**2.4 Ethnobotanical study of carnauba wax**

#### *2.4.1 Antioxidant activity*

Phenolic bioactive and polyphenolic compounds occur normally and significant segments of the human diet due to their antioxidant capacity that decreases oxidative stress-inducing cellular damage associated with severe pathologies such as cardiovascular, neurodegenerative diseases and cancers [17]. The simplest bioactive phytochemicals containing a single substituted phenolic ring, like cinnamic acid and caffeic acid. Cinnamic acid is a naturally proceeding organic acid in plants, has low toxicity and a broad spectrum of biological activities. However, cinnamic acid derivatives comprise a series of trans-3-phenylpropenoic acids which differ in their substituents on the aromatic ring. The presence of a benzene ring and a low unsaturated hydrocarbon chain determines its low polarity and solubility in water. The most common cinnamic acid derivatives in plants are *p*-coumaric, caffeic, and chlorogenic acids and hydroxybenzoic and hydroxycinnamic acids, respectively [18].

Claisa et al. [19] studied the antioxidant activity by ABTS and FRAP methods and *in-vivo* cellular antioxidant activity assay. The antioxidant activity of ethyl acetate and hexane extracts of *p*-methoxy cinnamic diester (**Figure 2**) (PCO-C) showed the values 107.27 ± 3.92 μM Trolox/g and 73.3 ± 1.83 μM iron sulfate/g, respectively. From these results showed significant antioxidant activity values due to the presence of derivative of cinnamic acid compounds [20]. In addition, the *in-vivo* antioxidant activity showed lower ROS values in PCO-C alone (50 and 250 μg/ mL). Accordingly, PCO-C did not produce any cellular oxidation significantly it produces low level of ROS, because of the oxidation of lymphocytes endured with

**77**

antioxidant capacity.

**Figure 2.**

*2.4.2 Anti-microbial and anti-fungal activities*

bacteria (*Escherichia coli* and *Proteus vulgaris*).

*DOI: http://dx.doi.org/10.5772/intechopen.90252*

*Chemical structure of p-methoxy cinnamic acid diesters.*

*The Phytochemical Composition of Medicinal Plants: Brazilian Semi-Arid Region (Caatinga)*

H2O2. However, PCO-C had a best antioxidant effect in high dose level (250 μg/mL) similar of Trolox (80 μM) and found an oxidation inhibition capacity in human peripheral blood lymphocytes (HPBLs). According to the authors, it is revealed that antioxidant activates arise from *p*-methoxy cinnamic diesters presence of phenolic compounds PCO-C. Therefore, the presence of these excellent antioxidant potentials of produce reflects its ability to deliver bioactive substances that neutralize reactive oxygen species (ROS) and scavenge free radicals produced by oxidative stress.

Rufino et al. [21] reported that significant antioxidant activities of polyphenolrich extracts from tropical fruits and dry fruits especially carnauba, both DPPH, ABTS, FRAP, β-Carotene oxidation methods and total phenolic contents were performed. The antioxidant activity of the methanolic and ethanolic extracts of fresh fruits of carnauba found decreased values DPPH values 3549 ± 184 g fruit/g DPPH· and increased ABTS values 10.7 ± 0.2 μmol Trolox/g, FRAP values 15.5 ± 0.4 μmol Fe2SO4/g and high β-Carotene bleaching values found 87.7 ± 2.7(% O.I) and extractable polyphenols values 830 ± 28.3 mg GAE/100 g, respectively. Additionally, the bioactive compounds values (mg/100 g fresh matter) such as, vitamin-C (78.1 ± 2.6), total anthocyanins (4.1 ± 0.1), yellow flavonoids (66.4 ± 2.3), and total carotenoids (0.6 ± 0.2), chlorophyll (4.2 ± 0.2), respectively. According to the authors, this study provides an adaptation of ABTS, DDPH, FRAP and β-carotene bleaching methods, along with an evaluation of the compounds related to antioxidant potential. The results showed promising perspectives for the exploitation of non-traditional tropical fruit species with considerable nutritional properties and

The prevention of the decomposition and assurance of the food safety can be attained by the use of compounds that act as preservatives of foodstuffs, by presenting antimicrobial properties, preventing the degradation by enzymatic and non-enzymatic reactions. The identification of new sources of compounds and increased demand for the prospection that has antimicrobial properties. However, its realization depends on some conditions, including solubility of the food and pH. Cinnamic acid derivatives (CAD) such as trans, hydroxy and methoxy cinnamic acid, 4-chlorocinnamate, cinnamic acid derived from oxazoline ions, antimicrobial and antifungal activities. These substances showed strong activity against Grampositive bacteria (*Staphylococcus aureus* and *Bacillus cereus*) and Gram-negative

