2.1 Plant materials

The roots, stems, leaves and fruits of I. lanceolatum were collected in August 2022 in a village near Guiyang (Latitude 26˚41΄5˝N, longitude 106˚30΄41˝E, altitude 1241 m a.s.l.) and naturally dried at room temperature in the shade for about three weeks before extraction of essential oils. The moisture contents in the roots, stems, leaves and fruits of I. lanceolatum were determined as 7.04 ± 0.15 %, 6.58 ± 0.09 %, 5.42 ± 0.12 % and 6.33 ± 0.08 %, respectively. Associate Prof. Yazhou Zhang from Guizhou University of Traditional Chinese Medicine made the identification of this species according to the Flora of China. Voucher specimens (MC015) of this plant were placed at Guizhou Institute of Technology.

2.2 Reagents

DPPH (2,2-diphenyl-1-picrylhydrazyl) and a mixed standard of n-alkanes C7-C30 were purchased from Sigma-Aldrich. ABTS [2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic Acid)], TPTZ(2,4,6-Tripyridyl-s-triazine) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were from Shanghai Yuanye Biotechnology company. Amphotericin B, gentamycin sulfate, tyrosinase from mushroom, levodopa, kojic acid, p-nitrophenyl-α-D-glucopyranoside (pNGP), acarbose, α-glucosidase from yeast, acetylthiocholine iodide (ATCI), butyryl thiocholine iodide (BTCI), 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB), acetylcholinesterase (AChE) from electric eel, butyrylcholinesterase (BChE) from horse serum and galanthamine were purchased from Shanghai Yuanye. For identification and activity evaluation of nine compounds in the root EO, terpinen-4-ol (95 %), linalool (98 %), α-terpineol (96 %) and borneol (98 %) were from Shanghai Yuanye; eucalyptol (99 %), camphene (96 %) and camphor (96 %) were from Shanghai Rhawn; estragole (98 %) was from Shanghai Aladdin; asaricin (98 %) was from Sichuan Weikeqi Biotech.

2.3 Extraction of essential oils

The dried roots, stems and leaves were firstly chopped into small pieces and then ground using a grinder. The dried fruits were directly ground. The powder of each organ was divided into three parts. The hydrodistillations of dry roots (300 g), stems (400 g), leaves (400 g) and fruits (150 g) of I. lanceolatum were performed in Clevenger-type apparatus for 5 h, according to the method listed in the Chinese Pharmacopoeia. The experiments were carried out in triplicates for each organ of I. lanceolatum to obtain three essential oils. 12 essential oils obtained in total were stored at −20 ℃ for further GC–MS and GC-FID analysis. For biological activity evaluation, three essential oils obtained from each organ were combined together to obtain one essential oil for each organ.

2.4 GC–MS and GC-FID analysis

The 12 essential oils from different parts of I. lanceolatum were diluted in n-hexane (1:50, v/v) and analyzed using a TG-5MS capillary column (30 m × 0.25 mm × 0.25 μm) on GC–MS (Shimadzu, TQ8040 NX) and GC (Thermo Scientific, Trace 1310) systems, according to the previous method (Zhao et al., 2021) with slight modifications. For GC–MS analysis, column temperature was initially maintained at 50 °C for 3 min, and raised to 140 ℃ at 3 ℃/min and kept for 2 min; then heated to 190 ℃ at 2 ℃/min and maintained for 2 min; finally raised up to 220 ℃ at 10 ℃/min. The carrier gas was helium with a flow rate of 1 mL/min and the injection volume was 1 μL in split mode (1:20). The temperature of injection port, interface and ion source was all set at 250 °C. The mass range was 45–550. A mixed standard of n-alkanes (C7-C30) was analyzed under the same conditions. Identification of each component in the I. lanceolatum EOs was based on the comparison of its calculated retention index and experimental mass spectra with those published in the literature (Adams, 2007) and NIST libraries. The quantification of essential oil components was carried out on GC with FID detector. The column and temperature programming conditions were the same as the GC–MS system. Nitrogen was used as the carrier gas with a flow rate of 1 mL/min. 1 μL of sample solution was injected with the split ratio as 1:20. The temperature of injection port and detector was 250 °C. The identification of asaricin, borneol, camphor, eucalyptol, camphene, estragole, terpinen-4-ol, linalool and α-terpineol were further confirmed by injection of authentic compounds on GC. The relative percentage (%) of each component in essential oils was based on peak area normalization of GC chromatogram.

