(Copper-curcumin) β-cyclodextrin vaginal gel: Delivering a novel metal-herbal approach for the development of topical contraception prophylaxis

Article abstract of DOI:10.1016/j.ejps.2014.09.019

Delivering a safe and effective topical vaginal contraceptive is the need of present era. We explored the potential of a metal (copper) and herbal moiety (curcumin) for this topical contraceptive prophylaxis. Complex of copper and curcumin (Cu-Cur) was synthesized and the concerns regarding its aqueous solubility was resolved by including it into the hydrophobic cavity of β-cyclodextrin (β-CD) as (Cu-Cur)CD inclusion complex. Dose assessment was made on the basis of in-vitro spermicidal assays and cell cytotoxicity studies. Finally the (Cu-Cur)CD loaded vaginal gel was prepared, characterized and evaluated for in-vitro spermicidal activity and preclinical toxicity studies. Spectral and morphological characterizations confirmed the synthesis of (Cu-Cur) and (Cu-Cur)CD inclusion complex. Spermicidal assays and Hela cell cytotoxic data revealed an optimized 1.5% (Cu-Cur)CD for further studies. 1.5% w/w (Cu-Cur)CD loaded carbopol 974p gel provided 100% motility even at 2-fold dilution and preclinical toxicity studies in Rats and Rabbits revealed its highly safe profile. The hypothesis of considering metal-herbal complex and its cyclodextrin complex has worked and the well planned strategy of including it in (β-CD) cavity provided a preeminent platform for vaginal delivery. In-vitro assays and preclinical toxicity analysis confirmed its potential to be used as highly safe and effective prophylaxis.

Full text of DOI:10.1016/j.ejps.2014.09.019

European Journal of Pharmaceutical Sciences 65 (2014) 183–191
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
(Copper–curcumin) b-cyclodextrin vaginal gel: Delivering a novel
metal–herbal approach for the development of topical contraception
prophylaxis
Chauhan Gaurav a, Rath Goutam a, Kesarkar N. Rohan b, Kothari T. Sweta b, Chowdhary S. Abhay b,
Goyal K. Amit a,
⇑
a DBT Lab, Indo-Soviet Friendship College of Pharmacy, Moga, Punjab, India
b Department of Virology, Haffkine Institute for Training Research and Testing, Parel, Mumbai, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 April 2014
Received in revised form 17 August 2014
Accepted 19 September 2014
Available online 28 September 2014
Delivering a safe and effective topical vaginal contraceptive is the need of present era. We explored the
potential of a metal (copper) and herbal moiety (curcumin) for this topical contraceptive prophylaxis.
Complex of copper and curcumin (Cu–Cur) was synthesized and the concerns regarding its aqueous sol-
ubility was resolved by including it into the hydrophobic cavity of b-cyclodextrin (b-CD) as (Cu–Cur)CD
inclusion complex. Dose assessment was made on the basis of in-vitro spermicidal assays and cell cyto-
toxicity studies. Finally the (Cu–Cur)CD loaded vaginal gel was prepared, characterized and evaluated for
in-vitro spermicidal activity and preclinical toxicity studies. Spectral and morphological characterizations
confirmed the synthesis of (Cu–Cur) and (Cu–Cur)CD inclusion complex. Spermicidal assays and Hela cell
cytotoxic data revealed an optimized 1.5% (Cu–Cur)CD for further studies. 1.5% w/w (Cu–Cur)CD loaded
carbopol 974p gel provided 100% motility even at 2-fold dilution and preclinical toxicity studies in Rats
and Rabbits revealed its highly safe profile. The hypothesis of considering metal–herbal complex and its
cyclodextrin complex has worked and the well planned strategy of including it in (b-CD) cavity provided
a preeminent platform for vaginal delivery. In-vitro assays and preclinical toxicity analysis confirmed its
potential to be used as highly safe and effective prophylaxis.
Keywords:
Copper–curcumin
b-Cyclodextrin
Nano-inclusion complex
Spermicidal gel
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
mer, polycarbophil with the most widely used spermicide available
i.e. nonoxynol-9 (Raymond et al., 2004). But deleterious effects of
An unintended pregnancy is a pregnancy that is mistimed,
unplanned, or unwanted at the time of conception (Santelli et al.,
2003). Worldwide, 38% of pregnancies were unintended in year
1999 and present scenario is touching the altitude of 41% (Singh
et al., 2010). Condoms are considered as the best way to tackle this
problem. But its denial, inconsistent or incorrect use and failure
favor this misfortune. Further, a large population is not comfort-
able with oral contraceptives; this creates a thought of having a
women oriented approach which can be easily used to avoid this
epidemic. Bioadhesive polymers based contraceptive gels in con-
nection with condoms could be an alternative to hormonal contra-
ception. Contraceptive gel is an example of such patented
technology which combines bioadhesive polymers such as carbo-
nonoxynol-9 on vaginal epithelium limit its use (Catalone et al.,
2004).
