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Antifeedant activities of methanol extracts of four Zanthoxylum species and benzophenanthridines from stem bark of Zanthoxylum schinifolium against Tribolium castaneum
Industrial Crops and Products 74 (2015) 407–411
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Antifeedant activities of methanol extracts of four Zanthoxylum species and benzophenanthridines from stem bark of Zanthoxylum schinifolium against Tribolium castaneum Cheng-fang Wang a,b , Chun-xue You a , Kai Yang a , Shan-shan Guo a , Zhu-feng Geng c , Li Fan b,∗ , Shu-shan Du a,∗∗ , Zhi-wei Deng c , Yong-yan Wang a a
Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Beijing Normal University, Beijing 100875, China China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, China c Analytical and Testing Center, Beijing Normal University, Beijing 100875, China b
a r t i c l e
i n f o
Article history: Received 20 January 2015 Received in revised form 27 March 2015 Accepted 21 May 2015 Keywords: Benzophenanthridines Antifeedant activity
a b s t r a c t Antifeedant activities of the methanol extracts from Zanthoxylum bungeanum, Zanthoxylum schinifolium, Zanthoxylum armatum and Zanthoxylum dissitum were assessed on Tribolium castaneum adults. It was found that the methanol extract of stem bark of Z. schinifolium had the highest antifeedant activity at 41.12% (antifeedant index). Based on bioactivity-guided fractionation, six benzophenanthridines norchelerythrine (1), decarine (2), 8-hydroxy-9-methoxy-2,3-(methylenedioxy) benzophenanthridine (3), 6-hydroxydihydrochelerythrine (4), 6-methoxy-7-hydroxydihydrochelerythrine (5) and oxychelerythrine (6) were isolated from the stem barks of Z. schinifolium. And their antifeeding activities were also evaluated against T. castaneum. All of them exhibited strong antifeeding activity in a concentrationdependant manner with EC50 of 62.67, 66.97, 151.39, 96.72, 141.61 and 192.32 ppm, respectively. The six bioactive compounds from the stem bark of Z. schinifolium might be used as antifeedants against T. castaneum. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The red flour beetle, Tribolium castaneum (Herbst), is considered to be causing serious damage to stored grain products throughout the world, which reduces the quantity and quality of the food economically (Buckman and Campbell, 2013). Fumigants and chemical insecticides are widely used to control of T. castaneum population (White and Leesch, 1995). As their intensive application for decades, the negative consequences of commercial chemical insecticides have exposed, such as disruption biological control by natural enemies and outbreaks of insect species, development of insect resistance, undesirable effects on non-target organisms (Zettler and Arthur, 2000; Isman, 2006). Therefore, to develop new
∗ Corresponding author. Tel.: +86 10 62389938; fax: +86 10 62389938. ∗∗ Corresponding author. Tel.: +86 10 62208022; fax: +86 10 62208022. E-mail addresses: [email protected] (C.-f. Wang), [email protected] (C.-x. You), yangk [email protected] (K. Yang), [email protected] (S.-s. Guo), [email protected] (Z.-f. Geng), msfl@sina.com (L. Fan), [email protected] (S.-s. Du), [email protected] (Z.-w. Deng), [email protected] (Y.-y. Wang). http://dx.doi.org/10.1016/j.indcrop.2015.05.045 0926-6690/© 2015 Elsevier B.V. All rights reserved.
alternatives replacement is needed and natural products are a potential source of safer insecticides. (Dayan et al., 2009). The genus Zanthoxylum (Rutaceae) comprises a plenty of trees and shrubs, until now about 250 species have been found in temperate and tropical regions around the world (Pirani, 1993). Many of them are often used as condiments (due to the pungent taste of fruits, seeds, leaves, and bark) and therapeutic remedies especially in Eastern Asian countries (Epifano et al., 2011). Studies have demonstrated that Zanthoxylum plants (Rutaceae) have a potential in insect control as some of their contained compounds possess significant fumigant, contact, repellent, larvicidal and deterrent activity against some insects (Wang et al., 2011; Liang et al., 2013; Japheth et al., 2014; Ge and Weston, 1995). In this work, the samples of four Zanthoxylum species distributed widely in China, namely, Zanthoxylum bungeanum, Zanthoxylum schinifolium, Zanthoxylum armatum and Zanthoxylum dissitum were collected and their antifeedant activities were assessed on the red flour beetle. Meanwhile, six benzophenanthridines were isolated from the methanol extract of Z. schinifolium stem bark and their antifeedant activities were tested against T. castaneum for the first time.
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Fig. 1. Structure of benzophenanthridines from Z. schinifolium.
