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Phytochemical Screening, Antibacterial, Antifungal, Antiviral, Cytotoxic, and Anti-Quorum-Sensing Properties of Teucrium polium L. Aerial Parts Methanolic Extract

Plants 2020, 9(11), 1418; https://doi.org/10.3390/plants9111418

by Mousa Alreshidi¹, Emira Noumi^1,2, Lamjed Bouslama³, Ozgur Ceylan⁴

, Vajid N. Veettil¹, Mohd Adnan¹

, Corina Danciu⁵, Salem Elkahoui^1,3, Riadh Badraoui^1,6,7

, Khalid A. Al-Motair⁸, Mitesh Patel⁹

, Vincenzo De Feo¹⁰

and Mejdi Snoussi^1,11,*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3: Anonymous

Plants 2020, 9(11), 1418; https://doi.org/10.3390/plants9111418

Submission received: 22 September 2020 / Revised: 14 October 2020 / Accepted: 19 October 2020 / Published: 23 October 2020

(This article belongs to the Special Issue Natural Compounds–Successful Solutions for the Medical Challenges of the 21st Century)

Round 1

Reviewer 1 Report

The novelty of the study is poor. It seem that Authors confirmed previously reported antibacterial and antiviral activity of T. polium. In lines 176-178 they stated: “In fact, our results are in agreement with previous studies which demonstrated that methanolic extract exhibited high antibacterial activity”.

Phytochemical part of investigation is poorly described and documented. Lack of chromatographic details, e.g. type of column, mobile phase composition, flow rate, temperature. Lack of details on identification of compounds (mass spectra and fragmentation patterns). Figure 1 is completely unclear.

Section 2.1. – is not clear. On what basis the % different classes of compounds was assessed. Moreover, the values for different classes are the same – is it correct? A lot of various compounds are listed in lines 80-82 but on chromatogram they are not presented.

Discussion is very chaotic and should be more focused on the subject of the study. It need strong reedition. Sometimes the information are not associated with the subject of the study, e.g. lines 149-158 (the optimization of extraction in context of yield is not the aim of the work) etc..

There are also some minor errors, e. g. line 265: ratio 1/4? (should be 1:10), unnecessary or lack of capital letters etc

The quality of Fig.1 and 2 is very poor.

I strongly agree with Authors that “Further analysis are needed to correlate between the obtained results with the phytochemical composition of organic extract (…).”

Author Response

Point-by-point response to Reviewer 1:

Dear Dr. (Reviewer 1): Thank you for your consideration of our manuscript. We found the comments helpful, and believe our revised manuscript represents a significant improvement over our initial submission.

Comments and Suggestions for Authors

1-The novelty of the study is poor. It seem that Authors confirmed previously reported antibacterial and antiviral activity of T. polium. In lines 176-178 they stated: “In fact, our results are in agreement with previous studies which demonstrated that methanolic extract exhibited high antibacterial activity”.

Dear colleague, Special thanks for this valuable comment. We modified this sentence and novelty of the present work is added in order to highlight the importance of our work and the main new findings added in the same topic using Teucrium polium plant species. In fact different techniques were used to characterize the active molecules from T. polium methanolic extract. We report in this paper the use of High resolution-Liquid chromatography coupled to mass spectrometry (HR-LCMS). Many compounds are described for the first time in T. polium methanolic extract. The antiviral activity was tested for the first time also against two viruses: CVB3 and HSV-2. Few studies reported the anti-quorum sensing activity of methanolic extract from Teucrium species.

For this purpose, the main objectives of the present investigation were to assess for the first time the phytochemical profile of T. polium methanolic extract from Hail region (Saudi Arabia) using High resolution-Liquid chromatography coupled to mass spectrometry (HR-LCMS). The antimicrobial activities of germander extract against a wide range and types of clinical bacterial and fungal isolates was also tested. The antiviral activity was tested for the first time against CVB3 and HSV-2. Inhibition of the production of some virulence factors in C. violaceum and P. aeruginosa PAO1 reporter strains was also investigated for the first time in the present work.

2-Phytochemical part of investigation is poorly described and documented. Lack of chromatographic details, e.g. type of column, mobile phase composition, flow rate, temperature. Lack of details on identification of compounds (mass spectra and fragmentation patterns). Figure 1 is completely unclear.

The HR-LCMS analysis of T. polium was analyzed by using UHPLC-PDA-Detector Mass spectrometer (HR-LCMS 1290 Infinity UHPLC System, 1260 Infinity Nano HPLC with Chipcube, 6550 iFunnel QTOFs), Agilent Technologies, USA. For chromatographic separation, an Agilent 1200 Series HPLC system (Agilent Technologies, USA) equipped with a binary gradient solvent pump, HiP Sampler, column oven and MS Q-TOF with Dual AJS ES Ion Source. Samples were separated on SB-C18 column (2.1 × 50 mm, 1.8-particle size; Agilent Technologies, USA) maintained at 25°C. The solvents used were: water containing 0.1% HCOOH and Acetonitrile containing 10% water and 0.1% HCOOH. The following gradient elution program at a flow rate of 0.3 mL min−1 was applied. MS detection was performed in MS Q-TOF Mass spectrometer (Agilent Technologies). Compounds were identified via their mass spectra and their unique mass fragmentation patterns. Compound Discoverer 2.1, ChemSpider and PubChem were used as the main tools for the identification of the phytochemical constituents.

All information regarding the phytochemical study of T. polium methanolic extract was added in details in Introduction and results and discussion sections.

Introduction part: Different techniques were used to identify the bioactive compounds in T. polium aerial parts extracted using different solvent (water, methanol, 65.5%- menthol-water, dichloromethane/methanol, acetone, ethanol 70%, and ethanol 95%) from different regions around the word [35-44]. A literature review of the phytochemical compounds isolated from T. polium aerial parts showed the identification of more than 130 molecules dominated by terpenoids (60%) [45]. The main identified compounds from T. polium roots, aerial parts and inflorescence are apigenin, luteolin, rutin, cirsiliol, cirsimaritin, salvigenin and eupatorin [45]. Five flavonoids were widely isolated from different Teucrium species from European countries namely: cirsiliol, cirsimaritin, cirsilineol, salvigenin and 5-hydroxy-6,7,3’,4’-tetramethoxyllavone [44]. Four new sesquiterpenes were identified in dichloromethane/methanol extract from T. polium, namely 4β,5α-epoxy-7αH-germacr-10(14)-en-6β-ol-1-one, 4β,5α-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol, 4β,5β-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol and 4α,5β-epoxy-7αH-germacr-10(14)-e [39].

Results parts: We added a table summarizing the list of compounds identified in

Our Results showed the identification of 29 molecules in T. polium methanolic extract by using HR-LCMS technique. The complete list of the identified compounds together with their respective chemical class are listed in table 1.

