Presentation of the antioxidant activity values of different plant extracts obtained with SFE, involving the plants under study (
This chapter will describe the antioxidant and biological activity of Cissus sicyoides and Rosmarinus officinalis leaf extracts, which represent an important natural source of antioxidants. These plants contain several bioactive compounds with high antioxidant activity, such as phenolic compounds, which are compounds that prevent or delay oxidative stress, acting as free radical scavengers (FRSs), and thus reduce the onset of cardiovascular disease, cancer, diabetes, epilepsy, stroke, among other diseases. The supercritical fluid extraction (SFE) has been studied to obtain antioxidant compounds from natural sources, without the drawbacks associated with conventional extraction processes, such as the use of organic solvents, which present toxicity and contaminate the extracts, is proposed.
- C. sicyoides
- R. officinalis
- antioxidant activity
- biological activity
- supercritical extraction
The Amazonian biodiversity presents a great source of foods and medicinal plants rich in antioxidant compounds whose study and conscious exploration contribute to the region sustainable development [1, 2]. The plants have a great importance due to their medicinal and nutritional properties. About 70–90% of the world population prefers the use of medicinal plants or plant extracts to treat common diseases [3, 4]. Plants have been extensively studied in recent years for their antioxidant activity. The main classes of plant chemicals are phenolic compounds, tocopherols, carotenoids, and alkaloids. Among these compounds, phenolic compounds are the most important. They prevent or delay oxidative stress, acting as free radical scavengers (FRSs), and thus reduce the onset of different chronic diseases [5, 6, 7, 8].
Antioxidants are a set of substances that can delay or inhibit oxidation reactions and act as a defense mechanism to neutralize the harmful effects of oxidation in biological systems and foods [6, 9, 10]. Oxidative stress is considered a state of imbalance where excessive amounts of reactive oxygen and nitrogen species (ROS/RNS, for example, superoxide anion, hydrogen peroxide, hydroxyl radical, peroxynitrite) exceed the capacity of endogenous antioxidants (uric acid, superoxide dismutase, catalase, glutathione peroxidase), leading to the oxidation of a biomacromolecule variety such as enzymes, proteins, DNA, and lipids. Exogenous antioxidants (phenolic compounds, carotenoids, tocopherols, and ascorbates) are consumed in the diet mainly of fruits, leaves, seeds, vegetables, and cereals, they have the function of increasing or protecting the antioxidant defense in biological systems and, therefore, they are important for endogenous oxidative stability [11, 12, 13].
It is conflicting that oxygen and nitrogen, considered essential for biological processes, are also cofactors for toxic and degenerative processes. In this sense, the antioxidant compounds act through different chemical mechanisms in order to minimize or maintain redox balance in vivo [9, 14]. There are several mechanisms by which oxidation can be inhibited. In general, the mechanisms involved include FRSs, ester bond enzymatic hydrolysis, transition metal ion sequestration, and enzyme-catalyzed peroxide reduction. The last three mechanisms mentioned do not cease reactive species action, but prevent the formation of molecules capable of promoting free radical chain reactions .
There is a growing interest in new sources of natural antioxidant compounds due to synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ) in the food industry being severely restricted, since they may show carcinogenic effects on living organisms [16, 17, 18]. In this sense, the scientific community and consumers are looking for new bioactive compounds of natural origin that can be used to develop new treatments against diseases. In addition, they may be employed in the food industry as functional food ingredients.
Supercritical fluid extraction (SFE) has already been studied to obtain bioactive compounds from natural sources. Salazar et al. and Carvalho et al. showed that the application of SFE technology is successful in obtaining extracts from
Supercritical CO2 (Sc-CO2) has a limitation in dissolving polar molecules. However, this disadvantage can be solved by polar solvent addition, called modifiers or cosolvents, which modify the supercritical fluid polarity and, consequently, improve polar fraction extraction rich in bioactive substances, such as phenolic compounds related to high antioxidant activity [37, 38]. The aim of this chapter is to describe the antioxidant and biological activity of
2.1 Botanical description
2.2 Chemical composition
The bioactive compounds present in the leaf and stem are represented by carotenoids (α-carotene and β-carotene)  and phenolic compounds such as flavonoids (quercetin 3-O-rhamnoside and kaempferol 3-O-rhamnoside) . But also, three new flavonoid glycosides were found, denominate cissosides I, II, and III (kaempferol 3-O-α-L-(5”-O-acetyl)-arabinofuranosyl-7-O-α-L-rhamnopyranoside, quercetin 3-O-α-L-arabinofuranosyl-7-O-α-L-rhamnopyranoside, and quercetin 3-O-α-L-(5”-O-acetyl)-arabinofuranosyl-7-O-α-L-rhamnopyranoside) .
