Antimildew Effects of Plectranthus amboinicus Leaf Essential Oil on Paper

Abstract

In this study, we evaluated the antimildew effects on paper of Plectranthus amboinicus (Lour.) Spreng leaf essential oil prepared through hydrodistillation. The oil was isolated with a Clevenger-type apparatus and then characterized through gas chromatography with flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 43 compounds were identified, representing 99.5% of the oil. The main ingredients were carvacrol (50.0%), γ-terpinene (13.1%), and β-caryophyllene (11.3%). The leaf oil had excellent antimildew effects; its minimum inhibitory concentration (MIC) values against Aspergillus clavatus, A. niger, Cladosporium cladosporioides, Chaetomium globosum, Myrothecium verrucaria, Penicillium citrinum, and Trichoderma viride were 100, 200, 75, 75, 100, 150, and 150 μg/cm² respectively. The leaf oil was further partitioned into 6 derivative fractions (PO1-PO6). PO2 had excellent antimildew effects on the abovementioned strains; at a concentration of 100 μg/cm², all were 100% inhibited, except A. niger, which was 75.8% inhibited. Carvacrol, the main ingredient of PO2, was then isolated and used for antimildew tests; carvacrol had excellent antimildew activities. Since both P. amboinicus leaf essential oil and carvacrol were proved to have excellent antimildew effects on paper, they are worth further research and development.

The climate in Taiwan is hot and rainy and such a climate is suitable for the growth of molds, including mildew. ¹ Modern advanced technology makes numerous antimildew methods possible. Physical methods include dry/moist heat sterilization, anoxic fumigation, freezing, and ultraviolet radiation. Chemical methods are also used, but since microorganisms can be drug-resistant, it is necessary to develop new antimildew products. ² Therefore, plant essential oils or compounds found in them have become a research focus for antimildew materials. Our previous study showed that Chamaecyparis formosensis heartwood essential oil had excellent antimildew activities; at a concentration of 100 µg/mL, it could fully inhibit the growth of Aspergillus clavatus, A. niger, Cladosporium cladosporioides, Chaetomium globosum, Myrothecium verrucaria, and Trichoderma viride. ³

Plectranthus amboinicus (Lour.) Spreng is a Lamiaceae perennial herb. It is small (30-90 cm high), aromatic, and has fleshy leaves. While Malaysia, Brazil, China, and India are its places of origin, it is also an indigenous species of Taiwan. ⁴ It is widely used in folk medicine to treat cold, asthma, constipation, headache, cough, fever, and skin diseases. ^5,6 There is a lot of literature on the ingredients and bioactivities (antibacterial and antioxidant) of P. amboinicus essential oil: Oliveira et al ⁷ reported that it could inhibit yeasts such as Candida albicans, Candida guilliermondii, and Candida stellatoidea, and Murthy et al ⁸ found that 60% growth of Aspergillus ochraceus could be inhibited with 10 µL oil. Given these studies, P. amboinicus is worth further research.

This present study consists of 2 parts: the first reports the process of extracting the essential oil from P. amboinicus indigenous to Taiwan through hydrodistillation and analyzing the ingredients through GC-FID and GC-MS, and the second part shows how the essential oil can be applied to paper for daily use; antimildew tests were carried out, and the compounds able to inhibit molds were isolated and identified. The purpose of this study was to establish a chemical basis for effective multipurpose utilization of this species.

Based on the dry weight of leaves, hydrodistillation of P. amboinicus produced yellow-colored oil with a yield of 0.58 ± 0.03 mL/100 g. All compounds detected are listed in order of their elution from a DB-5 column (Table 1); 43 compounds were identified. Oxygenated monoterpenes were predominant (52.5%), followed by monoterpene hydrocarbons (26.9%), sesquiterpene hydrocarbons (16.4%), and oxygenated sesquiterpenes (3.7%). Among the oxygenated monoterpenes, carvacrol (50.0%) was the chief compound. Of the monoterpene hydrocarbons, γ-terpinene (13.1%) was the main compound, whereas β-caryophyllene (11.3%) was the major sesquiterpene hydrocarbon.

Table 1

Chemical Composition of the Leaf Essential Oil of Plectranthus amboinicus.

