Antioxidant Activities of Commiphora leptophloeos (Mart.) J. B. Gillett) (Burseraceae) Leaf Extracts Using In Vitro and In Vivo Assays

Commiphora leptophloeos is widely used in folk medicine without any scientific basis. Considering this, the aim of this study was to evaluate the chemical profile and the antioxidant activity of C. leptophloeos leaf extracts using in vitro and in vivo assays. Six extracts were obtained from fresh leaves using a serial extraction (nonpolar to polar solvents). These extracts were first evaluated with the presence of phytochemical compounds using the methods thin layer chromatography (TLC), ultrahigh performance liquid chromatography (UHPLC-DAD), and high performance liquid chromatography, both with diode array detection (HPLC-DAD). Based on the compounds identified, it was used some bioinformatics tools in order to identify possible pathway and gene targets. After that, the antioxidant capacity from these extracts was analysed by in vitro assays and in vivo assays using Caenorhabditis elegans model. Phytochemical analyses showed the presence of polyphenols, such as rutin, vitexin, and quercetin diglycosides in all extracts, especially in ethanol extract (EE) and methanol extract (EM). Bioinformatics analysis showed these polyphenols linked to antioxidant pathways. Furthermore, EE and EM displayed a high antioxidant capacity in DPPH and superoxide radical scavenging assays. They also had no effect on cell viability for 3T3 nontumour cell. However, for B16-F10 tumour cell lines, these extracts had toxicity effect. In vivo assays using C. elegans N2 showed that EE was not toxic, and it did not affect its viability nor its development. Besides, EE increased worm survival under oxidative stress and reduced intracellular reactive oxygen species (ROS) levels by 50%. Thus, C. leptophloeos EE displayed an important in vitro and in vivo antioxidant capacity. The EE extract has polyphenols, suggesting that these compounds may be responsible for a myriad of biological activities having this potential to be used in various biotechnological applications.


Introduction
Medicine plants have a millenary practice in some cultures [1]. It is known that plants produce different intermediate compounds (phenolic compounds, terpens, and alkaloids), and these compounds may have a pharmacology activity like antioxidant role, anticancer agent, and dietary supplementation [2]. According to Newman and Cragg [3] from the period from 1981 to 2014, half of the new molecules approved were originated from medicinal plants. Due to this, medicinal plants have been receiving a special attention in order to identify new bioactive molecules that may be used in medicine for the different diseases such as antioxidant, antimicrobial, antiproliferative, antifungal, antiparasitic, and immunomodulatory [4,5].
Many age-related and chronic illnesses like diabetes, cardiovascular disease, Parkinson's disease, Alzheimer's disease, inflammatory diseases, and cancer have been associated with oxidative stress due to an imbalance between reactive oxygen species (ROS) production and degradation, consequently increasing the ROS levels [6,7]. Plant phenolic compounds are known as potent antioxidants, which are crucial for the maintenance of redox homeostasis [4,6,7].
Commiphora leptophloeos (Mart.) J. B. Gillett. (Burseraceae) is a native species from Caatinga, an exclusive Brazilian biome characterised by low water availability, high temperatures, and high light. Pereira et al. [8] reported on the antimicrobial activity of methanol extract obtained from barks of this plant. However, no data pertaining to its chemical profile and potential antioxidant effects exist. Therefore, the chemical profile of C. leptophloeos leaf extracts and the antioxidant activity was investigated in this study, by in vitro and in vivo assays using antioxidant assays, the 3T3 and B16F10 cell lines as well as the C. elegans worms.

