doi:10.14720/aas.2014.104.1.3
COBISS: 1.01 Agris category code: H01
NEMATICIDAL ISOCHROMANE GLYCOSIDE FROM Kigelia pinnata LEAVES
Olubunmi ATOLANI 1 2, 3, Oluwatoyin Adenike FABIYI 4, Gabriel Ademola OLATUNJI 2,
Received April 02, 2014; accepted June 23, 2014. Delo je prispelo 02. aprila 2014, sprejeto 23. junija 2014.
Nematicidal isochromaneglycoside from Kigelia pinnata leaves
Synthetic nematicides such as oxamyl and carbofuran play significant roles in the management of plant-parasitic nematodes. However, their negative environmental impacts have it imperative to search for safer alternatives. As part of our contribution in the search for bio-nematicides, compounds from plant extract were screened for possible potent nematicidal agent. A new isochromane carboxylic acid glycoside, isolated from the leaves of Kigelia pinnata (Lam.) Benth (Bignoniaceae) was evaluated for its nematicidal activity. The structure of the proposed compound was characterized by various spectro-scopic methods, which included UV, FTIR, 1D-, and 2D-NMR, FAB-MS, TOF-ESI-MS and TOF-ESI-MS/MS (TANDEM). The in vitro experiment conducted on the glycoside against Meloidogyne incognita juveniles and eggs indicated an induced mortality. Its activity can be compared favourably with oxamyl, when tested at 0.1 mg/mL concentration. At four hours of observation, no significant difference (P < 0.05) between oxamyl and the glycoside was observed. The present data sustains that natural glycoside is a promising oxamyl alternate for controlling nematode-induced plant root knots and may contribute to integrated pest management.
Key words: plant protection / nematodes / Kigelia pinnata / isochromane glycoside / nematicides
1 INTRODUCTION
Plant parasitic nematodes are important contributors to crop loss globally. Over 90 species of the genus Meloidogyne have been described worldwide (Sikora and Fernadez, 2005). Precisely, the root knot nematode Meloidogyne incognita, has been reported to infect about 232
Nematocidni i-kromanski glikozid iz listov rasline Kigelia pin-nata
Sintetični nematocidi, kot na primer oxamyl in karbo-furan igrajo pomembno vlogo pri zatiranju nematod, ki pa-razitirajo na rastlinah, zaradi njihovega negativnega vpliva na okolje pa je nujno iskanje varnejših alternativ. Naš prispevek na področju raziskav bioloških nematocidov je poskus osamitve potencialnih nematocidnih substanc rastlinskega izvora. Nov glikozid, izoliran iz listov rastline Kigelia pinnata (Lam.) Benth (Bignoniaceae) smo proeizkusili na nematocidno aktivnost. Strukturo predlagane spojine smo opredelili z različnimi metodami, vključno z UV, FTIR, 1D in 2D-NMR, FAB-MS, TOF--ESI-MS in TOF_ESI_MS/MS (TANDEM). In vitro poskus z glikozidom na jajčecih in juvenilnih osebkih Meloidogyne incognita je nakazal povečano smrtnost. Njegova aktivnost je primerljiva z oxamylom v koncentraciji 0,1 mg/mL. Po štirih urah opazovanja nismo opazili nobenih značilnih razlik med glikozidom in oxamylom. Dosedanji podatki kažejo, da je naravni glikozid obetavna alternativa za oxamyl za kontrolo z nemato-dami povzročenih koreninskih vozlov in da lahko prispeva k naravnemu načinu zatiranja škodljivcev.
