Terpenoid components, antioxidant and antibacterial activities of essential oils from fresh immature and mature leaves of Blumea balsamifera, extracted with different hydrodistillation periods

Volatiles and antioxidant capacities in essential oils (EOs) from fresh immature and mature leaves of Blumea balsamifera, extracted with different extraction periods of hydrodistillation, were investigated. There were seven major terpenoid compounds in the leaf extracts, including 2 monoterpenes of camphor and L-borneol, and 5 sesquiterpenes of silphiperfol-5-ene, 7-epi-silphiperfol-5-ene, ß-caryophyllene, ɤ-eudesmol, and α-eudesmol. Different hydrodistillation periods resulted in different quantitates and compositions of the terpenoids in EOs. The yield of EOs from the immature leaves was 1.4 times higher than the mature ones, whereas 73% of the yield was collected from the first 6 h of hydrodistillation. Camphor and L-borneol were almost collected in the first 6 h, while ß-caryophyllene, silphiperfolene, and 7-epi-silphiperfolene were above 80%, but ɤ-eudesmol and α-eudesmol were only 32 and 54% released. ß-Caryophyllene, ɤ-eudesmol, and α-eudesmol were found higher in the mature leaf EOs. Antioxidant capacities in EOs were positively related to terpenoid contents. Antibacterial activity of EOs from the immature leaves was subsequently tested. Although EOs from the hydrodistillation period of 12-18 h contained fewer terpenoid compositions, it showed the same minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) on Escherichia coli and Pseudomonas aeruginosa, compared to 0-6 h EOs.


Introduction
Blumea balsamifera (Linn.), belonging to the Family Asteraceae, is a locally grown traditional medicine plant in Thailand. The leaves have long been used for conventional therapy in terms of healing many conditions, including skin injury, skin bruises, beriberi, eczema, dermatitis, lumbago, menorrhagia, rheumatism, and some other diseases [1,2]. In Thailand, volatiles from B. balsamifera leaves have been traditionally used in heated/steamed incubation to heal women after childbirth and those who get injured. Furthermore, extracts of the leaves have been exhibited in physiological activities on free radical scavenging [3], plasmininhibitory [4,5], anti-obesity functions [6], antifungal activities [7], antimicrobial activities [8], anticancer [9], inhibition against the maize weevil (Sitophilus zeamais) [10], a reduction of blood pressure, an inhibition of sympathetic nervous system, curing over-excitement of insomnia [11].
From previous studies, 93 constituents of volatiles were reportedly found in B.
Many reports studied chemical compositions of EOs in various cultivated regions [14] and in different harvesting times [20]. However, there is no report yet, studying properties of EOs from the fresh leaves to varying maturities since B. balsamifera is such a bush plant containing young and mature leaves on any branch. Furthermore, there was an effort to improve the extraction efficiency and prepare and separate high purity of borneol by modifying hydrodistillation and sublimation [21]. So, in the present study, volatile compositions and antioxidant capacities were investigated in hydrodistillated oils from immature and mature leaves of B. balsamifera using a modified hydrodistillation with different extracting times was studied. Comparison of antibacterial activities of the selected leafy EOs was then investigated.

Plant materials
Fresh, bright green immature leaves (the 2 nd -4 th leaves from the shoot containing small soft trichomes and soft surface on upper epidermis), and dark green mature leaves (leaves containing tiny stiff trichomes and matted surface on upper epidermis) of B. balsamifera ( Figure S1)

Extraction of essential oil:
Fresh immature or mature leaves (500 g) were blended and then put in a 10-liter round bottom flask with deionized water (5 liters). Then, the flask was subjected to hydrodistillation using a Clevenger type apparatus ( Figure S2). Hydrodistillated oil was collected every 6 h until 24 h. Each partial oil was dried over anhydrous sodium sulfate and stored in sealed vials at -20 °C until analysis.

