Industrial Crops & Products xxx (xxxx) xxxx

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Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

Two new coumarins from Zanthoxylum dimorphophyllum spinifolium and their

feeding deterrent activities against Tribolium castaneum
Wen-juan Zhanga,b, Wei Guanc, Zhu-feng Gengb,d, Yang Wangb, Xue Pangb, Chun-xue Youa,
Shu-shan Dub,*
a
College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, No. 22 Jinjing Road, Tianjin, 300384, China
b
Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Faculty of Geographical Science, Beijing Normal University, No.19 Xinjiekouwai Street,
Haidian District, Beijing, 100875, China
c
Dandong Chinese Medicine Hospital, No. 22 Jinshan Street, Dandong, Liaoning, 118000, China
d
Analytical and Testing Center, Beijing Normal University, No. 19 Xinjiekouwai Street, Haidian District, Beijing, 100875, China

ARTICLE INFO ABSTRACT

Keywords: Zanthoxylum species and volatile oils have shown to have insect resistance. The present work was conducted to
Coumarins find alternative botanical source with significant activity on storage pests control. Chemical examination of
Feeding deterrent activity Zanthoxylum dimorphophyllum Hemsl. var. spinifolium Rehder et E. H. Wilson stem bark has led to fifteen cou-
Structure-effectiveness relationship marins, which including two were novel compounds named 6-(2′,3′-dihydroxy-3′-methylbutyl)-7-hydroxyl-8-
(3″-methyl-2″-butenyl)-2H-1-benzopyran-2-one (7) and 6-(2′-hydroxyl-3′-methyl-3′-butenyl)-7-methoxyl-8-(3″-
methyl-2″-butenyl)-2H-1-benzopyran-2-one (8). All the chemical structures were elucidated on the basis of
nuclear magnetic resonance spectroscopy and high resolution electrospray ionization mass spectrometry ana-
lysis. Additionally, dose-dependent feeding deterrent activities of fifteen coumarins against Tribolium castaneum
(Herbst) (Coleoptera: Tenebrionidae) were evaluated scientifically. Among them, compound 12 (phellopterin)
exhibited stronger feeding deterrent activity (EC50 = 64.00 ppm) than the positive control, toosendanin. While,
compound 11 (imperatorin) displayed the same level of feeding deterrent activity as the positive control with
median effect concentration of 73.10 ppm. The relationship between chemical structure and effectiveness
showed that compounds with epoxy structure or isopentenyl group exhibited higher feeding deterrent activities
and might have potential to be developed into novel feeding deterrent reagents or potential lead compounds on
storage insect prevention and control.

1. Introduction repeated usage of the synthetic insecticides has been demonstrated to

Storage pests cause both qualitative and quantitative loss to crops
and medicinal herbs, making them unsuitable for food, herbs and feed
sowing (Guru-Pirasanna-Pandi et al., 2018). As one of the foremost
destructive storage insects, Tribolium castaneum is used in various bio-
logical disciplines, including immunology, developmental biology,
evolutionary, and ecology (Gokhale et al., 2017; Kittelmann et al.,
2013; Richards et al., 2008). Nowadays, botanical pesticides have been
commonly used to protect stored grains and medicinal materials against
pests (Zhang et al., 2018). Also, some plant derivatives of secondary
metabolites exhibited more potent insecticidal activity than too-
sendanin, a commercial botanical insecticide isolated from Melia aze-
darach (Huang et al., 2018; Xu et al., 2019). However, massive and
have a series of negative impacts, including pesticide residue, en-
vironmental pollution, and insect resistance (Cheng et al., 2003; Zettler
and Arthur, 2000). Therefore, storage insects control has been given
more and more attention to develop alternative strategies which in-
clude using botanical compounds as effectively natural anti-insect
agents or leading compounds to control insects in storage (Arthur,
1996; Cheng et al., 2003; Zhang et al., 2018). Thus, botanical bioactive
compounds with little or no harmful effects on environment and non-
targeted organisms have potential to be developed into an alternative
source of insect-control agents (Adakole and Adeyemi, 2012; Tavares
et al., 2010).
As one of the traditional shrub, Zanthoxylum dimorphophyllum
Hemsl. var. spinifolium Rehder et E. H. Wilson, belonging to Rutaceae

