Chemical Composition,Antimicrobial and Antioxidant Properties of Essential Oil From the Leaves of Vernonia volkameriifolia DC.

Abstract

Objectives

The objective of this study was to investigate the essential oil (EO) extracted from the leaves of Vernonia volkameriifolia DC., a Vietnamese plant belonging to the Asteraceae family. The focus was on determining the chemical composition of the EO and assessing its antimicrobial and antioxidant activities.

Methods

The EO obtained from V. volkameriifolia underwent comprehensive analysis using gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) to identify its chemical constituents. Antimicrobial activity was determined against eight microorganisms using the broth microdilution susceptibility method, with minimum inhibitory concentration (MIC) values calculated. Additionally, antioxidant activity was assessed through DPPH and ABTS assays, with half maximal inhibitory concentration (IC₅₀) values indicating the potency of the EO.

Results

The main components of the V. volkameriifolia EO were identified as β-bisabolene (25.2%), β-pinene (19.1%), germacrene D (13.1%), and cis-β-elemene (11.4%). The EO exhibited significant antimicrobial activity, with a MIC value of 200 μg/mL against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Aspergillus niger, and Saccharomyces cerevisiae. Additionally, it displayed activity against Candida albicans with a MIC value of 100 μg/mL. In antioxidant tests, the EO demonstrated notable potential, with IC₅₀ values of 144.1 μg/mL and 179.8 μg/mL for DPPH and ABTS, respectively.

Conclusion

This study represents the inaugural investigation into the EO extracted from the leaves of V. volkameriifolia. The identified chemical components, along with the demonstrated antimicrobial and antioxidant activities, contribute valuable insights into the potential applications of V. volkameriifolia EO in pharmaceutical and therapeutic contexts. Further research can build upon these findings to uncover additional properties and applications of this EO.

Keywords

essential oil Asteraceae terpenes antimicrobial activity antioxidant activity MIC

Introduction

There are about 350 known species in the genus Vernonia of the Asteraceae family. These species usually exist as herbaceous flowering plants and shrubs. Vernonia volkameriifolia DC. is a small shrub tree with a height of about 2-6 m.¹ The leaves are oblanceolate or obovate. The inflorescences are terminal with capitula containing numerous flowers. According to indigenous knowledge, this species is used for treating malaria and gynecological diseases. A previous study confirmed the isolation of dihydrobenzofuran from the non-volatile extract of V. volkameriifolia,² while no study could be found on the volatile composition and pharmaceutical activities. Literature reports are available describing the essential oil (EO) and potential activities of some other Vernonia plants elsewhere. The EOs of some Vernonia species from Vietnam were dominated by high contents of sesquiterpenes. These include V. patula EO with β-caryophyllene (28.5%), caryophyllene oxide (16.6%), α-copaene (9.0%), and α-humulene (7.1%) as the main constituents.³ V. amygdalina leaf EO is dominated by caryophyllene oxide (29.51%), β-caryophyllene (19.95%), and α-humulene (8.50%), while the stem EO is made up of caryophyllene oxide (36.28%) and humulene epoxide II (10.63%).⁴ On the other hand, the V. solanifolia leaf EO was rich in a mixture of monoterpene and sesquiterpene, namely sabinene (49.2%), δ-cadinene (10.9%), and β-caryophyllene (7.8%).⁵

Also, various monoterpenes and sesquiterpene compounds have been reported in Vernonia EO samples from different parts of the world. The flowering aerial parts EO of Indian-grown V. cinerea contains sesquiterpenes, with β-caryophyllene (23.2%), δ-cadinene (10.3%), γ-amorphene (7.5%), cis-β-guaiene (6.8%), and premnaspirodiene (6.3%) being the major components.⁶ Analysis of V. chalybaea EO from Brazil revealed the presence of β-caryophyllene (39.06%) and bicyclogermacrene (19.69%).⁷ Similarly, V. scorpioides EO from Brazil exhibited β-caryophyllene (30.6%), germacrene D (27.3%), and bicyclogermacrene (8.5%) as its constituents.⁸ V. amygdalina leaves from Nigeria yielded an EO rich in α-muurolol (45.7%),⁹ while another sample from Nigeria contained thymol (27.0%), (E)-phytol (15.7%), o-cymene (12.7%), and β-selinene (8.1%).¹⁰ Additionally, V. amygdalina leaf EO from Kenya consisted mainly of 1,8-cineole (25.11%), β-pinene (14.54%), myrtenal (6.52%), and trans-pinocarveol (6.24%).¹¹ In conclusion, the main chemical compounds found in V. amygdalina EO samples vary. In the leaf and stem EO of V. perrottettii from Nigeria, β-caryophyllene (28.1% and 39.8%) and germacrene D (34.5% and 16.0%) dominate.¹² V. migeodii leaf EO from Nigeria contains germacrene D (26.8%) and β-caryophyllene (16.4%), while the stem EO consists of α-phellandrene (16.3%), germacrene D (9.8%), (E)-β-farnesene (9.7%), caryophyllene oxide (8.2%), and β-caryophyllene (8.0%).¹³ A report indicates that V. condensata EO from Brazil exhibits varying compositional patterns in its different organs. The leaf EO is abundant in β-caryophyllene (18.35%), γ-muurolene (16.41%), and α-humulene (6.03%), while the stem EO is dominated by linalool (12.13%), 10-epi-italicene ether (8.88%), myrcene (7.68%), and 2-δ-carene (6.86%).¹⁴ In contrast, the flower EO contains 1,8-cineole (11.03%), thymol (7.68%), and linalool (6.30%).¹⁴