2.5 Antioxidant activity assays

Three different methods, including DPPH radical scavenging assay, ABTS cation radical scavenging assay and ferric reducing/antioxidant power (FRAP) assay, were employed to evaluate the antioxidant activities of I. lanceolatum EOs. The experiments were conducted according to the previously reported method (Zhao et al., 2021). In brief, essential oil solution of 20 mg/mL in methanol was firstly prepared. For DPPH assay, 6 mL of DPPH in methanol (6 × 10^-5 mol/L) was mixed with 150 μL of essential oil solution. The mixed solution was put in the darkness for 30 min and then measured at 515 nm on the UV–vis spectrophotometer (UH5300, HITACHI). For ABTS assay, 50 μL of essential oil solution was mixed with 4 mL of diluted ABTS solution. After 6 min of reaction, the absorbance of mixture was recorded at 734 nm. For FRAP assay, 150 µL of essential oil solution was mixed with 450 µL of methanol, and then 4.5 mL of FRAP reagent. The mixture was incubated for 30 min at 37 ℃ and the absorbance was recorded at 593 nm. The antioxidant activities of nine major compounds in the root EO, including asaricin, borneol, camphor, eucalyptol, camphene, estragole, terpinen-4-ol, linalool and α-terpineol, were further evaluated using FRAP assay. The methanol solutions of Trolox with known concentrations ranging from 25 to 250 mg/L were used for calibration. The antioxidant activity was expressed in mg Trolox equivalent (TE)/g of EO. BHT (butylated hydroxytoluene), a synthetic antioxidant, was used as positive control.

2.6 Antimicrobial activity assays

The pathogenic microorganisms including 5 gram-positive bacteria, 3 gram-negative bacteria and 1 yeast, were purchased from BeNa Culture Collection company (Beijing, China). Bacillus cereus BNCC 103930 (B. cereus), Listeria monocytogenes BNCC 336877 (L. monocytogenes), Staphylococcus aureus BNCC 186335 (S. aureus), Staphylococcus epidermidis BNCC 102555 (S. epidermidis), Staphylococcus lentus BNCC 336683 (S. lentus), Escherichia coli BNCC 133264 (E. coli), Pseudomonas aeruginosa BNCC 337005 (P. aeruginosa) and Salmonella typhimurium BNCC 108207 (S. typhimurium) were cultured in Mueller-Hinton Broth medium. Candida albicans BNCC 186382 (C. albicans) was grown in yeast maltose medium.

The minimum inhibitory concentrations (MICs) and minimum microbiocidal concentrations (MMCs) of I. lanceolatum EOs were determined by the previous method with some modifications (Zhao et al., 2021). The concentration of microbial strain suspension was diluted to 10⁵ CFU/mL for bacteria or 10³ CFU/mL for yeast. 120 μL of essential oil solutions was firstly added and then serial concentrations were obtained by 2-fold dilution method. After that, 60 μL of diluted strain suspension was added. The plates were incubated at 37 °C for 16–24 h for the bacteria and at 28 °C for 48 h for the yeast. The lowest concentrations of essential oils corresponding to the wells in which the mixture remained clear were the MICs. After obtaining the MIC results, 60 μL of the suspension from the wells in which there was no visible growth was spread on nutrient agar plates. For the bacteria, the Petri dishes were incubated at 37 °C for 16–24 h, and for the yeast, they were at 28 °C for 48 h. The lowest essential oil concentrations of the MIC wells in which strains failed to grow were recorded as MMCs. The gentamycin sulfate and amphotericin B were used as positive controls for antibacterial and antifungal tests, respectively. The MICs and MMCs of nine compounds against S. aureus, B. cereus and L. monocytogenes, including asaricin, borneol, camphor, eucalyptol, camphene, estragole, terpinen-4-ol, linalool and α-terpineol, were further determined.

2.7 Enzyme inhibitory activity assays

Tyrosinase, α-glucosidase, acetylcholinesterase and butyrylcholinesterase inhibitory activity assays of I. lanceolatum EOs were conducted to evaluate their whitening effect, anti-diabetes effect and improvement effect on neurodegenerative diseases. Essential oil solutions in ethanol of 50 mg/mL were firstly prepared and diluted with buffer solution to the concentrations of 4, 2 and 1 mg/mL before the tests. All the enzymes and substrates were prepared daily, and kept in the ice bath for subsequent experiments. The inhibition effects on tyrosinase and butyrylcholinesterase of nine compounds including asaricin, borneol, camphor, eucalyptol, camphene, estragole, terpinen-4-ol, linalool and α-terpineol were further evaluated.