Gonçalves et al. [22] studied the effects on different concentration of carnauba wax (1, 2, 3 and 4.5%) on the brown rot, produced by *Monilinia fructicola* (G. Wint.) and Rhizopus rot, developed by *Rhizopus stolonifer* (Erhenb.:Fr.) *in vitro* *DOI: http://dx.doi.org/10.5772/intechopen.90252 The Phytochemical Composition of Medicinal Plants: Brazilian Semi-Arid Region (Caatinga)*

H2O2. However, PCO-C had a best antioxidant effect in high dose level (250 μg/mL) similar of Trolox (80 μM) and found an oxidation inhibition capacity in human peripheral blood lymphocytes (HPBLs). According to the authors, it is revealed that antioxidant activates arise from *p*-methoxy cinnamic diesters presence of phenolic compounds PCO-C. Therefore, the presence of these excellent antioxidant potentials of produce reflects its ability to deliver bioactive substances that neutralize reactive oxygen species (ROS) and scavenge free radicals produced by oxidative stress.

#### **Figure 2.**

*Phytochemicals in Human Health*

(NMR) methods.

1

respectively.

**2.3 Proteins**

flame ionization detections (GC-FID), 1

(campesterol, cholesterol, sitosterol and stigmasterol) were detected and identified. These finding isolated compounds were characterized by using Infrared (IR) spectra and confirmed by classical chromatographic techniques such as gas chromatography-

H and 13C NMR spectroscopy techniques have been applied for structural elucidation of dammarane triterpenoids [basic skeleton as carnaubadiol] in carnauba wax powder obtained from the leaves of *Copernicia cerifera* [13]. Totally four types of triterpenes were identified from hexane extract of carnauba wax. Four of these compounds were, structure1, (24RŁ)-24-methyldammara-21,25- diene-3ˇ-ol, structure of 2 and 3 was distinguished as (24RŁ)-24-methyldammara-25-ene-3-one. Furthermore, the structure of 4, illustrated as (E)-25-hydroperoxydammar-23 ene-3ˇ,20-diol. The chemical composition analyzed after successive column chromatography using silica gel hexane followed by ethanol at room temperature,

Cruz et al. [12] isolated the wax protein from "Carnauba" wax and the samples accomplished by SDS-Tricine-gel electrophoresis technique. It showed relative molecular masses of 26,000 (β-1,3-glucanase) and 24,000 Da (class III chitinase), respectively. However, these proteins have been involved in the resistance systems of plants against insects and pathogens. In addition, the authors found that proteins segregated from the different portions of carnauba wax have antifungal enzymatic action. These chemicals, chitinase and β-1,3-glucanases, can hinder early development of organisms and modify hyphal (threadlike fibers like mycelium of parasites)

Phenolic bioactive and polyphenolic compounds occur normally and significant segments of the human diet due to their antioxidant capacity that decreases oxidative stress-inducing cellular damage associated with severe pathologies such as cardiovascular, neurodegenerative diseases and cancers [17]. The simplest bioactive phytochemicals containing a single substituted phenolic ring, like cinnamic acid and caffeic acid. Cinnamic acid is a naturally proceeding organic acid in plants, has low toxicity and a broad spectrum of biological activities. However, cinnamic acid derivatives comprise a series of trans-3-phenylpropenoic acids which differ in their substituents on the aromatic ring. The presence of a benzene ring and a low unsaturated hydrocarbon chain determines its low polarity and solubility in water. The most common cinnamic acid derivatives in plants are *p*-coumaric, caffeic, and chlorogenic acids and hydroxybenzoic and hydroxycinnamic acids, respectively [18]. Claisa et al. [19] studied the antioxidant activity by ABTS and FRAP methods and *in-vivo* cellular antioxidant activity assay. The antioxidant activity of ethyl acetate and hexane extracts of *p*-methoxy cinnamic diester (**Figure 2**) (PCO-C) showed the values 107.27 ± 3.92 μM Trolox/g and 73.3 ± 1.83 μM iron sulfate/g, respectively. From these results showed significant antioxidant activity values due to the presence of derivative of cinnamic acid compounds [20]. In addition, the *in-vivo* antioxidant activity showed lower ROS values in PCO-C alone (50 and 250 μg/ mL). Accordingly, PCO-C did not produce any cellular oxidation significantly it produces low level of ROS, because of the oxidation of lymphocytes endured with

morphology of growths developing within the proteins.

