Physical and Chemical Stability of Curcumin in Aqueous Solutions

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Physical and chemical stability of curcumin in aqueous solutions and emulsions: Impact of pH, temperature, and molecular environment Mahesh Kharat, Zheyuan Du, Guodong Zhang, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04815 • Publication Date (Web): 09 Dec 2016 Downloaded from http://pubs.acs.org on December 12, 2016

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Journal of Agricultural and Food Chemistry

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Physical and chemical stability of curcumin in aqueous

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solutions and emulsions: Impact of pH, temperature, and

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molecular environment

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Mahesh Kharat1, Zheyuan Du1, Guodong Zhang1, and David Julian

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McClements1,2* 1

Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA 2

Department of Biochemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203 Jeddah 21589 Saudi Arabia

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Journal: Journal of Agricultural and Food Chemistry

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Submitted: October 2016

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*Contact Information for Corresponding Author: David Julian McClements, Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA; Tel 413 545 1019; Fax 413 545 1262; email [email protected].

1 ACS Paragon Plus Environment


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ABSTRACT

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The utilization of curcumin as a nutraceutical in food and supplement products is

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often limited because of its low-water solubility, poor chemical stability, and low oral

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bioavailability. This study examined the impact of pH, storage temperature, and

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molecular environment on the physical and chemical stability of pure curcumin in

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aqueous solutions and in oil-in-water emulsions. Unlike naturally occurring

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curcuminoid mixtures (that contain curcumin, demethoxy-curcumin and

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bisdemethoxy-curcumin), pure curcumin was highly unstable to chemical degradation

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in alkaline aqueous solutions (pH ≥ 7.0), and tended to crystallize out of aqueous

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acidic solutions (pH < 7). These effects were attributed to changes in the molecular

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structure of curcumin under different pH conditions. The curcumin crystals formed

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were relatively large (10-50 µm), which made them prone to rapid sedimentation. The

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incorporation of curcumin into oil-in-water emulsions (30% MCT, 1 mg curcumin/g

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MCT, d32 ≈ 298 nm) improved its water-dispersibility and chemical stability. After

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incubation at 37 oC for one month more than 85% of curcumin was retained by

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emulsions stored under acidic conditions (pH < 7), while 62, 60, and 53% was

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retained by emulsions stored at pH 7.0, 7.4, and 8.0, respectively.

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change in the color of curcumin-loaded emulsions when stored under acidic

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conditions, but their yellow color faded when stored under alkaline conditions. There

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was no evidence of droplet aggregation or creaming in emulsions stored for 15 days at

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ambient temperature. These results suggest that emulsion-based delivery systems may

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be suitable for improving the water-dispersibility and chemical stability of curcumin,

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which would facilitate their application in foods and supplements.

There was little

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Key words: Curcumin; stability; delivery; emulsion; crystallization


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INTRODUCTION

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Curcumin is the most abundant and biologically active of the curcuminoids

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present in turmeric.1 As a part of powdered turmeric, curcumin has been used since

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ancient times in India and other South Asian countries for its flavor, color, and

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medicinal properties. For example, it is widely used as a spice and colorant in

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curries and cosmetics, and as a nutraceutical in Ayurvedic remedies to treat numerous

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diseases. Recent in vitro and in vivo research has provided insights into the potential

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mechanisms underlying the health benefits of curcumin including antioxidant,

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anti-cancer, anti-rheumatic, and anti-inflammatory activities.2 Curcumin has

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numerous chemically reactive functional groups, which means that it can participate

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into many different biochemical pathways by interactions such as donating or

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accepting H-bonds, cation binding, and the Michael reaction.3 Various molecular

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targets of curcumin have been identified including transcription and growth factors,

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protein kinases, cytokines and others.4

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be effective against a range of different types of cancer cells.3 Moreover, curcumin is

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considered to be safe for consumption at the levels required to have a beneficial health

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effect. Clinical trials suggest that humans have a high tolerance for curcumin without

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any toxic effects if ingested at levels of 8 g/day,5 and even higher amounts (12 g/day)

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can still be tolerated with minimal toxicity.6 Despite its extraordinary potential as a

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bioactive agent , especially against cancer, there are major challenges in using

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curcumin as a therapeutic agent in medicines, and as a nutraceutical ingredient in

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foods and supplements.7 Firstly, the water-solubility of curcumin is extremely low

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(estimated to be 3×10-8 M)8, which makes it difficult to incorporate into many

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aqueous-based products.9 Moreover, its solubilization in the aqueous fluids within the

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gastrointestinal tract is also relatively poor, which means that its absorption by

Consequently, curcumin has been shown to



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epithelial cells is inefficient.