2. Materials and methods
2.4. Identification of the compounds
2.1. Plant material
Chemical structures were assigned by analysis of the MS, 1 H, and 2 D NMR spectra and by comparison with literature values. Accordingly, compound 1–6 were identified as norchelerythrine (Jaromir et al., 2004), decarine (Martin et al., 2005), 8-hydroxy-9methoxy-2,3-(methylenedioxy) benzophenanthridine (Oscar and Luis, 2004), 6-hydroxydihydrochelerythrine (Seckarova et al., 2002), 6-methoxy-7-hydroxydihydrochelerythrine (Tarus et al., 2006), and oxychelerythrine (He et al., 2002), respectively (Fig. 1). Norchelerythrine (1). C20 H15 NO4 , colorless needles, ESI-MS m/z: 334.1150 [M + H]+ . 1 H NMR(500 MHz, CDCl3 ) ıppm: 9.77(1H, s, H-6), 8.75(1H, s, H-4), 8.37(1H, d, J = 9.0 Hz, H-10), 8.35(1H, d, J = 9.0 Hz, H-11), 7.86(1H, d, J = 9.0 Hz, H-12), 7.61(1H, d, J = 9.0 Hz, H-9), 7.28(1H, s, H-1), 6.16(2H, s, H-13), 4.15(3H, s, 8-OCH3 ), 4.08(3H, s, 7-OCH3 ). 13 C NMR(125 MHz, CDCl3 ) ıppm: 149.37(C-8), 148.47(C-2), 148.23(C-3), 146.48(C-6), 145.26(C-7), 129.75(C-4b, 10b), 128.10(C-12a), 127.05(C-12), 121.81(C-4a), 120.00(C-10a), 118.80(C-9), 118.25(C-10), 118.18(C-11), 110.00(C-6a), 104.36(C1), 102.18(C-4), 101.32(C-13), 61.90(7-OCH3 ), 56.83(8-OCH3 ). Decarine (2). C19 H13 NO4. Pale yellow needles, ESI-MS m/z: 320.09 [M + H]+ . 1 H NMR(500 MHz, DMSO-d6 ) ıppm: 10.11(1H, s, 8OH), 9.59(1H, s, H-6), 8.55(1H, s, H-4), 8.53(1H, d, J = 9.0 Hz, H-11), 8.48(1H, d, J = 9.0 Hz, H-10), 7.97(1H, d, J = 9.0 Hz, H-12), 7.59(1H, d, J = 9.0 Hz, H-9), 7.53(1H, s, H-1), 6.23(2H, s, H-13), 4.03(3H, s, 7-OCH3 ). 13 C NMR(125 MHz, DMSO-d6 ) ıppm: 148.61(C-2), 148.37(C-3), 148.00(C-8), 146.28(C-6), 142.56(C-7), 139.10(C4b), 129.64(C-12a), 128.74(C-4a), 127.55(C-12), 126.83(C-10a), 124.03(C-9), 122.10(C-6a), 120.49(C-10b), 119.26(C-10), 119.14(C11), 104.99(C-1), 101.96(C-13), 101.34(C-4), 61.67(7-OCH3 ). 8-Hydroxy-9-methoxy-2,3-(methylenedioxy) benzophenanthridine (3). C19 H13 NO4 , yellow needles, ESI-MS m/z: 320.09 [M + H]+ . 1 H NMR(500 MHz, DMSO-d ) ıppm: 10.00(1H, s, -OH), 9.22(1H, 6 s, H-6), 8.62(1H, d, J = 9.0 Hz, H-11), 8.54(1H, s, H-4), 8.16(1H, s, H-10), 7.95(1H, d, J = 9.0 Hz, H-12), 7.52(1H, s, H-1), 7.49(1H, s, H-7), 6.21(2H, s, H-13), 4.11(3H, s, 9-OCH3 ). 13 C NMR(125 MHz, DMSOd6 ) ıppm: 153.28(C-9), 150.23(C-6), 148.41(C-3), 148.24(C-8), 148.23(C-2), 139.63(C-4b), 129.53(C-4a), 128.88(C-12a), 127.83(C6a), 126.63(C-12), 122.75(C-10a), 120.34(C-10b), 119.64(C-11), 111.37(C-7), 104.94(C-1), 103.09(C-10), 101.86(C-13), 101.48(C-4), 56.58(9-OCH3 ). 6-Hydroxydihydrochelerythrine (4). C21 H19 NO5 , pale yellow needles, ESI-MS m/z: 388.1 [M + Na]+ . 1 H NMR(500 MHz, CDCl3 ) ıppm: 7.81(1H, d, J = 8.5 Hz, H-11), 7.72(1H, s, H-4), 7.65(1H, d, J = 8.5 Hz, H-10), 7.50(1H, d, J = 8.5 Hz, H-12), 7.16(1H, s, H1), 7.05(1H, d, J = 8.5 Hz, H-9), 6.08(2H, s, H-13), 5.73(1H, s, H-6), 4.02(3H, s, 8-OCH3 ), 3.95(3H, s, 7-OCH3 ), 2.78(3H, s, NCH3 ). 13 C NMR(125 MHz, CDCl3 ) ıppm: 152.18(C-8), 147.90(C-3), 147.33(C-2), 146.63(C-7), 138.68(C-4b), 131.02(C-10b), 126.79(C13 C
Z. bungeanum (pericarps) was collected from Yanqing County of Beijing, China (40.53◦ N latitude; 115.92◦ E longitude); Z. schinifolium (pericarps, leaves, stem barks) was collected from Kuandian Manchu Autonomous County, Liaoning Province, China (40.43◦ N latitude; 124.47◦ E longitude); Z. armatum (leaves) was collected from Xishuangbanna, Yunnan Province, China (21.99◦ N latitude; 100.83◦ E longitude); Z. dissitum (leaves and roots) was collected from Yulin, Guangxi Province, China (22.67◦ N latitude; 110.14◦ E longitude). The voucher specimens were deposited at college of resources science & technology, Beijing Normal University. The fresh materials were air dried and then ground to a powder. 2.2. Extraction and isolation The powdered plant materials (100 g) were performed by ultrasonic extraction with methanol for 30 min at room temperature (three times). The extracts were filtered and concentrated under reduced pressure to residues. The methanol extract of stem barks from Z. schinifolium (15 g) was chromatographed on silica gel column (500 g, 160–200 mesh, Qingdao Marine Chemical Plant, Shandong Province, China), eluting with a gradient of chloroform–methanol (from 100/0 to 0/100). With the monitoring of thin-layer chromatography (TLC, precoated silica gel GF254 plates), 25 fractions were obtained. Among them fractions 5, 7, 13 and 15 showed considerable antifeedant activity. The fraction 5 (108 mg) was eluted with PE–EtOAc 85/15 on silica gel to afford 15 sub-fractions, and sub-fractions 5–9 (50 mg) were further purified by silica gel chromatography and recrystallized to get compound 1 (15 mg) and 2 (10 mg). Fraction 7 (62 mg) was rechromatographed on silica gel and eluted with CHCl3 –MeOH 95/5. The elutes (22 mg) were purified by gel permeation chromatography (GPC) on a Sephadex LH-20 column (Pharmacia, Sweden) by eluting with methanol to yield compound 3 (7 mg). The fraction 13 (115 mg) was repeatedly chromatographed by silica gel and Sephadex LH-20 to obtain compound 4 (12 mg) and 5 (17 mg) after recrystallization. The fraction 15 (85 mg) was repeatedly chromatographed by silica gel to obtain compound 6 (10 mg). 2.3. General information 1 H and 13 C NMR spectra were recorded on Bruker Avance DRX 500 instrument at room temperature. High-resolution mass spectra were obtained on a Bruker Q-TOF mass spectrometer, equipped with Apollo II electrospray ionization source, operated in the positive ion mode.