Leaf Extract Preparation.
After harvesting, the fresh leaves were transported to the laboratory where it was washed with water to remove impurities and crushed into small pieces with the aid of scissors. Commiphora leptophloeos leaves (100 g) were subjected to a serial extraction with hexane (EH), chloroform (EC), ethanol (EE), methanol (EM), and water (EAR-residual aqueous), with solvent proportion of 1 : 10 (w/v) (100 g fresh leaves to 1000 mL of solvent) for 24 h at 23°C, 150 rpm and under light protection. Another aqueous extract (EA) was prepared according to description of traditional use (100 g fresh leaves to 1000 mL of distilled water). All extracts were filtered on Whatman No. 1 filter paper and dried using a rotary evaporator

Ethanol Extract Analysis
Using HPLC-DAD. According to our biological results, the ethanol extract (EE) profile was determined using the Agilent 1260 chromatograph with DAD detector used. Phenylpropanoid analysis was performed using a C18 column (4:6 mm × 150 mm) with 3.5 μm diameter particle (Zorbax Eclipse Plus C18). The extract was diluted with methanol to a concentration of 1 mg/mL. The injection volume was 3 μL. The flow rate used in the column was 1 mL/min. The EE was analysed at a concentration of 1 mg/mL. The mobile phase used was an elution gradient containing 0.1% acetic acid (A) and acetonitrile (B), and the gradient composition was 10% B (0-6 min), 10-15% B (6-7 min), 15% B (7-22  The column temperature was 45°C, and detection was done at 352 nm. The identification of phenolic compounds was obtained by comparison of retention time and UV-visible absorption spectra with standards of this substance.

Bioinformatics Analysis.
Based on the possible bioactive molecules identified by UHPLC-DAD and HPLC-DAD analysis-rutin, vitexin, and quercetin diglycosides-the Traditional Chinese Medicine System Pharmacology (TCMSP) database was used [11]. The data obtained at TCMSP were used at Kyoto Encyclopedia Genes and Genome (KEGG database) [12]. Then, the pathways were listed in descending order according to p value. These data were used to build a network using String 10 version 11 (https://string-db.org/).

2.9.
In Vitro Antioxidant Activity 2.9.1. Reducing Power Assay. The reducing power of C. leptophloeos extracts was quantified by the reduction of potassium ferricyanide into potassium ferrocyanide [9,13]. The reaction mixture (4 mL) containing the plant extracts at 50, 100, and 250 μg/mL in phosphate buffer (0.2 M, pH 6.6) was incubated with potassium ferricyanide (1% w/v) at 50°C for 20 min. The reaction was terminated by the addition of 10% trichloroacetic (TCA) solution, distilled water, and 0.1% ferric chloride. The absorbance was measured at 700 nm, using phosphate buffer as a blank. Results were expressed as activity percentage reported by 100 μg/mL of ascorbic acid (standard-Sigma-Aldrich, Saint Louis, MO, USA) according to Athukorala, et al. [9] and Wang et al. [13].

DPPH Radical Sequestration
Assay. The DPPH assay was performed according to the method by Shimada et al. [14], using a 96-well plate. Aliquots of extracts (1, 2, and 5 μL) at final concentration of 50, 100, and 250 μg/mL were added, and 150 μM DPPH were added into each well, mixed, and the plate kept at room temperature for 30 min. The absorbance was measured at 517 nm. The DPPH freeradical scavenging activity was calculated as follows: DPPH scavenging activity : ð%Þ = ½1 -ðA1/A0Þ × 100, where A0 is the absorbance of the control (DPPH) and A1 is the absorbance of the samples.
2.9.3. Superoxide Radical Scavenging Activity Assay. This assay was based on the method by Dasgupta and De [15] inside a dissipating chamber of light. This assay was based on the extracts to inhibit the reduction of nitroblue 3 Oxidative Medicine and Cellular Longevity tetrazolium (NBT) in the riboflavin-light-NBT system. The extracts at different final concentrations (100, 250, and 500 μg/mL) were mixed to 3 mL reaction mixture (50 mM phosphate buffer (pH 7.8), 13 mM methionine, 2 mM riboflavin, 100 mM EDTA, and 75 mM NBT) and exposed to a fluorescent lamp for 10 min. Thereafter, absorption was measured at 560 nm-EDTA was used as a control and distilled water as the blank. Results were expressed as percent of radical scavenging: Percentage of radical scavenging = ð½Acontrol − Asample/ Acontrol − Ablank ½ Þ × 100: Acontrol: absorbance of the control; Asample: absorbance of the sample; Ablank: absorbance of the blank.  The other aspect analysed was the body length. The L1 synchronised worms were kept at NGM medium plus E. coli OP50 in the absence or presence of EE at 100, 250, 500, and 1000 μg/mL. These plates were kept at 20°C for 48 h. Approximately 25 animals were used per treatment, and each experiment was performed in triplicate. The animals were photographed, and their body length was measured using ImageJ software, version 1.8.

