Chemical Composition,in Vitro Antibacterial and Antifungal Activities of Different Parts Essential Oils of Neolitsea sericea var. aurata From Taiwan

Abstract

This study examined the chemical composition and antimicrobial activities of essential oils isolated from the leaves, twigs, and fruits of Neolitsea sericea var. aurata from Taiwan. The major compounds responsible for antimicrobial activity were isolated and identified. The leaf, twig, and fruit essential oils were obtained through hydrodistillation in a Clevenger-type apparatus and were characterized through gas chromatography (GC) with flame ionization detection (GC/FID) and GC/mass spectrometry (GC/MS). The major compounds were (E)-β-ocimene (49.3%) and sericenine (31.6%) in the leaf oil; (E)-β-ocimene (73.7%), α-cadinol (6.8%), and α-muurolol (4.0%) in the twig oil; and (E)-β-ocimene (84.7%) in the fruit oil. The twig oil exhibited the strongest antibacterial and antifungal activities. We isolated α-cadinol and α-muurolol, the main components of NTO4 fraction, and used them in antimicrobial tests; the two compounds exhibited excellent antimicrobial activities. The twig essential oil contains α-cadinol and α-muurolol and exhibited excellent antimicrobial activities against food-borne pathogens; thus, they are worthy of further research and development.

Keywords

food-borne pathogens antimicrobial activities essential oil terpene

Introduction

Microorganisms play a vital role in the daily life of humans. For example, microorganisms are used to brew wine and prepare soy sauce and fermented food. However, not all microorganisms are beneficial to humans. Some microorganisms can cause food-borne diseases. Food poisoning resulting from the consumption of pathogen-contaminated food has become a public health problem.¹ Therefore, controlling pathogen growth can reduce the incidence of food-borne illness and ensure the supply of safe, healthy, and nutritious food. Various methods have been developed to control microbial growth, and the use of synthetic antimicrobials can reduce the incidence of food poisoning.² However, synthetic antimicrobials lead to microbial resistance and adverse effects. Therefore, the use of antimicrobial compounds derived from plants as natural antimicrobials has become a crucial research direction.³ Our previous studies have demonstrated the strong antimicrobial activity of the essential oil isolated from the heartwood of Cunninghamia lanceolata var. konishii⁴ and that isolated from the leaves of Litsea kostermansii.⁵

Neolitsea is one of the main genera of Lauraceae family, its morphology as shrub or evergreen trees, including about 100 species, mainly distributed in tropical Asia. Neolitsea sericea var. aurata (Lauraceae), an evergreen large tree, is endemic to Taiwan. The leaves are known to be ovate to elliptic, with acuminate to at the apex, and beneath covered with yellowish-golden pilose.⁶ No study has examined the chemical composition and biological activity of essential oils isolated from this species. Therefore, we extracted leaf, twig, and fruit oils from this tree through hydrodistillation and analyzed these isolated oils through gas chromatography (GC) with flame ionization detection (GC/FID) and GC/mass spectrometry (GC/MS). In addition, we investigated the activities of the leaf, twig, and fruit oils against food-borne bacterial and fungal pathogens and isolated and identified antimicrobial compounds. The findings of this study can provide a chemical basis for the effective multipurpose utilization of N. sericea var. aurata.

Results and Discussion

The hydrodistillation of the leaves, twigs, and fruits of N. sericea var. aurata produced yellow essential oils with yields of 0.92 ± 0.03, 0.14 ± 0.01, and 0.17 ± 0.01 mL/100 g of the dry weight, respectively. Table 1 lists the identified compounds, percentages, and linear retention index (LRI) values in the order of elution from the DB-5 capillary column. A total of 29 compounds were identified in the leaf oil. The essential oils were characterized on the basis of the presence of monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and other compounds. The leaf oil consisted of 51.2% monoterpene hydrocarbons, 37.3% oxygenated sesquiterpenes, 11.0% sesquiterpene hydrocarbons, 0.3% oxygenated monoterpenes, and 0.2% other compounds. The main monoterpene hydrocarbon was (E)-β-ocimene (49.3%), and the main oxygenated sesquiterpene was sericenine (31.6%).

Table 1.

Chemical Composition of the Leaf, Twig, and Fruit Essential Oils of Neolitsea sericea var. aurata.

