Kinetic and mechanistic investigations on reductions of aflatoxins by lactic acid

Article abstract of DOI:10.1016/S0960-894X(02)00533-4

The kinetics of reduction of AFB₁ to AFB₂ and AFG₁ to AFG₂ by lactic acid has been investigated in dilute aqueous acidic solutions (pH 3.35-4.50) as a function of the concentrations of lactic acid, AFB₁, AFG₁ and hydrogen ion at 37°C. The rate of the reaction was found to be first order with respect to the concentrations of lactic acid and aflatoxins and independent on hydrogen ion concentration. The experimental results are interpreted in terms of mechanisms involving an initial formation of transient oxonium intermediate, which tends to polarize the olefinic (C=C) carbon, which in turn causes the hydride abstraction from α-carbon atom of lactic acid in rate determining step. The proposed mechanisms involve an overall transfer of two protons and two electrons from lactic acid to AFB₁ and AFG₁ to give the corresponding reduced less toxic products AFB₂ and AFG₂ and the oxidised product pyruvic acid.

Full text of DOI:10.1016/S0960-894X(02)00533-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2737–2741
Kinetic and Mechanistic Investigations on Reductions of
Aflatoxins by Lactic Acid
Ram S. Shukla,a Ramtej J. Vermab,* and Daxa N. Mehtab
aSilicates and Catalysis Division, Central Salt and Marine Chemicals Research Institute, Bhavnagar 364 002, India
bDepartment ofZoology, University School ofSciences, Gujarat University, Ahmedabad 380 009, India
Received 21 September 2001;accepted 22 June 2002
Abstract—The kinetics of reduction of AFB1 to AFB2 and AFG1 to AFG2 by lactic acid has been investigated in dilute aqueous
acidic solutions (pH 3.35–4.50) as a function of the concentrations of lactic acid, AFB1, AFG1 and hydrogen ion at 37 ꢀC. The rate
of the reaction was found to be first order with respect to the concentrations of lactic acid and aflatoxins and independent on
hydrogen ion concentration. The experimental results are interpreted in terms of mechanisms involving an initial formation of
transient oxonium intermediate, which tends to polarize the olefinic (C¼C) carbon, which in turn causes the hydride abstraction
from a-carbon atom of lactic acid in rate determining step. The proposed mechanisms involve an overall transfer of two protons
and two electrons from lactic acid to AFB1 and AFG1 to give the corresponding reduced less toxic products AFB2 and AFG2 and
the oxidised product pyruvic acid.
# 2002 Elsevier Science Ltd. All rights reserved.
Aflatoxins (B1, B2, G1 and G2) are food-borne second-
ary toxic fungal metabolites produced by Aspergillus
flavus and Aspergillus parasiticus. Consumption of afla-
toxin in many parts of the world varies from 0 to 30,000
ng/kg/day.1 Aflatoxins have been incriminated in hepa-
tocellular carcinoma, acute hepatic failure and Reye’s
Syndrome. Exposure to aflatoxin may begin pre-
natally2,3 persist during breast feeding4,5 and continue in
adult life. Visualizing the seriousness of aflatoxin prob-
lem in human and animal health, various methods were
tested to control aflatoxin production. Uses of pro-
pionic acid, butyric acid6 or lauric acid derivatives7 have
been shown to reduce fungal growth and aflatoxin pro-
duction by A. flavus. Our earlier studies have also
revealed that presence of lactic acid8 or l-ascorbic acid9
in culture medium significantly reduces mycelial growth
and aflatoxin production. Amongst different aflatoxins
present in culture medium, the concentrations of AFB2
and G2 were comparatively higher than B1 and G1.
system also seems to be efficiently operating in reduc-
tion of aflatoxins B1 and G1, to corresponding less toxic
products aflatoxins B2 and G2, respectively. In order to
understand the chemical nature of interaction of lactic
acid, which behaves as a source of donor of two protons
and two electrons during its interaction with aflatoxin,
present investigation was undertaken to study the
kinetic and mechanistic aspects of the redox reaction in
aqueous solution.
A. parasiticus (NRRL 3240) obtained from Indian
Agricultural Research Institute, New Delhi, was grown
on sucrose-magnesium sulphate-potassium nitrate-yeast
extract (SMKY) liquid medium at 28ꢁ2 ꢀC for 10
days.11 Culture filtrates were extracted with analytical
grade chloroform (1:2;v/v) which was passed through
anhydrous sodium sulphate. The chloroform extract
was evaporated to dryness. The residue was dissolved in
fresh chloroform, transferred to vials and labelled. All
the chemicals used were of A.R. grade and A.R. grade
lactic acid was used without further purification. All the
reactions were run in freshly prepared double distilled
water.