The stake of copper in the form of copper T has been in use for
over five decades, touting a greater than 99% pregnancy prevention
rate. Focusing on a particular aspect relating the deleterious effect
of copper on sperm explains the rationale behind this research
work. Copper ions prevent pregnancy by inhibiting the movement
of sperm, because the copper-ion-containing fluids are directly
toxic to sperm and inhibit the sperm’s motility and viability. As
mentioned in literature copper ions affect the integrity of sperm
in multiple ways. One aspect defines its mechanism by interacting
with the cells lipid bilayer, leaking the contents of the cell into the
surrounding environment. There are reports conforming the dele-
terious effect of copper ions on the biochemical structures and con-
found enzyme structures, making them useless (Ortiz and
Croxatto, 2007). Moreover, the studies explaining the overall effect
of copper ions revealed that copper causes the uterus and fallopian
⇑
Corresponding author at: Department of Pharmaceutics, ISF College of Phar-
macy, Moga, Punjab, India.
E-mail address: amitkumargoyal1979@gmail.com (G.K. Amit).
http://dx.doi.org/10.1016/j.ejps.2014.09.019
0928-0987/Ó 2014 Elsevier B.V. All rights reserved.

184
C. Gaurav et al. / European Journal of Pharmaceutical Sciences 65 (2014) 183–191
tubes to produce a fluid that contains white blood cells, copper
ions, enzymes, and prostaglandins, a combination that is toxic to
sperm.
the supernatant after centrifugation at 1500 rpm and then recov-
ered by lyophilizer (Alpha 1–2 LD plus, Martin Christ, Germany).
At constant process conditions (stirring time, stirring speed and
temperature), (Cu–Cur) loading inside (b-CD) cavity was analyzed
in all the cases and the saturation level was taken as the optimum
(Cu–Cur) percentage in complex formation for further studies. (Cu–
Cur) loading inside the complex was analyzed using DMSO based
extraction method. Optimized inclusion complex was character-
ized by IR, 1H NMR, morphology (Scanning electron microscope,
JSM-840 SEM, Jeol, Japan) and DSC.
Curcumin (diferuloyl methane) is the second component of our
study. Inhibitory effect of curcumin on sperm function, fertilization,
and fertility is reported in a study. (Naz, 2011) Curcumin also inhib-
its human sperm motility, function and fertility both in-vitro and
in-vivo. Total loss in forward motility as well as concentration
dependent inhibition of capacitation and acrosome reaction provide
a strong basis to use it in contraceptive microbicide formulation.
In this research we aimed to explore the potential of Metal
Ligand (M–L) complex of Copper (M) and curcumin (L) in the area
of vaginal contraception. Copper–curcumin (Cu–Cur) complex had
already been synthesized by many researchers (Barik et al., 2005;
Chauhan et al., 2013; Krishnankutty and Venugopalan, 1998;
Zhao et al., 2010). Aqueous solubility of the Cu–Cur complex pre-
sented a serious problem for its therapeutic research. The first task
of this research work is to solve the issues of poor aqueous solubil-
ity. Here we resolved this problem by including the (Cu–Cur) com-
plex in (b-CD) cavity. Lyophilized (Cu–Cur)CD nano-inclusion
complex is evaluated for vaginal epithelial cell cytotoxicity and
spermicidal activity. Optimum dose selected on the basis of the
cytotoxicity and efficacy data is then incorporated in carbopol-
974p gel. (Cu–Cur)CD loaded gel is then explored for vaginal safety
aspects by pre-clinical toxicology study on Wistar Rats, Rabbits
and vaginal lactobacillus bioflora. Spermicides capable of killing
100% human sperm almost instantaneously at physiological con-
centrations in vitro are likely to provide adequate pregnancy pro-
tection in vivo.
2.2. Spermicidal assays
2.2.1. Modified Sander-Cramer assay (sperm motility inhibition assay)
Human sperm were obtained from consenting three healthy
donors after 72 h of abstinence. The samples were washed once
in Ham’s F10 containing 0.1% human serum albumin (HSA) and
centrifuged. The sperm were resuspended to give a concentration
of 60 million motile sperm per milliliter. Spermicidal activity of
(Cu–Cur) complex, (b-CD) and (Cu–Cur)CD was evaluated. Briefly,
in case of (Cu–Cur) complex, sequential dilutions (5–100 lg/ml)
were prepared by dissolving the complex in a minimum volume
of DMSO and further diluting it with Ham’s F10. Ten sequential
dilutions of (b-CD) and (Cu–Cur)CD ranging (2–20) mg/ml were
prepared in Ham’s F10. Tergitol NP-9 was used as positive control,
while medium containing no test compounds was employed as
negative control. Aliquots of sperm 50
in the presence of 5% CO2) with various concentrations of test
ingredients in a final volume of 100 L. At different time points
ll were incubated (37 °C
l
of 0 s, 30 s, 60 s and 120 s the reaction was terminated with
1.5 mL of Ham’s F10. After 10 min centrifugation at 290g, the
sperm pellet was obtained, which was further resuspended in
2. Materials and methods
100 lL of Ham’s F10. Sperm motility was assessed manually under
inverted phase-contrast Microscope (MKX-41, Olympus, Japan)
(Gupta et al., 2005; Sander and Cramer, 1941).
