**3.2 Chemical composition**

*R. officinalis* chemical composition varies greatly, due to some factors that directly influence the quality, amount of oil, and extract produced. However, it is possible to verify, through the literature, that its main chemical constituents are flavones, diterpenes, steroids, and triterpenes [17, 26, 27, 31]. The phenolic compounds present in *R. officinalis* were grouped into three classes: (i) phenolic acids (vanillic, caffeic, ferulic, and rosmarinic acids), (ii) diterpenes (carnosol, rosmadial, carnosic acid, methyl carbonate, rosmanol, epirosmanol, epiisorosmanol, epirosmanol methyl ether, and epiisorosmanol ethyl ether), and (iii) flavonoids (hesperetin, apigenin, genkwanin, 4′-methoxytectochrysin, cirsimaritin, scutellarein, 4″,5,7,8-tetrahydroxyflavone, homoplantaginin, and 6-hydroxyluteolin 7-glucoside) [27]. Recently, a *R. officinalis* chromatographic analysis was carried out, which revealed two large groups: oxygenated monoterpenes and hydrocarbonated monoterpenes. The main constituents of these groups were 1,8-cineole followed by camphor, borneol, and α- and β-pinene. The oxygenated and hydrocarbonated sesquiterpenes were composed of caryophyllene and caryophyllene oxide [26]. In the supercritical extracts of *R. officinalis* leaves, chemical analysis confirmed the

**321**

*R. officinalis*.

**Figure 3.**

**3.3 Antioxidant and biological activity**

caffeic acid derivatives [61, 62].

*Antioxidant and Biological Activity of* Cissus sicyoides and Rosmarinus officinalis *Extracts*

presence of 1,8-cineole, camphor, carnosic acid, and rosmarinic acid [17, 33, 59]. **Figure 3** shows the chemical structures of the main antioxidant compounds found in

*Description of R. officinalis parts and the main chemical structure of the antioxidant compounds.*

*R. officinalis* has been recognized as one of the plants with high antioxidant activity [28–30]. Its antioxidant effect is due to the phenolic compounds present in the leaves and stems [60]. Among the most effective antioxidant constituents, cyclic diterpene diphenols, carnosic acid, and carnosol were identified. In addition, its extract contains epirosmanol, rosmanol, metilcarnosato, isorosmanol, and other

The action mechanism of these compounds has been widely discussed in several studies. The carnosic acid and carnosol act as potent sequesters of peroxyl radicals and are responsible for 90% of the antioxidant properties, where both are inhibitors of lipid peroxidation in liposomal and microsomal systems, besides being good sequestrants of hydroxyl radicals. Specifically, carnosic acid removes hydrogen peroxide but may also act as a substrate for its ability to increase or maintain the superoxide dismutase and glutathione peroxidase activities. The most important elements in the *R. officinalis* structure are the diterpenes containing the aromatic ring (C11–C12) in the catechol group, with the conjugation of three basic rings. The

*DOI: http://dx.doi.org/10.5772/intechopen.83733*

*Antioxidant and Biological Activity of* Cissus sicyoides and Rosmarinus officinalis *Extracts DOI: http://dx.doi.org/10.5772/intechopen.83733*

**Figure 3.** *Description of R. officinalis parts and the main chemical structure of the antioxidant compounds.*

presence of 1,8-cineole, camphor, carnosic acid, and rosmarinic acid [17, 33, 59]. **Figure 3** shows the chemical structures of the main antioxidant compounds found in *R. officinalis*.

#### **3.3 Antioxidant and biological activity**

*R. officinalis* has been recognized as one of the plants with high antioxidant activity [28–30]. Its antioxidant effect is due to the phenolic compounds present in the leaves and stems [60]. Among the most effective antioxidant constituents, cyclic diterpene diphenols, carnosic acid, and carnosol were identified. In addition, its extract contains epirosmanol, rosmanol, metilcarnosato, isorosmanol, and other caffeic acid derivatives [61, 62].

The action mechanism of these compounds has been widely discussed in several studies. The carnosic acid and carnosol act as potent sequesters of peroxyl radicals and are responsible for 90% of the antioxidant properties, where both are inhibitors of lipid peroxidation in liposomal and microsomal systems, besides being good sequestrants of hydroxyl radicals. Specifically, carnosic acid removes hydrogen peroxide but may also act as a substrate for its ability to increase or maintain the superoxide dismutase and glutathione peroxidase activities. The most important elements in the *R. officinalis* structure are the diterpenes containing the aromatic ring (C11–C12) in the catechol group, with the conjugation of three basic rings. The

*Antioxidants*

tea had antidiabetic activity [55].

