Nitrification inhibitors from the root tissues of Brachiaria humidicola, a tropical grass

Article abstract of DOI:10.1021/jf062593o

Nitrification inhibitory activity was found in root tissue extracts of Brachiaria humidicola, a tropical pasture grass. Two active inhibitory compounds were isolated by activity-guided fractionation, using recombinant Nitrosomonas europaea containing luxAB genes derived from the bioluminescent marine gram-negative bacterium Vibrio harveyi. The compounds were identified as methyl-p-coumarate and methyl ferulate, respectively. Their nitrification inhibitory properties were confirmed in chemically synthesized preparations of each. The IC₅₀ values of chemically synthesized preparations were 19.5 and 4.4 μM, respectively. The ethyl, propyl, and butyl esters of p-coumaric and ferulic acids inhibited nitrification, whereas the free acid forms did not show inhibitory activity.

Full text of DOI:10.1021/jf062593o

J. Agric. Food Chem. 2007, 55, 1385−1388 1385
Nitrification Inhibitors from the Root Tissues of Brachiaria
humidicola, a Tropical Grass
†
,†
SUBRAMANIAM GOPALAKRISHNAN, GUNTUR V. SUBBARAO,*
†
†
†
‡
KAZUHIKO NAKAHARA, TADASHI YOSHIHASHI, OSAMU ITO, IKUKO MAEDA,
HIROSHI ONO,‡ AND MITSURU YOSHIDA‡
Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi,
Tsukuba, Ibaraki 305-8686, Japan, and National Food Research Institute, 2-1-12 Kannondai,
Tsukuba, Ibaraki 305-8642, Japan
Nitrification inhibitory activity was found in root tissue extracts of Brachiaria humidicola, a tropical
pasture grass. Two active inhibitory compounds were isolated by activity-guided fractionation, using
recombinant Nitrosomonas europaea containing luxAB genes derived from the bioluminescent marine
gram-negative bacterium Vibrio harveyi. The compounds were identified as methyl-p-coumarate and
methyl ferulate, respectively. Their nitrification inhibitory properties were confirmed in chemically
synthesized preparations of each. The IC50 values of chemically synthesized preparations were 19.5
and 4.4 µM, respectively. The ethyl, propyl, and butyl esters of p-coumaric and ferulic acids inhibited
nitrification, whereas the free acid forms did not show inhibitory activity.
KEYWORDS: Brachiaria humidicola; nitrification inhibitor; methyl ferulate; methyl-p-coumarate
INTRODUCTION
minutiflora, Andropogon gayanus, and Brachiaria decumbens)
in the acid soils of Colombia; also, B. humidicola responded
less well to the application of inorganic fertilizer N than did
the other forage grasses (5, 6). Brachiaria humidicola inhibits
nitrification by suppressing the function of the nitrifying bacteria
Nitrification is an important biological process in global
nitrogen cycling whereby ammonia is converted to nitrite and
nitrate by nitrifying bacteria (species of Nitrosomonas and
Nitrobacter). The nitrification products are vulnerable to leach-
ing and denitrification: an estimated 45% of applied fertilizer
is lost by leaching (1) and 10-30% by denitrification (2). If
the nitrification process is inhibited or slowed, then plants have
adequate time to take up fertilizer N; N recovery and uptake
+
in the soil, and it keeps soil nitrogen in the NH4 form (7).
Recently, it was shown that compounds released from the roots
of B. humidicola are mainly responsible for its inhibitory effect
on soil nitrification (8) and that root tissue extracts of B.
humidicola have substantial inhibitory effects on nitrification.
Our investigation was aimed at isolating these nitrification
inhibitors from the root tissues of B. humidicola.
-
are substantially improved and NO3 pollution problems are
reduced (3). Considerable evidence shows that plants produce
secondary metabolites that inhibit nitrification; e.g., grasslands
display low nitrification rates and produce phenolic acids and
flavonoids that inhibit nitrification (3).
