Development of a gradient tube method for examining microbial population structures in floating sulphur biofilms
July 2013. July 2013

M L Bowker
Environmental Biotechnology Research Institute
Rhodes University
GrahamstownSouth Africa

J B Molwantwa
Digby Wells Associates
JohannesburgSouth Africa

J Gilfillan
Department of Biochemistry, Microbiology and Biotechnology
Rhodes University
GrahamstownSouth Africa

R Dorrington
Department of Biochemistry, Microbiology and Biotechnology
Rhodes University
GrahamstownSouth Africa

R Kirby
Department of Life Sciences and Institute of Genome Science
National Yang-Ming University
Taiwan

P D Rose email@example.com
Department of Biochemistry, Microbiology and Biotechnology
Rhodes University
GrahamstownSouth Africa

Development of a gradient tube method for examining microbial population structures in floating sulphur biofilms

= Water SA
On-line394July 2013. July 201310.4314/wsa.v39i4.11Received 23 July 2012; accepted in revised form 24 June 2013.523 * To whom all correspondence should be addressed.  +27 82 8101353; Technical notegradient tube methodfloating biofilmsfloating sulphur biofilmsmicrobial ecologysulphur biotechnologyacid mine drainage wastewaters
Floating biofilms occur in thin layers of between 50 μm and 500 µm on the surface of certain organic, sulphidic aquatic environments and, at times, may only be several cells deep. While these structures may be important in terms of energy flow pathways, and possibly also in wastewater treatment operations, little is known about their structural/functional properties. This is due, in part, to their flimsy nature but also to methodological constraints related to their sampling and manipulation. We have investigated floating sulphur biofilms that appear as white layers on the surface of anoxic sulphidic organic wastewaters and describe here the development of a novel gradient tube method for investigating these systems. This approach enables testing of the hypothesis that these floating sulphur biofilms are complex well-differentiated structures rather than disordered dispersions of microbial biomass as has been previously thought. Furthermore, if the former is correct, they would seem to resemble the structure and functionality of comparable complex bioflms that are attached to solid substrates. The gradient tube method involves the establishment of apposing gradients of sulphide and oxygen that are expanded across a tube of agarose 10 cm in length; this simulates the oxic/anoxic interface that occurs over only several micrometres in the natural biofilm system. A plug of sulphide-enriched agarose is first placed in the base of the tube. Samples of the floating sulphur biofilm are then mixed into agarose growth medium and, before it sets, this is overlaid on top of the plug. The tubes are then open capped and incubated. A variety of different microbial populations may thus become established in the separate physiological niches that are set up in this way within the gradient tube. The populations may be quite robustly sampled by extruding and then sectioning the agarose plug. This expansion of the biofilm enables more detailed molecular phylogenetic studies of the populations found in the various niches within the biofilm and also measurement of physico-chemical parameters within the system.

INTRODUCTION

The examination of the population structure of biofilms, and particularly attempts to correlate structure and function in a way that rigorously represents the actual situation within the biofilm, remains an exacting methodological task. The requirements are particularly acute when studying floating biofilms that occur as layers of around 50 μm to 500 µm on water surfaces in some aquatic environments and are, during the early stages of formation, possibly only several cells deep (Gilfillan, 2000).

We have examined floating sulphur biofilms that form at the oxic/anoxic interface on the surface of tannery evaporation ponds (Fig. 1A), which receive high organic and sulphide loads (Gilfillan, 2000;Bowker, 2002). Similar structures have been observed in other sulphidic aquatic environments such as brackish and salt marshes and these biofilms may be an important component in total energy flow pathways in these systems (Whitcomb, 1989). While it has been suggested that they are simple accumulations of microbial growth, we have found that these floating sulphur biofilms are composed of both obligate aerobic and anaerobic populations, including sulphide oxidising bacteria, which suggests rather that they are complex well-differentiated structures that are comparable to stratified biofilm growth found on solid substrates (Monds and O'Toole, 2009). While scanning electron microscopy of the floating sulphur biofilm (Fig. 2) clearly shows that the microbial population is embedded in an exopolysaccharide matrix that includes the production of ortho-rhombic crystals of elemental sulphur (Bowker, 2002), the possible functional differentiation of the population structure within the biofilm has remained undescribed up to the present.

The possible application of the floating sulphur biofilm system for sulphur recovery in the biological treatment of acid mine drainage wastewaters has been proposed and bioprocess development studies have been reported by Gilfillan (2000) 524 experimental manipulation of these thin and fragile structures. More clarity on the structural/functional attributes of these floating biofilm systems could be important to the future engineering of their use in wastewater treatment applications. We report here the development of a gradient tube method for expanding the spatial distribution of the various physiologically functional microbial groups that grow actively within these biofilms. This approach allows the effective sampling of the different components found within the biofilm population structure.

MATERIALS AND METHODS

Actively growing floating sulphur biofilm was sampled from both tannery ponding systems (Fig. 1A), and a laboratory reactor (Fig.1B) that was fed organic sulphide-enriched water generated in a degrading packed bed reactor as described by Molwantwa et al. (2007). Aliquots of these biofilms (500 µℓ) were mixed with 500 µℓ of glycerol and frozen at −70 o C until used. Gradient tubes were prepared by aseptically placing, at the bottom of a sterile 15 mℓ glass test tube, a 5 mℓ molten agarose plug containing 1% low-melt agarose, 0.5 g/ℓ sodium sulphide in lactate medium (1.75 mℓ 60% sodium lactate, 1 g MgSO 4 •7H 2 O, 0.5g yeast extract, 0.25g K 2 HPO 4 , 0.05 g CaCl 2 •6H 2 O, 0.5 g NH 4 Cl; made up in 250 mℓ with ddd H 2 O and sterilised). After the sulphide plug had set, an overlay of the same lactate medium, but without sodium sulphide, was prepared, to which the biofilm sample was added and thoroughly mixed; the biofilm inoculum was added while the medium was still liquid but as cool as possible. Then 10 mℓ of the mixture was pipetted aseptically onto the surface of the solid agarose sulphide plug while avoiding the introduction of air bubbles.

The tubes were open capped (agarose surface having access to air) and incubated in the dark at 20 o C for 7 days (Fig. 3).

After incubation had been completed, tubes were swabbed with ethanol, etched with a diamond glass cutter and then broken cleanly at the bottom. The agarose plug was then extruded onto an aseptic surface in a laminar flow hood. The plug was sliced into 5 mm lengths and each sample placed into a separate sterile Eppendorf tube that contained 1 mℓ sterile Tris-EDTA buffer (0.01 M Tris, 0.05M EDTA). Samples were heated to 50 o C, vortexed, and (with the exception of those to be used in sulphide analyses) were stored at 4 o C overnight to facilitate diffusion. They were then analysed to measure the levels of sulphate, sulphur and total organic carbon (TOC). Samples for molecular phylogenetic analysis (results not reported here) were obtained prior to the heating step.