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with digestible lipids that form mixed micelles in the small intestine so that it can be

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effectively solubilized and transported to the epithelium cells.10

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Consequently, curcumin usually has to be co-ingested

Another major factor that limits curcumin bioavailability is its chemical

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instability under physiological conditions.11, 12 At physiological pH, curcumin rapidly

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degrades to bicyclopentadione through autoxidation, with cleavage products like

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bicyclopentadione, vanillin, and ferulic acid being formed.13, 14 Curcumin that

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survives this form of chemical degradation may undergo biotransformation due to the

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presence of metabolizing enzymes to form reduction products and glucuronide in the

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liver and other organs.15, 16 As a result of these chemical and biochemical processes

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the fraction of ingested curcumin that actually reaches the blood is very low. For

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example, the serum concentration of curcumin has been reported to range from

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around 0 to 156 nM two hours after ingestion of 12 g of curcuminoids (8 g

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curcumin).6 This discussion clearly highlights some of the challenges associated with

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using curcumin for treating diseases and developing functional food and supplement

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products.

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Many approaches have been studied for their potential to improve the

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water-dispersibility, chemical stability, and bioavailability of curcumin. One of the

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most promising approaches has been to encapsulate the curcumin in delivery systems

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such as conjugates, molecular complexes, micelles, liposomes, suspensions, hydrogels,

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and emulsions.17 Each of these delivery systems has particular advantages and

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disadvantages for particular applications. In the food industry, oil-in-water

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emulsions are particularly suitable for the encapsulation of hydrophobic nutraceuticals

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because they can easily be manufactured using food-grade ingredients and widely

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used production equipment (homogenizers).18, 19

In addition, many food products


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already exist in the form of oil-in-water emulsions (such as beverages, dressings,

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soups, sauces, deserts, and yogurts), and so it is relatively easy to incorporate

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nutraceutical-loaded emulsions into them. Moreover, emulsions can easily be

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converted into a powdered form by freeze or spray drying, which means that

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encapsulated nutraceuticals can also be incorporated into solid foods (such as cereals,

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confectionary, and baked goods). For these reasons, the main objective of the current

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study was to examine the impact of incorporating curcumin into emulsion-based

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delivery systems on its water-dispersibility and chemical stability. Numerous

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previous studies have highlighted the potential of emulsion-based delivery systems for

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improving the stability and bioavailability of curcumin.20-27 However, to the author’s

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knowledge, the current study is the first to give a detailed comparison of the impact of

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storage pH on the physical and chemical stability of pure curcumin in aqueous

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solutions and emulsions.

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curcumin may be incorporated into commercial products with different pH values and

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because ingested curcumin experiences different pH conditions as it travels through

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the human gastrointestinal tract.

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This information is important because encapsulated

Most of the previous studies on encapsulated curcumin have used naturally

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occurring “curcumin”, which is actually a mixture of three major components:

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curcumin, demethoxy-curcumin, and bis-demethoxy-curcumin.3

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different physicochemical and physiological properties than demethoxy- and

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bis-demethoxy-curcumin,28, 29 which may impact the design of appropriate delivery

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systems for this nutraceutical.

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current study and then used to compare the impact of pH on the physical and chemical

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stability of curcumin in aqueous solutions and oil-in-water emulsions.

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knowledge obtained from this study may facilitate the rational design of

Pure curcumin has

For this reason, pure curcumin was synthesized in the

The



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emulsion-based delivery systems suitable for utilization in foods, supplements, and

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pharmaceuticals.

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MATERIALS AND METHODS

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Materials

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Pure curcumin was synthesized and purified in the Department of Food Science

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at the University of Massachusetts using the method described by Pabon.30 Medium

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chain triglyceride (MCT) oil was purchased from Warner Graham Co. (Cockeysville,

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MD). Dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), sodium phosphate

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anhydrous dibasic, and potassium phosphate monobasic were obtained from Fisher

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Scientific (Fair Lawn, NJ). Curcumin (≥ 65%), Hydrochloric acid (HCl), Tween 20,

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and Tween 80, were purchased from the Sigma-Aldrich Company (St. Louis, MO).

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All solvents and reagents were of analytical grade. Double distilled water from a

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water purification system (Nanopure Infinity, Barnstaeas International, Dubuque, IA)

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was used throughout the experiments.

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Dissolution and stability of curcumin in aqueous buffer solutions

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A stock curcumin solution was prepared by adding a weighed amount of

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curcumin (65%) or pure curcumin into DMSO and then dissolving it for 2 min using a

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vortex. This solution was stored at 4 °C and used throughout the experiments. An

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aliquot of the stock curcumin solution (4 mM) was diluted in a quartz cuvette (path

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length 1 cm) with phosphate buffer solution (10 mM; 0.25 g/L KH2PO4, and 1.15 g/L

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Na2HPO4). HCl and/or NaOH were added previously to adjust the buffer pH to values

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ranging from 2.0 to 8.0.

