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An Enhanced Bioavailable Formulation of Curcumin Using Fenugreek-Derived Soluble Dietary Fibre

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JOURNAL OF FUNCTIONAL FOODS 4 ( 2 0 1 2 ) 3 4 8 –3 5 7

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/jff

An enhanced bioavailable formulation of curcumin


using fenugreek-derived soluble dietary fibre

Krishnakumar IMa,*, Abhilash Ravia, Dinesh Kumara, Ramadasan Kuttanb,


Balu Maliakela
a
Akay Flavours & Aromatics Ltd., R&D Centre, Ambunadu, Malayidamthuruthu P.O., Cochin 683561, India
b
Amala Cancer Research Centre, Amala Nagar P.O., Thrissur 680 555, India

A R T I C L E I N F O A B S T R A C T

Article history: Poor intestinal absorption has been regarded as a major limiting factor for the therapeutic
Received 20 June 2011 use of curcumin, the primary active constituent of turmeric. Herein, we investigated the
Received in revised form bioavailability of a novel formulation of curcumin-impregnated soluble dietary fibre disper-
23 December 2011 sions (BR213 curcumagalactomannosides) comprising an extensive gel forming and non-
Accepted 4 January 2012 digestible soluble galactomannan fibre derived from the spice fenugreek. The dispersions
Available online 8 February 2012 were prepared as microgranulates of mean particle size 150 ± 20 lm by an ultrasound med-
iated gel-phase dispersion technique. In vitro release studies at pH 1.2 and 6.8 showed slow
Keywords: and prolonged release of colloidal curcumin from the amorphous microgranulates of cur-
Bioavailability cumin-impregnated soluble fibre. Enhanced bioavailability of the new formulation was fur-
Curcumin ther demonstrated in animals (Wistar rats) and human volunteers in comparison with
Controlled-release unformulated curcumin. It was observed that the relative absorption of curcumin from
Fenugreek
the novel fibre formulation, as evident from the area under curve calculations, was 20 times
Soluble fibre
higher in animals and 15.8 times higher in humans when supplemented orally. Maximum
Ultrasound
absorption was also found to be prolonged as compared to the unformulated curcumin.
 2012 Elsevier Ltd. All rights reserved.

1. Introduction 1996), arthritis (Funk et al., 2006), rheumatism (Deodhar, Sethi,


& Srimal, 1980), Alzheimer’s disease (Yang et al., 2005), Crohn’s
Turmeric (Curcuma longa L.), belonging to the family of disease (Holt, Katz, & Kirshoff, 2005), diabetes (Arun & Nalini,
Zingiberaceae, is a perennial herb native to India where its 2002) and in various types of cancer (Aggarwal, Kumar, &
rhizome is used as a yellow colorant curry spice and traditional Bharti, 2003; Dorai & Aggarwal, 2004; Hsu & Cheng, 2007;
medicine. The active principle in turmeric was identified as a Kunnumakkara, Anand, & Aggarwal, 2008; Ruby, Kuttan, Babu,
group of polyphenolic compounds, namely curcumin (74– Rajasekharan, & Kuttan, 1995). Curcumin is hepato- and neph-
78%), demethoxycurcumin (15–18%) and bisdemethoxycurcu- roprotective (Kiso, Suzuki, Watanabe, Oshima, & Hikino, 1983;
min (4–6%) commonly referred to as ‘curcumin’ (Aggarwal, Venkatesan, Punithavath, & Arumugam, 2000) with a strong
Kumar, Aggarwal, & Shishodia, 2004, chap. 23). Numerous pre- capacity to reduce proliferation of a variety of malignant cells,
clinical and clinical evaluations have confirmed many inter- to induce apoptosis and to suppress tumour initiation, promo-
esting bioactivities of curcumin in various disease states, tion and metastasis (Dorai & Aggarwal, 2004; Kunnumakkara
including thrombosis (Srivastava, Dikshit, Srimal, & Dhawan, et al., 2008). Curcumin has shown to act mainly by down-
1985), myocardial infarction (Nirmala & Puvanakrishnan, regulating the transcription factors like NF-jB, which leads to

