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Chemical Composition and Antifungal, Anti-Inflammatory, Antiviral, and Larvicidal Activities of the Essential Oils of Zanthoxylum acanthopodium DC. from China and Myanmar

Zanthoxylum acanthopodium DC. is a widely used traditional medicinal plant to treat fever, flu, stomachache, traumatic injury, and mosquito bite in tropical and subtropical Asia. This study aimed to investigate the antifungal, anti-inflammatory, antiviral, and larvicidal activities of its fruit essential oil. The essential oil sample from China (EOZC) was mainly composed of limonene (29.78%) and β-myrcene (26.65%), while the sample from Myanmar (EOZM) was dominated by Terpinen-4-ol (43.35%). Both essential oils showed antifungal activity, with 90% minimum inhibitory concentration (MIC90) values ranging from 26.3 to 499 μg/mL. By obviously inhibiting nitric oxide (NO) in RAW 264.7 cells, EOZC (IC50, 16 μg/mL) showed comparable anti-inflammatory activity to the positive control L-NMMA (IC50, 12.2 μg/mL). EOZM showed significant antiviral activity against the dengue virus with an IC50 value of 13 μg/mL. Additionally, both EOZC and EOZM demonstrated dose-dependent larvicidal activity against Aedes albopictus, with LC50 and LC90 values ranging from 45.8 to 144.0 μg/mL. Our results contribute a theoretical foundation for the further application of Zanthoxylum acanthopodium DC. as an antifungal and anti-inflammatory ingredient in the pharmaceutical industry and further indicate that it has the potential to be developed as a new source of natural and eco-friendly medicine for the prevention and treatment of dengue virus.


Introduction
The genus Zanthoxylum (Rutaceae), distributed mainly in tropical and subtropical regions of Asia, Africa, Oceania, and North America, consists of approximately 250 species, of which 39 species and 14 varieties were found in China [1]. Fruits from this genus are commonly used as spices, antiseptics, and insecticides worldwide. Earlier studies have shown that Zanthoxylum plants have antioxidant, antibacterial, insecticidal, immunomodulatory, anti-malignant cell proliferation, anti-inflammatory, and analgesic properties [2][3][4][5][6][7]. Zanthoxylum acanthopodium DC., mainly distributed in China, Myanmar, India, and Nepal, is used as a medicine to cure many diseases and as a spice in cooking by local people [1]. Ethnobotanical research revealed that it was called "ge ma ga", "mo zi la", or "za bu ga biu" by local Dai, Hani, and Yao people, and its roots and fruit were used to treat cold, stomach pain, bruising and injury in Yunnan and Guizhou, China [8][9][10][11][12][13]. In Myanmar, local Chin people name it "Dan bung hling" and use its crushed fruits to cure toothache, mosquito, and leech bites, or boil its fruits and leaves and then apply them as warm baths [14]. In northern Sumatra, Indonesia, it was named "andaliman" and used as a spice to prepare traditional fish dishes [15]. In India, it is also used as a spice and wild vegetable and its fruit is rich in fatty acids, and the leaves have high contents of protein, potassium, calcium, and phenolic compounds [16][17][18]. Its methanol, ether, and ethanol extracts showed antibacterial, antioxidant, and anti-inflammatory activities [19]. Its stem essential oil from Malaysia expressed larvicidal activity against Aedes aegypti larvae [20]. Ethanol extraction of its fruits presented anticancer activities [21]. It was reported that the essential oil extracted from the aerial part of Z. acanthopodium collected in May was rich in estragole (15.46%) and eucalyptol (10.94%) and showed larvicidal activity against the malaria mosquitoes Anopheles anthropophagus and Anopheles sinensis [22]. In addition to the explorations mentioned above, few studies have focused on the chemical composition and biological activity of the fruit essential oils of Z. acanthopodium collected from different countries.
Dengue fever is a predominantly viral disease by Aedes albopictus and Aedes aegypti. In 2019, World Health Orgnization (WHO) listed dengue fever as one of the top 10 global public health threats, noting that it can cause the death of 20% of severe cases [23]. According to the WHO report, the global incidence of dengue fever has increased eight-fold in the past two decades and there are approximately 1-4 million infection cases every year [24]. Nearly 70% of those at high risk of being infected are in the Asia-Pacific region [25]. Moreover, global climate change could exacerbate the crisis, and dengue outbreaks are likely to occur more frequently and spread further, even to higher elevations [26]. Recent research suggests that urbanization will drive dengue's global spread and estimates that by 2080, dengue fever will threaten 6 billion people in 197 countries or 60 percent of the world's population [27]. The global outbreak of dengue fever has strained public immunization systems and caused substantial economic losses. There are four different subtypes of dengue fever. A globally approved dengue vaccine is still under development, and there is no specific treatment for dengue fever [28]. In addition, studies have shown that residents in China and Southeast Asia also face potential threats from the Zika virus [29].
Recent studies found that essential oils demonstrated anti-mosquito and mosquito repellent effects [30][31][32][33]. Essential oil is a mixture of several compounds collected from aromatic plants and has been frequently used in pharmaceuticals, preservatives, households, and cosmetic products since ancient times [34]. Compared with synthetic insecticides, essential oils are likely to cause less pollution to the environment and less toxic to humans [35]. The mechanism by which essential oil is lethal to mosquitoes is complicated. Thus, it is difficult for mosquitoes to develop resistance to essential oils [36,37]. Without an effective vaccine, the best way to control dengue fever is to limit the population of mosquitoes [28,38,39]. Therefore, it is of great significance to develop eco-friendly insecticides from essential oils to eliminate mosquitoes.
To verify its traditional use and provide a scientific basis for the sustainable use and commercial development of Z. acanthopodium in the pharmaceutical field, we extracted its essential oils from fruits collected from China and Myanmar and investigated their antifungal, anti-inflammatory, antiviral, and larvicidal activities.

