Applications of Antioxidants from Natural Origin

### **Chapter 11**

## Elaboration of a Purple Corn Drink with Maximum Retention of Anthocyanins

*Genaro Christian Pesantes Arriola, Víctor Alexis Higinio Rubio, Carlos Enrique Chinchay Barragán, Enrique Gustavo García Talledo, César Ángel Durand Gonzales and Wilmer Huamani Palomino*

### **Abstract**

In the present work, the anthocyanin extraction process was characterized during the elaboration of a purple corn drink, using the response surface analysis method with temperature intervals between 47.57 and 132.43°C, and times from 11, 36 at 138.64 minutes. In addition, with the stationary point technique, the maximum retention of anthocyanin (33.99 mg/g) was determined at a temperature of 98.39°C at a time of 105.89 minutes of extraction. Since this time is too long and to reduce production costs, without resorting to considerable losses of anthocyanins, canonical analysis was used, redefining the optimal extraction parameters at a temperature of 100°C for 60 minutes with a reduction of the anthocyanin content of 2.49% (33.14 mg/g) concerning the maximum, a value that is within the optimum area of performance of the process. With the extract obtained under optimal conditions, a drink was prepared and, using the differential pH method and Student's t-test (p = 0.05), its anthocyanin content was quantified and compared with that of a commercial drink with typical characteristics. Similar, observing that the elaborated drink presents higher contents, whose difference varies within the range of 2.79 and 4.72 mg/mL. Finally, using a satisfaction test with a nine-point hedonic scale, it was determined that the beverage was "very well liked" by a semi-trained sensory panel.

**Keywords:** purple corn, anthocyanin, optimization, response surface, canonical analysis

### **1. Introduction**

The cultivation of purple corn is of growing economic importance in Peru, mainly for producers in the mountains who have few possibilities of generating economic income from the sale of agricultural products that they produce on their plots. In recent years, the consumption of purple corn has intensified, in the country and abroad, because the purple pigment that this type of corn has (anthocyanins) prevents diseases such as colon cancer, and reduces obesity and diabetes, among other

diseases; likewise, it is a natural colorant for the industry. Among the anthocyanins of purple corn, cyanidin-3-glucoside is found in greater quantity, constituting a powerful natural antioxidant [1].

Purple corn is a vegetable resource native to Andean Peru and to which an interesting biological activity as an antioxidant is attributed, due to the type of bioactive compounds it contains. Due to the increased demand for this plant resource and its derivatives in the national and international market, efforts have been made in the country to expand the cultivation areas and introduce improved varieties of purple corn that can be adapted to these new cultivation areas. and that they improve the production and commercialization of this resource [2].

Antioxidants are responsible for stabilizing free radicals by transferring electrons and hydrogen atoms, and they also have the ability to inhibit oxidative degradation such as lipoperoxidation. For this reason, they play an important role in the prevention of various degenerative diseases such as cancer, diabetes, obesity, high blood pressure [3–5].

Within this group of antioxidants are anthocyanins, natural dyes that belong to the group of flavonoids, because they have a characteristic structure of C6-C3-C6 (**Figure 1**). Its basic structure is the flavylium group (2-phenylbenzopyrylium). These pigments are responsible for giving the pink, red, blue, mauve and violet color of flowers, fruits and vegetables, they are polar compounds, which allows them to be soluble in ethanol and water. Anthocyanins are glycosides that have a sugar in position 3 linked by the ß-glycosidic bond that, when broken, forms the aglycone, known as anthocyanidin, the most common being: pelargonidine, cyanidin, delphinidin, peonidin, malvidin and petunidin [6–10]. On the other hand, pelargonidin-3-glucoside, peonidin-3-glucoside, cyanidin-3-glucoside and the acylated forms of each of them were found in purple corn from the Andean region of Peru [11].

In the Peruvian market, two commercial beverages based on purple corn are offered; however, the industry has prioritized the sanitary quality of the product before the beneficial effect on health provided by anthocyanins due to their antioxidant capacity and the phenolic compounds present in their chemical structure [12].

The extraction of anthocyanins depends on the temperature and extraction time, being favored by the 20% ethanolic medium and pH between 1 and 4 [13]. While, used solutions as solvents hydroalcoholic (ethanol or methanol) acidified with acetic acid, concluding that the acid methanol is more effective for extraction, although its toxicity prevents it from being used when the extracted substances will be used for human consumption [8]. On the other hand, recommend using water, methanol, or

**Figure 1.** *Basic structure of anthocyanins.*

*Elaboration of a Purple Corn Drink with Maximum Retention of Anthocyanins DOI: http://dx.doi.org/10.5772/intechopen.109142*

ethanol acidified with hydrochloric acid at a pH between 4 and 5 at a temperature between 70 and 100°C as solvents, to avoid pigment degradation [14]. The extraction using an ethanolic solution as solvent acidified with hydrochloric acid by immersion for 15 minutes in an ultrasound bath; finally, the appropriate extraction conditions take place in an aqueous medium with a contact time of 120 minutes and a limit temperature of 50°C [15, 16]. All the aforementioned extraction techniques were carried out under laboratory conditions using inorganic acids as acidifying agents and alcoholic solutions as solvents; conditions that cannot be reproduced on an industrial scale due to the high toxicity of said compounds.

In this sense, the present work aims to determine the optimal physical parameters of time and temperature for the extraction of anthocyanins from corn earns to fortify a commercial purple corn drink.

### **2. Materials and method**

A Central Composite Rotational Design (DCCR) was carried out with 4 factorial points, 4 axial points, and 5 repetitions in the central points, having a total of 13 treatments. The independent variables or "Factors" of the design were factor A (X1): Temperature, with a low level at 60°C, a high level at 120°C, the central point at 90°C and axial points at 47.57°C and 132.43°C); and factor B (X2): Time, with a low level at 30 min, high level at 120 min, a central point at 75 min and its axial points at 11.36 min and 138.64 min as shown in **Table 1**.

The purple corn cobs used came from the Majes district of the department of Arequipa, Peru, located between 200 and 800 meters above sea level, with an average annual temperature between 14 and 32°C.


### **Table 1.**

*Design of Experiments Matrix.*

The present research work was carried out in the Food Technology laboratories of the Chucuito Pilot Plant, of the Faculty of Fisheries and Food Engineering of the National University of Callao, and Bromatology of the Faculty of Pharmacy and Biochemistry of the University Inca Garcilaso de la Vega.

### **2.1 Obtaining the anthocyanin extract**

The ears were selected to discard those that have symptoms of deterioration or perceptible damage, then they were shelled to remain only with the shelled ears in the next stage of the process, later they were rolled in a circular shape with an approximate thickness of 3 mm and dried. in an oven at a temperature of 65°C for 2 hours, until reaching an approximate humidity of 8%.

The shelled and dehydrated cobs were ground with a manual mortar and diluted in a ratio of 2.5 g. in 100 mL of extraction solution (treated water adjusted to pH 2 with citric acid), applying the times and temperatures as indicated in **Table 1**, to develop the experimental model.

Subsequently, the temperature of the extract was brought below 30°C to filter it through a 1 mm diameter mesh.

### **2.2 Anthocyanin quantification**

The quantification of the content of anthocyanins was expressed as mg of cyanidin 3-glucoside/g of shelled cob, was used, where an aliquot of 0.3 ml of extract was diluted in 2.7 mL of buffer solution of potassium chloride (pH 1) and sodium acetate (pH 4.5), separately, leaving it to stand for 20 minutes [17]. Finally proceeding to read their respective absorbances as indicated in the following expression:

Total anthocyanins mg / L A x PM x FD x / n x l ( ) = 1000 ( )

Where:

TA = cyanidin 3-glucoside content; A = (A510 – A700) pH 1 - (A510 – A700) Ph 4.5; MW = molecular weight; DF = dilution factor; 1000 = conversion factor from grams to milligrams; ϵ = molar extinction factor (26900) for cyanidin 3-glucoside; l = cell length.

