Universidade Federal de Santa Maria

Ci. e Nat., Santa Maria, v. 46, e83759, 2024

DOI: 10.5902/2179460X83759

ISSN 2179-460X

Submitted: 12/05/2023 • Approved: 31/10/2024 • Published: 29/11/2024

1 INTRODUCTION

2 MATERIALS AND METHODS

3 RESULTS

4 DISCUSSION

5 CONCLUSION

ACKNOWLEDGMENTS

REFERENCES

Chemistry

Ageratum conyzoides, an invasive species with antioxidant and antifungal potential

Ageratum conyzoides, uma espécie invasiva com potencial antioxidante e antifúngica

Vanusa Souza Rocha PereiraI

Gabriela Catuzo Canônico SilvaI

Edneide Bezerra da Cruz OliveiraI

Mariane de Almeida MachadoI

Jéssica da Silva SenaI

Marisa Cássia Vieira de Araújo BentoI

Selma Alves RodriguesI

Rodrigo Sadao InumaroII

José Eduardo GonçalvesII

Maria Graciela Iecher Faria NunesI

Suelen Pereira Ruiz HerigI

Ranulfo Piau JuniorI

Zilda Cristiani GazimI

I Universidade Paranaense, Umuarama, PR, Brazil

II Centro Universitário Cesumar, Maringá, PR, Brazil

ABSTRACT

This study aimed to evaluate the chemical composition and the antioxidant and antifungal activities of the essential oil (EO) extracted from the aerial parts (leaves and flowers) of Ageratum conyzoides (Asteraceae). The EO was extracted by the hydrodistillation process (3h), and the compounds were identified by gas chromatography coupled to mass spectrometry (GC-MS). Antioxidant activity was performed by the β-carotene/linoleic acid co-oxidation system and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging and the iron reduction method (FRAP). The antifungal activity was performed by the broth microdilution (MIC) method using the strains Rhizopus oryzae ATCC 7560; Aspergillus flavus ATCC 1217; Aspergillus ochraceus ATCC 6787 and Penicillium verrucosum ATCC 7680. The results indicated a yield of 0.82 mg/kg. Sesquiterpenes hydrocarbons (33.28%) were the major class, and precocene I (48.19%), precocene II (7.38%) and β-caryophyllene (19.66%) were the major constituents. The co-oxidation system of β-carotene/linoleic acid showed 52.18% inhibition of oxidation in the concentration of 1.0 mg/mL. Of the four fungi evaluated, only Aspergillus ochraceus showed results, with a MIC of 1250 μL/mL, and the importance of finding activity on this fungus lies in the fact that it is a producer of ochratoxin A, infesting mainly green coffee beans. The results found open up new perspectives in valuing a species considered invasive.

Keywords: Precocenes; β-caryophyllene; Aspergillus ochraceus; Mentrast

RESUMO

Este estudo teve como objetivo avaliar a composição química e as atividades antioxidante e antifúngica do óleo essencial (OE) extraído das partes aéreas (folhas e flores) de Ageratum conyzoides (Asteraceae). O OE foi extraído pelo processo de hidrodestilação (3h), e os compostos identificados por cromatografia gasosa acoplada à espectrometria de massas (CG-EM). A atividade antioxidante foi avaliada pelo sistema de co-oxidação β-caroteno/ácido linoléico e pelo seqüestro de radicais livres 2,2-difenil-1-picril-hidrazil (DPPH) e pelo método de redução de ferro (FRAP). A atividade antifúngica foi realizada pelo método de microdiluição em caldo (MIC), utilizando as cepas Rhizopus oryzae ATCC 7560; Aspergillus flavus ATCC 1217; Aspergillus ochraceus ATCC 6787 e Penicillium verrucosum ATCC 7680. Os resultados indicaram um rendimento de 0,82 mg/kg. Hidrocarbonetos sesquiterpenos (33,28%) foram a classe majoritária e precoceno I (48,19%), precoceno II (7,38%) e β-cariofileno (19,66%) os constituintes majoritários. O sistema de co-oxidação β-caroteno/ácido linoléico apresentou 52,18% de inibição da oxidação na concentração de 1,0 mg/mL. Dos quatro fungos avaliados, apenas Aspergillus ochraceus apresentou resultado, com CIM de 1250 μL/mL, e a importância de encontrar atividade sobre esse fungo está no fato de ser produtor de ocratoxina A, infestando principalmente grãos de café verde. Os resultados encontrados abrem novas perspectivas na valorização de uma espécie considerada invasora..

Palavras-chave: Precocenos; β-cariofileno; Aspergillus ochraceus; Mentrasto

1 INTRODUCTION

Ageratum conyzoides L. belongs to the Asteraceae family. It is a species native to Central America and the Caribbean and distributed throughout the tropical and subtropical regions (Yadav et al., 2019; Karayat et al., 2024) which justifies the presence of 36 scientific synonyms (Powo, 2023).  The common name, “goat weed” or “Billy goat weed”, is derived from an Australian male goat due to a close resemblance in odor (Kaur et al., 2023).

In Brazil, it is popularly known as “mentrasto, catinga-de-bode, catinga-de-borrão, erva-de-são-joão, maria-preta, celestina, picão-roxo, erva-de-santa-luzia and camará-opela” (Dores et al., 2014). It is a very widespread weed in all agricultural regions of the country, infesting crops, vegetable gardens and vacant lots (Desai et al., 2024), being able to complete its life cycle in two months, even if its greatest propagation occurs in the summer (Sastry et al., 2019). The inflorescences last two months, have different colors (lilac and white) and undergo self-pollination, which produces about 94,000 seeds per plant (Dhami, 2018), justifying the rapid spread.