Curcumin, cupric acetate, Dimethyl sulfoxide (DMSO), b-Cyclo-
dextrin (b-CD), Tergitol, tetrahydrofuran (THF) and Triethanola-
mine, Propidium iodide was purchased from Sigma Aldrich, 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), Nutrient
Mixture F-10 Ham, Lactobacillus MRS Agar (MRS Agar) was
purchased from Himedia Laboratories, Carbopol 974p was pro-
cured as a gift sample from Lubrizol (Belgium), L. acidophilus and
L. jensenii were procured from IMTECH Chandigarh.
2.2.2. Hypo-osmotic swelling test
Hypo-osmotic swelling test is based on the loss of semi-perme-
ability of the intact cell membrane, after an exposure to membrane
attacking moiety (Jeyendran et al., 1984). (Cu–Cur)CD treated sper-
matozoas were exposed to a HOS solution (75 mM fructose and
20 mM sodium citrate) for at least 30 min at 37 °C to detect
changes in the sperm membrane integrity. The number of sperma-
tozoa showing characteristic tail curling or swelling was counted
under inverted phase-contrast Microscope (MKX-41, Olympus,
Japan).
2.1. Synthesis, optimization and characterization of copper–curcumin
(Cu–Cur) and copper–curcumin-b-cyclodextrin (Cu–Cur)CD inclusion
complex
25 ml Methanolic solution of cupric acetate (0. 220 g, 4 mmol)
was added into the 13.5 ml of methanolic solution of curcumin
(0.185 g, 2 mmol). Dark reddish brown precipitates were produced
immediately. The mixture was refluxed for 2 h under a nitrogen
atmosphere. The solid product was then filtered, washed with cold
methanol and water to remove the residual reactants, and then the
product was dried in vacuum overnight. The compound is
characterized by UV absorption spectroscopy (UV–Visible spectro-
photometer, Shimadzu, Japan), IR (FTIR Spectrophotometer,
Nicolet-380, Thermo, USA), 1H NMR (Bruker Avance II 400 NMR)
and DSC (Mettler Toledo DSC 822e). Inclusion complex was pre-
pared by solvent evaporation encapsulation method; with slight
modifications as reported in the previous research (Yallapu et al.,
2010). (b-CD) 100 mg was dissolved in 20 mL deionized water in
a 50 mL beaker containing a magnetic bar. (Cu–Cur) complex in
different concentrations, (10, 20, 30, 40 and 50)% was dissolved
in 1 ml tetrahydrofuran (THF) was added to (b-CD) solution under
stirring at 600–800 rpm. Stirring was done at ambient temperature
for 12 h in dark with a perforated aluminum foil covering for THF
evaporation. Highly water soluble (Cu–Cur)CD was separated from
2.3. In-vitro cell viability assay
HeLa cell line (cervical) was obtained from National Center for
Cell Sciences (NCCS), Pune, India. Cells were cultured in Dulbecco’s
modified eagle medium (DMEM) supplemented with 10% (v/v)
heat inactivated fetal bovine serum. Cells were maintained in 5%
CO2 humidified incubator at 37 °C. During subculture, cells were
detached by trypsinization when they reached 80% confluence
and split (1:4). Growth medium was changed every 3 days.
Two different assays were performed for the determination of
cytotoxicity of the above synthesized components i.e. optimized
(Cu–Cur)CD, (Cu–Cur) and (b-CD). Propidium iodide based selec-
tive non-viable cell sorting was done by using flow-cytometer.
MTS assay cell viability assay was performed to study both specific
on Hela cells and non-specific on HEL (Human embryonic lung),
VERO, CRFK (Crandell-Rees Feline Kidney cells), MDCK (Madin
Darby canine kidney) cells) toxicity. This experiment (MTS assay)
was performed in Belgium (Laboratory of Virology and Chemother-
apy, Department of Microbiology and Immunology, Rega Institute).

