Phytochemical profiling of Piper crocatum and its antifungal mechanism action as Lanosterol 14 alpha demethylase CYP51 inhibitor: a review

Mycoses or fungal infections are a general health problem that often occurs in healthy and immunocompromised people in the community. The development of resistant strains in Fungi and the incidence of azole antibiotic resistance in the Asia Pacific which reached 83% become a critical problem nowadays. To control fungal infections, substances and extracts isolated from natural resources, especially in the form of plants as the main sources of drug molecules today, are needed. Especially from Piperaceae, which have long been used in India, China, and Korea to treat human ailments in traditional medicine. The purpose of this review is to describe the antifungal mechanism action from Piper crocatum and its phytochemical profiling against lanosterol 14a demethylase CYP51. The methods used to search databases from Google Scholar to find the appropriate databases using Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Flow Diagram as a clinical information retrieval method. From 1.150.000 results searched by database, there is 73 final results article to review. The review shows that P. crocatum contains flavonoids, tannins, terpenes, saponins, polyphenols, eugenol, alkaloids, quinones, chavibetol acetate, glycosides, triterpenoids or steroids, hydroxychavikol, phenolics, glucosides, isoprenoids, and non-protein amino acids. Its antifungal mechanisms in fungal cells occur due to ergosterol, especially lanosterol 14a demethylase (CYP51) inhibition, which is one of the main target sites for antifungal activity because it functions to maintain the integrity and function of cell membranes in Candida. P. crocatum has an antifungal activity through its phytochemical profiling against fungal by inhibiting the lanosterol 14a demethylase, make damaging cell membranes, fungal growth inhibition, and fungal cell lysis.


Introduction
Mycoses or fungal infections are a general health problem that often occurs in healthy and immunocompromised people in the community (Ramírez et al. 2013). Fungi are divided into four classes: yeasts, filamentous, dimorphic, and dermatophytes; generally, and ubiquitous in the environment, and become pathogenic when immune cells decrease (Howard et al. 2020). Fungal cells have essentially dynamic structure walls for morphogenesis, pathogenesis, and cell viability and act as a dynamic organelle, and need one-fifth of the yeast genome for cell wall biosynthesis (Gow et al. 2017). The development of resistant strains in fungi and the incidence of azole antibiotic resistance in the Asia Pacific which reached 83% become a critical problem nowadays (Whaley and Rogers 2016;Whaley et al. 2017).
The Azoles are commonly used because cheaper and have a broad spectrum of antimicrobials (Rosam et al. 2021). To control fungal infections, substances, and extracts isolated from natural resources, especially in the form of plants as the main sources of drug molecules today, are needed (Balouiri et al. 2016). Natural products have limited or no side effects on human and animal antifungal activity (Tabassum and Vidyasagar 2013). Antifungal mechanisms in fungal cells occur due to ergosterol inhibition as a result of 5,6 desaturase (ERG3) downregulation which is the second final step of the ergosterol biosynthetic pathway (Alizadeh et al. 2017). Ergosterol at the fungal plasma membrane is the most common sterol and binding at lanosterol 14α demethylase, an ergosterol-specific enzyme that can cause lanosterol demethylation (Loeffler and Stevens 2003;Ashley et al. 2006;Emami et al. 2017).
Piperaceae plant extracts have long been used in India, China, and Korea to treat human ailments in traditional medicine (Jeon et al. 2019). The part of the Piperaceae family which has large species of up to 1000 is the Piper genus (Durant-Archibold et al. 2018). Piper can be found in temperate regions with tropical and sub-tropical (Lima et al. 2020). Indonesia is located on the equator, which has a tropical climate with high humidity and many natural and biological resources (Puspita et al. 2018). The seeds and leaves of Piper species are often cultivated and consumed for various REVISED Amendments from Version 2 The difference between versions 2 and 3 of this research is in the manuscript because the author had revised the English language and grammar.
There are also revisions in the result and discussion, from Table 1. The author explained the common name of P. crocatum -Red betel leaf in the introduction so that these synonyms name can be used further without confusing the reader. The explanation of the solvent used to extract secondary metabolites from P. crocatum has also been explained in Table 1, using ether 40-60°C, chloroforms, ethanol, water, and methanol extract from the type and polarity of the solvent used.
In the previous version, the author only explained the bioactive compound of P. crocatum from only one reference. For the latest version, 4 (four) new references have been added that support information about bioactive compounds in P. crocatum, becoming more complete.
Several sentences have been improved which have missing verbs so that they are easier to understand.
The title of Figure 3 revised also have been made as the reviewer suggested its grammar and added a new explanation regarding capital letters in Figure from A-F. The A-F explained about the differentiation of cell wall association in fungal adhesins in several fungi species, like C. albicans, C. cerevisiae, P. braziliensis, A. fumigatus, B. dermatitis, S. pombe, and C. neoformans.
The narrative in Figure 3 also has been revised and broken down into 3 new sentences that are simpler than one long and unclear sentence before.
The author also explained the antifungal activities by P. crocatum phytochemicals like phenols, polyphenols, tannins, saponins, and flavonoids to inhibit ergosterol by lanosterol 14 alpha demethylase inhibition.
The conclusion has been revised in the grammar and added a new explanation about the relationship between ergosterol and lanosterol 14 alpha demethylase.
Any further responses from the reviewers can be found at the end of the article diseases treatment such as antifungal, antibacterial, and disinfectant effects (Astuti et al. 2014;Mgbeahuruike et al. 2017).
Isolation of several secondary metabolites of Piper species shows that therapeutically molecules like lignans, flavones, alkaloids, unsaturated amides, long and short-chain esters, aristolactams, monoterpenes, sesquiterpenes, ketones, aldehydes, arylpropanoids, chalcones, propenylphenols, and amide alkaloids as a typical constituent (Gutierrez et al. 2013). However, no tests were found on specific compounds for antibacterial activity from Piper (Barh et al. 2013). Based on the literature, antifungal compounds are classified into flavonoids, amides, acid derivatives, lignans, prenylated benzoic, cyclopentanedione, butenolides, and phenylpropanoids (Xu and Li 2011). Of the various Piper species, the main constituent is an amide, which is classified as aristolactams, open-chain alkamides, amides with pyrrolidine, 4,5dioxoaporphines, Piperidine, and Piperidone groups, ceramides, cyclohexanamid, and cyclobutanamide (Do Nascimento et al. 2012). In this review, we summarize the antifungal activity properties, structural studies, and bio-mechanism of P. crocatum, commonly known as Red Betel leaf, which is found worldwide, against lanosterol 14 α demethylase CYP51 in fungi. This review is expected to allow us to find new alternative antifungal treatment agents from natural resources based on their active chemical compounds and activities to cure fungal infections, thus reducing the extensive and inappropriate use of antibiotics for antifungal treatment.

