Industrial Crops & Products 133 (2019) 295–303

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Industrial Crops & Products

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Wormwood (Artemisia absinthium L.) as a promising nematicidal and T

antifungal agent: Chemical composition, comparison of extraction
techniques and bioassay-guided isolation
⁎
Ting-ting Liua,b, Hai-bin Wuc, Hai-bo Wua, , Jing Zhanga
a
College of Life and Environmental Sciences, Minzu University of China, Beijing, 100081, China
b
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
c
Shandong Institute of Pomology, Tai'an, 271000, China

A R T I C LE I N FO A B S T R A C T

Keywords: The ethyl acetate, ethanol and water extracts of different organs (flowers, leaves, stems and roots) of wormwood
Artemisia absinthium L. (Artemisia absinthium L.) were investigated to examine nematicidal and antifungal activities in an effort to
Thiophenes promote future agricultural applications of the wormwood and its potential as an industrial crop. The results
Supercritical fluid extraction showed that highest concentration of total thiophene content (TTC), as well as nematicidal and antifungal ac-
Root-knot nematode
tivities were found in ethyl acetate extract of roots. TTC and biological activities were highly correlated, in-
Soil-borne fungi
dicating that thiophenes played an important role in nematicidal and antifungal activities. Furthermore, four
extraction techniques (Maceration, Soxhlet extraction, Supercritical fluid extraction and Ultrasound-assisted
extraction) were compared by evaluating the yield of thiophenes. Supercritical fluid extraction (SFE) appeared to
be the best method for extracting thiophenes with the highest TTC and superior activities. In addition, eight
thiophenes (1–8) including two new compounds (1 and 2) were isolated from the SFE extract by the bioassay-
guided method. All thiophenes showed potent nematicidal activities. In particular, 1–4 (50% lethal con-
centration (LC50) values of 2.69, 4.17, 6.13 and 7.65 mg/L, respectively) were more effective than commercial
nematicide abamectin (LC50, 9.47 mg/L). Also, these thiophenes showed antifungal activities against four soil-
borne fungi at different degrees, especially for compounds 1 and 2 which exhibited noticeable antifungal effects
(minimum inhibitory concentration (MIC) values ranging from 8–16 mg/L). Findings of this work suggest that
wormwood root and its thiophenes could be used in nematicide and fungicide agent industry.

1. Introduction To date, management of RKNs and SBFs primarily relies on the use
of artificially synthetized chemical products. While it is known that
Diseases and damages caused by plant pathogens, including nema- application of commercial nematicides and fungicides repeatedly would
todes and fungi, can greatly diminish quality and yield of crops (Bi lead to increased nematode and pathogenic fungi resistance (Bi et al.,
et al., 2018). Of the parasitic nematode species, the Meloidogyne in- 2018), and may have limited ability to cope with the disease induced by
cognita (Kofoid and White) Chitwood, which preferentially attacks the the combination of RKN and SBF (Akhtar et al., 2005). Given these
roots of host plants, is regarded as one of the most infective agricultural major drawbacks and the fact that some chemical nematicides and
root-knot nematode (RKN) species, and is responsible for yield reduc- fungicides are progressively banned in many countries (Caboni et al.,
tion of various crop species worldwide (D’Addabbo et al., 2017). 2013), demand for environmentally acceptable nematicides and fun-
Moreover, RKN can disorder plant physiology by assisting the invasion gicides that are applicable to organic farming has been soaring rapidly
of many other pathogenic soil-borne fungi (SBF) (Jang et al., 2014). The over the years (Oka et al., 2012; De Rodríguez et al., 2017). Searching
interaction of RKN and SBF can be synergistic, leading to greater ne- for novel, association virulence and low-toxicity alternatives has
gative consequences than either of them functions in isolation (Son emerged as a top urgency in agricultural industry (Tocco et al., 2017).
et al., 2009), especially in Meloidogyne spp. and Fusarium spp. disease Plant secondary metabolites defend plants against various herbi-
complex, consequently posing a great problem for the cultivation of vores and pathogenic microorganism (Luo et al., 2010). These phyto-
crops (Akhtar et al., 2005; Son et al., 2009). chemicals feature many unique properties compare to synthetized

⁎
Corresponding author.
E-mail address: whbnmr@muc.edu.cn (H.-b. Wu).

https://doi.org/10.1016/j.indcrop.2019.03.039
Received 8 December 2018; Received in revised form 15 March 2019; Accepted 15 March 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
T.-t. Liu, et al. Industrial Crops & Products 133 (2019) 295–303