Quantification of Intracellular ROS.
Wild-type animals synchronised at L1 were kept on NGM plates with E. coli OP50. These worms were treated with EE at concentrations of 0, 100, 500, and 1000 μg/mL for 48 h. Thereafter, worms were washed thrice with M9 buffer and incubated with 500 μL M9 buffer. Afterward, approximately 40 animals were transferred to 96-well plates containing 2 mM H2DCF-DA (2,7-dichlorofluorescein diacetate fluorescent probe), to measure ROS levels. Samples were read using a multilabel microplate reader GloMax®-Multi Detection System (Promega, Wisconsin, USA), with excitation at 490 nm and emission at 510-570 nm, and the mean values were calculated. Readings were done intermittently every 30 min during 4 h. This assay was performed in triplicate.
2.14. Survival Test under Oxidative Stress Conditions. Synchronised wild-type animal at L4 were treated with EE at concentrations of 0, 100, 500, and 1000 μg/mL for 48 h. Thereafter, approximately 50 animals were transferred to 24-well plates with NGM containing 10 mM t-BOOH (tertbutyl hydroperoxide). Survival was monitored every three hours until all animals were considered dead (no movement). Each group was made up of 10 animals, and each assay was analysed five times [18].
2.15. Analysis of sod-3::GFP Expression. The transgenic line CF1553 worms were treated with EE at 0, 100, 500, and 1000 μg/mL for 48 h. Thereafter, 25 worms were transferred to microscopic slides with 1% agarose and covered. These worms were photographed using an Olympus BX51 fluorescence microscope using a 10-fold eye magnification and 365 nm excitation filter. The GFP fluorescence intensity was analysed using ImageJ Software version 1.8 (https://imagej.net).
2.16. Statistical Analysis. The results were expressed as the mean ± standard deviations. Each assay was repeated 2-3 times and was performed in triplicate or quintuplet. Statistical analysis was performed using GraphPad Prism 6.0 (2014), and the results were obtained using one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis (P < 0:05).

4
Oxidative Medicine and Cellular Longevity

Commiphora leptophloeos Leaf Extracts Are Rich in
Phenolic Compounds. In this study, a serial approach using solvents from different polarity (nonpolar to polar) was done in order to obtain five extracts from C. leptophloeos leaves: hexane (EH), chloroform (EC), ethanol (EE), methanol (EM), and water (EAR-residual aqueous), and the sixth extract was prepared only with water-EA. The total phenolic content of compounds obtained ranged from 5:44 ± 0:4 mg/g to 126:41 ± 1:8 mg/g. The EH had 10:18 ± 0:4 mg/g, while EC had 42:47 ± 0:2 mg/g, EAR had 24:27 ± 0:6 mg/g, and EA had 5:44 ± 0:4 mg/g. The highest values were obtained from the EM (126:41 ± 1:8 mg/g) and EE (105:67 ± 1:7 mg/g). The presence of terpenes was observed in TLC analysis for the EH. The presence of terpenes, phenolic acids, and fla-vonoids was detected in the EC; terpenes, phenolic compounds, flavonoids, saponins, and tannins were observed in both the EE and EM. Phenolic compounds were detected for EAR and not observed for EA. Moreover, the fingerprinting analysis of extracts via UHPLC-DAD allowed the detection of several compounds similar to phenols at higher concentrations mainly in EE and EM according to UV spectra (see Figure 1(a)). In green represented the EE, and the peak #1 may correspond to the rutin flavonoid (tr = 9:6). Regrettably, the other peaks in Figure 1(a) may not be determined by our analysis.
Based on the biological results obtained with EE, then it was analysed using HPLC-DAD at 360 nm. The presence of vitexin-peak 1 (tr = 13,734); peak 2), and peak 2 suggested the presence of quercetin diglycosides (tr = 20,166) (see Figure 1(b)).  Figure 1: UHPLC chromatogram. The extracts: EH (hexane extracT), EC (chloroform extract, EE (ethanol extract), EM (methanol extract), EAR (residual aqueous extract), EA (aqueous extract) were analysed using UHPLC-DAD at 360 nm. In (a), the chromatogram obtained for each extract is shown. Green represents the chromatogram detail obtained for the EE. Peak number 1 corresponds to rutin. The x-axis corresponds to the retention time (min) of each peak. The y-axis corresponds to the intensity of the peaks in mAU. In (b), the EE analysis using HPLC-DAD was shown. The detection wavelength was set at 360 nm, the x-axis corresponds to the retention times (min) of each peak, and the y-axis corresponds to the intensity of the peaks in mAU. The peak absorption spectra #1 corresponds to vitexin #2 to quercetin diglycosides.