Biochemical oxidation of lactic acid to pyruvic acid by
lactate dehydrogenase is a well known enzymatic reac-
tion in metabolic pathways in which NAD is reduced to
NADH2 involving an overall transfer of two protons
and two electrons from lactic acid. A lactate dehy-
drogenase analogue of ascorbate dehydrogenase10
100 mL aflatoxin extract along with a pure aflatoxin
standard (a gift from the International Agency for
Research on Cancer, Lyon, France) were spotted on
silica gel G coated activated TLC plates. Thereafter, the
*Corresponding author. E-mail: zooldeptgu@satyam.net.in
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00533-4

2738
R. S. Shukla et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2737–2741
plates were developed in a solvent consisting of toluene/
12
part (after 6 h). After 6 h, the rates (ꢃd[AFB1]/dt) and
(d[AFB2]/dt) were 0.66ꢂ10ꢃ8 and 0.67ꢂ10ꢃ8 mol dmꢃ3
isoamyl alcohol/methanol (90:32:2;v/v).
The plates
were air-dried and observed under long wave UV light
(360 nm) for aflatoxins (B1, B2, G1 and G2). The afla-
toxins were chemically confirmed by spraying tri-
fluoroacetic acid or 25% sulphuric acid. Each spot was
eluted separately and dissolved in chilled methanol.
Aflatoxins were quantified using Shimadzu 160 UV–vis
spectrophotometer at 360 nm according to the method
of Nabney and Nesbitt.13 Analysis of standard as well
as mixture of aflatoxins were also done by HPLC in the
beginning while initiating the studies involved in this
work.
h
ꢃ1, respectively. The rates of formation of pyruvic acid
(PA), (d[PA]/dt), determined in the beginning (6 h) and
in the later part (after 6 h) were 6.18ꢂ10ꢃ8 and
0.64ꢂ10ꢃ8 mol dmꢃ3
h
ꢃ1, respectively. Similar results
were obtained for reduction of AFG1 to AFG2 also. In
beginning, during first 6-h rates (mol dmꢃ3 ꢃ1) corre-
h
sponding to (ꢃd[AFG1]/dt), (d[AFG2]/dt) and (d[PA]/
dt): 5.67ꢂ10ꢃ8, 5.68ꢂ10ꢃ8 and 5.65ꢂ10ꢃ8 were higher
than the respective rates 0.53ꢂ10ꢃ8, 0.55ꢂ10ꢃ8 and
0.53ꢂ10ꢃ8, determined after 6 h. These findings indi-
cated that the rates of the formation of the products are
almost identical with the rates of the decrease in the
concentration of the reactants. Hence, in the experi-
mental conditions, all the experimental rates were found
to be almost equal for a particular reaction indicating
that (ꢃd[AFB1]/dt)=(d[AFB2]/dt)=(d[PA]/dt) and
(ꢃd[AFG1]/dt)=(d[AFG2]/dt)=(d[PA]/dt). These rates
in turn will be also equal to the rate of decrease in the
concentration of lactic acid (LA), that is (ꢃd[LA]/dt).
Aqueous solution of aflatoxin (3.89ꢂ10ꢃ6 mol dmꢃ3
)
was mixed with 4.44ꢂ10ꢃ4 mol dmꢃ3 of lactic acid and
incubated at 37 ꢀC. In kinetic experiments the required
pH of aflatoxin and lactic acid reaction mixture was
maintained by adding dilute solution of HCl/carbonate
free NaOH.10 pH measurements were done with a digi-
tal pH meter (ꢁ0.01 pH) equipped with combined glass
and calomel electrodes. An appropriate quantity of
aflatoxin (solution A) and pre-equilibrated (ca. 30 min)
solutions containing required concentrations of lactic
acid and HCl/carbonate free NaOH (solution B) were
made in separate containers. These containers were kept
for sufficient time in thermostatic bath (temperature
37 ꢀC). Each of the kinetic runs were started by mixing
thoroughly solution A and B and the resulting solutions
were subjected for finding the rate of change of
concentrations of different aflatoxins with time.
