Metabolites of Pinang Yaki (Areca vestiaria) Fruit Extract: A Metabolite Profiling Study

Background: Pinang yaki has bioactive compounds that have potential as a new herbal supplement. 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 and in particular its potential for the therapy and prevention of Covid-19. 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 UPLC-MS. Results: Based on an untargeted metabolomic profiling study of pinang yaki, 2504 compounds in ESI− and 2645 compounds in ESI+ were successfully obtained. After the analysis, 356 compounds in ESI− and 543 compounds in ESI+ were identified successfully. Major compounds Alpha-Chlorohydrin (PubChem ID: 7290) and Tagatose (PubChem ID: 439312) were found in ESI+ and ESI−. Discussion: The Top 10 metabolites from pinang yaki extract (ESI+) juga have been indicated in preventing SARS Cov2 infection and have exhibited good neuroprotective immunity. Benzothiazole (PubChem ID: 7222), L-isoleucine (PubChem ID: 6306), D-glucono-delta-lactone (PubChem ID: 736), Diethylpyrocarbonate (PubChem ID: 3051), Bis(2-Ethylhexyl) amine (PubChem ID: 7791), Cinnamic acid (PubChem ID: 444539), and Trigonelline (PubChem ID: 5570) also had potential effects as an antiviral, anti-inflammatory, and anti-Covid19. Conclusion: Untargeted metabolomic profiling showed many bioactive compounds contained in pinang yaki ( Areca vestiaria) extract. The top 10 compounds have been identified and explored for their potential benefits as anti-Covid19 supplement products. This is a preliminary study which still needs further research such as preclinical and clinical trials.


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.

Graphs shown in
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).
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.The manuscript by Simbala et al. explores the metabolites of Pinang yaki fruit extract for various biological activities.The authors hypothesise that identified compounds from Pinang yaki fruit extract has antiviral and neuroprotective potential which warrants further studies.The following issues need to be addressed by authors: 1-Certain names such as COVID-19, SARS-CoV-2, should be capitalized throughout the manuscript.
Terminologies like In silico etc should be italicized.Ensure the consistency in terms throughout the manuscript.

2-Introduction.
More relevant papers should be cited.Tannins and flavonoids are broader chemical class of compounds, isolated from Pinang yaki fruit.Please specify some of the names of these chemical compounds which have been identified in prior studies.Similarly in the next line, certain biological activities of saponins have included.It would be better to include the name of saponin.
3-Methods: How was plant material cutting, pulverizing or drying done?.Need to include details in methods.
Please state if triplicates were done for UHPLC-Ms studies.

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

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Introduction: The authors just mentioned the classes of metabolites in pinang yaki fruit, such as flavonoids, tannins, saponins, etc.As these classes of metabolites are quite common in plants, the statement did not give much information.Are there any known specific bioactive metabolites in pinang yaki fruit?If so, this should be mentioned.
Also, are there any known properties or benefits of pinang yaki fruit or extract at all?

2.
The authors mentioned the use of UPLC-MS, but from the name of the instrument given, it's more like UHPLC system, rather than UPLC.

3.
How exactly were the botanical identification and authentication carried out? 4.
How many replicates of samples were carried out?I looked up the authors' raw data on the Excel spreadsheet, and the headings of the two columns are BH and PY.It's unclear what these abbreviations stand for.Are these the two replicates of the sample?In any case, more replicates are likely required to make sure that what the authors observed are not just some contaminants that got picked up by LC-MS.

5.
Injection volume for LC-MS analysis should be specified.6.
The details of the data analysis method are needed.In addition, as metabolite identification is the main content of this report, it should be discussed in detail and should be done rigorously.How exactly were metabolites identified, i.e. by accurate mass, MS/MS, the use of standard, or the combination thereof?With the identification of tagatose, for example, how can you know it's not glucose or other sugars?From the structure, it should ionize better in the positive ion mode than in the negative ion mode as well, so it's a surprise that the authors did not observe a higher peak of tagatose in the positive ion mode.

7.
Do the numbers of compounds detected in the positive and negative ion modes include isotopes and various adducts?How could you distinguish these from real compounds?8.
It's unclear how metabolites are ranked.Is it by peak area?However, the peak area in ESI-MS does not correlate with the abundance of metabolites, as different compounds have differing abilities to ionize in different modes.Thus, the levels of these different metabolites cannot be compared simply by peak areas.In addition, the statement that chelidonic acid could be found in the top 10 metabolites lists in both modes just means that it possesses the structure that allows it to ionize very well in both modes.It does not have any significance other than this.

9.
If the authors want to really investigate the abundance of these metabolites in the sample, they will have to acquire the standard for each compound, and compare its abundance to the constructed standard curve.

Are sufficient details of methods and analysis provided to allow replication by others? No
If applicable, is the statistical analysis and its interpretation appropriate?Zanariah Hashim School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, Skudai, Malaysia Please check the grammar.Specific names require capitalization (for example, department of pharmacology, faculty of mathematics and natural sciences, should be changed to Department of Pharmacology, Faculty of Mathematics and Natural Sciences), whereas compound names do not need to be capitalized (e.g.Chelidonic Acid --> chelidonic acid).

1.
Which part of the plant was used as the extract?How old is the plant?Include more specifically where the plant comes from (not just some forests in North Sulawesi).Additionally, the authors need to explain the sample preparation procedure more clearly (including cutting, washing, sorting, etc.) 2.
Explain how the compound identification was performed.This is the most vital part of this work.
What does "quantity" in Table 1 and Table 2 refer to?Is it peak area or peak height?Higher peak area/height does not necessarily mean that the compound is more abundant in the sample.It simply means the compound is easier (more sensitive) to be detected using the method.To confirm the abundance/concentration of the compound, the authors need to analyze standard compounds and draw calibration curves.

5.
This is a preliminary result of untargeted metabolomic analysis of pinang yaki extract (which part should be mentioned clearly).Although the discovered compounds may have the 6. potential to treat Covid-19, the discussion is based only on literature, without evidence from the authors themselves.As such, I think some parts are overly-discussed and the title is also misleading.Discussion can be improved by adding pathway analysis or other multivariate analysis.Reviewer Expertise: metabolomics, LC-MS, multivariate analysis I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Is
Please ensure the manuscript is proofread and edited as there is a significant number of grammatical errors throughout the manuscript.

2.
The abstract description is good, there is no need to rewrite to provide details.The most important result obtained from the study should be emphasized in this section.

3.
Conclusion: Please rewrite.It should not be the summary of the findings as this has been stated in the Results.The conclusion should be more concise and match the aim.

4.
It can be seen from the research, that this research gives us new insights into the world of science, especially on the problem of Covid-19.

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

Are sufficient details of methods and analysis provided to allow replication by others? 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
Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Health and Nutrition
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com 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.
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 Are sufficient details of methods and analysis provided to allow replication by others?Partly If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Partly Competing Interests: No competing interests were disclosed.
Figure 2. Total-Ion Current (TIC) was obtained from Pinang Yaki in ESIÀ mode.In ESIÀ, a total of 2504 compounds were obtained, and 356 compounds were successfully identified by name (see underlying) (Nurkolis