Essential Oil of Ipomoea carnea : Chemical Proﬁle, Chemometric Analysis, Free Radical Scavenging, and Antibacterial Activities

: Essential oils (EOs) have been reported as a promising group of naturally extracted compounds due to their various reported biological activities. Ipomoea carnea is a widely distributed plant with many traditional uses worldwide. However, although the EOs of various Ipomea species have been reported, I. carnea remains poorly studied. Therefore, the present investigation aimed to characterize the chemical proﬁle of the EO of I. carnea growing in Egypt via gas chromatogra-phy/mass spectroscopy (GC-MS) and correlate its proﬁle with other reported species via chemometric analysis using agglomerative hierarchical clustering (AHC) and principal component analysis (PCA). In addition, the aim was to determine the antioxidant and antibacterial activities of the extracted EO. Depending on the GC-MS analysis, 31 compounds were identiﬁed, mainly terpenes (94.82), with traces of carotenoid and apocarotenoid-derived compounds. The major compounds were tau-cadinol (35.68%), α -cadinol (26.76%), spathulenol (8.11%), and caryophyllene oxide (6.56%), which were assigned as major compounds. The chemometric studies showed that the Egyptian ecospecies of I. carnea differs in chemical proﬁle from those growing in Brazil, as well as those reported for other Ipomea species. The EO showed signiﬁcant DPPH and ABTS radical scavenging abilities, with IC 50 values of 33.69 and 40.86 mg L − 1 , respectively. Additionally, the I. carnea EO displayed signiﬁcant inhibition against the growth of all tested bacterial strains, where it showed an MIC range of 82–1442 mg mL − 1 . Based on the current results, the I. carnea EO, particularly the major identiﬁed compounds, could be used as a potential eco-friendly green resource for antioxidant and antimicrobial activities. Therefore, further study is recommended to evaluate the biological signiﬁcance of the main compounds, either individually or in combination, as well as assess their modes of action and safety.


Introduction
Throughout the ages, medicinal plants have been widely used in traditional medicine because of their viability, safety, low toxicity, and pharmacological potential [1]. Among the overall phytochemicals derived from plants, essential oils (EOs) are considered a promising using a GC-MS apparatus combined with the TRACE GC ultra gas chromatograph and Thermo Scientific™ EC quadrupole mass spectrometer unit (Waltham, MA, USA). The dimension and film thickness of the GC-MS column was 30 m × 0.32 mm × 0.25 µm. Helium as a transporter gas was used with a split ratio of 1 to 10 and a flow rate of 1.0 mL per min. The temperature was justified as usual according to the following: 60 • C (1 min), then increased to 240 • C within 4 • C/min. The EO samples were diluted in 1 µL of n-hexane with a ratio of 1:10 (v/v) and then injected in an injector and detector modified at 210 • C. The mass spectral data of components were obtained via electron ionization (EI) with m/z 40-450 as the spectral range at 70 eV. The chemical components were authenticated and identified using Automated Mass Spectral Deconvolution and Identification System (AMDIS) software, NIST library database, the Wiley Spectral Library Collection, and the retention indices closed to n-alkanes (C 8 -C 22 ).

EO Extraction Analysis and Characterization
From each sample of I. carnea, 250 g of EO was extracted by hydrodistillation over a Clevenger device for 3 h, separated by n-hexane, and dried with about 0.5 g of anhydrous Na2SO4. The three EO samples were stored in glass vials at 4 °C in a refrigerator for analysis. Chemical profiling of the EOs was established separately using gas chromatography/mass spectrometry (GC/MS) at the National Research Center of Egypt under the same protocols described previously [33]. In detail, the analysis of the extracted EO samples was performed using a GC-MS apparatus combined with the TRACE GC ultra gas chromatograph and Thermo Scientific™ EC quadrupole mass spectrometer unit (Waltham, MA, USA). The dimension and film thickness of the GC-MS column was 30 m × 0.32 mm × 0.25 µm. Helium as a transporter gas was used with a split ratio of 1 to 10 and a flow rate of 1.0 mL per min. The temperature was justified as usual according to the following: 60 °C (1 min), then increased to 240 °C within 4 °C/min. The EO samples were diluted in 1 µL of n-hexane with a ratio of 1:10 (v/v) and then injected in an injector and detector modified at 210 °C. The mass spectral data of components were obtained via electron ionization (EI) with m/z 40-450 as the spectral range at 70 eV. The chemical components were authenticated and identified using Automated Mass Spectral Deconvolution

