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molecules
Article
Chemical Composition, Algicidal, Antimicrobial,
and Antioxidant Activities of the Essential Oils of
Taiwania flousiana Gaussen
Hongmei Liu 1, Jiguang Huang 1, Sifan Yang 2, Jialin Li 1and Lijuan Zhou 1,*
1Key Lab of Natural Pesticides & Chemical Biology, Ministry of Education, South China Agricultural
University, Guangzhou, Guangdong 510642, China; liuhongmei@stu.scau.edu.cn (H.L.);
hnnyzx@scau.edu.cn (J.H.); 1945619358@stu.scau.edu.cn (J.L.)
2Organic Agriculture, Wageningen University and Research, Gelderland, 6708 PB Wageningen,
The Netherlands; sifan.yang@wur.nl
*Correspondence: zhoulj@scau.edu.cn
Received: 27 November 2019; Accepted: 14 February 2020; Published: 20 February 2020
Abstract:
Taiwania flousiana (T. flousiana) Gaussen is a precious wood in the family Taxodiaceae.
This study investigated the chemical components of the essential oil from the stem bark of T. flousiana
and its algicidal, antifungal, and antioxidant properties. Sixty-nine compounds representing 89.70%
of the stem bark essential oil were identified by GC-MS. The essential oil showed strong anti-algae,
anti-bacteria, and anti-fungus activities against the tested species, and antioxidant activities. The IC
50
values of the essential oil against chlorophyll a, chlorophyll b, and the total chlorophyll of Spirogyra
communis (a species of algae), 24–96 h after the treatment, ranged from 31.77 to 84.92
µ
g/mL, while the
IC
50
values of butachlor ranged from 40.24 to 58.09
µ
g/mL. Ultrastructure changes revealed by the
transmission electron microscopy indicated that the main algicidal action sites were the chloroplast
and cell wall. The essential oil showed antifungal activities on Rhizoctonia solani (
EC50 =287.94 µg/mL
)
and Colletotrichum gloeosporioiles (
EC50 =378.90 µg/mL
). It also showed bactericidal activities on
Ralstonia solanacearum and Staphylococcus aureus, with zones of inhibition (ZOIs) being 18.66 and
16.75 mm, respectively at 40
µ
g/disk. Additionally, the essential oil possessed antioxidant activity
estimated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method (IC
50
=33.51
µ
g/mL; IC
50
value of the
positive control ascorbic acid was 7.98
µ
g/mL). Thus, the essential oil of this plant might be used
as a possible source of natural bioactive molecules in agrochemical industry as well as in food and
cosmetic industries.
Keywords:
Taiwania flousiana Gaussen; essential oil; algicidal activity; antimicrobial activity;
antioxidant activity; algicidal mechanism
1. Introduction
T. flousiana (a synonym of Taiwania cryptomerioides Hayata), belonging to the family Taxodiaceae,
is endemic to China. T. flousiana has been considered as one of the most precious woods in
China for its outstanding quality. It has been used in construction industry, furniture industry,
and paper industry
[1–3]
. Thus, the cultivation and plantation of T. flousiana have received significant
attention
[4–8]
. The chemical constituents of the extract from the bark of T. flousiana have been
investigated [
9
] and there were patents on the use of the chemicals from T. flousiana in medicinal
industry [
10
]. Recently, a new chemical isolated from T. flousiana with strong herbicidal activity has
been patented also [
11
]. However, it has not been previously investigated chemically for its essential oil.
In the production process of farm produce, various undesirable biotic factors such as algae and
microbes can cause great loss of quantity and quality. Spirogyra (Zygnemataceae, Zygnematales) is a
Molecules 2020,25, 967; doi:10.3390/molecules25040967 www.mdpi.com/journal/molecules
Molecules 2020,25, 967 2 of 17
genus in the Class Zygnematophyceae (Conjugatophyceae), which is a member of the Infrakingdom
Streptophyta. Spirogyra communis (Hassall) Kuetzing in the genus is widely distributed in freshwater
habitats including flowing water, permanent ponds, and temporary pools and can cause great loss to
farm produce [
12
,
13
]. Microbes such as Rhizoctonia solani,Fusarium moniliforme Sheld, and Fusariun
oxysporun, etc., are common pathogens with a great diversity of host plants and can significantly
reduce the quantity and quality of farm produce [
14
–
16
]. Normally, synthetic chemicals are extensively
used to control them. However, the resistance issues caused by synthetic control agents are very
common. Meanwhile, synthetic control agents lack selectivity and are toxic to non-target organisms.
As an important part of pesticides, the algicide resistance has been reported. Meanwhile, synthetic
algicides such as diuron are toxic and have negative impact on the environment. Similarly, synthetic
antimicrobial chemicals have the same problems [
17
,
18
]. Therefore, as an alternative to these synthetic
control agents, natural compounds and extracts from plants are the main sources [
19
]. Among them,
essential oil has received significant attention [20,21].
Consumers need natural control agents not only in the production process of farm produce,
but also in food preservation process. Nowadays, antioxidants have been widely used as food additives
to provide protection against oxidative degradation of foods by free radicals. Normally, synthetic
antioxidants such as butylated hydroxyanisole have been widely used. However, the toxicity of
synthetic antioxidants has been questioned. Thus, the development of natural antioxidants is warmly
desired [
22
]. Plant-derived essential oil has also received significant attention in this field [
23
]. In this
work, we extracted the essential oil of T. flousiana and further (i) identified its chemical constituents;
(ii) investigated its algicidal, antifungal, antibacterial, and antioxidant activities; (iii) characterized its
mechanisms as an algicide.
2. Results
2.1. Chemical Components Identified in the Essential Oil
The major components of the essential oil identified from T. flousiana are listed in Table 1. The yield of the
essentialoilextractedfromT. flousianawas0.31% (v/w). TheGC-MSchromatogramof theessentialoilis shown
in Figure 1. In total, 69 components were identified and accounted for 89.70% of the total oil composition.
