Enantioselective separation of 4,8-DHT and phytotoxicity of the enantiomers on various plant species

Article abstract of DOI:10.3390/molecules21040528

As a candidate for bioherbicide, 4,8-dihydroxy-1-tetralone (4,8-DHT) was isolated from Caryospora callicarpa epicarp and its two enantiomers, S-(+)-isosclerone and R-(-)-regiolone, were separated by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OD column with chiral stationary phase (CSP)-coated cellulose-tris(3,5-dimethylphenylcarbamate). Then, the phytotoxicity of 4,8-DHT and its enantiomers toward the seeds germination and seedling growth of the five tested plant species, including lettuce (Latuca sativa), radish (Raphanus sativus), cucumber (Cucumis sativus), onion (Allium cepa), and wheat (Triticum aestivum), were investigated and the results indicated a hormesis at low concentration of 4,8-DHT and its enantiomers, but a retardant effect at high concentration. Between the two enantiomers of 4,8-DHT, the S-(+)-isosclerone was more toxic to seeds germination and seedling growth of the five tested plant species than the R-(-)-regiolone, and also the phytotoxicity of S-(+)-isosclerone varied with different plants. For example, S-(+)-isosclerone was the most active to seedling growth of lettuce, indicating that S-(+)-isosclerone had specific effects on different organisms. Thus, all of the chirality and concentration of 4,8-DHT, as well as the affected plant species, need to be taken into consideration in the development and utilization of 4,8-DHT.

Full text of DOI:10.3390/molecules21040528

molecules
Article
Enantioselective Separation of 4,8-DHT and
Phytotoxicity of the Enantiomers on Various
Plant Species
Li Yang 1,2, Xiao-Yan Ma 1,3, Xiao Ruan , De-An Jiang , Cun-De Pan and Qiang Wang 1,*
1
2
3
1
Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China; yangli0817@yeah.net (L.Y.);
1
5676230509@yeah.net (X.-Y.M.); ruanxiao@nit.net.cn (X.R.)
2
3
College of Life Sciences, Zhejiang University, Hangzhou 310058, China; dajiang@zju.edu.cn
College of Forestry and Horticulture, Xinjiang Agricultural University, Urumqi 830052, China;
pancunde@163.com
*
Correspondence: wangqiangsky@263.net; Tel.: +86-574-8813-4338; Fax: +86-574-8822-9545
Academic Editor: Derek J. McPhee
Received: 23 March 2016; Accepted: 19 April 2016; Published: 22 April 2016
Abstract: As a candidate for bioherbicide, 4,8-dihydroxy-1-tetralone (4,8-DHT) was isolated from
Caryospora callicarpa epicarp and its two enantiomers, S-(+)-isosclerone and R-( )-regiolone, were
´
separated by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OD column
with chiral stationary phase (CSP)-coated cellulose-tris(3,5-dimethylphenylcarbamate). Then, the
phytotoxicity of 4,8-DHT and its enantiomers toward the seeds germination and seedling growth
of the five tested plant species, including lettuce (Latuca sativa), radish (Raphanus sativus), cucumber
(Cucumis sativus), onion (Allium cepa), and wheat (Triticum aestivum), were investigated and the results
indicated a hormesis at low concentration of 4,8-DHT and its enantiomers, but a retardant effect at
high concentration. Between the two enantiomers of 4,8-DHT, the S-(+)-isosclerone was more toxic to
seeds germination and seedling growth of the five tested plant species than the R-( )-regiolone, and
´
also the phytotoxicity of S-(+)-isosclerone varied with different plants. For example, S-(+)-isosclerone
was the most active to seedling growth of lettuce, indicating that S-(+)-isosclerone had specific effects
on different organisms. Thus, all of the chirality and concentration of 4,8-DHT, as well as the affected
plant species, need to be taken into consideration in the development and utilization of 4,8-DHT.
Keywords: 4,8-dihydroxy-1-tetralone (4,8-DHT); chiral-selective separation; enantiomers; phytotoxicity
1
. Introduction
Chirality of a pair of molecules with a non-superposable mirror image is almost caused by
the presence of an asymmetric carbon atom. Chiral molecules are widely used as the mainstay of
pesticides. As many as 25% of all pesticide active ingredients are chiral, existing as two mirror images
called enantiomers [
enantiomers or racemates [
physical and chemical properties, they usually display different physiochemical and biochemical
properties in metabolism, excretion, side effects, and toxicity, even acting as an antagonist [ ]. One
enantiomer of a chiral pesticide may have the desired effects on a target species, whereas the other
enantiomer may not [ ]. In recent years, the enantioselective behavior of chiral pesticides has received
more attention at the forefront of chemistry and toxicology research [ ]. It was reported that the
enantiomers of many chiral pesticides showed different activity. For example, the toxicity to Daphnia
revealed that the ( )-enantiomer of leptophos showed a lower toxicity than its (+)-form and racemic
form [ ]; (+)-fenamiphos proved be about 20 times more toxic to Daphnia than ( )-fenamiphos [10];
1]. For economic reasons, chiral pesticides are primarily used as a mixture of
2
]. In vivo, although the enantiomers of chiral pesticides have identical
3–5
2
6–8
´
9
´
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www.mdpi.com/journal/molecules