Antioxidant Activity
The EO extracted from I. carnea was assayed for its antioxidant activity via the scavenging ability of the free radicals 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Sigma-Aldrich, Darmstadt, Germany). According to Miguel [34], serial concentrations of the EO (5-50 mg L −1 ) were prepared using 95% methanol as a solvent. In glass tubes, each concentration (2 mL) and freshly prepared DPPH (2 mL of 0.3 mM) were vigorously shaken and left for 30 min in dark conditions at room temperature. At 517 nm, the measurement of the absorbance was performed using a Spectronic 21D model spectrophotometer. For additional confirmation of the antioxidant activity, ABTS scavenging was tested according to Re et al. [35]. In brief, 0.2 mL of each concentration was mixed with 2 mL of 7 mM of freshly prepared ABTS. The mixtures were incubated for 6 min at room temperature, and the absorbance was immediately measured with a spectrophotometer at 734 nm. The positive control was prepared using ascorbic acid (standard antioxidant) with a range of 1-20 mg L −1 , while the negative control was treated using methanol similarly to the samples. The calculation of the scavenging potential was established by the following equation: The IC 50 (required EO concentration for 50% scavenging) was calculated based on the exponential curve of the absorbance and concentrations using Microsoft Excel 2019.

Antibacterial Activity
The EO extracted from the aerial parts of I. carnea was evaluated for its bacterial inhibitory potential via the agar diffusion method [36]. The activity was tested against four Gram-negative bacterial isolates (Escherichia coli ATCC 10536, Klebsiella pneumoniae ATCC 10031, Pseudomonas aeruginosa ATCC 9027, and Salmonella typhimurium ATCC 25566) and four Gram-positive bacterial isolates (Bacillus cereus EMCC 1080, Staphylococcus epidermidis ATCC 12228, Staphylococcus haemolyticus ATCC 29970, and Staphylococcus xylosus NCCP 10937). These bacterial isolates were purchased from the Cairo Microbiological Resources Centre (Cairo MIRCEN), Agriculture College, Ain Shams University, Egypt. To test the antibacterial activity of the EO, 10 mg mL −1 EO was prepared using 1% (CH 3 ) 2 SO (DMSO), and filter paper discs (0.5 cm) were impregnated with 50 µL of EO. Petri plates with the medium of nutrient agar were incubated with 1 × 10 8 colony forming units (CFU)/mL of each bacterial isolate, and the discs saturated with EO were placed over the medium within the plates. The plates were immediately sealed with Parafilm ® tape (Sigma, St. Louis, MO, USA) and incubated in an incubator modified at 37 • C. After 24 h, the diameter of the clear (inhibition) zone around the disc was measured in millimeters from three points. In addition, DMSO was used as a negative control, where it was prepared and treated as previously mentioned for the EO, as it did not show any antibacterial activity. Cephalexin, tetracycline, ofloxacin, and ampicillin were tested as the positive control (standard antibiotic). The minimum inhibitory concentration (MIC) was calculated for each bacterial isolate based on the exponential curve of the inhibition zone diameter and various concentrations of the EO.

Data Analysis
The antioxidant and antibacterial activity experiments were repeated with 3 replicas, and the data were expressed as average ± standard error. To test the significance among treatments, the data of either antioxidant or antibacterial activities, with replications, were subjected to a one-way ANOVA, followed by Duncan's post-hoc test at a 0.05 probability level using CoStat software (version 6.311, CoHort Software, Monterey, CA, USA). To correlate the ecospecies studied in this paper with other reported species, we used two chemometric analyses: agglomerative hierarchical clustering (AHC) and principal component analysis (PCA). A dataset of 22 Ipomea ecospecies was constructed, consisting of (1) I. carnea (leaf) collected from Egypt, (2) I. pes-caprae (fresh leaf) collected from Mauritius, (3) I. pes-caprae (dry leaf) collected from Mauritius [13], (4) I. obscura (dry leaf) collected from India [28], (5) I. aquatica (aerial parts) collected from Japan [27], (6) I. asarifolia (leaf) collected from Brazil, (7) I. setifera (leaf) collected from Brazil [29], (8) I. batatas (leaf) collected from Nigeria  Table S1). The dataset matrix was subjected to AHC and PCA.