Compound I.D.	LRILit^a	LRIExp^b	Concentration (%)			Identification^c
Compound I.D.	LRILit^a	LRIExp^b	Leaf	Twig	Fruit	Identification^c
Myrcene	991	988	0.1	0.7	0.3	MS,LRI,CO-ST
1,8-Cineole	1031	1033	-^d	-	0.1	MS,LRI,CO-ST
(Z)-β-Ocimene	1037	1036	1.8	3.3	3.0	MS,LRI,CO-ST
5-Methylhexanoic acid	1039	1038	0.2	-	-	MS,LRI,CO-ST
(E)-β-Ocimene	1050	1047	49.3	73.7	84.7	MS,LRI,CO-ST
Linalool	1096	1097	0.2	0.1	0.3	MS,LRI,CO-ST
(3Z)-Hexenyl butanoate	1186	1183	0.1	-	0.7	MS,LRI
(3Z)-Hexenyl 2-methylbutanoate	1232	1229	-	-	0.3	MS,LRI
δ-Elemene	1338	1329	0.2	-	0.2	MS,LRI
Linalool isobutanoate	1375	1370	-	0.1	0.2	MS,LRI
β-Elemene	1391	1389	0.8	0.4	0.7	MS,LRI,CO-ST
β-Caryophyllene	1419	1421	3.5	2.8	0.7	MS,LRI,CO-ST
trans-α-Bergamotene	1435	1433	-	0.3	-	MS,LRI
Aromadendrene	1441	1441	0.3	-	-	MS,LRI,CO-ST
α-Humulene	1455	1457	0.4	0.3	-	MS,LRI,CO-ST
γ-Muurolene	1479	1475	0.2	0.2	0.4	MS,LRI
Germacrene D	1485	1482	0.9	0.2	0.5	MS,LRI
β-Selinene	1490	1490	1.0	0.7	0.9	MS,LRI
Bicyclogermacrene	1500	1496	2.4	-	-	MS,LRI
α-Selinene	1498	1496	-	0.1	0.5	MS,LRI
(E,E)-α-Farnesene	1506	1501	0.3	-	-	MS,LRI,CO-ST
Bicyclogermacrene	1500	1501	-	-	0.3	MS,LRI
δ-Amorphene	1512	1517	-	0.4	-	MS,LRI
δ-Cadinene	1523	1518	0.3	0.1	0.2	MS,LRI
Germacrene B	1561	1561	0.5	0.3	-	MS,LRI
Spathulenol	1578	1577	1.3	-	-	MS,LRI
Caryophyllene oxide	1583	1583	0.4	1.2	0.5	MS,LRI,CO-ST
Viridiflorol	1593	1586	0.3	-	-	MS,LRI
Guaiol	1601	1595	0.9	0.3	-	MS,LRI
1-epi-Cubenol	1629	1627	0.5	0.8	0.6	MS,LRI
Selina-13,7(11)-trien-8-one	1634	1634	1.0	0.5	0.3	MS,LRI
α-Muurolol	1646	1643	0.3	4.0	-	MS,LRI,CO-ST
α-Cadinol	1654	1655	0.5	6.8	0.5	MS,LRI,CO-ST
Selin-11-en-4-α-ol	1659	1659	0.3	-	0.4	MS,LRI
7-epi-α-Eudesmol	1672	1665	-	0.2	-	MS,LRI
Germacrone	1694	1693	0.2	0.4	-	MS,LRI
Sericenine	1731^e)	1733	31.6	2.1	3.6	MS,LRI
Monoterpene hydrocarbons (%)			51.2	77.7	88.0
Oxygenated monoterpenes (%)			0.3	0.1	1.4
Sesquiterpene hydrocarbons (%)			11.0	5.9	4.4
Oxygenated sesquiterpenes (%)			37.3	16.3	6.2
Others (%)			0.2	-	-
Oil Yield (mL/100 g)			0.92 ± 0.03	0.14 ± 0.01	0.17 ± 0.01

LRILit = linear retention indices from a previous study.⁹

LRIExp = computed linear retention indices obtained for a mixture of a continuous series of n-alkane hydrocarbons (C8 to C30) run on a DB-5 capillary column;

Identification through MS = comparison of NIST and Wiley mass spectral libraries; LRI = linear retention index (LRI) is the same as the previous findings^9–11; CO-ST = co-injection/comparison with the LRI and MS standards.

= not detected.

