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Research Communication
Dose-dependent bioavailability indicators for
curcumin and two of its novel derivativesMohamed Abd el Aziz
Mohamed El-AsmerAmeen Rezek
Abdulrahman Al-Malki
Taha KumosaniHanan Fouad
Hanan AQ2 Ahmed
Fatma Taha *
Amira Hassouna
and Hafez Hafez

Abstract
Novel water-soluble curcumin derivatives have been devel-
oped to overcome low in vivo bioavailability of curcumin. The
aim of this work is to assess the potential utility of certain
downstream targets as bioavailability indicators of systemic
activity of pure curcumin and two novel water-soluble curcu-min derivatives (NCD) by constructing dose-dependent
response curves and to prove whether this novel curcumin
derivatives retained, improved, or abolished biological activityof pure curcumin when applied in vivo . Pure curcumin (CUR),
curcumin-carboxy derivative (NCD-1), and curcumin protein
conjugate (NCD-2) were administered orally to rats at escalat-ing doses: 37, 74, 148, and 296 lM/kg body weight, respec-
tively. Plasma levels of GST activity, cavernous tissue levels ofcGMP, andAQ3
enzymatic activity of both HO-1 and GST were
assessed one-and-half hour and 24 h AQ4 after oral administration
of curcumin formulae. This study showed that there was a
progressive elevation of cavernous tissue levels of cGMP and
enzymatic activity of both HO-1 and GST in a dose-dependentmanner that was maintained for 24 h with CUR, NCD-1, and
NCD-2. Except for the lowest doses on the curve, plasma GST
activity was decreased. The three dose-dependent bioavailabil-ity indicators as surrogates of curcumin and two of its novel
derivatives are valid in the studied range of concentration and
extended time. The novel curcumin derivatives still retain withimprovement the biological activity of natural curcumin when
applied in vivo . AQ5
VC2013 BioFactors, 00(00):000–000, 2013
Keywords: curcumin; NCD; GST; cGMP; HO-1; bioavailability
indicators
1. Introduction
Early clinical trials with molecular targeting agents should
include the measurement of biomarkers to assess the pharma-codynamic effect of the molecular target and the relevant
downstream components that may correlate with pharmaco-
logic response.
Curcumin, a hydrophobic polyphenol derived from the rhi-
zome of the herb Curcuma longa has a wide spectrum of bio-
logical and pharmacological activities [1]. Curcumin has beenshown to prevent cancer in the colon, skin, stomach,
duodenum, soft palate, and breasts of rodents after oral
administration [2–4]. Mechanisms by which curcumin prevents
cancer are thought to involve upregulation of carcinogen-detoxifying enzymes such as glutathione S-transferases [5,6],
antioxidation [7,8], and suppression of oxidative DNA adduct
(M1G) formation [9].
The pharmacokinetic properties of curcumin indicated
that curcumin undergoes metabolism by conjugation and
reduction, and it exhibits poor systemic bioavailability afteroral dosing [10,11]. Curcumin has been granted an acceptable
daily intake of up to 3 mg/kg based on genotoxic studies in two
generations of Wistar rats, with parenteral intake of 250–320mg/kg BW with no observed adverse effect level by the joint
FAO and WHO expert committee on food additives [12].
The systemic bioavailability of orally administered
curcumin is invariably low, as curcumin and/or its metabolites
could not be detected in plasma at oral doses lower than
3.6 g/day [13].VC2013 International Union of Biochemistry and Molecular Biology, Inc.
Volume 00, Number 00, Month/Month 2013, Pages 00–00
*Address for correspondence: Fatma Taha. E-mail:
fatmatahaus@yahoo.com. AQ12
Received 2 March 2013; accepted 7 May 2013
DOI 10.1002/biof.1118
Published online 00 Month 2013 in Wiley Online Library(wileyonlinelibrary.com)
BioFactors
1J_ID: BIOF Customer A_ID: BIOF1118 Cadmus Art: BIOF1118 Ed. Ref. No.: 13-0041.R1 Date: 29-May-13 Stage: Page: 1
ID:hariv Time: 17:45 I Path: N:/3b2/BIOF/Vol00000/130025/APPFile/JW-BIOF130025

The improvement of the bioavailability of curcumin is a
challenge. It could be improved by different techniques such
as heat treatment [14] or solubilizing in dilute alkali [15].