*Cladosporium cladosporioides* [57].

**3.** *Rosmarinus officinalis*

**3.1 Botanical description**

**3.2 Chemical composition**

possible alternative medicine in disease treatment.

blue, violet, and white flowers (**Figure 3**) [25, 26, 58].

was demonstrated that treatment of diabetic rats with *C. sicyoides* aqueous extract obtained by decoction, for 7 days (100 and 200 mg/kg), reduced blood glucose levels by 22 and 25%, respectively [49]. However, *C. sicyoides* leaf tea was used to investigate the plant therapeutic efficacy in volunteers who are diabetic and intolerant to glucose. A single dose of tea (1 g of dried leaf powder in 150 ml of water) was used for a period of 7 days. It was observed in people intolerant to glucose that the

*C. sicyoides* has antibacterial activity, showing inhibitory capacity against bacteria that cause food poisoning [56], which causes acute effects in the gastrointestinal tract and, in some cases, a high severity that patients come to death (*Bacillus cereus*, *Bacillus subtilis*, *Bacillus megaterium*, *Staphylococcus aureus*, and *Escherichia coli*). In addition, the antifungal activity of plant leaf and stem alcoholic extracts was demonstrated, inhibiting the growth of fungi *Cladosporium sphaerospermum* and

Recently, Salazar et al. carried out the biological activity determination of *C. sicyoides* supercritical extract; an in vivo test using a focal cerebral ischemia model was performed, and the extract had shown to have a neuroprotective and anti-inflammatory effect, justifying the use in traditional folk medicine for central nervous system diseases. These effects were associated to the presence of phenolic compounds in the extract [24]. Therefore, the results of these studies justify the traditional use of *C. sicyoides*, pointing to the plant extract potential benefit as a

*Rosmarinus officinalis* is an aromatic plant of the Lamiaceae family, native to the Mediterranean region, and is also cultivated in Central Asia, India, Southeast Asia, South Africa, Australia, the United States, and Brazil. Today, it has been grown in many parts of the world and is commonly known as rosemary. The plant is a bush that reaches from 0.50 to 1.50 m in height, with very pungent aroma leaves and

*R. officinalis* chemical composition varies greatly, due to some factors that directly influence the quality, amount of oil, and extract produced. However, it is possible to verify, through the literature, that its main chemical constituents are flavones, diterpenes, steroids, and triterpenes [17, 26, 27, 31]. The phenolic compounds present in *R. officinalis* were grouped into three classes: (i) phenolic acids (vanillic, caffeic, ferulic, and rosmarinic acids), (ii) diterpenes (carnosol, rosmadial, carnosic acid, methyl carbonate, rosmanol, epirosmanol, epiisorosmanol, epirosmanol methyl ether, and epiisorosmanol ethyl ether), and (iii) flavonoids (hesperetin, apigenin, genkwanin, 4′-methoxytectochrysin, cirsimaritin, scutellarein, 4″,5,7,8-tetrahydroxyflavone, homoplantaginin, and 6-hydroxyluteolin 7-glucoside) [27]. Recently, a *R. officinalis* chromatographic analysis was carried out, which revealed two large groups: oxygenated monoterpenes and hydrocarbonated monoterpenes. The main constituents of these groups were 1,8-cineole followed by camphor, borneol, and α- and β-pinene. The oxygenated and hydrocarbonated sesquiterpenes were composed of caryophyllene and caryophyllene oxide [26]. In the supercritical extracts of *R. officinalis* leaves, chemical analysis confirmed the

**320**

#### *Antioxidants*

catechol group is responsible for eliminating the radical electrons formed as an oxidation result. Lactone carnosol, rosmarinic acid, and hesperetin were cited in the literature as important FRSs [31, 63–65]. Rosmarinic acid has two aromatic rings, each with two OH groups that are capable of donating hydrogen and chelating metals [66]. Caffeic acid derivatives may act as metal ion chelators (Fe2+), thus reducing the formation of ROS [67].

The antioxidant of *R. officinalis* extracts obtained in SFE was confirmed. Carvalho et al. analyzed the plant antioxidant activity through a coupled reaction of β-carotene and linoleic acid; the results indicated that the extracts obtained at high pressures and low temperature (300 bar/40°C) exhibited the highest antioxidant activities, in comparison to extracts obtained in low pressures (150 bar/30°C). In any case, antioxidant activities were always above the control used (β-carotene and linolenic acid) as shown in **Figure 4**. The authors state that the antioxidant action remained approximately constant for the 3-h reaction for all extracts tested. The major compounds detected in the extracts were camphor (0.6% d.b.) and 1,8-cineol (0.043% d.b.). The extract obtained by hydrodistillation showed the highest yield of camphor (1.22% d.b.) and 1,8 cineol (0.23% d.b.) compared to other extraction methods (SFE and Soxhlet) [33].