In addition, Vernonia volatiles have demonstrated various biological activities. The EO from V. patula exhibited antibacterial and anti-yeast activity against Enterococcus faecalis, with a minimum inhibitory concentration (MIC) value of 8.67 µg/mL.³ It also showed activity against Staphylococcus aureus (MIC = 19.34 µg/mL), Bacillus cereus (MIC = 15.67 µg/mL), Pseudomonas aeruginosa (MIC = 23.45 µg/mL), and Candida albicans (MIC = 15.99 µg/mL).³ Similarly, V. solanifolia leaf EO exhibited activity against E. faecalis (MIC = 32.20 µg/mL), S. aureus (MIC = 33.56 µg/mL), B. cereus (MIC = 32.76 µg/mL), P. aeruginosa (MIC = 15.66 µg/mL), and C. albicans (MIC = 16.33 µg/mL).⁵ V. chalybaea EO and its major compound, β-caryophyllene, are toxic to Trichophyton rubrum and possess antioxidant properties, with a half maximal inhibitory concentration (IC₅₀) value of 35.87 ± 0.32 µg/mL.⁷ The EO of V. amygdalina from Kenya demonstrated insecticidal activity against the maize weevil, Sitophilus zeamais.¹¹

The aim of this research is to investigate the chemical composition, antimicrobial, and antioxidant properties of the EO extracted from the leaves of V. volkameriifolia. Although there are many studies on EOs of different Vernonia species, as analyzed above, the lack of data on V. volkameriifolia prompted us to conduct this study. Moreover, EOs play a crucial role as alternatives to synthetic antibiotics and antioxidants.¹⁵ Therefore, it is essential to understand the composition and potential medicinal properties of the EO extracted from V. volkameriifolia to expand our knowledge of natural products and explore their therapeutic applications. This current study, among a series on the analysis of chemical constituents and biological actions of EOs from plants in Vietnam,^16–18 provides the first information on the EO from V. volkameriifolia.

Results and Discussion

Chemical Compositions of V. volkameriifolia EO

A physical examination revealed that V. volkameriifolia produced a light-yellow EO. The yield obtained for the EO was 0.24% (v/w), determined on a dry weight basis. Mass spectrometry analysis of V. volkameriifolia identified 34 different compounds (Table 1), comprising 13 monoterpene hydrocarbons (29.5%), 1 oxygenated monoterpene (0.1%), 13 sesquiterpene hydrocarbons (58.6%), 6 oxygenated sesquiterpenes (5.6%), and 1 diterpene (0.2%). The main sesquiterpene compounds of V. volkameriifolia are β-bisabolene (25.2%), germacrene D (13.1%) and cis-β-elemene (11.4%). The other significant sesquiterpene compounds were 9-epi-(E)-caryophyllene (2.3%), spathulenol (2.2%), δ-elemene (1.6%), 1-epi-cubenol (1.6%), germacrene B (1.3%), and δ-cadinene (1.2%), all other sesquiterpene compounds were identified in amount less than 1%. The main monoterpene compound was β-pinene (19.1%). The EO also featured a sizeable quantity of α-pinene (2.9%), myrcene (1.9%), α-phellandrene (1.3%), limonene (1.3%), and sabinene (1.0%).

Table 1.

Chemical Compositions of Vernonia volkameriifolia EO.