Corresponding author.
⁎

E-mail addresses: zhangwj0729@163.com (W.-j. Zhang), guanwei1978@163.com (W. Guan), gengzhufeng@bnu.edu.cn (Z.-f. Geng),
wangyangjs@mail.bnu.edu.cn (Y. Wang), pangxue0924@mail.bnu.edu.cn (X. Pang), youchunxue@163.com (C.-x. You), dushushan@bnu.edu.cn (S.-s. Du).

https://doi.org/10.1016/j.indcrop.2019.111889
Received 30 June 2019; Received in revised form 22 September 2019; Accepted 19 October 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Wen-juan Zhang, et al., Industrial Crops & Products, https://doi.org/10.1016/j.indcrop.2019.111889
W.-j. Zhang, et al.
family, distributed in Shaanxi, Hubei, Guizhou, and Sichuan province of
China. In Folk, it is used as medicinal plants for aches and wet sores
(Tao et al., 2001, 2005). And, recent research has shown that its volatile
oil has obvious repellent effect on storage pests (Zhang et al., 2017).
According to literatures, chemical constituents of Z. dimorphophyllum
spinifolium are various, which including coumarins, lignans, alkaloids,
glycosides, sterols, and flavonoids (Du, 2015; Tao, 2002; Xiang et al.,
2000). In our present work, fifteen coumarins, including two novel
compounds were isolated from Z. dimorphophyllum spinifolium and some
of them exhibited notable feeding deterrent activity.
2. Materials and methods
2.1. General information
Bruker Avance III 500 instrument (Switzerland, Bruker A.G.) was
used to record NMR spectra at room temperature. Column chromato-
graphy was subjected on silica gel (160–200 mesh) and Thin Layer
Chromatography (TLC) analysis was carried out on silica gel G plates
which were purchased from Qingdao Marine Chemical Plant of China.
Sephadex LH-20 purchased from Amersham Pharmacia Biotech of
Beijing were used as well. Mitsubishi Chemical Ion (MCI) gel CHP20P
(75–150 μm) was supplied by Kaiteki Company of Japan. All the ana-
lytical reagents used in this project were purchased from Beijing
Chemical Factory of China. The deuterated solvents (CDCl3, Deuterated
ratio, 99.8%) with tetramethylsilane (TMS) were bought from
Cambridge Isotope Labo-ratories, Inc of the United States.
2.2. Plants
The fresh stem bark of Z. dimorphophyllum spinifolium collected in
Wen county, Gansu Province of China (32.95 °N, 104.70 °E) in 2
September 2015 was identified by Prof. Liu, Q.R. (College of Life
Sciences, Beijing Normal University, Beijing, China) and the voucher
specimen (BNU-CMH-DuSS-2015-09-02-003) was deposited at the
Herbarium of Faculty of Geographical Science, Beijing Normal
University.
2.3. Insects culture
Laboratory culture of red flour beetle, Tribolium castaneum identi-
fied by Prof. Liu, Z.L. (College of Plant Protection, China Agricultural
University, Beijing, China) were followed the same method mentioned