commercial products, including easily-biodegradable, low residual chemicals were of analytical grade.
toxicity and potential different mode of actions (Wu et al., 2016; De
Rodríguez et al., 2017). Therefore, these bioactive molecules represent 2.2. Plant material
a rich pool for the exploration and development of commercial in-
secticide, nematicide, herbicide and fungicide. Plant material of A. absinthium was collected in August 2016 in
Artemisia absinthium L. (Asteraceae), commonly known as worm- Jintas Steppe-Grassland Nature Reserve of Fuhai, Xinjiang Uygur
wood, is well-known as a medicinal, aromatic, edible and insecticidal Autonomous Region, China. It was identified by Haibo Wu (College of
plant (Tariku et al., 2011). It is widely distributed in Europe, Asia, Life and Environmental Sciences, Minzu University of China). A vou-
North and South America as well as New Zealand (Maw et al., 1985). A. cher specimen (NO. 20160822) was deposited in the herbarium of the
absinthium has long been used for medical purposes such as restoring College of Life and Environmental Sciences, Minzu University of China.
the declining mental function (Wake et al., 2000; Guarrera, 2005). In
addition, aerial parts of A. absinthium are used for manufacturing of 2.3. Preparation of organ extracts using different solvents by maceration
drink absinthe, and also forms the basic flavor of many popular spirits method
and wines (Strang et al., 1999; Ali and Abbasi, 2013). In particular,
leaves and stems of A. absinthium are recognized in folk as an insect Plants were separated into flowers, leaves, stems and roots, oven
repellent and insecticidal plant to fend off mosquitoes, flies and mites in dried for 72 h at 40 °C, then powdered until experiments initiated. Each
many countries (Beigh and Ganai, 2017). Therefore, it is widely culti- dried sample (10 g) was extracted with 250 mL ethyl acetate, ethanol
vated in temperate regions as an economically important plant species and distilled water for 48 h at the room temperature (20 °C). The ex-
(Tariku et al., 2011; Nibret and Wink, 2010). tracts were then filtered, and the ethyl acetate and ethanol phases were
Early studies have shown that extracts from A. absinthium exhibit removed using a rotary evaporator. Thereafter, the water extracts were
nematicidal activities against M. javanica (Julio et al., 2017) and ovine freeze-dried and stored at 4 °C until analyzed. All extracts were dis-
nematodes (Tariq et al., 2009), as well as antifungal activities against a solved at the concentration of 500 mg/L (DMSO as solvent of stock
wide range of agricultural pathogenic fungal species such as Plasmopara solutions and never exceeded 1% in working solutions) for the sub-
viticola (Kordali et al., 2005; Andreu et al., 2018). It has been known sequent nematicidal and antifungal assays.
that phenolic, flavonoid, thiophene and terpenoid are the main sec-
ondary metabolites of this species (Gonzalez-Coloma et al., 2012; 2.4. Determination of contents for total phenolic, flavonoid and thiophene
Aberham et al., 2010; Yamari et al., 2013), yet the effects of various
compounds on its nematicidal and antifungal activities remain unclear. 2.4.1. Total phenolic content (TPC) determination
In addition, the best approach for the extraction of nematicidal and The TPC determination from crude extract was measured color-
antifungal constituents for this species is still unestablished. imetrically using Folin-Ciocalteu method (Sarikurkcu et al., 2015).
Accordingly, the objective of this study was to examine the effects of Extract solution (0.5 mL) was mixed with diluted Folin–Ciocalteu re-
various extracts from different organs against RKNs and SBFs to further agent (2 mL, 1:9) and shaken vigorously for 3 min. Then, Na2CO3 so-
increase the use and availability of wormwood. The correlations be- lution (1.5 mL, 1%) was added and the sample absorbance was read at
tween constituents (total contents of phenolic, flavonoid and thio- 765 nm after 2 h incubation at room temperature. Standard curve of the
phene) and activities (nematicidal and antifungal effects) were de- assay was generated using gallic acid with mixture of water and re-
termined. Furthermore, four extraction techniques were compared in agents used as blank. The data on total phenolic content were expressed
terms of yield of active constituent, and bioactive compounds were as GAE (mg GAEs/g) equivalents.
isolated from the active extracts by the bioassay-guided method. The
results of this research will help to explore natural alternatives for the 2.4.2. Total flavonoid content (TFC) determination
synthesis of nematicides and fungicides, thereby promoting industrial The TFC of crude extract was measured using a modified aluminium
consumption of A. absinthium in the field of agriculture. chloride colorimetric method following Sarikurkcu et al. (2015).
Briefly, sample solution (2 mL) was mixed with the same volume of
2. Materials and methods aluminium trichloride (2%) in methanol. A blank was prepared by
adding sample solution (2 mL) to methanol (2 mL) without AlCl3. The
2.1. Instruments sample and blank absorbance were read at 415 nm after 10 min in-
cubation at room temperature. A standard curve of the assay was
Supercritical CO2 fluid extraction was performed in a HA-121-50-02 plotted using quercetin. The data on total flavonoid content were ex-
supercritical fluid CO2 extraction apparatus (Huaan Supercritical Fluid pressed as QE (mg QEs/g) equivalents.
Extraction Inc., Jiangsu, China). Ultrasound-assisted extraction in a KQ-
500DE (Kunshan Electronics Co, Ltd., China). HRESIMS and ESIMS data 2.4.3. Total thiophene content (TTC) determination
were collected from an Agilent HPLC-QTOF/MS 6520 system instru- The TTC of crude extract was measured by fluorospectrophotometry
ment equipped with an electrospray ionization source data and Agilent as described by Wang et al. (2005) with slight modifications. Different
6890N-5975N system instrument (Agilent Technologies Inc., Palo Alto, concentrations of extracts were prepared in acetone. Each extract so-
USA). UV spectra were recorded on Thermo-Genesys 10 s spectro- lution (0.5 mL) was measured, with excitation and emission wavelength
photometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). IR being 352 nm and 428 nm, respectively. The slit width was set at
spectra were obtained on a Nicolet iS5 FTIR spectrometer (Thermo 5.0 nm A standard curve for the assay was plotted by using α-terthienyl.
Fisher Scientific Inc., Waltham, MA, USA). Fluorescence measurements The data on total thiophene content were expressed as TE (μg TEs/g)
were performed on an F-2500 fluorospectrophotometer (Hitachi, equivalents.
Japan). 1D and 2D NMR spectra were recorded on a Bruker Avance
600 MHz spectrometer (Bruker Co., Karlsruhe, Germany) with TMS as 2.5. Preparation of the root extracts with different extraction method
an internal standard in chloroform-d (Sigma-Aldrich Co., Missouri,
USA). CC (column chromatography) was taken on silica gel (Qingdao 2.5.1. Soxhlet extraction (SE)
Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 10 g dried roots were extracted successively by 250 mL ethyl acetate
(Pharmacia Biotech Ltd, Uppsala, Sweden). Abamectin, α-terthienyl using a Soxhlet extractor. After 20 exchanges of the extract (5 h), which
and carbendazim were purchased from Shanghai Aladdin Biochemical was enough for discoloration, the extracts were filtered, and the ethyl
Polytron Technologies Inc. (Shanghai, China). All other reagents and acetate was then removed by using a rotary evaporator. All the