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12a), 126.04(C-4a), 124.98(C-10a), 123.34(C-12), 122.67(C-6a), 120.14(C-11), 119.02(C-10), 112.86(C-9), 104.63(C-1), 101.02(C13), 100.69(C-4), 84.52(C-6), 61.64(8-OCH3 ), 55.97(7-OCH3 ), 40.70(N-CH3 ). The 1 H and 13 C NMR data were in agreement with the reported data. 6-Methoxy-7-hydroxydihydrochelerythrine (5). C21 H19 NO5 , pale yellow powder. ESI-MS m/z: 388.1 [M + Na]+ . 1 H NMR(500 MHz, CDCl3 ) ıppm: 7.95(1H, s, H-4), 7.71(1H, d, J = 8.5 Hz, H-11), 7.51(1H, d, J = 8.5 Hz, H-10), 7.47(1H, d, J = 8.5 Hz, H-12), 7.19(1H, s, H-1), 6.87(1H, d, J = 8.5 Hz, H-9), 6.63(1H, s, H-6), 6.14(2H, s, H-13), 3.75(3H, s, 8-OCH3 ), 3.08(3H, s, N-CH3 ), 2.44(3H, s, 6OCH3 ). 13 C NMR(125 MHz, CDCl3 ) ıppm: 152.11(C-8), 148.06(C-2), 147.51(C-3), 146.34(C-7), 138.42(C-4b), 131.20(C-12a), 126.93(C4a), 126.16(C-6a), 125.52 (C-10a), 123.24(C-12), 123.01(C-10b), 119.83(C-11), 118.68(C-10), 112.32(C-9), 104.49(C-1), 101.10(C13), 100.85(C-4), 77.47(C-6), 60.40(6-OCH3 ), 55.66(8-OCH3 ), 40.85(N-CH3 ). The 1 H and 13 C NMR data were in agreement with the reported data. Oxychelerythrine (6). C21 H17 NO5 , white needles. ESI-MS m/z: 364.1 [M + H]+ . 1 H NMR(500 MHz, CDCl3 ) ıppm: 8.01(1H, d, J = 9.0 Hz, H-11), 8.00(1H, d, J = 9.0 Hz, H-10), 7.57(1H, s, H4), 7.55(1H, d, J = 9.0 Hz, H-12), 7.41(1H, d, J = 9.0 Hz, H-9), 7.19(1H, s, H-1), 6.12(2H, s, H-13), 4.11(3H, s, 7-OCH3 ), 4.01(3H, s, 8-OCH3 ), 3.93(3H, s, N-CH3 ). 13 C NMR(125 MHz, CDCl3 ) ıppm: 162.63(C = O), 152.71(C-8), 150.17(C-7), 147.50(C3), 147.07(C-2), 131.69(C-4b), 128.94(C-10a), 123.30(C-12), 121.03 (C-4a), 118.47(C-11), 117.85(C-10), 117.81(C-9), 117.20(C-10b), 114.39(C-12a), 111.45(C-6a), 104.67(C-1), 102.50(C-13), 101.50(C4), 61.77(8-OCH3 ), 56.62(7-OCH3 ), 40.82(N-CH3 ). The 1 H and 13 C NMR data were in agreement with the reported data. 2.5. Insects culture T. castaneum was obtained from laboratory cultures and maintained at 28–30 ◦ C, >70% relative humidity for the last 2 years in a dark incubator. The insects were reared in glass containers (0.5 L) containing wheat flour at 12–13% moisture content mixed with yeast (wheatfeed/yeast, 10:1, w/w). Adults used in the experiments were about two weeks old.
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Table 1 Antifeedant activity of the methanol extracts obtained from some Zanthoxylum species. Extract
Concentration (ppm)
Control Z. bungeanuma
– 500 1500 500 1500 500 1500 500 1500 500 1500 500 1500 500 1500
Z. schinifoliuma Z. schinifoliumb Z. schinifoliumc Z. armatumb Z. dissitumb Z. dissitumd a b c d
Antifeedant index%(mean ± SD) – 27.95 ± 39.42 ± 20.79 ± 38.17 ± 10.72 ± 33.09 ± 26.8 ± 41.12 ± 10.17 ± 11.81 ± – 2.52 ± – 6.95 ±
5.90 4.84 7.44 6.31 6.41 7.83 5.48 7.41 4.36 2.16 1.05 1.96
Pericarps. Leaves. Stem barks. Roots.
the control group. Fractions were tested feeding deterrent activity at a concentration of 1500 ppm in bioactivity-guided fractionation. The percentage antifeedant index was calculated from the following equation: Antifeedant index (%) = [(C − T)/C] × 100. Where C is the weight of diet consumed in control and T is the weight of diet consumed in the treatment. Five replicates of each concentration of the extracts or compounds and control were done for this experiment. The data were subjected to analysis of variance (ANOVA) and the differences between treatments (for each compound) were determined by the LSD test, which were conducted using SPSS 19.0 for Windows 2007. The concentration needed to inhibit insect feeding by 50% relative to controls (EC50 ) was analyzed by Logistic regression (Chen et al., 1996). 3. Results and discussion 3.1. Antifeedant activity of the crude extracts
2.6. Antifeedant bioassay A flour disk bioassay was used to evaluate the activities of methanol extracts of four Zanthoxylum species and isolated compounds from the stem bark of Z. schinifolium (Du et al., 2011). The testing solutions (1 mg/ml) were prepared with methanol extracts or pure compounds dissolved in ethanol. Serials of even flour–water suspensions (2 ml) were prepared with 0.4 g wheat flour, different volumes of testing solutions, one drop of DMSO (dimethylsulfoxide) and distilled water. Aliquots of 200 l of this stirred suspension were placed on the bottom of a polystyrene Petri dish to form disks. The pipette was fitted with a disposable tip that had an opening enlarged to about 2 mm internal diameter by cutting about 1 cm from the bottom of the tip with a razor blade. The same amounts of ethanol and DMSO were applied to produce the control flour disks. The flour disks were left in the fume-hood overnight to air dry. The flour disks were then transferred to an incubator to equilibrate at 28–30 ◦ C and 70–80% R.H. for 48 h. The moisture content of the disk was determined to be 13.5 ± 0.1% using the Kett’s Grain moisture tester (Model PB-1D2, Japan). The disks were placed in glass vials (diameter 2.5 cm, height 5.5 cm) for weighing. Twenty group-weighed, unsexed insects were then added to each vial prior for further weighing. All the insects were starved for 24 h before use. The experimental set-up was left in the incubator for 3 days. Finally, the uneaten parts of the flour disks were weighed. The insect consumption for the different test samples was compared to
