Enhanced absorption and pharmacokinetics of fresh turmeric (Curcuma Longa L) derived curcuminoids in comparison with the standard curcumin from dried rhizomes

Journal of Functional Foods 17 (2015) 55–65

Available online at www.sciencedirect.com

ScienceDirect j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff

Enhanced absorption and pharmacokinetics of fresh turmeric (Curcuma Longa L) derived curcuminoids in comparison with the standard curcumin from dried rhizomes Krishnakumar IM a,*, Dinesh Kumar a, Eapen Ninan a, Ramadassan Kuttan b, Balu Maliakel a a b

R&D Centre, Akay Flavours & Aromatics Ltd., Ambunadu, Malayidamthuruthu P.O., Cochin 683561, India Amala Cancer Research Centre, Amala Nagar P.O., Trichur 680555, India

A R T I C L E

I N F O

A B S T R A C T

Article history:

Despite the various attempts to overcome the poor oral bioavailability of curcuminoids iso-

Received 16 February 2015

lated from dried turmeric, no attempt has so far been reported on fresh turmeric derived

Received in revised form 15 April

curcuminoids. Herein, we report a novel preparation of curcuminoids from fresh turmeric

2015

rhizomes (FTC) as amorphous water soluble stable powder and its enhanced absorption and

Accepted 15 April 2015

pharmacokinetics in comparison with 95% pure curcuminoids isolated from dried tur-

Available online

meric (DTC). While the oral administration of FTC at 250 mg/kg body weight was found to offer significant levels of plasma curcumin in Wistar rats for longer duration, an equiva-

Keywords:

lent dose of curcuminoids as DTC failed to produce detectable plasma levels. Dose dependent

Bioavailability

human studies (100, 250 and 1000 mg doses of FTC) also showed significant (p < 0.001) ab-

Pharmacokinetics

sorption of total curcuminoids (46-fold) and free unconjugated curcuminoids from FTC with

Curcuma longa L

improved pharmacokinetics as compared to equivalent doses of curcuminoids as DTC.

Curcumin

© 2015 Elsevier Ltd. All rights reserved.

Fresh rhizomes Turmeric

1.

Introduction

Turmeric (Curcuma longa L) is a famous ancient spice having rich tradition of historical usage owing to its flavour characteristics and medicinal properties. Curcuminoids, the yellow pigment in turmeric rhizomes, have been identified as the most bioactive principle and were characterized as a

group of bis-α,β-unsaturated β-diketone polyphenols; namely, curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC). Commercially available natural form is a mixture of three curcuminoids: curcumin (72 to 78%), DMC (12 to 18%) and BDMC (3 to 8%) with a purity of ≥95% (commonly referred to as ‘standard curcumin’), and has been widely used as dietary supplement. A large number of preclinical and clinical studies on standard curcumin have

* Corresponding author. R&D Centre, Akay Flavours & Aromatics Ltd, Ambunadu, Malaidamthuruth P.O., Cochin-683561, India. Tel.: +91 484 2686111; fax: +91 484 2680891. E-mail address: [email protected] (K. IM). http://dx.doi.org/10.1016/j.jff.2015.04.026 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

56

Journal of Functional Foods 17 (2015) 55–65

demonstrated its extreme safety profile, beneficial pharmacological effects and pleiotropic mechanism of action suitable for further development as a plausible functional food ingredient or therapeutic agent for the prevention and/or treatment of many pro-inflammatory diseases including cancer and Alzheimer’s (Aditya et al., 2015; Goel, Jhurani, & Aggarwal, 2008; Kapakos, Youreva, & Srivastava, 2012; Lao et al., 2006; Ono, Hasegawa, Naiki, & Yamada, 2004; Prasad, Gupta, Tyagi, & Aggarwal, 2014). However, the poor bioavailability of curcumin emanating from its aqueous insolubility at physiological pH (~11 ng/L), hydrolytic instability due to highly labile bis-α,βunsaturated β-diketone structure, rapid metabolism and fast elimination has very often limited its applications (Jantarat, 2013; Prasad, Tyagi, & Aggarwal, 2014). Following the very first report by Wahlstrom and Blennow in 1978 that only negligible amounts of curcumin were observed in the plasma of rats upon oral administration of 1 g/kg body weight of curcumin, many studies have established the high metabolic turnover of curcumin and its in vivo fate to simply pass through the gastrointestinal tract and be excreted (Garcea et al., 2004; Jantarat, 2013; Prasad et al., 2014; Wahlstrom & Blennow, 1978). Consumption of even 10 to 12 g of curcumin was reported to provide only 50 ng/mL of curcumin in the human plasma (Lao et al., 2006). Many attempts have been reported to derive various formulations and delivery techniques to improve the systemic bioavailability of curcumin (Hamam & Al-Remawi, 2014; Prasad et al., 2014; Yallapu, Jaggi, & Chauhan, 2012). It was observed that all the attempts that have been reported to improve the bioavailability of curcumin have employed commercially available grade of natural curcuminoids isolated from the dried turmeric rhizomes by solvent extraction (Sahebkar, Serban, Ursoniu, & Banach, 2015). Since solvent extraction process employs organic solvents like dichloroethane, hexane, acetone, ethyl acetate, methanol, isopropanol etc., the complete removal of such solvents from the isolated curcumin was never achieved. Various regulatory agencies prevailing in each country have thus implemented a list of approved solvents and their allowable residual limits for the commercial grade curcuminoids intended to the market for food, dietary supplements or pharmaceutical use (USP 32-NF 27, General Chapter <467>, 2009). Thus, the present contribution was aimed at the solvent free preparation of significant levels of curcuminoids (curcumin, DMC and BDMC) containing turmeric extract powder from fresh turmeric rhizomes (hereinafter referred to as ‘FTC’). The relative absorption of curcuminoids from FTC and its pharmacokinetics were further investigated in comparison with equivalent doses of standard curcumin isolated from dried turmeric rhizomes by solvent extraction (hereinafter referred to as ‘DTC’). The dose dependence of curcuminoids absorption in human volunteers was also followed by the oral administration of 100, 250 and 1000 mg hard shell gelatin capsules of FTC in comparison with 1000 mg dose of standard curcumin from dried rhizomes (DTC), to confirm the significantly high absorption of FTC derived curcuminoids despite its very low curcumin content (4.2%) in comparison with DTC having 95% curcumin content. The term ‘fresh turmeric rhizome’ used in the present paper intends to mean the green turmeric rhizomes, either harvested from the plants or stored under suitable conditions without losing the moisture content.