No.	Compound	Molecular formula	RI^a	RI^b	Concentration (%)
1	β-Bisabolene	C₁₅H₂₄	1518	1505	25.2
2	β-Pinene	C₁₀H₁₆	985	974	19.1
3	Germacrene D	C₁₅H₂₄	1497	1484	13.1
4	cis-β-Elemene	C₁₅H₂₄	1403	1389	11.4
5	α-Pinene	C₁₀H₁₆	939	932	2.9
6	9-epi-(E)-Caryophyllene	C₁₅H₂₄	1479	1464	2.3
7	Spathulenol	C₁₅H₂₄O	1596	1577	2.2
8	Myrcene	C₁₀H₁₆	992	988	1.9
9	δ-Elemene	C₁₅H₂₄	1348	1335	1.6
10	1-epi-Cubenol	C₁₅H₂₆O	1644	1627	1.6
11	α-Phellandrene	C₁₀H₁₆	1010	1002	1.3
12	Limonene	C₁₀H₁₆	1034	1024	1.3
13	Germacrene B	C₁₅H₂₄	1578	1559	1.3
14	δ-Cadinene	C₁₅H₂₄	1537	1522	1.2
15	Sabinene	C₁₀H₁₆	979	969	1.0
16	α-Humulene	C₁₅H₂₄	1471	1452	0.9
17	β-Eudesmol	C₁₅H₂₆O	1664	1649	0.6
18	(E,Z)-Farnesol	C₁₅H₂₆O	1718	1714	0.5
19	(E)-β-Ocimene	C₁₀H₁₆	1048	1044	0.4
20	γ-Terpinene	C₁₀H₁₆	1061	1054	0.4
21	β-Caryophyllene	C₁₅H₂₄	1437	1417	0.4
22	γ-Elemene	C₁₅H₂₄	1445	1434	0.4
23	epi-α-Muurolol	C₁₅H₂₆O	1661	1640	0.4
24	o-Cymene	C₁₀H₁₄	1029	1022	0.3
25	Terpinolene	C₁₀H₁₆	1094	1086	0.3
26	α-Copaene	C₁₅H₂₄	1373	1374	0.3
27	β-Cubebene	C₁₅H₂₄	1386	1387	0.3
28	Guaiol	C₁₅H₂₆O	1615	1600	0.3
29	δ-3-Carene	C₁₀H₁₆	1016	1008	0.2
30	α-Terpinene	C₁₀H₁₆	1022	1014	0.2
31	γ-Muurolene	C₁₅H₂₄	1491	1478	0.2
32	Phytol	C₂₀H₄₀O	2117	2109	0.2
33	Camphene	C₁₀H₁₆	956	946	0.1
34	α-Terpinyl acetate	C₁₂H₂₀O₂	1357	1346	0.1
	Total				93.9
	Monoterpene hydrocarbons				29.4
	Oxygenated monoterpenes				0.1
	Sesquiterpene hydrocarbons				58.6
	Oxygenated sesquiterpenes				5.6
	Diterpenes				0.2

Retention indices on HP-5MS column.

Literature retention indices.

The current result is the first study on the EO constituents of V. volkameriifolia and so could not be compared with similar data on the plant. The EO of V. volkameriifolia was mostly composed of monoterpenes and sesquiterpenes, which have been previously found in other Vernonia plants. However, due to various factors such as the nature of the plant and climatic conditions, the compositional pattern of V. volkameriifolia EO was quite different from several previously reported Vernonia plants. In previous studies of EOs from Vernonia plants, some compositional patterns were discernible such as:

β-caryophyllene and an abundant of another sesquiterpenoid, as found in V. patula EO (with caryophyllene oxide) from Vietnam;³ V. amygdalina EO (with caryophyllene oxide) from Vietnam;⁴ V. cinerea EO (with δ-cadinene) from India;⁶ V. chalybaea EO (with bicyclogermacrene) from Brazil⁷; V. scorpioides EO (with germacrene D) from Brazil;⁸ V. perrottettii and V. migeodii EOs (with germacrene D) from Nigeria;^12,13 and V. condensata EO (with γ-muurolene) from Brazil;¹⁴

V. amygdalina EO from Vietnam with a lower amount of β-caryophyllene and a higher amount of caryophyllene oxide and humulene epoxide II;⁴

V. solanifolia EO from Vietnam with a lower amount of β-caryophyllene and a higher amount of sabinene and δ-cadinene;⁵

V. amygdalina EO from Nigeria with a high amount of α-muurolol⁹;

thymol and (E)-phytol rich EO of V. amygdalina from Nigeria¹⁰;

1,8-cineole and another compound of V. amygdalina EO (with β-pinene) from Kenya¹¹ and V. condensata EO (with thymol) from Brazil¹⁴;

V. migeodii EO from Nigeria which has a low amount of β-caryophyllene and a significant amount of α-phellandrene and germacrene D¹³; and

V. condensata EO from Brazil, which consist mainly of linalool.¹⁴

Therefore, the EO of Vernonia plants with a high amount of combination of β-bisabolene/β-pinene/germacrene D/cis-β-elemene series presented here for V. volkameriifolia was uncommon. This is evident from the fact that several compounds such as 1,8-cineole, linalool, thymol, caryophyllene oxide among others that were identified in abundant quantities were conspicuously absent in this EO sample. By chemotaxonomic consideration, the leaf EO of V. volkameriifolia belongs to the group characterized by an abundance of terpene hydrocarbons.

Antimicrobial Activity of V. volkameriifolia EO

The performance of the EO in relation to microbial tests was observed and recorded in Table 2. Notably, V. volkameriifolia EO exhibited varying effects against the microorganisms. It showed strong anti-fungal activity against C. albicans with a MIC value of 100 μg/mL. However, V. volkameriifolia EO only demonstrated moderate activity against Bacillus subtilis (Gram-positive), S. aureus (Gram-positive), and E. coli (Gram-negative), with a MIC of 200 μg/mL. This value was also obtained in tests against other fungi, namely Aspergillus niger and Saccharomyces cerevisiae. A comparative analysis of this antimicrobial data with results reported for other Vernonia plants in the literature revealed that V. volkameriifolia EO exhibited lower potency against comparable microorganisms. For example, V. patula EO was highly effective against S. aureus (MIC = 19.34 µg/mL) and C. albicans (MIC = 15.99 µg/mL).³ Similarly, V. solanifolia EO exhibited a MIC of 33.56 µg/mL against S. aureus and a MIC of 16.33 µg/mL against C. albicans,⁵ making it more active than V. volkameriifolia EO. Moreover, both V. patula and V. solanifolia EOs displayed promising antimicrobial activity against P. aeruginosa (MIC of 23.45 µg/mL and 15.66 µg/mL, respectively) and B. cereus (MIC of 15.67 µg/mL and 32.76 µg/mL, respectively).^3,5 However, both V. patula and V. solanifolia EOs were found to be ineffective against E. coli, in contrast to V. volkameriifolia EO.^3,5

Table 2.