Seven test samples from different parts of Z. bungeanum, Z. schinifolium, Z. armatum and Z. dissitum were collected. Antifeedant effects of their methanol extracts on T. castaneum adults were performed and the results are summarized in Table 1. Among the test samples, maximum antifeedant activity was observed in the MeOH extract of Z. schinifolium stem barks (41.12%), followed by the MeOH extract of Z. bungeanum pericarps (39.42%) with the concentration of 1500 ppm. While the extracts of Z. armatum and Z. dissitum caused very low or no antifeedant activity (Table 1). It has been reported that two significant feeding deterrents (schinifoline and skimmianine) were isolated from pericarps of Z. schinifolium by bioassay- guided fractionation (Liu et al., 2009). Thus, the potent antifeedant activity of Z. schinifolium has encouraged us to investigate the alkaloids from stem barks based on activity-guided chromatographic separations and six benzophenanthridines were isolated. 3.2. Antifeedant activity of the isolated compounds The results of antifeedant activity of six benzophenanthridines are listed in Table 2. The isolated alkaloids performed a significant deterrence of food consumption at various concentrations. All of the compounds exhibited strong antifeedant activity at the highest concentration (1500 ppm) with antifeeding index percentages ranging between 75.86% and 84.90%. At the lower concentrations
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Table 2 Antifeedant activity of benzophenanthridines isolated from stem bark of Z. schinifolium against T. castaneum adults. Compounds
Concentration (ppm)
Antifeedant index* (%)(mean ± SD)
EC50 (ppm)
95% fiducial limits
Chi square (2 )
Control Norchelerythrine (1)
0 15 50 150 500 1500
– 30.95 ± 1.56 a 52.85 ± 2.18 b 61.72 ± 2.74 c 68.85 ± 0.59 d 75.86 ± 2.28 e
– 62.67
– 37.97–103.43
2.51
Decarine (2)
15 50 150 500 1500
30.44 ± 1.46 a 47.21 ± 1.68 b 63.38 ± 2.14 c 73.91 ± 2.83 d 76.37 ± 1.82 d
66.97
43.15–103.95
2.34
15
17.22 ± 1.62 a
151.39
111.39–205.76
1.89
50 150 500 1500
32.27 ± 3.14 b 51.73 ± 2.71 c 69.35 ± 1.63 d 80.33 ± 1.12 e
15
23.56 ± 2.55 a
96.72
65.47–142.90
2.70
50 150 500 1500
44.27 ± 3.08 b 60.36 ± 2.64 c 69.18 ± 1.95 d 77.88 ± 3.11 e
15
13.43 ± 4.39 a
141.61
107.84–185.95
4.99
50 150 500 1500
31.05 ± 1.83 b 59.86 ± 2.10 c 66.34 ± 2.92 d 84.90 ± 2.20 e
15 50 150 500 1500
8.65 ± 2.90 a 29.09 ± 0.62 b 45.46 ± 2.22 c 69.62 ± 1.94 d 81.23 ± 2.95 e
192.32
149.19–247.92
2.43
8-Hydroxy-9-methoxy-2,3(methylenedioxy) benzo[c]phenanthridine (3)
6Hydroxydihydrochelerythrine (4)
6-Methoxy-7hydroxydihydrochelerythrine (5)
Oxychelerythrine (6)
*
Antifeedant index percentages within a column followed by the same letter are not significantly different (P > 0.05, based on the LSD test).
(15–150 ppm), compound 1 and 2 exhibited similar feeding deterrence and much more active than the other four compounds. Under the Logistic regression, it could be concluded that compounds 1–6 exhibited antifeeding activity against T. castaneum in a concentration-dependant manner (Table 2). Although the antifeedant activities of compounds 1 and 2 were weaker than the commercial feeding deterrent azadirachtin (EC50 = 3 ppm) (Ahond et al., 1979), they were similar with the activity of toosendanin (EC50 = 66 ppm) (Liu et al., 2009). These results revealed that the main antifeedant activity of Z. schinifolium stem bark could be attributed to the existed six benzophenanthridines and their relative distribution ratio. Consideration of the antifeedant activity of the isolated benzophenanthridines in relation to their structure led us to summarize some preliminary features of structure-activity relationships. For example, compounds 1, 2 or 4 with a methoxy group at C-7 were more potent antifeedants than other compounds with a methoxy group at C-8 or 9. However, the carbonyl group (oxidation of C-6) in compound 6 reduced the antifeedant effect. The presence of a methoxy group at C-8 slightly enhanced the antifeedant activity of 1 compared with the hydroxyl group in 2, indicating that the substituents at C-8 in this type of molecule had no marked effect on antifeedant activity. Moreover, the influence of the unsaturation or saturation in nitrogen-containing ring (position 5–6) for this type of activity was not obvious.