Chemical Profile of I. carnea EO
The hydrodistillation of the I. carnea aerial parts provided 0.034% (v/w) golden-yellow EO. The yield of EOs from other reported Ipomea species such as I. carnea, I. pes-caprae, I. alba, I. batatas, and I. indica was 0.01-0.13% for fresh leaves and 0.01-0.03 for fresh flowers [26]. The extracted oil was subjected to GC-MS analysis (Figure 2), where 31 compounds were identified with a relative concentration of 97.58%. The compound names, molecular formulas, retention times, relative concentrations (%), and Kovats indices (KIs) of all identified compounds are presented in Table 1.  Table S1). The dataset matrix was subjected to AHC and PCA.

Chemical Profile of I. carnea EO
The hydrodistillation of the I. carnea aerial parts provided 0.034% (v/w) golden-yellow EO. The yield of EOs from other reported Ipomea species such as I. carnea, I. pes-caprae, I. alba, I. batatas, and I. indica was 0.01-0.13% for fresh leaves and 0.01-0.03 for fresh flowers [26]. The extracted oil was subjected to GC-MS analysis (Figure 2), where 31 compounds were identified with a relative concentration of 97.58%. The compound names, molecular formulas, retention times, relative concentrations (%), and Kovats indices (KIs) of all identified compounds are presented in Table 1.   The identified compounds can be grouped into six classes: oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, diterpene hydrocarbons, carotenoid, and apocarotenoid-derived compounds (Table 1). Sesquiterpene represented the abundant EO components with a relative concentration of 92.71% comprising mainly oxygenated sesquiterpene (82.88%) and a low relative concentration of sesquiterpene hydrocarbons (9.83%). The abundance of the sesquiterpenoids was common in the EOs of several Ipomoea plants such as I. batatas (46.5%) [30], I. pes-caprae (70.4%) [13], and I. obscura (84.9%) [28].
On the other hand, monoterpenes were assigned as trace components and represented only by oxygenated compounds (1.75%) and no monoterpene hydrocarbons were detected (Table 1). Three oxygenated monoterpenes, 4-terpineol (0.69%), α-fenchyl alcohol (0.57%), and linalool (0.49%), were only assigned. The scarcity of the monoterpenes is common in the reported EO profiles of many studied Ipomoea species [13,27,28,30]. In the extracted EO of I. carnea in the present study, only one diterpene compound, beyerene, was identified with a very low relative concentration (0.36%). The scarcity of the diterpenes in the EOs derived from medicinal and aromatic plants is common, with a few exceptions [8]. In addition, the EOs of Ipomoea plants have been reported to contain a low relative concentration of diterpenoid compounds [13,27,28,30]. In addition, three nonterpenoid compounds were characterized that can be categorized into carotenoid (0.95%), including E-β-damascenone (0.77%), E-α-ionone (0.18%), and apocarotenoid (1.81%), comprising compounds derived from hexahydrofarnesyl acetone (1.81%). These three compounds were identified as trace components in the EOs of some I. pes-caprae [13]. The present chemical characterization of I. carnea EO deduced some similarities with the other Ipomoea species, which may be ascribed to the climatic, genetic, and environmental conditions [4,37,38].

Chemometric Analysis of the EOs of Ipomea Specie
The chemometric analysis of the EO profile of the Egyptian I. carnea ecospecies and the other reported Ipomea species revealed considerable variation (Figure 3). terpenoid compounds were characterized that can be categorized into carotenoid (0.95%), including E-β-damascenone (0.77%), E-α-ionone (0.18%), and apocarotenoid (1.81%), comprising compounds derived from hexahydrofarnesyl acetone (1.81%). These three compounds were identified as trace components in the EOs of some I. pes-caprae [13]. The present chemical characterization of I. carnea EO deduced some similarities with the other Ipomoea species, which may be ascribed to the climatic, genetic, and environmental conditions [4,37,38].

