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Year: 2016  |  Volume: 7  |  Issue: 4  |  Page No.: 193 - 201

In vitro Antioxidant Activity Investigation of Vanillomopsis arborea Baker Aqueous Extracts, Essential Oil and Isolated Compound: (-)-α-bisabolol

Gerlania de Oliveira Leite, Albys Ferrer Dubois, Rodrigo Lopes Seeger, Aline Augusti Boligon, Jose Galberto Martins da Costa, Thiago Henrique Lugokenski, Adriana Rolim Campos, Roselei Fachinetto, Jean Paul Kamdem, Joao Batista Teixeira da Rocha and Caroline Wagner    

Abstract: Background and Objective: Vanillosmopsis arborea Baker (candeeiro) is a native plant from the Northeast of Brazil. Recently, this plant attracted interest of researchers due to its pharmacological properties, however, there is no underling mechanism established for its properties. So, the aim of this study was to investigate the antioxidant potential of this plant, since oxidative stress is in the core of the development of the diseases that V. arborea shows to be efficient in counteract. Materials and Methods: For this purpose, used aqueous extracts from bark, trunk and leaves for the plant, as well as the essential oil from the truck, in a set of oxidative stress models. Results: The main results obtained here demonstrate that aqueous extract from leaves are able to reduce Fe(II)-induced lipid peroxidation. However, when tested for iron chelation, none of the extracts shows any effect. For this reason, performed free radical scavenging test, by the quenching of 1’1’-diphenil-2-picrylhydrazyl (DPPH). It was observed that both aqueous extract from leaves and essential oil from the trunk were capable to scavenge free radical, indicating a direct effect of the plant on free radicals. Due to high (-)-α-bisabolol content in the essential oil and in the aqueous extract from leaves, hypothesized that this compound could be a central character in the antioxidant activity of the plant. So, performed a 2’7’-dichlorodihydrofluorescein diacetate (DCFH-DA) oxidation test with the essential oil and the (-)-α-bisabolol, which confirm the suggestion that (-)-α-bisabolol could be a major responsible for the antioxidant activity of V. arborea. Conclusion: Thus, V. arborea Baker could be considered an effective agent in the prevention of various diseases associated with oxidative stress and (-)-α-bisabolol is suggested to have prominent role in the plant properties.

1. In folk medicine, Vanillosmopsis arborea is used as a repellent2.

In the past decade, more attention have been paid on the pharmacological activities of V. arborea essential oil (EOVA). Studies have demonstrated that EOVA exhibit anti-inflammatory3,4, antinociceptive4,5,6, gastroprotective7, larvicidal8, antibiotic9, antimalarial10 and antileishmanial activities11. To knowledge, there is however, no studies on the antioxidant activities of the EOVA that may at least it part, justify such activities.

The major chemical constituents found in different parts of Vanillosmopsis arborea include phenolic compounds (apigenin, quercetin, luteolin and their glucosides)1,12, while (-)-α-bisabolol (BISA) (Fig. 1) has been reported as the pharmacologically active principle of its essential oil4,12,13. The BISA is a monocyclic sesquiterpene alcohol found in chamomile (Matricaria recutita) and other plants and it has been widely used in dermatological and cosmetic formulations14. The BISA was shown to exhibit apoptosis-inducing action in malignant tumor cells15 and to inhibit the activities of major human drug-metabolizing enzymes16. It possesses antimutagenic14 and antipeptic17 properties and has the potential to modulate the activity of antibiotics9. Although, BISA has been reported to exhibit gastroprotective effect via diverse mechanisms of action including reduction of lipid peroxidation and superoxide dismutase activity, there is limited information on the antioxidant activity of BISA against hydrogen peroxide, a source of hydroxyl radicals18.

Free radicals are constantly produced in living organisms and detoxified by antioxidants. However, they can cause oxidative damage to important cellular compartments when present in excess. Indeed, consumption of foods antioxidants have been reported to have health-promotion and disease-prevention effects19, since they can delay the development human diseases, specially the Reactive Oxygen Species (ROS)-mediated ones20,21. In addition, natural and synthetic antioxidant compounds have been reported to afford protection in a variety of in vitro and in vivo models of toxicity22-28, highlighting the importance of evaluating the antioxidant activity of plant extracts and/or chemicals for potential therapeutic approach.

Given the interesting biological activities of V. arborea and that of the active principle of its essential oil, (-)-α-bisabolol (BISA) and considering the scarcity of information in regard to their antioxidant activity, the present study aimed to investigate the antioxidant activity of aqueous extract from different parts of V. arborea (leaves, trunk and bark) in chemical and biological models. In addition, the effect of EOVA and its active component were tested against H2O2-induced ROS generation in stomach mucus as well as the characterization of the chemical constituents of those aqueous extracts.