Subsequently, the data were analyzed to obtain the response surface, identifying the point of maximum performance through the stationary point methodology,

### **2.3 Response surface methodology**

The response surface methodology was applied to the response variable using the commercial statistical software Design Expert Version 5.0 (Stat-Ease, Minneapolis, USA). Second-order polynomials were fitted to the data to obtain regression equations for the response variables analyzed. The graph of the response surfaces, the variance analysis, and the determination coefficients (R2 ) was generated with the same software. Then, the canonical analysis of the data was performed to adjust the optimal point of the process.

### **2.4 Preparation of the drink and comparison of the anthocyanin content with that of a commercial drink**

For this, the purple corn drink was prepared, for which the purple corn extract obtained using the optimal parameters determined in the previous point was diluted

### *Elaboration of a Purple Corn Drink with Maximum Retention of Anthocyanins DOI: http://dx.doi.org/10.5772/intechopen.109142*

with treated water at a temperature of 78°C in a volumetric ratio of 2 of treated water and 1 of extract. Subsequently, the drink was standardized until it reached a pH of 3, an acidity content of 0.2% citric acid, and 13°Brix. The mixture was pasteurized at 72°C for 10 minutes and then bottled in glass bottles with a capacity of 250 mL, amber color, and screw cap.

Finally, the anthocyanin content was determined in triplicate, in the drink made under the aforementioned conditions, and in a commercial purple corn drink. The mean values were compared using the t-Student test (p = 0.05).

### **2.5 Satisfaction degree test**

It was carried out with 50 students of the eighth cycle of the Professional School of Food Engineering of the National University of Callao, who had completed the Sensory Analysis of Food subject, following the methodology [18].

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

### **3.1 Anthocyanin quantification and application of the response surface methodology**

The yield of anthocyanins in the extracts obtained with the different treatments tested is shown in **Table 2**.

To determine if the anthocyanin extraction levels were within the region of maximum yield, the fit was made to a second-order polynomial model [19] (**Table 3**).

Based on the fact that the Fc of the lack of fit is much lower than the Ft, for a significance level of 0.5%, it can be concluded with a statistical significance level


### **Table 2.**

*Design of Experiments Matrix.*

*Recent Developments in Antioxidants from Natural Sources*


### **Table 3.**

*Analysis of variance table for a second order model.*

of 99.5% that the second-order model is an adequate approximation to the actual behavior of the experiment. Therefore, the second-order equation was established to predict anthocyanin yields when the food matrix is subjected to the extraction factors that are the object of this study. The analysis of the yields of anthocyanins obtained with the Desing Expert Version 5.0 software, allowed us to calculate the values of the regression coefficients that are presented in **Table 4**.

The equation obtained to predict the content of anthocyanins was:

$$\mathbf{Y} = -\mathbf{g}.\mathbf{o}\mathbf{y} + \mathbf{o}.64\mathbf{X}1 + \mathbf{o}.\mathbf{n}\mathbf{1}\mathbf{X}2 - \mathbf{o}.\mathbf{o}\mathbf{o}\mathbf{o}\mathbf{2}4\mathbf{X}1\mathbf{X}2 - \mathbf{o}.\mathbf{o}\mathbf{o}\mathbf{3}\mathbf{X}12 - \mathbf{o}.\mathbf{8}\mathbf{2}\mathbf{X}22$$

Where: Y = yield of anthocyanins in mg of cyanidin 3-glucoside/g of shelled cob; X1 = temperature in °C; X2 = time in minutes. This corroborates that the established mathematical model describes the anthocyanin extraction process very closely to reality under the pre-established experimental conditions and within the study region.


### **Table 4.**

*Values of the regression coefficients obtained in the anthocyanin extraction process.*

*Elaboration of a Purple Corn Drink with Maximum Retention of Anthocyanins DOI: http://dx.doi.org/10.5772/intechopen.109142*

**Figure 2.** *Response surface of the experiment.*

This is important to highlight, since the equation obtained should not be used for extrapolate data outside the study range or for conditions other than those pre-established in the design. With this equation, we proceeded to characterize the response surface shown in **Figure 2**, for this the adjusted data of the experiment were used.

**Figure 2** shows the behavior of the process against the factors of temperature and time, where overexposure to high temperatures and times causes a decrease in | performance by degradation of anthocyanins. The auxiliary graph or contour graph was also made to facilitate the interpretation of the response surface and the region of the maximum performance of the process, which can be seen in green in **Figure 3**.

With the use of the stationary point methodology, it was obtained that the highest yield of anthocyanins (33.99 mg/g) is obtained at a temperature of 98.39°C and 105.89 minutes of extraction, maintaining preset pH values and the shelled cob/ solvent ratio. However, a time of approximately 106 minutes is excessive and represents high power consumption. From the analysis of **Figure 3**, it was deduced that the extraction process has a greater dependence on temperature than on extraction time, so it was decided to reduce the time to the minimum possible without leaving the zone of maximum yield and using parameters of the time and temperature variables that present operational ease in a process at an industrial level. For this reason, the canonical analysis of the results was performed, obtaining an extraction yield of 33.08 mg/g for a temperature of 96.29°C and a time of 59.50 min. These new extraction parameters are very advantageous compared to those with higher yields, however, in an industrial process, they are difficult to maintain constant and even to achieve exactly, so it was decided to explore the neighborhood of the new parameters obtained, in the order to establish as optimal points of the process, those that are also easy to operationalize.

Based on the results in **Table 5**, it was decided to operate the extraction process at a temperature of 100°C for 60 minutes, reaching an anthocyanin yield of 33,144 mg/g. Comparing these parameters with the highest yield initially obtained, it can be seen

### **Figure 3.**

*Contour plot of the response surface of the experiment.*


### **Table 5.**

*Exploration of the optimal point of the extraction process.*

that with a temperature increase of 2°C the extraction time was reduced by 45% with only a 2.49% decrease in the yield of anthocyanins.

### **3.2 Comparison of the anthocyanin content of the beverage made with a commercial beverage**

**Table 6** shows the average values and standard deviation of the anthocyanin content in the samples evaluated. The difference test showed that the elaborated beverage had a significantly higher average anthocyanin content (p = 0.05) than that of the commercial beverage, this difference is found in a range between 2.79 and 4.72 mg/mL.

### **3.3 Satisfaction degree test**

According to the hedonic scale of acceptability, the attribute color and flavor obtained average ratings of 8.28 and 7.64 (**Figure 4**), which is equivalent to saying *Elaboration of a Purple Corn Drink with Maximum Retention of Anthocyanins DOI: http://dx.doi.org/10.5772/intechopen.109142*


### **Table 6.**

*Summary of the comparison of means test.*

### **Figure 4.**

*Degree of satisfaction with the drink.*

that the color was "liked a lot" and the flavor "liked a lot", respectively. The average of the general acceptability was 8.18, that is, "liked a lot", which indicated that the flavor attribute does not significantly affect (p = 0.05) the general acceptability of the beverage made with the optimal parameters.

### **4. Discussions**

Carried out the extraction of anthocyanins from shelled ears of purple corn at different pH, solvents, temperatures, and times, observing that for a process at pH 2, and using water as the solvent, a maximum yield was obtained (33.509 mg/g) at a temperature of 90°C and a time of 240 minutes, yield very similar to that obtained in the present work (33.14 mg/g), using the same pH and solvent, but at a temperature

of 100°C and a time of 60 minutes [13]. That is, by increasing the extraction temperature by 10°C, it is possible to reduce the process by 3 hours, which allows for increasing the production volumes of a purple corn drink and a significant reduction in the cost of producing the product.

On the other hand, it has been shown that the anthocyanin extraction process depends mainly on the temperature, rather than on the extraction time; because in recent works, an anthocyanin content of 22.68 mg/g in an extract obtained from the same raw material (shelled cobs), using water as a solvent, but at a temperature of 50°C with contact times of 120 minutes [16].

An important factor to consider is the type of statistical treatment used in the study of the extraction process only carried out the variance analysis of the different factors considered, which allowed to select the best combination of the different levels of the tested factors; while in the present study the response surface analysis was carried out, the same one that allows characterizing the entire process within the range under study and optimizing it [13, 16].