Table 2. Bioactive compounds and their chemical class identified in T. polium methanolic extract using HR-LCMS technique.

RT	Identified Compound Name	Chemical class
9.262	Larixol Acetate	Bicyclic labdane diterpenes
5.948	Cepharanthine	Alkaloid
1.062	Bis (2-hydroxypropyl) amine	Amino Alcohol
1.447	9-amino-nonanoic acid	Amino Fatty Acid
3.807	10-Amino-decanoic acid	Amino Fatty Acid
6.329	CMP-N-acetylneuraminic acid	Amino Sugar
8.848	Selinidin	Coumarin Derivative
10.779	Dihydrosamidin	Coumarins
9.807	Triptonide	Diterpene triepoxide
11.022	10S,11R-Epoxy-punaglandin 4	Eicosanoid
6.376	Rhoifolin	Flavonoid
13.274	3-hydroxy-3',4'- Dimethoxyflavone	Flavonoid
7.838	Sericetin diacetate	Flavonol
5.96	Troxerutin	Flavonol
20.37	1-dodecanoyl-sn-glycerol	Glycerolipid
9.1	Harpagoside	Iridoid Glycoside
10.036	Koparin 2'-Methyl Ether	Isoflavonoid
12.149	Isotectorigenin, 7- Methyl ether	Isoflavonoid
0.945	10-Hydroxyloganin	Isoprenoid
4.739	7-Epiloganin tetraacetate	Isoprenoid
6.319	Deoxyloganin tetraacetate	Isoprenoid
9.126	8-Epiiridodial glucoside tetraacetate	Isoprenoid
11.28	16-Hydroxy-4-carboxyretinoic Acid	Isoprenoid
8.198	Carapin-8 (9)-Ene	Limonoid
18.427	Khayanthone	Limonoid
1.046	13R-hydroxy-9E,11Z octadecadienoic acid	Octadecanoid
5.342	b-D-Glucopyranoside uronic acid, 6-(3-oxobutyl)-2- naphthalenyl	Organic Acid, Phenol
11.279	16alpha,17beta-Estriol 3-(beta-D-glucuronide)	Steroidal glycosides
9.525	Valtratum	Terpene

In fat, T. polium methanolic extract was dominated by terpenes and their derivatives, fatty acids and their derivatives, polyphenols, flavonoids and derivatives, coumarines, amino acid derivatives, and alkaloids. The tested extract was dominated by 3R-hydroxy-9E,11Z-octadecadienoic acid, valtratum, dihydrosamidin, and cepharanthine. The chromatograms (figure 2) showed respectively major peaks indicating the presence of various phytochemical constituents.

Discussion section: A table summarizing different molecules isolated from Teucrium extracts are reported, especially from methanolic one.

Table 4 summarize some important bioactive molecules isolated from Teucrium species, mainly T. polium using various identification techniques and different extraction procedures [35-44].

Table 4. Literature review of the main identified bioactive compounds in Teucrium species and the solvent used.

*Teucrium* species /Origin	Solvent Used	Main identified compounds	References
T. polium L. Serbia	Methanol	Phenolic acids (×10^–4 µg/mL extract µg/mL extract): gallic acid (8.1±0.2), vanillic acid (2.1±0.3), caffeic acid (1.7 ± 0.2), chlorogenic acid (90.00±0.2), and p-coumaric acid (30.0±0.6). Flavonoids (×10^–4 µg/mL extract µg/mL extract): catechin (235.0±0.5), rutin (77.0±0.1), myricetin (55.0±0.1), luteolin (22.0±0.6), quercetin (24.0±0.4), and apigenin (157.0 ± 0.3).	[35]
T. scordium L. Serbia	Methanol	Phenolic acids (×10^–4 µg/mL extract µg/mL extract): vanillic acid (501.1 ± 0.6), p-coumaric acid (111.5 ± 0.1), and sinapinic acid (26.9 ± 0.2). Flavonoids (×10^–4 µg/mL extract µg/mL extract): catechin (32.5 ± 0.6) and rutin (24.7±0.1).	[35]
T. polium L. Greece	Methanol/Water 62.5%	Phenolic compounds (mg/100g dry sample): tyrosol (0.42 ± 0.01), caffeic acid (0.65 ± 0.01), ferulic acid (0.95 ± 0.02), and luteolin (0.48 ± 0.01)	[36]
T. polium L. Greece	Methanol/Water 62.5%	o-Hydroxybenzoic acid, hydroxytyrosol, p-Hydroxybenzoic acid, vanillic acid, gentisic acid, ferulic acid, caffeic acid, 3-Nitro-phthalic acid , and quercetin.	[36]
T. polium L. Egypt	Dichloromethane–methanol (1:1; v/v)	(1R,4S,10R) 10,11-dimethyl-dicyclohex-5 (6)-en-1,4-diol-7-one and (R)-mandelonitrile-b-laminaribioside.	[37]
T. polium L Italy	Methanol	Poliumoside B, poliumoside, teucardosid, 8-O-acetylharpagid, luteolin7-O-rutinoside, luteolin7-O-neohesperidoside, luteolin7-O-glucosied, luteolin 4´-O-glucoside, teulamifin, and teusalvin C.	[38]
T. stocksianum Bioss. Italy	Water	Flavonoids, Saponins, Reducing sugars, Terpenoids, and Tannins.	[39]
T. polium L. R. Macedonia	Methanol	Hydroxycinnamic acid derivatives (2 compounds), phenylethanoid glycosides (12 compounds), flavonoid glycosides (11 compounds) and flavonoid aglycones (6 compounds).	[40]
T. marum Italy	Acetone or ethanol 95%	Teumarin 1 and teumarin B 2.	[41]
T. polium L. Italy	Methanol	Poliumoside, apigenin, luteolin, montanin D, montanin E, teubutilin A, teuchamecrin C, teulamifin B, teupolin VI, teupolin VII, teupolin VIII, teupolin IX, teupolin X, teupolin XI, and teupolin XII.	[42]
T. polium L. Iran	Methanol	Rutin, apigenin, (3´, 6 dimethoxy apigenin, and 4´ ,7 dimethoxy apigenin.	[43]
T. polium L. Europe	Ethanol 70%	Rutin, apigenin, apigenin-4, 7-dimethylether, cirsimaritin, cirsiliol, luteolin, 6-hydroxyluteolin, luteolin-7-O-glucoside, salvigenin, apigenin 7-glucoside, eupatorin, apigenin-5 galloylglucoside, 3′,6- dimethoxy apigenin, 4′,7- dimethoxy apigenin and salvigenin.	[44]
T. polium L. Egypt	Dichloromethane–methanol (1:1; v/v)	Sesquiterpenes: 4β,5α-epoxy-7αH-germacr-10(14)-en-6β-ol-1-one, 4β,5α-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol, 4β,5β-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol 4α,5β-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6α-ol, 10a,1b;4b,5a-diepoxy-7aH-germacrm-6-ol, teucladiol, 4b,6b-dihydroxy-1a,5b(H)-guai-9-ene, oplopanone, oxyphyllenodiol A, eudesm-3-ene-1,6-diol, rel1b,3a,6b-trihydroxyeudesm-4-ene, arteincultone. Flavonoids: 7,40 -O-dimethylscutellar-ein(5,6-dihydroxy-7,40 -dimethoxyflavone) and salvigenin. Glycosides: teucardoside and poliumoside.	[90]
T. polium L. Saudi Arabia	Methanol	13R-hydroxy-9E,11Z-octadecadienoic acid, rhoifolin, sericetin diacetate, selinidin, harpagoside, valtratum, triptonide, koparin 2'-methyl ether, dihydrosamidin, 10S,11R-epoxy-punaglandin, khayanthone, 4, 16alpha, 17beta-Estriol 3-(beta-D-glucuronide), 10-hydroxyloganin, 7-epiloganin tetraacetate, cepharanthine, deoxyloganin tetraacetate, carapin-8 (9)-Ene, and 1-dodecanoyl-sn-glycerol.	This Study