See reference 8

A total of 25 compounds were identified in the twig oil. The twig oil contained 77.7% monoterpene hydrocarbons, 16.3% oxygenated sesquiterpenes, 5.9% sesquiterpene hydrocarbons, and 0.1% oxygenated monoterpenes. Among sesquiterpene hydrocarbons, the most abundant compound was (E)-β-ocimene (73.7%). Among oxygenated sesquiterpenes, the main compounds were α-cadinol (6.8%) and α-muurolol (4.0%).

A total of 23 compounds were identified in the fruit oil. The fruit oil contained 88.0% monoterpene hydrocarbons, 6.2% oxygenated sesquiterpenes, 4.4% sesquiterpene hydrocarbons, and 1.4% oxygenated monoterpenes. Among monoterpene hydrocarbons, (E)-β-ocimene (84.7%) was the most abundant component.

Few studies have examined the chemical composition of the essential oil of N. sericea var. aurata. However, we identified two studies reporting the composition of the leaf oil^7,8 and one study reporting the composition of the fruit oil⁷ of N. sericea. Nii et al⁷ reported that the main ingredients of the leaf essential oil were isosericenine (43.9%), trans-β-ocimene (13.6%), caryophyllene (7.6%), and sabinene (6.1%). Yoon et al.⁸ indicated that the leaf essential oil contained sericenine (32.3%), sabinene (21.0%), trans-β-ocimene (13.3%), β-caryophyllene (4.8%), and 4-terpineol (4.2%). These components are different from the ones we identified in the leaf oil of N. sericea var. aurata in this study. Nii et al⁷ reported that the main components in the fruit oil of N. sericea were isosericenine (56.0%–69.9%) and trans-β-ocimene (4.8%–17.5%). These compounds are different from the ones we identified in the fruit essential oil of N. sericea var. aurata in this study. Therefore, this is the first study to report the composition of the leaf, twig, and fruit oils of N. sericea var. aurata.

We examined the antibacterial activity of the leaf, twig, and fruit oils of N. sericea var. aurata against food-borne pathogenic bacteria by using disk diffusion method and serial dilution method, respectively. Six strains of food-borne pathogenic bacteria were selected: three Gram-positive bacterial strains, namely Bacillus cereus, Staphylococcus aureus, and S. epidermidis, and three Gram-negative bacterial strains, namely Escherichia coli, Enterobacter aerogenes, and Vibrio parahaemolyticus. Table 2 lists the inhibition zone (IZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) values of the leaf, twig, and fruit essential oils. The antibacterial activity of the twig oil was higher than that of the leaf and fruit oils (Table 2). The twig oil exhibited moderate to strong antibacterial activity against all the six food-borne pathogenic bacteria. The strongest inhibition was observed against B. cereus, S. aureus, and S. epidermidis, with their inhibition zones ranging from 28 to 34 mm, their MIC values ranging from 128 to 256 μg/mL, and their MBC values ranging from 256 to 512 μg/mL. However, the three essential oils exerted a stronger bacteriostatic effect on the Gram-positive bacteria than on the Gram-negative bacteria.

Table 2.

Antibacterial Activity of the Leaf, Twig, and Fruit Essential Oils of Neolitsea sericea var. aurata.

Bactrial species^a	Neolitsea sericea var. aurata									Compounds^e		Antibiotics^f
	Leaf			Twig			Fruit			α-Cadinol	α-Muurolol	Amoxicillin (25 μg/disk)
	IZ^b	MIC^c	MBC^d	IZ	MIC	MBC	IZ	MIC	MBC	IZ	IZ	IZ
I	15.8 ± 0.4	1024	>1024	28.2 ± 0.4	256	512	14.8 ± 0.4	1024	>1024	42.6 ± 0.5	45.8 ± 0.6	21.6 ± 0.5
II	18.2 ± 0.4	1024	>1024	32.8 ± 0.8	256	512	15.6 ± 0.5	1024	>1024	51.6 ± 0.8	48.6 ± 0.6	24.6 ± 0.5
III	20.6 ± 0.5	1024	>1024	34.4 ± 0.5	128	256	17.4 ± 0.5	1024	>1024	56.8 ± 0.8	51.2 ± 0.5	31.4 ± 0.5
IV	11.4 ± 0.5	>1024	>1024	24.2 ± 0.8	512	1024	11.4 ± 0.5	>1024	>1024	26.6 ± 0.4	32.6 ± 0.5	11.6 ± 0.5
V	10.4 ± 0.5	>1024	>1024	27.4 ± 0.5	512	512	10.2 ± 0.8	>1024	>1024	38.5 ± 0.5	42.3 ± 0.6	10.2 ± 0.8
VI	9.4 ± 0.5	>1024	>1024	20.6 ± 0.5	1024	1024	9.2 ± 0.4	>1024	>1024	23.8 ± 0.6	28.9 ± 0.5	9.4 ± 0.5