For the sulphide analysis, 20 µℓ samples were taken immediately from each of the 0.5 g sections and added to 100 µℓ of zinc acetate and made up to 5 mℓ with ddd H 2 O. The assay was performed as follows: the samples were vortexed briefly and 500 µℓ of ferric chloride solution (8 g FeCl 3 in 500 mℓ 6 M HCL) and 500 µℓ amide-sulphuric acid solution (2g N-N dimethyl-p-phenylene diamine dihydrochloride in 500 mℓ 6 M H 2 SO 4 ) were added. The samples were allowed to stand at room temperature for 1 h and then the absorbance was read on a Shimadzu UV-160A UV-visible recording spectrophotometer at 670 nm.   525 1 h and then filtered through a 0.45 µm nylon membrane to remove any particulate matter. The samples were analysed by HPLC using a Phenomenex LUNA 5µ C18 (2) size 150 x 4.6 mm column, mobile phase 95:5 methanol:water, run at a flow rate of 2 mℓ/min and using a Waters 484 Absorbance Detector. Sulphate analysis was performed by HPLC. Samples were diluted 1 in 10 and then 1 mℓ of this sample was filtered through a 0.45 µm nylon filter followed by a Waters C18 solidphase extraction cartridge. A Hamilton PRP-X100 10 µm 150 x 4.1 mm column with a para-hydroxybenzoic acid/methanol (5.52 g:250 mℓ) mobile phase was used at a flow rate of 2 mℓ/ min with a Waters 430 conductivity detector.

For total organic carbon (TOC) analysis, a ROSEMOUNT Dohrmann Total Organic Carbon Analyzer DC-180 was used. All reagents were supplied by Merck. Figure 3 illustrates the principle of the gradient tube method whereby apposing gradients, in reducing concentrations, are established, and where sulphide migrates from the bottom plug upwards and oxygen migrates from the top of the tube downwards. This is designed to replicate conditions at the oxic/ anoxic interface of the water surface in the sulphidic aquatic environments being investigated, where oxygen migrating from the air into the water column meets the underlying sulphide layer. It is in the presence of this type of gradient that the appearance of the floating sulphur biofilm structures is observed. In the tube method, gradients of high sulphide/low oxygen and low sulphide/high oxygen are established over a 10 cm length of the agarose plug; this provides a range of niches for the growth of any physiologically distinct microbial populations that are present in the original biofilm. These would possibly grow and be discernible as separate zones of population dominance within the tube, which has now been expanded across the 10 cm length of the agarose column, rather than the 50 μm to 500 µm of the original biofilm. The results detailed in Fig. 4    For sulphur analysis 200 µℓ of each 0.5 g sample was placed in an Eppendorf tube and microfuged at 13 000 r/min for 10 min. The supernatant was discarded and the pellet re-suspended in 100 µℓ of high performance liquid chromatography (HPLC) grade acetone. The sample was allowed to stand for 526 agarose column cut sections, and compare various biofilm inoculated samples with uninoculated controls. These results are plotted as depth profiles on the y-axis and report the results within the agarose column from the bottom of the tube upwards, which occurs against a background of increasing oxygen and decreasing sulphide concentrations.

RESULTS AND DISCUSSSION

Comparison of controls against experimental results reported in Fig. 4 shows that a sulphide gradient is established along the length of the agarose column as anticipated. In the biofilminoculated sample sulphide removal was found to be active across about 80% of the column length. Figures 4B and 4C report the sulphur and sulphate concentration profiles, respectively. These results indicate the presence of incomplete oxidation of sulphide with a predominant conversion to sulphur, rather than complete oxidation to sulphate. These findings indicate a careful poising of the redox environment (Rein 2002) which, if unregulated in some way, would expect to show the complete oxidation of sulphide in the upper oxic zones of the tubes. If a metabolic energy input is required to maintain the redox poising of the system, some TOC would be expected to be consumed in the process, and this is what was found in Fig. 4D, where up to 30% of TOC was removed in the upper layer of the tubes.  527 Figures 5B and 5C show the appearance of sulphur bands in the tubes after 1 day and 7 days of growth, respectively. Figure 6 shows scanning electron micrographs of samples from the top, middle and bottom of the gradient tubes and indicates differentiation of the microbial populations within separate bands. The upper layers consist mainly of cocci, while mainly bacilli and filamentous microorganisms occur in the middle and lower layers. Molecular phylogenetic analyses of the actual population structures established by this method were undertaken and are to be reported elsewhere.

This method can be used successfully to investigate many aspects of the complex population structure of floating sulphur biofilms. However, there are limitations that need to be taken into account. The most important relates to a long-standing problem in microbial ecology studies, highlighted by Amann et al. (1995) and others, that only those groups present in the biofilm that are able to grow under the conditions established in the gradient tube experimental system will be easily identified and therefore studied. These are the organisms used to present any descriptive account of the system. Other organisms present in the biofilm that are unable to grown in the medium present in the gradient tube will go unnoticed and this may bias any conclusions drawn.

CONCLUSIONS

The gradient tube method is a novel application that enables the study of microbial population structure and function in thin biofilm layers and does this by expanding the size of sample over which experimental investigation is possible.

The results show that gradients are effectively established in this system with respect to sulphide, sulphur and TOC and to a lesser extent also sulphate.

Organisms of different morphological types were observed at different points within the gradients that were established along the tube length.

Follow-up molecular phylogenetic studies of the microbial populations in floating sulphur biofilms, and correlation of these results with biofilm structure, as determined by microprobe measurements, have been undertaken and are to be reported elsewhere.

The gradient tube method provides an instrument that may be used in conjunction with other methodological approaches to throw light on the microbial ecology of floating sulphur biofilm systems. A better understanding of these structures may facilitate their development in bioprocess applications including acid mine drainage wastewater treatment.

Figure 2
2Scanning electron micrograph showing large amorphous structures which accumulate on the underside of the floating sulphur biofilm.These were shown elsewhere to be composed largely of sulphur, possibly in the long chain polysulphide form.

Figure 1
1A. Photograph of a tannery wastewater evaporation pond, an example of a high-sulphide organic-enriched aquatic environment, and showing formation of the white sulphur biofilm floating on the water surface. Sulphur produced in this system is blown into thick windrows on the sides of the levies but may also be deposited as a sediment within the pond. B. Field-scale floating sulphur biofilm reactor developed to enable the simulation of floating sulphur biofilm formation under experimental conditions.