2.

Materials and methods

2.1. Preparation of fresh and dry turmeric derived curcuminoids (FTC and DTC) Both fresh and dried turmeric rhizomes used in the present study were obtained from a selected farm in Cambodia and harvested during December 2011. An authenticated botanist identified the sample and a voucher specimen (AK-fTUR-02/ 12) was deposited in the herbarium of M/s Akay Flavours & Aromatics Ltd, Cochin, India. Freshly harvested turmeric rhizomes were water washed, wiped with tissue paper and packed in zip-locked polyethylene bags and stored below 15 °C without losing its moisture content. Fresh rhizomes (1 kg) were ground and treated with a mixture of 70 mg/kg each of pectinase and amylase (HiMedia Laboratories Pvt Ltd, Mumbai, India) at 50 ± 2 °C for 4 h at pH 4.5 to 5.5. Ultrasonication, using a 1000 W ultrasound generator fitted with a sonotrode (Hielscher Ultrasonics GmbH, Teltow, Germany), was then applied to the wet mass for 10 min as pulses of 30 to 60 s duration and the juice was expressed using an in-house built expeller. The juice was again subjected to ultrasonication for 5 min as pulses of duration 60 s, at below 50 °C. It was cooled to 25 °C and centrifuged. The supernatant juice thus obtained was finally converted to a free flowing powder by spray drying at an inlet temperature of 130 °C and outlet temperature of 60 °C (Labultima, Model No. LU 228 Advanced, Mumbai, India). Total curcuminoids content (as the sum of curcumin, DMC and BDMC) and the ratio of the individual curcuminoids were measured by following a reported reverse phase HPLC procedure (Jadhav, Mahadik, & Paradkar, 2007) employing a Shimadzu LC 20 AT system with M20A Photodiode array (PDA) detector (Shimadzu Analytical Pvt. Ltd., Mumbai, India) fitted with a reverse phase C18 column (250 × 4.6 mm, 3 µm) (Phenomenex India Pvt Ltd, Hyderabad, India). The identity of curcuminoids was confirmed by tandem mass spectrometric measurements employing Agilent 6460 tandem mass spectrometer fitted with an electrospray ionization (ESI) probe (Agilent India Pvt. Ltd, Bangalore, India), in comparison with the reference standards of curcumin (CAS# 458-37-7; purity >98%), DMC (CAS# 22608-11-3; purity >98%) and BDMC (CAS# 33171-05-0; purity >95%) obtained from SigmaAldrich, Bangalore, India. The commercial grade standard curcumin (DTC) with a total curcuminoids content of 95.1% (w/ w) was obtained from M/s Akay Flavours & Aromatics Pvt Ltd.

2.2.

Characterization of FTC and DTC

FTC and DTC were characterized by investigating the nutritional composition, stability, particle size, crystallinity and nature of entrapment of curcuminoids in the juice powder matrix. Nutritional composition was determined by analysing protein, carbohydrate, dietary fibre and the lipids content as per the standard methods of the Association of Official Analytical Chemists (AOAC) (Horwits & Latimer, 2006). Particle size was measured using a Melvern Zetasizer Nano ZS90 particle size analyser (Malvern Instruments Ltd, Worcestershire, UK). Temperature stability, crystallinity and nature of entrapment of curcuminoids were assessed by differential scanning calorimeter (DSC) (Mettler-Toledo India Pvt. Ltd, Mumbai, India), powder

Journal of Functional Foods 17 (2015) 55–65

X-ray diffraction (PXRD) (Bruker AXS GmbH, Karlsruhe, Germany), solid state Fourier-transform Infrared spectroscopy (FTIR) (Thermo Nicolet Corporation, Madison, WI, USA) and scanning electron microscopy (SEM) (JEOL Ltd, Tokyo, Japan) investigations.

2.3.

Stability studies

Storage stability studies were carried out using a protocol prepared by following the guidelines of International Conference on Harmonisation (ICH) of technical requirements for registration of pharmaceuticals for human use (Thakur, Ghodasra, Patel, & Dabhi, 2011). Briefly, the sample packets (10 g) of the extracts were incubated at 40 ± 2 °C and 70 ± 5% relative humidity for a period of 6 months in a stability chamber (Remi, Mumbai, India). The samples were withdrawn at 0, 1, 2, 3, and 6 months and subjected to analysis for various physical, chemical and microbial parameters. The pH stability of the aqueous solution was checked by preparing 5% (w/w) solutions at pH 2.0, 5.0, and 6.8 using hydrochloric acid and phosphate buffers. 5 mL volumes of the solutions were withdrawn at regular intervals of 2 h for a period of 24 h, and the curcumin content was checked by HPLC. Temperature stability was verified by maintaining the solution at 90 ± 2 °C for 30 min followed by HPLC analysis.

2.4.

Animals

Adult Wistar rats of both sexes, 180 to 200 g body weight, were purchased from National Institute of Nutrition, Hyderabad, India, and housed in an air-conditioned room at 22 ± 2 °C, and relative humidity 60 ± 5% with 12 h light and dark cycle, at the animal house facility of M/s Amala Cancer Research Centre, Kerala, India, in strict accordance with the ethical norms approved by the Institutional Animal Ethics Committee (IAEC) recognized by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (Registration No. 149/199/CPCSEA; approval number 012/2011). Rats were provided with standard pellet diet (M/s Sai Durga Feeds and Foods, Bangalore, India) having a composition (g/100 g) of proteins, 22.4; carbohydrates, 55.6; fat, 2.5; fibre, 4.2; vitamins, 0.75; minerals, 2.73; salt, 3.0; cinders, 3.5%; moisture, 5.4% and water ad libitum.

2.5.

Animal studies

Thirty six adult rats were divided into two groups, in such a way that each group contains eighteen animals of approximately the same body weight. The study was conducted by a crossover design, where the first group of eighteen animals received standard curcumin (DTC) and second group of eighteen animals received fresh turmeric derived curcuminoids (FTC). After completion of the initial cycle of experiments with DTC and FTC, the groups were reversed in such a way that the animals provided with DTC was given with FTC and viceversa. A minimum of ten days of wash out period was given for each animal for oral administration of FTC and DTC. The dose of FTC was fixed at 250 mg/kg body weight as a suspension in 0.1% carboxymethylcellulose solution by oral gavage. By considering the total curcuminoids content in 250 mg/kg

57

of FTC, an equivalent dose of DTC corresponding to the same curcuminoids content was provided to investigate the comparative absorption and pharmacokinetics of both FTC and DTC. A higher dose of 250 mg/kg body weight of DTC was also administered to check the dose response. At post-dose, animals were tail-veined under terminal anaesthesia (EMLA cream) and 1 mL of blood was collected after each time point, ca. 0.5, 1, 3, 5, 8 and 12 h respectively, into heparinized tubes, centrifuged (1008 × g, 10 min, at 4 °C) and the plasma was stored at −80 °C until analysis. The animals used for each time point study of the first group (DTC) were separately marked and the same animals were further used for the same post-administration time point for the second group (FTC). The average values (n = 3) were used for the plasma curcuminoids vs. time plot and area under curve (AUC) calculations.