The oil composition was dominated by the presence of hexadecanoic acid comprising 27.13% from
total, followed by 2-penten-1-ol, 3-methyl-5-[octahydro-4, 5-dimethyl-7a-(1-methylethenyl)-1H-inden-4-yl]-
(16.16%), linoleic acid (13.48%), podocarpa-6,8,11,13-tetraen-12-ol, 13-isopropyl-, acetate (7.56%),
ferruginol (6.52%), and
α
-linolenic acid (5.41%). The contents of abietatriene, tetradecanoic acid,
pentadecanoic acid ranged from 1.04 to 1.37%. The others were less than 1.00% (Table 1).
Figure 1. The GC-MS chromatogram of the essential oil.
Molecules 2020,25, 967 3 of 17
Table 1. Percent concentration (%) of chemical constituents of T. flousiana stem bark essential oils.
No. Compounds Molecular
Formula Percentage (%) RI aRI b(Reference) Methods of
Identification
Fatty Acids
1 Nonanoic acid C9H18O20.03 1264 2192 [24] a,c,d
2 Decanoic acid C10H20O20.03 1361 2298 [25] a,c,d
3 Dodecanoic acid C12H24O20.18 1560 2503 [24] a,c,d
4 Tridecanoic acid C13H26 O20.02 1657 2617 [25] a,c,d
5 Tetradecanoic acid C14H28O21.10 1764 2670 [25] a,c,d
6 14-Pentadecenoic acid C15H28O20.52 1848 3181 [26] a,c,d
7 Pentadecanoic acid C15H30O21.37 1868 2822 [25] a,c,d
8 Palmitoleic acid C16H30O20.81 1946 2948 [27] a,c,d
9 9-Hexadecenoic acid C16H30O20.09 1952 - a,c,d
10 Hexadecanoic acid C16 H32O227.13 1992 2931 [25] a,c,d
11 cis-10-Heptadecenoic acid C17H32 O20.08 2055 - a,c,d
12 Heptadecanoic acid C17H34O20.20 2068 2305 [28] a,c,d
13 Linoleic acid C18H32O213.48 2155 3157 [29] a,c,d
14 α-Linolenic acid C18H30O25.41 2160 3193 [29] a,c,d
Monoterpenes
15 α-Terpineol C10H18 O 0.03 1194 1706 [24] a,c,d
16 Carvacrol C10H14O 0.02 1301 2241 [30] a,c,d
Sesquiterpenes
17 (-)-Spathulenol C15 H24O 0.09 1599 2144 [25] a,c,d
18 Widdrol C15H26O 0.12 1611 2179 [31] a,c,d
19 Cedrol C15H26O 0.13 1614 2093 [32] a,c,d
20 6-Methyl-2-(4-methylcyclohex-3-en-1
-yl)hepta-1,5-dien-4-ol C15H24O 0.05 1631 - a,c,d
21 8-Cedren-13-ol C15H24O 0.11 1637 2199 [33] a,c,d
22 γ-Eudesmol C15H26O 0.12 1640 2193 [24] a,c,d
23 epi-α-Muurolol C15H26O 0.06 1650 1621 [34] a,c,d
24 β-Eudesmol C15H26O 0.06 1660 2257 [24] a,c,d
25 T-cadinol C15H26O 0.13 1662 2187 [31] a,c,d
26 Germacra-4(15),5E,10(14)-trien-1β-ol C15H24O 0.05 1667 - a,c,d
27 Humulenol-II C15 H24O 0.09 1681 - a,c,d
28 α-Bisabolol C15H26O 0.05 1688 2232 [35] a,c,d
29 Longifolaldehyde C15H24O 0.02 1692 - a,c,d
Molecules 2020,25, 967 4 of 17
Table 1. Cont.
No. Compounds Molecular
Formula Percentage (%) RI aRI b(Reference) Methods of
Identification
30 β-Acoradienol C15H24O 0.05 1787 - a,c,d
31 Drimenol C15H26O 0.04 1817 1772 [36] a,c,d
Diterpenes
32 Ambrial C16H26O 0.07 1809 - a,c,d
33 Biformene C20H32 0.32 1937 1907 [37] a,c,d
34 Cembrene C20H32 0.16 1958 - a,c,d
35 Manoyl oxide C20H34O 0.64 2000 2376 [31] a,c,d
36 13-epi-Manoyl oxide C20H34O 0.11 2021 2335 [38] a,c,d
37 Manool C20H34O 0.03 2027 2180 [39] a,c,d
38 Geranyl linalool C20H34O 0.06 2033 1912 [37] a,c,d
39 Abietatriene C20H30 1.04 2063 2065 [37] a,c,d
40 Thunbergol C20H34O 0.02 2076 - a,c,d
41 Abieta- 7, 13- diene C20H32 0.04 2088 - a,c,d
42
2-Penten-1-ol,
3-methyl-5-[octahydro-4,5-dimethyl-7a-
(1-methylethenyl)-1H-inden-4-yl-
C20H34O 16.16 2121 - a,c,d
43 Sandaracopimarinal C20H30O 0.17 2192 - a,c,d
44 Larixol C20H34O20.28 2211 - a,c,d
45 4-epi-Dehydroabietol C20H30O 0.77 2227 - a,c,d
46 Nimbiol C18H24O20.26 2258 - a,c,d
47 Isopimara-7,15-dien-3-one C20H30O 0.26 2261 - a,c,d
48 trans-Totarol C20H30O 0.19 2286 2280 [32] a,c,d
49 Dehydroabietal C20H28O 0.78 2305 - a,c,d
50 Podocarpa-6,8,11,13-tetraen-12-ol,
13-isopropyl-, acetate C22H30O27.56 2332 - a,c,d
51 Ferruginol C20H30O 6.52 2339 2327 [37] a,c,d
52 Hinokione C20H28O20.51 2463 - a,c,d
53 Dronabinol C21H30O20.11 2515 - a,c,d
Esters
54 Methyl pentadecanoate C15H30O20.22 1825 2099 [29] a,c,d
55 Diisobutyl phthalate C16H22O40.14 1870 - a,c,d
56 Cyperolactone C15H22O20.02 1873 2480 [40] a,c,d
57 Methyl hexadecanoate C17H34O20.46 1926 2226 [41] a,c,d
Molecules 2020,25, 967 5 of 17
Table 1. Cont.
