidase release from peritoneal mast cells stimulated by different triggers, acting then as

*P < 0.05 versus secretagogue, \*\*P < 0.01 versus* 

*Kinetic study related to the effect of hydroxytyrosol or oleuropein preincubation on mast cell β-hexosaminidase release, induced by concanavalin A, compound 48/80, or calcium ionophore A23187. Peritoneal mast cells were preincubated for increasing times with 100 μM hydroxytyrosol or oleuropein and then stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187 for 10 min at 37°C. β-Hexosaminidase release was measured by colorimetric reaction with the chromogenic substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide. Results are expressed as percentage release of β-hexosaminidase.* 

1,4,5-triphosphate, and diacylglycerol. Degranulation dependent on the influx of extracellular calcium may be associated to the members of the SNARE (soluble NSF attachment protein receptor) family, such as synaptosome-associated protein of 23 kDa (SNAP-23), syntaxin, synaptotagmin, and molecules of the vesicleassociated membrane protein (VAMP) family which regulate the granule-to-granule or granule-to-plasma membrane fusion process [28]. Sodium cromoglycate is a compound commonly used in the treatment of allergic diseases. The effect of sodium cromoglycate is due to its ability to stabilize the mast cell membrane and to prevent the release of histamine and inflammatory mediators [29]. Mechanisms of the action of sodium cromoglycate include blocking of the influx of calcium into mast cells, inhibition of phosphodiesterase, and regulation of phosphorylation of mast cell proteins [30].

The findings of this study suggest that hydroxytyrosol and oleuropein act at different molecular sites. It seems that the hydroxytyrosol inhibitory mechanism may be related, at least partially, to inhibition of the cross-linking of high-affinity receptors for IgE (FcεRI) or to a probable interaction of the polyphenol with concanavalin A. Reports show that polyphenols form soluble and insoluble complexes with proteins and may cause them to become hypoallergenic by either modifying the structure of the allergenic protein or making it less bioavailable [15]. Moreover, two mechanisms are proposed for the mast cell inhibitory action of quercetin and other polyphenols: one where polyphenols impact allergen-IgE complex formation and another one where the polyphenols impact on the complex binding to their receptor (FcεRI) on mast cells [15].

Our results also suggest that oleuropein seems to block signaling pathways downstream of cytosolic calcium increase. However, further research is needed in order to explain the exact molecular mechanisms of these actions.

Despite the strong biochemical evidence, we considered a morphological evaluation necessary to reinforce the validity of our initial findings. Thus, a second set of experiments was designed to analyze the effect of the hydroxytyrosol and oleuropein on mast cell morphology by light and electron microscopy.

Peritoneal mast cells were easily identified by the presence of a cytoplasm dominated by distinctive secretory granules which stain metachromatically (**Figure 5A–J**). A representative mast cell from the basal group is shown in **Figure 5A**. Tightly packed secretory granules dominate the cytoplasm. Characteristic mast cells stimulated with concanavalin A, compound 48/80, and calcium ionophore A23187 are shown in **Figure 5B–D**, respectively. Cell surface disruption, typical of degranulating mast cells, may be observed. **Figure 5E**–**G** show mast cells treated with 100 μM hydroxytyrosol, for 10 min, prior to the challenge with concanavalin A, compound 48/80, and calcium ionophore A23187, respectively. **Figure 5H**–**J** show mast cells treated with 100 μM oleuropein, for 10 min, prior to the challenge with concanavalin A, compound 48/80, and calcium ionophore A23187, respectively. The morphology of the cells treated with the polyphenols shows a lower degree of degranulation than that of secretagogue samples.

**Figure 6A–J** shows mast cells observed under transmission electron microscope. **Figure 6A** shows a characteristic view of the basal mast cell population. These cells are characterized by a non-segmented, irregular nucleus with only moderate nuclear chromatin condensation, narrow surface folds, and numerous secretory granules regularly distributed throughout the cell cytoplasm. Almost all granules display either round or oval profiles and appear homogenously dense. Representative mast cells from the concanavalin A-, compound 48/80-, and calcium ionophore A23187-treated group are shown in **Figure 6B**–**D**, respectively.

**61**

**Figure 5.**

secretagogue samples.

*secretagogue samples. 600×.*

those obtained by light and electron microscopy.

