Asia and Pacific Commission on Agricultural Statistics APCAS, 24th session

In addition, this study elucidated some of the important factors that could influence the bioactivity. Major factors that, to our best knowledge, have not been reported elsewhere are the carbohydrate to protein to sulfate composition and the sugar composition. In view of these influential factors, tailor-made oligosaccharides could be synthesized and designed as food supplements.

1. Chemical analysis and evaluation of the biological activities of these fractions will, thus, be employed to study this effect.
2. Furthermore, the band pertaining to the O–H bending vibration of phenolic groups appeared at 1365 cm−1 in F3 only, while that corresponding to the thiol stretching vibration (S–H) appeared only in F8 at 2583 cm−1 31,32.
3. 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.
4. By using a high-resolution x-ray spectrometer, the cross sections for the three processes are measured separately as a function of projectile energy in the range of 6 to 40 MeV.
5. In this experiment it was possible to determine the activity of Protein C, since the activity in the fractions collected in the CaCl2 linear gradient were lower than the detection limit of the chromogenic method.
6. The analysis of one of the fractions obtained in the HPLC analysis of sample 9 made it possible to identify a fragment of Protein Z, which is a vitamin K-dependent factor of 62 kDa and has a plasma concentration of 1.2 to 2.9 µg mL−1.
7. Therefore, the good separation of vitamin K dependent proteins and FVIII depends on the combination of the NaCl e CaCl2 concentrations.

It is therefore clear that by increasing the concentration of CaCl2 to 25 mM, the protein is displaced from the column by pseudoaffinity. During the study using different resins, we observed, in the non-reducing SDS-PAGE, that non-vitamin dependent proteins coeluted with the vitamin K dependent proteins, although their conformations are not expected to be affected by the variation of the calcium concentration. It was possible to observe high molecular mass proteins present in all collected fractions (Figure 7). The chromatogram of the purification with one calcium gradient step with 25 mM CaCl2 is presented in Figure 4b. The yield of FVIII eluted with buffer C was 67% with a purification factor of 250 (Table 1).

5.1. Antioxidant Activity

Figure 9 shows that the 3 non-vitamin K dependent proteins (C4bBP, C4 and Fibrinogen), identified by mass spectrometry, are the contaminant proteins present in the obtained PCC. Considering the SDS-PAGE analyses shown in Figure 3, bands of these proteins are present in other, if not all, gels. It seems that these proteins are present in the collected fractions of all resins and in the FVIII containing fractions.

These results indicate that the vitamin K dependent coagulation factors bind stronger on Fractogel EMD DEAE than on TMAE. Anion exchange resins are widely used in the plasma fractionation processes 3,12,13,14,15 due to their high capacity, good yields and low cost compared to other chromatographic techniques. Upon loading whole plasma directly onto an anion exchange column, without the cryoprecipitation step, FVIII and PCC are strongly adsorbed and cannot be eluted separately using NaCl gradient 16,17. A second column was needed to separate FVIII from PCC proteins, and it was successfully achieved by immobilized metal affinity chromatography 16,18. Conventional affinity chromatography involves the immobilization of a ligand for which the protein of interest has an affinity. Pseudoaffinity elution in ion exchange chromatography differs from affinity chromatography in that there is no ligand immobilized on the matrix for which the protein of interest has affinity, and this matrix can be a conventional ion exchange resin or hydrophobic interaction 19.

The above findings are in agreement with the antitumor results shown earlier, where F5 exhibited the highest percentage lethality on HePG2. Elution was carried out with a 5 CV linear gradient of 100 mM to 600 mM NaCl in Buffer A and 5 CV step of the Buffer A + 600mM NaCl. Ulva lactuca, or as commonly known by “sea lettuce”, is an edible green seaweed from which the SPs, ulvans, are extracted. Their molecular weight ranges from 189 to 8200 KDa and they are composed of units of mono or disaccharides such as rhamnose, xylose, and iduronic or glucuronic acid. The most abundant unit is ulvan biouronic acid with sulfate at C3 where the acid unit could be either glucuronic or iduronic acid 2. Twenty CV of FFP were applied to the column and the unbound proteins were washed with 14 CV of Buffer A. FT was collected in fractions of 2 CV.