### **5. Conclusion**

In this study, the response surface analysis was a useful technique to forecast a hight anthocyanin yield in an extraction process of this molecule from earns corn. We obtained the highest anthocyanin yield (33.99 mg/g) at 98.39°C after 105.89 minutes of extraction, maintaining preset pH values and the hulled ears/solvent ratio. In addition, the results showed that the extraction process has a higher dependence on temperature than the extraction time.

The canonical analysis of the anthocyanin retention results in the vicinity of the maximum retention temperature made it possible to select an extraction temperature of 100°C for a period of 60 minutes, with which an anthocyanin yield of 33.144 mg/g (2.49% below optimal yield), but with a 45% reduction in extraction time.

### **Author details**

Genaro Christian Pesantes Arriola1 \*, Víctor Alexis Higinio Rubio1 , Carlos Enrique Chinchay Barragán1 , Enrique Gustavo García Talledo1 , César Ángel Durand Gonzales1 and Wilmer Huamani Palomino2

1 National University of Callao, Callao, Perú

2 Facultad de Ingenieria Pesquera y de Aiimentos, Universidad Nacional del Callao, Lima, Perú

\*Address all correspondence to: gcpesantesa@unac.edu.pe

© 2022 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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[5] Sánchez V, Méndez N. Estrés oxidativo, antioxidantes y enfermedad. Revista de Investigación Médica Sur México. 2013;**20**(3):161-168

[6] Cuevas, E., Antezana, A., y Winterhalter, P. (2008). Análisis y caracterización de Antocianinas en diferentes variedades de Maíz Morado (*Zea Mays* L.) boliviano. Memorias del Encuentro Final Red-Alfa Lagrotech. 79-95.

[7] Guillén, J., Mori, S., y Paucar, L.M. (2014). Características y propiedades funcionales del maíz morado (*Zea mays* L.) var. Subnigroviolaceo. Revista Scientia Agropecuaria; 5, 211-217. DOI: 10.17268/sci.agropecu.2014.04.05

[8] Salinas Y, Rubio D, y Díaz, A. Extracción y uso de pigmentos del grano de maíz (*Zea mays* L.) como colorantes en yogur. Archivos Latinoamericanos de Nutrición. 2005;**55**(3):293-298

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[11] Pedreschi R, y Cisneros, L. Phenolics profiles of andean purple corn (*Zea mays* L.). Food Chemical. 2007;**100**:956-963. DOI: 10.1016/j.foodchem.2005.11.004

[12] Pesantes G, Paucar J, Franco J. Elaboración de una bebida de maíz morado con máxima retención de antocianinas. Revista Alpha Centauri. 2021;**2**(1):52-61. DOI: 10.47422/ac.v2i1.29

[13] Gorriti, A; Quispe, F; Arroyo, J; Córdova, A; Jurado, B; Santiago, I y Taype, E. Extracción de antocianinas de las corontas de *Zea mays* L. "Maíz Morado", Revista de Ciencia e Investigación de la Facultad de Farmacia y Bioquímica de la UNMSM. 2009; 12 (2):64-74. DOI: 10.15381/ci.v12i2.3395

[14] Elías, J. y Gamero, D. Obtención de colorante a partir del maíz morado. [Tesis de pregrado, Universidad Nacional de Ingeniería]. 1988.

[15] Mendoza C. Las antocianinas del maíz: su distribución en la planta y producción. [Tesis de maestría. Colegio de Posgraduados, Campus Montecillo; 2012

[16] Almeida J. Extracción y caracterización del colorante natural del maíz negro

(*Zea mays* L.) y determinación de su actividad. [Tesis de pregrado. Escuela Politécnica Nacional; 2012

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### **Chapter 12**

## Antioxidant Strategies to Improve Female Reproduction

*Tarique Hussain*

### **Abstract**

Animals are only productive once their reproductive cycle is continuously flown. There are several causes of stresses which interrupt animal physiology and make animal less productive. All factors involved in stress could eventually generate reactive oxygen species (ROS). Limited production of these reactive species performs several functions to maintain redox homeostasis. When these reactive oxidative metabolites are overwhelmed, it may generate oxidative stress. Disruption in oxidant/ antioxidant mechanism leads to cause oxidative stress. Naturally, the body system is equipped with an antioxidant defense system. Once this system is broken-down due to the overproduction of ROS, it may have a detrimental effect on lipids, proteins, DNA, and carbohydrates and eventually influence animal fertility and productivity. Antioxidants available in nature are of two types: natural and synthetic. These compounds endowed several properties in the mitigation of various animal stresses, starting from physiology to molecular level. This chapter elucidates oxidative stress, natural and synthetic antioxidants, and particular focus are emphasized that how antioxidant supplementation can help to improve animal fertility and productivity. Moreover, the mechanism by which antioxidants produce fruitful effects will also be highlighted.

**Keywords:** reactive oxygen species, oxidative stress, animal fertility, productivity, antioxidants

### **1. Introduction**

Oxidative stress is the condition in which the overproduction of free radicals is produced and the antioxidant system unable to neutralize them [1]. Limited quality of free radicals is compulsory to maintain physiological function, and accelerated production may lead to damage lipids, DNA, and proteins [2]. The efficacy of oxidative metabolites in female reproduction depends upon the site, amount and exposing levels to oxidant molecules [3].

In livestock, the appearance of the disease causes a reduction in the antioxidant status of the animals [4]. Oxidative stress is observed in several pathological conditions that influence animal health, welfare, and productive performance [5]. Indeed, in some productive phases, animals experience physiological alteration, such as

farrowing and lactation, weaning, high temperature, and different stresses, that may decline antioxidant status [6–8].

Oxidative stress may influence physiological function of various reproductive events which thus involved in problems associated with pregnancy [9]. Antioxidants are chemical compounds is known to suppress autoxidation *via* reduction in free radicals' production using several different mechanisms. They can be categorized as primary antioxidants, chelators, O2 •− quenchers, oxygen scavengers, and antioxidant regenerators [10]. The purpose of this chapter is to exploit the beneficial effect of antioxidant compounds various aspects of female reproduction and also discuss how antioxidant approaches improve animal productive performance and well-being.

### **2. Reactive oxygen species**

Reactive oxygen species (ROS) encompasses of superoxide anion, hydrogen peroxide and hydroxyl radical. Their production is possible due to natural oxygen leakage [11]. Other origin of ROS production are metabolic reactions that are sustainable for life, the exogenous sources include X-rays, ozone, cigarette smoking, air pollutants, certain drugs and pesticides, and industrial chemicals [12]. The endogenous site of free radicals' production may be from mitochondria, xanthine oxidase, peroxisomes, inflammation, phagocytosis, arachidonate pathways, exercise, and ischemia/ reperfusion injury [13]. Interestingly, the reactions of consists of enzymatic and nonenzymatic reactions within the body also produce free radicals. Many enzymatic reactions prevail in the respiratory chain, phagocytosis, prostaglandin synthesis, and cytochrome P-450 system [14], although nonenzymatic reactions are based on oxygen with organic compounds and ionizing reactions also generate free radicals [15].

ROS are comprised of oxygen ions, free radicals and peroxides. High level of ROS or reduce concentration of antioxidants induce oxidative stress that trigger cellular damage of macromolecules utilizing different ways. It has responsible for causing chronic diseases by interacting with molecular signaling pathways which alters gene expression [16]. The interaction between chemicals and signaling molecules are necessary to understand the involvement of ROS role in pathogenesis. Redox interaction with various proteins residues and ROS is the key component of inter-processes. Further reaction yields to produce reactive sulfenic acid and sulfonamide. Oxidation of these molecules leads to cause ultra-structural changes or functional alteration [17].

Pregnancy is a physiological phenomenon in which an ample amount of energy is required to balance the body's condition and combat fetal requirements. Thus, for this purpose more oxygen is required which in turn causes the overproduction of ROS. So, it is very crucial to maintain the balance between oxidative stress and the antioxidant system for perfect functioning of the body [18].

