Yield and chemical composition of Hesperozygis ringens (Benth.) Epling essential oil cultivated in different areas

Rendimento e composição química do óleo essencial de Hesperozygis ringens (Benth.) Epling cultivado em diferentes áreas

Abstract

The essential oil (EO) of Hesperozygis ringens (Benth.) Epling, which has several proven properties, is comprised mainly of monoterpenes and sesquiterpenes, and pulegone is the major compound. However, this is an endemic and endangered species. The aim of this study is to evaluate the viability of cultivating Hesperozygis ringens in different regions of Rio Grande do Sul and in a greenhouse. Seedlings obtained by using cuttings were transferred and cultivated for a year at each site. Then, leaves were collected and underwent EO extraction through steam distillation. During the same period, EO was also obtained from the natural population from which the cuttings were obtained. The chemical composition of EO samples in triplicates from each cultivation site and from the natural population were characterized in GC-MS. EO yield (%) was obtained by the ratio between leaf biomass (g) and EO mass (g) and it showed differences between areas; the yield obtained in the natural population was higher (3.14%) than the yield obtained in the cultivated areas (1.29%) and in the greenhouse (0.86%). The chemical compounds had low variation between areas, and most of them were oxygenated monoterpenes (74.82%). The commercial exploitation of H. ringens in different regions is feasible, as the plants tolerated climatic and edaphic conditions that were different from the ones of its natural occurrence area, and their EO yield remained high. The exception was the greenhouse, with lower yield yet with higher pulegone concentration. Furthermore, EO maintained its compounds, with few changes in their concentration.

Keywords:  Pampa biome; Lamiaceae; commercial production; pulegone; yield

Resumo

Epling cultivado em diferentes áreas) O óleo essencial (OE) de Hesperozygis ringens (Benth.) Epling, que possui diversas propriedades comprovadas, é composto principalmente de monoterpenos e sesquiterpenos, sendo a pulegona o composto majoritário. No entanto, esta é uma espécie endêmica e ameaçada de extinção. O objetivo deste estudo é avaliar a viabilidade do cultivo de Hesperozygis ringens em diferentes regiões do Rio Grande do Sul e em casa de vegetação. Mudas obtidas por meio de estacas foram transferidas e cultivadas durante um ano em cada local. Em seguida, as folhas foram coletadas e submetidas à extração do OE por destilação a vapor. No mesmo período, o OE também foi obtido da população natural da qual foram obtidas as estacas. A composição química das amostras de OE em triplicatas de cada local de cultivo e da população natural foi caracterizada em GC-MS. A produção de OE (%) foi obtida pela razão entre a biomassa foliar (g) e a massa de OE (g) e apresentou diferenças entre as áreas; o rendimento obtido na população natural foi superior (3,14%) ao rendimento obtido nas áreas cultivadas (1,29%) e na casa de vegetação (0,86%). Os compostos químicos apresentaram pouca variação entre as áreas, sendo a maioria monoterpenos oxigenados (74,82%). A exploração comercial de H. ringens em diferentes regiões é viável, pois as plantas toleraram condições climáticas e edáficas diferentes daquelas de sua área de ocorrência natural e sua produção de OE permaneceu elevada. A exceção foi a casa de vegetação, com menor produtividade, porém com maior concentração de pulegona. Além disso, o OE manteve seus compostos, com poucas alterações em sua concentração.

Palavras-chave:  bioma Pampa; Lamiaceae; produção comercial; pulegona; rendimento

1. Introduction

Characterized by being volatile and comprised of a complex mixture of chemical compounds, essential oils (EO) are aromatic compounds produced by plant secondary metabolism. They play an important role in protecting plants and attracting pollinator insects (Bakkali et al., 2008). Due to the high complexity of their chemical composition, they have been exploited by the industry for different purposes, e.g. developing herbicides (Roberts et al., 2022). As herbicides, their role is to inhibit seed germination, increase oxidative stress, break cell membranes, and promote metabolic damages (Kaur et al., 2021). Compared to synthetic herbicides, EO-based herbicides are biodegradable, have different action modes, cause less damage to human health, and frequently do not affect non-target species (Maes et al., 2021; Roberts et al., 2022). However, economic exploitation of essential oil depends on how possible it is to cultivate the plants outside their natural environment and on whether the cultivated plants will maintain their natural characteristics, such as EO yield similar to that of natural populations, as well as having low variation in chemical composition, an aspect considered instrumental for commercial purposes (Andi and Maskani, 2021).