Molecules 2016, 21, 528
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the R-(+)-enantiomer of the herbicide diclofop-methyl showed significantly higher herbicidal activity
than the S-( )-enantiomer [11]; and the (2S,3S)-( )-enantiomer of paclobutrazol was more active than
the (2R,3R)-(+)-enantiomer toward wheat seedling shoot or root growth inhibition [ ]. Moreover, the
´
´
8
enantioselectivity occurred in aquatic toxicity for chiral compound benalaxyl and S-(+)-benalaxyl was
less toxic than R-(+)-benalaxyl and its racemate to freshwater microalgae Scenedesmus obliquus [12],
and the significant enantioselective toxicity of imazapyr in Arabidopsis thaliana for greater toxicity
with (+)-imazapyr than (+)-imazapyr and ( )-imazapyr, suggesting that (+)-imazapyr had a more
´
herbicidal effect [13]. Therefore, research on stereoselective behaviors of such compounds is necessary
to promote the use of pure active enantiomers in order to reduce waste and undesirable effects.
In recent years, signficant progress has been made for the separation of chiral pesticides. Among
the many types of chiral separation methods, high-performance liquid chromatography (HPLC) with
a chiral stationary phase column (CSP column) and a specific mobile phase has been used most
widely for racemic resolution. HPLC is preferable to separate enantiomers because of its robust
enantioselectivity and applicability on both the analytical and the preparative scale [14]. Many of the
commercial pesticides used as racemates in agricultural practice can be separated by HPLC [
,8-DHT, as a natural product, was first isolated from a Scytalidium species [18]. Subsequently,
the dihydroxy naphthalenone has been found in both enantiomeric forms (named as regiolone and
isosclerone) in a number of fungi and plants [19 21]. Biological properties of 4,8-dihydroxy-1-tetralone
have been largely studied, including cytotoxic activities against human cancer cells [20 22 23]. It
9,15–17].
4
–
,
,
was found that 4,8-DHT isolated and purified from Caryospora callicarpa epicarp could significantly
inhibit the germination of lettuce and cucumber and decrease the radicle length, plumule length,
and fresh weight of lettuce and onion seedlings [21]. Based on our previous research, 4,8-DHT, as
an allelochemical, has the potential to become a natural herbicide [21,24]. In view of its molecular
structure, 4,8-DHT has an asymmetric carbon atom, typically consisting of two enantiomers. However,
very little information is available on the phytotoxicity of 4,8-DHT to plants and other non-target
organisms. Therefore, this study aimed to understand the toxicity of 4,8-DHT and its enantiomers
to the plants at the morphological level. We first developed a separation method by using HPLC
equipped with a chiral column to prepare two enantiomers of 4,8-DHT, and then comprehensively
assessed the phytotoxicity of the 4,8-DHT racemate and its enantiomers on five plant species, including
lettuce (Latuca sativa), radish (Raphanus sativus), cucumber (Cucumis sativus), onion (Allium cepa), and
wheat (Triticum aestivum).
2
. Results and Discussion
2
.1. Separation of 4,8-DHT Enantiomers
Enantiomeric separation of 4,8-DHT by HPLC on the Chiralcel OD column with CSP-coated
cellulose-tris(3,5-dimethylphenylcarbamate) could be easily performed. Among phenylcarbamate
derivatives, 3,5-dimethylphenylcarbamate affords the most useful CSP, which can separate a wide
range of racemates [25], and in view of the structure of 4,8-DHT containing a C=O group, phenyl ring,
and hydroxyl directly linking to the chiral center, the 3,5-dimethylphenylcarbamate was selected as
the CSP.
Alcohol in the mobile phase competes for chiral bonding sites with chiral solutes and can alter
the steric environment of the chiral cavities on the CSP by binding to achiral sites at or near the chiral
cavity [26]. There have been many studies to change the polarity of the mobile phase with various
alcohols, such as n-butanol, iso-butanol, n-propanol, iso-propanol, and ethanol [27
–29]. Considering
4
,8-DHT is a weak acidic material [30], a certain amount of acetic acid (AA) is added into mobile phase,
which can reduce the tailed of chromatographic peaks and increase the symmetry of peaks. Thus, in
this study, the optimized chromatographic condition was determined to be n-hexane/iso-propanol
´ ˝
1
(
95:5, v/v) as the mobile phase containing 0.1% AA with a flow rate of 0.8 mL
¨
min at 20 C and
UV detection at 254 nm. The capacity factor of regiolone and isosclerone (k and k ), the separation
1
2

