Correlation between Chemical Composition and Antifungal Activity of Clausena lansium Essential Oil against Candida spp.

Essential oils (EOs) have been shown to have a diversity of beneficial human health effects. Clausena is a large and highly diverse genus of plants with medicinal and cosmetic significance. The aim of this study was to analyze the composition of Clausena lansium EOs and to investigate their potential antifungal effects. The chemical compositions of Clausena lansium EOs obtained by hydrodistillation were analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 101 compounds were identified among the diverse extracts of C. lansium. EOs of leaves and pericarps from different cultivars (Hainan local wampee and chicken heart wampee) collected in Hainan (China) were classified into four clusters based on their compositions. These clusters showed different antifungal activities against five Candida species (C. albicans, C. tropicalis, C. glabrata, C. krusei and C. parapsilosis) using the disc diffusion method. Clausena lansium EOs of pericarps displayed noteworthy antifungal activitives against all the tested Candida strains with inhibition zone diameters in the range of 11.1–23.1 mm. EOs of leaves showed relatively low antifungal activities with inhibition zone diameters in the range of 6.5–22.2 mm. The rank order of antifungal activities among the four EO clusters was as follows: Cluster IV> Cluster III > Cluster I ≥ Cluster II. These results represent the first report about the correlation between chemical composition of C. lansium EOs and antifungal activity. Higher contents of β-phellandrene, β-sesquiphellandrene and β-bisabolene in EOs of pericarps were likely responsible for the high antifungal activity of Cluster IV EOs. Taken together, our results demonstrate the chemical diversity of Clausena lansium EOs and their potential as novel antifungal agents for candidiasis caused by Candida spp. Furthermore, the obtained results showing a wide spectrum of antifungal activities provide scientific evidence for the traditional use of these plants.


Introduction
Natural plant products, especially essential oils (EOs), have many beneficial biological effects, such as antibacterial, anti-inflammatory, antitumor and analgesic activities. EOs are volatile aromatic substances found in many plants. The EOs may exist in fruits, seeds, flowers and leaves. They have already significant attention because of their abundance, broad spectrum activities, and diverse mechanisms of action. They are among the most popular natural antimicrobial agents and they have recently gained a great popularity and scientific interest [1][2][3][4].