Bioavailable formulation of curcumin has been developed.
Two novel water-soluble curcumin derivatives with conserved
natural functional groups (NCD-1 and NCD-2) were developed inour laboratories through covalent modification of the curcumin
molecule on sites remote from its natural functional groups.
2. Aim of Work
This study was conducted to assess three dose-dependent
bioavailability indicators as surrogates of curcumin and two ofits water-soluble novel derivatives. These indicators include
cavernous cGMP levels, heme oxygenase-1 enzyme activity in
addition to cavernous, and plasma glutathione S-transferase
enzyme activities, determining whether these derivatives could
retain, improve, or abolish biological activity of natural curcu-
min when applied in vivo .
3. Materials and Methods
3.1. Reagents and Chemicals
Pure curcumin [l,7-bis(4-hydroxy-3-methoxyphenyl)-l,6-hepta-
diene-3,5-dione] was purchased from Sigma (St. Louis, Missouri).
Two novel water-soluble curcumin derivatives with con-
served natural functional groups (NCD-1 and NCD-2) were
developed and were internationally registered under the rights
of the Patent Cooperation Treaty [16,17].
NCD-1 [1,7-bis(5-carboxyphenylazo-4-hydroxy-3-
methoxyphenyl) 21,6-heptadiene-3,5-dione] was used for the
synthesis of the novel curcumin protein conjugate (NCD-2)
through the use of l-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide hydrochloride.
3.2. Experimental Animals
The study was carried on 170 male rats, of an average weight
150–200 gm from an inbred colony (Curl: HEL 1) at the Kasr
Al-Aini experimental animals unit, Faculty of Medicine, CairoUniversity, Giza, Egypt. Rats were bred and maintained in air-
conditioned pathogen-free conditions, with 12:12-h daylight/
darkness cycles and allowed free access to chow and water.Ethical protocols for animal treatment were followed with
approval from the Institutional Animal Care Committee.
The rats were divided into 17 groups, each of 10 rats: a
control group and four duplicated groups that received
orally pure curcumin at doses equivalent to 37, 74, 148, and
296 lM/kg body weights, respectively. In addition, another
four duplicated groups received orally the water-soluble cur-
cumin derivatives (NCD-1 and NCD-2) on the same equimolar
bases. One-and-half hour and 24 hours after oral administra-tion of the respective doses of each compound, venous blood
samples were withdrawn from the tail veins on tubes contain-
ing EDTA to measure plasma GST activity. Then, the experi-mental groups were sacrificed and individual cavernoustissues were obtained for measurement of cGMP, HO-1, and
GST activities.
3.2.1. Glutathione S-transferase enzyme activity
Plasma and cavernous tissue levels of GST were assessed by
the commercial kit supplied by Bio-Diagnostic (Giza, Egypt)according to the manufacturer’s recommendations. Glutathi-
one S-transferase assay kit was used to measured total GST
activity by measuring the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The conjugation is
accompanied by an increase in absorbance at 340 nm. The
rate of increase is directly proportional to the GST activity inthe sample [18].
3.2.2. cGMP assays
Frozen cavernous tissue samples (50 mg) stored in a specificcell lysis buffer were grounded with a stainless steel mortar,
homogenized, and centrifuged at 600 gat 4
/C14C for 10 min. The
supernatant was used for cGMP assay with the ELISA kit sup-plied by R&D Systems (Minneapolis, MN) [19].
3.2.3. HO enzyme activity assay
Cavernous tissue samples were homogenized with 3 volume
homogenization buffer. The homogenate was centrifuged at
3000 rpm for 4 min and then at 14,000 rpm for 5 min at 4/C14C
to produce the mitochondrial pellet. The supernatant was
withdrawn. The protein content was determined by the
method proposed by Lowry et al. [20]. The activity of HO inthe supernatant was determined as previously described
by Abraham et al. [21]. Briefly, the supernatants were
incubated at 37
/C14C for 1 h with heme (50 mmol/L), rat liver
cytosol (5 mg/mL), MgCl 2(2 mmol/L), glucose-6-phosphate
dehydrogenase (1 U), glucose-6-phosphate (2 mmol/L), and
reduced nicotinamide adenine dinucleotide phosphate (0.8mmol/L) in 0.5 mL of 0.1 mol/L phosphate buffer saline (pH
7.4). Reaction was stopped by placing the tubes on crushed
ice, then the bilirubin generated was extracted by chloroform,and its amount was determined with a scanning spectropho-
tometer and was defined as the difference between the absorb-
ance at 463 and 520 nm using a standard bilirubin curve.