In the present work, we reported the identification of a new undescribed compounds in T. polium aerial parts methanolic extract using HR-LCMS technique: 13R-hydroxy-9E,11Z-octadecadienoic acid, rhoifolin, sericetin diacetate, selinidin, harpagoside, valtratum, triptonide, koparin 2'-methyl ether, dihydrosamidin, 10S,11R-epoxy-punaglandin, 4, 16alpha, 17beta-Estriol 3-(beta-D-glucuronide), khayanthone, 10-hydroxyloganin, 7-epiloganin tetraacetate, cepharanthine, deoxyloganin tetraacetate, carapin-8 (9)-Ene, and 1-dodecanoyl-sn-glycerol. In previous work, Pacifico et al. [42] reported the identification of poliumoside, apigenin, luteolin, montanin D, montanin E, teubutilin A, teuchamecrin C, teulamifin B, teupolin VI, teupolin VII, teupolin VIII, teupolin IX, teupolin X, teupolin XI, and teupolin XII from T. polium methanolic extract from Italy. Using the same solvent, De Marino et al. [40] identified poliumoside B, poliumoside, teucardosid, 8-O-acetylharpagid, luteolin7-O-rutinoside, luteolin 7-O-neohesperidoside, luteolin 7-O-glucosied, luteolin 4´-O-glucoside, teulamifin, and teusalvin C as main bioactive compounds from T. polium aerial parts from Italy. While, Sharififar et al. [43] identified 3´,6 dimethoxy apigenin, rutin, apigenin, and 4´,7 dimethoxy apigenin as the main bioactive molecules in T. polium methanolic extract from Iran.

Dear Dr., special thanks for the comment: In fact, we discussed the yield of extraction because we have used only methanol as solvent for the extraction procedure. We previously reported that this solvent possess the highest yield of extraction and the best biological activities. Many works described the use of ethanol as solvent for the bioactive compounds isolation from Teucrium species. So, we decided to compare our findings in comparison with previous reports as summarized in Table 4.

Here are also some minor errors, e. g. line 265: ratio 1/4? (should be 1:10), unnecessary or lack of capital letters etc

The whole manuscript was deeply revised in order to avoid spelling and grammatical errors.

The quality of Fig.1 and 2 is very poor.

The figure 1 was deleted and figure 2 was updated.

I strongly agree with Authors that “Further analysis are needed to correlate between the obtained results with the phytochemical composition of organic extract (…).”

Author Response File: Author Response.docx

Reviewer 2 Report

Manuscript title: Phytochemical screening, antibacterial, antifungal, antiviral, cytotoxic and anti-quorum sensing properties of Teucrium polium L. aerial parts methanolic extract.

Authors: Mousa Alreshidi , Emira Noumi , Lamjed Bouslama , Ozgur Ceylan , Vajid N. Veettil , Mohd Adnan , Corina Danciu , Salem Elkahoui , Riadh Badraoui , Khalid A. Al-Motair , Mitesh Patel , Vincenzo De Feo , Mejdi Snoussi

The manuscript is fairly well written and includes a great deal of information, which is reflected in the significant number of references listed.

Abbreviations should be defined the first time they appear in the abstract.

The full name of the plant should be mention in the abstract.

All the Latin names of the plants, viruses, bacteria, and fungi should be written in italic. Check the whole manuscript!

The keywords should be specific to the article but not the same as in the title.

23 position for the first paragraph of the introduction part? Isn't that an unnecessary multiplication of reference positions?

More details on the bioactive compounds found in the studied plants should be mentioned in the introduction part.

The phytochemical part of the result section should be more described.

Figure 3 The description of the Figure 3 should contain the name of the viability test used.

Table 1 The description of the Table 1 is confusing… the extract or essential oil was used?

Table 2 The description of the Table 2 is confusing… the extract or essential oil was used? Bactericidal or fungicidal? Against bacteria or against fungi?

The geographical position of the plant habitat as well as the voucher specimen should be added to the experimental/ methodology part.

What was the positive and negative control in the cytotoxic activity experiment?

References should be described as recommended by the journal.

Moderate English changes are required.

Author Response

Dear Dr. (Reviewer 2): Thank you for your consideration of our manuscript. We found the comments helpful, and believe our revised manuscript represents a significant improvement over our initial submission.

Comments and Suggestions for Authors

Manuscript title: Phytochemical screening, antibacterial, antifungal, antiviral, cytotoxic and anti-quorum sensing properties of Teucrium polium L. aerial parts methanolic extract.

The manuscript is fairly well written and includes a great deal of information, which is reflected in the significant number of references listed.

Abbreviations should be defined the first time they appear in the abstract.

All abbreviations were checked and were defined the first time they appear in the text.

The full name of the plant should be mention in the abstract.

The full name of the studied plant was added in the abstract: Teucrium polium L.

All the Latin names of the plants, viruses, bacteria, and fungi should be written in italic. Check the whole manuscript!

All Latin names for viruses, bacteria, and plants were checked and rewritten in italic in whole manuscript.

The keywords should be specific to the article but not the same as in the title.

Keywords were modified as suggested. Teucrium polium L., aerial parts; bioactive compounds; HR-LCMS; biological activities.

23 position for the first paragraph of the introduction part? Isn't that an unnecessary multiplication of reference positions?

Dear Colleague, special thanks for your comment.

In fact, we used new references to describe the interest of natural compounds from plants in different countries around the word.

For this reason, we cited six references in the first three lines.

Aromatic and medicinal plants history is associated with the evolution of civilizations and occupied a crucial place in medicine, cosmetology and culinary preparations in China, India, the Middle East, Egypt, Saudi Arabia and Greece [1-6].

