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Identification and Antifeedant Activities of Limonoids from Azadirachta indica by Yu-Xin Yan a ), Jie-Qing Liu a ), Hong-Wei Wang a ), Jin-Xiong Chen a ), Jian-Chao Chen a ), Li Chen b ), Lin Zhou a ), and Ming-Hua Qiu* a ) a
) State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, P. R. China (phone: þ 86-871-65223327; fax: þ 86-871-65223255; e-mail: [email protected]) b ) Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, P. R. China
Four new limonoids, azadiraindins A – D (1 – 4, resp.), together with seven known analogs, were isolated from the MeOH extract of Azadirachta indica. The structures of 1 – 4 were elucidated by NMR and MS spectroscopic analyses, and the relative configuration of 1 was determined by single-crystal X-ray crystallography. The compounds isolated in comparatively large amount were evaluated for their antifeedant activities against Plutella xylostella; the antifeedant rate of 10 was 90.6% and the corrected mortality of 8 was 79.2%.
Introduction. – Azadirachta indica (neem tree), a plant of the Meliaceae family, is variously known as ÐSacred TreeÏ, ÐNatureÏs DrugstoreÏ, and ÐVillage PharmacyÏ. Previous phytochemical and pharmacological studies revealed that the main components isolated from this plant are limonoids [1] [2], which have attracted considerable interest because of their insecticidal and antifeeding activities [3 – 9]. Commercial products, such as Margosan-OÔ, AzitinÔ, and TurlexÔ, are applied as pesticides and insect repellents in many countries [10] [11]. To develop an environment-friendly biopesticide, A. indica has been introduced and also planted on a large scale since 2000 in Yunnan Province, P. R. China, where it has become a growing base of botanical pesticides. Due to our interest in insect control limonoids, we investigated fresh fruit of A. indica, which led to the isolation of four new limonoids, azadiraindins A – D (1 – 4, resp.), together with seven known analogs, desfuranoazadiradione (5) [12], (5a,7a,13a)-7-acetoxy-4,4,8-trimethyl-17-oxaandrosta-1,14-diene-3,16-dione (6) [12], (5a,7a)-7-acetoxy-4,4,8-trimethyl-17-oxaandrosta-1,14-diene-3,16-dione (7) [12], epoxyazadiradione (8) [13], 7-deacetyl-7-benzoylepoxyazadiradione (9) [13] [14], azadiradione (10) [15], 17b-hydroxyazadiradione (11) (Fig. 1) [16]. Herein, we describe the isolation and structural elucidation of the new limonoids and their antifeedant activities. Results and Discussion. – Structure Elucidation. Fresh fruit of A. indica were extracted with MeOH at room temperature, and after solvent evaporation the residue was fractionated by column chromatography on DIAION HP-20 using MeOH/H2O mixed-solvent system. Further purification by repeated normal-phase column chromatography and semipreparative HPLC yielded eleven limonoids, 1 – 11. Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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Fig. 1. Structures of 1 – 11
Compound 1 was obtained as colorless crystals (MeOH). Its molecular formula was established to be C26H34O7 by HR-ESI-MS (m/z 481.2207 ([M þ Na] þ ; calc. 481.2197)), which indicated ten degrees of unsaturation. The IR spectrum indicated the presence of OH (3488 cm ¢ 1) and three C¼O groups (1746, 1716, and 1649 cm ¢ 1). The 1H-NMR spectrum (Table 1) showed the presence of five Me groups (d(H) 1.53, 1.17, 1.08, 1.06, and 1.00), one Me group of AcO (2.14), and an AB system at 7.22 (d, J ¼ 10.2, 1 H) and 5.88 (d, J ¼ 10.2, 1 H). The 13C-NMR spectrum displayed 26 Catom signals which were classified by a DEPT experiment into six Me (d(C) 29.5, 27.4, 21.8, 21.4, 20.1, and 19.6), four CH2 (including an O-bearing one), and seven CH groups (including two olefinic ones), and nine Cq-atoms (including two ketones and one ester C¼O group). Comparison of the 1D- and 2D-NMR data with those of limonoids reported in the literature suggested that 1 had a skeleton similar to that of epoxyazadiradione (8) [13], with an 1-en-3-one system in ring A, an AcO group linked to C(7), and an epoxide ring bridging at C(14) and C(15). The difference between 1 and 8 was the absence of the furan ring signals in the case of 1. Since three C¼O groups, one C¼C bond, and five rings accounted for nine out of ten degrees of unsaturation, the remaining degree of unsaturation was assumed to be accounted for by another ring. In the HMBC spectrum of 1, correlations of H¢C(17) (d(H) 1.81) with C(16) (d(C) 112.2, Cq ), C(15) (62.2, CH), C(20) (208.2, Cq ), and C(21) (74.2, CH2 ), of H¢C(15) (d(H) 3.51) with C(17) (d(C) 60.4) and C(16), and of CH2(21) (d(H) 4.34 and 4.14) with C(16), C(17), and C(20), suggested the presence of a ring between C(16), C(17), C(20), and C(21). The chemical shift and HMBCs suggested C(16) to be a hemiketal C-atom. The structural assignments were confirmed