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measuring the change in absorbance at 433 nm over time using a UV-visible

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spectrophotometer (Cary 100 UV-Vis, Agilent Technologies). In some experiments,

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the effect of mixing was studied by agitating the samples continuously to ensure

Curcumin degradation was determined at 37 °C by


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homogeneity using a cross-type stir bar (FisherbrandTM) driven by a magnetic stirrer.

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Turbidity measurements

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Turbidity measurements were used to monitor the formation of curcumin crystals

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in aqueous systems. The turbidity (at 600 nm) of freshly prepared curcumin

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solutions was measured at regular time intervals using a UV-visible

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spectrophotometer, with or without sample mixing using a stir bar and magnetic stirrer.

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All samples were made uniform by gentle mixing using a spatula before measuring

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the turbidity.

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Oil-solubility of curcumin

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Excess curcumin was added to a weighed amount of MCT oil, the resulting

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mixture was then stirred at 60 °C and 1200 rpm for 2 h, and sonicated for 20 min to

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ensure dissolution. This process was repeated again if needed to ensure saturation of

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the curcumin in the oil phase. The mixtures were then centrifuged at 12,000rpm

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(accuSpin Micro 17, Fisher Scientific) for 20 min at room temperature to remove the

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curcumin crystals. The supernatant was collected and analyzed for curcumin content

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as described later.

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Preparation of curcumin emulsions

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An aqueous phase was prepared by mixing surfactant (either Tween 20 or Tween

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80) in phosphate buffer (10 mM, pH 5.0) at room temperature for at least 2 h. An oil

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phase was prepared by dissolving curcumin in MCT (1 mg/g MCT) with constant

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stirring at 60 °C for 2 h and then sonicating for 20 min. This whole procedure was

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repeated again if needed to completely dissolve the crystals. A stock emulsion was

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prepared by homogenizing the oil and aqueous phases together using a high-speed

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blender for 2 min (M133/1281-0, Biospec Products, Inc., ESGC, Switzerland). The

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resulting coarse emulsion was then passed through a microfluidizer (M110L,



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Microfluidics, Newton, MA) at 12,000 psi for 5 passes. The final composition of the

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stock emulsion produced was 40% oil phase, 2% Tween 20 or Tween 80, and 58%

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phosphate buffer by weight. Sample emulsions containing 30% w/w oil and a range of

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different pH values were then prepared by diluting the stock emulsion with pH

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adjusted buffer solutions. The sample emulsions were then incubated at 37 °C

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throughout the study. Additionally, some samples were maintained at 20 °C, and

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55 °C to study the effect of storage temperature.

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Curcumin concentration measurements

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Curcumin quantification was carried out using the simple spectrophotometric

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method described by Davidov-Pardo et al.31 A specific volume of emulsion (100 µL)

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was added to DMSO (5900 µL), and the contents were mixed well. The bottom layer

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was withdrawn after centrifugation (2000 rpm) for 15 min, and the absorbance was

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measured at 433 nm using a spectrophotometer. Preliminary experiments showed that

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the absorbance of the resulting mixtures was stable for at least 1 h for all pH values

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(data not shown). Linear regression (r2 = 0.9993) obtained from curcumin-DMSO

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standard solutions was used to quantify the amount of curcumin present (Supporting

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information, Figure S1).

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Color measurements

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The intensity of the yellow color of a sample is a measure of the curcumin

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stability, as well as the overall appearance. For this reason, changes in the color of the

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curcumin emulsion was measured using an instrumental colorimeter (ColorFlez EZ,

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HunterLab, Reston, VA) equipped with a tristimulus absorption filter. A measured

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volume of curcumin emulsion was transferred into a disposable petri dish and then

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analyzed with using a pure black plate as the background.


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Particle size and charge measurements The particle size distribution of the emulsions was determined using static light

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scattering (Mastersizer 2000, Malvern Instruments Ltd., Malvern, Worcestershire,

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UK), which utilizes measurements of the angular scattering pattern of emulsions.

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Samples were gently hand-mixed to ensure homogeneity. Phosphate buffer (10 mM)

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was used as diluent and had the same pH as the sample being measured to avoid

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multiple scattering effects. Average particle sizes are reported as the surface-weighted

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mean diameter (d32).