Among botanical species, those from the Lamiaceae family have high EO yield and have been exploited for the development of several products. This is the case of Salvia rosmarinus Schleid. (Maccioni et al., 2020), Rosmarinus officinalis L. (Mahdi et al., 2020), Lavandula dentada L. (Wagner et al., 2021), Anisomeles indica (L.) Kuntze (Batish et al., 2012), Origanum acutidens (Hand.-Mazz.) Ietsw. (Kordali et al., 2008), and Mentha × piperita L. CV. Mitcham (Mahdavikia and Saharkhiz, 2015). Aside from those, studies using Hesperozygis ringens (Benth.) Epling EO have indicated different potentials (Ribeiro et al., 2010; Silva et al., 2014; Pinheiro et al., 2017; Rosa et al., 2019; Giacomin, 2020).

H. ringens is native to Brazil and endemic in native fields of southern Rio Grande do Sul (RS) (Antar and Oliveira, 2020), a region that belongs to the Pampa Biome, according to the Brazilian classification of biomes (IBGE, 2019). Popularly known as “espanta-pulga” (“to keep fleas away”), it is classified as vulnerable by the Official List of Endangered Species of the Brazilian Flora (MMA, 2022). It is a woody bush with a quite branched stem (Fracaro and Echeverrigaray, 2006), and high EO yield due to the presence of glandular trichomes, distributed on leaf blades, petioles, and stem (Pinheiro et al., 2018).

Among EO chemical constituents, pulegone was described as its major compound (81.20%) (Bruxel et al., 2022), with variation in production and in its composition according to seasonality. Additionally, there is a wide array of potential uses for EO; it is used as bioherbicide of agricultural crop-infesting species (Von poser et al. 1996; Pinheiro et al., 2017; Lima et al., 2020; Bruxel et al., 2022), larvicide (Silva et al., 2014), antibacterial (Rosa et al., 2019), acaricide (Ribeiro et al., 2010; Giacomin, 2020), antioxidant, cytotoxic, and genotoxic (Dolwitsch et al., 2019). On the other hand, pulegone is a toxic substance with proven insecticidal and repellant properties (Ainane et al., 2022; Lee et al., 2003).

As this is an endemic and endangered species, its exploitation depends on the possibility to cultivate it in areas that are outside its natural occurrence area. In face of this information and due to the seasonal variation in EO and to this species many potential uses, it is necessary to evaluate EO production and its chemical composition when cultivated in different regions of RS, with edaphic and climatic variations, and in a greenhouse, with the purpose of checking the feasibility of its commercial exploitation. First, the greenhouse cultivation was considered, as it allows low variations in environmental conditions (temperature, humidity, and commercial substrate), and higher variations in yield and in composition were expected. São Pedro do Sul, where a natural population of this species has been reported, was chosen due to its geographical proximity and to the similarity in climatic conditions with the species natural occurrence area. Therefore, lower variations in yield and in EO constituents compared to the natural population were expected. In the municipality of Sério, on the other hand, lower yield and higher variation in constituent percentages were expected, due to its climatic conditions, with higher rainfall, lower temperatures, and a more clayish soil, which was quite different from the natural population. Arroio do Meio and Barra do Ribeiro were mostly chosen due to the cultivation systems found at these sites. At the former, the production system was agroforestry with a less sandy soil, while the latter had a monoculture of Eucalyptus sp. and a more sandy soil. Climatic conditions, however, were similar to the natural population area. Lower EO variations, both in yield and in constitution, were expected for these two crops.

2. Materials and Methods

2.1. Propagation of Hesperozygis ringens seedlings

Hesperozygis ringens seedling propagation was performed through cuttings in a greenhouse. Branches were collected from a natural population in the municipality of São Francisco de Assis, RS, Brazil, (29º36'50”S - 55º09'41”W) (Figure 1), and a specimen of this species was deposited at the Herbarium of the Taquari Valley (HVAT) of Universidade do Vale do Taquari, under registration number 6380. After sampling, apical cuttings with mean length of 12 cm were obtained from branches and established in Carolina Soil^® substrate (Siqueira et al., 2020) and were irrigated as required. Seedlings were cultivated for one year in greenhouses, and then, they were transferred to different cultivation areas.

Figure 1
Location of the cultivation areas and of the natural population (where the cuttings of Hesperozygis ringens (Benth.) Epling from which the essential oil were collected).

2.2. Location of the cultivation areas

Seedlings were planted (Figure 1) in Arroio do Meio (AM) (29°23’52”S - 51°56’14”W) in a soybean plantation area after a fallow period of 5 years; Barra do Ribeiro (BR) (30°36’87.03”S - 51°25’30.09”W) in a commercial plantation of Eucalyptus sp. (young seedlings); São Pedro do Sul (SP) (29°29’14”S - 54°11’18”W), beside a maize plantation after a fallow period of two years; (SE) (22°23’33.8”S - 52°16’45.2”W) in the vicinities of a residence, in an access road; and in Lajeado (LA) (29°27'01.3”S - 51°56'37.5”W), in a greenhouse. In addition, EO production in the population from which cuttings were collected was also evaluated, in São Francisco de Assis (SF).