Tyrosinase inhibition activity assay was conducted according to the previous method (Sharmeen Jugreet et al., 2021) with some modifications. In brief, 110 μL of phosphate buffer (pH6.8) was mixed with 40 μL of tyrosinase solution (110 U/mL) and 10 μL of essential oil solution in a 96-well microplate. After 15 min of incubation at 37 ℃, 40 μL of levodopa (5 mM) was added to start the reaction. After an additional 10 min of incubation at 37 ℃, absorbance was immediately measured at 492 nm using microplate reader (DNM-9606, Perlong, Beijing). A blank solution without essential oil was tested using the same procedure and then inhibition percentages of essential oils were calculated. Kojic acid was used as positive control and the tyrosinase inhibitory activities of essential oils were expressed as mg kojic acid equivalents (KAE)/g of EO.

α-glucosidase inhibitory activity assay was based on the previous method (Hong et al., 2021) with some modifications. 80 μL of phosphate buffer (pH6.8) was mixed with 20 μL of α-glucosidase solution (1 U/mL) and 10 μL of essential oil solution in a 96-well microplate. After 15 min of incubation at 37 ℃, 10 μL of p-NPG (1 mM) was added to initiate the reaction. The reaction lasted for 20 min and stopped by adding 80 μL of sodium carbonate solution (0.2 M). The absorbances were recorded at 405 nm using microplate reader. A blank solution without essential oil was tested using the same method. Acarbose was used as positive control and the results were expressed as mg acarbose equivalents (AE)/g of EO.

Acetylcholinesterase and butyrylcholinesterase inhibitory activity assays were carried out following the previous method (Hong et al., 2021) with modifications. 140 μL or 120 μL of Tris-HCl buffer (pH8.0) was firstly added to a 96-well microplate, followed by the addition of 20 μL of DTNB solution (1 mM), 10 μL of essential oil solution and 10 μL of acetylcholinesterase solution (2 U/mL) or 30 μL of butyrylcholinesterase solution (2 U/mL). After 15 min of incubation at 37 ℃, 20 μL of ATCI (1 mM) or BTCI (1 mM) was added to start the reaction. After an additional 15 min of incubation at 37 ℃, absorbance was immediately measured at 405 nm. A blank solution without essential oil was tested using the same method. Galanthamine was used as positive control and the results were expressed as mg galanthamine equivalents (GE)/g of EO.

2.8 Cytotoxic activity assays

Cytotoxic activities of I. lanceolatum EOs were evaluated by using the MTT assay against three cell lines, including HK-2 cells (proximal tubular cell line of normal kidney), HepG2 cells (human liver carcinoma cell line) and MCF-7 cells (human breast cancer cell line). Cytotoxicity assays were conducted according to the previous method (Zhang et al., 2017) with some modifications. Essential oil solutions in DMSO of 100 mg/mL were firstly prepared and six final concentrations (500, 200, 100, 50, 20 and 2 μg/mL) were evaluated for each essential oil. The tested cells were cultured in a humidified 5 % CO₂ atmosphere. When the cells were cultivated to the logarithmic growth stage, they were seeded at 1 × 10⁴ cells/well in 100 µL of growth medium and incubated at 37 ℃ for 24 h. The microplates were treated with the essential oil solutions and incubated for 24 h. Then, 20 µL of MTT solution (5 g/L) was added to each well and the microplate were incubated for an additional 4 h. 100 µL of DMSO was added to each well. The absorbance was measured at 570 nm. The percentages of cell viability were calculated by the formula: cell viability (%) = [A_sample/A _control] × 100 %. Doxorubicin was used as the positive control.

2.9 Molecular docking study

Molecular docking studies of selected compounds with better enzyme inhibitory activities in the root EO were carried out according to a previous method (Sun et al., 2023) with some modifications. The interactions between these compounds and enzymes were simulated using AutoDock Vina 1.1.2 (Trott and Olson, 2010). The protein structures of tyrosinase (Mauracher et al., 2014) and butyrylcholinesterase (Knez et al., 2018) were obtained from the Protein Data Bank database, with PDB ID codes as 4OUA and 6F7Q, respectively. Docking results were given as binding affinity values (kcal/mol). PyMOL and online tool named Protein-Ligand Interaction Profiler (PLIP) (Salentin et al., 2015) were used to analyze the interactions between the ligands and the active sites of enzymes.