2.6.

Human studies

Human study was conducted as per the institutional ethical committee (IHEC) approved protocol, in accordance with the regulations by the Government of India. After detailing the protocol and purpose of the study, written informed consent was obtained from all the fifteen human volunteers (10 male and 5 female; aged between 25 and 50 years), who were healthy and not involved in any medication or dietary supplementation. Generally, subjects having history of gastrointestinal problems, gallbladder issues, hyperacidity, hypertension, diabetes, hyperlipidaemia etc. were excluded from the study. The volunteers were not allowed to take alcohol and turmericcontaining food for three days prior to the test. The study was followed by a single-blind crossover design with a minimum of one week washout period between the FTC and DTC administrations. Each of the volunteer reported to the lab at 6.0 to 6.30 AM following a 10 h overnight fasting (except water), and the capsules of FTC or DTC at respective doses were provided after comfortable resting time. Blood samples at various intervals of post-administration (0, 0.5, 1, 3, 5, 8, 12 h) were collected via a catheter inserted into the forearm vein. A standardized south Indian meal containing no turmeric was given to the volunteers after 3 h and 8 h during the study. The first meal consisted of a typical south Indian breakfast made of rice (having an approximate nutritional composition of fat: 20 to 24%, carbohydrates: 35 to 40%, protein: 25 to 30%; energy 400 to 500 calories), and the second meal was the lunch with rice and vegetable/non-vegetable curries (having an approximate nutritional composition of fat: 25 to 30%, carbohydrates: 35 to 42%, protein: 32 to 35%; energy 500 to 580 calories). FTC was administered as hard gelatin capsules at 100 mg (100 mg × 1), 250 mg (250 mg × 1) and 1000 mg (250 mg × 4) doses to investigate the dose dependence on curcuminoids absorption and pharmacokinetics. An equivalent concentration of curcuminoids containing DTC corresponding to each of the above doses of FTC was also administered as hard gelatin capsules to gather comparative absorption data. An inert filler (microcrystalline cellulose) was used to match the total weight of each of the capsule. In addition to these doses, DTC was also administered at a dose of 1000 mg (250 mg × 4 capsules), to compare its absorption and pharmacokinetics with various doses of FTC, as mentioned above. The blood at various inter-

58

Journal of Functional Foods 17 (2015) 55–65

vals was collected using an indwelling venous cannula and plasma was stored at −80 °C for analysis.

2.7.

Estimation of curcuminoids in blood plasma

Curcuminoids in plasma was extracted using a previously reported procedure (Krishnakumar, Ravi, Kumar, Kuttan, & Maliakel, 2012) and subjected to HPLC-DAD-ESI-MS/MS analysis to determine the total curcumin content and free unconjugated curcuminoids in plasma at various post-dose time intervals (Krishnakumar et al., 2015). Briefly, 1 mL of plasma was extracted with 3 × 5 mL of 5% (v/v) methanol containing ethyl acetate and evaporated to dryness under nitrogen atmosphere at 45 °C. The residue was then made up to 1 mL with methanol, filtered through a 0.45 µm syringe filter and further used for quantification. The identification and measurements of free curcuminoids was confirmed by tandem mass measurements in multiple reaction monitoring (MRM) using a triple quadruple mass spectrometer operated in positive ionization mode. Agilent JetStream source was operated with a capillary voltage of 3500 V, sheath gas temperature 375 °C and drying gas temperature of 325 °C. MRM transitions for each of the curcuminoids were recorded at collision energies 48, 24 and 20 V respectively for curcumin, DMC and BDMC with the corresponding fragmentor energies of 113, 103 and 94 V. The free curcuminoids in plasma were also resolved into three separate peaks corresponding to curcumin, DMC and BDMC using a reverse phase C18 column (150 × 4.6 mm, 2.6 µm) (Phenomenex India Pvt Ltd, Hyderabad, India) and the mobile phase consisting of 3.25% (v/v) acetonitrile, 61.75% (v/v) methanol and 35% (v/v) water (adjusted to pH 3.0 with perchloric acid). The detection was performed with a variable wavelength fluorescence detector (Waters India Pvt. Ltd, Bangalore, India), set at excitation and emission wavelengths of 426 and 536 nm respectively (Schiborr et al., 2014).

2.8.

Statistical analyses

Statistical analyses were performed on all data and values are reported as mean ± SD. Observed differences were evaluated using t-test and p < 0.05 was selected to indicate statistical significance. AUC was calculated using Graph Pad prism 5.04 software (Graph Pad, San Diego, CA, USA) and the effect of FTC upon the baseline characteristics of DTC was computed by student’s t-test.

3.

Results and discussion

Although the pharmacodynamics of curcumin was found to be attractive, the oral absorption and pharmacokinetics were regarded as not significant (Jantarat, 2013; Krishnakumar et al., 2015). Despite the various attempts to enhance the oral bioavailability of dry turmeric derived standard curcuminoids, no report was available on the fresh turmeric derived curcuminoids or its absorption; though medicinal preparations of fresh turmeric were well documented in the ancient texts of Ayurveda. The present study was an attempt to develop a method for the preparation of organic quality curcuminoids

extract from fresh turmeric rhizomes and further to investigate its relative absorption and pharmacokinetics in comparison with the dried turmeric derived standard curcumin (DTC). Thus, herein we report for the first time, a novel food grade and water soluble preparation of curcuminoids from fresh turmeric (without using any solvents or synthetic excipients), in a free flowing stable powder form and its enhanced plasma absorption compared to the standard curcumin.