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil*

These cells display obvious morphological changes and show evidence of increased granule release by exocytosis compared to basal cells. Mast cells exhibit a cytoplasm

*Light microscopic photographs of peritoneal mast cells (toluidine blue stain). Mast cells were preincubated with 100 μM hydroxytyrosol or oleuropein for 10 min and then stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187, for 10 min at 37°C. After incubations, cells were fixed and stained for light microscopy. (A) Basal. The cytoplasm is dominated by closely packed secretory granules. (B–D) Concanavalin A (ConA), compound 48/80 (48/80), and calcium ionophore A23187 (A23187), respectively. Degranulating cells may be seen. (E–G) hydroxytyrosol + ConA, hydroxytyrosol + 48/80, and hydroxytyrosol + A23187, respectively. (H–J) Oleuropein + ConA, oleuropein + 48/80, and oleuropein + A23187, respectively. The morphology of the polyphenol-treated cells shows a lower degree of degranulation than that of* 

Our biochemical results about β-hexosaminidase release are consistent with

with irregular secretory granules showing various degrees of electron densities. Perigranular dilated and electron-lucent spaces surround some granules. Some of these spaces look fused, forming multiple cavities and intracytoplasmic channels. **Figure 6E**–**G** show mast cells treated with 100 μM hydroxytyrosol, for 10 min, prior to the challenge with concanavalin A, compound 48/80, and calcium ionophore A23187, respectively. **Figure 6H**–**J** show mast cells treated with 100 μM oleuropein, for 10 min, prior to the challenge with concanavalin A, compound 48/80, and calcium ionophore A23187, respectively. The morphology of the polyphenol-treated cells shows a lower degree of degranulation than that of

*DOI: http://dx.doi.org/10.5772/intechopen.84595*

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84595*

#### **Figure 5.**

*Technological Innovation in the Olive Oil Production Chain*

tion of mast cell proteins [30].

receptor (FcεRI) on mast cells [15].

1,4,5-triphosphate, and diacylglycerol. Degranulation dependent on the influx of extracellular calcium may be associated to the members of the SNARE (soluble NSF attachment protein receptor) family, such as synaptosome-associated protein of 23 kDa (SNAP-23), syntaxin, synaptotagmin, and molecules of the vesicleassociated membrane protein (VAMP) family which regulate the granule-to-granule or granule-to-plasma membrane fusion process [28]. Sodium cromoglycate is a compound commonly used in the treatment of allergic diseases. The effect of sodium cromoglycate is due to its ability to stabilize the mast cell membrane and to prevent the release of histamine and inflammatory mediators [29]. Mechanisms of the action of sodium cromoglycate include blocking of the influx of calcium into mast cells, inhibition of phosphodiesterase, and regulation of phosphoryla-

The findings of this study suggest that hydroxytyrosol and oleuropein act at different molecular sites. It seems that the hydroxytyrosol inhibitory mechanism may be related, at least partially, to inhibition of the cross-linking of high-affinity receptors for IgE (FcεRI) or to a probable interaction of the polyphenol with concanavalin A. Reports show that polyphenols form soluble and insoluble complexes with proteins and may cause them to become hypoallergenic by either modifying the structure of the allergenic protein or making it less bioavailable [15]. Moreover, two mechanisms are proposed for the mast cell inhibitory action of quercetin and other polyphenols: one where polyphenols impact allergen-IgE complex formation and another one where the polyphenols impact on the complex binding to their

Our results also suggest that oleuropein seems to block signaling pathways downstream of cytosolic calcium increase. However, further research is needed in

Despite the strong biochemical evidence, we considered a morphological evaluation necessary to reinforce the validity of our initial findings. Thus, a second set of experiments was designed to analyze the effect of the hydroxytyrosol and oleuro-

Peritoneal mast cells were easily identified by the presence of a cytoplasm dominated by distinctive secretory granules which stain metachromatically (**Figure 5A–J**). A representative mast cell from the basal group is shown in **Figure 5A**. Tightly packed secretory granules dominate the cytoplasm.

Characteristic mast cells stimulated with concanavalin A, compound 48/80, and calcium ionophore A23187 are shown in **Figure 5B–D**, respectively. Cell surface disruption, typical of degranulating mast cells, may be observed. **Figure 5E**–**G** show mast cells treated with 100 μM hydroxytyrosol, for 10 min, prior to the challenge with concanavalin A, compound 48/80, and calcium ionophore A23187, respectively. **Figure 5H**–**J** show mast cells treated with 100 μM oleuropein, for 10 min, prior to the challenge with concanavalin A, compound 48/80, and calcium ionophore A23187, respectively. The morphology of the cells treated with the polyphenols shows a lower degree of degranulation than that of secretagogue

**Figure 6A–J** shows mast cells observed under transmission electron microscope. **Figure 6A** shows a characteristic view of the basal mast cell population. These cells are characterized by a non-segmented, irregular nucleus with only moderate nuclear chromatin condensation, narrow surface folds, and numerous secretory granules regularly distributed throughout the cell cytoplasm. Almost all granules display either round or oval profiles and appear homogenously dense. Representative mast cells from the concanavalin A-, compound 48/80-, and calcium ionophore A23187-treated group are shown in **Figure 6B**–**D**, respectively.

order to explain the exact molecular mechanisms of these actions.

pein on mast cell morphology by light and electron microscopy.