MATERIALS AND METHODS
Creeping signal grass or false creeping paspalum (Brachiaria
humidicola [Rendle] Schweick) is a native pasture grass of
Africa and is found from southern Sudan and Ethiopia in the
north to South Africa and Namibia in the south. It is also grown
widely in the humid-tropical countries of South America, the
Pacific Islands, and South East Asia and in coastal Northern
Australia (4). This tropical grass is grown widely not only as
permanent pasture and as ground cover for the control of erosion
and weeds but also for its adaptability to acid infertile soils
and waterlogged habitats, particularly in the South American
Cerrados (5). Lower levels of NO3-N were found in fields of
B. humidicola than in fields of other forage grasses (Melinis
Chemicals. Ferulic acid and p-coumaric acid were purchased from
Sigma (St. Louis, MO) and ICN Biomedicals (Aurora, OH), respec-
tively. All the solvents and other chemicals used were purchased from
Wako Pure Chemical Industries Ltd. (Osaka, Japan) unless otherwise
stated.
Plant Materials. Seeds of B. humidicola (Rendle) Schweick (CIAT
6
79) were germinated, at 25 °C with RH 75%, in sand-vermiculite
mixture (3:1) in trays and watered with distilled water. Two-week-old
plants were transferred to aerated nutrient solution. The composition
-1
of the nutrient solution (mg L ) was 38.31:31.02:10.5:36.93:15.1:0.57:
0.078:2.35:0.126:0.220 KH PO :K SO :CaCl ‚2H O:MgSO ‚7H O:
FeEDTA:H BO :CuSO ‚5H O:MnSO ‚6H O:Na MoO ‚2H O:ZnSO
O. Nitrogen at 1 mmol L was added as (NH SO to the nutrient
2
4
2
4
2
2
4
2
3
3
4
2
-
4
2
2
4
2
4
‚
1
7H
2
4
)
2
4
solution. The pH of the nutrient solution was adjusted to 5.0, and the
solution was constantly aerated. The nutrient solution was replaced with
fresh solution at weekly intervals. The plants were grown in 50 L tanks
on styrofoam blocks with 10 holes and 4 plants per hole, supported
with sponge. Actively growing roots were cut from the plants once in
*
To whom correspondence should be addressed. Tel./Fax: +81-29-
8
38-6354. E-mail: subbarao@jircas.affrc.go.jp.
†
JIRCAS.
‡
National Food Research Institute.
1
0.1021/jf062593o CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/23/2007

1386 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Gopalakrishnan et al.
3
months for 1 year, washed twice with ion-exchange water, and
allowed to drain at room temperature for 30 min before being stored
in a freezer at -20 °C until use.
Extraction of Nitrification Inhibition Activity. Roots of B.
humidicola (from the freezer) were cut into small pieces (approximately
1
cm long) and blended in a mixer with 70% methanol (MeOH) in
ultrapure water. The extracts were filtered through Whatman No. 1
filter paper, and the MeOH was removed in vacuo on a rotary evaporator
for 45 min at 35 °C. The aqueous remainder was centrifuged at 15000g
for 20 min, and the supernatants were partitioned repeatedly against
ethyl acetate (EtOAc). The organic phases were combined, and the
EtOAc was removed in vacuo on a rotary evaporator at 35 °C. The
residues were dissolved in a minimum volume of MeOH and stored in
a freezer at -20 °C; aliquots of these samples (100 µL) were further
concentrated on a centrifugal evaporator (model CVE-200D, Eyela,
Tokyo, Japan) to 25 µL in dimethyl sulfoxide (DMSO) for bioassay.
Nitrification Inhibition Bioassay. The nitrification inhibition (NI)
activity of the samples was determined using a modified bioassay that
utilizes recombinant luminescent Nitrosomonas (8, 9). The detailed
methodology for the detection, quantification, and expression of NI
activity has been described previously (8). The NI activity of the
samples is expressed in units defined in terms of the action of a standard
Figure 1. Chemical structures of methyl-p-coumarate (1) and methyl
ferulate (2). Arrows indicate important NMR correlations. Solid arrows
are selected HMBC correlations (from C to H), and the broken arrow is
a NOESY correlation.
chromatography, definite activity was detected in the 60%
MeOH effluent. This active fraction was further purified by
reversed-phase HPLC using a 10-30% acetonitrile gradient
mobile phase system. Finally, two compounds (1 and 2) were
isolated as having NI activity. The dry weights of 1 and 2 were
inhibitor, allylthiourea (AT); the inhibitory effect of 0.22 µM AT in
+
an assay containing 18.9 mM of NH
4
is defined as one AT unit of
activity (8).
