Synthesis of amino acid conjugates of tetrahydrocurcumin and evaluation of their antibacterial and anti-mutagenic properties

Article abstract of DOI:10.1016/j.foodchem.2013.01.081

Tetrahydrocurcumin (THC), the hydrogenated and stable form of curcumin, exhibits physiological and pharmacological activities similar to curcumin. A protocol has been developed for the synthesis of novel conjugates of THC with alanine (2a), isoleucine (2b), proline (2c), valine (2d), phenylalanine (2e), glycine (2f) and leucine (2g) in high yields (43-82%). All the derivatives of THC exhibited more potent anti-microbial activity than THC against Bacillus cereus, Staphylococcus aureus, Escherichia coli and Yersinia enterocolitica. The MIC values of the derivatives were 24-37% of those for THC in case of both Gram-positive and Gram-negative bacteria. Derivatives 2g and 2d exhibited maximum anti-mutagenicity against Salmonella typhimurium TA 98 and TA 1538, respectively at a low concentration of 313 μg/plate, with comparable activity for THC evident only at 3750 μg/plate. These results clearly demonstrated that the conjugation of THC at the phenolic position with amino acids led to significant improvement of its in vitro biological attributes.

Full text of DOI:10.1016/j.foodchem.2013.01.081

Food Chemistry 139 (2013) 332–338
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Synthesis of amino acid conjugates of tetrahydrocurcumin and evaluation
of their antibacterial and anti-mutagenic properties
J.R. Manjunatha a, B.K. Bettadaiah a, P.S. Negi b, P. Srinivas a,
⇑
a Department of Plantation Products, Spices and Flavour Technology, CSIR-Central Food Technological Research Institute, Mysore 570 020, India
b Department of Fruits and Vegetable Technology, CSIR-Central Food Technological Research Institute, Mysore 570 020, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 August 2012
Received in revised form 20 December 2012
Accepted 29 January 2013
Available online 6 February 2013
Tetrahydrocurcumin (THC), the hydrogenated and stable form of curcumin, exhibits physiological and
pharmacological activities similar to curcumin. A protocol has been developed for the synthesis of novel
conjugates of THC with alanine (2a), isoleucine (2b), proline (2c), valine (2d), phenylalanine (2e), glycine
(2f) and leucine (2g) in high yields (43–82%). All the derivatives of THC exhibited more potent anti-micro-
bial activity than THC against Bacillus cereus, Staphylococcus aureus, Escherichia coli and Yersinia enterocol-
itica. The MIC values of the derivatives were 24–37% of those for THC in case of both Gram-positive and
Gram-negative bacteria. Derivatives 2g and 2d exhibited maximum anti-mutagenicity against Salmonella
Keywords:
Tetrahydrocurcumin
Amino acids
Conjugates
Synthesis
typhimurium TA 98 and TA 1538, respectively at a low concentration of 313
lg/plate, with comparable
activity for THC evident only at 3750 g/plate. These results clearly demonstrated that the conjugation
l
of THC at the phenolic position with amino acids led to significant improvement of its in vitro biological
attributes.
Antimutagenicity
Antibacterial activity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
to be more stable than curcumin in buffer solutions of physiologi-
cal pH of 7.2 and also at basic pH, as well as in plasma.
Curcumin is the major colour constituent of turmeric (Curcuma
longa, Zingiberaceae) used in Indian cuisine mainly for its colouring
and flavoring attributes. Curcumin is highly valued for its medici-
nal and nutraceutical attributes (Ravindran, Babu, & Sivaraman,
2007). Although curcumin has promising therapeutic potential
(Corson & Crews, 2007), various studies have highlighted its insta-
bility under physiological conditions (Anand, Kunnumakkara,
Newman, & Aggarwal, 2007). It is soluble in organic solvents like
acetone and ethyl acetate but insoluble in water at acidic or neutral
pH. At pH 7.2, curcumin is sparingly soluble in aqueous media
THC is derived from curcumin by selective reduction of olefinic
bonds alpha to the carbonyl group in a diferuloyl backbone. THC is
an effective natural antioxidant (Osawa, Sugiyama, Inayoshi, &
Kawakishi 1995; Sugiyama, Kawakishi, & Osawa, 1996) and anti-
inflammatory compound (Rao, Basu, & Siddiqui 1982), employed
in many formulations in the field of cosmetics and nutrition. THC
offers protection to the skin against damage due to ultraviolet radi-
ation and other factors which are essential for sun protection prep-
arations and cosmetics for aged skin. Tetrahydrocurcuminoids
have superoxide scavenging ability and inhibitors of fat oxidation
and offers protection against rancidity of several fat components.