C. Gaurav et al. / European Journal of Pharmaceutical Sciences 65 (2014) 183–191
185
2.3.1. Nonviable cells sorting assay (Propidium iodide staining)
Propidium iodide (PI) is a membrane impermeable dye that is
generally excluded viable cells from non-viable ones. It binds to
double stranded DNA by intercalating between base pairs. PI stain-
lowing the similar procedure the sperm motility was assessed
manually under inverted phase-contrast Microscope (MKX-41,
Olympus, Japan).
ing solution was prepared using 10 lg/ml PI in PBS stored at 4 °C in
the dark. Hela cells were treated with sequential dilutions of
2.5. Pre-clinical toxicology study of 1.5% (Cu–Cur)CD gel
(Cu–Cur)CD, (Cu–Cur) and (b-CD) for 24 h maintaining the final cell
Study was performed in 18 female Wistar rats and 6 female
Albino rabbits according to the protocol recommended by the US
Food and Drug Administration (US-FDA) for products meant for
vaginal use (Stone, 2009; Talwar et al., 2008).
concentration of 1 Â 106 cells/100
untreated as well as treated Hela cells were taken in FACS tubes
and washed with PBS. Cells were then resuspended in 100 L of
flow-cytometry buffer. 5 l Of PI staining solution to a control tube
l
l. After that 100
l
l
of
l
l
of otherwise unstained cells and a gentle mixing is followed by
1 min incubation. Finally dead cell sorting was done by using (BD
Accuri C6) flowcytometer.
2.5.1. On female Wistar rats
Animals were divided in 3 groups G-1, G-2 and G-3. Each group
consist 6 animals with avg. weight in the range of 160–220 g. G-1
animals were treated with the 1.5% (Cu–Cur)CD gel while G-2 was
treated with placebo gel and G-3 was kept as control group.
300 mg Of intravaginal application was done twice a day for
21 days in G-1 and G-2. Vagina was examined macroscopically
for signs of irritation, inflammation, ulceration, vaginal histology
and hematology (Stone, 2009).
2.3.2. Cell viability evaluation (MTS assay)
The (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazolium) dye reduction assay in
the presence of phenazinemethosulfate (PMS), produces a forma-
zan product that has an absorbance maximum at 490–500 nm in
phosphate-buffered saline. The MTS assay is often described as a
‘one-step’ MTT assay was performed to determine the cytotoxicity
of the optimized (Cu–Cur)CD, Cu–Cur and (b-CD) in Hela, HEL,
VERO, CRFK, MDCK cell cultures.
2.5.2. Standard rabbit vaginal irritation test
Animals were divided in two groups each comprised of 3 ani-
mals. One group is treated with 1.5% (Cu–Cur)CD gel and other
with placebo once daily for 10 days. A standard procedure and
scoring system was used to assess the irritating properties of the
formulation in the rabbit model. Vagina was examined macroscop-
ically for signs of irritation, inflammation and ulceration (Dhondt
et al., 2005).
2.4. Preparation and characterization of (Cu–Cur)CD loaded gel
Nano-herbal gel was formulated using 1.5% w/v of (Cu–Cur)CD
in 1% w/v carbopol 974p gel (pH of the gel is maintained at 4.5
using triethanolamine). The developed nano-herbal gel formula-
tion was characterized for macroscopic properties, rheological
analysis using (Brookfield Rheometer), mucoadhession, spreadibil-
ity using (Brookfield Texture analyzer CT3 10K) (Garg et al., 2003).
2.5.3. Lactobacillus toxicity screening
This study is a part of toxicity screening, for the acceptability of
any intra-vaginal product (Fichorova and Anderson, 1999;
Fichorova et al., 2001a,b). Using two lactobacilli strains i.e. L. aci-
dophilus and L. jensenii, anti-lactobacillus effect of 4 serial dilutions
of 1.5% (Cu–Cur)CD gel was seen. Firstly, lactobacillus strains were
2.4.1. Rheological analysis
Rheology of gels will affect both their distribution and retention
in the vaginal cavity. The study is carried out using R/S-CPS + Rhe-
ometer, Brookfield. This study is performed under the thixotropic
protocol, shear rate was set in the range of 100–500 (1/s) for a total
time interval of 10 min (5 min for both ascending and descending
mode) using C75-1 measuring system at 37 °C.
cultured on sterilized MRS broth. Then 50 ll of broth was trans-
ferred to sterilized plates containing MRS agar. After 30 min, wells
were made and 4 serial dilutions of 1.5% (Cu–Cur)CD gel were
transferred to these wells. Plates were incubated at 32 °C, after
48 h. Anti-Lactobacillus activity was expressed in terms of diame-
ter of zone of inhibition (in mm).