in literature (Ma et al., 2004). The unsexed insect adults used in the
experiments were about 7 days old.
2.4. Extraction and isolation
The fresh stem bark (4.5 kg) of Z. dimorphophyllum spinifolium were
roughly crushed and extracted with methanol under ultrasound for three
times. Crude extracts (280.0 g) were collected under vacuum. The extract
was crudely fractionated by silica gel column chromatography (160–200
mesh, 10.0 × 63 cm, 1300.0 g), eluting with a stepwise gradient of PE
(petroleum ether) / EtOAc (ethyl acetate) (60:1, 40:1, 20:1, 10:1, 5:1 and
1:1), then CHCl3/CH3OH (30:1, 15:1, 5:1 and 1:1) to receive 36 fractions.
Totally, fifteen coumarins were isolated from Z. dimorphophyllum spini-
folium. All purified products were analyzed by 1H and 13C NMR spectra
and compared with literature. Coumarins 1-15 were characterized and
identified as umberlliferone (1, 10.6 mg) (Duan et al., 2015), 5,7,8-tri-
methoxycoumarin (2, 34.3 mg) (Shen et al., 2014), 8-methoxyl-6-(3-
methybut-2-enyl)-2H-1-benzopyran-2-one (3, 20.0 mg) (Schmidt and
Riemer, 2016), 7-geranoxycoumarin (4, 24.5 mg) (Girennavar et al.,
2007), 6-(3′-methyl-2′,3′-oxiranylbutyl)-7-methoxyl-8-(3″-methyl-2″-bu-
tenyl)-2H-1-benzopyran-2-one (5, 207.0 mg) (Tao et al., 2005), 6-(3′-
methyl-2′,3′-dihydroxylbutyl)-7-methoxyl-8-(3″-methyl-2″-butenyl)-2H-
1-benzopyran-2-one (6, 3601.0 mg) (Shi et al., 2006), 6-(2′,3′-dihydroxy-
2
Industrial Crops & Products xxx (xxxx) xxxx
3′-methylbutyl)-7-hydroxyl-8-(3″-methyl-2″-butenyl)-2H-1-benzopyran-
2-one (7, 11.8 mg), 6-(2′-hydroxyl-3′-methyl-3′-butenyl)-7-methoxyl-8-
(3″-methyl-2″-butenyl)-2H-1-benzopyran-2-one (8, 19.8 mg), xantho-
toxin (9, 13.4 mg) (Duan et al., 2015), isopimpinellin (10, 12.6 mg) (Kim
et al., 2015), imperatorin (11, 1600.1 mg) (Zhang et al., 2009), phel-
lopterin (12, 17.6 mg) (Li et al., 2015), marmesin (13, 15.6 mg)
(Ludemann and Nimz, 1974), 2′,3′-dihydro-2′-(4′- hydroxymethyl-ethyl)-
8-(3″-methyl-2″-buten-1″-yl)-2H-1-benzopyran-2-one (14, 5400.3 mg)
(Rao et al., 1980), and pimpinellin (15, 15.6 mg) (Li et al., 2015), re-
spectively.
2.4.1. Feeding deterrent assay
Feeding deterrent assay was performed by a modified method de-
scribed in literature (Du et al., 2011; Wang et al., 2015; Zhang et al.,
2018). Precisely weighed compounds (2.0 mg) were diluted in ethanol
to make the mother liquor (1.0 mg/mL). Then, mother liquor was di-
luted in water to obtain a series of concentration gradients of 25, 74,
222, 667, 2000 ppm. After that, the final solution was mixed well with
flour (0.4 g). While, the blank control was followed by the mixture of
pure water (2.0 mL) and flour (0.4 g). Six small cookies were made drop
by drop on clean dishes. After air dried overnight, the small cookies
with different concentrations were transferred into the incubator for