2.3. Cultivation of Hesperozygis ringens seedlings

Seedlings were planted in the mornings, between March 20 and 29, 2022. In each area, 90 seedlings were planted and cultivated. The cultivation in greenhouse occurred in vases placed on benches containing Carolina Soil^® substrate. After one year of cultivation, H. ringens branches were collected from each area (including from the natural population that provided the cuttings) in the mornings, during the same week, and stored in plastic containers sealed with lids and transported to the Laboratório de Botânica (Botanical Laboratory) of the Universidade do Vale do Taquari (University of Taquari Valley) – UNIVATES. After that, leaves with no signs of insect attacks and no debris were clipped and stored under ambient temperature until EO extraction.

Environmental conditions of temperature and rainfall in the BR and SF areas were obtained from the fixed meteorological stations near the areas. Climatic conditions in AM, SE, and SP were calculated using a rain gauge (Incoterm^®) and a meter for temperature and humidity (AKSO AK174^®). In LA, only temperature data were obtained.

2.4. Soil analysis in the planting area and in the natural population

One composite soil sample was obtained in each plantation and in the natural population area, by collecting soil in 10 random points at a 10 cm depth, with the help of a Dutch auger. They were then homogenized and stored in a sterilized plastic bag identified with the name of the area and transferred to the Central Analítica (Analytical Center) of Universidade de Santa Cruz do Sul (University of Santa Cruz do Sul) for physical and chemical characterization. A sample of the commercial substrate used for plants cultivated in greenhouse was also sent for analysis. Physical characteristics were determined using the Bouyoucos Densimeter method and classified using the soil texture triangle. The chemical components calculated were phosphorus (P), copper (Cu), zinc (Zn), boron (B), potassium (K), and organic matter percentage (OM%), determined using the method of oxidation by sulphochromic solution and densimeter; calcium (Ca), magnesium (Mg), and manganese (Mn), determined by the Ammonium Oxalate method; and sulfur (S), determined using the Ammonium Acetate method. Soil pH (H₂O) was defined using the potentiometric method (Tedesco et al., 1995).

2.5. Obtaining the essential oil

The leaves collected in each area were submitted to steam stripping, using an EO distillator, with capacity for 10 L (Marconi^®). In each extraction, 500 g of fresh leaves were used, placed in a basket, and inserted in the boiler of the equipment with water. The extraction process lasted two hours in each area at a level five of the potentiometer. When the extraction process was completed, the hydrolite obtained was decanted and the EO obtained was separated and stored in an amber flask in a refrigerator at -5 °C. Yield was defined for each area using the Formula 1:

$Y\%:\frac{Moil}{mleaves}*100$ (1)

where Y (%) corresponds EO yield, M_oil corresponds to the mass of oil obtained (g), and m_leaves corresponds to the mass of leaves used in the distillation (g). EO was extracted in triplicates for each cultivation area and for the natural population.

2.6. Characterization of chemical constituents

EO samples were characterized at the Central Instrumental do Centro Tecnológico de Pesquisa e Produção de Alimentos (Instrumental Center of the Technological Center for Research and Production of Foodstuffs) - CTPPA - of Univates. EO aliquots of 10 µL were solubilized in 990 µL of bi-distilled n-hexane and submitted to gas chromatography (Shimadzu - model GC2010) coupled to a Shimadzu mass detector (GCMS-QP2110 Ultra), operating at 70 eV, in a Rtx®-5MS fused silica capillary column (30 m x 0.25 mm x 0.25 μm). Helium was used as carrier gas. Sample injections were approximately 1 μL, using a Shimadzu AOC-5000 Plus auto injector. For this analysis, the following were employed: an injector temperature of 220 ºC; split injection ratio of 36.5 with purge of 3 mL.min-1; gas flow control by linear velocity; carrier gas flow of 0.93 mL.min-1; with a program of 60 ºC-280 ºC (Bruxel et al., 2022). Most constituents were identified by comparing Kovats index with a mixture of n-alkanes, mass specters of pure patterns, and literature data (Adams, 2017).

2.7. Data analysis

The diversity of chemical compounds in the EO obtained was determined by a principal coordinate analysis (PCoA). SIMPER test identified the chemical compounds that most influenced the dissimilarity of the areas evaluated. The chemical compounds that contributed with more than 1% of total dissimilarity (determined by the SIMPER test) and soil physical and chemical characteristics (pH, OM%, clay, and sand) were selected for a canonical correspondence analysis (CCA). These analyses were conducted using PAST version 4.11 (Hammer et al., 2001). After that, chemical constituent data were normalized and displayed in a heat map format, using Heatmapper (Babicki et al., 2016).