Thus, the antioxidant capacity of *R. officinalis* leaf and stem extract obtained with supercritical CO2 was also evaluated by the ORAC method. The extract showed a high antioxidant capacity (1.9 mol Trolox/mg) similar to that of BHT and vitamin E (2.8–3.0 mol Trolox/mg). In addition, the extract presented a high percentage of lipid oxidation inhibition (88%) of fatty acids present in an analyzed cosmetic foundation. The extract volatile fraction was characterized by compounds such as camphor, 1,8-cineol, and trans-β-caryophyllene present in relative amounts of less than 25% [59].

Due to antioxidant properties, *R. officinalis* has been used in food preservation and in disease treatment. In food preservation, components such as rosmanol and carnosol prevent oxidation and microbial contamination and also can be up to four times more effective than BHA and equal to BHT as an antioxidant [27, 68, 69]. Different studies have demonstrated the potent activity of *R. officinalis* in inhibiting the formation of hydroperoxides, reducing carotenoid color loss, and retarding lipid oxidation in corn [78] and hazelnut oils [70].

The *R. officinalis* extract has been successfully commercially exploited as a natural antioxidant, for its use as synthetic antioxidants such as BHA, BHT, and TBHQ, in the food industry and is severely restricted as they may have carcinogenic effects on living organisms [5, 16, 17]. In this way, *R. officinalis* extract may be useful to

**323**

*Antioxidant and Biological Activity of* Cissus sicyoides and Rosmarinus officinalis *Extracts*

replace or even decrease the synthetic antioxidants in foods. As preservatives, the extracts offer several technological advantages and benefits to consumers [71]. Health problems derived from lipid oxidation have attracted consumers' and researchers' attention, since numerous diseases are linked to dietary and biological lipid oxidation products. Therefore, *R. officinalis* extracts have been related to several biological activities, such as anticancer, antidiuretic, anti-inflammatory, antibacterial, antidiabetic, antiangiogenic, antioxidant, and hepatoprotective [28, 71–74], besides allowing the use of the plant in the treatment and/or prevention of Alzheimer's disease, urinary and gastrointestinal infections, diabetes, aging, ischemia, and

Among the most important groups of compounds isolated from the plant, phenolic diterpenes account most of their biological activity. These compounds have been indicated in recent years as inhibitors of neuronal cell-induced death by a variety of agents both in vitro and in vivo, confirming the therapeutic potential of these compounds for Alzheimer's disease, due to the compounds multifunctional nature in the neuronal protection mediated by the plant antioxidant activity [32]. Several studies show that *R. officinalis* has pharmacological activity for chemoprevention and cancer therapy. In the extract antiproliferative activity evaluation against human ovarian cancer cells, it was corroborated that the extract inhibited the proliferation of cancer cell lines, affecting the cell cycle in multiple phases. In addition, it induced apoptosis by modifying the multiple gene expression that regulates apoptosis. Thus, the extract can be considered as an adjuvant to chemotherapy [62]. Also, the antiangiogenic effect of the carnosic acid present in *R. officinalis* extract was corroborated in angiogenesis models using human umbilical vein endothelial cells in relation to the tube formation in the reconstituted basement membrane, chemotaxis, and proliferation. Carnosic acid from the extract may be useful in preventing disorders due to angiogenesis, and its

antiangiogenic effect may contribute to a neuroprotective effect [72].

n-butanol showed a high anti-hypercholesterolemic activity [76].

tions to inflammatory disease prevention [77].

The anti-inflammatory activity of *R. officinalis* supercritical extracts was studied. Absorptions of extract fractions were tested on monolayers of Caco-2 cells (2–12 h of incubation). Human macrophages were treated with basolateral fractions, and TNF-α, IL-1β, IL-6, and IL-10 secretions were measured by ELISA. Fractions obtained after 8 and 12 h in absorption experiments caused a considerable reduction in the excretion of pro-inflammatory cytokines. This reduction in cytokine secretion levels was associated with the amounts of carnosol and carnosic acid. Thus, the *R. officinalis* supercritical extract can be used in formula-