THC produces the protective effect to cells against oxidative stress
by scavenging free radicals and reactive oxygen species (Nakam-
ura, Ohto, Murakami, Osawa, & Ohigashi, 1998). THC is a potent
antioxidant under the conditions where the radical initiators are
produced in the polar water medium (Khopde et al., 2000). Admin-
istration of THC to streptozotocin (STZ)-nicotinamide-induced dia-
betic male Wistar rats significantly improves specific insulin
binding to the receptors, with the receptor numbers and affinity
binding reaching near-normal levels resulting in a significant
increase in plasma insulin with the effect of THC being more
prominent than that of curcumin (Murugan, Pari, & Rao, 2008).
THC-inhibits lipoxygenase (LOX) enzyme implicated in inflamma-
tory conditions by preventing the activation of LOX-1 (Sneharani,
Sridevi, Srinivas, & Rao, 2011). It is also effective in inhibiting
(ꢀ20
lg per ml) but degrades rapidly with the half-life of 30 min.
The stability further decreases with increase in pH (Wang et al.,
1997). Tetrahydrocurcumin (THC, 1) is the major and stable metab-
olite of curcumin. Its glucuronidation constitutes the reported
pathway of metabolism of curcumin (Pan, Huang, & Lin, 1999).
Glucuronides of curcumin and THC can serve as bio-available
forms of curcumin in vivo. It exhibits physiological and pharmaco-
logical activities similar to curcumin and, in some systems, does
show higher antioxidant activity than curcumin. THC is reported
⇑
Corresponding author. Address: Department of Plantation Products, Spices and
Flavour Technology, CSIR-Central Food Technological Research Institute, Chel-
uvamba Mansion, Mysore 570 020, India. Tel.: +91 821 2512352; fax: +91 821
2517233.
E-mail address: psrinivas@cftri.res.in (P. Srinivas).
0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.01.081

J.R. Manjunatha et al. / Food Chemistry 139 (2013) 332–338
333
cyclooxygenase-2 and phospholipase A2 (Hong et al., 2004). THC
inhibits cancer (HT1080) cell migration and invasion by down reg-
ulation of extracellular matrix (ECM) degradation enzymes and the
inhibition of cell adhesion to ECM proteins (Yodkeeree, Garbisa, &
Limtrakul, 2008).
Na2SO4 and concentrated. Further, the product was purified by
triturating using ethyl acetate and petroleum ether (60–80 °C)
mixture to afford t-Boc protected amino acid derivatives of curcu-
min in 75–95% yield.
Recent attempts at preparing sugar and amino acid conjugates
of curcumin led to the preparation of a number of water-soluble
curcumin derivatives, which exhibited potent antioxidant, antimi-
crobial and antimutagenic properties comparable to and in several
cases superior than curcumin (Kumar, Narain, Tripathi, & Misra,
2001; Parvathy, Negi, & Srinivas, 2009, 2010; Parvathy & Srinivas,
2008). The authors demonstrated that curcumin–b-diglucoside
2.3. General method for deprotection of t-Boc group in curcumin–t-Boc
amino acid conjugates
Each of the curcumin–amino acid t-Boc conjugates (ꢀ3 g) was
dissolved in CH2Cl2 (20 ml). To this solution, trifluoroacetic acid
(TFA, 4 ml) in CH2Cl2 (20 ml) was added slowly under ice cold con-
dition. The reaction mixture was allowed to stir at 25 °C till the
completion of the reaction (3–5.5 h). It was followed by neutralisa-
tion of TFA by gradual addition of solid K2CO3. The product gets
separated from the mixture and settled at the bottom of the flask.
It was filtered, washed with water, dried and then kept in a vac-
uum desiccator over KOH pellets for 12 h. The pure products were
obtained in the 60–90% yield. The structure of curcumin–amino
acid conjugates (1a–1g) was confirmed by 1D and 2D-NMR and
mass spectral analyses.
prevented oligomer formation and inhibited fibril formation of a-
synuclein, whose aggregation is centrally implicated in Parkinson’s
disease (Bharathi, Parvathy, Srinivas, Indi, & Rao, 2012). Curcumin
is unstable and is metabolised to THC and other reduced forms
in vivo. Hence, the bioavailability of curcumin is limited. Tetrahy-
drocurcumin (THC) is a stable metabolite of curcumin in physio-
logical systems and more lipophilic than curcumin. Also, in
several studies THC is shown to exhibit a wide array of bioactive
properties more potent than curcumin. Hence, it was envisaged
that it would be of great interest to synthesise new amino acid
derivatives of THC and explore their bioactive attributes. In the
present study, we attempted the syntheses of selected novel amino
acid conjugates of THC and evaluated their in vitro antimicrobial
and antimutagenicity potential.