Figure 4
4A. Sulphide concentration profile through the depth of the gradient tube as measured in each of the 5 mm agarose cut sections. An inoculated tube is compared with the uninoculated control. B. Sulphur concentration profile through the depth of the gradient tube as measured in each of the 5 mm agarose cut sections. An inoculated tube is compared with the uninoculated control. C. Sulphate concentration profile through the depth of the gradient tube as measured in each of the 5 mm agarose cut sections. An inoculated tube is compared with the uninoculated control. D. Total organic carbon concentration profile through the depth of the gradient tube as measured in each of the 5 mm agarose cut sections. An inoculated tube is compared with the uninoculated control.

Figure 3
3Diagram showing the setup of the gradient tubes with a plug of sulphide in the base and overlaid with agarose in which a composite sample of biofilm had been suspended. The opposing gradients of oxygen and sulphide that are set up in this way are shown.

Figure 6
6Scanning electron micrograph of inoculated agarose samples from the top (A), middle (B) and bottom (C) sections of separate gradient tubes. The presence of different microbial morphological types occurring at different levels is apparent, with coccoid forms predominating in the upper layers, rods in the middle layers and filamentous forms appearing also in the bottom layers.

, Bowker (2002), Bowker et al. (2002), Molwantwa et al. (2007), Molwantwa (2008), Rose and Rein (2007), Molwantwa et al. (2009), Mack et al. (2009) and Van Hille et al. (2011). However, uncertainty about a biological role, if any, for sulphur formation in these films, the possible presence of differentiation within the microbial population structure and, importantly, the possibility of physiological functional differentiation within this type of system has remained unresolved. The main reasons for this are methodological constraints related to sampling and http://dx.doi.org/10.4314/wsa.v39i4.11 Available on website http://www.wrc.org.za ISSN 0378-4738 (Print) = Water SA Vol. 39 No. 4 July 2013 ISSN 1816-7950 (On-line) = Water SA Vol. 39 No. 4 July 2013

http://dx.doi.org/10.4314/wsa.v39i4.11 Available on website http://www.wrc.org.za ISSN 0378-4738 (Print) = Water SA Vol. 39 No. 4 July 2013 ISSN 1816-7950 (On-line) = Water SA Vol. 39 No. 4 July 2013

report analyses for sulphide, sulphur, sulphate and TOC for the 5 mmLow Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

overlay column

Sulphide plug

Low Oxygen
High Sulphide

overlay column

Sulphide plug

Low Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

10cm agarose
overlay column

Sulphide plug

Low Oxygen
High Sulphide

overlay column

Sulphide plug

Low Oxygen
High Sulphide

overlay column

Sulphide plug

http://dx.doi.org/10.4314/wsa.v39i4.11 Available on website http://www.wrc.org.za ISSN 0378-4738 (Print) = Water SA Vol. 39 No. 4 July 2013 ISSN 1816-7950 (On-line) = Water SA Vol. 39 No. 4 July 2013Figure 5
Photographs of
uninoculated gradient
tube controls (A) and
inoculated tubes (B)
showing the appearance
of visible bands and a
change in colour of the
neutral red indicator
after 24 h incubation.
The sulphide plug may
be seen in the base of
the tube. Photograph of
inoculated tubes after
incubation for 7 days
showing white bands
of sulphur forming at
different levels within the
gradient tubes (C).

A
B
C

Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Ludwig W Amann R, Schleifer K-H , Microbiol. Rev. 59AMANN R, LUDWIG W and SCHLEIFER K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59 143-169.

The biology and molecular ecology of floating sulphur biofilms. Bowker M, GrahamstownRhodes UniversityPhD thesisBOWKER M (2002) The biology and molecular ecology of floating sulphur biofilms. PhD thesis, Rhodes University, Grahamstown.

The molecular microbial ecology of floating sulphur biofilms. Molwantwa Bowker M, Rose Jb, Pd, Proc. WISA Biennial Conference. WISA Biennial ConferenceDurbanBOWKER M, MOLWANTWA JB and ROSE PD (2002) The molecular microbial ecology of floating sulphur biofilms. Proc. WISA Biennial Conference, 19-23 May 2002, Durban.

The structure and microbiology of floating sulphide oxidizing biofilms. Gilfillan J, GrahamstownRhodes UniversityMasters ThesisGILFILLAN J (2000) The structure and microbiology of floating sulphide oxidizing biofilms. Masters Thesis, Rhodes University, Grahamstown.

Application of the linear flow channel reactor (LFCR) for the removal of sulphide from semi-treated acid mine drainage (AMD). Mack Cl, Raja Sa, Pulles Wb, Muhlbauer Rc, Heath Ra, Abstracts of the International Mine Water Conference. PretoriaMACK CL, RAJA SA, PULLES WB, MUHLBAUER RC and HEATH RA (2009) Application of the linear flow channel reactor (LFCR) for the removal of sulphide from semi-treated acid mine drain- age (AMD). Abstracts of the International Mine Water Conference, 19-23 October 2009, Pretoria.

Development of the floating sulphur biofilm reactor for sulphide oxidation in biological water treatment systems. Coetser Molwantwa Jb, Heath R Se, Rose Pd, Water SA. 305MOLWANTWA JB, COETSER SE, HEATH R and ROSE PD (2007) Development of the floating sulphur biofilm reactor for sulphide oxidation in biological water treatment systems. Water SA 30 (5) 655-657.

Investigation and development of the biotechnology of sulphur biofilms in the beneficiation and treatment of wastewater. Molwantwa Jb ; Grahamstown, Molwantwa Jb, Bowker M, J Gilfillan, Rein R, Hart Dorrington R, Rose Oo, Pd, No. TT 411/09Floating sulphur biofilms: Structure, function and biotechnology. PretoriaWater Research CommissionRhodes University,WRC ReportMOLWANTWA JB (2008) Floating sulphur biofilms: Structure, function and biotechnology. PhD Thesis, Rhodes University, Grahamstown. MOLWANTWA JB, BOWKER M, GILFILLAN J, REIN R, DORRINGTON R, HART OO and ROSE PD (2009) Investigation and development of the biotechnology of sulphur biofilms in the beneficiation and treatment of wastewater. WRC Report No. TT 411/09. Water Research Commission, Pretoria.

The developmental model of microbial biofilms: ten years of a paradigm up for review. Monds Rd, O'toole Ga, Trends Microbiol. 172MONDS RD and O'TOOLE GA (2009) The developmental model of microbial biofilms: ten years of a paradigm up for review. Trends Microbiol. 17 (2) 73-87.