No. Compounds Molecular
Formula Percentage (%) RI aRI b(Reference) Methods of
Identification
58 Methyl linoleate C19H34 O20.35 2099 2490 [42] a,c,d
59 Methyl linolenate C19H32O20.17 2105 2478 [42] a,c,d
Phenols
60 2-Allyl-4-methylphenol C10H12O 0.05 1373 - a,c,d
61 2,2-Methylene-bis(4-
methyl-6-tert-butylphenol) C23H32O20.10 2421 - a,c,d
Alkanes
62 Docosane C22H46 0.07 2200 2196 [34] a,c,d
63 Pentacosane C25H52 0.05 2499 2500 [25] a,c,d
64 Heptacosane C27H56 0.06 2698 2700 [25] a,c,d
65 Nonacosane C29H60 0.03 2895 2900 [25] a,c,d
Others
66
2,5,5,8a-Tetramethyl-4-methylene-6,7,8,8a-tetrahydro
-4H,5H-chromen-4a-yl hydroperoxide C14H22O30.02 1740 - a,c,d
67 cis-9-Hexadecenal C16H30O 0.03 1750 - a,c,d
68 1-Hexadecanol C16H34O 0.17 1881 2384 [27] a,c,d
69 13-Heptadecyn-1-ol C17H32O 0.03 2041 - a,c,d
Total 89.70
a Kov
á
ts retention indices (RI) calculated from the retention time in relation to those of a series of C7–C30 n-alkanes on a HP-5 column. b Kov
á
ts retention indices (RI) on HP-Innowax
column from literature. c Values compared with [
43
]. d The mass spectra of authentic reference compounds where possible and by reference to NIST 17 database. “-” Kov
á
ts retention
indices (RI) on HP-Innowax column was not found from the literature.
Molecules 2020,25, 967 6 of 17
2.2. Algicidal Activity and Algicidal Mechanism of Action of T. flousiana Essential Oil
Algicidal activity of the essential oil extracted from stem bark of T. flousiana on S. communis
was tested for the first time. The algicidal effects of T. flousiana essential oil on S. communis were
dose-dependent at the concentrations from 12.5 to 200 µg/mL 24 to 72 h after the treatment.
The IC
50
values of the essential oil on the inhibition of chlorophyll a ranged from 40.64 to
90.10
µ
g/mL 24–96 h after the treatment. As a contrast, those of butachlor ranged from 36.60 to 55.28
µ
g/mL. The IC
50
values of the essential oil on the inhibition of chlorophyll b ranged from 53.39 to
106.91
µ
g/mL 24–96 h after the treatment. As a contrast, those of butachlor ranged from 47.29 to
79.12
µ
g/mL. Specially, 48 h after the treatment, The IC
50
values of the essential oil was 47.49
µ
g/mL,
while that of butachlor was 62.95
µ
g/mL, indicating that the essential oil showed a better algicidal
effect at 48 h after the treatment based on the inhibition of chlorophyll b. The IC
50
values of the
essential oil on the inhibition of the total chlorophyll ranged from 31.77 to 84.92
µ
g/mL 24–96 h after
the treatment. As a contrast, those of butachlor ranged from 40.24 to 58.09
µ
g/mL. Specially, 72 h after
the treatment, the IC50 values of the essential oils was 31.77 µg/mL, while that of butachlor was 40.91
µ
g/mL, suggesting that the essential oil showed a better algicidal effect at 48 h after the treatment based
on the inhibition of chlorophyll b.
In summary, the algicidal activity of the essential oil was comparable to or even better than that of
butachlor (Table 2).
Table 2.
IC
50
of T. flousiana essential oil and butachlor on the content of chlorophyll a, chlorophyll b,
and total chlorophyll of S. communis.
Pigment Treatment Time
(h) Regression Equation r IC50
(µg/mL)
95%CL
(µg/mL)
Chlorophyll
a
Essential oil
24 y =1.2251 +1.9312x 0.9327 90.10 74.01–109.68
48 y =−0.0821 +3.0372x 0.9896 47.13 42.09–52.76
72 y =1.6596 +2.0762x 0.9437 40.64 34.98–47.21
96 y =2.6742 +1.3534x 0.9536 52.29 42.40–64.50
Butachlor
24 y =0.7963 +2.4091x 0.9459 55.58 48.57–63.60
48 y =0.9345 +2.5763x 0.9794 37.85 33.08–43.30
72 y =1.5056 +2.2331x 0.9946 36.71 31.66–42.58
96 y =1.4714 +2.2568x 0.9174 36.60 31.49–42.54
Chlorophyll
b
Essential oil
24 y =0.7342 +2.1024x 0.9438 106.91 86.86–131.58
48 y =−1.2225 +3.7115x 0.9759 47.49 42.73–52.77
72 y =1.9149 +1.7859x 0.9560 53.39 45.22–63.05
96 y =2.7763 +1.2659x 0.9584 57.09 45.60–71.47
Butachlor
24 y =0.6332 +2.3004x 0.9186 79.12 67.10–93.00
48 y =−0.0687 +2.8175x 0.9467 62.95 55.74–71.10
72 y =1.5328 +2.0981x 0.9424 44.93 38.67–52.20
96 y =1.8578 +1.8762x 0.9530 47.29 40.24–55.57
Total
chlorophyll
Essential oil
24 y =1.2976 +1.9193x 0.9309 84.92 70.19–102.75
48 y =−0.0821 +3.0372x 0.9623 46.62 41.67–52.15
72 y =1.6945 +2.2008x 0.9200 31.77 27.16–37.17
96 y =2.7412 +1.3035x 0.9482 54.05 43.48–67.18
Butachlor
24 y =0.6609 +2.4596x 0.9436 58.09 50.76–66.49
48 y =0.7453 +2.6025x 0.9719 43.14 37.87–49.14
72 y =1.7028 +2.0456x 0.9566 40.91 35.07–47.74
96 y =1.7113 +2.0495x 0.9256 40.24 34.43–47.04
2.3. The Effect of Light on the Algicidal Activity of T. flousiana Essential Oil in S. communis
Further study revealed that light could affect the algicidal activity of T. flousiana essential oil
in S. communis. 96 h after the essential oil treatment with light, the IC
50
values of the essential oil
Molecules 2020,25, 967 7 of 17
ranged from 71.58 to 87.89
µ
g/mL, which were much lower than the values without light ranging from
1156.28 to 1229.24
µ
g/mL (Table 3). This result indicated that some active ingredients of the essential
oil were photo-activated.