13 14 15 16 17 18 19 20 21 22 23 28 29 30 MeCO MeCO
12
7 8 9 10 11
1 2 3 4 5 6
Position
2.14 (s)
1.06 (s) 1.08 (s) 1.00 (s)
4.34, 4.14 (2d, J ¼ 16)
1.81 (s) 1.53 (s) 1.17 (s)
3.51 (s)
2.00 – 2.03 (m), 1.86 – 1.87 (m) 2.50 – 2.52 (m), 1.90 – 1.91 (m)
2.50 – 2.57 (m)
2.16 – 2.20 (m) 1.94 – 1.96 (m), 1.84 – 1.85 (m) 4.74 – 4.76 (m)
7.22 (d, J ¼ 10.2) 5.88 (d, J ¼ 10.2)
21.8 27.4 19.6 170.0 20.1
49.0 74.0 62.2 112.2 60.4 29.5 21.4 208.2 74.2
24.8
74.2 42.6 40.1 40.1 16.2
158.3 126.1 204.9 44.7 47.3 24.6
1.92 (s)
4.46, 4.31 (2dd, J ¼ 18.8, 4.0) 1.09 (s) 1.09 (s) 1.31 (s)
2.88 (s) 1.14 (s) 1.24 (s) 2.84 – 2.86 (m), 2.17 – 2.18 (m)
5.82 (s)
2.08 – 2.11 (m), 1.85 – 1.86 (m) 2.06 – 2.07 (m), 1.81 – 1.83 (m)
2.44 – 2.48 (m)
2.19 – 2.20 (m) 1.99 – 2.10 (m), 1.90 – 1.94 (m) 5.28 – 5.31 (m)
7.14 (d, J ¼ 10.2) 5.89 (d, J ¼ 10.2)
2 d( H)
d( H )
d(C )
1
208.6 68.6 21.2 26.9 26.4 169.5 20.9
46.5 193.7 122.7 206.3 58.5 25.3 19.0 33.8
30.2
73.9 44.7 37.9 39.9 15.7
156.7 125.8 204.0 44.0 46.1 23.4
d(C )
1.95 (s)
1.10 (s) 1.10 (s) 1.29 (s)
3.88 (s) 1.21 (s) 1.24 (s)
5.85 (s)
2.17 – 2.18 (m), 1.86 – 1.88 (m) 2.19 – 2.20 (m), 1.72 – 1.74 (m)
2.43 – 2.46 (m)
2.21 – 2.24 (m) 2.00 – 2.02 (m), 1.91 – 1.93 (m) 5.29 – 5.33 (m)
7.16 (d, J ¼ 10.2) 5.91 (d, J ¼ 10.2)
d( H )
3
Table 1. 1H- and 13C-NMR Data (in CDCl3 ) of 1 – 4. d in ppm, J in Hz.
21.0 26.7 25.3 169.6 20.6
47.6 192.0 120.4 206.5 84.7 23.2 18.6
29.0
73.5 39.6 37.8 44.1 15.2
157.0 125.5 204.2 43.7 45.7 23.1
d(C )
2.00 (s)
1.10 (s) 1.07 (s) 1.41 (s)
3.97 (s) 1.26 (s) 1.20 (s)
5.58 (s)
1.93 – 1.95 (m), 1.85 – 1.87 (m) 2.16 – 2.19 (m), 1.58 – 1.60 (m)
1.78 – 1.80 (m)
2.15 – 2.19 (m) 2.04 – 2.07 (m), 1.88 – 1.89 (m) 5.19 – 5.22 (m)
7.21 (d, J ¼ 10.2) 5.92 (d, J ¼ 10.2)
d( H )
4
21.2 27.4 22.7 170.1 21.2
48.6 186.9 120.2 206.7 84.6 24.3 19.5
37.7
73.0 44.0 46.3 40.0 17.3
157.3 126.2 204.3 44.8 45.3 22.9
d(C )
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Fig. 2. X-Ray crystal structure of 1
by X-ray structure analysis (Fig. 2), which established the relative configuration of 1. Thus, 1 was elucidated as shown in Fig. 1, and was named azadiraindin A. Compound 2 has the molecular formula C27H36O6 as determined by positive-ionmode HR-ESI-MS (m/z 479.2403 ([M þ Na] þ ; calc. 479.2404)). 1H- and 13C-NMR data of 2 (Table 1) were similar to those of azadiradione (10), the only difference was the absence of the furan ring signals in the case of 2. The HMBCs of H¢C(17) (d(H) 2.88) with C(13) (d(C) 46.5), C(16) (206.3), C(20) (33.8), and C(22) (208.6), of CH2(20) (d(H) 2.84 – 2.86 and 2.17 – 2.18) with C(17) (d(C) 58.5) and C(22), and of CH2(23) (d(H) 4.46 and 4.31) with C(22) suggested the presence of a C3 side chain attached to C(17). The side chain at C(17) in 2 was similar to that of the known compound 21,24,25,26,27-pentanor-7,23-dihydroxy-15,22-oxoapotirucalla(eupha)-1-en-3-one [17], and 2 was therefore identified as shown in Fig. 1 and named azadiraindin B. Compounds 3 and 4 were isolated as a pair of epimers, both having the molecular formula C24H32O5 according to their HR-ESI-MS. Their 13C-NMR signals (Table 1) were similar to those of desfuranoazadiradione (5), except that the CH2(17) group (d(C) 55.6) of 5 was oxygenated to a CH group in 3 and 4 (84.7 and 84.6, resp.). Compounds 3 and 4 have one more OH group (at C(17)) than 5, which was supported by HMBCs of H¢C(17) with C(13), C(15), and C(16), and of H¢C(15) with C(17) and C(16). The key ROESY correlations (Fig. 3) of 3 (Hb¢C(12) correlated with H¢C(17) (d(H) 3.88) and Me(30) (1.29), Ha¢C(12) correlated with Me(18) (1.21) and H¢C(9) (2.43 – 2.46)) and 4 (H¢C(17) (3.97) correlated with Me(18) (1.26) and Ha¢C(12) (1.58 – 1.60), Ha¢C(12) correlated with H¢C(9) (1.78 – 1.80)) indicated that HO¢C(17) was a-oriented in 3 and b-oriented in 4, respectively. Compounds 3 and 4 were determined to be azadiraindins C and D, respectively.
