Localities and dates of collection of individuals of S. serrata.
Stevia serrata Cav. (Eupatorieae, Asteraceae) grows in Central America and Mexico usually over 1500 m. In this study, essential oils of aerial parts from three populations of western Guatemala were obtained yielding 0.17–0.27% of oil by hydrodistillation. Chamazulene (42–62%) was the most abundant compound in the oil analyzed GC/MS, also presenting germacrene D (4.4–15.3%), caryophyllene oxide (3.2–11.8%), (E)-nerolidol (3.9–7.1%), spathulenol (2.3–7.9%), and (E)-caryophyllene (2.5–6.6%). Besides, a propagation trial was carried out on seeds of plants collected in Santa Lucía Utatlán, as the first step for the domestication of the plant, obtaining approximately 75% survival in the transplanting of the germinated seedlings. After the flowering of the individuals, a greenish essential oil was obtained from the roots yielding 0.2% of oil. This oil did not present chamazulene, but α-longipinene (23.5%), germacrene D (22.2%), santolina triene (12.6%), and (E)-caryophyllene (8.1%) as major components. As conclusion, it was confirmed that the aerial parts of the essential oil of S. serrata from western Guatemala presents a high content of chamazulene and that there is feasibility for the domestication of the plant through the germination of seeds.
- Stevia serrata
The high biodiversity of Guatemala, caused by the great variety of microclimates and the convergence of the flora of North and South America, presents plants that have developed a large number of secondary metabolites to fulfill functions of defense and interaction with the environment. Many of these metabolites have biological and pharmacological activities that are used by communities, through the use of plants for the treatment of different diseases . In this way, many investigations have been carried out aimed at determining the composition and biological activity of the metabolites of different medicinal plants used in Guatemala [2, 3, 4, 5].
One of the biodiverse plants of Guatemala, which also grows in neighboring countries and for which no medicinal uses have been reported in Guatemala, is Stevia serrata Cav.  whose essential oil presents chamazulene in high proportions. Chamazulene is a substance of intense blue color of high economic value, which has been shown to have a high anti-inflammatory activity .
The genus Stevia belongs to the Asteraceae family within the Eupatorieae tribe . It is a New World genus distributed from the south of the United States to Argentina and the highlands of Brazil, passing through Mexico, the Central American countries, and the South American Andes [9, 10]. The records indicate that the genus is not represented in the Antilles or the Amazon. The members of the Stevia genus are found mainly at altitudes between 500 and 3500 m. Although they usually grow in semidry mountainous terrain, their habitats range from meadows, leafy forests, forested mountain slopes, coniferous forests, to subalpine vegetation .
The genus Stevia consists of between 220 and 230 accepted species. Of these, only about 34 (15%) have some type of ethnobotanical record that relate uses with common names of the species. Of these 34 species, only the South American species Stevia rebaudiana (Bertoni) Bertoni presents records of outstanding use because its sweet leaves are used for imparting sweetness to beverages and foods [8, 12]. Due to this, S. rebaudiana is of great economic importance internationally, given its intensive commercialization due to its use as a natural low calorie sweetener .
The sesquiterpenoids are by far the majority and characteristic constituents of the aerial parts and roots of the Stevia genus. The overwhelming majority of these compounds belong to the guaiane, longipinane, and germacrene groups . Derivatives of longipinene have been isolated and elucidated mainly in roots of S. eupatoria, S. porphyria, and S. pilosa in Mexico, in S. triflora from Venezuela, and in S. lucida of Colombia [13, 14, 15, 16, 17, 18]. Diterpene glycosides have been isolated from commercial extracts of S. rebaudiana leaves in Malaysia [19, 20]. The composition of the essential oil of plants of the genus Stevia has been determined in leaves of S. urticifolia in Brazil being the main components found the oxygenated sesquiterpene α-cadinol (8.6%) and the sesquiterpene hydrocarbon germacrene D (10.4%) .
On the other hand, the composition of the essential oil of S. rebaudiana leaves analyzed in Nigeria showed carvacrol (67.89%), caryophyllene oxide (23.50%), spathulenol (15.41%), cardinol (5.59%), α-pinene (3.75%), ibuprofen (1.79%), isopinocarveol (1.26%), and α-caryophyllene (1.15%) as the main components found .