3.1. Preparation of fresh turmeric derived curcuminoids (FTC) The primary objective of the present study was to develop a suitable method to extract significantly high levels of curcuminoids (curcumin, DMC and BDMC) from fresh turmeric and further to formulate into a water soluble free flowing powder form suitable for functional and medicinal applications. An ultrasound-aided enzymatic digestion process was developed to extract curcuminoids into the juice along with the other natural components of rhizomes such as carbohydrates, proteins and lipids. Enzyme treatment followed by ultrasonication was found to provide better yield (72 mL juice with 8.2% total dissolved solids) and concentration of curcuminoids in the juice (4.4% (w/w) of the dissolved solids), as compared to the juice obtained by simple mechanical processes (44 mL juice with 6.3% total dissolved solids, and 2.6% (w/w) curcumin). In spite of the fact that some quantity of curcumin can always be present in the juice of fresh turmeric rhizomes, the better yield and curcumin content in the ultrasonication-assisted enzymatic digested juice can be attributed to the rupture of cell walls leading to better leaching of curcuminoids into the juice along with its other natural components. Further steps in the preparation process comprising sonication, filtration and spray drying of the juice could produce free flowing water soluble powder with a total curcuminoids content of 4.2% (w/w). The percentage composition of curcuminoids in FTC was found to be curcumin: 57% (w/w), DMC: 20% (w/w) and BDMC: 23% (w/w). Ultrasonication of the juice to reduce the particle size and further conversion of the resulting homogeneous juice into a spray dried powder form were found to have no significant effect upon the curcuminoids content. The relative distribution of curcuminoids in the standard curcumin derived from dried turmeric rhizomes (DTC) was found to be: curcumin (76%), DMC (15%) and BDMC (4.1%), with a total curcuminoids content of 95.1% when analysed by HPLC. When tested for storage stability, FTC was found to cause only 3.2% loss in curcumin content and other parameters like colour, appearance, odour, taste, moisture content, bulk density, carbohydrates, proteins, lipids, and microbial status remained within ±2% of the initial value indicating its safe storage stability for a period of two years in closed containers under ambient conditions of less than 30 °C, in dark cool place without direct sunlight or moisture (Table 1). DTC, the golden yellow powder, was also found to possess comparable storage stability with only 2.1% of curcumin content loss during the study period. The other physicochemical properties and microbial status of DTC also remained with no significant change (Table 1).

59

Journal of Functional Foods 17 (2015) 55–65

Table 1 – Storage stability data of FTC performed as per an in-house protocol based on ICH guidelines. The samples were incubated at 40 ± 2 °C and 75 ± 5% RH for a period of 6 months. Parameter

0 month

1 month

2 months

3 months

6 months

Appearance

Brownish yellow powder Characteristic 4.2 2.3 0.49 80.34 6.0 4.5 0

Brownish yellow powder Characteristic 4.2 2.5 0.49 80.14 6.0 4.5 0

Brownish yellow powder Characteristic 4.2 2.6 0.49 80.26 5.8 4.5 0

Brownish yellow powder Characteristic 4.1 2.5 0.51 80.31 6.0 4.5 0

Brownish yellow powder Characteristic 4.1 2.6 0.50 80.36 6.0 4.5 0

3000 22 <3 Absent/g Absent/25 g

3200 30 <3 Absent/g Absent/25 g

3800 36 <3 Absent/g Absent/25 g

3500 42 <3 Absent/g Absent/25 g

3500 44 <3 Absent/g Absent/25 g

Odour Curcumin content (%)a Moisture (%)a Bulk density (g/mL)a Carbohydrate (g/100 g)a Protein (g/100 g)a Total fat (g/100 g)a Cholesterol (mg/100 g)a Microbiology Total plate count (cfu/g)a Yeast and mould (cfu/g)a Coliforms (MPN/g)a E. colia Salmonellaa a

Each value was presented as an average of three measurements.

3.2. Characterization of fresh turmeric derived curcuminoids (FTC) FTC was further characterized as an intimate homogeneous blend of turmeric derived proteins (12%), carbohydrates (62%), dietary fibre (13%) and lipids (4.6%) with a total curcumin content of 4.2% and moisture of 3%. Despite the high volatile oil content of dry turmeric (5 to 7%), the present juice powder was found to contain only 0.8% (v/w) of volatile oil. The standard curcumin, DTC, on the other hand was found to contain 95.1% (w/w) of curcuminoids with no fibre, proteins, carbohydrates or fixed oil content. The physical nature, thermal stability and nature of entrapment of curcuminoids in the juice powder matrix as compared to the standard curcumin (DTC) were investigated by DSC, PXRD, FTIR and SEM analyses (Fig. 1). DSC studies of standard curcumin (DTC) produced a sharp endotherm at 186 °C corresponding to its melting, whereas FTC showed no such peaks characteristic of amorphous nature. PXRD studies of DTC showed sharp and intense peaks between 7 and 27° 2θ, whereas the juice powder produced a pattern typical of amorphous character with broad and less intense peaks. Thus, a considerable reduction in crystallinity of curcumin was observed in the juice powder. This was further evident from the shift of the characteristic sharp FTIR peaks of curcumin observed at 3510.1, 1628.7 and 1278.2 cm−1 to broad and less intense peaks at 3380.2, 1640.9, 1156.9 and 1005.3 cm−1 in fresh turmeric juice powder. SEM photographs demonstrated a smooth, continuous and porous surface of the juice powder particles, as compared to the highly crystalline powder of standard curcuminoids (DTC). Thus, the fresh turmeric juice powder (FTC) prepared in the present study was found to have a matrix of turmeric derived carbohydrates, proteins, and lipids in which the crystalline curcumin was found buried to form amorphous, water soluble and stable juice powder as demonstrated by DSC, PXRD and SEM studies. The carbohydrates and proteins of turmeric have already been reported with varying pharmacological effects such as antioxidant, antidiabetic, anti-depressant, anti-tumour, hepatoprotective and

immunomodulatory effects (Deshpande, Ingle, & Maru, 1998; Yu, Kong, & Chen, 2002). Further particle size measurements revealed uniform distribution of curcumin particles of size 1 ± 0.2 µm as compared to the crystalline and insoluble standard curcumin (DTC) particles ranging from 200 to 600 µm. Since it has already been proven that the conversion of crystalline curcumin particles to amorphous water soluble nanoparticles can significantly enhance the bioavailability (Krishnakumar et al., 2015; Sasaki et al., 2011; Schiborr et al., 2014), FTC was further investigated for oral bioavailability.

3.3.

