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Monday Article #62: A brief review of the effects of Antrodia Camphorata against Sars-CoV-2


Figure 1: Antrodia Camphorata mushroom (Maca. ph., 2020)


Plants are widely regarded as a rich source of natural products which are capable of preventing diseasse while lowering health-care costs (Tu et al., 2022). Various effects which include anti-microbial, anti-oxidative, anti-inflammatory, anti-diabetic, anti-aging, anti-carcinogenic, neuroprotective, hepatoprotective, cardioprotective and immunomodulatory effects have been discovered in a mushroom called Antrodia camphorata (Tu et al., 2022). According to Lee et al., 2002, it was found that the Antrodia camphorata polysaccharides exhibit anti-HBV activity. In another study, it was discovered that the active extracts in Antrodia camphorata notably reduced the viral replication when added to Vero-cells infected with Herpes-Simplex Virus (HSV) (He et al., 2015). This is illustrated in Figure 2:


Figure 2: The plague reduction results of (A) Herpes-Simples Virus (HSV) and (B) HSV-2 with Fraction A, Antrodin A and Acyclovir (He et al., 2015)


This suggests that Antrodia camphorata possesses antiviral property, which can potentially be effective on SARS-CoV-2. Unique steroid-like compounds isolated from Antrodia camphorata, known as antcins, have been reported to be novel anti-angiotensin-converting enzyme 2 (ACE2) agents (Tu et al., 2022). Therefore, this highlights its potential to be used as a treatment alternative for Covid-19. According to Senthil Kumar et al., 2021, except for antcin-M, all of the treated antcins which include antcin-A, antcin-B, antcin-C, antcin-I and actcin-H inhibited ACE2 activity significantly (Figure 3). Antcin-A, antcin-B, antcin-C and antcin-I reduced human ACE2 levels in HT-29 cells from 11.23 ng/mL (control) to 4.39 ng/mL, 4.22 ng/mL and 4.19 ng/mL respectively, while antcin-H treated cells showed a moderate reduction (5.91 ng/mL) (Senthil Kumar et al., 2021).


Figure 3: An illustration of the effect of Antcins on the human ACE2 levels in HT-29 cells (Senthil Kumar et al., 2021).


Experimental Design


MTT Assay The SARS-CoV-2 Omicron Variant (BA.1) will be propagated in Vero cells and stored in -80 °C until further usage. Normal Human Bronchial Epithelial (NHBE) cells will be cultured and maintained. Upon reaching confluence, the cells will be seeded in 3 microplates. Each microplate will be labelled 24, 48 and 72 hours respectively. The NHBE cultured in a transwell should be inoculated with SARS-CoV-2 Omicron Variant (BA.1) and incubated for 24 hours. This will be done to infect the NHBE cells with the SARS-CoV-2 Omicron Variant (BA.1). Next, the media in all wells will be replaced and proceeded with treatment with different concentrations of Antrodia Camphorata mushroom extract. There will also be some uninfected cells which will be treated. This serves as the control as well as a method to evaluate the toxicity of the extract. All the plates will then be incubated at 37 °C and 5% CO2. After 24 hours, the 24-hour plate will be taken out from the incubator and 2,5-diphenyl-2H-tetrazolium bromide (MTT) solution will be added to each well of interest. The plate will then be incubated for 3 hours, after which all liquid from the wells will be removed and Dimethyl sulfoxide (DMSO) will be added to each well of interest to solubilize the formazan crystals. The absorbance will be measured at 570 nm using a microplate reader. This will then be repeated for the other 2 microplates at 48 and 72-hour time points respectively. The viability will then be calculated to determine the effect of each concentration of the Antrodia Camphorata mushroom extract. Data will presented using graphs and analysed.


qRT-PCR

The viral RNA kit (Macherey-Nagel) will be used to extract viral RNA from 50 uL culture supernatant of SARS-CoV-2 infected Vero E6 according to the manufacturer's instructions

(Mösbauer, 2021). Quantitative RT-PCR [LightCycler 480 Real-Time PCR System and Software version 1.5 (Roche)] will be used to detect SARS-CoV-2 genome equivalents (GE), with the following primers targeting the SARS-CoV-2 E gene:

  1. E gene-F (5′-ACAGGTACGTTAATAGTTAATAGCGT-3′)

  2. E gene-R (5′-ATATTGCAGCAGTACGCACACA-3′).

Figure 4: Positions of amplicon targets relative to the 2019 novel coronavirus genome and the SARS coronavirus (Corman, 2020)

Data obtained from both assays will then be compared to verify if they direct to the same conclusion. The figure below shows an example of expected result to be obtained from the experiment.