* Corresponding author: Tel.: +91 484 2686111; fax: +91 484 2680891.
E-mail address: krishnakumar.im@akay-group.com (Krishnakumar IM).
1756-4646/$ - see front matter  2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jff.2012.01.004
JOURNAL OF FUNCTIONAL FOODS 4 ( 20 1 2) 3 4 8–35 7 349

decreased gene expression of cytokines (TNF-a, IL-1b, iNOS), dia. MilliQ Plus (Millipore) purified water was employed for
chemokines (MCP-1) and other inflammatory proteins (COX- all experiments. Sonication was performed using 1000 W
2) that are critical mediators in inflammatory diseases (Abe, ultrasound generator fitted with a sonotrode (Hieilscher,
Hashimoto, & Horie, 1999; Huang, Lysz, & Ferraro, 1991; Jobin, Germany). UV/vis analysis was performed on Varian-Cary 5000
Bradham, & Russo, 1999; Surh, Chun, & Cha, 2001). Curcumin’s UV–vis–NIR spectrophotometer (Varian Inc., Chennai, India).
activity as an anticancer, chemosensitisation and radiosensiti- Fourier-transformed infrared spectra (FTIR) was recorded on
sation agent has been shown to be by down-regulating the Shimadzu spectrophotometer 8700 using potassium bromide
MDM2 oncogene through the PI3K/mTOR/ETS2 pathway (Li, pellets prepared by compressing the powder at 20 psi for
Zhang, Hill, Wang, & Zhang, 2007). In the case of Alzheimer’s 10 min on KBr press (Shimadzu Analytical Pvt. Ltd., Mumbai,
disease curcumin offers its benefits by effectively chelating India). The spectra were scanned over the wavenumber range
with amyloid plaques forming peptides, chelating metal ions of 3600–400 cm1. Thermogram was recorded using Q10 DSC,
that cause lipid, protein and DNA oxidative damage and differential scanning calorimeter (DSC Metler Toledo 822e),
inhibiting NF-jB transcription and the production of TNF-a, Mettler-Toledo India Pvt. Ltd., Mumbai, India. Samples (3–
under conditions of oxidative damage and inflammation 5 mg) were sealed in the aluminium crimp pan, and heated
(Chan, 1995). at a rate of 10 C/min from 30 to 300 C under a nitrogen
Curcumin is extremely safe even at very high doses of atmosphere. Powder X-ray diffraction studies (PXRD) were
8–12 g/day (Lao et al., 2006). Despite the safety, efficacy and performed on a Bruker D8 Advance instrument: target Cu,
well-tracked mechanisms of action, poor systematic bioavailabil- k – 1.54 Å, filter – Ni, voltage 40 kV, time constant 5 min/s;
ity upon oral administration (low plasma levels, limited tissue scanning rate 1/min (Bruker AXS GmbH, Karlsruhe, Germany).
distribution, rapid metabolism and short half-life), is limiting Scanning electron microscopic analysis was done on SEM Jeol
its approval as a therapeutic agent (Anand, Kunnumakkara, 6390 LA equipment (JEOL Ltd., Tokyo, Japan). HPLC analyses
Newman, & Aggarwal, 2007). Though a minimum concentration were carried out on a Shimadzu LC 20 AT system, with
of 0.1 lM curcumin in plasma was established for the in vitro effi- M20A Photo diode array (PDA) detector ((Shimadzu Analytical
cacy, the oral delivery of even 3.6 g to humans could detect only Pvt. Ltd., Mumbai, India), fitted with a reverse phase C18 Phe-
nanomolar in vivo levels an hour after consumption (Sharma nomenex column (250 · 4.6 mm, 3 lm) using methanol as the
et al., 2004). Marczylo et al. (2007) have reported a plasma concen- mobile phase. LC/MS was performed on Waters HPLC (Alli-
tration of 6.5 ± 4.5 nM, 30 min after oral administration at ance system, with MassLynx software) fitted with 3100 ESI
340 mg/kg on rats. Many attempts based on adjuvant, liposomes, mass detector (Waters Ltd., Bangalore, India).
miscelles, phospholipid complexes and nanoparticles have also Curcuminoids were isolated by solvent extraction. Dried
been reported to circumvent the problems of poor bioavailability rhizomes of turmeric were powdered (2 mm) and extracted
of curcuminoids (Anand et al., 2007). with a mixture of hexane and acetone (30:70, v/v) and solvent
In the present study, the bioavailability of a novel formula- was evaporated under vacuum to produce a crude turmeric
tion of curcumin, namely BR213 curcumagalactomannosides extract, from which curcuminoids were crystallised using
(hereinafter referred to as CGM), using fenugreek (Trigonella ethanol (1:1, w/v) to more than 90% purity, as determined by
foenum-graecum)-derived soluble dietary fibre composed of gal- the method of JECFA (2003) method (Joint Expert Committee
actose and mannose units (galactomannan) was investigated on Food Additives), by measuring the absorbance at 420 nm
in animals and humans. Soluble dietary fibre is an extensive in acetone.
gel forming non-digestible hydrocolloid which undergoes Soluble fibre was isolated from fenugreek seeds by an in-
fermentation in the colon by the action of b-mannanase and house built technology (unpublished data). Briefly, fenugreek
may provide protection to curcumin from the degrading seeds were flaked and successively extracted with ethanol
enzymes of the upper gastrointestinal tract (Sinha & Rachna, to remove bitter taste and its characteristic odour. The resi-
2001). Ultrasound mediated gel-phase dispersion was em- due was dried under vacuum and then extracted with water.
ployed to produce highly amorphous curcumin-impregnated Water extract was concentrated and ethanol (1:2, v/v) was
soluble fibre microgranulates, BR213 (CGM) with enhanced added to precipitate soluble fibre. The precipitate was filtered
aqueous solubility, stability, compatibility and slow release and the process of dissolution in water and re-precipitation
kinetics under physiological pH. In vitro release of the formu- with ethanol was repeated two times to get minimum 85%
lation was assessed at pH 1.2 and 6.8 in comparison with soluble fibre content upon enzymatic–gravimetric analysis
unformulated curcumin. The enhanced bioavailability of (AOAC Official methods of Analysis, 2000, chap. 45). It was
CGM upon oral administration was then established on Wistar ground to 125 lm and dissolved in 10 times water, with the
rats and human volunteers. aid of sonication, to a uniform viscous solution.

