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Insecticidal Metabolites from the Rhizomes of Veratrum album against Adults of Colorado Potato Beetle, Leptinotarsa decemlineata by Tuba Aydin a ), Ahmet Cakir* b ), Cavit Kazaz c ), Neslihan Bayrak d ), Yasin Bayir e ), and Yavuz Tas¸kesenligil f ) ) Agri I˙brahim Cecen University, Health Services Vocational School, Department of Paramedic, TR-04100, Agri b ) Kilis 7 Aralik University, Faculty of Sciences & Arts, Department of Chemistry, TR-79000, Kilis (phone: þ 90-348-8222350; fax: þ 90-348-8222351; e-mail: [email protected]; [email protected]) c ) Atatrk University, Faculty of Science, Department of Chemistry, TR-25240, Erzurum d ) Bozok University, Faculty of Agriculture, TR-66900, Yozgat e ) Atatrk University, Faculty of Pharmacy, Department of Biochemistry, TR-25240, Erzurum f ) Atatrk University, Kazim Karabekir Education Faculty, Department of Chemistry, TR-25240, Erzurum a
The dried rhizomes of Veratrum album were individually extracted with CHCl3 , acetone, and NH4OH/benzene to test the toxic effects against the Colorado potato beetle, Leptinotarsa decemlineata, which is an important agricultural pest. Fifteen compounds in various amounts were isolated from the extracts using column and thin-layer chromatography. The chemical structures of 14 compounds were characterized as octacosan-1-ol (1), b-sitosterol (2), stearic acid (3), diosgenin (4), resveratrol (5), wittifuran X (6), oxyresveratrol (7), b-sitosterol 3-O-b-d-glucopyranoside (8), diosgenin 3-O-a-lrhamnopyranosyl-(1 ! 2)-b-d-glucopyronoside (9), oxyresveratrol 3-O-b-d-glucopyranoside (10), jervine (11), pseudojervine (13), 5,6-dihydro-1-hydroxyjervine (14), and saccharose (15) using UV, IR, MS, 1 H- and 13C-NMR, and 2D-NMR spectroscopic methods. However, the chemical structure of 12, an oligosaccharide, has not fully been elucidated. Compounds 4, 6, 9, and 10 were isolated from V. album rhizomes for the first time in the current study. The toxic effects of three extracts (acetone, CHCl3 , and NH4OH/benzene) and six metabolites, 2, 2 þ 4, 5, 7, 8, and 11, were evaluated against the Colorado potato beetle. The assay revealed that all three extracts, and compounds 7, 8, and 11 exhibited potent toxic effects against this pest. This is the first report on the evaluation of the toxic effects of the extracts and secondary metabolites of V. album rhizomes against L. decemlineata. Based on these results, it can be concluded that the extracts can be used as natural insecticides.
Introduction. – Veratrum (Hellebore) is an important plant genus belonging to the family Liliaceae. The crude extracts and compounds isolated from Veratrum plants have been reported to possess various pharmacological activities such as hypotensive, antithrombotic, and antitumor activities [1] [2]. In particular, their alkaloid constituents showed significant anticancer activities by blocking the hedgehog signal pathway [2 – 4]. Veratrum species are not consumed by herbivores due to their toxic steroidal alkaloids [5]. V. album, which is among Turkish flora, is a poisonous type of plant because of its steroidal alkaloids and glycosides. V. album is known by the local names çpleme, Ak Åçpleme, Beyaz Åçpleme, and emah in Turkey [6]. All parts of V. album have a bitter taste, and its powders have sternutatory effects. It has been used as a traditional medicine in Turkey. However, due to its irritant properties, its infusion was only used externally. It was used in the treatment of scabies and body interference. Its 2014 Verlag Helvetica Chimica Acta AG, Zrich
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powdered rhizomes have been used to remedy nasal congestion during cold and sinusitis [6]. Veratrum preparations were used for centuries in the treatment of circulatory disorders and the control of insects [7]. The insect repellent effect of V. album has been known since ancient times [8] [9]. Leptinotarsa decemlineata, Colorado potato beetle, known as potato beetle in Turkey is an insect that damages the leaves of potato, eggplants, and tomatoes. L. decemlineata spread over an area of 16 million km2 in North America, Europe, and Asia, and continues to spread. Larvae and adults of L. decemlineata feed on the leaves of potato and remain for long periods on the potato plant. Therefore, no control technique has been developed against L. decemlineata so far, leading to a decrease in development and production of the potato. This situation requires chemical control against this insect [10] [11]. L. decemlineata can develop resistance to many insecticides. In the last 60 years, L. decemlineata has developed resistance against 52 different compounds which were considered important insecticides. Therefore, L. decemlineata is highly important in the development of modern pesticides. Since 1864, hundreds of compounds were tested against this insect, and various drugs have been developed to reduce its harms [11 – 14]. Extracts of some plant species and pyrrolizidine alkaloids, cardiopetamins, terpenoids, cucurbitacins, silphinens, and limonoids isolated from some plants have been shown to be toxic against L. decemlineata [15 – 20]. There is no report in the literature on the toxicity of the extracts and metabolites of Veratrum species against adults of L. decemlineata. Therefore, the aim of the present study was to assess the toxicity of V. album extracts, its pure metabolites, and lambda cyhalothrin, an insect control reactive against adults of L. decemlineata. Herein, we describe the isolation and chemical characterization of 15 compounds. The insecticidal effects of some extracts and some compounds isolated from V. album against adults of L. decemlineata are also reported. Results and Discussion. – The Chemical Composition of the Extracts. The acetone, NH4OH/benzene, and EtOH extracts of V. album was subjected to fractionation by column chromatography (CC) and TLC. One aliphatic alcohol 1, one fatty acid 3, four steroids and steroid glycosides, 2 and 4, and 8 and 9, respectively, four stilbenoids and their glycosides, 5, 6, 7, and 10, three steroidal alkaloids, 11, 13, and 14, and one carbohydrate, 15, were isolated. The chemical structures of these compounds were characterized by UV, IR, MS, 1H- and 13C-NMR, 1D-(DEPT), and 2D-NMR (1H,1HCOSY, 1H,13C-COSY, and HMBC) analyses (Fig. 1). However, the chemical structure of compound 12, an oligosaccharide, has not been fully characterized. The chemical structures of the known compounds were also confirmed by comparison of their spectral data with those reported in the literature, i.e., as octacosan-1-ol (1), b-sitosterol (2) [21] [22], stearic acid (3), diosgenin (4) [23 – 29], resveratrol (5) [30] [31], wittifuran X (6) [32 – 34], oxyresveratrol (7) [35], b-sitosterol 3-O-b-d-glucopyranoside (8), diosgenin 3-O-a-l-rhamnopyranosyl-(1 ! 2)-b-d-glucopyronoside (9) [23 – 29] [36 – 39], oxyresveratrol 3-O-b-d-glucopyranoside (10) [33] [40] [41], jervine (11) [42 – 44], pseudojervine (13) [42 – 44], 5,6-dihydro-1-hydroxyjervine (14) [43] [45] [46], and saccharose (15) [47 – 49]. Compounds 4, 6, 9, and 10 were isolated from V. album rhizomes for the first time.
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Fig. 1. Chemical structures of the compounds 2, 4 – 11, 13, and 14
Insecticidal Effects of the Extracts and Pure Metabolites. The toxicities of the extracts and the pure metabolites of V. album were determined against adults of L. decemlineata. The extracts and pure metabolites at three different concentrations, 10, 20 and 40 mg/Petri dish, were investigated, and their toxicities were compared with toxicity of the reference, lambda cyhalothrin, a commercial insecticide at the same concentrations (Figs. 2 and 3). The data show that the extracts and pure metabolites display various toxicities against the adults depending on exposure time and tested substances. Generally, the mortality increased with increasing doses of the substances and exposure times (Figs. 2 and 3). The extracts were found to be more toxic than the pure compounds, the NH4OH/benzene extract being the most toxic sample. All doses of this extract caused 100% mortality against the adult within the first 12 h (Fig. 2). Acetone and CHCl3 extracts led to complete mortality for the adult in 24 h (Fig. 3). Among the tested metabolites, b-sitosterol 3-O-b-d-glucopyranoside (8) was more effective against adults as compared to other metabolites. Figs. 2 and 3 show that 20 and 40 mg/ml concentrations of this compound caused 100% mortality in the first 24 h, and the remaining doses within 48 h. Another compound which caused 100% mortality against the adult at 24 h was oxyresveratrol (7), which, at 40 mg/ml concentration, caused 100% mortality after 24 h (Fig. 3). Jervine (11) and b-sitosterol (2) þ diosgenin (4) at 40 mg/ml concentration displayed low toxicities as compared with those of the extracts, b-sitosterol 3-O-b-d-glucopyranoside (8) and oxyresveratrol (7; Figs. 2 and 3).
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Fig. 2. Mortality [%] of the adults of L. decemlineata exposed to the extracts and pure metabolites after 12 h
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Fig. 3. Mortality (%) of the adults of L. decemlineata exposed to the extracts and pure metabolites after 24 h
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When compared with the toxicity of b-sitosterol (2), the mixture of b-sitosterol (2) þ diosgenin (4) was more effective than 2 against adults of the insect. Hence, it can be concluded that 4 is more toxic than 2. Furthermore, among the pure metabolites, resveratrol (5) was less toxic against the adults of Colorado potato beetle (Figs. 2 and 3). The LC50 value is dose that kills 50% of the test animals when applied through the skin and mouth, and it is given in mg/kg. If the LC50 value of a substance is low, it is highly toxic [50]. The LC50 values in 12 h of the tested substances are compiled in Table 1. NH4OH/Benzene extract and commercial insecticide, lambda cyhalothrin, were the most toxic substances against the Colorado potato beetle. As can be seen from Table 1, LC50 values of NH4OH/benzene, CHCl3 , and acetone extracts were found to be low as compared with other treatments; therefore, they were classified as highly toxic insecticides against the beetle. Furthermore, based on the present results, b-sitosterol 3O-b-d-glucopyranoside (8), oxyresveratrol (7), jervine (11), b-sitosterol (2) þ diosgenin (4), and resveratrol (5; LC50 values 10 – 60 mg/ml) were classified as toxic insecticides, whereas b-sitosterol (2; LC50 501.5 mg/ml) was classified as a mediumtoxic insecticide [50]. The durations, calculated by the probit analyses for the 50 and 90% mortality of the adults of the L. decemlineata exposed to the extracts and the pure metabolites are collected in Table 2. Durations of the applications for 50 and 90% mortality of the adults of Colorado potato beetle decreased depending on the concentration increase. These data indicate that the toxicities of the applications were concentrationdependent. Among all applications, the shortest exposure time for 50 and 90% mortality of the pest were of NH4OH/benzene extract and positive control, lambda cyhalothrin. These results suggested that of all treatments, NH4OH/benzene extract is the most toxic reagent for the adults of the pest. Also CHCl3 extract, b-sitosterol 3-O-bd-glucopyranoside (8), and oxyresveratrol (7) turned out to be toxic (Table 2). Based on the durations for 50 and 90% mortality of the insect, b-sitosterol (2) þ diosgenin (4) was more toxic than 2. This can be attributed to the synergistic effect of 4 on the toxicity of 2. Our results also indicated that resveratrol (5) and jervine (11) were the less toxic metabolites as compared with other compounds tested.