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Fig. 3. Key ROESY correlations of 3 and 4 Table 2. Antifeedant Activities for Selected Compounds a ) Compound
Antifeedant rate [%]
Corrected mortality
1 5 8 10 Azadirachtin
28.0 37.2 39.6 90.6 64.9
0.0 16.7 79.2 33.3 66.7
a ) All test compounds and azadirachtin (positive control) were tested at a concentration of 2000 mg ml ¢ 1. The antifeedant rate was calculated after 48 h, and the corrected mortality was calculated after 6 d.
Antifeedant Activity. Compounds 1, 5, 8, and 10 (purity > 90%) were tested for their antifeedant activities against the third-instar larvae of Plutella xylostella (Table 2). Compared with the positive control azadirachtin (purity > 97%, Yunnan Zhongke BioIndustry Limited Company), 8 and 10 showed a relatively potent effect against P. xylostella. The antifeedant rate of 10 was 90.6% (azadirachtin 64.9%), and the corrected mortality of 8 was 79.2% (azadirachtin 66.7%). Conclusions. – Limonoids are known as tetranortriterpenoids with an a-oriented furan ring at C(17). Up to now, more than 1000 limonoids have been isolated from the Meliaceae family [2]. These include four basic skeletons, ring intact, ring-seco, rearranged, and degraded. Compounds 5 – 7 are known degraded 17a-furan ring limonoids, 3 and 4 are possibly derived from 5 (one more OH group at C(17)). Compounds 1 and 2 are C24- and C25-limonoids. We presume that 1 and 2 are biogenetically generated before the formation of the furan ring. However, the specific biogenetic pathway is still unclear and needs further investigation. This project was supported by the National Special Program of Basic Research (SB2007FY400), the National Science and Technology Underpin Program (2007BAD32B01-03), the Knowledge Innovation Program of CAS (Grant No. KSCX2-YW-G-038, Qian-2011), as well as the Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2010-ZZ14).
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Experimental Part General. Column chromatography (CC): silica gel (SiO2 ; 200 – 300 mesh, Qingdao Marine Chemical, Inc.), LiChroprep RP-18 gel (40 – 63 mm, Merck), DIAION HP-20 (75 – 150 mm, Mitsubishi), and Sephadex LH-20 (Pharmacia). Semiprep. HPLC: Agilent 1100 apparatus. IR Spectra: Shimadzu IR-450 instrument; KBr pellets; ˜n in cm ¢ 1. 1H- and 13C-NMR spectra: Bruker AV-400 (400 and 100 MHz, resp.) or DRX-500 (500 and 125 MHz, resp.) instrument (Bruker, Zîrich, Switzerland); in CDCl3 ; d in ppm rel. to Me4Si as internal standard, J in Hz. ESI- and HR-ESI-MS: VG Autospec-3000 spectrometer; in m/z. Plant Material. A. indica fresh fruits were collected at Yuanmou, Yunnan Province, P. R. China, in June 2010. The sample was identified by Prof. Hua Peng of the Kunming Institute of Botany, and a voucher specimen (KIB 20100618) has been deposited with the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and Isolation. Fresh fruit of A. indica (11.0 kg) were extracted with MeOH (3 25 l, each for 2 d) at r.t. and concentrated in vacuo to give a crude extract (1060 g), which was partitioned between AcOEt and H2O. The AcOEt part (440 g) was subjected to CC (DIAION HP-20 (2000 g, 10 140 cm); MeOH/H2O 40 : 60, 60 : 40, 80 : 20, and 100 : 0) to afford four fractions, Frs. 1 – 4. Fr. 2 (160 g) was subjected to CC (RP-18 (500 g, 8 100 cm); MeOH/H2O 6 : 4 ! 9 : 1) to afford four subfractions, Frs. 2.1 – 2.4. Fr. 2.1 (13 g) was subjected to CC (RP-18 (200 g, 4 50 cm); MeOH/H2O 5 : 5 ! 6 : 4) to afford 5 (300 mg), and then purified by semiprep. HPLC (52% MeOH, flow rate 3 ml min ¢ 1) to yield 3 (20 mg), 4 (6 mg), 5 (56 mg), 6 (13 mg), and 7 (12 mg). Fr. 2.2 (36 g) was subjected to CC (RP-18 (200 g, 4 50 cm); MeOH/H2O 6 : 4 ! 7 : 3), and then further subjected to CC (Sephadex LH-20 (200 g, 2 200 cm); MeOH) to give 1 (110 mg) and 2 (18 mg). Fr. 2.3 (50 g) was subjected to CC (RP-18 (200 g, 4 50 cm); MeOH/H2O 65 : 35 ! 75 : 25), and then purified by repeated CC (SiO2 (500 g, 6 100 cm); CHCl3/MeOH 10 : 1 ! 9 : 1) to give 8 (12.8 g), 9 (6 mg), 10 (3.1 g), and 11 (8 mg). Azadiraindin A ( ¼ (5a,7a,13a,14b,15b,16b)-14,15 : 16,21-Diepoxy-16-hydroxy-4,4,8-trimethyl-3,20dioxopregn-1-en-7-yl Acetate; 1). Colorless needles (MeOH). [a] 24 D ¼ þ 17.5 (c ¼ 1.06, CHCl3 ). IR: 3531, 3488, 3239, 2977, 1746, 1716, 1649. 1H- and 13C-NMR: see Table 1. HR-ESI-MS: 481.2207 ([M þ Na] þ , C26H34NaO þ7 ; calc. 481.2197). Crystallographic Data for 1 1). Crystal system, triclinic; space group, P1; Z ¼ 1; a ¼ 6.7216(10), b ¼ 8.7403(13), c ¼ 10.3335(15) è; V ¼ 588.44(15) è3 ; Dx ¼ 1.345 g cm ¢ 3. The data were collected on a Bruker APEX DUO diffractometer, with graphite-monochromated MoKa radiation using a colorless crystal of dimensions 0.52 0.33 0.08 mm3, maximum 2q value of 55.348. The crystal structure of 1 was solved by direct methods with SHELXS-97 and expanded using difference Fourier techniques, refined by the program and method full-matrix, least-squares calculations. The final indices were R1 ¼ 0.0364, wR2 ¼ 0.0940. Azadiraindin B ( ¼ (5R,7R,8R,9R,10R,13S,17S)-4,5,6,7,8,9,10,11,12,13,16,17-Dodecahydro-17-(3hydroxy-2-oxopropyl)-4,4,8,10,13-pentamethyl-3,16-dioxo-3H-cyclopenta[a]phenanthren-7-yl Acetate; 1 2). Colorless needles (MeOH). [a] 24 D ¼ ¢ 42.7 (c ¼ 1.27, CHCl3 ). IR: 3455, 1736, 1719, 1660. H- and 13 þ þ C-NMR: see Table 1. ESI-MS: 479 ([M þ Na] ). HR-ESI-MS: 479.2403 ([M þ Na] , C27H36NaO þ6 ; calc. 479.2404). Azadiraindin C ( ¼ (5a,7a,13a,17a)-17-Hydroxy-4,4,8-trimethyl-3,16-dioxoandrosta-1,14-dien-7-yl Acetate; 3). Colorless needles (MeOH). 