In the leaves, flowers and branches of this species are found coumarin, chromene, flavonoids (kaempferol, quercetin, quercetin-3-rhamnopyranoside), caffeic acid, phytol, echinatine (pyrrolizidine alkaloids), sterols (stigmasterol, β-sitosterol, and friedelin) (Satija et al., 2018; Erida et al., 2023). It also contains an essential oil composed of benzofurans (precocene I, precocene II, and ageratochromene dimer), terpenes (α-pinene, β-pinene) and phenylpropanoids (eugenol) possessing diverse herbicidal properties (Erida et al., 2023).

These phytoconstituents have shown diverse pharmacological properties including antimicrobial, anti-inflammatory, analgesic, antioxidant, anticancer, antiprotozoal, antidiabetic, spasmolytic, allelopathy (Yadav et al., 2019), hypoglycemic, analgesic, anti-diarrheal, diuretic, antitussive, antirheumatic (ChabI-Sika et al., 2023), anti-fungal, anti-bacterial, anti-ulcerogenic, anti-malarial, antioxidant, anti-protozoal, anti-Ehrlichia and anti-insecticidal activity (Kouame et al., 2018; Erida et al., 2023).

The pharmacological activities of this species justify its use in folk medicine to treat diseases in central Africa such as pneumonia and burns. The leaves or stems of this plant are utilized against inflammatory stomach or intestine diseases (Quoc & Pham, 2020). Additionally, A. conyzoides is used in traditional medicine in Asia, South America and Africa in healing wounds, in rheumatic states, and in fighting fever (Jomba & Kumar, 2023).

The chemical composition of plants is influenced by several external factors, including climate, as some compounds can be accumulated over a given period in response to environmental changes (Hussain et al., 2010). Variability in chemical composition remains a challenge to accurately determine whether these changes will have a positive or negative impact on the chemical constituents of plants (Dobhal et al., 2024). This change may constitute difficulty in the commercialization of essential oils, as they provide color changes, odor and mainly pharmacological changes (Linde et al., 2016), highlighting the antioxidant action, the focus of this research.

There is a lot of interest in discovering natural antioxidants from plants. According to studies on medicinal herbs, the majority of them have substantial antioxidant activity. The medicinal plants include a wide range of natural antioxidants and are used to cure and prevention a variety of diseases such as cancer, diabetes, atherosclerosis, heart disease, nephrotoxicity, hepatotoxicity, cognitive and vision loss (Makhammra et al., 2023).  

Antioxidant compounds are essential for maintaining the balance of the organism, acting in the scavenging of free radicals produced in excess during the metabolic process, where they are important mediators of biochemical reactions (Rahaman et al., 2023).  In some cases, the production of these molecules increases significantly, which can lead to oxidative stress (Barbosa et al., 2010). Exogenous types of antioxidants such as vitamins, flavonoids, anthocyanins, and some mineral compounds are derived from natural sources but also obtained in synthetic forms, like butylhoxyanisole (BHA), butylhydroxytoluene (BHT), and gallates which are primarily synthetic (Rahaman et al., 2023). The industry has widely used synthetic antioxidants such as butyl hydroxyanisole (BHA) and butyl hydroxytoluene (BHT). However, studies indicate possible toxicity and carcinogenic potential related to these substances (Oyetayo, 2009). In this sense, there is a growing interest in the investigation of antioxidants from natural sources such as extracts and essential oils from plants.

Some species of Aspergillus and Penicillium are associated with the production of mycotoxins. The main mycotoxin studied in coffee is ochratoxin A (OTA) and its presence has been reported to be associated with the presence of the fungus A. ochraceus. Fungi that produce mycotoxins are present in coffee plantations, preparation and storage environments and their relationship with the quality and safety of the final product depends on environmental conditions, crop management and post-harvest processing (Ferreira et al., 2011).

The need for new compounds to be used as therapeutic alternatives, such as those obtained from medicinal plants, can be considered powerful resources in the development of antioxidant and antimicrobial agents (Rossato Viana et al., 2023). In this context, the objective of the present experiment consisted of the chemical analysis and determination of the antioxidant and antifungal activity of the essential oil of the mixture of flowers and leaves of Ageratum conyzoides cultivated in southern Brazil.

2 MATERIALS AND METHODS

2.1 Botanical identification

The culture of A. conyzoides is implanted in the Medicinal Garden of Universidade Paranaense – Unipar, located in the city of Umuarama, State of Paraná- Brazil at the coordinates (latitude 23 °C 45’ 59” S, longitude 53 °C 19’ 30” W and altitude of 442 m). 

One specimen was authenticated and deposited in the herbarium of Universidade Educacional Paranaense (HEUP), under number 63. This species is registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under registration number AD5B6A4.

2.2. Obtaining Plant Material and Essential Oil Extraction

Leaves and flowers were collected in June and July 2018 and 2019, in the morning (7:00-9:00 am). The average temperature in the months of the collection was 18.67 ± 0.71 ºC, where the air humidity remained at 70.69 ± 5.96 (%) and the average rainfall index was 1.46 mm in June and 0.03 mm in July.