Table 1. The LC50 Values of the Tested Extracts and Metabolites Substance Extracts
NH4OH/benzene CHCl3 Acetone
Metabolites
b-Sitosterol 3-O-b-d-glucopyranoside (8) Oxyresveratrol (7) Jervine (11) b-Sitosterol (2) þ diosgenin (4) Resveratrol (5) b-Sitosterol (2)
Positive control
Lambda cyhalothrin
LC50 Value [mg/ml] 0.025 0.14 8.30 10.48 14.32 26.69 26.96 57.68 501.46 0.025
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Table 2. Durations for 50 and 90% Mortality of the Adults of the Pest Exposed to Different Concentrations of the Extracts and Metabolites Extracts and metabolites
Extracts
Acetone
CHCl3
NH4OH/benzene
Metabolites
b-Sitosterol (2)
b-Sitosterol (2) þ diosgenin (4)
Resveratrol (5)
Oxyresveratrol (7)
b-Sitosterol 3-O-b-d-Glc (8)
Jervine (11)
Control
Lambda cyhalothrin
Concentration
Duration [h]
[mg/ml]
50% mortality
90% mortality
10 20 40 10 20 40 10 20 40
9.99 7.48 2.64 7.33 7.33 4.70 2.64 2.64 2.64
18.08 16.07 7.49 13.88 13.88 11.62 7.49 7.49 7.49
10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40
56.74 26.87 23.13 43.00 24.01 6.46 99.38 29.41 15.60 22.82 9.71 2.64 13.78 7.33 2.64 69.76 26.85 7.99
130.63 45.81 52.17 95.10 51.15 14.99 365.22 118.37 51.19 58.70 21.58 7.49 29.92 13.88 7.45 490.5 61.01 21.88
10 20 40
2.64 2.64 2.64
7.49 7.49 7.49
Veratrum species and their metabolites, steroidal alkaloids, are well-known to display toxic properties. Insecticidal effect of Veratrum extracts and some steroidal alkaloids has been reported against mosquito, silkworm, and fruit fly [51] [52]. Toxic effects of V. album extract and some fractions have also been determined against Pseudococcus longispinus and Pseudococcus affinis (Momoptera: Pseudococcidae) [53]. An acetone extract of V. album exhibited toxic effect against Drosophila melanogaster [51]. In another study, toxic effects of steroidal alkaloids, veratradine, cevadine, veracevine, protoveratrine A, and protoverotrine, isolated from V. album, were investigated against the housefly (Musca domestica: Diptera) and Oncopeltus fasciatus, and it was documented that of all compounds with strong toxicities against both species, protoveratrine A was the most toxic compound with the lowest LD50 value (for housefly, LD50 of 0.10 mg/kg and for Oncopeltus fasciatus LD50 of 0.09 mg/kg) [7].
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However, according to our literature survey, insecticidal activities of Veratrum extracts and pure metabolites against L. decemlineata have not been investigated. The result of the present study revealed that, in particular, NH4OH/benzene, CHCl3 , and acetone extracts of V. album rhizomes are more toxic against L. decemlineata. Considering the insect mortality in 12 h, clearly the most toxic substance is the NH4OH/ benzene extract (Table 2 and Fig. 2). As shown in Table 2 and Fig. 2, NH4OH/benzene extract included major amounts of jervine (11). As compared to the toxic effects of NH4OH/benzene extract and jervine (11) at 12 h (Fig. 2, and Tables 1 and 2), NH4OH/ benzene extract was more toxic than 11. Low toxic effect of 11 as compared to that of the NH4OH/benzene extract can be attributed to other minor substances in the NH4OH/benzene extract. Among the tested pure metabolites, b-sitosterol 3-O-b-dglucopyranoside (8) and oxyresveratrol (7) showed potent toxic effects against L. decemlineata. Conclusions. – Developing natural or biological insecticides will help to decrease some negative features of synthetic chemicals, including residues in products, insect resistance, and environmental pollution. In this respect, the use of natural insecticides can also be effective, selective, and easily bio-degradable, causing relatively low pollution for environment. In the present study, the toxic effects of the extracts and some pure metabolites were tested against Colorado potato beetle, and NH4OH/ benzene (rich in alkaloids), CHCl3 , and acetone extracts, and b-sitosterol 3-O-b-dglucopyranoside (8) and oxyresveratrol (7) were found to be toxic. Based on the present study, the extracts as well as some pure metabolites can be used as natural insecticides against Colorado potato beetle. However, further studies are needed to evaluate the economic and safety aspects of the extracts and pure metabolites. The authors would like to thank the Atatrk University Research Fund for financial support (grant BAP: 2010/191, University Research Fund). Experimental Part General. TLC and prep. TLC: silica gel 60 F-254 (Merck, precoated plates); visualization by UV254 and UV365 , and spraying with 1% vanillin-H2SO4 , followed by heating (1058). Column chromatography (CC): silica gel 60 (Merck, 70 – 230 and 230 – 400 mesh). M.p.: Thermo Scientific 9200 apparatus. Optical rotations: Bellingham þ Stanley Ltd. ADP 220 polarimeter (equipped with a Na lamp and a 10-cm microcell in a suitable solvent at 208). UV/VIS: Jasco V-530 spectrophotometer; lmax in nm. IR Spectra: Perkin Elmer FTIR-1600 spectrophotometer; ˜n in cm 1. 1D and 2D-NMR spectra: Bruker 400 MHz (1H: 400 and 13C: 100 MHz) spectrometer; CDCl3 , (D6 )DMSO, (D6 )acetone, (D5 )pyridine and CD3OD solns.; d in ppm rel. to Me4Si as an internal standard, J in Hz. MS: Thermofinnigan Trace GC/Trace DSQ/ A1300 (70 eV, EI Quadrapole); in m/z. Materials and Chemicals. Rhizomes of V. album were collected from forest areas near the Hamsi village in the northern foothills of Zigana Mountains at northeastern Anatolia in July and August, 2009. Plant sample was deposited with the Herbarium of Kazım Karabekir Education Faculty, Atatrk University. Insect adults were collected from unsprayed potato fields in the eastern Anatolia (Erzurum) and kept in the laboratory in the Department of Plant Protection at Atatrk University. They were brought to the laboratory in plastic containers. The pure chemicals were purchased from Fluka, Merck, Aldrich, and Alfa. Vanillin and KI were used as solid chemical substances. Extraction and Isolation. Dried and powdered rhizomes of V. album were extracted with acetone (5 4 l), CHCl3 (5 4 l), and EtOH (5 1 l) at r.t. To obtain an enriched alkaloid extract, the rhizomes were
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extracted with benzene (5 l) with a Soxhlet apparatus after treatment with 7.5% NH4OH (3.4 mol). For acetone, CHCl3 , EtOH, and benzene extracts, 4000, 2150, 1300, and 200 g, resp., of plant samples were used. The org. solvents were evaporated under reduced pressure. The acetone extract (76 g) was fractionated by CC (SiO2 (385 g, 70 – 230 mesh); CHCl3/AcOEt 9 : 1, 8 : 2, 7 : 3, ... 