Lessons in passive treatment: Towards efficient operation of a sulphate reduction -sulphide oxidation system. Rose Pd, Rein Nb ; Van Hille, Rp, Motleleng Van Wyk N, Mooruth , Abstracts of the International Mine Water Conference. Aachen, GermanyWater Treatment. US Patent 7285216ROSE PD and REIN NB (2007) Water Treatment. US Patent 7285216. VAN HILLE RP, VAN WYK N, MOTLELENG L and MOORUTH N (2011) Lessons in passive treatment: Towards efficient operation of a sulphate reduction -sulphide oxidation system. Abstracts of the International Mine Water Conference, 4-11 September 2011, Aachen, Germany.

Chemical oxidation of sulfide to elemental sulphur: its possible role in marsh energy flow. Delaune Whitcomb Jh, Patrick Rd, Jr, Mar. Chem. 26WHITCOMB JH, DELAUNE RD and PATRICK JR (1989) Chemical oxidation of sulfide to elemental sulphur: its possible role in marsh energy flow. Mar. Chem. 26 205-214.

. 10.4314/wsa.v39i4.11http://dx.doi.org/10.4314/wsa.v39i4.11

. 1816-7950Print) = Water SA. 394= Water SAAvailable on website http://www.wrc.org.za ISSN 0378-4738 (Print) = Water SA Vol. 39 No. 4 July 2013 ISSN 1816-7950 (On-line) = Water SA Vol. 39 No. 4 July 2013


Diverse effects of a low dose supplement of lipidated curcumin in healthy middle aged people

Robert A Disilvestro
Elizabeth Joseph
Shi Zhao
Joshua Bomser
Diverse effects of a low dose supplement of lipidated curcumin in healthy middle aged people
R E S E A R C H Open Access
Background: Curcumin extracts of turmeric are proposed to produce health benefits. To date, human intervention studies have focused mainly on people with existing health problems given high doses of poorly absorbed curcumin. The purpose of the current study was to check whether in healthy people, a low dose of a lipidated curcumin extract could alter wellness-related measures.Methods: The present study was conducted in healthy middle aged people (40-60 years old) with a low dose of curcumin (80 mg/day) in a lipidated form expected to have good absorption. Subjects were given either curcumin (N = 19) or placebo (N = 19) for 4 wk. Blood and saliva samples were taken before and after the 4 weeks and analyzed for a variety of blood and saliva measures relevant to health promotion. Results: Curcumin, but not placebo, produced the following statistically significant changes: lowering of plasma triglyceride values, lowering of salivary amylase levels, raising of salivary radical scavenging capacities, raising of plasma catalase activities, lowering of plasma beta amyloid protein concentrations, lowering of plasma sICAM readings, increased plasma myeloperoxidase without increased c-reactive protein levels, increased plasma nitric oxide, and decreased plasma alanine amino transferase activities.Conclusion: Collectively, these results demonstrate that a low dose of a curcumin-lipid preparation can produce a variety of potentially health promoting effects in healthy middle aged people.

Background

Curcumin extracted from the spice turmeric shows many different actions in cell cultures and experimental animals [1][2][3]. However, the applicability of this work to human health has been questioned due to the low absorption of curcumin from extract supplement products [1,[4][5][6]. Even so, some studies of high doses of curcumin preparations have had some effects in people with established health problems rev in [1,2]. These doses are called high because the 1 g or more quantities used in most studies exceed what can be typically obtained by people using turmeric related spices. At least one study has tried a much lower dose of curcumin, but this study uses whole turmeric products that cannot really be called a concentrated curcumin extract ie [7]. A few studies have also tried 500 mg curcumin/day as concentrated extract ie [8,9]. Yet, this dose is still fairly high and has not always been given without another active agent ie [9]. In the latter case, the effects of the curcumin versus those of the other agent cannot easily be distinguished. One exception to this high dose approach comes from a recent study that used 180 mg/day of curcumin, but the curcumin by itself did not affect the measures under consideration [10]. In contrast to these studies, the present study examined a fairly concentrated extract at 80 mg curcumin/day, which is a much lower dose than has generally been tried in previous work. Also in contrast to most previous work, the present study used a lipid-curcumin mixture that was projected to be relatively well absorbed.

Another difference between the present study and previous human work on curcumin is that the present study examined healthy subjects rather than people with a health problem. Nearly all of the previous human studies on curcumin extracts have studied people with established health problems. One exception is a study on 500 mg curcumin/day in relatively healthy people, but the intervention lasted just 1 week [8]. More studies of healthy people are needed because the curcumin effects seen in cell cultures and experimental animals could reduce the risk of diseases not yet present.

In studies on cell cultures and experimental animals, curcumin has shown a wide range of effects. For example, curcumin has shown actions that affect lipid metabolism [11][12][13] various anti-inflammatory pathways [3], antioxidant reactions [1][2][3]14], endogenous antioxidant levels [15][16][17][18], endogenous pro-oxidant molecule concentrations [3], neurological processes [19][20][21] and cardiovascular physiology [1,3]. Therefore, the present study covered a wide range of potential mechanisms of action for curcumin and a wide range of health implications.

Methods

Subjects

The protocol was approved by The Ohio State University Human Subjects Biomedical Institutional Review Board. Apparently healthy adult males and postmenopausal females, 40-60 years old, were recruited from the Columbus, Ohio area. Potential subjects were excluded for: current major health problems, cigarette smoking, previous cardiovascular incidents, history of cancer other than small sections of skin, regular use of turmeric, dentist confirmed gingivitis, and use of supplemental phytochemical concentrates.

Research design

Accepted subjects were assigned to either starch placebo or curcumin (N = 19/group, female/male split of 17/2, mean age ± SEM of 48 ± 6 years for the placebo and 47 ± 5 years for the curcumin). The curcumin was Longvida W Optimized Curcumin from Curcuma Longa root given at 400 mg powder per day containing 80 mg curcumin with each of the following ingredients as a proprietary blend: vegetable-derived stearic acid dextrin, hydroxypropylmethylcellulose (vegetarian capsule), soy lecithin, ascorbyl palmitate and silicon dioxide. Longvida W is a trademark of Verdure Sciences, Noblesville, IN, USA. Subjects consumed the assigned product for 4 weeks, with blood samples taken before and after the supplementation period. Subjects were instructed to maintain their previous dietary and exercise practices during participation.

Laboratory analysis

After a fast of about 8 h or more, blood was collected into a tube with heparin, centrifuged at 3000 x g for 30 min to obtain plasma and erythrocytes, and the erythrocytes were washed with phosphate buffered saline, and then extracted with ethanol: chloroform as described earlier [22]. Plasma was stored at −70°C and the erythrocyte extract was stored at −20°C. Saliva was obtained without acute stimulation and stored at −70°C until assayed. Before assay, the saliva samples were briefly centrifuged in an Eppendorf Microfuge to remove solid material and precipitates.

