Chemical Compositions and Anti-Mildew Effects of Cinnamomum micranthum Leaf and Twig Essential Oils on Paper

Abstract

In this study, we evaluated the anti-mildew effects of paper treated with essential oils of leaves, twigs, and their main constituents from Cinnamomum micranthum. The main ingredients with the greater anti-mildew effects on paper capability were also purified and identified. Fresh leaves and twigs of C. micranthum were hydrodistillated in a Clevenger-type apparatus, and the resulting oil characterized using GC-FID and GC-MS instruments. The leaf essential oil consisted principally of n-decanal (50.1%), (E)-β-ocimene (7.9%), (E)-nerolidol (6.5%), and (E)-β-caryophyllene (3.8%), and the twig oil's main components were τ-cadinol (18.3%), (E)-β-ocimene (16.4%), α-cadinol (13.6%), n-decanal (10.6%), and β-selinene (5.8%). Comparing the mildew resistance of the oils on paper exhibited that twig oil was the best anti-mildew activity; at 200 μg/cm², the twig oil completely inhibited the growth of Aspergillus clavatus, Cladosporium cladosporioides, Chaetonium globosum, Myrothecium verrucaria, and Penicillium citrinum. The twig oil was further divided into 8 fractions (TO1-TO8). TO4 fraction had moderate anti-mildew effects; at the concentration of 200 μg/cm², all fungi strains were totally inhibited, except A niger, and Trichoderma viride, which were 83.5%, and 93.2% inhibited, respectively. The main ingredients of TO4 fraction were τ-cadinol, and α-cadinol, so we isolated and used the for anti-mildew effect tests; τ-cadinol, and α-cadinol showed moderate anti-mildew activities. Since C. micranthum twig essential oil, τ-cadinol, and α-cadinol were exhibited a great anti-mildew effects on paper, they are worth further investigations and utilization.

Keywords

anti-mildew effects essential oil τ-cadinol α-cadinol

Introduction

The climate in Taiwan is warm and humid, which is ideal for mold growth. Moreover, many molds are harmful microorganisms. Mycotoxins produced by mycelium are harmful to human health, such as with food poisoning caused by aflatoxins, and produce pathogenic microorganisms that cause human diseases, such as tinea pedis, ringworm, and allergies. In addition to being detrimental to medical hygiene and food preservation, mold in the environment also causes serious harm to building materials, textiles, and paper products.^1–3 Nowadays, several methods of anti-mildew and sterilization, including sterilization with dry or steam heat, and treatment with infrared rays, ultraviolet rays, microwaves, radiation, etc. In addition, chemical agents are commonly used for sterilization and disinfection. However, the use of synthetic chemicals will cause problems such as carcinogenicity, acute toxicity, and teratogenicity to the human body, and they degrade slowly in the environment and cause problems such as environmental pollution, so the use is restricted.⁴ Therefore, the use of plant essential oils or compounds as natural antifungal agents has gradually attracted many researchers’ attention in recent years.

Cinnamomum micranthum (Hayata) is an evergreen broad-leaved tree mainly found in Taiwan, China, and Vietnam.⁵ The wood of C. micranthum can be used in furniture- and ship-making due to its straightness and good quality.⁶ Three reports have identified leaf, trunk, and root essential oil constituents, respectively, but these reports did not assess the biological activity.^7–9 There are no studies have investigated the twig essential oil chemical composition and biological activity. Therefore, this report is made up of 2 parts: the first part, used hydrodistillation to obtain the leaf and twig essential oil of C. micranthum indigenous to Taiwan and its chemical compositions were determined using GC-FID and GC-MS. The second part determines the application of the essential oils of leaf and twig to paper for daily use and carries out anti-mildew tests. Then, compounds capable of inhibiting the mold were isolated and identified. The purpose of this study was to build a chemical foundation for the efficient multipurpose utilization strategies of the native species.