0 : 10 and AcOEt/MeOH 9 : 1, 8 : 2, 6 : 4, 4 : 6). The fractions (50 ml each) were checked by TLC (SiO2 ; CHCl3/hexane 8 : 2, 9 : 1, CHCl3 , CHCl3/AcOEt 9 : 1, 8 : 2, 7 : 3, 6 : 4, 5 : 5, and CHCl3/MeOH 9 : 1, 8 : 2, 7 : 3), and the fractions with the same Rf value were combined. Ten fractions, A – J, were finally obtained, their amounts being 4.46, 4.2, 4.34, 2.2, 3.95, 4.15, 6.45, 2.54, 11.5, and 22.0 for Frs. A, B, C, D, E, F, G, H, I, and J, resp. Fr. B was submitted to CC (SiO2 (100 g, 230 – 400 mesh)) to yield compounds 1 (120 mg) and 2 (150 mg). Precipitation occurred when MeOH was added to Fr. C, the precipitate was separated, and the liquid portion was concentrated and purified by CC (SiO2 (110 g, 230 – 400 mesh)) to yield 3 (70 mg) and 4 (65 mg). The precipitate (1.22 g) contained a mixture of 2 and 4 as determined by TLC. Fr. D was subjected to CC (SiO2 (175 g, 230 – 400 mesh)) to yield 4 (170 mg). When CHCl3/MeOH 95 : 5 was added to the Fr. E, an amorphous material separated. Compound 5 (975 mg) was isolated washing this material with MeOH. The liquid portion (2.70 g) of Fr. E was submitted to CC (SiO2 (170 g, 230 – 400 mesh)) to yield compound 6 (20 mg) besides compound 5 (345 mg). Frs. F and G were individually separated by CC (SiO2 (160 g, 230 – 400 mesh)) to yield compound 7 (1.3 g and 460 mg, resp.). Fr. H was submitted to CC (SiO2 (100 g, 230 – 400 mesh)) to afford compound 8 (65 mg). Fr. I was separated by CC (SiO2 (175 g, 230 – 400 mesh)) to yield compound 9 (160 mg). Fr. J was subjected to CC (SiO2 (160 g, 230 – 400 mesh)) to give compounds 11 (2.27 g) and 12 (2.95 g). Compound 11 (5.40 g) was further isolated from the NH4OH/benzene extract (24 g) by CC (SiO2 (350 g, 70 – 230 mesh); CHCl3 and CHCl3/MeOH 95 : 5). The CHCl3 extract (30 g) of the plant samples were fractioned by CC (SiO2 (365 g, 70 – 230 mesh); CH2Cl2/AcOEt (80 : 20, 70 : 30, ..., 0 : 100 and AcOEt/MeOH 90 : 10, 80 : 20, 60 : 40, 40 : 60, 50 : 50). The fractions (50 ml each) were checked by TLC, and those with similar stains were combined. A total of eight fractions, A’ – H’, were finally obtained, with amounts being 2.9, 1.5, 2.6, 3.5, 3.6, 4.8, 4.7, and 1.7 g for Frs. A’, B’, C’, D’, E’, F’, G’, and H’, resp. These fractions were subjected to CC (SiO2 ) and yielded compounds 5 (240 mg), 7 (200 mg), 8 (100 mg), 10 (120 mg), 11 (2.4 g), 13 (130 mg), and 14 (30 mg). Compound 15 was crystallized from CHCl3/MeOH of the EtOH extract (5 g). The crystals were washed with the same solvent mixture to yield 70 mg of 15. Octacosanol (1). White needles. M.p. 72 – 738. IR (CHCl3 ): 3264 (OH), 2920 and 2845 (CH), 1462, 1427, 1061 (CO). 1H-NMR (400 CDCl3 ): 3.63 (t, J ¼ 6.6, CH2OH); 1.33 (quint., MeCH2 ); 1.22 (br. s, CH2 ); 0.84 (t, J ¼ 6.6, MeCH2 ). 13C-NMR (CDCl3 ): 14.30 (q); 29.91 (26 t); 63.30 (t). MS: 410 (M þ ; not observed), 211 (6), 196 (8), 183 (10), 168 (16), 155 (23), 139 (41), 125 (95), 111 (100). b-Sitosterol ( ¼ (3b)-Stigmast-5-en-3-ol; 2). White needles. M.p. 136 – 1388. [a] 20 D ¼ 35 (c ¼ 1, CHCl3 ). IR (CHCl3 ): 3359 (OH), 2950, 2933, and 2868 (aliph. CH), 1464 and 1376 (C¼C), 1061 (CO). 1 H-NMR (CDCl3 ): 5.35 (d, J ¼ 5.1, HC(6)); 3.56 – 3.48 (m, HC(3)); 1.00 (s, Me(19)); 0.91 (d, J ¼ 6.6, Me(27)); 0.83 (d, J ¼ 6.6, Me(29)); 0.80 (d, J ¼ 7.0, Me(21)); 0.67 (s, Me(18)). 13C-NMR (CDCl3 ): 141.0 (C(5)); 122.0 (C(6)); 72.0 (C(3)); 57.0 (C(14)); 56.3 (C(17)); 50.4 (C(9)); 46.1 (C(24)); 42.5 (C(4)); 42.5 (C(13)); 40.0 (C(12)); 37.5 (C(1)); 36.7 (C(10)); 36.4 (C(20)); 34.2 (C(22)); 32.1 (C(7)); 32.1 (C(8)); 32.0 (C(2)); 29.4 (C(25)); 28.5 (C(16)); 26.4 (C(23)); 24.5 (C(15)); 23.3 (C(28)); 21.3 (C(11)); 20.0 (C(26)); 19.6 (C(19)); 19.3 (C(27)); 19.0 (C(21)); 12.2 (C(29)); 12.1 (C(18)). Stearic Acid ( ¼ Octadecanoic Acid; 3). White needles. M.p. 70 – 718. IR (CHCl3 ): 3264 (COOH), 2920 and 2848 (aliph. CH). 1H-NMR (CHCl3 ): 2.34 (t, J ¼ 7.5, CH2(2)); 1.63 (quint., CH2(3)); 1.25 (s, CH2 ); 0.88 (t, J ¼ 6.8, Me(18)). 13C-NMR (CHCl3 ): 179.7 (C(1)); 34.1 (C(2)); 24.9 (C(3)); 14.3 (C(18)). Diosgenin ( ¼ (3b,25R)-Spirost-5-en-3-ol; 4). White needles. M.p. 191 – 1938. [a] 20 D ¼ 127 (c ¼ 1, CHCl3 ). IR (CHCl3 ): 3318 (OH), 2930 and 2850 (aliph. CH), 1450 (C¼C), 1065 (CO). 1H-NMR (CDCl3 ): 5.35 (br. s, HC(6)); 4.44 (dt, J ¼ 7.1, 7.6, HC(16)); 3.98 (dd, J ¼ 11.0, 2.7, HaC(26)); 3.54 – 3.48 (m, HC(3)); 3.32 (d, J ¼ 10.9, HbC(26)); 1.10 (d, J ¼ 7.1, Me(27)); 1.05 (s, Me(22)); 1.02 (d, J ¼ 6.8, Me(20)); 0.81 (s, Me(21)). 13C-NMR (CDCl3 ): 140.8 (C(5)); 121.4 (C(6)); 109.8 (C(19)); 80.9 (C(16)); 71.8 (C(3)); 65.1 (C(26)); 61.9 (C(17)); 56.5 (C(14)); 50.1 (C(9)); 42.3 (C(12)); 42.1 (C(18)); 40.3 (C(4)); 39.8 (C(13)); 37.2 (C(1)); 36.7 (C(10)); 32.1 (C(7)); 31.8 (C(15)); 31.6 (C(23)); 31.5 (C(8)); 27.1 (C(25)); 25.9 (C(2)); 25.8 (C(24)); 20.8 (C(11)); 19.4 (C(22)); 16.3 (C(21)); 16.1 (C(27)); 14.4 (C(20)).
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Resveratrol ( ¼ 5-[(E)-2-(4-Hydroxyphenyl)ethenyl]benzene-1,3-diol; 5). Light-brownish needles. M.p. 255 – 2568 (dec.). UV (CHCl3 ): 305. IR (solid): 3270 (OH), 1587, 1514, 1325 (C¼C). 1H-NMR ((D6 )DMSO): 7.37 (d, J ¼ 8.6, HC(2’), HC(6’)); 6.91 (d, J ¼ 16.4, HC(b)); 6.78 (d, J ¼ 16.4, HC(a)); 6.73 (d, J ¼ 8.6, HC(3’), HC(5’)); 6.36 (d, J ¼ 2.2, HC(2), HC(6)); 6.10 (t, J ¼ 2.2, HC(4)). 13 C-NMR ((D6 )DMSO): 159.2 (C(3), C(5)); 157.8 (C(4’)); 139.9 (C(1)); 128.8 (C(1’)); 128.6 (C(b)); 128.5 (C(2’), C(6’)); 126.4 (C(a)); 116.2 (C(3’), C(5’)); 104.9 (C(2), C(6)); 102.5 (C(4)). MS: 229 (100, M þ ), 211 (36), 228 (65), 230 (13), 257 (5). Wittifuran X ( ¼ 5-(6-Hydroxy-5-methoxy-1-benzofuran-2-yl)benzene-1,3-diol; 6). Light-brown, amorphous solid. UV (DMSO): 330. IR (solid): 3241 (OH), 1594 and 1450 (C¼C), 1141 (CO). 1 H-NMR ((D6 )acetone): 8.43 (s, 2 OH); 7.74 (s, OH); 7.14 (s, HC(3)); 7.03 (s, HC(4), HC(7)); 6.86 (d, J ¼ 1.5, HC(2’), HC(6’)); 6.37 (t, HC(4’)); 3.91 (s, MeO). 13C-NMR ((D6 )DMSO): 159.0 (C(3’), C(5’)); 154.2 (C(2)); 149.4 (C(7a)); 146.1 (C(5)); 145.8 (C(6)); 132.3 (C(1’)); 120.5 (C(3a)); 103.5 (C(4’)); 103.0 (C(4)); 102.8 (C(2’), C(6’)); 102.6 (C(3)); 98.4 (C(7)); 56.6 (MeO). Oxyresveratrol ( ¼ 4-[(E)-2-(3,5-Dihydroxyphenyl)ethenyl]benzene-1,3-diol; 7). Dark-brown, amorphous solid. M.p. 199 – 2008. UV (CHCl3 ): 289, 327. IR (CHCl3 ): 3694 (OH), 3041 (CH), 1599, 1457, 1305 (C¼C). 1H-NMR ((D6 )DMSO): 7.32 (d, J ¼ 8.2, HC(6’)); 7.13 (d, J ¼ 16.4, HC(a)); 6.77 (d, J ¼ 16.4, HC(b)); 6.40 (br. s, HC(2), HC(6)); 6.30 (s, HC(3’)); 6.28 (d, J ¼ 8.2, HC(5’)); 6.10 (s, HC(4)). 13 C-NMR ((D6 )DMSO): 158.1 (C(3), C(5)); 157.8 (C(4’)); 156.2 (C(2’)); 140.6 (C(1)); 128.0 (C(6’)); 125.3 (C(a)); 123.9 (C(b)); 116.0 (C(1’)); 107.9 (C(5’)); 104.7 (C(2), C(6)); 103.0 (C(3’)); 101.9 (C(4)). b-Sitosterol 3-O-b-d-Glucopyranoside ( ¼ (3b)-Stigmast-5-en-3-yl b-d-Glucopyranoside; 8). Lightbrown, amorphous solid. M.p. 297 – 2998. [a] 20 D ¼ 30 (c ¼ 1, pyridine). UV (MeOH): 224. IR (solid): 3364 (OH), 2932 (CH), 1366 (C¼C), 1060 (CO). 1H-NMR ((D5 ) pyridine): 5.34 (d, J ¼ 2.6, HC(6)); 5.04 (d, J ¼ 7.7, HC(1’)); 4.55 (dd, J ¼ 11.8, 2.4, 2.3, HaC(6’)); 4.40 (dd, J ¼ 11.8, 5.2, 6.5, HbC(6’)). 13 C-NMR ((D5 )pyridine): 140.7 (C(5)); 121.7 (C(6)); 102.4 (C(1’)); 78.4 (C(5’)); 78.2 (C(3’)); 77.9 (C(3)); 75.1 (C(2’)); 71.5 (C(4’)); 62.6 (C(6’)); 56.6 (C(14)); 56.1 (C(17)); 50.2 (C(9)); 45.9 (C(24)); 42.3 (C(13)); 39.8 (C(12)); 39.1 (C(4)); 37.3 (C(1)); 36.7 (C(10)); 36.2 (C(20)); 34.0 (C(22)); 32.0 (C(7)); 31.9 (C(8)); 30.0 (C(2)), 29.3 (C(25)); 28.3 (C(16)); 26.2 (C(23)); 24.3 (C(15)); 23.2 (C(28)); 21.1 (C(11)); 19.8 (C(27)); 19.2 (C(26)); 19.0 (C(19)); 18.8 (C(21)); 12.0 (C(29)); 11.8 (C(18)). Diosgenin 3-O-a-l-Rhamnopyranosyl-(1 ! 2)-b-d-glucopyronoside ( ¼ (3b,25R)-Spirost-5-en-3-yl 2O-(a-l-Rhamnopyranosyl)-b-d-glucopyranoside; 9). Light-brown, amorphous solid. M.p. 248 – 2508. [a] 20 D ¼ 60 (c ¼ 1, pyridine). UV (MeOH): 216, 285. IR (solid): 3354 (OH), 2929 (CH), 1050 (CO). 