3. Results

3.1. Meteorological data

Over the cultivation period, there were oscillations in rainfall between plantation areas (Figure 2A), ranging from 50 mm in March 2022 to 350 mm in May of the same year in BR, which was the highest rainfall. The other areas had lower volumes reported and were similar to each other. Temperature measurements had little variation between areas, with a minimum of 13 °C and a maximum of 30 °C in June 2022 and February 2023, respectively (Figure 2B)

Figure 2
(A) Rainfall (mm) and (B) temperature (º C) in the cultivation area and in the natural population of Hesperozygis ringens (Benth.) Epling. AM: Arroio do Meio; BR: Barra do Ribeiro; SF: São Francisco de Assis; SP: São Pedro do Sul; SE: Sério.

3.2. Soil characterization

The physical analysis of soil samples (Table 1) showed variation in clay among areas, going from 10% (SF) to 42% (SE), while sand ranged from 88% (SF) to 13% (AM). On the other hand, silt content was the highest in AM (49%) and the lowest (2%) in SF. Moreover, texture class showed difference among areas.

Table 1
Physical characteristics and texture class of the soil derived from each cultivation area and from the natural population area of Hesperozygis ringens (Benth.) Epling.

Experimental area	Physical analysis
	Clay %	Sand %	Silt %	Texture class
AM	38	13	49	Silty-Clay Loam
BR	22	73	5	Silty-Sand Loam
SP	32	42	26	Clay Loam
SE	42	14	44	Silty Clay
SF	10	88	2	Loam sand

• AM: Arroio do Meio; BR: Barra do Ribeiro; SP: São Pedro do Sul; SE: Sério; SF: São Francisco de Assis.

The pH (H₂O) value was similar among areas, ranging from 4.7 (BR) to 5.6 (AM). On the other hand, OM% had a higher variation and its values ranged between 0.7% (BR) and 3.6% (SE). Similarly, nutrients P, K, Mg, Zn, and B had higher values in SE; Ca and Cu, on the other hand, were higher in AM, and S was higher in SF (Table 2). The commercial substrate Carolina Soil^®, used in the cultivation of seedlings in greenhouses (LA) was comprised of 78% sphagnum moss and 22% vermiculite, dolomitic limestone, and macro- and micronutrients derived from fertilizers. The pH (H₂O) of this substrate was 6.15 and electric conductivity lay around 0.35 mS cm^-1.

Table 2
Chemical characterization of soil samples derived from each cultivation area and from the natural area of Hesperozygis ringens (Benth.) Epling. population.

Experimental Area	Chemical Analysis
	pH H20	OM%	P mg.dm-3	K mg.dm-3	Ca cmolc.dm-3	Mg cmolc.dm-3	S mg.dm-3	Zn mg.dm-3	Cu mg.dm-3	B mg.dm-3	Mn mg.dm-3
AM	5.6	2.3	9.7	100	11.1	3.1	3.9	12.4	4.6	0.5	23
BR	4.7	0.7	4.3	52	1.3	0.5	6.5	1.4	1.2	0.4	30
SP	5.4	2	5	94	8.3	2.5	3.1	5	4.1	0.51	29
SE	5.1	3.6	64.1	132	9.9	3.2	6	18.8	1.3	0.89	17
SF	4.7	0.9	2.4	16	0.8	0.5	8.6	0.4	0.8	0.23	18

• AM: Arroio do Meio; BR: Barra do Ribeiro; SP: São Pedro do Sul; SE: Sério; SF: São Francisco de Assis; pH H₂0: water hydrogenionic potential; OM%: organic matter; P: phosphorus; K: potassium, Ca: calcium; Mg: magnesium; S: sulfur; Zn: zinc; Cu: copper; B: boron; Mn: manganese.

3.3. Essential oil yield

Mean yield of H. ringens EO was 1.21% in the cultivated areas, and 3.14% in the natural area. The plants collected in SE had a yield of 1.34%, followed by SP, with 1.31%, and yield in AM and BR was 1.28% and 1.24%, respectively. On the other hand, LA plants, which were cultivated in greenhouse, had the lowest yield (0.86%). In the natural occurrence area of H. ringens, SF, yield was 3.14% (Table 3).

Table 3
Chemical composition of essential oil of Hesperozygis ringens (Benth.) Epling Derived from the cultivation areas and in the natural population.

