Chemometrics of the Composition and Antioxidant Capacity of Hyptis crenata Essential Oils from Brazil

Hyptis crenata (Pohl) ex Benth is used in traditional medicine as an analgesic to treat general pain. Six Hyptis crenata samples (Hc-1 to Hc-6) were collected in Pará state, Brazil. The leaf essential oils were obtained by hydrodistillation, and GC-MS and GC-FID were used to analyze their chemical compositions. The antioxidant capacity was measured in vitro using DPPH and carotene/linoleic acid assays. Chemometrics analysis (PCA, HCA, and clustered heat map) were used to identify the sample relationships between those collected in this study and those from the literature (Hc-7 to Hc-16) samples. According to the main chemical constituents identified in the samples described in this work and the literature, the sixteen samples were classified into ten groups. Group I was characterized by 1,8-cineole (31.0%), α-pinene (13.6%), (E)-caryophyllene (7.8%), and β-pinene (7.6%); and Group IV was characterized by 1,8-cineole (17.4–23.5%), α-pinene (15.7–23.5%), β-pinene (10.5–13.4%), and limonene (8.5–9.7%). Both groups are described for the first time. The total antioxidant capacity was expressed in Trolox Equivalent Antioxidant Capacity values (TEAC): TEAC of Hc-5 (551.9 mg.TE/g) and Hc-6 (475.1 mg.TE/g). In the β-carotene/linoleic acid assay, the highest inhibition was from Hc-2 (40.0%), Hc-6 (39.0%), and Hc-3 (29.4%).


Introduction
Lamiaceae comprises over 200 genera and 7000 species as the sixth largest family of angiosperms. Its species have economic, medicinal, and ecological importance [1]. Hyptis Jacq. is the largest genus of the subtribe Hyptidinae, which includes 19 genera, with 144 species occurring mainly in tropical America [2]. Hyptis species have constituents with pharmacological potential, with antibacterial, antifungal, anti-inflammatory, antioxidant, and cytotoxic properties [3].
Hyptis crenata (Pohl) ex Benth (syn. Hyptis crenata var. hirsuta Pohl ex J.A.Schmidt, Hyptis crenata var. microphylla Pohl ex J.A.Schmidt, Mesosphaerum crenatum Kuntze, Mesosphaerum crenatum var. albiflorum Kuntze, and Mesosphaerum crenatum var. subviolacum Kuntze) [4] is popularly known as "salva," "salva-do-marajó," "salsa-do-campo," "hortelã-brava," "hortelã-do-campo," and "hortelãzinha" [5]. Hyptis crenata (Figure 1) can be shrub or subshrub with erect stems that are branched and densely villous. The leaves are spreading ( Figure 1) can be shrub or subshrub with erect stems that are branched and densely villous. The leaves are spreading or sometimes slightly deflexed, sessile, membranous, rugose, ovate to ovate-oblong, apex obtuse to acute, and base rounded to subcordate. Its inflorescence appear with pedunculate capitula, most of which positions near the apex and sub-corymbose. Capitula are semiglobose, involucral bracteoles, and linear-subulate to lanceolate. Flowers have fruiting calyx tubes with lobes that are lanceolate-acuminate to subulate. It is native to Brazil, mainly distributed in the north, northeast, midwest, and southeast regions [6]. Ethnopharmacological studies carried out in the Brazilian Tropical Atlantic Forest reported that the leaf decoction of this species is used as an analgesic, and the infusion of the roots is used to treat general pains, bad cold, rheumatism, and menstrual colic [7]. In the Brazilian Pantanal region, leaf tea made from the plant is used for antiulcer and antiinflammatory treatments [8].
The biological properties of essential oils are very likely influenced by the plant s phytochemical composition. For example, there are six chemotypes of tea tree (Melaleuca alternifolia Cheel), but commercial tea tree essential oils are always the terpinen-4-ol chemotype, which is used to treat several skin conditions such as acne, eczema, herpes simplex, wounds, burns, insect bites, and mycoses [9]. The thymol/carvacrol chemotypes of Thymus vulgaris L. essential oils demonstrated significantly greater antioxidant activity than chemotypes with low thymol and carvacrol concentrations [10]. Similarly, the Piper betle L. essential oil with the highest chavibetol concentration also exhibited the best DPPH and ABTS radical-scavenging activity [11].
Therefore, due to the pharmacological and biological potential presented by Hyptis crenata, this work aimed to investigate the chemical variability of H. crenata essential oils and their antioxidant capacity by applying chemometric analysis. We hypothesize that H. crenata will exhibit variation in volatile chemical profiles and that these variations are likely to affect biological properties such as antioxidant activities.