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experiments were performed in triplicates. All extracts were dissolved were diluted with distilled water containing Tween-80. Final con-
at 200 mg/L (DMSO as solvent of stock solutions and never exceeded centrations of organic solvent (DMSO, methanol or acetone) and
1% in working solutions) for the nematicidal and antifungal assays. Tween-80 were controlled lower than 1 and 0.1% v/v, respectively (Bi
et al., 2018). Abamectin was used as the positive control. Distilled
2.5.2. Supercritical fluid extraction (SFE) water and a mixture of the organic solvent and aqueous Tween-80 were
10 g dried roots were placed in cylindrical extractor. The conditions served as controls. Treatments were performed in 96-well plates with
of SFE were as follows: extraction pressure of 30 MPa, extraction tem- 50–100 juveniles in each replication. To avoid solvent evaporation,
perature of 40 °C, CO2 flow rate of 20 L/h, extraction time was 60 min. plates were covered and kept in darkness at 28 °C. Juveniles were ob-
These parameters were selected based on previous report focusing on served under a light microscope after 24 h treatment. Nematodes were
the optimization of SFE conditions for extracting thiophenes (Szarka considered as dead if their bodies were straight without movements
et al., 2010). The SFE extracts were collected in glass vials, weighed, even if physically stimulated with a fine needle. Then J2s corrected
transferred into opaque bottles and kept refrigerated at 4 °C until fur- mortality of each sample was calculated based on the data of nemati-
ther treatments. All the experiments were performed in triplicates. All cidal bioassays as: corrected mortality % = 100 × [(mortality % in
extracts were dissolved at 200 mg/L (DMSO as solvent of stock solu- treatment − mortality % in control)/(100 − mortality % in control)].
tions and never exceeded 1% in working solutions) for the nematicidal
and antifungal assays.
2.8. Antifungal assay
2.5.3. Ultrasound-assisted extraction (UAE)
Evaluation of antifungal activitiy against four soil-borne fungi were
10 g dried roots were mixed in 250 mL ethyl acetate. The conditions
performed on Fusarium oxysporum, Fusarium solani, Phytophthora in-
of UAE were as follows: ultrasonic power 300 W, extraction time 30 min
festans and Phytophthora capsici, which represent major plant pathogens
and extraction temperature of 40 °C. These parameters were selected
of economically important crops. These fungi were obtained from the
based on previous report on thiophenes extraction by UAE (Qiao et al.,
State Key Laboratory for Biology of Plant Diseases and Insect Pests,
2010). All the experiments were performed in triplicates. All extracts
Chinese Academy of Agricultural Sciences, Beijing, China.
were dissolved at 200 mg/L (DMSO as solvent of stock solutions and
Antifungal activities of extracts were examined using in vitro assay
never exceeded 1% in working solutions) for the nematicidal and an-
following Kordali et al. (2005). Stock solutions of different extracts
tifungal assays.
were prepared in DMSO. The final concentration of DMSO was kept less
than 1% of the total volume (Xiao et al., 2014). Carbendazim was used
2.6. Bio-guided isolation
as the positive control. Briefly, PDA plates were prepared with 9 cm
diameter glass Petri dishes. Different extracts were added to each of the
500 g dried roots were extracted by SFE method. Then the dried SFE
PDA plates containing 20 mL agar. A 5-mm diameter disk of the fungal
extract (SER, 30 g) was suspended in water and partitioned sequentially
species was cut from a 1-week-old culture within PDA plates, and then
with petroleum ether, dichloromethane and acetone (Dehghan et al.,
the mycelial surface of the disk was placed upside down on the center of
2018). Each subextract was concentrated under reduced pressure to
the dish, thus ensuring the fungal species was in contact with the
yield a petroleum ether subextract, dichloromethane subextract,
growth medium on the dish. Then, the plates were incubated in dark-
acetone subextract and a remainder of water subextract.
ness at 28 ± 0.5 °C for 6 days. PDA plates treated with distilled water
The dichloromethane subextract (10 g) was subjected onto silica gel
were used as negative control. IC50 values was taken as the con-
CC eluting with PE–Acetone (15:1–1:1, gradient system). For the TLC
centration of the extract inhibiting radial growth by 50%.
analysis, six fractions (F1–F6) were obtained. F2 and F3 showed higher
The micro-dilution method was used to evaluate the antifungal ac-
activities and were selected for further isolation. F2 (1.8 g) was frac-
tivity of the compounds with 96-well microplates using a PD medium
tionated with CC (silica gel; n-hexane–CHCl3 5:1), further purified on a
(Xiao et al., 2014). Stock solutions of pure compounds were prepared in
Sephadex LH-20 column to afford 2 (3.6 mg), 6 (4.7 mg), 1 (11.5 mg)
methanol or acetone, and were kept below 1% of the working solutions.
and 8 (2.6 mg). F3 (2.3 g) was fractionated with CC (silica gel; CHCl3-
Carbendazim was used as the positive control. The fungi were in-
ethyl acetate 4:1), further purified on a Sephadex LH-20 column to
cubated in PD medium for 24 h at 28 ± 0.5 °C while rotating at
afford 3 (11.4 mg), 4 (7.3 mg), 9 (2.6 mg), 5 (3.2 mg) and 7 (4.4 mg).
150 rpm. Spore concentrations of different microorganism were diluted
to approximately 1 × 106 colony-forming units/mL (CFU/mL) with PD
2.7. Nematicidal bioassay
medium. In flat microtiter plates, tested compounds, fungal suspension,
and sterile water were added to make up final concentrations of the
A population of M. incognita was obtained from pepper (Capsicum
compounds in the range of 0.5–256 mg/L. After incubation for 48 h at
annuum L.) roots collected from a greenhouse at Shandong Institute of
28 ± 0.5 °C, minimum inhibitory concentration (MIC) was taken as the
Pomology, Tai'an, China. Dentification of M. incognita was carried out
lowest concentration of the tested compounds in the wells of the 96-
based on the perineal pattern morphology (Naz and Khan, 2013).
well plate, in which no microbial growth could be observed.
Perineal patterns were excised from 10 to 15 mature females following
the technique described by Eisenback et al. (1985) and were observed
using a Nikon Diaphot 200 inverted microscope (Nikon Corporation, 2.9. Statistical analysis
Tokyo, Japan) at 100X magnification. To obtain clean eggs from M.
incognita, infected roots of pepper were extracted using 1% NaClO so- The experiments were performed in triplicate and experimental
lution for 4 min (Hussey, 1973), and were then rinsed with distilled results were expressed as the mean ± standard deviation of mean (SD).
water for three times. Surface-sterilized eggs were then placed into a The data of TPC, TFC and TTC for different extracts as well as nema-
Petri dish filled with water and incubated in darkness at 25 °C to pre- ticidal and antifungal assays were statistically analyzed using one-way
pare second-stage juveniles (J2s). Newly-emerged J2s were collected ANOVA followed by post hoc Tukey’s multiple range test, and was
daily after egg hatching. considered statistically significant when p < 0.05. The degree of cor-
All samples were tested against the J2s of M. incognita using 96- relation between different extracts and their activities were evaluated
microwell plates for their nematicidal activities using the method de- based on Pearson correlation coefficient (r) and was classified as very
scribed in Tocco et al. (2017). Stock solutions of different extracts were high (r = 0.9–1), high (r = 0.7–0.89), moderate (r = 0.4–0.69), low
prepared by diluting with DMSO while pure compounds were prepared (r = 0.2–0.39) and very low (r < 0.2). All statistical analyses were
in methanol or acetone to overcome the insolubility. Working solutions performed in SPSS 19.0 (SPSS Inc., Chicago, IL, USA).

