Curcumin analogues have been shown to inhibit the COX-1 enzyme, which is linked to inflammation and cancer. This process is described in the study titled Synthesis of novel curcumin analogues and their evaluation as selective cyclooxygenase-1 (COX-1) inhibitors, published in the Chemical and Pharmaceutical Bulletin in January, 2007:

April 21 2008 post: This morning I read something that I did not know about curcumin in the “Times of India” Researchers at New Delhi’s Jamia Hamdard University have successfully used curcumin – extracted from turmeric and broken into nano form – to control and cure cirrhosis of liver in an animal model experiment. “Nano,” huh? How “nano” were these nanoparticles, I wonder? Well, even though the jury is still out on the nano-stuff, in this case the nanocurcumin appears to have worked. Interesting.

Loading of curcumin

Curcumin was a kind gift of Indsaff, Inc. (Batala, Punjab, India). Curcumin loading in the polymeric nanoparticles was done by using a post-polymerization method. In this process of loading, the drug is dissolved after the co-polymer formation has taken place. The physical entrapment of curcumin in NIPAAM/VP/PEG-A polymeric nanoparticles was carried out as follows: 100 mg of the lyophilized powder was dispersed in 10 ml distilled water and was stirred to re-constitute the micelles. Free curcumin was dissolved in chloroform (CHCl₃; 10 mg/ml) and the drug solution in CHCl₃ was added to the polymeric solution slowly with constant vortexing and mild sonication. Curcumin is directly loaded into the hydrophobic core of nanoparticles by physical entrapment. The drug-loaded nanoparticles are then lyophilized to dry powder for subsequent use.

Entrapment efficiency (E %)

The entrapment efficiency (E %) of curcumin loaded in NIPAAM-VP-PEG-A nanoparticles was determined as follows: the nanoparticles were separated from the un-entrapped free drug using NANOSEP (100 kD cut off) membrane filter and the amount of free drug in the filtrate was measured spectrophotometrically using a WALLAC plate reader at 450 nm. The E% was calculated by

E% = ([Drug]_tot - [Drug]_free)/[Drug]_tot × 100

Fourier Transform Infrared (FT-IR) studies of polymeric nanoparticles

Mid infra red (IR) spectrum of NIPAAM, VP and PEG-A monomers, as well as the void polymeric nanoparticles were taken using Bruker Tensor 27 (FT-IR) spectrophotometer (Bruker Optics Inc., Billerica, MA, USA).

^1-H Nuclear Magnetic Resonance (NMR) studies

The NMR spectrum of monomers NIPAAM, VP and PA, as well as void polymeric nanoparticles were taken by dissolving the samples in D₂O as solvent using Bruker Avance 400 MHz spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA).

Dynamic light scattering (DLS) measurements

DLS measurements for determining the average size and size distribution of the polymeric micelles were performed using a Nanosizer 90 ZS (Malvern Instruments, Southborough, MA). The intensity of scattered light was detected at 90° to an incident beam. The freeze-dried powder was dispersed in aqueous buffer and measurements were done, after the aqueous micellar solution was filtered with a microfilter having an average pore size of 0.2 mm (Millipore). All the data analysis was performed in automatic mode. Measured size was presented as the average value of 20 runs, with triplicate measurements within each run.

Transmission electron microscopy (TEM)

TEM pictures of polymeric nanoparticles were taken in a Hitachi H7600 TEM instrument operating at magnification of 80 kV with 1 K × 1 K digital images captured using an AMT CCD camera. Briefly, a drop of aqueous solution of lyophilized powder (5 mg/ml) was placed on a membrane coated grid surface with a filter paper (Whatman No. 1). A drop of 1% uranyl acetate as immediately added to the surface of the carbon coated grid. After 1 min excess fluid was removed and the grid surface was air dried at room temperature before loaded in the microscope.

In vitro release kinetics of nanocurcumin

A known amount of lyophilized polymeric nanoparticles (100 mg) encapsulating curcumin was dispersed in 10 ml phosphate buffer, pH 7.4, and the solution was divided in 20 microfuge tubes (500 μl each). The tubes were kept in a thermostable water bath set at room temperature. Free curcumin is completely insoluble in water; therefore, at predetermined intervals of time, the solution was centrifuged at 3000 rpm for 10 minutes to separate the released (pelleted) curcumin from the loaded nanoparticles. The released curcumin was redissolved in 1 ml of ethanol and the absorbance was measured spectrophotometrically at 450 nm. The concentration of the released curcumin was then calculated using standard curve of curcumin in ethanol. The percentage of curcumin released was determined from the equation

where, [Curcumin]_rel is the concentration of released curcumin collected at time t and [Curcumin]_tot is the total amount of curcumin entrapped in the nanoparticles.

In vitro and vivo toxicity studies with void polymeric nanoparticles

In order to exclude the possibility of de novo toxicity from the polymeric constituents, we utilized void nanoparticles against a panel of eight human pancreatic cancer cell lines (MiaPaca2, Su86.86, BxPC3, Capan1, Panc1, E3LZ10.7, PL5 and PL8). These cells were exposed to void nanoparticles for 96 hours across a 20-fold concentration range (93 – 1852 μg/mL) and cell viability measured by MTS assay, as described below. Further, limited in vivo toxicity studies were performed in athymic (nude) mice by intraperitoneal injection of void polymeric nanoparticles at a considerably high dosage of 720 mg/kg twice weekly, for a period of three weeks. Mice receiving intra-peritoneal nanocurcumin (N = 4) were weighed weekly during the course of therapy and average weight compared to that of control littermate nude mice (N = 4). At the culmination of the three week course, mice were euthanized and necropsy performed to exclude any intraperitoneal deposition of polymers, or gross organ toxicities.