Molecules 2016, 21, 528
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factor (
α
), and the resolution (Rs) were calculated as follow: k = 4.09, k = 4.77,
α
= 1.16, Rs = 1.70.
1
2
The elution order of 4,8-DHT enantiomers was distinguished at 254 nm of optical rotation detection.
The first eluted enantiomer was S-(+)-isosclerone at 24.4 min and the second was R-( )-regiolone at
7.7 min (Figure 1). Additionally, a 20.8 mg 4,8-DHT sample was separated through preparative HPLC,
´
2
and the elution of enantiomers were collected. The contents of S-(+)-isosclerone and R-( )-regiolone
´
were 10.2 mg (yield 49.0%) and 10.0 mg (48.2%).
Figure 1. The elution order of 4,8-DHT enantiomers. Conditions: mobile phase, n-hexane/
´1
˝
iso-propanol/AA (95:5:0.1, v/v/%); flow rate, 0.8 mL¨ min ; column temperature, 20 C.
2
.2. Effects of Rac-4,8-DHT, S-(+)-Isosclerone, and R-(´)-Regiolone on Germination of the Tested Plants
Crop seeds are commonly selected for use in phytotoxic bioassays because they satisfy a
number of selection criteria: readily available, affordable, repeatable and reliable; and they germinate
quickly, completely, and uniformly [18]. In this study, we selected five crops as test species. The
enantioselective toxicity of 4,8-DHT on plants can be estimated by several indices. Germination vigor
and germination rate are two important indices to determine the seeds’ germination [21
,31]. In this
study, rac-4,8-DHT, S-(+)-isosclerone, and R-( )-regiolone on germination of the tested plants were
´
measured in terms of these parameters (Table 1). Compared with the control, germination of the
five tested plants was significantly affected after they were exposed to different concentrations of
rac-4,8-DHT, S-(+)-isosclerone, and R-(
hormesis to the germination vigor when exposed to the racemate.
S-(+)-isosclerone and R-( )-regiolone at the 2 mM level, whereas the germination vigor was lower
than the control at high concentration of 4,8-DHT and its enantiomers, and the retardant effect of
S-(+)-isosclerone on the germination vigor was higher than that of the racemate or R-( )-regiolone.
´)-regiolone (Table 1). For lettuce, there was a pronounced
´
´
Regarding to the germination rate of lettuce exposed to 4,8-DHT and its enantiomers, a similar
pattern was displayed. For cucumber, a pronounced hormesis on the germination vigor appeared
after treatment with 4,8-DHT and its enantiomers at the concentration of 1–3 mM except for 1 mM
R-(
´
)-regiolone, and the germination vigor was at 2 and 3 mM levels of 4,8-DHT and its enantiomers,
except for 2 mM R-(
´)-regiolone. After treatment with the highest concentration of 4,8-DHT
and its enantiomers (4 mM), the germination rate was inhibited, compared with the control, and
S-(+)-isosclerone caused the most inhibition, whereas only S-(+)-isosclerone had an inhibitory effect
on germination vigor. After exposure to 4,8-DHT and its enantiomers, the 1–3 mM concentrations