Other types of compounds isolated in plants of this genus include four flavonoids isolated from the aerial parts of S. urticifolia in Brazil , two triterpenes isolated from the roots of S. viscida and S. eupatoria from Mexico , the breviarolide and guaianolide isolated from the aerial parts of S. breviaristata from Argentina , and the stephalic acid isolated from the whole plant of S. polycephala from Mexico . Nineteen hydroxycinnamic acid derivatives were successfully characterized in S. rebaudiana leaves: three monocaffeoylquinic acids (Mr354), seven dicaffeoylquinic acids (Mr516), one p-coumaroylquinic acid (Mr338), one feruloylquinic acid (Mr368), two caffeoyl-feruloylquinic acids (Mr530), three caffeoylshikimic acids (Mr336), and two tricaffeoylquinic acids (Mr678) .
Likewise, two new stevia amino acid sweeteners have been synthesized from natural stevioside: stevia glycine ethyl ester and stevia L-alanine methyl ester. The sweetness intensity rate of the new sweeteners was higher than sucrose, and they also had a clean sweetness without the unpleasant bitter aftertaste of stevioside . Stevia products have been elaborated as an infusion with suitable organoleptic characteristics using a formulation of 80–85% of leaves + dried flowers of anise (Tagetes filifolia Lag.) and 15–20% of dried stevia leaves (S. rebaudiana) .
As for the S. serrata plant, it is distributed from southern Arizona, New Mexico and Texas to northern Oaxaca, and from Chiapas to Honduras, Colombia, Venezuela, and Ecuador. In Guatemala it is found in the departments of Chimaltenango, Guatemala, Huehuetenango, Quetzaltenango, El Quiché, Sacatepéquez, and Sololá [6, 11].
The species grows along pastures and roadsides in various habitats from Yucca-Opuntia scrub, sand pine woods, steep rock outcrops in Quercus-Acacia grasslands, and pastured slopes, usually between 900 and 2800 m. The plants prefer sunny, stony, well-drained places but also grow in moist pastures and other flat areas [6, 11]. They grow as erect perennial herbs to 0.6–1 m, the stems single to many, puberulent to densely pilose. Leaves alternate, scattered and often crowded, sessile to subsessile, serrate toward the apex, 2.5–6.5 cm long, 0.2–1.5 cm wide, apex rounded to acute. Capitula 5–9 mm, phyllaries 3.5–6 mm long, 0.7–1 mm wide, puberulent with numerous glandular dots. Corollas white, 3–5 mm long, often gland-dotted, lobes 1–1.5 mm long, puberulent. Achenes are usually heteromorphic, 2.2–4.2 mm long, hispid. Pappus of the four adelphocarps with 3–5 awns equaling the corolla and alternating with 3–5 scales, 0.2–0.7 mm long [6, 11].
As for the chemistry of S. serrata, five new derivatives of longipinene have been isolated and elucidated from the roots of the plant in Mexico, these being 7β,9α-diangeloyloxy-8α-hydroxylongipinan-1-one; 8β,9α-diangeloyloxy-9α-hydroxylongipinan-1-one; 7β,9α-diangeloyloxy-8α-acetyloxylongipinan-1-one; 7β,9α-diangeloyloxy-8α-acetyloxylongipin-2-en-1-one; and 7β-angeloyloxy-8α-isobutyryloxylongipin-2-en-1-one . Likewise, in Mexico, two new prochamazulene sesquiterpene lactones from the dried leaves of S. serrata from Mexico were isolated and identified: steviserrolide A and steviserrolide B . The presence of the R enantiomer of chamazulene carboxylic acid (Figure 1) of S. serrata from Central America was determined .
Regarding studies of the essential oil of the plant, the distillation of 178 g of flowers of S. serrata from Mexico provided 700 mg of the blue essential oil, which yielded 320 mg of chamazulene . The compounds found in highest concentration in the essential oil of S. serrata from Guatemala were the sesquiterpenes chamazulene (60.1%), (E)-nerolidol (7.3%), caryophyllene oxide (6.3%), and germacrene D (5.4%) , which are shown in Figure 2. Chamazulene is produced from prochamazulenic sesquiterpenlactones. Among these precursors, matricine (Figure 2) and the carboxylic acid of chamazulene, among others, have been identified, which are present in the plant and are transformed into chamazulene by the action of the temperature during the steam extraction process . Other compounds isolated from the plant include the methyl-ripariochromene A from the dried leaves of S. serrata cultivated in Japan .