48 h to achieve equilibration at 29–30 °C and 70–80% relative humidity
(RH). Twenty unsexed insects were selected and moved into glass vials
(diameter 2.5 cm, height 5.5 cm) and starved for 24 h in the incubator
for further weighing. Then, small cookies were added into vials and
weighed. Five duplications were performed for each concentration.
After 72 h incubate, the total weight was weighed. Finally, all the in-
sects were picked out immediately, and weighed again. The value of
percent feeding deterrent index was calculated as follows: feeding de-
terrent index (%) = [(C-T)/C] × 100. Where C represents the weight of
diet consumed in blank control groups and T represents the treated
groups. The feeding deterrent index and EC50 were calculated by SPSS
(IBM) and Origin software. Statistical analysis was carried out using
Student’s t-test to compare two groups, with p < 0.05 being indicative
of significance. A commercial feeding deterrent reagent, toosendanin
(Batch No.: 79304-40-8), as a positive control, was purchased from
National Institutes for Food and Drug Control.
3. Results and discussion
3.1. Chemical compounds isolated from Z. dimorphophyllum spinifolium
Chemical isolation has led to thirteen known coumarins, including
umberlliferone (1), 5,7,8-trimethoxycoumarin (2), 8-methoxyl-6-(3-
methybut-2-enyl)-2H-1-benzopyran-2-one (3), 7-geranoxycoumarin
(4), 6-(3′-methyl-2′,3′-oxiranylbutyl)-7-methoxyl-8-(3″-methyl-2″-bu-
tenyl)-2H-1-benzopyran-2-one (5), 6-(3′-methyl-2′,3′-dihydroxylbutyl)-
7-methoxyl-8-(3″-methyl-2″-butenyl)-2H-1-benzopyran-2-one (6), xan-
thotoxin (9), isopimpinellin (10), imperatorin (11), phellopterin (12),
marmesin (13), 2′,3′-dihydro-2′-(4′-hydroxymethyl-ethyl)-8-(3″-me-
thyl-2″-buten-1″-yl)-2H-1-benzopyran-2-one (14), and pimpinellin (15)
and two novel compounds, including 6-(2′,3′-dihydroxy-3′-methyl-
butyl)-7-hydroxyl-8-(3″-methyl-2″-butenyl)-2H-1-benzopyran-2-one
(7) and 6-(2′-hydroxyl-3′-methyl-3′-butenyl)-7-methoxyl-8-(3″-methyl-
2″-butenyl)-2H-1-benzopyran-2-one (8). All the chemical structures
were shown in Fig. 1. As it shown in Fig. 1, the structural characteristics
of fifteen coumarins are obvious. Compounds 1-8 were simple cou-
marins, while compounds 9-15 were furocoumarin. However, com-
pounds 3-8, 11-12, and 14 have the same substituent of isopentenyl.
Therefore, different chemical structure with different substituent might
result in different bioactivity.
As it displayed in Fig. 1, compounds 1-8, and compounds 9-5 have
the same basic skeletons. Compared with compounds 1-8, compounds
9-15 have furan rings on the basic skeletons. Among compounds 9-15,
compounds 9-14 are linear furocoumarins, while compound 15 is
W.-j. Zhang, et al. Industrial Crops & Products xxx (xxxx) xxxx