2.4. General method for the synthesis of THC–amino acid conjugates
(2a–2g)
The solution of curcumin–amino acid conjugates (1 g, 1a–1g) in
methanol (15 ml) was taken in hydrogenation flask and charged
with Pd/BaSO4 (10 mg, 10% w/w). The mixture was agitated at
30 psi H2 pressure till completion of the reaction (0.5–1.5 h), as in-
ferred by disappearance of golden yellow colour. Additionally, it
was confirmed by TLC analysis. After completion of the reaction,
the catalyst was filtered off and the filtrate was concentrated under
reduced pressure to afford pure products (2a–2g) in quantitative
yields. The products were characterised by 1D and 2D-NMR and
high resolution mass spectral analyses. The physical and spectro-
scopic data of the novel THC–alanine conjugate (2a) are presented
below with numbering of the carbon atoms as depicted in Table 1.
The physical and spectroscopic data of other conjugates (2b–2g)
are provided in the Supplementary information.
2. Materials and methods
2.1. Apparatus and materials
All the solvents and reagents used for the synthesis were of ana-
lytical grade. Curcumin (ꢀ95% purity) was procured from Ms. Spi-
cex Chemicals Pvt. Ltd., Mysore, India. Palladium on barium
sulphate catalyst, t-Boc amino acids, 4-dimethylaminopyridine
and N,N0-dicyclohexylcarbodiimide were purchased from Sigma Al-
drich Chemical Co. (St. Louis, MO, USA). Dry 1,4-dioxane, trifluoro-
acetic acid, chloroform and dichloromethane were purchased from
Merck Specialty Chemicals, Mumbai, India. 1H and 13C NMR spectra
for the compounds were recorded on a 500 MHz NMR spectrome-
ter (Bruker Avance, Reinstetten, Germany), using CD3OD solvent.
Coupling constants (J values) are given in Hz. High resolution mass
spectral analyses of the compounds were carried out using MS
(Waters Q-Tof Ultima, Manchester, UK) in the ESI positive mode.
Thin-layer chromatographic (TLC) analysis was done on silica gel
60 F254 (Merck, Germany) coated on an alumina sheet and 3%
methanol in chloroform as the developing solvent. The products
were purified by triturating using ethyl acetate and hexane mix-
ture. All the chemicals and Petri plates used for microbial studies
were procured from Hi Media Ltd., Mumbai, India.
(2a): [(Z)-5-hydroxy-1,7-bis(4-O-L-alaninoyl-3-methoxyphenyl)
hept-4-en-3-one]; off-white solid; m. p. 74–76 °C; 1H NMR
(500 MHz, CD3OD): d 1.70 (d, J = 7.2 Hz, 6H, H-17 & H-170), 2.55–
2.62 (m, 2H, H-6). 2.72–2.83 (m, 4H, H-1 & H-2), 2.84–2.89 (m,
2H, H-7), 3.76 (s, 6H, H-14 & H-140), 4.35 (q, J = 7.3 Hz, 2H, H-16
& H-160), 5.56 (s, 1H, H-4), 6.72–6.81 (m, 2H, H-13 & H-130),
6.88–6.94 (m, 2H, H-9 & H-90), 6.95–7.01 (m, 2H, H-12 & H-120).
13C NMR (125 MHz, CD3OD): d 16.52 (C-17 & C-170), 30.11 (C-1),
32.33 (C-7), 40.59 (C-6), 45.70 (C-2), 49.89 (C-16 & C-160), 56.47
(C-14 & C-140), 100.86 (C-4), 113.99 (C-9 & C-90), 121.54 (C-13 &
C-130), 123.12 (C-12 & C-120), 138.61 (C-11), 138.70 (C-110),
142.14 (C-8), 142.31 (C-80), 151.92 (C-10 & C-100), 169.65 (C-15 &
C-150), 194.15 (C-5), 206.01 (C-3). Mass: Calculated for formula
C
27H34N2O8: 514.2315; Found: [M++1] = 515.4629.