Ključne besede: varstvo rastlin / nematode / Kigelia pinnata / i-kromanski glikozid / nematocidi
genera of plant species (Swarup et al., 1989). The problem of root-knot nematode in Nigeria is well established. Severe growth reduction, wilting, unfilled spikelets and poor yield of crop are attributed to root-knot nematode (Babatola, 1984). Several other crops such as yam, maize, cassava and vegetables have also been affected by Meloidogyne spp. The use of synthetic nematicides in the
1	Redeemer's Univ., Dept. of Chemical Sciences, Mowe, Nigeria
2	Univ. of Ilorin, Dept. of Chemistry, Ilorin, Nigeria
3	Corresponding author: tolanvent@yahoo.com, atolanio@run.edu.ng, tel.: +2348034467136
4	Univ. of Ilorin, Dept. of Crop Protection, Ilorin, Nigeria
control of soil nematodes is a major concern because of the health and environmental hazards. The use of synthetic nematicides, such as oxamyl and carbofuran, could produces toxic metabolites in ground water and aquatic bodies. Oxamyl, which belongs to a group of pesticides called carbamates is reported to be rapidly absorbed and excreted in mammals where the highest concentration has been reported to be in blood, heart, liver, kidney, lungs, spleen and the gastro-intestinal tract (EFSA, 2005; USEPA, 2007). Several nematicides are phytotoxic and require a long time for degassing and decomposition. The increasing incidence of pesticide poisoning and mortality (Kottegoda, 1985) highlights the risks associated with their use. Natural products have been investigated as alternatives to synthetic nematicides (Abdel-Rahman et al., 2012). Kigelia pinnata (Lam.) Benth (Bignoniaceae) is a rich source of diverse phytochemicals including naphthoquinones and essential fatty acids (Atolani et al., 2011, 2012) with antioxidant and anticancer properties (Atolani et al, 2009, 2013; Olatunji and Atolani, 2009). We here describe the isolation of a new glycoside from Kigelia pinnata and its nematicidal activity.
2 MATERIAL AND METHODS
2.1 GENERAL EXPERIMENTAL PROCEDURES
UV Spectra were recorded on a Hitachi U-3200 spectrophotometer. FTIR Spectra (KBr) were measured on a JASCO-320-A spectrophotometer. Bruker AMX 500 instrument (300 MHz for 1H and 75 MHz for 13C) was used to acquire (1D and 2D) NMR data. FAB-MS was recorded on MAT 312 mass spectrometer using glycerol-water (1:1) in the presence of KI as matrix, while EI-MS spectral data was obtained on a Finnigan MAT 312 double-focusing mass spectrometer. ESI-MS and ESI-MS/ MS data were obtained on QqTOFMS/MS instrument (QSTAR XL mass spectrometer Applied Biosystem/ MDS Sciex, Darmstadt, Germany) at room temperature. Samples were dissolved in appropriate polar solvent, and working dilutions were prepared in acetonitrile-water containing 0.1 % trifluoroacetic acid. Analysis was carried out by electrospray ionization (ESI) and collision-induced dissociation (CID) MS, positive ion mode on the instrument at room temperature. MS/MS Experiment was conducted by selecting the product ion. TLC Analyses were carried out on silica gel 60 F254 precoated plates (Merck, Darmstadt); detection by ceric sulphate/H2SO4. Silica gel 60 (0.063D 0.200 mm; Merck, Darmstadt, Germany) was used for open column chromatography (CC).
2.2 PLANT MATERIAL
Kigelia pinnata (Lam) Benth. leaves were collected from Ado Ekiti, in Ekiti State, Nigeria, and identified by Mr Ajayi, a plant taxonomist at the Herbarium of the Department of Plant Biology, University of Ilorin, Ilorin, Nigeria. A voucher specimen (UIH 958) was previously deposited at the Herbarium.
2.3 EXTRACTION AND ISOLATION
The air-dried and powdered leaves (205 g) were sequentially extracted with adequate volume of hexane, dichloromethane, ethyl acetate, methanol and water at room temperature for five days each. The aqueous extract was concentrated and partitioned with butanol. The resulting aqueous fraction was filtered and concentrated under reduced pressure to obtain a crude extract, which was further subjected to a silica gel column chromatography (CC). The column was eluted with the increasing polarity of the mixture of DCM, ethyl acetate, methanol and water. Forty-seven fractions were obtained and pooled to a group of eight (fractions A to H), based on their TLC profile. Further purification of fraction C (CC, MeOH) afforded a yellow syrup, a glycoside tagged "tola-side" with Rf 0.5 (DCM : MeOH, 1:1)
Tolaside (1): yellow syrup (95 mg) MF: C16H20O14; MW: 436.09; FAB-MS m/z 459 [M+Na]+; EI-MS m/z 256 [M+ - 180]; TOF-ESI-MS m/z 437 [M + H]+; 1D and 2D NMR ppm (Results and discussion); UV Amax (MeOH, nm): 208, 275. IR v (KBr, cm-1) 3383, 2927,7598, 1403,
'	max v	'
1051, 718, 669. 1H-NMR: 500 MHz, CDCl3, 8 2.90 (s, 1H, OH, H-2), 2.91 (s, 1H, OH, H-5), 3.0 - 3.18 (m, 2H, CH H-3, H-4'), 3.31 - 3.43 (m, 3H, CH, H-2', H-6'), 3.55 -3.79 (m, 4H, OH, H-2, 5, 2', 3' 4' 6'), 3.98 (d, 2H, CH, H-1, H-1'), 4.26 (d, 1H, CH, H-2), 4.43 (sss, 3H, ArOH, H-6, 8, 9), 4.88 (s, 1H, CH, H-5), 8.39 (s, 1H, OH, H-10). 13C-NMR: (125 MHz, CDCl3): 8 173.1 (C-10), 104.0 (C-6), 97.0 (C-8), 92.4 (C-3), 89.3 (C-9), 76.9 (C-4), 76.7 (C-1), 75.3 (C-1'), 74.0 (C-7), 73.5 (C-5), 72.9 (C-5'), 72.8 (C-3'), 72.4 (C-2'), 71.9 (C-2), 56.52 (C-4'), 52.6 (C-6').