Plasma total cholesterol, triglyceride, LDL, HDL and alanine aminotransferase (ALT) were measured using the Roche Cobas C111 Clinical Chemistry Analyzer (Indianapolis, Indiana, USA). Salivary amylase was determined using an ELISA kit from Salimetrics (State College, Pennsylvania, USA). Plasma catalase and nitric oxide were assayed using kits From Cayman Chemical Company (Ann Arbor, Michigan, USA). Plasma β amyloid protein (1-40) was measured with a commercial ELISA kit from Wako Pure Chemical Industries (Osaka, Japan). Plasma soluble intercellular adhesion molecule (sICAM) was assayed by an ELISA kit from Invitrogen (Frederick, Maryland, USA). Salivary antioxidant status was assessed as free radical scavenging capacity by a kit from Oxford Biomedical Research (Oxford, Michigan, USA). Plasma c-reactive protein was determined by a high sensitivity ELISA kit from MP Biomedicals (Solon, Ohio, USA). Plasma myeloperoxidase was measured by an ELISA kit from Assay Designs (Ann Arbor, Michigan, USA). Plasma ceruloplasmin activity was determined colorimetrically by oxidation of p-phenylenediamine as described earlier [22]. Erythrocyte superoxide dismutase was assayed by a spectrophotometric kinetic assay described before [22].

Results

Curcumin supplementation produced a variety of effects in plasma and saliva measures that are relevant to health promotion. Supplementation, but not placebo, lowered plasma triglyceride values ( Figure 1). The curcumin effect did not extend to plasma lipids in general since no significant effects were seen for plasma total cholesterol ( Figure 1) nor for LDL or HDL cholesterol (data not shown).

Curcumin also affected two non-lipid related measures relevant to cardiovascular health ( Figure 2). One of these effects was an increase in plasma contents of nitric oxide, a molecule that can work against high blood pressure [23]. The other cardiovascular-relevant effect was a lowering of plasma concentrations of sICAM, a molecule linked to atherosclerosis [24].

Curcumin supplementation, but not placebo, raised plasma myeloperoxidase concentrations (Figure 3), a part of both normal and inflammation-related neutrophil function [25,26]. This effect was not accompanied by a rise in plasma levels of c-reactive protein (Figure 3), nor by a rise in ceruloplasmin values (data not shown), both of which can be markers of inflammation [1,22,27]. In addition, curcumin supplementation, but not placebo, lowered salivary amylase activities (Figure 4), which can mark sympathetic nervous system stress [28]. Curcumin also raised salivary radical scavenging capacities ( Figure 4). The latter effect, a boosting of one determinant of antioxidant protection, was complimented by raising activities of the plasma antioxidant enzyme catalase ( Figure 5). However, this last effect did not extend to all antioxidant enzymes as no effect was seen for erythrocyte superoxide dismutase activities ( Figure 5) nor for plasma glutathione peroxidase (data not shown). Curcumin supplementation, but not placebo, reduced plasma contents of beta amyloid protein ( Figure 6), a maker of brain aging, especially in relation to Alzheimer's disease [29]. Curcumin also reduced plasma alanine amino transferase activities ( Figure 6), a liver injury marker [30].

Discussion

This study demonstrated that in apparently healthy individuals, a relatively low dose of a specific curcumin preparation can exert a variety of health promoting effects. Previous human intervention studies had generally emphasized much larger doses in people with existing health problems rev in [1,2,6]. Since the relatively low dose used here did produce various bio-actions, presumably a good amount of curcumin was absorbed.

However, this was not tested directly. The variety of actions seen here were tested for only one fairly narrow age range, but future studies can examine other ages. Certain dietary bioactive compounds only show very distinct effects when counteracting a physiological stress. This is likely why curcumin has been tested largely in the presence of health problems in people and in animal models for human disease. However, the wide variety of potentially health promoting effects seen in the present study suggests that curcumin can produce benefits in people without immediate disease states. This diversity of effects seen here conforms to the diverse range of mechanisms that can be affected by curcumin in experimental animals and cell cultures [1][2][3]. For example, curcumin affected readings for both nitric oxide and sICAM. Both of these molecules hold relevance for cardiovascular disease risk, but from completely different perspectives. Nitric oxide relates to blood pressure [23] while sICAM relates to atherosclerosis [24]. The current nitric oxide results may seem to contradict two previous studies where curcumin reduces nitric oxide [31,32]. However, the previous studies examined inflammatory states where nitric oxide levels can be raised by an inducible synthesis enzyme. The curcumin effect seems to result from actions on this enzyme [31], which would not show much activity in the healthy, non-stressed subjects studied here.

Another effect of curcumin was reduction of plasma ALT activities, which are generally used to mark liver injury [30]. This result for curcumin is consistent with work in experimental animals with various chemicallyinduced liver injuries ie [3,33]. In these animals, a rise in ALT activities can be limited by administering curcumin. However, two major differences exist between those studies and the present work. First, the present study utilized a much lower curcumin dose than in the animal work, and second, the current study did not include any overt chemical injury. Thus, the animal work raises the possibility of using high dose curcumin for drug type effects, but the current work raises the possibility of using low dose curcumin for liver health maintenance. This contrasts some suggestion that high dose curcumin doses can produce liver toxicity [34].

In the present study, curcumin effects on blood lipids gave mixed results compared to experimental animal studies. Specifically, in the present study, curcumin lowered triglyceride readings but did not affect various types of cholesterol readings. In experimental animals, curcumin can affect all these readings ie [11][12][13][14]. However, it should be noted that these experimental animal models induce high cholesterol and triglyceride levels via oral intake of agents such as cholesterol, fat or alcohol. Conversely, in the present study, neither plasma cholesterol nor triglycerides were elevated by any extreme dietary intervention. Curcumin may regulate cholesterol through mechanisms that only become major effectors under certain stress conditions. In opposition to this proposition, one study finds cholesterol lowering by 500 mg curcumin/day in relatively healthy people. However, this study lasted just 1 week [8]. In one other study [35], curcumin failed to lower either cholesterol or triglyceride readings. The exact dose given cannot be ascertained since the percent curcumin of the extracts were not analyzed. The study did provide evidence that some curcumin was absorbed, but the amount absorbed may not have been enough to lower triglyceride readings.

Curcumin raised plasma myeloperoxidase, an effect often associated with neutrophil mediated inflammation [26]. Yet, in the present study, no change was seen for either c-reactive protein or ceruloplasmin concentrations, both of which rise with inflammation [22,27]. Possibly, the curcumin effect on myeloperoxidase indicated strengthened cellular immune function, not an inflammatory reaction. Curcumin is known to strengthen some aspects of cellular immunity even though it also suppresses pro-inflammatory aspects of immune function [rev in 36].

Research in experimental animals and carried out in vitro have raised the possibility that curcumin could work against development of Alzheimer's disease rev in [21]. In the present study, curcumin decreased plasma beta amyloid protein concentrations, which relates to one mechanism by which curcumin may impact Alzheimer's disease development. Although the percent decrease was not big, the decrease could become larger with a longer intervention.