Antimicrobial Activity of Vernonia volkameriifolia EO (MIC, μg/mL).

Microorganisms	Essential oil	Streptomycin	Tetracycline	Nystatin
Bacillus subtilis	200	6.25	−	−
Staphylococcus aureus	200	12.5	−	−
Escherichia coli	200	−	12.5	−
Pseudomonas aeruginosa	−	−	12.5	−
Aspergillus niger	200	−	−	12.5
Fusarium oxysporum	−	−	−	25.0
Candida albicans	100	−	−	12.5
Saccharomyces cerevisiae	200	−	−	12.5

It is widely believed and known that the chemical contents of natural products, including EOs, have a direct bearing on their potential activities.^19,20 Also worth mentioning is that the antibacterial activity of EOs is generally lower against Gram-negative bacteria than against Gram-positive bacteria.²⁰ Moreover, EOs contain a large number of compounds, and it is imperative that their mode of action against targeted microorganisms involves various target points within the microorganism cells.²⁰ The compounds responsible for eliciting the antimicrobial and other biological activities of EOs vary from plant to plant and are widely regarded as the major constituents, while also noting, in several instances, the presence of both synergies and antagonistic effects between the major and some minor constituents.²⁰

In effect, the antimicrobial effect of an EO is attributed to the combined actions of many substances.²⁰ For example, β-bisabolene showed selective activity and synergistic bactericidal effects with ampicillin against S. aureus.²¹ EOs hydrodistilled from Bocageopsis pleiosperma and Psammogeton canescens, which contained a large amount of β-bisabolene, elicited antimicrobial activities and inhibited the growth of C. albicans and E. coli, respectively.^22,23 The compound germacrene D was reported to be active against several bacteria and possesses antibacterial properties.²⁴ The report showed that β-caryophyllene was selective in its activity, specifically against S. aureus, and exhibited pronounced anti-fungal activity more than kanamycin.²⁵ In another study, β-caryophyllene exhibited activity towards B. cereus,²⁶ in addition to potentiating the action of norfloxacin against S. aureus, P. aeruginosa, and E. coli.²⁷ Results have shown that, among other microbes, α-pinene has inhibited the growth of S. aureus, B. cereus, E. coli, and C. albicans.²⁸ The antimicrobial activities of EOs from some plants were most strongly correlated with the content of (Z)-β-ocimene, δ-cadinene, camphene, β-elemene, γ-cadinene, among others.²⁹ β-Pinene^30,31 and β-ocimene^30,32 have been implicated in the antimicrobial potential of several plant extracts. Spathulenol, a sesquiterpenoid, is known to have exhibited a killing effect on some microbes.^33,34

Antioxidant Activity of V. volkameriifolia EO

Most often, the process of antioxidant activity involves multiple mechanisms.¹⁷ This may necessitate using multiple testing methods in order to accurately determine the antioxidant activity of natural products.¹⁷ In the study of the antioxidant activity of V. volkameriifolia EO, the assays of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging activity were employed. The EO exhibited good antioxidant activity. Specifically, the IC₅₀ value for the EO was 144.1 μg/mL in the DPPH assay and 179.8 μg/mL in the ABTS assay. As seen in Table 3, however, the activity was lower than that of the standard antioxidant compound, butylated hydroxytoluene (BHT), which exhibited IC₅₀ values of 20.1 μg/mL and 27.3 μg/mL for DPPH and ABTS, respectively. The result presented here represents the first antioxidant study of V. volkameriifolia EO. There are few reports available on the antioxidant data for EOs from other Vernonia species in the literature. However, V. chalybaea EO displayed antioxidant activity in the β-carotene/linoleic acid system, with an IC₅₀ value of 35.87 ± 0.32 µg/mL.⁷

Table 3.

Antioxidant Activity of Vernonia volkameriifolia EO.

Concentration (μg/mL)	DPPH assay		ABTS assay
Concentration (μg/mL)	Scavenging (%)^a	IC₅₀ (μg/mL)	Scavenging (%)^a	IC₅₀ (μg/mL)
25	21.5 ± 0.95^D	144.1	17.3 ± 0.91^D	179.8
50	35.4 ± 1.24^C		25.1 ± 1.04^C
100	41.8 ± 1.42^B		35.9 ± 1.31^B
200	60.4 ± 1.67^A		53.1 ± 1.52^A

Data represent means ± SD (n = 3); different letters within the column mean a significant difference at P < .05.