Methods
The author searches databases from Google Scholar to find the appropriate databases using the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Flow Diagram as a clinical information retrieval method. The search screening occurred through four stages. The first stage was screening by the keywords, the second stage was screening by the criteria, the third stage was screening by relevance and duplicates, and the fourth stage was screening by eligibility. The keyword of this search was 'Piper' which yielded 1,150,000 results. The second step was screening by inclusion criteria of the keyword 'Piper' that reported articles published from 2003 until 2022, which yielded 326,000 results. For the third step, screening by relevance and duplicate with the keyword of this search is 'Piper crocatum' AND 'antifungal', which yielded about 498 results. The final screening results included 73 articles to review. This search was conducted from February to May 2022. The criteria for this research were clinical trials in animal testing and humans, books, laboratory tests, case studies, article reviews, systematic reviews, narrative reviews, and meta-analyses. The study was conducted with a true-experimental (Double-Blind RCT), quasi-experimental, study protocol, or pilot study. Articles were published in English. The flow chart of the Literature Review showed in Figure 1.
The bioactive compounds of P. crocatum are as follows in Table 1 and Figure 2.
The heated leaves are often used to relieve asthma, sore throats, coughs, and vaginal discharge, while the essential oil is used as catarrh and diphtheria inhalation and mouthwash, with antifungal activity ability against Candida sphaerospermum and Candida cladospoiroides with strong MIC of 10 μg/ml (Safithri et al. 2020;Suri et al. 2021). The antimicrobial compounds include phenolics and flavonoids in P. crocatum by its hydroxyl group at 5 positions causing inner and outer bacterial cell membrane fluidity reduction (Puspita et al. 2018). Alkaloid in P. crocatum by its aromatic substitution, carbon rings, and oxidation nature caused inhibition of bacterial growth and cell lysis. Tannins also have antibacterial activity by slowing the fungal cell's growth, and shrinking cell membranes thereby limiting the development of cell membrane synthesis, distorting permeability, breakdown, and cell lysis while saponins can dilute lipids (lipophilic) and then reducing cell surface pressure with their ability to attract water (hydrophilic) and caused cell damage. Terpenoids in P. crocatum cause permeability decreased and rupture of cell membranes so the nutrients and enzymes leave the cytoplasm, decrease metabolism, reduce ATP production, and inhibit bacterial growth and reproduction (Cowan 1999; Rinanda et al. 2012).
Polyphenol inactivates protein and inhibits enzymes on the surface of bacterial cells; flavonoids form complexes that interfere with the function of the bacterial cell wall, inactivating microbial adhesion, enzymes, and cell protein transport by binding to bacterial extracellular proteins through hydrogen and covalent bonds; saponins have hydrophilic molecules and lipid thinning molecules (lipophilic) so that they can make lower cell surface pressure; tannins functions to form complex compounds with enzymes and substrates, thereby disrupting cell membranes, and phenol has hydroxyl and carbonyl groups that can interact with fungal cells through hydrogen bonds, thereby increasing protein coagulation and fungal cell membranes which will cause fungal cells to lyse (Januarti et al. 2019). Inhibition of fungal activity can be done by bothering cell membranes, the activity of enzymes, and fungi genetic mechanisms (Ejele et al. 2012).
