Terpenoids and Bio-Functions of Essential Oils Hydrodistilled Differently from Freshly Immature and Mature Blumea balsamifera Leaves

The volatiles and antioxidant capacity of essential oils (EOs) extracted from freshly immature and mature leaves of Blumea balsamifera at various hydrodistillation times were investigated. Seven major terpenoids were identified: two monoterpenes, camphor and L-borneol, and five sesquiterpenes, silphiperfol-5-ene, 7-epi-silphiperfol-5-ene, ß-caryophyllene, ɤ-eudesmol, and α-eudesmol. The quantity and terpenoid composition of the EOs were impressed by leaf maturity and hydrodistillation times. The yield of EOs from the immature leaves was 1.4 times that of mature leaves, with 73% of the yield acquired within the first 6 hours (hrs) of hydrodistillation. Approximately 97% of camphor and L-borneol, 80% of ß-caryophyllene, silphiperfolene, and 7-epi-silphiperfolene, 32% of ɤ-eudesmol, and 54% α-eudesmol were collected in the first 6 hrs of hydrodistillation. More ß-caryophyllene, ɤ-eudesmol, and α-eudesmol were found in the mature leaf EOs. The antioxidant capacity of the EOs was proportionally related to their terpenoid contents. The EOs extracted from immature leaves at 0–6 hrs of hydrodistillation demonstrated distinctive antibacterial activity against Staphylococcus aureus, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 0.5 mg/mL and 1 mg/mL, respectively.


Introduction
Blumea balsamifera (Linn.), a traditional herb locally grown in Tailand, belongs to the family Asteraceae. Based on medical concerns such as cost-efectiveness and widespread use in the local community, its leaves have been utilized in traditional medicine to treat bruising, beriberi, eczema, dermatitis, lumbago, menorrhagia, and rheumatism [1]. In Tailand, the leaves of B. balsamifera have long been employed as a favoring agent in traditional foods and beverages. Furthermore, the leaf extract exhibits free-radical scavenging [2], antiobesity [3], anticancer [4], anti-insomnia activities [5], insecticidal action against the maize weevil and ß-caryophyllene are three primary bioactive chemicals found in B. balsamifera leaves, promoting the percutaneous absorption of salbutamol sulfate [11] and having antimutagenic properties [8,12]. Additionally, research has been conducted to improve extraction efciency and optimize the preparation and separation of high-purity borneol by modifying the hydrodistillation and sublimation processes [13]. Te plant production of B. balsamifera has progressively increased in South Asia as a healthy dietary component. Diferent stages of leaf maturation may accumulate varying levels of bioactive chemicals that may be suited for specifc human health purposes [14,15]. To date, there is no ofcial report, but a bioRxiv preprint [16] on the characteristics of EOs extracted from fresh leaves of B. balsamifera at various maturity stages and the extraction efciency of certain volatiles. Terefore, we explored the volatile compounds in immature and mature leaves of B. balsamifera using a hydrodistillation approach at diferent extraction times and analyzed the EOs for antioxidant and antibacterial activities.

Extraction of Essential Oils.
Freshly immature and mature leaves (500 g) were blended and then put in a 10-liter round bottom fask with deionized water (5 L). Next, the fask was subjected to hydrodistillation using a clevengertype apparatus. Sampling was carried out every 6 hrs until 24 hrs. Te recovered EOs were dried over anhydrous sodium sulfate ( Figure S1) and stored in sealed vials at −20°C [17] until analysis.

Determination of Volatile Components by GC-MS.
Volatile compounds were analyzed using gas chromatography-mass spectroscopy (GC-MS) [18] on an Agilent 6890N (Agilent Technologies, USA) gas chromatograph equipped with an HP-5MS (5% phenyl dimethylpolysiloxane) fused silica capillary column (30 m length × 0.25 mm internal diameter × 0.25 μm flm thickness) and an Agilent 5973 Network mass selective detector (Agilent, USA). One μL of the EOs was injected, and helium was used as the carrier gas. Te column temperature was initially held at 120°C for 5 mins and then increased to 250°C at 10°C/min. Te temperatures of the injector, detector, manifold, and transfer line were 250°C, 200°C, 70°C, and 240°C, respectively, and the ionization energy was 70 eV. Te mass spectra ranged from 30-500 amu. Te obtained spectra matched to a mass spectral library (the NIST V.14L library values, Palisade Corp., USA) were compared with an internal standard (100 μg/mL thiophene). Camphor, Lborneol, and ß-caryophyllene authentic chemicals were subsequently used for quantitative analysis of the compounds. All experiments were analyzed in triplicates.