I. B. cereus, II. S. cereus. III. S. epidermidis, IV. E. coli, V. E. aerogenes, VI. V. parahaemolyticus.

Inhibition zone diameter (mm), including the diameter of the sterile disk (6 mm); values are presented as the mean ± SD.

The minimum inhibitory concentration is presented as μg/mL.

The minimum bactericidal concentration is presented as μg/mL.

α-Cadinol (100%) and α-Muurolol (100%) were isolated in this study.

Positive control: Amoxicillin (25 μg/disk).

Previous studies have reported that essential oils or antibacterial compounds are more effective against Gram-positive bacteria than against Gram-negative bacteria^12–15; this finding is in accordance with that of the present study. The penetration of hydrophobic compounds in Gram-negative microorganisms is more difficult because of the presence of a second physical barrier formed by the outer membrane.^15,16 Thus, the hydrophobicity of essential oils enables them to disrupt the lipid layer of bacterial cell membranes and mitochondria, thereby making the bacterial structure more permeable. This results in the leakage of ions and other cellular contents,¹⁷ leading to lysis and death when limits are exceeded.¹⁸ This mechanism interferes with the cytoplasmic membrane, enzyme activity, and electron flow, disrupting the proton motive force, active transport, ADP phosphorylation, and coagulation of cellular contents.^17–20

In the anti-food-borne fungal pathogen experiment, we selected five fungi, namely Aspergillus flavus, A. niger, Chaetomium globosum, Penicillium citrinum, and Trichoderma viride. These fungi cause food spoilage and poisoning. Table 3 lists the MIC and IC₅₀ values of the three essential oils derived from N. sericea var. aurata against the food-borne pathogenic fungi. The results revealed that the twig essential oil exhibited the highest inhibitory activity against the food-borne pathogenic fungi. The concentrations of the twig essential oil that completely inhibited the growth of A. flavus, A. niger, C. globosum, P. citrinum, and T. viride were 256, 256, 128, 512, and 256 μg/mL, respectively.

Table 3.

MIC and IC₅₀ Values (μg/mL) of the Leaf, Twig, and Fruit Essential Oils of Neolitsea sericea var. aurata. Against Five Food-Borne Pathogenic Fungi.

Fungal species	Leaf		Twig		Fruit		Nystatine
Fungal species	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀
Aspergillus flavus	>1024	>1024	256	103.1	>1024	>1024	<16	<16
Aspergillus niger	>1024	>1024	256	98.2	>1024	>1024	<16	<16
Chaetomium globosum	1024	958.3	128	52.3	>1024	>1024	<16	<16
Penicillium citrinum	>1024	>1024	512	198.2	>1024	>1024	<16	<16
Trichoderma viride	>1024	>1024	256	120.1	>1024	>1024	<16	<16

The aforementioned results indicate that the twig essential oil of N. sericea var. aurata exhibited the highest antibacterial and antifungal activities. Therefore, to determine the antimicrobial active components of the twig essential oil, we performed silica gel column chromatography of the twig oil, and the eluent consisted of n-hexane (n-hex) and ethyl acetate (EA) mixed in different ratios (n-hexane/EA = 100/0 to 0/100). The eluents were divided into seven fractions (NTO1 to NTO7). Then, we used the seven fractions to conduct antibacterial and antifungal tests separately. The results are presented in Figures 1 and 2.

Figure 1.

Inhibitory effects of the seven fractions of the twig essential oil against six food-borne pathogenic bacteria. Each experiment was performed five times, and the data were averaged (n = 5).

Figure 2.

Inhibitory effects of the seven fractions of the twig essential oil against five food-borne pathogenic fungi. Each experiment was performed five times, and the data were averaged (n = 5).