Isolation of NI Activity. Crude extracts containing NI activity were
dissolved in 5% MeOH before being loaded onto a reversed-phase
column (25 cm × 2.8 cm, Wakosil 40C18, Wako). The column was
eluted with 200 mL each of 5%, 10%, 20%, 40%, 60%, 80%, and 100%
MeOH. All the above fractions were dried in vacuo at 35 °C and the
residues were collected in MeOH (1 mL); aliquots of these fractions
0
2
.8 and 0.4 mg, respectively, and in the bioassay 1 mg of 1 and
was equivalent to 885 and 535 AT units of NI activity (21%
and 6% recovery), respectively.
The physicochemical properties of compounds 1 and 2 were
as follows:
Compound 1: pale yellow gum; UV λmax (CH3OH) nm 308;
(50 µL) were used for bioassay. The active fractions were further
1
H NMR (500.1 MHz, CD3OD) δ 3.76 (3H, s, OCH3), 6.33
purified by an HPLC on a Jasco Gulliver HPLC system consisting of
a PU-1580 reciprocal pump, UV-1570/1575 UV detector, and 807-IT
integrator with TSKgel Super-ODS (4.6 mm × 100 mm or 10 mm ×
(1H, d, J ) 16.0 Hz, H-2′), 6.80 (2H, brd, J ) 9.0 Hz, H-3 and
H-5), 7.45 (2H, brd, J ) 9.0 Hz, H-2 and H-6), 7.61 (1H, d, J
1
3
1
00 mm) columns (Tosoh, Japan); monitoring was performed at a
) 16.0 Hz, H-1′); C NMR (125.8 MHz, CD OD) δ 51.7
3
wavelength of 220 nm. The column was eluted with a linear-gradient
mobile-phase system (10-30% acetonitrile), and all the peaks and
troughs were checked for activity.
Instrumental Analyses. The UV absorption spectra of the com-
pounds in methanol were recorded on a UV-1600 spectrophotometer
(OCH3), 114.6 (C-3 and C-5), 116.5 (C-2′), 126.8 (C-1), 130.9
(C-2 and C-6), 146.3 (C-1′), 161.1 (C-4), 169.5 (CdO); EIMS
+
m/z (%) 178 [M] (66), 147 (100), 119 (41), 91 (34), 65 (21).
1
13
Assignments of the chemical shifts of H and C NMR spectra
were confirmed by 2D-NMR analyses, NOESY, HMBC, and
HSQC. These data agreed well with the data as previously
reported (11, 12, 13). Therefore, compound 1 was identified as
methyl (E)-3-(4-hydroxyphenyl)prop-2-enoate [methyl-p-cou-
marate, 1, Figure 1].
(Shimadzu, Kyoto, Japan). The mass (MS) spectra were recorded on a
GCMS-QP2010 spectrometer (Shimadzu) by direct electron ionization
1
13
(
EI) at an ionization energy of 70 eV. The H NMR, C NMR, nuclear
overhauser enhancement spectroscopy (NOESY), heteronuclear single
quantum correlation (HSQC), and heteronuclear multiple bond cor-
relation (HMBC) spectra at 298 K were recorded on an Avance 500
spectrometer (Bruker Biospin, Karlsruhe, Germany) equipped with a
CryoProbe; the pulse sequences and software provided by the manu-
facturer were used.
Compound 2: Pale yellow gum; UV λ (CH OH) nm 325
max
3
1
and 295 (shoulder); H NMR (500.1 MHz, CD3OD) δ 3.76 (3H,
s, COOCH3), 3.89 (3H, s, ArOCH3), 6.36 (1H, d, J ) 15.9 Hz,
H-2′), 6.80 (1H, d, J ) 8.1 Hz, H-5), 7.07 (1H, dd, J ) 2.0, 8.1
Hz, H-6), 7.18 (1H, d, J ) 2.0 Hz, H-2), 7.61 (1H, d, J ) 15.9
NI Activity of Free Acids and Synthesized Esters. C
1
4
-C alkyl
esters of p-coumaric acid and ferulic acid were synthesized by acid
esterification (10). Briefly, p-coumaric acid or ferulic acid (0.5 g) was
dissolved in 10 mL of acidified (0.1 N HCl) methanol, ethanol,
1
3
Hz, H-1′); C NMR (125.8 MHz, CD3OD) δ 52.0 (COOCH3),
6.0 (ArOCH3), 111.5 (C-2), 115.0 (C-2′), 116.3 (C-5), 123.9
5
(C-6), 127.8 (C-1), 146.8 (C-1′), 149.2 (C-3), 150.4 (C-4), 169.8
1
-propanol, or 1-butanol. The solution was incubated at 37 °C for 24
+
(CdO); EIMS m/z (%) 208 [M] (100), 177 (66), 145 (55),
h. The volatiles were removed thoroughly with a centrifugal evaporator.