2. Materials and methods 2.2. Preparation of curcumin-impregnated fenugreek


soluble fibre microgranulates (CGM)
2.1. Materials
Various percentage CGM were prepared by ultrasound medi-
Standard curcumin (CAS Registry No. 458-37-7) was pur- ated gel-phase dispersion of curcuminoids in fibre matrix.
chased from Sigma–Aldrich, Bangalore, India. All solvents Briefly, 1 g curcumin powder was uniformly suspended in
and reagents for analysis were of HPLC grade and that for 100 ml of water containing 0.1% weight of hydroxypropylm-
extraction were of analytical grade from Merck, Mumbai, In- ethyl cellulose and 10% (w/v) glycerine under sonication for
350 JOURNAL OF FUNCTIONAL FOODS 4 ( 2 0 1 2 ) 3 4 8 –3 5 7

20 min, at 60 C. Soluble dietary fibre powder (2.5 g) was sepa- The study protocol and comprehensive written informed con-
rately dissolved in 100 ml water with sonication to a homoge- sent were approved by the Institute Ethics Committee. The
neous viscous solution. Curcumin solution was then slowly volunteers were not allowed to take turmeric-containing food
added to the fibre solution. Care was taken to keep the tem- for 2 days prior to the test. Each volunteer was first given two
perature below 50 C during ultrasound-mediated mixing. capsules of 500 mg (500 mg · 2) of unformulated curcumin
Cloudy yellow aqueous solution thus obtained was dried un- and 2 ml blood sample were withdrawn at 0, 0.5, 1, 3, 5, 8
der vacuum at 60 C and the resulting yellow flakes were and 24 h post-dose; plasma was separated and frozen at
milled and sifted to produce uniform microgranulates of 20 C till analysis. After 1 week of curcumin feeding, the
150 ± 20 lm in size. The procedure was repeated to produce subjects were given three capsules of 500 mg (500 mg · 3) con-
CGM containing various percentages of curcumin, ca. 20, 40 taining newly formulated CGM and the protocol was repeated
and 60. exactly the same as above for collection of plasma samples at
various time intervals. The dosage of three capsules was
2.3. In vitro release studies of CGM equivalent to 600 mg of curcumin administration. The same
was repeated again after 1 week with 250 mg CGM and the
In vitro release of CGM was evaluated in PBS at pH 6.8 and in blood samples were withdrawn for analysis as mentioned
0.1 M HCl at pH 1.2 for 24 h. A known amount of CGM (50 mg) above. Two-hundred and fifty milligrams of CGM was equiva-
was dispersed in 10 ml phosphate buffer, pH 6.8, and 0.1 M lent to 100 mg of unformulated curcumin.
HCl solution, pH 2.0 and kept in a sealed thermostated water
bath set at 37 ± 0.5 C under constant shaking. Five hundred 2.5. Estimation of curcumin content in plasma samples
microlitres of solution was carefully withdrawn from the mix-
ture without contamination from the microgranulate parti- Curcumin and its metabolites from plasma were extracted
cles and made up to 50 ml with acetone and the absorbance and quantified by the procedure of Antony (2008). Briefly,
was read at 420 nm using UV/vis spectrophotometer. The 1 ml of plasma was transferred to a polypropylene tube and
concentration of the released curcumin was then calculated to this 10 ml of ethyl acetate was added and vortexed for
using a standard curve of curcumin in acetone. From the cal- 5 min, followed by centrifugation at 1957g for 15 min. The
ibration curve plotted for absorbance verses concentration, extraction was repeated three times and the combined ethyl
the release kinetics of entrapped curcumin with time was acetate layer was evaporated under vacuum. The residue