The actions of *R. officinalis* leaf ethanolic extract obtained with Soxhlet extraction were tested in glucose homeostasis and antioxidant defense in rabbits. Serum levels, glucose levels and insulin levels were studied in diabetic rabbits (alloxan was used to induce diabetes); it was shown that at a dose of 200 mg/kg was possible to reduce the blood glucose level and to increase the serum insulin concentration. In addition, during 1 week of animal treatment with the extract, it was demonstrated that it had an ability to inhibit lipid peroxidation and to activate the antioxidant enzymes. Due to its potent antioxidant properties, the plant extract has a remarkable antidiabetogenic effect [75]. Recently, the antidiabetic and antihypercholesterolemic action of flavonoid-rich fractions of *R. officinalis* (fractions obtained with n-butanol and diethyl ether) in diabetic mice induced by streptozotocin was evaluated. Both fractions showed a decrease in the glucose level at a dose of 400 mg/kg, especially the fraction obtained with diethyl ether; plasma glucose levels decreased up to 60.38%. The pancreas histopathological study showed that both fractions regenerated the pancreatic β cells and increased the mass of islets. *R. officinalis* fractions exhibited a potent antidiabetic effect, while the fraction obtained with

*DOI: http://dx.doi.org/10.5772/intechopen.83733*

atherosclerosis [25, 32].

**Figure 4.** *Antioxidant activity of R. officinalis extracts obtained with supercritical CO2 [38].*

#### *Antioxidant and Biological Activity of* Cissus sicyoides and Rosmarinus officinalis *Extracts DOI: http://dx.doi.org/10.5772/intechopen.83733*

replace or even decrease the synthetic antioxidants in foods. As preservatives, the extracts offer several technological advantages and benefits to consumers [71].

Health problems derived from lipid oxidation have attracted consumers' and researchers' attention, since numerous diseases are linked to dietary and biological lipid oxidation products. Therefore, *R. officinalis* extracts have been related to several biological activities, such as anticancer, antidiuretic, anti-inflammatory, antibacterial, antidiabetic, antiangiogenic, antioxidant, and hepatoprotective [28, 71–74], besides allowing the use of the plant in the treatment and/or prevention of Alzheimer's disease, urinary and gastrointestinal infections, diabetes, aging, ischemia, and atherosclerosis [25, 32].

Among the most important groups of compounds isolated from the plant, phenolic diterpenes account most of their biological activity. These compounds have been indicated in recent years as inhibitors of neuronal cell-induced death by a variety of agents both in vitro and in vivo, confirming the therapeutic potential of these compounds for Alzheimer's disease, due to the compounds multifunctional nature in the neuronal protection mediated by the plant antioxidant activity [32].

Several studies show that *R. officinalis* has pharmacological activity for chemoprevention and cancer therapy. In the extract antiproliferative activity evaluation against human ovarian cancer cells, it was corroborated that the extract inhibited the proliferation of cancer cell lines, affecting the cell cycle in multiple phases. In addition, it induced apoptosis by modifying the multiple gene expression that regulates apoptosis. Thus, the extract can be considered as an adjuvant to chemotherapy [62]. Also, the antiangiogenic effect of the carnosic acid present in *R. officinalis* extract was corroborated in angiogenesis models using human umbilical vein endothelial cells in relation to the tube formation in the reconstituted basement membrane, chemotaxis, and proliferation. Carnosic acid from the extract may be useful in preventing disorders due to angiogenesis, and its antiangiogenic effect may contribute to a neuroprotective effect [72].

The actions of *R. officinalis* leaf ethanolic extract obtained with Soxhlet extraction were tested in glucose homeostasis and antioxidant defense in rabbits. Serum levels, glucose levels and insulin levels were studied in diabetic rabbits (alloxan was used to induce diabetes); it was shown that at a dose of 200 mg/kg was possible to reduce the blood glucose level and to increase the serum insulin concentration. In addition, during 1 week of animal treatment with the extract, it was demonstrated that it had an ability to inhibit lipid peroxidation and to activate the antioxidant enzymes. Due to its potent antioxidant properties, the plant extract has a remarkable antidiabetogenic effect [75]. Recently, the antidiabetic and antihypercholesterolemic action of flavonoid-rich fractions of *R. officinalis* (fractions obtained with n-butanol and diethyl ether) in diabetic mice induced by streptozotocin was evaluated. Both fractions showed a decrease in the glucose level at a dose of 400 mg/kg, especially the fraction obtained with diethyl ether; plasma glucose levels decreased up to 60.38%. The pancreas histopathological study showed that both fractions regenerated the pancreatic β cells and increased the mass of islets. *R. officinalis* fractions exhibited a potent antidiabetic effect, while the fraction obtained with n-butanol showed a high anti-hypercholesterolemic activity [76].