Extraction Results and Chemical Composition of EOs
Clausena lansium EOs extracted by hydrodistillation were light yellow to yellow in color. The yields ranged from 0.23 to 0.51% (v/w). The source, cultivar, part, color and yield of EO details are listed in Table 1. There were differences in the color and yield of the eight EOs, which may be related to the geographic source, cultivar, and part of plants, but it is uncertain which is the most important of these factors. It is possible to come to a conclusion from the specific differences of the components. To compare the compositional differences, each EO extract was injected into GC-MS for analysis. Figure 1 shows GC-MS chromatograms of C. lansium EOs. The chemical compositions and contents results are shown in Table 2.       .39% of the total components, respectively. These components were monoterpenes, sesquiterpenes, alcohols, esters, etc. Some samples such as MCL and RCL were dominated by sesquiterpenes, with β-caryophyllene and β-sesquiphellandrene as the main components. MHL and RHL were rich in sesquiterpenes with cis-α-santalol and santalol being the most abundant species. There were a large number of monoterpenes in some samples, such as MCP, RCP, MHP and RHP. In all the combined samples, we detected 101 chemicals in the EO extracts. However, the distributions of the 101 chemicals differed widely among the extracts. To investigate the similarity and differences among these extracts in EO profiles, a cluster analysis was conducted using SPSS (version 17.0). The clustering result is shown in the Figure 2.
According to the GC-MS analysis, components and contents were different in leaves and pericarps of HLW and CHW. A total of 69, 63, 65, 63, 70, 71, 69 and 66 compounds were identified in MCL, MCP, RCL, RCP, MHL, MHP, RHL and RHP, amounting to 98.87%, 96.90%, 99.19%, 98.18%, 98.10%, 98.60%, 96.87% and 96.39% of the total components, respectively. These components were monoterpenes, sesquiterpenes, alcohols, esters, etc. Some samples such as MCL and RCL were dominated by sesquiterpenes, with β-caryophyllene and β-sesquiphellandrene as the main components. MHL and RHL were rich in sesquiterpenes with cis-α-santalol and santalol being the most abundant species. There were a large number of monoterpenes in some samples, such as MCP, RCP, MHP and RHP. In all the combined samples, we detected 101 chemicals in the EO extracts. However, the distributions of the 101 chemicals differed widely among the extracts. To investigate the similarity and differences among these extracts in EO profiles, a cluster analysis was conducted using SPSS (version 17.0). The clustering result is shown in the Figure 2. Samples were clustered into two clusters for the first time, leaves and pericarps. This result indicates that most of the differences were caused by plant parts. The next contributor was plant cultivar, followed by geographic location of where the plants were grown. The difference between Samples were clustered into two clusters for the first time, leaves and pericarps. This result indicates that most of the differences were caused by plant parts. The next contributor was plant cultivar, followed by geographic location of where the plants were grown. The difference between Meixiao Village and Rulin Village was the smallest, which is probably because the two places are geographically close (both in Haikou city). Table 3 shows the variation of some important volatile components (%) of C. lansium in four clusters. The domonant EOs in each of the four clusters are listed below: Cluster I: β-caryophyllene and β-sesquiphellandrene. MCL and RCL belonged to this cluster. These samples were leaves of CHW. The components were rich in β-caryophyllene (21.13%, 23.48%), β-sesquiphellandrene (18.52%, 21.80%), and accompanied by α-bergamotene (9.49%, 10.63%).
Cluster IV: β-phellandrene and β-sesquiphellandrene. Pericarps of CHW such as MCP and RCP in this cluster. β-Phellandrene in this cluster had a percentage higher than 30%. The percentages of β-phellandrene were 32.43% and 45.15% in MCP and RCP, respectively. β-Sesquiphellandrene was also relative high, with percentages of 10.89% and 7.35%, respectively.
In previous studies, EOs of C. lansium from China [12], Thailand [13], and Cuba [14] were obtained by hydrodistillation and analyzed by GC-MS. Thirty-two components were identified in leaves samples from China. The main components identified in the EO of leaves were β-santalol (35.20%) and bisabolol (13.70%). In both fresh and dried fruit samples from Thailand, fifty-three components were identified, and the main components were sabinene (33.68-66.73%), α-pinene (9.57-13.35%) and 1-phellandrene (5.77-10.76%). For leaf samples from Cuba, seventy compounds were identified. The most prominent components were caryophyllene oxide (16.80%) and (Z)-α-santalol (11.70%). The components and percentages were different from different areas, and the differences were much greater in different parts of C. lansium. There were some common components in EOs of leaves in the present study and the above reports, such as cis-α-santalol, β-bisabolene, and caryophyllene oxide. In contrast, the components of pericarps in our study were very different from those of the fruit samples in previous studies. The reason may be that fruit contains not only pericarp, but also pulp and seed.
In addition to the hydrodistillation method, supercritical fluid extraction (SFE) was used in the extraction of C. lansium leaves from Guangdong, China [18]. Thirty-six components were identified and the main components were 4-terpineol (26.94%) and γ-terpinene (14.39%). Chokeprasert et al. [19] analyzed the volatile components of fresh leaves and pericarps in Thailand by headspce (HS) GC-MS. Thirty-nine components were identified in the leaves, and the major components were sabinene (15%) and β-bisabolene (9.88%); thirty components were identified in the pericarps, and the major components were sabinene (69.07%) and α-phellandrene (10.63%). The components and percentages obtained by SFE and HS were quite different from that by hydrodistillation.
In the present study, the differences of the compositions of Clausena lansium leaves and pericarps from different cultivars were analyzed and compared for the first time. Cluster analysis result showed that the differences in the components were most significantly contributed by plant parts, followed by cultivar, and with geographic origin contributing the least to the variation.

