The influence of the chemical composition of essential oils of Clausena lansium seeds on the growth of Candida strains

Clausena lansium (Lour.) Skeels seeds have been shown to have diverse beneficial medical value due to their unique active components. This study analysed the composition of essential oils (EOs) of C. lansium seeds and investigated their potential antifungal effects against Candida strains. A total of forty-six components were identified in all samples by gas chromatography-mass spectrometry (GC–MS). The main components were sabinene, β-phellandrene and 4-terpineol. Thirteen EOs of C. lansium seeds were classified into three clusters based on their components. Cluster analysis showed that the difference between the tropics and subtropics was the greatest. These EOs and the three main chemicals showed different antifungal activities against five Candida species (C. albicans, C. tropicalis, C. glabrata, C. krusei and C. parapsilosis). The antifungal activity against C. glabrata and C. krusei was higher than that against other Candida strains. EOs of C. lansium seeds displayed noteworthy antifungal activity against both sensitive and fluconazole-resistant strains, with inhibition zone diameters in the range of 9.4–23.4 mm. Comprehensive analysis illustrated the importance of sabinene, β-phellandrene and 4-terpineol to antifungal activity, and there may be some synergistic effects with other components. These results represent the first report about the correlation between the chemical composition of EOs of C. lansium seeds and antifungal activity. Taken together, the results obtained provide scientific evidence for the traditional use of C. lansium seeds waste.


Results
Extraction results and chemical composition of Eos. C. lansium EOs extracted by hydrodistillation were white to light yellow in colour. The yields ranged from 0.43 to 0.91% (v/w). The colour and yield of EOs are listed in Table 1. There were some differences in the colour and yield of the eleven EOs, which may be related to the geographic source and cultivar, but it is uncertain which is the most important of these factors. Through the statistical analysis, there were no significant differences (P > 0.05). It is impossible to come to a conclusion from the colour and yield of EOs. To compare the compositional differences, each EO was subjected to gas chromatography-mass spectrometry (GC-MS) for analysis. Figure 1 shows the GC-MS chromatograms of EOs of C. lansium seed. The chemical composition and content results are shown in Table 2.
According to the GC-MS analysis, almost thirty components were identified in each EO, amounting to 97.10% to 98.64% of the total components. Differences existed in the components and contents of EOs of C. lansium seeds from different cultivars and different areas in Hainan. These components were monoterpene hydrocarbons, oxygen-containing monoterpenes, sesquiterpene hydrocarbons and other compounds. The main components were monoterpene hydrocarbons, such as sabinene and β-phellandrene, and oxygen-containing monoterpenes represented by 4-terpineol. The differences and relationships of these EOs cannot be easily seen from these complex data. Thus, a hierachical cluster analysis was conducted using SPSS (version 17.0) to investigate the similarities and differences among these extracts in EO profiles. The variables were the chemical components and contents of EOs of C. lansium seeds. The clustering results are shown in Fig. 2.
Samples were clustered into two clusters: QZ1, QZ2 and other samples. Geographically, QZ1 and QZ2 were both located at 110.02°E, 18.90°N, which were closest to the tropics. This result indicates that the difference Table 1. Extraction results of EOs of C. lansium seeds. a Values represent the means of three independent replicates ± SD, *P = 0.07. www.nature.com/scientificreports/ between the tropics and subtropics is the greatest. Based on the cluster analysis, three clusters were classified. Table 3 shows the variations in some important volatile components (%) of EOs of C. lansium seeds in the three clusters.
The dominant EOs in each of the three clusters are listed below: Cluster I: sabinene. HK3, HK4, HK1, HK2, QH1 and TC2 belonged to this cluster. Sabinene in this cluster had a high percentage of almost 50%. The percentages of β-phellandrene ranged from 13.39% to 16.19%. HK3, HK4, HK1 and HK2 were classified into a small cluster, which contained more 4-terpineol with relative percentages of 9.37%, 9.92%, 11.28% and 11.83%, respectively. This result is probably because the four places are geographically close (both are in Haikou city). QH1 and TC2, which are both Hainan local wampees, were classified into a small cluster.