More details on the bioactive compounds found in the studied plants should be mentioned in the introduction part.

More details were added in the new version of the manuscript. All these modifications are highlighted in yellow in the text.

Different techniques were used to identify the bioactive compounds in T. polium aerial parts extracted using different solvent (water, methanol, 65.5%- menthol-water, dichloromethane/methanol, acetone, ethanol 70%, and ethanol 95%) from different regions around the word [35-44]. A literature review of the phytochemical compounds isolated from T. polium aerial parts showed the identification of more than 130 molecules dominated by terpenoids (60%) [45]. The main identified compounds from T. polium roots, aerial parts and inflorescence are apigenin, luteolin, rutin, cirsiliol, cirsimaritin, salvigenin and eupatorin [45]. Five flavonoids were widely isolated from different Teucrium species from European countries namely: cirsiliol, cirsimaritin, cirsilineol, salvigenin and 5-hydroxy-6,7,3’,4’-tetramethoxyllavone [44]. Four new sesquiterpenes were identified in dichloromethane/methanol extract from T. polium, namely 4β,5α-epoxy-7αH-germacr-10(14)-en-6β-ol-1-one, 4β,5α-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol, 4β,5β-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol and 4α,5β-epoxy-7αH-germacr-10(14)-e [39].

The phytochemical part of the result section should be more described.

More details were added in the new version of the manuscript. All these modifications are highlighted in yellow in the text.

In a systematic review, Harborne et al. [44] reported the distribution of free flavone aglycones and flaoonoid glycosides from 42 European taxa of the genus Teucrium (75% ethanol extract from aerial parts). These authors reported the identification of 5,7-Dihydroxytkvones, 6-hydroxyBavones; 6-methoxytIavoncs; flavonols; 8-hydroxyflavones; cirsiliol; cirsimaritin; citsilineol; 5-hydroxy-6,7,3’,4’-tetramethoxyllavone; salvigenin; luteolin; apigenin; diosmetiq, hydroxyluteolin; cirsimariti, quercetin; isorhamnetin; hypolaetin; isoscutellarein, vicenin-2; 5,7dihydroxytlavone glycosides; 6-hydroxytIavone glycosides; I-hydroxyflavone glycoside, 6-methoxyflavone glycosides; tlavonol glycoside, apigenin-7-glucoside, apigenin 7-rutinoside; luteolin 7-glucoside; lutcolin 7-rutinoside; luteolin 7-sambubiosidc; diosmetin 7-rutinosidc; 6-OH-luteolin 7-glucoside; 6-OH-luteolin 7-rhamnoside; isoscutellarein 7-allosylglucoside, hypolaetin 7-allosylglucoside; cirsimaritin 4´-glucoside; quercetin 3-glucoside; quercetin 3-rutinoside; isorhanmetin 3-glucoside; and isorhamnetin 3-rutinoside.

Table 4 summarize some important bioactive molecules isolated from Teucrium species, mainly T. polium using various identification techniques and different extraction procedures [35-44].

Table 4. Literature review of the main identified bioactive compounds in Teucrium species and the solvent used.

*Teucrium* species /Origin	Solvent Used	Main identified compounds	References
T. polium L. Serbia	Methanol	Phenolic acids (×10^–4 µg/mL extract µg/mL extract): gallic acid (8.1±0.2), vanillic acid (2.1±0.3), caffeic acid (1.7 ± 0.2), chlorogenic acid (90.00±0.2), and p-coumaric acid (30.0±0.6). Flavonoids (×10^–4 µg/mL extract µg/mL extract): catechin (235.0±0.5), rutin (77.0±0.1), myricetin (55.0±0.1), luteolin (22.0±0.6), quercetin (24.0±0.4), and apigenin (157.0 ± 0.3).	[35]
T. scordium L. Serbia	Methanol	Phenolic acids (×10^–4 µg/mL extract µg/mL extract): vanillic acid (501.1 ± 0.6), p-coumaric acid (111.5 ± 0.1), and sinapinic acid (26.9 ± 0.2). Flavonoids (×10^–4 µg/mL extract µg/mL extract): catechin (32.5 ± 0.6) and rutin (24.7±0.1).	[35]
T. polium L. Greece	Methanol/Water 62.5%	Phenolic compounds (mg/100g dry sample): tyrosol (0.42 ± 0.01), caffeic acid (0.65 ± 0.01), ferulic acid (0.95 ± 0.02), and luteolin (0.48 ± 0.01)	[36]
T. polium L. Greece	Methanol/Water 62.5%	o-Hydroxybenzoic acid, hydroxytyrosol, p-Hydroxybenzoic acid, vanillic acid, gentisic acid, ferulic acid, caffeic acid, 3-Nitro-phthalic acid , and quercetin.	[36]
T. polium L. Egypt	Dichloromethane–methanol (1:1; v/v)	(1R,4S,10R) 10,11-dimethyl-dicyclohex-5 (6)-en-1,4-diol-7-one and (R)-mandelonitrile-b-laminaribioside.	[37]
T. polium L Italy	Methanol	Poliumoside B, poliumoside, teucardosid, 8-O-acetylharpagid, luteolin7-O-rutinoside, luteolin7-O-neohesperidoside, luteolin7-O-glucosied, luteolin 4´-O-glucoside, teulamifin, and teusalvin C.	[38]
T. stocksianum Bioss. Italy	Water	Flavonoids, Saponins, Reducing sugars, Terpenoids, and Tannins.	[39]
T. polium L. R. Macedonia	Methanol	Hydroxycinnamic acid derivatives (2 compounds), phenylethanoid glycosides (12 compounds), flavonoid glycosides (11 compounds) and flavonoid aglycones (6 compounds).	[40]
T. marum Italy	Acetone or ethanol 95%	Teumarin 1 and teumarin B 2.	[41]
T. polium L. Italy	Methanol	Poliumoside, apigenin, luteolin, montanin D, montanin E, teubutilin A, teuchamecrin C, teulamifin B, teupolin VI, teupolin VII, teupolin VIII, teupolin IX, teupolin X, teupolin XI, and teupolin XII.	[42]
T. polium L. Iran	Methanol	Rutin, apigenin, (3´, 6 dimethoxy apigenin, and 4´ ,7 dimethoxy apigenin.	[43]
T. polium L. Europe	Ethanol 70%	Rutin, apigenin, apigenin-4, 7-dimethylether, cirsimaritin, cirsiliol, luteolin, 6-hydroxyluteolin, luteolin-7-O-glucoside, salvigenin, apigenin 7-glucoside, eupatorin, apigenin-5 galloylglucoside, 3′,6- dimethoxy apigenin, 4′,7- dimethoxy apigenin and salvigenin.	[44]
T. polium L. Egypt	Dichloromethane–methanol (1:1; v/v)	Sesquiterpenes: 4β,5α-epoxy-7αH-germacr-10(14)-en-6β-ol-1-one, 4β,5α-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol, 4β,5β-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol 4α,5β-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6α-ol, 10a,1b;4b,5a-diepoxy-7aH-germacrm-6-ol, teucladiol, 4b,6b-dihydroxy-1a,5b(H)-guai-9-ene, oplopanone, oxyphyllenodiol A, eudesm-3-ene-1,6-diol, rel1b,3a,6b-trihydroxyeudesm-4-ene, arteincultone. Flavonoids: 7,40 -O-dimethylscutellar-ein(5,6-dihydroxy-7,40 -dimethoxyflavone) and salvigenin. Glycosides: teucardoside and poliumoside.	[90]
T. polium L. Saudi Arabia	Methanol	13R-hydroxy-9E,11Z-octadecadienoic acid, rhoifolin, sericetin diacetate, selinidin, harpagoside, valtratum, triptonide, koparin 2'-methyl ether, dihydrosamidin, 10S,11R-epoxy-punaglandin, khayanthone, 4, 16alpha, 17beta-Estriol 3-(beta-D-glucuronide), 10-hydroxyloganin, 7-epiloganin tetraacetate, cepharanthine, deoxyloganin tetraacetate, carapin-8 (9)-Ene, and 1-dodecanoyl-sn-glycerol.	This Study

