Cytotoxic and Antioxidant Properties of Natural Bioactive Monoterpenes Nerol, Estragole, and 3,7-Dimethyl-1-Octanol

The therapeutic potential of medicinal plants is noted because of the presence of varieties of biochemicals. The monoterpenes, like nerol, estragole, and 3,7-dimethyl-1-octanol, have been reported for antimicrobial, antifungal, anthelmintic, and antioxidant activities. This study evaluated the toxic, cytotoxic, and oxidant/antioxidant effects of these compounds by several in vitro (DPPH and ABTS radical scavenging, and ferric reducing potential), ex vivo (hemolysis), and in vivo (Artemia Salina and Saccharomyces cerevisiae) assays. Results suggest that estragole and 3,7-dimethyl-1-octanol at 31.25–500 μg/mL did not exhibit significant cytotoxic effects in the A. Salina and hemolysis tests. Nerol showed significant cytotoxic effects on these test systems at all test concentrations. The monoterpenes showed radical (ABTS•+ and DPPH•) scavenging capacities in a concentration-dependent manner in vitro tests. However, they did not oxidize the genetic material of S. cerevisiae (SODWT, Sod1Δ, Sod2Δ, Sod1/Sod2Δ, Cat1Δ, and Cat1Δ/Sod1Δ) lines. Among the three monoterpenes, nerol may be a good candidate for antioxidant and anti-tumor therapies.


Introduction
At present, many studies have focused on fnding alternatives to traditional treatments for many diseases in plants [1][2][3]. Active compounds extracted from plants show their efcacy in many pathologies [4][5][6][7][8][9][10], even cancers [11,12], but their application in the clinic is many times limited by low availability in the target organs [13,14]. Te evolution of nanomedicine has tried to solve some of these bioavailability problems of natural bioactive compounds [15], but things are still in the testing step due to the potential toxicity of these nanoformulations [16][17][18][19]. Aromatherapy is part of phytotherapy, which uses essential oils extracted from aromatic plants for the treatment or as an adjuvant in the traditional treatment of several clinical pathologies [20][21][22]. Te composition of essential oils is complex and depends on a series of factors, like the species of plant from which they are extracted, the technique used for the extraction, the geographic location where the plant is growing, and also the harvest time [23,24]. Te main components of essential oils are terpenoids and phenylpropanoids [25,26]. Monoterpenes are chemical compounds found in medicional plant-derived essential oils. Tey have a pleasant aroma and are used commercially as favorings, fragrances, and cleaning agents. Tey are also well-known for their signifcant biological functions, including their analgesic, antispasmodic, antibacterial, anti-infammatory, antifungal, and schistosomicidal properties [27]. Nerol, estragole, and 3,7-dimethyl-1-octanol ( Figure 1) are monoterpenes that exist in essential oils extracted from various plant species, such as Croton zehntneri, Cymbopogon citrates, Cymbopogon nardus, Citrus bergamani, Zingiber ofcinalle, and Lavandulasp., and have sedative efects, stimulatory efects on appetite, and benefcial efects on intestinal disorders. Tey are also known for their attractive, repellent, and toxic efects on insects and microorganisms [10,28].
Oxidative stress is involved in the onset and/or development of many chronic diseases. Certain defence mechanisms act to maintain the levels of reactive species (e.g., oxygen/nitrogen; ROS/RNS) under normal conditions and to prevent oxidative damage as the action of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH). Many natural compounds have been investigated for their antioxidant efects through inhibiting free radicals/chemicals that could inhibit oxidative damage [33,34].

Preparation of Test Compounds.
Te tested monoterpenes (nerol, estragole, and 3,7-dimethyl-1-octanol) and standards (trolox and K 2 Cr 2 O 7 ) were dissolved in the abovementioned vehicle to obtain the desired concentration for each assay.

Artemia salina Bioassay.
Tis test was carried out with a slight modifcation, as described by Amaral et al. [35]. Briefy, shrimp cysts were incubated in a small aquarium containing a brine solution (50 mg of cysts and 300 mL of brine) for 48 h with direct lighting. Test samples (nerol, 3,7dimethyl-1-octanol, and estragole) were diluted in the brine solution to get a concentration in the range of 31.25 to 500 μg/mL with a fnal volume of 5 mL. Ten larvae were added to each tube, and after 24 h, manual observation was done to count the number of live naupli. In the negative (NC) and positive (PC) control groups, saline solution and potassium dichromate (K 2 Cr 2 O 7 ) were added, respectively. Te concentration range used in the case of the PC group was 31.25 to 500 μg/mL.