Table 3.
IC
50
T. flousiana essential oil and Butachlor on the content of chlorophyll a, chlorophyll b, and
total chlorophyll of S. communis with or without light (96 h).
Treatment Regression
Equation r IC50 (µg/mL) 95%CL
(µg/mL)
With Light
chlorophyll a
y=
−
1.9294 +3.7360x
0.9062 71.58 57.66–88.84
chlorophyll b
y=
−
1.5076 +3.3476x
0.9310 87.89 67.42–114.59
total chlorophyll
y=
−
2.0191 +3.6402x
0.9473 84.77 65.94–108.97
Without Light
chlorophyll a y =2.1333 +0.9279x 0.9814 1229.24
121.24–12463.23
chlorophyll b y =1.6774 +1.0713x 0.9071 1156.28
156.28–10205.66
total chlorophyll y =2.1286 +0.9302x 0.9328 1221.58
120.76–12357.62
2.4. Antifungal Activities of T. flousiana Essential Oil
The effects of the essential oil of T. flousiana on Rhizoctonia solani Kuhn, Colletotrichum
gloeosporioiles, Fusarium moniliforme Sheld, Thanatephorus cucumeris (Frank) Donk.,
Fusariun oxysporun f. sp. cubense, and Didymella bryoniae (Auersw.) Rehm. mycelial growth
are listed in Table 4. The antifungal activity estimated by the EC
50
value of the oil indicated a high
variation of EC
50
values among the fungal species (Table 4). The lowest EC
50
value was observed
against R. solani (287.94
µ
g/mL), while the highest was detected against D. bryoniae (3162.34
µ
g/mL).
Generally, the oil showed better inhibitory activities on R. solani (287.94
µ
g/mL) and C. gloeosporioiles
(378.90
µ
g/mL). The oil possessed activities on F. moniliforme, T. cucumeris, and F. oxysporun f. sp.
cubense and the EC50 values were 923.03, 623.36, and 809.07 µg/mL, respectively.
Table 4. The antifungal activities of the essential oil of T. flousiana (EC50).
Fungus Time
(h)
Regression
Equation rEC50
(µg/mL) 95%CL(µg/mL)
Rhizoctonia solani 24 y =1.8819 +1.2679x 0.9876 287.94 195.23–424.68
Colletotrichum
gloeosporioiles 48 y =2.8088 +0.8498x 0.9261 378.90 205.68–698.17
Fusarium moniliforme 72 y =2.4418 +0.8627x 0.9865 923.03 529.24–1609.80
Thanatephorus
cucumeris 72 y =1.4164 +1.2822x 0.9716 623.36 453.38–857.06
Fusariun oxysporun 72 y =1.5112 +1.1997x 0.9754 809.07 545.56–1199.86
Didymella bryoniae 72 y =1.5316 +0.9910x 0.9122 3162.34
1187.37–8422.33
2.5. Antibacterial Activities of T. flousiana Essential Oil
The
in vitro
antibacterial activities of T. flousiana essential oil, against four species of microorganisms
were estimated by measuring the diameter of inhibition zone and varied by the sample types and
bacteria strains. The T. flousiana essential oil showed obvious activity against Ralstonia solanacearum
Yabuuhi et al. (ATCC 11696) and Staphylococcus aureus, S. aureus (ATCC 25923) strains. The growth
of the two bacteria species was inhibited by the essential oil in a dose-dependent manner under the
exposure of increasing concentrations (0, 5, 10, 20, 30, and 40
µ
g/disk). At 40
µ
g/disk, the diameters of
the inhibition zone (ZOI, mm) caused by the essential oil to R. solanacearum and S. aureus were 18.66
and 16.75 mm, respectively. However, the essential oil had not exhibited significant growth inhibition
against Escherichia coli (Migula) Castellani and Chalmers (ATCC 8739) and Bacillus subtilis (Ehrenberg)
Cohn. (ATCC 23857), with the ZOIs being 7.23 and 7.91 mm, respectively, at 40 µg/disk (Figure 2).
Molecules 2020,25, 967 8 of 17
Figure 2.
Antibacterial activity of T. flousiana essential oil estimated by diameter of inhibition zone.
Diameter of inhibition zone includes diameter of discs (6 mm). Bacteria were cultured for 12 h at 37
◦
C.
Different letters represent values that differed significantly in the Duncan’s multiple range test (
p<0.05)
.