Chemicals: (-)-α-bisabolol, tris-HCl, thiobarbituric acid (TBA), 1,1-diphenil-2-picrylhydrazyl (DPPH), gallic acid and malonaldehyde bis-dimethyl acetal (MDA) were obtained from Sigma (St. Louis, MO, USA). Iron sulfate (Fe2SO4), ascorbic acid, rutin, caffeic acid, gallic acid, chlorogenic acid, chloridric acid and acetic acid were obtained from Merck (Rio de Janeiro, RJ, Brazil). The CH3CN and MeOH (HPLC grade) were from Merck (Darmstadt, Germany); 85% formic acid was provided by Carlo Erba (Milan, Italy). All laboratory chemicals used in this study were of analytical grade.

Plant material and extracts preparation: Vanillomopsis arborea was collected in Crato City, state of Ceará, Brazil. The plant material was identified by Dr. Arlene Pessoa and a voucher specimen was deposited under the number 18639 at the Herbarium “Dardano de Andrade Lima’’ of the Universidade Regional do Cariri (URCA). The aqueous extracts of leaves, bark and trunk were obtained by infusion in hot water at 100°C and they were prepared prior to use.

Essential oil analysis: Oil analysis was performed using a Shimadzu GC-17 A/MS QP5050A (GC/MS system): DB-5HT capillary column (30 m×0.251 mm, 0.1 μm film thickness); helium carrier gas at 1.7 mL min–1, injector temperature 270°C, detector temperature 290°C, column temperature 60°C (2 min) to 180°C (1 min) and at 4°C min–1. Then 180-260°C; at 10°C min–1 (10 min). Scanning speed was 0.5 scan/sec from 40-450 m/z. Split ratio (1:30). Injected volume: About 1 μL of 5 mg mL–1 solution ethyl acetate. Solvent cut time = 3 min. The mass spectrometer was operated using 70 eV of ionization energy. Identification of individual components was based on their mass spectral fragmentation based on two computer library MS searches (Wiley 229), retention indices and comparison with published data. The EOVA was extracted from chopped plant trunk by steam distillation and analyzed at the Natural Products Research Laboratory of the Regional University of Cariri (URCA). Freshly chopped trunk was placed in a glass flask connected at one end to a glass vessel with water and at the other end to a water-cooled condenser. When the water was boiled, steam percolated through the barks and was collected in the condenser. After condensation, the essential oil was separated from the aqueous phase with its solutes.

Animals: Male Wistar rats (3.0-3.5 months of age and weighing (270-320 g)) had free access to food and water and were maintained in a room with controlled temperature (22±3°C) and on a 12 h light/dark cycle. The animals were maintained and used in accordance to the guidelines of the National Council for the Control of Animal Experimentation (CONCEA).

Tissue preparation: Rats were killed by decapitation and the encephalic tissue-brain was rapidly dissected and placed on ice. Tissue was immediately homogenized in cold 10 mM tris-HCl, pH 7.4 (1/10, w/v). The homogenate was centrifuged for 10 min at 4000×g to yield a pellet that was discarded and a low-speed supernatant (S1) was used for the lipid peroxidation assay29.

Lipid peroxidation assay: Lipid peroxidation was determined by measuring the production of TBARS according to the method of Ohkawa30 with slights modifications29. The homogenate (100 μL) was pre-incubated with or without 50 μL of freshly prepared pro-oxidant (iron sulfate, 10 μM) agent and different concentrations of the plant extracts together with an appropriate volume 10 mM tris-HCl, pH 7.4 to give a total volume of 300 μL at 37°C. After 1 h of incubation at 100°C, the color reaction was developed by adding subsequently, 200 μL of 8.1% Sodium Dodecyl Sulphate (SDS), 500 μL of 1.33 M acetic acid buffer (pH 3.4) and 500 μL of 0.6% TBA (thiobarbithuric acid). After cooling the tubes, the absorbance was read at 532 nm in a spectrophotometer. The results were expressed as nmol of MDA/g tissue.

Iron chelation assay: The ability of the aqueous extracts from the leaves, trunk and bark of V. arborea to chelate Fe (II) was determined using a modified method of Puntel et al.31. Briefly, 20 μL of freshly prepared 150 μM FeSO4 were added to a reaction mixture containing 168 μL of 0.1 M tris-HCl (pH 7.4), 218 μL saline and different concentrations of aqueous extract of the plant. The reaction mixture was incubated for 5 min, before the addition of 13 μL of 0.25% 1,10-phenanthroline (w/v). The absorbance was subsequently measured at 510 nm in the spectrophotometer.

DPPH radical scavenging activity: The potential of essential oil from the trunk of V. arborea and aqueous extracts from the leaves, the trunk and the bark of V. arborea to scavenge the DPPH radical was evaluated according to Hatano32. The absorbance was measured at 517 nm using spectrophotometer and the percent inhibition was calculated in relation to the control. Ascorbic acid known as standard antioxidant was used as reference.