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Table 1
Extraction yields, total phenolic contents (TPC), total flavonoid contents (TFC), total thiophene contents (TTC), nematicidal activity against M. incognita and
antifungal effect against F. oxysporum of the different organs extracts of wormwood.
Organs Solvents Yield (%) TPC (mg GAEs/g) TFC (mg QEs/g) TTC (μg TEs/g) Nematicidal activities (%, Mycelial growth inhibition (%,
500 mg/L) 500 mg/L)

Flowers Ethyl acetate 8.4 96.17 ± 3.41 cd 88.92 ± 2.21 de 143.78 ± 1.78 g 56.32 ± 2.62 e 45.82 ± 1.73 bc

Ethanol 13.5 105.42 ± 2.51 de 126.43 ± 3.59 g 92.45 ± 4.44 f 54.35 ± 1.14 e 65.69 ± 5.45 fg

Water 11.3 104.17 ± 3.21 d 96.45 ± 4.17 f 53.55 ± 2.27 c 35.28 ± 4.91 ab 53.43 ± 2.33 e

Leaves Ethyl acetate 11.2 86.35 ± 2.11 b 45.47 ± 1.15 a 56.32 ± 2.54 c 47.41 ± 2.25 de 42.67 ± 3.27 ab

Ethanol 16.6 108.42 ± 2.62 de 59.46 ± 2.97 b 31.25 ± 0.57 ab 38.52 ± 1.22 bc 62.69 ± 1.67 f

Water 12.7 88.49 ± 5.53 b 43.58 ± 5.42 a 26.42 ± 2.02 a 29.46 ± 3.33 a 51.33 ± 2.37 de

Stems Ethyl acetate 6.3 78.42 ± 3.55 a 58.45 ± 2.91 b 65.78 ± 3.33 d 36.49 ± 1.59 ab 38.38 ± 1.83 a

Ethanol 11.7 91.12 ± 2.66 bc 92.39 ± 1.01 ef 53.56 ± 2.94 c 42.45 ± 2.21 cd 42.71 ± 2.04 ab

Water 9.6 75.30 ± 2.77 a 83.21 ± 2.47 de 36.36 ± 1.91 b 33.00 ± 2.02 a 38.87 ± 3.73 a

Roots Ethyl acetate 8.8 81.28 ± 5.42 ab 68.22 ± 1.03 c 478.65 ± 5.23 i 87.31 ± 2.84 g 72.45 ± 3.12 g

Ethanol 9.4 103.36 ± 2.14 d 86.00 ± 1.74 de 320.18 ± 4.35 h 77.24 ± 2.31 f 64.63 ± 2.81 f

Water 14.2 106.36 ± 2.09 de 73.04 ± 3.39 c 76.46 ± 0.95 e 42.89 ± 3.09 cd 47.91 ± 1.19 cd

Abamectin 100.00 ± 0.00 h

h
Carbendazim 100.00 ± 0.00

Values are shown as mean ± standard deviation of mean (SD) (n = 3) and were compared using one-way ANOVA followed by post hoc Tukey’s multiple range test.
Different letters in same columns indicate significant differences (p < 0.05).