1 H-NMR ((D5 )pyridine): 6.42 (s, HC(1’)); 5.32 (d, J ¼ 4.8, HC(6)); 5.08 (d, J ¼ 7.3, HC(1’)); 3.35 (d, J ¼ 11.0, CH2(26)); 2.10 – 2.20 (m, CH2(15)); 1.78 (d, J ¼ 6.2, HC(6’)); 1.15 (d, J ¼ 6.9, Me(27)); 1.08 (d, J ¼ 7.1, Me(20)); 1.06 (s, Me(22)); 0.83 (s, Me(21)). 13C-NMR ((D5 )pyridine): 140.7 (C(5)); 121.5 (C(6)); 109.5 (C(19)); 101.9 (C(1’’)); 100.2 (C(1’)); 81.0 (C(16)); 79.5 (C(2’)); 78.1 (C(3)); 77.8 (C(3’)); 77.7 (C(5’)); 74.0 (C(4’’)); 72.7 (C(3’’)); 72.4 (C(2’’)); 71.7 (C(4’)); 69.3 (C(5’’)); 64.9 (C(26)); 62.5 (C(17)); 62.5 (C(6’)); 56.4 (C(14)); 50.1 (C(9)); 42.3 (C(18)); 40.2 (C(12)); 39.7 (C(13)); 38.8 (C(4)); 37.3 (C(10)); 36.9 (C(1)); 32.1 (C(7)); 32.0 (C(8)); 31.5 (C(23)); 30.0 (C(15)); 27.3 (C(25)); 26.2 (C(2)); 26.0 (C(24)); 20.9 (C(11)); 19.2 (C(22)); 18.5 (C(6’)); 16.1 (C(21)); 16.1 (C(20)); 14.6 (C(27)). Oxyresveratrol 3-O-b-d-Glucopyranoside ( ¼ 3-[(E)-2-(2,4-Dihydroxyphenyl)ethenyl]-5-hydroxyphenyl b-d-Glucopyranoside; 10). Dark-yellow, amorphous solid. [a] 20 D ¼ 27 (c ¼ 1, MeOH). UV (DMSO): 333. IR (CHCl3 ): 3411 (OH), 2923 and 2846 (CH), 1659 (C¼C), 1050 (CO). 1H-NMR (CD3OD): 7.36 (d, J ¼ 8.9, HC(6’)); 7.33 (d, J ¼ 16.4, HC(b)); 6.90 (d, J ¼ 16.4, HC(a)); 6.77 (t, J ¼ 1.6, HC(2)); 6.64 (t, J ¼ 1.6, HC(6)); 6.46 (t, J ¼ 2.1, HC(3’)); 6.30 – 6.35 (m, HC(4), HC(5’)); 3.95 (dd, J ¼ 12.0, 1.7, HaC(6’’)); 3.76 (dd, J ¼ 12.0, 4.9, HbC(6’’)); 3.40 – 3.55 (m, HC(4’’), HC(2’’), HC(5’’), HC(3’’). 13C-NMR (CD3OD): 159.0 (C(5)); 158.2 (C(3)); 157.9 (C(4’)); 156.0 (C(2’)); 140.9 (C(1)); 127.2 (C(6’)); 124.8 (C(a)); 124.0 (C(b)); 116.3 (C(1’)); 107.0 (C(5’)); 106.6 (C(6)); 105.7 (C(2)); 102.3 (C(3’)); 102.2 (C(4)); 101.0 (C(1’’)); 76.7 (C(3’’)); 76.6 (C(5’’)); 73.5 (C(2’’)); 69.9 (C(4’’)); 61.3 (C(6’’)). Jervine ( ¼ (3b,17b,22S,23R)-3-Hydroxy-9-methyl-17,23-epoxyveratraman-11-one; 11). Light-brown, amorphous solid. M.p. 246 – 2478. [a] 20 D ¼ 167 (c ¼ 1, CHCl3 ). UV (CHCl3 ): 251. IR (solid): 3297 (NH), 3300 (OH), 2800 – 2936 (CH), 1708 (C¼O), 1463 (C¼C), 1060 (CO). 1H-NMR (CDCl3 ): 5.35 (d, J ¼ 4.8, HC(6)); 3.44 – 3.55 (m, HC(3)); 3.29 (dt, J ¼ 10.3, 4.0, HC(23)); 3.06 (dd, J ¼ 12.4, 4.0,
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HaC(26)); 2.65 (t, J ¼ 9.2, HC(22)); 2.31 (t, J ¼ 12.5, HbC(26)); 2.15 (s, Me(18)); 0.98 (s, Me(19)); 0.94 (d, J ¼ 6.0, Me(21)); 0.93 (d, J ¼ 6.2, Me(27)). 13C-NMR (CDCl3 ): 207.0 (C(11)); 145.9 (C(12)); 142.6 (C(5)); 137.4 (C(13)); 121.1 (C(6)); 85.8 (C(17)); 76.6 (C(23)); 71.8 (C(3)); 66.8 (C(22)); 62.8 (C(9)); 54.8 (C(26)); 45.1 (C(20)); 41.7 (C(4)); 40.6 (C(14)); 39.1 (C(24)); 38.2 (C(8)); 37.3 (C(10)); 37.0 (C(1)); 31.7 (C(25)); 31.4 (C(16)); 31.2 (C(2)); 30.9 (C(7)); 24.6 (C(15)); 18.9 (C(27)); 18.7 (C(19)); 12.3 (C(18)); 10.9 (C(21)). Pseudojervine ( ¼ (3b,17b,22S,23R)-9-Methyl-11-oxo-17,23-epoxyveratraman-3-yl b-d-Glucopyranoside; 13). White amorphous solid. M.p. 291 – 2938. [a] 20 D ¼ 160 (c ¼ 1, pyridine). UV (MeOH-CHCl3 ): 357, 279, 277, 275. IR (solid): 3297 (NH), 2937 (CH), 1708 (C¼O), 1463 (C¼C), 1060 (CO, CN). 1 H-NMR ((D5 )pyridine): 5.28 (d, J ¼ 5.1, HC(6)); 5.07 (d, J ¼ 7.4, HC(1’)); 4.57 (dd, J ¼ 11.7, 2.3, HaC(6’)); 4.40 (dd, J ¼ 11.7, 5.5, HbC(6’)); 4.32 – 4.24 (m, HC(3), HC(5’)); 3.41 (dt, J ¼ 10.1, 3.1, HC(23)); 3.07 (dd, J ¼ 12.1, 3.9, HaC(26)); 2.41 (s, Me(18)); 2.31 (t, J ¼ 11.7, HbC(26)); 1.04 (d, J ¼ 7.4, Me(21)); 1.00 (s, Me(19)); 0.83 (d, J ¼ 6.6, Me(27)). 13C-NMR ((D5 )pyridine): 206.5 (C(11)); 146.2 (C(12)); 141.9 (C(5)); 136.9 (C(13)); 121.4 (C(6)); 102.3 (C(1’)); 85.2 (C(17)); 78.4 (C(5’)); 78.3 (C(3’)); 77.7 (C(4’)); 76.6 (C(23)); 75.1 (C(2’)); 71.5 (C(3)); 67.2 (C(22)); 62.6 (C(6’)); 62.4 (C(9)); 54.9 (C(26)); 44.6 (C(14)); 40.8 (C(20)); 39.2 (C(4)); 38.3 (C(24)); 38.2 (C(8)); 37.3 (C(10)); 37.0 (C(1)); 31.4 (C(25)); 30.9 (C(16)); 30.6 (C(2)); 29.5 (C(7)); 24.6 (C(15)); 18.8 (C(27)); 18.2 (C(19)); 12.2 (C(18)); 10.9 (C(21)). 5,6-Dihydro-1-hydroxyjervine ( ¼ (1b,17b,22S,23R)-1-Hydroxy-9-methyl-17,23-epoxyveratraman-11one; 14). Light-brown, amorphous solid. M.p. 239 – 2408. [a] 20 D ¼ þ 78 (c ¼ 1, CHCl3 ). IR (solid): 3300 (OH), 3297 (NH), 2857 – 2930 (CH), 1706 (C¼O), 1457 (C¼C), 1059 (CO), 754 (CN). 1H-NMR ((D5 )pyridine): 5.54 (br. s, HC(1)); 4.94 – 4.85 (m, CH2(3)); 3.65 (dt, J ¼ 10.1, 3.1, HC(23)); 3.26 (dd, J ¼ 12.4, 4.0, HaC(26)); 2.97 (t, J ¼ 9.3, HC(22)); 2.37 (d, J ¼ 2.0, Me(18)); 1.41 (s, Me(19)); 1.23 (d, J ¼ 7.4, Me(21)); 0.84 (d, J ¼ 6.6, Me(27)). 13C-NMR ((D5 )pyridine): 208.6 (C(11)); 146.7 (C(13)); 138.2 (C(12)); 87.2 (C(17)); 77.0 (C(23)); 72.3 (C(1)); 67.7 (C(22)); 66.9 (C(3)); 55.8 (C(9)); 55.3 (C(26)); 46.0 (C(14)); 43.8 (C(5)); 42.1 (C(10)); 41.7 (C(20)); 41.0 (C(2)); 40.1 (C(24)); 39.1 (C(4)); 37.9 (C(8)); 32.3 (C(6)); 31.8 (C(25)); 29.1 (C(16)); 26.5 (C(7)); 25.7 (C(15)); 20.0 (C(27)); 19.5 (C(19)); 13.6 (C(18)); 12.8 (C(21)). Saccharose ( ¼ b-d-Fructofuranosyl a-d-Glucopyranoside; 15). Brownish needles. M.p. 182 – 1838. [a] 20 D ¼ þ 66 (c ¼ 1, MeOH). IR (solid): 3324 (OH), 2800 – 2936 (CH), 1343 (C¼C), 1066, 1049 (CO). 1 H-NMR ((D6 )DMSO): 5.21 (br. s, HOC(4’)); 5.15 (d, J ¼ 3.5, HC(1)); 5.06 (br. s, HOC(2)); 4.84 – 4.76 (m, HOC(4)); 4.54 (d, J ¼ 8.5, HOC(3’)); 4.46 – 4.38 (m, HOC(5’), HOC(6’)); 3.86 (t, J ¼ 7.7, HC(3’); 3.78 – 3.72 (m, HC(4’)). 13C-NMR ((D6 )DMSO): 104.5 (C(2’)); 92.2 (C(1)); 82.9 (C(5’)); 77.5 (C(3’)); 74.7 (C(4’)); 73.3 (C(3)); 73.2 (C(5)); 72.1 (C(2)); 70.3 (C(4)); 62.6 (C(6’)); 62.5 (C(1’)); 60.9 (C(6)). Evaluation of Insecticidal Activities. Glass Petri dishes (9-cm-wide 1.5-cm-deep; corresponding to 120 ml volume) were used to test the toxicities of the acetone, NH4OH/benzene, and CHCl3 extracts of V. album, and of 2, 2 þ 4, 5, 7, 8, and 11 against adults of L. decemlineata. The solns. (10, 20, and 30 mg/ml) of each substance was prepared by suspending and/or dissolving in DMSO/dist. H2O 1 : 10. Randomly, ten potato beetle adults were placed in the Petri dishes without distinguishing between male and female. One ml of the prepared suspension, corresponding to 10, 20, and 40 mg/ml concentrations, was sprayed into each Petri dish to wet the insects using an atomizer. The adults were placed on filter paper, containing the appropriate amounts of potato leaves. Petri dishes were closed with adhesive tape and transferred into an incubator, and then kept under standard conditions at 25 18 and 64 5% relative humidity, and for 16 : 8 (light: dark) photoperiod for 4 d. Lambda cyhalothrin (10, 20, and 40 mg/ml concentrations) was used as a positive control under the same conditions. After treatments, the mortality of the adults was counted after 12, 24, 48, and 96 h, resp. Control treatments with DMSO/dist. H2O 1 : 10, and without the extracts and compounds were conducted in the same way. All experiments were run in triplicate. Statistical Analyses. For comparison of data, Variance Analysis method was applied, and differences between means were tested through LSD, and values of p < 0.05 were considered significantly different. In addition, durations for 50 and 90% mortality of the adults and LC50 values were determined in the first 12 h, in order to determine the toxic effects of substances used as insecticide by probit analysis.
