Chemical composition of essential oil and antioxidant activity of the essential oil and methanol extracts of Knema globularia (Lam.) Warb. from Vietnam

Abstract This study is the first to investigate the chemical composition of essential oil and antioxidant activity of the essential oil and methanol extracts from the leaves of Knema globularia (Lam.) Warb. from Vietnam. According to gas chromatography and gas chromatography-mass spectrometry analysis, the major constituents of K. globularia essential oil were β-elemene (25.48%), α-copaene (17.05%), β-caryophyllene (9.37%), and α-humulene (8.42%). The antioxidant activity of the samples was determined using DPPH and ABTS methods. In both assays, the polar subfraction of the methanolic extract showed better antioxidative capacity than the nonpolar subfraction and the essential oil. In addition, the amounts of total phenol value in the polar subfraction and the nonpolar subfraction were determined to be 113.84 μg/mg and 47.52 μg/mg, respectively. The findings demonstrate that the essential oil and methanol extracts of K. globularia possess significant antioxidant activities and may be a new potential source of natural antioxidants. Graphical Abstract


Introduction
The genus Knema (Myristicaceae) comprises over 60 species found in tropical and subtropical regions of Asia, Africa, and Australia (Pham 1999;Salleh and Ahmad 2017). About 13 species of this genus can be found in evergreen forests throughout Vietnam. Some Knema species are used to treat cancer, sores, pimples, and skin diseases (Perry 1980;Chi 2012). Previous phytochemical studies of Knema species have led to the isolation of compounds including acetophenones, lignans, flavonoids, alkyl and acyl resorcinols, and phenylalkylphenol derivatives (Salleh and Ahmad 2017). The compounds and extracts isolated from Knema species exhibited cytotoxicity, antimicrobial, antioxidant, anti-apoptotic protein, antinematodal, antituberculosis, antiviral, antimalarial, and acetylcholinesterase inhibition activities (Salleh and Ahmad 2017).
Antioxidants are compounds that neutralize chemically active products of metabolism, such as free radicals that can damage the body (Safaei-Ghomi et al. 2009). However, the application of synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, and citric acid have been shown to have toxic and mutagenic effects (Tepe et al. 2004;Ebrahimabadi et al. 2010). Recently, essential oils and various extracts of plants have provoked interest as natural sources of antioxidant compounds. Knema globularia (Lam.) Warb. is a species of the genus Knema, distributed in Southeast Asia, China and India. In traditional medicine, the bark of this plant has been used in a blood tonic, while oil from the seeds has been employed to treat scabies (Pham 1999). Previously, flavonoids, polyketides, and steroids were isolated from this species (Sriphana et al. 2016;Chuenban et al. 2021;Le et al. 2022;Sriphana et al. 2022;Pham et al. 2022). The biological activities of K. globularia such as inhibition of a-glucosidase, antimalarial, and cytotoxicity activities have been reported (Sriphana et al. 2016;Chuenban et al. 2021;Le et al. 2022;Sriphana et al. 2022;Pham et al. 2022).
To the best of our knowledge, there is no information on the chemical composition of essential oil and antioxidant activity of the essential oil and methanol extracts of K. globularia. Thus, the present research reports; (1) the chemical composition of the essential oil of K. globularia growing in the wild in Vietnam, (2) antioxidant activity profiles of this plant products (essential oil and extracts) using DPPH and ABTS assays, and (3) total phenolic compounds content of the plant extracts as gallic acid equivalents.

Chemical composition of essential oil
A pale-yellow essential oil was obtained from K. globularia leaves using hydrodistillation, with a yield of 0.07 ± 0.01% (w/w). The composition of the K. globularia essential oil was analyzed by gas chromatography and gas chromatography-mass spectrometry. The total number of chemical constituents identified in K. globularia essential oil was 38, representing 96.31% of the total oil content ( Table 1). The essential oil of K. globularia was composed of sesquiterpene hydrocarbons (81.20%), oxygenated sesquiterpenes (10.38%), and oxygenated monoterpenes (4.73%). The major components of the essential oil were b-elemene (25.48%), a-copaene (17.05%), b-caryophyllene (9.37%), and a-humulene (8.42%). In addition, considerable amounts of d-cadinene (4.07%), germacrene D (3.72%), a-cadinol (3.45%), bicyclogermacrene (2.94%), 1,8-cineole (2.87%), humulene oxide II (2.17%), and a-cubebene (2.12%) were also detected. To the best of our knowledge, the essential oil composition of K. globularia has not been reported before and therefore our results could be considered as the first report about the composition of the essential oil of this plant.  Adams (2007) or NIST (2018). d Relative percentage values are means of three determinations ± SD. e Identification: Std, comparison with analytical standard; RI, coherence of the temperature-programmed retention index with respect to those reported in Adams (2007); MS, matching with mass libraries (Adams 2007;FFNSC 2012;NIST 2018).
Until now, studies on the chemical composition of essential oils from Knema species are limited. We only found a report on the essential oil composition of K. kunstleri from Malaysia. b-Caryophyllene (23.2%), bicyclogermacrene (9.6%), d-cadinene (7.3%), a-humulene (5.7%), and germacrene D (4.3%) were major components of K. kunstleri essential oil (Salleh et al. 2021). The high content of sesquiterpene hydrocarbons compounds in the K. globularia essential oil observed in our study agrees with the levels found in K. kunstleri oil; however, there is variation in the major components. Indeed, although bicyclogermacrene was one of the main components in the K. kunstleri oil (Salleh et al. 2021), this component was found in lower amounts in the K. globularia oil in the present study. Furthermore, b-elemene was found in high amounts in the K. globularia oil in the present study (Table 1), while this component was found in lower amounts in the K. kunstleri oil (Salleh et al. 2021). The observed chemical variability may be due to the various geographical and ecological conditions between the sites of analyses, the harvest time, as well as the age and nature of the different plant species (Figueiredo et al. 2008).