2.2. General method for synthesis of curcumin–t-Boc amino acid
conjugates
2.5. Antibacterial studies of tetrahydrocurcumin–amino acid
conjugates
A solution of curcumin (2 g, 5.43 mmol) in dioxane (25 ml) was
taken in
a
round-bottomed flask. t-Boc amino acid (a–g,
The antibacterial activity of THC–amino acid conjugates was
tested essentially by the method described by Negi, Jayaprakasha,
Rao, and Sakariah (1999). Bacillus cereus (F 4810, Public Health Lab-
oratory, London, UK), Staphylococcus aureus (FRI 722, Public Health
Laboratory, The Netherlands), Escherichia coli (MTCC 108, Microbial
Type Culture Collection, Institute of Microbial Technology, Chandi-
garh, India) and Yersinia enterocolitica (MTCC 851, Microbial Type
Culture Collection, Institute of Microbial Technology, Chandigarh,
India) were sub-cultured in BHI broth and incubated for 24 h at
37 °C. After incubation cells were harvested by centrifugation
13.58 mmol) was added gradually to ensure its complete solubility.
4-Dimethylaminopyridine (5.43 mmol) was added to reaction mix-
ture and stirred for 5 min. After this N,N0-dicyclohexylcarbodiimide
(13.58 mmol) was added and set for stirring until the completion
of reaction (ꢀ7–12 h) under nitrogen atmosphere. The progress
of the reaction was monitored by TLC analysis. After the comple-
tion of reaction dicyclohexylurea was filtered off. The filtrate was
mixed with chloroform (25 ml) and subjected to aqueous workup.
The organic layer was separated, passed through anhydrous

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J.R. Manjunatha et al. / Food Chemistry 139 (2013) 332–338
Table 1
Amino acid conjugates of tetrahydrocurcumin.
Entrya
Product
Time in h
Yield (%)b
2a
(i) 11
(ii) 05
(iii) 01
85
80
>95
14′
14
O
OH
1
7
9
9′
5
3
O
O
O
O
O
10′
10
11
8
8′
13′
6
2
4
17′
17
13
16
16′
15
15′
11′
O
12
12′
NH2
NH2
2b
(i) 12
(ii) 4.5
(iii) 1.5
90
85
>95
O
OH
O
O
O
O
O
O
NH2
O
NH2
2c
(i) 08
(ii) 05
(iii) 0.5
75
71
>95
O
O
OH
OH
O
O
O
O
O
O
H
N
H
N
2d
(i) 10
(ii) 04
(iii) 01
92
90
>95
O
O
O
O
O
NH2
NH2
NH2
2e
(i) 07
95
O
OH
OH
(ii) 03
(iii) 0.5
90
>95
O
O
O
O
O
O
NH2
2f
(i) 12
75
O
(ii) 5.5
(iii) 0.5
60
>95
O
O
O
O
H2N
NH2
O
O
2g
(i) 12
(ii) 5
(iii) 1.5
90
86
>95
O
OH
O
O
O
O
O
O
NH2
NH2
a
Conjugates of THC with alanine (2a), isoleucine (2b), proline (2c), valine (2d), phenylalanine (2e), glycine (2f) and leucine (2g).
Isolated yields of products of steps (i), (ii) and (iii) as specified in Fig. 1.
b
(5000 rpm, 10 min) and serially diluted in saline solution (0.85%
NaCl, w/v) for use in antibacterial assay. Test samples were pre-
pared by dissolving the THC and its amino acid conjugates in
DMSO. To flasks containing 20 ml of melted warm agar, different
concentrations of test material (equivalent amount of DMSO in
TA 1538, Microbial Type Culture Collection, Institute of Microbial
Technology, Chandigarh, India) through the standard plate incor-
poration test as described by Maron and Ames (1983). In the anti-
mutagenicity studies on THC–amino acid conjugates, the inhibition
of mutagenicity of the sodium azide by the test samples was calcu-
lated by determining the number of His+ revertants in the plate.
The test samples (313, 625, 1250, 2500, 3750, 5000, 6250 and
control) and 100 l
l (about 103 cfu/ml) of the test organism were
added and the contents poured onto sterilised Petri plates after
thorough mixing. The plates were incubated at 37 °C for 24 h.
The colonies developed after incubation were counted and the
inhibitory effect calculated using the following formula:
7500 lg) were assayed by plating with molten soft agar (0.6%,
2 ml) containing 0.5 mM of histidine/biotin (0.2 ml) and 0.1 ml of
10 h old culture of either S. typhimurium TA 98 or S. typhimurium
TA 1538 on minimal glucose agar plates. Sodium azide was used
% inhibition ¼ ð1 ꢁ T=CÞ ꢂ 100
as a diagnostic mutagen (1.5 lg/plate) in positive control and
where T is cfu/ml of test sample and C is cfu/ml of control.