Bioinformatics Analysis.
Bioinformatics may be a powerful tool to make a data mining from potential targets using the bioactive molecules presented in the extracts. The pharmacological analysis was done in order to identify the potential targets from the three bioactive molecules presens at C. leptophloeos leaf extract: rutin, vitexin, and quercetin diglycosides. It may be observed that these molecules may be associated to pathways that were related to anti-inflammatory, cardiovascular disease, neurodegenerative disease, cancer, and others. It was observed more than 56 targets (see Table 1). Moreover, when the overlap was analysed, then some gene argets were PTGS2, PIK3CG, CASP3, MAPK7, SOD, CAT, ETNK1, ENTK2, HSP90, TNF, IL1β, and IL6 (see Table 1, Figure 2) Furthermore, the bioinformatics approach proposed that the bioactive molecules identified at C. leptophloeos may have an antioxidant activity.
In Figure 2, it may be observed how the genes identified at Table 1 may interact. Although many of these genes are correlated to the immune system, there is also a correlation to oxidative stress as the SOD2 (Sodium Dismutase) and CAT (Catalase) was observed for example. Then, this approach showed that it was important to analyse if these extracts (complex mixture of bioactive molecules) may have antioxidant activities and others activities.

In Vitro Antioxidant Assays and In Vivo Cell Viability.
Considering the presence of phenolic compounds in the order EM > EE > CE > EAR > EH > EA, the in vitro antioxidant capacity of these extracts were then evaluated using seven different assays. The extract concentrations that were analysed were 50, 100, and 250 μg/mL. The assays that had activity were presented. The reducing power potential increased in a dose-dependent manner (see Figure 3(a)). A 100% reducing power potential was observed at 250 μg/mL for both EE and EM (see Figure 3(a)). The DPPH assay displayed saturation as the value reached 100% of DPPH scavenging at the lowest concentration (50 μg/mL) (see Figure 3(b)). Moreover, the EE and EM exhibited the highest superoxide radical scavenging potential (see Figure 3(c)). The EE reached 65% at 100 μg/mL.
Since EE presented the highest antioxidant activity amongst all extracts, its effect on NIH/3T3 cell viability was analysed (see Figure 3(d)). As expected, no effect on cell viability was observed. Furthermore, higher concentrations of EE seemed to increase cell metabolism and viability when measured by the MTT assay (see Figure 3(d)). On the other hand, when it was evaluated, the EE effect on B16F10 tumour cell line was observed a cytotoxicity effect as it may be observed a reduction on cell viability in a range of 30% at 100 μg/mL up to 45% at 500 μg/mL extract concentration.