Antifungal properties and structure Treatment of vaginal discharge due to Candida gave the best response to a combination of the intravaginal vulva and topical therapy (Mitchell 2004). Antifungals for the treatment of vaginal discharge caused by C. albicans are fluorinated pyrimidine cytosine (5-FC) which targets RNA synthesis and DNA replication, polyenes which affect the integrity of cell membranes, azoles which affect the target of the ergosterol biosynthetic pathway, and echinocandins which affect cell wall biosynthesis, while the use of broad-spectrum antibiotics increases cases of immunocompromise (Yücesoy and Marol 2003;Sendid et al. 2007). Antifungals that damage cell membrane permeability work by binding to ergosterol in the polyene group, inhibiting the synthesis of ergosterol in squalene monooxygenase or epoxidase in the allylamines group, and inhibiting the synthesis of ergosterol in 14-α-demethylase or fungal cytochrome P450 in the azoles group; antifungal destroying cell walls works by inhibiting the synthesis of 1,3-β-glucan by binding to the glucan synthase enzyme which functions to form glucan in the echinocandin group; and antifungal inhibitors of DNA synthesis from fungal cells by inhibiting the synthesis of thymidylate or pyrimidine analogs in flucytosine (5-Fluorocytosine) and mitotic inhibitors in griseofulvin (Cannon et al. 2007; Lewis 2011).
Fungal cell walls, fungal-specific, serve as protection from harmful environments and are aggressive because of its toxic and hydrolytic molecules. Ninety percent consist of polysaccharides, with Saccharomyces and Candida subphylum as the well-characterized fungal adhesins ( Figure 3) (Latgé 2007). The core of the central fungal cell wall consists of chitin that is linked to branched β-1,3-glucan, combined with galactomannan, galactosamynoglycan, and β-1,3-1,4-glucan in A. fumigatus and β-1,6-glucan in C. albicans (Adams 2004;Latgé 2010). Chitin, with a weight of 1-2% from dry cell wall yeast, is an important structure that consists of a homopolymer of β-1,4-linked N-acetylglucosamine that is long and linear, while disrupted chitin synthesis will cause the fungal cell wall to be lysis and unstable in osmotic (Bowman and Free 2006).
Ergosterol is a bitopian endoplasmic reticulum protein, which spans the entire length of the lipid bilayer ( Figure 4) (Emami et al. 2017;Rosam et al. 2021). Ergosterol is a key enzyme in fungal-specific sterols, cytochrome P450 enzyme in fungi derived from S. cerevisiae, belonging to the CYP51 (lanosterol 14-α-demethylase) family. The biosynthesis inhibition of ergosterol will be caused by intermediates toxic sterol accumulation (14-α-methyl-3,6-diol) by ERG3. By binding the nitrogen atom containing heterocyclic moiety in the core ring to the iron atom of the heme domain group in the active site and preventing the formation of lanosterol demethylation, the cell membranes will get damaged, and lysis  lanosterol to 4,4 0 -dimethyl cholesta-8,14,24-triene-3-β-ol, sterol 14-reductase then reductases to episterol, which in turn is converted to ergosta-5,7,24(28)-trienol by sterol 5,6 desaturase (ERG3), and with sterol 8-isomerase converted it into ergosterol ( Figure 5). The fungistatic mechanism by inhibiting lanosterol 14α demethylase (encoded by ERG11), which leads to a block in ergosterol synthesis and the accumulation of toxic sterol intermediates, including 14-α-methyl-3,6-diol produced by Erg3 (Lee et al. 2021). This toxic sterol gave the membrane cell heavy stress and impairs cell membrane permeability, thereby ergosterol biosynthesis becomes inhibited and cell membranes get damaged, leading to fungal lysis