Determination of Antioxidant Activity
2.5.1. DPPH Radical Scavenging Assay. Te free radical scavenging capacity of the EOs was estimated following the DPPH free radical method [19] with slight modifcations. Te DPPH solution was prepared by dissolving 2.4 mg of DPPH in 100 mL of absolute ethanol. Te test solution (50 μL) was added to 1.950 mL of the ethanolic DPPH. Te mixture was shaken vigorously and kept at room temperature for 30 min in the dark. Te absorbance of the mixture was measured at 517 nm using a UV spectrophotometer (UV-1800, Shimadzu, Japan).

ABTS Radical Scavenging Assay.
Free radical scavenging activity of the EOs was also determined using the ABTS radical cation decolorization assay [20]. Te ABTS solution was obtained by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate at a ratio of 1 : 0.5 and was stored in the dark at room temperature for 12-16 hrs. Te ABTS solution was then diluted with absolute ethanol to an absorbance of 0.700 at 734 nm. Te B. balsamifera extract (50 μL) was mixed with 1.950 mL of the diluted ABTS solution. Te absorbance of the mixture was spectrophotometrically measured at 734 nm.
2.6. Determination of Antibacterial Activity. Te antibacterial activities of EOs extracted from immature leaves at 0-6 hrs and 12-18 hrs of hydrodistillation ( Figure S2) were tested in vitro. Te broth dilution approach described in modifed CLSI M7-A7 [21], which is briefy detailed in the next paragraph, was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Te MIC and MBC of EOs obtained against three diferent species, namely, Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 9027), were tested and certifed by the Expert Centre of Innovative Herbal Products (Inno Herb), Tailand Institute of Scientifc and Technological Research (TISTR).
Te inoculums were prepared by growing each of the three bacteria in Mueller-Hinton broth (MHB) at 35 ± 2°C for 18-24 hrs. Te bacterial suspensions were adjusted to a turbidity of approximately 1 × 10 8 CFU/mL, and then 50 μL of each inoculum was put in a tube containing 5 mL of MHB to achieve the fnal concentrations of each EO in the media. Te fnal concentrations of each EO in MHB were 0, 0.5, 1, and 5 mg/mL. Te cultures were incubated at 35 ± 2°C for 18-24 hrs before being assessed for the MIC ( Figure S3). Subsequently, the cultures in diluted-EOs broth with zero turbidity (efective inhibition of bacterial growth) were streaked on sterilized MHA plates containing the same concentration of EOs, incubated at 35 ± 2°C for 18-24 hrs, and MBC was determined by observing the presence or absence of bacterial growth ( Figure S4). Te MBC value was defned as the concentration of EOs in the absence of bacterial colony development.

Statistical Analysis.
All experiments were carried out in three replications (3 trees for a replication), and the data were expressed as mean values ± standard deviation. Te data were statistically analyzed by variance analysis (ANOVA) at P < 0.05-P < 0.01 using SPSS software version 18. Mean comparisons were performed using Duncan's multiple range test (DMRT).