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Insecticidal Metabolites from the Rhizomes of Veratrum album against Adults of Colorado Potato Beetle, Leptinotarsa decemlineata by Tuba Aydin a ), Ahmet Cakir* b ), Cavit Kazaz c ), Neslihan Bayrak d ), Yasin Bayir e ), and Yavuz Tas¸kesenligil f ) ) Agri I˙brahim Cecen University, Health Services Vocational School, Department of Paramedic, TR-04100, Agri b ) Kilis 7 Aralik University, Faculty of Sciences & Arts, Department of Chemistry, TR-79000, Kilis (phone: þ 90-348-8222350; fax: þ 90-348-8222351; e-mail: [email protected]; [email protected]) c ) Atatrk University, Faculty of Science, Department of Chemistry, TR-25240, Erzurum d ) Bozok University, Faculty of Agriculture, TR-66900, Yozgat e ) Atatrk University, Faculty of Pharmacy, Department of Biochemistry, TR-25240, Erzurum f ) Atatrk University, Kazim Karabekir Education Faculty, Department of Chemistry, TR-25240, Erzurum a
The dried rhizomes of Veratrum album were individually extracted with CHCl3 , acetone, and NH4OH/benzene to test the toxic effects against the Colorado potato beetle, Leptinotarsa decemlineata, which is an important agricultural pest. Fifteen compounds in various amounts were isolated from the extracts using column and thin-layer chromatography. The chemical structures of 14 compounds were characterized as octacosan-1-ol (1), b-sitosterol (2), stearic acid (3), diosgenin (4), resveratrol (5), wittifuran X (6), oxyresveratrol (7), b-sitosterol 3-O-b-d-glucopyranoside (8), diosgenin 3-O-a-lrhamnopyranosyl-(1 ! 2)-b-d-glucopyronoside (9), oxyresveratrol 3-O-b-d-glucopyranoside (10), jervine (11), pseudojervine (13), 5,6-dihydro-1-hydroxyjervine (14), and saccharose (15) using UV, IR, MS, 1 H- and 13C-NMR, and 2D-NMR spectroscopic methods. However, the chemical structure of 12, an oligosaccharide, has not fully been elucidated. Compounds 4, 6, 9, and 10 were isolated from V. album rhizomes for the first time in the current study. The toxic effects of three extracts (acetone, CHCl3 , and NH4OH/benzene) and six metabolites, 2, 2 þ 4, 5, 7, 8, and 11, were evaluated against the Colorado potato beetle. The assay revealed that all three extracts, and compounds 7, 8, and 11 exhibited potent toxic effects against this pest. This is the first report on the evaluation of the toxic effects of the extracts and secondary metabolites of V. album rhizomes against L. decemlineata. Based on these results, it can be concluded that the extracts can be used as natural insecticides.
Introduction. – Veratrum (Hellebore) is an important plant genus belonging to the family Liliaceae. The crude extracts and compounds isolated from Veratrum plants have been reported to possess various pharmacological activities such as hypotensive, antithrombotic, and antitumor activities [1] [2]. In particular, their alkaloid constituents showed significant anticancer activities by blocking the hedgehog signal pathway [2 – 4]. Veratrum species are not consumed by herbivores due to their toxic steroidal alkaloids [5]. V. album, which is among Turkish flora, is a poisonous type of plant because of its steroidal alkaloids and glycosides. V. album is known by the local names çpleme, Ak Åçpleme, Beyaz Åçpleme, and emah in Turkey [6]. All parts of V. album have a bitter taste, and its powders have sternutatory effects. It has been used as a traditional medicine in Turkey. However, due to its irritant properties, its infusion was only used externally. It was used in the treatment of scabies and body interference. Its 2014 Verlag Helvetica Chimica Acta AG, Zrich
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powdered rhizomes have been used to remedy nasal congestion during cold and sinusitis [6]. Veratrum preparations were used for centuries in the treatment of circulatory disorders and the control of insects [7]. The insect repellent effect of V. album has been known since ancient times [8] [9]. Leptinotarsa decemlineata, Colorado potato beetle, known as potato beetle in Turkey is an insect that damages the leaves of potato, eggplants, and tomatoes. L. decemlineata spread over an area of 16 million km2 in North America, Europe, and Asia, and continues to spread. Larvae and adults of L. decemlineata feed on the leaves of potato and remain for long periods on the potato plant. Therefore, no control technique has been developed against L. decemlineata so far, leading to a decrease in development and production of the potato. This situation requires chemical control against this insect [10] [11]. L. decemlineata can develop resistance to many insecticides. In the last 60 years, L. decemlineata has developed resistance against 52 different compounds which were considered important insecticides. Therefore, L. decemlineata is highly important in the development of modern pesticides. Since 1864, hundreds of compounds were tested against this insect, and various drugs have been developed to reduce its harms [11 – 14]. Extracts of some plant species and pyrrolizidine alkaloids, cardiopetamins, terpenoids, cucurbitacins, silphinens, and limonoids isolated from some plants have been shown to be toxic against L. decemlineata [15 – 20]. There is no report in the literature on the toxicity of the extracts and metabolites of Veratrum species against adults of L. decemlineata. Therefore, the aim of the present study was to assess the toxicity of V. album extracts, its pure metabolites, and lambda cyhalothrin, an insect control reactive against adults of L. decemlineata. Herein, we describe the isolation and chemical characterization of 15 compounds. The insecticidal effects of some extracts and some compounds isolated from V. album against adults of L. decemlineata are also reported. Results and Discussion. – The Chemical Composition of the Extracts. The acetone, NH4OH/benzene, and EtOH extracts of V. album was subjected to fractionation by column chromatography (CC) and TLC. One aliphatic alcohol 1, one fatty acid 3, four steroids and steroid glycosides, 2 and 4, and 8 and 9, respectively, four stilbenoids and their glycosides, 5, 6, 7, and 10, three steroidal alkaloids, 11, 13, and 14, and one carbohydrate, 15, were isolated. The chemical structures of these compounds were characterized by UV, IR, MS, 1H- and 13C-NMR, 1D-(DEPT), and 2D-NMR (1H,1HCOSY, 1H,13C-COSY, and HMBC) analyses (Fig. 1). However, the chemical structure of compound 12, an oligosaccharide, has not been fully characterized. The chemical structures of the known compounds were also confirmed by comparison of their spectral data with those reported in the literature, i.e., as octacosan-1-ol (1), b-sitosterol (2) [21] [22], stearic acid (3), diosgenin (4) [23 – 29], resveratrol (5) [30] [31], wittifuran X (6) [32 – 34], oxyresveratrol (7) [35], b-sitosterol 3-O-b-d-glucopyranoside (8), diosgenin 3-O-a-l-rhamnopyranosyl-(1 ! 2)-b-d-glucopyronoside (9) [23 – 29] [36 – 39], oxyresveratrol 3-O-b-d-glucopyranoside (10) [33] [40] [41], jervine (11) [42 – 44], pseudojervine (13) [42 – 44], 5,6-dihydro-1-hydroxyjervine (14) [43] [45] [46], and saccharose (15) [47 – 49]. Compounds 4, 6, 9, and 10 were isolated from V. album rhizomes for the first time.
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Fig. 1. Chemical structures of the compounds 2, 4 – 11, 13, and 14
Insecticidal Effects of the Extracts and Pure Metabolites. The toxicities of the extracts and the pure metabolites of V. album were determined against adults of L. decemlineata. The extracts and pure metabolites at three different concentrations, 10, 20 and 40 mg/Petri dish, were investigated, and their toxicities were compared with toxicity of the reference, lambda cyhalothrin, a commercial insecticide at the same concentrations (Figs. 2 and 3). The data show that the extracts and pure metabolites display various toxicities against the adults depending on exposure time and tested substances. Generally, the mortality increased with increasing doses of the substances and exposure times (Figs. 2 and 3). The extracts were found to be more toxic than the pure compounds, the NH4OH/benzene extract being the most toxic sample. All doses of this extract caused 100% mortality against the adult within the first 12 h (Fig. 2). Acetone and CHCl3 extracts led to complete mortality for the adult in 24 h (Fig. 3). Among the tested metabolites, b-sitosterol 3-O-b-d-glucopyranoside (8) was more effective against adults as compared to other metabolites. Figs. 2 and 3 show that 20 and 40 mg/ml concentrations of this compound caused 100% mortality in the first 24 h, and the remaining doses within 48 h. Another compound which caused 100% mortality against the adult at 24 h was oxyresveratrol (7), which, at 40 mg/ml concentration, caused 100% mortality after 24 h (Fig. 3). Jervine (11) and b-sitosterol (2) þ diosgenin (4) at 40 mg/ml concentration displayed low toxicities as compared with those of the extracts, b-sitosterol 3-O-b-d-glucopyranoside (8) and oxyresveratrol (7; Figs. 2 and 3).
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Fig. 2. Mortality [%] of the adults of L. decemlineata exposed to the extracts and pure metabolites after 12 h
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Fig. 3. Mortality (%) of the adults of L. decemlineata exposed to the extracts and pure metabolites after 24 h
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When compared with the toxicity of b-sitosterol (2), the mixture of b-sitosterol (2) þ diosgenin (4) was more effective than 2 against adults of the insect. Hence, it can be concluded that 4 is more toxic than 2. Furthermore, among the pure metabolites, resveratrol (5) was less toxic against the adults of Colorado potato beetle (Figs. 2 and 3). The LC50 value is dose that kills 50% of the test animals when applied through the skin and mouth, and it is given in mg/kg. If the LC50 value of a substance is low, it is highly toxic [50]. The LC50 values in 12 h of the tested substances are compiled in Table 1. NH4OH/Benzene extract and commercial insecticide, lambda cyhalothrin, were the most toxic substances against the Colorado potato beetle. As can be seen from Table 1, LC50 values of NH4OH/benzene, CHCl3 , and acetone extracts were found to be low as compared with other treatments; therefore, they were classified as highly toxic insecticides against the beetle. Furthermore, based on the present results, b-sitosterol 3O-b-d-glucopyranoside (8), oxyresveratrol (7), jervine (11), b-sitosterol (2) þ diosgenin (4), and resveratrol (5; LC50 values 10 – 60 mg/ml) were classified as toxic insecticides, whereas b-sitosterol (2; LC50 501.5 mg/ml) was classified as a mediumtoxic insecticide [50]. The durations, calculated by the probit analyses for the 50 and 90% mortality of the adults of the L. decemlineata exposed to the extracts and the pure metabolites are collected in Table 2. Durations of the applications for 50 and 90% mortality of the adults of Colorado potato beetle decreased depending on the concentration increase. These data indicate that the toxicities of the applications were concentrationdependent. Among all applications, the shortest exposure time for 50 and 90% mortality of the pest were of NH4OH/benzene extract and positive control, lambda cyhalothrin. These results suggested that of all treatments, NH4OH/benzene extract is the most toxic reagent for the adults of the pest. Also CHCl3 extract, b-sitosterol 3-O-bd-glucopyranoside (8), and oxyresveratrol (7) turned out to be toxic (Table 2). Based on the durations for 50 and 90% mortality of the insect, b-sitosterol (2) þ diosgenin (4) was more toxic than 2. This can be attributed to the synergistic effect of 4 on the toxicity of 2. Our results also indicated that resveratrol (5) and jervine (11) were the less toxic metabolites as compared with other compounds tested.