Peak no.	Constituents	KI a	KI b	Concentration (%)	Identification c
1	α-Thujene	930	931	0.7	KI, MS, ST
2	α-Pinene	939	939	0.3	KI, MS, ST
3	Camphene	954	954	0.1	KI, MS, ST
4	Sabinene	975	976	t d	KI, MS, ST
5	β-Pinene	979	980	0.1	KI, MS, ST
6	Myrcene	991	992	1.1	KI, MS, ST
7	α-Phellandrene	1003	1003	0.4	KI, MS, ST
8	α-Terpinene	1017	1017	2.8	KI, MS, ST
9	p-Cymene	1025	1026	7.8	KI, MS, ST
10	Limonene	1029	1031	t	KI, MS, ST
11	β-Phellandrene	1030	1033	t	KI, MS
12	cis-Ocimene	1037	1038	0.2	KI, MS
13	γ-Terpinene	1060	1061	13.1	KI, MS, ST
14	Terpinolene	1089	1090	0.3	KI, MS, ST
15	p-Cymenene	1091	1093	0.1	KI, MS, ST
16	Linalool	1097	1100	0.3	KI, MS, ST
17	1,3,8-p-Menthatriene	1110	1112	0.1	KI, MS
18	Camphor	1146	1148	0.3	KI, MS, ST
19	Borneol	1169	1170	0.1	KI, MS, ST
20	Terpinene-4-o1	1177	1180	1.3	KI, MS, ST
21	α-Terpineol	1189	1191	0.2	KI, MS, ST
22	Thymol	1290	1291	0.4	KI, MS, ST
23	Carvacrol	1299	1301	50.0	KI, MS, ST
24	β-Caryophyllene	1419	1420	11.3	KI, MS, ST
25	cis-Muurola-3.5-diene	1450	1452	0.1	KI, MS
26	α-Himachalene	1451	1453	0.1	KI, MS
27	α-Caryophyllene	1455	1457	3.5	KI, MS, ST
28	γ-Muurolene	1480	1481	0.2	KI, MS
29	α-Amorphene	1485	1486	0.1	KI, MS
30	β-Selinene	1490	1491	t	KI, MS
31	α-Muurolene	1500	1502	0.3	KI, MS
32	γ-Cadinene	1514	1516	0.1	KI, MS
33	δ-Cadinene	1523	1525	0.1	KI, MS
34	trans-Calamenene	1529	1531	0.1	KI, MS
35	trans-Cadina-1(2),4-diene	1535	1537	0.3	KI, MS
36	cis-Cadinene	1554	1556	0.3	KI, MS
37	Caryophyllene oxide	1583	1582	0.9	KI, MS, ST
38	Guaiol	1601	1603	0.3	KI, MS
39	β-Cedrene epoxide	1623	1625	0.4	KI, MS
40	10-epi-γ-Eudesmol	1624	1627	0.2	KI, MS
41	γ-Eudesmol	1632	1634	0.4	KI, MS
42	β-Eudesmol	1651	1653	0.3	KI, MS, ST
43	α-Eudesmol	1654	1656	1.3	KI, MS
	Identified compound (%)			99.5
	Monoterpene hydrocarbons			26.9
	Oxygenated monoterpenes			52.5
	Sesquiterpene hydrocarbons			16.4
	Oxygenated sesquiterpenes			3.7
	Yield (mL/100 g)			0.58 ± 0.03

KI, Kovats index; ST, authentic standard compounds.

a

Kovats indices on a DB-5 column with reference to n-alkanes. ⁹

b

Relative retention indices, experimental: n-alkanes (C9-C24) were used as reference points in the calculation of relative retention indices.

c

MS, National Institute of Standards and Technology and Wiley library spectra, and the literature.

d

Trace <0.1%.

In reviewing the literature on P. amboinicus leaf oil, there are numerous chemotypes, for instance, Indian oils were found to have the following as their main components: carvacrol, thymol, eugenol, and chavivol ¹⁰ ; thymol ¹¹ ; thymol, carvacrol, and 1,8-cineole ¹² ; carvacrol, p-cymene, and β-caryophyllene ^8,13 ; eugenol and methyl eugenol ¹⁴ ; carvacrol, β-caryophyllene, and patchoulane ¹⁵ ; carvacrol, thymol, undecanal, and humulene ¹⁶ ; and thymol, γ-terpinene, and p-cymene. ¹⁷ In Mauritius, the P. amboinicus leaf oil was found to have carvacrol and camphor as the main components, ¹⁸ whereas in Martinique leaf oil, carvacrol was the main component, and in Cambodia, thymol, carvacrol, and p-cymene. ¹⁹ In Cuba and Venezuela, the P. amboinicus leaf oils mainly contained carvacrol, ^20,21 whereas in the Archipelago of Comoros, the oil contained mainly carvacrol, camphor, and δ-3-carene. ²² In Uganda, the main components were linalool, carvacrol, nerol acetate, and geranyl acetate, ¹⁷ while the Malaysian oil had carvacrol, δ-3-carene, and camphor ²³ ; and thymol, 1,8-cineole, and carvacrol as the main components. ²⁴ In the present study of Taiwan P. amboinicus leaf oil, the main constituents were carvacrol, γ-terpinene, and β-caryophyllene. These results are different from the aforementioned literature and represent a first publication of such a P. amboinicus leaf oil.