In this study, curcumin showed signs of both direct and indirect antioxidant actions. The curcumin-induced increase in salivary radical scavenging capacity is consistent with a direct antioxidant action (elimination of free radicals by curcumin and/or its metabolites). Curcumin has shown this type of activity in vitro [2,37]. In the present study, curcumin also showed indirect antioxidant action by elevating plasma activities of the endogenous antioxidant enzyme catalase. Interestingly, low plasma catalase is associated with a high risk for one form of cardiovascular disease [38]. Experimental animal studies provide precedent for curcumin stimulation of increases in antioxidant enzyme activities [14][15][16][17]. In the present study, plasma catalase activities increased after curcumin treatment, but readings for two other antioxidant enzymes, plasma glutathione peroxidase and erythrocyte superoxide dismutase, did not change. In contrast, curcumin is reported to increase all three enzymes in humans exposed to arsenic [39]. However, this increase is not from normal activities to above normal, which is what was observed for catalase activities in the present study. In the people with arsenic exposure, low activities are partially returned to normal. Thus, the work with arsenic exposed subjects pertains to protection against toxininduced reduction in activity, but the present study considered activity elevations above normal for three antioxidant enzymes. Although only the catalase activity showed such elevation in blood samples, activities of the other two antioxidant enzymes may have increased in other body sites.

Conclusions

In summary, a low dose of a lipidated curcumin product produced a range of potentially health promoting actions in healthy middle aged people.

Figure 1 Figure 2
12Curcumin effects on plasma triglycerides and cholesterol concentrations (mg/dl). Values are means ± SEM for N = 19 pre-and post-treatment of 4 weeks. *Significantly different from pre-value, paired t-test, p < 0.05. Curcumin effects on plasma concentrations of nitric oxide (μM x 10) and soluble intercellular adhesion molecule (sICAM) (ng/ml). Values are means ± SEM for N = 19 pre-and post-treatment of 4 weeks. *Significantly different from pre-value, paired t-test, p < 0.05.

Figure 3 Figure 4
34Curcumin effects on plasma concentrations of myeloperoxidase (ng/ml) and c-reactive protein (mg/L x 10). Values are means ± SEM for N = 19 pre-and post-treatment of 4 weeks. *Significantly different from pre-value, paired t-test, Curcumin effects on saliva activities of amylase (U/L) and antioxidant status (μM of copper reducing equivalents). Values are means ± SEM for N = 19 pre-and post-treatment of 4 weeks. *Significantly different from pre-value, paired t-test, p < 0.05.

Figure 5 Figure 6
56Curcumin effects on plasma activities of catalase (U/ml) and erythrocyte superoxide dismutase (U/ml packed cells x 10 -2 ).Values are means ± SEM for N = 19 pre-and post-treatment of 4 weeks. *Significantly different from pre-value, paired t-test, Curcumin effects on plasma activities of alanine aminotransferase (ALT) (U/L) and beta amyloid protein (pmoles/L). Values are means ± SEM for N = 19 pre-and post-treatment of 4 weeks. *Significantly different from pre-value, paired t-test, p < 0.05.
http://www.nutritionj.com/content/11/1/79
AcknowledgementsThis work was supported by a grant to RAD from Verdure Sciences. The authors acknowledge Milena Micu for her assistance in contacting and screening potential subjects.Competing interestsThe authors declare that they have no competing interests.Authors' contributions RAD secured the funding, oversaw the study, and had the main writing responsibility. EJ had the main responsibility for the laboratory analysis including adapting methods to this study's need. EJ also made some decisions on the assay methodology to be used, formatted the paper, and gave input on the study design and manuscript writing. SZ did some of the laboratory work and made decisions on some of the assay methodology to be used. SZ also gave input on the study design and manuscript writing. JB was the lead investigator for human subject research regulation compliance and contributed to study design and manuscript writing. All authors read and approved the final manuscript.Submit your next manuscript to BioMed
Curcumin: preventive and therapeutic properties in laboratory studies and clinical trials. A Strimpakos, R Sharma, Food Chem Toxicol. 10Strimpakos A, Sharma R: Curcumin: preventive and therapeutic properties in laboratory studies and clinical trials. Food Chem Toxicol 2008, 10:511-545.

Curcumin as a therapeutic agent: the evidence from in vitro, animal and human studies. J Epstein, I Sanderson, T Macdonald, Br J Nutr. 103Epstein J, Sanderson I, Macdonald T: Curcumin as a therapeutic agent: the evidence from in vitro, animal and human studies. Br J Nutr 2010, 103:1545-1557.

Protective effect of curcumin on experimentally induced inflammation, hepatotoxicity and cardiotoxicity in rats: evidence of its antioxidant property. S Naik, V Thakare, S Patil, Exp Toxicol Pathol. 63Naik S, Thakare V, Patil S: Protective effect of curcumin on experimentally induced inflammation, hepatotoxicity and cardiotoxicity in rats: evidence of its antioxidant property. Exp Toxicol Pathol 2011, 63:419-431.

Oral bioavailability of curcumin in rat and the herbal analysis from curcuma Longa by LC-MS/MS. K Yang, L Lin, T Tseng, S Wang, T Tsai, J Chromatogr B Analy Technol Biomed Life Sci. 853Yang K, Lin L, Tseng T, Wang S, Tsai T: Oral bioavailability of curcumin in rat and the herbal analysis from curcuma Longa by LC-MS/MS. J Chromatogr B Analy Technol Biomed Life Sci 2007, 853:183-189.

Pharmacokinetics of curcumin conjugate metabolites in healthy human subjects. S Vareed, M Kakarala, M Ruffin, Cancer Epidemiol Biomarkers Prev. 17Vareed S, Kakarala M, Ruffin M, et al: Pharmacokinetics of curcumin conjugate metabolites in healthy human subjects. Cancer Epidemiol Biomarkers Prev 2008, 17:1411-1417.

Systemic delivery of curcumin: 21st century solutions for an ancient conundrum. S Bisht, A Maitra, Curr Drug Discov Technol. 6Bisht S, Maitra A: Systemic delivery of curcumin: 21st century solutions for an ancient conundrum. Curr Drug Discov Technol 2009, 6:192-199.

Oral supplementation of turmeric attenuates proteinuria, transforming growth factor-β and interleukin-8 levels in patients with overt type 2 diabetic nephropathy: a randomized, double-blind and placebo-controlled study. P Khajehdehi, M Pakfetrat, K Javidnia, Scand J Urol Nephrol. 45Khajehdehi P, Pakfetrat M, Javidnia K, et al: Oral supplementation of turmeric attenuates proteinuria, transforming growth factor-β and interleukin-8 levels in patients with overt type 2 diabetic nephropathy: a randomized, double-blind and placebo-controlled study. Scand J Urol Nephrol 2011, 45:365-370.