### **3. Antioxidants**

Antioxidants are substances that overcome adverse effect of oxidative damages. They are available as natural and synthetic compounds. Natural antioxidants are derived from natural sources like food, cosmetics, and pharmaceutical industries. While the synthetic one is created artificially through chemical reactions [19].

The antioxidant system consists of enzymatic and non-enzymatic. The first one is also referred as natural antioxidants. They consist of superoxide dismutase (SOD)

### *Antioxidant Strategies to Improve Female Reproduction DOI: http://dx.doi.org/10.5772/intechopen.110639*

catalase and glutathione peroxidase (GSH-Px). Antioxidant enzymes are endowed to protect living cells against oxidant products. The SOD is an enzyme district superoxide anion radical into hydrogen peroxide. Another enzyme called catalase is in charge of catalysing the breakdown of hydrogen peroxide into water and oxygen. GSH-Px employs glutathione as a co-substrate and is composed of selenium. An enzyme found in the cytoplasm, it excludes hydrogen peroxide. However, in comparison with catalase, it has various ranges of substrates comprising lipid peroxides. The prime function of the Glutathione peroxidase is to decontaminate low levels of hydrogen peroxide in the cell.

Non-enzymatic antioxidants include dietary supplements or synthetic antioxidants. The complex nature of the body antioxidant system is impaired by consumption of dietary antioxidant such as vitamins and minerals [20]. Vitamin C, vitamin E, plant polyphenol, carotenoids, and glutathione are non-enzymatic antioxidants, they causes inhibition of free radicals reactions. Antioxidants can be classified as watersoluble or lipid-soluble depending on how potent they are. A water-soluble vitamin called vitamin C is found in cellular fluids such the cytosol and cytoplasmic matrix.

Antioxidants can be divided into small-molecule and large-molecule antioxidants depending on their size. The small one neutralizes ROS through scavenging process. The example includes Vitamin C, vitamin E, carotenoids, and glutathione (GSH). A large size of the molecule antioxidants comprises SOD, CAT, and GPx and albumin that captivate ROS and the attack form essential proteins. The mechanism by which antioxidants are utilized which offer protection against inhibition of free radical formation, scavenging free radicals, involved in repair-damage via free radicals, help to establish an environment that is conducive for the antioxidants to function effectively [21].

### **4. Synthetic antioxidants**

Synthetic antioxidants are phenolic compounds responsible for eliminating free radicals and suppressing chain-reaction. They consist of butylated hydroxyl anisole (BHA), butylated hydroxyltoluene (BHT), propyl gallate (PG), metal chelating agent (EDTA), tertiary butyl hydroquinone (TBHQ ), and nordihydroguaiaretic acid (NDGA) [21].

### **5. Oxidative stress and selenium**

In commercial dairy and beef farming, various stresses influence economic benefit that is linked with declined productive and reproductive performance in cattle. It has been observed that diverse endogenous and exogenous sources of ROS lead to stresses that cause over generation of free radicals and eventually result in oxidative stress [22, 23]. It is well recognized that the consequences of oxidative stress have deleterious effects on immune system reproductive function, animal growth, development, and on general health [24, 25]. Hence, the antioxidant network is responsible for the preservation and maintenance of animal redox status in cells and tissues and is thus responsible for neglecting the harmful effect of stresses. In animals' body, the antioxidant system work either individually or in combination to exert particular function. Considering this mechanism, selenium has its own importance [26, 27]. It is noted that 25 selenoproteins have been identified in animal tissues; most of them are

contributed in the conservation of body redox balance and antioxidant defense [27]. A deep theoretical knowledge of Se uptake toward the body/cells is required and its particular utilization for the balance of animal health. It is well-recognized in some animal species that the bioavailability of the Se relies on the dietary source of Se provided [28–30]. The Se integration relies on the rumen environment, which gradually declines depending upon the particular source of Se [31]. Selenium is present in two forms, inorganic and organic [32]. These forms may be a vital source of selenium [33].

It has been known that Se supplementation enhances female fertility but the exact mechanism is still not unknown. The progesterone hormone derived from the corpus luteum is a dominant hormone of pregnancy. This hormone is synthesized from cholesterol *via* several enzymatic reactions in which molecular oxygen is utilized for its reactions. These reactions generate oxygen radicals and different peroxides which are detrimental to cells [34]. In *in vivo* study, indicated that the inclusion of luteinizing hormone in luteal cells culture concurrently enhanced progesterone level in the medium and also the lipid peroxides in cells [35]. For luteal regression, the accretion of H2O2 [36] or lipid peroxides [37] in the corpus luteum has been documented. These findings show that corpus luteum requires antioxidant defense toward peroxides to stabilize normal functions. The significance of the corpus luteum has also been projected by Ref. [38]. Moreover, the inclusion of Se in luteal cells reduced the concentration of lipid peroxides in a cell [35]. Se as the part of glutathione peroxidase may destroy peroxides, in connection with superoxide dismutase, vitamin E, and beta-carotene.

### **6. Oxidative stress during pregnancy and antioxidants**

Pregnancy is a normal mechanism in which overburden metabolic rate disrupts antioxidant status and energy balance. During first phase of pregnancy, 25% of the embryos die or reabsorbed within two weeks of pregnancy before implantation [39]. Once the zona pellucida is separated from the embryo, it enhances the production of ROS [40]. The imbalance between oxidant/antioxidant systems during early pregnancy may lead to disturbances in molecules which eventually compromise growth and development of embryo [41].

The nutritional requirement during early pregnancy is increased to maintain animal health and pregnancy, which is in turn generation of oxidative stress [42]. Although, malnutrition is also common around the globe in small ruminants because of the high price of the feed, especially in developing countries. Hence, small ruminants are easy to keep due to several reasons [43]. Malnutrition during pregnancy has deleterious effect on the conception rate and on fetus development [44]. Animal supplementation in a diet with plant source have sufficient nutrition and has been assumed to be the potential source of antioxidants to attenuate early pregnancy stress in goats [45, 46]. The plant compounds exert diverse nutrition comprises of rich source of antioxidants and immune-modulatory properties, which act as a potential feed supplement for ruminants [47, 48].

*Moringa oleifera* (MO) is a multifaceted medicinal tree with high nutritional values [49]. Its leaves are rich sources of several nutritious compounds, such as proteins, amino acids, minerals, and vitamins [50]. Apart of that, MO is also a rich source of antioxidant compounds, such as phenolic acids, vitamin E, vitamin C, selenium, zinc, and β carotene. These compounds have more robust antioxidant potential than synthetic ones [51]. The basal diet supplemented with 3.2% MOLP increased antioxidant

*Antioxidant Strategies to Improve Female Reproduction DOI: http://dx.doi.org/10.5772/intechopen.110639*

index and blood biochemical in early pregnancy in Beetal goats. It also promoted progesterone profile, improved conception rate, and attenuated ROS production in early pregnancy of goats.

In another study, by Ref. [52] reported the use of herbal antioxidants during pregnancy and their effect on piglet performance. The supplementation profoundly enhanced number of live-born piglets, total litter weight, and reducing the chance of low-weight piglets. Moreover, supplementation declined MDA levels in sows and piglets. The mothers who had supplementation showed a higher trend of weaning weight. The results conclude from 1000 pregnancies that offering maternal supplementation with herbal antioxidants in pregnancy profoundly enhanced reproductive efficiency, litter traits, and piglet performance.

