Then this mixture is immediately vigorously shaken to prevent the formation of lumps. After 2 hr, the thick suspension has been filtered off; the dark red cake was shaken for another 15 min with 75 ml mixture of equal volume of methanol and carbon tetrachloride and separated by filtration. The carbon tetrachloride phase was transferred to a separating funnel; added one volume of water and shaked well. After phase separation, the carbon tetrachloride phase was evaporated and the residue was diluted with about 2ml of benzene. Using a dropper, 1 ml of boiling methanol was added in portion, then crystals of crude lycopene were appeared immediately and the crystallization was completed by keeping the liquid at room temperature and ice bath, respectively.
The solvents DMF and methanol were distilled for purification. Other chemicals were used as obtained. 2.2 Preparation of polystyrene (PS) Polystyrene prepared by free radical polymerization of styrene monomer. Styrene (1 mole) was taken in a round bottom flask (RBF) fitted with a reflux condenser. DMF was used as a solvent and AIBN (0.5% w/w of total monomer) as free radical initiator .The reaction was carried out at 70±2° C for 6 hour with constant stirring.
The natural agar consist of Yeast Extract, Tryptone , Lactose , Manniiol, Sodium Chloride , Dip otassium Hydrogen Phosphate ,Gelatin and Agar .Then , in a conical flask 14g of nutrient agar was mixed into 500 ml of distilled water and , The mixture was stirred and dissolved until most of the agar dissolve. By non-absorbent cotton wool plug the mouth of the flask was closed. By using the autoclave the agar was sterilized for 15 minutes.The cotton wool was removed. By the flame the mouth of the flask was heated before and after pouring the agar into the Petri dishes. And, the left hand the lid of the Petri dish was lift, just enough to enter the mouth of the flask and quickly was poured in agar (about 15 cm3).
Pepsin (1 wt. % of the estimated collagen) was added to the supernatant, stirred for another 2 days. Soluble collagen was purified by a repeated process of salting-out with 1 M NaCl, condensation by centrifugation at 14000 ×g for 20 minutes and subsequent solubilization in 0.5 M acetic acid. The collagen solution thus obtained was dialyzed in dialysis tubes (VISKING dialysis tubing, MWCO 12 000 - 14 000 RC, diameter 21 mm) against sterile 0.1 M acetic acid and sterile distilled water, respectively and
Dialysis membrane having a pore size 2.4 nm and molecular weight cut off between 12,000 and 14,000 was used. The membrane was soaked in double distilled water for 12 hr. before mounting in a Franz diffusion cell. About 1ml of semisolid preparation of NLC was applied to the donor compartment, and the receptor compartment was filled with phosphate buffer, pH 7.4 (14 ml). During the experiment, the solution in the receptor side was maintained at 370C and stirred at 800 rpm with Teflon coated magnetic stirrer bar.
The substrate was hydrolyzed separately using various concentrations (10, 25, and 50 mg/mL) of Alcalase, Flavourzyme, Neutrase and papain. The samples were activated with the individual enzyme conditions listed in Table 1. After the hydrolysis process, the SBP were immediately heated at 80°C for 20 min to inactivate the enzyme, followed by centrifugation at 5,000×g for 15 min and collection of the supernatants for further use. DPPH radical scavenging assay The antioxidant activity of SBP was measured with DPPH radicals according to the method of Chantaranothai et al.  Appropriate concentrations of SBP hydrolysate were mixed 1: 4 (v/v) with 200 M DPPH solution in anhydrous methanol for 30 min in the dark.
Then, the solution was loaded into the well. The gel then was run at 100 volt for 30 minutes. After that, the agar was cleaned and put into the EtBr solution for 10 minutes. The agar then washed by using tap water. Next, the agar was observed under UV transilluminator to see the formation of band.
The ground plant powder (300 gm) was extracted with (80%) methanol by soxhelation for 48 hours. The extract was filtered through Whatman filter paper and concentrated under reduce pressure and transferred to pre weighed China dish and stored in vacuum desiccators until constant weight was obtained. The antimicrobial screening of extracts was laid down for further purification of potent extract. The potent extract was successively macerated with petroleum ether, ethyl acetate, chloroform, n-butane and water (according to solvent polarity). The fractions of these two potent anti acne extracts were subjected to antimicrobial testing using serial dilution to ascertain the most effective anti method against P. acnes.
Dried starch was powdered with morter and pestle and passed through 200 mm sieve and the powdered oxidized starch was kept in a polythene bag. Determination of carboxyl content Carboxyl content was determined by using the modified procedure of Chattopadhyay et al. (1997). First, 2g (dry basis) of oxidized taro starch was dispersed in 25 ml of 0.5M HCl. The starch slurry was stirred for 30 min followed by filtration through a medium porosity fritted glass funnel (we used bottman filter paper).
It has been prepared with some modifications. ZP was prepared as follows: typically, 5 g ZrOCl2.8H2O was refluxed with 50 ml of 12 M H3PO4 at 100 °C for 24 h. The obtained precipitate was filtered off and washed with 0.1 M H3PO4 until free of chloride ion. Finally, the solid was washed with distilled water several times until the neutral pH and dried in an oven at 110 °C for 24 h. The final product was ground into fine powders and confirmed by XRD (Fig.1). The ZPFe was prepared through an ion-exchange reaction[50, 51] (. 3 g of ZP was dispersed into the 50 ml deionized water at 50 °C.