2.6. Antioxidant Activity
Essential oils have been proposed as potential substitutes for synthetic antioxidants in food
preservation because of their antioxidant activity [
44
]. In this work, the antioxidant activity of
T. flousiana oil was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay. In this
assay, the antioxidant reacts with the stable free radical 2,2-diphenyl-1-picrylhydrazyl with a deep violet
color and produces 2,2-diphenyl-1-picrylhydrazine with no color [
45
]. The free radical scavenging
activity is usually expressed either as percentage of DPPH inhibition or by the antioxidant consumption
for a 50% DPPH reduction (IC
50
). The amount of essential oil needed to decrease the initial DPPH by
50% (IC
50
) is a parameter widely used to measure the antioxidant activity. The lower the IC
50
value,
the more potent the antioxidant is. In our results, positive control ascorbic acid had the strongest
antioxidant activity with IC
50
value of 7.98
µ
g/mL. The IC
50
value of the essential oil of T. flousiana was
33.51 µg/mL (Table 5), indicating it scavenged the free radical DPPH.
Table 5.
IC
50
values of T.flousiana essential oil and ascorbic acid measured by 2,2-diphenyl-1-picrylhydrazyl
(DPPH).
Treatment Regression Eq IC50 (µg/mL) r 95% CL (µg/mL)
Essential oil y =1.6712 +2.1903x33.51 0.9766 26.15–42.95
Ascorbic acid y =3.5608 +1.6001x7.98 0.9808 5.76–11.04
All treatments were performed in triplicates and repeated at least three times. IC
50
: concentration (
µ
g/mL) for a
50% inhibition.
2.7. Effects of Essential Oil on Algal Internal Structure
We found that the chloroplast disintegration and the shrink of plane transverse cell walls became
more apparent with the increase of the essential oil concentration. With the treatment of 50
µ
g/mL, the
chloroplast started to disintegrate. Meanwhile the structure of the chloroplast shrank. Severe damage of
cell structure was observed at higher essential oil concentrations (100–200
µ
g/mL). In these treatments,
plasmolysis occurred and damage to basic cell structure was severe, as revealed by the fact that the
chloroplast disintegration became more apparent, and as well by damage to the plasma membrane and
was accompanied by increased cell wall opacity. As a contrast, intact cell wall and normally distributed
chloroplast were present in the control cells (Figure 3).
Molecules 2020,25, 967 9 of 17
Figure 3.
Effect of the essential oil of T. flousiana on the morphology of S. communis.S. communis was
treated with the essential oil at different concentrations for 96 h, the morphological changes were
imaged with microscope (Nikon Eclipse Ti) at a magnification of 10×.
2.8. Alteration of the Cell Ultrastructure of S. communis by the Essential Oil of T. flousiana
The treated S. communis cells were analyzed by transmission electron microscope (TEM). Specifically,
it was found that essential oil at 200
µ
g/mL could damage the cell wall and the chloroplast, leaving the
cells vacuolated, with only some organelle remained in the cell (Figure 4G–I). This result indicated that the
main action site of the essential oil of T. flousiana might be the cell wall and chloroplast. It was noteworthy
that the chloroplast is an important target of the essential oil of T. flousiana, this finding further supported
our result that the light contributed to the algicidal activity of the oils presented in Table 3.
Figure 4.
Effect of the essential oil of T. flousiana on the ultrastructure of S. communis.S. communis was
treated with the essential oil at different concentrations (50 and 200
µ
g/mL) for 96 h and the ultrastructure
changes were assessed by TEM). The control cells (
A
–
C
), treatment with the essential oil of T. flousiana
(
D
–
F
) treated with the essential oil of T. flousiana at 50
µ
g/mL and (
G
–
I
) treated with the essential oil of
T. flousiana at 200 µg/mL). a: cell wall; b: nucleus; c: chloroplast; d: thylakoid; e: starch grain.
Molecules 2020,25, 967 10 of 17
3. Discussion
In 2001, research interests in the components of the essential oil of T. cryptomerioides were reported
for
α
-cadinol, ferruginol, and cedrol isolated from the essential oils of sapwood and heartwood of
Taiwania cryptomerioides Hayata [
46
], and a-cadinol, T-muurolol, ferruginol, and T-cadinol obtained from
T. cryptomerioides heartwood [
47
]. Further study in 2012 identified 35 compounds from the twig essential
oil of T. cryptomerioides, of which cadinol (45.9%), ferruginol (18.9%), and
β
-eudesmol (10.8%) were the
major compounds [
48
]. In our study, 69 components were identified from the bark of T. flousiana and
accounted for 89.70% of the total oil composition. The main components were hexadecanoic acid (27.13%),
2-penten-1-ol, 3-methyl-5-[octahydro -4,5-dimethyl-7a-(1-methylethenyl)-1H-inden-4-yl]-(16.16%),
linoleic acid (13.48%), podocarpa- 6,8,11,13-tetraen-12-ol, 13-isopropyl-, acetate (7.56%), ferruginol
(6.52%), and
α
-linolenic acid (5.41%). The contents of abietatriene, tetradecanoic acid, pentadecanoic
acid ranged from 1.04 to 1.37%. The others were less than 1.00%. The diterpene ferruginol was also
one of the major components of bark essential oil of T. flousiana, cedrol and
β
-eudesmol were also
present in the bark essential oil of T. flousiana, but with quite less contents. The contents of ferruginol,
cedrol, and
β
-eudesmol were 6.52%, 0.13%, and 0.06%, respectively. The differences of our results
from those in the literatures were probably because of the different parts of the test material and its
collection locations.
Further, we found that T. flousiana essential oil possessed inhibition of chlorophyll content on the
S. communis, which is a species of algae. Harmful algae blooms have increased globally and many
researchers are focusing on the development of the effective control agents. Plant-derived chemicals
are important sources of selective and biodegradable algicides [
49
]. Recently, algicidal polyphenolic
p-hydroxybenzoic acid, coumarin, and fatty acids have been isolated from different plants [
50
–
52
].