Results and Discussions

Hydrodistillation of C. micranthum leaf and twigs gave yellow essential oils with yields of 0.59 ± 0.01 and 0.38 ± 0.01 mL/100 g, based on the dry weight of leaves and twigs, respectively. All identified compounds, their percentages, and their linear retention index (LRI) values listed in order of elution from the DB-5 column (Table 1). Fifty-two compounds were identified, representing 99.6% of the leaf oil. Among the groups, non-terpenoids were predominated (57.8%), followed by oxygenated sesquiterpenes (19.5%), sesquiterpene hydrocarbons (11.7%), monoterpene hydrocarbons (9.2%), and oxygenated monoterpenes (1.4%). The main compound of the non-terpenoids was n-decanal (50.1%). Among the oxygenated sesquiterpenes, (E)-nerolidol (6.5%) was the chief component, and (E)-β-caryophyllene (3.8%) was the major compound of sesquiterpene hydrocarbons. (E)-β-ocimene (7.9%) was the major compound of the monoterpene hydrocarbons. After an exhaustive search, we have found 3 reports showed that the leaf oil of C. micranthum and their components.^7–9 In these reports, the major component of the leaf essential oil was n-decanal, which agrees with our present results.

Table 1.

Composition of the Leaf and Twig Essential Oils From Cinnamomum micranthum.

Compounds identified	LRILit^a	LRIExp^b	Concentration(%)		Identification^c
Compounds identified	LRILit^a	LRIExp^b	Leaf	Twig	Identification^c
n-Hexanal	801	801	-^d	0.2	MS, LRI, CO-ST
n-Nonane	900	900	-	0.2	MS, LRI, CO-ST
α-Thujene	930	928	0.4	0.2	MS, LRI, CO-ST
α-Pinene	939	938	-	0.5	MS, LRI, CO-ST
Camphene	954	954	0.4	0.7	MS, LRI, CO-ST
Sabinene	975	975	0.1	-	MS, LRI, CO-ST
n-Octanal	988	1002	0.5	-	MS, LRI, CO-ST
α-Terpinene	1017	1018	-	0.3	MS, LRI, CO-ST
p-Cymene	1024	1026	-	0.3	MS, LRI, CO-ST
1,8-Cineole	1031	1034	0.2	0.4	MS, LRI, CO-ST
(Z)-β-Ocimene	1037	1037	0.4	1.1	MS, LRI, CO-ST
(E)-β-Ocimene	1050	1051	7.9	16.4	MS, LRI, CO-ST
Linalool	1096	1098	0.6	1.6	MS, LRI, CO-ST
n-Nonanal	1100	1101	0.7	0.9	MS, LRI, CO-ST
allo-Ocimene	1132	1132	-	0.8	MS, LRI, CO-ST
trans-Pinocarveol	1139	1138	-	0.4	MS, LRI
n-Nonanol	1169	1172	-	1.0	MS, LRI
Terpinen-4-ol	1177	1180	-	0.4	MS, LRI, CO-ST
n-Decanal	1201	1203	50.1	10.6	MS, LRI, CO-ST
Bornyl acetate	1288	1285	0.5	0.7	MS, LRI, CO-ST
2-Undecanone	1294	1293	0.2	-	MS, LRI
n-Undecanal	1306	1306	0.2	-	MS, LRI, CO-ST
Decanoic acid	1366	1366	0.7	-	MS, LRI, CO-ST
α-Copaene	1376	1377	-	0.7	MS, LRI
(E)-β-Damascenone	1384	1383	0.1	-	MS, LRI
β-Elemene	1390	1389	0.1	0.5	MS, LRI
n-Dodecanal	1408	1408	1.4	-	MS, LRI, CO-ST
α-cis-Bergamotene	1412	1413	0.4	-	MS, LRI
(E)-β-Caryophyllene	1419	1421	3.8	0.5	MS, LRI
Aromadendrene	1441	1441	0.3	0.6	MS, LRI, CO-ST
α-Humulene	1454	1453	0.6	1.5	MS, LRI, CO-ST
(E)-β-Famesene	1456	1454	0.2	-	MS, LRI
β-Acoradiene	1470	1469	-	0.4	MS, LRI
trans-Cadina-1(6),4-diene	1477	1476	-	1.6	MS, LRI
γ-Muurolene	1479	1478	0.6	-	MS, LRI
α-Curcumene	1480	1480	-	1.1	MS, LRI
α-Amorphene	1484	1483	0.3	-	MS, LRI
β-Selinene	1490	1490	1.8	5.8	MS, LRI
α-Selinene	1498	1496	0.8	2.7	MS, LRI
β-Bisabolene	1505	1506	0.9	1.9	MS, LRI
γ-Cadinene	1513	1513	0.2	0.6	MS, LRI
δ-Cadinene	1523	1521	0.6	2.1	MS, LRI
Calamenene^e	1529	1528	-	0.9	MS, LRI
Zonarene	1529	1529	-	0.4	M5S, LRI
trans-γ-Bisabolene	1531	1531	0.3	0.3	MS, LRI
α-Cadinene	1538	1535	0.8	0.5	MS, LRI, CO-ST
α-Calacorene	1545	1545	-	0.5	MS,LRI
(E)-Nerolidol	1563	1563	6.5	0.6	MS, LRI, CO-ST
Dodecanoic acid	1566	1568	-	0.5	MS, LRI, CO-ST
Dendrolasin	1571	1571	0.5	-	MS, LRI
Caryophyllenyl alcohol	1572	1572	-	1.9	MS, LRI, CO-ST
Spathulenol	1578	1580	-	0.2	MS, LRI, CO-ST
Caryophyllene oxide	1583	1582	0.7	1.4	MS, LRI, CO-ST
Octanedioic acid, diethyl ester	1585	1584	3.7	0.5	MS, LRI
Globulol	1590	1588	0.5	-	MS, LRI, CO-ST
Carotol	1594	1594	0.5	-	MS, LRI
Cubeban-11-ol	1595	1596	0.3	0.2	MS, LRI
Khusimone	1604	1603	0.2	-	MS, LRI
Humulene epoxide II	1608	1608	0.3	0.1	MS, LRI
Junenol	1619	1619	0.2	-	MS, LRI
10-epi-γ-Eudesmol	1623	1624	1.3	0.8	MS, LRI
1-epi-Cubenol	1628	1629	1.0	0.3	MS,LRI
τ-Cadinol	1640	1638	0.9	18.3	MS, LRI, CO-ST
α-Cadinol	1654	1653	1.2	13.6	MS, LRI, CO-ST
Selin-11-en-4-α-ol	1660	1659	-	0.5	MS, LRI
epi-β-Bisabolol	1671	1670	0.6	-	MS, LRI
α-Bisabolol	1685	1685	1.9	-	MS, LRI
2,3-dihydro-Farnesol	1689	1689	0.3	0.1	MS, LRI
Eudesm-7(11)-en-4-ol	1700	1699	-	0.4	MS, LRI
(E)-Nerolidyl acetate	1717	1712	0.2	0.1	MS, LRI
γ-Costol	1746	1745	1.0	0.2	MS, LRI
dihydro-Columellarin	1900	1903	1.4	0.3	MS, LRI
n-Hexadecanoic acid	1960	1960	0.3	0.3	MS, LRI, CO-ST