1H- and 13C-NMR: see Table 1. ESI-MS: 423 ([M þ Na] þ ). HR-ESI-MS: 423.2153 ([M þ Na] þ , C24H32NaO þ5 ; calc. 423.2142). Azadiraindin D ( ¼ (5a,7a,13a,17b)-17-Hydroxy-4,4,8-trimethyl-3,16-dioxoandrosta-1,14-dien-7-yl Acetate; 4). Colorless needles (MeOH). 1H- and 13C-NMR: see Table 1. ESI-MS: 423 ([M þ Na] þ ). HR-ESI-MS: 423.2145 ([M þ Na] þ , C24H32NaO þ5 ; calc. 423.2142). Bioassay. The antifeedant activities of test compounds were determined against the third instar larvae of P. xylostella by conventional leaf disk method as previously reported [18]. Briefly, the test compounds were dissolved in acetone at concentrations of 2000 mg ml ¢ 1. 2000 mg ml ¢ 1 of azadirachtin 1)
Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre (Deposition No. CCDC-922226). These data can be obtained free of charge via www.ccdc.cam.ac.uk.
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was used as standard control. Leaf disks of Brassica oleracea L. (1.1 cm diameter) were dipped in the test solutions, and the control disks were dipped in acetone for 1 s. All leaf disks were air-dried before being presented to the third instar larvae of P. xylostella. Three Petri dishes, each containing ten randomly selected larvae and five leaf disks, were used for each sample. After 48 h, the areas eaten were measured, and the antifeedant rate was calculated from [(C – T)/C] · 100%, where C and T are control disk areas eaten and treated disk areas eaten, resp. After 6 d, the mortality of the test insects was calculated. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
B. S. Siddiqui, Ghiasuddin, S. Faizi, M. Rasheed, J. Nat. Prod. 1999, 62, 1006. Q.-G. Tan, X.-D. Luo, Chem. Rev. (Washington, DC, U.S.A.) 2011, 111, 7437. E. S. Garcia, H. Rembold, J. Insect Physiol. 1984, 30, 939. S. Siddiqui, S. Faizi, T. Mahmood, B. S. Siddiqui, J. Chem. Soc., Perkin Trans. 1 1986, 1021. B. S. Siddiqui, F. Afshan, T. Gulzar, R. Sultana, S. N.-H. Naqvi, R. M. Tariq, Chem. Pharm. Bull. 2003, 51, 415. M. C. Carpinella, M. T. Defago, G. Valladares, S. M. Palacios, J. Agric. Food Chem. 2003, 51, 369. M. Defago, G. Valladares, E. Banchio, C. Carpinella, S. Palacios, Fitoterapia 2006, 77, 500. N. K. Neoliya, D. Singh, R. S. Sangwan, Curr. Sci. 2007, 92, 94. M. J. Gualtieri, N. Malafronte, A. Vassallo, A. Braca, R. Cotugno, M. Vasaturo, N. De Tommasi, F. Dal Piaz, J. Nat. Prod. 2014, 77, 596. G. Chianese, S. R. Yerbanga, L. Lucantoni, A. Habluetzel, N. Basilico, D. Taramelli, E. Fattorusso, O. Taglialatela-Scafati, J. Nat. Prod. 2010, 73, 1448. C.-X. He, W.-W. Wu, K.-S. Yin, Z.-X. Guo, Y.-J. Luo, Southwest China J. Agric. Sci. 2006, 19, 552. B. S. Siddiqui, Ghiasuddin, S. Faizi, S. Siddiqui, Phytochemistry 1992, 31, 4275. W. Kraus, R. Cramer, G. Sawitzki, Phytochemistry 1981, 20, 117. T. Akihisa, T. Noto, A. Takahashi, Y. Fujita, N. Banno, H. Tokuda, K. Koike, T. Suzuki, K. Yasukawa, Y. Kimura, J. Oleo Sci. 2009, 58, 581. W. Kraus, R. Cramer, Tetrahedron Lett. 1978, 19, 2395. S. Siddiqui, S. Fuchs, J. Lîcke, W. Voelter, Tetrahedron Lett. 1978, 19, 611. D. A. G. Cortez, J. B. Fernandes, P. C. Vieira, M. F. das G. F. da Silva, A. G. Ferreira, Phytochemistry 2000, 55, 711. S.-H. Qi, D.-G. Wu, L. Chen, Y.-B. Ma, X.-D. Luo, J. Agric. Food Chem. 2003, 51, 6949. Received April 29, 2014
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Identification and Antifeedant Activities of Limonoids from Azadirachta indica by Yu-Xin Yan a ), Jie-Qing Liu a ), Hong-Wei Wang a ), Jin-Xiong Chen a ), Jian-Chao Chen a ), Li Chen b ), Lin Zhou a ), and Ming-Hua Qiu* a ) a
) State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, P. R. China (phone: þ 86-871-65223327; fax: þ 86-871-65223255; e-mail: [email protected]) b ) Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, P. R. China
Four new limonoids, azadiraindins A – D (1 – 4, resp.), together with seven known analogs, were isolated from the MeOH extract of Azadirachta indica. The structures of 1 – 4 were elucidated by NMR and MS spectroscopic analyses, and the relative configuration of 1 was determined by single-crystal X-ray crystallography. The compounds isolated in comparatively large amount were evaluated for their antifeedant activities against Plutella xylostella; the antifeedant rate of 10 was 90.6% and the corrected mortality of 8 was 79.2%.