2.4.2. Mucoadhession and spreadibility
Mucoadhession and spreadibility was evaluated using Brook-
field Texture analyzer CT3 10K. Mucoadhession study was per-
formed on a special testing assembly with TA10 cylinder probe
(12.7 mm D, 35 mm L) and maintains the temperature of 37 °C.
Fresh got vaginal epithelium was placed both inside the mucoad-
hession assembly and on the bottom of the probe. 1 ml Gel was
placed in the cavity of assembly and the test was run in the com-
pression mode setting the target of 100 g and holds time of 15 s
and the trigger load of 3 g. Test and return speed was kept
0.5 mm/s. Spreadibility is assessed by using a male and female
cone assembly setup. Male cone was completely filled with the
gel formulation and the test was run at ambient temperature. Test
was run in the compression mode setting the target of 4 mm and
holds time of 2 s, trigger load of 3 g, test and return speed was kept
0.5 mm/s.
2.6. Statistical analysis
Each experiment was repeated three times using sperm sample
from three different donors and the data were analyzed by one-
way analysis of variance using the GraphPad Prism software (Ver-
sion 3.0). P-value less than 0.05 were considered statistically
significant.
3. Results
3.1. Synthesis, optimization and characterization of copper–curcumin
(Cu–Cur) and copper–curcumin-b-cyclodextrin (Cu–Cur)CD inclusion
complex
Reddish brown colored (Cu–Cur) complex was obtained and
this synthesized complex was characterized for various parame-
ters. UV spectral study in DMSO revealed a single peak for Cur at
432 nm and two peaks for Cu–Cur complex at 433 and 456 nm.
IR (KBr) cmÀ1: spectral data of curcumin and (Cu–Cur) reveals
the formation of new bands at 535 cmÀ1 and 477 cmÀ1 in (Cu–
Cur) spectra indicating the interaction between copper(II) and oxy-
gen (O) atom of curcumin (Kolev et al., 2005). 1H NMR (DMSOd):
2.4.3. Modified Sander-Cramer assay (sperm motility inhibition assay)
for 1.5% (Cu–Cur)CD gel
The study was performed in similar manner as described above
for (Cu–Cur)CD. 4 Serial dilutions of 1.5% (Cu–Cur)CD gel in Ham’s
F10 were tested for the spermicidal activity.
Tergitol NP-9 was used as positive control while medium con-
taining no test compounds was employed as negative control. Fol-

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C. Gaurav et al. / European Journal of Pharmaceutical Sciences 65 (2014) 183–191
spectral data of curcumin and (Cu–Cur) revealed the absence of
characteristic peaks of curcumin (6–8 ppm) in the spectra of the
metal complex, indicating the chelation with the metallic ion.
Metal ion interaction with the keto-enolic region of curcumin moi-
ety is an evident observation from new IR peaks; moreover the
absence of some key hydrogen peaks justified the tampering of
keto-enolic group. DSC thermogram showed a sharp peak for the
melting of (Cu–Cur) around 197.3 °C, which indicates the forma-
tion of crystalline metal complex.
(Cu–Cur)CD inclusion was prepared according to the procedure
mentioned and further it was optimized on the basis of loading of
(Cu–Cur) inside the b-CD hydrophobic cavity. (Cu–Cur) loading
was estimated for each batch of inclusion complex. Table 1
explains the preparation and optimization of inclusion complex
on the basis of (Cu–Cur) inclusion. Optimization study revealed
that lyophilized batch (MC-40) and (MC-50) have shown compar-
atively higher as well as identical (Cu–Cur) loading (around
(Cu–Cur)CD complex (MC-40) appears as light greenish yellow
colored fluffy powder. This fluffy appearance points toward its
amorphous nature. Aqueous saturation solubility of the (MC-40)
at ambient conditions was found to be around 38.5 1.4 mg/ml.
UV spectra in deionized water showed an absorption maxima at
265 nm.
IR (KBr) cmÀ1 spectra revealed the major existence of (b-CD)
specific peaks with a slight shift to higher/lower wave numbers
while only few characteristic peaks of (Cu–Cur) were observed.
1H NMR spectra again displayed the same dominance as (MC-40)
showed all the peaks relevant to (b-CD). But inclusion of (Cu–
Cur) results in the shifting of proton signals to high field region.
DSC thermograms are indicative of complexation of (Cu–Cur)
within cyclodextrin cavity. DSC thermograms of (MC-40) com-
plexes showed a substantial reduction in the intensity of the peak,
broadening of peak and a shift to lower temperature (136–143 °C)
in comparison to sharp melting point of pure (Cu–Cur). Marked
reduction in intensity accompanied by broadening and shift to a
lower temperature of the endotherm indicates the substantial
inclusion of (Cu–Cur) in the (b-CD) cavities. Scanning electron
microscopy clearly justifies the above observation of having a
highly amorphous nature of (MC-40). Fig. 1 displays a descriptive
view of the FT-IR, 1H NMR, DSC analysis of (Cu–Cur), (b-CD) and
(Cu–Cur)CD as well as UV spectral and SEM image of (Cu–Cur)CD.