Monoterpene hydrocarbons (%)			9.2	20.3
Oxygenated monoterpenes (%)			1.4	3.5
Sesquiterpene hydrocarbons (%)			11.7	22.6
Oxygenated sesquiterpenes (%)			19.5	39.0
Non-terpenoids (%)			57.8	14.2
Oil yield (mL/100 g)			0.59 ± 0.01	0.38 ± 0.01

^aLRILit, linear retention indices from a previous study.¹⁰

^bLRIExp, computed linear retention indices against a mixture of a continuous series of n-alkane hydrocarbons n-alkanes (C8-C30) run on DB-5 capillary column.

^cIdentification by: MS, comparison of NIST, and Wiley libraries mass spectra databases; LRI, linear retention index (LRI) is the same as the previous findings^10–12; CO-ST, co-injection/comparison to LRI and MS of standards.

^dNot detected.

^eCorrect isomer not determined.

In total, 55 compounds were identified, representing 99.6% of the twig essential oil. Among the twig oil compounds, the most dominant was oxygenated sesquiterpenes (39.0%), next in order were sesquiterpene hydrocarbons (22.6%), monoterpene hydrocarbons (20.3%), non-terpenoids (14.2%), and oxygenated monoterpenes (3.5%). Of the oxygenated sesquiterpenes, τ-cadinol (18.3%) and α-cadinol (13.6%) were the dominant compounds. The main component of the sesquiterpene hydrocarbons was β-selinene (5.8%), and the monoterpene hydrocarbons (E)-β-ocimene (16.4%) was the principal compound. Of the non-terpenoids, n-decanal (10.6%) was the main component. According to the literature, there is no report about the twig oil composition of C. micranthum. Therefore, this study is the first to report the composition of twig oil from C. micranthum in the literature.