As illustrated in Figure 1, the seven fractions were used for the antibacterial test, and the concentration of each fraction was 10 μL/disc. The NTO4 fraction exhibited the largest inhibition zones against the six food-borne pathogenic bacteria. In the antifungal test, the seven fractions were used, and the concentration of each fraction was 100 μg/mL. The results are presented in Figure 2. The NTO4 fraction exhibited the strongest inhibitory activity and the highest antifungal index against the five food-borne pathogenic fungi. Among the food-borne pathogenic fungi, the NTO4 fraction completely inhibited the growth of A. niger and C. globosum.

The aforementioned findings indicated that the NTO4 fraction displayed excellent inhibitory activity against food-borne pathogens. Therefore, we performed GC and GC/MS to identify the components of the NTO4 fraction. We identified 13 compounds in the NTO4 fraction, and its main components were α-muurolol (33.5%) and α-cadinol (46.2%) (Table 4).

Table 4.

Composition of the NTO4 Fraction Isolated from the Twig Essential oil of Neolitsea sericea var. aurata.

Compound I.D.	LRILit^a	LRIExp^b	Concentration (%)	Identification^c
Compound I.D.	LRILit^a	LRIExp^b	NTO4	Identification^c
γ-Muurolene	1479	1475	1.5	MS,LRI
Germacrene D	1485	1482	1.6	MS,LRI
β-Selinene	1490	1490	1.3	MS,LRI
α-Selinene	1498	1496	0.6	MS,LRI
δ-Cadinene	1523	1518	0.8	MS,LRI
Caryophyllene oxide	1583	1583	1.5	MS,LRI,CO-ST
Guaiol	1601	1595	2.1	MS,LRI
1-epi-Cubenol	1629	1627	2.3	MS,LRI
Selina-13,7(11)-trien-8-one	1634	1634	2.1	MS,LRI
α-Muurolol	1646	1643	33.5	MS,LRI,CO-ST
α-Cadinol	1654	1655	46.2	MS,LRI,CO-ST
7-epi-α-Eudesmol	1672	1665	3.1	MS,LRI
Germacrone	1694	1693	2.3	MS,LRI
Identified components(%)			98.9

LRILit = linear retention indices from a previous study.⁹

LRIExp = computed linear retention indices obtained against a mixture of a continuous series of n-alkane hydrocarbons (C8 to C30) run on a DB-5 capillary column;

Identification through MS = comparison of NIST and Wiley mass spectral libraries; LRI = linear retention index (LRI) is the same as those in previous studies^9–11; CO-ST = co-injection/comparison with LRI and MS of standards.

We used the isolated and purified α-muurolol and α-cadinol to conduct antibacterial and antifungal tests against the six bacterial strains and five fungal strains separately. We used the disc diffusion method (a concentration of 10 μL/disc) to examine antibacterial activity. The two compounds inhibited the six bacterial strains, with their inhibition zones ranging from 23.8 to 56.8 mm (Table 2). Furthermore, both the compounds exhibited strong antifungal activity (Figure 3). At a concentration of 50 μg/mL, the two compounds completely inhibited A. flavus, A. niger, and C. globosum. Previous studies have indicated that these two compounds strongly inhibit the growth of bacteria and fungi.^16,21

Figure 3.

Antifungal index (%) of two compounds (50 μg/mL) from the Neolitsea sericea var. aurata twig oil against five food-borne fungal pathogens. Each experiment was performed five times, and the data were averaged (n = 5).

Conclusion

This study investigated the leaf, twig, and fruit oils of N. sericea var. aurata. The major components of the leaf oil were (E)-β-ocimene (49.3%) and sericenine (31.6%). The major components of the twig oil were (E)-β-ocimene (73.7%), α-cadinol (6.8%), and α-muurolol (4.0%). The major component of the fruit oil was (E)-β-ocimene (84.7%). The twig oil exhibited the strongest inhibitory activity against the food-borne bacterial and fungal pathogens. The twig oil was purified through fractionation. Among the seven fractions, NTO4 fraction displayed the strongest inhibitory activity against the pathogens. The main components of the NTO4 fraction were α-muurolol (33.5%) and α-cadinol (46.2%). These two compounds displayed excellent antibacterial and antifungal activities. The findings of this study can facilitate the development of diverse natural anti-food-borne pathogen products.

Experimental

Plant Materials

We collected the fresh leaves, twigs, and fruits of N. sericea var. aurata in March 2022 from Taimalee Research Center, Taiwan Forestry Research Institute (TFRI; Taitung County, Taiwan, elevation: 250 m, 22°35′59.2′′N, 120°58′48.9′′E). The plant was identified by Dr Hsu Chun-Kai from TFRI. A voucher specimen (HCCLH-093) was deposited at the Division of Wood Cellulose, TFRI, Taiwan.