Table 1. The LC50 Values of the Tested Extracts and Metabolites Substance Extracts
NH4OH/benzene CHCl3 Acetone
Metabolites
b-Sitosterol 3-O-b-d-glucopyranoside (8) Oxyresveratrol (7) Jervine (11) b-Sitosterol (2) þ diosgenin (4) Resveratrol (5) b-Sitosterol (2)
Positive control
Lambda cyhalothrin
LC50 Value [mg/ml] 0.025 0.14 8.30 10.48 14.32 26.69 26.96 57.68 501.46 0.025
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Table 2. Durations for 50 and 90% Mortality of the Adults of the Pest Exposed to Different Concentrations of the Extracts and Metabolites Extracts and metabolites
Extracts
Acetone
CHCl3
NH4OH/benzene
Metabolites
b-Sitosterol (2)
b-Sitosterol (2) þ diosgenin (4)
Resveratrol (5)
Oxyresveratrol (7)
b-Sitosterol 3-O-b-d-Glc (8)
Jervine (11)
Control
Lambda cyhalothrin
Concentration
Duration [h]
[mg/ml]
50% mortality
90% mortality
10 20 40 10 20 40 10 20 40
9.99 7.48 2.64 7.33 7.33 4.70 2.64 2.64 2.64
18.08 16.07 7.49 13.88 13.88 11.62 7.49 7.49 7.49
10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40
56.74 26.87 23.13 43.00 24.01 6.46 99.38 29.41 15.60 22.82 9.71 2.64 13.78 7.33 2.64 69.76 26.85 7.99
130.63 45.81 52.17 95.10 51.15 14.99 365.22 118.37 51.19 58.70 21.58 7.49 29.92 13.88 7.45 490.5 61.01 21.88
10 20 40
2.64 2.64 2.64
7.49 7.49 7.49
Veratrum species and their metabolites, steroidal alkaloids, are well-known to display toxic properties. Insecticidal effect of Veratrum extracts and some steroidal alkaloids has been reported against mosquito, silkworm, and fruit fly [51] [52]. Toxic effects of V. album extract and some fractions have also been determined against Pseudococcus longispinus and Pseudococcus affinis (Momoptera: Pseudococcidae) [53]. An acetone extract of V. album exhibited toxic effect against Drosophila melanogaster [51]. In another study, toxic effects of steroidal alkaloids, veratradine, cevadine, veracevine, protoveratrine A, and protoverotrine, isolated from V. album, were investigated against the housefly (Musca domestica: Diptera) and Oncopeltus fasciatus, and it was documented that of all compounds with strong toxicities against both species, protoveratrine A was the most toxic compound with the lowest LD50 value (for housefly, LD50 of 0.10 mg/kg and for Oncopeltus fasciatus LD50 of 0.09 mg/kg) [7].
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However, according to our literature survey, insecticidal activities of Veratrum extracts and pure metabolites against L. decemlineata have not been investigated. The result of the present study revealed that, in particular, NH4OH/benzene, CHCl3 , and acetone extracts of V. album rhizomes are more toxic against L. decemlineata. Considering the insect mortality in 12 h, clearly the most toxic substance is the NH4OH/ benzene extract (Table 2 and Fig. 2). As shown in Table 2 and Fig. 2, NH4OH/benzene extract included major amounts of jervine (11). As compared to the toxic effects of NH4OH/benzene extract and jervine (11) at 12 h (Fig. 2, and Tables 1 and 2), NH4OH/ benzene extract was more toxic than 11. Low toxic effect of 11 as compared to that of the NH4OH/benzene extract can be attributed to other minor substances in the NH4OH/benzene extract. Among the tested pure metabolites, b-sitosterol 3-O-b-dglucopyranoside (8) and oxyresveratrol (7) showed potent toxic effects against L. decemlineata. Conclusions. – Developing natural or biological insecticides will help to decrease some negative features of synthetic chemicals, including residues in products, insect resistance, and environmental pollution. In this respect, the use of natural insecticides can also be effective, selective, and easily bio-degradable, causing relatively low pollution for environment. In the present study, the toxic effects of the extracts and some pure metabolites were tested against Colorado potato beetle, and NH4OH/ benzene (rich in alkaloids), CHCl3 , and acetone extracts, and b-sitosterol 3-O-b-dglucopyranoside (8) and oxyresveratrol (7) were found to be toxic. Based on the present study, the extracts as well as some pure metabolites can be used as natural insecticides against Colorado potato beetle. However, further studies are needed to evaluate the economic and safety aspects of the extracts and pure metabolites. The authors would like to thank the Atatrk University Research Fund for financial support (grant BAP: 2010/191, University Research Fund). Experimental Part General. TLC and prep. TLC: silica gel 60 F-254 (Merck, precoated plates); visualization by UV254 and UV365 , and spraying with 1% vanillin-H2SO4 , followed by heating (1058). Column chromatography (CC): silica gel 60 (Merck, 70 – 230 and 230 – 400 mesh). M.p.: Thermo Scientific 9200 apparatus. Optical rotations: Bellingham þ Stanley Ltd. ADP 220 polarimeter (equipped with a Na lamp and a 10-cm microcell in a suitable solvent at 208). UV/VIS: Jasco V-530 spectrophotometer; lmax in nm. IR Spectra: Perkin Elmer FTIR-1600 spectrophotometer; ˜n in cm 1. 1D and 2D-NMR spectra: Bruker 400 MHz (1H: 400 and 13C: 100 MHz) spectrometer; CDCl3 , (D6 )DMSO, (D6 )acetone, (D5 )pyridine and CD3OD solns.; d in ppm rel. to Me4Si as an internal standard, J in Hz. MS: Thermofinnigan Trace GC/Trace DSQ/ A1300 (70 eV, EI Quadrapole); in m/z. Materials and Chemicals. Rhizomes of V. album were collected from forest areas near the Hamsi village in the northern foothills of Zigana Mountains at northeastern Anatolia in July and August, 2009. Plant sample was deposited with the Herbarium of Kazım Karabekir Education Faculty, Atatrk University. Insect adults were collected from unsprayed potato fields in the eastern Anatolia (Erzurum) and kept in the laboratory in the Department of Plant Protection at Atatrk University. They were brought to the laboratory in plastic containers. The pure chemicals were purchased from Fluka, Merck, Aldrich, and Alfa. Vanillin and KI were used as solid chemical substances. Extraction and Isolation. Dried and powdered rhizomes of V. album were extracted with acetone (5 4 l), CHCl3 (5 4 l), and EtOH (5 1 l) at r.t. To obtain an enriched alkaloid extract, the rhizomes were
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extracted with benzene (5 l) with a Soxhlet apparatus after treatment with 7.5% NH4OH (3.4 mol). For acetone, CHCl3 , EtOH, and benzene extracts, 4000, 2150, 1300, and 200 g, resp., of plant samples were used. The org. solvents were evaporated under reduced pressure. The acetone extract (76 g) was fractionated by CC (SiO2 (385 g, 70 – 230 mesh); CHCl3/AcOEt 9 : 1, 8 : 2, 7 : 3, ... 