Plasma curcuminoids absorption measurements

Despite the various reports on the health benefits of fruit/ vegetable juices, the bioavailability of phytonutrients from the juice powder matrix have not been investigated much (Jaganathan, Vellayappan, Narasimhan, & Supriyanto, 2014). In the present study, the oral absorption of biologically active forms of curcuminoids (curcumin, DMC and BDMC) from the juice powder matrix (FTC) was investigated in comparison with the standard curcumin isolated from dried rhizomes (DTC) to better understand the significance of fresh turmeric juice powder as a source of dietary curcumin. Measurement of curcuminoids levels in plasma after administration of fresh turmeric juice powder was carried out by a standardized HPLC-DAD-ESI-MS/ MS procedure and the pharmacokinetic parameters were deducted from the plasma curcumin concentration–time plot as reported earlier (Cuomo et al., 2011; Krishnakumar et al., 2012). The relative absorption of curcumin from FTC in comparison with the standard curcumin DTC, was expressed in terms of the area under curve (AUC), since it is regarded as the most reliable method of expressing the absorption unlike the maximum plasma concentration of the drug (Cmax) which is just a single time point measurement (Kocher, Schiborr, Behnam, & Frank, 2015). Range and linearity for curcumin extracted from plasma was confirmed by spiking the standard curcumin and the internal standard, salbutamol. The extraction recovery of curcuminoids from plasma was found to be

60

Journal of Functional Foods 17 (2015) 55–65

Fig. 1 – Differential Scanning Calorimetry (DSC), Powder X-ray diffraction (PXRD), solid state Fourier transform spectra (FTIR) and Scanning Electron Micrograph (SEM) of fresh turmeric rhizome juice powder (FTC) and standard regular curcumin with 95% purity isolated from dried turmeric rhizomes (DTC).

61

c

b

The dose of FTC was fixed at 250 mg/kg body weight for each animal. Since FTC contains only 4.2% of curcumin content, an equivalent dose of DTC corresponding to the same curcumin content was also administered. For example, for a rat weighing 200 g body weight, 50 mg of FTC (2.1 mg curcuminoids) corresponding to 250 mg/kg body weight were given. For the same animal, 2.2 mg of DTC containing 2.1 mg curcuminoids were also administered. Since equivalent dose of curcuminoids for DTC failed to provide detectable levels of curcuminoids in rat plasma, further supplementation at 250 mg/kg b.wt of DTC was also performed to study the dose dependence on pharmacokinetic parameters. 1000 mg of DTC contains 951 mg of total curcuminoids. a

486,000 ± 91,800 27,378,000 ± 901,800 36,855,000 ± 1,107,000 48,438,000 ± 1,587,600 N.D N.D 54,600 ± 880 1,053,000 ± 148,500 N.D 101.115 ± 3.4 293.058 ± 25.7 616.032 ± 76.6 N.D N.D N.D N.D 0.5 1.3 1.3 1.3 N.D N.D 0.5 0.5 20 15 15 15 15 15 15 15 Humans

DTC FTC FTC FTC DTC DTC DTC DTCc

31.0 ± 4.4 2171.016 ± 75.1 2758.428 ± 153.1 3748.456 ± 249.2 N.D N.D 14.86 ± 0.54 80.109 ± 8.8

0.7 3.1 3.1 3.1 N.D N.D 0.6 0.7

23,085,000 ± 769,500 N.D 361.8 ± 54 N.D 1.6 N.D 1483.7 ± 119.2 N.D 20 20

250 mg/kga Equivalent dose of curcuminoids corresponding to 250 mg/kg b.wt of FTCa 250 mg/kgb 4.2 mg 10.5 mg 42 mg 4.2 mg 10.5 mg 42 mg 950 mg

4.1 N.D

AUC (nmol/L) C12max (nmol/L) T1/2max h) Tmax (h) Cmax (nmol/L)

FTC DTC

Human studies

Dose-dependent human oral absorption of curcuminoids upon consumption of hard gelatin capsules of 1000 mg, 250 mg and 100 mg doses of FTC have shown significant (p < 0.001) absorption of curcuminoids into the blood stream as compared to standard curcumin (DTC) at equivalent doses. Since FTC contains only 4.2% of total curcumin content as measured by curcumin, DMC and BDMC levels, the above doses of FTC were found to contain only 42, 10.5 and 4.2 mg respectively of total curcuminoids. The equivalent doses of standard curcumin (DTC) were 42, 11.0 and 4.4 mg respectively, since the total curcumin content of DTC used in the present study was 95.1%. Although a dose dependence in absorption of curcuminoids was observed (as measured by the various pharmacokinetic pa-

Animals

3.3.2.

Dosea (Curcumin)

Animal studies

Oral administration of newly prepared FTC to Wistar rats (250 mg/kg b. wt.) was found to significantly enhance (p < 0.001) the absorption of curcuminoids, as compared to an equivalent dose of curcuminoids derived from dry turmeric (DTC), which even failed to provide detectable levels of curcuminoids in rat plasma. Further supplementation at 250 mg/kg b. wt. of DTC produced a maximum curcumin concentration of 31.0 ± 4.4 nmol/L after 30 min post-administration. The pharmacokinetic parameters comprising the maximum curcumin concentration in the plasma (Cmax), the time at which maximum curcumin concentration was found in plasma (Tmax), the time taken for 50% of absorbed curcumin to degrade (T1/2max), concentration of curcumin found in plasma after 12 h of ingestion (C12max) and the area under curve (AUC0–12 h) of plasma curcumin concentration vs. time plot for various doses of FTC and DTC were given in Table 2. Administration of DTC at equivalent dose of curcuminoids corresponding to the 250 mg/kg body weight of FTC failed to produce detectable plasma curcuminoids levels. However, total curcuminoids absorption, including both free and conjugated curcuminoids, was about 48-fold higher for FTC when compared to a higher dose of DTC at 250 mg/kg body weight, as observed with Cmax and AUC0–12 h values (Fig. 2A). Tmax for FTC was around 1.6 h as compared to 30 min for DTC. Moreover, the absorbed curcumin was found to stay in the blood for longer duration, as evident from the T1/2max values of 4.1 h and C12max of 361.8 ± 54 nmol/L, as compared to DTC, which could not be even detected after 2 h of ingestion (Fig. 2A and Table 2).

Subjects (n)

3.3.1.

Sample administered

88.9%. Individual curcuminoids retention time was confirmed by repeated six analyses at 1.0 mg/mL concentration on the same column under identical conditions. The identity of curcuminoids in plasma was further confirmed by spiking standard curcumin in animal plasma at 1.0 µg/mL followed by extraction and electrospray tandem mass spectrometric analysis which could detect m/z 369 (369 → 116.9 for curcumin), m/z 339 (339 → 146.9 for DMC) and m/z 309 (309 → 146.9 for BDMC) when operated under multiple reaction monitoring mode. The absorption of unconjugated free curcuminoids (curcumin, DMC and BDMC) into the blood plasma was also verified by an HPLCfluorescence detection method (Schiborr et al., 2014), which could resolve the plasma curcuminoids into three distinct peaks of curcumin, DMC and BDMC, without interference from any of the curcumin metabolites.