Molecules 2016, 21, 528
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had no effect on the germination rate of radish, except for 3 mM S-(+)-isosclerone. In addition, the
highest concentration (4 mM) had a retardant effect, and the most retardant effect was observed in
S-(+)-isosclerone. The hormesis on the germination vigor of radish induced by S-(+)-isosclerone at
1
–3 mM was higher than that by the racemate and R-( )-regiolone. The germination rate of radish
´
exposed to the highest concentration of 4,8-DHT and its enantiomers was similar to the germination
vigor of radish. For the germination of onion and wheat, the hormesis was only observed at the lowest
concentration of 4,8-DHT and its enantiomers. The hormesis on onion was significantly different
among 4,8-DHT and its enantiomers, in the order of S-(+)-isosclerone > racemate > R-(
germination vigor, and the germination rate exposed to S-(+)-isosclerone and racemate was higher
than R-( )-regiolone. For wheat, only R-( )-regiolone had a significant hormesis on the germination
vigor and germination rate.
´)-regiolone for
´
´
Table 1. Effect of rac-4,8-DHT, S-(+)-isosclerone, and R-(
seed germination of five plant species.
´
)-regiolone at different concentrations on
Plant
Species
Germination Vigor
Germination Rate
Forms
Control
1 mM
2 mM
3 mM
4 mM
Control
1 mM
2 mM
3 mM
4 mM
a
a
a
a
a
a
a
a
a
a
a
a
rac
S
76 ˘ 1
76 ˘ 2
76 ˘ 2
83 ˘ 1
85 ˘ 2
82 ˘ 2
86 ˘ 2
87 ˘ 2
86 ˘ 1
69 ˘ 2
60 ˘ 1
86 ˘ 1
87 ˘ 1
86 ˘ 2
88 ˘ 2
89 ˘ 1
88 ˘ 2
87 ˘ 2
88 ˘ 2
88 ˘ 1
94 ˘ 1
96 ˘ 1
92 ˘ 1
83 ˘ 2
82 ˘ 2
83 ˘ 2
79 ˘ 1
a,b
a
a,b
a
a,b
a
Lettuce
75 ˘ 2
65 ˘ 2
57 ˘ 2
85 ˘ 1
62 ˘ 1
R
69 ˘ 2
90 ˘ 2
90 ˘ 2
89 ˘ 1
63 ˘ 2
81 ˘ 2
80 ˘ 1
82 ˘ 2
a
a
a
a
a
a
a
a
a
a
a
a
rac
S
R
85 ˘ 1
90 ˘ 1
93 ˘ 2
a
a
a,b
a
Cucumber
Radish
83 ˘ 1
82 ˘ 1
40 ˘ 4
68 ˘ 2
86 ˘ 2
79 ˘ 2
87 ˘ 2
88 ˘ 2
45 ˘ 4
80 ˘ 2
90 ˘ 1
93 ˘ 2
80 ˘ 2
a,b
84 ˘ 2
83 ˘ 1
81 ˘ 1
88 ˘ 1
89 ˘ 1
90 ˘ 1
90 ˘ 1
91 ˘ 1
83 ˘ 1
83 ˘ 2
a
a
a
rac
S
R
83 ˘ 1
86 ˘ 2
79 ˘ 1
91 ˘ 1
a
a
a,b
a
a,b
a,b
a,b
a
84 ˘ 1
84 ˘ 2
89 ˘ 1
74 ˘ 1
93 ˘ 1
90 ˘ 2
77 ˘ 2
a
82 ˘ 1
83 ˘ 1
85 ˘ 1
80 ˘ 2
82 ˘ 2
40 ˘ 1
a,b
a
a
a
a
a
a
a,b
a
a
rac
S
R
63 ˘ 3
67 ˘ 4
59 ˘ 2
36 ˘ 3
34 ˘ 2
32 ˘ 2
34 ˘ 2
38 ˘ 5
35 ˘ 4
39 ˘ 3
32 ˘ 1
30 ˘ 1
33 ˘ 2
27 ˘ 2
24 ˘ 3
28 ˘ 3
68 ˘ 1
43 ˘ 1
42 ˘ 1
39 ˘ 1
42 ˘ 1
