The Chemical Composition and Biological Activities of Essential Oils from Zanthoxylum rhetsa Grown in Son La, Northwest Vietnam

Essential oils (EOs) from the stem barks, leaf petioles, fruit petioles, fresh leaves, and fresh and dried fruits of Zanthoxylum rhetsa were extracted by hydrodistillation. The volatile compounds of the products were analyzed by gas chromatography (GC-FID) and gas chromatography/mass spectrometry (GC/MSD). Monoterpene hydrocarbons formed the predominant fraction of all six EO samples, of which sabinene is one of the major components (from 12.37% to 41.13%). For the leaf petiole EO, limonene (25.01%), sabinene (14.56%), and linalool (12.63%) are the major constituents, while the main constituents of fruit petiole EO were terpinolene (19.66%), terpinen-4-ol (19.07%), and sabinene (17.83%). The major components of stem bark EO are terpinen-4-ol (18.23%), sabinene (12.37%), α -phellandrene (7.34%), β -phellandrene (6.32%), and c -terpinene (6.12%), while sabinene (38.35%), terpinen-4-ol (13.71%), c -terpinene (6.47%), and limonene (6.02%) are the major constituents of fresh leaf EO. For the EOs of dried fruits and fresh fruits, sabinene, terpinolene, limonene, and terpinen-4-ol are the major constituents. The essential oils were also tested for their cytotoxic and antimicrobial activities. The results revealed that six EOs at concentrations of 50 μ g/mL exhibited inhibitory activity against at least one tested cancer cell line but were nontoxic on Vero normal cells. Most EOs showed moderate antimicrobial activity against F. oxysporum ; however, there were no obvious activity against B. subtilis and S. aureus .


Introduction
Zanthoxylum rhetsa (Roxb.) DC (Z. rhetsa) is a flowering plant of the Rutaceae family found in India, Myanmar, ailand, Lao, and Vietnam. e tree has a medium size (about 14-18 meter in height) with a straight body, thorny branches, and 10-15 cm lanceolate leaves. e Z. rhetsa flowering season is between June and July with clusters of gray-white flowers and fruiting in October and November [1,2]. Z. rhetsa is an indigenous plant in the northwest of Vietnam, where the Son La province accounted for 71% of the total production [3]. Fruit and seed powders of Z. rhetsa are used as spices for cooking or for meat preservation by ethnic minorities such as ai and H'Mong. Moreover, the plant is also used as traditional treatment for toothache, abdominal and stomach pain, and improving digestion [3,4]. e chemical composition of the essential oil of Z. rhetsa grown in India and ailand has been reported. For instance, the seed EO of Z. rhetsa grown in Kerala (South India) contained mostly monoterpenes [5], while sesquiterpenes were predominant in the leaf EO, with the major components include caryophyllene oxide, β-caryophyllene, β-copaene, and spathulenol [6]. e phytochemical profile of the seed EO, i.e., the presence of sabinene, α-pinene, α-terpinene, β-pinene, c-terpinene, myrcene, terpinolene, and limonene, was also varied according to the pH of environment [7]. Meanwhile, the seed coat of Z. rhetsa collected from Senapati (the northeast of India) mainly consisted of terpinen-4-ol (32.1%), α-terpineol (8.2%), sabinene (8.1%), along with β-phellandrene and 2-undecanone at 7.4% and 7.1%, respectively [8]. However, in some areas of ailand such as Nan and Chiang Rai, the dried and the fresh fruits of Z. rhetsa contained different levels of limonene (27.10%-59.68%), β-phellandrene (10.88%-19.40%), and sabinene (25.03%-31.21%) [9]. On the other hand, sabinene (22.51%) and terpinene-4-ol (32.33%) were the major components of the EO extracted from fresh fruits of Z. rhetsa collected from Phayao ( ailand) [10].
Numerous studies have reported the interesting biological activities of Z. rhetsa EOs. e fresh fruit EO of Z. rhetsa grown in Phayao, ailand, has showed antiproliferative activity against breast cancer cells, and thus it was proposed as a potential food preservative and anticancer drug [10]. Meanwhile, terpinen-4-ol, which is the main constituent of pericarp EO, has the ability to inhibit the stress and diseases related to stomach and intestines [11].
In this paper, the EOs obtained from the different parts of Z. rhetsa (e.g., stem bark, leaf petiole, fruit petiole, fresh leaves, and fresh and dried fruit) grown in Son La, Northwest Vietnam, were extracted by hydrodistillation and its chemical composition was analyzed by GS/MS. In addition, these EOs have been evaluated for their biological activities, which included antibacterial and antiproliferative activities.