Pyruvic acid obtained as the oxidation product of lactic
acid was measured spectrophotometrically at 456 nm by
using Salicyaldehyde.14 In order to understand the stoi-
chiometry of the reaction, experiments were conducted
by maintaining slight excess of aflatoxins in the reaction
mixtures and after completion of the reactions the
amounts of aflatoxins (B1 and G1), respectively reduced
to aflatoxins (B2 and G2), were estimated. The data
revealed that the total concentration of the reduced
products of aflatoxins (B2+G2) was almost identical
with the initial concentrations of lactic acid allowed in
the reaction mixtures. Experiments conducted by main-
taining excess of lactic acid in the reaction mixture also
indicated that the concentration of the consumed lactic
acid during the reaction, was almost equal to, the total
initial concentration of AFB1 and AFG1 which was
identical with the total concentration of the reduced
products AFB2 and AFG2.The amount of pyruvic acid
spectrophotometrically measured, after the completion
of the reaction, was also found to be identical with the
total concentration of the reduced aflatoxins B2 and G2.
These results indicated that one equivalent of lactic acid
reduces one equivalent of AFB1 and AFG1. Thus, the
overall reactions between lactic acid and aflatoxins B1
Aqueous solution of 3.89ꢂ10ꢃ6 mol dmꢃ3 aflatoxin
containing mixtures of aflatoxin B1, B2, G1 and G2
(3:2:3:2) were thoroughly mixed with 4.44ꢂ10ꢃ4 mol
dmꢃ3 of lactic acid and incubated at 37 ꢀC and aflatox-
ins were quantified at regular suitable intervals. Chan-
ges in concentrations of AFB1 to AFB2 (Fig. 1) and
AFG1 to AFG2 (Fig. 2) revealed a significant decrease
in AFB1 and AFG1 followed with increase in con-
centrations of AFB2 and AFG2. For reduction of AFB1
to AFB2 the rates (mol dmꢃ3
h
ꢃ1) of decrease in the
concentration of AF B1 (ꢃd [AFB1]/dt=7.34ꢂ10ꢃ8
)
and increase in the concentration of AFB2 (d [AFB2]/
dt=6.21ꢂ10ꢃ8), determined in the beginning, during
first 6 h, were found to be higher than that of in the later
Figure 1. Time-dependent changes in the concentrations of AFB1 and
AFB2 during interactions with lactic acid.
Figure 2. Time dependent changes in concentrations of AFG1 and
AFG2 during interaction with lactic acid.

R. S. Shukla et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2737–2741
2739
and G1 are outlined as in eqs 1 and 2, where the oxida-
tion product of lactic acid is pyruvic acid and the
reduction product of aflatoxins B1 and G1 are aflatoxins
B2 and G2, respectively.
Based on kinetic and experimental evidences the mechan-
ism proposed for reduction of AFB1 to AFB2 and AFG1
to AFG2 by lactic acid are given in Schemes 1 and 2
respectively in which for brevity the whole structures of
AFB1 and AFG1 are given in the first and last steps
only. Formation of pyruvic acid as the oxidation pro-
duct of lactic acid overruled the C–C bond scission of
lactic acid in the activated state. Hence the cleavage of a
C–H bond from the carbon bearing the functional
group OH in lactic acid, seems to be reasonable to pro-
pose to be operative in the activation state for the two
electron oxidation. Based on experimentally observed
large kinetic isotope effect values, cleavage of a C–H
bond during oxidation of lactic acid to pyruvic acid is
already reported.15
CH3CH OH COOH þ AFB1
! CH3CO COOH þ AFB2
ð1Þ
CH3CH OH COOH þ AFG1
! CH3CO COOH þ AFG2
ð2Þ
Studies on reduction of AFB1 to AFB2 and AFG1 to
AFG2 were performed in presence of lactic acid by per-
forming a series of kinetic experiments at different initial
concentrations of lactic acid and hydrogen ion at con-
stant concentrations of AFB1 and AFG1 at 37 ꢀC. An
excess of lactic acid was used in all kinetic runs to
ensure pseudo first order kinetic conditions and the
corresponding rate constants (kobs) in terms of con-
centration of aflatoxin were computed from the slopes
of the plots of log ÁC versus time (multiplied by 2.303)
where ÁC is the difference in concentrations at zero and
experimental times. The reduction of aflatoxins studied
at different concentrations of lactic acid (Table 1) indi-
cated a linear increase in kobs with increasing initial
concentration of lactic acid. The plot of log kobs versus
log [lactic acid] was found to be linear with unit slopes,
indicating that the reaction is first order with respect to
concentration of lactic acid. First order kinetic plots of
log ÁC versus time, in terms of aflatoxins concentra-
tions, were linear. Also these first order rate constants
kobs were found to be constant on varying the initial
concentrations of aflatoxins, which obviously indicates
that the reaction is first order with respect to aflatoxin
concentrations. No dependence on variation of the
initial concentration of hydrogen ion on the rate of
reduction of aflatoxins was observed in the said pH
range (Table 1). Thus the rate of reduction of aflatoxins
by lactic acid is first order in both [lactic acid] and
[aflatoxins] following an overall second order kinetics.