The plant material was dried at room temperature. 150g of dry material was subjected to essential oil extraction, using a hydrodistillation process in a modified Clevenger apparatus for 3 hours (Ekundayo et al., 1987); afterwards, the essential oil was removed and stored at 4 °C (Pereira et al., 2016).

The essential oil yield (%) was calculated by the mass (g) of essential oil per mass (g) of dry aerial parts (leaves and flowers). Absolute density was determined in graduated capillaries (5.0 µL), determining the ratio between mass (g) and volume (mL) of essential oil at 20 oC. The refractive index was determined using an Abbe refractometer Q767B (model RL3) that was calibrated with distilled water (refractive index 1.3330) at 20 ºC (Farmacopeia Brasileira, 2010).

2.3. Chemical Characterization

The chemical identification of the essential oil was performed by GC-MS (Agilent 7890B-5977A MSD). The capillary column was HP-5MS IU 5% (30 m x 0.25 mm x 0.25 µm), with initial temperature of 80 °C and heating to 185 °C (2 °C min) maintained for 1 minute, followed by heating to 275 °C (9 °C min) maintained for 2 minutes and final heating to 300 °C (25 °C min) maintained for 1 min. Helium was used as carrier gas at a linear velocity of 1 mL/min. The injector temperature was 280 °C; the injection volume was 1 µL; the injection took place in Split mode (2:1). The transfer line was maintained at 280 °C, the ionization source and quadrupole at 230 °C and 150 °C, respectively. The EM detection system was used in “scan” mode, at a mass/charge ratio (m/z) of 40-600, with a “solvent delay” of 3 min.  Compounds were identified by comparing mass spectra found in NIST 11.0 libraries and comparing retention indices (RI) obtained by a standard homologous series (C7-C28) (Adams, 2017).

2.4 Antioxidant activity

2.4.1 Determination of antioxidant activity by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) scavenging assay

DPPH• assays were performed according to Rufino et al. (2007). A 10 µL aliquot of essential oil from A. conyzoides leaves and flowers at different concentrations (1,00; 0,75; 0,50; 0,25; 0,125; 0,062; 0,03; 0,01 mg/mL) was added to 290 µL of methanolic DPPH• solution (60 µM). The negative control was 10 µL of methanolic DPPH• solution (60 µM). The mixtures were kept in the dark at room temperature for 30 min. The reduction in absorbance was measured at 515 nm by using a Spectra Max Plus 384 microplate reader. The total antioxidant capacity of essential oil was calculated by using a standard solution of quercetin (60 µM) as a 100% reference. The concentration required to scavenge 50% of free radicals (IC50) was determined from absorbance versus sample concentration curves.

2.4.2 Determination of antioxidant activity by the β-carotene/linoleic acid co-oxidation assay

The ability of essential oil from A. conyzoides leaves and flowers to inhibit β-carotene/linoleic acid co-oxidation was assessed according to Rufino et al. (2006). To a beaker were added 20 µL of linoleic acid, 265 µL of Tween 40, 25 µL of β-carotene solution (20 mg/mL), and 0.5 mL of chloroform. The solvent was removed using a dryer. Then, the emulsion was dissolved in 20 mL of hydrogen peroxide. The antioxidant activity was determined by adding 280 µL of emulsion and 20 µL of essential oil at different concentrations (1.00, 0.75, 0.50, and 0.25 mg/mL), incubating the samples for 120 min, and measuring the absorbance at 470 nm. A Trolox solution was a used as control. The results are expressed as a percentage of oxidation inhibition, as given by Eqs. (1), (2), and (3).

Ared = Ai – Af (1)

O = [(Aredsample × 100]/(Aredsystem) (2)

I = 100 − (O) (3)

Where Ared is the reduction in absorbance, Ai the initial absorbance, Af the final absorbance, O the oxidation percentage, and I the inhibition percentage.

2.4.3 Determination of antioxidant activity by the ferric reducing antioxidant power (FRAP) assay

The FRAP assay was performed according to the procedures described by Benzie and Strain (1996) and modified by Rufino et al. (2006). Briefly, the FRAP reagent was prepared by mixing 25 mL of acetate buffer (0.3 M), 2.5 mL of an aqueous solution of 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ, 10 mM), and 2.5 mL of an aqueous solution of ferric chloride (20 mM). Then, 10 µL of essential oil from A. conyzoides leaves and flowers at different concentrations (1.00, 0.75, 0.50, and 0.25 mg/mL) and 290 µL of FRAP reagent were added to the wells of a 96-well microplate. The microplate was placed on a Spectra Max Plus 384 reader, homogenized by vigorous shaking, and kept at 37 °C for 30 min. The change in absorbance was read at 595 nm. Antioxidant activity was calculated against a standard curve of ferrous sulfate (1000 µM).

2.5. Microbiological tests and fungal used

For this test, four fungal strains were used: Rhizopus oryzae ATCC 7560; Aspergillus flavus ATCC 1217; Aspergillus ochraceus ATCC 6787 and Penicillium verrucosum ATCC 7680.

2.5.1 Fungal Suspension

For each fungal sample, a standardized suspension was made from a 24-hour culture in Müeller Hinton broth (MHB) (DIFCO®). In a tube containing sterile saline (NaCl 0.85%), the fungal suspension was added drop by drop until a turbidity identical to that compared to the 0.5 tube on the McFarland scale (BaSO4 suspension corresponding to 1.0 x 108 CFU/mL). Subsequently, a 1:10 dilution was performed in an eppendorf tube containing CMH, in order to obtain a bacterial suspension of 107 CFU/mL, whose inoculum was used in the assay to determine the (MIC) (NCCLS, 2002).