**60**

samples.

*Light microscopic photographs of peritoneal mast cells (toluidine blue stain). Mast cells were preincubated with 100 μM hydroxytyrosol or oleuropein for 10 min and then stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187, for 10 min at 37°C. After incubations, cells were fixed and stained for light microscopy. (A) Basal. The cytoplasm is dominated by closely packed secretory granules. (B–D) Concanavalin A (ConA), compound 48/80 (48/80), and calcium ionophore A23187 (A23187), respectively. Degranulating cells may be seen. (E–G) hydroxytyrosol + ConA, hydroxytyrosol + 48/80, and hydroxytyrosol + A23187, respectively. (H–J) Oleuropein + ConA, oleuropein + 48/80, and oleuropein + A23187, respectively. The morphology of the polyphenol-treated cells shows a lower degree of degranulation than that of secretagogue samples. 600×.*

These cells display obvious morphological changes and show evidence of increased granule release by exocytosis compared to basal cells. Mast cells exhibit a cytoplasm with irregular secretory granules showing various degrees of electron densities. Perigranular dilated and electron-lucent spaces surround some granules. Some of these spaces look fused, forming multiple cavities and intracytoplasmic channels. **Figure 6E**–**G** show mast cells treated with 100 μM hydroxytyrosol, for 10 min, prior to the challenge with concanavalin A, compound 48/80, and calcium ionophore A23187, respectively. **Figure 6H**–**J** show mast cells treated with 100 μM oleuropein, for 10 min, prior to the challenge with concanavalin A, compound 48/80, and calcium ionophore A23187, respectively. The morphology of the polyphenol-treated cells shows a lower degree of degranulation than that of secretagogue samples.

Our biochemical results about β-hexosaminidase release are consistent with those obtained by light and electron microscopy.

#### **Figure 6.**

*Transmission electron micrographs showing peritoneal mast cells. Mast cells were preincubated with 100 μM hydroxytyrosol or oleuropein for 10 min and then stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187, for 10 min at 37°C. After incubations, cells were processed for electron microscopy. (A) Basal. A non-segmented nucleus (N), narrow surface folds (red arrows), and numerous secretory granules (G) distributed throughout the cell cytoplasm are observed. (B–D) Concanavalin A (ConA), compound 48/80 (48/80), and calcium ionophore A23187 (A23187), respectively. Granules surrounded by perigranular dilated and electron-lucent spaces are evident. Some granules are seen out of mast cell cytoplasm, and others appear fused, forming cavities and intracytoplasmic channels (asterisk). (E–G) Hydroxytyrosol + ConA, hydroxytyrosol + 48/80, and hydroxytyrosol + A23187, respectively. (H–J) Oleuropein + ConA, oleuropein + 48/80, and oleuropein + A23187, respectively. Polyphenol-treated cells show minimal degranulation. 5000×.*

#### **4. Conclusions**

In conclusion, our present findings reveal for the first time that hydroxytyrosol and oleuropein, the major phenolic compounds in olive, inhibit mast cell degranulation triggered by both immune and nonimmune pathways. Our discoveries also suggest that olive polyphenols, especially hydroxytyrosol and oleuropein, may provide insights to develop useful tools to prevent and treat mast cell-mediated disorders. These findings may be of interest to the immunopharmacology industry or could even lead in determining the ideal concentrations of each component in

**63**

provided the original work is properly cited.

\*Address all correspondence to: apenissi@yahoo.com.ar

Aconcagua - Mendoza, Argentina

© 2019 The Author(s). Licensee IntechOpen. 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,

"Dr. Mario H. Burgos" (IHEM-CCT Mendoza-CONICET), Universidad Nacional de

2 Facultad de Ciencias Médicas, Instituto de Investigaciones, Universidad del

1 Facultad de Ciencias Médicas, Instituto de Histología y Embriología

*Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil*

virgin olive oils to be able to label them as healthy oils. However, further tests are needed in order to generate hypoallergenic products via polyphenol treatment.

I thankfully acknowledge Dr. Fabio A. Persia, Dra. María Laura Mariani, Ing. Norberto Domizio, and Ing. Elisa Bocanegra for their technical support. Funding for this work was provided by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (PIP-CONICET 5128) and Secretaría de Ciencia, Técnica y Postgrado de la Universidad Nacional de Cuyo (Research Proyects SeCTyP UNCuyo

06/J268-06/J395 and Research Program CS-453-2010), Argentina.