Antifungal Activity
The antifungal activity of C. lansium EOs was examined using the filter paper disc diffusion method. As shown in Table 4, the EOs inhibited the growth of all seven yeast strains tested in our study. The inhibition zone diameters were in the range of 6.5-23.1 mm. The EOs of Clausena lansium pericarps showed significant activity against all the Candida strains, whereas, EOs of leaves showed relatively poor activity. The RCP EO exhibited the greatest antifungal effect against C. glabrata, with an inhibition zone diameter of 23.1 mm. Notably, C. albicans 27, a yeast strain which is resistant to FLZ and AMB, can be inhibited by Clausena lansium EOs with an inhibition zone diameter of 11.3-15.3 mm. This observation suggests that Clausena lansium EOs is active towards certain FLZ and AMB resistant strains. It can be seen from Table 4, the antifungal activitives efficacy order was as follows: Cluster IV ≥ Cluster III > Cluster I ≥ Cluster II. The two most active EOs against C. albicans, C. glabrata, C. krusei and C. parapsilosis, which were pericarps of CHW (Cluster IV) and were all rich in β-phellandrene (32.43%, 45.15%) and β-sesquiphellandrene (10.89%, 7.35%). Inhibition zone diameters were in the range of 12.0-23.1 mm. EOs of pericarps of HLW (Cluster III) were the most active against C. tropicalis and C. albicans 27 (a clinical strains resistant to FLZ and AMB), with the most predominant components being β-phellandrene (26.13%, 23.15%) and β-bisabolene (7.74%, 10.21%). Inhibition zone diameters were in the range of 11.1-22.1 mm. The percentages of the monoterpene β-phellandrene in the two clusters were all high. There were some differences of compositions between Cluster I and II, but they were all dominated by sesquiterpenes as the main components. The antifungal activities against C. glabrata were almost equal to Cluster IVand III with inhibition zone diameters 22.0-22.2 mm. However, the antifungal activities against other six Candida strains were relatively low, with inhibition zone diameters 6.5-13.0 mm. Antifungal effect of EOs of CHW was higher than that of HLW in both leaves and pericarps. Comprehensive analysis illustrates the importance of monoterpenes to antifungal activities and there were differences among different types of Candida strains. The results agree with those of Białon et al. [20] who suggested that the antifungal potential of lemon essential oils against Candida yeast strains was related to the high content of monoterpenoids and the type of Candida strains.
There are few studies reporting the antifungal activity of Clausena lansium extracts against infectious yeasts. XU et al. [21] reported that the extracts of pericarps of Clausena lansium using 95% alcohols had the significant inhibitory effect on C. albicans with an inhibition zone diameter of 16.8 mm by the filter paper disc diffusion method. However, the components of this extracts are not yet clear. In the present study, we obtained Clausena lansium EOs by hydrodistillation and determined the chemical compositions by GC-MS. In all the tested EOs of pericarps, β-phellandrene was determined to be present at the highest percentage which could be mainly responsible for the antifungal effects. β-Sesquiphellandrene and β-bisabolene were also important compositions which were possible to have antifungal activities. Perigo et al. [22] studied the chemical compositions and antibacterial activities of Piper species from distinct rainforest areas in Southeastern Brazil, and found that higher contents of β-phellandrene were positively correlated to wide spectrum antibacterial activity. Antibacterial activity of essential oils of Tripleurospermum disciforme was reported that the reason for higher antibacterial effect is the presence of β-farnesene and β-sesquiphellandrene [23]. The essential oils of Bocageopsis pleiosperma Maas rich in β-bisabolene were reported to have high antifungal activities [24]. Their results are consistent with ours. Taken together, we speculate that antifungal activities in our study may be attributed to C. lansium EOs of pericarps rich in β-phellandrene, β-sesquiphellandrene and β-bisabolene.
However, antifungal activity may not entirely depend on the main chemical components. Other chemical components in lower concentrations may also have antifungal activity or have synergistic effects with other components. Therefore, it is necessary to study antifungal activity of the component alone or in combination with others, in order to further elucidate the material basis of antifungal activity of C. lansium EOs against Candida spp. It may provide a basis for the development of C. lansium EOs as new antifungal agents with high efficiency, broad spectrum, low toxicity and low cost.