In our previous research paper, essential oils and the ingredients have been shown to have inhibitory activities against bacteria, foodborne pathogens, mildew, and wood-decay fungi.^13–15 Mildew damage to paper products is very serious, it will not only destroy the appearance of paper products but also reduce the mechanical strength of paper products, making paper brittle, thinner, and more easily damaged. Therefore, it is necessary to evaluate the feasibility of anti-mildew treatment of paper when applying essential oils to food wrapping paper, kitchen paper, and wallpaper. In this experiment, according to the CNS2690 and TAPPI T487 cm-93 methods, 7 strain fungi were used, viz. Aspergillus clavatus, A. niger, Chaetonium globosum, Cladosporium cladosporioides, Myrothecium verrucaria, Penicillium citrinum, and Trichoderma viride. These pathogens are extremely harmful to paper products, cellulosic materials, leather, and wooden product.^16–18

Figure 1 displays the antifungal activities of leaf and twig parts of C. micranthum oil. At the same essential oil concentration of 200 μg/cm² on paper, the twig oil demonstrated clearly that the antifungal indices on paper are better than that of the leaf oil (Figure 1). At this concentration, twig oil suppressed totally the growth of A. clavatus, Ch. globosum, Cl. cladosporioides, M. verrucaria, and P. citrinum.

Figure 1.

Inhibitory effects of leaf and twig parts essential oils (200 μg/cm²) from Cinnamomum micranthum against 7 mildew fungi. A. c., Aspergillus clavatus; A. n., A. niger; Ch. g., Chaetonium globosum; Cl. c., Cladosporium cladosporoides; M. v., Myrothecium verrucaria; P. c., Penicillium citrinum; T. v., Trichoderma viride.

To evaluate the half-maximal inhibitory concentration (IC₅₀) values of the oils of leaf and twig, regression analysis was performed on the antifungal indices of the 2 oils versus fungi. The results are indicated in Table 2. The table displays that the IC₅₀ of twig oil resistant 7 mold pathogens was between 56.8 and 136.5 μg/cm²; for leaf oil was >200 μg/cm². According to the above results, the anti-mildew experiments showed that twig oil has the moderate antifungal activity on paper. Therefore, the twig oil was selected for further examine its composition.

Table 2.

The MIC and IC₅₀ Values (μg/cm²) of Leaf and Twig Essential Oils From Cinnamomum micranthum Against 7 Mildew Fungi.

Parts	A. c.		A. n.		Cl. c.		Ch. g.		M. v.		P. c.		T. v.
Parts	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC
Leaf	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200
Twig	56.8	100	136.5	>200	63.9	125	105.8	125	82.3	125	103.2	200	109.3	>200

Abbreviations: A. c., Aspergillus clavatus; A. n., A. niger; Ch. g., Chaetonium globosum; Cl. c., Cladosporium cladosporoides; M. v., Myrothecium verrucaria; P. c., Penicillium citrinum; T. v., Trichoderma viride.

The 2 chemotypes of Cinnamomum osmophloeum essential oils, cinnamaldehyde, and cinnamaldehyde–cinnamyl acetate oils were shown to require a concentration of 175 μg/cm² to inhibit the growth of half of T. viride strain.¹⁹ Our research shows that C micranthum essential oil is equally good at inhibiting molds.

To further understand the anti-mildew fungi activities of ingredients of the oil extracted from the twig of C. micranthum, we performed antifungal experiments using the 8 separate fractions (TO1-TO8) of the twig essential oil obtained in the above experiments. The results are shown in Figure 2. TO4 fraction had the best anti-mildew fungi activity with the highest antifungal indices against 8 mildew fungi. Among the mildew fungi, at a concentration of 200 μg/cm², A. clavatus, Ch. globosum, Cl. cladosporioides, M. verrucaria, and P. citrinum were completely inhibited by this fraction.

Figure 2.

Inhibitory effects of 8 fractions of twig essential oil against mildew fungi. Each experiment was performed 5 times and the data were averaged (n = 5). A. c., Aspergillus clavatus; A. n., A. niger; Ch. g., Chaetonium globosum; Cl. c., Cladosporium cladosporioides; M. v., Myrothecium verrucaria; P. c., Penicillium citrinum; T. v., Trichoderma viride.