Determination of volatile components by GC-MS
Analysis of volatile components was followed as previously using gas chromatographymass spectroscopy (GC-MS) [22]. An Agilent 6890N (Agilent Technologies, USA) gas chromatograph was equipped with an HP-5MS (5 % of phenyl dimethylpolysiloxane) as a fused-silica capillary column (30 m length × 0.25 mm i.d., 0.25 μm film thickness), and an Agilent 5973 Network mass selective detector (Agilent, USA). One L of 100 ppm thiophene (v/v) (as internal standard) and 1 L of EOs were injected. The carrier gas was Helium. The column's temperature was 120ºC, with 5 min initial hold and then increased to 250ºC at a 10ºC/min rate. Temperatures of injector and detector were 250ºC and 200ºC, respectively, manifold at 70ºC with line transfer at 240ºC the ionization energy was 70eV. Scan the mass spectra were in the range of 30-500 amu. Authentic standards of camphor, L-borneol, and ßcaryophyllene were used for the quantitative calculation of the compounds. The standard curves of camphor (0-2000 mg/mL; y = 0.017x -1.6243; R 2 = 0.9926), L-borneol (200-1000 mg/mL; y = 0.0131x -2.1567; R 2 = 0.9959) and ß-caryophyllene (200-800 mg/mL; y = 0.0342x -2.9789; R 2 = 0.9926) were prepared. Camphor, L-borneol, and ß-caryophyllene contents were expressed as microgram per one-hundred-gram fresh weight (μg /100g FW). All experiments were analyzed in triplicate.
Individual constituents of EOs were identified by comparing their Kovats retention indices (RI) relative to (C 8 -C 30 ) n-alkanes on nonpolar and polar columns and comparison with literature data. Furthermore, Identification mass spectrums were matched with mass spectral library (NIST databases) or comparison with authentic compounds [23].

Determination of antioxidant activity
DPPH radical scavenging assay: The total free radical scavenging capacity of the EOs was estimated according to the previously reported method [24] with slight modification using DPPH radical. The radical solution is prepared by dissolving 2.4 mg of DPPH in 100 mL of absolute ethanol. A test solution (50 μL) was added to 1.950 mL of ethanolic DPPH. The mixture was shaken vigorously and kept at room temperature for 30 min in the dark. Blank was prepared by replacing 50 μL of EOs with 50 μL of ethanol. The mixture's absorbance was measured at 517 nm using a UV spectrophotometer (UV-1800, Shimadzu, Japan). Trolox solution was used to calibrate the standard curve at the range of 20-100 mg/mL, obtaining y = 0.0041x + 0.0262 (R 2 = 0.9993). DPPH values were expressed as microgram of Trolox equivalent per one-hundredgram fresh weight (μg Trolox/100g FW).
ABTS radical scavenging assay: The free radical scavenging activity of EOs was determined by ABTS radical cation decolorization assay [25]. ABTS cation radical was produced by the reaction between 7mM ABTS in water and 2.45 mM potassium persulfate (1:0.5) stored in the dark at room temperature for 12-16 h. ABTS solution was then diluted with ethanol to obtain an absorbance of 0.700 at 734 nm by diluting absolute ethanol before it was used. Fifty μL of B. balsamifera extract was mixed with 1.950 mL of ABTS radical solution. Blank was prepared by replacing 50 μL of EOs with 50 μL of ethanol. The absorbance of the mixture was spectrophotometrically measured at 734 nm. Trolox solution (20-100 mg/mL) was used to calibrate the standard curve with y = 0.006x + 0.0249 and R 2 = 0.9996. ABTS values were expressed as microgram of Trolox equivalent per one-hundred-gram fresh weight (μg Trolox/100g FW).

Determination of antibacterial activity
In this study, EOs from immature leaves at the hydrodistillation period of 0-6 h and 12-

Statistical analysis
All experiments were carried out in three replicates, and the data were expressed as mean values ± standard deviation of three replication of each EOs. The data was statistically analyzed by variance analysis (ANOVA) using SPSS software version 18. Mean comparisons were operated using Duncan's multiple range test (DMRT) at P<0.05 and P<0.01.