3.3. Calculations and Statistical Analysis
All mathematical and statistical analyses were performedusing the software package “Statistica-8” of Statsoft (Tulsa,OK). Results of the one-and-half-hour and the 24-h postdosing
response curves of curcumin (CUR) and its novel derivatives
(NCD-1 and NCD-2) for all studied parameters were based onmean6SD percentage change relative to the basal normal
control levels.
Cubic (third-order) polynomial curve fitting was applied to
construct the nonlinear dose-response curves of percentage
changes of the selected bioavailability indicators to each of
CUR and the NCDs. The general form of the equation isy5a1bx1cx
21dx3. The inputs are x(the dependent variable
or dose of the test compound in lM/kg body weight) and y(the
independent variable or dose-response parameter as percent-age change from basal normal control levels).J_ID: BIOF Customer A_ID: BIOF1118 Cadmus Art: BIOF1118 Ed. Ref. No.: 13-0041.R1 Date: 29-May-13 Stage: Page: 2
ID:hariv Time: 17:45 I Path: N:/3b2/BIOF/Vol00000/130025/APPFile/JW-BIOF130025BIOFACTORS
2 Dose-dependent bioavailability indicators for curcumin 1

4. Results
The results are presented in four graphs, each accompanied
with its relevant table of curve fitting equations. The three
tested compounds had shown one-and-half-hour and 24-h
postdosing response relationships to all suggested bioavailabil-ity markers, namely, cavernous and plasma GST activities,
cavernous cGMP levels, and cavernous HO-1 activity. The
dose-response relationships followed the cubic (third-order)polynomial equation, as presented in Tables T1-T4 (1–4). In general,
the results point out to the superiority of both NCD-1 and
NCD-2 over pure curcumin in advancing the dose responsesfor all the tested bioavailability markers in both the one-and-
half-hour and the 24-h postdosing studies. It is clear from
Fig.F1 1 that a maximal percent increase of 3803.8% in cavern-
ous GST is most evident with NCD-2 in the one-and-half-hour
postdosing study. The twenty-four-hour postdosing study still
shows the same superiority with 1877.1% over NCD-1 and cur-cumin for all the studied bioavailability markers. As for Fig.F2 2,
a maximal percent increase of 77.3% in plasma GST is most
evident with NCD-2, and 47.4% with NCD-1, whereas curcu-
min followed with a score of 30.4% in the one-and-half-hour
postdosing study. The twenty-four-hour postdosing study stillshows almost the same pattern for all the studied bioavailabil-
ity markers. Except for NCD-2 in both the one-and-half-hour
and the 24-h postdosing studies, a decrease in plasmaGST has been observed with the lowermost doses on the
curve. Of special interest, in the one-and-half-hour postdosing
study, curcumin effect showed an increase of 26.6% in themiddle of the curve, but did not increase anymore on doubling
the dose to the maximum used; however, it maintained a more
or less a low effect near the basal level in the 24-h postdosingstudy.Perhaps, Fig. 3 is the simplest to comment on. Again,
NCD-2 is the most effective in inducing increases in cavernous
cGMP of 1151.7% and 825.0% for the one-and-half-hour and
the 24-h postdosing studies, respectively. However, NCD-1showed lower percentage increase of 594.6% for the one-and-
half-hour postdosing study and 511.7% for the 24-h postdosing
study. The maximal response to curcumin was 421.4% and233.7% for the one-and-half-hour and the 24-h postdosing
studies, respectively. Finally, Fig. 4 is not so different from Fig.