In order to ascertain the nature of cleavage of C–H
bond, conducted experiments revealed that the oxida-
tion of lactic acid was not affected by adding radical
scavangers like allyl acetate. Also the oxidation of lactic
acid, under Argon, failed to induce polymerization of
acrylonitrile. These experiments suggest that a hydrogen
abstraction mechanism is unlikely in view of the nil
effect of radical scavangers on the reaction rate. Radical
mechanism also could not be considered because radical
formation could not be demonstrated by acrylonitrile
polymerization during oxidation of lactic acid.
The above results are best explained by assuming an
initial formation of transient oxonium ion, which is
formed by the interaction of weakly dissociable proton
of –COOH group in lactic acid16 (pK=3.862 at 25 ꢀC)
with the active oxygen of the alkenyl ether. The oxo-
nium intermediate tends to polarize the olefinic (C¼C)
carbon, which in turn causes the hydride abstraction
from a-carbon atom of lactic acid. The rate determining
steps k1 (Scheme 1) and k2 (Scheme 2) are proposed to
involve, hydride ion (Hꢃ) abstraction from a-carbon
atom of lactic acid by the polarized (C¼C) carbon at the
b-position. The next step then involves the rearrange-
ments of the protons of, oxonium ion and alcoholic
position of lactic acid to yield the reduced products
AFB2 (Scheme 1) and AFG2 (Scheme 2) and the oxi-
dised product pyruvic acid. Hence an overall transfer of
Table 1. Kinetic data for the reduction of AFB1 and AFG1 by lactic acid at 37 ꢀC
[Lactic acid]ꢂ104
pH
kobsꢂ102
k1ꢂ10ꢃ2 molꢃ1 dm3 hꢃ1
kobsꢂ102 hꢃ1
k2ꢂ10ꢃ2 molꢃ1
b
mol dmꢃ3
h
ꢃ1 for AFB1
for AFB1
for AFG1
dm3 hꢃ1 for AFG1
a
a
b
0.25
0.50
1.00
2.20
4.44
4.44
4.44
4.44
4.44
4.44
3.35
3.35
3.35
3.35
3.35
3.80
4.05
4.50
3.35
3.35
0.30
0.59
1.17
2.56
5.15
5.18
5.28
5.34
5.18c
5.17e
1.20
1.18
1.17
1.16
1.16
1.17
1.19
1.20
1.17c
1.16e
0.29
0.57
1.13
2.48
4.97
4.98
5.00
5.02
4.97d
5.00f
1.16
1.14
1.13
1.11
1.12
1.12
1.13
1.13
1.12d
1.13f
a[AFB1]=1.20ꢂ10ꢃ6 mol dmꢃ3
.
.
b[AFG1]=1.14ꢂ10ꢃ6 mol dmꢃ3
c[AFB1]=0.60ꢂ10ꢃ6 mol dmꢃ3
.
d[AFG1]=0.57ꢂ10ꢃ6 mol dmꢃ3
.
e[AFB1]=1.45ꢂ10ꢃ6 mol dmꢃ3
.
f[AFG1]=1.37ꢂ10ꢃ6 mol dmꢃ3
.

2740
R. S. Shukla et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2737–2741
Scheme 1.
Scheme 2.

R. S. Shukla et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2737–2741
2741
two protons and two electrons take place during the
reduction of AFB1 to AFB2 and AFG1 to AFG2 by
lactic acid. Mechanism involving transfer of a hydride
ion from lactic acid to the oxidant sodium N-chlor-
obenzene sulphonamide is also reported17 in aqueous
acidic medium.
reduction of AFB1 to AFB2 and AFG1 to AFG, are
9.70ꢂ102 and 5.90ꢂ102 molꢃ1 dm3 hꢃ1, respectively, in
presence of the reductant ascorbic acid. The nature of
transfer of electrons and protons from the reductants to
aflatoxins seems to be the major factor responsible for
making the ascorbic acid system relatively more effi-
cient. The alteration of C–OH bond in ene-diol struc-
ture of ascorbic acid during reaction is easier than the
cleavage of C–H bond of lactic acid which is involved in
the mechanism.