Isolation of Leaf, Twig, and Fruit Essential Oils

We performed the hydrodistillation of 10 kg each of the leaves, twigs, and fruits by using a Clevenger-type apparatus for 3 h. Essential oils derived from the leaves, twigs, and fruits were dried using anhydrous sodium sulfate and stored in a specimen bottle at 4 °C until their use. The essential oil yield and all experimental data are expressed as the mean ± standard deviation of triplicate analyses.

Essential Oil Analysis

The N. sericea var. aurata leaf, twig, and fruit oils were analyzed through GC/FID and GC/MS. GC/FID was performed using a Hewlett–Packard 6890 gas chromatograph with FID fitted with a DB-5 fused silica capillary column (5% phenyl, 95% methylpolysiloxane, length: 30 m, inner diameter: 0.25 mm, film thickness: 0.25 μm). The oven temperature was maintained at 50 °C for 2 min and then gradually increased to 250 °C at a rate of 5 °C/min. The injector temperature was 270 °C, and the detector temperature was 250 °C. Hydrogen was used as a carrier gas at a rate of 1.0 mL/min and a split ratio of 1:60. The injection volume was 1 μL (1/100, v/v, in EA). LRI values of all compounds were calculated using the homologous series of n-alkanes (C8 to C30) as reference.^9,10 The relative proportions of essential oil compositions were calculated using the peak normalization method. The results are summarized in Table 1 and listed in the order in which compounds were eluted from the DB-5 capillary column.

GC/MS was performed using a Hewlett-Packard 6890/5973 GG–MS system equipped with a DB-5 fused silica capillary column. Helium (1 mL/min, 99.995% purity) was used as the carrier gas. The parameters were identical to those used in GC/FID. The MS conditions were as follows: full scan mode (scan time: 0.3 s) and mass range = m/z 30–500 at 70 eV in the electrospray ionization mode.

Component Identification

Essential oils components were identified by comparing LRIs, retention time, and mass spectra with those obtained from authentic standards, NIST and Wiley mass spectral libraries, and previous studies.^8,10,11

Isolation and Purification of Twig Oil Components

The twig oil of N. sericea var. aurata (20 g) mixed with silica gel (60 g) (Merck 7734, Merck Co., Germany) was analyzed using a silica gel column (600 g) and eluted with n-hex/EA. Gradient elution was performed by changing the ratio from 100/0 to 0/100. Then, each collected fraction was examined through thin-layer chromatography (TLC), and fractions with similar profiles were combined to generate seven subfractions (NTO1 to NTO7). The yield of each fraction was 18.5% (NTO1), 29.3% (NTO2), 20.8% (NTO3), 15.5% (NTO4), 10.5% (NTO5), 3.1% (NTO6), and 2.3% (NTO7). According to the antimicrobial activity assay, the NTO4 fraction exhibited the strongest antibacterial and antifungal activities. Next, we isolated and purified α-muurolol and α-cadinol from the NTO4 fraction through HPLC. The conditions were as follows: semipreparative HPLC Si-60 column, mobile phase: EtOAc:n-C6H14 = 30:70, and flow rate: 1 mL/min. α-Muurolol was isolated at retention time (R.t.) = 23.2 min, and α-cadinol was isolated at R.t. = 31.6 min. The chemical structures of the isolated compounds were determined by through MS, and ¹H NMR spectroscopy. Two sesquiterpenoids, α-muurolol and α-cadinol, were isolated and identified through spectral analysis.^16,22,23 α-Cadinol: Colorless needle crystal, m.p. 74 °C to 75 °C, EI-MS for C₁₅H₂₆O (EI-MS: 222), ¹H NMR (in CDCl₃): δ (ppm) 0.75 (3H, d, J = 7.0 Hz, H-12), 0.88 (3H, d, J = 7.0 Hz, H-13), 1.06 (3H, s, H-14), 1.63 (3H, s, H-15), 2.14 (1H, m, H-11), 5.46 (1H, br s, H-14). α-Muurolol: White solid, m.p. 137 to 138.5 °C, EI-MS for C₁₅H₂₆O (EI-MS: 222), 1H-NMR (in CDCl₃): δ (ppm) 0.81 (3H, d, J = 6.9 Hz, H-14), 0.88 (3H, d, J = 6.9 Hz, H-13), 1.29 (3H, s, H-15), 1.65 (3H, s, H-11), 1.97 (1H, m, H-12), 5.51 (1H, m, H-5).