a,b
ab
a
a,b
a
a,b
a
a
a,b
a
Onion
33 ˘ 2
69 ˘ 3
40 ˘ 1
33 ˘ 1
a
36 ˘ 2
63 ˘ 1
83 ˘ 2
43 ˘ 1
40 ˘ 1
a
a
a
a
a
a
a
a
a
a
a
a
rac
S
R
71 ˘ 2
64 ˘ 2
75 ˘ 4
43 ˘ 5
27 ˘ 2
25 ˘ 1
29 ˘ 1
a
a,b
a
a,b
Wheat
a
74 ˘ 3
58 ˘ 3
85 ˘ 1
65 ˘ 1
79 ˘ 3
40 ˘ 4
a,b
70 ˘ 1
65 ˘ 1
83 ˘ 1
50 ˘ 4
b
denotes a significant difference between control and treatments (p < 0.05); denotes a significant difference
among rac-4,8-DHT, S-(+)-isosclerone, and R-(´)-regiolone (p < 0.05).
The germination vigor and rate of onion and wheat incubated with 2–4 mM of 4,8-DHT and
its enantiomers were significantly decreased, especially at 4 mM, the inhibition rate of germination
vigor and rate was the highest. At the concentration of 2 mM, S-(+)-isosclerone inhibited germination
vigor and germination rate in wheat more than the racemate and R-(
germination rate in onion treated by 3 mM and 4 mM of S-(+)-isosclerone were significantly more
than that of the racemate and R-( )-regiolone. The results of the seeds germination indicated that
there was a significant toxicity of rac-4,8-DHT, S-(+)-isosclerone, and R-( )-regiolone to the five plants.
)-regiolone was found to be
´)-regiolone. In addition, the
´
´
Furthermore, the phytotoxicity of rac-4,8-DHT, S-(+)-isosclerone, and R-(
dependent on their concentration and chirality.
´
2
.3. Effects of Rac-4,8-DHT, S-(+)-Isosclerone, and R-(
Radicle length, pumule length, and fresh weight are important traits for measuring plant
growth [18 26] and, thus, these parameters were used to determine enantioselective activities of
,8-DHT and its enantiomers in the five tested plants’ seedlings. As shown in Figures 2–6 the growth
trends of the plants seedlings exposed to different concentration of rac-4,8-DHT, S-(+)-isosclerone,
and R-( )-regiolone were similar to those of the germination in the tested plants. There was a
´
)-Regiolone on Seedlings Growth of the Tested Plants
,
4
´
hormesis on radicle length, pumule length, and fresh weight when exposed to 4,8-DHT and its
enantiomers at concentrations of 1–2 mM for lettuce, at 1–3 mM for cucumber and radish, and at
the lowest concentration of 1 mM for onion and wheat, while the retardant effect was observed
at concentrations of 3–4 mM for lettuce, at 2–4 mM for onion and wheat, and at the highest