N°	Chemical Constituent	IKa	IKb	Cultivation areas and natural population
				AM	BR	SP	SE	LA	SF	Classification of compounds
1	α-Thujene	934	930	0.76	0.66	0.73	0.75	0.19	0.55	MH
2	Sabinene	974	975	0.58	0.51	0.55	0.51	0.24	0.68	MH
3	β-Pinene	979	979	0.88	0.76	0.8	0.84	0.37	0.77	MH
4	Myrcene	991	990	0.93	0.79	0.83	0.88	0.53	0.9	MH
5	Limonene	1029	1029	4.99	3.49	3.72	3.17	4.24	4.85	MH
6	1,8-Cineole	1032	1031	0.14	0.13	0.14	0.12	0.04	0.12	OM
7	(Z)-β-Ocimene	1037	1037	0.12	0.1	0.15	0.24	0.07	0.15	MH
8	(E)-β-Ocimene	1047	1050	0.96	0.98	1.19	2.47	0.91	1.16	MH
9	Linalool	1102	1096	1.88	1.85	1.82	1.98	2.03	1.53	OM
10	1-Octen-3-yl acetate	1113	1112	0.08	-	-	0.1	0.11	0.03	OM
11	p-Mentha-3-en-8-ol	1150	1150	0.11	-	-	-	0.05	0.23	OM
12	Menthone	1155	1152	0.05	0.05	0.07	-	0.04	0.08	OM
13	iso-Isopulegone	1158	1159	0.12	0.03	0.07	0.03	-	0.15	OM
14	iso-Menthone	1166	1162	0.06	0.05	-	-	-	0.13	OM
15	α-Terpineol	1193	1188	0.5	0.53	0.42	0.48	0.36	0.4	OM
16	Pulegone	1246	1237	72.15	73.14	70.69	70.98	74.05	70.59	OM
17	Carvone	1247	1243	-	-	0.10	-	-	-	OM
18	Piperitenone	1344	1343	0.13	0.21	0.16	0.19	0.25	0.21	OM
19	α-Terpinyl acetate	1351	1349	0.28	0.29	0.52	0.12	0.17	0.26	OC
20	Phenyl ethyl isobutanoate	1398	1393	0.05	0.07	-	-	0.06	0.05	OC
21	(E)-Caryophyllene	1422	1419	5.67	6.11	5.15	6.13	6.86	5.6	SH
22	α-Humulene	1456	1454	0.13	0.15	0.11	0.13	0.19	0.12	SH
23	Germacrene D	1483	1481	0.42	0.53	0.49	0.94	0.73	0.45	SH
24	Bicyclogermacrene	1499	1500	2.97	3.93	3.55	5.91	5.13	3.74	SH
25	Spathulenol	1581	1578	0.17	0.17	0.22	0.12	0.44	0.13	OS
26	Caryophyllene oxide	1586	1583	0.26	-	0.2	0.12	0.47	0.15	OS
Total (%)
Total identified per area (%)				94.39	94.53	91.68	96.21	97.53	93.03	94.56
Total monoterpenes hydrocarbons (MH)				9.22	7.29	7.97	8.86	6.55	9.06	8.15
Total oxygenated monoterpenes (OM)				75.22	75.99	73.47	73.88	76.93	73.47	74.82
Total sesquiterpenes hydrocarbons (SH)				9.19	10.72	9.3	13.11	12.91	9.91	10.85
Total oxygenated sesquiterpenes (OS)				0.43	0.17	0.42	0.24	0.91	0.28	0.40
Other oxygenated compounds (OC)				0.33	0.36	0.52	0.12	0.23	0.31	0.31
Yield (%)				1.28	1.24	1.31	1.34	0.86	3.14

• IK^a: retention index related to the mixture of n-alkanes; IK^b: ratio obtained from Adams (2017); AM: Arroio do Meio; BR: Barra do Ribeiro; SP: São Pedro do Sul; SE: Sério; LA: Lajeado; SF: São Francisco de Assis; MH: monoterpene hydrocarbon; OM: oxygenated monoterpene; SH: sesquiterpene hydrocarbon; OS: oxygenated sesquiterpene; OC: other compounds.

3.4. Chemical constituents of the essential oil

On average, 94.56% of the chemical constituents of EO samples from the areas were identified. Among them, LA had the highest percentage identified (97.53%), followed by SE (96.21%). Chemical constituents of the EO samples in each area were classified into monoterpenes hydrocarbons (8.15%), oxygenated monoterpenes (74.82%), sesquiterpenes hydrocarbons (10.85%), oxygenated sesquiterpenes (0.40%), and other compounds (0.31%), with variations according to cultivation area. AM had the highest percentage of monoterpenes hydrogens (9.22%), while LA (6.55%) had the lowest. On the other hand, oxygenated monoterpenes occurred at higher amounts and LA had the highest percentage (76.93%) among the plantations, followed by SP and SF, both with the same percentage (73.47%). Sesquiterpenes hydrocarbons had the highest percentage in SE (13.11%) and the lowest in AM (9.91%). The compounds classified as oxygenated sesquiterpenes and other compounds, in turn, had the lowest percentages in all areas investigated, with 0.17% in BR and 0.12% in SE, respectively. Among the 26 chemical constituents found, the majority were pulegone, (e)-caryophyllene, limonene, bicyclogermacrene, and linalool (Table 3). Pulegone was the chemical constituent with the highest percentage in all areas, with values ranging between 74.05% (LA) and 70.59% (SF). The second most abundant compound, (e)-caryophyllene, varied from 6.86% (LA) to 5.15% (SP). After that, bicyclogermacrene varied between 5.91% (SE) and 2.97% (AM), while limonene ranged from 4.99% (AM) to 3.17% (SE), and linalool varied from 2.03% (LA) to 1.53% (SF). The other chemical constituents had low percentage, e.g. phenyl ethyl isobutanoate, which ranged from 0.07% (BR) to 0.05% (AM and SF, respectively) (Table 3).