The best and potentially most effective natural products with antioxidant activity have IC₅₀ values that range from 8.3 µg/mL to 278 µg/mL.^35,36 These values vary due to the nature of their chemical compositions. The EO from V. volkameriifolia falls within this range of IC₅₀ values. EOs with antioxidant properties naturally inhibit the formation of free radicals and free radical ions. The data on the antioxidant activity of V. volkameriifolia EO can be explained from the perspective of several constituents it contains, including β-bisabolene,²³ germacrene D,³⁷ β-pinene,³⁰ among others, which exhibit potent antioxidant activities. Additionally, the antioxidant properties may also involve the synergistic actions of minor constituents, including β-caryophyllene,³⁸ α-pinene,^30,39 limonene,³⁹ and myrcene.⁴⁰ These compounds are inclined to neutralize free radicals by either contributing hydrogen atoms or electrons, thus stabilizing the radicals and impeding the oxidation process.⁴¹ This mechanism plays a vital role in shielding cells and tissues from oxidative stress, which is linked to a range of illnesses and the aging process. Additionally, both the DPPH and ABTS assays are commonly employed in tandem to evaluate the antioxidant capacity of EOs.¹⁷ These tests offer valuable insights into the EOs’ capability to counteract free radicals and safeguard against oxidative harm.

Conclusions

In summary, this study has provided valuable insights into the composition and properties of V. volkameriifolia leaf EO. The EO was found to contain significant amounts of β-bisabolene, β-pinene, germacrene D, and cis-β-elemene. Importantly, this EO exhibited notable antimicrobial activity against a range of microorganisms, including B. subtilis, S. aureus, E. coli, A. niger, S. cerevisiae, and C. albicans. Furthermore, the EO displayed promising antioxidant capabilities, effectively scavenging free radicals as evidenced by its IC₅₀ values in DPPH and ABTS assays. These findings highlight the potential of V. volkameriifolia EO as a valuable natural resource with dual properties as an antimicrobial and antioxidant agent. The results of this study lend scientific support to the traditional ethnomedical uses of this plant, suggesting that it may have practical applications in pharmaceuticals, cosmetics, or food industries for its beneficial properties. Further research and exploration of the therapeutic potential of V. volkameriifolia EO are warranted to harness its full range of benefits for various applications.

Materials and Methods

Plant material and EO extraction

Leaves of V. volkameriifolia were collected in July 2022 from Pu Luong Nature Reserve, located in Thanh Hoa Province, Vietnam. The identification of the plant sample was conducted by Dau B. Thin, a botanist affiliated with Hong Duc University, Vietnam. A voucher specimen (TH2022-50) was deposited in the herbarium of that university. After collection, the plant material was air-dried at room temperature for one week. EO extraction was carried out via hydrodistillation, following the guidelines outlined in the Vietnamese Pharmacopoeia.⁴² This extraction process spanned 3 hours, employing a Clevenger-type apparatus, with distilled water serving as the collection solvent. After the removal of water from EO by anhydrous sodium sulfate, the EO sample was stored in a vial at 4 °C for future use.

Analysis of V. volkameriifolia EO

The chemical composition of the EO extracted from V. volkameriifolia was analyzed using gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS), following established procedures.^43,44 The GC analyses were conducted on an Agilent Technologies GC apparatus, model HP 7890A Plus, equipped with a flame ionization detector and an HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness). The GC oven temperature program initiated at 60 °C for 2 minutes, then increased to 220 °C at a rate of 4 °C/min. The injector and detector temperatures were maintained at 250 °C and 260 °C, respectively. Helium served as the carrier gas at a flow rate of 1.0 mL/min.

For GC–MS analyses, the same equipment and experimental parameters as the GC analyses were applied, connected to an HP 5973 MSD mass spectrometer. A split ratio of 10:1, an ionization energy of 70 eV, and an emission current of 40 mA were set. The sampling rate was 1.0 scan/s, and the mass range extended from 35 amu to 350 amu. Component identification relied on comparing GC retention times with known compounds and mass spectra with those stored in the computer database and published spectra.^45,46 The percentage composition was determined by normalizing peak areas without incorporating corrective factors.

Antimicrobial assay of V. volkameriifolia EO

The EO extracted from V. volkameriifolia was evaluated for its antimicrobial activity against eight microorganisms sourced from the American Type Culture Collection (ATCC, Manassas, USA). This collection comprised two Gram-positive bacteria (B. subtilis ATCC 6633 and S. aureus ATCC 6538), two Gram-negative bacteria (E. coli ATCC 8739 and P. aeruginosa ATCC 9027), two filamentous fungi (A. niger ATCC 9763 and F. oxysporum ATCC 48112), and two yeasts (C. albicans ATCC 10231 and S. cerevisiae ATCC 16404). The bacteria and fungi were cultured on Tryptic Soy Agar and Sabouraud Dextrose Agar, respectively. The determination of the MIC value was conducted using the broth microdilution susceptibility method, following established procedures.^16,17

For this purpose, the EO was dissolved in 1% dimethylsulfoxide (DMSO) and subsequently diluted to the highest concentration. Serial doubling dilutions of the EO were then prepared in a 96-well microtiter plate. Overnight broth cultures of each microbial strain were prepared, and the final concentration in each well was adjusted to 5 × 10⁵ CFU/mL for bacteria and 1 × 10³ CFU/mL for fungi. Subsequently, the bacteria and fungi were incubated for 24 hours at 37 °C and 30 °C, respectively. Positive controls, including Streptomycin for Gram-positive bacteria, Tetracycline for Gram-negative bacteria, and Nystatin for fungi, were prepared under the same experimental conditions. Additionally, a negative control using the vehicle (DMSO) was included. The MIC value was defined as the lowest concentration at which no visible growth of the microorganism occurred.