Fig. 1. Chemical structures of coumarins from Z. dimorphophyllum spinifolium stem bark.

angular furocoumarin. In addition, compounds 9-12 are dihydro fur- Table 1

1
ocoumarins, however, compounds 13 and 14 are saturated fur- H- and 13C-NMR data of compounds 7 and 8 in CDCl3.
ocoumarins. From the perspective of substituent groups, compounds 3- Position 7 8
8, 11, 12, and 14 have same substituent group of isopentenyl group.
Hence, different chemical structure with different substituent group δH δC δH δC
might results in different bioactivity.
2 161.2 161.0
3 6.32 (d, J =9.5 Hz) 114.8 6.35 (d, J =9.5 Hz) 115.0
4 7.65 (d, J =9.5 Hz) 143.7 7.64 (d, J =9.5 Hz) 143.5
3.2. Identification of two new compounds 5 7.35 (s) 127.5 7.27 (s) 127.5
6 129.9 128.8
Compound 7 was isolated as light yellow oil and its molecular for- 7 159.7 159.6
8 123.5 123.7
mula (C21H28O5) was determined on the basis of HR-ESI-MS, which 9 152.1 152.3
indicted an pseudo-molecular peak at m/z 383.1850 [M + Na]+ (calc. 10 115.5 115.5
360.1937) and NMR data (Table 1), requiring eight degrees of un- 1' 2.79 (dd, J = 10.5, 31.8 2.99 (dd, J = 3.5, 14.0 Hz); 2.83 36.4
saturation. A total of 21 carbon resonances were exhibited in the 13C- 2.0 Hz) (dd, J = 8.5, 14.0 Hz)
2' 3.74 (dd, J = 10.5, 77.2 4.34 (dd, J = 3.5, 8.5 Hz) 75.8
NMR spectrum. Characteristic peaks of coumarin skeleton were re-
2.0 Hz)
vealed at δC 161.2, δC 159.7, δC 152.1, δC 143.7, δC 114.8, δC 129.9, δC 3' 71.8 147.1
127.5, δC 123.5, δC 115.5 in the 13C-NMR spectrum. Isopentenyl carbon 4' 1.27 (s) 19.6 5.02 (s), 4.89 (s) 111.2
signals were displayed at δC 132.7, δC 121.6, δC 25.7, δC 23.1, δC 18.1 in 5' 1.25 (s) 20.5 1.85 (s) 18.1
the 13C-NMR spectrum. At δH 6.32, δH 7.65 of 1H-NMR spectrum, a pair 6' 3.83 (s) 61.7
7' 3.30 (s) 49.3
of characteristic double peaks with a coupling constant of 9.5 Hz de- 1” 3.58 (t, J =7.0 Hz) 23.1 3.58 (d, J =7.0 Hz) 23.1
monstrated that the compound belonged to coumarin. Moreover, che- 2” 5.26 (m) 121.6 5.26 (t, J =7.0 Hz) 121.4
mical shifts at δH 5.26, δH 3.58, δH 1.86, and δH 1.70 proved the ex- 3” 132.7 132.9
istence of isopropyl group. The 1H- and 13C-NMR data featured a 4” 1.86 (s) 18.1 1.86 (s) 18.1
5” 1.70 (s) 25.7 1.71 (s) 25.7
coumarin skeleton. The COSY correlations between H-1′ (δH 2.79) and
7- OCH3 3.85 (s) 61.8
H-2′ (δH 3.74), as well as the HMBC correlations arising from H-1′ (δH
2.79) to C-5 (δC 127.5), C-6 (δC 129.9), C-2′ (δC 77.2), from H-4′ (δH
1.27) to C-5′ (δC 20.5), C-2′ (δC 77.2), from H-5′ (δH 1.25) to C-4′ (δC compound 7 was identified as 6-(2′,3′-dihydroxy-3′-methylbutyl)-7-
19.6), C-2′ (δC 77.2) and from H-7′ (δH 3.30) to C-2′ (δC 77.2) confirmed hydroxyl-8-(3′'-methyl-2′'-butenyl)-2H-1-benzopyran-2-one.
the structure of 6-substituted group. While, the COSY correlations be- Compound 8 was obtained as light yellow needles and its molecular
tween H-2′' (δH 5.26) and H-1′' (δH 3.58), H-2′' (δH 5.26) and H-4′' (δH formula (C20H24O4) was determined on the basis of HR-ESI-MS, which
1.86), H-2′' (δH 5.26), and H-5′' (δH 1.70), as well as the HMBC corre- indicted an pseudo-molecular peak at m/z 329.1791 [M+H]+ (calc.
lations arising from H-1′' (δH 3.58) to C-7 (δC 159.7), C-9 (δC 152.1), C- 328.1791) and NMR data (Table 1), requiring nine degrees of un-
2′' (δC 121.6), and C-3′' (δC 132.7), from H-2′' (δH 5.26) to C-5′' (δC saturation. This was verified by 20 carbon resonances in the 13C-NMR.
25.7), from H-4′' (δH 1.86) to C-1′' (δC 23.1), C-5′' (δC 25.7), and from H- Like compound 7, nine characteristic peaks, including δC 161.0, δC
5′' (δH 1.70) to C-2′' (δC 121.6), C-3′' (δC 132.7), C-4′' (δC 18.1) allowed 159.6, δC 152.3, δC 143.5, δC 128.8, δC 127.5, δC 123.7, δC 115.5 and δC
the establishment of the substitution of isopentenyl group. The key 115.0 revealed a coumarin skeleton. There was an isopentenyl sub-
COSY and HMBC correlations were presented in Fig. 2. Finally, on the stitution which carbon signals were displayed at δC 132.9, δC 121.4, δC
basis of NMR spectrum and HR-ESI-MS analysis, the structure of

3
W.-j. Zhang, et al. Industrial Crops & Products xxx (xxxx) xxxx

Fig. 2. The key COSY and HMBC correlations of compounds 7 and 8.