Materials.
e stem bark, leaf petiole, fruit petiole, leaves, and fruits of Z. rhetsa were collected from the uan Chau district, Son La province, Vietnam. Plant identification was performed by Dr. Nguyen Quoc Binh, the Vietnam Museum of Nature (VMN), Vietnam Academy of Science and Technology (VAST). All the plant parts were washed with tap water three times, air-dried at room temperature, and then stored in a refrigerator. 500 g of each fresh sample of stem bark, leaf petiole, fruit petiole, and leaves was chopped into pieces, and 200 g of fresh fruits was crushed as samples for EO isolation. 500 g of fresh fruits was dried at room temperature and then were ground as samples for EO isolation.

Isolation of Essential Oils.
e oil extraction was performed by hydrodistillation in the Clevenger-type apparatus for 3 h at normal pressure. e collected EOs were dehydrated with anhydrous sodium sulfate, weighted, and refrigerated until analysis. e samples were labeled as SB: stem bark; LP: leaf petiole; FP: fruit petiole; FL: fresh leaves; DF: dried fruit; and FF: fresh fruit.

GC-MSD and GC-FID Analysis.
e chemical compositions of EOs were analyzed by Agilent 7890A gas chromatography (GC) equipped with an MSD Agilent 5975C detector and a HP-5MS column (60 m × 0.25 mm, 0.25 µm film thickness) (Agilent Technologies, CA, USA). Other conditions were set as follows: 250°C as injector temperature, helium as the carrier gas, 1 mL·min −1 as flow rate, and temperature program from 60°C to 240°C (4°C/min). e split ratio was 100 : 1, and the injection volume of EO was 1 μL. e MSD full-scan mode was applied under 70 eV of ionization voltage, 40 mA of emission current, and 35-450 amu of acquisition scan mass range. e constituents were identified by comparing their mass spectrum with the W09N08 libraries and NIST Chemistry WebBook (http://webbook.nist.gov/chemistry/) database. e retention indices (RIs) of EO components were calculated by MassFinder 4.0 software base on homologous n-alkanes with same conditions. e relative content of each phytochemical component was estimated based on the GC-FID peak area with same conditions.

Cell Proliferation Assays.
Five human cancer cell lines (e.g., HeLa, Hep-G2, A-549, MCF-7, and HGC-27) and a normal cell line Vero were obtained from ATCC and maintained in suitable media (RPMI 1640, MEM, DMEM; Sigma Aldrich Inc., Saint. Louis, MO, USA) at 37°C in 5% CO 2 . MTT assay was performed to investigate the viability of cancer cells [13,14]. Dilution was performed in a 96-well microplate to obtain a density of 5 × 10 4 cells per well. e samples (0.63-50 µg/mL), DMSO as the negative control (Merck KGaA) and ellipticine as the positive control (Merck KGaA), were added to the wells and incubated at 37°C for 48 h. A total of 20 µL of MTT (Sigma-Aldrich, Saint. Louis, MO, USA) was then added, and incubation was continued for another 4 h at 37°C. Absorbance was measured at 540/ 720 nm using a Spark multimode reader (Tecan, Männedorf, Switzerland). All experiments were performed in triplicate. e growth inhibition (%) was calculated by using the formula: Inhibition rate (%) � (1 − OD sampl /OD con ) × 100%, where OD sampl and OD con are the optical densities of the sample groups and control, respectively.

Chemical Composition of Z. rhetsa Essential Oils.
Essential oils from the different parts of Z. rhetsa were obtained by hydrodistillation and analyzed for phytochemical profile by GC-MSD/GC-FID. e results are shown in Table 1 and Figure 1.