The plant, known in Mexico as “tlachichinole,” was used in decoction of the aerial parts for the washing of infected pimples , while the “donkey chili” or “sheep tail” is used as medicine to treat intestinal discomforts in Honduras ; the decoction of the “October flower” is used by the midwives to accelerate the contractions of the parturients during childbirth . Oral administration of S. serrata essential oil from Guatemala produced a marked antinociceptive activity in mice in the formalin test .
The purpose of the study was to determine the composition of the essential oil of aerial parts of S. serrata from different localities of the Guatemalan highlands, to evaluate the variability of the content of chamazulene. The capability of propagation of plants of S. serrata was also determined by a seed propagation trial. Finally, the composition of the essential oil of the roots of the propagated plants was determined to compare it with the composition of the oil extracted from aerial parts of the plant.
2.1 Collection and preparation of plant material
Aerial parts of S. serrata were collected from populations in different localities (Table 1) during 2018. The plant material was dried in a solar dryer at a temperature between 30 and 35°C and immediately extracted. Figure 3 shows pictures of the population in Santa Cruz del Quiché, Quiché, and details of floral button of the plant.
2.2 Seed germination
Seeds of S. serrata were collected in the surroundings of Santa Lucia Utatlán, Sololá (N 14° 46 40.4″ W 091° 14 41.5″/2430 m), in December 2015. Seeds were stored in trays inside a solar dryer at a temperature between 30 and 35°C for 2 months.
After drying, seeds were manually removed from the flower receptacles and subsequently placed for germination in peat moss previously moistened into plastic strainers (Figure 4).
2.3 Transplantation of seedlings and root obtention
The seedlings obtained were transplanted to 4-gallon flowerpots containing potting soil. The plants were placed in direct sunlight and watered daily. After the seed production by the individuals grown in pots, their roots were removed, washed, and dried in a solar dryer. Then, the roots were pulverized in a forage mill for the extraction of the essential oil.
2.4 Extraction of essential oil
The oil from 50.0 g of aerial parts of S. serrata was extracted by hydrodistillation using a Clevenger-type apparatus for 2 h. It was then weighed with an analytical scale. The extraction of the essential oil of 100 g of powdered roots was carried out in the same Clevenger-type apparatus for 2 h. The essential oils of the aerial parts and of the roots were collected in pentane which was later removed in a rotatory evaporator at 40°C. All the extractions were made in triplicate, and the reported yield corresponds to the average of the three extractions.
2.5 Gas chromatography coupled to mass spectrometry analyses (GC/MS)
GC/MS analyses were performed using a chromatograph Shimadzu 2010 Plus system coupled with a Shimadzu QP-2010 Plus selective detector (MSD) and equipped with a DB5-MS capillary fused silica column (60 m, 0.25 mm I.D., 0.25 μm film thickness). The oven temperature program initiated at 60°C, then was raised by 3°C/min to 246°C, and then was held for 20 min. Other operating conditions were as follows: carrier gas, He (99.999%), with a flow rate of 1.03 mL/min; injector temperature, 220°C; split ratio of 1:50; and injection volume of 1 μL. Mass spectra were taken at 70 eV. The m/z values were recorded in the range of m/z 40–700 Da.
Tables 2 and 3 present the results of yields and chemical composition of the essential oils of the three sampled populations of S. serrata and roots of plants obtained by seed propagation, respectively. Chamazulene was the major component of the essential oils of the aerial parts meanwhile α-longipinene was the compound found in major proportion in the essential oil of the roots.
4.1 Essential oil of aerial parts of S. serrata
Table 2 shows the yield and composition results of the intense blue essential oil obtained from the aerial parts of individuals of S. serrata collected in three different populations distinct of the population sampled in a previous study of the chemical composition of oil of S. serrata from Guatemala . The three populations are located in the highlands of western Guatemala. Extraction yields were between 0.2 and 0.3% (w/w) (Table 3), corresponding the highest yield to the SS4 oil from Santa Cruz del Quiché. A probable explanation for the difference in yields among the sampled populations is that the production of essential oil depends on the phenological stage, so that there is a greater production of oil in the flowering stage and lower production in the fruiting stage.
Another probable explanation could be edaphic factors affecting the production of secondary metabolites in general, but only after new investigations could the relationship between these factors and the production of essential oil and other metabolites be determined.