The benzophenanthridines are a relatively small class of plant products conspicuous for their bright colors and interesting for their chemical properties. The distribution of these alkaloids in higher plants is restricted to the families Papaveraceae, Fumariaceae and Rutaceae (Dostal and Slavik, 2002; Dvorak et al., 2006). They have a variety of medicinal effects on organisms, including antimicrobial, anti-inflammatory, antiviral, cytotoxicity and other effects (Obiang-Obounou et al., 2011; Hu et al., 2006; Kerry et al., 1998; Mansoor et al., 2013). In previous reports, benzophenanthridines are also found to possess insecticidal activity against larvae of mosquito and flies (Ueno, 2008). However, very little information exists with antifeedant effects of benzophenanthridines on the stored-product insects. Our investigation enriched more biological information of benzophenanthridines and provided experimental basis for the potential application of Z. schinifolium in the control of storage pests. 4. Conclusions The results of this work demonstrate the potent antifeedant activities of six benzophenanthridines isolated from Z. schinifolium stem bark against T. castaneum adults, and particularly the activities of compounds 1 and 2. These benzophenanthridine alkaloids could be a promising alternative in the control of stored-product insects.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Antifeedant activities of methanol extracts of four Zanthoxylum species and benzophenanthridines from stem bark of Zanthoxylum schinifolium against Tribolium castaneum Cheng-fang Wang a,b , Chun-xue You a , Kai Yang a , Shan-shan Guo a , Zhu-feng Geng c , Li Fan b,∗ , Shu-shan Du a,∗∗ , Zhi-wei Deng c , Yong-yan Wang a a
Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Beijing Normal University, Beijing 100875, China China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, China c Analytical and Testing Center, Beijing Normal University, Beijing 100875, China b
a r t i c l e
i n f o
Article history: Received 20 January 2015 Received in revised form 27 March 2015 Accepted 21 May 2015 Keywords: Benzophenanthridines Antifeedant activity
a b s t r a c t Antifeedant activities of the methanol extracts from Zanthoxylum bungeanum, Zanthoxylum schinifolium, Zanthoxylum armatum and Zanthoxylum dissitum were assessed on Tribolium castaneum adults. It was found that the methanol extract of stem bark of Z. schinifolium had the highest antifeedant activity at 41.12% (antifeedant index). Based on bioactivity-guided fractionation, six benzophenanthridines norchelerythrine (1), decarine (2), 8-hydroxy-9-methoxy-2,3-(methylenedioxy) benzophenanthridine (3), 6-hydroxydihydrochelerythrine (4), 6-methoxy-7-hydroxydihydrochelerythrine (5) and oxychelerythrine (6) were isolated from the stem barks of Z. schinifolium. And their antifeeding activities were also evaluated against T. castaneum. All of them exhibited strong antifeeding activity in a concentrationdependant manner with EC50 of 62.67, 66.97, 151.39, 96.72, 141.61 and 192.32 ppm, respectively. The six bioactive compounds from the stem bark of Z. schinifolium might be used as antifeedants against T. castaneum. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The red flour beetle, Tribolium castaneum (Herbst), is considered to be causing serious damage to stored grain products throughout the world, which reduces the quantity and quality of the food economically (Buckman and Campbell, 2013). Fumigants and chemical insecticides are widely used to control of T. castaneum population (White and Leesch, 1995). As their intensive application for decades, the negative consequences of commercial chemical insecticides have exposed, such as disruption biological control by natural enemies and outbreaks of insect species, development of insect resistance, undesirable effects on non-target organisms (Zettler and Arthur, 2000; Isman, 2006). Therefore, to develop new
∗ Corresponding author. Tel.: +86 10 62389938; fax: +86 10 62389938. ∗∗ Corresponding author. Tel.: +86 10 62208022; fax: +86 10 62208022. E-mail addresses: [email protected] (C.-f. Wang), [email protected] (C.-x. You), yangk [email protected] (K. Yang), [email protected] (S.-s. Guo), [email protected] (Z.-f. Geng), msfl@sina.com (L. Fan), [email protected] (S.-s. Du), [email protected] (Z.-w. Deng), [email protected] (Y.-y. Wang). http://dx.doi.org/10.1016/j.indcrop.2015.05.045 0926-6690/© 2015 Elsevier B.V. All rights reserved.
alternatives replacement is needed and natural products are a potential source of safer insecticides. (Dayan et al., 2009). The genus Zanthoxylum (Rutaceae) comprises a plenty of trees and shrubs, until now about 250 species have been found in temperate and tropical regions around the world (Pirani, 1993). Many of them are often used as condiments (due to the pungent taste of fruits, seeds, leaves, and bark) and therapeutic remedies especially in Eastern Asian countries (Epifano et al., 2011). Studies have demonstrated that Zanthoxylum plants (Rutaceae) have a potential in insect control as some of their contained compounds possess significant fumigant, contact, repellent, larvicidal and deterrent activity against some insects (Wang et al., 2011; Liang et al., 2013; Japheth et al., 2014; Ge and Weston, 1995). In this work, the samples of four Zanthoxylum species distributed widely in China, namely, Zanthoxylum bungeanum, Zanthoxylum schinifolium, Zanthoxylum armatum and Zanthoxylum dissitum were collected and their antifeedant activities were assessed on the red flour beetle. Meanwhile, six benzophenanthridines were isolated from the methanol extract of Z. schinifolium stem bark and their antifeedant activities were tested against T. castaneum for the first time.
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Fig. 1. Structure of benzophenanthridines from Z. schinifolium.