Antioxidant assay of V. volkameriifolia EO

The EO extracted from V. volkameriifolia was evaluated for its antioxidant activity using DPPH and ABTS assays, following established procedures.^17,47 In the DPPH assay, a 0.1-mM DPPH solution in methanol was prepared, and 3 mL of this solution was mixed with 1 mL of the EO sample, previously dissolved in methanol at varying concentrations. The resulting mixture underwent agitation and was then incubated for 30 minutes at 37 °C in darkness. Subsequently, absorbance readings were recorded at 517 nm using a UV spectrophotometer (Thermo Scientific, Waltham, MA, USA).

For the ABTS assay, a 7-mM ABTS solution was created by dissolving ABTS powder in distilled water, and this solution was mixed with a potassium persulfate solution (2.45 mM) in a 1:1 ratio (v/v). The resulting blend was incubated in darkness at room temperature for 16 hours to generate free radicals. Following this, a 3-mL volume was selected from the solution and adjusted to an absorbance of 0.708 ± 0.025 at 734 nm by adding ethanol. Then, 0.2 mL of the EO sample was combined with 2 mL of the ABTS reagent and allowed to incubate for 6 minutes. Post-incubation, the absorbance at 734 nm was measured.

The radical scavenging activity of each sample in both DPPH and ABTS assays was determined by calculating the percentage inhibition (I%) using the formula: I% = (A₁ – A₂/A₁) × 100. Where, A₁ is the absorbance of the control reaction, and A₂ is the absorbance of the test sample. The results of the DPPH and ABTS scavenging activities were expressed as IC₅₀ values, representing the concentration of the antioxidant necessary to scavenge 50% of the DPPH or ABTS present in the test solution. The experiments were conducted independently three times. The IC₅₀ values for both DPPH and ABTS assays were calculated through linear regression analysis using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA) as the analytical tool. BHT served as a positive control for reference purposes.

Statistical Analysis

The data were statistically analyzed using SPSS Statistics software (version 24.0 for Windows, SPSS Inc.). Differences between means were assessed using the least significant difference test at a significance level of P < .05. The results are presented as the mean value ± standard deviation.

Footnotes

Acknowledgments

The authors would like to thank the editor and anonymous reviewers for their thoughtful comments and efforts toward improving our manuscript.

Author's Note

Bui B. Thinh is also affiliated at Biotechnology Center of Ho Chi Minh City, Ho Chi Minh City, Vietnam.

Authors Contributions

BBT conducted experiments, analyzed data, wrote the first draft, and edited the manuscript. DBT conceived the idea, designed the research, and analyzed data. IAO analyzed data, interpreted the results, and wrote the first draft. All authors read and approved the final draft of the manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Bui B. Thinh

References

. An illustrated the flora of Vietnam, Vol. 3. Tre Publishing House, Ho Chi Minh; 1999.

Bai

Lou

Liu

. Studies on the chemical constituents of Vernonia volkameriifolia (Wall.) Dc. Zhongcaoyao. 1985;16(3):530‐532.

Hoi

Chung

Huong

Ogunwande

. Studies on Asteraceae: chemical compositions of essential oils and antimicrobial activity of the leaves of Vernonia patula (Dryand.) Merr. and Grangea maderaspatana (L.) Poir. from Vietnam. J Essent Oil Bearing Plants. 2021;24(3):500‐509. doi:10.1080/0972060X.2021.1947390

Thuc

Mai

VTH

. Chemical composition of essential oils from the leaves and stems of Vernonia amygdalina Del. collected in Vietnam. Am J Essent Oil Nat Prod. 2021;9(1):39‐42.

Huong

Hoi

Chung

Binh

Son

Ogunwande

. Chemical composition and antimicrobial activity of the leaf essential oil of Vernonia solanifolia. Chem Nat Compd. 2022;58(5):943‐946. doi:10.1007/s10600-022-03836-1

Joshi

. Sesquiterpene rich essential oil of Vernonia cinerea Less. from India. S Afr J Bot. 2014;95:129‐130. doi:10.1016/j.sajb.2014.09.005

Sobrinho

ACN

Morais

SMD

Souza

EBD

, et al. Antifungal and antioxidant activities of Vernonia chalybaea Mart. ex DC. essential oil and their major constituent β-caryophyllene. Braz Arch Biol Technol. 2020;63:e20190177. doi:10.1590/1678-4324-2020190177

Albuquerque

MRJR

Lemos

TLG

Pessoa

ODL

Nunes

Nascimento

Silveira

. Chemical composition of the essential oil from Vernonia scorpioides (Asteraceae). Flavour Fragr J. 2007;22(4):249‐250. doi:10.1002/ffj.1782

Ogunbinu

Flamini

Cioni

Ogunwande

Okeniyi

. Essential oil constituents of Eclipta prostrata (L.) L. and Vernonia amygdalina Delile. Nat Prod Commun. 2009;4(3):421‐424. doi:10.1177/1934578X0900400321

10.