Introduction. – Azadirachta indica (neem tree), a plant of the Meliaceae family, is variously known as ÐSacred TreeÏ, ÐNatureÏs DrugstoreÏ, and ÐVillage PharmacyÏ. Previous phytochemical and pharmacological studies revealed that the main components isolated from this plant are limonoids [1] [2], which have attracted considerable interest because of their insecticidal and antifeeding activities [3 – 9]. Commercial products, such as Margosan-OÔ, AzitinÔ, and TurlexÔ, are applied as pesticides and insect repellents in many countries [10] [11]. To develop an environment-friendly biopesticide, A. indica has been introduced and also planted on a large scale since 2000 in Yunnan Province, P. R. China, where it has become a growing base of botanical pesticides. Due to our interest in insect control limonoids, we investigated fresh fruit of A. indica, which led to the isolation of four new limonoids, azadiraindins A – D (1 – 4, resp.), together with seven known analogs, desfuranoazadiradione (5) [12], (5a,7a,13a)-7-acetoxy-4,4,8-trimethyl-17-oxaandrosta-1,14-diene-3,16-dione (6) [12], (5a,7a)-7-acetoxy-4,4,8-trimethyl-17-oxaandrosta-1,14-diene-3,16-dione (7) [12], epoxyazadiradione (8) [13], 7-deacetyl-7-benzoylepoxyazadiradione (9) [13] [14], azadiradione (10) [15], 17b-hydroxyazadiradione (11) (Fig. 1) [16]. Herein, we describe the isolation and structural elucidation of the new limonoids and their antifeedant activities. Results and Discussion. – Structure Elucidation. Fresh fruit of A. indica were extracted with MeOH at room temperature, and after solvent evaporation the residue was fractionated by column chromatography on DIAION HP-20 using MeOH/H2O mixed-solvent system. Further purification by repeated normal-phase column chromatography and semipreparative HPLC yielded eleven limonoids, 1 – 11. Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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Fig. 1. Structures of 1 – 11
Compound 1 was obtained as colorless crystals (MeOH). Its molecular formula was established to be C26H34O7 by HR-ESI-MS (m/z 481.2207 ([M þ Na] þ ; calc. 481.2197)), which indicated ten degrees of unsaturation. The IR spectrum indicated the presence of OH (3488 cm ¢ 1) and three C¼O groups (1746, 1716, and 1649 cm ¢ 1). The 1H-NMR spectrum (Table 1) showed the presence of five Me groups (d(H) 1.53, 1.17, 1.08, 1.06, and 1.00), one Me group of AcO (2.14), and an AB system at 7.22 (d, J ¼ 10.2, 1 H) and 5.88 (d, J ¼ 10.2, 1 H). The 13C-NMR spectrum displayed 26 Catom signals which were classified by a DEPT experiment into six Me (d(C) 29.5, 27.4, 21.8, 21.4, 20.1, and 19.6), four CH2 (including an O-bearing one), and seven CH groups (including two olefinic ones), and nine Cq-atoms (including two ketones and one ester C¼O group). Comparison of the 1D- and 2D-NMR data with those of limonoids reported in the literature suggested that 1 had a skeleton similar to that of epoxyazadiradione (8) [13], with an 1-en-3-one system in ring A, an AcO group linked to C(7), and an epoxide ring bridging at C(14) and C(15). The difference between 1 and 8 was the absence of the furan ring signals in the case of 1. Since three C¼O groups, one C¼C bond, and five rings accounted for nine out of ten degrees of unsaturation, the remaining degree of unsaturation was assumed to be accounted for by another ring. In the HMBC spectrum of 1, correlations of H¢C(17) (d(H) 1.81) with C(16) (d(C) 112.2, Cq ), C(15) (62.2, CH), C(20) (208.2, Cq ), and C(21) (74.2, CH2 ), of H¢C(15) (d(H) 3.51) with C(17) (d(C) 60.4) and C(16), and of CH2(21) (d(H) 4.34 and 4.14) with C(16), C(17), and C(20), suggested the presence of a ring between C(16), C(17), C(20), and C(21). The chemical shift and HMBCs suggested C(16) to be a hemiketal C-atom. The structural assignments were confirmed
13 14 15 16 17 18 19 20 21 22 23 28 29 30 MeCO MeCO
12