### **7. Oxidative stress during lactation and antioxidants**

Reproductive performance is a main indicator related to maternal nutrition. The periparturient period causes reduced feed intake, and endocrine and metabolic alterations which disrupt energy balance and antioxidant index [22, 53]. In this period, increased nutritional requirements, such as digestion rate, mammary development, and fetus growth have been reported [54]. Pasture grazing and feeding on crop residues have a diverse nutritional profile and feeding on such sources is not adequate to meet the energy requirement of lactating animals [55]. In this scenario, pregnant animals are vulnerable to oxidative stress [56], which threatens to biomolecules and eventually affect productive and reproductive parameters [57]. Colostrum is the composition of immunoglobulins, minerals and other biological substances which transfer form colostrum to the young ones [58]. The quality of the colostrum depends upon maternal nutrition [59, 60]. The diet supplemented with phytobiotics has been assumed to be the main source of managing nutrition-induced oxidative stress in pregnancy and lactation in livestock [45, 46, 61]. In a recent study by Ali et al., (2022) using 2% and 3.5% *M. oleifera* leaf powder (MOLP) during periparturient period. He reported the increased biochemical and antioxidant indices of colostrum and milk. The milk yield, weight gain of the kids, and reproductive performance were enhanced with 2 and 3.5% MOLP. Further, the findings suggested that the diet supplemented with 3.5% MOLP promotes antioxidant index, milk yield, and reproductive performance in goats.

### **8. Effect of oxidants and antioxidants on embryo production**

The *in vitro* embryo production (IVEP) technique is employed to combat infertility-related problems in mammalian species [62]. This tool has been known to be utilized for the production of large scale offspring from elite animals. The IVM prognosis relies on diverse factors consisting of oocyte quality and culture conditions [63]. The source of antioxidants from female organs has been reported to reduce ROS production [64]. The main hurdle which decides the fate of oocyte success during IVM is oxidative stress [64]. Accelerated ROS production might result in oocyte death and embryonic loss [65, 66]. An antioxidant approach during IVM has been proposed to govern oocytes from the deleterious effect of oxidative stress by maintaining a basal level of ROS [67, 68]. Presently, different antioxidants are utilized during IVM to confirm balanced intracellular redox status, resulting in good-quality

of oocytes [69, 70]. The inclusion of antioxidants, such as thiols, polyphenols, melatonin, carotenoids, resveratrol, and vitamins C and E, to the IVM medium has been verified in different studies to increase oocyte quality and attenuate exceeding ROS damage [71, 72].

Previous evidence has reported that the balance amount of antioxidants and ROS in IVEP media which may be favorable for embryonic development [73, 74]. At present, the widely employed antioxidant in IVEP is cysteamine; its efficiency is mostly associated with the stage of IVM. It has been found to stimulate the embryonic process and secreting of glutathione (GSH), which is prevalent in male and female gametes from harmful effect of ROS [75]. Moreover, cysteine and glutathione have been implied in IVEP protocols with good results [73, 76]. The application of quercetin (2 μM), resveratrol (2 μM), vitamin C (50 μg/mL), carnitine (0.5 mg/mL), and cysteamine (100 μM) were determined to prove the vibrant antioxidant toward deleterious effects of ROS during IVM of bovine oocytes [71]. The positive effect of antioxidants is illustrated in **Table 1**.


### **Table 1.**

*The beneficial effect of antioxidant supplementation in different animal species.*

*Antioxidant Strategies to Improve Female Reproduction DOI: http://dx.doi.org/10.5772/intechopen.110639*

### **Author details**

Tarique Hussain Animal Sciences Division, Nuclear Institute for Agriculture and Biology College (NIAB-C), Pakistan Institute of Engineering and Applied Sciences (PIEAS), Faisalabad, Pakistan

\*Address all correspondence to: drtariquerahoo@gmail.com

© 2023 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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## Dietary Antioxidants and Bioactive Compounds in Food Processing

*Veerapandi Loganathan and Lakshmi Mohan*

### **Abstract**

The antioxidants available in fresh organic materials could vary significantly from all those we consume through diet, as it has historically been recognized. Plants contain several phytochemicals, which possess strong antioxidant activities. A large variety of phytochemicals have been isolated and characterized from familiar sources, including vegetables, such as onion and broccoli; fruits, such as apples and grapes; spices, such as nutmeg, pepper, and turmeric; and brews, such as green tea, oolong tea, and red wine; which possess strong antioxidant properties. This is typically affected by the usage of thermal and nonthermal food processing methods. This chapter deals with various traditional and unconventional techniques that can be utilized to recover bioactive constituents. Any traditional method's extraction effectiveness is primarily influenced by the solvents utilized. Among the most effective approaches, notably pressurized solvent extraction, supercritical fluid extraction, pressurized low-polarity water extraction, enzyme-assisted extraction, pulsed electric field extraction, ultrasound-assisted extraction, and microwave-assisted extraction were reviewed. The contrasting antioxidant activities of various extraction techniques were emphasized, as well as the processing techniques and industrial applications for unconventional ways of antioxidant extraction. How well this varies throughout absorption, how this impacts gastrointestinal function, and subsequent accumulation into the plasma, but which *in vivo* biological consequences it has on the internal organs all are aspects to consider.

**Keywords:** antioxidants, dietary antioxidants, bioactive compounds, food industry, food processing, BHT: Butylated hydroxytoluene; BHA: Butylated hydroxy anisole, extraction, microwave-assisted extraction

### **1. Introduction**

Antioxidants are elements, which are derived from natural and chemical substances, which is having the potent ability to scavenge free radicals by losing an electron and neutralize or slowdown the autooxidation process. Several free radicals are unbalanced and extremely reactive. Antioxidants are ready to contribute to an electron either by oxidizing or reducing other molecules [1]. Also, these antioxidants are capable of preventing and inhibiting cell membrane, structural, DNA, Lipids, carbohydrates, and cellular protein damage. During our body's metabolism and diet, it produces lighter and strong antioxidants, such as uric acid, ubiquinol, glutathione

micronutrients α-tocopherol, and ascorbic acid. Even though numerous amounts of antioxidants are there in the form of macro and micronutrients, such as vitamin E, Vitamin C, and beta-carotene to scavenge free radicals [2]. Both ionizing processes and nonenzymatic reactions involving oxygen and organic molecules can lead to the form of free radicals. Few organs are internally produced by some free radicals, such as mitochondria, peroxisomes, xanthine oxidase and some pathways arachidonate, phagocytosis, reperfusion injury, and other external factors.

Fruits and vegetables are one of the best sources of antioxidants. Consuming fresh juices, pastes, and canned foods gives an enormous quantity of antioxidants to our body [3, 4]. Whatever, during food processing least number of antioxidants are loosed and hence, might impact the final product to stimulate health properties [5]. During the twentieth century, food industries introduced antioxidants to inhibit the oxidation process of packed and stored foods [6]. While the impact of antioxidants as loss and gains of bioavailability in food processing have been studied before [7]. It is very important to develop augmented approaches for the preservation of food and the development of activity and bioavailability of antioxidants. It is also used to study about significant of the functional elements of food materials that we used in our daily diet and the changes in the composition of food during processing [8]. Especially performing thermal and nonthermal processing, determining the bioavailability of dietary antioxidants level and quality of dietary antioxidants [9–11].

### **2. Classification of antioxidants**

Generally, antioxidants are present in various foods in various forms. Those antioxidants are classified depending on their functions, mode of action, characteristics, and type of nature [12]. The known major antioxidants are natural and synthetic antioxidants (based on the type), dietary, and endogenous and exogenous antioxidants (based on the function). Furtherly it is classified as enzymatic and nonenzymatic antioxidants. The enzymatic antioxidants are catalase, glutathione, and dismutase, and nonenzymatic antioxidants are tocopherols, melatonin, ascorbic acid, vitamin E, and uric acid. These antioxidants play a very crucial role in food processing and preservation [13].

### **2.1 Endogenous antioxidants**

The only antioxidants with the ability to synthesize their own antioxidant compounds are called an endogenous antioxidants. Further, they can be classified based on their structural characteristics as enzymatic and nonenzymatic antioxidants. The body utilizes a variety of endogenous protective mechanisms alongside dietary antioxidants to support and protect against various consequences. Those enzymes utilized nutrient cofactors copper, zinc, selenium, iron, and manganese to react with toxic intermediate oxidative complexes for maximum catalytic activity [12]. The amino acids are glycine, glutamate, and cysteine synthesized glutathione is an essential water-soluble antioxidant.