**2.4 Ethnobotanical study of carnauba wax**

*2.4.1 Antioxidant activity*

H and 13C nuclear magnetic resonance

**76**

*Chemical structure of p-methoxy cinnamic acid diesters.*

Rufino et al. [21] reported that significant antioxidant activities of polyphenolrich extracts from tropical fruits and dry fruits especially carnauba, both DPPH, ABTS, FRAP, β-Carotene oxidation methods and total phenolic contents were performed. The antioxidant activity of the methanolic and ethanolic extracts of fresh fruits of carnauba found decreased values DPPH values 3549 ± 184 g fruit/g DPPH· and increased ABTS values 10.7 ± 0.2 μmol Trolox/g, FRAP values 15.5 ± 0.4 μmol Fe2SO4/g and high β-Carotene bleaching values found 87.7 ± 2.7(% O.I) and extractable polyphenols values 830 ± 28.3 mg GAE/100 g, respectively. Additionally, the bioactive compounds values (mg/100 g fresh matter) such as, vitamin-C (78.1 ± 2.6), total anthocyanins (4.1 ± 0.1), yellow flavonoids (66.4 ± 2.3), and total carotenoids (0.6 ± 0.2), chlorophyll (4.2 ± 0.2), respectively. According to the authors, this study provides an adaptation of ABTS, DDPH, FRAP and β-carotene bleaching methods, along with an evaluation of the compounds related to antioxidant potential. The results showed promising perspectives for the exploitation of non-traditional tropical fruit species with considerable nutritional properties and antioxidant capacity.

#### *2.4.2 Anti-microbial and anti-fungal activities*

The prevention of the decomposition and assurance of the food safety can be attained by the use of compounds that act as preservatives of foodstuffs, by presenting antimicrobial properties, preventing the degradation by enzymatic and non-enzymatic reactions. The identification of new sources of compounds and increased demand for the prospection that has antimicrobial properties. However, its realization depends on some conditions, including solubility of the food and pH. Cinnamic acid derivatives (CAD) such as trans, hydroxy and methoxy cinnamic acid, 4-chlorocinnamate, cinnamic acid derived from oxazoline ions, antimicrobial and antifungal activities. These substances showed strong activity against Grampositive bacteria (*Staphylococcus aureus* and *Bacillus cereus*) and Gram-negative bacteria (*Escherichia coli* and *Proteus vulgaris*).

Gonçalves et al. [22] studied the effects on different concentration of carnauba wax (1, 2, 3 and 4.5%) on the brown rot, produced by *Monilinia fructicola* (G. Wint.) and Rhizopus rot, developed by *Rhizopus stolonifer* (Erhenb.:Fr.) *in vitro* evaluation on infection of nectarine and plums. The authors, distinguished that no mycelial development of *M. fructicola* at any wax concentrations in post-contamination tests, however, *R. stolonifer* was totally restrained via by carnauba wax at all concentrations except at 1%. Additionally, *in vitro* evaluation for both *M. fructicola* and *R. stolonifera* no germination occurred of spores at any carnauba wax concentrations. There was 50% inhibition observed in spore germination for *M. fructicola* by utilizing 9% carnauba wax concentration and covered with nectarines 90% for *R. stolonifera*. The carnauba wax concentrations (4.5% and 9%) were applied to the protections with essentially reduced frequencies of both diseases in nectarines and plums. Nevertheless, the utilization of wax control was ineffective after infection by both diseases.

According to Jo et al. [23] studied quality and microbial safety of Fuji apples coated with CSW/LO (Carnauba-shellac wax nanoemulsion containing lemongrass oil). In this work, carnauba wax incorporated into shellac wax (Carnauba-shellac wax) with essential oils like lemongrass oil coating formulations and their effects on the coating and shelf life of the Fuji apples were evaluated. Total soluble solid content to titratable acid ratio, hardness, weight loss and color, sensory quality and microbial growth of fresh Fuji apples were studied during 5 months of storage at room temperature. According to the authors, results showed that carnauba extracts incorporated to shellac wax-based coatings together with lemongrass oil successfully maintained the firmness and color of coated freshly harvested apples in comparison with uncoated control samples, which presented severe texture softening. During storage conditions, the hardness of the uncoated apples exhibited the lowest conditions by 3.3 N and the weight loss was found by 7.7%. Interestingly, the weight loss was found to be 5.2% and the hardness of the coated apples did not change at any conditions, respectively. The total soluble solids and titratable acidity revealed that not significantly different between coated and uncoated apples.

Hence, the application of CSW-LO coated apples had better sensory scores with the sensory acceptability threshold for any attributes evaluated. In addition, the total aerobic bacteria population on the coated apples were deteriorated (1.4 log CFU/g) compared with uncoated apples after 5 months of storage. Additionally, the population of yeast and molds of the uncoated apples were found 2.2 log CFU/g after 5 months of storage, although yeast and molds were not detected on the coated apples, respectively. The results achieved demonstrate the feasibility of the addition of carnauba wax coating formulations for increasing the nutritional value of fresh apples without compromising their fresh-like quality attributes.