The minimum inhibitory concentration (MIC) was determined
as the lowest concentration of the compound inhibiting the com-
plete growth of bacterium being tested. The growth inhibition
studies for each compound were done in duplicate and the exper-
iments were repeated three times.
plates without sodium azide and without test samples were con-
sidered as negative controls. His+ revertants were counted after
incubation of the plates at 37 °C for 48 h. Each sample was assayed
using duplicate plates and the data were presented as mean SD of
three independent assays. The mutagenicity of sodium azide in the
absence of test samples was defined as 100% or 0% inhibition. The
calculation of percentage inhibition was done according to the fol-
lowing formula
2.6. Antimutagenicity tetrahydrocurcumin–amino acid conjugates by
Ames test
% Inhibition ¼ ½1 ꢁ T=Mꢃ ꢂ 100
The antimutagenicity of THC–amino acid conjugates was stud-
ied using the tester strains of Salmonella typhimurium (TA 98 and
where T is number of revertants per plate in presence of mutagen
and test sample, and M is number of revertants per plate in positive

J.R. Manjunatha et al. / Food Chemistry 139 (2013) 332–338
335
control. The number of spontaneous revertants (negative controls)
was subtracted from the numerator and the denominator. The anti-
mutagenic effect was considered weak when the inhibitory effect
was less than 25%, medium when the inhibitory effect was 25–
40% and strong when the inhibitory effect was more than 40% (Ik-
ken et al., 1999).
proline, valine, phenylalanine, glycine and leucine were prepared
in 68%, 77%, 53%, 83%, 86%, 45% and 77% yields, respectively. The
corresponding THC conjugates (2a–2g) were obtained from the
reduction of respective curcumin–amino acid conjugates in high
yields (43–82%, Table 1). The compounds were characterised by
their 1D and 2D 1H and 13C NMR and high resolution mass spectral
data. The distinguishing feature of all the 1H NMR spectra in THC
conjugates of the various amino acids was the presence of the
methylene protons in the hepta-3,5-dione portion of the molecule
as multiplet for protons attached to C-1, C-2, C-6 and C-7 in the
2.10–2.93 ppm range. This clearly indicated the reduction of the
double bond of curcumin–amino acid conjugate. In few cases
methylene protons of C-2 and C-6 registered as triplets, while pro-
tons of C-1 and C-7 collectively appeared as multiplet integrating
to 4 protons. In case of the THC–alanine conjugate (2a), the 13C
NMR spectra exhibited the signals at 30.11, 32.33, 40.59 and
45.70 corresponding to methylene carbons. Signals related to
vinylic carbon (C-5) and carbonyl (C-3) appeared at 194.15 and
206.01 respectively. The carbon peaks at 16.52, 49.89 and 169.65
corresponds to methyl (C-17, 170), methine (C-16, 160) and car-
bonyl (C-15, 150) of alanine. The magnetically equivalent carbons
of aromatic and amino acid moieties appeared together indicating
the symmetric nature of the molecule. However, the quaternary
carbon signals of C-8 & C-80 and C-11 & C-110 registered small dif-
ferences in their chemical shift values. The structure of the com-
pound was further ascertained by 2D NMR spectral correlations
and high resolution mass spectral studies.
2.7. Statistical analysis
All the experiments were repeated three times and the data
were calculated as mean SD. The data of all the assays were ana-
lysed by one-way ANOVA. Duncan’s multiple range test (DMRT)
was used to make the comparisons among various treatments.
3. Results and discussion
3.1. Synthesis of amino acid conjugates of THC
The reaction of THC (1, Fig. 1) with t-Boc amino acids was inves-
tigated as per the protocol for synthesis of conjugates of curcumin
with t-Boc amino acids reported earlier (Parvathy et al., 2010). It
was observed that the reactions were slower and the yields of
the products were relatively low. More importantly, the efforts to
de-protect the t-Boc group from the THC–t-Boc amino acid conju-
gates were not successful as the reaction led to the cleavage of the
ester group resulting in the formation of THC. Hence, an alternative
route had to be delineated for the synthesis of amino acids conju-
gates of tetrahydrocurcumin (THC). In the present study, a path-
way has been envisaged to THC conjugates via the selective
reduction of the double bonds alpha to carbonyl groups in the cur-
cumin moiety in the curcumin–amino acid conjugates (Fig. 1).