The anti-inflammatory activity of *R. officinalis* supercritical extracts was studied. Absorptions of extract fractions were tested on monolayers of Caco-2 cells (2–12 h of incubation). Human macrophages were treated with basolateral fractions, and TNF-α, IL-1β, IL-6, and IL-10 secretions were measured by ELISA. Fractions obtained after 8 and 12 h in absorption experiments caused a considerable reduction in the excretion of pro-inflammatory cytokines. This reduction in cytokine secretion levels was associated with the amounts of carnosol and carnosic acid. Thus, the *R. officinalis* supercritical extract can be used in formulations to inflammatory disease prevention [77].

*Antioxidants*

the formation of ROS [67].

methods (SFE and Soxhlet) [33].

oxidation in corn [78] and hazelnut oils [70].

*Antioxidant activity of R. officinalis extracts obtained with supercritical CO2 [38].*

catechol group is responsible for eliminating the radical electrons formed as an oxidation result. Lactone carnosol, rosmarinic acid, and hesperetin were cited in the literature as important FRSs [31, 63–65]. Rosmarinic acid has two aromatic rings, each with two OH groups that are capable of donating hydrogen and chelating metals [66]. Caffeic acid derivatives may act as metal ion chelators (Fe2+), thus reducing

The antioxidant of *R. officinalis* extracts obtained in SFE was confirmed. Carvalho et al. analyzed the plant antioxidant activity through a coupled reaction of β-carotene and linoleic acid; the results indicated that the extracts obtained at high pressures and low temperature (300 bar/40°C) exhibited the highest antioxidant activities, in comparison to extracts obtained in low pressures (150 bar/30°C). In any case, antioxidant activities were always above the control used (β-carotene and linolenic acid) as shown in **Figure 4**. The authors state that the antioxidant action remained approximately constant for the 3-h reaction for all extracts tested. The major compounds detected in the extracts were camphor (0.6% d.b.) and 1,8-cineol (0.043% d.b.). The extract obtained by hydrodistillation showed the highest yield of camphor (1.22% d.b.) and 1,8 cineol (0.23% d.b.) compared to other extraction

Thus, the antioxidant capacity of *R. officinalis* leaf and stem extract obtained with

The *R. officinalis* extract has been successfully commercially exploited as a natural antioxidant, for its use as synthetic antioxidants such as BHA, BHT, and TBHQ, in the food industry and is severely restricted as they may have carcinogenic effects on living organisms [5, 16, 17]. In this way, *R. officinalis* extract may be useful to

supercritical CO2 was also evaluated by the ORAC method. The extract showed a high antioxidant capacity (1.9 mol Trolox/mg) similar to that of BHT and vitamin E (2.8–3.0 mol Trolox/mg). In addition, the extract presented a high percentage of lipid oxidation inhibition (88%) of fatty acids present in an analyzed cosmetic foundation. The extract volatile fraction was characterized by compounds such as camphor, 1,8-cineol, and trans-β-caryophyllene present in relative amounts of less than 25% [59]. Due to antioxidant properties, *R. officinalis* has been used in food preservation and in disease treatment. In food preservation, components such as rosmanol and carnosol prevent oxidation and microbial contamination and also can be up to four times more effective than BHA and equal to BHT as an antioxidant [27, 68, 69]. Different studies have demonstrated the potent activity of *R. officinalis* in inhibiting the formation of hydroperoxides, reducing carotenoid color loss, and retarding lipid

**322**

**Figure 4.**

In relation to the antibacterial activity, *R. officinalis* essential oils obtained by hydrodistillation exhibited antibacterial activity against *Escherichia coli*, *Salmonella typhi*, *S. enteritidis*, and *Shigella sonnei*; this activity was associated with the oil ability to reduce DPPH radical formation (CI50 = 3.82 μg/ml) [61]. However, the antibacterial and antifungal activities of *R. officinalis* leaf extracts obtained by SFE extraction were confirmed, and the extracts showed antibacterial activity against Gram-positive bacteria (*Staphylococcus aureus* and *Bacillus cereus*) and Gram-negative bacteria (*Escherichia coli* and *Pseudomonas aeruginosa*) and antifungals against *Candida albicans*. Obtaining *R. officinalis* extracts by SFE has been shown to be a promising extraction with respect to its incorporation into various foods, cosmetics, and pharmaceuticals products that a natural aroma, color, and antioxidant/antimicrobial additive are desired. These properties are also necessary for the food industry in order to find possible alternatives to synthetic preservatives [17].