Fluorescence microscopy for nanocurcumin uptake by pancreatic cancer cells

Curcumin is naturally fluorescent in the visible green spectrum. In order to study uptake of curcumin encapsulated in nanoparticles, BxPC3 cells were plated in 100 mm dishes, and allowed to grow to sub-confluent levels. Thereafter, the cells were incubated with nanocurcumin for 2–4 hours, and visualized in the FITC channel.

Cell viability (MTS) assays in pancreatic cancer cell lines exposed to nanocurcumin

Growth inhibition was measured using the CellTiter 96^® A_queous Cell Proliferation Assay (Promega), which relies on the conversion of a tetrazolium compound (MTS) to a colored formazan product by the activity of living cells. Briefly, 2000 cells/well were plated in 96 well plates, and were treated with 0, 5, 10, 15 and 20 μM concentrations of free curcumin and equivalent nanocurcumin, for 72 hours, at which point the assay was terminated, and relative growth inhibition compared to vehicle-treated cells measured using the CellTiter 96^®reagent, as described in the manufacturer's protocol. A panel of ten human pancreatic cancer cell lines were examined (BxPC3, AsPC1, MiaPaca, XPA-1, XPA-2, PL-11, PL-12, PL-18, PK-9 and Panc 2.03) in the MTT assays; the sources and culture conditions of these ten lines have been previously described [58]. All experiments were set up in triplicates to determine means and standard deviations.

Colony assays in soft agar

Colony formation in soft agar was assessed for therapy with free curcumin and equivalent dosage of nanocurcumin. Briefly, 2 ml of mixture of serum supplemented media and 1 % agar containing 5, 10 or 15 μM of free curcumin and equivalent nanocurcumin was added in a 35 mm culture dish and allowed to solidify (base agar) respectively. Next, on top of the base layer was added a mixture of serum supplemented media and 0.7 % agar (total 2 mL) containing 10,000 MiaPaca2 cells in the presence of void polymer, free or nano-curcumin, and was allowed to solidify (top agar); a fourth set of plates contained MiaPac2 cells without any additives. Subsequently, the dishes were kept in tissue culture incubator maintained at 37°C and 5 % CO₂ for 14 days to allow for colony growth. All assays were performed in triplicates. The colony assay was terminated at day 14, when plates were stained and colonies counted on ChemiDoc XRS instrument (Bio-Rad, Hercules, CA).

Electrophoretic mobility shift assay (EMSA)

Nuclear extracts were prepared as described [59]. Briefly, double-stranded oligonucleotides containing a consensus binding site for c-Rel (5'-GGG GAC TTT CCC-3') (Santa Cruz Biotechnology) were 5' end-labeled using polynucleotide kinase and [³²P]dATP. Nuclear extracts (2.5–5 μg) were incubated with ≈1 μl of labeled oligonucleotide (20,000 c.p.m.) in 20 μl of incubation buffer (10 mM Tris-HCl, 40 mM NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol, 2% glycerol, 1–2 μg of poly dI-dC) for 20 min at 25°C. DNA-protein complexes were resolved by electrophoresis in 5% non-denaturing polyacrylamide gels and analyzed by autoradiography.

Determination of IL-6, IL-8 and TNF-alpha synthesis

IL-6, IL-8 and TNF-alpha mRNA levels were assessed as described previously [55]. Briefly, peripheral blood mononuclear cells (PBMC) from a healthy donor were isolated by centrifugation on a Ficoll Hypaque density gradient (GE Healthcare Biosciences) and washed twice with phosphate buffered saline (PBS; Invitrogen, Carlsbad, CA). Next, 500,000 cells per well of a 24 well plate in 1 ml of RPMI (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1× Pen/Strep (Biofluids, Camarillo, CA) were co-stimulated with 2% phytohaemagglutinin (PHA M-Form, liquid; Invitrogen) and 1 μg/ml lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO) in the presence of free or nanocurcumin, using solvent and void nanoparticles as controls, respectively. Cells were lysed after 24 hours incubation at 37°C and 5% CO₂ and RNA extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA). Relative fold steady-state mRNA levels were determined on a 7300 Real time PCR System (Applied Biosystems, Foster City, CA,) by RT-PCR as described.

It's easy to understand the excitement. Protein is an important component of every cell in the body. Hair and nails are mostly made of protein. Your body uses protein to build and repair tissues. You also use protein to make enzymes, hormones, and other body chemicals. Protein is an important building block of bones, muscles, cartilage, skin, and blood.

Along with fat and carbohydrates, protein is a "macronutrient," meaning that the body needs relatively large amounts of it. Vitamins and minerals, which are needed in only small quantities, are called "micronutrients." But unlike fat and carbohydrates, the body does not store protein, and therefore has no reservoir to draw on when it needs a new supply.

So you may assume the solution is to eat protein all day long. Not so fast, say nutritionists.

The truth is, we need less total protein that you might think. But we could all benefit from getting more protein from better food sources.

How Much Protein Is Enough?

We've all heard the myth that extra protein builds more muscle. In fact, the only way to build muscle is through exercise. Bodies need a modest amount of protein to function well. Extra protein doesn't give you extra strength. According to the U.S. Department of Health and Human Services:

• Teenage boys and active men can get all the protein they need from three daily servingsfor a total of seven ounces.

• For children age 2 to 6, most women, and some older people, the government recommends two daily servings for a total of five ounces.

• For older children, teen girls, active women, and most men, the guidelines give the nod to two daily servings for a total of six ounces.

Everyone who eats an eight-ounce steak typically served in restaurants is getting more protein that their bodies need. Plus they're getting a hefty amount of artery-clogging saturated fat as well.