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In addition, F4 has the highest protein content while F7 and F8 contain traces of proteins. It can also be deduced from the table that the algal extract (S1) contains comparable contents of carbohydrates and sulfates. Prothrombin (FII) was identified in all preparations because this protein is in greater abundance. While the concentration of FII in plasma is 90 µg mL−1, the second most abundant vitamin K dependent protein is Protein S, with 30 µg mL−1 and is complexed with the C4b binding protein.

The main reason for introducing the calcium gradient step with lower ionic strength was the loss of FVIII. Therefore, much higher concentrations of CaCl2 were required to elute PCC(FIX) (Figure 2b,c,e). In the purification of FVIII from cryoprecipitate carried out by Mori et al. 3, the yield and purity of this coagulation factor was much higher using TMAE than DEAE.

Previous studies reported that better lung cancer A549 antitumor activity was exhibited with the more complex polysaccharides that contained different kinds of monosaccharides. The more complex ones comprised eight kinds of monosaccharides as opposed to other samples that contained three and five types only 38. However, the algal extract S1 contains five monomers but is active on one cell line only, which suggests that the molecular weight along with the complexity of the molecule should be taken into consideration.

Figure 9.

Proteins identified by mass spectrometry of fractions collected from plasma purification on ANX Sepharose FF using linear 5 to 25 mM CaCl2 gradient. The samples that showed 75% or more lethality at 100 ppm were further investigated for lower concentrations. The cancer cells used were all from the American type culture collection ATCC (USA) and were four types (HCT116, colon cell line; A549, lung carcinoma cell line; HePG2, human hepatocellular carcinoma cell line; MCF7, breast carcinoma). Cell culture was conducted in batch for 10 days, and cells were afterwards seeded at a concentration of 10 × 103 cells/well in fresh complete growth medium placed in 96-well microtiter plastic plates.

1. Doxorubicin was used as a positive control at 100 µg/mL since it is a known cytotoxic natural agent, which gives 100% lethality under the same conditions as per Thabrew et al. 55,56.
2. Infinium-8 (INF8) is a privacy-centric cryptocurrency using the CryptoNote protocol.
3. The vitamin K dependent proteins remain in the supernatant (called cryo-poor plasma), along with albumin, IgG and other proteins.
4. This is because they were more strongly bound to the column by virtue of their charge and therefore required an eluent with higher ionic strength.
5. This suggests that ligands in the same fraction possibly interact with different sets of target molecules from one cell line to another.

Figure 2.

They are frequently captured by anion exchange chromatography, resulting in a mixture of intermediate purity, called the prothrombin complex concentrate (PCC) 9,10. Highly purified FIX, used in hemophilia B replacement therapy, is isolated from PCC 9,11. The chromatogram of the purification with calcium gradient step with CaCl2 10 mM and 25 mM is shown in Figure 4c. In the insert of Figure 4c, it is possible to see that the two peaks were well resolved. The coagulation proteins Prothrombin, FIX and Protein C were recovered in the 25 mM CaCl2 fraction.

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In this test, cell viability was measured, as per Mosmann assay 53,54, by reduction of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan via mitochondria. Separation and quantitation were carried out with deionized water as the mobile phase. Samples were analyzed for mono- and disaccharides by high-performance liquid chromatography (HPLC) according to AOAC 51. Sugars were extracted using double distilled water for 3 h; the extract was passed through C18 Sep-Pakcartridge, refrigerated, and stored for further analysis. Standard solutions of glucose, glucuronic acid, rhamnose, galactose, and xylose sugars were prepared by diluting each analyzed sugar with deionized water. These sugars were chosen for analysis as they are the most abundant sugars in Ulva lactuca.

Green Ulva lactuca algae were collected in summer time from the Mediterranean Sea in Egypt, specifically Alexandria in Abou Kir region (N 31°19′ E 30°03′). Algae were washed with tap water followed by deionized water, then dried and ground to start extraction. Dose–response curves for fractions S1 on HCT116 cells (A), F4 on HePG2 cells (B), F5 on HePG2 cells (C), F5 on A549 cells (D), and F5 and F8 on MCF7 cells (E). The dose–response curves were used to determine LC50, LC90, and Hill coefficient values. Total protein concentration was determined by the Bradford method 28, using bovine serum albumin (Pierce, Rockford, IL, USA) as standard.

Fractions S1, F4, F5, and F8 showed promising antioxidant and antitumor activities in vitro. F5 also possessed the highest Hill coefficient among the inf8 exchange four promising fractions indicating a higher degree of cooperativity in ligand binding. Other influencing factors including DP, composition, and type of characteristic functional groups were also discussed. The implications of this work could potentially benefit the industries of food supplements and pharmaceuticals. In addition to sugar composition, the complexity of the polysaccharide in terms of the variety of sugar units constituting its chain is another important influencing factor.

1. Therefore, much higher concentrations of CaCl2 were required to elute PCC(FIX) (Figure 2b,c,e).
2. Columns were equilibrated with five column volumes (CV) of Buffer A (25 mM sodium citrate, 85 mM NaCl and 5 mM CaCl2, pH 6.0).
3. Fractions S1, F4, F5, and F8 showed promising antioxidant and antitumor activities in vitro.
4. Maximum elution was observed with F8–F10, where Ca2+ was between 31.0 mM and 42.0 mM (Figure 2e).
5. Antioxidant activity has been reported in the literature to be directly related to the phenolic content 21,36.

The loading profiles of FVIII and FIX in the purification of plasma on ANX are quite different (Figure 5). While the FVIII starts leaking from the column after the loading of 10 CV of plasma, the FIX activity measured after loading 20 CV of plasma was negligible. This result indicates that using plasma as starting material, the binding capacity of FIX is higher than the binding capacity of FVIII. The antitumor cell test depends basically on the cytotoxic effect of each of the samples on the cancer cells.

SDS-PAGE were performed on a 7.5% polyacrylamide gel under reducing (β-mercaptoethanol) and non-reducing conditions, on a 6% gel under non-reducing conditions, and on a 5 to 15% gradient under reducing conditions following Laemmli’s procedure 29. In these experiments, instead of the citrate buffer, Bis-Tris or MES buffers were used, except in the elution of FVIII, which was carried out with citrate (Buffer C). All other experimental conditions followed the procedure described in item Section 4.3.1. Activity of FVIII was observed in all collected fractions, the maximum being roughly the same as that observed for FIX in F8, where the NaCl concentration was 440 mM and conductivity 45 mS cm−1 (Figure 6). FIX has a sharp peak between F5 and F12, that is, between 320 mM NaCl and 600 mM NaCl, respectively. FVIII and FIX dynamic binding capacity and NaCl elution in purification of plasma on ANX Sepharose FF.