Cluster II: sabinene and β-phellandrene. These samples were LG1, TC1, WC1, QZ3 and LG2. Although sabinene was the most predominant component in this cluster, with a relative percentage of approximately 40%, the β-phellandrene percentage was relatively higher than that in cluster I (more than 20%). LG1 and TC1, chicken heart wampees were classified into a small cluster. WC1, QZ3 and LG2, Hainan local wampees, were classified into a small cluster. Hence, we infer that although the cultivar was not a very clear cause of the different components and contents, some relationships were evident.
Cluster III: β-phellandrene. QZ1 and QZ2 were classified into this cluster. They clustered differently from other samples, possibly because QZ1 and QZ2 are closest to the tropics. The percentages of β-phellandrene were the highest in all samples, at 33.24% and 25.91%, respectively. They contained similar percentages of sabinene, with 27.70% in QZ1 and 33.24% in QZ2.
Antifungal activity. The antifungal activity of EOs of C. lansium seeds was examined using the filter paper disc diffusion method. Since sabinene, β-phellandrene and 4-terpineol are the main components in these EOs, the antifungal activities of three pure chemicals were also examined. As shown in Table 4, the EOs inhibited the growth of all seven strains tested in our study. The inhibition zone diameters of EOs, sabinene, β-phellandrene, 4-terpineol and FLZ were in the ranges of 9.4-23.4 mm, 13.7-25.7 mm, 15.6-22.1 mm, 26.7-35.6 mm, and 0-35.3 mm, respectively. Statistical analysis results showed that there was significant difference between FLZ and EOs of C. lansium seeds (P < 0.05). In all samples and chemicals, 4-terpineol exhibited the greatest antifungal effect against all strains. Table 4 shows that there were differences in the antifungal activity efficacy among different types of strains. The antifungal activity of EOs against C. glabrata and C. krusei was higher than that of the other five Candida strains and there were signifcant differences (P < 0.05).
For C. albicans, the antifungal activity efficacies of both EOs and the constituent chemicals against the FLZresistant strain (C. albicans 27) were better than those against the FLZ-sensitive strain (C. albicans ATCC 10231). The efficacy order of the three pure chemicals was as follows: 4-terpineol > β-phellandrene > sabinene. Among the EOs, HK1, HK2, HK3 and HK4 in cluster I exhibited the greatest antifungal effects and were rich in 4-terpineol.   The antifungal activity of QZ3 in cluster II was also higher, which may be because it contained higher contents of 4-terpineol and β-phellandrene. Cluster III showed the weakest effect against C. albicans. The EOs C. lansium seeds exhibited the greatest antifungal effect against C. krusei, with inhibition zone diameters of 17.7-23.4 mm. The efficacy order was 4-terpineol > sabinene > β-phellandrene > cluster I > cluster II > cluster III > FLZ. In EOs, cluster I exhibited the greatest antifungal effect, which may be due to the high contents of 4-terpineol and sabinene. The percentage of 4-terpineol and sabinene in cluster III were relatively lower. Therefore, even if the percentage of β-phellandrene was the highest, the antifungal effect of cluster III was still the worst against C. krusei.
For C. glabrata, the inhibition zone diameters of FLZ, sabinene, β-phellandrene, and 4-terpineol were 23.0 mm, 22.3 mm, 22.1 mm, and 28.1 mm, respectively. Statistical analysis results showed that the antifungal activity efficacies of sabinene and β-phellandrene were similar (P = 0.687). The antifungal activity of 4-terpineol   Considering their compositions, we infer that these differences may be related to the contents of monoterpenes containing oxygen: the higher the contents of monoterpenes containing oxygen, the higher the antifungal effect. The antifungal activity efficacy order was 4-terpineol > β-phellandrene > sabinene against C. parapsilosis, with inhibition zone diameters of 33.6 mm, 15.6 mm, and 15.0 mm, respectively. Among the EOs of C. lansium seeds, the antifungal activities of clusters I and II were similar, and cluster III showed the weakest effect against C. parapsilosis.