Table 2 – Pharmacokinetic parameters calculated from plasma curcumin concentration vs time plot for animals and humans after administration of turmeric juice powder (FTC) and standard curcumin (DTC).

Journal of Functional Foods 17 (2015) 55–65

62

Journal of Functional Foods 17 (2015) 55–65

Fig. 2 – (A) Average concentration of curcumin observed in animal plasma after oral administration of fresh turmeric rhizome juice powder (FTC) containing 4.2% curcuminoids and standard curcumin with 95% purity isolated from dried turmeric rhizomes (DTC) at 250 mg/kg body weight. The data are expressed as the mean ± SD (n = 10); *p < 0.05. (B) Average concentration of curcumin observed in human plasma after oral administration of fresh turmeric rhizome juice powder (FTC) containing 4.2% curcuminoids at 100, 250 and 1000 mg dose capsules. Standard curcumin with 95% purity isolated from dried turmeric rhizomes (DTC) was administered at 1000 mg/kg body weight. The data are expressed as the mean ± SD (n = 15); *p < 0.05.

rameters), the relative absorption of curcuminoids even from 100 mg dose of FTC containing only 4.2 mg of curcuminoids was significantly high (Cmax 2171.016 ± 75.1 nmol/L) as compared to DTC at equivalent concentrations (Table 2). While the consumption of 44.2 mg of DTC corresponding to 42 mg of total curcuminoids (equivalent to 1000 mg dose of FTC) produced a maximum absorption of 14.86 ± 0.54 nmol/L of plasma curcumin content at 30 min post-administration, no curcumin was found to be detected in plasma after 4.4 and 11.0 mg doses of DTC administration (Table 2). Though the absorbed curcumin from DTC at 44 mg dose disappeared after 60 min of consumption, the FTC was found to stay in the blood for longer duration, as evident from the T 1/2max values of 3.1 h and C 12max of 616.032 ± 76.6 nmol/L at 1000 mg dose (Table 2). Tmax for FTC was around 1.3 h as compared to the 30 min for DTC. Thus,

the relative absorption of curcuminoids from 1000 mg FTC was found to be about 46-fold higher, as compared to the absorption from DTC containing an equivalent dose of curcuminoids (Fig. 2B and Table 2). Further, 1000 mg dose of DTC containing 951 mg of total curcumin content was also administered to check the pharmacokinetics, since ≥1000 mg is a commonly used oral dose in previous studies (Antony et al., 2008; Cuomo et al., 2011; Jager et al., 2014; Krishnakumar et al., 2012; Sahebkar et al., 2015). It was observed that all the pharmacokinetic parameters (Cmax, Tmax, T1/2max, C12max and AUC0–12 h), followed by the administration of even the low dose of 100 mg FTC containing only 4.2 mg of curcuminoids was significantly higher than DTC at 1000 mg oral dose (Fig. 2B and Table 2), leading to the conclusion that higher plasma curcumin concentrations for longer duration can be observed with even

Journal of Functional Foods 17 (2015) 55–65

low doses of the novel FTC preparation when compared to the standard curcumin (DTC) from dried rhizomes. The above results demonstrated the better absorption of the amorphous and water soluble submicron particles of the juice powder derived curcuminoids (FTC) under gastrointestinal tract conditions to provide significantly higher concentrations of curcuminoids in the plasma of both animals and humans. Earlier studies have shown better absorption of curcumin when converted to amorphous water soluble sub-micron particles using various hydrocolloids (Jager et al., 2014; Krishnakumar et al., 2012; Sasaki et al., 2011). Co-administration of turmeric oil along with curcumin was also reported to be effective to enhance the bio absorption through phase II metabolizing enzyme inhibition (Antony et al., 2008). Thus, the native state of existence of curcuminoids in the complex natural lap of proteins, carbohydrates and lipids of juice as an amorphous, water soluble submicron-sized particle can be considered as one of the factor for enhanced absorption of curcuminoids from FTC. Further, the significant presence of turmeric lipids in FTC could also be a protecting factor for curcuminoids against their rapid enzymatic metabolism of curcuminoids, unlike the highly purified standard curcuminoids, DTC.

3.4.

Absorption of free unconjugated curcuminoids

HPLC-fluorescence method coupled with ESI-MS/MS measurements employed in the present study was found to be a useful and reliable tool for the better resolution and measurement of plasma curcuminoids into unconjugated curcumin, DMC and BDMC (Krishnakumar et al., 2015). Since the oral administration of standard curcumin and most of its current formulations could not contribute measurable levels of free curcuminoids in plasma, a protocol involving the preliminary treatment of plasma with β-glucuronidase (type H-1 from Helix pomatia) enzyme has generally been used to convert all the glucuronide and sulfate metabolites of curcuminoids in plasma into free curcuminoids for further chromatographic separation and estimation (Cuomo et al., 2011; Jager et al., 2014; Sasaki et al., 2011; Schiborr et al., 2014). In the present study, the human plasma was separately collected after oral administration of 1000 mg of FTC and an equivalent concentration of standard curcumin (DTC) corresponding to the same dose of curcuminoids. The collected plasma was directly processed without treatment with β-glucuronidase to check whether free curcuminoids (curcumin, DMC and BDMC) have absorbed as such in significantly high levels and stably exist in the plasma for longer duration (Krishnakumar et al., 2015). It was observed from the pharmacokinetic studies that significantly high levels of free curcuminoids were absorbed from FTC, and existed in plasma for longer duration as compared to those from DTC, which did not provide any detectable concentration of plasma curcuminoids. All the three peaks corresponding to curcumin, DMC and BDMC were also observed in the HPLC chromatogram of plasma collected after the oral administration of FTC, as confirmed by tandem mass measurements. However, the relative enhancement in the absorption of free curcuminoids upon FTC administration was not possible since DTC at the same level was not detected. Further, 1000 mg of DTC containing 951 mg of total curcuminoids with a respective distribution of 760 mg curcumin, 160 mg DMC and 80 mg BDMC