2.5.2 Sensitivity tests to determine the Minimum Inhibitory Concentration (MIC)

The antifungal activity of the essential oil of A. conyzoides was evaluated by the broth microdilution method (MIC), determining the minimum inhibitory concentration (MIC) of the essential oil for the strains. This methodology was performed according to the M27-A2 broth microdilution reference protocols (NCCLS, 2002).

The essential oil of A. conyzoides (40 mg/mL) was diluted in a 2% aqueous solution of polysorbate 80. In a 96-well plate, 95 µL of Müeller Hinton broth was added to each well; 100 µL of essential oil (dissolved in 2% polysorbate) and 5 µL of inoculum, resulting in a final volume of 200 µL in each well. Microplates were incubated at 37 °C for 24 h. As a positive control, Levofloxacin was used. MIC was defined as the lowest concentration that resulted in the inhibition of microbial growth. 2,3,5-triphenyltetrazolium chloride was used as a developer (Beloti et al., 1999).

2.6 Statistical analysis

All experiments were performed in triplicate. Data were subjected to analysis of variance (ANOVA) and differences between means were determined by Tukey’s test at the 5% significance level using Minitab version 17 software.

3 RESULTS

The essential oil of A. conyzoides showed a light yellow color and a strong floral odor. The yield of essential oil from aerial parts (leaves and flowers) was 0.82 ± 0.14% (dry basis), density 0.95 ± 0.04 g/mL, and refractive index 1.5355. 

The essential oil from aerial part presented 54 compounds, predominantly sesquiterpene hydrocarbons (33.28%) and chromenes (55.57%) (Table 1). The major compounds were precocene I (48.19%), precocene II (7.38%) and β –caryophyllene (19.66%) (Table 1 and Figure 1). Figure 1 represents the mass spectra of the major compounds precocene I, precocene II and β-caryophyllene found in the essential oil of A. conyzoides, thus indicating a higher concentration of Precocene in the EO of A. conyzoides.

Table 1 – Chemical composition of Ageratum conyzoides aerial parts (leaves and flowers) essential oil

Ret Time	Compounds	Relative area %	RI calc	RI Lit.	MF	MM	Identification Methods
3.672	ni	0.36	858				a,b,c
5.188	α-pinene	0.16	938	939	C10H16	136	a,b,c
5.552	Camphene	0.57	953	952	C10H16	136	a,b,c
6.258	β-pinene	0.07	980	980	C10H16	136	a,b,c
6.613	β-myrcene	0.1	992	992	C10H16	136	a,b,c
6.912	α-terpinene	0.47	1002	1003	C10H16	136	a,b,c
7.762	D-limonene	0.2	1031	1030	C10H16	136	a,b,c
9.791	Terpinolene	0.03	1089	1088	C10H16	136	a,b,c
10.222	Linalool	0.05	1099	1100	C10H18O	154	a,b,c
10.834	ni	0.07	1117				a,b,c
12.774	Borneol	0.03	1167	1166	C10H18O	154	a,b,c
15.281	Endo fenchyl acetate	1.58	1228	1222	C12H20O2	196	a,b,c
17.762	ni	0.71	1287				a,b,c
19.392	α-longipinene	0.1	1327	1330	C15H24	204	a,b,c
20.297	α-cubebene	0.63	1349	1349	C15H24	204	a,b,c
21.382	α-copaene	0.56	1374	1376	C15H24	204	a,b,c
21.739	β-cuvebene	3.95	1382	1388	C15H24	204	a,b,c
22.061	β-caryophyllene	19.66	1390	1399	C15H24	204	a,b,c
24.015	α-bergamotene	0.34	1439	1433	C15H24	204	a,b,c
24.388	γ-elemene	0.06	1457	1458	C15H24	204	a,b,c
24.753	Humulene	2.1	1457	1456	C15H24	204	a,b,c
25.799	Precocene I	48.19	1482	1471	C12H14O2	190	a,b,c
27.508	α-farnesene	3.73	1526	1515	C15H24	204	a,b,c
27.876	β –sesquiphellandrene	0.15	1536	1523	C15H24	204	a,b,c
28.166	δ-cadinene	1.89	1544	1541	C15H24	204	a,b,c
28.345	Trans-cadina- 1, 4- diene	0.11	1548	1548	C15H24	204	a,b,c
28.634	Trans-sesquisabinene hydrate	0.2	1556	1565	C15H26O	222	a,b,c
28.936	Nerolidol	0.74	1564	1565	C15H26O	222	a,b,c
29.125	ni	0.06	1568				a,b,c
29.692	Germacrene D-4-ol	0.4	1583	1579	C15H26O	220	a,b,c
30.037	Caryophyllene oxide	1.06	1591	1589	C15H24O	220	a,b,c
30.172	Widdrol	0.19	1594	1592	C15H26O	222	a,b,c
30.603	Humulene epoxide II	0.16	1606	1605	C15H24O	222	a,b,c
30.840	Patchoulane	0.17	1613	1618	C15H26O	222	a,b,c
31.107	ni	0.47	1620				a,b,c
31.494	α-cadinol	0.47	1631	1632	C15H26O	220	a,b,c
32.009	Precocene II	7.38	1645	1656	C13H16O3	220	a,b,c
32.266	ni	0.09	1652				a,b,c
32.452	Acorenone	0.48	1657	1655	C15H24O	222	a,b,c