The reaction product was then purified using a Sep-Pack C18 cartridge
117 (24), 89 (20), 77 (13). Assignments of the chemical shifts
1
13
(
Waters, Milford, MA; purity >95%, HPLC), and the molecular mass
was confirmed by EI-MS. The NI activity of the free acids (p-coumaric
acid and ferulic acid) and various ester forms (C -C alkyl esters of
of H and C NMR spectra were confirmed by 2D-NMR
analyses, NOESY, HMBC, and HSQC. These data agreed well
with the data as previously reported (12, 13). Hence, compound
1
4
p-coumaric acid and ferulic acid) was assayed, and the inhibitory
concentration 50% (IC50) values (mean results of three replications)
were calculated.
2
was identified as methyl (E)-3-(4-hydroxy-3-methoxyphenyl)-
prop-2-enoate [methyl ferulate, 2, Figure 1].
Compounds 1 and 2 exhibited definite NI activity, but it was
not possible to estimate their IC50 values because of the small
amounts purified. To confirm the NI properties of these two
phenolic compounds, we chemically synthesized both through
esterification of the corresponding free acids. These preparations
were also found to have definite NI activity, with IC50 values
of 19.5 and 4.4 µM, respectively. Thus, it was proved that
compounds 1 and 2 were both NI-active.
RESULTS AND DISCUSSION
Roots of B. humidicola were extracted initially with 70%
MeOH. This was followed by solvent partitioning to obtain 35-
-1
4
0 AT units of NI activity g of dried roots. The crude extracts
with NI activity were further fractionated by activity-guided
fractionation. Upon fractionation by reversed-phase column

Nitrification Inhibitors from B. humidicola Root Tissues
Table 1. IC50 (in mol) of the Chemically Synthesized
Methyl-p-coumarate, Methyl Ferulate, and Other Ester Forms and Free
Acids
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1387
To our knowledge, this is the first time that specific
nitrification inhibitors have been isolated and characterized from
B. humidicola; moreover, no previous authors have ever reported
the NI activity of compounds 1 and 2. There have been reports
of some natural products that act as nitrification inhibitors;
examples are neem (Azadirachta indica Juss.) cake or an
acetone/alcohol extract of neem (26, 27) and karanj (Pongamia
glabra vent.) seed, bark, and leaves (28). Nitrification inhibitors
such as gallocatechin, epigallocatechin, catechin, and epicatechin
have also been isolated from the roots of Leucaena leuco-
cephala, a multipurpose tree from tropical America introduced
into most of the tropics (29). Crude extracts from the roots of
Astragalus mongholicus have been reported to inhibit nitrifica-
tion in soil (30).
µ
compound
IC50 (in µ
M)a
p-coumaric acid
methyl-p-coumarate
ethyl-p-coumarate
propyl-p-coumarate
butyl-p-coumarate
ferulic acid
methyl ferulate
ethyl ferulate
propyl ferulate
>10000
19.5
16.0
16.0
36.0
>10000
4.4
±
0.50
0.50
1.00
1.50
±
±
±
±
±
±
±
0.10
0.08
0.10
0.06
0.2
2.2
4.2
butyl ferulate
a
Mean
±
standard deviation.
Nitrification is an essential biological process during which
nitrifying bacteria (species of Nitrosomonas and Nitrobacter)
convert ammonia to hydroxylamine by ammonia monooxyge-
nase (AMO); the hydroxylamine is converted into nitrite by
hydroxylamine oxidoreductase (HAO, 3). Many synthetic
nitrification inhibitors, such as allylthiourea, and some phenolic
nitrification inhibitors, such as diphenyliodonium (known to
irreversibly inactivate flavoproteins) and phenylacetylene (known
for its high solubility and low volatility but high potency), inhibit
the AMO activity of N. europaea but do not inhibit HAO
activity (14, 31, 32, 33). Compounds 1 and 2, also phenolic
compounds having similar chemical structure to diphenyliodo-
nium and phenylacetylene, perhaps also inhibit AMO activity
and thus inhibit the process of nitrification. However, to support
this hypothesis, further experiments on, for example, the effects
of compounds 1 and 2 against isolated AMO activity are
required.