Effect of Commiphora leptophloeos EE on
Caenorhabditis elegans Development. The nematode C. elegans was used to Figure 2: Gene network from Commiphora leptophloeos bioactive molecules. The data obtained from KEEG were used to build a network using SRTING10 version 11. Each circle corresponds to a gene; the lines are how these genes are connected between each other. The blue line corresponds to curate database; pink determined experimentally; yellow data from text mining; black coexpression; green colour means gene neighbourhood.  All assays (a-e) were done in triplicate for each extract, and the data obtained were analysed using ANOVA and Tukey's test (P ≤ 0:05). Different letters (a, b, c, d, e, and f) correspond to significant differences. 7 Oxidative Medicine and Cellular Longevity analyse the EE effect on animal development as well as its antioxidant capacity using this in vivo model. The EE concentration used were 0, 100, 250, 500, and 1000 μg/mL. The 1000 μg/mL concentration was used due to the C. elegans cuticle. The effect of EE on the development C. elegans was evaluated, and any effects on egg hatching at any of the concentrations analysed were not observed (see Figure 4(a)). Moreover, any modifications in body length (see Figure 4(b)) nor in animal development (see Figure 4(c)) were not observed.

In Vivo Antioxidant Effect of EEs Using Caenorhabditis elegans.
Since EE exhibited an in vitro antioxidant capacity and had any cytotoxic effects on cell viability and on C. elegans development, it was essential to evaluate the effect of EE treatment on ROS production using the C. elegans model. The EE treatment at 500 and 1000 μg/mL was able to reduce approximately 50% of the intracellular ROS level when compared to the untreated control (see Figure 5(a)). Furthermore, it was also observed that the EE concentration of 1000 μg/mL increased survival under oxidative stress induced by t-BOOH (see Figure 5(b)). The mean survival time of 1000 μg/mL of EEtreated animals increased by 5% compared to the untreated control. Moreover, it was observed that the expression level of sod-3::GFP transgenic animals was reduced by approximately 50% on GFP expression, when treated with any EE concentration (see Figures 5(c) and 5(d)).

