Chemical Composition and Variability of the Volatile Components of Myrciaria Species Growing in the Amazon Region

Myrciaria (Myrtaceae) species have been well investigated due to their chemical and biological relevance. The present work aimed to carry out the chemotaxonomic study of essential oils of the species M. dubia, M. floribunda, and M. tenella, sampled in the Brazilian Amazon and compare them with the volatile compositions from other Myrciaria species reported to Brazil and Colombia. The leaves of six Myrciaria specimens were collected (PA, Brazil) during the dry season, and their chemical compositions were analyzed by gas chromatography-mass spectrometer (GC-MS) and gas chromatography-flame ionization detector (GC-FID). The main compounds identified in the essential oils were monoterpenes with pinane and menthane skeletons, followed by sesquiterpenes with caryophyllane and cadinane skeletons. Among the sampled Myrciaria specimens, five chemical profiles were reported for the first time: profile I (M. dubia, α-pinene, 54.0–67.2%); profile II (M. floribunda, terpinolene 23.1%, α-phellandrene 17.7%, and γ-terpinene 8.7%); profile III (M. floribunda, γ-cadinene 17.5%, and an unidentified oxygenated sesquiterpene 15.0%); profile IV (M. tenella, E-caryophyllene 43.2%, and α-humulene 5.3%); and profile V (M. tenella, E-caryophyllene 19.1%, and caryophyllene oxide 41.1%). The Myrciaria chemical profiles showed significant variability in extraction methods, collection sites, plant parts, and genetic aspects.


Introduction
Myrciaria comprises 31 species growing in Argentina, Paraguay, Peru, Bolivia, Brazil, and Australia [1], belonging to the Myrtaceae, which has a significant botanic representation, with 142 genera and about 3500 tropical and subtropical species [2].

Myrciaria dubia
Myrciaria dubia (Kunth) McVaugh, popularly known as camu-camu, is native to the Brazilian and Peruvian Amazon regions. This species' leaves and fruit peels are used in Brazilian traditional medicine to treat diarrhea, female diseases, and labyrinthitis [14], while in Peru, M. dubia leaves are used to treat colds and arthritis [15]. Moreover, M. dubia fruits are considered a natural source of antioxidants by their significant content of ascorbic acid and phenolic compounds. Thus, this species has socioeconomic and nutritional potential [1,16].
Mdub-1 and Mdub-2 (Table 1) samples of M. dubia in this work, and those reported in the literature, Mdub-3 to Mdub-10 (Table A1), were classified in seven distinct chemical profiles according to the composition of their essential oils. The first profile (Mdub-1 and Mdub-2) was characterized by the highest amount of α-pinene (54.4-67.2%), from hydrodistilled specimens growing in Pará state, Brazil. Profile II was characterized by limonene (74.3%) and α-pinene (10.8%), which comprises the leaves' volatile concentrate

Myrciaria dubia
Myrciaria dubia (Kunth) McVaugh, popularly known as camu-camu, is native to the Brazilian and Peruvian Amazon regions. This species' leaves and fruit peels are used in Brazilian traditional medicine to treat diarrhea, female diseases, and labyrinthitis [14], while in Peru, M. dubia leaves are used to treat colds and arthritis [15]. Moreover, M. dubia fruits are considered a natural source of antioxidants by their significant content of ascorbic acid and phenolic compounds. Thus, this species has socioeconomic and nutritional potential [1,16].

Myrciaria floribunda
Myrciaria floribunda (H. West ex Willd.) O. Berg is known elsewhere as rumberry, camboim, and cambuí, growing naturally in the Central and South American continents [21,22]. Their fruits contain rutin, phenolic acids, and β-cryptoxanthin (pro-vitamin A) and are consumed in nature as jelly and in distilled beverages [23].
As the specimen samples (Mten-1 to Mten-7) were all obtained by the same extraction method (hydrodistillation), it does not seem to have been the factor that influenced the variability of the chemical profiles and may be associated with the genetic aspects of their different locations of collection.