While, such an approach was successful in obtaining THC from cur-
cumin, the reduction in the presence of a labile ester group in cur-
cumin–amino acid conjugates has never been tried earlier.
Interestingly, the selective reduction of double bonds in the pres-
ence of carbonyl moiety occurred smoothly when the reaction of
curcumin–amino acid conjugates with hydrogen was carried out
under pressure in presence of palladium on barium sulphate cata-
lyst. Accordingly, conjugates of curcumin with alanine, isoleucine,
These structural features along with signals for other carbons
and protons of the aliphatic and aromatic moieties of the respec-
tive amino acid and aryl groups were clearly discernable in the
spectra for conjugates of THC with other amino acids. These new
compounds were fully characterised by the concurrence of the cal-
culated masses with those obtained by high resolution mass spec-
tral analyses.
3.2. Antibacterial properties of THC–amino acid conjugate
THC and its amino acid conjugates (2a–2g) were tested for their
antimicrobial potential against two Gram-positive (B. cereus and S.
O
OH
OH
O
O
O
O
O
O
O
O
O
O
R
i
2
HO
+
R
R
NHBoc
HO
OH
NH-Boc
NH-Boc
a - g
ii
Curcumin
t-Boc amino acids
H2 Pd/BaSO4
O
OH
O
O
O
O
O
OH
R
R
O
O
O
O
1a - 1g
iii
NH2
NH2
HO
OH
1
Tetrahydrocurcumin
O
OH
O
O
O
a,1a, 2a: R = -CH3 ; b, 1b, 2b: R = -CH(CH3)C2H5
c, 1c, 2c: R = -(CH2)3- as part of pyrrolidine ring;
d, 1d, 2d: R = -CH(CH3)2 ; e, 1e, 2e: R = -CH2(C6H5);
f, 1f, 2f: R = -H; g, 1g, 2g: R = -CH2CH(CH3)2
;
O
R
R
O
O
2a - 2g
NH2
NH2
oC, 3-5.5 h, 60-90%;
i
ii
Reagents and condition: ( ) DCC, DMAP, Dioxane, RT, 7-12 h, Inert, 75-95%; (
) 10% TFA in DCM, 0
iii
(
) H2, Pd/BaSO4, 30 psi, 0.5-1.5 h, > 95%
Fig. 1. Synthesis of THC–amino acid conjugates.

336
J.R. Manjunatha et al. / Food Chemistry 139 (2013) 332–338
Table 2
teria, they were still much lower than the corresponding values for
THC (1723 and 2114 mol, respectively).
MIC of THC–amino acid conjugates against Gram-positive and Gram-negative
bacteria.
l
Fig. 2(A–D) depicts the growth inhibition of bacteria by THC (1)
and its amino acid conjugates. In case of B. cereus (Fig. 2A), 2f
showed highest inhibition and 1 showed least inhibition of growth
at both concentrations (100 and 150 mg/l). The growth inhibition
pattern by all the compounds was similar (2f > 2d > 2a > 2c >
2b > 2e > 2g > 1) at both concentrations, however, statistically
(p < 0.05) similar inhibition of growth was shown by 2a and 2c,
2b and 2e at 100 mg/l; and 2e and 2g at 150 mg/l. As 150 mg/l
was higher than the MIC values for 2d and 2f, both showed 100%
inhibition of growth at this concentration.
Compounds
MIC (lMol)
B. cereus
S. aureus
E. coli
Y. enterocolitica
1
1066
340
334
309
263
337
257
334
1329
437
459
353
482
487
360
376
1723
583
543
485
526
600
514
501
2114
777
585
618
657
750
617
585
2a
2b
2c
2d
2e
2f
2g
Compound 2f also showed highest inhibition of growth of S.
aureus (Fig. 2B), however, minimum inhibition was shown by 2g
(which was statistically [p < 0.05] lower than 1) at both concentra-
tions. At 100 mg/l, 2f and 2c showed highest but statistically
(p < 0.05) similar inhibition of growth followed by 2a, which was
followed by 1, 2b, 2d and 2e (all showed statistically (p < 0.05) sim-
ilar inhibition of growth) and least inhibition was shown by 2g,
whereas the trend for growth inhibition was 2f > 2c > 2d > 2a =
2b = 2e > 1 > 2g at 150 mg/l. For E. coli (Fig. 2C) and Y. enterocolitica
(Fig. 2D), highest inhibition of growth was shown by 2f and mini-
mum inhibition was shown by 1 at 200 and 300 mg/l, however,
growth inhibition shown by 1 against Y. enterocolitica was statisti-
cally (p < 0.05) similar to 2d and 2g at 200 mg/l (Fig. 2D). In case of
E. coli, 2a showed statistically (p < 0.05) similar inhibition as 2f at
200 mg/l. At 300 mg/l, 2a, 2c, 2d, 2f and 2g showed 100% inhibition
of growth (Fig. 2C). The trend of growth inhibition shown by vari-
ous compounds at two different concentrations was in concurrence
with their MIC values (Table 2).