Figure 3 The description of the Figure 3 should contain the name of the viability test used.

The name of the viability test used was added.

Table 1 The description of the Table 1 is confusing… the extract or essential oil was used?

*Inhibition zone around the discs impregnated with the methanolic extract (10 µl/disk) expressed as mean of three replicates (mm ± SD). SD: standard deviation. a: Minimal Inhibitory Concentration. b: Minimal Bactericidal Concentration. The letters (a–e) indicate a significant difference between the inhibition zones of the sample according to the Duncan test (p < 0.05).

Table 2 The description of the Table 2 is confusing… the extract or essential oil was used? Bactericidal or fungicidal? Against bacteria or against fungi?

*Inhibition zone around the discs impregnated with the methanolic extract (10 µl/disk) expressed as mean of three replicates (mm ± SD). SD: standard deviation. a: Minimal Inhibitory Concentration. b: Minimal Fungicidal Concentration. The letters (a–e) indicate a significant difference between the inhibition zones according to the Duncan test (p < 0.05).

The geographical position of the plant habitat as well as the voucher specimen should be added to the experimental/ methodology part.

The plant material was collected in October 2019 from a nursery belonging to the Ministry of Agriculture in Hail region, Saudi Arabia. A voucher specimen (AN 01) was deposited in the Department of Biology (College of Science, University of Hail, KSA).

What was the positive and negative control in the cytotoxic activity experiment?

Cell culture without extract was used as negative control. The positive control is not required for the cytotoxicity assay.

The cytotoxicity of the T. polium extract was evaluated on VERO (African green monkey kidney) cells line. 10⁴ Vero cells per well were seeded in 96-well plates and grown in Eagle’s minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS), 100 units/ml of penicillin, 100 g/mL of streptomycin, 0.25 g/mL of Amphotericin B at 37◦C under 5% CO₂ atmosphere. After 24 h incubation, confluent monolayers of Vero cells were exposed to decreasing concentration of the extract (from 3333 µg/mL to 1 g/mL) diluted in MEM supplemented with 2% FBS. After 48 h incubation, cell viability was observed in an inverted microscope. The 50% cytotoxic concentration (CC₅₀), defined as the concentration of the extract able to reduce of 50% the cell viability, was determined by regression analysis in comparison to negative control.

References should be described as recommended by the journal.

All references were checked and updated according to the journal guidelines.

Moderate English changes are required.

The whole manuscript was revised in order to avoid spelling and grammatical errors.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript described the antimicrobial, antiviral and citotoxic activity of Teucrium polium L. aerial parts. Many studies have already studied this effect so the authors should underline the novelty of this work, especially in the discussion section, In fact the authors mentioned several previous studies but the comparison withobtained results should be better explained in the relative section. Moreover, the choice of extraction solvent is not clear: previous studies reported a higher yield with ethanol, please justify the use of methanol. On the other hand, about the extraction procedure, time and temperature should be indicated as well as in other experiments like cytoxic and antibacterial activity. The method used to compound identification is missing. The abbreviations should be explain when mentioned the first time in the paper. The use of VERO cell lines should be motivated. Results of cytotoxic effect should be better discussed and possibly correlated with other activities.

Author Response

Dear Dr. (Reviewer 3): Thank you for your consideration of our manuscript. We found the comments helpful, and believe our revised manuscript represents a significant improvement over our initial submission.

The manuscript described the antimicrobial, antiviral and cytotoxic activity of Teucrium polium L. aerial parts.

Many studies have already studied this effect so the authors should underline the novelty of this work, especially in the discussion section, In fact the authors mentioned several previous studies but the comparison with obtained results should be better explained in the relative section.

More details were added in the new version of the manuscript in the Itroduction part. All these modifications are highlighted in yellow in the text.

More details were added in the new version of the manuscript in the Discussion part. All these modifications are highlighted in yellow in the text.

Moreover, the choice of extraction solvent is not clear: previous studies reported a higher yield with ethanol, please justify the use of methanol.

Dear Colleague, Yes I agree that many reports have tested ethanol as solvent (extraction). But there is also many reports describing the use of methanol for the extraction of bioactive molecules from Teucrium polium aerial parts.

Table 4 summarize some important bioactive molecules isolated from Teucrium species, mainly T. polium using various identification techniques and different extraction procedures [35-44].

Table 4. Literature review of the main identified bioactive compounds in Teucrium species and the solvent used.