Recently, three different flavonoids were identified (quercetin-3-O-hexoside, quercetin-3-O-deoxyhexoside, and kaempferol-3-O-deoxyhexoside) . In addition, resveratrol (3,5,4′-trihydroxystilbene)  and a new benzofuran-type stilbene (cissusin) , tannins, coumarins (glycoside 5,6,7,8-tetrahydroxycoumarin-5β-xylopyranoside and sabandin), and steroids (β-sitosterol and 3β-O-β-D-glucopyranosyl sitosterol) were found . The presence of essential oils was also detected . In the supercritical extracts of
2.3 Antioxidant and biological activity
Figure 2 shows the results of the qualitative analysis of antioxidant activity by HPTLC of C. sicyoides extracts obtained by supercritical extraction (essays 1–15), hexane extract (HE), and ethanolic extract (EE) obtained with conventional extraction by Soxhlet. The plaque was derivatized with DPPH (2,2-diphenyl-1-picrylhydrazyl), and it was possible to detect the presence of yellow spots on the plaque purple bottom resulting from the reduction of the DPPH•; in the presence of antioxidant substances, 2,2-diphenyl-picryl-hydrazine is reduced, losing its purple coloration. The study results confirmed the presence of chemical constituent characteristic of this plant, with antioxidant activity. In the same study, a quantitative determination of the antioxidant activity by the DPPH method was carried out. It was demonstrated that with the extractive methodologies (SFE and Soxhlet) used it was possible to extract with low EC50 values, related to a high antioxidant activity; for EE, the value of EC50 (325.67 g of extract/g of DPPH) is similar to the value of EC50 (404.81 g of extract/g of DPPH) obtained with SFE .
In the in vitro antioxidant activity determination by the ABTS method of the
Due to antioxidant properties,
Several studies point to the application of
Regarding the plant anti-inflammatory activity, it has been demonstrated that the oral administration of 300 and 500 mg/kg of the
In addition, pharmacological effects were detected in the treatment and/or prevention of dysfunctions such as hypertension and vasoconstriction of arteries, veins, and capillaries with aqueous extract of
In the evaluation of the antidiabetic potential of
Recently, Salazar et al. carried out the biological activity determination of
3.1 Botanical description
3.2 Chemical composition
3.3 Antioxidant and biological activity
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
The antioxidant of
Thus, the antioxidant capacity of
Due to antioxidant properties,
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,
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 .
Several studies show that
The actions of
The anti-inflammatory activity of
In relation to the antibacterial activity,
4. Supercritical fluid extraction (SFE) of antioxidant compounds from plant matrices
When a new extract from a natural source is tested, the most important aspects to take into account are the extraction method and the type of solvent used, as this will affect the antioxidant properties. Several extraction methods for the selective extraction from plant matrices such as
Thus, the bioactive compound extraction has been considered one of the most important steps in the approach of obtaining or recovering bioactive compounds. Conventional extractions have been the most used technology for these compound recovery. It is based on the extraction power of different solvents and the application of high temperatures, promoting mass transfer. However, there are drawbacks associated with conventional extraction processes such as the use of large amounts of organic solvents, toxic to human health and the environment, extraction time, and the use of high temperatures that can degrade the thermosensitive compounds [6, 8]. They motivated the search for environmentally safe extraction techniques such as microwave-assisted extraction (MAE), ultrasonic-assisted extraction (UAE), pressurized liquid extraction (PLE), and supercritical fluid extraction (SFE) [36, 78]. SFE has already been studied to obtain antioxidant compounds from natural sources [24, 33, 77, 79]. Table 1 presents the antioxidant activity values of different plant extracts obtained with SFE, involving the plants under study (
4.1 SFE procedure
A solvent is considered a supercritical fluid when the pressure and temperature of the system are above its critical point. This point is defined as the highest temperature and pressure at which a substance can exist in equilibrium between the liquid and vapor phases. Above its critical temperature (Tc) and critical pressure (Pc), the supercritical fluid can be considered as an expanded liquid or as a compressed gas, whose density (ρ) is relatively high and consequently has a high solvency power. This effect gives the solvent a certain degree of selectivity, in addition to allowing easy separation of the solvent from the solute, which can be achieved by a simple system depressurizing, resulting in products totally solvent-free and without thermal degradation of the compounds of interest, due to low operating temperatures [35, 38].
