Nematicidal Activity of 3,4-Dihydroxybenzoic Acid Purified From Terminalia Nigrovenulosa Bark Against Meloidogyne
Nematicidal Activity of 3,4-Dihydroxybenzoic Acid Purified From Terminalia Nigrovenulosa Bark Against Meloidogyne
Nematicidal Activity of 3,4-Dihydroxybenzoic Acid Purified From Terminalia Nigrovenulosa Bark Against Meloidogyne
Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath
a r t i c l e i n f o a b s t r a c t
Article history: In this study, the 3,4-dihydroxybenzoic acid (3,4-DHBA) from Terminalia nigrovenulosa bark (TNB) was
Received 21 January 2013 purified and its in vitro nematicidal activity was investigated against Meloidogyne incognita. The purifi-
Received in revised form cation of 3,4-DHBA used a silica gel column and Sephadex LH-20 chromatography combined with thin-
5 April 2013
layer chromatography and high performance liquid chromatography. Structural identification of the 3,4-
Accepted 7 April 2013
Available online 18 April 2013
DHBA was conducted using 1H nuclear magnetic resonance (NMR), 13C NMR, and liquid chromatography
time-of-flight mass spectrometry. Nematicidal activity bioassays revealed that 3,4-DHBA treatment
resulted in 33.3, 47.5, 72.5 and 94.2% J2 mortality at 0.125, 0.25, 0.5 and 1.0 mg/ml, respectively after 12 h
Keywords:
Egg morphology
incubation. J2 mortality was increased significantly (P < 0.0001) with increasing incubation time in the
Fluorescence staining range of 54.2e94.2% from 3 to 9 h after incubation with 3,4-DHBA (1.0 mg/ml), but with no significant
Nematicidal compound difference observed where the incubation time was increased from 9 to 12 h. The 3,4-DHBA treatment
Plant extract resulted in 33.3, 65.0, 76.7 and 85.0% hatch inhibition at 0.125, 0.25, 0.5 and 1.0 mg/ml, respectively, 3
days after incubation. Changes in the shape of the eggs were determined after incubation for 1 day with a
3,4-DHBA concentration of 1.0 mg/ml.
Ó 2013 The authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license.
0882-4010 Ó 2013 The authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license.
http://dx.doi.org/10.1016/j.micpath.2013.04.005
D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59 53
often act at multiple and novel target sites, thereby reducing the
potential of plant-parasitic nematodes becoming resistant to them
[22,23]. The nematicidal principles of plants, based on materials
such as isothiocyanates, thiophenics, glucosides, alkaloids, pheno-
lics, thianins and fatty acids, have been identified and reviewed
[9,13,24,25].
Plants in the genus Terminalia, family Combretaceae, comprising of
some 250 species, are widely distributed in tropical areas of the world
[26]. A methanol extract of Terminalia arjuna bark showed nematicidal
activity against Haemonchus contortus [27]. Also, ethanol extract of
Terminalia chebula fruit was toxic to both Meloidogyne spp. and
Cephalobus litoralis [28]. 3,4-dihydroxybenzoic acid (3,4-DHBA, pro-
tocatechuic acid), a phenolic compound, has been isolated from
Camellia sinensis [29], Hibiscus sabdariffa L. [30], Eucommia ulmoides
Oliv. [31] and Smilacis chinae [32]. Currently there are very few reports
regarding the nematicidal activity effects of 3,4-DHBA from Terminalia
nigrovenulosa bark (TNB) against root-knot nematode M. incognita. The
objectives of this study were 1) to purify the nematicidal compounds
from TNB, and 2) to evaluate the in vitro nematicidal activity of 3,4-
DHBA purified from TNB against M. incognita.
M. incognita was collected from the roots of infected cucumber Elution was carried out using 200 ml of chloroform: methanol at
plants. Briefly, the galled root samples were washed with water, cut varying ratios (100:0 (F1), 90:10 (F2), 80:20 (F3), 70:30 (F4), 60:40
into pieces (4e8 mm long) and poured into a conical flask con- (F5) and 50:50 (F6)). Nematicidal activity assays were conducted
taining a mixture of distilled water (950 ml) and 10% NaOCl using all six fractions. The fraction that showed the high nemati-
(50 ml). The flask was then shaken vigorously to dislodge the eggs cidal activity was further separated using chloroform: ethyl acetate
from the roots. Next, the egg suspension was passed through a at varying ratios (90:10 (F4-1), 70:30 (F4-2) and 50:50 (F4-3)). The
series of sieves with pore sizes of 45 and 25 mm, respectively. fraction with the strongest nematicidal activity was further purified
Sterilized eggs were retained on a 25 mm sieve, refrigerated over- by column (65 cm 1.8 cm) chromatography on Sephadex LH-
night at 4 C in sterile distilled water, and were later used for 24 h 20 (stepwise gradient of 50e100% methanol) to yield 28 fractions
assays [33]. Sterilized eggs were incubated for 3e5 days using a (1e28). The solvent-fractionized compounds were confirmed
modified Baermann funnel method under sterile conditions, to by thin-layer chromatography (TLC) on silica gel plates F254
obtain J2 [34,35]. Next, the number of nematode juveniles was (20 cm 20 cm) [37] with chloroform: ethyl acetate: formic acid
adjusted to 250 J2/ml and the number of J2 was counted under a (70:30:0.5, v/v/v), and the compound bands were observed under
microscope (40). ultraviolet light at 254 nm (Vilber Lourmat).