*Teucrium* species/ Origin	Solvent Used	Main identified compounds	Reference
T. polium L. Serbia	Methanol	Phenolic acids (×10^–4 µg/mL extract µg/mL extract): gallic acid (8.1±0.2), vanillic acid (2.1±0.3), caffeic acid (1.7 ± 0.2), chlorogenic acid (90.00±0.2), and p-coumaric acid (30.0±0.6). Flavonoids (×10^–4 µg/mL extract µg/mL extract): catechin (235.0±0.5), rutin (77.0±0.1), myricetin (55.0±0.1), luteolin (22.0±0.6), quercetin (24.0±0.4), and apigenin (157.0 ± 0.3).	35
T. scordium L. Serbia	Methanol	Phenolic acids (×10^–4 µg/mL extract µg/mL extract): vanillic acid (501.1 ± 0.6), p-coumaric acid (111.5 ± 0.1), and sinapinic acid (26.9 ± 0.2). Flavonoids (×10^–4 µg/mL extract µg/mL extract): catechin (32.5 ± 0.6) and rutin (24.7±0.1).	35
T. polium L. Greece	Methanol/Water 62.5%	Phenolic compounds (mg/100g dry sample): tyrosol (0.42 ± 0.01), caffeic acid (0.65 ± 0.01), ferulic acid (0.95 ± 0.02), and luteolin (0.48 ± 0.01)	36
T. polium L. Greece	Methanol/Water 62.5%	o-Hydroxybenzoic acid, hydroxytyrosol, p-Hydroxybenzoic acid, vanillic acid, gentisic acid, ferulic acid, caffeic acid, 3-Nitro-phthalic acid , and quercetin.	36
T. polium L. Egypt	Dichloromethane–methanol (1:1; v/v)	(1R,4S,10R) 10,11-dimethyl-dicyclohex-5 (6)-en-1,4-diol-7-one and (R)-mandelonitrile-b-laminaribioside.	37
T. polium L Italy	Methanol	Poliumoside B, poliumoside, teucardosid, 8-O-acetylharpagid, luteolin7-O-rutinoside, luteolin7-O-neohesperidoside, luteolin7-O-glucosied, luteolin 4´-O-glucoside, teulamifin, and teusalvin C.	38
T. stocksianum Bioss. Italy	Water	Flavonoids, Saponins, Reducing sugars, Terpenoids, and Tannins.	39
T. polium L. R. Macedonia	Methanol	Hydroxycinnamic acid derivatives (2 compounds), phenylethanoid glycosides (12 compounds), flavonoid glycosides (11 compounds) and flavonoid aglycones (6 compounds).	40
T. marum Italy	Acetone or ethanol 95%	Teumarin 1 and teumarin B 2.	41
T. polium L. Italy	Methanol	Poliumoside, apigenin, luteolin, montanin D, montanin E, teubutilin A, teuchamecrin C, teulamifin B, teupolin VI, teupolin VII, teupolin VIII, teupolin IX, teupolin X, teupolin XI, and teupolin XII.	42
T. polium L. Iran	Methanol	Rutin, apigenin, (3´, 6 dimethoxy apigenin ), and 4´ ,7 dimethoxy apigenin.	43
T. polium L. Egypt	Dichloromethane–methanol (1:1; v/v)	Sesquiterpenes: 4β,5α-epoxy-7αH-germacr-10(14)-en-6β-ol-1-one, 4β,5α-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol, 4β,5β-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6β-ol 4α,5β-epoxy-7αH-germacr-10(14)-en,1β-hydroperoxyl,6α-ol, 10a,1b;4b,5a-diepoxy-7aH-germacrm-6-ol, teucladiol, 4b,6b-dihydroxy-1a,5b(H)-guai-9-ene, oplopanone, oxyphyllenodiol A, eudesm-3-ene-1,6-diol, rel1b,3a,6b-trihydroxyeudesm-4-ene, arteincultone. Flavonoids: 7,40 -O-dimethylscutellar-ein(5,6-dihydroxy-7,40 -dimethoxyflavone) and salvigenin. Glycosides: teucardoside and poliumoside.	44
T. polium L. Europe	Ethanol 70%	Rutin, apigenin, apigenin-4, 7-dimethylether, cirsimaritin, cirsiliol, luteolin, 6-hydroxyluteolin, luteolin-7-O-glucoside, salvigenin, apigenin 7-glucoside, eupatorin, apigenin-5 galloylglucoside, 3′,6- dimethoxy apigenin, 4′,7- dimethoxy apigenin and salvigenin.	45

On the other hand, about the extraction procedure, time and temperature should be indicated as well as in other experiments like cytotoxic and antibacterial activity.

The methods used were described in details.

Extraction procedure

The fresh aerial flowering parts were dried at room temperature for seven to ten days. For the bioactive compounds extraction, 4g of the plant powder material were macerated in 40 mL absolute methanol (ratio: 1:10, w/v). Methanolic extracts were pooled, filtered, and the solvent was removed at 60◦C in the incubator chamber.

Evaluation of the cytotoxicity

The cytotoxicity of the T. polium extract was evaluated on VERO (African green monkey kidney) cell lines using the MTT colorimetric method. A decreasing concentration of the extract (from 3333 µg/mL to 1 µg/mL) diluted in MEM supplemented with 2%-FBS was applied on VERO cell in a 96-well plate. Cell controls were incubated under the same conditions without extracts. After 72 h of incubation at 37°C, the extract dilutions were substituted with the MTT solution (5 mg/mL), incubated for 2 h at 37°C, and then dissolved by the dimethyl sulfoxide (DMSO). The plate was read on an ELISA reader at a 570 nm wavelength to measure the optical density. The 50% cytotoxic concentration (CC50) was determined by regression analysis in comparison to negative control [83].

Antibacterial and antifungal activity using disc diffusion assay

The disc diffusion assay was performed on Mueller-Hinton agar plates for all bacteria, Sabouraud chloramphenicol agar for yeasts, and Potato Dextrose agar for Aspergillus strains. 10 μL of methanolic extract mother solution (300 mg/ml) was used to impregnate 6 mm-sterile discs. The experiment was done in triplicate. All Petri dishes were incubated overnight at 37°C and the diameter of growth inhibition zone was recorded using 1 cm- flat ruler. The mean diameter was recorded and expressed as (GIZ mm±SD). Results obtained are interpreted using the scheme proposed by Parveen et al. [95]: no activity (GIZ 0), low activity (GIZ:1–6 mm), moderate activity (GIZ :7–10 mm), high activity (GIZ:11–15 mm), and very high activity (GIZ:16–20 mm). Ampicillin and Amphotericin B were used as control.

4.4.3. MICs and MBCs/MFCs determination using the microdilution assay

MICs and MBCs/MFCs values were determined by a microtitre broth dilution method as previously described by Snoussi et al. [96]. The test medium was Mueller–Hinton Broth (MHB) for bacterial strains, and Sabouraud dextrose broth for fungal isolates. Bacterial/fungal suspensions (100 μL) were inoculated into the wells of 96-well microtitre plates in the presence of samples with different final concentrations (ranging from 0.039 mg/ml to 100 mg/ml). The inoculated microplates were incubated at 37°C for 24 h. The lowest concentration of T. polium methanolic extract, which did not show any visual growth of tested organisms was recorded as MIC value (expressed in mg/mL). The MBCs/MFCs values are determined by streaking all wells after the MICs values on the correspondent agar media of the tested microorganisms. MBC/MIC ratio and MFC/MIC ratio were used to interpret the activity of the essential oil as described by Gatsing et al., [97].

