Curcuminoid

Pulsed laser deposition technic (PLD) was performed on both glass slab and hemp twill fabric in order to study the physico-chemical properties of the obtained thin films, as well as the physico-chemical processes and mechanisms developed under laser interaction with the natural biocomposite of oyster shells that led to obtaining the thin films of a certain chemical composition. Neither preliminary preparation nor chemical treatments were performed on the oyster shells used as targets in pursuing the purpose of the study to find new uses for such raw materials, and also to investigate the natural biocomposite behavior as a whole and by its components under laser irradiation and the results of the ablation and deposition process.
The investigation of the new procedure that we purpose herein was assisted and completed by COMSOL numerical simulation with finite element method (FEM) of heat effects induced by the laser beam on the composite structure where chitosan and calcium carbonate are considered, in thermal interaction, as being the main components of the oyster shell.
For oyster shell irradiation, the YG 981E/IR-10 laser system (Quantel, Les Ulis, France) in the installation of Figure 1 was used with the following parameters: τ = 10 ns pulse width, λ = 532 nm wavelength, α = 45° incident angle, ν = 10 Hz pulse repetition time, r = 336 μm spot radius, F = 25 J/cm² fluence, d = 3.5 cm distance between target and support.
The laser beam wavelength of 532 nm (in visible) was chosen because CaCO₃, one of the main components in oyster shell [16 (link),18 (link)], does not absorb the laser beam of 532 nm [22 (link),23 ,24 ] as opposed to chitin, the component of interest for deposition and which will absorb the laser radiation of 532 nm [25 (link)]. Therefore, a high ablation rate of chitin was expected. The process is a selective extraction of one component of the composite material due to the different response of the components to the laser irradiation. Further, the 532 nm wavelength is preferred, being expected to be more suitable for a less aggressive interaction with the polymerized structures of the components in the oyster shell, same reason as reported by I. Cocean et al., 2019 and A. Cocean et al., 2021 on pulsed laser extraction and deposition of alpha-keratin [26 (link)] and of curcuminoids, respectively [27 (link)].
The analyses consisting of Fourier-transform infrared (FTIR) spectroscopy, laser-induced fluorescence (LIF) spectroscopy and Energy Dispersive X-ray coupled with Scanning Electron Microscope (SEM-EDS) were performed for the oyster shell used as target and for the deposited thin film. Atomic Force Microscopy (AFM) analysis provides information on the chitosan thin film morphology and topography. UV-Vis spectrum of the thin film obtained is also presented.

Treatment was administered following the protocols previously used by the group [6 (link)]. Briefly, 10 mg/kg/day for VER treatment (equivalent to 480 mg/day in human patients) and 60 mg/kg/day CUR (Super Bio-Curcumin^®, from Life Extension^®, Lauderdale Lakes, FL, USA, total curcuminoids complex with essential oils of turmeric rhizome by HPLC 400 mg) were freshly prepared once per week. Both compounds were dissolved in drinking water with 1% DMSO. The vehicle control group (VEH) consisted of WT and CD mice drinking water containing 1% DMSO. Experimental groups were: 1) WT and CD mice drinking verapamil (VER), 2) WT and CD mice drinking curcumin (CUR), and 3) WT and CD mice drinking a combination of both compounds (CURVER). Treatment was started at eight weeks of age (young mice). All animals drank their treatments for four weeks before measuring arterial pressure and were then sacrificed at 12 weeks of age (young adult).
Two-way ANOVA of repeated measures did not show any significant interaction between time and treatment (Males: F_{(45, 240)} = 0.7963, p = 0.8189; Females: F_{(45, 240)} = 0.7335, p = 0.8936) (Figure S5A). Also, there were no significant differences in the treatment consumption in relation to genotype (F_{3, 24} = 0.1347, p = 0.9384) (Figure S5B).
In agreement with previous reports [4 (link)], both male and female CD mice had significantly lower body weights compared with WT mice (effect of genotype F_{1, 102} = 92.76, p < 0.0001 in males; F_1,62 = 38.57, p < 0.0001 in females), without any significant effect of treatment (Figure S5C,D). Supplementary Table S6 shows a detailed statistical analysis.