aureus) and two Gram-negative bacteria (E. coli and Y. enterocoliti-
ca). The minimum inhibitory concentration (MIC) values of THC
and its amino acid conjugates are presented in Table 2. All the
compounds showed appreciably higher activity than THC. The
THC–glycine and THC–valine conjugates (2f and 2d) were the most
potent against B. cereus as they exhibited the lowest MIC values of
257 and 263
values in the range of 309–340
much lower than that of THC (1066
with 2c and 2g also showed higher antibacterial activity (353–
376 mol) against S. aureus, which was much lower than that of
THC (1329 mol). In case of Gram-negative bacteria, E. coli and Y.
enterocolitica, MIC values for all the compounds were in the range
of 485–583 and 585–777 mol, respectively. Although these MIC
values were higher than the values against the Gram-positive bac-
l
mols, respectively. Other compounds showed MIC
mol against B. cereus, which was
mol). The compound 2f, along
l
l
l
l
l
100
100
90
80
70
60
50
40
30
20
10
0
a
a
b
a
A
a
B
90
c
b
80
d
a
b
c
e
e
70
60
50
40
30
20
10
0
a
c
c
d
d
de
e
b
d
d
f
c
c
c
c
f
e
d
f
100 mg/l
150 mg/l
100 mg/l
150 mg/l
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
a
a
a
a
a
a
D
C
b
a
a
b
c
b
b
b
a
b
bc
b
c
c
c
c
c
c
c
d
d
d
d
d
d
e
200 mg/l
300 mg/l
200 mg/l
300 mg/l
Fig. 2. Antibacterial activity of tetrahydrocurcumin and tetrahydrocurcumin–amino acid conjugates against (A) Bacillus cereus, (B) Staphylococcus aureus, (C) Escherichia coli
and (D) Yersinia enterocolitica at two different concentrations.
not significantly (p < 0.05) different.
1
2a 2b 2c 2d 2e 2f 2g. Values at each concentration followed by same letter in each strain are

J.R. Manjunatha et al. / Food Chemistry 139 (2013) 332–338
337
a
a
a
a
a
a
a
a
(a)100
a
a
a
a
100
80
60
40
20
0
a
b
ab
abc
b
b
b
bc
80
60
40
20
0
c
b
c
a
d
ab
b
bc
d
b
bc
c
c
d
e
d
e
313 625 1250 2500 3750 5000 6250 7500
Concentration (µg / plate)
313 625 1250 2500 3750 5000 6250 7500
Concentration (µg / plate)
a
a
a
a
(b)100
a
a
a
a
a
100
80
60
40
20
0
a
ab
b
b
a
80
60
40
20
0
c
ab
b
b
c
c
b
d
e
d
d
e
313
625
1250
2500
3750
5000
313
625
1250
2500
3750
5000
Concentration (µg / plate)
Concentration (µg / plate)
Fig. 3. Inhibitory effect of tetrahydrocurcumin and tetrahydrocurcumin–amino acid conjugates against the sodium azide induced mutagenicity in (a) Salmonella typhimurium
TA 98 and (b) Salmonella typhimurium TA 1538. (2a) (2b) (2c) (2d) (2e) (2f) (2g) THC (1). Values at each concentration
followed by same letter in each strain are not significantly (p < 0.05) different.