Antiviral activity

The antiviral activity of T. polium methanolic extract was evaluated on Coxsakievirus B-3 (CVB3) and Herpes Simplex Virus type 2 (HSV-2). The viruses were propagated on Vero cells in MEM medium supplemented with 2% FBS at 37◦C under humidified 5% CO2 atmosphere. The infectious titer of the stock solution was 2x107 TCID50/mL (50% tissue culture infectious doses/mL) for CVB-3 and 8x105 PFU/mL (plaque formation units/mL, PFU) for HSV-2. A MOI (multiplicity of infection) of 0.01 for CVB-3 and 0.1 for HSV-2 was seeded into cells monolayers cultivated in 96-well culture plates (2x104 cells/wells), with different concentrations of the extract starting from the CC50 value. Plates without virus or extract were used as negative and positive controls, respectively. After 48 h incubation, the inhibition of the cytopathic effect (CPE) for CVB-3 and the plaque virus for HSV-2 was observed in an inverted microscope [94].

The method used to compound identification is missing.
Phytochemical analysis of polium extract.

The HR-LCMS analysis of T. polium was analyzed by using UHPLC-PDA-Detector Mass spectrometer (HR-LCMS 1290 Infinity UHPLC System, 1260 Infinity Nano HPLC with Chipcube, 6550 iFunnel QTOFs), Agilent Technologies, USA. For chromatographic separation, an Agilent 1200 Series HPLC system (Agilent Technologies, USA) equipped with a binary gradient solvent pump, HiP Sampler, column oven and MS Q-TOF with Dual AJS ES Ion Source. Samples were separated on SB-C18 column (2.1 × 50 mm, 1.8-particle size; Agilent Technologies, USA) maintained at 25°C. The solvents used were: water containing 0.1% HCOOH and Acetonitrile containing 10% water and 0.1% HCOOH. The following gradient elution program at a flow rate of 0.3 mL min⁻¹ was applied. MS detection was performed in MS Q-TOF Mass spectrometer (Agilent Technologies). Compounds were identified via their mass spectra and their unique mass fragmentation patterns. Compound Discoverer 2.1, ChemSpider and PubChem were used as the main tools for the identification of the phytochemical constituents [93].

The abbreviations should be explain when mentioned the first time in the paper.

All abbreviations were explained in whole manuscript.

The use of VERO cell lines should be motivated.

VERO cell lines used for the antiviral activity because they are considered as permissive cell lines.

Results of cytotoxic effect should be better discussed and possibly correlated with other activities.

In fact, we discussed the result obtained with other published papers and with with chemical profile of T. polium extract.

Nematollahi-Mahani et al. [51] studied the toxicity of T. polium 96%-ethanolic extract on different known malignant cell lines like A549 (human lung adenocarcinoma), BT20 (human breast ductal carcinoma), MCF-7 (human breast adenocarcinoma), and PC12 (mouse pheochromocytoma). The results reported showed that IC50 values were 90 µg/ml (A549), 106 µg/ml (BT20), 140 µg/ml (MCF-7) and 120 µg/ml (PC12). The cytotoxic properties of T. polium aerial parts can be attributed the presence in the organic extracts of some diterpenoids and their acyl derivatives (teucvin and teucvidin), flavonoids (cirsiliol, cirsimaritin, cirsilineol, salvigenin and 5-hydroxy-6,7,3’,4’-tetramethoxyllavone), saponin poliusaposide [52], and selenium [53-55].

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The novelty of the study in the context given by Authors is poor and unclear. Authors stated that (…) the main objectives of the present investigation were to assess for the first time the phytochemical profile of T. polium methanolic extract from Hail region (Saudi Arabia) using High resolution-Liquid chromatography coupled to mass spectrometry (..)” – it is hardly to assess the novelty of the paper. New methodology? Or new site of the plant?

It is hardly to agree with statement: “assess for the first time the phytochemical profile” – because only 29 compounds (according to Tab. 1) were found in extracts and I think it is not enough to use the term “phytochemical profile”. Moreover, on chromatogram (Fig.1) only 12 peaks are shown (what with the others?).

If the compounds in Table are new, MS identification is not enough to confirm their structure.

The method is not new and it was used in plant investigation. Moreover the term - liquid chromatography coupled with high resolution mass spectrometry is more adequate.

On what basis the Authors stated “ The tested extract was dominated by 3R-hydroxy-9E,11Z-octadecadienoic acid, rhoifolin, sericetin diacetate, dihydrosamidinand cepharanthine”. It is difficult to agree with this on the basis of the chromatogram. To confirm it quantitative analysis should be done.

On chromatogram (fig.1) peak no. (6). – Valtratum has retention time about 20 min, while in table 1 it is 9,5 min.

Why were the other species (not related with present study e.g. T. scordium, T. stocksianum) given in Table 5?

There are still many errors, e. g. line 100: “in fat”?; line 102 “3R-hydroxy-9E” ? (in table is: 13R-hydroxy-9E…), lack of capital letters, doubled phrase in Table 5: μg/mL extract μg/mL extract), erroneous format of fonts e.g. in legend for Fig. 1……etc

The quality of Fig.1 is still very poor and the figure is unreadable (the same comment for Supplementary data)

In my opinion, it is only preliminary study. Lack of correlation of extract composition with activity. I agree with Authors that “Further analysis are needed to correlate between the obtained results with the phytochemical composition of organic extract (…).”

Author Response

Point-by-point response to Reviewer 1:

Dear Dr. (Reviewer 1): Thank you for your second round consideration of our manuscript. We found the comments helpful, and believe our revised manuscript represents a significant improvement over our initial submission.

Open Review

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( ) English language and style are fine/minor spell check required
(x) I don't feel qualified to judge about the English language and style

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	( )	(x)	( )
Is the research design appropriate?	( )	( )	(x)	( )
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Comments and Suggestions for Authors

Special thanks for your remarks: In fact, the main goal of the present study was the identification of bioactives molecules from T. polium methanolic extract collected from Hail region (Saudia Arabia) using HR-LCMS technique.

In fact, we added more details about the retention time, the chemical structure, area the pics obtained and chemical structure of the identified 29 compounds in the revised paper.