As demonstrated in Table 1, various alkyl esters (C1-C4) of
p-coumarate and ferulate exhibited NI activity. Among the esters
tested, ethyl and/or propyl esters showed the highest NI activity,
but their free acids were not active. It is well-known that in
Nitrosomonas europaea, the nitrification enzymes and related
electron carrier system are located in the periplasm or inner
membrane (14). Ethyl or propyl esters may have suitable
structures or polarity for penetrating the bacterial cell. In
contrast, the presence of a polar -COOH group in the free acids
might make their permeability too low to approach the action
point. Similarly, Daayf et al. has reported that the methyl esters
of phenolic acids are more toxic to fungi than are their
corresponding free acids (15), and Kikuzaki et al. observed an
increase in antioxidant activity when ferulic acid was esterified
(16).
Our findings demonstrate that B. humidicola roots produce
two methylated phenolic acids, methyl-p-coumarate and methyl
ferulate, that have inhibitory effects on nitrification. The isolation
of these nitrification inhibitors from roots has many implications
in our quest for understanding the biologically produced
nitrification inhibitors in B. humidicola pastures. It is estimated
that nearly 30% of the root mass is turned over annually in B.
humidicola pastures, and this amounts to an annual addition of
Ferulic acid and p-coumaric acid play important roles in the
structural integrity of the plant cell wall matrix and represent
factors that partly limit the biodegradability of nonlignified plant
cell-wall polysaccharides (17). Both of these acids are found
widely distributed in nature, e.g., in root exudates of Arabidopsis
thaliana (18) and in the rhizomes and roots of black cohosh
(
Actaea racemosa L., 19), as secondary metabolites of plants,
and exhibit considerable antimicrobial effects against fungi,
bacteria, and viruses. However, the methyl ester form is rare
and is found only occasionally in some herbaceous and
medicinal plants (20).
-
1
1
t of root tissue ha (on a dry weight basis) to the soils in
these systems (34). Thus, substantial amounts of nitrification
inhibitors are added annually to the soil during the process of
root turnover (i.e., through decomposition of the old roots), and
this could have important additive effects over time in influenc-
ing soil nitrification in these pastoral systems. This is perhaps
one of the main reasons for the observed low nitrification rates
in soils where B. humidicola is grown.
Nevertheless, some literature is available on compounds 1
and 2. For example, Seifert and Unger (21) isolated compound
from the aerial parts of woad (Isatis tinctoria L., a medicinal
plant) and showed that it has insecticidal activity against termites
Reticulitermes santonensis) and the larvae of the house
1
(
longhorn beetle (Hylotrupes bajulus) and fungicidal activity
against brown-rot fungus (Coniophora puteana). Rhizomes of
crepe ginger (Costus speciosus, a herbaceous and medicinal
plant) and leaves of cucumber (Cucumis satiVus L.) also contain
compound 1, which has been shown to have antifungal activity
against many plant pathogens, including Aspergillus niger,
Cladosporium cladosporioides, Colletotrichum gloeosporioides,
CurVularia sp., Penicillium sp., Cladosporium cucumerinum,
Botrytis cinerea, Pythium ultimum, and Pythium aphaniderma-
tum (15, 20, 22). Compound 1 has also been reported in aloe
leaves (Aloe Vera, 23), the aerial parts of the herb Psoralea
plicata (a grazing and medicinal plant, 24), and the leaves of
Lonchocarpus yucatanensis and Lonchocarpus xuul (endemic
trees growing on the Yucatan Peninsula of Mexico, 25).
Nuntanakorn et al. (19) reported compound 2 in extracts of the
rhizomes and roots of black cohosh, used as antioxidants and
for their anticancer activity.
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Received for review September 8, 2006. Revised manuscript received
December 8, 2006. Accepted December 17, 2006. We thank the Japan
Society for the Promotion of Science (JSPS) and the Japan International
Research Center for Agricultural Sciences (JIRCAS) for funding this
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