33.142	α –bisabolol	0.61	1676	1681	C15H26O	222	a,b,c
33.489	Cis-farnesol	0.15	1685	1697	C15H26O	222	a,b,c
33.630	ni	0.06	1689				a,b,c
33.807	Farnesol	0.23	1693	1695	C15H26O	222	a,b,c
34.564	Eudesm-7(11)-en-4α-ol	0.24	1715	1700	C15H26O	222	a,b,c
37.030	ni	0.81	1785				a,b,c
39.917	Kaur-16-ene	0.02	2071	2061	C20H32	272	a,b,c
44.849	ni	0.13	2164				a,b,c
	Total identified	97.23
	Monoterpene hydrocarbons	1.60
	Oxygenated monoterpenes	0.08
	hydrocarbon sesquiterpenes	33.28
	oxygenated sesquiterpenes	5.10
	hydrocarbon diterpenes	0.02
	Chromens	55.57
	not identified	2.76
	Other compounds	1.58

aCompounds listed according to HP-5MS elution order; bretention rate (RR) calculated using C7 to C26 n-alkanes in capillary column (HP-5MS); cIdentification based on comparison of mass spectra from Wiley 275 libraries; Relative Area (%): Percentage of the area (%) that the compound occupies within the chromatogram; RI: Retention Index, MF: Molecular Formula and MM: Molecular mass of chemical compounds from Ageratum conyzoides; n.i = Not identified

The results of the antioxidant activity of the essential oil of A. conyzoides are detailed in Tables 2 and 3 and Figure 2. The results of antifungal activity indicated that of the four fungi used, only Aspergillus ochraceus presents a result, with a MIC of 1250 μL/mL; the other fungi did not show a viable result.

Figure 1 – Mass spectra obtained by GC-MS from the Precocene I (m/z = 190), Precocene II (m/z = 220) and caryophyllene (m/z = 204) found in Ageratum conyzoides aerial parts (leaves and flowers) essential oil

Table 2 – Antioxidant activity of Ageratum conyzoides aerial parts (leaves and flowers) essential oil by DPPH• and FRAP methods

Antioxidant activity	DPPH•  IC50 (mg/mL)	FRAP (µM Fe2+/mg de amostra)
Essential oil	5.01 ± 0.99b	0.46 ± 0.05b
Quercetin	0.03 ± 0.001a	–
Trolox	–	9.18 ± 0.83a

Values are the mean ± standard deviation (n = 3). The statistical analysis used was analysis of variance (ANOVA), and differences between means determined by Tukey’s test (p ≤ 0.05). Values in the same column with different letters show significant difference (p ≤ 0,05). IC50: required amount of the sample to reduce 50% of the free radical DPPH (2,2 diphenyl-1-picrylhydrazyl); FRAP: iron-reducing antioxidant power. Positive control: quercetin (for DPPH) and trolox (for FRAP)

Table 3 – Percent inhibition of oxidation of Ageratum conyzoides aerial parts (leaves and flowers) essential oil by the β-carotene/linoleic acid co-oxidation system (BCLA). Results are expressed as β-carotene color protection (%) during 120 minutes of oxidative reaction

Samples (mg/mL)	Protective effect (%)
Trolox	79.85a ± 6.27
1.00	52.18b ± 7.81
0.75	50.97b ± 6.29
0.50	47.38b ± 7.16
0.25	46.13b ± 7.72
0.125	45.23b ± 5.89
0.625	44.63b ± 4.70
0.03	43.91b ± 6.64
0.01	40.24b ± 7.69
Negative control	0.00c ± 0.00

Values are the mean ± standard deviation (n = 3). Data were subjected to analysis of variance (ANOVA), and differences between means were assessed by Tukey’s test (p ≤ 0.05). Values in the same column followed by different letters are significantly different (p ≤ 0.05)

Figure 2 - Absorbance at 470 nm over time (120 minutes) of β-carotene co-oxidation reaction / linoleic acid in Ageratum conyzoides aerial parts (leaves and flowers) essential oil at concentrations of 1.00; 0.75; 0.50; 0.25; 0.12; 0.06; 0.03 and 0.01 mg/mL

4 DISCUSSION

The collection of A. conyzoides in June and July (which corresponds to the winter season in the southern hemisphere) occurred when the plant suffered interference in the rainfall regime, since, according to climatological data, the average rainfall index was 1.46 mm in June and 0.03 mm in July, indicating a period with low rainfall where the crop is installed. A. conyzoides requires low irrigation however, it is observed that plants in more humid places, as in the vicinity of treetops and mountain regions, they are more developed, thus indicating their preference for more humid places (Ming, 1999). 

The average temperature in the months of the collection was 18.67 ± ٠.٧١ºC, where the air humidity remained at ٧٠.٦٩ ± ٥.٩٦ (٪). A. conyzoides is considered an annual species, invasive in pastures, wastelands and cultivated areas, as it adapts to both wet and dry areas (Castro et al., 2016). For fresh parts of the plant essential oil yields of 0.11% are reported for forming aerial parts (Liu & Liu, 2014), 0.22% and 0.19% for flowers and stems (Kouame et al., 2018), 0.16 to 0.26% for flowers (Dung et al., 1989) and 1.60% for flowers (Kasali et al., 2002). For dry parts, the essential oil yield was: 2,8% for inflorescences, 1.0% for flowers, 0.6% for roots and 0.5% for stems (Zoghbi et al., 2007). The yield of the essential oil of A. conyzoides obtained in our study (0.82% of dry plants) is within the variability of results described in the literature for this plant.