N°	RT	Identified Compound Name	Chemical class	Chemical formula
1	0.945	10-Hydroxyloganin	Isoprenoid	C₁₇H₂₆O₁₁
2	1.046	13R-hydroxy-9E,11Z octadecadienoic acid	Octadecanoid	C₁₈H₃₂O₃
3	1.062	Bis (2-hydroxypropyl) amine	Amino alcohol	C₆H₁₅NO₂
4	1.447	9-amino-nonanoic acid	Amino fatty acid	C₉H₁₉NO₂
5	3.807	10-Amino-decanoic acid	Amino fatty acid	C₁₀H₂₁NO₂
6	4.739	7-Epiloganin tetraacetate	Isoprenoid	C₂₅H₃₄O₁₄
7	5.342	b-D-Glucopyranoside uronic acid, 6-(3-oxobutyl)-2- naphthalenyl	Organic acid, Phenol	C₂₀H₂₂O₈
8	5.948	Cepharanthine	Alkaloid	C₃₇H₃₈N₂O₆
9	5.96	Troxerutin	Flavonol	C₂₇H₃₀O₁₄
10	6.319	Deoxyloganin tetraacetate	Isoprenoid	C₂₉H₂₈O₇
11	6.329	CMP-N-acetylneuraminic acid	Amino sugar	C₃₃H₄₂O₁₉
12	6.376	Rhoifolin	Flavonoid	C₂₅H₃₄O₁₃
13	7.838	Sericetin diacetate	Flavonol	C₂₀H₃₁N₄O₁₆P
14	8.198	Carapin-8 (9)-Ene	Limonoid	C₂₇H₃₀O₇
15	8.848	Selinidin	Coumarin derivative	C₁₉H₂₀O₅
16	9.1	Harpagoside	Iridoid glycoside	C₂₄H₃₀O₁₁
17	9.126	8-Epiiridodial glucoside tetraacetate	Isoprenoid	C₂₄H₃₄O₁₁
18	9.262	Larixol Acetate	Bicyclic labdane diterpenes	C₂₂H₃₆O₃
19	9.525	Valtratum	Terpene	C₂₂H₃₀O₈
20	9.807	Triptonide	Diterpene triepoxide	C₂₀H₂₂O₆
21	10.036	Koparin 2'-Methyl Ether	Isoflavonoid	C₁₇H₁₄O6
22	10.779	Dihydrosamidin	Coumarins	C₂₁H₂₄O₇
23	11.022	10S,11R-Epoxy-punaglandin 4	Eicosanoid	C₂₅H₃₅ClO₉
24	11.279	16alpha,17beta-Estriol 3-(beta-D- glucuronide)	Steroidal glycosides	C₂₄H₃₂O₉
25	11.28	16-Hydroxy-4-carboxyretinoic Acid	Isoprenoid	C₂₀H₂₄O₅
26	12.149	Isotectorigenin, 7- Methyl ether	Isoflavonoid	C₁₈H₁₆O₆
27	13.274	3-hydroxy-3',4'- Dimethoxyflavone	Flavonoid	C₁₇H₁₄O₅
28	18.427	Khayanthone	Limonoid	C₃₂H₄₂O₉
29	20.37	1-dodecanoyl-sn-glycerol	Glycerolipid	C₁₄H₂₂N₂O₃

Table 1. Bioactive compounds and their chemical class identified in T. polium methanolic extract using HR-LCMS technique.

In fat, T. polium methanolic extract was dominated by terpenes and their derivatives, fatty acids and their derivatives, polyphenols, flavonoids and derivatives, coumarines, amino acid derivatives, and alkaloids. The tested extract was dominated by 3R-hydroxy-9E,11Z-octadecadienoic acid, rhoifolin, sericetin diacetate, dihydrosamidin and cepharanthine. The chromatograms (figure 1) showed respectively major peaks indicating the presence of various phytochemical constituents. Data related to MS spectra, MS/MS spectrum peak list, chemical formula, and m/z values are listed in the supplementary file (Supplementary data S1).

Figure 1. Main identified phytocompounds in T. polium methanolic extract using HR-LCMS technique. (2A): +ve Chromatogram – (1). 13R-hydroxy-9E,11Z-octadecadienoic acid, (2). Rhoifolin, (3). Sericetin diacetate, (4). Selinidin, (5). Harpagoside, (6). Valtratum, (7). Triptonide, (8). Koparin 2'-Methyl Ether, (9). Dihydrosamidin, (10). 10S,11R-Epoxy-punaglandin, (11). 4, 16alpha, 17beta-Estriol 3-(beta-D-glucuronide), (12). Khayanthone. (2B): -ve Chromatogram – (1). 10-Hydroxyloganin, (2). 7-Epiloganin tetraacetate, (3). Cepharanthine, (4). Deoxyloganin tetraacetate, (5). Carapin-8 (9)-Ene, (6). 1-dodecanoyl-sn-glycerol.

The chemical structure of the identified 29 compounds are listed in Figure 2.

Figure 2. Chemical structure of the 29 compounds identified in T. polium methanolic extract byg HR-LCMS. The name of compounds (1-29) are listed in table 1.

If the compounds in Table are new, MS identification is not enough to confirm their structure.

In fact, the reported compounds were identified according to their spectrum peak list in both MS and MSⁿ as reported in the Supplementary data. These compounds are not new identified ones, but are well known compounds found in diverse plant species. The importance of our work is that we report for the first time the identification of these components in T. polium methanolic extract from the aerial parts using HR-LCMS technique.

The method is not new and it was used in plant investigation.

Previously, different techniques were used to identify and screen methanolic extracts from the same plant organs in different countries around the word. As summarized in table 5, the identified compounds are reported for the first time in this plant material (same plant, same organs, same protocol of extraction and same solvent used) but using another analytical technique (HR-LCMS).

Moreover the term - liquid chromatography coupled with high resolution mass spectrometry is more adequate.

We also used the suggested term: liquid chromatography coupled with high resolution mass spectrometry in the new revised version of our manuscript.

In fact, the dominance was estimated according the area of pic obtained for each compound in the chromatograms obtained.

On chromatogram (fig.1) peak no. (6). – Valtratum has retention time about 20 min, while in table 1 it is 9,5 min.

Why were the other species (not related with present study e.g. T. scordium, T. stocksianum) given in Table 5?

In fact, the cited Teucrium species other than polium species in Table 5 were used for the comparison and correlation between species. But we removed these non polium species from the table 5.

The whole manuscript was entirely revised for grammatical and spelling errors. We hope that the new submitted version of the manuscript is correct.

The quality of Fig.1 is still very poor and the figure is unreadable (the same comment for Supplementary data)

The figure 1 was entirely changed to increase its readability.

In fact, as we suggested the correlation between the composition and biological activity needs further studies and new experiments to can use results as proof for a possible correlation between the suggested phytochemical composition and all obtained biological properties of the tested extract. In fact, in silico approach is now running in order to correlate between composition/biological properties.

Finally, special thanks for your important comments made on our manuscript helping us to reorganize and format the presented results in the present study.

All the best

Prof. Dr. Mejdi Snoussi

Author Response File: Author Response.docx

Reviewer 2 Report

The Authors clearly addressed all previous reviewer' comments.

Author Response

Dear colleague

Special thanks for your previous remarks about our manuscript

Special thanks for accepting our paper.

All the best

Round 3

Reviewer 1 Report

I accept the manuscript in present form

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Phytochemical Screening, Antibacterial, Antifungal, Antiviral, Cytotoxic, and Anti-Quorum-Sensing Properties of Teucrium polium L. Aerial Parts Methanolic Extract

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