Our previous study showed that the essential oil and its ingredients had excellent antibacterial activities. ^{25 –27} In order to apply them to paper for daily use, the feasibility of developing an antimildew treatment of paper needed to be evaluated. Regulations CNS2690 and TAPPI T487 cm-93 were adopted for this experiment, with A. clavatus, A. niger, Cl. cladosporioides, Ch. globosum, M. verrucaria, Penicillium citrinum, and T. viride as the target strains. Table 2 lists the antimildew activities on paper of different concentrations of P. amboinicus essential oil. For full inhibition, a concentration of 75 µg/cm² was required for Cl. cladosporioides and Ch. globosum; 100 µg/cm² for A. clavatus and M. verrucaria; 150 µg/cm² for P. citrinum and T. viride; and 200 µg/cm² for A. niger.

Table 2

Inhibitory Effects of Plectranthus amboinicus Leaf Essential Oil Against Mildew Fungi.

Mildew	Concentration (μg/cm2)
	50	75	100	125	150	200
A. c.	38.9	85.2	100	100	100	100
A. n.	0	38.6	68.3	86.5	93.2	100
Cl. c.	48.1	100	100	100	100	100
Ch. g.	38.3	100	100	100	100	100
M. v.	49.8	83.6	100	100	100	100
P. c.	0	58.6	85.2	93.2	100	100
T. v.	0	61.3	81.3	89.3	100	100

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.

Cinnamomum osmophloeum essential oil, which mainly consisted of cinnamaldehyde, required a concentration of 75 µg/cm² to inhibit fully Ch. globosum, ²⁸ and so our research shows that P. amboinicus essential oil is equally good at inhibiting molds.

Since the MIC values of P. amboinicus leaf essential oil for each type of strain used in this experiment were high, we isolated the compounds from the oil by silica gel column chromatography. The eluents were separated into 6 fractions (PO1-PO6) with solvents of different polarities.

Figure 1 illustrates the antimildew effects of the fractions on the selected strains. PO2 had the best performance: at a concentration of 100 µg/cm², it completely inhibited all the strains except A. niger (75.8% inhibition). We therefore isolated and identified the ingredients from PO2 by GC and GC-MS; the results are shown in Table 3. Nine compounds were found, the predominant ones being carvacrol (88.0%), γ-terpinene (6.3%), p-cymene (3.1%), and α-terpinene (1.3%).

Figure 1

Inhibitory effects of 6 fractions of leaf essential oil against mildew fungi. Each experiment was performed 5 times and the data 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; and T. v.: Trichoderma viride.

Table 3

Constituents and Contents of PO2 Fraction From Leaf Essential Oil of Plectranthus amboinicus.

Constituents	KI a	KI b	Concentration (%)	Identification c
			PO2
Myrcene	991	992	0.3	KI, MS, ST
α-Terpinene	1017	1017	1.3	KI, MS, ST
p-Cymene	1025	1026	3.1	KI, MS, ST
γ-Terpinene	1060	1061	6.3	KI, MS, ST
Terpinene-4-o1	1177	1180	0.3	KI, MS, ST
α-Terpineol	1189	1191	0.1	KI, MS, ST
Thymol	1290	1291	0.3	KI, MS, ST
Carvacrol	1299	1301	88.0	KI, MS, ST
β-Caryophyllene	1419	1420	0.3	KI, MS, ST
Identified compound (%)			100.0

KI, Kovats index; ST, authentic standard compounds.

a

Kovats indices on a DB-5 column with reference to n-alkanes. ⁹

b

Relative retention indices, experimental: n-alkanes (C9-C24) were used as reference points in the calculation of relative retention indices.

c

MS, National Institute of Standards and Technology and Wiley library spectra, and the literature.

Accordingly, we continued to isolate the compounds from subfraction PO2 by high performance liquid chromatography (HPLC). The individual compounds were identified from infrared radiation (IR), Carbon-13 nuclear magnetic resonance (¹³C NMR) and proton nuclear magnetic resonance (¹H NMR) spectroscopic, and mass spectrometric data. Carvacrol was identified through spectral analyses. ²⁹

Finally, we used 4 isolated compounds to carry out antimildew tests. The MIC and IC₅₀ values are shown in Table 4. Carvacrol had excellent antimildew activities, which confirmed some previous studies. ^{30 –32} Hence, we concluded that P. amboinicus essential oil and carvacrol had excellent antimildew activities, and they could be applied to paper products. In addition, the oil is aromatic enough to serve multiple purposes.