### **2.2 Exogenous antioxidants**

Vitamins, polyphenols, carotenoids, as well as certain mineral complexes are examples of naturally occurring sources through which exogenous antioxidants could be synthesized [12]. Antioxidants are getting more prominent, particularly in those

intended to avoid the anticipated adverse consequences of the existence of reactive oxygen species in the internal organs and the degeneration of additional dietary constituents, such as fats [13].

### **2.3 Dietary antioxidants**

Ascorbate, tocopherols, carotenoids, and bioactive plant phenols are types of dietary antioxidants. The antioxidant vitamins in fruits and vegetables, some of which possess more potent antioxidant properties than others, are mainly accountable for their health benefits [14–16]. Among the most exhaustively researched dietary antioxidants include vitamins C and E, ß-carotene, other carotenoids, and oxycarotenoids, such as lycopene and lutein [13]. Vitamin C is thought to be the most significant water-soluble antioxidant in extracellular fluids. Before lipid per oxidation commences, it has the potential to remove free radicals in the aqueous environment. The most potent chain-breaking antioxidant in the cell membrane, wherever it protects cell wall fatty acids from lipid peroxidation, is vitamin E, a prominent lipidsoluble antioxidant. Vitamin C has allegedly demonstrated the ability to stimulate vitamin E [17].

It is also hypothesized that ß-carotene and certain other pigments protect lipidrich tissues from damaging free radicals. According to studies, several vitamins and ß-carotene may enhance the other's actions [18]. Although carotenoids seem to function as "pathogen-associated molecular enhancers" in individuals, flavonoids protect plants from either a wide range of environmental stresses. The anti-inflammatory, anti-allergic, antimicrobial, antiaging, and anti-carcinogenic characteristics of flavonoids have been demonstrated [13].

### **2.4 Natural antioxidants**

Those oxidants known as naturally occurring antioxidants could be present in foods, including fruit and vegetables and livestock [19]. All-natural compounds, especially berries, plants, nuts, beans, branches, stems, and barks, possess antioxidant compounds [19]. Vitamin C, E, and A (ascorbic acid, tocopherols, and carotenoids), different polyphenols, comprising quercetin, proanthocyanidins, and lutein, and ubiquitin known as a coenzyme Q10, a type of dietary protein, are among the most abundant vitamins in food products [13]. Plants namely the vitamins as well as other originating molecules in our food create natural antioxidants. The majority of fresh fruit and vegetable include ubiquitin, a specific type of protein, which is a potent antioxidant [20].

Living beings get all these elements mostly through plant-derived substances [11]. The excellent sources of antioxidant substances, comprising vitamins A, C, and E, ß-carotene, and essential minerals, are fruits, vegetables, and medicinal plants [21]. The total amount of phenol in various parts of plants, or even of the same vegetables and fruit varies greatly [22]. Enzymatic antioxidants and nonenzymatic oxidants make up the two main categories of the biological antioxidant system [23].

### *2.4.1 Enzymatic antioxidants*

Both primary and secondary metabolic inhibitors are even further characterized as catalytic antioxidants. The basic protection is composed of three essential enzymes that prevent the production of free radicals or neutralize them: glutathione peroxidase, catalase, and superoxide dismutase [23]. Glutathione reductase and glucose-6-phosphate dehydrogenase are two supplementary metabolic inhibitors [24]. Even though these two enzymes really should not directly counteract free radicals, they might enhance existing endogenous inhibitors in their abilities to achieve this.

### *2.4.2 Nonenzymatic antioxidants*

The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are certainly scavengers by nonenzymatic antioxidants, which include proteins (glutathione), vitamins E and C (that also inhibit the oxidative damage of cellular membrane), nitrogenous compounds, including uric acid, which inherently acts as an antioxidant against peroxynitrite in bloodstream, albumin, bilirubin, N-Acetylcysteine (NAC), and melatonin [17, 25, 26].

### **2.5 Synthetic antioxidants**

Antioxidants that are chemically synthesized and added to perishable foods as preserves to contribute to the inhibition of peroxidation are termed synthetic antioxidants, since they cannot appear in nature [27]. Synthetic antioxidants were created in order to provide a consistent catalase activity analysis method to correlate with antioxidant properties and to be incorporated into foods. Such bioactive molecules are included in the food to enhance storability and to assist it to resist alternative treatments and environments. Consequently, synthetic antioxidants are incorporated into essentially all packaged products, which are supposedly acceptable [25]. The two most commonly used synthetic scavengers are butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). Considering the daily allowance and effective dose for the synthesis of various inhibitors, contradictory results were obtained [14]. Moreover, mixed data exist on the influence of antioxidant compounds on people's well-being. BHT, BHA, propyl gallate (PG), dodecyl gallate (DG), and tertiary butylhydroquinone are among the chemical additives currently authorized to be used in foods [2, 27].

### **3. Sources of antioxidants**

The majority of natural antioxidants in use today are derived from plant sources, such as fruits, vegetables, spices, and herbs [26]. These foods are particularly rich in plant secondary metabolites, such as phenolic compounds, terpenoids, alkaloids, vitamins, and carotenoids [28].

The most popular antioxidants are the essential oils extracted from culinary spices and herbs, such as thyme, oregano, marjoram, basil, lavender, and rosemary. They have been reported to be good sources of natural antioxidant-rich molecules, but have limited applications due to their strong aroma and flavor characteristics [29, 30].

Another common source of dietary antioxidants is the brewed tea used around the world. They include non-fermented (green), semi-fermented (Oolong), aged (Pu'er), and fermented (black). Tea is very rich in polyphenols. Fresh green tea leaves have been reported to contain around 36% polyphenols, on a dry-weight basis [26]. Aqueous tea extracts are also good sources of natural antioxidants owing to a large number of metabolites, such as catechins, tannins, and other flavonoids, in a fresh brew. They have the additional advantage of not presenting a strong flavor when compared to essential oils [29].

### *Dietary Antioxidants and Bioactive Compounds in Food Processing DOI: http://dx.doi.org/10.5772/intechopen.109557*

The polyphenols in green tea have been observed to bring about programmed cell death otherwise known as apoptosis in numerous cancer cells, such as prostate, lymphoma, colon, and lung cancer cells. Black tea, on the other hand, was found to inhibit DNA synthesis while enhancing the apoptosis of benign and malignant tumor cells. The antioxidant activity of green tea catechins is normally in the order of EGCG (epigallocatechin gallate) almost equal to ECG (epicatechin gallate) activity, which is better than EGC (epigallocatechin) and greater than EC (epicatechin), while that of theaflavins of fermented teas are theaflavin digallate (TF-2) greater than theaflavin monogallate (TF-1 A & B) than theaflavin (TF). The antioxidant activity of green tea almost often surpasses that of oxidized black tea and its extracts [31].

Gallic acid is a recognized natural antioxidant that induced fragmentation of DNA in THP-1, HL-60, U-937, and ML-1; four diverse human myelogenous leukemia cell lines, but not in erythroleukemia (K- 562) cell lines and human T-cell leukemia (MOLT-4). Gallic acid was found to induce apoptosis in HL-60 RG cells through ROS (reactive oxygen species) generation, an influx of Ca2+ leading to the activation of calmodulin. The primary pigment in turmeric, curcumin, and phenolic compound was found to induce apoptosis in transformed human and rodent cells in culture. Curcumin mediates this chemopreventive action by inhibiting the formation of cyclooxygenase metabolites, which provides a mechanism for the induction of apoptosis. Quercitrin, quercetin, and kaempferol are flavonoids present widely in around 70% of all plants. Flavonoids differ notably in their antioxidative effectiveness, depending on their structure [31].

The most effective dietary antioxidants belong to the family of phenolics and polyphenolics. Phenolic compounds occurring in foods belong to the phenylpropanoid (C6-C3) family and are derivatives of cinnamic acid. These compounds are formed from phenylalanine, and to a lesser extent in some plants from tyrosine, via the action of phenylalanine lyase, or its corresponding tyrosine lyase. Edible oils and oilseeds provide a rich source of unsaponifiable matter that contains a variety of active ingredients that may be used to prevent or control deteriorative processes. The non-triacylglycerol constituents in oils and oilseeds belong primarily to the tocols (tocopherols and tocotrienols) family, phenolics and flavonoids, sterols, phospholipids, carotenoids, and triterpene alcohols as well as the phytic acid family of compounds [27]. Carotenoids play a major role in the prevention of various health disorders, such as cancer, metabolic disease, and cardiovascular diseases [32].