7 8 9 10 11
1 2 3 4 5 6
Position
2.14 (s)
1.06 (s) 1.08 (s) 1.00 (s)
4.34, 4.14 (2d, J ¼ 16)
1.81 (s) 1.53 (s) 1.17 (s)
3.51 (s)
2.00 – 2.03 (m), 1.86 – 1.87 (m) 2.50 – 2.52 (m), 1.90 – 1.91 (m)
2.50 – 2.57 (m)
2.16 – 2.20 (m) 1.94 – 1.96 (m), 1.84 – 1.85 (m) 4.74 – 4.76 (m)
7.22 (d, J ¼ 10.2) 5.88 (d, J ¼ 10.2)
21.8 27.4 19.6 170.0 20.1
49.0 74.0 62.2 112.2 60.4 29.5 21.4 208.2 74.2
24.8
74.2 42.6 40.1 40.1 16.2
158.3 126.1 204.9 44.7 47.3 24.6
1.92 (s)
4.46, 4.31 (2dd, J ¼ 18.8, 4.0) 1.09 (s) 1.09 (s) 1.31 (s)
2.88 (s) 1.14 (s) 1.24 (s) 2.84 – 2.86 (m), 2.17 – 2.18 (m)
5.82 (s)
2.08 – 2.11 (m), 1.85 – 1.86 (m) 2.06 – 2.07 (m), 1.81 – 1.83 (m)
2.44 – 2.48 (m)
2.19 – 2.20 (m) 1.99 – 2.10 (m), 1.90 – 1.94 (m) 5.28 – 5.31 (m)
7.14 (d, J ¼ 10.2) 5.89 (d, J ¼ 10.2)
2 d( H)
d( H )
d(C )
1
208.6 68.6 21.2 26.9 26.4 169.5 20.9
46.5 193.7 122.7 206.3 58.5 25.3 19.0 33.8
30.2
73.9 44.7 37.9 39.9 15.7
156.7 125.8 204.0 44.0 46.1 23.4
d(C )
1.95 (s)
1.10 (s) 1.10 (s) 1.29 (s)
3.88 (s) 1.21 (s) 1.24 (s)
5.85 (s)
2.17 – 2.18 (m), 1.86 – 1.88 (m) 2.19 – 2.20 (m), 1.72 – 1.74 (m)
2.43 – 2.46 (m)
2.21 – 2.24 (m) 2.00 – 2.02 (m), 1.91 – 1.93 (m) 5.29 – 5.33 (m)
7.16 (d, J ¼ 10.2) 5.91 (d, J ¼ 10.2)
d( H )
3
Table 1. 1H- and 13C-NMR Data (in CDCl3 ) of 1 – 4. d in ppm, J in Hz.
21.0 26.7 25.3 169.6 20.6
47.6 192.0 120.4 206.5 84.7 23.2 18.6
29.0
73.5 39.6 37.8 44.1 15.2
157.0 125.5 204.2 43.7 45.7 23.1
d(C )
2.00 (s)
1.10 (s) 1.07 (s) 1.41 (s)
3.97 (s) 1.26 (s) 1.20 (s)
5.58 (s)
1.93 – 1.95 (m), 1.85 – 1.87 (m) 2.16 – 2.19 (m), 1.58 – 1.60 (m)
1.78 – 1.80 (m)
2.15 – 2.19 (m) 2.04 – 2.07 (m), 1.88 – 1.89 (m) 5.19 – 5.22 (m)
7.21 (d, J ¼ 10.2) 5.92 (d, J ¼ 10.2)
d( H )
4
21.2 27.4 22.7 170.1 21.2
48.6 186.9 120.2 206.7 84.6 24.3 19.5
37.7
73.0 44.0 46.3 40.0 17.3
157.3 126.2 204.3 44.8 45.3 22.9
d(C )
1042 CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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Fig. 2. X-Ray crystal structure of 1
by X-ray structure analysis (Fig. 2), which established the relative configuration of 1. Thus, 1 was elucidated as shown in Fig. 1, and was named azadiraindin A. Compound 2 has the molecular formula C27H36O6 as determined by positive-ionmode HR-ESI-MS (m/z 479.2403 ([M þ Na] þ ; calc. 479.2404)). 1H- and 13C-NMR data of 2 (Table 1) were similar to those of azadiradione (10), the only difference was the absence of the furan ring signals in the case of 2. The HMBCs of H¢C(17) (d(H) 2.88) with C(13) (d(C) 46.5), C(16) (206.3), C(20) (33.8), and C(22) (208.6), of CH2(20) (d(H) 2.84 – 2.86 and 2.17 – 2.18) with C(17) (d(C) 58.5) and C(22), and of CH2(23) (d(H) 4.46 and 4.31) with C(22) suggested the presence of a C3 side chain attached to C(17). The side chain at C(17) in 2 was similar to that of the known compound 21,24,25,26,27-pentanor-7,23-dihydroxy-15,22-oxoapotirucalla(eupha)-1-en-3-one [17], and 2 was therefore identified as shown in Fig. 1 and named azadiraindin B. Compounds 3 and 4 were isolated as a pair of epimers, both having the molecular formula C24H32O5 according to their HR-ESI-MS. Their 13C-NMR signals (Table 1) were similar to those of desfuranoazadiradione (5), except that the CH2(17) group (d(C) 55.6) of 5 was oxygenated to a CH group in 3 and 4 (84.7 and 84.6, resp.). Compounds 3 and 4 have one more OH group (at C(17)) than 5, which was supported by HMBCs of H¢C(17) with C(13), C(15), and C(16), and of H¢C(15) with C(17) and C(16). The key ROESY correlations (Fig. 3) of 3 (Hb¢C(12) correlated with H¢C(17) (d(H) 3.88) and Me(30) (1.29), Ha¢C(12) correlated with Me(18) (1.21) and H¢C(9) (2.43 – 2.46)) and 4 (H¢C(17) (3.97) correlated with Me(18) (1.26) and Ha¢C(12) (1.58 – 1.60), Ha¢C(12) correlated with H¢C(9) (1.78 – 1.80)) indicated that HO¢C(17) was a-oriented in 3 and b-oriented in 4, respectively. Compounds 3 and 4 were determined to be azadiraindins C and D, respectively.