3. Results (76.07 ± 2.62%, mortality at 200 mg/L), followed by the UAE extract
(72.14 ± 0.92%, mortality at 200 mg/L) and SE extract
3.1. Phytochemical contents (TPC, TFC and TTC) across organs and their (52.69 ± 0.55%, mortality at 200 mg/L). Maceration extract exhibited
correlations with biological effects (nematicidal and antifungal activities) the lowest nematicidal activity (43.87 ± 1.12%, mortality at 200 mg/
L). Also, strong correlation between TTC and nematicidal activities was
Ethanol extract prepared from the leaves exhibited the highest TPC identified (r = 0.962, p < 0.01).
(108.42 ± 2.62 mg GAEs/g), while water extract prepared from the in vitro evaluation of antifungal activities indicated that all the
stems contained the lowest TPC (75.30 ± 2.77 mg GAEs/g) (Table 1). extracts were active against the fungi tested here (Table 2). Antifungal
Ethanol extract prepared from the flowers showed the highest TFC activities, expressed as the concentration causing 50% mycelial growth
(126.43 ± 3.59 mg QEs/g), while water extract prepared from the inhibition (IC50), was found to the highest in SFE extract for F. oxy-
leaves showed the lowest TFC (43.58 ± 5.42 mg QEs/g). TPC and TFC sporum and F. solani (IC50 = 94.14 and 61.57 mg/L, respectively). The
of ethanol extracts were consistently higher than that of water extract. highest antifungal activity against P. infestans was found for UAE ex-
Ethyl acetate extract of the roots possessed the highest TTC tract (IC50 = 105.12 mg/L). SE extract showed the highest antifungal
(478.65 ± 5.23 μg TEs/g), followed by the ethanol extract activity against P. capsici (IC50 = 71.74 mg/L). Accordingly, SFE extract
(320.18 ± 4.35 μg TEs/g). For other roots extracts, the TTC ranged with the highest TTC and superior activities were chosen for bioassay-
from 26.42–143.78 μg TEs/g. guided isolation.
All organ extracts of A. absinthium showed nematicidal activities.
Across extracts, highest nematicidal activity was observed in ethyl 3.3. Bioassay-guided isolation
acetate extract of roots (87.31 ± 2.84%, mortality at 500 mg/L), fol-
lowed by the ethanol extract of the roots (77.24 ± 2.31%, mortality at To isolate nematicidal and antifungal compounds, the SFE extract
500 mg/L). Across organs, roots extract invariably showed higher ne- from wormwood roots (SER) was partitioned with various organic
maticidal activity than other organs. Similarly, highest antifungal ac- solvents including petroleum ether, dichloromethane, acetone to yield
tivity was observed in the ethyl acetate extract of roots four solvent-soluble fractions. Nematicidal and antifungal activities of
(72.45 ± 3.12%, mycelial growth inhibition at 500 mg/L). the four layers, including an aqueous layer, were tested at concentra-
Results of Pearson correlation analysis showed that both TPC and tions of 200 mg/L against M. incognita and F. oxysporum. Overall, the
TFC were not significantly correlated with nematicidal activity (p = dichloromethane subextract of SER (86.42 ± 1.58%) showed higher
0.875 and p = 0.528, respectively). On the other hand, strong corre- nematicidal activity than that of petroleum ether subextract
lation was found between TTC and nematicidal activity (r = 0.950, (59.47 ± 2.33%), acetone subextract (43.21 ± 3.65%) and water
p < 0.01). In addition, antifungal activity was strongly correlated with subextract (32.16 ± 2.74%) (Table 3). The highest antifungal activity
TTC (r = 0.658, p < 0.05) but not of TPC (r = 0.452, p = 0.140) or was also found for dichloromethane subextract (51.22 ± 2.96%, my-
TFC (r = 0.245, p = 0.442). Accordingly, the roots of A. absinthium celial growth inhibition at 200 mg/L). Based on these results, di-
with the highest TTC and activities were chosen for isolating nemati- chloromethane subextract of SER was chosen for further isolation.
cidal and antifungal compounds. The dichloromethane subextract of SER was separated by column
chromatography (CC) to six fractions (F1 to F6). Across fractions, F2
3.2. Comparison of nematicidal and antifungal activities of TTC extracted (nematicidal activity, 100.00 ± 0.00%; antifungal effect,
by different methods (SFE, SE, UAE and maceration) 64.62 ± 3.56%) and F3 (nematicidal activity, 94.53 ± 1.02%; anti-
fungal effect, 68.48 ± 2.17%) showed the best nematicidal and anti-
Another objective of this work was to compare different extraction fungal results (Table 3), and therefore were selected to isolate and
techniques for the isolation of thiophenes from wormwood. The results identify the potent active phytochemicals.
showed that SFE, SE and UAE methods gave higher total thiophene than
maceration (Fig. 1A). In addition, the SFE extract showed approxi- 3.4. Structure elucidation of compounds 1–9
mately 2%, 28% and 93% higher TTC (923.18 ± 11.19 μg TEs/g)
compare to the UAE, SE and maceration extracts, respectively. Compound 1, obtained as a pale yellow oil, was found to have the
All extracts exhibited nematicidal activities (Fig. 1B). Among others, molecular formula C14H12O2S2, as determined through HRESIMS
SFE extract presented the highest nematicidal activity ([M + Na]+ peak at m/z 299.0176, calc for C14H12NaO2S2, 299.0175).

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Fig. 1. Total thiophene contents (a) and nematicidal activities (b) of root extracts from four extraction techniques at 200 mg/L. Identical letters above the bars
indicate no statistically significant differences according to Tukey’s test post hoc multi comparison (p < 0.05).

IR spectrum indicated carbonyl group (1740 cm−1). The 1H NMR 3.4.1. Artabsithiophene A (1)
spectrum (Table 4) exhibited two group of double doublets signals at δH Yellow oil; [α ]20
D = + 0.38° (c 0.04, MeOH); HRESIMS at m/z
6.99 (1H, d, J = 3.6 Hz, H-3) and δH 6.97 (1H, d, J = 3.6 Hz, H-4), δH 299.0176 [M + Na]+ (calcd for C14H12NaO2S2, calc. Mass for
6.96 (1H, d, J = 3.5 Hz, H-3′) and δH 7.00 (1H, d, J = 3.6 Hz, H-4′) for a 299.0175); UV (MeCN) λmax (logε): 244 (2.6), 339 (4.2) nm; IR (KBr)
typical 2,2′-bithiophene moieties with two substituents at δC 137.1 (C- Vmax: 1740, 1225, 1023 cm−1; 1H NMR and 13C NMR (see Table 4).
5) and δC 123.4 (C-5′) in its 13C NMR spectrum (Ibrahim et al., 2016).
Furthermore, there was an acetoxymethyl moiety (δH 2.09, δC 170.7 3.4.2. Artabsithiophene B (2)
and δC 20.9) in 1 (Fig. 2). Moreover, the characteristic 13C NMR che- Yellow oil; [α ]20
D = + 1.48° (c 0.05, MeOH); HRESIMS at m/z
mical shifts at δC 72.9 (C-6′), δC 91.4 (C-7′) and δC 4.7 (C-8′), indicating 341.0640 [M + Na]+ (calcd for C17H18NaO2S2, calc. Mass for
the presence of a prop-1-ynyl group in this compound. In the HMBC 341.0646); UV (MeCN) λmax(logε): 245 (1.1), 339 (2.6) nm; IR (KBr)
spectrum, long-range correlations were observed from H2-6 (δH 5.20) to Vmax: 1735, 1163, 796 cm−1; 1H NMR and 13C NMR (Table 4)
C-4 (δC 129.0), C-5 and the carbonyl of acetoxymethyl (δC 170.7), as The six known thiophene compounds including methyl (E)-3-(5-
well as from H3-8′ (δH 2.08) to C-6′, C-7′, C-5′ and C-4′ (δC 131.8) (prop-1-yn-1-yl) thiophen-2-yl) acrylate (3) (Greger, 1978), trans-de-
(Fig. 3), implying that a acetoxymethyl group and a prop-1-ynyl group hydromatricaria ester (4) (Greger, 1978), rhapontiynethiophenes A (5)
were attached to the bithiophene ring at C-5 and C-5′, respectively. As a (Liu and Guo, 2008), 5-(3-hydroxmethyl-3-isovaleroyloxyprop-1-ynyl)-
result, the structure of compound 1 was elucidated as artabsithiophene 2,2′-bithiophene (6) (Liu and Guo, 2008), 5-(3,4-diacetoxybut-1-ynyl)-
A. 2,2′-bithiophene (7) (Lin et al., 1999) and 5-(3-acetoxy-4-iso-
Compound 2, obtained as a pale yellow powder, was found to have valeroyloxybut-1-ynyl)-2,2′-bithiophene (8) (Wang et al., 2006), as well
the molecular formula C17H18NO2S2, as determined through HRESIMS as one flavonoid artemisetin (9) (Aberham et al., 2010) were identified
([M + Na]+ peak at m/z 341.0640, calc for C17H18NaO2S2, 341.0646). by comparing their spectroscopic and physical data (see Supporting
IR spectrum indicated carbonyl group (1735 cm−1). The 1H NMR Information) to that reported in the literature. The structures of thio-
spectrum (Table 4) and 13C NMR spectra for compound 2 (Table 4) phenes were shown in Fig. 2.
exhibited close similarity to that of compound 1 except for a iso-
pentanoyl group [δH 2.25 (2H, d, J = 6.5 Hz, H2-2′'), 2.14 (1H, m, H-3′')
and 0.98 (6H, d, J = 6.7 Hz, H3-4′' and H3-5′'); δC 172.7 (C-1′'), 43.3 (C- 3.5. Nematicidal activities of compounds
2′'), 25.7 (C-3′'), 22.4 (C-4′') and 22.4 (C-5′')] (Fig. 2, Table 4). In the
HMBC spectrum, long-range correlations were observed from H2-6 (δH The 9 purified compounds were tested for in vitro nematicidal ac-
5.26) to C-4 (δC 128.8), C-5 (δC 137.5) and C-1′', and from H3-8′ (δH tivities against J2s of RKN. All thiophene compounds showed effective
2.11) to C-6′ (δC 72.9), C-7′ (δC 91.3), C-5′ (δC 123.4) and C-4′ (δC nematicidal activities against M. incognita with the exception of flavo-
131.7) (Fig. 3), suggesting a isopentanoyl group and a prop-1-ynyl noid artemisetin (9) (LC50 > 100 mg/L, Table 5). Of these, the four
group were attached to the bithiophene ring at C-5 and C-5′, respec- thiophenes 1–4 exhibited remarkable nematicidal activities, with LC50
tively. Thus, the structure of 2 was elucidated as artabsithiophene B. of 2.69, 4.17, 6.13 and 7.65 mg/L, respectively. In addition, moderate
nematicidal activities was observed for thiophenes 5–8, with the LC50