4.2. DEAE Sepharose FF Chromatography with Ca2+ 5 mM to 50 mM Linear Gradient

All the following procedures were done in a sterile area using a Laminar flow cabinet biosafety class II level (Baker, SG403INT, Sanford, ME, USA). Cells were suspended in Roswell Park Memorial Institute RPMI 1640 medium for HePG2, MCF7, and HCT116, and in Dulbecco’s Modified Eagle Medium DMEM for A549. Where Q is the absorbance ratio (A1/A2), and A1 and A2 are the respective absorbances of SPs samples before and after reduction. In the past decades, marine seaweeds have gained much interest as wealthy resources of bioactive compounds.

Table 5.

1. Hill coefficients (Table 5) were determined for fractions that showed lethality ≥75% and were all above 1.
2. Equilibration, sample, and washing with Buffers A were performed according to Section 4.1, Section 4.2.1 and Section 4.5.
3. The anion exchange resins Q, ANX and DEAE Sepharose FF resins were purchased from Cytiva (Uppsala, Sweden).
4. The results presented in this work showed that the proposed purification method could be successfully applied to all five resins tested, provided that the CaCl2 and NaCl concentrations in citrate buffer are carefully determined.
5. The presence of these proteins with similar concentrations along the entire gradient (Figure 7) suggests that these proteins did not elute due to the increase in CaCl2 concentration.
6. The samples that showed 75% or more lethality at 100 ppm were further investigated for lower concentrations.

SDS-PAGE 7.5% analysis under non-reducing conditions of the purification of plasma on ANX Sepharose FF with CaCl2 5 to 25 mM linear gradient in 25 mM citrate, 200 mM NaCl, pH 6.0 buffer. Interestingly, F6 and F7 showed no antitumor activity and relatively low antioxidant activity relative to F4 and F5, although they share the same DP of 3 with these two fractions. This could be attributed to their low protein content and hence their possession of less functional amide groups as compared to fractions 4 and 5. This is in addition to their highest sulfate contents amongst other fractions, which could have affected their antitumor activity as was the case with their antioxidant activity.

Our results indicate inf8 exchange that prothrombin complex could be eluted with CaCl2 without affecting FVIII activity using all five tested resins. Fourier transform infrared spectroscopy was performed for S1, V45, and all 8 column fractions. Samples were mixed with potassium bromide in the form of 1-mm pellets and analyzed in a TGA/FT-IR Nicolet 380 spectrometer for a range of wavenumbers between 500 and 4000 cm−1.

1. Chemical Analysis

Equilibration, sample, and washings with Buffers A and B were carried out as indicated in Section 4.1, Section 4.2.1, and Section 4.3. Next, 10 CV of linear gradient 5 mM to 25 mM CaCl2 (in Buffer B) and 5 CV stepwise Buffer B + 25 mM CaCl2 were carried out. They were then washed with Buffer B (200B) and the elution with Buffer C was performed as in Section 4.2.1. Proteins identified by mass spectrometry of the fraction “25” from plasma purification on ANX Sepharose FF using stepwise 10 mM and 25 mM CaCl2 gradient.

The open source reference implementation of CryptoNote was coded from scratch based on the CryptoNote reference implementation, and is not a fork of Bitcoin. Infinium-8 aims to be a fungible and untraceable digital medium of exchange. The volume of the Fractogel EMD DEAE column was 24 mL and flow rates were as indicated in Section 4.5. The resin was regenerated by sequentially washing with 2 CV 0.5M NaOH (with 1 h pause), 5 CV 2M NaCl and 10 CV purified water and stored in 10 mM NaOH. Equilibration, sample, and washings with Buffers A and B were carried out according to Section 4.1, Section 4.2.1 and Section 4.4. The KαL 1 /KαL 0 intensity ratio of fluorine is measured in five fluorine compounds with a crystal spectrometer.

The plates were left for 24 h in a water-jacketed carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA) at 37 °C and under 5% CO2. Cells were incubated either alone to determine the negative control, or with different concentrations of the sample to yield final concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 μg/mL. After 48 h of incubation, the medium was aspirated and 40 μL of 2.5 μg/mL of MTT salt were added to each cell and incubated for further 4 h at 37 °C under 5% CO2.

The total protein, carbohydrate, and sulfate contents in each collected fraction are listed in Table 1, along with the degree of polymerization (DP). Equilibration, sample, and washing with 10 CV of Buffer A were performed according to Section 4.1 and Section 4.4. Next, elution with 10 CV linear gradient of 5 mM to 50 mM CaCl2 (in Buffer A) was performed. Then a second wash with 5 CV Buffer A and a second elution with Buffer D (25 mM sodium citrate, NaCl 400 mM and 5 mM CaCl2, pH 6.0) was carried out.