For C. tropicalis, the antifungal activity of 4-terpineol was also greatest in these samples and chemicals. The inhibition zone diameters of EOs were in the range of 12.1-13.6 mm against the sensitive strain (C. tropicalis CMCC(F) c2f.) and 9.5-11.4 mm against the resistant strains (C. tropicalis 13). There was no significant difference in the antifungal effects among the three clusters (P > 0.05).

Discussion
In a previous study, EOs of C. lansium from Changjiang County, Hainan, China were obtained by hydrodistillation and were analysed by GC-MS 24 . The oil yield was 0.5% (v/w). Twenty-six components were identified, and the main components identified in the seed EO were phellandrene (54.8%), limonene (23.6%) and p-menth-1-en-4-ol (7.5%). Using the same hydrodistillation method, fifty-three components were identified in both fresh and dried fruit samples from Thailand 25 . The main components were sabinene (33.68-66.73%), α-pinene (9.57-13.35%) and 1-phellandrene (5.77-10.76%). Comparison revealed that the components and percentages were different from different areas. The reasons for the differences may be due to the fruit containing not only seeds but also pulp and pericarp.
In the present study, the differences in the compositions of EOs of C. lansium seeds from different areas and cultivars were analysed and compared for the first time. Cluster analysis results showed that the differences in the components were most significantly contributed by different harvest areas, followed by different cultivar. The main components were sabinene, β-phellandrene, and 4-terpineol.
Few studies have reported the antifungal activity of C. lansium extracts against Candida strains. Xu et al. 28 reported that extracts of pericarps of C. lansium using 95% alcohols had a 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 these extracts have not yet been elucidated. In our previous study 29 , we determined the chemical compositions of C. lansium EOs of pericarps and leaves by GC-MS. Their potential antifungal effects against five Candida species (C. albicans, C. tropicalis, C. glabrata, C. krusei and C. parapsilosis) were evaluated using the disc diffusion method. C. lansium EOs of pericarps displayed higher antifungal activities than EOs of leaves. Higher contents of β-phellandrene, β-sesquiphellandrene and β-bisabolene in EOs of pericarps were likely responsible for the high antifungal activity.
In previous reports, Abdelli et al. 30 studied the chemical compositions and antibacterial activities of the leaves and berries of Algerian Juniperus phoenicea EOs and found that higher contents of β-phellandrene and α-pinene were positively correlated with the antibacterial activity. A study of the antibacterial activity of Piper species EOs from distinct rainforest areas in southeastern Brazil showed that was β-phellandrene was responsible for the higher antibacterial effects 31 . Melaleuca alternifolia (tea tree) EO was particularly rich in 4-terpineol (40.44%) Table 4. Antifungal activity of EOs of C. lansium seeds and three pure chemicals. a Values represent the means of three independent replicates ± SD; concentrations: EOs:10 μL/disc; b FLZ: Fluconazole, 25 μg/disc. In this study, the EOs of C. lansium seeds showed significant activity against all the strains. There were differences among the different types of Candida strains. Notably, they were active towards FLZ-resistant strains (C. albicans 27, C. tropicalis 13). Moreover, the antifungal activity of EOs against C. glabrata and C. krusei was higher than that of the other five Candida strains, and C. glabrata and C. krusei were the main Candida spp. that might have fluconazole resistance (with lower inhibition zone diameters). Therefore, it comes to a conclusion that the natural EOs of C. lansium seeds might be more effective against FLZ-resistant strains. Comprehensive analysis illustrated the importance of sabinene, β-phellandrene and 4-terpineol to antifungal activity, and there might be synergism between the pure chemicals. In addition, antifungal activity may not entirely depend on the main chemical components. Other chemical components present at lower concentrations may also have antifungal activity or may have synergistic effects with other components. More detailed in vivo studies are still necessary.
