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Metabolites of Pinang Yaki (Areca vestiaria) Fruit Extract: A Metabolite Profiling Study [version 3; peer review: 1 approved, 1 approved with reservations, 1 not approved] titled: New Discovery of Covid-19 Natural-Based Antivirus Herbal Supplement Products from Pinang Yaki (Areca vestiaria) Extract by Untargeted Metabolomic Profiling

Background: Pinang yaki has bioactive compounds that have potential as a new herbal supplement, but their metabolites profil is lack of data. A better understanding of the bioactive compounds of pinang yaki using untargeted metabolomic profiling studies will provide clearer insight into the health benefits of pinang yaki in further. Methods: Fresh samples of pinang yaki ( Areca vestiaria ) are obtained from forests in North Sulawesi Province, Indonesia. Samples were used for untargeted metabolomics analysis by UHPLC-MS. Results: Based on an untargeted metabolomic profiling study of pinang yaki, 2504 compounds extract. The top 10 compounds capable to ionize well have been identified and explored for their potential benefits as antiviral supplement products by literature study. This is a preliminary study which still needs further research such as in vitro, preclinical, and clinical trials. In this manuscript, the authors identified metabolites in pinang yaki fruit extract using LC-MS and tried to link these metabolites to their anti-COVID19 properties. However, in my opinion, many points need to be improved before this manuscript can be indexed. Title: The title does not represent accurately what the authors did. The word “untargeted metabolomics profiling” should be reserved for comparative studies. In addition, the authors did not perform experiments to show the relevance of these metabolites or even the pinang yaki itself on Covid-19 prevention or treatment at all, and thus, this should not be in the title. The appropriate title should be more like “Metabolite profiling of pinang yaki ( Areca vestiaria ) fruit” or something similar.


Introduction
Coronavirus disease 19 (COVID-19) is a highly communicable and deadly virus caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first appeared in Wuhan, China in December 2019 and has since spread around the world (Mouffouk et al., 2021). The SARS-CoV-2 virus is a β-coronavirus, a non-segmented enveloped positive-sense RNA virus with a crown-like appearance and symmetric helical nucleocapsid (Astuti & Ysrafil, 2020). Unlike the previous outbreaks of coronavirus, SARS-CoV-2 is more transmittable and the majority of those infected remain asymptomatic, resulting in ineffective containment and mitigation (Andersen et al., 2020). It has become a major cause of mortality and morbidity worldwide (Das et al., 2021). Therefore, finding an optimal therapeutical solution is vital.
Medicinal plants and natural products can be promising alternatives to supplements or drugs to treat and prevent diseases (Benarba & Pandiella, 2020). Pinang yaki (Areca vestiaria), also known as crown shaft palm, is an endemic palm plant that grows in North Sulawesi that is traditionally utilized for the treatment of diabetes and diarrhoea, and as a contraceptive (Gosal et al., 2017;Herny et al., 2010). Pinang yaki fruit extract has bioactive compounds such as flavonoids, tannins, saponins, triterpenoids, and hydroquinones, which are known primarily as natural antioxidants (Simbala et al., 2017). Tannins and flavonoids are known for their antitumor, antiallergic, antihepatotoxic, and antioxidant activities (Londok et al., 2017), while triterpenoids exert antibacterial, anticancer, anti-inflammation, and wound care properties (Herny et al., 2010), as well as inhibiting viral replication (Das et al., 2021). Saponins also show antifungal, cytotoxic, antibacterial, and antiviral properties (Kregiel et al., 2017). A recent review also stated that flavonoids may inhibit viral replication and translation (Ahmad et al., 2015), enhance immune activity, antiviral protection, and reduce respiratory problems (Yang et al., 2020); since phenols inhibit the fusion of the virus to the host cell (Das et al., 2021). Further separation and analysis of pinang yaki fruit extract also identified pentadecane and hexadecanoic acid (Simbala et al., 2017), which were known for their ability in downregulating pro-inflammatory cytokines (Chuah et al., 2018;Aparna et al., 2012). Experimental animal model also revealed lipid-lowering properties of ethanol extract of pinang yaki fruit (Sagay, Simbala, & De Queljoe, 2019).
Despite all those health-beneficial properties, pinang yaki is still underutilized. Pinang yaki and its bioactive compounds have the potential as a novel herbal supplement. A better understanding of pinang yaki's bioactive compounds using an untargeted metabolomic profiling study will provide clearer insight into the health benefits of pinang yaki, in particular its potential for therapy and prevention of viral infection. It is hoped that through this study, the metabolites that may play important roles in sustaining life and health can be explored as stated in the article of Vinayavekhin and Saghatelian (2010) and Vinayavekhin (2013). More specifically, the possible potential of the metabolites of areca yaki as antiviral.

Methods
Fresh samples of pinang yaki (Areca vestiaria) fruit with the age of 3.5 months were obtained from Bolaang Mongondow forest in North Sulawesi Province, Indonesia. The samples were then cleaned using distilled water (Aquades) and then stored in a cooling box to be sent to the intended metabolomic testing laboratory. Samples were used for untargeted metabolomics analysis by UHPLC-MS. The botanical identification and authentication were confirmed at the Department of Pharmacology, Faculty of Mathematics and Natural Sciences, Sam Ratulangi University, Indonesia by comparing the specimen with previously identified reference specimens (Applequist & Miller, 2013). Untargeted metabolomics analysis and identification of compounds were conducted at Apical Scientific Sdn. Bhd. 43300 Seri Kembangan laboratory, Selangor, Malaysia with registration number #CPMO08102001a.