Chemometric Analysis of the EOs of Ipomea Specie
The chemometric analysis of the EO profile of the Egyptian I. carnea ecospecies and the other reported Ipomea species revealed considerable variation (Figure 3). However, the present results showed a positive correlation with the profile of the EO of the I. pes-caprae collected from Mauritius [13]. The cluster analysis showed that each However, the present results showed a positive correlation with the profile of the EO of the I. pes-caprae collected from Mauritius [13]. The cluster analysis showed that each Ipomea ecospecies has its own specific chemical profile signature of the EO compounds. These data could be ascribed to the genetic characteristics of each species [38,39]. In addition, the only studied I. carnea from Brazil did not correlate with those reported in the Egyptian ecospecies of the present study. This could be attributed to the variation in the environmental and climatic conditions [40]. The extraction techniques have been reported to have a great effect on the composition of the extracted EO [41][42][43]; therefore, this variation could be a reasonable factor for the determined variation between the Egyptian and Brazilian ecospecies of I. carnea, where, for the Brazilian ecospecies, the EO was extracted with steam distillation, while for the Egyptian ecospecies, the EO was extracted upon hydrodistillation.
The application of PCA on the dataset of the 22 Ipomea ecospecies showed that present Egyptian I. carnea and I. pes-caprae collected from Mauritius are correlated with each other, where they did not show specific correlation with specific compounds. On the other side of the PCA, the Brazilian ecospecies of Ipomoea amnicola, I. indica, I.alba, I. pes-caprae, I. carnea, I. tiliacea, and I. batatas were separated at the right side of the PCA and showed a correlation with β-caryophyllene, cis-cadina-1(6),4-diene, germacrene, β-elemene, bicyclogermacren, and δ-cadinene. On the other hand, the Japanese ecospecies of I. aquatica were separated alone and revealed a correlation with the phytol compound.

Free Radical Scavenging Activity of I. carnea EO
The free radical scavenging potentiality of I. carnea EO was evaluated using DPPH and ABTS assays with respect to ascorbic acid as a reference antioxidant agent. The present data showed that the EO of I. carnea has strong antioxidant capability in comparison to the standard antioxidant (ascorbic acid) ( Table 2). The activity of scavenging increased with the increment of the concentration. At a concentration of 20 mg L −1 , the EO showed 31.05% and 24.15% DPPH and ABTS scavenging, respectively, while ascorbic acid showed 73.17% and 69.06% scavenging, respectively. According to the IC 50 values, the EO of I. carnea displayed IC 50 of 33.69 mg L −1 for DPPH and 40.86 mg L −1 for ABTS, whereas ascorbic acid showed an IC 50 of 11.51 and 12.94 mg L −1 . The current findings were in total agreement with the fact of the significant antioxidant activities of the different extracts of the Ipomoea species. The alcoholic extract of the Indian I. carnea was reported to exhibit significant in vitro antioxidant activity using DPPH and ABTS assays [20]. In addition, the different extracts of the leaves of I. batatas, including EOs, were described to have strong antioxidant potential [13][14][15]. Alam  compared with ascorbic acid [44]. Moreover, the phenolic-enriched alcoholic extract of the halophyte, I. pes-caprae, was reported to have strong reducing power on DPPH radicals, better than butylated hydroxyanisole and butylated hydroxytoluene [45].
The antioxidant activity of I. carnea EO might be ascribed to the chemical constituents characterized mainly as terpenes. The reproducible free radical scavenging potential of the EOs was documented to be strongly correlated with the overbalance of the terpenoid compounds [6,8,46]. In addition, the oxygenated compounds are responsible for more antioxidant activity due to their functions in the oxygenation of the components and scavenging the free radicals [8,47,48]. The chemical profiling of the I. carnea EO showed that 83.63% of overall oil mass are oxygenated compounds that could cause significant observed antioxidant activity.
Among the oxygenated compounds, the oxygenated sesquiterpene compounds, taucadinol, α-cadinol, spathulenol, and caryophyllene oxide, represented the main compounds in the I. carnea EO. These compounds might have played an effective role as antioxidant agents either individually or synergistic with others. The EO derived from Cullen plicata has been reported to possess a strong antioxidant ability due to the abundance of tau-cadinol, α-cadinol, and caryophyllene oxide [49]. In addition, the EO of Algerian Teucrium polium was reported to have significant antioxidant activity due to its high content of spathulenol and tau-cadinol [50]. Caryophyllene oxide represented one of the most common compounds in the plants' EOs with important biological actions, especially antioxidant activity, due to the presence of lone pairs of electrons that increase the free radicals trapping [51]. This compound was reported to be of the main contributors to the increase in the antioxidant activity of the EOs of Cannabis sativa [51], Salvia palaestina, S. ceratophylla [52], and Heliotropium curassavicum [53]. Additionally, α-cadinol was promoted to act as a significant antioxidant mediator in the EO of several herbs such as Tabernaemontana catharinensis [54] and Xenophyllum poposum [55]. Furthermore, the minor constituents could be contributed to the antioxidant activity via the synergetic effect [56].