Determination of ROS formation in stomach: The levels of ROS in stomach mucus were measured by the oxidation of 2’,7’-dichlorofluorescein (DCFH) as described by Wang and Joseph33. The mucus obtained from rat was homogenized in cold 5 mM tris-HCl, pH 7.4 (1/20, w/v) and then was pre-incubated with different concentrations of essential oil of V. arborea, (-)-α-bisabolol or vitamin E, as a positive control. Experiments were carried out in a standard reaction medium containing H2O2 (20 μL/30%), 20 μL of mucus, tris-HCl (5 mM), pH 7.4, DCFH-DA (5 μM). The fluorescence emission of DFC resulting from DCFH oxidation was monitored in three Independent experiment for 600 sec (10 min) at 480 and 525 nm, excitation and emission wavelengths, respectively, using spectrofluorimeter (Fluorescence spectrophotometer, Hitachi F-2000). The results were expressed as percent of controls.

Quantification of primary constituents: Chromatographic analyses were carried out in isocratic conditions using RP-C18 column (4.6×250 mm) packed with 5 μm diameter particles. The mobile phase consisted of methanol-acetonitrile-water (45:10:45, v/v/v) containing 1.0% acetic acid. The flow rate was 0.8 mL min–1, injection volume 50 μL and the wavelength 257 nm. The mobile phase was filtered through a membrane filter 0.45 μm and then degassed by an ultrasonic sound before use. The solutions of standards (rutin, caffeic acid, chlorogenic acid, gallic acid and (-)-α-bisabolol) were prepared in the same mobile phase of HPLC. The concentration range for the standard curves used was 0.0125-0.200 mg mL–1. The chromatographic peaks were confirmed by comparing its retention time with those of reference standards and quantification was performed by peak integration using the external standard method. The calibration curve for caffeic acid was: Y = 12153x-21513 (r = 0.9983), the curve of gallic acid was: Y = 109130x-526314 (r = 0.9998) and the curve of rutin was: Y = 102171x-16949 (r = 1). All chromatographic operations were performed at room temperature and in triplicate.

Statistical analysis: The results are expressed as Mean±SEM (standard error of mean). Statistical analysis was performed using a one or two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison or Bonferroni posttests multiple comparison test when appropriated. Data from TBARS, DPPH and Iron chelation assays were analyzed by one-way ANOVA, while, that of DCFH oxidation was analyzed by two-way ANOVA. The results were considered statistically significant for p<0.05.


Quantification of primary constituents: The quantification of the constituents of the extracts by HPLC showed that in the aqueous extract of the bark the major constituents are gallic acid (7.25%) and BISA (2.08%); in the aqueous extract of the leaves are gallic acid (11.03%), chlorogenic acid (5.23%) and BISA (4.12%) and in the trunk aqueous extract of the trunk are gallic acid (6.56%) and BISA (2.34%) (Fig. 2a-c).

Lipid Peroxidation (LP): The iron-induced lipid peroxidation was reduced by aqueous extract from leaves of V. arborea as shown in Fig. 2. Statistical analyzes revealed that Fe2+ induced a significant stimulation in brain LP levels (p<0.05), which were reduced by aqueous extract of V. arborea Baker leaves in a concentration-dependent manner p<0.05 (Fig. 3a). In contrast, the aqueous extracts from trunk and bark are not able in prevent lipid peroxidation in brain homogenates in tested conditions (Fig. 3b and c).

Iron chelation assay: The extracts tested did not showed any properties on the chelation of iron (II) chelation as showed in the Fig. 4.

DPPH radical scavenging: The leaves aqueous extract and EOVA inhibited DPPH radical, with the maximal observable effect of 40 and 60% radical scavenging for essential oil trunk and aqueous leaves extract (Fig. 5a, b). In contrast, aqueous extract from trunk did not reduce DDPH radical (Fig. 5c). Bark aqueous extract moderately reduced DPPH radical and its maximal effects was about 40% inhibition (Fig. 5d).

Determination of ROS formation in stomach: Data show ROS formation in the mucus was markedly increased in the presence of H2O2, as expected. The EOVA or (-)-α-bisabolol added in the presence of H2O2 caused a reduction in ROS production in a concentration-dependent manner (Fig. 6).


In this study, tested the effect of the plant V. arborea against well-known pro-oxidant agents, to investigate the effects of this plant and search for new potential antioxidants from natural sources. The results suggested that V. arborea leave extract and EOVA presented a markedly antioxidant capacity by scavenging free radicals. In addition EOVA and their major compound (-)-α-bisabolol reduced ROS formation in stomach mucous exposed to H2O2.