25.7, δC 23.1, δC 18.1 in the 13C-NMR spectrum. As a coumarin, char- methoxyl-8-(3′'-methyl-2′'-butenyl)-2H-1-benzopyran-2-one.

acteristic hydrogen proton signals were displayed as a pair of double
peaks with a coupling constant of 9.5 Hz at δH 7.64, δH 6.35 in 1H-NMR
spectrum. Moreover, the establishment of isopropyl group were proved
at δH 5.26, δH 3.58, δH 1.86 and δH 1.71. In 2D-NMR spectrum, COSY
correlations between H-1′ (δH 2.99, 2.83) and H-2′ (δH 4.34), and
HMBC correlations arising from H-1′ (δH 2.99, 2.83) to C-5 (δC 127.5),
C-2′ (δC 75.8), from H-2′ (δH 4.34) to C-6 (δC 128.8), C-1′ (δC 36.4), C-2′
(δC 75.8), C-3′ (δC 147.1), C-4′ (δC 111.2), from H-4′ (δH 5.02, 4.89) to
C-2′ (δC 75.8), C-5′ (δC 18.1), from H-5′ (δH 1.85) to C-4′ (δC 111.2), C-2′
(δC 75.8) confirmed the structure of 6-substituted group. The sub-
stitution of isopentenyl group was allowed by COSY correlations be-
tween H-1′' (δH 3.58) and H-2′' (δH 5.26), H-2′' (δH 5.26), and H-5′' (δH
1.71), H-4′' (δH 1.86) and H-5′' (δH 1.71) as well as HMBC correlations
from H-1′' (δH 3.58) to C-9 (δC 152.3), C-2′' (δC 121.4), C-3′' (δC 132.9),
from H-2′' (δH 5.26) to C-5′' (δC 25.7), from H-4′' (δH 1.86) to C-2′' (δC
121.4), C-5′' (δC 25.7), from H-5′' (δH 1.71) to C-4′' (δC 18.1). The key
COSY and HMBC correlations were presented in Fig. 2. Finally, on the
basis of NMR spectrum and HR-ESI-MS analysis, the structure of com-
pound 7 was identified as 6-(2′-hydroxyl-3′-methyl-3′-butenyl)-7-
3.3. Feeding deterrent activity
Concentration-dependent feeding deterrent activities against T. cas-
taneum adults of fifteen coumarins isolated from Z. dimorphophyllum
spinifolium stem bark were displayed in Table 2. As it shown in Table 2,
compounds 10 (isopimpinellin) and 12 (phellopterin) exhibited strong
feeding deterrent activities with FDIs more than 38% at the lowest
concentration of 25 ppm. Moreover, compounds 11 (imperatorin), 12
(phellopterin), 14 (2′,3′-dihydro-2′-(4′-hydroxymethyl-ethyl)-8-(3″-me-
thyl-2″-buten-1″-yl)-2H-1-benzopyran-2-one), and 15 (pimpinellin) dis-
played high feeding deterrent activities against T. castaneum adults with
FDIs more than 80% at the highest concentration of 2000 ppm. The EC50
of fifteen coumarins were shown in Fig. 3. As it shown in Fig. 3, com-
pound 12 (phellopterin) exhibited the highest feeding deterrent activity
against T. castaneum adults with an EC50 of 64.00 ppm and there was a
significant difference compared with the positive control of toosendanin
at p < 0.05. Moreover, compound 11 (imperatorin) showed the same
feeding deterrent activity as the positive control.