Discussion
Over the years, medicinal plants have received growing attention in research for the identification and characterization of new intermediate metabolites that may have a different biological effect [4]. Illnesses like cancer, diabetes, cardiovascular disease, inflammatory disease, aging, Parkinson's disease, and Alzheimer's disease have been associated with oxidative stress. The antioxidant effect of plant intermediate metabolites have an important impact on the maintenance of cellular redox homeostasis and could potentially influence the mechanism of these diseases [5,[19][20][21][22][23].
Commiphora leptophloeos is used in folk medicine for gastritis, anti-inflammatory, asthma, and bronchitis [24,25] but no scientific information. The data in this study showed that all the extracts displayed antioxidant activity at in the DPPH assay. Considering that the antioxidant activity has three steps: initiation, propagation, and termination [26][27][28]; our data showed that the EE and EM had activity at the initiation (DPPH and reducing power) and termination phase (superoxide radical scavenging).
Pereira et al. [8] worked with Commiphora leptophloeos stem bark, and they also identified the presence of phenolic compounds, flavonoids, and sugars. Our TLC, UHPLC, and HPLC data showed the presence of phenolic compounds such as rutin, vitexin, and possible quercetin diglycosides, especially in EE C. leptophloeos. The bioinformatics analysis  Oxidative Medicine and Cellular Longevity associated to the phenolic compounds identified possible gene targets that were associated to antioxidant and antiinflammatory pathways. It has been observed that misbalance between ROS production and degradation may produce an oxidative stress, which may be associated to inflammatory process as well as to different diseases [19]. Moreover, it has been shown that phenolic compounds as rutin, quercetin, and others may acts in signalling pathways involved in the inflammatory process [19]. Soukhtanloo et al. [22] showed that NF-kb signalling pathway may be a target for some natural products. Choudhari et al. [5] also showed the use of phytochemicals were promising at preclinical treatments. Moreover, He et al. [29] showed that vitexin and isovitexin, active phytochemicals in medicinal plants, have a broad spectrum effect including antioxidant, antiproliferative, antiinflammatory, and antihyperalgesic effects [29,30]. Furthermore, a neuroprotective effect that increases viability and impacts LDH release was observed. This could potentially influence Alzheimer's disease due to its neurological protection. The results showed the presence of rutin and probably the presence of quercetin diglycosides in EE C. leptophloeos extracts. These flavonoids showed promising antioxidant, anti-inflammatory, and immunoprotective activities and may be used to treat cardiovascular diseases, diabetes, asthma, and cancer [6,31,32]. The other flavonoid identified in C. eptophloeos extracts was rutin. It is commonly found in dark-coloured fruits that are known to have antioxidant, anti-inflammatory, neuroprotective, and other biological activities [33]. Furthermore, the chemical composition of C. leptophloeos is similar to other species from the genus [34,35]. The evaluation of cell toxicity is an important step in the discovery and application of new drugs. Thus, the data of cell lines and the C. elegans model showed that the EE had no in vivo cytotoxic effects on the NIH/3T3 cell line, only for B16F10 tumour cell line. Moreover, EE did not affect C. elegans egg hatching, body length, and development, which are  (3 to  30 h), and the y-axis, to the survival fraction. Three independent experiments were performed, with three replicates per treatment. The EE treatment was compared to the control by the Log-rank test (P < 0:0001). (c) sod-3::GFP expression in C. elegans after EE treatment 0, 100, 500, or 1000 μg/mL. The images were taken using the Olympus BX51 microscope. (d) sod-3::GFP expression was quantified. The x -axis corresponds to different concentrations of C. leptophloeos EE. The y-axis corresponds to GFP expression quantification. For all assays, three independent experiments were performed, with three replicates per treatment. Different letters correspond to significant differences. Statistical analysis was performed using ANOVA and Tukey test (P ≤ 0:05).
9 Oxidative Medicine and Cellular Longevity all important parameters for toxicity [36,37]. Literature, it showed that the flavonoids identified did not affect cell viability. However, as plant extracts contain a mixture of phytocompounds, it was important to analyse this aspect using cell lines and the C. elegans model [4,38,39].
Furthermore, C. elegans data reinforce the outcome of the bioinformatics and in vitro antioxidant assays as it verified that EE have the potential to reduce ROS production, even when animals were treated with t-BOOH (oxidative agent). Moreover, during oxidative stress, excessive production of ROS may promote lesions on DNA as well as promote cell damage. The compounds present in extracts reduced or neutralised ROS effects; thus, they are important for redox homeostasis [29,32]. Kampkotter et al. [40] showed that treatment with 100 μM of rutin reduced ROS levels by 15% in C. elegans. The data presented here also showed an effect on life span after EE treatment in animals that have been associated with the antioxidant capacity.
In this study, a reduction of sod-3::GFP expression (reporter gene) was observed in animals treated with EE. This reduction may be associated with the ROS scavenging effect of EE. Similar results were also observed when animals were treated with quercetin or Anacardium occidentale extracts [41,42]. The data suggest that these extracts may act synergistically with phenolic compounds to protect cells and C. elegans from oxidative stress by assisting in ROS reduction, consequently helping to maintain cellular homeostasis [28,38,39,[42][43][44].

Conclusions
The results presented here showed the antioxidant capacity of C. leptophloeos leaf extracts, especially for ethanol extract (EE). Our data showed the presence of phenolic compounds (rutin, vitexin, and quercetin diglycosides, amongst others), and no cytotoxic effects were observed on cell viability and C. elegans. Furthermore, potential antioxidant activity was observed using bioinformatics, in vitro and in vivo assays. The intermediate metabolites observed in ethanol extract (EE) may potentially reduce ROS effects and consequently maintain redox homeostasis. Thus, Commiphora leptophloeos is a promising plant species for future studies on biological activity as oxidative stress is involved in different chronic diseases.

Data Availability
The data obtained in this study are available from the corresponding author upon request.

Conflicts of Interest
The authors have no conflicts of interest to declare.