The brain is particularly susceptible to free radical damage due its high consumption of oxygen and its relative low concentration of antioxidants enzymes and free radicals scavengers. Considering this, used cerebral tissue for the TBARS assay. The iron-induced lipid peroxidation was reduced by aqueous extract from leaves of V. arborea. In contrast, the aqueous extracts from trunk and bark are not able in prevent lipid peroxidation in brain homogenates in tested conditions. These results are probably due to the minor content of caffeic acid, rutin, chlorogenic acid and (-)-α-bisabolol in these plant structures compared, which the extract from the leaves (Fig. 2). Take into account that free iron in the cytosol and in the mitochondria can cause considerable oxidative damage by increasing ROS production34 via stimulation of fenton reaction35, attribute the V. arborea effect in decrease/prevent lipid peroxidation to a possible free radical scavenging capacity of the plant contents, once this data do not show any iron chelation properties of all plant extracts, as shown in Fig. 4.

The 1,1- diphenil-2-picrylhydrazyl (DPPH) radical has been widely used to test the free radical scavenging ability of various natural products and has been accepted as a model compound for free radicals32,36. This hypothesis gathered strength when tested the plant extracts in a radical scavenging model, with the DPPH radical scavenging test. Data demonstrated that that leaves aqueous extract presented higher scavenging activity than bark and trunk. Here, introduced to our study essential oil of the plant (EOVA); this extract from the truck by practical laboratorial reasons, as well as because of the increased oil yield.

Additionally, the inhibition of lipid peroxidation by V. arborea extracts showed a relation with its phenol content (Fig. 2), suggesting once more that the effects are related with these compounds, which is also confirmed in the literature37. Especially important was the effect of EOVA from the trunks in the DPPH radical scavenging test.

Since, the EOVA showed an increased amount of (-)-α-bisabolol, we hypothesized whether this compound could be a major responsible for the antioxidant effects of plant, once it occurs in relatively high concentrations in the aqueous extract from leaves.

In order to test if (-)-α-bisabolol is a main responsible for the antioxidant effects observed used a model of oxidative stress in stomach mucous of rats. This choice was due to the major role of reactive oxygen species in the development of stomach pathogeneses, especially in gastric mucosal lesion associated with water immersion stress, anti-inflammatory drugs and ethanol-induced ulcers38. Under these conditions, there is an imbalance between formation and degradation of these species. The enzymatic antioxidant defenses and non-enzymatic cannot restrain the ROS increase, thus, may exert deleterious actions on the gastric mucosal epithelium.

Gastrointestinal tract cells have an antioxidant defense system capable of preventing the cytotoxicity of ROS through mechanisms that involve the action of enzymes and compounds with potential to scavenge free radicals. In the list of enzymes involved in this action are superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (Cat). The mucosa is also protected in a non-enzymatic mechanisms such as reduced glutathione (GSH), alpha-tocopherol (vitamin E), vitamin C, carotenoids, methionine and taurine, which can bind or reduce the oxygen radicals and prevent their harmful actions39.

Choose here the DCFHDA oxidation test to measure the amount of ROS generated. Briefly, DCFH-DA is cleaved intracellular by nonspecific esterases to form DCFH, which is further oxidized by ROS to form the fluorescent compound DCF. Data show ROS formation in the mucus was markedly increased in the presence of H2O2, as expected. The EOVA or (-)-α-bisabolol added in the presence of H2O2 caused a reduction in ROS production in a concentration-dependent manner (Fig. 6). Vitamin E, used here as a positive control, presented a markedly protection against ROS production. Taken together, the data suggest a major role of the (-)-α-bisabolol for the therapeutic properties of the V. arborea, acting as an antioxidant molecule and may be the responsible for the pharmacological properties previously observed to the plant here studied. This idea is reinforced by the previously showed gastroprotective effect of the (-)-α-bisabolol against ethanol- and indomethacin-induced ulcer in a model in mice, which effect as related to its antioxidant activity40. In addition, a previous study from the same research group make a relation between the gastroprotective effects of (-)-α-bisabolol and the reduction in lipid peroxidation in the gastric mucosa41 and the gastroprotective activity presented by EOVA7 can be related to antioxidant capacity, preventing the cytotoxicity of ROS.



In conclusion, work demonstrates that V. arborea, aqueous extracts and essential oil, present significant antioxidant activity, acting through scavenging radical directly. In addition, demonstrated here that (-)-α-bisabolol is, at least in part, responsible for the therapeutic properties of V. arborea. Consequently, this plant could be used as a potential antioxidant agent for the prevention of diseases associated with oxidative damage.


The financial support by CAPES/SAUX/PROCARD, VITAE Fundation, CNPq, FAPERGS, ICTP and FINEP and special thank to Margareth Linde Athayde, UFSM.

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