In summary, this study identified a total of forty-six components in the EOs of C. lansium (Lour.) Skeels seeds by GC-MS. The main components were sabinene, β-phellandrene, and 4-terpineol. Cluster analysis results showed that the differences in the components were most significantly contributed by different harvest area, followed by different cultivar. EOs of C. lansium seeds showed significant antifungal activity against all seven tested Candida strains. Through comprehensive comparison and analysis of chemical composition and antifungal activity, higher contents of sabinene, β-phellandrene and 4-terpineol may be positively correlated with antifungal activity. There were differences in the antifungal activity efficacies among the different types of strains. Notably, EOs of C. lansium seeds were active towards FLZ-resistant strains. This study may provide a basis for the development of EOs C. lansium seeds as new antifungal agents with high efficiency, broad-spectrum activity, low toxicity and low cost. It may also provide new solutions for clinical infections caused by drug resistance problems.

Materials and methods
Plant materials and chemicals. The fruits of C. lansium were collected from Hainan, China, in May and June 2020. The details of the source, location, and cultivar of C. lansium samples employed in this study are listed in Table 5. Experimental research and field studies on plants were carried out in accordance with relevant institutional, national, and international guidelines and legislation. The permissions for collection of seed specimens were approved by the planters.  Table 5. Source, location, and cultivar of C. lansium samples. a C. lansiumwith globose fruits is named Hainan local wampee (HLW): the berry fruit is approximately 1.5-2.5 cm in diameter. C. lansium with broadly ovoid fruits is named chicken heart wampee (CHW), with berries approximately 1.5-2.5 × 2.5-3.5 cm in diameter.  (1:20). The column temperature was maintained at 60 °C for 3 min and then programmed to 140 °C at a heating rate of 5 °C·min −1 , where it was held for 2 min, after which it was increased to 210 °C at a rate of 10 °C·min −1 for the remaining 7 min. The temperatures of both the injector and the connector were maintained at 250 °C. The operating parameters of MS were EI mode at 70 eV with a mass scanning range of 50-500 amu and a source temperature of 280 °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). The percentage of composition was computed from peak areas without applying correction factors 29 .
Paper disc diffusion method. The antifungal activity of the EOs of C. lansium seeds was first investigated using the paper disc diffusion method 33 . All strains were streaked onto the SDA plates. A single colony was streaked again to ensure the viability and purity of the strains. Then about 5 single colonies were picked up into 5 mL saline solution. Microbial suspensions of 0.5 McFarland standard were prepared equivalent to approximately 10 6 CFU·mL −1 yeast and evenly dispensed onto the MHA plates to form the microbe lawn. Pieces of papers were placed on the medium with sterile tweezers and keep the space between the paper more than 24 mm. The diameters of MHA plates and the paper were 120 mm and 6 mm, respectively. The antifungal drug FLZ (2.5 mg·mL −1 ) was used as a positive control. Ten microlitres of EOs of C. lansium seeds, the constituent chemicals (sabinene, β-phellandrene and 4-terpineol) and positive control were added onto the paper. The plates were incubated at 30 °C for 24 to 48 h. The antifungal activity of the samples was evaluated by measuring zones of inhibition of microbial growth surrounding the paper discs. The zones of inhibition corresponding to diameters were measured with an antibiotic zone scale (Cecon-Brasil) in mm. All the experiments were carried out in triplicate.
Statistical analysis. Data analysis was performed using SPSS software (version 17.0). Data represent the mean ± standard deviation (SD) from at least 3 distinct experiments. Statistical differences were determined using a 2-sample equal variance Student's t test. Statistical significance was defined as *P < 0.05. GC-MS data and chromatograms were processed with Shimadzu GC-MS Solution software. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.