Figure 5: An example of the expected result to be obtained for both assays (Driouich, 2022)


Firstly, treatment with Antrodia Camphorata mushroom extract will cause a reduction in the viability of the Sars-CoV-2 infected Normal Human Bronchial Epithelial (NHBE) cells. The higher the concentration of extract used, the lower the viability of the cells. This will indicate that the mushroom extracts possess cytotoxic effects against the Sars-CoV-2 infected cells. A similar trend is also expected for the non-infected cells as high concentrations would indeed also exert cytotoxic effects on normal cells. However, what is more important is that the half-maximal inhibitory concentration (IC50) of the infected cells should be lower than the non-infected cells. This will indicate that the same concentration will exert more cytotoxic effect on the infected cells as compared to the uninfected cells, thus exerting minimal toxicity.

Next, the treatment of the Antrodia Camphorata mushroom extract will also result in viral RNA inhibition. If the Sars-CoV-2 E-gene is present in the isolated RNA samples, it would indicate that they are viral RNA. The amount of this will then be quantified to determine if the treatment inhibits the viral RNA. It is expected that the higher concentration of extract used will cause higher viral RNA inhibition. This will indicate that the mushroom extracts possess antiviral effects against the Sars-CoV-2 virus.

In conclusion, this is a simple and brief experimental design to evaluate the cytotoxic and antiviral effects of Antrodia Camphorata on Sars-CoV-2 infected Normal Human Bronchial Epithelial (NHBE) cells. At the end of both these assays, it would provide an indication and primary verification if the Antrodia Camphorata mushroom actually has the capability to be considered as an alternative treatment option for Covid-19. However, a lot more tests and assays would need to be carried out following this to ensure that all the results actually point at the same direction. Further toxicity studies are also indeed crucial to ensure that the effects exerted by the mushroom extracts have minimal cytotoxic effects on normal cells.


References:

  1. Corman, V. M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D. K., Bleicker, T., Brünink, S., Schneider, J., Schmidt, M. L., Mulders, D. G., Haagmans, B. L., van der Veer, B., van den Brink, S., Wijsman, L., Goderski, G., Romette, J. L., Ellis, J., Zambon, M., Peiris, M., Goossens, H., Reusken, C., Koopmans, M. P. G. and Drosten, C. (2020) ‘Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR’, Eurosurveillance, 25(3), p.2000045. [Online] DOI: 10.2807/1560-7917.ES.2020.25.3.2000045 (Accessed: 20 June 2023)

  2. Driouich, J. S., Cochin, M., Touret, F., Petit, P. R., Gilles, M., Moureau, G., Barthélémy, K., Laprie, C., Wattanakul, T., Chotsiri, P., Hoglund, R. M., Tarning, J., Fraisse, L., Sjö, P., Mowbray, C. E., Escudié, F., Scandale, I., Chatelain, E., de Lamballerie, X., Solas, C. and Nougairède, A. (2022) ‘Pre-clinical evaluation of antiviral activity of nitazoxanide against SARS-CoV-2’, EBioMedicine, 82, p.104148. [Online] DOI: 10.1016/j.ebiom.2022.104148 (Accessed: 20 June 2023)

  3. He, Y.-C., Lu, Z.-H., Shi, P., Hao, J.-C., Zhao, Z.-J., Xie, H.-T., Mao, P. and Chen, S.-J. (2015) ‘Anti-Herpes Simplex virus Activities of Bioactive Extracts from Antrodia Camphorata Mycelia’, Antiviral Therapy, 21(5), pp.377–383. [Online] DOI: https://doi.org/10.3851/imp2988. (Accessed: 20 June 2023)

  4. Maca. ph. (2020). What is Antrorata® and Antrodia Camphorata Ingredient of Dr. Vita Maca. Retrieved from https://maca.ph/what-is-antrorata-and-antrodia-camphorata-ingredient-of-dr-vita-maca/

  5. Mösbauer, K., Fritsch, V.N., Adrian, L., Bernhardt, J., Gruhlke, M.C.H., Slusarenko, A.J., Niemeyer, D. and Antelmann, H. (2021) ‘The effect of allicin on the proteome of SARS-CoV-2 infected Calu-3 Cells’, Frontiers in Microbiology, 12, p.746795. [Online] DOI: 10.3389/fmicb.2021.746795 (Accessed: 20 June 2023)

  6. Senthil Kumar, K.J., Gokila Vani, M., Hsieh, H.-W., Lin, C.-C. and Wang, S.-Y. (2021) ‘Antcins from Antrodia cinnamomea and Antrodia salmonea inhibit Angiotensin-Converting Enzyme 2 (ACE2) in epithelial cells: Can be potential candidates for the development of SARS-CoV-2 prophylactic agents’, Plants, 10(8), pp. 1736. [Online] DOI: 10.3390/plants10081736 (Accessed: 20 June 2023).

  7. Tu, P.C., Jiang, W.P., Lin, M.K., Huang, G.J., Li, Y.J. and Kuo, Y.H. (2022) ‘Anti-inflammatory constituents of Antrodia camphorata on RAW 264.7 cells induced by polyinosinic-polycytidylic acid’, Molecules, 27(16), pp. 5320. [Online] DOI: 10.3390/molecules27165320 (Accessed: 20 June 2023).

 

This article was prepared by Thiiben A/L Krishnan Sami

 

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