Phenol
Phenol (carbolic acid) are secondary metabolites that can be found widely in Piper species, but rarely found in algae, fungi, and bacteria; organic compounds with low molecular weight, have one or more substituents hydroxyl group in aromatic phenyl ring, especially benzene, formed from phenylpropanoid or shikimate that produce phenylpropanoids and acetate or malonate polyketide pathway that produce simple phenols or with phenylpropanoids, identified by UV-Vis Spectra and retention times compared with the literature and reference compounds that available (Waniska 2000;Lattanzio 2013;Ferreira et al. 2016).
Phenolic compounds have hydroxyl and carbonyl groups that can interact with fungal cells through hydrogen bonds, thereby increasing protein and cell membranes of pathogen fungal coagulation which will cause the damage and lysis of the fungal cells, and make the next fungal ergosterol growth anomaly and malformation (

Polyphenol
Polyphenols, known as an antifungal that has been isolated in Piper species, are water-soluble, have at least two phenolic rings, usually have 12-16 groups of phenolic hydroxyl at aromatic rings, and have a molecular weight from 500 to 3,000  the Piper species and have been classified as natural polyphenol groups, water-soluble, a molecular weight of 500-3000 daltons, condensed and hydrolyzed to polymerization that reaches high degrees and have two or three phenolic hydroxyl and carboxyl functional groups on a phenyl ring, known as antimicrobial, against various types of microorganisms, including bacteria, yeasts, fungi, and virus (Chung et al. Tannins have the best antifungal activity in C. albicans at a concentration above 7.80 mg/L, similar to nystatin, slightly

Flavonoid
Flavonoids (bioflavonoids), flavus in Latin, yellow powder, low molecular weight, are secondary metabolites that are very useful as antimicrobials by making bacterial damage, contain diphenylpropane (C 6 -C 3 -C 6 ), and have a three-carbon bridge with phenyl groups, as the core structure of 2-phenylbenzopyra, less toxic and low cost (Baum et al. 2001;Galeotti et al. 2008;Can et al. 2015;Kurnia et al. 2018;Khalid et al. 2019;Herdiyati et al. 2020). As an important polyphenol class, flavonoids divided by C-ring oxidation degree, with the three-carbon segment oxidation degree and unsaturation degree, and the major classes are flavonols (3-hydroxy with the different site at OH group of phenolic), flavanones (C-4keto-group with double-blind of C-2 and C-3), flavanols or flavan-3-ols (C 3 -hydroxyl group and carbon ring that fully saturated), isoflavones (act like phytoestrogens), aurones, chalcones (two aromatic rings by three-unit carbons to make the group of α,β unsaturated carbonyl), and anthocyanidins (Pourcel et al. 2007;Corradini et al. 2011;Seleem et al. 2017). Flavonoids have activities such as antibacterial, antioxidant, anti-inflammation, and antifungal properties through its ability to form complexes with extracellular proteins and interfere with microbial membrane activity because of its lipophilic properties (Candiracci et al. 2011).
Research showed that flavonoids isolated, like 3,4-dihydroxy-5,6,7-trimethoxyflavone, cirsiliol, cirsimaritin, and hispidulin showed antifungal activity to C. sphaerospermum (Alcerito et al. 2002). Research also showed that flavonoids in honey have an antifungal activity to C. albicans growth inhibition, but not kill the yeasts (Candiracci et al. 2011), The new antifungal should be safer, have minimal side effects, be cheaper, easier to get, and more potent against fungal infections. Based on the results of the review, it was found that P. crocatum contains compounds that have antifungal activity. By its secondary metabolites, P. crocatum has the opportunity to become a new antifungal agent as an alternative non-pharmacological antifungal treatment.