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Fig. 3. Key ROESY correlations of 3 and 4 Table 2. Antifeedant Activities for Selected Compounds a ) Compound
Antifeedant rate [%]
Corrected mortality
1 5 8 10 Azadirachtin
28.0 37.2 39.6 90.6 64.9
0.0 16.7 79.2 33.3 66.7
a ) All test compounds and azadirachtin (positive control) were tested at a concentration of 2000 mg ml ¢ 1. The antifeedant rate was calculated after 48 h, and the corrected mortality was calculated after 6 d.
Antifeedant Activity. Compounds 1, 5, 8, and 10 (purity > 90%) were tested for their antifeedant activities against the third-instar larvae of Plutella xylostella (Table 2). Compared with the positive control azadirachtin (purity > 97%, Yunnan Zhongke BioIndustry Limited Company), 8 and 10 showed a relatively potent effect against P. xylostella. The antifeedant rate of 10 was 90.6% (azadirachtin 64.9%), and the corrected mortality of 8 was 79.2% (azadirachtin 66.7%). Conclusions. – Limonoids are known as tetranortriterpenoids with an a-oriented furan ring at C(17). Up to now, more than 1000 limonoids have been isolated from the Meliaceae family [2]. These include four basic skeletons, ring intact, ring-seco, rearranged, and degraded. Compounds 5 – 7 are known degraded 17a-furan ring limonoids, 3 and 4 are possibly derived from 5 (one more OH group at C(17)). Compounds 1 and 2 are C24- and C25-limonoids. We presume that 1 and 2 are biogenetically generated before the formation of the furan ring. However, the specific biogenetic pathway is still unclear and needs further investigation. This project was supported by the National Special Program of Basic Research (SB2007FY400), the National Science and Technology Underpin Program (2007BAD32B01-03), the Knowledge Innovation Program of CAS (Grant No. KSCX2-YW-G-038, Qian-2011), as well as the Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2010-ZZ14).
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Experimental Part General. Column chromatography (CC): silica gel (SiO2 ; 200 – 300 mesh, Qingdao Marine Chemical, Inc.), LiChroprep RP-18 gel (40 – 63 mm, Merck), DIAION HP-20 (75 – 150 mm, Mitsubishi), and Sephadex LH-20 (Pharmacia). Semiprep. HPLC: Agilent 1100 apparatus. IR Spectra: Shimadzu IR-450 instrument; KBr pellets; ˜n in cm ¢ 1. 1H- and 13C-NMR spectra: Bruker AV-400 (400 and 100 MHz, resp.) or DRX-500 (500 and 125 MHz, resp.) instrument (Bruker, Zîrich, Switzerland); in CDCl3 ; d in ppm rel. to Me4Si as internal standard, J in Hz. ESI- and HR-ESI-MS: VG Autospec-3000 spectrometer; in m/z. Plant Material. A. indica fresh fruits were collected at Yuanmou, Yunnan Province, P. R. China, in June 2010. The sample was identified by Prof. Hua Peng of the Kunming Institute of Botany, and a voucher specimen (KIB 20100618) has been deposited with the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and Isolation. Fresh fruit of A. indica (11.0 kg) were extracted with MeOH (3 25 l, each for 2 d) at r.t. and concentrated in vacuo to give a crude extract (1060 g), which was partitioned between AcOEt and H2O. The AcOEt part (440 g) was subjected to CC (DIAION HP-20 (2000 g, 10 140 cm); MeOH/H2O 40 : 60, 60 : 40, 80 : 20, and 100 : 0) to afford four fractions, Frs. 1 – 4. Fr. 2 (160 g) was subjected to CC (RP-18 (500 g, 8 100 cm); MeOH/H2O 6 : 4 ! 9 : 1) to afford four subfractions, Frs. 2.1 – 2.4. Fr. 2.1 (13 g) was subjected to CC (RP-18 (200 g, 4 50 cm); MeOH/H2O 5 : 5 ! 6 : 4) to afford 5 (300 mg), and then purified by semiprep. HPLC (52% MeOH, flow rate 3 ml min ¢ 1) to yield 3 (20 mg), 4 (6 mg), 5 (56 mg), 6 (13 mg), and 7 (12 mg). Fr. 2.2 (36 g) was subjected to CC (RP-18 (200 g, 4 50 cm); MeOH/H2O 6 : 4 ! 7 : 3), and then further subjected to CC (Sephadex LH-20 (200 g, 2 200 cm); MeOH) to give 1 (110 mg) and 2 (18 mg). Fr. 2.3 (50 g) was subjected to CC (RP-18 (200 g, 4 50 cm); MeOH/H2O 65 : 35 ! 75 : 25), and then purified by repeated CC (SiO2 (500 g, 6 100 cm); CHCl3/MeOH 10 : 1 ! 9 : 1) to give 8 (12.8 g), 9 (6 mg), 10 (3.1 g), and 11 (8 mg). Azadiraindin A ( ¼ (5a,7a,13a,14b,15b,16b)-14,15 : 16,21-Diepoxy-16-hydroxy-4,4,8-trimethyl-3,20dioxopregn-1-en-7-yl Acetate; 1). Colorless needles (MeOH). [a] 24 D ¼ þ 17.5 (c ¼ 1.06, CHCl3 ). IR: 3531, 3488, 3239, 2977, 1746, 1716, 1649. 