3 or the general trend of all other figures. The highest effect ininducing increases in cavernous HO-1 activity is referred to
Cubic polynomial AQ11 curve fitting equations for Fig. 1
StudyCubic polynomial dose-response
fitting equation
CUR 1.5 h y5395.6961 14.5366 3×10.0102 3
x224.4297E-5 3×3
NCD-1 1.5 h y5151.8554 19.6113 3×20.0337 3
x216.1654E-5 3×3
NCD-2 1.5 h y5341.0482 119.0092 3×20.0961 3
x210.0002 3×3
CUR 24 h y5560.7623 27.2835 3×10.0731 3
x220.0002 3×3
NCD-1 24 h y52 157.8142 111.6241 3×20.0635 3
x210.0001 3×3
NCD-2 24 h y52 94.3538 119.3715 3×20.1219 3
x210.0003 3x3Cubic polynomial curve fitting equations for Fig. 2
StudyCubic polynomial dose-response
fitting equation
CUR 1.5 h y518.8016 20.8282 3×10.0093
x222.0425E-5 3×3
NCD-1 1.5 h y59.3278 20.6711 3×10.0081 3
x221.7982E-5 3×3
NCD-2 1.5 h y58.8911 20.3804 3×10.0073
x221.6571E-5 3×3
CUR 24 h y57.5801 20.2758 3×10.0025 3
x225.3202E-6 3×3
NCD-1 24 h y52 9.9361 10.0947 3×10.0003 3
x226.8144E-7 3×3
NCD-2 24 h y52 1.7445 20.2115 3×10.0043
x229.268E-6 3×3
Cubic polynomial curve fitting equations for Fig. F3 3
StudyCubic polynomial dose-response
fitting equation
CUR 1.5 h y52 82.824 13.1103 3×20.0149 3
x213.4174E-5 3×3
NCD-1 1.5 h y568.9193 20.3697 3×10.0183 3
x23.7502E-5 /C2x3
NCD-2 1.5 h y5111.9849 11.2744 3×10.0125 3
x221.6775E-5 3×3
CUR 24 h y553.553 21.9343×10.0218 3
x224.4506E-5 3×3
NCD-1 24 h y534.9622 10.7833×10.0029 3
x224.1499E-7 3×3
NCD-2 24 h y56.3155 12.6973 3×20.0053 3
x211.884E-5 3x3TABLE 1TABLE 2
TABLE 3J_ID: BIOF Customer A_ID: BIOF1118 Cadmus Art: BIOF1118 Ed. Ref. No.: 13-0041.R1 Date: 29-May-13 Stage: Page: 3
ID:hariv Time: 17:45 I Path: N:/3b2/BIOF/Vol00000/130025/APPFile/JW-BIOF130025Aziz et al. 3

NCD-2 for up to 226.6% for the one-and-half-hour postdosing
study, but scored 89.7% only, which is lower than NCD-1 of
140.0% in the 24-h postdosing study. However, NCD-1 gavelower percentage increases not exceeding 203.3% with curcu-
min at the 141.4% mark for the one-and-half-hour study.
5. Discussion
Traditional bioavailability data are difficult to obtain for many
pharmacologically active phytochemicals. Bioavailability indi-
cators could provide indexes for target tissues/organ accessi-
bility for such compounds. The bioavailability indicators shouldbe selected to be directly associated with one or more pharma-
cological effect of the active phytochemical and should be eval-
uated in a dose-dependent relationship.
In this study, cavernous cGMP levels, heme oxygenase-1
activity, and cavernous and plasma glutathione S-transferase
activities were dose dependently assessed as surrogates of cur-cumin and two of its novel derivatives. The outcome shows
that the novel curcumin derivatives have retained, with
improvements, the investigated biological activities of naturalcurcumin when applied in vivo . AQ6This, in part, confirms theCubic polynomial curve fitting equations for Fig. F4 4
StudyCubic polynomial dose-response
fitting equation
CUR 1.5 h y524.5974 20.1878 3×10.0064 3
x221.4993E-5 3×3
NCD-1 1.5 h y52 82.2253 13.6343 3×20.0209 3
x214.0161E-5 3×3
NCD-2 1.5 h y550.5404 20.4007 3×10.0124 3
x223.0683E-5 3×3
CUR 24 h y52 2.2162 10.4008 3×20.0019 3
x213.6791E-6 3×3
NCD-1 24 h y52 8.8344 10.633×20.0014 3
x213.1925E-6 3×3
NCD-2 24 h y52 9.40310.5643 3×20.0009 3
x214.3003E-7 3×3
C
OL
O
R
Percent increase in plasma glutathione S-transferase
activity (U/L).
C
O
L
OR
Percent increase in cavernous cGMP (pmol/mg
protein).