Table 4

MIC and IC₅₀ Values (mg/cm²) of 4 Major Compounds From Plectranthus amboinicus Leaf Oil Against Mildew Fungi.

Compounds	A. c.		A. n.		C. c.		C. g.		M. v.		P. c.		T. v.
	IC50	MIC	IC50	MIC	IC50	MIC	IC50	MIC	IC50	MIC	IC50	MIC	IC50	MIC
α-Terpinene	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200
p-Cymene	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200
γ-Terpinene	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200	>200
Carvacrol	26.3	50	48.3	150	18.6	37.5	39.6	75	32.8	50	28.6	50	31.8	50
Nystatin	<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

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.

Experimental

Plant Materials

Fresh leaves of P. amboinicus were collected in March 2017 from Taipei Botanical Garden (Taipei County, elevation 1050 m, N 24°39′86″, 121°40′68″) for subsequent extraction and analysis. The samples were compared with specimen No. ou-0189 from the herbarium of National Chung Hsing University (NCHU) and were positively identified by Prof. Yen-Hsueh Tseng of NCHU. The voucher specimen (CLH-061) was deposited in the NCHU herbarium.

Isolation of Leaf Essential Oil

The air-dried leaves of P. amboinicus (1 kg) were hydrodistilled for 3 hours using a Clevenger-type apparatus. After distillation, the volume of oil obtained was measured, and the oil stored in a glass container hermetically sealed with rubber lids and aluminum foil to shield the contents from light. The container was kept refrigerated at <4°C until use. The oil yielded and all the test data are the average of triplicate analyses.

Essential Oil Analysis

A Hewlett-Packard HP 6890 gas chromatograph equipped with a DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 μm film thickness, J&W Scientific) and a FID detector were used for the quantitative determination of oil components. Oven temperature was programmed as follows: 50°C for 2 minutes, rising to 250°C at 5°C/min. Injector temperature: 270°C. Carrier gas: Helium with a flow rate of 1 mL/min. Detector temperature: 250°C, split ratio: 1:10. Diluted samples (1.0 µL, 1/100, v/v, in ethyl acetate, EA) were injected manually in the split mode. Identification of the oil components was based on their retention indices and mass spectra obtained from GC/MS analysis on a Hewlett-Packard HP 6890/HP5973 equipped with a DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 μm film thickness, J&W Scientific). The GC analysis parameters are listed above and the MS ones were obtained (full scan mode: scan time: 0.3 s, mass range was m/z 30-500) in the electron ionization mode at 70 eV. All the data were the average of triplicate analyses.

Component Identification

Identification of the leaf oil constituents was based on comparisons of Kovats index, ³² retention times, and mass spectra with those obtained from authentic standards and/or the National Institute of Standards and Technology and Wiley libraries spectra, and the literature, ^9,33 respectively.

Antimildew Assays

The mold strains used in this experiment were selected according to CNS2690 and TAPPI T487 cm-93; they were 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). Each strain was cultured in potato dextrose agar. Microbial strains were obtained from the Bioresource Collection and Research Center (BCRC) of Taiwan. The method by CNS2690 was used. The strains were cultured at 28°C for 10 days in incubators. Then, they were put into sterilized boxes and platinum wires were used to scratch down the spores onto the culture medium. Next, the spores were oscillated in 10 mL sterilized water until they were evenly distributed. Spore suspensions containing 1 × 10⁵ CFU/mL were prepared; these needed to be used within 24 hours. Filter paper pieces measuring 5 × 5 cm² were prepared and sterilized. Then, the essential oil (0-200 µg/cm²), purified compounds (0-200 µg/cm²), and 0.5 mL spore suspension were evenly applied onto the paper. Finally, the pieces were spread on the culture medium and put into incubators for 14 days. The growing conditions and areas of the strains were observed and measured, and the antimildew activities were evaluated based on the inhibition areas, MIC and IC₅₀ values. Each test was repeated 5 times, and the data were averaged.

Isolation and Purification of Leaf Oil Components

The leaf oil of P. amboinicus (50 g) was applied to a silica gel column and eluted with n-hexane/EA (gradient elution was performed from 100/0 to 0/100), and then each fraction was controlled by thin-layer chromatography (TLC). The compounds on TLC were visualized with UV 254 nm and UV 366 nm, iodine vapor, and 1% vanillin/H₂SO₄. The band fractions from the leaf oil containing the same compounds were combined to produce 6 subfractions (PO1-PO6). Subsequently, HPLC (column: diol, mobile phase: n-C₆H₁₄/EtOAc/ acetone = 80/15/5, flow rate: 1 mL/min) was engaged to separate and purify the components from PO2 with superior antimildew properties. Individual chemical structures were determined through MS, IR, and ¹³C NMR and ¹H NMR spectroscopic methods.