Sonibare

Adesanya

. Essential oil composition of Vernonia amygdalina Del. from Southwestern Nigeria. J Essent Oil Bearing Plants. 2009;12(1):55‐58. doi:10.1080/0972060X.2009.10643691

11.

Asawalam

Hassanali

. Constituents of the essential oil of Vernonia amygdalina as maize weevil protectants. Trop Subtrop Agroecosystems. 2006;6(2):95‐102.

12.

Ogunbinu

Okeniyi

Flamini

Cioni

Ogunwande

Olayinka

. Essential oil-bearing plants from Nigeria: studies on Vernonia perrottettii (leaf and stem bark), young leaves from Eucalyptus decaisneana and immature leaves of Hyptis suaveolens. J Essent Oil Res. 2009;21(2):154‐158. doi:10.1080/10412905.2009.9700137

13.

Ogunbinu

Okeniyi

Ogunwande

Flamini

Cioni

. Terpenoid composition of the leaf and stem bark essential oils of Vernonia migeodii S. Moore (Asteraceae). J Essent Oil Res. 2009;21(3):246‐248. doi:10.1080/10412905.2009.9700160

14.

Lopes

da Camara

CAG

de Moraes

. Composition of essential oils from the leaves, stems, and flowers of Vernonia condensata of Pernambuco, Brazil. Chem Nat Compd. 2019;55:756‐758. doi:10.1007/s10600-019-02802-8

15.

Mutlu-Ingok

Devecioglu

Dikmetas

Karbancioglu-Guler

Capanoglu

. Antibacterial, antifungal, antimycotoxigenic, and antioxidant activities of essential oils: an updated review. Molecules. 2020;25(20):4711. doi:10.3390/molecules25204711

16.

Thin

Korneeva

Thinh

Ogunwande

. Chemical composition, antimicrobial and antioxidant activities of essential oil and methanol extract from the stems of Dasymaschalon rostratum Merr. & Chun. Russ J Bioorg Chem. 2023;49(4):815‐822. doi:10.1134/S1068162023040209

17.

Thinh

Thin

. Essential oil composition, antimicrobial and antioxidant properties of Pluchea eupatorioides Kurz collected from Vietnam. J Essent Oil Bearing Plants. 2023;26(3):653‐663. doi:10.1080/0972060X.2023.2234398

18.

Huong

Dai

Thin

Hung

Thinh

. Essential oils of Distichochlamys benenica: chemical constituents, mosquito larvicidal and antimicrobial activities. Nat Prod Commun. 2023;18(8):1‐10. doi:10.1177/1934578X231193541

19.

Holetz

Pessini

Sanches

Cortez

DAG

Nakamura

Dias Filho

. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz. 2002;97:1027‐1031. doi:10.1590/S0074-02762002000700017

20.

Lang

Buchbauer

. A review on recent research results (2008–2010) on essential oils as antimicrobials and antifungals. A review. Flavour Fragr J. 2012;27(1):13‐39. doi:10.1002/ffj.2082

21.

Nascimento

Brandao

Oliveira

Fortes

Chartone-Souza

. Synergistic bactericidal activity of Eremanthus erythropappus oil or β-bisabolene with ampicillin against Staphylococcus aureus. Antonie van Leeuwenhoek. 2007;92:95‐100. doi:10.1007/s10482-006-9139-x

22.

Soares

da Silva

de Almeida

, et al. Chemical composition and antimicrobial evaluation of the essential oils of Bocageopsis pleiosperma Maas. Nat Prod Res. 2015;29(13):1285‐1288. doi:10.1080/14786419.2014.996148

23.

Kazemi

Rostami

. Chemical composition, antimicrobial and antioxidant activities of the essential oil of Psammogeton canescens. Nat Prod Res. 2015;29(3):277‐280. doi:10.1080/14786419.2014.951357

24.

Olajuyigbe

Ashafa

. Chemical composition and antibacterial activity of essential oil of Cosmos bipinnatus Cav. leaves from South Africa. Iran J Pharm Res. 2014;13(4):1417‐1423.

25.

Dahham

Tabana

Iqbal

, et al. The anticancer,antioxidant and antimicrobial properties of the sesquiterpene β-caryophyllene from the essential oil of Aquilaria crassna. Molecules. 2015;20(7):11808‐11829. doi:10.3390/molecules200711808

26.

Moo

Yang

Osman

, et al. Antibacterial activity and mode of action of β-caryophyllene on Bacillus cereus. Pol J Microbiol. 2020;69(1):49‐54. doi:10.33073/pjm-2020-007

27.