Yield and Chemical Composition of the Essential Oil
The essential oils of six Hyptis crenata specimens evaluated in this work showed chemical variability. The yield ranged from 1.1 to 3.1%, as shown in Table 1. The quantification and identification of 88 constituents in the analyzed oils represent an average of 97.7% of the total oil content. Ethnopharmacological studies carried out in the Brazilian Tropical Atlantic Forest reported that the leaf decoction of this species is used as an analgesic, and the infusion of the roots is used to treat general pains, bad cold, rheumatism, and menstrual colic [7]. In the Brazilian Pantanal region, leaf tea made from the plant is used for antiulcer and anti-inflammatory treatments [8].
The biological properties of essential oils are very likely influenced by the plant's phytochemical composition. For example, there are six chemotypes of tea tree (Melaleuca alternifolia Cheel), but commercial tea tree essential oils are always the terpinen-4-ol chemotype, which is used to treat several skin conditions such as acne, eczema, herpes simplex, wounds, burns, insect bites, and mycoses [9]. The thymol/carvacrol chemotypes of Thymus vulgaris L. essential oils demonstrated significantly greater antioxidant activity than chemotypes with low thymol and carvacrol concentrations [10]. Similarly, the Piper betle L. essential oil with the highest chavibetol concentration also exhibited the best DPPH and ABTS radical-scavenging activity [11].
Therefore, due to the pharmacological and biological potential presented by Hyptis crenata, this work aimed to investigate the chemical variability of H. crenata essential oils and their antioxidant capacity by applying chemometric analysis. We hypothesize that H. crenata will exhibit variation in volatile chemical profiles and that these variations are likely to affect biological properties such as antioxidant activities.

Yield and Chemical Composition of the Essential Oil
The essential oils of six Hyptis crenata specimens evaluated in this work showed chemical variability. The yield ranged from 1.1 to 3.1%, as shown in Table 1. The quantification and identification of 88 constituents in the analyzed oils represent an average of 97.7% of the total oil content. RI C = calculated retention index using an n-alkane standard solution (C 8 -C 40 ) in Rtx-5MS column; RI L = literature retention index; Main constituents in bold, n = 2 (standard deviation was less than 2.0); tr = traces (% < 0.1); * = The percentage composition of the oil samples was computed from the GC-FID peak areas; a = Adams library [15]; b = FFNCS library [16].
The essential oils from dry and fresh aerial parts of Hyptis crenata sampled in Marajó Island (Brazilian Amazon) presented yields of 0.9 and 1.4%, respectively, the highest yield reported in the literature [17]. Another study that evaluated the chemical variability of essential oils from aerial parts, collected in Pará state and Tocantins (Brazilian Amazon), reported that the yield ranged from 0.2 to 0.9% [5], values lower than those of this work. Another sample from the Brazilian Cerrado exhibited an oil content of 0.6% [12]. Therefore, the yields reported in the literature (0.2-1.4%) were lower than those described in this study (1.1-3.1%).
Therefore, according to the main chemical constituents identified in the samples described in this work (Hc-1 to -6) and the literature (Hc-7 to -16), the sixteen samples were classified in ten chemical groups. Two chemical groups (group I and V) are described for the first time.

Multivariate Analyses of Hyptis crenata Specimens
The chemical variability of Hyptis crenata oil samples was evaluated by multivariate statistical analyses (PCA, Principal Components Analysis; HCA, Hierarchical Cluster Analysis). The total percentage of monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), sesquiterpene hydrocarbons (SH), oxygenated sesquiterpenes (OS), and other compounds (OT) were obtained from oil samples, according to the original citations (Tables 1  and A1). The data were used as variables (see Table A2, Appendix B).
Analysis). The total percentage of monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), sesquiterpene hydrocarbons (SH), oxygenated sesquiterpenes (OS), and other compounds (OT) were obtained from oil samples, according to the original citations (Tables 1 and A1). The data were used as variables (see Table A2, Appendix B).
Analyzing the mean contents and standard deviations of the compound classes p sent in Hyptis crenata oil chemotypes (Figure 4) showed that Group I was statistically ferent (Tukey test, p < 0.05) from Group II by the content of monoterpene hydrocarbon = 28.6 ± 9.3%; II = 55.0 ± 12.3%) and oxygenated monoterpenes (I = 49.9 ± 6.9%; II = 31 8.6%). Furthermore, Group III was distinguished from the other groups by the conten oxygenated monoterpenes (1.6 ± 0.0%), oxygenated sesquiterpenes (9.8 ± 0.0%), and ot compounds (5.3 ± 0.0%). Applying additional multivariate analyses in the heatmap analysis combined with hierarchical clustering analysis with the compound classes, the color pattern varied with color intensity and increased gradually, from lowest to the highest grade. The clustered heatmap ( Figure 5) confirmed the above clustering results for HCA and PCA.
Nonpolar antioxidants exhibit stronger antioxidative properties in emulsions because they are concentrated in the lipid phase, thus, ensuring high protection to the emulsion. On the other hand, polar antioxidants remaining in the aqueous phase are more diluted and, therefore, less effective in protecting the lipid phase [33]. Moreover, the DPPH assay is performed in a polar system, and the β-carotene/linoleic acid assay is performed in an apolar system [34].
Rebelo et al. [17] performed the DPPH assay on essential oil samples from fresh leaves and methanolic extract of H. crenata at different concentrations. The inhibition values obtained were 42.6-79.9%, 24.5-71.4%, and 14.2-94.0%, respectively. In another assay, using the ABTS method, the inhibition of leaves and flowers of H. crenata at different extraction times was 26.0-65.8% for the leaves and 55.6-84.4% for the flowers [35].