Results and Discussion
3.1. Volatile Compounds of Immature and Mature B. balsamifera Leaf Extract. Te EOs of B. balsamifera's freshly immature, and mature leaves were yellowish in hue (Figures S1 and S5). When compared to mature leaves (352.4 mg/100 g FW), immature leaves produced more EOs (501.9 mg/100 g). Te EO yield from immature leaves was 29.8% higher than the amount from mature leaves.
According to the research on Syzygium aromaticum, a considerable proportion of essential oil is collected in the juvenile stages of leaves, with the largest output being in young leaves (5.1%), followed by expanded leaves (4.5%), expanded leaves (4.1%), and the mature leaves (3.8%) [22]. Previous research has found a link between canopy structure, sunshine exposure, and the resulting plant bioactive compounds [23]. Immature leaves receive more sunlight and acquire more secondary phytochemical compounds due to their outer position, as previously reported in Zingiber ofcinale [24], Cistus ladanifer [25], and Moringa oleifera [26]. Plants are protected from abiotic and biotic stress by secondary metabolites such as phenolics and favonoids [27]. Most of the EOs (73%) were extracted during the frst 6 hrs of hydrodistillation (Table 1), with the latter stage (18-24 hrs) accounting for only 6.5% of the total yield. Te EOs comprised Seven essential volatile compounds, namely, two monoterpenoids, camphor and L-borneol, and fve sesquiterpenoids, silphiperfol-5-ene, 7-epi-silphiperfol-5ene, ß-caryophyllene, ɤ-eudesmol, and α-eudesmol (Table 2, Figure 2, Figures S6-S7). Te contents of camphor, borneol, and 7-epi-silphiperfol-5-ene in the EOs from immature and mature leaves were not signifcantly diferent (Table 3), even though monoterpenes are biosynthesized in chloroplasts and mature leaves generally contain a higher chlorophyll content. Light has been shown to greatly stimulate monoterpene synthesis in plants while having little efect on sesquiterpene hydrocarbon concentrations [28]. Tis is probably due to diferent precursors of exogenous origin in photosynthesis [29]. Moreover, over 90% of the camphor, borneol, sesquiterpenoids of silphiperfolene, 7-epi-silphiperfolene, and ß-caryophyllene were eluted from the leaves within the frst 6 hrs, whereas only 32.4% of ɤ-eudesmol and 54.6% of α-eudesmol extracted in the same period.
Camphor, borneol, and ß-caryophyllene contents were subsequently determined using authentic standards that corresponded to the compounds (Table 4). Camphor and borneol are both important ingredients used in traditional Tai medicine [30], while ß-caryophyllene is an important sesquiterpene in the pharmaceutical industry for nervous system-related disorders such as depression, pain, anxiety, and Alzheimer's disease [12]. Despite the decline in yield during the later stages of the hydrodistillation, yields of sesquiterpenoids like ɤ-eudesmol and α-eudesmol remained considerably high at 12-18 hrs (Figure3), in both immature and mature leaves. All eudesmol isomers (α-, ß-, and ɤeudesmol) are cytotoxic to cancer cells [31]. Moreover, sesquiterpenoids have a unique tricyclopentane ring      Journal of Tropical Medicine structure and a variety of intriguing biomedical and pharmaceutical properties [32]. At 6 hrs of hydrodistillation, the immature leaves' EOs contained more ß-caryophyllene and borneol than mature leaves. Camphor was the most prevalent constituent of the EOs, followed by borneol and ßcaryophyllene ( Figure 4). Other studies have identifed Lborneol as the primary component, followed by camphor, ßcaryophyllene, eudesmol, isoborneol, and 1,8-cineole [8,30]. L-borneol, camphor, and ß-caryophyllene contents of 42.06, 1.07, and 12.24% were reported for EOs from the leaves of B. balsamifera grown in China [10], indicating a possible role of environmental conditions on the chemical composition and yield.

Antioxidant Activity of Extracts from Immature and
Mature Leaves. Terpenes and their OH and NH 2 functional groups of plant volatile compounds have been associated with antioxidant capacity (DPPH and ABTS) [33]. L-borneol, ɤeudesmol, and α-eudesmol containing some OH hydroxyl groups are all greater in immature leaves (5.73, 2.60, and 1.68 g thiophene/100 g FW, respectively) than in mature leaves (4.00, 2.03, and 1.35 g thiophene/100 g FW, respectively). Te bioactive compounds have a favorable link with antioxidant capacity, with DPPH capacities of 67.89 g Trolox/100 g FW in immature leaves and 60.39 g Trolox/100 g FW in mature leaves, and ABTS capacities of 224.86 g Trolox/100 g FW in immature leaves and 190.12 g Trolox/100 g FW in mature leaves. Te EOs from immature leaves contained higher amounts of these compounds (Table 3) and showed higher DPPH and ABTS activities than EOs from mature leaves, indicating that they were more active (Table 5). Extracts obtained at 0-6 hrs had the highest DPPH and ABTS activities, and the antioxidant activity decreased with extended hydrodistillation periods, mirroring the EOs yield pattern. DPPH is only soluble in organic solvents [34], while ABTS can determine both the hydrophilic and lipophilic antioxidant capacities of samples [35]. Hence, the DPPH value was generally lower, whereas the ABTS assay revealed greater treatment-related diferences in antioxidant activity, both in terms of hydrodistillation time and leaf maturity. Tis implies that the extracted volatile compounds are predominantly lipophilic rather than hydrophilic in nature.