Wang et al. [
53
] reported the algicidal activities of essential oils from six plant species, namely,
Potamogeton cristatus,Potamogeton maackianus, Potamogeton lucens, Vallisneria spinulosa, Ceratophyllum
demersum, and Hydrilla verticillata. The inhibition rates of essential oils on M. aeruginosa were 30.2–41.7%
at a concentration of 50.0
µ
g/mL. Phenolic and fatty acids were found to be the algicidal chemicals [
54
].
Normally, phenolic and fatty acids were the common components of essential oils, which provide useful
information for further study of these chemicals in control of the submerged weeds. Notably, we found
that light contributed to the algicidal activity of T. flousiana essential oil in S. communis. This indicated
that some active ingredients of the essential oil were photo-activated, which deserves further study.
In 2017, Chen et al. reported that phytochemicals (ferruginol, T-cadinol, alpha-cadinol,
and T-muurolol) of T. cryptomerioides heartwood had the potential to be used as environmentally benign
antifungal agents against brown root rot fungus Phellinus noxius in place of synthetic or inorganic
fungicides. Their results showed that ferruginol, T-cadinol, alpha-cadinol, and T-muurolol were
found to exhibit excellent antifungal activities against P. noxius, with IC
50
values 16.9, 25.8, 33.8 and
50.6
µ
g/mL, respectively [
55
]. In our study, the content of ferruginol in T. flousiana essential oil was
3.94% and the oil also showed antifungal activity. Thus, the antifungal activity of the active ingredient
ferruginol deserves more attention.
In 2002, Wang et al. demonstrated that ferruginol exhibited the strongest antioxidant activity
among the diterpenes isolated from T. cryptomerioides heartwood [
56
]. In 2012, Ho et al. also reported
that T. cryptomerioides twig essential oil showed antioxidant activity against DPPH. The IC
50
of
the DPPH free radical scavenging capability of the essential oil was 90.80
µ
g/mL and ferruginol
(
IC50 =48.0 µg/mL
) was identified to be the main active ingredient for the free radical scavenging [
57
].
In our study, the content of ferruginol was 3.94% in T. flousiana oil while the IC
50
value of the
essential oil was 33.51
µ
g/mL. As a contrast, in the report of Ho et al. the content of ferruginol in
T. cryptomerioides twig oil was 18.9% while the IC
50
value of the essential oils was 90.80
µ
g/mL [
57
].
In general, the antioxidant activity of essential oils is the product of additive, synergistic, and/or
antagonistic effects from a complex mixture of several classes of compounds. These suggested that
other components of the T. flousiana essential oil may possess the antioxidant activity. The biological
functions of each component of the essential oil need to be further investigated.
Molecules 2020,25, 967 11 of 17
Additionally, our results indicated that the main action site of the essential oil of T. flousiana might
be the cell wall and chloroplast. Mechanism study plays a very important role in the development of
new algicidal chemicals. Further mechanism studies should focus on cell wall and chloroplast.
4. Material and Methods
4.1. Plant Material
Whole T. flousiana plant was collected from Enshi autonomous prefecture in central China in July,
2019 (Enshi, Hubei province, China). The plant was further identified as T. flousiana (a synonym of
Taiwania cryptomerioides Hayata) and a voucher specimen was deposited in the Key Laboratory of
Natural Pesticides & Chemical Biology, Ministry of Education, South China Agricultural University,
China. The plant material was air-dried for up to 3 weeks at the ambient temperature and 24 h in a
50–60 ◦C incubator prior to pulverization.
4.2. Isolation of Essential Oil
The air-dried stem bark powder (250 g) of T. flousiana was subjected to hydrodistillation for 3 h
using a Clevenger-type apparatus. The oil was dried with anhydrous sodium sulphate. The yield (v/w,
dry weight basis) was calculated as volume (mL) of extracted essential oil per 250 g of plant material.
Then, the essential oil was stored in hermetically sealed dark-glass at 4 ◦C until further analysis.
4.3. Analysis of the Essential Oil
Analysis of the essential oils was carried out with an Agilent Technologies 7693A Gas
Chromatograph with 5977B Mass Spectrometer. A HP-5 MS capillary column (30 m
×
0.25 mm
×
0.25
µ
m; Agilent Technologies Inc., Santa Clara, USA) was employed. Analyses were carried out
using helium as the carrier gas at a flow rate of 1.0 mL/min, split ratio: 15:1. Oven temperature was
programmed as follows: 40
◦
C initially rising to 150
◦
C at a rate of 6
◦
C/min; rising to 270
◦
C at a rate
of 3
◦
C/min; rising to 300
◦
C at a rate of 10
◦
C/min and held for 3 min. The injector and detector were
held at 325
◦
C. The mixtures of the normal alkane of C
7
–C
30
(1000
µ
g/mL) and EO samples dissolved
in hexane of 0.8
µ
L were injected and all samples were filtered through a 0.22
µ
m organic phase filter.
The Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of 30–550
AMU in full scan mode. The MS (Agilent Technologies Inc., Santa Clara, USA) transfer line was set at
250 ◦C.
4.4. Identification of the Essential Oil Chemical Constituents
The essential oil constituents identification was carried out by comparing their recorded mass
spectra with those stored in the National Institute of Standards and Technology Mass Spectral database
(NIST 17 database) or with authentic compounds and confirmed by comparison of their retention index
with authentic compounds reported in the literature [
58
]. The relative percent of each component in
essential oil was counted by the area normalization method. The retention index was defined by the
following:
RI =100n +100(tx−tn)/(tn+1−tn) (1)
where tn, tn+1, and txwere net retention times [59].