estimated. Studies were performed in triplicate and mean was then made up to 10 ml with methanol, vortexed for
cumulative percentage of released curcumin was calculated 2 min and again centrifuged at 10,350g for 5 min. The super-
and plotted against time. natant (20 ll) was injected to HPLC. Separation was effected
on a Phenomenex C18 column (250 · 4.6, 3 lm) using metha-
2.4. Animal and human studies nol as mobile phase at 1 ml/min flow rate and detection at
420 nm. Extraction efficiency of curcumin was confirmed by
Both animal and human studies were conducted at Amala spiking 10 lg/ml of standard curcumin solution in 1 ml of
Cancer Research Institute, Kerala, India, following the institu- rat plasma followed by HPLC analysis. Curcumin retention
tional and international guideline for the care and use of lab- time was confirmed by 10 repeated analyses at 20 lg/ml level
oratory animals (Registration No. 149/199/CPCSEA). Wistar on the same column under identical conditions. Measure-
rats 200–250 g body weight purchased from National Institute ment of curcumin content in plasma was validated by spiking
of Nutrition, Hyderabad, India, were housed at the animal standard curcumin in plasma at 10 and 20 lg/ml concentra-
house facility of the institute in well ventilated polypropylene tions. Extraction efficiency from plasma was 92% with a linear
cages under controlled temperature (27 ± 2 C), pressure and response of R2 value of 0.999. HPLC peak identity of curcumin
relative humidity (60–80%) and under light–dark cycle of was established by negative ion electrospray mass spectrom-
12 h. Rats were provided with pellet diet (Sai Durga Feeds etry. Analysis was performed using 3100 electrospray ionisa-
and Foods, Bangalore, India) and water ad libitum. The ani- tion mass detector coupled to a Waters high performance
mals were fasted overnight and divided into two groups for liquid chromatograph.
convenience. The first group of eight animals received unfor-
mulated curcumin and second group of eight animals re- 2.6. Toxicity studies
ceived CGM as a suspension in 0.1% carboxymethylcellulose
solution, at 250 mg/kg by oral gavage. At post-dose, animals Wistar rats (20 males and 20 females) were randomly divided
were exsanguinated under terminal anaesthesia (EMLA into four groups consisted of five male and five female rats.
cream) and blood was collected after each time point, ca. 0, Formulated curcumin BR213 (CGM) was suspended in distilled
0.5, 1, 3, 5, 8 and 24 h, respectively, into heparinised tubes water in such a way that all the animals will receive same vol-
and centrifuged at 1957g for 15 min. Plasma was decanted ume of vehicle. CGM was administered orally once per day
and stored at 20 C until analysis. After 1 week, the first using oral needle and continued for 28 days. Rats were mon-
group animals were given CGM and second group received itored, during this period for any type of clinical symptoms,
unformulated curcumin at same dosage and blood samples mortality and adverse reaction of the administered drug.
for analysis at various time points were withdrawn as before. Body weight and food consumption were determined every
Human study was conducted on eight male human volun- 5 days. The food consumption was determined by the follow-
teers, aged between 25 and 50 years, who were healthy and ing method. A known quantity of pelletised food was kept in
not involved in any medication or health supplementation. the cages and after 24 h the left over feed was weighed and
JOURNAL OF FUNCTIONAL FOODS 4 ( 20 1 2) 3 4 8–35 7 351