2.10 Statistical analysis

Hydrodistillation, GC–MS and GC-FID analysis of essential oils were performed in triplicates. For antioxidant, antimicrobial and cytotoxic activity assays, three parallel experiments were performed. For enzyme inhibitory activity assays, four parallel experiments were performed in a 96-well plate. The data were expressed as the mean ± SD. Statistical analysis was performed by use of SPSS 25.0 software. One-way ANOVA analysis was used to evaluate the differences among samples and statistical significance is indicated by a value of p < 0.05.

3.1 Chemical composition of I. lanceolatum EOs

I. lanceolatum EOs were light yellow oily liquids. Essential oil yields in different parts of I. lanceolatum ranged from 0.09 % to 1.02 %. The leaves showed the highest essential oil yield (1.02 ± 0.05 %), followed by the fruits (0.30 ± 0.02 %), stems (0.21 ± 0.01 %) and roots (0.09 ± 0.01 %). The results seemed to be much lower than the previous studies (Huang et al., 2012; Liang et al., 2012) in which essential oil yields in the roots and fruits were 0.31 ± 0.03 % and 1.37 ± 0.11 %, respectively.

Total ion chromatograms of the root EO, stem EO, leaf EO and fruit EO could be seen in Figs. 1-4. Chemical composition of these four EOs were presented in Table 1. In the essential oils from the roots, stems, leaves and fruits, 45, 63, 57 and 82 chemical compounds were identified, accounting for 91.85 %, 93.19 %, 94.14 % and 92.62 % of the total composition of the essential oils, respectively. The major compounds in the root EO were asaricin (50.52 %), borneol (7.01 %), camphor (5.71 %), eucalyptol (5.44 %) and camphene (3.29 %). The stem EO had eucalyptol (27.63 %), linalool (14.42 %), caryophyllene oxide (6.02 %), α-cadinol (5.30 %) and α-terpineol (4.11 %) as the major compounds. The major compounds in the leaf EO included eucalyptol (42.42 %), caryophyllene oxide (9.77 %), α-calacorene (3.77 %), linalool (3.35 %) and caryophylla-4(12),8(13)-dien-5-ol (3.08 %), while the dominant compounds in the fruit EO were eucalyptol (12.23 %), linalool (10.22 %), terpinen-4-ol (5.02 %), α-terpineol (4.41 %) and α-pinene (4.32 %).In terms of chemical structure, oxygenated monoterpenes dominated in the stem, leaf and fruit EOs, representing 51.84 %, 51.06 % and 40.93 % of the total composition, respectively. However, the most abundant compound in the root EO was asaricin, a phenylpropanoid compound which accounted for 50.52 %.

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Fig. 1

Total Ion Chromatogram of the root EO of I. lanceolatum.

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Fig. 2

Total Ion Chromatogram of the stem EO of I. lanceolatum.

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Fig. 3

Total Ion Chromatogram of the leaf EO of I. lanceolatum.

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Fig. 4

Total Ion Chromatogram of the fruit EO of I. lanceolatum.

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A total of 110 chemical compounds were identified, but only 18 compounds existed in all four essential oils. These 18 compounds accounted for 19.49 %, 67.54 %, 59.79 % and 54.06 % in the root, stem, leaf and fruit EOs, respectively. Five compounds, including eucalyptol, linalool, terpinen-4-ol, α-terpineol and α-cadinol, had the relative percentages of more than 1 % in all four essential oils and could be regarded as representative compounds in I. lanceolatum EOs. In particular, eucalyptol was the most abundant compound in the stem, leaf and fruit EOs. As the most abundant compound in the root EO, asaricin accounted for less than 1 % in the stem and leaf EOs, and even not detected in the fruit EO. Therefore, there were more differences between the root EO and other three EOs.

Of four EOs of I. lanceolatum, the chemical composition of the stem EO was firstly reported. The previous study (Huang et al., 2012) reported myristicin (17.6 %), α-asarone (17.2 %) and methyl isoeugenol (11.2 %) as the major compound in the root EO, which was quite different from our results. α-asarone was not detected and myristicin accounted for only 0.15 % in the current research. However, eucalyptol or 1,8-cineole (24.5 %) was previously reported as the most abundant compound in the fruit EO (Huang et al., 2012), which were in accordance with our results. For leaf EO, linalool (16.2 %), elemicin (14.9 %) and eucalyptol (14.8 %) were major compound in the previous study (Huang et al., 2012) while our study gave eucalyptol (42.42 %), caryophyllene oxide (9.77 %), α-calacorene (3.77 %) and linalool (3.35 %). The geographic location of I. lanceolatum could be the reason for the significant differences in chemical composition of essential oils.

