ProFX Mouth Spray and its degradation ability on spike glycoprotein of SARS CoV-2 Virus

Our proprietary ingredient, Hydrolyzed Collagen Trypsin (HCT), is a protein and proteolytic enzyme derived from marine collagen. It is extracted from Crustacean sources through a proprietary extraction method to yield hydrolyzed collagen that contains trypsin. HCT has various benefits that could be used in biomedical applications, supplements, industrial use, and cosmetic applications.

The current formulation that contains HCT is ProFX Mouth Spray. There is approximately 2% of HCT used in ProFX Mouth Spray along with other natural ingredients such as eucalyptus globulus oil and mentha arvensis oil, to aid in the functionality of HCT and as a mouth spray. The intended use of ProFX Mouth Spray is to act as a protective barrier in the oral mucosa from microbial penetration, promote wound healing and rehydrate and moisturize the oral cavity.

In the light of SARS CoV-2 Virus and the pandemic, there is a need in understanding the infection of the virus and ways to prevent and/or treat them. The attachment of spike glycoprotein (S-protein) of SARS-CoV-2 to angiotensin-converting enzyme 2 (ACE2) receptor of the host cell is a critical step in the infection route of this virus. Blocking this attachment is therefore considered a promising strategy to prevent the virus replication inside the host cells. In this respect, the degradation of S-protein by proteases should prevent the attachment, which puts the proteases as a promising compound for combating COVID-19. Reports by Sagar et al. (2020) convincingly showed that the degradation of S-protein by bromelain protease prevented the infection of SARS-CoV-2 towards the VeroE6 cells line. This also was supported by Akhter et al. (2020), who reported that bromelain protease disintegrates the spike and envelope protein from SARS-CoV-2 and may render it non-infective. 

PRO-FX is a mouth spray containing trypsin protease (0.6 mg/mL) extracted from marine collagen to serve the purpose of killing bacteria and promoting wound healing in the oral cavity. The presence of trypsin protease in this product leads to an assumption that this product might also be able to degrade the S-protein of SARS-CoV-2 virus. Hence, prompted the research on degradation of spike glycoprotein of SARS CoV-2 Virus by ProFX Mouth Spray.

*The concentration of trypsin used in this research is (0.02mg/mL) which is approximately 0.07% due sample tested has reached its expiry date.

Methodology:

In this research, 5 μl (concentration = 0.0067 mg/mL), 10 μl (concentration = 0.01 mg/mL) and 20 μl (concentration = 0.013 mg/mL) of ProFX solution was added into 10 μl of S1 and S2 subunit (final concentration was 0.5 mg/ml each) at 37 ̊C. Initial assay of 0 hour of incubation was run on SDS PAGE and then it was incubated for 2 hours. The reaction was stopped by adding 2X SDS-Sample buffer and heated to 100 ̊C for 10 minutes and the mixture is loaded onto SDS-PAGE. The gel was stained with Coomassie Blue solution for 30 minutes followed by acetic acid staining for another 30 minutes. The protein fragments can be seen on the band visualized on Bio-rad Gel Doc.

Result

1. Degradation of S1 and S2 subunits of spike glycoprotein of SARS CoV-2 Virus by ProFX  Mouth Spray in various concentrations and time.

Figure 1. SDS-Page of S1 and S2 subunits in the presence of various concentration /volume of PRO-FX 

Figure 1 showed the thickness of the band corresponding to S1 and S2 subunit was decreased proportionally with the increase of PRO-FX concentrations. This indicated that degradation activity of PRO-FX was proportional with the concentration of PRO-FX given in this study. 

Table 1. Percentage of Degradation of S1 and S2 subunits by PRO-FX per concentration unit

 

Table 1 showed that the % of degradation of PRO-FX against S1 and S2 subunits significantly increased by the increase of the concentration. At PRO-FX’s trypsin concentration of 0.013 mg/mL, PRO-FX was able to degrade the S1 subunit by 97.81% and 61.74% for S2 subunit. The volume for each spray of ProFX Mouth spray is 100 µl, hence it can be assumed that the volume and concentration of ProFX in one spray can degrade at least 99% of S1 and S2 subunits. 

2. Stability of ProFX and proteolytic activity of S1 subunits of spike glycoprotein of SARS CoV-2 Virus after additional 2 hours of incubation

In this test, only 0.025 mg/mL of S1 subunits was added after the 2 hour incubation and proteolytic activity of additional 2 hour incubation was taken.

Figure 2. Degradation of S1 subunit by PRO-FX with addition of S1 subunit after 2h incubation 

Figure 2 showed that PRO-FX retains its degradation activity after 2h incubation. PRO-FX was able to degrade the addition of S1 subunit after 2-hour incubation which indicated that PRO-FX’s activity is still active and present. 

3. Comparison of degradation of S1 and S2 subunits of spike glycoprotein of SARS CoV-2 virus by ProFX and Product X

Figure 3. SDS-Page of S1 and S2 subunits in the presence of Product X and PROFX

Figure 3 shows the thickness of the band corresponding to S1 and S2 subunit in the presence of PRO-FX was remarkably lower than that of Product X. This indicated that more subunits were degraded by PRO-FX than the Product X during 2h incubation time. Accordingly, PRO- FX exhibits higher apparent degradation activity than the Product X.

 

Table 2 Degradation rate of ProFX and Product X against S1 and S2 subunits

Table 2 clearly indicated that during 2h incubation, PRO-FX was able to degrade S1 and S2 subunit at the rates of 21.38% and 10.99% per hour, respectively. It is approximately two times higher than Product X which has a degradation percentage of 8.31% for S1 subunits and 0.98% for S2 subunits.

Limitations:

  • Concentration of Hydrolyzed Collagen Trypsin found in the sample are lesser than actual product; sample contains only 0.007% of HCT whereas the actual product contains 2% of HCT. Hence, this is not a direct comparison to the actual product due to difference of concentration. However, an implication can be made that the actual product would outperform the current concentration and yield higher degradation activity.
    • Due to limited lab equipment in the biotech lab, spectrophotometers were not used in this test for spectrophotometric enzyme assay. Instead, SDS-PAGE was used to identify the protein degradation fragments. This would result in limited data and result due to the methodology used.