Samples and Chemicals
The leaves and fruits of Clausena lansium were collected from Haikou City in Hainan, China, in June 2018. Figure 3 shows the leaves and fruits of HLW (a) and CHW (b). All samples were identified by Professor Jianping Tian and were deposited in Public Research Center, Hainan Medical University (Hainan, China). Table 1

Hydrodistillation
The collected leaves of Clausena lansium were dried in shade. After removing pulps and seeds of fruits, pericarps were washed and then dried in an oven at 40-50 • C. The leaves and pericarps were milled into powder with a grinder. The EOs were extracted by hydrodistillation for 2.5-3.0 h in a Clevenger-type apparatus. The obtained EOs were stored at 4 • C in an air-tight container and dried using anhydrous sodium sulfate before being analyzed.

GC-MS Analysis
Samples were analyzed by the GC-MS EI method with column ZB-5MS. Helium was used as a carrier gas at a flow rate of 1.0 mL·min −1 in split mode (1:20). The column temperature was maintained at 60 • C for 5 min and then programmed to 120 • C at a heating rate of 10 • C·min −1 , further increased to 170 • C at a rate of 2 • C·min −1 , and finally increased to 210 • C at a rate of 10 • C·min −1 for remaining 10 min. Temperatures of both injector and connector were maintained at 270 • C. Operating parameters of MS were: EI mode at 70 eV with a mass scanning range of 50-500 amu and source temperature of 250 • C. C8-C40 n-alkanes were used as reference points in the calculation of relative retention indexes (RIs). The percentages of compositions were obtained from the electronic integration of peak areas. The identity of each compound was determined by comparing its RI to n-alkanes and the NIST library database (NIST08 and NIST08s). Percentage of composition was computed from peak areas without applying correction factors.

Candida Strains and Culture Media
The Candida strains include the standard reference strains (C. albicans ATCC 10231, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. tropicalis CMCC(F) c2f, and C. glabrata CMCC(F) c6e), and the clinical strains (C. albicans 53, C. albicans 27). The clinical strains were from oral mucosa of patients with oral candidiasis in the Hainan Wenchang General Hospital located in Wenchang, China. Two clinical isolates were previously identified based on ITS gene sequence analysis. The obtained yeast isolates were stored at −80 • C freezer until use. All strains were streaked onto plates containing the sabouraud dextrose agar (SDA) plate at 35 • C for 24 h. SDA plate composed of 2% (w/v) agar powder, 2% glucose, and 1% tryptone. A single colony was streaked again to ensure the viability and purity of the strains and the colonies were incubated at 35 • C for 24 h. The colonies with the diameter of about 1 mm were selected and microbial suspensions were prepared in a saline solution. The MH agar plate was used for testing antifungal susceptibilities by the filter paper disc diffusion method.

Probing the Antifungal Activity of the EO Sample by the Filter Paper Disc Diffusion Method
The antifungal activity of the EOs of C. lansium was first investigated using the filter paper disc diffusion method following the protocol described in CLSI M44-A2 [25]. Microbial suspensions were prepared in a saline solution and standardized to a turbidity equivalent to that of the tube No. 0.5 on the McFarland scale, corresponding to approximately 1-5 × 10 6 CFU·mL −1 . The antifungal drug FLZ (2.5 mg·mL −1 ) and AMB (1.0 mg·mL −1 ), which are commonly used to treat candidiasis, were used as positive controls. 10 µL of Clausena lansium EOs and positive controls was respectively added onto a piece of paper placed on the medium in a plate containing the fungal lawn. The plates were incubated at 35 • C for 24 to 48 h, respectively. The antifungal activity the samples was evaluated by measuring zones of inhibition of fungal growth surrounding the paper discs. The zones of inhibition were measured with antibiotic zone scale (Cecon-Brasil) in mm. All the experiments were carried out in triplicate. When the inhibition zone diameters of FLZ and AMB against Ca ATCC 10231 were within the prescribed range 28-39 mm and 20-27 mm, respectively, it is considered that the operation of this test was effective.