Antibacterial Activity

The following food-borne pathogenic bacteria were used in this study: three Gram-positive bacteria, namely B. cereus (ATCC 11778), S. aureus (ATCC 6538P), and S. epidermidis (ATCC 12228), and three Gram-negative bacteria, namely E. coli (IFO 3301), E. aerogenes (ATCC 13048), and V. parahaemolyticus (ATCC 17803). The bacterial strains were obtained from the Culture Collection and Research Center of the Food Industry Research and Development Institute, Hsinchu City, Taiwan.

The antibacterial activities of the leaf, twig, and fruit oils of N. sericea var. aurata were evaluated using the paper disc diffusion method, and their MIC and MBC were determined. In addition, the antibacterial activities of the main compounds obtained after the isolation of the twig essential oil were analyzed using the paper disc diffusion method, which was reported by Bauer et al.²⁴ In brief, the bacterial inoculum was seeded on solidified Mueller Hinton agar (MHA) in a Petri dish to produce uniform growth throughout the dish. After preparing the dishes, 6-mm-diameter discs of the filter paper containing 10 μL of the undiluted oil were placed on the plates inoculated with the test microorganisms. The zone of growth inhibition (including a disc diameter of 6 mm) was measured after 24 h incubation at 37 °C. Amoxicillin was used as the positive control. Each test was conducted with three replicates.

The MIC values were determined by performing a microdilution susceptibility test, which was conducted in accordance with the National Committee of Clinical Laboratory Standards,²⁵ as reported previously.²⁶ The essential oils were diluted to 10.24 mg/mL in dimethyl sulfoxide. A series of 2-fold dilutions of each individual essential oil, ranging from 1024 to 16 μg/mL, was tested.

The MBC was determined using the method reported by Chikezie.²⁷ Aliquots (100 μL) of each dilution without visible growth were spread on MHA. The inoculated Petri dishes and controls were incubated at 37 °C for 24 h. The number of colonies was counted and compared with those on control dishes. The MBC was defined as the lowest concentration that killed >99.9% of the initial inoculum. Data are expressed as the mean ± standard deviation of three independent experiments.

Anti-Food-Borne Fungal Pathogen Assays

The antifungal assay method reported by Su et al.²⁸ was adopted. The food-borne pathogenic fungi were obtained from the Culture Collection and Research Center of the Food Industry Research and Development Institute, Hsinchu City, Taiwan. The fungi used were A. clavatus (ATCC 1007), A. niger (ATCC 6275), C. globosum (ATCC 6205), P. citrinum (ATCC 9849), and T. viride (ATCC8678). The assays were performed in triplicate, and the data were averaged. Different concentrations of the essential oils and main constituents of the twigs oil (16 to 1024 μg/mL) were added to sterilized potato dextrose agar (PDA). The test Petri dishes were incubated at 27 °C. When the mycelium of the fungi reached the edge of the control plate, the anti-food-borne fungal pathogen index (%) was calculated as (1 − Da/Db) × 100, where Da is the mycelium diameter (cm) of the experimental groups, and Db is the mycelium diameter (cm) of the control group. Nystatin was used as the positive control. Each test was repeated five times, and the data were averaged. The concentration that inhibited 50% of mycelial growth (IC₅₀) was calculated in probit analysis.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Ethical approval is not applicable for this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iD

Chen-Lung Ho

References

Bajpai

Baek

Kang

. Control of Salmonella in foods by using essential oils: a review. Food Res Int. 2012;45(2):722-734.

Burt

. Essential oils: their antibacterial properties and potential applications in food sea review. Int J Food Microbiol. 2004;94(3):223‐253.

Cakir

Kordali

Kilic

Kaya

. Antifungal properties of essential oil and crude extracts of Hypericum linarioides Bosse. Biochem Syst Ecol. 2005;33(3):245‐256.

Hsu

Wang

EIC

. Composition, anticancer, and antimicrobial activities in vitro of the heartwood essential oil of Cunninghamia lanceolata var. konishii from Taiwan. Nat Prod Commun. 2012;7(9):1245‐1247.