the difference was considered as the amount of food con- hydrocolloid to provide texture, gelling, thickening, emulsify-
sumed. This was expressed for a single cage of five animals. ing, stabilizing and encapsulating properties (Srivastava &
Animals were sacrificed on day 29 by ether anaesthesia and Kapoor, 2005). Highly purified soluble fibre has also been
blood was collected into EDTA and non-EDTA vials by direct found to offer better hardness, greater degree of cohesion over
heart puncture method. The blood was further used for assay- a longer period of time, better hydration and a controlled-
ing haematological parameters, and serum biochemistry. All release matrix when used as a pharmaceutical excipient in tab-
the organs were examined visibly for any type of abnormali- lets and capsules (Nokhodchi et al., 2008).
ties in the structure. Photographs of necropsy were taken
and selected organs, liver, kidney, brain and spleen were dis- 3.1. Preparation and characterisation of BR213 (CGM)
sected out and washed thoroughly in ice-cold normal saline
and weights were recorded. Blood was analysed for haemato- Being an efficient tool for the particle size reduction in disper-
logical parameters such as heamoglobin, red blood cells (RBC) sions and emulsions, ultrasound was employed in the present
and platelet using Haematology Diatron analyzer (Abacus Jr., study to disperse the hydrophobic curcumin in water contain-
Wein, Austria). Biochemical parameters were analysed using ing the surfactants glycerine and hydroxypropylmethyl cellu-
Hitachi 902 analyzer (Roche Diagonostics, Minato-Ku, Tokyo, lose (Zheng et al., 2010). The reduction in particle size by the
Japan). Total white blood cells (WBC) were measured after force of cavitations was followed by a Zeiss Stemi 2000-C
diluting the blood in Turk’s fluid and counting on a haemocy- Trinocular zoom stereomicroscope (Texas Nautical Repairs
tometer. For differential count, blood was spread on a clean Inc., Houston, TX, USA) of 100· magnifications, by spreading
slide and treated with Leishman’s stain. Various types of cells a drop of solution over a glass slide, at regular intervals of
were counted using a 100· microscope (Labomed LX 400, time. Curcumin particles were initially observed as crystalline
Springfield, NJ, USA). lumps, which were broken up and eventually turned into col-
loidal dispersions within 30 min sonication as pulses of 2 min
2.7. Statistics duration. Ultrasound also helped to dissolve the fibre powder
in water to form homogeneous free flowing gel phase, which
Statistical analyses were performed on all data and values are otherwise form a thick paste-like mass due to its extreme
reported as mean ± SD. Observed differences were evaluated water holding capacity. The microturbulances of ultrasonic
using t-test and p < 0.05 were required to indicate statistical cavitation also helped the colloidal curcumin to uniformly
significance. Toxicity study data was analysed by one-way disperse in the fibre matrix during mixing. A yellow viscous
ANOVA followed by post hoc test (Dunnett test) using graph solution of curcumin in fibre thus obtained was dried under
pad In Stat 3 software. All the values were compared with that vacuum at 60 C to produce curcumin-impregnated soluble fi-
of untreated control animals as well as with standard range bre dispersions as flakes, which on pulverisation and sifting
for rats. gave uniform microgranulates with mean particle size of
150 ± 20 lm. Various relative percentages of fibre were thus
3. Results and discussion incorporated ca. 20%, 40% and 60% to produce respective
CGM. The entrapped curcumin content in CGM was estimated
Commercial grade curcumin (isolated from dried rhizomes of by acetone extraction and measuring the absorbance at
turmeric by solvent extraction followed by crystallisation) 420 nm (JECFA, 2003). Curcumin was found to bind strongly
with minimum purity of 90% was used in the present study. to fibre, since the yellow colour of curcumin could not be re-
Soluble fibre was isolated from debitterised and deodorised moved completely from microgranulates even after seven
fenugreek seeds by aqueous extraction and alcoholic precipi- washings with acetone.
tation. Both methanol and ethanol were found to be suitable Stability, crystallinity and curcumin–fibre interactions
for debitterisation of fenugreek seeds. The water soluble fibre were assessed by differential scanning calorimeter (DSC),
powder thus obtained was found to contain 87% soluble fibre powder X-ray diffraction (PXRD) and Fourier-transformed
content and 8.3% protein. It swelled extensively in water with infrared spectra (FTIR) investigations of unformulated curcu-
a water holding capacity of 20 ml/g and an oil holding capac- min, fenugreek-derived soluble fibre, CGM with 20%, 40% and
ity of 3 ml/g. 60% (w/w) fibre and the corresponding physical mixtures.
Many studies have demonstrated the positive health ef- Physical mixtures were prepared by mixing appropriate per-
fects such as hypoglycemic, hypolipidemic, appetite sup- centage of curcumin and fibre powder in water without soni-
pressing and gastrointestinal benefits of fenugreek fibre cation and drying under vacuum at 60 C. DSC studies showed
(Boban, Nambisan, & Sudhakaran, 2006; Evans, Hood, Oaken a sharp endotherm at 186 C for pure curcumin due to its
full, & Sidhu, 1992; Hannan et al., 2003). Fenugreek’s soluble melting, whereas the soluble fibre exhibited no peaks. Neither
fibres were composed of linear chains of (1 ! 4) linked the nature nor the position of endotherm showed a change
b-D-mannopyranosyl residues to which a-D-galactopyranosyl when formulated as physical mixture, though a decrease in
groups are attached via (1 ! 6) glycosidic linkages (Srivastava intensity proportional to the percentage composition was ob-
& Kapoor, 2005). The physiological effects of soluble fibre were served (data not shown). All samples except pure curcumin,
attributed to its non-digestibility, fermentability and ability to showed an endothermic shift around 95 C due to the residual
swell extensively to increase the viscosity of the digest. In the moisture content in the fibre matrix. In the case of CGM, the
modern functional food segment, it was considered as a good endothermic peak broadened, reduced intensity and was
352 JOURNAL OF FUNCTIONAL FOODS 4 ( 2 0 1 2 ) 3 4 8 –3 5 7