The coordinate 1 of PCoA (Figure 3) explained 51.9% of total data variability, while the coordinate 2 explained 34.8%, totaling 86.7% of total variability. This beta-diversity analysis showed differences among the experimental areas, as coordinate 1 separated LA and BR samples from AM, SF, and SP samples, while coordinate 2 separated SE from the other areas, which shows that the total composition of EO extracted was different according to site and type of cultivation. Overall, SF and SP had a similar EO chemical composition.

Figure 3
Principal Coordinate Analysis (PCoA) of cultivated areas and of the natural occurrence area of Hesperozygis ringens (Benth.) Epling. Caption: AM: Arroio do Meio; BR: Barra do Ribeiro; LA: Lajeado; SP: São Pedro do Sul; SE: Sério; SF: São Francisco de Assis.

The canonical correspondence analysis (CCA) excluded the LA samples, as the plants in this system were cultivated under different conditions from those of the other areas (Figure 4). Soil characteristics (sand, clay, pH, and OM%) and the chemical constituents that most affected data dissimilarity (determined by the SIMPER test) were used to build the CCA. The major constituent, pulegone, was located at the center of the analysis, which shows that the soil conditions evaluated did not affect its production. The compounds p-mentha-3-em-8-ol, caryophyllene oxide, and limonene were related to AM, SF, and SP, areas where soil pH values were higher. On the other hand, α-thujene and germacrene D were related to plants cultivated in SE, the area with the highest OM% and clay percentage. This analysis also showed that (E)-β-ocimene, bicyclogermacrene, and germacrene D are inversely related to sand percentage, and positively related to clay and OM%.

Figure 4
Canonical Correlation Analysis (CCA) of cultivated areas and natural occurrence area of Hesperozygis ringens (Benth.) Epling. AM: Arroio do Meio; BR: Barra do Ribeiro; LA: Lajeado; SP: São Pedro do Sul; SE: Sério; SF: São Francisco de Assis; OM: organic matter.

The heat map divided the five experimental areas studied in three different groups (Figure 5). One group comprised of the samples from AM and SF, another group comprised of BR and SP, and the third group with the SE sampled, separated from the others. The map also showed variations in constituents that were more evident in each area. In AM, the most evident compound was myrcene, while p-mentha-3-en-8-ol, sabinene, and iso-menthone were the most evident in SF, which belongs to the same group. In the other group, the compounds α-humulene and pulegone were the highest in BR, while the highest in SP were spathulenol, α-terpinyl acetate, and carvone. Finally, in SE, the compounds (E)-β-ocimene, (Z)-β-ocimene, bicyclogermacrene, and germacrene D were the highest.

Figure 5
Heat map showing the chemical constituents of Hesperozygis ringens (Benth.) Epling essential oil, classified according to the occurrence area in each municipality. SE: Sério, AM: Arroio do Meio, SF: São Francisco de Assis, BR: Barra do Ribeiro; and SP: São Pedro do Sul.

4. Discussion

EO yield of H. ringens and of some Lamiaceae species was high, compared to other species, whether it derived from the natural population or from those cultivated under favorable conditions for their development. In the present study, mean EO yield in the cultivated areas was higher (1.21%) than the findings of other studies on species of the same family, yet lower than that reported in the naturally occurring population (3.14%). Ocimum gratissimum L. and Ocimum basilicum L., for instance, had yields of 0.44 and 0.38%, respectively (Vasconcellos et al., 2023). On the other hand, the yield of natural populations of Origanum elongatum L. varied from 0.81% to 3.12% (Bakha et al., 2020), whereas Lavandula dentata L., also belonging to Lamiaceae, had a lower yield (0.08%) (Wagner et al., 2021). The percentage of EO produced by the plants in the present study, both cultivated and from the natural population, was higher than that reported by other studies, conducted with species from the same family and from other families, as is the case of Zingiber zerumbet (L.) Sm. (Zingiberaceae) (0.02%) (Rawat et al., 2023), Ferula tunetana Pomel ex Batt. (Apiaceae) (0.06%) (Baccari et al., 2023), Baccharis vulneraria Baker (0.08%) (Rodrigues et al., 2023), Baccharis dracunculifolia DC. (Asteraceae) (0.85%) (Rigotti et al., 2023), among others. Variation in yield and in the quality of the EO obtained, observed in studies with the same species yet derived from different populations or plantation sites, might be a result of management, which includes the use of fertilizers and harvest period, as well as edaphic and climatic conditions (Sales et al., 2009). In the present study, seedlings were cultivated with no interference, e.g. the use of fertilizers and soil preparation, and they were collected in the same week. However, the cultivated areas had different environmental characteristics from each other and from the natural occurrence area of the species. These conditions might affect EO production differently, as shown, for instance, by Tursun (2022), who studied Ocimum basilicum and found higher yield when it was cultivated in sandy soils, and conversely, lower yield when cultivated in clayish soils. In spite of that, the author emphasizes that there were no changes in EO chemical constituents. In our study, on the other hand, H. ringens EO yield had little variation among the four cultivation areas, even with differences in the mineral contents and in the texture class between areas (clay and sand percentages). This is the case, for instance, of SE and BR, as the clay and sand amounts were quite different between them. SE had the highest clay percentage, whereas BR had the lowest, and the opposite occurred with sand, which shows that texture class did not affect yield, and neither did minerals.