2. Materials and methods
2.4. Identification of the compounds
2.1. Plant material
Chemical structures were assigned by analysis of the MS, 1 H, and 2 D NMR spectra and by comparison with literature values. Accordingly, compound 1–6 were identified as norchelerythrine (Jaromir et al., 2004), decarine (Martin et al., 2005), 8-hydroxy-9methoxy-2,3-(methylenedioxy) benzophenanthridine (Oscar and Luis, 2004), 6-hydroxydihydrochelerythrine (Seckarova et al., 2002), 6-methoxy-7-hydroxydihydrochelerythrine (Tarus et al., 2006), and oxychelerythrine (He et al., 2002), respectively (Fig. 1). Norchelerythrine (1). C20 H15 NO4 , colorless needles, ESI-MS m/z: 334.1150 [M + H]+ . 1 H NMR(500 MHz, CDCl3 ) ıppm: 9.77(1H, s, H-6), 8.75(1H, s, H-4), 8.37(1H, d, J = 9.0 Hz, H-10), 8.35(1H, d, J = 9.0 Hz, H-11), 7.86(1H, d, J = 9.0 Hz, H-12), 7.61(1H, d, J = 9.0 Hz, H-9), 7.28(1H, s, H-1), 6.16(2H, s, H-13), 4.15(3H, s, 8-OCH3 ), 4.08(3H, s, 7-OCH3 ). 13 C NMR(125 MHz, CDCl3 ) ıppm: 149.37(C-8), 148.47(C-2), 148.23(C-3), 146.48(C-6), 145.26(C-7), 129.75(C-4b, 10b), 128.10(C-12a), 127.05(C-12), 121.81(C-4a), 120.00(C-10a), 118.80(C-9), 118.25(C-10), 118.18(C-11), 110.00(C-6a), 104.36(C1), 102.18(C-4), 101.32(C-13), 61.90(7-OCH3 ), 56.83(8-OCH3 ). Decarine (2). C19 H13 NO4. Pale yellow needles, ESI-MS m/z: 320.09 [M + H]+ . 1 H NMR(500 MHz, DMSO-d6 ) ıppm: 10.11(1H, s, 8OH), 9.59(1H, s, H-6), 8.55(1H, s, H-4), 8.53(1H, d, J = 9.0 Hz, H-11), 8.48(1H, d, J = 9.0 Hz, H-10), 7.97(1H, d, J = 9.0 Hz, H-12), 7.59(1H, d, J = 9.0 Hz, H-9), 7.53(1H, s, H-1), 6.23(2H, s, H-13), 4.03(3H, s, 7-OCH3 ). 13 C NMR(125 MHz, DMSO-d6 ) ıppm: 148.61(C-2), 148.37(C-3), 148.00(C-8), 146.28(C-6), 142.56(C-7), 139.10(C4b), 129.64(C-12a), 128.74(C-4a), 127.55(C-12), 126.83(C-10a), 124.03(C-9), 122.10(C-6a), 120.49(C-10b), 119.26(C-10), 119.14(C11), 104.99(C-1), 101.96(C-13), 101.34(C-4), 61.67(7-OCH3 ). 8-Hydroxy-9-methoxy-2,3-(methylenedioxy) benzophenanthridine (3). C19 H13 NO4 , yellow needles, ESI-MS m/z: 320.09 [M + H]+ . 1 H NMR(500 MHz, DMSO-d ) ıppm: 10.00(1H, s, -OH), 9.22(1H, 6 s, H-6), 8.62(1H, d, J = 9.0 Hz, H-11), 8.54(1H, s, H-4), 8.16(1H, s, H-10), 7.95(1H, d, J = 9.0 Hz, H-12), 7.52(1H, s, H-1), 7.49(1H, s, H-7), 6.21(2H, s, H-13), 4.11(3H, s, 9-OCH3 ). 13 C NMR(125 MHz, DMSOd6 ) ıppm: 153.28(C-9), 150.23(C-6), 148.41(C-3), 148.24(C-8), 148.23(C-2), 139.63(C-4b), 129.53(C-4a), 128.88(C-12a), 127.83(C6a), 126.63(C-12), 122.75(C-10a), 120.34(C-10b), 119.64(C-11), 111.37(C-7), 104.94(C-1), 103.09(C-10), 101.86(C-13), 101.48(C-4), 56.58(9-OCH3 ). 6-Hydroxydihydrochelerythrine (4). C21 H19 NO5 , pale yellow needles, ESI-MS m/z: 388.1 [M + Na]+ . 1 H NMR(500 MHz, CDCl3 ) ıppm: 7.81(1H, d, J = 8.5 Hz, H-11), 7.72(1H, s, H-4), 7.65(1H, d, J = 8.5 Hz, H-10), 7.50(1H, d, J = 8.5 Hz, H-12), 7.16(1H, s, H1), 7.05(1H, d, J = 8.5 Hz, H-9), 6.08(2H, s, H-13), 5.73(1H, s, H-6), 4.02(3H, s, 8-OCH3 ), 3.95(3H, s, 7-OCH3 ), 2.78(3H, s, NCH3 ). 13 C NMR(125 MHz, CDCl3 ) ıppm: 152.18(C-8), 147.90(C-3), 147.33(C-2), 146.63(C-7), 138.68(C-4b), 131.02(C-10b), 126.79(C13 C
Z. bungeanum (pericarps) was collected from Yanqing County of Beijing, China (40.53◦ N latitude; 115.92◦ E longitude); Z. schinifolium (pericarps, leaves, stem barks) was collected from Kuandian Manchu Autonomous County, Liaoning Province, China (40.43◦ N latitude; 124.47◦ E longitude); Z. armatum (leaves) was collected from Xishuangbanna, Yunnan Province, China (21.99◦ N latitude; 100.83◦ E longitude); Z. dissitum (leaves and roots) was collected from Yulin, Guangxi Province, China (22.67◦ N latitude; 110.14◦ E longitude). The voucher specimens were deposited at college of resources science & technology, Beijing Normal University. The fresh materials were air dried and then ground to a powder. 2.2. Extraction and isolation The powdered plant materials (100 g) were performed by ultrasonic extraction with methanol for 30 min at room temperature (three times). The extracts were filtered and concentrated under reduced pressure to residues. The methanol extract of stem barks from Z. schinifolium (15 g) was chromatographed on silica gel column (500 g, 160–200 mesh, Qingdao Marine Chemical Plant, Shandong Province, China), eluting with a gradient of chloroform–methanol (from 100/0 to 0/100). With the monitoring of thin-layer chromatography (TLC, precoated silica gel GF254 plates), 25 fractions were obtained. Among them fractions 5, 7, 13 and 15 showed considerable antifeedant activity. The fraction 5 (108 mg) was eluted with PE–EtOAc 85/15 on silica gel to afford 15 sub-fractions, and sub-fractions 5–9 (50 mg) were further purified by silica gel chromatography and recrystallized to get compound 1 (15 mg) and 2 (10 mg). Fraction 7 (62 mg) was rechromatographed on silica gel and eluted with CHCl3 –MeOH 95/5. The elutes (22 mg) were purified by gel permeation chromatography (GPC) on a Sephadex LH-20 column (Pharmacia, Sweden) by eluting with methanol to yield compound 3 (7 mg). The fraction 13 (115 mg) was repeatedly chromatographed by silica gel and Sephadex LH-20 to obtain compound 4 (12 mg) and 5 (17 mg) after recrystallization. The fraction 15 (85 mg) was repeatedly chromatographed by silica gel to obtain compound 6 (10 mg). 2.3. General information 1 H and 13 C NMR spectra were recorded on Bruker Avance DRX 500 instrument at room temperature. High-resolution mass spectra were obtained on a Bruker Q-TOF mass spectrometer, equipped with Apollo II electrospray ionization source, operated in the positive ion mode.