Volatile components in extracts with different leaf maturities and hydrodistillation times
Hydrodistillated EOs of fresh immature and mature leaves of B. balsamifera afforded yellowish color. The yield of EOs from the immature leaves (501.9 mg/100g FW) was higher than that from the mature leaves (352.4 mg/100g FW). This could be caused by the higher biosynthetic rates of metabolites in young laves, resulting in increased biomass accumulation.
The immature leaves raising in the positioning are intensely exposed by sunlight, which induces many secondary phytochemical compounds. Thus, the immature leaves can perceive more sunlight, affecting the efficient production of bioactive compounds in the leaves, as found in Cistus ladanifer [27]. The result was similar to a previous study with B. balsamifera in China, which studied different plant organs and other growth times [16]. Phenolics and flavonoids are a group of secondary metabolites of plants used to protect against abiotic or biotic stresses. Furthermore, secondary metabolites are from different metabolite families [28].
Thus, mature leaves could use some of the secondary metabolites used to protect themselves from the environment. Hydrodistillated EOs from the first 6 h contained the highest portion (73%) of the yields ( Table 1). The longer extracting time resulted in reducing portions of the yield when the 18-24 h contained only 6.5% of the total yield. However, there was an interaction between leaf maturities and hydrodistillation times. So, EOs extracted by hydrodistillation from this plant could be collected by 6 h concerning the yield compared to the operating time.
Seven essential volatile oils were found in the EOs from both immature and mature leaves of B. balsamifera, including 2 monoterpenoids of camphor, L-borneol, and 5 sesquiterpenoids of silphiperfol-5-ene, 7-epi-silphiperfol-5-ene, ß-caryophyllene, ɤ-eudesmol, and α-eudesmol (Table 2, Figure 1, Figure S3-S17). From the chemical content in Table 3, compared with thiophene, the internal standard, camphor, borneol, and 7-epi-silphiperfol-5ene were not a significant difference between EOs from the immature and mature leaves. Since monoterpenes are biosynthesized in chloroplasts of plant cell, but camphor and borneol contents in the immature and mature leaves were not different. This evidence could bring the attention of chloroplast metabolisms between these two maturities. Although the mature leaves could contain higher chlorophylls and chloroplasts in the cells, the top of branches located of young leaves could be activated by high intense sunlight. Light was reported to activate monoterpenes synthesis in plants strongly [29] but did not affect the content of the sesquiterpene hydrocarbon. This was probably due to the different precursors of exogenous origin from photosynthesis [30]. Moreover, for hydrodistillation periods, 6 h extraction conducted the highest content of the seven selected terpenoids, compared with other extracting times. When 5 compounds of camphor, borneol, sesquiterpenoids of silphiperfolene, 7-episilphiperfolene, and ß-caryophyllene were above 90% eluted from the leaves at the first 6 h, ɤeudesmol and α-eudesmol were extracted only 32.4 and 54.6%, respectively.
Three main components, including camphor, borneol, and ß-caryophyllene, were further quantified with the corresponding authentic standards in Table 4. Camphor and borneol are essential ingredients used in many traditional medicines in Thailand, having therapeutic efficacies of many conventional and indigenous against diseases [31]. On the other hand, ßcaryophyllene is a sesquiterpene that has important pharmacological activities. Its exhibits a protective role of nervous system-related disorders such as depression, pain, anxiety, and Alzheimer's disease [19]. Hydrodistillation period at 12-18 h in both immature and mature leaves obtained high amounts of ɤ-eudesmol and α-eudesmol, which are sesquiterpenoid alcohols. All eudesmol isomers (α-, ß-and ɤ-eudesmol) perform a cytotoxic effect on cancer cells [32]. Furthermore, sesquiterpenoid has a unique structure of a tricyclopentane ring, exhibiting a wide range of bioactive, biomedical, and pharmaceutical properties [33]. At the period of 6 h, EOs from the immature leaves contained quantitative of ß-caryophyllene and borneol higher than those from the mature leaves. B. balsamifera leaves in this study had camphor in the highest composition, followed by borneol and ß-caryophyllene ( Figure 2). In contrast to some previous studies, the main components in EOs were L-borneol, followed by camphor, ß-caryophyllene, ɤ-eudesmol, isoborneol, and 1,8-cineole [10,12,13,31]. The comparison between young and mature leaves of B. balsamifera in China found that L-borneol, camphor, and ß-caryophyllene in the young leaves was 42.06, 1.07, and 12.24% (% relative content), whereas in mature leaves, L-borneol, camphor, and ß-caryophyllene was at 40.73, 1.12, and 11.36%, respectively [15]. Consequently, factors affecting chemical variability and yield include physiological variations, environmental conditions, and geographic variations [34].