Rainfall during the cultivation period was different in the areas and might have affected EO produced by the plants of this species. In SE, for instance, rainfall volume in the period from May to September 2022 was higher than that reported in other areas, and this area corresponded to the cultivation with the highest EO yield. In SF, on the other hand, the lowest rainfall volumes were reported in May and August 2022, while the rainfall in all study areas was low in the period prior to harvesting (summer), conducted in March 2023. In this regard, Silva et al. (2018) observed a negative correlation between EO content in Lippia thymoides Mart. & Schauer (Lamiaceae) with periods of little rainfall in the experimental area of the municipality of Abaetetuba (PA).

On the other hand, variation in temperature was low among areas, as can be seen in Figure 2A, and this factor possibly did not affect the differences in EO yield and composition. Nevertheless, Santos et al. (2023) planting Allophylus edulis (A.St.-Hil.) Hieron. ex Niederl. (Sapindaceae) in two municipalities in the Cerrado (Brazilian biome), reported a gradual increase in EO yield with increased temperature, which emphasized that temperature does affect its production.

Yield was lower when cultivated in vases, under more controlled conditions in a greenhouse, containing commercial substrate, most likely due to lower exposure to sunlight, which differs from the native fields where the species naturally occurs (Antar and Oliveira, 2020). These results corroborate Botrel et al. (2010), who found that Hyptis marrubioides Epling (Lamiaceae) cultivated in vases and maintained in greenhouses had a low EO yield. The authors believe that this low yield might be associated to the limited space in the vases for the development of the root system. On the other hand, our results differ from those obtained by Luz et al. (2014), who cultivated Melissa officinalis L. (Lamiaceae) both in a protected environment (greenhouse) and in the field, and did not obtain significantly different results in EO yield, nor any changes in EO chemical composition between the studied areas. Despite the lower yield, chemical composition did not change, and the major compound in this cultivation condition had a higher concentration than the cultivated areas and the natural population. This result is important for studies in which the study object is to obtain EO with a higher amount of pulegone, for instance.

The 26 chemical compounds found in our EO samples were classified mostly as oxygenated monoterpenes (74.82%), corroborating Phatak and Heble (2002), who also found oxygenated monoterpenes as the major EO components in Lamiaceae. On the other hand, Caputo et al. (2021), investigating the chemical composition of Mentha pulegium L. (Lamiaceae) cultivated at three sites, observed that oxygenated monoterpenes were the most abundant in the six EO samples obtained, ranging from 92.2% to 97.7%.

Among the chemical compounds identified, whether in plants from the cultivation areas, from the greenhouse, or from the natural population, the five compounds with the highest concentrations were the same, and pulegone had the highest percentage, with 71.93% in all samples. This compound is present in high amounts in EO of other Lamiaceae species, such as M. pulegium (60.2%) (Caputo et al., 2021), Ziziphora tenuior L. (46.28%) (Bakhtiar et al., 2021), and Cunila angustifolia Benth. (29.55%) (Sousa et al., 2020), but always in amounts lower than that of H. ringens.

Among the cultivated areas, the highest percentage of pulegone was reported in LA (74.05%), which emphasizes that the more controlled conditions of temperature and humidity in the greenhouse stimulate the production of pulegone despite the lower exposure to sunlight. This percentage exceeded that reported for the population (70.59%), which suggests that the increase might have been favored by the lower variation of some condition that stimulates pulegone production. If the objective of a study is to increase pulegone percentage in EO, the plants can therefore be maintained in a greenhouse. However, this variation in composition might be normal for the species, since other studies have reported quite different values, e.g. Bruxel et al. (2022) (81.20%), Pinheiro et al. (2018) (63.86%), and Rosa et al. (2019) (47%). Variation in production environments might possibly cause one or another compound to have either higher or lower quantities, or it might even maintain its original characteristics, which thus increases the potential for the economic exploitation of that species.