Intestinal pig epithelial cells (IPEC) J2 cells were used and grown for 8 days to confluency in Corning™ 6 well Transwell™ multi-well plates with permeable polycarbonate membrane inserts, using Dulbecco’s Modified Eagle (DME) medium and 5% fetal calf serum [32 (link),33 (link)]. The density of IPEC J2 cells was ∼2 × 10⁶ cells/well initially and was grown to confluence. The plates had a polyester membrane thickness of 10 μm, and 0.4 μm pore size. The cells were washed with Dulbecco phosphate-buffered saline. One milliliter of saline was present in the bottom wells at all times. Twenty milligrams of curcuminoids, THC, or TurmiZn complex were solubilized separately in 10 mL saline with 5.0% dimethylsulfoxide (DMSO), and 1 mL of the respective solutions was placed in the top of six replicate wells per treatment, and incubated for 2 h at 37 °C. Optical density (OD) at 220 nm and 425 nm was then measured in the top and bottom wells by ultraviolet/visible (UV/Vis) spectrophotometry (Figure 1A,B). The percent net average absorbance was calculated by dividing the bottom well absorbance by the total absorbances of the upper and bottom wells combined. High-performance liquid chromatography (HPLC) analysis was conducted on the upper and bottom wells at 280 nm on pooled samples (Figure 2A). HPLC analysis was carried out with a Waters 2965 HPLC coupled with a photodiode array (PDA) detector (model 2996) along with Empower software. Column: Phenomenex Luna C18 250 × 4.6 mm, 5 µM. Isocratic: 50:50 acetonitrile: 2% acetic acid in water. Flow rate 1 mL/min for 20 min. The extraction solvent for analytes was acetone. The column temperature was 25 °C. PDA wavelengths 425 nm and 220 nm were used. The mean values ± standard error are reported.

Animal trials were conducted to determine the bioavailability of the TurmiZn complex in rats and chickens. Rats are a more suitable mammalian model than mice to perform kinetic studies that require blood withdrawal for longer durations. Chickens are suitable models for bioavailability studies for researchers interested in avian production. Doses were selected based on the ability to readily monitor TurmiZn metabolites analytically in plasma and were not based on therapeutic levels.
For the avian bioavailability trials, chickens (Rhode Island Red, 3 chickens/group) that were each 2 years old and weighing 5 pounds were selected, and 1 mL of blood was drawn from the wing vein for baseline measurements. The blood was allowed to clot and the plasma was removed by centrifugation for subsequent baseline analysis. The chickens were allowed to rest overnight. One gram of TurmiZn, one gram of curcumin, and one gram of THC were suspended in 5 mL of water and placed into respective syringes. The contents of each of the respective syringes were then administered orally ensuring that the contents were consumed. Blood was drawn at 2, 4, and 24 h from each bird and allowed to clot. The recovered serum was mixed with equal volumes of acetone via vortexing for 30 s and then centrifuged to remove proteins [34 (link),35 (link)] in the pellet while recovering curcuminoids, THC, or elements of TurmiZn in the supernatant. The supernatants from each time point were analyzed by HPLC methodology as described for the presence of curcuminoids, THC, or TurmiZn at 425 nm.
Bioavailability study in rats was approved by IAEC (Institutional Animal Ethics Committee) of the Institute of Pharmacy, Nirma University (Ahmedabad, India) as per the CPCSEA (Committee for Control and Supervision of Experiments on Animals) guidelines, Department of Animal Husbandry and Dairying (DAHD), Ministry of Fisheries, Animal Husbandry and Dairying (MoFAH&D), Govt. of India. The protocol number was IP/PCOL/FAC/29/2021/32. The bioavailability was evaluated in adult healthy male Wistar rats with two different studies: oral administration and intravenous administration of TurmiZn complex as a single dose. For orally administered trials in mammals, baseline blood samples were taken and three animals were administered TurmiZn 5 g by mouth. Blood samples were then withdrawn at 15-minute intervals up to 60 min and then hourly for up to 4 h and allowed to clot. The recovered serum was mixed with equal volumes of acetone via vertexing for 30 s and then centrifuged to remove proteins in the pellet while recovering elements of TurmiZn in the supernatant. The samples were analyzed by HPLC and liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS). In another study, intravenous (IV) injection of TurmiZn in a single healthy adult Wistar rat was used to obtain metabolic profiles of TurmiZn over time. This was based on a protocol [36 (link)] for the pharmacokinetics and tissue distribution of curcumin in mice. TurmiZn was injected (100 mg/Kg, IV) and blood was collected at 2, 5, 15, 30, 45, and 60 min and allowed to clot and extracted as previously described in acetone for ultra-fast liquid chromatography (UFLC), LC-MS/MS analysis.