Fig. 1 – DSC thermograms of (A) soluble fibre from fenugreek (B) curcumin-impregnated soluble fibre microgranulate
containing 60% (w/w) fibre, CGM (C) curcumin.

shifted to lower temperature with increase in fibre content. at 3415.1, 1594.2, 1689.5 and 1278.3 cm1 when formulated
The change was very evident at 60% (w/w) fibre containing to 60% (w/w) fibre containing amorphous CGM (Fig. 4).
CGM, where the endotherm appeared at 174 C, indicating
its predominantly amorphous nature (Fig. 1). 3.2. In vitro release study
PXRD studies of curcumin showed sharp and intense
peaks between 7 and 27 2h, whereas the soluble fibre gave In vitro release studies were conducted on CGM containing
typical amorphous pattern with just a broad less intense hill 60% (w/w) soluble fibre and unformulated curcumin powder
at 21 2h scattered angle. The physical mixtures showed char- at pH 1.2 and 6.8 to simulate stomach and colonic conditions
acteristic pattern of curcumin, though with an elevated base- (Fig. 5). Cumulative percentage of release of curcumin from
line and less intensity. In the case of CGM, a decrease in unformulated curcumin powder was only 0.08% (pH 6.8) and
number and intensity of the peaks with a general broadening 0.06% (pH 1.2) after 24 h, due to insolubility of curcumin in
was observed with increase in percentage of fibre. In the case water. The release testing of curcumin from CGM containing
of 60% (w/w) fibre containing CGM, characteristic sharp peaks 60% (w/w) fibre, showed percentage release of only
of curcumin observed at 15.1, 17.0, 19.3, 20.2, 21.5, 25.7 and 5.1 ± 1.2% at pH 6.8 and 3.7 ± 0.9% at pH 1.2 in the initial 5 h;
26.6 2h were disappeared and the peaks at 9.3, 12.5, 17.4, indicating an increased solubility and water compatibility of
18.5 and 23.4 2h scattered angles becomes broad and less in- curcumin when impregnated and uniformly dispersed in fibre
tense (Fig. 2). Thus it is clear that a considerable reduction in matrix, as compared to unformulated curcumin. On continu-
crystallinity of curcumin occurred in fibre matrix confirming ation of the release study, curcumin release was prolonged
the encapsulation effect and more amorphous nature of cur- and found to increase with time in a steady and sustained
cumin-impregnated soluble fibre microgranulates. This was manner, such that 28.6 ± 1.8% was observed at pH 6.8 and
further evident from SEM photographs. Curcumin showed 22.3 ± 2.1% at pH 1.2 (Fig. 5). Thus the release of curcumin
various shapes with well defined edges whereas the microg- from CGM was very little during the initial hours with a lag
ranulates prepared with 60% (w/w) fibre formed smooth, con- time of more than 5 h and the total release was less than
tinues, highly porous surface in which amorphous curcumin 30% even after 24 h. This could be attributed to the very slow
was uniformly imbibed and dispersed in continues fibre ma- initial swelling of polysaccharide chains in water and its fur-
trix (Fig. 3). The characteristic FTIR peaks of curcumin ob- ther dissolution to allow the leaching of entrapped curcumin
served as sharp peaks at 3510.1, 1699.5 and 1282.5 cm1 into the solution in the dissolution flask. Delayed lag time has
were found to be changed into broad and less intense peaks shown to provide protection to the CGM in the gastrointesti-
JOURNAL OF FUNCTIONAL FOODS 4 ( 20 1 2) 3 4 8–35 7 353

Fig. 2 – PXRD spectra of soluble fibre from fenugreek (dark) curcumin (grey – sharp peaks) and curcumin-impregnated soluble
fibre microgranulate containing 60% (w/w) fibre, CGM (dark – broad peaks).