Molecules 2016, 21, 528
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concentration of 4 mM for cucumber and radish, respectively. These results were consistent with the
previous observations [ 13], in where paclobutrazol and its enantiomers at low concentrations of
induced hormesis on root growth of rice seedlings, whereas high concentration had
]. The enantioselective toxicity of imazapyr on Arabidopsis thaliana showed that the
lowest concentration of herbicides had no effect on plant shoots, but the shoots exposed to a high
concentration of (+)-imazapyr were smaller than those exposed to ( )-imazapyr and ( )-imazapyr [13].
8
,
´
L
1
0
.125–0.5 mg
¨
retardant effects [
8
´
˘
Thus, the toxic effect of 4,8-DHT and its enantiomers on the growth of the five tested plants seedlings
was related to their concentration.
Figure 2. Effect of rac-4,8-DHT, S-(+)-isosclerone, and R-(
´
)-regiolone at different concentrations on
radicle length ( ); plumule length ( ); and fresh weight ( ) in lettuce. a denotes a significant difference
A
B
C
between control and treatments (p < 0.05); b denotes significant difference among rac-4,8-DHT,
S-(+)-isosclerone, and R-(´)-regiolone (p < 0.05).

Molecules 2016, 21, 528
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Figure 3. Effect of rac-4,8-DHT, S-(+)-isosclerone, and R-(
´
)-regiolone at different concentrations on
) in cucumber. a denotes a significant
radicle length ( ); plumule length ( ); and fresh weight (C
A
B
difference between control and treatments (p < 0.05); b denotes a significant difference among
rac-4,8-DHT, S-(+)-isosclerone, and R-(´)-regiolone (p < 0.05).

Molecules 2016, 21, 528
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Figure 4. Effect of rac-4,8-DHT, S-(+)-isosclerone, and R-(
´
)-regiolone at different concentrations on
radicle length ( ); plumule length ( ); and fresh weight ( ) in radish. a denotes a significant difference
A
B
C
between control and treatments (p < 0.05); b denotes a significant difference among rac-4,8-DHT,
S-(+)-isosclerone and R-(´)-regiolone (p < 0.05).

Molecules 2016, 21, 528
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Figure 5. Effect of rac-4,8-DHT, S-(+)-isosclerone, and R-( )-regiolone at different concentrations on
´
radicle length ( ); plumule length ( ); and fresh weight (C) in onion. a denotes significant difference
A
B
between control and treatments (p < 0.05); b denotes significant difference among rac-4,8-DHT,
S-(+)-isosclerone and R-(´)-regiolone (p < 0.05).

Molecules 2016, 21, 528
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Figure 6. Effect of rac-4,8-DHT, S-(+)-isosclerone, and R-( )-regiolone at different concentrations on
´
radicle length ( ); plumule length ( ); and fresh weight (C) in wheat. a denotes a significant difference
A
B
between control and treatments (p < 0.05); b denotes a significant difference among rac-4,8-DHT,
S-(+)-isosclerone and R-(´)-regiolone (p < 0.05).
It has been reported that enantioselective effect of paclobutrazol on Microcystis aeruginosa and
Anabaena sp. was different. The (2S,3S)-( )-enantiomer showed stronger stimulatory activity on
´
M. aeruginosa cyanobacteria than the (2R,3R)-(+)-enantiomer, whereas the latter was a more potent
stimulator of Anabaena sp. growth, indicating that enantioselectivity has specific effects on different
organisms [
,8-DHT and its enantiomers, we found that 4,8-DHT and its enantiomers could improve the quality
of the five tested plants seedling at low concentration, while pure S-(+)-isosclerone was more effective
8]. However, in this study, compared with the parameters of seedlings growth between
4