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An electrophoresis instrument was used to measure the -potential of the droplets

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in the emulsions (Zetasizer Nano ZS series, Malvern Instruments Ltd. Worcestershire,

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UK). Before analysis, emulsions were diluted with phosphate buffer (10 mM) having

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the same pH as the sample to avoid multiple scattering effects.

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Microstructure analysis

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The microstructures of aqueous solutions and emulsions containing curcumin

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were recorded at various pH values using optical microscopy with a 60× oil

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immersion objective lens and 10× eyepiece (Nikon D-Eclipse C1 80i, Nikon, Melville,

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NY, US.). For this, an aliquot of sample was placed on a microscope slide, covered by

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a cover slip, and then microstructure images were acquired using image analysis

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software (NIS-Elements, Nikon, Melville, NY). Polarized light microscopy was

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carried out using a cross-polarized lens (C1 Digital Eclipse, Nikon, Tokyo, Japan).

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Statistical analysis

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Experiments were carried out using two or three freshly prepared samples. The

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results are reported as averages and standard deviations. The significant differences

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among treatments were evaluated using the Tukey multiple comparison test at a

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significance level of p ≤ 0.05 (SPSS ver.19, SPSS Inc., Chicago, IL).



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RESULTS AND DISCUSSIONS

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Physical and chemical stability of curcumin in aqueous solutions

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Initially, the physical and chemical stability of curcumin added to aqueous buffer

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solutions with different pH values was examined. In the absence of continuous

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stirring, the absorbance of curcumin-buffer solutions decreased over time at all pH

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values studied, with the rate of decrease depending on pH (Figure 1A).

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alkaline conditions (pH ≥ 7.0), the solutions remained clear throughout storage, but

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there was a rapid decrease in absorbance during the first 7 minutes with calculated

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degradation rates of 92, 135, and 125 cm-1/min at pH 7.0, 7.4, and 8.0 respectively.

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The degradation rates were calculated as the slope of absorbance versus time in the

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initial period, when the change in absorbance was approximately linear. This result is

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in agreement with previous studies that have reported that curcumin is highly unstable

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to chemical degradation around physiological pH.12 It was also noted that when

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curcumin-DMSO stock solution was first added to the buffers, a distinct red-brown

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color appeared, but that this initial color rapidly changed. As a result, the alkaline

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curcumin solutions had slightly higher initial absorbance values than the acid

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curcumin solutions. A possible explanation for this effect could be the formation of

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condensation products (such as feruloylmethane) under alkaline conditions, which are

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yellow in color and therefore increase the overall absorbance.32 Interestingly, a

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commercial curcumin sample, which actually consisted of a mixture of curcuminoids,

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appeared to have better stability at pH 7 than the pure curcumin sample (Figure 1A).

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This result is in agreement with previous studies28 and suggests that the different

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constituents of curcuminoids have different pH stabilities, which is important to

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understand when developing effective delivery systems for this bioactive component.

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The decrease in absorbance over time occurred much more slowly for the acidic

Under


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curcumin solutions than for the alkaline ones in the absence of stirring (Figure 1A),

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which is in agreement with previous studies that have shown that curcumin is more

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chemically stable under acidic conditions.11, 33 Interestingly, the decrease in

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absorbance over time for the acidic curcumin solutions was much faster when they

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were continuously stirring, than in the absence of stirring (Figure 1B).

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suggest that stirring altered either the physical or chemical stability of the curcumin.

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Crystallization of curcumin in aqueous solutions

These results

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During the spectrophotometric analysis of curcumin stability in aqueous solutions,

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it was observed that yellow colored crystals appeared in solution and on the surface of

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the stir bar. This observation suggested that mixing promoted the nucleation and

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crystallization of curcumin in solution, which reduced the measured absorbance

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because some of the curcumin molecules were no longer in the path of the UV-visible

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light beam used to analyze the samples.

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poor solubility in water, especially at lower pH values due to a change in its molecular

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structure under acidic conditions 8, 11. The formation of crystals was confirmed using

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spectrophotometry to measure the turbidity (at 600 nm) of the samples (since

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curcumin does not absorb light strongly at this wavelength). At this wavelength, a

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decrease in the intensity of the transmitted light (corresponding to an increase in

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measured absorbance) is due to light scattering by any crystalline curcumin rather

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than absorption by the functional groups on curcumin.

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7.0), there was an increase in turbidity over time corresponding to crystal formation

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(Figure 2A). At alkaline pH values (pH ≥ 7.0), there was little change in turbidity

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over time, which can be attributed to the fact that no visible crystals were seen under

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these conditions.

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were formed under mixing conditions (Figure 5, C), but small uniformly dispersed

It is known that curcumin has a relatively

At acidic pH values (pH

Physical and Chemical Stability of Curcumin in Aqueous Solutions

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