Antioxidant activity of extracts from the immature and mature leaves
The results of antioxidant capacity assays (DPPH and ABTS) were related to major terpenes from immature and mature leaves of B. balsamifera. Radical scavenging of DPPH and ABTS assays is related to the compound structure, which dependable on the number of active groups (OH or NH 2 ) [35]. From the main composition in this study, L-borneol, ɤ-eudesmol, and α-eudesmol have hydroxyl group (OH), so that most antioxidant capacities of DPPH and ABTS could be correlated with the quantity of these 3 components. The EOs of the immature leaves comprised main components more than that of the mature leaves, and at 0-6 h of hydrodistillation, the EOs contained most compositions of the terpenes (Table 5). Extracts from the leaves at 0-6 h had the highest DPPH and ABTS activities, but the activities of EOs from the immature leaves were higher. DPPH assay has a limitation to detect only those soluble in organic solvents [36]. On the other hand, ABTS can determine both hydrophilic and lipophilic antioxidant capacity of samples [37]. So, DPPH values showed less difference in each treatment, but ABTS values have different significance between the maturity and hydrodistillation period. This implies that the photo-components extracted by the hydrodistillation method were likely to be as lipophilic higher than hydrophilic compounds.

Antibacterial activity of extracts from the immature leaves
Two different fractions of 0-6 h and 12-18 h hydrodistillated from immature B. balsamifera leaves were selected to evaluate in vitro antibacterial activity with 3 pathogenic bacteria. EOs in the 0-6 h contained high camphor, l-borneol, and ß-caryophyllene, whereas EOs in the 12-18 h had two significant elements of ɤ-eudesmol and α-eudesmol ( Figure 3). The EOs were examined against a Gram-positive bacterium (S. aureus) and 2 Gram-negative bacteria (E. coli and P. aeruginosa). When S. aureus causes skin and soft tissue infections, bone and joint infections, bacteremia, and endocarditis [38], E. coli results in diarrheal disease, sepsis, and urinary tract infections [39], and P. aeruginosa causes gastrointestinal infection, keratitis, otitis media, and pneumonia [40][41][42]. Both fractions of hydrodistillation at 0-6 h and 12-18 h showed the best antibacterial effect against S. aureus at 0.5 mg/mL and 1 mg/mL MIC, respectively (Table 6). A previous study of EOs of B. balsamifera leaves from Thailand showed antimicrobial activity against S. aureus, Bacillus cereus, and Candida albicans, but there was no effect against Salmonella enterica, Enterobacter cloacae, Klebsiella pneumoniae, E. coli, and P. aeruginosa [8]. EOs of B. balsamifera leaves from Luodian, China had antibacterial activity against S. aureus and E.coli, whereas EOs from Hainan China performed against S. aureus, C. albicans, Aspergillus flavus, E. coli, Listeria monocytogenes, and S. enterica [14].
As a result, the EOs showed different antimicrobial activities due to various chemical compositions and different key compounds, and the extraction methods [14]. Furthermore, EOs from 0-6 h hydrodistillation conducted MBC at 1 mg/mL on S. aureus. Camphor is the main component of EOs, whose camphor's aqueous solubility is more extensive than other terpenoid components in EOs [43]. Camphor's property could probably elucidate these to penetrate through the outer membrane of bacteria [44]. The gram-negative bacteria are generally more resistant to EOs than gram-positive ones. This result could be due mainly to the outer layer of gram-negative bacteria comprised of lipopolysaccharide, which restricts hydrophobic compounds' diffusion through its lipopolysaccharide covering [45].
Hydrodistillation time at 0-6 h mostly extracted 7 major terpenoids from the leaves.
Antioxidant capacity assays (DPPH and ABTS) were related to L-borneol, ɤ-eudesmol, and αeudesmol. EOs extracts from immature leaves at 0-6 h of hydrodistillation period exhibited the most antioxidant activity. When the minimum inhibitory concentration (MIC) of EOs extracted from immature leaves against S. aureus at 0-6 h hydrodistillation period was at 0.5 mg/mL, the minimum bactericidal concentration (MBC) was at 1 mg/mL.

Conflict of interest
We do not have a conflict of interest. Table 1. Yields of essential oils extracted from immature and mature leaves of B. balsamifera with different hydrodistillation periods Data are expressed as mean ± SD (n = 3). Values in the same column followed by different letters indicate significant differences at p < 0.05 according to Duncan's multiple range test; * = significant at p < 0.05; ** = significant at p < 0.01 level.