63

was also administered to check whether this higher dose of DTC can provide detectable levels of free curcuminoids in plasma. It was observed that only curcumin at 8.1 nmol/L was found in human plasma without producing detectable levels of DMC and BDMC and the detected curcumin disappeared after 1 h of consumption. Thus, the present data demonstrated significant human hepatic absorption of free curcuminoids from fresh turmeric juice powder over 12 h of post-dose time span, even though 1000 mg contains only 42 mg of total curcuminoids with a relative distribution of 23.5 mg curcumin, 8.4 mg DMC and 9.2 mg BDMC. It was reported that relative absorption of curcumin from standard unformulated curcumin with 95% purity is only less than 1% and the absorbed fraction quickly metabolizes into glucuronides and sulfates (Pan, Huang, & Lin, 1999), leading to the hypothesis that these metabolites are responsible for bioactivity of curcuminoids. However, a recent study on cell lines demonstrated no anti-proliferative and anti-inflammatory effects to curcumin glucuronide metabolites (Pal et al., 2014). Moreover, curcumin glucuronides and other metabolites of curcumin such as tetrahydrocurcumin, hexahydrocurcumin, octahydrocurcumin, curcumin sulfate etc. have also shown less bioactivity as compared to the free native curcumin (Ireson et al., 2001; Sandur et al., 2007), albeit a few studies with enhanced activity for metabolites (Lin et al., 2015; Okada et al., 2001). Thus, the current understanding of the pharmacological aspects indicates the significance of the absorption and retention of free curcuminoids over curcumin metabolites (Krishnakumar et al., 2015), which leads towards the plausible importance of fresh turmeric derived curcuminoids, in addition to its enhanced absorption and organic quality.

4.

Conclusion

In conclusion, significant levels of curcuminoids containing extract can be expressed from fresh turmeric rhizomes without using any solvents and can be conveniently converted to a stable water soluble powder in which the curcuminoids were characterized to be encapsulated in the complex natural matrix of proteins, carbohydrates and lipids of turmeric. Upon oral administration, fresh turmeric derived curcuminoids (FTC) were found to deliver significantly high levels of plasma curcuminoids in both animals and humans and exist for longer duration, as compared to the standard curcumin isolated from dried turmeric rhizomes (DTC). Dose dependent pharmacokinetic studies of FTC at 100, 250 and 1000 mg doses have clearly demonstrated the enhanced plasma absorption of curcuminoids from native juice powder than from even higher doses of standard curcumin. The enhanced absorption of FTC derived curcuminoids was attributed to its water solubility, amorphous nature and the presence of turmeric lipids as homogeneous colloidal submicron sized particles suitable for the better intestinal absorption and inhibition of metabolizing enzymes. Further, the facts that the fresh turmeric juice powder can be prepared without using solvents or excipients, and the waste after turmeric juice extraction can be further used for extraction of standard curcumin of commercial use, indicate the significance of FTC and its novel process of

64

Journal of Functional Foods 17 (2015) 55–65

preparation as demonstrated in the present study. Thus, FTC is a value added form of turmeric which otherwise would have been lost during the commonly practiced process of preparation of dried turmeric.

Conflict of interest Authors disclose the conflict of interest. FTC is a patented pending formulation of M/s Akay Flavours & Aromatics Pvt Ltd, Cochin, India. All animal studies and interpretation of the results have been carried out by RK, Director of research at M/s Amala Cancer Research Centre, Trichur, India, a non-profitable research organization. RK has no known conflict of interest.

Acknowledgements The authors express sincere thanks to SIFC, Cochin, India for instrumental analysis and also thank Mr. Shibu Anandarajan of M/s Akay Flavours & Aromatics Pvt. Ltd, Cochin, India for arranging the fresh rhizome samples of turmeric with farm level traceability. The study was financially supported by M/s Akay Flavours & Aromatics, Pvt. Ltd as a part of the research program AK/R&D/NPP-02/2010 for the development of ‘Spiceuticals®’ – the nutraceutical ingredients from spices.

REFERENCES

Aditya, N. P., Aditya, S., Yang, H.-J., Kim, H. W., Park, S. O., Lee, J., & Ko, S. (2015). Curcumin and catechin co-loaded water-in-oilin-water emulsion and its beverage application. Journal of Functional Foods, 15, 35–43. Antony, B., Merina, B., Iyer, V. S., Judy, N., Lennertz, K., & Joyal, S. (2008). A pilot cross-over study to evaluate human oral bioavailability of BCM-95CG (Biocurcumax), a novel bioenhanced preparation of curcumin. Indian Journal of Pharmceutical Sciences, 70, 445–449. Cuomo, J., Appendino, G., Dern, A. S., Schneider, E., McKinnon, T. P., Brown, M. J., Togni, S., & Dixon, B. M. (2011). Comparative absorption of a standardized curcuminoid mixture and its lecithin formulation. Journal of Natural Products, 74, 664–669. Deshpande, S. S., Ingle, A. D., & Maru, G. B. (1998). Chemopreventive efficacy of curcumin-free aqueous turmeric extract in 7, 12-dimethylbenz[a]anthraceneinduced rat mammary tumorigenesis. Cancer Letters, 123, 35– 40. Garcea, G., Jones, D. J., Singh, R., Dennison, A. R., Farmer, P. B., Sharma, R. A., Steward, W. P., Gescher, A. J., & Berry, D. P. (2004). Detection of curcumin and its metabolites in hepatic tissue and portal blood of patients following oral administration. British Journal of Cancer, 90, 1011– 1015. Goel, A., Jhurani, S., & Aggarwal, B. B. (2008). Multi-targeted therapy by curcumin: How spicy is it? Molecular Nutrition and Food Research, 52, 1010–1030. Hamam, F., & Al-Remawi, M. (2014). Novel delivery system of curcumin through transdermal route using sub-micronized particles composed of mesoporous silica and oleic acid. Journal of Functional Foods, 8, 87–99.