In comparison with other species in the genus Illicium, many compounds in common could be found, such as eucalyptol, estragole, pinene and linalool in the essential oils from I. verum (Li et al., 2020), safrole, myristicin and eucalyptol in the essential oils from I. henryi (Liu et al., 2015). However, significant differences were also observed. Anethole, the most important compound in the I. verum essential oil was not detected in our study. Safrole (54.09 %) and myristicin (22.24 %), which were the major compounds in the I. henryi essential oil, accounted for only 2.01 % and 0.15 % in this study, respectively.

Of chemical compounds of I. lanceolatum EOs, attention should be paid to several compounds with similar structure, including asaricin, myristicin, elemicin and safrole. Myristicin, elemicin and safrole have been extensively studied. Myristicin could be found in nutmeg and parsley, and has shown several promising activities, such as anti-inflammatory, anticancer and insecticidal activities (Seneme et al., 2021). The toxicities of myristicin and elemicin have been reported in some studies, and safrole has been classified as genotoxic carcinogens based on extensive toxicological evidence (Götz et al., 2022). In this work, safrole was found in the fruit EO with relative percentage of 2.01 % and its existence could limit the application of the fruit EO in food industry. Asaricin accounted for 50.52 % in the root EO and has been previously reported in the essential oils of some plants, such as Beilschmiedia miersii (Carvajal et al., 2016) and several Piper species (Soares et al., 2022). Due to its insecticidal activity, this compound has been structurally modified to obtain a series of asaricin analogues (Guo et al., 2016). Many biological activities of this compound remained unknown. As asaricin has a similar structure with myristicin, it is likely to have some degree of toxicity. The root EO of I. lanceolatum could be a natural source of asaricin for further studies. Due to the existence of these toxic compounds, the application of I. lanceolatum essential oils in food industry may be restricted.

3.2 Antioxidant activity of I. lanceolatum EOs

In order to give a comprehensive evaluation on antioxidant activities of I. lanceolatum EOs, three methods with different mechanisms were employed. The antioxidant activity results were displayed in Table 2. In the DPPH assay, the fruit EO had the highest Trolox equivalent value (6.36 ± 0.10 mg TE/g of EO), while the highest Trolox equivalent value (10.24 ± 0.11 mg TE/g of EO) was observed for the root EO in the ABTS assay. In the DPPH and ABTS assays, I. lanceolatum EOs showed very weak radical scavenging capacity when they were compared with BHT. However, the root EO showed moderate antioxidant activity in the FRAP assay, with the Trolox equivalent value as 176.33 ± 4.52 mg TE/g of EO. Other three essential oils displayed very weak antioxidant activity in the FRAP assay.

Strong radical scavenging capacity of essential oil is closely connected with phenolic compounds, like thymol, carvacrol and eugenol, which can react with radicals by donating hydrogen atom (Zeb, 2020). Thymol and carvacrol existed in the root and fruit EOs, however, their relative percentages were only 0.10 % and 0.06 %, respectively. Very low relative percentages of phenolic compounds in the I. lanceolatum EOs could be the reason for their low radical scavenging capacities in the DPPH and ABTS assays.

As the root EO showed moderate antioxidant activity in the FRAP assay, its major compounds were further studied. There were 11 compounds which had the relative percentages of more than 1 % in the root EO. 9 purchased compounds could be in Table 3 and 2 compounds including τ-cadinol (1.35 %) and α-cadinol (1.41 %) were not available. These nine compounds were further evaluated by FRAP assay and results were given in Table 3. Seven compounds, such as borneol and eucalyptol, showed no activities. Camphene had very weak antioxidant activity in the FRAP assay. However, antioxidant activity with 311.76 ± 3.64 mg TE/g of TC was noticed for asaricin which had higher Trolox equivalent value than the root EO. Considering its high relative percentage (50.52 %), this molecule could be the main antioxidant compound in the root EO. Certainly, some other compounds which had lower relative percentages in the root EO could contribute together to the antioxidant activity. Synergistic and antagonistic effects between these compounds could exist.

