Phytochemical Analysis of Bixa orellana L. Seeds by GC-MS and Evaluation of Antioxidant Activity

1. Introduction

Oxidative stress occurs when there is an imbalance in cells between the production of reactive oxygen species (ROS) and an organism’s ability to eliminate them through endogenous or exogenous antioxidant systems [1]. Free radicals are molecules characterized by having unpaired electrons in their outer orbital, a condition that makes them highly reactive; therefore, they are also called reactive oxygen and nitrogen species (ROS/RNS) [2,3]. Their accumulation can induce oxidative damage to a variety of biomolecules, which is a precursor to neurological diseases such as Alzheimer’s, Parkinson’s, and multiple sclerosis [4,5]. It also contributes to aging, cancer, diabetes, high blood pressure disorders, and cardiovascular diseases [4,6].

Antioxidants are chemical compounds capable of neutralizing reactive oxygen species through electron donation, hydrogen atom transfer, or metal ion chelation, thereby attenuating oxidative stress induced damage [3]. Among the natural sources of antioxidants, phenolic compounds, flavonoids, terpenoids, and carotenoids stand out, and their potential has been extensively studied [7,8,9]. These metabolites provide protection against biotic and abiotic stress in plants, and in humans, they exhibit antioxidant, anti-inflammatory, and chemoprotective properties [7,10,11].

Bixa orellana L. is a shrub belonging to the Bixaceae family, characterized by producing reddish, triangular seeds contained within a fruit capsule (Figure 1). It is cultivated in the tropics worldwide [12]. Its commercial importance lies in the dye obtained from the seed, primarily bixin and norbixin [13,14]. In addition to these carotenoids, the presence of terpenoids, flavonoids, alkaloids, and glycosides have also been reported [15,16,17], to which antimicrobial and antiproliferative activity is attributed [14,15,17,18,19].

Despite advances in phytochemical research on B. orellana L., comparative studies integrating specialized metabolite analysis with antioxidant activity using standardized methods are still needed. This will help to achieve a better understanding of the relationship between phytochemical composition and the ability to neutralize free radicals. Therefore, the present study purpose to perform a phytochemical analysis of B. orellana seeds and determine their antioxidant activity using in vitro assays.

2. Materials and Methods

2.1. Biological Material

Bixa orellana L. seeds collected in San Juan Comaltepec, Oaxaca, were used to obtain crude extracts from 50 g of dried and crushed seeds. An extraction train was performed in increasing order of polarity with hexane (Mc. Meyer), ethyl acetate (Mc. Meyer), and methanol (Mc. Merck), macerating for one week with solvent changes every 24 hours. The extract was filtered using Whatman No. 1 filter paper and evaporated in a rotary evaporator (Buchi B-480, Equipar, Switzerland) at 45 °C to concentrate and recover the extract for each solvent. The yield of the resulting extracts was calculated, and a pool was prepared to obtain a sample containing the mixture of metabolites extracted with the three solvents. This sample was stored in amber bottles at 4 °C until analysis.

2.2. Phytochemical Analysis

Qualitative identification of the compounds in the seed of B. orellana L. was performed using an Agilent 7890B gas chromatograph (GC-MS), and quantification of their relative abundances was performed using an Agilent 5977a mass selective detector (MSD).

An amount of 50 mg of the sample was dissolved in 2 mL of HPLC grade methanol (Meyer brand). The sample was passed through a PTFE microfilter and injected into the gas chromatograph in splitless mode. The chromatographic conditions were as follows: DB-WAX Ultra Inert column 60m x 250 µm x 0.25 µm, column temperature was 40 °C for 9 °C/min to 240 °C for 10 min, carrier gas was helium, flow rate was 1 mL/min, and the injection of temperature was 220 °C. The conditions of the mass selective detector were as follows: the electron ionization energy was 70 Ev, the scan range between 30 and 550 amu, the reading speed of 13.8 spectra/s, the temperature of the ionization chamber of 200 °C and that of the transfer line of 250 °C.

Data processing was performed using MassHunter Workstation software (Agilent Technologies, Inc. 2012). Compounds were identified based on mass spectral fragmen-tation patterns, which were compared with chemical compound information in the NIST (National Institute of Standards and Technology) database and the Flavor fragrance da-tabase [20,21].

2.3. Antioxidant Activity by DPPH

The determination of antioxidant activity was performed by DPPH (2,2-Diphenyl-1-picrylhydrazyl) according to the methodology followed by Liu et al. [22] and Ibarra Estrada et al. [23].

The DPPH radical scavenging assay provides an approximation for evaluating the antioxidant potential of an extract. The radical exhibits a violet coloration, and the reduced form is yellow; therefore, the decrease in absorbance at 517 nm determines the activity of the compound compared to the DPPH control solution [3].

A 0.1 mM methanolic solution of DPPH was prepared. The initial absorbance of DPPH in methanol was measured at 517 nm, and no changes were recorded during the assay with the samples. A standard curve was constructed from the absorbance measurements of the DPPH radical at different DPPH concentrations (2.0, 1.6, 1.2, 0.08, 0.04, 0.02, and 0 mM). The crude extract of B. orellana L. seed was dissolved in methanol (5mg in 1 mL) (Mc. Merk), from which three concentrations were established (0.05, 0.5 and 1 mg/mL-1).