Horwits, W., & Latimer, G. W., Jr. (Eds.), (2006). Official methods of analysis of AOAC International (18th ed.). Gaithersburg, Maryland, USA: AOAC International. Ireson, C. R., Orr, S., Jones, D. L., Verschoyle, R., Lim, C. K., Luo, J. L., Howells, L., Plummer, S. M., Jukes, R., Williams, M., Steward, W. P., & Gescher, A. (2001). Characterization of metabolites of the chemopreventive agent curcumin in human and rat hepatocytes and in rat plasma and evaluation of their ability to inhibit cyclooxygenase-2 expression. Cancer Research, 61, 1058–1064. Jadhav, B. K., Mahadik, K. R., & Paradkar, A. R. (2007). Development and validation of improved reversed phaseHPLC method for simultaneous determination of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Chromatographia, 65, 483–488. Jaganathan, S. K., Vellayappan, M. V., Narasimhan, G., & Supriyanto, E. (2014). Role of pomegranate and citrus fruit juices in colon cancer prevention. World Journal of Gastroenterology, 20, 4618–4625. Jager, R., Lowery, R. P., Calvanese, A. V., Joy, J. M., Purpura, M., & Wilson, J. M. (2014). Comparative absorption of curcumin formulations. Nutrition Journal, 13, 11. Jantarat, C. (2013). Bioavailability enhancement technologies of herbal medicine: A case example of curcumin. International Journal of Pharmacy and Pharmaceutical Sciences, 5, 493–500. Kapakos, G., Youreva, V., & Srivastava, A. K. (2012). Cardiovascular protection by curcumin: Molecular aspects. Indian Journal of Biochemistry and Biophysics, 49, 306–315. Kocher, A., Schiborr, C., Behnam, D., & Frank, J. (2015). The oral bioavailability of curcuminoids in healthy humans is markedly enhanced by micellar solubilisation but not further improved by simultaneous ingestion of sesamin, ferulic acid, naringenin and xanthohumol. Journal of Functional Foods, 14, 183–191. Krishnakumar, I. M., Abhilash, M., Gopakumar, G., Kumar, D., Maliakel, B., & Kuttan, R. (2015). Improved blood–brain-barrier permeability and tissue distribution following the oral administration of a food-grade formulation of curcumin with fenugreek fibre. Journal of Functional Foods, 14, 215–225. Krishnakumar, I. M., Ravi, A., Kumar, D., Kuttan, R., & Maliakel, B. (2012). An enhanced bioavailable formulation of curcumin using fenugreek derived soluble dietary fibre. Journal of Functional Foods, 4, 348–357. Lao, C. D., Ruffin, M. T., 4th., Normolle, D., Heath, D. D., Murray, S. I., Bailey, J. M., Boggs, M. E., Crowell, J., Rock, C. L., & Brenner, D. E. (2006). Dose escalation of a curcuminoid formulation. BMC Complementary and Alternative Medicine, 6, 10. Lin, H.-Y., Lin, J.-N., Ma, J.-W., Yang, N.-S., Ho, C.-T., Kuo, S.-C., & Way, T.-D. (2015). Demethoxycurcumin induces autophagic and apoptotic responses on breast cancer cells in photodynamic therapy. Journal of Functional Foods, 12, 439–449. Okada, K., Wangpoengtrakul, C., Tanaka, T., Toyokuni, S., Uchida, K., & Osawa, T. (2001). Curcumin and especially tetrahydrocurcumin ameliorate oxidative stress-induced renal injury in mice. The Journal of Nutrition, 131, 2090–2095. Ono, K., Hasegawa, K., Naiki, H., & Yamada, M. (2004). Curcumin has potent anti-amyloidogenic effects for Alzheimer’s betaamyloid fibrils in vitro. Journal of Neuroscience Research, 75, 742– 750. Pal, A., Sung, B., Bhanu Prasad, B. A., Schuber, P. T., Jr., Prasad, S., Aggarwal, B. B., & Bornmann, W. G. (2014). Curcuminglucuronides: assessing the proliferative activity against human cell lines. Bioorganic and Medicinal Chemistry, 22, 435–439. Pan, M. H., Huang, T. M., & Lin, J. K. (1999). Biotransformation of curcumin through reduction and glucuronidation in mice. Drug Metabolism and Disposition: The Biological Fate of Chemicals, 27, 486–494.

Journal of Functional Foods 17 (2015) 55–65

Prasad, S., Gupta, S. C., Tyagi, A. K., & Aggarwal, B. B. (2014). Curcumin, a component of golden spice: From bedside to bench and back. Biotechnology Advanced, 32, 1053–1064. 14, S0734-9750 00051-2. Prasad, S., Tyagi, A. K., & Aggarwal, B. B. (2014). Recent developments in delivery, bioavailability, absorption and metabolism of curcumin: The golden pigment from golden spice. Cancer Research and Treatment: Official Journal of Korean Cancer Association, 46, 2–18. Sahebkar, A., Serban, M.-C., Ursoniu, S., & Banach, M. (2015). Effect of curcuminoids on oxidative stress: A systematic review and meta-analysis of randomized controlled trials. Journal of Functional Foods, http://dx.doi.org/10.1016/j .jff.2015.01.005. (in press). Sandur, S. K., Pandey, M. K., Sung, B., Ahn, K. S., Murakami, A., Sethi, G., Limtrakul, P., Badmaev, V., & Aggarwal, B. B. (2007). Curcumin, Demethoxycurcumin, Bisdemothoxycurcumin, Tetrahydrocurcumin, and Turmerones Differentially Regulate Antiinflammatory and Antiproliferative Responses Through a ROSIndependent Mechanism. Carcinogenesis, 28, 1765–1773. Sasaki, H., Sunagawa, Y., Takahashi, K., Imaizumi, A., Fukuda, H., Hashimoto, T., Wada, H., Katanasaka, Y., Kakeya, H., Fujita, M.,

65

Hasegawa, K., & Morimoto, T. (2011). Innovative preparation of curcumin for improved oral bioavailability. Biological and Pharmaceutical Bulletin, 34, 660–665. Schiborr, C., Kocher, A., Behnam, D., Jandasek, J., Toelstede, S., & Frank, J. (2014). The oral bioavailability of curcumin from micronized powder and liquid micelles is significantly increased in healthy humans and differs between sexes. Molecular Nutrition and Food Research, 58, 516–527. Thakur, L., Ghodasra, U., Patel, N., & Dabhi, M. (2011). Novel approaches in stability improvement of natural medicines. Pharmacognosy Reviews, 5, 48–54. USP 32-NF 27, General Chapter <467>. (2009). Organic volatile impurities, US Pharmacopeia, Rockville, MD, 8/2009. Wahlstrom, B., & Blennow, G. (1978). A study on the fate of curcumin in the rat. Acta Pharmacologica et Toxicologica, 43, 86– 92. Yallapu, M. M., Jaggi, M., & Chauhan, S. C. (2012). Curcumin nanoformulations: A future nanomedicine for cancer. Drug Discovery Today, 17, 71–80. Yu, Z. F., Kong, L. D., & Chen, Y. (2002). Antidepressant activity of aqueous extracts of Curcuma longa in mice. Journal of Ethnopharmacology, 83, 161–165.