Molecules 2016, 21, 528
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than the others. Therefore, pure S-(+)-isosclerone can be used as a regulator of plant growth to reduce
the environmental load. Meanwhile, S-(+)-isosclerone also inhibited seedlings growth of the five
tested plants more than R-(
toxicity of the chiral herbicide diclofop-methyl and its enantiomers, in where S-(
´
)-regiolone at the higher concentration. This result was paralleled to
´)-enantiomer of
diclofop-methyl had a stronger toxicity to both Chlorella ulgaris and Scenedesmus obliquus than the
R-(+)-enantiomer [32]. It is clear that the toxicity of 4,8-DHT and its enantiomers has specific effects
on different organisms, but the mechanism for differential enantiomers specific responses in these
species is uncertain and deserves further research. Among the five tested plants, seedling growth of
lettuce was the most sensitive to S-(+)-isosclerone treatments. At the treatment concentration of 1 mM
the radicle length and fresh weight of lettuce treated with S-(+)-isosclerone were 26.8% and 25.0%
higher than those of lettuce treated with R-(
of 2 mM S-(+)-isosclerone enhanced the fresh weight of lettuce by 44.8% higher than the racemate
and R-( )-regiolone. In addition, the radicle length and fresh weight of lettuce treated by 4 mM
S-(+)-isosclerone were 21.8% and 26.3% lower than those of lettuce treated by the 4 mM racemate and
R-( )-regiolone, respectively. However, the plumule length of lettuce cultivated with all different
groups showed no significant differences between the S-(+)-isosclerone and R-( )-regiolone treatment,
except for S-(+)-isosclerone at the higher concentration level of 4 mM. Different enantiomer-specific
responses in radicle and plumule length have also been studied for other chiral herbicides [33 34].
´)-regiolone, respectively. At the treatment concentration
´
´
´
,
For instance, (2S,3S)-paclobutrazol reduced the shoot growth more effectively than the root growth,
while (2R,3R)-paclobutrazol reduced the root growth more effectively than the shoot growth [33]. The
R-enantiomer of diclofop acid was more active toward root growth while the S-enantiomer was more
active toward leaf growth [34]. However, the mechanism is still uncertain and requires further research
in the future. As indicated by the above results for the five tested plants, all of the factors including
concentration and configuration forms of 4,8-DHT, as well as the plant species, must be considered in
order to estimate the toxic behaviors of 4,8-DHT.
3
. Materials and Methods
3
.1. Chemicals, Plant Materials and Reagents
4
,8-DHT was isolated and purified from C. callicarpa epicarp in our laboratory (purity
ě
98%).
1
,3,5-tri-tert-butylbenzene was purchased from Sigma Aldrich (St. Louis, MO, USA). Seeds of lettuce
(
(
Latuca sativa), radish (Raphanus sativus), cucumber (Cucumis sativus), onion (Allium cepa), and wheat
Triticum aestivum) were purchased from the market in Ningbo, China, and used for bioassay. HPLC
grade of iso-butanol, n-butanol, iso-propanol, n-propanol, and ethanol were purchased from Tedia
Company Inc. (Fairfield, IA, USA). All other chemicals and solvents in analytical grade were purchased
from commercial sources.
3
.2. Chromatographic Separation and Analysis
Analytical HPLC: Enantioseparation of 4,8-DHT was conducted on the HPLC system consisting
of two LC-10AT HPLC pumps, an SPD-10A UV detector monitored at 254 nm, and an FCV-12AH
-PortValve, controlled by a CBM-10A module (all items obtained from Shimadzu, Duisburg, Germany).
Column temperature was controlled by an AT-930 heater column attemperator (Tianjin Automatic
Science Instrument, Tianjin, China). The column was a Chiralcel OD column (250 mm 4.6 mm, 5 m)
Guangzhou Research and Creativity Biotechnology Co. Ltd., Guangzhou, China) with chiral stationary
6
ˆ
µ
(
phase (CSP)-coated cellulose-tris(3,5-dimethylphenylcarbamate). The mobile phase consisted of a
mixture of n-hexane and iso-propanol (95:5, v/v) containing 0.1% acetic acid (AA) under a flow rate of
´ ˝
sample injection volume was 20 µL.
1
0
.8 mL min at 20 C. The injection of samples was performed using a 7725i manual injector and the
¨
Preparative HPLC: The column was Chiralcel OD column (250 mm
Research and Creativity Biotechnology Co. Ltd., Guangzhou, China). The mobile phase consisted
ˆ
20 mm, 5 µm) (Guangzhou