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12a), 126.04(C-4a), 124.98(C-10a), 123.34(C-12), 122.67(C-6a), 120.14(C-11), 119.02(C-10), 112.86(C-9), 104.63(C-1), 101.02(C13), 100.69(C-4), 84.52(C-6), 61.64(8-OCH3 ), 55.97(7-OCH3 ), 40.70(N-CH3 ). The 1 H and 13 C NMR data were in agreement with the reported data. 6-Methoxy-7-hydroxydihydrochelerythrine (5). C21 H19 NO5 , pale yellow powder. ESI-MS m/z: 388.1 [M + Na]+ . 1 H NMR(500 MHz, CDCl3 ) ıppm: 7.95(1H, s, H-4), 7.71(1H, d, J = 8.5 Hz, H-11), 7.51(1H, d, J = 8.5 Hz, H-10), 7.47(1H, d, J = 8.5 Hz, H-12), 7.19(1H, s, H-1), 6.87(1H, d, J = 8.5 Hz, H-9), 6.63(1H, s, H-6), 6.14(2H, s, H-13), 3.75(3H, s, 8-OCH3 ), 3.08(3H, s, N-CH3 ), 2.44(3H, s, 6OCH3 ). 13 C NMR(125 MHz, CDCl3 ) ıppm: 152.11(C-8), 148.06(C-2), 147.51(C-3), 146.34(C-7), 138.42(C-4b), 131.20(C-12a), 126.93(C4a), 126.16(C-6a), 125.52 (C-10a), 123.24(C-12), 123.01(C-10b), 119.83(C-11), 118.68(C-10), 112.32(C-9), 104.49(C-1), 101.10(C13), 100.85(C-4), 77.47(C-6), 60.40(6-OCH3 ), 55.66(8-OCH3 ), 40.85(N-CH3 ). The 1 H and 13 C NMR data were in agreement with the reported data. Oxychelerythrine (6). C21 H17 NO5 , white needles. ESI-MS m/z: 364.1 [M + H]+ . 1 H NMR(500 MHz, CDCl3 ) ıppm: 8.01(1H, d, J = 9.0 Hz, H-11), 8.00(1H, d, J = 9.0 Hz, H-10), 7.57(1H, s, H4), 7.55(1H, d, J = 9.0 Hz, H-12), 7.41(1H, d, J = 9.0 Hz, H-9), 7.19(1H, s, H-1), 6.12(2H, s, H-13), 4.11(3H, s, 7-OCH3 ), 4.01(3H, s, 8-OCH3 ), 3.93(3H, s, N-CH3 ). 13 C NMR(125 MHz, CDCl3 ) ıppm: 162.63(C = O), 152.71(C-8), 150.17(C-7), 147.50(C3), 147.07(C-2), 131.69(C-4b), 128.94(C-10a), 123.30(C-12), 121.03 (C-4a), 118.47(C-11), 117.85(C-10), 117.81(C-9), 117.20(C-10b), 114.39(C-12a), 111.45(C-6a), 104.67(C-1), 102.50(C-13), 101.50(C4), 61.77(8-OCH3 ), 56.62(7-OCH3 ), 40.82(N-CH3 ). The 1 H and 13 C NMR data were in agreement with the reported data. 2.5. Insects culture T. castaneum was obtained from laboratory cultures and maintained at 28–30 ◦ C, >70% relative humidity for the last 2 years in a dark incubator. The insects were reared in glass containers (0.5 L) containing wheat flour at 12–13% moisture content mixed with yeast (wheatfeed/yeast, 10:1, w/w). Adults used in the experiments were about two weeks old.
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Table 1 Antifeedant activity of the methanol extracts obtained from some Zanthoxylum species. Extract
Concentration (ppm)
Control Z. bungeanuma
– 500 1500 500 1500 500 1500 500 1500 500 1500 500 1500 500 1500
Z. schinifoliuma Z. schinifoliumb Z. schinifoliumc Z. armatumb Z. dissitumb Z. dissitumd a b c d
Antifeedant index%(mean ± SD) – 27.95 ± 39.42 ± 20.79 ± 38.17 ± 10.72 ± 33.09 ± 26.8 ± 41.12 ± 10.17 ± 11.81 ± – 2.52 ± – 6.95 ±
5.90 4.84 7.44 6.31 6.41 7.83 5.48 7.41 4.36 2.16 1.05 1.96
Pericarps. Leaves. Stem barks. Roots.
the control group. Fractions were tested feeding deterrent activity at a concentration of 1500 ppm in bioactivity-guided fractionation. The percentage antifeedant index was calculated from the following equation: Antifeedant index (%) = [(C − T)/C] × 100. Where C is the weight of diet consumed in control and T is the weight of diet consumed in the treatment. Five replicates of each concentration of the extracts or compounds and control were done for this experiment. The data were subjected to analysis of variance (ANOVA) and the differences between treatments (for each compound) were determined by the LSD test, which were conducted using SPSS 19.0 for Windows 2007. The concentration needed to inhibit insect feeding by 50% relative to controls (EC50 ) was analyzed by Logistic regression (Chen et al., 1996). 3. Results and discussion 3.1. Antifeedant activity of the crude extracts
2.6. Antifeedant bioassay A flour disk bioassay was used to evaluate the activities of methanol extracts of four Zanthoxylum species and isolated compounds from the stem bark of Z. schinifolium (Du et al., 2011). The testing solutions (1 mg/ml) were prepared with methanol extracts or pure compounds dissolved in ethanol. Serials of even flour–water suspensions (2 ml) were prepared with 0.4 g wheat flour, different volumes of testing solutions, one drop of DMSO (dimethylsulfoxide) and distilled water. Aliquots of 200 l of this stirred suspension were placed on the bottom of a polystyrene Petri dish to form disks. The pipette was fitted with a disposable tip that had an opening enlarged to about 2 mm internal diameter by cutting about 1 cm from the bottom of the tip with a razor blade. The same amounts of ethanol and DMSO were applied to produce the control flour disks. The flour disks were left in the fume-hood overnight to air dry. The flour disks were then transferred to an incubator to equilibrate at 28–30 ◦ C and 70–80% R.H. for 48 h. The moisture content of the disk was determined to be 13.5 ± 0.1% using the Kett’s Grain moisture tester (Model PB-1D2, Japan). The disks were placed in glass vials (diameter 2.5 cm, height 5.5 cm) for weighing. Twenty group-weighed, unsexed insects were then added to each vial prior for further weighing. All the insects were starved for 24 h before use. The experimental set-up was left in the incubator for 3 days. Finally, the uneaten parts of the flour disks were weighed. The insect consumption for the different test samples was compared to