Conclusions
Natural products are important resources in the discovery and development of new medicinal raw materials. P. crocatum has antifungal activities that are against fungal by its compounds and inhibit ergosterol as a key enzyme in fungal-specific sterols. The inhibition of ergosterol can be induced by the inhibition of lanosterol 14 α demethylase in the biosynthesis which will cause integrity and function damage to fungal cell membranes. Damaged cell membranes will cause fungal growth inhibition, morphological changes, and fungal cell lysis. Based on the review data, it is hoped that it can be used as a reference regarding information of new potential bioactive compounds as an alternative treatment for fungal infections by their lanosterol 14 α demethylase CYP51 inhibition effect other than the use of antibiotics or currently used drugs.

Data availability
No data are associated with this article. I have received the following manuscript for review from F1000Research: "Phytochemical profiling of Piper crocatum and its antifungal mechanism action as Lanosterol 14 alpha demethylase CYP51 inhibitor".

General comments:
I think overall the manuscript need a proof reading for the English language and grammar.

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The following sections required revisions: Results and Discussion; and Conclusion ○ Results and Discussion Table 1: -The name of the species P. crocatum suddenly was changed into betel plant? In the introduction section can be mentioned that betel plant is a common name for P. crocatum.

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The betel leaf extract was extracted using which solvent (alcohol or other organic solvents?) ○ I suggest the bioactive compounds contained in P. crocatum should be derived from more than 1 references. ○ A sentence below Figure 2 here: "The bioactive compound of P. crocatum as follows Table 1 and Figure 2".
This sentence is missing a verb (grammatically incorrect). -"Ergosterol is a key enzyme in fungal-specific sterols, cytochrome P450 enzyme in fungi derived from S. cerevisiae, belonging to the CYP51 (lanosterol 14-α-demethylase) family, so with inhibition of biosynthesis in ergosterol, caused intermediates toxic sterol accumulation (14-α-methyl-3,6diol) by ERG3, by binding the nitrogen atom containing heterocyclic moiety in the core ring to the iron atom of the heme domain group in the active site and preventing the formation of lanosterol demethylation, will damage cell membranes (Arthington-Skaggs et al.1999;Flowers et al. 2015)." This sentence is too long and unclear. Authors should break this sentence into 2-3 sentences.

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All the phytochemical content in P. crocatum, do they have activities as Lanosterol 14 alpha demethylase CYP51 inhibitor? This is because in the explanations of phytochemicals like polyphenols, tannins, saponins, it seems they don't have activities as inhibitor for demethylase CYP51. Or these are different activities as anti fungi?
These sentences before conclusion section: -"Based on the results of the review, it was found that P. crocatum contain compounds that have activity as an antifungal agent so it is expected to become a new antifungal agent that can slowly replace the extensive use of antibiotics. Therefore, new antifungal agents that safer, few side effects, cheaper, easier to get, and more potent against fungal infections are needed." I think these sentence need to be revised, since the sentences are unclear in particular the last sentence.

Conclusions
The conclusion section need to be revised since there are grammatical error and unclear. The authors have revised and followed the suggestions.
2. Table 1 The name of the species P. crocatum suddenly was changed into betel plant? In the introduction section can be mentioned that betel plant is a common name for P. crocatum Answer: Thank you for your suggestion.
The authors have added the common name of P. crocatum as Red Betel plant in the introduction.
3. Table 1 The Red Betel leaf extract was extracted using which solvent (alcohol or other organic solvents?) Answer: Thank you for the correction and suggestion.
The authors have revised and added an explanation about this sentence in the narrative.
4. Table 1 I suggest the bioactive compounds contained in P. crocatum should be derived from more than 1 references Answer: Thank you for your correction and suggestion.
The authors have added 4 (four) new references to P. crocatum bioactive compounds.
5. Table 1 A sentence below Figure 2 here: "The bioactive compound of P. crocatum as follows Table 1 and Figure 2". This sentence is missing a verb (grammatically incorrect). Answer: Thank you for your correction and suggestion. The authors have revised the grammar in the sentence.
6. Figure  7. Narrative in Figure 3 This sentence is too long and unclear. Authors should break this sentence into 2-3 sentences. Answer: Thank you for your correction and suggestion.
The authors have revised this sentence into 3 sentences as the reviewer suggested.