0 : 10 and AcOEt/MeOH 9 : 1, 8 : 2, 6 : 4, 4 : 6). The fractions (50 ml each) were checked by TLC (SiO2 ; CHCl3/hexane 8 : 2, 9 : 1, CHCl3 , CHCl3/AcOEt 9 : 1, 8 : 2, 7 : 3, 6 : 4, 5 : 5, and CHCl3/MeOH 9 : 1, 8 : 2, 7 : 3), and the fractions with the same Rf value were combined. Ten fractions, A – J, were finally obtained, their amounts being 4.46, 4.2, 4.34, 2.2, 3.95, 4.15, 6.45, 2.54, 11.5, and 22.0 for Frs. A, B, C, D, E, F, G, H, I, and J, resp. Fr. B was submitted to CC (SiO2 (100 g, 230 – 400 mesh)) to yield compounds 1 (120 mg) and 2 (150 mg). Precipitation occurred when MeOH was added to Fr. C, the precipitate was separated, and the liquid portion was concentrated and purified by CC (SiO2 (110 g, 230 – 400 mesh)) to yield 3 (70 mg) and 4 (65 mg). The precipitate (1.22 g) contained a mixture of 2 and 4 as determined by TLC. Fr. D was subjected to CC (SiO2 (175 g, 230 – 400 mesh)) to yield 4 (170 mg). When CHCl3/MeOH 95 : 5 was added to the Fr. E, an amorphous material separated. Compound 5 (975 mg) was isolated washing this material with MeOH. The liquid portion (2.70 g) of Fr. E was submitted to CC (SiO2 (170 g, 230 – 400 mesh)) to yield compound 6 (20 mg) besides compound 5 (345 mg). Frs. F and G were individually separated by CC (SiO2 (160 g, 230 – 400 mesh)) to yield compound 7 (1.3 g and 460 mg, resp.). Fr. H was submitted to CC (SiO2 (100 g, 230 – 400 mesh)) to afford compound 8 (65 mg). Fr. I was separated by CC (SiO2 (175 g, 230 – 400 mesh)) to yield compound 9 (160 mg). Fr. J was subjected to CC (SiO2 (160 g, 230 – 400 mesh)) to give compounds 11 (2.27 g) and 12 (2.95 g). Compound 11 (5.40 g) was further isolated from the NH4OH/benzene extract (24 g) by CC (SiO2 (350 g, 70 – 230 mesh); CHCl3 and CHCl3/MeOH 95 : 5). The CHCl3 extract (30 g) of the plant samples were fractioned by CC (SiO2 (365 g, 70 – 230 mesh); CH2Cl2/AcOEt (80 : 20, 70 : 30, ..., 0 : 100 and AcOEt/MeOH 90 : 10, 80 : 20, 60 : 40, 40 : 60, 50 : 50). The fractions (50 ml each) were checked by TLC, and those with similar stains were combined. A total of eight fractions, A’ – H’, were finally obtained, with amounts being 2.9, 1.5, 2.6, 3.5, 3.6, 4.8, 4.7, and 1.7 g for Frs. A’, B’, C’, D’, E’, F’, G’, and H’, resp. These fractions were subjected to CC (SiO2 ) and yielded compounds 5 (240 mg), 7 (200 mg), 8 (100 mg), 10 (120 mg), 11 (2.4 g), 13 (130 mg), and 14 (30 mg). Compound 15 was crystallized from CHCl3/MeOH of the EtOH extract (5 g). The crystals were washed with the same solvent mixture to yield 70 mg of 15. Octacosanol (1). White needles. M.p. 72 – 738. IR (CHCl3 ): 3264 (OH), 2920 and 2845 (CH), 1462, 1427, 1061 (CO). 1H-NMR (400 CDCl3 ): 3.63 (t, J ¼ 6.6, CH2OH); 1.33 (quint., MeCH2 ); 1.22 (br. s, CH2 ); 0.84 (t, J ¼ 6.6, MeCH2 ). 13C-NMR (CDCl3 ): 14.30 (q); 29.91 (26 t); 63.30 (t). MS: 410 (M þ ; not observed), 211 (6), 196 (8), 183 (10), 168 (16), 155 (23), 139 (41), 125 (95), 111 (100). b-Sitosterol ( ¼ (3b)-Stigmast-5-en-3-ol; 2). White needles. M.p. 136 – 1388. [a] 20 D ¼ 35 (c ¼ 1, CHCl3 ). IR (CHCl3 ): 3359 (OH), 2950, 2933, and 2868 (aliph. CH), 1464 and 1376 (C¼C), 1061 (CO). 1 H-NMR (CDCl3 ): 5.35 (d, J ¼ 5.1, HC(6)); 3.56 – 3.48 (m, HC(3)); 1.00 (s, Me(19)); 0.91 (d, J ¼ 6.6, Me(27)); 0.83 (d, J ¼ 6.6, Me(29)); 0.80 (d, J ¼ 7.0, Me(21)); 0.67 (s, Me(18)). 13C-NMR (CDCl3 ): 141.0 (C(5)); 122.0 (C(6)); 72.0 (C(3)); 57.0 (C(14)); 56.3 (C(17)); 50.4 (C(9)); 46.1 (C(24)); 42.5 (C(4)); 42.5 (C(13)); 40.0 (C(12)); 37.5 (C(1)); 36.7 (C(10)); 36.4 (C(20)); 34.2 (C(22)); 32.1 (C(7)); 32.1 (C(8)); 32.0 (C(2)); 29.4 (C(25)); 28.5 (C(16)); 26.4 (C(23)); 24.5 (C(15)); 23.3 (C(28)); 21.3 (C(11)); 20.0 (C(26)); 19.6 (C(19)); 19.3 (C(27)); 19.0 (C(21)); 12.2 (C(29)); 12.1 (C(18)). Stearic Acid ( ¼ Octadecanoic Acid; 3). White needles. M.p. 70 – 718. IR (CHCl3 ): 3264 (COOH), 2920 and 2848 (aliph. CH). 1H-NMR (CHCl3 ): 2.34 (t, J ¼ 7.5, CH2(2)); 1.63 (quint., CH2(3)); 1.25 (s, CH2 ); 0.88 (t, J ¼ 6.8, Me(18)). 13C-NMR (CHCl3 ): 179.7 (C(1)); 34.1 (C(2)); 24.9 (C(3)); 14.3 (C(18)). Diosgenin ( ¼ (3b,25R)-Spirost-5-en-3-ol; 4). White needles. M.p. 191 – 1938. [a] 20 D ¼ 127 (c ¼ 1, CHCl3 ). IR (CHCl3 ): 3318 (OH), 2930 and 2850 (aliph. CH), 1450 (C¼C), 1065 (CO). 1H-NMR (CDCl3 ): 5.35 (br. s, HC(6)); 4.44 (dt, J ¼ 7.1, 7.6, HC(16)); 3.98 (dd, J ¼ 11.0, 2.7, HaC(26)); 3.54 – 3.48 (m, HC(3)); 3.32 (d, J ¼ 10.9, HbC(26)); 1.10 (d, J ¼ 7.1, Me(27)); 1.05 (s, Me(22)); 1.02 (d, J ¼ 6.8, Me(20)); 0.81 (s, Me(21)). 13C-NMR (CDCl3 ): 140.8 (C(5)); 121.4 (C(6)); 109.8 (C(19)); 80.9 (C(16)); 71.8 (C(3)); 65.1 (C(26)); 61.9 (C(17)); 56.5 (C(14)); 50.1 (C(9)); 42.3 (C(12)); 42.1 (C(18)); 40.3 (C(4)); 39.8 (C(13)); 37.2 (C(1)); 36.7 (C(10)); 32.1 (C(7)); 31.8 (C(15)); 31.6 (C(23)); 31.5 (C(8)); 27.1 (C(25)); 25.9 (C(2)); 25.8 (C(24)); 20.8 (C(11)); 19.4 (C(22)); 16.3 (C(21)); 16.1 (C(27)); 14.4 (C(20)).
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Resveratrol ( ¼ 5-[(E)-2-(4-Hydroxyphenyl)ethenyl]benzene-1,3-diol; 5). Light-brownish needles. M.p. 255 – 2568 (dec.). UV (CHCl3 ): 305. IR (solid): 3270 (OH), 1587, 1514, 1325 (C¼C). 1H-NMR ((D6 )DMSO): 7.37 (d, J ¼ 8.6, HC(2’), HC(6’)); 6.91 (d, J ¼ 16.4, HC(b)); 6.78 (d, J ¼ 16.4, HC(a)); 6.73 (d, J ¼ 8.6, HC(3’), HC(5’)); 6.36 (d, J ¼ 2.2, HC(2), HC(6)); 6.10 (t, J ¼ 2.2, HC(4)). 13 C-NMR ((D6 )DMSO): 159.2 (C(3), C(5)); 157.8 (C(4’)); 139.9 (C(1)); 128.8 (C(1’)); 128.6 (C(b)); 128.5 (C(2’), C(6’)); 126.4 (C(a)); 116.2 (C(3’), C(5’)); 104.9 (C(2), C(6)); 102.5 (C(4)). MS: 229 (100, M þ ), 211 (36), 228 (65), 230 (13), 257 (5). Wittifuran X ( ¼ 5-(6-Hydroxy-5-methoxy-1-benzofuran-2-yl)benzene-1,3-diol; 6). Light-brown, amorphous solid. UV (DMSO): 330. IR (solid): 3241 (OH), 1594 and 1450 (C¼C), 1141 (CO). 1 H-NMR ((D6 )acetone): 8.43 (s, 2 OH); 7.74 (s, OH); 7.14 (s, HC(3)); 7.03 (s, HC(4), HC(7)); 6.86 (d, J ¼ 1.5, HC(2’), HC(6’)); 6.37 (t, HC(4’)); 3.91 (s, MeO). 13C-NMR ((D6 )DMSO): 159.0 (C(3’), C(5’)); 154.2 (C(2)); 149.4 (C(7a)); 146.1 (C(5)); 145.8 (C(6)); 132.3 (C(1’)); 120.5 (C(3a)); 103.5 (C(4’)); 103.0 (C(4)); 102.8 (C(2’), C(6’)); 102.6 (C(3)); 98.4 (C(7)); 56.6 (MeO). Oxyresveratrol ( ¼ 4-[(E)-2-(3,5-Dihydroxyphenyl)ethenyl]benzene-1,3-diol; 7). Dark-brown, amorphous solid. M.p. 199 – 2008. UV (CHCl3 ): 289, 327. IR (CHCl3 ): 3694 (OH), 3041 (CH), 1599, 1457, 1305 (C¼C). 1H-NMR ((D6 )DMSO): 7.32 (d, J ¼ 8.2, HC(6’)); 7.13 (d, J ¼ 16.4, HC(a)); 6.77 (d, J ¼ 16.4, HC(b)); 6.40 (br. s, HC(2), HC(6)); 6.30 (s, HC(3’)); 6.28 (d, J ¼ 8.2, HC(5’)); 6.10 (s, HC(4)). 13 C-NMR ((D6 )DMSO): 158.1 (C(3), C(5)); 157.8 (C(4’)); 156.2 (C(2’)); 140.6 (C(1)); 128.0 (C(6’)); 125.3 (C(a)); 123.9 (C(b)); 116.0 (C(1’)); 107.9 (C(5’)); 104.7 (C(2), C(6)); 103.0 (C(3’)); 101.9 (C(4)). b-Sitosterol 3-O-b-d-Glucopyranoside ( ¼ (3b)-Stigmast-5-en-3-yl b-d-Glucopyranoside; 8). Lightbrown, amorphous solid. M.p. 297 – 2998. [a] 20 D ¼ 30 (c ¼ 1, pyridine). UV (MeOH): 224. IR (solid): 3364 (OH), 2932 (CH), 1366 (C¼C), 1060 (CO). 1H-NMR ((D5 ) pyridine): 5.34 (d, J ¼ 2.6, HC(6)); 5.04 (d, J ¼ 7.7, HC(1’)); 4.55 (dd, J ¼ 11.8, 2.4, 2.3, HaC(6’)); 4.40 (dd, J ¼ 11.8, 5.2, 6.5, HbC(6’)). 13 C-NMR ((D5 )pyridine): 140.7 (C(5)); 121.7 (C(6)); 102.4 (C(1’)); 78.4 (C(5’)); 78.2 (C(3’)); 77.9 (C(3)); 75.1 (C(2’)); 71.5 (C(4’)); 62.6 (C(6’)); 56.6 (C(14)); 56.1 (C(17)); 50.2 (C(9)); 45.9 (C(24)); 42.3 (C(13)); 39.8 (C(12)); 39.1 (C(4)); 37.3 (C(1)); 36.7 (C(10)); 36.2 (C(20)); 34.0 (C(22)); 32.0 (C(7)); 31.9 (C(8)); 30.0 (C(2)), 29.3 (C(25)); 28.3 (C(16)); 26.2 (C(23)); 24.3 (C(15)); 23.2 (C(28)); 21.1 (C(11)); 19.8 (C(27)); 19.2 (C(26)); 19.0 (C(19)); 18.8 (C(21)); 12.0 (C(29)); 11.8 (C(18)). Diosgenin 3-O-a-l-Rhamnopyranosyl-(1 ! 2)-b-d-glucopyronoside ( ¼ (3b,25R)-Spirost-5-en-3-yl 2O-(a-l-Rhamnopyranosyl)-b-d-glucopyranoside; 9). Light-brown, amorphous solid. M.p. 248 – 2508. [a] 20 D ¼ 60 (c ¼ 1, pyridine). UV (MeOH): 216, 285. IR (solid): 3354 (OH), 2929 (CH), 1050 (CO). 