The antioxidant activity of asaricin in the FRAP assay was firstly reported in this research, however, antioxidant mechanism of asaricin was still unknown. Subsequent investigations could be performed to find out how asaricin react with ferric and if asaricin could exert real antioxidant protection in the application experiments.

3.3 Antimicrobial activity of I. lanceolatum EOs

Antimicrobial activities of essential oils from the roots, stems, leaves and fruits of I. lanceolatum were evaluated against 5 gram-positive bacteria, 3 gram-negative bacteria and 1 yeast. The MICs and MMCs of four essential oils were determined and presented in Tables 4-6. I. lanceolatum EOs showed some degree of antimicrobial activities against all tested microorganisms, with MIC values ranging from 3.13 to 25 mg/mL. According to the MIC criteria of essential oils (Van Vuuren and Holl, 2017), if the essential oil displayed an MIC of less than 1000 µg/ml, the antimicrobial activity could be considered to be noteworthy; from 101 to 500 μg/mL, the antimicrobial activity was strong; from 500 to 1000 µg/ml, the antimicrobial activity was moderate. In this work, I. lanceolatum EOs showed very weak antibacterial or antifungal activities, which was also confirmed by comparing their MICs with those of gentamycin sulfate or amphotericin B.

The root EO showed better antimicrobial activities than other three EOs, with MIC values as 3.13 mg/mL against three gram-positive bacteria including S. aureus, B. cereus and L. monocytogenes. MICs and MMCs of nine major compounds in the root EO against S. aureus, B. cereus and L. monocytogenes were further determined and displayed in Table 7. Two compounds (eucalyptol and estragole) showed no antimicrobial activities against three bacteria. MIC of asaricin against S. aureus was 12.5 mg/mL, which was higher than the MIC of the root EO. Of nine compounds, camphor showed the best antimicrobial activities, followed by borneol. MICs of camphor against three bacteria ranged from 0.78 to 3.13 mg/mL while MICs of borneol ranged from 1.56 to 3.13 mg/mL. These two compounds accounted for 7.01 % and 5.71 % in the root EO, while their highest relative percentage in other three EOs was only 0.26 %. Therefore, camphor and borneol could be the reason for better antibacterial activities of the root EO than other three EOs, and were two important antimicrobial compounds in the root EO. Besides the compounds listed in Table 7, many other compounds in the root EO, such as p-cymene (Marchese et al., 2017), α-pinene, β-pinene (Rivas da Silva et al., 2012) and thymol (Escobar et al., 2020) have been reported with antimicrobial activities. In general, essential oils with high content of thymol and other phenolic compounds have strong antimicrobial activities, as in the case of oregano essential oil. The relative percentage of thymol in the root EO was only 0.10 %, which means little contribution to antimicrobial activity. Only by looking at the antimicrobial activities of these compounds in the root EO, it was difficult to fully explain antimicrobial activity of this essential oil as synergistic and antagonistic effects existed in the essential oils. In previous studies, camphor has been reported to have synergistic antimicrobial effects with Lavandula latifolia essential oil (Karaca et al., 2021), and three essential oils from cinnamon, manuka, and winter savory have demonstrated synergistic antimicrobial activities (Fratini et al., 2019). Synergistic effects could be another factor for better antimicrobial activities of the root EO.

3.4 Enzyme inhibitory activities of I. lanceolatum EOs

Tyrosinase, α-glucosidase, acetylcholinesterase and butyrylcholinesterase inhibitory activities of I. lanceolatum EOs were determined to evaluate their whitening effect, anti-diabetes effect and improvement effect on neurodegenerative disease, and the results expressed as mg kojic acid equivalent (KAE)/g of EO, mg acarbose equivalent (AE)/g of EO and mg galanthamine equivalent (GE)/g of EO were presented in Table 8. In the antidiabetic drugs, one important category is α-glucosidase inhibitor, like acarbose and miglitol. Four essential oils showed no inhibitory activity on α-glucosidase, which indicated no potential of I. lanceolatum EOs as antidiabetic agents. However, I. lanceolatum EOs displayed inhibitory activities on other three enzymes. For tyrosinase and butyrylcholinesterase, the highest KAE value (30.34 ± 0.40 mg KAE/g of EO) and GE value (43.25 ± 1.50 mg GE/g of EO) were observed for the root EO, followed by the fruit EO.