2.4 NEMATICIDAL ASSAY
Meloidogyne incognita 'race one' eggs were extracted from Solanum melongena L. roots by agitating in 0.06 % sodium hypochlorite solution for three minutes. The eggs were collected and rinsed with tap water on nested mesh 75 (m and 25 (m. Root particles were retained in 75 (m sieve, while the eggs were collected in the 25 (m sieve. The eggs were later standardized to 250 eggs per mL. The
O
(1)
extracted eggs were incubated at room temperature for twenty-four hours (24 hrs) to hatch out the second stage juveniles. The juveniles were poured into a 500 mL beaker for standardization and 1 mL was standardized to contain 250 juveniles. The experimental design was a 3 by 4 by 3 factorial experiment in a completely randomised design (CRD) involving three treatments at four levels and each replicated three times. 4 mg of the isolate was dissolved in 40 mL distilled water which is equivalent to 0.1 mg per mL concentration. 250 eggs and juveniles were used for each of the assay. Different doses of treatments were prepared by diluting the stock solution as follows: 6 mL fraction with 2 mL water to afford 75 %; 4 mL fraction with 4 mL water to afford 50 % and 2 mL fraction with 6 mL water affording 25 %, while distilled water served as control (Fabiyi et al., 2012). Data obtained were subjected to analysis of variance. Significant means were separated using Duncan's multiple range test at 5 % level of probability (P < 0.05).
3 RESULTS AND DISCUSSION
The glycoside, tolaside (1) was obtained as yellow syrup (95 mg). The molecular formula was determined as C16H20O14 by using the combination of various mass spectral data. FAB-MS showed m/z 459 [M + 23]+, calculated for C,,H.nO, .Na, m/z 258 calculated for C,nH,nOa
16 2U 14	1U 1U 8
(aglycone) and m/z 180 for the sugar unit; EI-MS showed m/z 256 [M+. - 180 (one glucose unit)]+, 236 (1), 185 (4), 157 (4), 103 (8), 97 (8), 85 (7), 73 (100), 60 (49), 57 (20), 44 (45); TOF-ESI-MS showed m/z 437 [M + H]+ calculated for C16H21O14. In order to obtain structural feature of the compound, TOF-ESI-MS/MS (TANDEM) for m/z 437 [M+H]+ was carried out. MS/MS indicated major peaks at m/z 359, calculated for C16H23O9 (indicating loss of five hydroxyl groups on the aglycone). Based on the spectroscopic data obtained, the structure 1 was proposed for the new isochromane glycoside, tolaside.
The UV spectrum exhibited maxima at 207 and 275 nm. The FTIR spectrum showed absorption bands
"Oh j Oh Oh
i/z = 437 [M+H]+
C10H10O7 m/z = 243 [M+H]+ m/z = 265 [M+Na]+
----C16H22O9 m/z = 359 [M+H]+
C16H22O8 m/z = 343 [M+H-OH]+
Figure 1: Proposed ESI-MS-MS fragmentation pattern for tolaside (1) Slika 1: Predlagani ESI-MS-MS fragmentacijski vzorec za tolazid (1)
for hydroxy! at 3383 cm-1, C-H stretching of aliphatic at 2927 cm-1, aromatic C=C stretching at 1599 cm-1, characteristic O-H bonding of the carboxylic at 1402 cm-1, C-O stretching of alcohol at 1051 cm-1 (broad) and aromatic CH bending at 718 cm-1. 1H-NMR spectrum displayed three hydroxyl protons on the aromatic ring at 8H 4.43, and the carboxylic proton at 8 8.39. The methine protons germinal to -OH and ether in ring A appeared at 8H 3.55-3.79. Methine protons of the glycone appeared between 8H 3.0-3.18 and 3.31-3.43, while hydroxyl protons of the glycone moiety resonated between 8H 3.55-3.79. Three me-thine proton signals of the aglycone appeared at 8h 3.98, 4.26, and 4.88. The 13C NMR spectrum displayed 16 signals of which 10 were ascribed to the aglycone. The carbonyl appeared at 8C 173.07. DEPT 90° and 135° indicated corresponding eight signals representing eight methine carbons, while one signal at 8C 61.4 for one methylene of the sugar. HMBC experiment also indicated the long range heteronu-clear coupling.