Essential Oil Composition
The essential oil yields were 2.62% and 4% (w/w) on a dry weight basis for Z. acanthopodium fruits from China (EOZC) and the sample from Myanmar (EOZM) respectively. GC/MS analyzed both essential oils, and their chemical compositions are shown in Table 1. There were 36 chemical components identified in the two samples, and the identification rates of EOZC and EOZM were 96.96% and 98.17%, respectively. Both essential oils had 17 chemical components in common, while the content of their major components varied widely. Specifically, the EOZC was mainly composed of limonene (29.78%) and β-myrcene (26.65%), while the EOZM was dominated by terpinen-4-ol (43.35%). The steam distillation extracted essential oils of Z. acanthopodium fruits from Medan, Indonesia, had 29 chemical compositions with geranyl acetate (23.18%), citronella (11.23%), and β-citronelol (10.64%) as the main chemical components and had different influences in terms of locomotor activity dependent on doses given to mice [40]. The n-hexane extracted essential oils of Z. acanthopodium fruits from Sumatra Island, Indonesia, had 32 chemical compositions, with carveol (47.70%) and myrtenyl acetate (12.55%) as the dominant components and expressed antidiabetic activity [41]. Steam distillation extracted essential oils of Z. acanthopodium fruits from Meghalaya state, India, had 21 chemical compositions, with eucalyptol (36.56%), limonene (16.90%), and δ-3-carene (13.53%) as the primary components and showed promising antibacterial activity against Staphylococcus aureus [42]. Thus, we found that the essential oils of Z. acanthopodium fruits, collected from different geographical sites and extracted by different methods, had significant differences in their major components and biological activities, indicating that the exploration of the essential oil composition of the same species collected from different regions remains both very important and enlightening.

Antifungal Activity
Two essential oils displayed significant activity against four tested fungi (Table 2). EOZC showed the best antifungal activity against Epidermophyton floccosum, with 90% minimun inhibitory concentration (MIC 90 ) values of 26.3 µg/mL. Generally, EOZC exhibited better antifungal activity than EOZM. The antifungal activity of EOZC may be due to its higher content of limonene, which has shown significant antimicrobial activity [43]. The results showed that essential oils had sound inhibitory effects on the four tested fungi, which could cause skin infections and itching, and may partly explain why local people like to boil the fruit of Z. acanthopodium for bathing.

Anti-Inflammatory Activity
Nitric oxide (NO) has an essential role in inflammation, tumors, and the cardiovascular system [44]. Therefore, the inhibition of NO production is a direct indicator of the compound's anti-inflammatory activity. At a concentration without an effect on the viability of the cells (Figures 1 and 2), the results showed that the two essential oils had significant NO inhibitory activity (Table 3). EOZC showed better anti-inflammatory activity with IC 50 values of 16 µg/mL, similar to the positive control drug L-NMMA, with IC 50 values of 12.2 µg/mL. The anti-inflammatory activity of the two essential oils might be attributed to their high contents of limonene, β-myrcene, and terpinen-4-ol, which all demonstrated apparent anti-inflammatory activity [45][46][47]. The inhibitin of EOZM on LPS-induced NO production in RAW 264.7 cells. Results were presented as mean ± standard deviation (SD) of three independent tests, **** p < 0.0001. Table 3. Anti-inflammatory activity of essential oils from Z. acanthopodium fruits.