One of the challenges for using essential oils in food preservation is the high cost, which can be up to six times higher than chemical fungicides (Kouassi et al., 2012). Burt (2004) reports that the increased demand for essential oil for uses mainly in the chemical, cosmetic and food industries may lead to bioengineering of its synthesis in plants. According to the European Pharmacopoeia, for the development of applications with essential oils, a minimum of 2 mL/kg (0.2%) of plants is required (Nemeth & Bernath, 2008). A. conyzoides has a high essential oil extraction yield of up to 2.8% of dry plants (Zoghbi et al., 2007), and in our results, the essential oil of A. conyzoides presented a yield of 0.82%, indicating that it is a species with high potential for the development of applications due to its high yield of essential oil.

Regarding the color of the oil, the reports in the literature are also varied from reddish orange (Kasali et al., 2002; Ekundayo et al., 1988) to light yellow (Liu & Liu, 2014; Kouame et al., 2018). Differences in yield and characteristics of the essential oil obtained from A. conyzoides may be related to sample processing before extraction. In our study, dried flowers and leaves were used; while Liu and Liu (2014) used fresh aerial parts, and Dung et al. (1989) and Kasali et al. (2002) used fresh leaves. The use of different parts of A. conyzoides results in different EO yields as demonstrated by Zoghbi et al. (2007). Sample preparation and other biotic and abiotic factors can also affect the yield and chemical composition of essential oils (Bettiol, 2009).

One of the important factors in the chemical composition of essential oils are the genetic characteristics that generate the chemotypes and these directly influence the biological activities of the essential oil. Chart 1 shows 3 chemotypes identified in the essential oil of A. conyzoides, which may suggest that the essential oil identified in our experiment belongs to chemotype I.

For Kasali et al. (2002), the species A. conyzoides has insecticidal, healing, bactericidal, fungicidal and anti-gonadotropic properties. The insecticidal action occurs in chemotypes that have precocene as the majority, because according to Binder et al. (1991), precocenes have caused a direct impact on the insects, and according to Kafi-Farashah et al. (2018), act as anti-juvenile hormones and cause genotoxicity. Ekundayo et al. (1988), also demonstrated the juvenileizing hormonal action of precocene I and II in insects, the most common effect being precocious metamorphosis, producing sterile or dying adults.

Chart 1 – Main chemotypes of Ageratum conyzoides essential oil and biological activities

Chemotypes	Regions	Temperature and Rainfall	Part of the plant	Majority (%)			EO Yield (%)	Biological Activities/ References
I	Umuarama northwest region of Paraná - Brazil	20.7 °C and 1512 mm.	Dried leaves and flowers	Precocene I	Precocene II	β- caryophyllene
	Northwest Karnataka, India	25°C.	Fresh leaves	48.19	7.38	19.66	0.82%	Antioxidant Our results
			Fresh flowers	72.30	3.10	12.10	0.15%	Test not performed Joshi, (2014)
			Fresh stem	66.50	10.50	10.20	0.17%
			Fresh root	50.30	0.30	14.60	0.5%
	Belém and Santarém Novo, state of Pará- Brazil	25.9 °C and 2150 mm.	Leaves	79.30	0.40	6.00	0.08%
			Twigs	69.60	-	14.40	1.0%	Test not performed Zoghbi et al. (2007)
			Roots	71.60	-	12.80	0.5%
			Inflorescences	67.40	-	15.30	0.6%
	Ivory Coast-Africa	Semi-humid tropical	Fresh flowers	55.50	-	19.40	2.8%
			Fresh twigs	58.80	-	15.20	0.22% 0.19%	Antimicrobial moderate against gram positive bacteria
	Ribeirão Pires, State of São Paulo – Brazil	17.2°C and 2159 mm.	Fresh leaves	76.50	-	8.10		Kouame et al. (2018)
	Badagry Lagos Nigeria	33 ºC and 100mm	Fresh mature leaves hydrodistillation 4 hours	79.11	10.39	-	0.11 a 0.19%	Antifungal Esper et al. (2015)
	Kumaun Himalaya	20.9ºC and 389.52mm	Fresh flowering aerial part	63.08	5.04	-	1.6 %	Kasali et al. (2002)
II	Minas Gerais - Brasil	21°C and 1341 mm.	Dry aerial part - Access Piranga-MG	16.7	42.5	20.7	0.30%	Padalia et al. (2010)
	Minas Gerais - Brasil	25°C	Dry aerial part –Viçosa-MG	76.5	-	-	0.48%	Test not performed Castro et al. (2014)
			Dry aerial part-Mariana MG	15.63	76.71	-	0.49%
III	Nova Santa Rita, Rio Grande do Sul –Brasil	19.5°C and 1419 mm.	Fresh aerial part	10.46	70.77	-	0.49%	Test not performed Barros et al. (2015) Dung et al. (1989)
	Hanoi, Vietnam	23.8°C and 1684 mm.	Fresh leaves	28.24	28.55	-	0.13%
	Pakistan	Tropical to temperate	Fresh flowers	29.00	31.10	-	0.16 to 0.26%
			Fresh leaves	30.30	34.90	14.40	0.5%	Antifungal namaticide Riaz et al. (1995)