Identification of the individual components was based on: (i) Comparison with the mass spectra
of authentic reference compounds possible and by reference to NIST 17 database, and Adams terpene
library [
43
]; (ii) comparison of their retention indices (RI) on a HP-5, calculated relative to the retention
times of a series of C-7 to C-30 n-alkanes, with linear interpolation, with those of authentic compounds
or literature data [43].
Molecules 2020,25, 967 12 of 17
4.5. Evaluation of the Algicidal Activity with Light
S. communis was collected from the Southern China Botanical Garden, Chinese Academy of
Sciences. S. communis was incubated in a modified Bold basal medium composed of NaNO
3
(250 mg/L), K
2
HPO
4
(75 mg/L), CaCl
2·
H
2
O (25 mg/L), MgSO
4·
7H
2
O (75 mg/L), NaCl (25 mg/L),
KH
2
PO
4
(175 mg/L), Na
2
EDTA
·
2H
2
O (4.5 g/L), FeCl
3·
6H
2
O (0.582 g/L), MnCl
2·
4H
2
O (0.246 g/L), ZnCl
2
(0.030 g/L), Na
2
MoO4
·
2H
2
O (0.024 g/L), CoCl
2·
6H
2
O (0.012 g/L), vitamin B1 (1.1 g/L), vitamin B6 (0.025
g/L), and vitamin B12 (0.135 g/L) [60].
The alga was cultured in 500 mL of sterilized culture medium in 1000 mL conical flasks under
an irradiance of 4000 lux, 12 h light/12 h dark (12:12), at 25
±
1
◦
C for 5 days. Then the 10 mL of
the algal cultures (0.1 g) was transferred to a 6-well plate. The stock solution of the essential oil was
prepared in acetone. The final concentrations of the essential oil in the test solution were 12.5, 25, 50, 100,
and 200
µ
g/mL, respectively. Acetone in the test solution was lower than 0.2% (v/v). The commercial
herbicide, butachlor (12.5, 25, 50, 100, and 200
µ
g/mL), was used as a control. The plates were sealed
with polyethylene wrapping film and incubated in a growth chamber at 25
±
1
◦
C, RH 50–60%, and a
photoperiod of 12:12. Each treatment has three replicates. All experiments were repeated at least
three times.
The algicidal activity was determined by the chlorophyll content as described by Dere et al. [
61
]
with some modifications. Specifically, after being dried with absorbent paper, S. communis in each
replicate was grounded in 2 mL of 80% acetone. Then the mixture was transferred into a centrifugal
tube (2 mL) followed by centrifugation for 10 min at 4000 rpm. The supernatant was transferred into a
tube and diluted with acetone to 4 mL. Then 2 mL of the solution was mixed with 80% acetone to make
a final volume of 10 mL and the absorbances were recorded at 663 nm and 645 nm. Acetone (80%)
alone was used for the blank control. The amount of the pigments was calculated according to the
following formulas:
Ca=12.7 ×A663 −2.69 ×A645 (2)
Cb=22.9 ×A645 −4.68 ×A663 (3)
Cab =8.02 ×A663 +20.21 ×A645 (4)
The content of the chlorophyll was calculated according to the following formula:
The chlorophyll content (mg/g) =(Chlorophyll concentration ×the volume of the tested
solution ×the dilution factor)/the sample mass) ×100 (5)
The inhibition rate of chlorophyll was calculated according to the following formula:
The inhibition rate of chlorophyll (%) =(([C] −[S])/[C]) ×100 (6)
where [C] means the chlorophyll content of control and [S] means the chlorophyll content of sample.
4.6. Evaluation of the Algicidal Activity without Light
The same method as described above in 4.5 was followed except the plates were incubated
under a photoperiod of 24:0 (dark:light). The inhibition rate of chlorophyll was evaluated 96 h after
the treatment.
4.7. Morphological Changes of S. communis Treated with the Essential Oil of T. flousiana
The morphological changes of S. communis treated with the essential oil of T. flousiana for 24, 48,
and 72 h were evaluated with light microscopy (Leica DMLB2, Leica Microsystems, Wetzlar, Germany).
Transmission electron microscopy (TEM) (FEI Tecnai 12, FEI company, Hillsboro, USA) evaluation was
further performed in order to examine the effect of the essential oil of T. flousiana (at 12.5, 25, 50, 100 and
Molecules 2020,25, 967 13 of 17
200
µ
g/mL) on the ultra-structure of S. communis. The TEM samples were processed as previously described
by Houot et al. [62]. Samples were examined by TEM (FEI Tecnai 12, FEI company, Hillsboro, USA).
4.8. Determination of Antimicrobial Effects of the Essential Oils on Mycelial Growth
Fungi including R. solani,C. gloeosporioiles,F. moniliforme,T. cucumeris,F. oxysporun f. sp. cubense,
and D. bryoniae were obtained from the Department of Plant Pathology, South China Agricultural
University.
In vitro
antifungal assays were conducted according to the method of Boubaker et al. [
63
],
with slight modifications. Briefly, sterile molten potato-dextrose-agar (PDA) supplemented with
essential oil of T. flousiana, at final concentrations of 62.5, 125, 250, 500, and 1000
µ
g/mL, was poured
into Petri plates (6-cm-diameter). All tests were performed in PDA supplemented with 0.5% (v/v)
DMSO to enhance oil solubility. Afterwards, plates were inoculated with fungal cultures with 5-mm
diameter agar disks from one-week-old cultures, with mycelia surface facing down. The agar plates
were then incubated at 25
◦
C for 2 days. Plates with medium supplemented with 0.5% DMSO only
was used as the control. The antifungal activity was expressed as percent of mycelial radial growth
inhibition and calculated according to the following formula: MGI (%) =((C
−
T)/C)
×
100, where C and
T represent mycelial growth diameter in control and EO treated plates, respectively. Each treatment
has three replicates. All experiments were repeated at least three times.