### **4. Extraction of antioxidants from natural sources**

Antioxidants are isolated and purified from different parts of plants, such as roots, stems, leaves, fruits, seeds, and peels. The attributes of antioxidants from natural sources and their antioxidant potential depends not only on the quality of the extract concerning its geographic origin, nutritional aspects, and storage, but also on the methodologies used for their extraction.

The methods for analyzing antioxidants include the estimation of total antioxidant activity by electrochemical or spectrophotometric methods. The specific detection and assessment of different antioxidant molecules are facilitated by various chromatographic—TLC, HPLC, LC, GC, MS, NMR, capillary electrophoresis, and NIR methods. However, before quantification can be done, it is required to extract the different components from the food matrix. This involves technologies such as the use of organic solvents for solvent extraction, subcritical water extraction, supercritical fluids, high hydrostatic pressure, microwave procedures, pulsed electric fields, or ultrasonics.

The yield of antioxidants extracted from the plant material is affected mainly by the environment under which the process of extraction occurs. Every plant material is unique in terms of its structure and composition; therefore, the behavior of the resulting material-solvent system is unpredictable upon combining it with solvents. Assisted techniques using original fluids, which are referred to as nonconventional methods of extraction, such as supercritical fluids, supercritical water, ultrasoundassisted extraction, enzymatic-assisted extraction (EAE), microwave-assisted extraction (MAE), supercritical fluid, and pulsed electric field (PEF) have become more efficient and popular in recent times [33].

### **4.1 Solvent extraction**

Solvent extraction is a technique that involves applying a solvent to extract or separate the desired component, called the solute from solid food material. The separation factor in solvent extraction is the chemical equilibrium that exists between the solid and solvent phases. And the concentration difference of the component between the two phases is the driving force for solvent extraction. An ideal solvent for extraction should have a high affinity for the solute being separated, it should be selective and dissolve the component of interest to a large extent while having a minimum capacity for the other undesirable components. It should be chemically stable, forming no irreversible reactions with contacting components, regenerable, and have low viscosity values for easy pumping and transportation [33].

The most efficient solvents used in extracting anthocyanins, being polar molecules, are aqueous mixtures of methanol, ethanol, and acetone. Among the most frequent solvent extraction methods are the ones that use acidified ethanol or methanol as solvents. The acids in the solvent system rupture the cell membranes and release anthocyanins. As this can cause damage to the anthocyanin structure, it is advised to acidify the solvents with organic acids, such as formic or acetic acid, rather than mineral acids, such as 0.1% HCl, to minimize damage [33].

Alcoholic solvents are used to extract antioxidant phenolic compounds from various sources. Ethanol, a polar solvent has been shown to effectively extract several secondary metabolites, including flavonoids, catechol, glycosides, and tannins, from raw plant materials. It is also to be noted that in food processing industries, ethanol is preferred over methanol due to its inherent toxicity. Lycopene, a fat-soluble antioxidant present in large quantities in tomatoes, is extracted with organic solvents, such as benzene, acetone, petroleum ether, ethanol, hexane, and chloroform.

### **4.2 Extraction using supercritical fluids**

Extraction with the help of supercritical fluids (SCF) has gained popularity in the food processing domain. Similar to conventional solvent extraction, SCF extractions use fluids in their supercritical states, which have desirable transport properties that enhance their potential as solvents for extraction processes [34]. CO2 is one such example that is nontoxic, noninflammable, and requires only a bare minimum amount of solvent for the process. Extraction is quicker, takes about 10–60 min, is selective, and requires only small quantities of sample and no additional cleanup. An improvement over this method is the use of enhanced solvent extraction, which uses carbon dioxide, organic solvents, or water at high temperatures and pressure. SCF extraction has been used successfully for extracting anthocyanins and polyphenols from grapes, wine, and some herbs [34].

### **4.3 Ultrasonics**

Ultrasonics is one of the commonly used techniques in the food and beverage industry, which enhances the mass-transfer phenomena. It has been successfully applied for extracting anthocyanins, polyphenols, and flavonoids from various plant sources.

### **4.4 Microwave-assisted extraction (MAE)**

Microwave-assisted extraction (MAE) helps reduce the time needed for extraction and the quantity of solvent used. MAE involves extraction under controlled conditions of temperature and pressure with or without the addition of a solvent. It has been reported that using closed vessels cuts down the extraction time and increases the efficiency of extraction. MAE has been used to extract phenolics in a very effective manner [35]. Recent advances in this domain include microwave hydrodiffusion and gravity (MHG) and solvent-free microwave extraction (SFME).

### **4.5 Subcritical water extraction (SWE)**

Subcritical water extraction uses subcritical water or pressurized hot water below the critical pressure of 22 MPa to extract natural compounds from herbs, plants, and food materials, such as pomegranate seed residues, red grapes, potato, and citrus peels [8].

### **4.6 High hydrostatic pressure (HHP)**

High hydrostatic pressure (HHP) works by improving mass transfer rates, thereby increasing cell permeability and secondary metabolite diffusion by changes in phase transitions. HPP has been reported to be utilized in extracting anthocyanins and polyphenols from grapes, red fruits, and grape skins [35].

### **4.7 Enzyme-assisted extraction (EAE)**

Enzyme-assisted extraction (EAE) has been used efficiently to release and recover bioactive molecules from several algal and plant sources, such as lemon balm, red algae, alfalfa, and pumpkins. Enzymes are capable of catalyzing the degradation of plant cell walls, thereby releasing the bioactive compounds stored inside the cells. Examples of enzymes used for this procedure are cellulases, hemicellulases, pectinases, etc. [35].

Other major techniques, such as pulsed electric fields and high voltage electrical discharges, are also gaining popularity as noninvasive techniques to extract secondary metabolites from plant sources.

### **5. Processing of antioxidants**

Despite the knowledge that there are numerous antioxidants in nature, typically just a few amounts of basic ingredients, especially vegetable fatty acids and oils and rosemary leaves, are utilized to synthesize extracts with antioxidant potential [31]. Considering these substances have enough potential to cause serious harm, cytotoxic,


**Table 1.**

*Antioxidant amounts in different by-products.*

or neurotoxic to people, the desire for bioactive components has resulted in a reduction in their utilization [15]. It is, therefore, argued that synthetic substances, such as BHT, are hazardous for ingestion when used in therapeutics because they could have negative health consequences for humans [34]. The unfavorable effects of antioxidant compounds on well-being have been significantly reduced through investigation.

The amount of production of natural antioxidants from waste vegetables and other fruits has generated a significant amount of attention. Huge quantities of waste products, particularly peels and nuts, are generated during the process of processing fruits, vegetables, and grains [34]. Regulatory constraints mean handling these substances is a challenge that is already complex. As a result, unique perspectives on utilizing these materials as by-products for further utilization on the development of additives or supplement with high amounts of nutrients have drawn increasing attention considering that these are greater commodities and their recovery may be economically feasible. According to the original source, the production of fruit and vegetables and oilseeds generates different quantities of by-product (**Table 1**) [17].

### **6. Antioxidant composition varies during food processing**

Consumption of natural antioxidant compounds from food products that are already abundant in all of these bioactive substances [36]. Although food processing is proven that it has a significant impact on nutritional properties and pharmacological activities [31]. Food processing involves both thermal and nonthermal procedures, including storing, sorting, washing, packaging, and transportation, to produce the desired final product [18]. Antioxidants are lost during the processing of fruits and vegetables, and processed foods have significantly lower bioavailability when compared to fresh foods, which leads to rapid oxidation, enzymatic reaction, and degradation of enzymes due to thermal processing [37]. However, there is significant information that shows food processing might not even necessarily have such a negative influence on the effectiveness of dietary ingredients [33].