Determination of Hemolytic Activity in Mice Erythrocytes.
Tis test was performed on adult Swiss albino mouse (Mus musculus) erythrocytes. Te mouse was collected from the Central Animal House of the Federal University of Piaui, Brazil. It was allowed free access to standard food and water ad libitum and was kept under controlled lighting (12 h dark/ light cycles) at 24 ± 2°C. Blood was collected from the retroorbital plexus of the mouse (male, 24 gm body weight). Hemolytic activity in mouse erythrocytes was performed, according to Jamialahmadi et al. [36]. Briefy, 300 μL of erythrocyte suspension (10% suspension in PBS, pH 7.4) was mixed with 500 μL of each monoterpene (31.25-500 μg/mL) and placed in a water bath at 37°C for 2 h. After the reaction time, the reaction mixture was centrifuged at 2000 rpm for 5 min, and the absorbance of the supernatant was measured at 540 nm and the results were compared to the triton X-100 (31.25-500 μg/mL). . Te absorbance was measured using a spectrophotometer at 515 nm.

Ferric Reducing Potential Assay.
For this assay, the method described earlier by Singhal et al. [39] was followed. Briefy, a mixture of 500 μL of sodium phosphate bufer (0.2 M, pH 6.6) and 500 μL of 1% potassium ferricyanide was added to the tubes containing monoterpenes (0.9-14.4 μL/ mL) and was subsequently placed in a water bath at 50°C for 20 min. After that, 500 μL of 10% trichloroacetic acid, 500 μL of distilled water, and 250 μL of 0.1% ferric chloride were added, followed by an absorbance measurement at 700 nm using a spectrophotometer. Te NC and PC groups were carried out with the vehicle (0.05% tween-80 dissolved in 0.9% NaCl solution) and Trolox, respectively.

In Vivo Antioxidant (S. cerevisiae) Test.
Te central disc test was done according to the method presented by Islam et al. [40]. Te S. cerevisiae strains used in the assay with their genotypes are shown in Table 1. Superoxide dismutase (CuZnSOD-Sod1) is a cytosolic enzyme that is faulty in strain EG118, whereas mitochondrial SOD is faulty in strain EG110 (MnSOD-Sod2). Te strain EG133 has both Sod1 and Sod2 defects, while EG223 has a cytosolic catalase defciency (Cat1). While EG103 is a competent strain that is identical to the wild-type (SODWT), EG is a dual isogenic strain for the Sod1 and Cat1 genes.

Statistical Analysis.
Te results are expressed as the mean ± standard deviation (SD). Te results were evaluated by analysis of variance (ANOVA) followed by a post hoc Newman-Keuls test using GraphPad Prism (version: 6.00; Windows, Graph Pad Software, San Diego California, USA). A P-value <0.05 was considered signifcant criterion.

Brine Shrimp Lethality Bioassay.
In the brine shrimp lethality bioassay (BSLB), all studied monoterpenes except nerol exhibited low toxicity in 24 hours (Figure 2(a)). After 48 h of exposure, the nerol showed increased toxicity, more specifcally, at the dose of 62.5 μg/mL the mortality was 100% similar to that of the positive control group (Figure 2(b)). Estragole toxicity increased in a dose-dependent manner from the concentration of 250 μg/mL, while 3,7-dimethyl-1-octanol toxicity increased in a dose-dependent manner from the concentration of 125 μg/mL.