Wang

EIC

Hsu

Lee

. Composition and antimicrobial activity of the leaf essential oil of Litsea kostermansii from Taiwan. Nat Prod Commun. 2009;4(8):1123‐1126.

Yang

Liu

. Manual of Taiwan vascular plants. Council of Agriculture, Executive Yuan. 1999.

Nii

Furukawa

Iwakiri

Kubota

. The constituents of the essential oil from Neolitsea sericea (Blume) Koidz. Fruit. Nippon Nogeikagaku Kaishi. Journal of the Agricultural Chemical Society of Japan. 1981;55(4):345‐348.

Yoon

Moon

Kang

Kim

Lee

Hyun

. Neolitsea sericea essential oil attenuates LPS-induced inflammation in RAW 264.7 macrophages by suppressing NF-kappa B and MAPK activation. Nat Prod Commun. 2010;5(8):1311‐1316.

Adams

. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. 4th ed. Allured; 2007.

10.

Van den Dool

Kratz

. A generalization of the retention index system including linear temperature programmed gas liquid partition chromatography. J Chromatogr A. 1963;11(2):463‐471.

11.

NIST. NIST Chemistry WebBook: NIST Standard Reference Database Number 69. Available: http://webbook.nist.gov/chemistry/.

12.

Pintore

Usai

Bradesi

, et al. Chemical composition and antimicrobial activity of Rosmarinus officinalis L. Oils from sardinia and Corsica. Flavour Frag J. 2002;17(1):15‐19.

13.

Harpaz

Glatman

Drabkin

Gelman

. Effects of herbal essential oils used to extend the shelf life of freshwater reared Asian sea bass fish (Lates calcarifer). J Food Protect. 2003;66(3):410‐417.

14.

Bhattacharjee

Chatterjee

Chandra

. Antibacterial potentiality of Argemone mexicana solvent extracts against some pathogenic bacteria. Mem Inst Oswaldo Cruz. 2006;101(6):645‐648.

15.

Al-Reza

Rahman

Lee

Kang

. Potential roles of essential oil and organic extracts of Zizyphus jujuba in inhibiting food-borne pathogens. Food Chem. 2010;119(3):981‐986.

16.

Kalemba

Kunicka

. Antibacterial and antifungal properties of essential oils. Curr Med Chem. 2003;10(10):813‐829.

17.

Mann

Cox

Markham

. The outer membrane of Pseudomonas aeruginosa NCTC6749 contributes to its tolerance to the essential oil of Melaleuca alternifolia (tea tree oil). Lett Appl Microbiol. 2000;30(4):294‐297.

18.

Lambert

RJW

Skandamis

Coote

Nychas

GJE

. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. J Appl Microbiol. 2001;91(3):453‐462.

19.

Denyer

Hugo

. Mechanisms of antibacterial action - A summary. In: Mechanisms of action of chemical biocides. Wiley; 1991: 331‐334.

20.

Cosentino

Tuberoso

CIG

Pisano

, et al. In vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Lett Appl Microbiol. 1999;29(2):130‐135.

21.

Nabeta

Katayama

Matsubara

Hatakeyama

Shimada

Tazaki

. Oxygenated sesquiterpenes from needles of Korean pine (Pinus koraiensis Sieb.). Mokuzai Gakkaishi. 1992;38(10):963‐971.

22.

Chang

Wang

Chen

Kuo

. Comparison of the antifungal activity of Cadinane skeletal sesquiterpenoids from Taiwania (Taiwania cryptomerioides Hayata) heartwood. Holzforschung. 2000;54(3):241‐245.

23.

Borg Karlson

Norin

Talvitie

. Configurations and conformations of torreyol (δ-cadinol), α-cadinol, T-muurolol and T-cadinol. Tetrahedron. 1981;37(2):425‐430.

24.

Bauer

Kirby

WMM

Sherris

Turck

. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 1966;45(4):493‐496.

25.

NCCLS National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. 9th International Supplement, Wayne PA., M100-S9; 1999.

26.

Wang

EIC

Wei

. Composition and bioactivities of the leaf essential oils of Cinnamomum subavenium Miq. from Taiwan. J Essent Oil Res. 2008;20(4):328‐334.

27.

Chikezie

. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a novel dilution tube method. Afr J Microbiol Res. 2017;11(23):977‐980.

28.

Wang

EIC

Chang

. Antifungal activities and chemical compositions of essential oils from leaves of four eucalypts. Taiwan J For Sci. 2006;21:49‐61.