C
O
L
O
R
Percent increase in cavernous glutathione S-transfer-
ase (U/mg protein/min).TABLE 4
FIG 2
FIG 3 FIG 1J_ID: BIOF Customer A_ID: BIOF1118 Cadmus Art: BIOF1118 Ed. Ref. No.: 13-0041.R1 Date: 29-May-13 Stage: Page: 4
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4 Dose-dependent bioavailability indicators for curcumin AQ1

concept of conserving all the natural functional groups upon
covalent modification of the curcumin molecule.
These findings coincided with previous studies that
assessed curcumin downstream signaling pathways. Abdel
Aziz et al. [22–25] reported similar findings. The authorsstated that water-soluble curcumin protein conjugate could
mediate erectile function via induction of HO-1 enzyme with
subsequent upregulation of cGMP levels in cavernous tissue.The inducible enzyme HO-1 metabolizes heme to biliverdin
and carbon monoxide (CO), which exhibits physiological prop-
erties similar to NO mediated in part by the ability of CO to actas an activator of soluble guanylate cyclase (sGC) [11]. AQ7 Abdel
Aziz et al. [26–29] proved that HO and its product CO domi-
nates nitric oxide (NO) as a signaling molecule in erectilefunction and could partially mediate the actions of phosphodi-
esterase inhibitors.
Meanwhile, Schini-Kerth et al. [30] stated that polyphenols
are able to induce NO, which is a well-known activator of
soluble sGC that leads to elevation of cGMP, and later, Xu et al.
[31] reported similar findings with curcumin. Abusnina et al.[32] found that curcumin treatment decreased PDE1 and PDE4
activities and dose dependently increased intracellular cGMP
levels, whereas cAMP levels were unchanged.
Several studies suggest that phase II detoxifying enzyme
genes AQ8 such as GST and HO-1 could be used as markers of sys-
temic activity of curcumin. Yang et al. [33] stated that selectiveNrf2-Keap1-antioxidant-response element (ARE) signaling
pathway activators induce the synthesis of phase II detoxifying
enzyme genes. Lee et al. [34] and Giudice et al. [35] statedthat phase II detoxifying enzyme genes are ARE-regulated
genes, and they include glutathione S-transferase and
heme oxygenase-1. The Nrf2-Keap1-ARE signaling pathwaycan be modulated by several upstream kinases, includingphosphatidylinositol 3-kinase and mitogen-activated protein
kinases which are activated by curcumin. These studies con-
firm our findings that demonstrated significant progressivedose-dependent elevation of cavernous tissue levels of heme
oxygenase activity as well as plasma levels of GST with all cur-
cumin formulae where water-soluble curcumin derivativeswere superior to pure curcumin with maintained effect for
24 h at certain previously specified dosages. AQ9
In the current study, plasma GST activity was decreased
by the lowest doses of curcumin and its two derivatives, these
results agree with the published observation that GST enzyme
activity has been shown to be upregulated or downregulatedin rat tissues after oral consumption of curcumin, depending
on the dose and route of administration [5,6,8].
GST activity was one of two biomarkers of the efficacy of
curcumin, which were evaluated in the blood of patients with
advanced colorectal cancer who received up to 180 mg of cur-
cumin per day for up to 4 months [36]. In three patients on 36mg of curcumin daily, lymphocytic activity of GST was
decreased with time to reach 41% of control (untreated) on
day 29 of treatment. This decline was not observed at thehigher dose levels and was not reproduced in a subsequent
study of higher doses in the patients with the same disease [9].
A structure–activity study of the potency of curcumin ana-
logs suggested that their ability to induce phase II enzymes
might be linked to the presence of the phenolic hydroxy and
b-diketone groups [37].
6. Conclusion
This study shows that the selected three dose-dependent bioa-
vailability indicators as surrogates of curcumin and two of itsnovel water-soluble derivatives are valid in the studied range
of concentration and extended time. This work points out to
that the novel curcumin derivatives have retained, withimprovement, the biological activity of natural curcumin when
applied in vivo . Hence, bioavailability indicators should pro-
vide indexes for target tissues/organ accessibility of curcuminand its novel water-soluble derivatives when applied in vivo .
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6 Dose-dependent bioavailability indicators for curcumin AQ1

AQ1: Please confirm whether the short title is OK as given.
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