Determination of Hemolytic Activity in Mice Erythrocytes.
In the hemolysis test, all the monoterpenes showed <25% hemolysis, up to a concentration of 250 μg/mL. However, nerol at 500 μg/mL exhibited hemolysis by 39.12% (Table 2).
Te data presented in Figures 3(a) and 3(b) show a variation in the inhibition of DPPH and ABTS radicals in a concentration-dependent manner by the test samples. An increased dose was found to augment an increase in the scavenging capacity of the radicals. Although the activity of the monoterpenes was less than that of the standard drug, Trolox, nerol, and estragole showed the highest scavenging activity against ABTS •+ and DPPH • , Advances in Pharmacological and Pharmaceutical Sciences respectively. Te presence of allylic hydrogens (number: 13) in nerol (Figure 4(a)), which forms a resonance structure, may be responsible for its antioxidant capacity. Possible hydrogen abstraction and formation of R + is shown in Figure 4(b).
In the ferric reduction assay, all the monoterpenes showed a low reduction capacity of iron (Fe +3 to Fe +2 ) compared with the standard drug, Trolox. In this test, all the monoterpenes had almost the same activity at all concentrations tested ( Figure 5).
Data indicate that nerol ( Figure 6), estragole (Figure 7), and 3,7-dimethyl-1-octanol ( Figure 8) showed signifcant antioxidant activity in S. cerevisiae, as they inhibited oxidative damage caused by the stressor, H 2 O 2 in all strains and concentrations tested. Te percentage modulation of oxidative damage was found to be more than 60%. Te strains were cultured in YEPD medium (Yeast extract 0.5%, bacto-peptone 2% and dextrose 2%) at 28°C HYPERLINK [51]. Suspended cells were seeded in the center of a Petri dish in a continuous cycle. H2O2 (10 mM) was used as an inducer of oxidative stress and formed the positive control (PC) group. Saline 0.9% and DMSO 0.05% groups are the negative control (NC) groups.

Discussion
BSLB is a cheap, rapid, and sensitive test to assay a wide variety of toxicants, including bioactive molecules [42,43]. In our study, we found a dose-response relationship for all tested monoterpenes along with time-dependent cytotoxicity. Te EC 50 > 1000 μg/mL was classifed as inactive (or nontoxic), while the EC 50 < 100 μg/mL for highly active (very toxic) in the A. Salina test [44]. In our study, nerol showed prominent cytotoxicity, which is concurrent with an earlier study [45]. On the other hand, 3,7-dimethyl-1-octanol has been noted for its higher cytotoxic potential compared with estragole.
According to Carvalho [38], toxicity tests should be considered indispensable in the biomonitoring of plant extracts. Another test for toxicological screening of xenobiotics is the in vitro evaluation of hemolytic capacity estimated by erythrocyte damage [46]. Tere are several variations of this test using human or animal erythrocytes such as rabbits and mice [46][47][48][49]. Te hemolytic activity in the erythrocytes of mice has a great similarity to the human erythrocytes [44]. Tis test evaluates the potential of xenobiotics to cause damage to the plasma membrane of the cell, either by the formation of pores or a total rupture of the membrane. Tis test is very important and can serve as a preliminary test before preclinical and clinical studies [46]. In this study, we observed that of the three tested compounds, nerol has the highest potential to produce hemolysis in a dose-dependent manner, with the highest hemolysis appearing at a concentration of 500 μg/mL.     Concentration (μg/mL) Ferric reduction potential at 700 nm (mV) Figure 5: Ferric reduction potential of estragole, nerol, 3,7-dimethyl-1-octanol and Trolox. Notes: Values are mean ± SD of triplicate value. * p < 0.05 vs. NC (ANOVA followed by t-Studentpost-hocNeuman-Keuls test). NC showed negligible ferric reduction capacity (1.33 ± 0.78); therefore, the value has not shown in the fgure. 6 Advances in Pharmacological and Pharmaceutical Sciences A persistent radical with a modest rate of deterioration and interactions with the majority of substances, DPPH•. Only powerful reducing chemicals can interact with DPPH to stabilize it in a stoichiometric phase [50,51]. As opposed to this, 2, 2-azobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) is oxidized in the presence of K 2 S 2 O 8 solution and results in the ABTS• + radical. It is very stable and soluble in polar and nonpolar solvents. It remains unafected by the ionic strength of many solvents. Terefore, this radical is suitable for both polar and nonpolar extracts [44]. In a study, Oliveira [41] also reported a mild antioxidant capacity of estragole at concentrations from 7.83 to 500 μg/mL. In this study, we also observed a concentration-dependent (0.9-14.4 μg/mL) anti-radical activity against DPPH and ABTS radicals. However, the activity was not strong in comparison to the PC group. Estragole showed that scavenger propertied in a dose-dependent manner also in previous studies [30,52]. In a previous study, we reported the inhibition of lipid peroxidation and protein oxidation by substances having anti-radical and strong reducing capacity [53]. Tat is why we use the ferric reducing potential technique for evaluating the antioxidant efects of our compounds. Te reduction of iron ions (Fe 3+/ Fe 2+ ) is infuenced by the solubility of the reducing compounds, which in turn is guided by the presence of other acyclic, monocyclic, and bicyclic monoterpene components, derived from phenyl propane and sesquiterpene lactones [44,54]. Tus, the reducing capacity of the tested monoterpenes may be afected by this consequence. In the case of our compounds, there were no diferences between the 3 monoterpenes tested, but all showed low reduction capacity of iron.