24 lg/mg of complex) and % yield (88–90 mg). This clearly indi-
cates the saturation of (Cu–Cur) inclusion inside the available
b-CD cavities after 40% (Cu–Cur) trial. Rationally, formulation
(MC-40) was considered as optimum for further characterization
and other studies.
Table 1
Optimization table of (Cu–Cur)CD complex (n = 3).
3.2. Spermicidal assays
Batch
b-CD (mg) (Cu–Cur)% Yield (mg)
Loading of (Cu–Cur)
(Cu–Cur)CD per mg of (Cu–Cur)CD (
l
g)
3.2.1. Modified Sander-Cramer assay (sperm motility inhibition assay)
Scoring the motile and immotile sperm under the phase con-
trast microscope revealed that (Cu–Cur) and (Cu–Cur)CD complex
presented both concentration and time dependent effect as
mentioned in Fig. 2. (Cu–Cur) potentially immobilized human
sperms irreversibly at minimum effective concentration (MEC) of
MC-10 100
MC-20
MC-30
MC-40
MC-50
10
20
30
40
50
63
76
81
88
90
6.2 1.4
7.8 0.4
17.4 1.1
24.2 2.6
24.5 1.7
Fig. 1. Characterization of copper–curcumin (Cu–Cur) and copper–curcumin-b-cyclodextrin (Cu–Cur)CD inclusion complex. (A) IR spectral overlay of copper II acetate,
curcumin and (Cu–Cur), (B) IR spectral overlay of b-CD and (Cu–Cur)CD inclusion complex, (C) 1H NMR spectras of curcumin and (Cu–Cur), (D) characterization parameters
for (Cu–Cur)CD including UV spectra, DSC, NMR, 1H NMR spectras and SEM picture.

C. Gaurav et al. / European Journal of Pharmaceutical Sciences 65 (2014) 183–191
187
Fig. 2. Spermicidal assays reports (A, B and C) displays the sperm mobility inhibition assay data of (Cu–Cur), b-CD and (Cu–Cur)CD respectively. (D) Reports the HOS positive
sperms percentage for control (untreated), (Cu–Cur) and (MC-40) treated sperms (n = 3).
2.2 0.4
90 s. In comparison to the (Cu–Cur), (MC-40) showed relatively
higher MEC of 290 15 g/ml and complete immobilization at
14 mg/ml after 90 s. b-CD displayed MEC of 680 15 g/ml with
l
g/ml and complete immobilization at 40
l
g/ml after
MCC (minimum cytotoxic concentration) of 1.8 0.2 and CC50
(concentration for 50% cell cytotoxicity) of 14.5 0.75 g/ml.
While both (MC-40) and b-CD both showed a highly nontoxic nat-
ure with MCC of 400 0.20 g/ml and 750 0.12 g/ml. Fig. 3 dis-
l
l
l
l
l
only around 15% inhibition at relatively high concentration of
plays the cell death pattern revealed after this study.
20 mg/ml.
3.3.2. Cell viability evaluation (MTS assay)
Irrespective of the specificity view the CC50 of 13.4 2.9 lg/ml
3.2.2. Hypo-osmotic swelling test
marks the innate toxicity of (Cu–Cur) on all challenged cells. Unre-
latedly, the (b-CD) and (MC-40) showed negligible toxicity describ-
ing their highly safe nature at both specific and non-specific levels.
Table 2 provides the descriptive view of the MTS assay on five dif-
ferent cell cultures.
Percentage of tail curling observed in control is quite high
(83.8 2.5)%, while after treatment with 40 lg/ml of (Cu–Cur)
and 14 mg/ml (MC-40) the tail curling of spermatozoa was signif-
icantly reduced to (18.0 1.8)% and (26.0 2.2)%. The loss of HOS
responsiveness after these treatments indicated the compromised
sperm membrane integrity, suggesting an overall loss of sperm
membrane physiology. Fig. 2 shows the HOS responsiveness of
the sperm toward all three abovementioned observations, indi-
cated by tail curling.
3.4. Characterization of (MC-40) loaded gel
The prepared gel was homogenous greenish-yellow clear gel,
with good lubrication when textured on hand but non-greasy.