Regarding the chemical composition analyzed by GC/MS, 22 compounds were identified in the SS3 (94.7% of the total area) and SS4 (97.6% of the total area) oils and 18 compounds in the SS5 oil (98.4% of the total area). A chromatogram of the essential oil of SS4 is shown in Figure 5. The most abundant compound was the chamazulene in area percentages between 42 and 62%, with the highest percentage corresponding to the SS5 essential oil. The mass spectrum of chamazulene from the essential oil of sample SS4 is shown in Figure 6. The other compounds found in high percentage in the oil were germacrene D (4.4–15.3%), caryophyllene oxide (3.2–11.8%), (E)-nerolidol (3.9–7.1%), spathulenol (2.3–7.9%) and (E)-caryophyllene (2.5–6.6%). The α-longipinene, frequently found in Stevia genus plants  that had not been reported in the essential oil of S. serrata, was found in the SS4 oil in 0.4%.
The results confirm that essential oil of S. serrata with high content of chamazulene can be obtained from the different populations of the Guatemalan highlands. The authors consider that although the extraction yield in all the samples has been lower than 0.3%, the plant presents economic potential for its domestication for oil production in view of its high content of chamazulene and the presence in it of other components for which pharmacological activity has been reported. When comparing this source of essential oil with chamazulene content in the oil of Matricaria recutita L. (Asteraceae), which is obtained only from the flowers of this species , S. serrata is shown as a promising species because all aerial parts (leaves, stems, and flowers) produce essential oil with high chamazulene content.
It is worth noting that the composition of the three oils is in congruence with the composition obtained by Simas et al.  of S. serrata from a population in the department of Sololá, presenting the same major compounds with some percentage variations and the majority of compounds such as sesquiterpenoids.
4.2 Essential oil of roots of propagated plants of S. serrata
A seed propagation trial was carried out with seeds of plants of S. serrata collected from a population of Santa Lucía Utatlán, Sololá, from where the composition of essential oil with a high content of chamazulene had been previously reported . The purpose of the trial was to evaluate the capability of propagation of the plants, generate new seeds, and extract and analyze the essential oil from the root. The interest in analyzing the root oil was due to the fact that in interviews with residents of the region, the authors had received information that previously the root of the plant had been used in traditional medicine for the treatment of stomach pain . The seeds were germinated in peat moss, and then seedlings were transplanted to pots where they developed well with approximately 75% survival reaching 1 m height after 6 months. It is important to note that the cultivation experiment was carried out in Guatemala City, at an altitude of 1495 m, this being a lower altitude than in the region where the plant grows naturally.
After obtaining the seeds during a plant vegetative stage, the roots were collected from which an essential oil with a light green color was obtained with a yield of 0.2% (w/w), and 25 compounds representing 95.8% of the total chromatographic area were identified (Table 3). The chromatogram of the essential oil of the roots is shown in Figure 7. Due to the green coloration of the oil, it was supposed that the chamazulene was absent in the oil, which was confirmed after the analysis by GC/MS. The major components of the root oil corresponded to α-longipinene (23.5%), germacrene D (22.2%), santolina triene (12.6%), and (E)-caryophyllene (8.1%). The mass spectrum of α-longipinene is shown in Figure 8.
The common components between the root and the aerial parts oils were germacrene D and (E)-caryophyllene. The α-longipinene (Figure 9) was only found in one of the oils of the aerial parts in low percentage (0.4%), while the santolina triene (Figure 9) was not found in any of the oils of the aerial parts. As in the oil of aerial parts, sesquiterpenoids predominated in the root oil. Since the plant has been used in the past for the treatment of stomach pain, the authors consider it of value to carry out pharmacological activity tests with this oil in the near future.
It was found in this study that the essential oil of aerial parts of wild S. serrata from different populations of the highlands of Guatemala showed high concentrations of chamazulene. In addition, the essential oil of roots of the plant was analyzed for the first time, which presented a composition very different from that of the aerial parts, as it did not present chamazulene and presented α-longipinene as the major component. It was also verified that the seeds of S. serrata present a high viability and that the seedlings obtained from seeds also have a high percentage of survival. Therefore, S. serrata can be considered as a plant with high potential for domestication and cultivation for the production of essential oil with high content of chamazulene.
The present research was partially funded by the General Directorate of Research of the University of San Carlos of Guatemala, project 18.104.22.168.06, within the framework of the University Program of Interdisciplinary Research in Health. The authors would like to agree to CAPES, CNPq, and FAPERJ from Brazil.
Conflict of interest
The authors declare that they have no conflict of interest with respect to this publication.