Advances in Pharmacological and Pharmaceutical Sciences
S. cerevisiae is a popular test system to estimate the oxidative stress capacities of a wider number of biological components. Te metabolism of S. cerevisiae is quite similar to that of higher eukaryotes, featuring the activation of cytochrome P450 and detoxifcation [55]. Te yeasts, mainly the S. cerevisiae species, are widely used in the food and beverage industries and research laboratories [56]. Being a eukaryotic system, S. cerevisiae has similarities with mammalian cells at the level of its organelles and macromolecular components, and many of its proteins can be functionally interchangeable with homologous human proteins. Teir genetic manipulation is also cheaper compared to other models. In addition, the antioxidant response of S. cerevisiae is similar to that of mammalian cells, and 30% of genes related to human diseases are their functional homologues, which makes this test system quite popular [57,58]. Organisms, in general, are protected against damage caused by oxidative stress by endogenous enzymes such as SOD, CAT, GPx, and glutathione reductase (GR), which can remove free radicals and other oxidizing species and/or repair oxidative damage. Tese enzymes remove superoxide and peroxides before reacting with catalytic metals to form reactive compounds. However, this inhibition may promote an imbalance in the levels of free radicals, which thus causes DNA damage, protein denaturation, lipid peroxidation, and even cell death.
Medicinal plants rich in monoterpenes are commonly used to treat various diseases. In our study, estragole and 3,7dimethyl-1-octanol showed low cytotoxicity in A. salina and mouse erythrocytes. However, nerol showed high cytotoxic efects at all the doses tested on these test systems. Te studied monoterpenes showed a low antioxidant efect in the ferric reducing potential test, but signifcant inhibition of the oxidative damage caused by H 2 O 2 in all the S. cerevisiae strains was seen at all concentrations of the tested monoterpenes. Regarding the scavenging properties of the monoterpenes, nerol showed a prominent anti-radical capacity in ABTS and DPPH assays, followed by estragole and 3,7-dimethyl-1-octanol.
Antioxidants are of two types, such as primary and secondary. Primary types scavenge free radicals, thereby inhibiting or slowing initiation and/or propagation steps by releasing electrons or hydrogen. Tis class of antioxidants can react with lipid and peroxyl radicals (e.g., ROO • , RO•, and R • ) and convert them into more stable products (e.g., ROOH, ROH, and RH), for example, phenolic compounds, tocopherols, and synthetic antioxidants (e.g., PG, BHA,     Terpenes, including monoterpenes, play vital roles in plants as they provide defense against various bacteria, fungi, and viruses [60]. Tese have promising cytotoxic efects against these kinds of pathogens. For example, linalool (a naturally occurring terpene alcohol) is known to exert signifcant cytotoxic efects by inducing oxidative stress and apoptotic mechanisms [61]. In this study, we also observed that the alcoholic monoterpene nerol exerted better cytotoxic efects on mouse erythrocytes and S. cerevisiae.

Conclusion
Te monoterpenes estragole and 3,7-dimethyl-1-octanol did not exhibit signifcant cytotoxic efects on the test systems (A. Salina and mouse erythrocytes). Nerol exhibited signifcant cytotoxic efects on both test systems in a concentration-dependent manner. All the tested monoterpenes scavenged free radicals' concentration dependently. However, nerol showed a signifcantly higher antioxidant capacity than estragole and 3,7-dimethyl-1octanol. Te monoterpenes did not oxidize the genetic material of the profcient and defcient S. cerevisiae strains. Among the three monoterpenes, nerol may be a good candidate for antioxidant and anti-tumor therapies. Further research is necessary to evaluate the mechanism of action behind the antioxidant and cytotoxic efects in vivo test models.

Data Availability
Te datasets used to support the fndings of this study are available from the corresponding authors upon request.

Conflicts of Interest
Tere are no conficts of interest. Advances in Pharmacological and Pharmaceutical Sciences 9