3.3. In-vitro cell viability assay
3.4.1. Rheology
3.3.1. Nonviable cells sorting assay (Propidium iodide staining)
Specific toxicity studies on cervical Hela cells revealed some
interesting outcomes. (Cu–Cur) showed potential cell death with
Thixotropic analysis of gel formulation revealed its pseudoplas-
tic flow with a very narrow deformation at the end of the descend-
ing protocol. Viscosity values at shear rate of 100–500 (1/s) ranges

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C. Gaurav et al. / European Journal of Pharmaceutical Sciences 65 (2014) 183–191
Fig. 3. Specific Hela cell cytotoxicity data of (Cu–Cur), b-CD and (Cu–Cur)CD. (Done by flowcytometry based non-viable cell sorting assay (n = 3).
Table 2
Nonspecific cell cytotoxicity data on Hela, HEL, VERO, CRFK, MDCK cells as determined by measuring the cell viability with the colorimetric formazan-based MTS assay. (MCC is
the challenged concentration required to cause a microscopically detectable alteration of normal cell morphology and CC50 is 50% cytotoxic concentration) (n = 3).
Compound
Hela
MCC
HEL
VERO
MCC
CRFK
MCC
MDCK
MCC
CC50
MCC
CC50
CC50
CC50
CC50
l
g/ml
(Cu–Cur)
(MC-40)
b-CD
4
>100
>100
16.3
>100
>100
1.8
>100
>100
13.5
>100
>100
2.9
>100
>100
15.2
>100
>100
2.6
>100
>100
11.4
>100
>100
3.2
52.3
65.7
10.6
>100
>100
Table 3
from 0.1 Pa s to 0.29 Pa s. Viscosity falls in the range of most of the
vaginal products such as KY JellyÒ Lubricant), Lacta-GynecogelÒ
Acidifying gel and CanastenÒ Antimycotic (Brouwers et al., 2008).
Sperm motility inhibition study of different dilutions of 1.5% (MC-40) loaded gel
(n = 3).
Dillution
% Inhibition
time (0 s)
% Inhibition
time (30 s)
% Inhibition
time (60 s)
% Inhibition
time (90 s)
3.4.2. Mucoadhession and spreadibility
(No dillution) Â
68 ( 8)
43 ( 3)
26 ( 2)
23 ( 8)
22 ( 5)
100
100
100
100
96 ( 6)
87 ( 3)
81 ( 4)
Mucoadhession detachment force of (Cu–Cur)CD (MC-40)
loaded gel was found to be 17.5 0.4 g/cm2. Spreadibility of the
loaded gel was found to be 23.5 (gm cm/s).
2Â
78 ( 5)
44 ( 4)
37 ( 2)
25 ( 7)
92 ( 2)
72 ( 4)
54 ( 12)
49 ( 3)
4Â
8Â
16Â
3.4.3. Modified Sander-Cramer assay (sperm motility inhibition assay)
for 1.5% (MC-40) gel
as after 4Â dilution the complete inhibition has not been observed
even after the time span of 90 s.
Four serial dilutions of 1.5% (Cu–Cur)CD gel in Ham’s F10 were
tested for the spermicidal activity at different time intervals after
exposure. Table 3 represents the percentage of motility inhibition
of different dilutions after different time intervals. Study revealed
that undiluted formulation is providing complete inhibition of
sperm motility within the time span of 30 s; while the two fold
dilution i.e. 2Â took up to 90 s to render the sperms completely
immotile. Increase in dilution have a direct impact on the activity
3.5. Pre-clinical toxicology study
3.5.1. Pre-clinical toxicology study on female wistar rats
Examining the structural integrity of the vaginal epithelium
after 21 days experimental protocol revealed that there was no
macroscopic sign of any kind of redness, edema and inflammation,

C. Gaurav et al. / European Journal of Pharmaceutical Sciences 65 (2014) 183–191
189
Fig. 4. Biopsy and histopathology images of vaginal epithelium. G-1 animals were treated with the 1.5% (MC-40) gel while G-2 was treated with placebo gel and G-3 was kept
as control group.
Moreover during the protocol period, no sign of irritation was
observed. Microscopic examination of histopathology samples
shows the presence of stratified squamous epithelium in all sam-
ples with no sign of any type of damage. Fig. 4 shows the biopsy
images and microscopic images of histopathology samples (G-1,
G-2, G-3 single tan colored tissue). This study revealed the absolute
safety of formulation regarding blood chemistry. Haematogram
results from animal of each group showed similarity in CBC (com-
plete blood count) and DLC (differential leucocyte count).
two peak UV spectra (as a result of p ?
p⁄ electronic transitions
and charge transfer) indicated the chelation of copper with the
1,3-diketone moiety (which transform automatically to a more sta-
ble keto-enol tautomeric form) of curcumin as explained in
Fig. 5(a). IR, 1H NMR study further confirms this chelation and
DSC provides a conformation of its crystalline nature with melting
point slightly higher than pure curcumin.
This metal–ligand complex possesses a serious issue regarding
its aqueous solubility. Affinity of this hydrophobic chelate toward
the hydrophobic b-CD cavity is utilized to render it water soluble.