The abovementioned results prove that TO4 exerted the best antibacterial activity. We further identified the ingredients from the TO4 fraction using GC and GC-MS. The results are shown in Table 3. Eight compounds were identified from the TO4 fraction; τ-cadinol (56.8%) and α-cadinol (36.6%) were the main ones. Finally, we used the 2 isolated and purified compounds (τ-cadinol and α-cadinol) to conduct antifungal tests against 7 mildew fungi. The MIC and IC₅₀ values are shown in Table 4. The results indicated that the active source compounds were α-cadinol and τ-cadinol. Previous studies support the contention that these compounds have significant activity for suppressing microbial growth.^20–24

Table 3.

Constituents and Contents of TO4 Fraction From Twig Essential Oil of Cinnamomum micranthum.

Compounds identified	LRILit^a	LRIExp^b	TO4	Identification^c
Compounds identified	LRILit^a	LRIExp^b	Concentration(%)	Identification^c
β-Selinene	1490	1490	2.1	MS, LRI
α-Selinene	1498	1496	1.2	MS, LRI
β-Bisabolene	1505	1506	0.9	MS, LRI
δ-Cadinene	1523	1521	0.7	MS, LRI
Caryophyllenyl alcohol	1572	1572	0.8	MS, LRI, CO-ST
Caryophyllene oxide	1583	1582	0.9	MS, LRI, CO-ST
τ-Cadinol	1640	1638	56.8	MS, LRI, CO-ST
α-Cadinol	1654	1653	36.6	MS, LRI, CO-ST

^aLRILit, linear retention indices from a previous study.¹⁰

^bLRIExp, computed linear retention indices against mixture of a continuous series of n-alkane hydrocarbons n-alkanes (C8-C30) run on DB-5 capillary column.

Table 4.

The MIC and IC₅₀ Values (μg/cm²) of 2 Major Compounds From Cinnamomum micranthum Against 7 Mildew Fungi.

Compounds	A. c.		A. n.		Cl. c.		Ch. g.		M. v.		P. c.		T. v.
Compounds	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC	IC₅₀	MIC
τ-Cadinol	23.8	37.5	53.1	75	31.8	50	45.8	75	33.9	50	50.1	75	51.9	75
α-Cadinol	20.2	37.5	51.8	75	13.6	25	21.9	50	16.8	37.5	41.2	75	31.2	50
Nystatine	<12.5	<12.5	<12.5	<12.5	<12.5	<12.5	<12.5	<12.5	<12.5	<12.5	<12.5	<12.5	<12.5	<12.5

Abbreviations: A. c., Aspergillus clavatus; A. n., A. niger; Ch. g., Chaetonium globosum; Cl. c., Cladosporium cladosporioides; M. v., Myrothecium verrucaria; P. c., Penicillium citrinum; T. v., Trichoderma viride.

Conclusion

This study investigated the leaf and twigs essential oils extracted from C. micranthum, with the leaf oil main components of n-decanal, (E)-β-ocimene, (E)-nerolidol, and (E)-β-caryophyllene, and the twig oil's major components were τ-cadinol, (E)-β-ocimene, α-cadinol, n-decanal, and β-selinene, by which the twig oil possesses pronounced anti-mildew effects on paper. The twig essential oil is mainly composed of oxygenated sesquiterpenes. Among these compounds, τ-cadinol and α-cadinol have been proved to have moderate anti-mildew activity on paper. In the future, studies should focus on determining the half-life of the essential oil on paper and developing formulations to extend the longevity of effective essential oil concentrations on paper to prevent mildew.

Experimental

Plant Materials

The plant material of leaves and twigs of C. micranthum were harvested from Lienhuachih Experimental Forest of the Taiwan Forestry Research Institute (TFRI) (elevation 350 m, N 23° 93′ 30″, E 120° 92′ 25″) in July 2021. The plant was identified by Dr Wu Chia-Chen from the TFRI. A voucher specimen (WCCLH-092) has been deposited at the department of wood cellulose, TFRI, Taiwan.

Isolation of the Leaf and Twigs Essential Oil

The fresh leaves and twigs of C. micranthum (10 kg) were using a Clevenger-type apparatus for 3 h. The leaf and twigs essential oils were then dried using anhydrous sodium sulfate and stored in a sealed vial at 4 °C until needed, respectively. The yields of essential oils and all experiment data were calculated and shown as the mean ± standard deviation of triplicate analyses.