Table 2
Feeding deterrent activities of coumarins from stem bark of Zanthoxylum dimorphophyllum spinifolium.
Compound Feeding Deterrent Index (FDI)(%, Mean ± SDa) EC50b (ppm)

25 ppm 74 ppm 222 ppm 667 ppm 2000 ppm

1 21.38 ± 1.56 34.56 ± 2.18 50.15 ± 1.51 59.99 ± 1.48 70.05 ± 2.01 289.05 ± 3.73
2 25.64 ± 1.01 33.42 ± 1.94 49.38 ± 1.29 60.27 ± 1.03 70.76 ± 1.42 273.81 ± 3.20
3 21.05 ± 2.36 43.66 ± 2.95 57.13 ± 2.48 67.45 ± 2.25 74.05 ± 1.12 175.26 ± 11.17
4 30.93 ± 1.12 44.50 ± 1.20 56.98 ± 1.67 67.90 ± 2.42 70.43 ± 1.62 140.69 ± 6.53
5 28.65 ± 1.17 43.66 ± 2.65 58.74 ± 2.56 67.39 ± 1.43 69.45 ± 2.05 151.31 ± 3.14
6 29.48 ± 0.53 45.38 ± 0.99 55.78 ± 1.59 63.03 ± 1.54 68.52 ± 1.65 166.92 ± 2.79
7 28.63 ± 1.23 43.46 ± 1.95 58.56 ± 0.70 67.89 ± 2.03 66.52 ± 1.59 158.35 ± 4.64
8 29.93 ± 1.48 45.12 ± 1.45 57.93 ± 1.63 62.00 ± 1.20 72.09 ± 1.17 151.88 ± 1.37
9 28.34 ± 1.86 46.30 ± 1.12 59.21 ± 2.73 69.83 ± 1.26 73.60 ± 1.11 129.03 ± 5.23
10 38.65 ± 2.17 49.99 ± 1.51 50.35 ± 1.84 61.44 ± 1.17 68.33 ± 2.28 123.59 ± 2.82
11 36.50 ± 2.78 52.27 ± 0.81 60.85 ± 1.17 75.80 ± 1.70 83.77 ± 0.96 73.10 ± 8.58
12 38.62 ± 1.81 54.64 ± 1.19 59.08 ± 1.21 79.31 ± 1.69 85.33 ± 1.60 64.00 ± 4.16
13 35.47 ± 1.57 40.53 ± 4.24 56.52 ± 0.84 66.98 ± 1.29 71.41 ± 1.51 137.76 ± 6.18
14 24.62 ± 2.71 46.85 ± 0.59 58.89 ± 2.57 67.84 ± 2.00 81.30 ± 1.53 133.73 ± 7.29
15 35.50 ± 1.54 49.27 ± 2.12 60.85 ± 1.46 65.80 ± 1.43 89.75 ± 1.45 88.38 ± 7.26
Toosendaninc 32.32 ± 2.28 52.45 ± 3.27 69.52 ± 2.47 76.54 ± 3.62 86.27 ± 3.51 71.69 ± 3.13

a
Means standard deviation.
b
Means concentration for 50% of maximal effect.
a
Data from reference (Zhang et al., 2018).