Plant Material
The leaves of the six Hyptis crenata specimens were collected in Marajó Island, Pará state, Brazil, during the rainy season (August-December). The collection site, herbarium voucher number, and geographic coordinates are listed in Table 2. 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, then pulverized. The leaves were submitted to essential oil hydrodistillation in duplicate using a Clevenger-type apparatus (2 h). The oils obtained were dried over anhydrous sodium sulfate, and total oil yields were expressed as mL/100 g of the dried material [36].

Analysis of Essential Oil Composition
GC-MS and GC-FID were performed to analyze the oil composition. A Shimadzu instrument Model QP 2010 ultra (Shimadzu, Tokyo, Japan) was used. An Rtx-5MS (30 m × 0.25 mm; 0.25 µm film thickness) fused silica capillary column (Restek, Bellefonte, PA, USA) was used as stationary phase. Helium was the carrier gas adjusted to 1.0 mL/min at 57.5 kPa with a split injection mode (split ratio 1:20) of 1 µL of n-hexane solution (oil 5 µL: 500 µL n-hexane); 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. With EIMS (Electron Ionization Mass Spectrometry) at 70 eV, the ion source temperature was 200 • C.
The mass spectra were obtained by automatic 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 spectra and retention indices, calculated for all volatile components using a linear equation of Van Den Dool and Kratz [37], with the data present in the commercial libraries FFNSC-2 [16] and Adams [15]. The retention index was calculated using n-alkane standard solutions (C8-C40, Sigma-Aldrich, St. Louis, MO, USA) in the same chromatographic conditions.
The GC-FID analysis was carried out on a Shimadzu QP-2010 instrument (Shimadzu, Tokyo, Japan), 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.

DPPH Radical Scavenging Assay
The antioxidant activity of the oils samples was evaluated by the DPPH radical scavenging method as described by Figueiredo et al. [33]. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a stable dark-violet free radical with maximum absorption at 517 nm, which is reduced in the presence of antioxidants.
The DPPH was diluted to initial absorbance of 0.62 ± 0.02 at 517 nm and room temperature. Each essential oil sample (50 µL, 10 mg/mL) was mixed with Tween 20 solution (0.5%, 50 µL, w/w) and then added to DPPH (0.5 mM, 1900 µL) in ethanol. For each sample, an ethanol blank was also measured. The absorbance was measured at the start of the reaction (time zero), each 5 min during the first 30 min, and then at continuous intervals of 30 min up to constant absorbance (plateau of reaction, 2 h) in Ultrospec™ 7000 spectrophotometer (Biochrom US, Holliston, MA, USA). The standard curves were prepared using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 60, 120, 240, and 480 µg/mL. The results were expressed as milligrams of Trolox (mg TE/g) equivalents per gram of the sample.

β-Carotene/linoleic Acid Assay
The amount of 0.2 mg of β-carotene was dissolved in 1 mL of chloroform (HPLC grade), and 25 µL of linoleic acid and 200 µL of Tween 20 were added. Chloroform was wholly evaporated using a vacuum evaporator. Then, 50 mL of oxygen-saturated water was added with vigorous shaking. An aliquot of 2300 µL of this reaction mixture was dispensed into test tubes, and 200 µL portions of the oil samples (1.0 mg/mL in ethanol) were added. This emulsion system was incubated at 50 • C. The same procedure was repeated with Trolox and a blank of ethanol.

Multivariate Statistical Analysis
The multivariate statistical analysis was carried out to discern any relationship among Hyptis crenata oil samples (described in Appendix A). The total percentage of the monoterpene hydrocarbons (MH), oxygenated monoterpenes (OM), sesquiterpene hydrocarbons (SH), and oxygenated sesquiterpenes (OS) of each oil was extracted from the literature (Table A1). The 17 × 6 data matrix was used for variables (see Appendix B). The matrix was standardized for the multivariate analysis by subtracting the mean and then dividing it by the standard deviation.
Hierarchical grouping analyses (HCA) were performed considering the Euclidean distance and the Ward linkage (Minitab free 390 version, Minitab Inc., State College, PA, USA). The principal component analysis (PCA) was applied to verify the interrelation (OriginPro trial version, OriginLab Corporation, Northampton, MA, USA) [38]. A clustered heat map was constructed using Euclidean distance via the Ward linkage (OriginPro trial version, OriginLab Corporation, Northampton, MA, USA).
The antioxidant capacity was calculated in triplicate, and the data were expressed as mean± SD. Statistical differences were evaluated by Tukey's test (p < 0.05) using the software GraphPad Prism 6.0.
Further research on essential oils from other plant parts (stems, roots, flowers) should also be explored in future study. Since there is intraspecific chemical variability in H. crenata, prior chemical knowledge must be available before suggesting its use for phytomedicinal purposes.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. - [19] Appendix B * Data showed in Table 2.