Earlier workers reported that certain curcumin bio-conjugates
containing esters and peptides showed enhanced antifungal and
antibacterial activities, which was attributed to better cellular
uptake, increased cellular concentration and better receptor
binding (Kapoor, Narain, & Misra, 2007). We also observed that
THC–amino acid conjugates had antimicrobial activities much
higher than THC against all the bacteria tested in the present
study. Apparently, the amino acid portion of the derivatives ren-
ders the THC conjugates hydrophilic which assists in the cellular
uptake of the covalently bound THC by the bacterial cells. The
higher antimicrobial activities of THC conjugates could thus be
attributed to their higher concentration per se in the bacterial
cell or THC that could be liberated in vivo from breakage of
the ester bond. While comparing the activity of individual com-
pounds against bacteria, it was observed that all the compounds
showed higher MIC values against Gram-negative bacteria than
3.3. Antimutagenicity studies of THC–amino acid conjugates
THC–amino acid conjugates showed significantly (p < 0.05)
higher antimutagenic activity against sodium azide induced muta-
tion in both the tester strains (S. typhimurium TA 98 and TA 1538)
than THC (1) at all the tried concentrations (313–7500 lg/plate,
Fig. 3). The highest antimutagenic activity was shown by 2g
against TA 98 strain, whereas 2d was most effective against
TA1538 strain. THC showed strong antimutagenic activity at
3750 and 1250
respectively, whereas similar activity was observed for 2c and 2d
at 313 g/plate concentration in both the strains. The antimuta-
lg/plate concentration in TA 98 and TA 1538
l
genic activity of THC and its conjugates was concentration depen-
dent, and significant (p < 0.05) variation among conjugates was
observed (up to 2500
in TA-1538).
lg/plate in TA 98 and up to 1250 lg/plate
Gram-positive ones.
A
similar trend of higher resistance to
Several natural compounds act as potent antimutagenic agents
(Jayaprakasha, Negi, & Jena, 2006; Jayaprakasha, Negi, Jena, & Rao,
2007; Negi, Jayaprakasha, & Jena, 2010). Curcumin (Goud, Polasa, &
Krishnaswamy, 1993) and its amino acid conjugates (Parvathy
et al., 2010) are also reported to be good antimutagens. In the pres-
ent study, it was observed that the conjugation of amino acids in-
creases the antimutagenic efficacy of THC, as its conjugation with
amino acids may facilitate its transport across the membrane bar-
rier. This would improve its intracellular uptake in bacteria as ob-
served with the curcumin derivatives (Kumar, Dubey, Tripathi,
Fujii, & Misra, 2000; Mishra, Narain, Mishra, & Misra, 2005).
Gram-negative bacteria than Gram-positive bacteria has been
observed earlier (Nostro, Germano, D’Angelo, Marino & Cannatel-
li, 2000). This could be due to their differences in cell structure
as the Gram-positive bacteria contain an outer peptidoglycan
layer, which is not a good permeability barrier (Scherrer and
Gerhardt, 1971). In case of Gram-negative bacteria, the outer
phospholipidic membrane is impermeable to lipophilic solutes,
and porins present in membrane act as a selective barrier to
the hydrophilic solutes making them comparatively more resis-
tant to antibacterial compounds (Nikaido and Vaara, 1985).

338
J.R. Manjunatha et al. / Food Chemistry 139 (2013) 332–338
Khopde, S. M., Priyadarshini, K. I., Guha, S. N., Satav, J. G., Venkatesan, P., & Rao, M. N.
4. Conclusion
A. (2000). Inhibition of radiation-induced lipid peroxidation by
tetrahydrocurcumin: Possible mechanism by pulse radiolysis. Bioscience
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In conclusion, a facile and efficient synthetic procedure has
been outlined for the synthesis of several novel amino acid conju-
gates of tetrahydrocurcumin (THC). The results of their in vitro bio-
logical evaluation established that these amino acid conjugates of
THC exhibited significant antibacterial and antimutagenic proper-
ties. The effects revealed that the conjugation of the phenolic group
to amino acid moiety via an ester linkage improved the antibacte-
rial and antioxidant attributes of THC. The molecules may have po-
tential food and pharmacological applications.
Kumar, S., Dubey, K. K., Tripathi, S., Fujii, M., & Misra, K. (2000). Design and synthesis
of curcumin-bioconjugates to improve systemic delivery. Nucleic Acids
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Kumar, S., Narain, U., Tripathi, S.,
& Misra, K. (2001). Synthesis of curcumin
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Maron, D. M.,
& Ames, B. N. (1983). Revised methods for the Salmonella
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Mishra, S., Narain, U., Mishra, R., & Misra, K. (2005). Design, development and
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Acknowledgements
receptor status in type
2 diabetic rats: Studies on insulin binding to
erythrocytes. Journal of Bioscience, 33(1), 63–72.
JRM thanks the Indian Council of Medical Research, New Delhi,
India for the scholarship. The authors are grateful to Dr. G. Venk-
ateswara Rao, Director, CFTRI and Dr. M. C. Varadaraj, Head, Hu-
man Resources Development, CFTRI for their support and
constant encouragement for the work. The help of Mr. Padmere
Mukund Lakshman in the MS studies of the compounds is grate-
fully acknowledged.
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