Antibacterial Activity of I. carnea EO
The inhibitory effects of the EO derived from I. carnea were estimated against eight bacterial strains (four Gram-negative and four Gram-positive strains). The results showed that the EO displayed significant inhibitory effects against all strains. For Gram-negative bacteria, the activity can be ordered as follows: K. pneumoniae > E. coli > P. aeruginosa > S. typhimurium, where they showed MIC values of 84, 94, 124, and 158 mg mL −1 , respectively (Table 3). However, the EO exhibited inhibition against the four Gram-positive bacteria strains in the following order: S. xylosus > B. cereus > S. epidermidis > S. haemolyticus, with MIC values of 82, 84, 1442, and 82 mg mL −1 , respectively. Previously, different extracts and EOs of several Ipomoea plants were stated to have inhibitory effects against different bacterial strains. The crude alcoholic extract of I. mauritiana was described to significantly inhibit the growth of several bacteria strains [44]. Moreover, the whole plant extracts of I. pes-caprae have been reported to inhibit a set of Gram-positive and Gram-negative bacterial strains [57]. In addition, some compounds isolated from different extracts and oils of I. pes-caprae, such as (-) mullein [58] and E-phytol [59], have been described to have the potential for inhibiting some bacterial strains such as Escherichia coli and Staphylococcus sp. In agreement with our data, Yuan et al. [60] described that the EO derived from the waste materials of I. batatas can strongly stop the growth of Pseudomonas aeruginosa, Escherichia coli, and Bacillus cereus.
The bacterial inhibition capability of the I. carnea EO was stronger than that described for other plants' EOs as Kickxia aegyptiaca [46], Teucrium polium [41], and Deverra tortuosa [6]. The observed antibacterial activity of the I. carnea EO in the present study could be attributed to the major compounds such as caryophyllene oxide, tau-cadinol, α-cadinol, and spathulenol. The two main compounds, tau-cadinol and α-cadinol, in the current study were also reported in high concentrations of the EO of various plants that showed strong bacterial growth inhibition such as Eugenia chlorophylla [61], Litsea acutivena [62], Diospyros discolor [63], and Teucrium polium [41]. Spathulenol and caryophyllene oxide represented common compounds in EOs derived from the plant kingdom [4]. Many EOs were documented to have strong antibacterial activity due to the preponderance of spathulenol and caryophyllene oxide such as Baccharis dracunculifolia [64], Salvia hydrangea [65], Satureja coerulea [66], and Kickxia aegyptiaca [46]. Schmidt and coworkers described that caryophyllene oxide is one of the most active antibacterial agents as a single compound against Escherichia, Klebsiella, and Salmonella strains [67]. The major compound, tau-cadinol, in the present study was described as a potent antibacterial agent against S. aureus [68]. In addition, caryophyllene and its oxygenated derivatives were documented to have bactericidal effects against several bacterial strains, including B. cereus, B. subtilis, E. coli, S. aureus, and P. aeruginosa [69]. Spathulenol has been reported in high concentrations (36.6%) of Helichrysum amorginum EO, where it showed strong inhibitory effects against S. aureus and S. epidermidis [70]. Moreover, the other minor components might act in synergetic interaction with other compounds as antibacterial agents [4]. Table 3. Antibacterial activity of the Ipomoea carnea essential oil and some selected reference antibiotics (10 mg mL −1 ). # Value is an average of three replicas of the inhibition zone (IZ) diameter expressed as mm ± standard error. Within each column, different superscript letters mean significant variation. LSD: least significant difference. *** p < 0.001.
The Gram-negative bacteria were well-known bacteria with strong resistance to antibiotics due to their outer rigid membrane with a high content of lipopolysaccharide [9]. Several documented data have revealed that the EOs and their bioactive constituents could attach to the bacterial cell surface and therefore perforate the cell membrane phospholipid bilayer, then accumulate with mischievous effects on the cell metabolism, causing bacterial cell death [71].

Conclusions
The present study showed for the first time the chemical profile of the EO extracted from the Egyptian ecospecies of I. carnea. The identified compounds were mainly sesquiterpenes with caryophyllene oxide, tau-cadinol, α-cadinol, and spathulenol as major compounds. The chemometric analysis revealed that the EO of the presently studied I. carnea is different from that reported from other species of Ipomoea. The extracted EO exhibited substantial antioxidant activity and strong inhibition against the tested Gram-negative and Gram-positive bacteria. The observed activities of this oil in the current study might be ascribed to the major identified constituents, which could be integrated as eco-friendly natural resources with antioxidant and antibacterial potential. Further study is recommended to evaluate the various activities of the major identified compounds, either individually or in combination, and determine their potential, modes of action, and safety.