Bibliometric Network Data
The bibliometric network map represents data of scientific bases, identifying the degree of connection among the various elements through the distance between their nodes. The smaller the distance, the greater the degree of connection. In addition, the node's size indicates its relevance in the analyzed universe [39]. The co-occurrence of similar terms in titles, abstracts, and keywords of nineteen articles in the Scopus database from 2010 to 2021 were analyzed to relate and identify the most widespread themes about Myrciaria essential oils. Figure 4 exhibits the generated map and its associations. The terms "essential oils", "Myrtaceae", "Myrciaria floribunda", and "Myrciaria tenella" were the most frequent. Moreover, terms were grouped into five clusters. The largest cluster is represented in red and includes terms related to biological assays, such as "animal cell", "mice" and "cytotoxic-ity", and terms referring to chemical composition analysis, as "chemical analysis", "mass fragmentography", "α-cadinol" and "copaene".
node's size indicates its relevance in the analyzed universe [39]. The co-occurrenc similar terms in titles, abstracts, and keywords of nineteen articles in the Scopus data from 2010 to 2021 were analyzed to relate and identify the most widespread themes a Myrciaria essential oils. Figure 4 exhibits the generated map and its associations. The terms "essential o "Myrtaceae", "Myrciaria floribunda", and "Myrciaria tenella" were the most frequ Moreover, terms were grouped into five clusters. The largest cluster is represented in and includes terms related to biological assays, such as "animal cell", "mice" "cytotoxicity", and terms referring to chemical composition analysis, as "chem analysis", "mass fragmentography", "α-cadinol" and "copaene". The second-largest cluster, in green, includes terms related to chemical composition analysis instrumentation, such as "gas chromatography" and "mass spectrometry"; and related terms to the chemical composition of essential oils, such as "essential oil composition", "volatile organic compound", "hydrocarbons", "α-pinene".
The third-largest cluster, in blue, has terms related to the chemical classes, such as "sesquiterpene" and "monoterpenes", and terms referring to the insecticide activity, such as "insecticide" and "insect growth regulator". The yellow cluster encompasses terms related to the extraction of essential oils, such as "distillation" and "hydrodistillation". Finally, in purple, the fifth cluster shows terms related to the topic of this article, such as "essential oils" and "chemical variability".

Plant Material
The leaves of the six Myrciaria specimens were collected in Pará state, Brazil, month-bymonth, during the dry season (August-December). The collection site, herbarium voucher number, and geographic coordinates are listed in Table 2. After identification, the plant specimens were deposited in the Herbarium of Museu Paraense Emílio Goeldi (MG) in the city of Belém, Brazil. The leaves were dried for three days at room temperature, ground, and then submitted to essential oil hydrodistillation in duplicate using a Clevenger-type apparatus. The oils obtained were dried over anhydrous sodium sulfate, and total oil yields were expressed as mL/100 g of the dried material [40,41].

Analysis of Essential Oil Composition
The oil composition analysis was performed by GC-MS, using a Shimadzu instrument Model QP-2010 ultra (Shimadzu, Tokyo, Japan), equipped with a Rtx-5MS (30 m × 0.25 mm; 0.25 µm film thickness) fused silica capillary column (Restek, Bellefonte, PA, USA). Helium was used as carrier gas adjusted to 1.0 mL/min at 57.5 KPa; split injection (split ratio 1:20) of 1 µL of n-hexane solution (oil 5 µL: n-hexane 500 µL); injector and interface temperature were 250 • C; oven programmed temperature was 60 to 240 • C (3 • C/min), followed by an isotherm of 10 min. EIMS (electron impact mass spectrometry): electron energy, 70 eV; ion source temperature was 200 • C. The mass spectra were obtained by automatically scanning every 0.3 s, with mass fragments in the range of 35-400 m/z. The compounds present in the samples were identified by comparison of their mass spectrum and retention index calculated for all volatile components using a linear equation by Van den Dool and Kratz [42], with the data present in the commercial libraries FFNSC-2 [13] and Adams [12]. The retention index was calculated using n-alkane standard solutions (C8-C40, Sigma-Aldrich, St. Louis, MO, USA) under the same chromatographic conditions. The GC-FID analysis was carried out on a Shimadzu QP-2010 instrument, equipped with an FID detector, in the same conditions, except that hydrogen was used as the carrier gas. The percentage composition of the oil samples was computed from the GC-FID peak areas. The analyses were carried out in triplicate.

Bibliographic Research Criteria
Bibliographic research was performed using Google Scholar, PubMed, Science Direct, Medline, and Scopus. Applied keywords were "Myrciaria", "essential oil" and "volatile compound". Some unusual or incorrect botanical names were updated based on "The Plant List" (http://www.theplantlist.org, accessed on 20 November 2021).
Bibliometric data analysis was done using more keywords to search for articles on the theme proposed in this review, using the VOSviewer software (version 1.6.15) [43]. The articles were downloaded from the databases in a supported format by the software. The primary data retrieved from the databases included information related to the article title, authors' names, keywords, and citation information, including the reference lists. In this way, a cluster was generated relating the main keywords and their links with others used less frequently in the searches [4].

Multivariate Statistical Analyses
The multivariate statistical analysis was carried out to discern any relationship among Myrciaria oil samples (described in Appendix A). The total percentage of the compound classes monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), sesquiterpene hydrocarbons (SH), and oxygenated sesquiterpenes (OS), to each oil, was extracted from the original citations (Table A1). The data were used as variables (see Appendix B). The data matrix was standardized for the multivariate analysis by subtracting the mean and then dividing it by the standard deviation. Principal component analysis (PCA) was applied to verify the interrelation (free 390 version, Minitab Inc., State College, PA, USA). Hierarchical grouping analysis (HCA), considering the Euclidean distance and the complete linkage, was used to verify the similarity between the oil samples (OriginPro trial version, OriginLab Corporation, Northampton, MA, USA) [44].

Conclusions
The profiles of Myrciaria species showed significant chemical variability. This variability is related to different extraction methods, collection sites, plant parts, and genetic variability. Among the collected samples, five chemical profiles were reported for the first

Conflicts of Interest:
The authors declare no conflict of interest.