Results and discussion
The explanations of phytochemicals like polyphenols, tannins, saponins, it seems they don't have activities as inhibitor for demethylase CYP51. Or these are different activities as anti fungi? Answer: Thank you for your question and suggestion. The author will try to answer this question. Not all of the secondary metabolites in the phytochemicals of P. crocatum have antifungal activity. Based on the literature, only secondary metabolites are included in a group of compounds such as phenols, polyphenols, tannins, saponins, and flavonoids that have antifungal activity. Antifungal activity has inhibitory mechanisms in several places such as inhibiting cell wall synthesis, destroying cell membrane permeability, inhibiting nucleic acid synthesis, and damaging fungal microtubules. The inhibition of lanosterol 14a demethylase in ergosterol biosynthesis will cause inhibition of ergosterol, a key enzyme that functions to maintain the integrity and function of cell membranes in fungal and main target to antifungal sites thereby impairing the permeability of the cell membrane. The antifungal activity contained in these compounds of P. crocatum will cause fungal cell lysis.
9. Results and discussion "Based on the results of the review, it was found that P. crocatum contain compounds that have activity as an antifungal agent so it is expected to become a new antifungal agent that can slowly replace the extensive use of antibiotics. Therefore, new antifungal agents that safer, few side effects, cheaper, easier to get, and more potent against fungal infections are needed". I think these sentence need to be revised, since the sentences are unclear in particular the last sentence. Answer: Thank you for your correction and suggestion.
The authors have revised this sentence.

Conclusions
The conclusion section need to be revised since there are grammatical error and unclear. For example: Natural products are an important resource…", should be "Natural products are important resources…" Answer: Thank you for your correction and suggestion. The authors have revised and followed the suggestions.

Conclusions
The conclusion should emphasize the antifungal activities of P. crocatum. Does P. crocatum have antifungal activities by inhibiting ergosterol synthesis or as a lanosterol 14α demethylase CYP51 inhibitor. Answer: Thank you for your correction and suggestion. The authors have revised and followed the suggestions.

Dikdik Kurnia
Thank you for the decision.
We are pleased to get a lot of valuable knowledge from the revisions suggested by the reviewer.
It is an honor for us to be reviewed by you.
Thank you. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Zubaida Yousaf
Lahore College for Women University, Lahore, Pakistan I have received the following manuscript for review from F1000Research: "Phytochemical profiling of Piper crocatum and its antifungal activity as Lanosterol 14 alpha demethylase CYP51 inhibitor: a review". The following sections required revisions and modifications: Title, abstract, introduction, results and discussion and references.

Title:
Please ensure that you title clearly specifies whether your study focuses on fungal enzyme inhibition assay, mechanism of action or another topic. Or change your title according to the results and discussion. Your abstract and introduction is somewhat related to your title but not according to the results and discussion.

Abstract:
There is no information found on these lines of abstract in your results and discussion: "Its antifungal mechanisms in fungal cells occur due to ergosterol especially lanosterol 14 alpha demethylase CYP51 inhibition as a result of 5,6 desaturase (ERG3) downregulation. P. crocatum has an antifungal activity by its phytochemical profiling that act against fungi by inhibiting the fungal cytochrome P 450 pathway, make damaging cell membranes, fungal growth inhibition, morphological changes, and fungal cell lysis".

Introduction:
Difference in statement of study idea in introduction and abstract.

Results and discussion:
In heading "Ethno-botany and ethno-pharmacology of P. crocatum". Add ethno botanical data related to plant like traditional uses and ethno-pharmacological aspects of plant.

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In heading "The phytochemical and chemical structures of P. crocatum". Sentence structure needs improvement. Don't repeat prepositions in sentence making. In heading "Antifungal properties and structure" these highlighted lines "Ergosterol is a key enzyme in fungal specific sterols, cytochrome P450 enzyme in fungi derived from S. cerevisiae, belonging to the CYP51 (lanosterol 14-α-demethylase) family, so with inhibition of biosynthesis in ergosterol, will damage cell membranes". I think these are the exact lines that have reflection of title statement. So, you should provide references of this activity from different published articles here in results and discussion and also in introduction and abstract.

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In figure 3: Spelling mistakes in caption.

References:
Following amendments are required in reference list and citations: Check references according to journal format.