### **6.1 Thermal treatments**

The majority of commercially available food processing techniques include one or even more than one thermal process in order to achieve a variety of final products.

### *Dietary Antioxidants and Bioactive Compounds in Food Processing DOI: http://dx.doi.org/10.5772/intechopen.109557*

The chemical content and nutritive values of the food material could change after the thermal process in addition to the desired consequence because of quantitatively or qualitatively changes in the amount of antioxidant properties, among several other factors. One of the main antioxidants, carotenoids, are widely distributed in tomatoes, watermelons, guava, papaya, and apricots [24]. Carotenoids are degraded if these food products are exposed to various treatments. Many vegetables and fruits contain phenols and phenolic substances. The majority of these food processing technologies include thermal treatments, which are reported that the plants do not have the ability to retain phenolic acids [38]. The outcomes of phenolic acids in food production can be significantly influenced by the product's composition and the processing methods, but the dietary substrate has also been shown to be an even more important factor [7].

### **6.2 Nonthermal treatments**

Cutting, blending, peeling, and crushing, as well as other nonthermal food processing technologies, could all have an influence on the antioxidant characteristics of food products. Additional "emerging" or "progressive" nonthermal food processing techniques have recently been developed, including high pressure, pulsed electric field, and ultrasonic processing [20]. Excessive temperatures could lead to their breakdown or polymerization, and that has negative consequences on some of these bioactive constituents, but they might also assist to extract higher carotenoids from the plant source. Various nonthermal processing technologies were suggested as alternative approaches to yield a product of a higher quality.

### **7. Changes in antioxidant bioavailability during food processing**

Antioxidant compounds must be released from the food material through metabolism in the gastrointestinal tract and thereafter biologically modified into ingestible components in order to demonstrate their nutrition properties or to be active compounds [16]. In order to be used in metabolic processes, substances can then gradually enter the circulatory system and be delivered to the blood circulation, becoming "bioavailable" [39]. "Bioavailability" is a word that describes the transportation and diffusion of active ingredients to specific cells and tissues, enabling those cells and tissues to demonstrate a range of antioxidant actions [40]. Oral bioavailability has frequently been assessed through *in situ* digestive assays. These methodologies mimic digestion in the small intestine and gastrointestinal tract, and in certain cases, Caco-2 cell absorption simulation was achieved initially [41]. The concentration of these molecules, their intermolecular interactions, and the molecular structure of the plant and food products are the characteristics that have a substantial impact on the oral bioavailability and digestibility of natural antioxidant substances. The concentrations of various bioactive constituents in the respective food are influenced by pre and postharvest handling, resulting in a significant impact on plant-based product are composed. As an outcome, processed food products may have varying levels of dietary and possibly bioavailable antioxidant compounds. Furthermore, modifying the molecular structure of the essential constituents during food processing seems to have the chance to have a significant favorable or detrimental effect on availability [40].

Research on the contribution of food processing technologies on the bioavailability of antioxidants is completely lacking overall.

### **8. Applications of antioxidants**

Natural and synthetically derived antioxidants have found wide applications in the food processing sector as food additives in meat, fruits, vegetables, beverage, spices, fats, and oil industries to enhance the appearance, taste, and color, and help prolong the shelf life. The addition of dietary antioxidants to meat and derived products has been observed to be effective in lipid oxidation and metmyoglobin formation. These compounds include plant phenolics as natural antioxidants, for instance, vitamin C ascorbic acid and vitamin E—α-tocopherol (E306), culinary herbs, and spices, such as oregano, rosemary, basil, thyme, sage, pepper, nutmeg, clove, cinnamon, and extracts from tea and grape seed. The potential applications of natural extracts with antioxidant activity in food are being thoroughly investigated for potential uses, including health paybacks, nutritional profile improvement, and shelf-life extension [1].

Ascorbic acid, E300 is added to cut fruits, beers, jams, dried potato, and other foods to prevent foods from going brown due to oxidation reactions that cause discoloration. It is also added to replace the vitamin C lost during processing.

Rosemary oleoresin has proven to be as successful as polyphosphate and a combination of BHT and BHT-citric acid in automatically deboned poultry meat and sausages made from automatically deboned poultry meat [16, 34] worked on ethanolic extracts of rosemary and demonstrated that it improved the stability of butter and that this effect was concentration-dependent. The research study also assessed the ability of rosemary extract to inhibit copper-catalyzed oxidation and proved that the extract could chelate metal ions.

Pepper nigrum extract isolated using supercritical carbon dioxide extractions was found to be efficient in preventing lipid oxidation in ground pork samples. The potent antioxidant activity of pepper has been credited to piperine and piperine isomers, such as chavicine, isopiperine, isochavicine, and some monoterpenes.

Farag et al. showed that the essential oils of Cuminum cyminum and Thymus vulgaris inhibited the oxidation in butter that was stored at room temperature. At a concentration of 200 ppm, these essential oils were more efficient than Butylated hydroxytoluene in inhibiting the oxidation of lipids.

It is a fact that all emulsified products tend to have a shorter shelf life when compared to edible oils, due to their lesser resistance to microbial spoilage. They are, therefore, stored under refrigerated conditions, so that autoxidation is low and the naturally present tocopherols are stabilized them. Some edible oils, such as sunflower oil, are less resistant due to high polyunsaturation, often requiring the addition of natural or synthetic antioxidants. When it comes to frying oils, the best way to add antioxidants is just before their operation. It has been observed that adding rice bran oil with inherent natural antioxidants enhanced the shelf life of nuts processed in oils, such as soybean or rapeseed [20].

The application of synthetic antioxidants has to be reduced further and replaced by safer alternatives, such as natural or nature-identical antioxidants. Prolongation of the shelf life of highly processed foods has to be accomplished by modifying existing recipes, introducing culinary herbs and spices, which contain a high concentration of inherent antioxidants, using high-oleic edible oils requiring lower added antioxidant levels, and by use of natural protein hydrolysates, which have good synergistic activity.

### **9. Conclusions**

There is increasing evidence to prove that consuming a range of dietary antioxidants available in natural foods reduces the risk of major health issues due to their antioxidant capability through several mechanisms. Care must be taken to choose optimal processing methods to ensure the quality of antioxidants from fresh fruits and vegetables and their products in order to achieve the objectives. The mode of action of these antioxidants in the body needs more research. Due to safety concerns, regarding the use of synthetic antioxidants and natural antioxidants acquired from edible sources, their by-products and coproducts are in the spotlight today. Further studies on the isolation of antioxidant compounds using nondestructive methods and their effects in animal models and human subjects are necessary to evaluate their potential benefits. Additionally, it is mandatory to confirm the bioavailability and lack of toxicity of such compounds. Delivery of isolated antioxidant metabolites as functional food ingredients or dietary supplements will help in promoting good health and reducing the risk of disease. In the last few decades, there has been considerable interest in the food as well as the pharmaceutical industry, for extracting and purifying antioxidants from natural sources. A sensible selection of appropriate food-handling methods right from the farm to the consumer for every type of product will make sure that the health-related benefits of specific antioxidants are maximized.

### **Author details**

Veerapandi Loganathan\* and Lakshmi Mohan Saintgits College of Engineering, Kottayam, Kerala, India

\*Address all correspondence to: veerapandi.l@saintgits.org

© 2023 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 Paz Otero Fuertes and María Fraga Corral*

Plants, fruits, and vegetables contain antioxidants that can be used as nutraceuticals or pharmaceuticals due to their perceived ability to reduce the risk of developing certain chronic diseases. This book includes thirteen chapters that discuss potential sources of new antioxidants from the fruits of South America and the flora of African countries, how to improve the production of antioxidants and methods to ensure the quality of antioxidants from fresh fruits and vegetables.

### *Miroslav Blumenberg, Biochemistry Series Editor*

Published in London, UK

© 2023 IntechOpen © monsitj / iStock

Recent Developments in Antioxidants from Natural Sources

IntechOpen Series

Biochemistry, Volume 41

Recent Developments

in Antioxidants from

Natural Sources

*Edited by Paz Otero Fuertes* 

*and María Fraga Corral*