One of the most commonly used solvents in SFE is carbon dioxide (CO2) because its critical points are moderate (Tc = 31.1°C, Pc = 73.8 bar, and critical density (ρc) = 0.468 g/cm3), nontoxic, non-flammable, affordable, chemically inert, and apolar and has an ideal behavior for thermosensitive compound extraction [34, 37]. In addition to the supercritical CO2 (Sc-CO2), there are other substances that are also used as supercritical fluids, as shown in Table 2.
|Plants||Extraction conditions||Solvents||Method of determination||Antioxidant capacity||Biological Activity||Refs.|
|40°C/400 bar||CO2 + 10% of ethanol||DPPH||404.81 ± 2.78 EC50: g of extract/g of DPPH||Neuroprotective and anti-inflammatory effect|||
|40°C/300 bar||CO2||DPPH||12.85 ± 0.10 IC50: μg.ml−1||Antioxidant, antibacterial, and antifungal|||
|100°C/350 bar||CO2||DPPH||0.23 ± 0.01 IC50: mg/ml||Antioxidant|||
|50°C/300 bar||CO2||ORAC||1.9 ± 0.10 μmol Trolox/mg extract||Antioxidant|||
|55°C/100 bar||CO2 + 20% of ethanol||DPPH||2.13 ± 0.24 EC50: DPPH μg/μg dry extract||Antioxidant|||
|60°C/400 bar||CO2 + ethanol||DPPH||>200 EC50 (μg/ml)||Antioxidant|||
|35°C/400 bar||CO2||DPPH||359 mg TE/100 g dry extract||Antioxidant and anti-inflammatory|||
|40°C/300 bar||CO2||DPPH||103.28 EC50: of μg.ml−1||Antioxidant|||
|Fluid||Tc (°C)||Pc (bar)||ρc (g/cm3)|
|Nitrous oxide (N2O)||36.5||71.0||0.457|
Due to its low polarity, Sc-CO2 presents a limitation to dissolve polar molecules. However, this disadvantage can be solved by the addition of polar solvents, called modifiers or cosolvents, which modify the supercritical fluid polarity and, consequently, improve the extraction of polar fractions rich in bioactive substances, such as phenol compounds related to high antioxidant activity [37, 38]. Methanol is the solvent most used as a modifier for various plant matrices, but it is toxic and different from ethanol, which is an environmentally safe solvent being a good choice for SFE processes, and can be used in the extraction of natural products [80, 81]. Water is also a very attractive cosolvent for natural product extraction due to its high polarity, which considerably increases the polarity of Sc-CO2 .
For antioxidant compound extraction and recovery by SFE, several vegetable matrices were used, such as seeds, fruits, leaves, flowers, rhizomes, roots, fruit peels, and tree branches. The SFE process consists basically in the extraction of soluble compounds present in the solid matrix by a supercritical solvent and then separates these compounds from the solvent after depressurizing the system. In order to achieve an efficient and adequate extraction, several factors must be taken into account, having a careful control of the operating conditions and process step optimization [35, 36, 82].
Initially, the raw material must pass through a pretreatment stage before being fed into the fixed bed extractor; this procedure is performed to prepare the solid particles, allowing a greater efficiency to be achieved in the extraction process . As shown in Figure 5, after the raw material is collected, one of the first stages of its pretreatment is the solid matrix moisture reduction, for example, drying leaves in an oven with air circulation. Generally, the plant matrix moisture should not exceed 14% (wet basis). Another important step is the moisture content determination by the distillation method of the Jacobs immiscible solvent, with the purpose of knowing if the quantity of water in the sample is adequate for the supercritical extraction process. The sieving stage is applied to standardize and determine the average particle size of the solid particles. The real and apparent density and bed porosity determination is also very important as they affect the particles packaging in the extraction vessel and consequently the solvent flow and the mass and heat transfer processes [35, 82].
After a suitable pretreatment, the solid matrix is placed in an extraction vessel forming a fixed bed. Depending on the compounds of interest, the supercritical solvent (Sc-CO2) or solvent + cosolvent is fed by the solvent pump and/or cosolvent into the extraction vessel, where it continuously flows through the fixed bed and dissolves the extractable components from the solid matrix. The mixture of solutes that is removed from the solid matrix is called extract. In the separation step, the mixture formed by the solvent extraction + extract leaves the vessel and feeds the separator (collection flask) where the mixture is separated by rapid reduction of pressure (ambient pressure). The extract precipitates in the separator, and the solvent is removed from the system [38, 83].
The identification of new natural antioxidant compounds is of great interest to the food, pharmaceutical, and cosmetic industry in order to find possible alternatives to synthetic antioxidants. In this way, plants such as
Mar Salazar (Process Number: 1777277) thanks CAPES for the doctorate scholarship.
Conflict of interest
The authors have no conflict of interest to declare and are responsible for the content and writing of the manuscript.