Comparing EO yield between our study and other reports of the same species, there is similarity in percentages between cultivation areas, with no changes in chemical compounds. However, PCoA, with a high percentage of diversity (86.7%), dissociated experimental areas, regardless of the characteristics of each location. This percentage was higher than that reported by Ray et al. (2019) (71.90%) studying morphological, phytochemical, and molecular diversity of 50 accessions of Hedychium coronarium J. Koenig (Zingiberaceae) in different geographical areas of India. Leontaritou et al. (2020), whose PCoA coordinates explained 52.30% of variability between individuals from 10 Salvia fruticosa Mill. (Lamiaceae) populations observed a high variability of EO morphological characteristics.

LA and BR were separated from the others by coordinate 1, likely because they had the lowest percentages of monoterpenes hydrocarbons (6.55% and 7.29%, respectively). Conversely, coordinate 1 clustered AM, SF, and SP, separating them from the others as they had higher percentages of monoterpenes hydrocarbons (9.22% and 9.06%, respectively), although this percentage in SP was a little lower (7.97%). The three areas are located in the central region of RS, and their environmental conditions are similar. On the other hand, coordinate 2 separated SE from the others due to the higher percentage of sesquiterpenes hydrocarbons (13.11%). In addition, SE was also among the areas with the lowest oxygenated monoterpenes (73.88%), higher only than SP and SF, both with 73.47%.

CCA showed that soil and the chemical constitution of the EO samples affected the dissimilarity of data collected in each area, as some compounds were related to some soil characteristics. This is the case of p-mentha-3-on-8-ol, caryophyllene oxide, and limonene, related to pH and to the percentage of sand in the soil, which brings together SF, SP, and AM, as shown by the PCoA. On the other hand, α-thujene, germacrene D, are related to OM and clay, responsible for distinguishing SE from the other areas. Pulegone was the compound reported at the center of the CCA, thus indicating that its production was not affected by any variable related to soil. However, clay, sand, and OM percentages and pH affected the production of the other EO compounds, and were responsible for the dissimilarity between areas. The heat maps provide a visual representative alternative for the data (Rasekh et al., 2023). In the present study, the heat map divided the five experimental areas in three main groups, with SE comprising a separate group, confirming the results of the PCoA and CCA. Although the other four areas were divided in two groups (AM-SF and BR-SP), they are similar. Excluding the EO obtained from the plants in the greenhouse (LA), the heat map also showed that among the areas, the EO composition in AM was closer to the EO of the natural population (SF). Moreover, it showed that the highest pulegone production, notwithstanding LA, occurred in the plants in BR (73.14%), followed by AM (72.15%), both with higher amounts than SF (70.59%), and marked in red in the heat map. Similarly, Moshrefi-Araghi et al. (2021), studying Mentha longifolia L. EO from 20 areas through a heat map, observed differences between cultivated areas, as there were four groups formed, and pulegone, one of the major compounds in the species, stood out in three of the four groups.

5. Conclusion

This study showed that H. ringens is tolerant to climate changes and to edaphic conditions that are different from its natural habitat. EO yield was lower in the greenhouse (LA), just as initially hypothesized; however, pulegone percentage was higher than in the other areas, including the natural population, which was not expected. Despite a lower yield, composition in SP was similar to that of the EO of the natural population, as expected. The same occurred in AM, which was more similar to the natural population. On the other hand, EO from BR lay in an intermediate position between LA and AM, as it had more differences from the natural population, which contradicted our initial hypothesis and showed that edaphic and climatic conditions had no impact on EO yield and its composition. Finally, EO from SE was different from the others, as expected, and in this case, environmental conditions likely affected both yield and composition.

Nonetheless, the commercial exploitation of H. ringens in different regions is feasible, as this species maintains a high oil yield, with little variation in its composition, and with pulegone – its major compound – reaching its highest percentage in all areas. Although yield was lower in the greenhouse, this cultivation condition favored the production of the highest percentage of pulegone compared to all the other areas, even the natural population. Thus, it can be a cultivation option when the aim is to have the highest percentage of the major component. Variation in composition, reported in the areas, indicates the possibility of producing a certain compound with higher amount in the EO. This can be done with the purpose of isolating that compound or using the EO for a specific biocidal activity, which can be determined in further studies.

Acknowledgements

We are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the scholarship granted through the Programa Doutorado Acadêmico para Inovação (Academic Doctorate Program for Innovation) – DAI (Process: 140567/2021-6); we also thank Universidade do Vale do Taquari – Univates for all the structure provided. Thanks are also due to the owners of the experimental areas, to the company CMPC Celulose Riograndense and to the scholarship students of Laboratório de Botânica of Univates.

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Publication Dates

• Publication in this collection
  06 Dec 2024
• Date of issue
  2024

History

• Received
  31 July 2024
• Accepted
  10 Oct 2024