GC/MS:
Unlike other tests, 0.04 g of both invertase and albumin cleavage products were taken and dissolved in 50 methanol in an ultrasonic bath. The solution was filtered on filter paper. The filtrate was used for GC/MS analysis.
As can be seen in Fig. 2-3 and Table 1-2, the list of chemicals with 95% and more matching were given from the GC/MS library of cleavage products. Both substances were found to break down into carboxylic acid esters and straight chain amides. Considering the structure of invertase and albumin, they were expected to give these products after a series of cycle mechanisms at high temperature in the acidic environment in which they are put into reaction.
Bacterial Test:
All dilutions were performed by comparing the optical density of the tubes where the microorganism load was not reduced. The microorganism introduced into the medium was expected to inhibit the chemicals ???, such as the main antibiotic agent in the control group (It cannot be called as antimicrobial/antifungal unless it shows an inhibition over 10%) [9] An antimicrobial agent is expected to reduce the burden of microorganisms in the environment after a certain period of time, which can be measured in many different ways and the accuracy of the result can be checked. As a result, the used sample did not act as an antimicrobial and antifungal agent, in Figure 4.
Viruses are very small and don’t metabolize by themselves, therefore, they don’t have the ability to reproduce on their own. They enter into the cells by force and use them to produce new viruses. They then keep proliferating rapidly in the patient's body by destroying the host cell. Antibiotics have no effect on them. On the other hand, bacteria are living small organisms that consists of a single cell. They can survive in any environment such as air, water, soil, etc. and reproduce very quickly. Antibiotics prevent the growth of bacteria [11]. Unlike the treatment of “pneumonia”, which is a bacterial disease that can be treated using antibiotics, an antiviral agent should be preferred in the treatment of “Viral Hepatitis”, which is a disease caused by viruses [10].
Cancer Test:
In the study, living cells were accepted as 100% control. No change was observed in the DNA contents of the cells compared to the control. The fluid obtained from its albumin killed more cells than any other substance, whereas all substances kill the cancerous cell around 100% [12], in Figure 5. The chemical mixture obtained did not show much effect on cancer cells. Because this mixture does neither have a toxic effect on cancer cells, nor supports the proliferation of cancer cells. It is understood that it does not have an effect on the cell death mechanism in a molecular sense and does not cause a change in the mitochondrial pathway [13].
Hepatitis Test:
The obtained cleavage products were dissolved in pure water in an ultrasonic bath and again filtered on filter paper. The filtrate was used for hepatitis test. As seen in the figure 6, in the control group 9,340-6,790-12,200 IU/mL hepatitis B virus were detected for 90-120-180 minute-cultivations, respectively, while 554,000-714,000-667,000 IU/mL hepatitis B virus were detected in cleavage products prepared with Invertase enzyme; in other words, the medium leaded 100 times more virus growth, especially in 120 minutes. Regarding the cleavage product of Albumin enzyme, 421,000-459,000-334,000 IU/mL hepatitis B virus were detected for 90-120-180 minute-cultivations, respectively, which means that it caused almost 60-70 times more virus growth especially in 120 minutes. Normally, when invertase and albumin enzymes are used as a nutrient medium without putting them in the supercritical environment, they cause 5-10 times more growth than the control group, whereas their products, which were disintegrated in the supercritical environment, caused the growth of almost 100 times more hepatitis B virus. This interesting result is because of the nucleic acid that the virus needs to reproduce. Viruses cannot reproduce on their own, they need other cells. They need nucleotides after being attached to a cell. Glycine/glutamine with a single carbon or amine is needed in the formation of nucleic acids. The concentration (0.003-0.004 g/50 ml) of the amino acids formed by the proteins disintegrated when exposed to high pressure and methanol correspond to an average of 0.5-1 mM. Having 10 times more methanol in a single carbon leads to ionization of the side chain and increases the disintegration of proteins but when faced with high pressure it mostly affects the length. Ultimately, these cleavage products obtained in the supercritical medium, provided the virus a good medium for nucleic acid synthesis.
In current study two distinct protein were used: albumin and invertase. Mild acidic conditions in methanol (pH: 5) along with high pressure and temperature breaks the proteins into smaller residues. Cleavage patterns are similar, and this suggest that the disintegration is independent of protein sequence. The process creates key precursors for nucleotide synthesis, protein coat, and single carbon metabolism. As known, in the replication of Hepatitis B virus nucleotides are needed for RNA synthesis. The virus also needs residues for protein coat of the viral genome. The replication is essential in the life cycle of the virus. Glycine, aspartic acid, glutamine, and formate are important for de novo pathway. All living organisms seem to have almost identical de novo pathways as a result of pyrimidine and purine biosynthesis. The free bases may not be the intermediates of the de novo pathways, but they form the intermediates of some salvage pathways. De novo pathways share various precursors in the synthesis of purines and pyrimidines. Each pathway type has an amino acid as an important precursor, namely purines have glycine and pyrimidines have aspartate. The most important source of amino groups is Glutamine, which is used in five different steps of de novo pathways. Purine pathways use aspartate as the source of an amino group in two steps. Phosphoribosyl pyrophosphate (PRPP) is important in both of them, and in these pathways the structure of ribose is retained in the product nucleotide [14-15].
Nucleotide synthesis determine the replication rate and may stop as the synthesis lag behind. Therefore, the cleavage of the proteins supplies the intermediates for salvage pathway and precursor for de novo synthesis. Supplying single carbon-formate (in Figure 7) and protein coat components through the process augments nucleotide content.
This process accelerated the replication effectively because cleavage pattern produced proper precursors and intermediates for Hepatitis B replication. Moreover, the addition of this soup at proper concentration (0.003 g/50 ml) accelerates virus replication cycle as well. Optimization of the process for different viruses may be the subject of clinical studies.