Santos

Freitas

Araújo

ACJ

, et al. Enhanced antibacterial effect of antibiotics by the essential oil of Aloysia gratissima (Gillies & Hook.) Tronc. and its major constituent beta-caryophyllene. Phytomed Plus. 2021;1(4):100100. doi:10.1016/j.phyplu.2021.100100

28.

Allenspach

Steuer

. α-Pinene: a never-ending story. Phytochemistry. 2021;190:112857. doi:10.1016/j.phytochem.2021.112857

29.

Pellegrini

Alonso-Salces

Umpierrez

Rossini

Fuselli

. Chemical composition, antimicrobial activity, and mode of action of essential oils against Paenibacillus larvae, etiological agent of American foulbrood on Apis mellifera. Chem Biodivers. 2017;14(4):e1600382. doi:10.1002/cbdv.201600382

30.

Salehi

Upadhyay

Erdogan Orhan

, et al. Therapeutic potential of α-and β-pinene: a miracle gift of nature. Biomolecules. 2019;9(11):738. doi:10.3390/biom9110738

31.

Bendifallah

Merah

. Phytochemical and biocidal properties of Artemisia campestris subsp. campestris L. (Asteraceae) essential oil at the southern region of Algeria. J Nat Pestic Res. 2023;4:100035. doi:10.1016/j.napere.2023.100035

32.

Farré-Armengol

Filella

Llusià

Peñuelas

. β-Ocimene, a key floral and foliar volatile involved in multiple interactions between plants and other organisms. Molecules. 2017;22(7):1148. doi:10.3390/molecules22071148

33.

Chen

Liu

Wang

, et al. Environmentally friendly plant essential oil: liquid gold for human health. Adv Agron. 2021;170:289‐337. doi:10.1016/bs.agron.2021.06.005

34.

Şener

Özkınalı

Gür

Güney

Özkan

Khalifa

. Determination of antimicrobial activity and chemical composition of pimento & ginger essential oil. Ind J Pharm Edu Res. 2017;51(3):S230‐S232. doi:10.5530/ijper.51.3 s.19

35.

Amiri

. Essential oils composition and antioxidant properties of three Thymus species. Evid Based Complement Alternat Med. 2012;2012:728065. doi:10.1155/2012/728065

36.

Pandey

Mohan

Singh

Palni

Tripathi

. Chemical composition, antibacterial and antioxidant activity of essential oil of Eupatorium adenophorum Spreng. from Eastern Uttar Pradesh, India. Food Biosci. 2014;7:80‐87. doi:10.1016/j.fbio.2014.06.001

37.

Victoria

Lenardão

Savegnago

, et al. Essential oil of the leaves of Eugenia uniflora L.: antioxidant and antimicrobial properties. Food Chem Toxicol. 2012;50(8):2668‐2674. doi:10.1016/j.fct.2012.05.002

38.

Montanari

Barbosa

Demuner

Silva

Carvalho

Andrade

. Chemical composition and antibacterial activity of essential oils from Verbenaceae species: alternative sources of (E)-caryophyllene and germacrene-D. Quim Nova. 2011;34:1550‐1555. doi:10.1590/S0100-40422011000900013

39.

Dowom

Abrishamchi

Asili

. Essential oil (EO) composition and antioxidant activity of two Salvia leriifolia Benth. (Lamiaceae) populations from Iran. N Biol Reperta. 2016;3(2):108‐117. doi:10.21859/acadpub.nbr.3.2.108

40.

Wang

Chen

Hou

. Antioxidant and antibacterial activity of seven predominant terpenoids. Int J Food Prop. 2019;22(1):230‐238. doi:10.1080/10942912.2019.1582541

41.

Brewer

. Natural antioxidants: sources, compounds, mechanisms of action, and potential applications. Compr Rev Food Sci Food Saf. 2011;10(4):221‐247. doi:10.1111/j.1541-4337.2011.00156.x

42.

Ministry of Health. Vietnamese pharmacopoeia. Medical Publishing House; 2009.

43.

Thin

Thinh

Igoli

. Chemical composition and biological activity of essential oil of Knema pierrei. Chem Nat Compd. 2023;59(3):584‐586. doi:10.1007/s10600-023-04061-0

44.

Chac

Hoi

Thinh

. Chemical composition and antimicrobial activity of the essential oil of Fibraurea tinctoria. Chem Nat Compd. 2023;59(3):597‐599. doi:10.1007/s10600-023-04065-w

45.

Adams

. Identification of essential oil components by gas chromatography/mass spectroscopy. Allured Publishing Corporation, Carol Stream; 2007.

46.

NIST. Chemistry Web Book. Data from NIST Standard Reference Database, 69; 2018.

47.

Thinh

Khoi

Doudkin

Thin

Ogunwande

. Chemical composition of essential oil and antioxidant activity of the essential oil and methanol extracts of Knema globularia (Lam.). Warb. from Vietnam. Nat Prod Res. 2023;37(10):1625‐1631. doi:10.1080/14786419.2022.2103698