One milliliter of each treatment was added to 3 mL of methanolic DPPH solution. The resulting mixture was incubated for 30 minutes in the dark at room temperature. Absorbance was measured at 517 nm using a spectrophotometer (Spectronic Genesis 5). Ascorbic acid was used as a reference control at the same concentrations as the seed sample. Measurements were performed in triplicate. Results were expressed as IC50 and as the percentage of DPPH inhibition compared to the control, according to the following formula:

% DPPH inhibited= (A _control – A _sample) x 100/A _control

where A _control is the absorbance of the control and A _sample is the absorbance of the sample

The IC₅₀ was determined from the graph of the percentage of inhibition using the linear regression method with GraphPad Prism version 10.0 [24] and corresponds to the concentration at which 50% of the free radicals were neutralized.

2.4. Statistical Analysis

The evaluations were done in triplicate. To determine differences between treatments, a two-way ANOVA was performed followed by Dunnett’s multiple comparisons test using GraphPad Prism version 10.0 [24].

3. Results and Discussion

3.1. Extract Yield

The pool of extracts had a total yield of 16.03% per gram of crude B. orellana L. seed extract.

3.2. Phytochemical Profile

Gas chromatography-mass spectrometry (GC-MS) analysis of the crude extract of B. orellana L. seeds identified 141 volatile and semi-volatile compounds (Figure 2) between 8.5 and 38 minutes. Few compounds were observed between 8.5 and 13 minutes. Between 14 and 20 minutes, a more complex region was observed with numerous small and medium peaks, and between 30 and 38 minutes, high molecular weight peaks were observed. Fifteen compounds had a relative abundance greater than 1%, while the rest had lower abundances.

Table 1 presents the 141 identified compounds. In general, the phytochemical profile is grouped into terpenes, esters, alcohols, ketones, and fatty acids. Fifty-five compounds were detected within the terpene group, with sesquiterpenes predominating at 43 compounds, representing a total relative abundance of 19.74%. Only six diterpenes were present, with a total relative abundance of 28.61%. Among the terpenes, geranylgeraniol (27.50%), ishwarane (11.016%), and geraniol acetate (7.76%) stand out with the highest relative abundance. Figure 3 shows the molecular structures of these compounds.

The phytochemical profile obtained shows agreement with previous research on the composition of B. orellana L. seeds. This species has been documented to contain higher levels of sesquiterpenes, diterpenes, and fatty acid esters, as well as prenylated alcohols such as geraniol, geranylgeraniol, and their derivatives [13,25,26]. Raju et al. [26] reported the presence of ishwarane as one of the sesquiterpenes present in various parts of the plant. On the other hand, geranylgeraniol, a diterpene associated with carotenoid metabolism and the biosynthesis of bixin and norbixin [27], has been reported as one of the most abundant constituents in the volatile fraction of the seed [14,18].

A considerable proportion of fatty acids was also present, including linoleic acid at 3.06%, palmitic acid at 3.66%, and oleic acid at 0.948% relative abundance. This is consistent with the oily nature of the seeds and with research describing that up to 30-445% of the seed weight corresponds to unsaturated oils [13,26].

The profile obtained confirms that the seeds of B. orellana L. contain a mixture of volatile metabolites, highlighting the abundance of sesquiterpenes, diterpenes and lipophilic compounds.

3.3. Antioxidant Activity

Table 2 presents the inhibitory concentrations 50 (IC₅₀) of the crude extract of B. orellana L. seed and ascorbic acid, both at three concentrations (1.0, 0.5 and 0.05 mg/mL^-1).

The seed extract shows clear, dose dependent antioxidant activity, with inhibition reaching 59.70% at 1 mg/mL^-1, decreasing to 47.50% at 0.5 mg/mL^-1. At 0.05 mg/mL^-1, activity barely reaches 37.45%. Ascorbic acid, on the other hand, exhibits greater antioxidant activity, which is expected given its high reducing capacity as a pure compound.

Regarding the IC₅₀, the value obtained for ascorbic acid (0.2266 mg/mL^-1) indicates that it has a high antioxidant potential, as very little is required to achieve 50% inhibition. The IC₅₀ of the crude seed extract (0.5108 mg/mL^-1) shows that more than twice the concentration of ascorbic acid is needed to inhibit 50% of DPPH. The larger standard deviation indicates greater variability in the extract’s behavior, which is typical of complex mixtures.

Figure 4 graphically represents the percentage of DPPH radical inhibition and confirms the trend of the values in Table 2. Therefore, the seed extract of B. orellana L. exhibits significant, concentration dependent antioxidant activity, suggesting that some secondary metabolites can donate hydrogens or electrons to neutralize free radicals. According to the molecular structure of geranylgeraniol (Figure 3), which contains -OH groups, it has the capacity to donate H to neutralize free radicals; furthermore, the presence of double bonds may enhance its radical scavenging capacity, thus promoting antioxidant activity [3].

4. Conclusions

Gas chromatography-mass spectrometry (GC-MS) analysis of B. orellana L. seeds reveals a diverse phytochemical composition of volatile and semi-volatile compounds, predominantly terpenes and fatty acids. The abundance of specialized metabolites such as geranylgeraniol and ishwarane suggests their contribution to the antioxidant activity observed in the DPPH assay.

Although the crude extract of B. orellana L. seed showed a lower antioxidant capacity than ascorbic acid, its action demonstrates that the compounds present in the volatile fraction can participate in free radical neutralization mechanisms.

The results obtained indicate that the seed of B. orellana L. could be a potential source of cosmetic or pharmaceutical applications, although further studies are needed to explore its mechanism of action and its stability in biological systems.