Essential Oils Analysis

The C. micranthum leaves and twigs oils were analyzed by GC-FID and GC-MS. For GC-FID analysis was carried out on a Hewlett-Packard 6890 gas chromatograph with a flame ionization detector (FID), fitted with a DB-5 capillary columns (5% phenyl 95% methylpolysiloxane, length = 30 m, ID = 0.25 mm, film thickness = 0.25 μm). The oven temperature was programmed from 50 °C constant for 2 min, and then increased at 5 °C/min until reached 250 °C. The injector and detector temperatures were programmed at 270 °C and 250 °C, respectively. The carrier gas was hydrogen (H₂) with a flow rate of 1 mL/min. The split ratio was 1:10, and 1 μL of samples (1/100, vol/vol, in ethyl acetate) were injected. Linear retention indices (LRI) values for all compounds were calculated with reference to the C8-C30 n-alkanes homologous series.¹⁶ The relative proportions of essential oil compositions were computed by internal normalization from the GC-FID peak areas without using correction for response factors. Results are summarized in Table 1 and are reported according to their eluted in order of the compounds on the DB-5 capillary column.

GC-MS analyses were performed on a Hewlett-Packard 6890/5973 GG-MS system, equipped with a DB-5 capillary columns, the carrier gas was helium (He) (1 mL/min, 99.995% purity) with the same parameters as for analytical GC-FID. The MS conditions were full scan mode (scan time: 0.3 s), and the mass range was m/z 30-500 in the electrospray ionization mode at 70 eV.

Component Identification

Compounds identification were based on calculated LRI comparison with either those in the literature^15–17 or with the standard pure compounds and by matching their MS with those obtained in the “NIST 17” and “WILEY 11” libraries and in some components by co-injection with standard pure compounds.

Anti-Mildew Assays

According to the regulations of CNS2690 and TAPPI T487 cm-93 methods, A. clavatus (ATCC 1007), A. niger (ATCC 6275), Ch. globosum (ATCC 6205), Cl. cladosporioides (ATCC 13276), M. verrucaria (ATCC 9095), P. citrinum (ATCC 9849), and T. viride (ATCC 8678) molds were tested. The mildew fungi were obtained from the Culture Collection and Research Center of the Food Industry Research and Development Institute, Hsinchu, Taiwan. These fungal strains were inoculated on potato-dextrose agar (PDA) medium, respectively, and placed in an incubator at 28 °C for 10 days. Then, they were placed in sterilized boxes and the spores are scraped onto the cultural medium using a platinum wire. Next, spores were shaken in 10 mL sterile water until they were distributed evenly, and then filter to complete the preparation of a single spore suspension with a concentration of 1 × 10⁵ CFU/mL. The spore suspension must be used within 24 h. Prepared 5 × 5 cm² filter paper pieces and sterilized them. The different concentrations (0-200 μg/cm²) of essential oils, purified compounds, and 0.5 mL of the spore suspension were evenly spread on the filter paper, respectively. Finally, spread the filter paper pieces on the cultural medium and placed in the incubator and cultivated for 14 days. The growth of fungi and the area of hyphae were measured. The anti-mildew effects were estimated according to the inhibition areas, IC₅₀ and MIC values. Each test was repeated 5 times, and the data were averaged.

Isolation and Purification of Twig Oil Components

The crude twig essential oil of C. micranthum (20 g) mixed with silica gel 60 (60 g) (200-300 mesh, Fluka) was chromatographed on a silica gel column (600 g) by gradient elution with n-hexane and ethyl acetate (the proportion of 2 solvents changed from 100:0 to 0:100). Then, collected fractions were monitored by TLC and those with similar profile were combined to produce 8 subfractions (TO1-TO8). The yields of each fraction were 5.3% (TO1), 8.2% (TO2), 18.3% (TO3), 42.8% (TO4), 10.3% (TO5), 8.2% (TO6), 4.1% (TO7), and 2.7% (TO8). According to the anti-mildew assay, the TO4 fraction shown the best antifungal activity. The pure τ-cadinol (RT: 22.1 min) and α-cadinol (RT: 31.2 min) compounds were separately obtained from fraction TO4 by semipreparative HPLC (column: Si-60 column, mobile phase: EtOAc/n-C₆H₁₄ = 30/70, flow rate: 1 mL/min). The structures of 2 compounds were confirmed by comparing ¹H-NMR, ¹³C-NMR, and EI-MS spectral data with the our previously literature values.²⁵

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.

Funding

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

ORCID iD

Chen-Lung Ho

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