3.1 EI-MS AND TANDEM-MS FRAGMENTATIONS
The ejection of a single electron from the O-linkage oxygen of the glycoside induced the production of the molecular ion [M+]. The M+ was unstable due to its high polarity as a result of the multiple hydroxyl ends. Cleavage of the C-O bond of the ether-linkage followed by double deprotonation, induced the formation of the cation, m/z 256. This fragment further undergoes multiple cleavages to produce the ethyl ethyl sec-buthyl ether ion at m/z 102. The a C-C bond of the fragment cleaved to eliminate (-CH3CH2) ethyl radical yielding an intermediate cation, which rearranges to the ether cation (CH3CHOCH2CH3+) at m/z 73 as the base peak. This confirms the presence of oxygen-containing fragment cation. The previous intermediate oxygen containing cation at m/z 102 decomposes with hydrogen migration and elimination of ethene to form an eno-late, which rearranges to produce an alcohol cation with m/z 45 (Scheme 1).
The ESI-MS spectrum of the compound 1 showed the [M+H]+ at m/z 437, with other major ions at m/z 386, 341, 308, 292, 252, 199,
OH OH HO. ^ JL O
O
O
OH OH	| ho
HO	-O'"
^h^OH^OH
O
m/z =180
m/z =102
O
- ch3ch2'
-H2C=CH2
H
Scheme 1: Proposed EI-MS fragmentation pattern for tolaside 1 Shema 1: Predlagani EI-MS fragmentacijski vzorec za tolazid
+
Table 1: Effect of tolaside (1), oxamyl and Kigelia pinnata crude extract on Meloidogyne incognita juveniles Preglednica 1: Učinek tolazida (1), oxamyla in ekstrakta Kigelie pinnate na juvenilne osebke Meloidogyne incognita
Exposure	time of juveniles to various agents					
30 min	1 hr	4 hrs	8 hrs	Day 1	Day 2	Day 3
Treatments						
Tolaside (1) 29.43a	38.08a	52.71a	74.18a	100.00a	100.0a	100.00a
Crude 10.00c	19.19b	32.74b	40.29b	51.10b	60.23b	72.00b
OXML 20.22b	37.62a	53.24a	73.62a	100.00a	100.0a	100.00a
S.E.M. 0.91	1.12	1.23	1.42	1.59	1.76	2.06
Treatment level, %						
0 0.00d	0.00d	0.00d	0.00d	0.97d	1.26d	2.63b
25 7.37c	12.61c	22.11c	41.39c	61.22c	83.29c	100.00a
50 11.09b	18.11b	37.33b	54.08b	70.01b	90.07b	100.00a
75 16.33a	23.00a	42.00a	60.23a	78.26a	96.18a	100.00a
S.E.M. 0.24	0.41	0.56	0.70	0.81	1.12	1.36
Crude = Kigelia pinnata crude methanolic extract / surovi metanolni ekstrakt Kigelie pinnate; Oxml = Oxamyl; S.E.M. = Standard error of mean / standardna napaka sredine;
Values with different alphabets along the same column are statistically different at P < 0.05 / Vrednosti, označene z različnimi črkami v isti koloni so statistično različne (p < 0,05)
157, 129, 118 and 101. The full scan of the MS-MS spectrum at m/z 437 [M+H]+ produced daughter ions at m/z 377, 359, 343, 283, 265, 189 and 171. The loss of five -OH on the aglycone produced the ion m/z 359, while further loss of another -OH produced ion at the m/z 343. The dehydroxylation of carboxylic hydroxyl on the aglycone
fragment yielded the ion m/z 243 [M+H]+ and the corresponding fragment at m/z 265 [M+Na]+ (Fig. 1).