Effect of different curcuminoid supplement dosages on total in vivo antioxidant capacity and cholesterol levels of healthy human subjects. K Pungcharoenkul, P Thongnopnua, Phytother Res. 25Pungcharoenkul K, Thongnopnua P: Effect of different curcuminoid supplement dosages on total in vivo antioxidant capacity and cholesterol levels of healthy human subjects. Phytother Res 2011, 25:1721-1726.

A pilot study of the antioxidant effect of curcumin in tropical pancreatitis. S Durgaprasad, C G Pai, J F Vasanthkumar Alvres, S Namitha, Indian J Med Res. 122Durgaprasad S, Pai CG, Vasanthkumar Alvres JF, Namitha S: A pilot study of the antioxidant effect of curcumin in tropical pancreatitis. Indian J Med Res 2005, 122:315-318.

Effect of endurance exercise training and curcumin intake on central arterial hemodynamics in postmenopausal women: pilot study. J Sugawara, N Akazawa, A Miyaki, Am J Hypertens. in pressSugawara J, Akazawa N, Miyaki A, et al: Effect of endurance exercise training and curcumin intake on central arterial hemodynamics in postmenopausal women: pilot study. Am J Hypertens 2012, in press.

Curcumin attenuates diet-induced hypercholesterolemia in rats. H Arafa, Med Sci Monit. 11Arafa H: Curcumin attenuates diet-induced hypercholesterolemia in rats. Med Sci Monit 2005, 11:BR228-BR234.

Comparative effects of curcumin and its analog on alcohol-and polyunsaturated fatty acid-induced alterations in circulatory lipid profiles. R Rukkumani, K Aruna, P Varma, K Rajasekaran, V Menon, J Med Food. 8Rukkumani R, Aruna K, Varma P, Rajasekaran K, Menon V: Comparative effects of curcumin and its analog on alcohol-and polyunsaturated fatty acid-induced alterations in circulatory lipid profiles. J Med Food 2005, 8:256-260.

Attenuation of fatty liver and prevention of hypercholesterolemia by extract of curcuma Longa through regulating the expression of CYP7A1, LDL-receptor, HO-1, and HMG-CoA reductase. W Yiu, P Kwan, C Wong, J Food Sci. 76Yiu W, Kwan P, Wong C, et al: Attenuation of fatty liver and prevention of hypercholesterolemia by extract of curcuma Longa through regulating the expression of CYP7A1, LDL-receptor, HO-1, and HMG-CoA reductase. J Food Sci 2011, 76:H80-H89.

Antioxidant activities of curcumin and combinations of this curcuminoid with other phytochemicals. N Aftab, A Vieira, Phytother Res. 24Aftab N, Vieira A: Antioxidant activities of curcumin and combinations of this curcuminoid with other phytochemicals. Phytother Res 2010, 24:500-502.

Dietary supplementation of curcumin enhances antioxidant and phase II metabolizing enzymes in ddY male mice: possible role in protection against chemical carcinogenesis and toxicity. M Iqbal, S Sharma, Y Okazaki, M Fujisawa, S Okada, Pharmacol Toxicol. 92Iqbal M, Sharma S, Okazaki Y, Fujisawa M, Okada S: Dietary supplementation of curcumin enhances antioxidant and phase II metabolizing enzymes in ddY male mice: possible role in protection against chemical carcinogenesis and toxicity. Pharmacol Toxicol 2003, 92:33-38.

Antioxidant status of red blood cells and liver in hypercholesterolemic rats fed hypolipidemic spices. R Kempaiah, K Srinivasan, Int J Vitam Nutr Res. 74Kempaiah R, Srinivasan K: Antioxidant status of red blood cells and liver in hypercholesterolemic rats fed hypolipidemic spices. Int J Vitam Nutr Res 2004, 74:199-208.

Protective effect of curcumin, ellagic acid and bixin on radiation induced toxicity. K Thresiamma, J George, R Kuttan, Indian J Exp Biol. 34Thresiamma K, George J, Kuttan R: Protective effect of curcumin, ellagic acid and bixin on radiation induced toxicity. Indian J Exp Biol 1996, 34:845-847.

Comparative study of natural antioxidants curcumin, resveratrol and melatonin -in cadmium-induced oxidative damage in mice. V Eybl, D Kotyzova, J Koutensky, Toxicology. 225Eybl V, Kotyzova D, Koutensky J: Comparative study of natural antioxidants curcumin, resveratrol and melatonin -in cadmium-induced oxidative damage in mice. Toxicology 2006, 225:150-156.

Neuroprotective effects of the polyphenolic antioxidant agent, curcumin, against homocysteine-induced cognitive impairment and oxidative stress in the rat. A Ataie, M Sabetkasaei, A Haghparast, A Moghaddam, B Kazeminejad, Pharmacol Biochem Behav. 96Ataie A, Sabetkasaei M, Haghparast A, Moghaddam A, Kazeminejad B: Neuroprotective effects of the polyphenolic antioxidant agent, curcumin, against homocysteine-induced cognitive impairment and oxidative stress in the rat. Pharmacol Biochem Behav 2010, 96:378-385.

Curcumin decreases amyloid-beta peptide levels by attenuating the maturation of amyloid-beta precursor protein. C Zhang, A Browne, D Child, R Tanzi, J Biol Chem. 285Zhang C, Browne A, Child D, Tanzi R: Curcumin decreases amyloid-beta peptide levels by attenuating the maturation of amyloid-beta precursor protein. J Biol Chem 2010, 285:28472-28480.

Curcumin and Alzheimer's disease. T Hamaguchi, K Ono, M Yamada, CNS Neurosci Ther. 16Hamaguchi T, Ono K, Yamada M: Curcumin and Alzheimer's disease. CNS Neurosci Ther 2010, 16:285-297.

Effects of inflammation and copper intake on rat liver and erythrocyte Cu-Zn superoxide dismutase activity levels. R A Disilvestro, J T Marten, J Nutr. 120DiSilvestro RA, Marten JT: Effects of inflammation and copper intake on rat liver and erythrocyte Cu-Zn superoxide dismutase activity levels. J Nutr 1990, 120:1223-1227.

Imbalance of central nitric oxide and reactive oxygen species in the regulation of sympathetic activity and neural mechanisms of hypertension. Y Hirooka, T Kishi, K Sakai, Am J Physiol Regul Integr Comp Physiol. 300Hirooka Y, Kishi T, Sakai K: Imbalance of central nitric oxide and reactive oxygen species in the regulation of sympathetic activity and neural mechanisms of hypertension. Am J Physiol Regul Integr Comp Physiol 2011, 300:R818-R826.