The antioxidant capacity values vary depending on the methodology used, and vegetables that contain a higher concentration of phenolic compounds do not always display a greater activity to counteract free radical effects (Viana et al., 2023). In this sense, it could be observed in the present experiment that of the three methods used to evaluate the antioxidant potential of A. conyzoides EO, the best results were obtained by the β-carotene/linoleic acid co-oxidation system (Table 3) indicating a moderate activity (52.18%) of oxidation inhibition at the concentration of 1.0 mg/mL and (40.24%) at the concentration of lowest concentration tested (0.01 mg/mL), with no significant difference between the highest and lowest concentration tested (Table 3) whose difference was 100 times. This protective action found in the essential oil of A. conyzoides is positive and may suggest the use of this oil in products that are susceptible to oxidative effects during storage, mainly in products susceptible to lipid peroxidation. The β-carotene/linoleic acid co-oxidation system determines the activity of a sample or compound in protecting a lipid substrate such as essential oils from oxidation, evaluating the level of inhibition of free radicals generated during linoleic acid peroxidation (Duarte-Almeida et al., 2006).

As for the moderate action found, we followed the parameters established by Rufino et al. (2010) who consider high antioxidant activity above 70% and moderate between 40 and 70% of oxidation inhibition.

Low antioxidant activities were observed by the DPPH (IC50= 5.01 mg/ mL) and FRAP (0.46 µM Fe2+/mg sample) methods (Table 2). These results are related to the methods employed since the DPPH method is routinely used for hydroorganic extracts containing hydrophilic and lipophilic compounds, whose mechanism of action consists of the ability of antioxidant compounds to sequester or donate hydrogen. The FRAP method is indicated for hydrophilic compounds, which analyzes the ability of the sample to donate electrons. Another point that must be considered is that the DPPH and FRAP methods correlate and are related to the presence of phenolic compounds (Pérez-Jiménez et al., 2008; Rufino et al., 2010) and this correlation was observed in the results of the oil essential A. conyzoides.

For chromenes, precocone I and II there are no reports of antioxidant activity, since these compounds are recognized as potent natural insecticides (Edwin & Kester, ٢٠١٨), being base compounds to produce synthetic bioactive analogues (Ekundayo et al., 1988).

The second most abundant compound in the essential oil of A. conyzoides was β-caryophyllene (19.66 %) (Table 1) and for this compound, there are conflicting results in the literature, because according to Ruberto and Baratta (2000), β-caryophyllene showed low antioxidant efficacy (only 8.9% protection against 93.5% for α-tocopherol, both at 1.0 mg/mL) using the non-polar method of thiobarbituric acid reactive species. Calleja et al. (2012) tested the antioxidant activity of β-caryophyllene and the positive control α-tocopherol using different methods. For the DPPH radical, the IC50 values were 132 and 1.8 mg/mL, respectively. For the inhibition of lipoxygenase activity, β-caryophyllene inhibited antioxidant activity higher than α-tocopherol (27.9% inhibition compared to 12.9% for α-tocopherol).

Patil et al. (2010) evaluated the antioxidant potential of the essential oil A. conyzoides, whose chemical composition indicated the presence of 26.22% of β-caryophyllene and 52.18% of Precocene I. The results showed better results for the non-polar methods, with the β-carotene/linoleic acid co-oxidation system method with a maximum inhibition of lipid peroxidation of the essential oil IC50 of 0.015 ± 0.005 mg/mL, while butyl hydroxy anisole (BHA), a standard antioxidant, had an IC50 of 0.023 ± 0.011 mg/mL. Lower antioxidant potential was verified for the more polar methods, DPPH (IC50 0.57 mg/mL) and FRAP (3.21 mg/mL). The high concentration of β-caryophyllene found by these authors in the essential oil suggests that β-caryophyllene may have influenced the antioxidant response found.

β-caryophyllene is a naturally occurring volatile bicyclic sesquiterpene present in numerous herbs, spices and foods (Basha & Sankaranarayanan, 2016). Due to its weak aromatic taste, β-caryophyllene is commercially used as a food additive and in cosmetics. The Flavoring and Extract Manufacturers Association has granted β-caryophyllene “generally recognized as safe” status, and it has been approved by the U.S. Food and Drug Administration for food use due to its low toxicity (Calleja et al., 2012). Sivakumar and Bautista - Banos (2014), recommend the use of essential oils in food preservation and Burt (2004), recommends concentrations from 0.1 to 6%. The antioxidant concentrations of the essential oil of A. conyzoides, reported in our study for antioxidant action, are from 1.00 to 0.01 mg/mL, that is, from 0.1 to 0.001%. Thus, the values found in our study are within the concentration range of essential oils applied in food preservation (Sivakumar & Bautista-Banos, 2014), making the essential oil of our study an alternative for the development of applications in food preservation. Jiang and Xiong (2016) conducted a review on the use of natural antioxidants for meat conservation, for these authors the production of meat and meat products using chemical components potentially impacting health is a major challenge for meat and food scientists. 