REVISED Amendments from Version 2
The authors made several improvements/revisions to the article according to the reviewer's constructive comments (Especially Dr. Nawaporn Vinayavekhin): -The title has been changed to be more appropriate, this is based on consideration of reviewer comments.
-Improved abstract at the request of reviewers.
-Improvement of Introduction, Method, and Discussion, including addition of references that are appropriate or related to this study.
Any further responses from the reviewers can be found at the end of the article

Sample preparation
Samples were thawed, and 50 mg of each sample was precisely weighed into a tube, add with 800 μL of 80% methanol with vortex for 90 s, and followed by sonication for 30 min, 4°C. Then all samples were kept at À40°C for 1 h. After that, samples were vortexed for 30 s, kept for 0.5 h, and centrifuged at 12000 rpm and 4°C for 15 mins. Finally, 200 μL of supernatant was transferred to a vial for LC-MS analysis.
Mass spectrometry parameters in positive ion mode (ESI+) and negative ion mode (ESIÀ) mode are listed as follows:

Results
Graphs shown in Figures 1 and 2 were used to determine the peak representing the number of annotated ions, retention time, and relative abundance of the ions. This data was then used to identify the compounds contained in the sample extract. The results of identification and mass of compounds were carried out by the laboratory of Apical Scientific Sdn. Bhd and the results provided in an Excel spreadsheet (Microsoft Excel, RRID:SCR_016137) (see data availibity statement) (Nurkolis & Simbala, 2021). Based on an untargeted metabolomic profiling study of pinang yaki, 2504 compounds in ESIÀ and 2645 compounds in ESI+ were successfully obtained (Nurkolis & Simbala, 2021). After the analysis, 356 compounds in ESIÀ and 543 compounds in ESI+ were identified successfully (Nurkolis & Simbala, 2021). From each chromatogram, the strength of the various adducts was roughly measured.  (Nurkolis & Simbala, 2021). After the analysis, 543 compounds in ESI+ were successfully identified (Nurkolis & Simbala, 2021). Figure 1 shows that the major compound was Alpha-chlorohydrin (PubChem ID: 7290).  (Nurkolis & Simbala, 2021). Figure 2 shows that the identified major compound was tagatose (PubChem ID: 439312). After 543 compounds in ESI+ were successfully identified, those compounds were ranked into the top 10 metabolites present in pinang yaki extract (ESI+) which have best capability to ionize in positive modes, as is shown in Table 1.
In Table 2, the ranking is also shown for ESIÀ, providing the top 10 metabolite compounds which have best capability to ionize in negative we have modes. Tables 1 and 2 show that chelidonic acid (PubChem ID: 7431) was present in both top 10 metabolites.

Discussion
The richness of Indonesia's natural ingredients and their active compounds still needs to be revealed and explored, in connection with the discoveries of new natural-based drug candidates. This untargeted metabolomic profiling study was conducted to clarify the content of compounds contained in pinang yaki (Areca vestiaria) and to see its potential as a Covid-19 and antiviral herbal remedy by literature study. But of course, this study is a basic study that does not necessarily represent efficacy in animals (preclinical studies) and clinical trials in humans. However, the content of compounds that have been successfully identified in this study can be used as a basic reference in determining the dose of trials in animals (preclinical study).
Tables 1 and 2 show that chelidonic acid (PubChem ID: 7431) is one of metabolites identified to ionize well using either ESI technique. A study conducted by Shin et al. (2011), showed the inhibitory effects of chelidonic acid on IL-6 production by blocking NF-κB and caspase-1 in HMC-1 cells that can be a potential therapy for inflammatory diseases, including Covid-19 (Shin et al., 2011;Zhang et al., 2020). In addition, Tagatose (PubChem ID: 439312) which occupies the highest peak of ESIÀ mode (Figure 2), indicates that its presence in pinang Yaki (Areca vestiaria) is also quite high. Studies have shown that it can be a functional antidiabetic food for diabetes, which according to meta-analysis, is comorbid for Covid-19 patients (Guerrero-Wyss et al., 2018;Kun'ain et al., 2020). Alpha-chlorohydrin, also topped the highest position in ESI+ mode (Figure 1), which has a potential immunostaining effect in people with declining viral infections such as SARS Cov-2 (Soliman et al., 2014). However, there are negative effects of the use of Alphachlorohydrin on epididymis rats, therefore a comprehensive follow-up study is needed to look at the beneficial effects as well as their toxicity (Soliman et al., 2014).
In addition, choline (PubChem ID: 305) which is the metabolite from pinang yaki extract (ESI+) has also been widely researched for its effects in preventing SARS Cov-2 Infection and has good neuroprotective immunity (Chowdhury & Pathak., 2020). Other contents in pinang yaki (Areca vestiaria) such as Benzothiazole ( The above compounds have been identified as contained in pinang yaki extract, which shows its potential as an antiviral herbal supplement product. Of course, it needs further study, researchers will conduct an in vitro, in vivo or preclinical study to find out the effect of pinang yaki extract as an antiviral herbal supplement (based on the results of the compounds or metabolomic profiling in this paper). In addition, with reference to the metabolite data from this study, it is highly recommended that further studies be carried out in the future, namely the abundance of these metabolites in the sample and molecular mechanisms against several viral infection receptors through computational studies or in silico molecular docking with molecular dynamics simulations.

Conclusion
This untargeted metabolomic profiling study shows many bioactive compounds are contained in pinang yaki (Areca vestiaria) extract. The top 10 compounds which can ionize well and their potential benefits as antiviral supplement products have been identified and explored by literature study. The study of in vitro, preclinical, and clinical trials of Pinang yaki metabolites with biological mechanism is urgently needed. Open Peer Review I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes