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This PREreview is the result of a virtual, live-streamed preprint journal club organized and hosted by PREreview and ASAPbio Preprint Reviewer Recruitment Network.

In their preprint, YC Hwang et al. characterize some of the biophysical and biochemical properties of extracts from the mushroom Agaricus bisporus. Using quantitative photometric assays, as well as a qualitative phytochemical assay, they determined the antioxidant activity, tyrosinase inhibition activity, and phytochemical composition of ethanolic extracts of A. bisporus. This characterization of the extract gives new insights into the phytochemical composition of A. bisporus. The motivation for the study is to explore new extracts for applications to UV-protection and/or melanin production inhibition.

The study involves assays to test for radical scavenging, sun protection factor (SPF), and anti-tyrosinase properties of the ethanol extract of A. bisporus. The assays show that the extract absorbs in the UV spectrum, has antioxidant properties, and is a potent inhibitor of tyrosinase in vitro. The study reports quantitative assays to identify the total phenolic and flavonoid contents in the extract, and a qualitative phytochemical assay that shows the presence of terpenoids in the extract. 

In order to improve the quality of this work, we suggest a few changes outlined below. To emphasize the strength of the findings, we recommend to briefly talk in the introduction about A. bisporus, the current state-of-the art is regarding UV-protection compounds, including those employed in the industry, and those from natural sources, and the potential benefits of characterization studies of extracts for compound discovery. The link between the biochemical activity measured in the study and the eventual health applications as well as possible toxic effects on human health could also be made clearer. In the discussion, the authors could talk a bit about future experiments that could help further characterize these extracts and their potential biochemical activities.

Major concerns and feedback

1. While the study offers valuable information about the phytochemical characteristics of A. bisporus extract, the title, abstract, and introduction imply UV protective qualities relevant for human subjects or assessment of toxic effects of these extracts on human subjects, which are best assessed with in vivo or clinical experiments. We suggest revising the manuscript to prevent overreaching. One potential approach would be to narrow the introduction and give a slightly more technical and descriptive background of the mushroom (and why this species was chosen for the study) and its extracts in general and why they are important for novel compound discovery. In the discussion, the authors can connect their findings to future outlooks, like further characterization of the composition of the extract and its biochemical activities, or in vivo studies (including assessments of toxicity) to suggest how these compounds might actually be useful. More specifically, statements such as “further supporting the conclusion that A.bisporus mushrooms are superior tyrosinase inhibitors and thus hyperpigmentation or other melanin-related disorder correctors” could either be backed up with a comparison to the in vivo activity of kojic acid as well as other tyrosinase inhibitors—for example the “gold standard” (source) hydroquinone, or the statements toned down to better align to the data shown in this manuscript. In the absence of opportunities to conduct in vivo studies, an inclusion of activity difference between in vitro and in vivo activities for different typically used tyrosinase inhibiting compounds might help the readers understand the usefulness of the mushroom extracts.
2. There is some confusion regarding the connection between the extract´s properties in tyrosinase inhibition and UV-induced-radical scavenging. From the introduction, it seems that both properties are connected, since the paragraph describing radical scavenging is immediately followed by the tyrosinase inhibition properties, both mentioning melanin. It can be misread that the inhibition of melanogenesis is UV-protecting. It could help to make a clear distinction between both properties, and that the interest comes from two very different applications (UV protection vs dark spot whitening). The authors could emphasize that the extract has a multitude of unknown compounds, some of which they think have UV-protecting properties (Fig.1 and Fig. 2), and other compounds that are inhibiting tyrosinase (Fig. 3). 
3. In order to establish the potential of the extracts for further investigation, it would be important to include positive controls in the tests, i.e., test the activity against that of a validated compound. For example, the DPPH radical scavenging assay could benefit from a positive control. 
4. In the Introduction and in the Materials and Methods, it would be important to back up statements and references to previous work and methodologies with relative citations in the literature. 

Minor concerns and feedback

1. It would be valuable to perform a complete chemical characterization of the extracts, e.g., by HPLC and fractionation, to clarify whether the activity comes from a compound, a synergy of different compounds, vehicle etc. Analysis (LC/GC-MS) of the different fractions could help to precisely determine the active compound´s structure, and help other researchers benchmark their own extracts. We think that these experiments would significantly strengthen the conclusions of the work, but we recognize that such experiments may require equipment that may not be readily accessible to the researchers. 
2. The tables include comparisons to other results from the literature, but the manuscript also discusses how different methodologies for extract preparation could result in variability. It would be important to perform activity tests on extracts obtained through methodology as reported in literature. Results from the literature can then be compared in discussion, rather than in the tables. Other studies with the same mushroom and their findings can be taken as reference to support Author’s claim. 
3. While the preprint includes replicates for the tyrosinase inhibition activity (Fig. 3), the manuscript could benefit from inclusion of replicates for other activity measurements. A statistical analysis of the measurements wherever appropriate would also help the reader to assess the significance of the measured quantities.
4. It would be helpful to include more descriptive Figure and Table captions and labels. For example, Figure 6 needs labels for the rows, and units are missing from concentration measurements in Table 2. Furthermore, we recommend that figures are appropriately described in the results section as they appear in the manuscript.
5. A key methodological reference for SPF determination does not seem to be the appropriate paper (Romulo et al., #14).
6. Sources for the chemicals/reagents are not provided, but would help other researchers to reproduce the work. 
7. The format that mixes results and discussion may somewhat mask what the novel contribution is from the study. It may be worth considering a revision to the text to have a result-focused section that highlights the new data collected by the authors.

We thank the authors for sharing their work as a preprint. We hope our feedback above will be helpful as they consider any revisions to the manuscript or future lines of work.