Larvicidal Activity
The essential oil from Myanmar or China expressed obvious larvicidal activity against Aedes albopictus larvae in a dose-dependent manner at concentrations of 18-150 μg/mL (Figure 3). The LC50 values of EOZC and EOZM were 45.8 μg/mL and 87 μg/mL, respectively. The LC90 values of EOZC and EOZM were 59.4 μg/mL and 144.0 μg/mL, respectively. Their larvicidal activities were lower than the positive control chlorpyrifos, with LC50/LC90 values of 0.0037 and 0.0064 μg/mL, respectively.
The variation in the main components of essential oils resulted in different lethal rates for Aedes albopictus larvae. On the one hand, limonene, β-myrcene, and terpinen-4-ol have been reported to have larvicidal activity against various mosquitoes, including Aedes albopictus. On the other hand, limonene and β-myrcene showed better larvicidal activity than terpinen-4-ol in previous reports [48]. Thus, EOZC, which had much higher contents of limonene and β-myrcene, showed more vigorous larvicidal activity than EOZM. Aedes albopictus is one of the main vectors for transmitting dengue fever and the NO is one of the critical determinants in infection and inflammation, so the significant NO inhibition effect supports the traditional use of Z. acanthopodium fruits as a remedy for inflammation-related diseases, such as toothache and stomachache, and swelling and pain caused by injury or mosquito and leech bites.

Larvicidal Activity
The essential oil from Myanmar or China expressed obvious larvicidal activity against Aedes albopictus larvae in a dose-dependent manner at concentrations of 18-150 µg/mL Zika virus [49]. Due to the lack of effective vaccines for dengue fever, the primary method to control or prevent dengue virus transmission is to combat mosquito vectors [24]. Our result could be a positive example of the traditional usage of Zanthoxylum plants as insecticides. Furthermore, EOZC and EOZM showed good larvicidal activity against Aedes albopictus larvae at relatively low concentrations, indicating that Z. acanthopodium fruits have the potential to be developed as a novel and natural agent for the control of mosquitoes.

Antiviral Activity
Two essential oil samples were active against the dengue virus, while only EOZM showed an inhibitory effect against the Zika virus with an IC50 value of 94 μg/mL (Table  4). EOZM showed significant inhibition of DENV with an IC50 value of 13 μg/mL, which was approximately 5-fold lower than that of the positive control (NIT008). The selective index (SI) of EOZM against DENV was 14, indicating a significant safety range [50]. Although scientists in many countries have made many efforts and discovered a variety of plant extracts that have inhibitory ability against the dengue virus [51,52], specific medicines to cure dengue fever are still being explored. Our findings might provide a new natural and eco-friendly agent for developing highly effective medicine to inhibit the dengue virus. As the mechanism for essential oils' larvicidal and antiviral activity is detailed, further studies are needed to apply this plant better. The variation in the main components of essential oils resulted in different lethal rates for Aedes albopictus larvae. On the one hand, limonene, β-myrcene, and terpinen-4-ol have been reported to have larvicidal activity against various mosquitoes, including Aedes albopictus. On the other hand, limonene and β-myrcene showed better larvicidal activity than terpinen-4-ol in previous reports [48]. Thus, EOZC, which had much higher contents of limonene and β-myrcene, showed more vigorous larvicidal activity than EOZM. Aedes albopictus is one of the main vectors for transmitting dengue fever and the Zika virus [49]. Due to the lack of effective vaccines for dengue fever, the primary method to control or prevent dengue virus transmission is to combat mosquito vectors [24]. Our result could be a positive example of the traditional usage of Zanthoxylum plants as insecticides. Furthermore, EOZC and EOZM showed good larvicidal activity against Aedes albopictus larvae at relatively low concentrations, indicating that Z. acanthopodium fruits have the potential to be developed as a novel and natural agent for the control of mosquitoes.

Antiviral Activity
Two essential oil samples were active against the dengue virus, while only EOZM showed an inhibitory effect against the Zika virus with an IC 50 value of 94 µg/mL (Table 4). EOZM showed significant inhibition of DENV with an IC 50 value of 13 µg/mL, which was approximately 5-fold lower than that of the positive control (NIT008). The selective index (SI) of EOZM against DENV was 14, indicating a significant safety range [50]. Although scientists in many countries have made many efforts and discovered a variety of plant extracts that have inhibitory ability against the dengue virus [51,52], specific medicines to cure dengue fever are still being explored. Our findings might provide a new natural and eco-friendly agent for developing highly effective medicine to inhibit the dengue virus. As the mechanism for essential oils' larvicidal and antiviral activity is detailed, further studies are needed to apply this plant better.

Chemicals and Reagents
Dimethylsulfoxide (DMSO), fetal bovine serum (FBS), penicillin-streptomycin, Lglutamine, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Thermo Scientific (Logan, UT, USA). CellTiter 96 ® AQueous One Solution Reagent for the MTS assay and the Griess reagent system for NO measurement were purchased from Promega Corporation and purchased from Promega Corporation (Madison, WI, USA). Ceftazidime was purchased from CAS mart. Penicillin G Na was purchased from Biosharp company. Terbinafine hydrochloride and amphotericin B were purchased from Sigma Company. Candida albicans (ATCC 10231) was purchased from the American Microbiologics Company. Epidermophyton floccosum (CBS 566.94), Trichophyton rubrum (ATCC 4438), and Microsporum gypseum (CBS 118893) were purchased from the China Center for Medical Culture Collection (CMCC). Standard Mueller-Hinton agar and broth (MHA and MHB) and Sabouraud agar and broth (SA and SB) were purchased from Huankai Microbial Technology Company (Guangdong, China). All reagents were analytical standards.

Plant Materials and Essential Oil Extraction
With the help of local villagers, Z. acanthopodium fruit samples were collected from Nat ma Taung National Park, Chin State, Myanmar, and Ailao Mountain, Yunnan, China, in September 2019 and were identified by Mr. Shi-shun Zhou from Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (no. 135658) was deposited in the herbarium (HITBC). The essential oils were extracted following the reproted procedure with minor modification [34]. Briefly, the samples (100 g), which were dried in shade and ground with a laboratory mill, were extracted with 2000 mL boiling water in a Liens-Nickerson simultaneous steam distillation continuous extraction (SDE) with 30 mL ether as the solvent for 3 h. The essential oil and solvent were collected together, and then ether was removed using a rotary evaporator to yield the essential oil at room temperature and room pressure. The collected essential oils were stored at −18 • C in the dark for further testing.

Analysis of Chemical Composition
Essential oils were characterized and quantified by GC-MS analysis on an Agilent 7890 gas chromatograph (GC) instrument equipped with an Agilent 5975 mass spectrometer (MS) and an HP-5MS capillary column (30 m × 250 µm × 0.25 µm, 5% Pheny Methyl Silox). The loading gas was helium at a 1.0 mL/min current flow rate. The temperature program for the oven is as follows: 40 • C for 1 min, then 40 • C to 150 • C at 3 • C/min, and increased to 250 • C at 10 • C/min, then hold for 10 min, the total running time was 57.667 min. The components were identified by comparing calculated experiment GC retention indices, which were determined concerning homologous series of n-alkanes C7-C30 under identical experimental conditions, with the GC retention indices reported in NIST Standard Reference Database (NIST Chemistry Web Book, 2014), by matching their mass spectra with those recorded in the NIST 08 database (National Institute of Standards and Technology, Gaithersburg, MD, USA) and mass spectra with published data. The relative percentage composition of individual components of the essential oils was calculated according to the GC peak area by normalization without the use of correction factors.

Antifungal Test
Candida albicans, Microsporum gypseum, Epidermophyton floccosum, and Trichophyton rubrum were tested for susceptibility. Candida albicans was prepared to a final concentration of 1 × 10 5 CFU/mL, and the others were prepared to a final concentration of 5 × 10 5 CFU/mL. They were incubated in different concentrations of both essential oils in 96-well plates. Candida albicans were incubated at 37 • C for 24 h, and the others were incubated at 25 • C for 5 day. Amphotericin B was the positive control drug for Candida albicans, and terbinafine hydrochloride was the positive control drug for other types of fungi. Media without antimicrobial agents were used as negative controls, and sterile culture medium without drugs and fungi was used as a blank control. The absorbance was measured at 625 nm using a microplate reader. The MIC 90 was calculated by Reed & Muench [53].