Fig. 3 – SEM photograph of (A) curcumin (B) curcumin-impregnated soluble fibre microgranulate containing 60% (w/w) fibre,
CGM.

nal tract, as already proved in high viscosity hydroxypropylm- using an electrospray ionisation mass spectrometer in nega-
ethyl cellulose coated systems with slow drug release colonic tive ionisation mode, which could detect molecular masses
delivery (Patel, Patel, Bhadani, Shah, & Amin, 2009). corresponding to protonated curcumin at 368. Concentration
of curcumin in plasma at each time point was calculated as
3.3. Animal study an average of sixteen HPLC measurements using methanol
mobile phase which eluted total curcumin (free curcuminoids
Rats received formulated curcumin CGM containing 40% (w/ and its metabolites) as a single peak at retention time 2.9 min
w) of curcumin and unformulated curcumin at a dose of (Antony, 2008). Fig. 6 depicts the respective concentration ver-
250 mg/kg by oral gavage. Plasma samples at various time sus time curve of curcumin in rat plasma after oral consump-
points up to 24 h post-administration were subjected to tion of CGM and unformulated curcumin at a dose of 250 mg/
HPLC-UV analysis, after confirming the detected peak identity kg. The analyses of pharmacokinetic parameters such as
354 JOURNAL OF FUNCTIONAL FOODS 4 ( 2 0 1 2 ) 3 4 8 –3 5 7

of curcumin in plasma, for CGM group was 5 h as compared to


the unformulated curcumin, Tmax of 3 h. Furthermore, curcu-
min was found in the plasma even after 24 h of administra-
tion with a C24
max of 0.21 lg/g for the new formulation, CGM
group. Unformulated curcumin group, on the other hand de-
tected a residual concentration of 0.008 lg/g only (Fig. 6).

3.4. Toxicity studies

Lethal dose studies conducted on Wistar rats at a single oral


dose of 2 g/kg body weight showed no mortality, clinical and
behavioural symptoms and none of the adverse reactions
during the study period of 28 days. The body weight and food
consumption of the animals were unchanged, indicating non-
toxicity of formulated curcumin, BR213 (CGM) after single
administration. Acute toxicity studies conducted for 28 days
also indicated that CGM administration (2, 1 and 0.5 g/kg body
weight) produced no change in food consumption, water con-
sumption and body weights. Biochemical parameters related
to hepatic and renal function and haematological parameters
such as WBC, RBC, platelet, haemoglobin, cholesterol levels,
Fig. 4 – Solid state FTIR spectra of (A) soluble fibre from high density lipoprotein (HDL), low density lipoprotein
fenugreek (B) curcumin-impregnated soluble fibre (LDL), very low density lipoprotein (VLDL), triglycerides and
microgranulate containing 60% (w/w) fibre, CGM (C) differential count were also remained unchanged after
curcumin. 28 days of consumption. Necropsy of the treated animals also
showed normal appearance of various tissues (unpublished
maximum curcumin concentration in plasma (Cmax), the time data). Further studies to check the effect of newly formulated
taken to reach the maximum concentration (Tmax) and con- curcumin, BR213 (CGM) on long term administration for
centration of curcumin in plasma after 24 h (C24
max ) suggested 90 days is going on in our laboratory.
a significant enhancement in the bioavailability of curcumin
released from the new formulation CGM, as compared to 3.5. Human study
unformulated curcumin when orally administered as a sus-
pension in methyl cellulose. It was observed that the relative The enhancement of bioavailability was also tested on eight
enhancement in the absorption of curcumin from the new male human volunteers of age group 25–50 at 250 mg and
formulation BR213 curcumagalactomannoside (CGM) is 20 1.5 g oral dosage. Curcumin content in plasma, at various
times higher than the absorption from unformulated curcu- time intervals were analysed as before. Fig. 7 shows the plas-
min, when the tentative area under curve (AUC) calculations ma concentration–time profiles of curcumin after the oral
of the plasma concentration–time plot was considered (Table administration of formulated curcumin, CGM and unformu-
1). Tmax, the time taken to reach the maximum concentration lated curcumin. Pharmacokinetic parameters at various

35
% Release of curcumin at pH 6.8 % Release of curcumin at pH 1.2
30
% Release of Curcumin