Antibacterial Activity of Extract from Immature Leaves.
Te antibacterial activity of EOs extracted from immature B. balsamifera leaves at 0-6 hrs and 12-18 hrs was determined in vitro using three pathogenic bacteria, Gram-positiveS. aureus (ATCC 6538), Gram-negative E. coli (ATCC 8739), and P. aeruginosa (ATCC 9027). By 0-6 hrs, the EOs included larger concentrations of camphor, L-borneol, and ß-caryophyllene, whereas at 12-18 hrs, ɤ-eudesmol and α-eudesmol were prevalent (Figure 3). S. aureus causes skin and soft tissue infections, bone and joint infections, bacteremia, and endocarditis [36], E. coli causes diarrheal disease, sepsis, and urinary tract infections [37], and P. aeruginosa causes gastrointestinal infection, keratitis, otitis media, and pneumonia [38]. Both hydrodistillation fractions (0-6 hrs and 12-18 hrs) were efective against S. aureus, with MIC values of 0.5 mg/mL and 1 mg/ mL, respectively. Te broth inoculated with S. aureus showed zero turbidity of culture starting from 0.5 to 5 mg/mL of the frst 6 hrs fraction, while it was from 1 to 5 mg/mL of 12-18 hrs fraction. All of the concentrations of EOs (0.5-5 mg/mL) were inefective against E. coli and P. aeruginosa (Table 6, Figure S3). Sakee et al. [39] reported that leaf-derived EOs of B. balsamifera in Tailand showed efective antimicrobial activity against S. aureus (1.2 mg/mL MIC), Bacillus cereus (150 μg/mL MIC), and Candida albicans (1.2 mg/mL MIC), but there was no efect against Salmonella enterica, Enterobacter cloacae, Klebsiella pneumoniae, E. coli, and P. aeruginosa [39]. EOs of B. balsamifera leaves from Luodian, China, had antibacterial activity against S. aureus (9.6 mg/mL MIC) and E. coli (4.8 mg/ mL MIC), whereas EOs from Hainan, China, inhibited S. aureus (2 mg/mL MIC), C. albicans (1.2 mg/mL MIC), P. aeruginosa (1 mg/mL MIC), and good antifungal activity (62.5-250 μg/mL MIC) [9]. Variation in antimicrobial activity is probably due to diferences in the chemical and extraction methods [9]. Furthermore, EOs obtained within the frst 6 hrs had an MBC value of 1 mg/mL against S. aureus (Table 6, Figure S4). Camphor is a major component of EOs and exhibits the highest aqueous solubility of other terpenoids in EOs [40]. Tis property probably enables it to penetrate through the outer membrane of bacteria [41]. Gram-negative bacteria are generally more resistant to EOs than Gram-positive ones, which is mainly because the outer layer of Gram-negative bacteria comprises lipopolysaccharides, which restrict the difusion of hydrophobic compounds through the lipopolysaccharide covering [42].

Conclusions
From our study, camphor, L-borneol, silphiperfol-5-ene, 7epi-silphiperfol-5-ene, ß-caryophyllene, ɤ-eudesmol, and α-eudesmol were the primary terpenoids discovered in the essential oils isolated from B. balsamifera leaves. In Eos, extracted from immature leaves, ß-caryophyllene, ɤeudesmol, and α-eudesmol were found to be more prevalent, but silphiperfol-5-ene was abundant in mature leaves. Camphor, L-borneol, and 7-epi-silphiperfol-5-ene  concentrations in essential oils isolated from immature and mature leaves were not signifcantly diferent. Te majority of the compounds (>90%) were extracted during the frst 6 hrs of hydrodistillation, including camphor, borneol, sesquiterpenoids of silphiperfolene, 7-epi-silphiperfolene, and ß-caryophyllene, while only 32.4% of ɤ-eudesmol and 54.6% of α-eudesmol were extracted during this period. At 6 hrs hydrodistillation, the essential oils isolated from immature leaves had the highest antioxidant activity (DPPH and ABTS). Te antioxidant capacity was related to the Lborneol, ɤ-eudesmol, and α-eudesmol contents. EOs extracted from immature leaves after 0-6 hrs of hydrodistillation also showed antibacterial action against S. aureus, with MIC and MBC values of 0.5 mg/mL and 1 mg/mL. Tis study demonstrates that it is advantageous to both alternative and traditional therapies Young leaves of B. balsamifera with high bioactive components, antioxidant activity, and antibacterial capabilities are suitable to be chosen for local food or therapy. In addition, 6-hour hydrodistillation of immature leaves can give a signifcant yield of Eos, and the bioactive chemicals required for further relevant product improvement for modern pharmacists or the cosmetic and food industries.

Data Availability
Te authors confrm that the data supporting the fndings of this study are available within the article and its supplementary material. Raw data that support the fndings of this study are available from the corresponding authors upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Sirinapha Jirakitticharoen was primarily responsible for conceiving the study, the experiments' design, and implementation. Technical analysis and experimental progress were advised by Wudtichai Wisuitiprot and Pongphen Jitareerat. Chalermchai Wongs-Aree provided conceptual and experimental work support, project management, and statistical interpretation assistance. Sirinapha Jirakitticharoen evaluated the data and wrote, managed, and revised the paper draft. As the correspondent, Chalermchai Wongs-Aree advised, corrected, and approved the manuscript.