Association of sICAM-1 and MCP-1 with coronary artery calcification in families enriched for coronary heart disease or hypertension: the NHLBI Family Heart Study. W Tang, J S Pankow, J J Carr, BMC Cardiovasc Disord. 730Tang W, Pankow JS, Carr JJ: Association of sICAM-1 and MCP-1 with coronary artery calcification in families enriched for coronary heart disease or hypertension: the NHLBI Family Heart Study. BMC Cardiovasc Disord 2007, 7:30.

How human neutrophils kill and degrade microbes: an integrated view. W M Nauseef, Immunol Rev. 219Nauseef WM: How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 2007, 219:88-102.

Association between myeloperoxidase levels and risk of coronary artery disease. R Zhang, M L Brennan, X Fu, JAMA. 286Zhang R, Brennan ML, Fu X: Association between myeloperoxidase levels and risk of coronary artery disease. JAMA 2001, 286:2136-2142.

CRP and the risk of atherosclerotic events. P Calabrò, E Golia, E Yeh, Semin Immunopathol. 31Calabrò P, Golia E, Yeh E: CRP and the risk of atherosclerotic events. Semin Immunopathol 2009, 31:79-94.

Determinants of salivary alpha-amylase in humans and methodological considerations. N Rohleder, U Nater, Psychoneuroendocrinology. 34Rohleder N, Nater U: Determinants of salivary alpha-amylase in humans and methodological considerations. Psychoneuroendocrinology 2009, 34:469-485.

Alzheimer's disease and amyloid beta-peptide deposition in the brain: a matter of 'aging'?. M Moro, M Collins, E Cappellini, Biochem Soc Trans. 38Moro M, Collins M, Cappellini E: Alzheimer's disease and amyloid beta-peptide deposition in the brain: a matter of 'aging'? Biochem Soc Trans 2010, 38:539-544.

Diagnostic approach to liver enzyme elevation. A Craxı, P Almasio, J Hepatol. 25Craxı A, Almasio P: Diagnostic approach to liver enzyme elevation. J Hepatol 1996, 25S:47-51.

Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-kB-dependent genes. A A Nanji, K Jokelainen, G Tipoe, A Rahemtulla, P Thomas, A J Dannenberg, Am J Physiol Gastrointest Liver Physiol. 284Nanji AA, Jokelainen K, Tipoe G, Rahemtulla A, Thomas P, Dannenberg AJ: Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-kB-dependent genes. Am J Physiol Gastrointest Liver Physiol 2003, 284:G321-G327.

Deleterious effect of nitric oxide inhibition in chronic hepatopulmonary syndrome. J A Almeida, S M Riordan, J Liu, S Galhenage, R Kim, D Bihari, E A Wegner, G B Cranney, P S Thomas, Eur J Gastroenterol Hepatol. 19Almeida JA, Riordan SM, Liu J, Galhenage S, Kim R, Bihari D, Wegner EA, Cranney GB, Thomas PS: Deleterious effect of nitric oxide inhibition in chronic hepatopulmonary syndrome. Eur J Gastroenterol Hepatol 2007, 19:341-346.

Hepatoprotective activity of picroliv, curcumin and ellagic acid compared to silymarin on paracetamol induced liver toxicity in mice. C Girish, B Koner, S Jayanthi, Ramachandra Rao, K Rajesh, B Pradhan, S , Fundam Clin Pharmacol. 23Girish C, Koner B, Jayanthi S, Ramachandra Rao K, Rajesh B, Pradhan S: Hepatoprotective activity of picroliv, curcumin and ellagic acid compared to silymarin on paracetamol induced liver toxicity in mice. Fundam Clin Pharmacol 2009, 23:735-745.

Toxicity prediction of compounds from turmeric (curcuma Longa L). S Balaji, B Chempakam, Food Chem Toxicol. 48Balaji S, Chempakam B: Toxicity prediction of compounds from turmeric (curcuma Longa L). Food Chem Toxicol 2010, 48:2951-2959.

Curcumin effects on blood lipid profile in a 6-month human study. L Baum, S K Cheung, V C Mok, L C Lam, V P Leung, E Hui, C C Ng, M Chow, P C Ho, S Lam, J Woo, H F Chiu, W Goggins, B Zee, A Wong, H Mok, W K Cheng, C Fong, J S Lee, M H Chan, S S Szeto, V W Lui, J Tsoh, T C Kwok, I H Chan, C W Lam, Pharmacol Res. 56Baum L, Cheung SK, Mok VC, Lam LC, Leung VP, Hui E, Ng CC, Chow M, Ho PC, Lam S, Woo J, Chiu HF, Goggins W, Zee B, Wong A, Mok H, Cheng WK, Fong C, Lee JS, Chan MH, Szeto SS, Lui VW, Tsoh J, Kwok TC, Chan IH, Lam CW: Curcumin effects on blood lipid profile in a 6-month human study. Pharmacol Res 2007, 56:509-514.

Immunomodulatory and therapeutic activity of curcumin. R Srivastava, S Singh, S Dubey, K Misra, A Khar, Int Immunopharmacol. 11Srivastava R, Singh S, Dubey S, Misra K, Khar A: Immunomodulatory and therapeutic activity of curcumin. Int Immunopharmacol 2011, 11:331-341.

Antioxidant and radical scavenging properties of curcumin. T Ak, I Gülçin, Chem Biol Interact. 174Ak T, Gülçin I: Antioxidant and radical scavenging properties of curcumin. Chem Biol Interact 2008, 174:27-37.

Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal aortic aneurysm progression. M P Ramos, M J Madrigal, P R Martinez, Arterioscler Thromb Vasc Biol. 31Ramos MP, Madrigal MJ, Martinez PR, et al: Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal aortic aneurysm progression. Arterioscler Thromb Vasc Biol 2011, 31:3011-3019.

Curcumin protects DNA damage in a chronically arsenic-exposed population of West Bengal. J Biswas, D Sinha, S Mukherjee, S Roy, M Siddiqi, M Roy, 10.1186/1475-2891-11-79Hum Exp Toxicol. 29Biswas J, Sinha D, Mukherjee S, Roy S, Siddiqi M, Roy M: Curcumin protects DNA damage in a chronically arsenic-exposed population of West Bengal. Hum Exp Toxicol 2010, 29:513-524. doi:10.1186/1475-2891-11-79

Diverse effects of a low dose supplement of lipidated curcumin in healthy middle aged people. Disilvestro, Nutrition Journal. 1179Cite this article asCite this article as: DiSilvestro et al.: Diverse effects of a low dose supplement of lipidated curcumin in healthy middle aged people. Nutrition Journal 2012 11:79.