The
in vitro
antibacterial activity of the essential oil from T. flousiana was carried out by using
filter paper disc diffusion assay [
64
,
65
]. Two Gram-positive bacteria S. aureus and B. subtilis and two
Gram-negative bacteria E. coli and R. solanacearum, provided by the Department of Plant Pathology,
South China Agricultural University, were tested. Typically, 500
µ
l of a suspension of the tested
microorganisms (approx. 10
6
colony-forming units (CFU)/mL) was spread with a sterile cotton swab
on the surface of Mueller-Hinton agar (MHA) plates at 37
◦
C and allowed to dry for 10 min. A stock
solution of the essential oil was prepared by dissolving 20 mg in 1 mL of DMSO. Then the stock
solution was diluted with 0.1% aqueous Tween solution to get series solution of 4000, 3000, 2000, 1000,
and 500
µ
g/mL. Each sterile filter paper disc (6 mm in diameter Whatman disks) was impregnated
with 10
µ
L solutions, respectively. The Petri dishes were kept at 4
◦
C for 2 h to allow the diffusion of
the oil, and further incubated at 37
◦
C for 24 h. Activity was expressed as percent of zone of inhibition
(ZOI, mm). The net zone of inhibition was determined by subtracting the disc diameter (i.e., 6.0
mm) from the total zone of inhibition shown by the test disc in terms of clear zone around the disc.
The control was the aqueous solution of 0.1% Tween +8% DMSO. Each treatment had three replicates.
All experiments were repeated at least three times.
4.9. Determination of Antioxidant Activity
The method of El-Gawad [
66
] was used. The antioxidant activity of T. flousiana essential oil
was measured in terms of radical scavenging activity, using the stable radical DPPH (Sigma-Aldrich,
Darmstadt, Germany) [
48
]. A reaction mixture of 1 mL of a hexane solution of the essential oil with
different concentrations (5, 10, 20, 40, and 80
µ
g/mL) and equal volume of the methanolic solution
of 0.3 mM DPPH was prepared, mixed well and incubated under dark condition for 15 min at room
temperature. Ascorbic acid (5, 10, 20, 40, and 80
µ
g/mL) was used as the reference. The decrease
in absorbance at 517 nm was determined using a spectrophotometer (UV-8500PC, Shanghai, China).
The IC
50
(the amount of sample necessary to decrease the absorbance of DPPH by 50%) was calculated
graphically. Each treatment has three replicates. All experiments were repeated at least three times.
The percent of the inhibition of the DPPH radical was calculated as following:
The inhibition (%) =1−(Absorbance of sample/Absorbance of control) ×100 (7)
4.10. Statistical Analysis
Statistical analysis (ANOVA) by means of the IBM SPSS Statistics, Version 19.0 (International
Business Machines Corporation, New York, USA) was applied to the data to determine the differences
Molecules 2020,25, 967 14 of 17
(p<0.05). Then, the estimation of the median effective concentration (EC
50
), median inhibition
concentration (IC
50
), and their 95% confidence limits were obtained. All quantitative data were
presented as the mean
±
SD of at least three independent experiments using the Duncan’s multiple
range test or Student’s ttest for group differences. A p<0.05 was considered as statistically significant.
5. Conclusions
Current investigation highlights the detailed chemical composition of EO extracted from
T. flousiana and their bioactive potential. Sixty-nine compounds representing 89.70% of the stem
bark essential oil of T. flousiana were identified. The main components were hexadecanoic acid (27.13%),
2-penten-1-ol, 3-methyl-5-[octahydro-4,5-dimethyl-7a-(1-methylethenyl)-1H-inden-4-yl]- (16.16%),
linoleic acid (13.48%), podocarpa-6,8,11,13-tetraen-12-ol, 13-isopropyl-, acetate (7.56%), ferruginol
(6.52%), and α-linolenic acid (5.41%).
The algicidal activity of the essential oil on S. communis was comparable to that of butachlor.
The IC
50
values of the essential oil ranged from 31.77 to 84.92
µ
g/mL, while the IC
50
values of butachlor
ranged from 40.24 to 58.09
µ
g/mL. Thus the essential oil from T. flousiana could be considered as a
potential algicidal substitute of synthetic ones. In addition, ultrastructure changes revealed by the
transmission electron microscopy indicated that the main action sites of the essential oil of T. flousiana
on S. communis cell were the chloroplast and cell wall.
The EO showed antifungal activities on Rhizoctonia solani (EC
50
=287.94
µ
g/mL) and Colletotrichum
gloeosporioiles (EC
50
=378.90
µ
g/mL). Meanwhile, The EO also showed bactericidal activities against
Ralstonia solanacearum and Staphylococcus aureus, with ZOIs being 18.66 and 16.75 mm, respectively at
40 µg/disk.
The level of antioxidant activity estimated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method
showed that the essential oil of T. flousiana demonstrated obvious antioxidant activity with IC
50
value of 33.51
µ
g/mL. While the IC
50
value of the positive control ascorbic acid was 7.98
µ
g/mL.
Thus, the essential oil of this plant could be used as a potential source of natural bioactive molecules in
agrochemical industry as well as in food and cosmetic industries.
Author Contributions:
H.L. contributed to GC-MS experiment, literature search, figures, and data analysis. J.H.
contributed to GC-MS experiment and identification. S.Y. contributed to literature search and bioassay. J.L.
contributed to data collection and bioassay. L.Z. contributed to the design of the work, interpretation of data for
the work, and writing. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by Common Key Technology R&D and Innovation of Modern Agricultural
Industry of Guangdong Province, China (2019KJ134), and Science and Technology Planning Projects of Guangdong
Province, China (2018A0303130093) and (2015A020209151).
Acknowledgments:
The authors also appreciate the help of the analysis of essential oil by Dr Huining Lu,
Instrumental Analysis and Research Center, Sun Yat-Sen University.
Conflicts of Interest: Authors declare no conflict of interest.
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