Seven test samples from different parts of Z. bungeanum, Z. schinifolium, Z. armatum and Z. dissitum were collected. Antifeedant effects of their methanol extracts on T. castaneum adults were performed and the results are summarized in Table 1. Among the test samples, maximum antifeedant activity was observed in the MeOH extract of Z. schinifolium stem barks (41.12%), followed by the MeOH extract of Z. bungeanum pericarps (39.42%) with the concentration of 1500 ppm. While the extracts of Z. armatum and Z. dissitum caused very low or no antifeedant activity (Table 1). It has been reported that two significant feeding deterrents (schinifoline and skimmianine) were isolated from pericarps of Z. schinifolium by bioassay- guided fractionation (Liu et al., 2009). Thus, the potent antifeedant activity of Z. schinifolium has encouraged us to investigate the alkaloids from stem barks based on activity-guided chromatographic separations and six benzophenanthridines were isolated. 3.2. Antifeedant activity of the isolated compounds The results of antifeedant activity of six benzophenanthridines are listed in Table 2. The isolated alkaloids performed a significant deterrence of food consumption at various concentrations. All of the compounds exhibited strong antifeedant activity at the highest concentration (1500 ppm) with antifeeding index percentages ranging between 75.86% and 84.90%. At the lower concentrations
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Table 2 Antifeedant activity of benzophenanthridines isolated from stem bark of Z. schinifolium against T. castaneum adults. Compounds
Concentration (ppm)
Antifeedant index* (%)(mean ± SD)
EC50 (ppm)
95% fiducial limits
Chi square (2 )
Control Norchelerythrine (1)
0 15 50 150 500 1500
– 30.95 ± 1.56 a 52.85 ± 2.18 b 61.72 ± 2.74 c 68.85 ± 0.59 d 75.86 ± 2.28 e
– 62.67
– 37.97–103.43
2.51
Decarine (2)
15 50 150 500 1500
30.44 ± 1.46 a 47.21 ± 1.68 b 63.38 ± 2.14 c 73.91 ± 2.83 d 76.37 ± 1.82 d
66.97
43.15–103.95
2.34
15
17.22 ± 1.62 a
151.39
111.39–205.76
1.89
50 150 500 1500
32.27 ± 3.14 b 51.73 ± 2.71 c 69.35 ± 1.63 d 80.33 ± 1.12 e
15
23.56 ± 2.55 a
96.72
65.47–142.90
2.70
50 150 500 1500
44.27 ± 3.08 b 60.36 ± 2.64 c 69.18 ± 1.95 d 77.88 ± 3.11 e
15
13.43 ± 4.39 a
141.61
107.84–185.95
4.99
50 150 500 1500
31.05 ± 1.83 b 59.86 ± 2.10 c 66.34 ± 2.92 d 84.90 ± 2.20 e
15 50 150 500 1500
8.65 ± 2.90 a 29.09 ± 0.62 b 45.46 ± 2.22 c 69.62 ± 1.94 d 81.23 ± 2.95 e
192.32
149.19–247.92
2.43
8-Hydroxy-9-methoxy-2,3(methylenedioxy) benzo[c]phenanthridine (3)
6Hydroxydihydrochelerythrine (4)
6-Methoxy-7hydroxydihydrochelerythrine (5)
Oxychelerythrine (6)
*
Antifeedant index percentages within a column followed by the same letter are not significantly different (P > 0.05, based on the LSD test).
(15–150 ppm), compound 1 and 2 exhibited similar feeding deterrence and much more active than the other four compounds. Under the Logistic regression, it could be concluded that compounds 1–6 exhibited antifeeding activity against T. castaneum in a concentration-dependant manner (Table 2). Although the antifeedant activities of compounds 1 and 2 were weaker than the commercial feeding deterrent azadirachtin (EC50 = 3 ppm) (Ahond et al., 1979), they were similar with the activity of toosendanin (EC50 = 66 ppm) (Liu et al., 2009). These results revealed that the main antifeedant activity of Z. schinifolium stem bark could be attributed to the existed six benzophenanthridines and their relative distribution ratio. Consideration of the antifeedant activity of the isolated benzophenanthridines in relation to their structure led us to summarize some preliminary features of structure-activity relationships. For example, compounds 1, 2 or 4 with a methoxy group at C-7 were more potent antifeedants than other compounds with a methoxy group at C-8 or 9. However, the carbonyl group (oxidation of C-6) in compound 6 reduced the antifeedant effect. The presence of a methoxy group at C-8 slightly enhanced the antifeedant activity of 1 compared with the hydroxyl group in 2, indicating that the substituents at C-8 in this type of molecule had no marked effect on antifeedant activity. Moreover, the influence of the unsaturation or saturation in nitrogen-containing ring (position 5–6) for this type of activity was not obvious.
The benzophenanthridines are a relatively small class of plant products conspicuous for their bright colors and interesting for their chemical properties. The distribution of these alkaloids in higher plants is restricted to the families Papaveraceae, Fumariaceae and Rutaceae (Dostal and Slavik, 2002; Dvorak et al., 2006). They have a variety of medicinal effects on organisms, including antimicrobial, anti-inflammatory, antiviral, cytotoxicity and other effects (Obiang-Obounou et al., 2011; Hu et al., 2006; Kerry et al., 1998; Mansoor et al., 2013). In previous reports, benzophenanthridines are also found to possess insecticidal activity against larvae of mosquito and flies (Ueno, 2008). However, very little information exists with antifeedant effects of benzophenanthridines on the stored-product insects. Our investigation enriched more biological information of benzophenanthridines and provided experimental basis for the potential application of Z. schinifolium in the control of storage pests. 4. Conclusions The results of this work demonstrate the potent antifeedant activities of six benzophenanthridines isolated from Z. schinifolium stem bark against T. castaneum adults, and particularly the activities of compounds 1 and 2. These benzophenanthridine alkaloids could be a promising alternative in the control of stored-product insects.
C.-f. Wang et al. / Industrial Crops and Products 74 (2015) 407–411
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