25

20

15

10

0
0 2 4 6 8 10 15 20 24
Time (h)

Fig. 5 – In vitro release of curcumin from curcumin-impregnated soluble fibre microgranulate containing 60% (w/w) fibre
(CGM) at pH 2.0 and 6.8, at 37 C. Each data point is an average of three measurements (n = 3) performed under identical
conditions.
JOURNAL OF FUNCTIONAL FOODS 4 ( 20 1 2) 3 4 8–35 7 355

Average plasma concentration (µg/g)


0.9

0.8 ** Formulated curcumin Normal curcumin


**
0.7

0.6
**
0.5 *

0.4

0.3 *

0.2

0.1

0
0 5 10 15 20 25 30
Time (h)

Fig. 6 – Average concentration of curcumin observed in rat plasma after oral administration curcumin-impregnated soluble
fibre microgranulate containing 60% (w/w) fibre (CGM) and unformulated curcumin at 250 mg/kg dosage. The data is
expressed as the mean ± SD from n = 16 rats used as two groups of eight animals each. *p < 0.05 and **p < 0.01, (250 mg/kg
CGM versus 250 mg/kg unformulated curcumin).

Table 1 – Pharmacokinetic parameters of formulated curcumin (CGM) and unformulated curcumin in animals and
humans.

Sample administered Subjects Dose Cmax Tmax C24


max AUC
(n) (mg) (lg/g) (h)d (lg/g) (lg/g h)

Animals Unformulated curcumin 16 250 mg/kg 0.02 ± 0.006 3.0 0.008 ± 0.006 1039 ± 118
Formulated curcumin (CGM)a 16 250 mg/kg 0.70 ± 0.13 5.0 0.21 ± 0.07 20777 ± 867

Humans Unformulated curcumin 8 1000 mg 0.022 ± 0.01 0.5 0.004 ± 0.002 510 ± 123
Formulated curcumin (CGM)b 8 250 mg 0.29 ± 0.11 1.0 0.042 ± 0.008 6587 ± 234
Formulated curcumin (CGM)c 8 1500 mg 0.37 ± 0.18 1.0 0.048 ± 0.004 8100 ± 287
a
CGM (250 mg/kg) was equivalent to only 100 mg/kg curcumin.
b
CGM (250 mg) was equivalent to 100 mg curcumin.
c
CGM (1500 mg) was equivalent to 600 mg curcumin.
d
Tmax was deduced from the plasma concentration–time plots.

0.65
Average plasma concentration (µg/g)

0.55 1 g Unformulated curcumin


**

0.45 250 mg Formulated curcumin (CGM)

1.5 g Formulated curcumin (CGM)


0.35 **
**
**
0.25 **
**
0.15 ** * *
*
**
0.05 *

-0.05 0 0.5 1 3 6 8 24

-0.15
Time (h)

Fig. 7 – Average concentration of curcumin observed in human plasma after oral administration curcumin-impregnated
soluble fibre microgranulate containing 60% (w/w) fibre (CGM) at doses of 250 and 1500 mg. Unformulated curcumin was
administered at 1000 mg dose. The data is expressed as the mean ± SD (n = 8); *p < 0.05 and **p < 0.01, (250 mg and 1.5 g CGM
versus 1 g unformulated curcumin).
356 JOURNAL OF FUNCTIONAL FOODS 4 ( 2 0 1 2 ) 3 4 8 –3 5 7

dosages were given in Table 1. It can be observed that the Acknowledgements


plasma concentrations of curcumin were significantly higher
for BR213 curcumagalactomannoside (CGM) group than the The authors dedicate the present work to Prof. H Junjappa,
unformulated curcumin, as evident from the area under former professor and head of the department of Chemistry,
curve (AUC) calculations of the plasma concentration–time North Eastern Hill University, Shillong, India, on the occasion
plot, which was 15.8 times higher at oral dosages of of his 70th birthday. The authors thank SIFC, Cochin, India for
1500 mg (equivalent to 600 mg curcumin) and 12.9 times high- instrumental analysis and also thank M/S Spices Board of In-
er at 250 mg (equivalent to 100 mg curcumin) of CGM as com- dia for financial support on Fenugreek value-addition project
pared to 1000 mg of unformulated curcumin, indicating a (MKT/04/09-10/4687).
dose dependency. The time taken to reach the maximum cur-
cumin concentration in plasma, Tmax, was 1 h for CGM, and R E F E R E N C E S
30 min for unformulated curcumin. C24 max , the observed con-
centration of curcumin in plasma after 24 h of consumption
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