Table 2
Concentration causing 50% mycelial growth inhibition (IC50) values of different extracts from four extraction techniques against four soil-borne fungi F. oxysporum, F.
solani, P. infestans and P. capsici.
Extracts F. oxysporum F. solani P. infestans P. capsici

IC50 (mg/L) (CI95) a

χ2 IC50 (mg/L) (CI95) a
χ2 IC50 (mg/L) (CI95) a
χ2 IC50 (mg/L) (CI95) a
χ2

Maceration 377.05 (285.64–626.80) 6.48 192.70 (173.71–218.67) 3.09 394.69 (293.52–747.34) 8.01 283.27 (241.09–375.11) 7.32
SE 103.17 (91.45–123.06) 1.58 147.78 (117.74–185.64) 7.16 239.06 (203.18–309.23) 6.24 71.74 (66.27–77.31) 4.37
SFE 94.14 (87.46–102.76) 2.72 61.57 (48.74–72.56) 7.78 108.88 (98.47–125.37) 4.16 73.31 (59.47–88.20) 12.46
UAE 254.57 (217.54–335.86) 5.89 77.95 (71.86–84.70) 5.16 105.12 (96.39–116.43) 3.22 231.38 (217.29–245.49) 3.59
Carbendazim 0.88 (0.58–1.50) 1.86 0.39 (0.23–0.67) 3.08 > 500 > 500

a
Numbers in parentheses stand for the upper and lower bounds of 95% confidence interval.

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T.-t. Liu, et al. Industrial Crops & Products 133 (2019) 295–303

Table 3
Nematicidal activity against M. incognita and antifungal effect against F. oxysporum of the subextracts of SFE extract for roots (SER).
Test samples Nematicidal activities (%, 200 mg/L) Mycelial growth inhibition (%, 200 mg/L)

c c
Petroleum ether subextract of SER 59.47 ± 2.33 46.36 ± 1.17
Dichloromethane subextract of SER 86.42 ± 1.58 e 51.22 ± 2.96 d

Acetone subextract of SER 43.21 ± 3.65 b 33.54 ± 1.08 ab

Water subextract of SER 32.16 ± 2.74 a 27.35 ± 3.33 a

F1 of dichloromethane subextract of SER 78.43 ± 2.76 d 47.25 ± 1.61 cd

F2 of dichloromethane subextract of SER 100.00 ± 0.00 g 64.62 ± 3.56 e

F3 of dichloromethane subextract of SER 94.53 ± 1.02 f 68.48 ± 2.17 e

F4 of dichloromethane subextract of SER 82.29 ± 3.57 de 42.25 ± 1.06 c

F5 of dichloromethane subextract of SER 61.90 ± 1.92 c 35.88 ± 2.10 b

F6 of dichloromethane subextract of SER 58.42 ± 2.20 c 37.49 ± 3.55 bc

Abamectin 100.00 ± 0.00 g

f
Carbendazim 98.38 ± 1.21

Values are shown as mean ± standard deviation of mean (SD) (n = 3) and were compared using one-way ANOVA followed by post hoc Tukey’s multiple range test.
Different letters in same columns indicate significant differences (p < 0.05).