A comparison of the EI and ESI mass spectra indicated that ESI-MS2 produced [M+H]+ as the main peak, while the M+ produced in the EI quickly decomposed losing H-, CH3-, R'-O-R- and OH The FAB-MS was also indicative of the presence of the glycone and aglycone, as
Table 2: Effect of tolaside, oxamyl and Kigelia pinnata crude extract on Meloidogyne incognita eggs Preglednica 2: Učinek tolazida (1), oxamyla in ekstrakta Kigelie pinnate na jajčeca Meloidogyne incognita
Exposure time of eggs to treatment
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Treatments Tolaside (1) Crude OXML S.E.M. Treatment level, % 0
25 50 75
S.E.M.
0.00a 0.00a 0.00a 0.00
13.33b 0.00a 0.00a 0.00a 0.00
0.00a 0.00a 0.00a 0.00
19.30b 0.00a 0.00a 0.00a 0.00
0.00a 0.03a 0.00a 0.00
26.15b
0.00a
0.00a
0.00a
0.10
0.00a 0.07a 0.00a 0.03
39.02b
0.03a
0.00a
0.00a
0.15
0.00a 0.12b 0.00a 0.05
47.28b
0.09a
0.00a
0.00a
0.18
0.00a 0.19b 0.00a 0.09
56.22c 0.11b 0.00a 0.00a 0.26
Crude = Kigelia pinnata crude methanolic extract / surovi metanolni ekstrakt Kigelie pinnate; Oxml = Oxamyl; S.E.M. = Standard error of mean / standardna napaka sredine;
Values with different alphabets along the same column are statistically different at P < 0.05 / Vrednosti, označene z različnimi črkami v isti koloni so statistično različne (p < 0,05)
well as the identification of pseudo molecular ion [M+] at m/z 459 [M+Na]+ of the glycoside.
3.2 NEMATICIDAL ACTIVITY OF TOLASIDE (1)
The effect of oxamyl, tolaside and Kigelia pinnata crude extract on Meloidogyne incognita juveniles is presented in Table 1. Tolaside (1) was significantly (P < 0.05) more active than the oxamyl at 30 minutes of exposure with a percentage mortality of 29.43 %, while oxamyl showed 20.22 % mortality. From one hour to the end of the observation on day three, there was however no significant difference in the action of the glycoside and oxamyl on Meloidogyne incognita juveniles. All the levels of concentration induced mortality, but the 75 % concentration was significantly (P < 0.05) more effective with a 60.23 % mortality at 8 hours of observation. The glycoside and oxamyl are both effective inhibitors of egg hatch as there was no record of hatching in the two treatments throughout the period of study (Table 2). The crude extract of Kigelia pinnata showed only a few hatches. The lowest concentration (25 %) allowed some hatching, while the higher doses (50 and 75 %) completely inhibited the hatching of eggs.
Previous studies revealed that natural glycosides have nematicidal activity. Green manures of Sudangrass (Sorghum sudanense, Poaceae) are being examined as nematode suppressants. It contains the active glycoside, dhurrin (Widmer and Abawi, 2000). Natural glycosides; asparanin I and asparanin B, from Asparagus adescend-ens seed, as well as two triterpenoid glycosides albichinin II from Albizia chinensis and sonunin III from Acacia concinna have been found to be active at doses as low as 200 (g/mL against M. incognita nematodes (Meher et al., 1988). A mixture of two furastanol glycosides, proto-dioscin and deltoside from Dioscorea deltoidea, (Diosco-reaceae) inhibited M. incognita motility at 5000 ( g/mL but decreased nematode infection of tomato roots at much lower doses through a host-mediated effect (Zinovieva et al., 1997). Other natural glycoside with nematicidal properties included 3-^-[0-^-D-glucopyranosyl-(1^3)-0-[a-L-arabinofuranosyl-(1^2)]-0-^-D-arabinopyranosyl) oxy] jujubogenin active against C. elegans (Renukappa et al., 1999) and flavone glycosides linaroside and lantano-side from L. camara, which were lethal to M. incognita juveniles at 1.0 % (Begum et al., 2000).
4 CONCLUSION
The mass spectrometry analyses with the 1D, 2D NMR and FTIR spectroscopic analyses has unequivocal-
ly enabled, the establishment of the proposed structure of newly isolated isochromane carboxylic glycoside. The nematicidal activity of the glycoside 1 can be compared favourably with that of oxamyl, a standard nematicidal agent. This result therefore indicates that natural glyco-sides holds promise as potential nematicides and can successfully replace the toxic synthetic counterpart.
5 ACKNOWLEDGEMENT
A.O. is grateful to the Third World Academy of Sciences for the 2012 award of fellowship to visit the International Centre for Chemical and Biological Sciences, Karachi, Pakistan, where part of this work was carried out.
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