1H- and 13C-NMR: see Table 1. HR-ESI-MS: 481.2207 ([M þ Na] þ , C26H34NaO þ7 ; calc. 481.2197). Crystallographic Data for 1 1). Crystal system, triclinic; space group, P1; Z ¼ 1; a ¼ 6.7216(10), b ¼ 8.7403(13), c ¼ 10.3335(15) è; V ¼ 588.44(15) è3 ; Dx ¼ 1.345 g cm ¢ 3. The data were collected on a Bruker APEX DUO diffractometer, with graphite-monochromated MoKa radiation using a colorless crystal of dimensions 0.52 0.33 0.08 mm3, maximum 2q value of 55.348. The crystal structure of 1 was solved by direct methods with SHELXS-97 and expanded using difference Fourier techniques, refined by the program and method full-matrix, least-squares calculations. The final indices were R1 ¼ 0.0364, wR2 ¼ 0.0940. Azadiraindin B ( ¼ (5R,7R,8R,9R,10R,13S,17S)-4,5,6,7,8,9,10,11,12,13,16,17-Dodecahydro-17-(3hydroxy-2-oxopropyl)-4,4,8,10,13-pentamethyl-3,16-dioxo-3H-cyclopenta[a]phenanthren-7-yl Acetate; 1 2). Colorless needles (MeOH). [a] 24 D ¼ ¢ 42.7 (c ¼ 1.27, CHCl3 ). IR: 3455, 1736, 1719, 1660. H- and 13 þ þ C-NMR: see Table 1. ESI-MS: 479 ([M þ Na] ). HR-ESI-MS: 479.2403 ([M þ Na] , C27H36NaO þ6 ; calc. 479.2404). Azadiraindin C ( ¼ (5a,7a,13a,17a)-17-Hydroxy-4,4,8-trimethyl-3,16-dioxoandrosta-1,14-dien-7-yl Acetate; 3). Colorless needles (MeOH). 1H- and 13C-NMR: see Table 1. ESI-MS: 423 ([M þ Na] þ ). HR-ESI-MS: 423.2153 ([M þ Na] þ , C24H32NaO þ5 ; calc. 423.2142). Azadiraindin D ( ¼ (5a,7a,13a,17b)-17-Hydroxy-4,4,8-trimethyl-3,16-dioxoandrosta-1,14-dien-7-yl Acetate; 4). Colorless needles (MeOH). 1H- and 13C-NMR: see Table 1. ESI-MS: 423 ([M þ Na] þ ). HR-ESI-MS: 423.2145 ([M þ Na] þ , C24H32NaO þ5 ; calc. 423.2142). Bioassay. The antifeedant activities of test compounds were determined against the third instar larvae of P. xylostella by conventional leaf disk method as previously reported [18]. Briefly, the test compounds were dissolved in acetone at concentrations of 2000 mg ml ¢ 1. 2000 mg ml ¢ 1 of azadirachtin 1)
Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre (Deposition No. CCDC-922226). These data can be obtained free of charge via www.ccdc.cam.ac.uk.
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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
was used as standard control. Leaf disks of Brassica oleracea L. (1.1 cm diameter) were dipped in the test solutions, and the control disks were dipped in acetone for 1 s. All leaf disks were air-dried before being presented to the third instar larvae of P. xylostella. Three Petri dishes, each containing ten randomly selected larvae and five leaf disks, were used for each sample. After 48 h, the areas eaten were measured, and the antifeedant rate was calculated from [(C – T)/C] · 100%, where C and T are control disk areas eaten and treated disk areas eaten, resp. After 6 d, the mortality of the test insects was calculated. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
B. S. Siddiqui, Ghiasuddin, S. Faizi, M. Rasheed, J. Nat. Prod. 1999, 62, 1006. Q.-G. Tan, X.-D. Luo, Chem. Rev. (Washington, DC, U.S.A.) 2011, 111, 7437. E. S. Garcia, H. Rembold, J. Insect Physiol. 1984, 30, 939. S. Siddiqui, S. Faizi, T. Mahmood, B. S. Siddiqui, J. Chem. Soc., Perkin Trans. 1 1986, 1021. B. S. Siddiqui, F. Afshan, T. Gulzar, R. Sultana, S. N.-H. Naqvi, R. M. Tariq, Chem. Pharm. Bull. 2003, 51, 415. M. C. Carpinella, M. T. Defago, G. Valladares, S. M. Palacios, J. Agric. Food Chem. 2003, 51, 369. M. Defago, G. Valladares, E. Banchio, C. Carpinella, S. Palacios, Fitoterapia 2006, 77, 500. N. K. Neoliya, D. Singh, R. S. Sangwan, Curr. Sci. 2007, 92, 94. M. J. Gualtieri, N. Malafronte, A. Vassallo, A. Braca, R. Cotugno, M. Vasaturo, N. De Tommasi, F. Dal Piaz, J. Nat. Prod. 2014, 77, 596. G. Chianese, S. R. Yerbanga, L. Lucantoni, A. Habluetzel, N. Basilico, D. Taramelli, E. Fattorusso, O. Taglialatela-Scafati, J. Nat. Prod. 2010, 73, 1448. C.-X. He, W.-W. Wu, K.-S. Yin, Z.-X. Guo, Y.-J. Luo, Southwest China J. Agric. Sci. 2006, 19, 552. B. S. Siddiqui, Ghiasuddin, S. Faizi, S. Siddiqui, Phytochemistry 1992, 31, 4275. W. Kraus, R. Cramer, G. Sawitzki, Phytochemistry 1981, 20, 117. T. Akihisa, T. Noto, A. Takahashi, Y. Fujita, N. Banno, H. Tokuda, K. Koike, T. Suzuki, K. Yasukawa, Y. Kimura, J. Oleo Sci. 2009, 58, 581. W. Kraus, R. Cramer, Tetrahedron Lett. 1978, 19, 2395. S. Siddiqui, S. Fuchs, J. Lîcke, W. Voelter, Tetrahedron Lett. 1978, 19, 611. D. A. G. Cortez, J. B. Fernandes, P. C. Vieira, M. F. das G. F. da Silva, A. G. Ferreira, Phytochemistry 2000, 55, 711. S.-H. Qi, D.-G. Wu, L. Chen, Y.-B. Ma, X.-D. Luo, J. Agric. Food Chem. 2003, 51, 6949. Received April 29, 2014