Table 4 showed moderate effects (MIC of 64 mg/L) (Table 6). For F. solani,
1 13
H (600 MHz) and C (150 MHz) NMR data of compounds 1 and 2 (CDCl3). significant antifungal activities were found for compound 1 and 2 (MIC
Position 1 2
of 8 mg/L), while compounds 3, 4, 6 and 8 showed moderate effects
δH (ppm) δC δH (ppm) δC (MIC of 64 mg/L). For P. infestans and P. capsici, thiophenes showed
antifungal activities, with MIC values ranging from 8 to 64 mg/L.
2 138.4 138.3 Overall, compounds 1 and 2 showed markedly antifungal effects (MIC
3 6.99 (1H, d, J = 3.6 Hz) 123.5 7.02 (1H, d, J = 3.6 Hz) 123.4
4 6.97 (1H, d, J = 3.6 Hz) 129.0 6.99 (1H, d, J = 3.6 Hz) 128.8
values ranging from 8–16 mg/L) and compounds 3–4 and 6–8 showed
5 137.1 137.5 moderate antifungal effects (MIC values ranging from 32–128 mg/L) on
6 5.20 (2H, s) 60.6 5.24 (2H, s) 60.3 all four plant pathogens while compounds 5 and 9 exhibited no anti-
2' 137.2 137.1 fungal activities (MIC values ≥256 mg/L).
3' 6.96 (1H, d, J = 3.5 Hz) 123.5 6.99 (1H, d, J = 3.5 Hz) 123.4
4' 7.00 (1H, d, J = 3.6 Hz) 131.8 7.02 (1H, d, J = 3.5 Hz) 131.7
5' 123.4 123.4
4. Discussion
6' 72.9 72.9
7' 91.4 91.3
8' 2.08 (3H, s) 4.7 2.11 (3H, s) 4.7 It has been known that contents of total phenolic and total flavo-
1” 170.7 172.7 noids are positively related to the antiradical and antioxidative activ-
2” 2.09 (3H, s) 20.9 2.25 (2H, d, J = 6.5 Hz) 43.3 ities of wormwood extracts (Canadanovic-Brunet et al., 2005). The
3” 2.14 (1H, m) 25.7
4” 0.98 (3H, d, J = 6.7 Hz) 22.4
fairly good levels of phenolic and flavonoid extracted from various
5” 0.98 (3H, d, J = 6.7 Hz) 22.4 organs of wormwood in the present study thus suggest that this species
has the potential to be utilized in pharmaceutical and food industry.
Moreover, ethanol extract of leaves showed the highest amount of total
values being 27.83, 12.25, 16.37 and 22.45 mg/L, respectively. phenol, while ethanol extract of flowers showed the highest total fla-
vonoid quantity, suggesting that leaves and flowers of wormwood have
3.6. Antifungal activities of compounds the ability to reduce or scavenge free radicals.
Wormwood has been widely used in pharmaceutical, food, and
For F. oxysporum, compounds 1 and 2 displayed significant anti- pesticidal industries (Beigh and Ganai, 2017). The huge demand for
fungal activities (MIC of 16 mg/L), while compounds 3, 4, 6 and 7 wormwood has led to its broad cultivation in many contraries (Bailen

Fig. 2. The structures of thiophenes 1–8 from A. absinthium.

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Fig. 3. Key HMBCs (H→C) correlations of compounds 1 and 2.a.

Table 5 provides experimental evidence for the potential application of the

Concentration of the extract inhibiting radial growth by 50% (LC50) against M. roots of A. absinthium in management of root-knot nematode.
incognita for compounds 1–9. The cross-technique comparison revealed that the novel extraction
Compounds Nematicidal activities Compounds Nematicidal activities methods (i.e. SFE and UAE) can produce more thiophene extracts than
(mg/L) (mg/L) traditional extraction methods (i.e. SE and maceration). Additionally,
the SFE extract showed good antifungal activities, especially against P.
1 2.69 ± 0.23 a 6 12.25 ± 3.34 ef
infestans, which is a difficult pathogen to control that causes late blight
2 4.17 ± 0.41 b 7 16.37 ± 2.11 f
3 6.13 ± 0.19 c 8 22.45 ± 3.09 g in tomato (Lee et al., 2009). Previous reports on extraction techniques
4 7.65 ± 0.22 d 9 > 100 for wormwood also showed that the supercritical extracts exhibited
5 27.83 ± 1.17 g Abamectin 9.47 ± 0.46 e stronger insecticidal activities against a wide range of insects than the
traditional ones, and moderate phytotoxic effects on root growth of
Values are shown as mean ± standard deviation of mean (SD) (n = 3) and
Lolium perenne (< 50% inhibition) (Martín et al., 2011a, 2013). SFE has
were compared using one-way ANOVA followed by post hoc Tukey’s multiple
been previously applied for selectively extracting some compounds
range test. Different letters indicate significant differences (p < 0.05).
from wormwood (Stahl and Gerard, 1983; Martín et al., 2011b). Ad-
ditionally, several studies have reported that SFE is a suitable method
Table 6
for potential industrial applications to obtain lipophilic (non-polar)
Minimum inhibitory concentration (MIC) values of compounds 1–9 against four
compounds (Fornari et al., 2012). Indeed, thiophenes 1–8 isolated from
soil-borne fungi.
supercritical fluid extracts in the present study were all lipophilic
Compounds F. oxysporum F. solani P. infestans P. capsici compounds with reduced polarity. Compare to hydrophilic compounds,
(mg/L) (mg/L) (mg/L) (mg/L)
lipophilic compounds can easily enter nematode cells through their
1 16 8 16 16 membranes which have lower lipid solubility. These results demon-
2 16 8 16 8 strate that SFE might be considered as a potential method to obtain
3 64 64 32 64 total thiophene from the wormwood in the agricultural industry, while
4 64 64 32 64
SFE extracts could be utilized as the raw materials in the nematicide
5 256 256 > 256 > 256
6 64 64 32 32 and fungicide industry.
7 64 128 64 64 Bioassay-guided fractionation and isolation on active ingredients
8 128 64 32 64 from SFE extract of wormwood led to the discovery of eight thiophenes
9 > 256 > 256 > 256 > 256
including two new compounds. All thiophenes showed potent nemati-
Carbendazim 4 1 256 128
cidal and antifungal activities. Natural thiophenes are a relatively small
class of plant products that are conspicuous in their sulfur atoms
et al., 2013; Kordali et al., 2005; Rezaeinodehi and Khangholi, 2008). structure chemical properties (Ibrahim et al., 2016). Thiophenes are
Often, the aerial parts of wormwood are used for medicinal and edible shown to be toxic to a number of pathogens, including nematodes, in-
purposes (Beigh and Ganai, 2017; Mahmoudi et al., 2009), while roots sects, fungi, and bacteria (Champagne et al., 1984; Gil et al., 2002).
were commonly wasted without effective development and utilization. These findings add to the information with respect to the structural and
Such findings demonstrate that the roots of wormwood should be biological properties of thiophenes. Furthermore, thiophenes isolated
considered as valuable by-products that bear application value and from wormwood maybe the leading compounds for the development of
development prospects, as indicated by their high thiophene content as agrochemical agents. The nematicidal activities of the isolated thio-
well as nematicidal and antifungal activities. phenes in relation to their structure led us to provide some preliminary
Nematotoxic properties have been identified in several plant ex- interpretations for the structure-activity relationships. Given com-
tracts (Ntalli and Caboni, 2012). For instance, azadirachtin, which is pounds 1–4 with a propynyl group at one side were more nematically
isolated from the extract of Azadirachta indica, is a commercial nema- potent than the compounds without this group (i.e. 6–8), we suggest
ticides registered for nematode control. (Akhtar, 2000). We found that that propynyl group play an important role in nematicidal activity.
ethyl acetate extract of wormwood roots was also extremely toxic to M. However, when a chorine group attached to the other side, nematocidal
incognita, with the nematicidal activity (mortality, 87.31% at 500 mg/ effect of compound 5 was significantly reduced compare with com-
L) being significantly higher than the extract of A. indica (mortality, pounds 1–4 and 6–8 even if a propynyl group was present. Likely,
55.5% at 500 mg/L) (Elbadri et al., 2008). We therefore suggest that the compound 1 with acetyl group showed more potent nematicidal than
crude extract of wormwood roots processes nematicidal activity and compound 2 with isovaleryl group and compound 5 with chlorine
thus could be utilized as the raw materials in the nematicide industry. atom, a similar result was observed 7 > 8, thus indicting that acetyl is
Results of present study indicated that thiophenes plays a more an important active group in terms of nematicidal activity. The isola-
important role in nematicidal activity than the other compounds. This is tion of constituents from wormwood, its nematicidal and antifungal
supported by the observation that isolated thiophenes (1–8) showed activities will enhance the future agricultural applications of this crop
good nematicidal activities compare to flavonoid artemisetin (9). It has and its potential as an industrial crop.
been reported that natural thiophenes originated exclusively from roots
in many species (Ibrahim et al., 2016; Marotti et al., 2010). For ex-
ample, Yamari et al. (2013) found that thiophene could be isolated from 5. Conclusion
the roots of A. absinthium but not from other organs. This finding
In conclusion, results of this study showed that thiophenes played