Leaf Petiole EO.
e chemical constituents accounted for 99.69% of the total oil ( Figure 1(b)). Monoterpene hydrocarbons were the most abundant in leaf EOs, with limonene being the major compound (25.01%). Oxygenated monoterpenes represented 24.07% of the EO, with linalool (12.63%) as the major compound. A minor quantity (2.7%) of sesquiterpene hydrocarbons was represented with β-caryophyllene (1.23%) as the major compound. Oxygenated sesquiterpenes represented 1.11% of the oil.
ese differences are displayed in Figure 2. Some compounds are only present in a certain EO and therefore are assumed to have properties specific to a certain EO: δ-3-carene, 2-undecanone, α-cubebene, β-cubebene, α-copaene, cis-β-elemene, β-selinene, elemol, spathulenol, 1-epi-cubenol, epi-α-cadinol, α-muurolol, and neo-    Table 2). Particularly, the EO of fresh leaves (FL) exhibited stronger cytotoxicity against four tested cancer cell lines, while the EO of stem bark (SB) and of fresh fruit (FF) exhibited cytotoxicity against HGC-27 and A-549, respectively. Significantly, these EOs demonstrated no cytotoxicity against the normal Vero cell line at the final concentration of samples up to 100 μg/mL. Naik et al. suggested that the EO from Z. rhetsa fruits could inhibit the cell viability and proliferation of breast cancer [10]. It was found the EO obtained from dried fruits collected from Nan of ailand exhibited inhibitory effect on the growth of human lung cancer cell line (H460) with an EC 50 value of 1.79 μL/mL. Meanwhile, the dried Z. rhetsa fruits collected from some districts of ailand (Nan, Phayao, and Chiang Rai) revealed a wide range of EC 50 values from 2.03 μg/mL to 7.07 μg/mL against human lung cancer cells (MRC-5) [9].

Antimicrobial Activity.
Six EO samples from different parts of Z. rhetsa collected from the Son La province in Vietnam were also tested for their antimicrobial activities (Table 3). e results demonstrated that most of the EOs showed moderate antimicrobial activity against F. oxysporum yet did not inhibited bacteria B. subtilis and S. aureus.
Vanden Bergher and Vlietinck also observed various degrees of inhibition of the fresh leaf EO of Z. rhetsa at different concentrations against the test fungal isolates. e obtained results have shown that the concentration of 12.5% exhibited the highest activity against A. niger, A. fumigatus, A. flavus, and Penicillium italicum in agar dilution tests [19]. Pham et al. suggested that terpinen-4-ol that is the main active constituent in Z. rhetsa pericarp EOs had the ability to inhibit stomach and intestine diseases [11]. Some other studies have also shown that essential oils obtained from plants exhibited potential antibacterial and antifungal activities [20][21][22].

Conclusions
Six EO samples were obtained by hydrodistillation from different parts of Z. rhetsa (e.g., stem barks, fresh leaves, leaf and fruit petioles, fresh and dried fruits) collected in the Son La province in Vietnam. Monoterpene hydrocarbons were found to be the predominant compound of all six EO samples, of which sabinene is one of the major components (from 12.37% to 41.13%) followed by limonene (from 4.18% to 25.01%). Oxygenated monoterpenes is present in quite high content in six EO samples, in which terpinen-4-ol was found to be the main compound of this fraction (from 5.35% to 19.07%). Sesquiterpene hydrocarbons and oxygenated sesquiterpenes were present at a relatively high concentration in stem bark EO (12.61% and 6.93%, respectively) but only in a trace amount in other samples. Especially, aliphatic ketones were found only in stem bark EO (2.77%) and completely absent in the remaining five EO samples. Some compounds were present in all six EO samples but at different concentrations, such as terpinolene is present in high content in dried fruits, fresh fruits, fruit petioles, and leaf petioles (30.37%, 27.05%, 19.66%, and 6.86%, respectively) but is in low content in fresh leaves and stem barks (1.91% and 1.57%, respectively); linalool is present in relatively high content in leaf petioles and fruit petioles (12.63% and 11.64%, respectively), but it is only present in trace amounts in fresh leaves, fresh fruits, stem barks, and dried fruits (1.80%, 1.71%, 1.61%, and 0.84%, respectively). e cytotoxicity results have shown that six EOs at a concentration of 50 μg/mL exhibited inhibitory activity against at least one tested cancer cell line but were nontoxic on Vero normal cells. For the antimicrobial activity, most EOs showed moderate inhibitory effect against F. oxysporum, yet no effects were observed against B. subtilis and S. aureus.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.