Supramolecular chemistry approach has been utilized for prepar-
ing this inclusion complex via solvent evaporation encapsulation
technique. Mass transfer process of hydrophobic (Cu–Cur) moiety
inside the hydrophobic cyclodextrin cavities was dependent on
the concentration of (Cu–Cur) presented during the inclusion pro-
cess. But the flux for the (Cu–Cur) inclusion and the amount of
inclusion is directly proportional to the availability of the inclusion
space. At constant process conditions (stirring speed stirring time,
temperature and b-CD concentration) and inclining (Cu–Cur) con-
centration, there must be a saturation level for the further inclu-
sion of (Cu–Cur). Optimization process clarified that 40% (Cu–
Cur) is required for the saturation of available b-CD cavities.
(MC-40) is thus selected as the optimized batch for further charac-
terization. Absence of (Cu–Cur) specific peaks in IR, 1H NMR spec-
tras confirmed the inclusion of (Cu–Cur) inside the b-CD cavities.
Lyophilization of the highly soluble (Cu–Cur)CD effects the crysta-
linity and finally results in a highly amorphous product. Fig. 5(b)
explains the probable structure of this inclusion complex and it
is given on the supposition that this complex will fits the same
conformation as mentioned for curcumin-b-CD complexes.
(Yallapu et al., 2010).
3.5.2. The standard rabbit vaginal irritation test
No macroscopic alteration in the vaginal morphology was
observed. No sign of redness, inflammation and edema confirms
the safety of vaginal gel.
3.5.3. Lactobacillus toxicity screening
Study conducted on two lactobacillus strains i.e. L. acidophilus
and L. jensenii assure the safety margins of the gel formulation.
Table 4 reveals the data of zone of inhibition against the two
strains. Minor signs inhibition was seen only in the case of undi-
luted formulation while afterward dilution fails to make any
impact on any of the two strains. Keeping the safety aspects in
mind this formulation is very much safe for the vaginal contracep-
tive use.
4. Discussion
Spectral characterization confirmed the synthesis of metal–
ligand Cu–Cur complex of copper with curcumin. Characteristic
Table 4
The facts emerged in our study points toward the direct effect of
(Cu–Cur) and (MC-40) on the intact sperm membrane. The
expected depletion in the potency measures is the virtue of
limiting the exposure of (Cu–Cur) by including it in dextrin cavity.
Comparing the spermicidal potential of inclusion complex with
plain b-CD clarified this point. Further HOS test clarifies the impact
of both (Cu–Cur) and (MC-40) on disturbing the normal sperm
membrane functioning. Literature provides multiple approaches
Zone of inhibition study conducted on two lactobacillus strains i.e. L. acidophilus and
L. jensenii (n = 3).
Formulation dilution
Zone of inhibition
Zone of inhibition
(L. acidophilus)
(L. jensenii)
No dilution
1.9 ( 0.4 mm)
No-zone
No zone
1.4 ( 0.2 mm)
No-zone
No zone
2Â
4Â
8Â
No-zone
No-zone

190
C. Gaurav et al. / European Journal of Pharmaceutical Sciences 65 (2014) 183–191
Fig. 5. Synthesized (Cu–Cur) and (Cu–Cur)CD. (a) Structure of (Cu–Cur) where metal is expected to bind with 1,3-diketone moiety of curcumin. (b) Probable inclusion of
(Cu–Cur) inside the hydrophobic b-CD cavities.
by which curcumin and copper ions displays spermicidal nature.
Moreover, reports are also available displaying the effect of b-CD
on membrane structure and function of sperm. Thus, exact mech-
anism responsible or the spermicidal activity of both these moie-
ties needs further studies and explanations.
Toxicity study on Hela cells presents a perfect profile for a prod-
uct intended to be used in vagina. (Cu–Cur) showed potential tox-
icity to these cells with CC50 value of 14.5 0.75 lg/ml. From the
safety and activity point of view this curcumin chelate is having
the toxic concentration lower than the effective concentration.
The case is different during the exposure of (MC-40), which the
quite similar toxicity profile as compared to b-CD (a GRAS excipi-
ent). Non-specific cell cultures responded in similar manner with
Acknowledgement
Author acknowledges Department of Biotechnology (DBT) India
and Punjab State Council of Science and Technology (PSCST) India
for providing the financial base for this research. Author also
acknowledges the insightful discussions provided by Dr Jitender
Bhariwal (Department of Medicinal Chemistry, ISF-CP, Moga, Pun-
jab, India) and technical support from Prof. R. Snoeck & Prof. G.
Andrei (Laboratory of Virology and Chemotherapy, Department of
Microbiology and Immunology, Rega Institute, Belgium.
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