الفهرس 
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Abstract
1- Grinding or milling the amorphous turmeric powder by The Ball milling mechanical method breaks down particles to finer ones. Similar amounts (100 gm) were conducted for different periods 30 and 90 minutes comparing to the non-grinded turmeric (0 min) before the curcumin extraction.
2- Extraction: Powder of each grinded curcuma (100 g) has been stirred and extracted with 250 ml ethanol alcohol 95% using magnetic stirrer at room temperature for 3 hours. The whole extracts were filtered in whatman paper and the supernatant was concentrated until dryness using rotary evaporator at 40°C. The yield was 6.66% for the grinded curcuma powder extract. Tubes were wrapped with dark paper maintaining the sensitive curcumin.
3- The Chemical structure was detected for dried extracts using HPLC-MS in Faculty of Pharmacy, Ain Shams University for 0 and 90 min oily extracts to follow their main components structure and ratios.
Mass spectrum has identified compounds related to that for Kunati’s research (2018). Curcumin and derivatives such as curcumin glucuronid (COG) and curcumin sulfate (COS), have been followed.
(A) Curcumin in the negative mode appeared at Rt 16.09 min with MW 367, and the fragmentation has been occurred as follows; one fragment represent the removal of one methoxy group (m/z 337) and the second is the removal of bis methoxy groups (m/z 307) as been mentioned in the MS fragments.
(B) The second identified compound; curcumin glucuronid (COG) with MW 543 has been found in the negative mode at Rt 19.89. This compound has been found both in the control and the highly blended sample, and as shown contains carbohydrates moieties. . Mass fragments were showed m/z 293 and 250, and it seems that the bond cleavage has been occurred to give the glucuronic attached with the methoxy aromatic ring.
(C) In the positive ionization mode, MW 448 curcumin derivative known as curcumin sulfate (COS) has been found at Rt 20.89 min both in control and grinded sample. Sulfonic group might be involved in sulfonic amino acid structures as in cysteine, cysteine and methionine. Toluenic rings fragments have been showed in mass spectrums as m/z 125, and 219 fragments. As has been detected in chromatogram spectrum, negative and positive modes showed slight difference between control and the highest grinded sample for curcuma powder.
4- Concentrated grinded sample extracts have been equally divided for encapsulation using sodium Alginate and CaCl2 in micro-encapsulation and sodium Alginate homogenizing with Tween and ultra-sonic in nano-encapsulation Comparing to the non-capsulated sample.
Microencapsulation was conducted using emulsion extrusion technique, Sodium Alginate was dissolved in distilled water to produce polymer solutions with a concentration of 2 % w/v; the solution was left standing for 3 hr. to disengage bubble before use, polymer Alg solution (100 ml) and curcumin extract (1 ml) were homogenized into a 200 ml beaker with stirring at a speed of 300 rpm for 10 hr using magnetic stirrer. The curcumin extract was gradually added to the polymer solution during mixing until the desired curcumin extract loading was obtained. Alginate - curcumin extract emulsion (50 ml) were then sprayed into a collecting water bath containing Calcium chloride solution (2 w/v%) using an Inotech Encapsulator with a 450- m nozzle.
Nano-encapsulation was carried out using homogenization (Homogenizer PRO 400 PC, Germany) model in a matrix comprising of sodium
Alginate and Tween 20 (T20), Sodium Alginate was dissolved in deionized water (3 g/100 ml) to which sodium Alginate and emulsifier Tween 20 were added at a final concentration of 100 ml). One gram of curcumin extract was added to 100 ml solution containing 3 g sodium Alginate gel and 1 % T20 in a high-pressure homogenizer at 18.000 rpm for 30 min, Emulsion was created by mixing the solution, and temperature was kept at 35°C and storage at 4°C until used.