Molecules 2016, 21, 528
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of a mixture of n-hexane and iso-propanol (90:10, v/v) monitored at 254 nm under a flow rate of
2 mL¨ min´ at 20 C. The sample injection volume was 5 mL.
1
˝
1
3
.3. Effects of 4,8-DHT on Seed Germination
One hundred grains of surface-sterilized lettuce seeds were placed in each sterile Petri dish (15 cm
diameter) lined with two layers of filter paper. Then, 10 mL of various concentrations of racemate,
S-(+)-isosclerone, or R-( )-regiolone (1, 2, 3, and 4 mM), and distilled water as a control, were added
into each Petri dish, respectively. Seeds were incubated in an artificial intelligence simulation incubator
´
´
2
´1
under an 8/16 h (day/night) photo period with a photon flux density of 40
µmol
¨
m
¨
s at a
˝
day/night temperature of 25/15 C. Experiments of seed germination were conducted according to
ISTA [35]. Germination (radicle emergence) was measured at four and seven days after treatment for
lettuce, four and 10 days for radish, four and eight days for cucumber and wheat, and six and 12 days
for onion.
3
.4. Effects of 4,8-DHT on Seedling Growth
Pre-germination of the tested seeds were preceded in plastic boxes (20
lined with two layers of filter paper for 3–4 days until radicle emergence. One hundred
successfully-germinated seeds were placed in Petri dishes and then 10 mL of various concentrations
ˆ
15
ˆ
10 cm)
of racemate, S-(+)-isosclerone, or R-(
were added into the dishes. Seedlings were incubated in an artificial intelligence simulation incubator
incubation conditions were the same as seed germination). Five seeds were randomly removed,
´)-regiolone (1, 2, 3, and 4 mM), and distilled water as a control,
(
and the length of the plumule and radicle were measured with a vernier caliper (GB/T 1214.2-1996,
Measuring Instrument Ltd., Shanghai, China). The fresh weight of seedlings was also recorded (Mettler
Toledo Instrument Ltd., Boston, MA, USA). The plumule length, radicle length, and fresh weight of
each tested seedlings were measured at 10 days after incubation.
3
.5. Statistic Analysis
All the above tests and measurements were performed in four replicates. All data were statistically
analyzed using Sigmaplot software. Values were considered significant when the results of one-way
ANOVA using least significant difference showed p < 0.05 using SPSS software. All other values in the
text are presented as the mean ˘ SD.
4
. Conclusions
In this study, enantiomers of 4,8-DHT were effectively separated by HPLC on the Chiralcel OD
column with CSP-coated cellulose-tris(3,5-dimethylphenylcarbamate) and showed different toxicity
towards seed germination and seedling growth of the five tested plant species. Low concentrations of
racemate, S-(+)-isosclerone, and R-( )-regiolone induced hormesis on seed germination and seedling
´
growth of the tested plant species, whereas high concentrations had a retardant effect. By comparison,
pure S-(+)-isosclerone was more toxic to seed germination and seedling growth of the five plants
and, thus, could be potentially applied as a herbicide to reduce the environmental load. Among
the five tested plants, seedling growth of lettuce was most sensitive to S-(+)-isosclerone treatments.
Additionally, it is clear that S-(+)-isosclerone has specific effects on different organisms. These results
indicated that the toxic effects of 4,8-DHT in the five tested plants were different, and all of the
concentrations and configuration forms of 4,8-DHT, as well as plants species, must be taken into
consideration in order to develop and utilize 4,8-DHT.
Acknowledgments: The authors are grateful to the Science Foundation of Ningbo (SFN, Project No.: 2015C50046)
for the financial support of the work.
Author Contributions: Conceived and designed the experiments: Qiang Wang. Performed the experiments and
analyzed the data: Li Yang, Xiao-Yan Ma, Xiao Ruan, De-An Jiang and Cun-De Pan. Contributed reagents/

Molecules 2016, 21, 528
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materials/analysis tools: Xiao-Yan Ma and Xiao Ruan. Contributed to the writing of the manuscript: Li Yang and
Qiang Wang.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
4
,8-DHT
4,8-dihydroxy-1-tetralone
high-performance liquid chromatography
chiral stationary phase
acetic acid
HPLC
CSP
AA
SD
standard deviation
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´
from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
(