The author declares no conflict of interest.

*DOI: http://dx.doi.org/10.5772/intechopen.84595*

**Acknowledgements**

**Conflict of interest**

**Abbreviations**

**Author details**

Cuyo, Argentina

Alicia Beatriz Penissi1,2

ConA concanavalin A 48/80 compound 48/80

IgE immunoglobulin E

A23187 calcium ionophore A23187 FcεRI high-affinity receptor for IgE *Regulation of Immune and Nonimmune Mast Cell Activation by Phenols from Olive Oil DOI: http://dx.doi.org/10.5772/intechopen.84595*

virgin olive oils to be able to label them as healthy oils. However, further tests are needed in order to generate hypoallergenic products via polyphenol treatment.

### **Acknowledgements**

*Technological Innovation in the Olive Oil Production Chain*

**62**

**4. Conclusions**

*minimal degranulation. 5000×.*

**Figure 6.**

In conclusion, our present findings reveal for the first time that hydroxytyrosol and oleuropein, the major phenolic compounds in olive, inhibit mast cell degranulation triggered by both immune and nonimmune pathways. Our discoveries also suggest that olive polyphenols, especially hydroxytyrosol and oleuropein, may provide insights to develop useful tools to prevent and treat mast cell-mediated disorders. These findings may be of interest to the immunopharmacology industry or could even lead in determining the ideal concentrations of each component in

*Transmission electron micrographs showing peritoneal mast cells. Mast cells were preincubated with 100 μM hydroxytyrosol or oleuropein for 10 min and then stimulated with 200 μg/ml concanavalin A, 10 μg/ml compound 48/80, or 50 μg/ml calcium ionophore A23187, for 10 min at 37°C. After incubations, cells were processed for electron microscopy. (A) Basal. A non-segmented nucleus (N), narrow surface folds (red arrows), and numerous secretory granules (G) distributed throughout the cell cytoplasm are observed. (B–D) Concanavalin A (ConA), compound 48/80 (48/80), and calcium ionophore A23187 (A23187), respectively. Granules surrounded by perigranular dilated and electron-lucent spaces are evident. Some granules are seen out of mast cell cytoplasm, and others appear fused, forming cavities and intracytoplasmic channels (asterisk). (E–G) Hydroxytyrosol + ConA, hydroxytyrosol + 48/80, and hydroxytyrosol + A23187, respectively. (H–J) Oleuropein + ConA, oleuropein + 48/80, and oleuropein + A23187, respectively. Polyphenol-treated cells show* 

I thankfully acknowledge Dr. Fabio A. Persia, Dra. María Laura Mariani, Ing. Norberto Domizio, and Ing. Elisa Bocanegra for their technical support. Funding for this work was provided by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (PIP-CONICET 5128) and Secretaría de Ciencia, Técnica y Postgrado de la Universidad Nacional de Cuyo (Research Proyects SeCTyP UNCuyo 06/J268-06/J395 and Research Program CS-453-2010), Argentina.

### **Conflict of interest**

The author declares no conflict of interest.

### **Abbreviations**


### **Author details**

Alicia Beatriz Penissi1,2

1 Facultad de Ciencias Médicas, Instituto de Histología y Embriología "Dr. Mario H. Burgos" (IHEM-CCT Mendoza-CONICET), Universidad Nacional de Cuyo, Argentina

2 Facultad de Ciencias Médicas, Instituto de Investigaciones, Universidad del Aconcagua - Mendoza, Argentina

\*Address all correspondence to: apenissi@yahoo.com.ar

© 2019 The Author(s). Licensee IntechOpen. 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.

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## *Edited by Innocenzo Muzzalupo*

Technological innovation has undergone unprecedented development; this evolution can offer extraordinary opportunities for product qualification, which today has not been adequately exploited due to a lack of vision. It took a disaster such as "mad cow" disease to accelerate the traceability plan; today, 36% of the analyzed agri-food companies, thanks to digital solutions, achieved a reduction in the times and costs connected with harvest processes, data management, and transmission. Digital solutions permit interventions aimed at food safety along the food chain, thus avoiding financial damage. But they can also be used to combat counterfeiting to protect the Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) systems for greater information for the consumer.

Published in London, UK © 2019 IntechOpen © MarcBruxelle / iStock

Technological Innovation in the Olive Oil Production Chain

Technological Innovation

in the Olive Oil Production

Chain

*Edited by Innocenzo Muzzalupo*