Cell Culture
The murine macrophage cell line RAW 264.7 was obtained from the Kunming Institute of Zoology, Chinese Academy of Sciences (KCB200603YJ), maintained in DMEM containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine at 37 • C in a 5% CO 2 incubator and incubated every 2 day.

Cell Viability Assay
As previously reported, cell viability was evaluated by MTS assay [54]. In the MTS assay, 100 µL cell suspensions (1.5 × 10 5 cells/well) were cultivated in 96-cell plates for 18 h, as described. Then, the cells were conditioned with different concentrations of essential oils for 30 min and incubated for an additional 24 h with 1 µg/mL LPS. Finally, 20 µL of CellTiter 96 ® AQueous One Solution Reagent, prepared by MTS in the presence of phenazine ethosulfate (PES), was added to each well and incubated for 1 h at 37 • C in a 5% CO 2 incubator. A multifunctional microplate reader measured each well's absorbance at 490 nm. The results are expressed as a percentage of MTS production by control cells maintained in a culture medium. A range of concentrations with no effect on cell viability was selected to test the inhibitory activity against NO.

Measurement of NO Production
NO production in LPS-stimulated RAW 264.7 cells was measured by nitrite accumulation in the culture supernatant using the Griess reagent system according to the manufacturer's directions. Cells were inoculated at 1.5 × 10 5 cells/well in 96-well culture plates and then incubated with medium (control) for 18 h. The cells were pretreated with different concentrations of essential oils for 30 min, stimulated with 1 µg/mL LPS, and cultured for 24 h. Briefly, 50 µL of culture supernatants were collected and mixed with the Griess reagent system and then incubated in the shade for 10 min. A multifunctional microplate reader measured each well's absorbance at 570 nm. L-NMMA, a well-known nitric oxide synthase (NOS) inhibitor, was used as a positive control, and untreated cells incubated in culture medium were employed as blank control. The nitrite concentration was determined from a sodium nitrite standard curve.

Larvicidal Activity
The eggs of Aedes albopictus were provided by the Centers for Disease Control of Yunnan Province, China. The mosquitoes were reared at 27 ± 2 • C and 70 ± 10% relative humidity with a 14 h light and 10 h dark photoperiod. Both essential oils were tested for larvicidal activity based on the recommendations of the WHO with slight modifications [55]. Briefly, 25 mosquito larvae of 4th instar were introduced with 99 mL dechlorinated water into the beaker. The essential oils were dissolved in 1 mL ethanol and prepared at different concentrations. The chemical larvicide chlorpyrifos was used as a positive control, and solvent ethanol served as a blank control. During the test, no food was supplied to the larvae. All the tests were replicated four times, and larval mortality was calculated after the 24 h exposure period.

Antiviral Activity
Antiviral activity was evaluated for Zika and dengue viruses in BHK cells. BHK cells were seeded into 96-well culture plates at 5 × 10 3 cells/well and cultured overnight. Both essential oils were gradient diluted using 2% DMEM and added to a 96-well culture plate. Then, BHK cells were inoculated with 100 TCID 50 of Zika or dengue virus. In this assay, NITD008 was used as a positive control. At 8 days post-infection (dpi), cell viability was evaluated by CellTiter-Glo ® Luminescent Cell Viability Assay following the manufacturer's instructions. The results were expressed as the 50% inhibitory concentration (IC 50 ) and 50% cytotoxic concentration (CC 50 ).

Statistical Analysis
All experiments were performed at least three times and expressed as the mean values ± standard deviation (SD). The 50% inhibition concentration (IC 50 ) was calculated by probit regression analysis using SPSS 17.0 for Windows (SPSS Inc. Chicago, IL, USA). One-way ANOVA with Dunnett's multiple comparison test and least significant difference (LSD) test were performed using SPSS 17.0 for statistical evaluation. Differences were accepted as significant at p < 0.05.

Conclusions
The results showed that the essential oils of Z. acanthopodium fruits collected from China and Myanmar were rich in limonene, β-myrcene, and terpinen-4-ol, had suitable inhibitory activities against four kinds of human epidermal fungi, and showed prominent anti-inflammatory activities by significantly inhibiting NO content in RAW264.7 cells. Our results verified its traditional medicinal value and supported its further development in the pharmaceutical industry. For the first time, our results demonstrated that Z. acanthopodium