The root EO showed better tyrosinase and butyrylcholinesterase inhibitory activities than other three EOs, its nine major compounds were further evaluated and the results could be seen in Table 9. Tyrosinase play an important role in the biogenesis of melanin and tyrosinase inhibitors could find their use in skin-whitening. All nine tested compounds showed tyrosinase inhibitory activities. Asaricin showed the highest KAE value (33.42 ± 2.11 mg KAE/g of TC), followed by estragole (14.68 ± 0.17 KAE/g of TC). Considering its high relative percentage (50.52 %), asaricin could be main contributor to tyrosinase inhibitory activity of the root EO. Estragole has been reported to have skin-whitening effect and compounds synthesized from this molecule showed good tyrosinase inhibitory activity (Motoki et al., 2003). In the previous studies, polyphenols like flavonoids and phenolic acids as natural tyrosinase inhibitor (Zolghadri et al., 2019) have attracted a lot of attention. There were also numerous studies on the tyrosinase inhibitory activities of different essential oils, however, the reports on the tyrosinase inhibitory activities of individual compounds in the essential oils were limited. In this study, tyrosinase inhibitory activities of some compounds like asaricin were firstly reported. Synergistic strategy is often used to improve the inhibitory activities of tyrosinase inhibitors (Zolghadri et al., 2019), the synergistic effect could be one reason for the better tyrosinase inhibitor activity of the root EO.

For butyrylcholinesterase, only five tested compounds showed inhibitory activities. α-terpineol showed the best butyrylcholinesterase inhibitory activity (128.14 ± 2.85 mg GE/g of TC), followed by terpinen-4-ol (55.58 ± 3.41 mg GE/g of TC) and asaricin (29.01 ± 2.60 mg GE/g of TC). Eucalyptol (Costa et al., 2012a), camphor (Costa et al., 2012b) and terpinen-4-ol (Bonesi et al., 2010) have been reported to have butyrylcholinesterase inhibitory activities in previous studies. Though α-terpineol and terpinen-4-ol showed higher GE values than the root EO (43.25 ± 1.50 mg GE/g of EO), their relative percentages were only 1.34 % and 1.59 %, respectively, counterbalancing their contribution to the butyrylcholinesterase inhibitory activity of the essential oil. Asaricin, with relative percentage of 50.52 %, was still the most important contributor to the butyrylcholinesterase inhibitory activity of the root EO. Certainly, the activity of the root EO could not be explained by these five compounds. There were other active compounds in the root EOs, like α-pinene and β-pinene (Orhan et al., 2008), which have been reported with activities. On the other hand, there were also the synergistic and antagonistic effects between these compounds.

For acetylcholinesterase, the leaf EO displayed better inhibitory activity (17.79 ± 0.32 mg GE/g of EO) than other three EOs. Chemical compounds of leaf EO were represented by eucalyptol (42.42 %) and caryophyllene oxide (9.77 %). The acetylcholinesterase inhibitory activity of eucalyptol (or 1,8-cineole) has been reported in many studies (Burcul et al., 2020) and that of caryophyllene oxide was also displayed in previous study (Savelev et al., 2004). In this study, eucalyptol was further evaluated for its acetylcholinesterase inhibition activity, with GE value as 32.59 ± 0.57 mg GE/g of TC. It could be concluded that eucalyptol was important active compound in the leaf EO. If considering both acetylcholinesterase and butyrylcholinesterase inhibitory activities values, the leaf EO seemed to be a better one which could exert improvement on neurodegenerative diseases.

3.5 Cytotoxic activity of I. lanceolatum EOs

Cytotoxic effects of I. lanceolatum EOs on HK-2 (proximal tubular cell line of normal kidney), MCF-7 (human breast cancer cell line) and HepG2 (human liver carcinoma cell line) cells could be seen in Figs. 5-7, respectively. Doxorubicin was used as the positive control. At concentrations of 2, 20, 50 and 100 μg/mL, the cell viabilities of HK-2 were found to be more than 95 %, which means that I. lanceolatum EOs were nontoxic to HK-2 at these concentrations. When the concentrations reached to 200 μg/mL, the cell viabilities of HK-2 were nearly 90 %. At the concentration of 500 μg/mL, the cell viabilities of HK-2 treated with the fruit EO, were still 88.57 %. The fruit was highly toxic due to the existence of anisatin, however, only weak toxicity to normal kidney cells was observed for the fruit EO at the tested concentrations.

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Fig. 5

Cytotoxic effects on HK-2 treated with root, stem, leaf and fruit EOs at six concentrations.