4
W.-j. Zhang, et al.
Fig. 3. Feeding deterrent EC50 of coumarins against T. castaneum. Ctrl: the positive control; ***means that there’s significant difference between tested compound
and the positive control at p < 0.05.
From the structural analysis of the tested coumarins, the different
basic skeletons and different substituents of coumarins have directly led
to different feeding deterrent activities against T. castaneum adults.
From the perspective of basic skeletons, the feeding deterrent activities
of furocoumarins on T. castaneum adults were significantly stronger
than that of simple coumarins. Furocoumarins form a large class of
naturally occurring compounds, which possess surprising pharmacolo-
gical activity including antioxidants, anti-inflammatory and they are
also used for the treatment of skin diseases, hyperproliferative dis-
orders, viral infections, and hypertension (Carbone et al., 2019; Guillon
et al., 2019; Olomola et al., 2014; Niu et al., 2019; Wang et al., 2016).
Seven furocoumarins with strong feeding deterrent activities were ob-
tained from the stem bark of Z. dimorphophyllum spinifolium. Among the
feeding deterrent activities of angular coumarins were higher than the
activities of linear coumarin. After the alkenyl bond between 2′ and 3′
positions saturated, the feeding deterrent activities were slightly re-
duced. In addition, it is interesting that the presence of isopentenyl
group could enhance the feeding deterrent activities of coumarin
against T. castaneum adults.
Compared with compound 9 (xanthotoxin, EC50 = 129.03 ppm) and
compound 10 (isopimpinellin, EC50 = 123.59 ppm), as linear fur-
ocoumarins, both compound 11 (imperatorin) and compound 12
(phellopterin) containing an oxygen isopentenyl exhibited strong
feeding deterrent activities with EC50 values of 73.10 ppm, 64.00 ppm,
respectively, which were close to the activity of the positive control,
toosendanin (EC50 = 71.06 ppm). Moreover, the feeding deterrent ac-
tivity of compound 12 (phellopterin) even exceeded that of too-
sendanin. In the absence of the isopentenyl substituent, the linear fur-
ocoumarins (compounds 9, 10, 13) were less active than the angular
fururofurocoumarin (compound 15) during feeding deterrent assay. In
addition, when the 2′ and 3′ positions of furocoumarin are saturated,
the π-π conjugation of the compound structure is weakened, such as
compound 13 (marmesin, EC50 = 137.76 ppm) and compound 14
(2′,3′-dihydro-2′-(4′-hydroxymethyl-ethyl)-8-(3″-methyl-2″-buten-1″-
yl)-2H-1-benzopyran-2-one, EC50 = 133.73 ppm), and the feeding de-
terrent activities against T. castaneum adults weakened as well.
From the analysis of the substituents of simple coumarins, the
coumarins containing a isopentenyl group have a significantly higher
feed deterrent activities against T. castaneum adults than other sub-
stituent groups, and the deterrent activities increase as the number of
5
Industrial Crops & Products xxx (xxxx) xxxx
isopentenyl substituents increases. For instance, compound 4 (7-ger-
anoxycoumarin, EC50 = 140.69 ppm) containing two isopentenyls dis-
played slightly higher feeding deterrent activity against T. castaneum
adults than that of compounds containing one or none isopentenyl.
Besides, compounds 5-8 have the same isopentenyl substituents at the
same position of R4. While, compared with compounds 6-8, compound
5 (6-(3′-methyl-2′,3′-oxiranylbutyl)-7-methoxyl-8-(3″-methyl-2″-bu-
tenyl)-2H-1-benzopyran-2-one, EC50 = 151.31 ppm) with epoxy struc-
ture displayed stronger feeding deterrent activity against T. castaneum
adults. Moreover, the appearance of alkenyl bond of 8-substituent on
compound 8 enhanced feeding deterrent activity on T. castaneum
adults. Recent research results indicated that isopentenyl groups were
critical to increase the membrane permeability and to dissipate the
proton motive force (Wu et al., 2019).
4. Conclusions
From the analysis of the substituents of simple coumarins, the
coumarins containing a isopentenyl group have a significantly higher
feed deterrent activities against T. castaneum adults than other sub-
stituent groups, and the deterrent activities increase as the number of
isopentenyl substituents increases. For instance, compound 4 (7-ger-
anoxycoumarin, EC50 = 140.69 ppm) containing two isopentenyls dis-
played slightly higher feeding deterrent activity against T. castaneum
adults than that of compounds containing one or none isopentenyl.
Besides, compounds 5-8 have the same isopentenyl substituents at the
same position of R4. While, compared with compounds 6-8, compound
5 (6-(3′-methyl-2′,3′-oxiranylbutyl)-7-methoxyl-8-(3″-methyl-2″-bu-
tenyl)-2H-1-benzopyran-2-one, EC50 = 151.31 ppm) with epoxy struc-
ture displayed stronger feeding deterrent activity against T. castaneum
adults. Moreover, the appearance of alkenyl bond of 8-substituent on
compound 8 enhanced feeding deterrent activity on T. castaneum
adults.
Acknowledgment
This project was supported by the Research Project of Tianjin
Municipal Education Commission (2018KJ191).
W.-j. Zhang, et al.
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