The antifungal potential of A. conyzoides EO on the fungus A. ochraceus indicates the potential that essential oil of this species in controlling ochratoxin A, an mycotoxin which mainly attacks green coffee beans during the drying process, which is one of the routes of infestation by ochratoxigenic Aspergillus species and consequently of contamination by ochratoxin A (Ferreira et al., 2011). The importance of finding activity in this fungus lies in the fact that it is a food contaminant, and A. conyzoides EO can act as a natural preservative.

5 CONCLUSIONS

The essential oil of Ageratum conyzoides presented high concentrations of hydrocarbon sesquiterpenes (33.28%), with the majority being β-caryophyllene (19.66%), and Chromens (55.57%), with emphasis on Precocene I (48.19%) and Precocene II (7.38%). The essential oil provided a protective effect of 52.18 to 44.63% at concentrations ranging from 1.00 to 0.01 mg/mL through the β-carotene/linoleic acid co-oxidation system, suggesting that the essential oil has promising potential to increase the shelf life of pharmaceutical, cosmetic and food products. The action against the fungus Aspergillus ochraceus, suggests the fungicidal action of the essential oil, controlling ochratoxin A, a mycotoxin that contaminates food, indicating that this oil can also act as a natural preservative.

ACKNOWLEDGMENTS

The authors would like to thank Universidade Paranaense, grant number 31165/2017; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)—finance code 001; and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant number 310105/2021-8 for supporting the research carried out.

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Authorship contributions

1 – Vanusa Souza Rocha Pereira

Mestrado em Biotecnologia Aplicada à Agricultura pela Universidade Paranaense

https://orcid.org/0000-0002-3401-3067 • vanusarocha@prof.unipar.br

Contribution: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Writing – original draft, Writing - review & editing

2 – Gabriela Catuzo Canônico Silva

Mestrado em Biotecnologia Aplicada à Agricultura pela Universidade Paranaense

https://orcid.org/0000-0002-4123-9507 • gabriela.canonico@edu.unipar.br

Contribution: Assisted in the execution of the project

3 – Edneide Bezerra da Cruz Oliveira

Graduação em Farmácia pela Universidade Paranaense

https://orcid.org/0000-0003-1977-2847 • edneide.cruz@edu.unipar.br

Contribution: Methodology

4 – Mariane de Almeida Machado

Graduação em Farmácia pela Universidade Paranaense

https://orcid.org/0000-0001-6729-3598 • mariane.machado@edu.unipar.br

Contribution: Methodology, Writing - original draft

5 – Jéssica da Silva Sena

Mestrado em Biotecnologia Aplicada à Agricultura pela Universidade Paranaense

https://orcid.org/0000-0002-8236-4013 • jessica_seena@hotmail.com

Contribution: Methodology

6 – Marisa Cássia Vieira de Araújo Bento

Mestrado em Ciência Animal pela Universidade Paranaense

https://orcid.org/ 0000-0002-2932-0732 • marisa.bento@edu.unipar.br

Contribution: Methodology

7 – Selma Alves Rodrigues

Graduação em Medicina Veterinária pela Universidade Paranaense

https://orcid.org/0000-0002-6670-5578 • selma.rod@edu.unipar.br

Contribution: Methodology

8 – Rodrigo Sadao Inumaro

Mestrado em Tecnologias Limpas pelo Centro de Ensino Superior de Maringá

https://orcid.org/0000-0003-1662-6748 • rodrigoinumaro@gmail.com

Contribution: Methodology, Software

9 – José Eduardo Gonçalves

Pós-Doutorado em Química pela UNICAMP

https://orcid.org/0000-0002-2505-0536 • jose.goncalves@unicesumar.edu.br

Contribution: Methoidology, Project administration, Software, Supervision, Validation, Visualization, Writing – original draft

10 – Maria Graciela Iecher Faria Nunes

Doutorado em Biotecnologia Aplicada à Agricultura pela Universidade Paranaense

https://orcid.org/0000-0003-4006-3527 • gracielaiecher@prof.unipar.br

Contribution: Methodology, Project administration, Supervision, Validation, Visualization Writing – original draft and finalization

11 – Suelen Pereira Ruiz Herrig

Doutorado em Ciência de Alimentos pela Universidade Estadual de Maringá

https://orcid.org/0000-0002-1094-174X • suelenruiz@prof.unipar.br

Contribution: Methodology, Supervision, Validation, Visualization, Writing – original draft

12 – Ranulfo Piau Junior

Doutorado em Biomedicina pela Universidad de Léon, Espanha

https://orcid.org/0000-0003-4765-6544 • piau@prof.unipar.br

Contribution: Methodology, Supervision, Validation, Visualization, Writing – original draft

13 – Zilda Cristiani Gazim

Doutorado em Ciências Farmacêuticas pela Universidade Estadual de Maringá

https://orcid.org/0000-0003-0392-5976 • cristianigazim@prof.unipar.br

Contribution: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing – original draft, Writing - review & editing

How to quote this article

Pereira, V. S. R., Silva, G. C. C., Oliveira, E. B. da C., Machado, M. de A., Sena, J. da S., Bento, M. C. V. de A., Rodrigues, S. A., Inumaro, R. S., Gonçalves, J. E., Nunes, M. G. I. F., Herig, S. P. R., Piau Junior, R. & Gazim, Z. C. (2024). Ageratum conyzoides, an invasive species with antioxidant and antifungal potential. Ciência e Natura, Santa Maria, v. 46, e83759. DOI: https://doi.org/10.5902/2179460X83759.