5- Differential Scanning Calorimetry DSC: The thermal Stability of curcuma non-encapsulated 30 min grinded as a model time, micro-encapsulated and nano-encapsulated at the same grinded time was determined using a Differential Scanning Calorimeter (DSC). Ten milligram samples were placed in aluminium crucibles, and the samples were analyzed under a flow of nitrogen gas (40 ml min-1), A dynamic scan was performed at a heating rate of 10ºC/min over a temperature ranged from 0-300ºC. The high thermal stability for nano-encapsulated maintained the antioxidant and anticancer activities (as will be explained later) and the melting point didn’t showed until 300ᴼC.
1- Electron microscopes for micro and nano-capsules:
(A) TEM: Transmission TEM was measured only for nano capsulated after milling 90 min. The pictures of spherical micelles confirmed the size of nano-capsules ranged between 7 – 42 nm. Curcumin nano-capsules exhibited a smooth surface and spherical shape.
(B) SEM: The morphology and particle size of the microparticles curcuma at 90 min was investigated using scanning electron microscopy. Micrographs of curcuma micro particles showed that microparticles were approximated spherical and appeared in a homogenous size distribution with larger particles. Average particle size was more than 450-550 µm and in the cutting across the capsules, holes have been detected between 10-50 µm.
All the samples of 0, 30 and 90 grinded minutes as their uncapsulated, micro and nanocapsulated, representing 9 samples which were measured as follows;
6-Antioxidant activity tests:
(A) The DPPH radical scavenging assay: The percentages of inhibition free radical DPPH was the highest value in microuncapsulated curcuma at abs 0 min (100 µl, 2.7 µg; 200 µl, 5.4 µg) M0, M90, then followed by M30 and after that for nanoencapsulated N0, N30, then N90. The lowest value was the grinded uncapsulated samples in the trend G30, G0, then G90 beginning from the highest value.
(B) Reducing Power: Uncapsulated samples showed the lowest reducing power comparing to micro and nano capsulation. The reduction in the particle size from micro- to nano-encapsulation with tween 80 did not improve the curcumin antioxidant activity. It was more obvious the highest activity for the micro capsules in reducing power than that for DPPH.
7- Total Phenolic Compounds Assay: Total phenolic compounds have increased about 16% from control to micro encapsulation. After that, the amount of phenolic compounds has increased 17.5% from micro encapsulation to nano encapsulation, in general. While at 90 min grinding time, the increasing in phenolic amount was dramatically (227%) from control to micro encapsulation, and 55% from the micro capsules to nano encapsulated curcuma.
8-Anticancer cell line activity: Anticancer activity of nano-encapsulation was the most superior pioneer against HepG2 cancer cell line, especially for that previously grinded at 90 min. That was followed by micro-encapsulation, and again the most powerful one is that grinding at 90 min. In the same while, noncapsulated curcuma was the lowest IC50 against hepato cancer cell line. In general view, toxicity against MCF7 appeared mostly beginning from 20 µg for non-, micro- and nano-capsules
Sulphorhodamine B (SRB) dye:
SRB detected that the toxicity against the human cancerous cells is potent in micro- and nano-encapsulation at 90 min grinding time comparing to grinded non-capsulated curcumin.
Human tumor cell line:
(A) Human HepG2 liver carcinoma cell line: Results declared higher antitumor activity in vitro against HepG2 cells for capsulated samples comparing with uncapsulated curcumin. This pattern also increased intracellular ROS level during the carcinoma cells treatment. The toxicity for capsules against human carcinoma cells is increased at 20 µg/ml non-capsulated, micro- and 5 µg/ml nano-encapsulation comparing to 90 min grinded curcumin for HepG2.
(B) MCF7 breast carcinoma cell line: IC50 for grinded curcumin is 14-19.8 µg/ml in MCF7 cells, 9-12 µg/ml for micro-encapsulated and 4-8 µg/ml for nano-encapsulated. Toxicity against MCF7 appeared beginning from 20 µg for non-, micro- and nano-capsules.
Our data revealed as well that the antitumor activity was up to 1.3 and 4 µg/ml for nano (grinded for 90 min) against HepG2 and MCF7, respectively. The prospected main result for nano-encapsules encourages successful food applications without any change in its characters and no loss of its antioxidant and anticancer activities. The reason is the ability of nanomaterial to protect and mix well those compounds either nutritional or pharmaceutical.