The deduced rate equations describing the mechanisms
of Schemes 1 and 2 are given from eqs 3–8.
-d½AFB1Š
The reaction of dilute solutions of lactic acid and afla-
toxin leads the reduction of AFB1 to AFB2 and AFG1
and AFG2 involving an initial formation of transient
oxonium intermediate, which tends to polarize the ole-
finic (C¼C) carbon, which in turn causes the hydride
abstraction from a-carbon of lactic acid. The overall
reduction process involves the transfer of two protons
and two electrons from lactic acid to the aflatoxins B1
and G1.
¼ k1 ½AFB1Š ½CH3CHOH COOHŠ
ð3Þ
dt
kobs ¼ ðꢀd½AFB1Š=dtÞ=½AFB1Š
¼ k1½CH3CHOH COOHŠ
ð4Þ
ð5Þ
k1 ¼ kobs=½CH3CHOH COOHŠ
where rate=(ꢃd[AFB1]/dt)=(d[AFB2]/dt)=(d[PA]/
dt)=(ꢃd[LA]/dt).
References and Notes
1. Denning, D. W. Adverse Drug React. Acute Pois. Rev. 1987,
4, 175.
k1 is the second order rate constant corresponding to
the rate determining step in Scheme 2. Similarly, for rate
constant k2 (Scheme 2), the rate equations are given (eqs
6–8),
2. Wild, C. P.;Rasheed, F. M.;Jawla, M. F. B.;Hall, A. J.;
Jansen, L. A. M.;Montesano, R. Lancet 1991, 337, 1602.
3. Pohland, A. E. Food Addit. Contam. 1993, 10, 17.
4. Wild, C. P.;Pionneau, F. A.;Montesano, R.;Mutino,
C. F.;Chetscaga, C. J. Trop. Dis. Bull. 1988, 85, 908.
5. Lamplugh, S. M.;Hendrickse, R. G.;Apeagyei, F.;Muan-
met, D. D. Br. Med. J. 1988, 296, 968.
ꢀd½AFG1Š
¼ k2½AFG1Š ½CH3CHOH COOHŠ
ð6Þ
dt
6. Ghosh, J.;Haggblom, P. Int. J. Food Microbiol. 1985, 2,
323.
7. Ramadevi, G.;Polasa, H. J. Stored Prod. Res. 1985, 21,
195.
8. Verma, R. J.;Mehta, D. N.;Raval, P. J.;Dube, H. C. In
Proc. DAE Symp. on Stress and Adaptive Responses in Biolo-
gical Systems, Baroda, India, 1994;p 360.
9. Mehta, D. N. PhD Thesis, Banvnagar University;Bavna-
gar, India, 1993.
kobs ¼ ðꢀd½AFG1Š=dtÞ=½AFG1Š
¼ k1½CH3CHOH COOHŠ
ð7Þ
ð8Þ
k2 ¼ kobs=½CH3CHOH COOHŠ
10. Verma, R. J.;Shukla, R. S.;Mehta, D. N. Natural Toxins
1999, 7, 1.
where rate=(ꢃd[AFG1]/dt)=(d[AFG2]/dt)=(d[PA]/
dt)=(ꢃd[LA]/dt).
11. Diener, U. L.;Davis, N. D. Phytopathology 1966, 56, 1390.
12. Reddy, T. V.;Vishwanathan, L.;Venkitasubramanian,
T. A. Anal. Biochem. 1970, 38, 568.
13. Nabney, J.;Nesbitt, B. F. Analyst 1965, 90, 155.
14. Snell, F. D.;Snell, C.T.;Colorimetric methods of analysis,
Van Nostrand: New York, 1961;Vol. IIIB, p 336.
15. Haight, G. P.;Jurisch, G. M.;Kelso, M. T.;Marrill, P. J.
Inorg. Chem. Year 1985, 24, 2740.
16. Encyclopedia ofChemical Technology , IV ed.;John Wiley
and Sons: New York, 1995;p 1044.
17. Mathur, A. K.;Banerji, K. K. Indian J. Chem. 1981, 20B,
529.
The rate constants k1 and k2 computed from eqs 5 and
8, respectively, were found to be constant verifying these
rate equations and proposed mechanisms. The values of
the second order rate constant k1 and k2 are 1.18ꢂ102
and 1.13ꢂ102 molꢃ1 dm3 hꢃ1, respectively. These rate
constants are appreciable but found to be less in com-
parison with the reductant l-ascorbic acid.10 The
reported10 values of the rate constants k1 and k2 for