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Fig. 6

Cytotoxic effects on MCF-7 treated with root, stem, leaf and fruit EOs at six concentrations.

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Fig. 7

Cytotoxic effects on HepG2 treated with root, stem, leaf and fruit EOs at six concentrations.

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As shown in Fig. 6, the cell viabilities of MCF-7 cells ranged from 77.84 % to 94.74 % at six tested concentrations. At the concentration of 500 μg/mL, I. lanceolatum EOs showed some degree of anticancer activities to MCF-7. Compared with doxorubicin which can inhibit almost 50 % of MCF-7 at the concentration of 1.36 μg/mL, anticancer activities to MCF-7 of I. lanceolatum EOs were very weak, indicating no potential as anticancer drugs.

For HepG2 cells treated with the I. lanceolatum EOs at concentrations of 200 μg/mL and below 200 μg/mL, the cell viabilities were more than 92 %. However, when the concentration reached up to 500 μg/mL, anticancer activities to HepG2 cells were observed for all four essential oils. The root and fruit EOs almost inhibited 50 % of HepG2 cells. The leaf EO inhibited even 75 % of HepG2 cells, indicating potential use in anticancer therapy.

A number of studies have been performed on the anticancer activities of the essential oils (Bhalla et al., 2013) and their constituents (Silva et al., 2021). Many compounds in the I. lanceolatum EOs have been reported with anticancer activities to HepG2 cells, such as eucalyptol (Rodenak-Kladniew et al., 2020), linalool (Rodenak-Kladniew et al., 2018), α-pinene (Xu et al., 2018) and β-elemene (Dai et al., 2013). The leaf EO was represented by high percentage of eucalyptol (42.42 %), which could be the reason for its better anticancer activity. Caryophyllene oxide, which accounted for 6.02 %, 9.77 % and 2.85 % in the stem, leaf and fruit EOs, respectively, were found to have no inhibition effects on HepG2 in previous study (Xiu et al., 2022). The synergistic effects between anticancer compounds may exist in the inhibition of HepG2 cells.

3.6 Molecular docking studies of selected compounds in the root EO

In the root EO, asaricin, estragole and α-terpineol showed better tyrosinase inhibitory activities while α-terpineol, terpinen-4-ol and asaricin showed better butyrylcholinesterase inhibitory activities. These compounds were selected for molecular docking to investigate the interactions between ligands and enzymes. The binding affinities (kcal/mol) of these compounds against tyrosinase and butyrylcholinesterase were presented in Table 10. Asaricin showed the lowest binding affinities (-6.3 kcal/mol) with tyrosinase, indicating the strongest inhibitory activity of these three compounds, which was in accordance with the experimental results. The butyrylcholinesterase inhibitory activity of α-terpineol (128.14 ± 2.85 mg GE/g TC) was much higher than those of terpinen-4-ol (55.58 ± 3.41 mg GE/g TC) and asaricin (29.01 ± 2.60 mg GE/g TC), however, no significant differences could be observed among binding affinities of these three compounds (p < 0.05). It is difficult to accurately predict the activity of one molecule only by using binding affinity. Generally, different molecular docking software should be employed together for screening or comparison of active compounds.

The interactions of asaricin, estragole and α-terpineol with amino acid residues in the binding pocket of tyrosinase could be seen in Fig. 8. The main interactions of asaricin and estragole with tyrosinase were hydrophobic interaction, hydrogen bond and π-stacking. α-terpineol formed two hydrogen bonds with ASP-344 and THR-345, and had hydrophobic interactions with three residues including THR-345, PHE-355 and VAL-366. The interactions of α-terpineol, terpinen-4-ol and asaricin with amino acid residues of butyrylcholinesterase were shown in Fig. 9. α-terpineol formed one hydrogen bond with TRP-82 and had hydrophobic interactions with three residues including PRO-285, TYR-332 and TRP-430. Terpinen-4-ol mainly interacted with butyrylcholinesterase by hydrophobic interaction. Asaricin had hydrophobic and π-stacking interactions with TRP-82. From the interactions of these three compounds, it can be seen that TRP-82 was one of important amino acid residues in the active site of butyrylcholinesterase.

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Fig. 8

The interactions of tyrosinase with asaricin (A), estragole (B) and α-terpineol (C).

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Fig. 9

The interactions of butyrylcholinesterase with α-terpineol (A), terpinen-4-ol (B) and asaricin (C).

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