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an important role in nematicidal and antifungal activities. Moreover, identification of the four most common species of root-knot nematodes (Meloidogyne
extracts using SFE method showed the highest TTC with strong nema- spp.). Adv. Treat. Meloidogyne 1, 95–112.
Elbadri, G.A., Lee, D.W., Park, J.C., Yu, H.B., Choo, H.Y., 2008. Evaluation of various
ticidal and antifungal activities. In addition, eight nematicidal and plant extracts for their nematicidal efficacies against juveniles of Meloidogyne in-
antifungal thiophenes including two new were isolated and identified. cognita. J. Asia-Pac. Entomol. 11 (2), 99–102.
From the perspective of industrial applications, we suggest that: (1) Fornari, T., Vicente, G., Vázquez, E., García-Risco, M.R., Reglero, G., 2012. Isolation of
essential oil from different plants and herbs by supercritical fluid extraction. J.
roots of wormwood could be regarded as a promising high potential by- Chromatogr. A 1250, 34–48.
product for the bio-nematicide and bio-fungicide agent industries; (2) Gil, A., Ghersa, C.M., Perelman, S., 2002. Root thiophenes in Tagetes minuta L. accessions
SFE is the most efficient method for the extraction of thiophenes from Argentina: genetic and environmental contribution to changes in concentration
and composition. Biochem. Syst. Ecol. 30 (1), 1–13.
compared with the conventional methods; (3) SFE extract from roots of Gonzalez-Coloma, A., Bailen, M., Diaz, C.E., Fraga, B.M., Martínez-Díaz, R., Zuñiga, G.E.,
wormwood could be utilized as the raw materials to produce nemati- Contreras, R.A., Cabrera, R., Burillo, J., 2012. Major components of Spanish culti-
cide and fungicide industrially; (4) thiophenes from wormwood roots vated Artemisia absinthium populations: antifeedant, antiparasitic, and antioxidant
effects. Ind. Crops Prod. 37, 401–407.
are valuable in agricultural industry as a good alternative to synthetize
Greger, H., 1978. A new acetylenic ester from Artemisia absinthium. Phytochemistry 17
nematicide and fungicide. (4), 806.
Guarrera, P.M., 2005. Traditional phytotherapy in central Italy (Marche, Abruzzo, and
Acknowledgements Latium). Fitoterapia 76 (1), 1–25.
Hussey, R.S.A., 1973. Comparison of methods of collecting inocula of Meloidogyne spp.,
including a new technique. Plant Dis. Rep. 57, 1025–1028.
This work was financially supported by the National Natural Science Ibrahim, S.R., Abdallah, H.M., El-Halawany, A.M., Mohamed, G.A., 2016. Naturally oc-
Foundation of China [31800278], the Young Teachers’ Scientific curring thiophenes: isolation, purification, structural elucidation, and evaluation of
bioactivities. Phytochem. Rev. 15 (2), 197–220.
Research Ability Promotion Program of Minzu University of China Jang, J.Y., Le Dang, Q., Choi, Y.H., Choi, G.J., Jang, K.S., Cha, B., Luu, N.H., Kim, J.C.,
[2019QNTS65] and the National College Students Innovation and 2014. Nematicidal activities of 4-quinolone alkaloids isolated from the aerial part of
Entrepreneurship Training Program [URTP2019110040]. Triumfetta grandidens against Meloidogyne incognita. J. Agric. Food Chem. 63 (1),
68–74.
Julio, L.F., González-Coloma, A., Burillo, J., Diaz, C.E., Andrés, M.F., 2017. Nematicidal
Appendix A. Supplementary data activity of the hydrolate byproduct from the semi industrial vapor pressure extraction
of domesticated Artemisia absinthium against Meloidogyne javanica. Crop Prot. 94,
33–37.
Supplementary material related to this article can be found, in the Kordali, S., Kotan, R., Mavi, A., Cakir, A., Ala, A., Yildirim, A., 2005. Determination of the
online version, at doi:https://doi.org/10.1016/j.indcrop.2019.03.039. chemical composition and antioxidant activity of the essential oil of Artemisia dra-
cunculus and of the antifungal and antibacterial activities of Turkish Artemisia ab-
sinthium, A. dracunculus, Artemisia santonicum, and Artemisia spicigera essential oils. J.
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