1 H-NMR ((D5 )pyridine): 6.42 (s, HC(1’)); 5.32 (d, J ¼ 4.8, HC(6)); 5.08 (d, J ¼ 7.3, HC(1’)); 3.35 (d, J ¼ 11.0, CH2(26)); 2.10 – 2.20 (m, CH2(15)); 1.78 (d, J ¼ 6.2, HC(6’)); 1.15 (d, J ¼ 6.9, Me(27)); 1.08 (d, J ¼ 7.1, Me(20)); 1.06 (s, Me(22)); 0.83 (s, Me(21)). 13C-NMR ((D5 )pyridine): 140.7 (C(5)); 121.5 (C(6)); 109.5 (C(19)); 101.9 (C(1’’)); 100.2 (C(1’)); 81.0 (C(16)); 79.5 (C(2’)); 78.1 (C(3)); 77.8 (C(3’)); 77.7 (C(5’)); 74.0 (C(4’’)); 72.7 (C(3’’)); 72.4 (C(2’’)); 71.7 (C(4’)); 69.3 (C(5’’)); 64.9 (C(26)); 62.5 (C(17)); 62.5 (C(6’)); 56.4 (C(14)); 50.1 (C(9)); 42.3 (C(18)); 40.2 (C(12)); 39.7 (C(13)); 38.8 (C(4)); 37.3 (C(10)); 36.9 (C(1)); 32.1 (C(7)); 32.0 (C(8)); 31.5 (C(23)); 30.0 (C(15)); 27.3 (C(25)); 26.2 (C(2)); 26.0 (C(24)); 20.9 (C(11)); 19.2 (C(22)); 18.5 (C(6’)); 16.1 (C(21)); 16.1 (C(20)); 14.6 (C(27)). Oxyresveratrol 3-O-b-d-Glucopyranoside ( ¼ 3-[(E)-2-(2,4-Dihydroxyphenyl)ethenyl]-5-hydroxyphenyl b-d-Glucopyranoside; 10). Dark-yellow, amorphous solid. [a] 20 D ¼ 27 (c ¼ 1, MeOH). UV (DMSO): 333. IR (CHCl3 ): 3411 (OH), 2923 and 2846 (CH), 1659 (C¼C), 1050 (CO). 1H-NMR (CD3OD): 7.36 (d, J ¼ 8.9, HC(6’)); 7.33 (d, J ¼ 16.4, HC(b)); 6.90 (d, J ¼ 16.4, HC(a)); 6.77 (t, J ¼ 1.6, HC(2)); 6.64 (t, J ¼ 1.6, HC(6)); 6.46 (t, J ¼ 2.1, HC(3’)); 6.30 – 6.35 (m, HC(4), HC(5’)); 3.95 (dd, J ¼ 12.0, 1.7, HaC(6’’)); 3.76 (dd, J ¼ 12.0, 4.9, HbC(6’’)); 3.40 – 3.55 (m, HC(4’’), HC(2’’), HC(5’’), HC(3’’). 13C-NMR (CD3OD): 159.0 (C(5)); 158.2 (C(3)); 157.9 (C(4’)); 156.0 (C(2’)); 140.9 (C(1)); 127.2 (C(6’)); 124.8 (C(a)); 124.0 (C(b)); 116.3 (C(1’)); 107.0 (C(5’)); 106.6 (C(6)); 105.7 (C(2)); 102.3 (C(3’)); 102.2 (C(4)); 101.0 (C(1’’)); 76.7 (C(3’’)); 76.6 (C(5’’)); 73.5 (C(2’’)); 69.9 (C(4’’)); 61.3 (C(6’’)). Jervine ( ¼ (3b,17b,22S,23R)-3-Hydroxy-9-methyl-17,23-epoxyveratraman-11-one; 11). Light-brown, amorphous solid. M.p. 246 – 2478. [a] 20 D ¼ 167 (c ¼ 1, CHCl3 ). UV (CHCl3 ): 251. IR (solid): 3297 (NH), 3300 (OH), 2800 – 2936 (CH), 1708 (C¼O), 1463 (C¼C), 1060 (CO). 1H-NMR (CDCl3 ): 5.35 (d, J ¼ 4.8, HC(6)); 3.44 – 3.55 (m, HC(3)); 3.29 (dt, J ¼ 10.3, 4.0, HC(23)); 3.06 (dd, J ¼ 12.4, 4.0,
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HaC(26)); 2.65 (t, J ¼ 9.2, HC(22)); 2.31 (t, J ¼ 12.5, HbC(26)); 2.15 (s, Me(18)); 0.98 (s, Me(19)); 0.94 (d, J ¼ 6.0, Me(21)); 0.93 (d, J ¼ 6.2, Me(27)). 13C-NMR (CDCl3 ): 207.0 (C(11)); 145.9 (C(12)); 142.6 (C(5)); 137.4 (C(13)); 121.1 (C(6)); 85.8 (C(17)); 76.6 (C(23)); 71.8 (C(3)); 66.8 (C(22)); 62.8 (C(9)); 54.8 (C(26)); 45.1 (C(20)); 41.7 (C(4)); 40.6 (C(14)); 39.1 (C(24)); 38.2 (C(8)); 37.3 (C(10)); 37.0 (C(1)); 31.7 (C(25)); 31.4 (C(16)); 31.2 (C(2)); 30.9 (C(7)); 24.6 (C(15)); 18.9 (C(27)); 18.7 (C(19)); 12.3 (C(18)); 10.9 (C(21)). Pseudojervine ( ¼ (3b,17b,22S,23R)-9-Methyl-11-oxo-17,23-epoxyveratraman-3-yl b-d-Glucopyranoside; 13). White amorphous solid. M.p. 291 – 2938. [a] 20 D ¼ 160 (c ¼ 1, pyridine). UV (MeOH-CHCl3 ): 357, 279, 277, 275. IR (solid): 3297 (NH), 2937 (CH), 1708 (C¼O), 1463 (C¼C), 1060 (CO, CN). 1 H-NMR ((D5 )pyridine): 5.28 (d, J ¼ 5.1, HC(6)); 5.07 (d, J ¼ 7.4, HC(1’)); 4.57 (dd, J ¼ 11.7, 2.3, HaC(6’)); 4.40 (dd, J ¼ 11.7, 5.5, HbC(6’)); 4.32 – 4.24 (m, HC(3), HC(5’)); 3.41 (dt, J ¼ 10.1, 3.1, HC(23)); 3.07 (dd, J ¼ 12.1, 3.9, HaC(26)); 2.41 (s, Me(18)); 2.31 (t, J ¼ 11.7, HbC(26)); 1.04 (d, J ¼ 7.4, Me(21)); 1.00 (s, Me(19)); 0.83 (d, J ¼ 6.6, Me(27)). 13C-NMR ((D5 )pyridine): 206.5 (C(11)); 146.2 (C(12)); 141.9 (C(5)); 136.9 (C(13)); 121.4 (C(6)); 102.3 (C(1’)); 85.2 (C(17)); 78.4 (C(5’)); 78.3 (C(3’)); 77.7 (C(4’)); 76.6 (C(23)); 75.1 (C(2’)); 71.5 (C(3)); 67.2 (C(22)); 62.6 (C(6’)); 62.4 (C(9)); 54.9 (C(26)); 44.6 (C(14)); 40.8 (C(20)); 39.2 (C(4)); 38.3 (C(24)); 38.2 (C(8)); 37.3 (C(10)); 37.0 (C(1)); 31.4 (C(25)); 30.9 (C(16)); 30.6 (C(2)); 29.5 (C(7)); 24.6 (C(15)); 18.8 (C(27)); 18.2 (C(19)); 12.2 (C(18)); 10.9 (C(21)). 5,6-Dihydro-1-hydroxyjervine ( ¼ (1b,17b,22S,23R)-1-Hydroxy-9-methyl-17,23-epoxyveratraman-11one; 14). Light-brown, amorphous solid. M.p. 239 – 2408. [a] 20 D ¼ þ 78 (c ¼ 1, CHCl3 ). IR (solid): 3300 (OH), 3297 (NH), 2857 – 2930 (CH), 1706 (C¼O), 1457 (C¼C), 1059 (CO), 754 (CN). 1H-NMR ((D5 )pyridine): 5.54 (br. s, HC(1)); 4.94 – 4.85 (m, CH2(3)); 3.65 (dt, J ¼ 10.1, 3.1, HC(23)); 3.26 (dd, J ¼ 12.4, 4.0, HaC(26)); 2.97 (t, J ¼ 9.3, HC(22)); 2.37 (d, J ¼ 2.0, Me(18)); 1.41 (s, Me(19)); 1.23 (d, J ¼ 7.4, Me(21)); 0.84 (d, J ¼ 6.6, Me(27)). 13C-NMR ((D5 )pyridine): 208.6 (C(11)); 146.7 (C(13)); 138.2 (C(12)); 87.2 (C(17)); 77.0 (C(23)); 72.3 (C(1)); 67.7 (C(22)); 66.9 (C(3)); 55.8 (C(9)); 55.3 (C(26)); 46.0 (C(14)); 43.8 (C(5)); 42.1 (C(10)); 41.7 (C(20)); 41.0 (C(2)); 40.1 (C(24)); 39.1 (C(4)); 37.9 (C(8)); 32.3 (C(6)); 31.8 (C(25)); 29.1 (C(16)); 26.5 (C(7)); 25.7 (C(15)); 20.0 (C(27)); 19.5 (C(19)); 13.6 (C(18)); 12.8 (C(21)). Saccharose ( ¼ b-d-Fructofuranosyl a-d-Glucopyranoside; 15). Brownish needles. M.p. 182 – 1838. [a] 20 D ¼ þ 66 (c ¼ 1, MeOH). IR (solid): 3324 (OH), 2800 – 2936 (CH), 1343 (C¼C), 1066, 1049 (CO). 1 H-NMR ((D6 )DMSO): 5.21 (br. s, HOC(4’)); 5.15 (d, J ¼ 3.5, HC(1)); 5.06 (br. s, HOC(2)); 4.84 – 4.76 (m, HOC(4)); 4.54 (d, J ¼ 8.5, HOC(3’)); 4.46 – 4.38 (m, HOC(5’), HOC(6’)); 3.86 (t, J ¼ 7.7, HC(3’); 3.78 – 3.72 (m, HC(4’)). 13C-NMR ((D6 )DMSO): 104.5 (C(2’)); 92.2 (C(1)); 82.9 (C(5’)); 77.5 (C(3’)); 74.7 (C(4’)); 73.3 (C(3)); 73.2 (C(5)); 72.1 (C(2)); 70.3 (C(4)); 62.6 (C(6’)); 62.5 (C(1’)); 60.9 (C(6)). Evaluation of Insecticidal Activities. Glass Petri dishes (9-cm-wide 1.5-cm-deep; corresponding to 120 ml volume) were used to test the toxicities of the acetone, NH4OH/benzene, and CHCl3 extracts of V. album, and of 2, 2 þ 4, 5, 7, 8, and 11 against adults of L. decemlineata. The solns. (10, 20, and 30 mg/ml) of each substance was prepared by suspending and/or dissolving in DMSO/dist. H2O 1 : 10. Randomly, ten potato beetle adults were placed in the Petri dishes without distinguishing between male and female. One ml of the prepared suspension, corresponding to 10, 20, and 40 mg/ml concentrations, was sprayed into each Petri dish to wet the insects using an atomizer. The adults were placed on filter paper, containing the appropriate amounts of potato leaves. Petri dishes were closed with adhesive tape and transferred into an incubator, and then kept under standard conditions at 25 18 and 64 5% relative humidity, and for 16 : 8 (light: dark) photoperiod for 4 d. Lambda cyhalothrin (10, 20, and 40 mg/ml concentrations) was used as a positive control under the same conditions. After treatments, the mortality of the adults was counted after 12, 24, 48, and 96 h, resp. Control treatments with DMSO/dist. H2O 1 : 10, and without the extracts and compounds were conducted in the same way. All experiments were run in triplicate. Statistical Analyses. For comparison of data, Variance Analysis method was applied, and differences between means were tested through LSD, and values of p < 0.05 were considered significantly different. In addition, durations for 50 and 90% mortality of the adults and LC50 values were determined in the first 12 h, in order to determine the toxic effects of substances used as insecticide by probit analysis.
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