Antioxidant activity
Antioxidant activities of the essential oil and methanol extracts from K. globularia leaves were tested using DPPH and ABTS radical scavenging. The IC 50 values of the essential oil and methanol extracts were compared with butylated hydroxytoluene (BHT). A lower IC 50 value indicates a greater antioxidant activity.
Specifically, the effect of antioxidants on DPPH radical scavenging was conceived due to their hydrogen-donating ability or radical-scavenging activity (Gholivand et al. 2010;Tahar et al. 2021). When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form 1,1diphenyl-2-picryl hydrazine (non-radical) accompanied by the loss of its violet color (Molyneux 2004;Oke et al. 2009). The results from Table 2 show that the polar subfraction of K. globularia methanol extract provided the highest DPPH radical-scavenging activity with the IC 50 value of 75.26 ± 1.38 lg/mL, compared with the nonpolar subfraction, with an IC 50 value of 121.77 ± 1.79 lg/mL, and the essential oil, with an IC 50 value of 198.13 ± 2.16 lg/mL. However, these activities were lower than that found for the synthetic antioxidant BHT, which had an IC 50 value of 45.02 ± 0.74 lg/mL.
The ABTS assay is often used in evaluating the total antioxidant power of single compounds and complex mixtures of various plants (Ye et al. 2013). Although the DPPH and ABTS methods are based on the same principle, the activity of essential oils and extracts detected using the ABTS assay is generally lower than that detected Values are expressed as mean ± standard deviation (SD) (n ¼ 3); -: not tested.
using the DPPH assay. This may be due to steric factors and the double bond in the side chain that stabilizes the DPPH radical by resonance (Jing et al. 2012;Ud-Daula et al. 2016). In the ABTS assay, the polar subfraction of the methanol extract of K. globularia provided the highest ABTS radical scavenging activity, with the IC 50 value of 54.09 ± 1.07 (Table 2). However, the ABTS scavenging abilities of the methanol extracts and essential oil were lower than that of the synthetic antioxidant BHT (IC 50 ¼ 39.26 ± 0.52 lg/mL). To our knowledge, the results obtained in this study are the first published data concerning the antioxidant activity of the essential oil and methanol extracts from K. globularia. In general, the DPPH and ABTS scavenging activity increased in the order of essential oil < nonpolar subfraction < polar subfraction < BHT. The low antioxidant activity found for the K. globularia essential oil can be explained by a low abundance of oxygenated monoterpenes and sesquiterpenes. This finding also contributed to the results of previous studies, confirming that polar subfraction extracts exhibited better antioxidant activity than nonpolar subfraction extracts and essential oils (Gholivand et al. 2010;Rahimi-Nasrabadi et al. 2012). Moreover, the antioxidant activity of K. globularia essential oil and extracts was weaker than that of the Knema species previously reported (Phadungkit et al. 2010;Vinayachandra and Chandrashekar 2014;Raja and Suku 2019;Salleh et al. 2021). These differences may be explained by different chemotypes of the plant species.

Total phenolic contents
The amounts of total phenolics compounds in the extracts from K. globularia were determined spectrometrically according to the Folin-Ciocalteu procedure and calculated as gallic acid equivalents (Oke et al. 2009;Gholivand et al. 2010). Gallic acid is a water-soluble polyhydroxy phenolic compound that can be found in various natural plants. The standard curve equation is as follows: y (absorbance) ¼ 0.0067 Â (lg gallic acid) þ 0.3005, R 2 ¼ 0.9999. The absorbance value was inserted in the above equation and the total amount of phenolic compound was calculated. The data presented in Table 2 indicates that the total phenol content in the polar subfraction (113.84 ± 1.13 lg/mg as gallic acid equivalents) was higher than in the nonpolar subfraction (47.52 ± 0.91 lg/mg). Phenolic compounds are important plant constituents because of their scavenging ability on free radicals due to their hydroxyl groups (Ahmed et al. 2019). Therefore, the high phenolic content in the polar subfraction contributes to its increased antioxidant potential in comparison with other extracts. Several investigations of the antioxidant activity of plant extracts have confirmed a correlation between total phenolic content and antioxidant activity (Oke et al. 2009;Gholivand et al. 2010;Ahmed et al. 2018).

Experimental
See Supplementary Material.

Conclusions
This study provides the first investigation of the chemical composition of essential oil and antioxidant activity of the essential oil and methanol extracts of K. globularia from Vietnam. The major components of the essential oil were b-elemene (25.48%), a-copaene (17.05%), b-caryophyllene (9.37%), and a-humulene (8.42%). The essential oil and methanolic extracts were found to be effective antioxidants using in vitro assays DPPH and ABTS which can be proposed as natural additives in the food and pharmaceutical industries. There was a good correlation between total phenol content and the antioxidant capacity of the extracts. The phenol content and antioxidant capacity were the highest in the polar subfraction of the methanol extract. According to the results of this study, the essential oil or the methanolic extracts of K. globularia may be a new potential source of natural antioxidants.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.