Chemical shifts are reported in ppm (d), using CD3OD as the solvent DHBA with 1.0 ml of nematode egg solution (approximately
(unless otherwise indicated) and trimethylsilane as the internal 250 eggs/ml) was incubated at 37 C with shaking. After 1 day,
standard [32]. nematode eggs were stained with Fluorescence Brightener 28
(Sigma) at a final concentration of 0.1 mg/ml for 5 min. The eggs
2.7. Liquid chromatography time-of-flight mass spectrometry were washed twice with distilled water by centrifugation and then
(LC-TOF-MS) observed under an Olympus Fluoview FV1000 confocal laser
scanning microscope with a 455-nm argon laser and appropriate
LC-TOF-MS analysis was performed as described earlier [39], filter sets. Images were taken with confocal fluorescent microscopy
with minor modifications. Chromatographic separation was per- using Program FV10-ASW.
formed on an Agilent 1100 trap mass spectrometer equipped with
an electrospray (ESI) interfere, a binary pump, column oven, and a 2.10. Statistical analyses
diode array detector (Agilent Technology, Santa Clara, CA, USA).
Chromatographic separation of the analyte of interest was achieved Data were compared using Tukey’s Studentized range (HSD)
on a C18 column (particle size 5 mm, 250 4.6 mm, Agilent Tech- test, with a P 0.05 indicating statistical significance. All data were
nology). The mobile phase was 5.0% acetonitrile/water at a flow rate analyzed using Statistical Analysis System 9.1 [42] and are pre-
of 1.0 ml/min. The post-column splitting ratio was set to 3:1 before sented as mean standard deviation.
the elution was transferred to ESI. Injection volume was 20 ml for all
analyses. TOF-MS was performed in negative mode using multiple 3. Results
reactions monitoring (MRM). The optimum conditions of the
interface were as follows: ion spray voltage of 24,500 V, collision 3.1. Purification of 3,4-DHBA from TNB
gas (He) pressure of 1.3 m Torr and 40 psi, and flow rates of the
nebulizer gas (N2) and dry gas (N2) of 40 psi and 8.0 l/min, Of the five partitioned fractions tested, the ethyl acetate frac-
respectively. The dry temperature was set to 350 C. Analysis was tion showed the highest nematicidal activity. Silica gel column
detected by trap MS using MRM with a 100 ms dwell time. Data chromatography of the crude ethyl acetate extract yielded six
were analyzed at the Korea Basic Science Institute, Chonnam Na- fractions (F1eF6). Among these, fraction 4 (F4) showed the
tional University, Korea. highest nematicidal activity significantly (93.3%, P < 0.0001)
compared to the remaining fractions (Fig. 2A). Further purification
2.8. Nematicidal activity of 3,4-DHBA from the TNB
Fig. 3. TLC plates of 28 fractions (Fr 1e28) obtained from F4-2 by Sephadex LH-20 chromatography (A); J2 mortality of M. incognita reduced by fractions from Sephadex LH-20
chromatography (B). Values are mean SD of three observations after 12 h incubation with 2 mg/ml. The different letters on the error bars indicate significant differences
based on Tukey’s Studentized range at p 0.05; HPLC of 3,4-DHBA purified from TNB (C).
of F4 yielded three fractions (F4-1, F4-2 and F4-3) and treatment Sephadex LH-20 chromatography, fractions 6 to14 showed a sin-
with two fractions (F4-1 and F4-2) resulted in >93% J2 mortality, gle spot and the same Rf value (0.74) on TLC plates (Fig. 3A). The
whereas treatment with F4-3 resulted in a much lower activity nematicidal activity of fractions 6e14 (98.3%) was significantly
(23.3%) (Fig. 2B). Among the 28 fractions obtained from F4-2 by higher than those of fractions 1e5 (81.7%) and fractions 15e28
Table 1
1 13
H- and C- NMR spectroscopic data of 3,4-DHBA from TNB.
s: singlet, d: doublet.
Fig. 5. Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) of 3,4-DHBA. (A) TOF-MS of 3,4-DHBA at library source. (B) TOF-MS of 3,4-DHBA purified from TNB.
TOF-MS was performed in negative mode for m/z 153.
D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59 57
Fig. 7. Staining of M. incognita eggs with Fluorescence Brightener 28 after various treatments. Egg shapes viewed under light microscope (A); egg shapes in the water control
stained with Fluorescence Brightener 28 and viewed under fluorescence microscope (B); egg shapes treated with 3,4-DHBA viewed under light microscopy (C); egg shapes treated
with 3,4-DHBA and stained with Fluorescence Brightener 28 viewed under fluorescence microscopy (D).
58 D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59
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