Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi

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The function of other components, such as α-guaiene, remains unknown. Therefore, this study reports the antifungal activity of α-guaiene isolated from patchouli oil against for pathogenic fungi: Aspergillus niger, Candida albicans ATCC 7102, Microsporum gypseum ATCC 14683, and Trichophyton mentagrophytes ATCC 16404. Methods The material from fraction (249°C-254°C) had the highest α-guaiene. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined using the microdilution technique to evaluate antifungal activity, with n-hexane and medium serving as negative controls, and ketoconazole and fluconazole serving as positive controls. Results The results showed that the MIC value was determined at 45%, 50% for C. albicans, 55%, 60% for A. niger, 50%, 60% for M. gypseum, and 95%, 100% for T. mentagrophytes, respectively. Positive and medium controls demonstrated no microbial growth, whereas negative and growth controls revealed the presence of microorganisms. Fungus resistance to α-guaiene T. mentagrophytes exhibited the highest MIC value. Conclusions Overall, this study reveals that α-guaiene is a promising agent effective against the studied pathogenic fungi. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/14-4/v1", "name": "Alpha-guaiene isolated from patchouli oil exhibits antifungal activity..." } } ] } Home Browse Alpha-guaiene isolated from patchouli oil exhibits antifungal activity... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Nurjanah S, Alhafidz Z, Maulani M et al. Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.12688/f1000research.154021.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] Sarifah Nurjanah https://orcid.org/0000-0002-5391-2260 1 , Zhaqqu Alhafidz 1 , Maghfira Maulani 1 , Tita Rialita 2 , Elazmanawati Lembong 2 Sarifah Nurjanah https://orcid.org/0000-0002-5391-2260 1 , Zhaqqu Alhafidz 1 , [...] Maghfira Maulani 1 , Tita Rialita 2 , Elazmanawati Lembong 2 PUBLISHED 02 Jan 2025 Author details Author details 1 Department of Agricultural and Biosystem Engineering, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, West Java, 40600, Indonesia 2 Department of Food Industry Technology, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, West Java, 40600, Indonesia Sarifah Nurjanah Roles: Conceptualization, Data Curation, Funding Acquisition, Investigation, Resources, Writing – Original Draft Preparation, Writing – Review & Editing Zhaqqu Alhafidz Roles: Data Curation, Investigation, Methodology, Resources, Writing – Original Draft Preparation Maghfira Maulani Roles: Investigation, Methodology, Resources, Writing – Original Draft Preparation, Writing – Review & Editing Tita Rialita Roles: Conceptualization, Formal Analysis, Methodology, Validation Elazmanawati Lembong Roles: Data Curation, Methodology, Project Administration, Software, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Pathogens gateway. This article is included in the Plant Science gateway. Abstract Background The major component of patchouli oil, patchouli alcohol, is used as fixative and has antimicrobial activity. The function of other components, such as α-guaiene, remains unknown. Therefore, this study reports the antifungal activity of α-guaiene isolated from patchouli oil against for pathogenic fungi: Aspergillus niger , Candida albicans ATCC 7102, Microsporum gypseum ATCC 14683, and Trichophyton mentagrophytes ATCC 16404. Methods The material from fraction (249°C-254°C) had the highest α-guaiene. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined using the microdilution technique to evaluate antifungal activity, with n-hexane and medium serving as negative controls, and ketoconazole and fluconazole serving as positive controls. Results The results showed that the MIC value was determined at 45%, 50% for C. albicans , 55%, 60% for A. niger , 50%, 60% for M. gypseum , and 95%, 100% for T. mentagrophytes , respectively. Positive and medium controls demonstrated no microbial growth, whereas negative and growth controls revealed the presence of microorganisms. Fungus resistance to α-guaiene T. mentagrophytes exhibited the highest MIC value. Conclusions Overall, this study reveals that α-guaiene is a promising agent effective against the studied pathogenic fungi. READ ALL READ LESS Keywords α-guaiene, patchouli oil, antifungal activity, Minimum Inhibitory Concentration, Minimum Fungicidal Concentration Corresponding Author(s) Sarifah Nurjanah ( [email protected] ) Close Corresponding author: Sarifah Nurjanah Competing interests: No competing interests were disclosed. Grant information: Universitas Padjadjaran-RKDU (Riset Kompetensi Dosen UNPAD) grant - 1427/UN6.3.1/LT/2020 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Nurjanah S et al . This is an open access article 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. How to cite: Nurjanah S, Alhafidz Z, Maulani M et al. Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.12688/f1000research.154021.1 ) First published: 02 Jan 2025, 14 :4 ( https://doi.org/10.12688/f1000research.154021.1 ) Latest published: 13 Oct 2025, 14 :4 ( https://doi.org/10.12688/f1000research.154021.2 )  There is a newer version of this article available. Suppress this message for one day. Introduction Patchouli oil is an essential oil extracted from various plant parts such as flowers, leaves, stems, and roots ( Pandey et al., 2022 ; van Beek & Joulain, 2018 ). This oil has promising export potential, as it is regularly used in perfume, soap, pharmaceutical, cosmetic, and other industries. Patchouli oil is renowned for its active constituents and therapeutic benefits ( Leong et al., 2019 ). Patchouli is a crucial aromatic plant in the perfume industry ( Jain et al., 2022 ). Furthermore, patchouli oil accounts for approximately 85% of Indonesia’s essential oil exports, with a current annual value of 1,200-1,500 tons. Indonesia exports 90% of the world’s patchouli oil ( Rahmayanti et al., 2018 ). Notably, patchouli oil’s constituent components, such as patchouli alcohol, α-guaiene, δ-guaiene, α-patchoullene, and seychellene, have several benefits ( Pandey et al., 2021 ). Pressure, temperature, reflux ratio, and fractionation column all play crucial roles in the fractionating process used to separate these constituents ( Almeida et al., 2018 ; Nurjanah et al., 2020 ). Essential oils have antiviral, antiparasitic, antifungal, bactericidal, insecticidal, and nematocidal properties ( Khaledi & Zahani, 2018 ). They can be used as bactericides and fungicides against various human-infecting fungus and bacterium types. A pathogenic fungus Candida albicans causes candidiasis, whereas Microsporum gypseum and Trichophyton mentagrophytes cause dermatophytosis ( Moskaluk & VandeWoude, 2022 ). Aspergillus niger is another fungal pathogen that affects the respiratory system by causing different diseases such as aspergillosis ( Fiema et al., 2022 ). Fortunately, essential oils can be developed as antifungal agents for preventing these. Most compounds derived from essential oils are terpenes and their metabolites derivatives ( D’agostino et al., 2019 ), such as α-guaiene, a sesquiterpene comprising an average of 11% of the total mass ( Orf et al., 2021 ). Patchouli oil’s main constituents are patchouli alcohol (27.0%-35%), bulnezen (13.0%-21.0%), and α-guaiene (11.0%-16.0%) ( Górski et al., 2021 ). Terpenes can inhibit protein and DNA synthesis and promote cell rupture in antibiotic-susceptible and antibiotic-resistant bacteria ( Masyita et al., 2022 ). The antimicrobial activity of essential oils, notably patchouli oil, has been reported. Patchouli essential oil has been shown to inhibit Malassezia furfur and to be effective as an antimicrobial and anti-inflammatory agents ( Srivastava et al., 2022 ). Treatment of C. albicans and T mentagrophytes with patchouli oil fraction 8 containing 55.59% patchouli alcohol content yielded inhabitation zone of 9.24 mm and 7.70 mm in both fungi, respectively ( Setyaningrum et al., 2017 ). Patchouli alcohol is the primary antifungal component of patchouli oil and can be used as a fixative in perfume and related industries. After patchouli alcohol extraction, other components such as α-guaiene can increase patchouli oil’s utilization. Studies on α-guaiene antifungal activity are few, necessitating more investigations on this compound. While patchouli alcohol (PA) exhibited antimicrobial properties against pathogenic bacteria ( Escherichia coli , Pseudomonas aeruginosa , Bacillus proteus , Shigella dysenteriae , Typhoid bacillus , Staphylococcus aureus ) ( Yang et al., 2013 ). In addition, PA proved effective in treating some germs that were resistant to antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA) ( Wan et al., 2021 ). Therefore, this study explores the antifungal activity of fraction 1 patchouli oil containing 38.8% α-guaiene against C. albicans , A. niger , M. gypseum , and T. mentagrophytes. Methods Materials Patchouli oil was obtained from distiller in Subang, West Java, Indonesia. The nutrient agar culture used was Potato Dextrose Agar (PDA), whereas the liquid medium was Potato Dextrose Broth (PDB). Other chemicals such as 1% BaCl 2 , 1% H 2 SO 4 , sterile distilled water, 70% alcohol, lactophenol cotton blue (LPCB), 0.85% NaCl, ketoconazole, fluconazole, and n-hexane were also utilized. Chemical compound were obtained from Bratachem, while drugs were obtained from Kimia Farma, Bandung, Indonesia. The instruments used included Spinning Band Distillation System Model 36-100 from B/R Instrument USA, autoclave, Erlenmeyer flask, beaker glass, scotch bottle, Bunsen, ose needle, Petri dish, cuvette, spectrophotometer, micropipette, microplate, fin pipet, microscope, laminar airflow, oven, spatula, and vortex. The study was conducted using a laboratory experimental method with descriptive analysis, with ketoconazole serving as the positive control for all fungi tests, fluconazole for M. gypseum and T. mentagrophytes , and n-hexane serving the negative control for the four fungi. α-guaiene preparation To obtain the dominant α-guaiene, the sample was fractionated with a pressure of 10 mmHg, column length of 90 cm, and reflux ratio of 20:1. Patchouli oil was divided into five fractions: 1 at 249 o C-254 o C, 2 at 254 o C-259 o C, 3 at 259 o C-264 o C, 4 at 264 o C-269 o C and 5 at 269 o C-274 o C. Fraction 4 was suspected to contain α-guaiene as the most predominant content. Gas Chromatography Mass Spectroscopy (GCMS) GCMS analysis of the sample was carried out on a Agilent ® 6890 GC-MS, equipped with a split-spitless injector, attached to an Agilent HP-5MS capillary column (30 m x 250 μm, 0.25 μm film thickness). The carries gas was helium at a flowrate of 1.0 mL/min, split ratio 400:1, injector temperature was 280 o C, pressure was 10.48 psi. The transfer line was heated to 280 o C. Identification of the oil components was accomplished by comparison of retention times with standard substances. Antifungal activity: Microdilution method The modified microdilution technique was used to determine the antifungal activity of the sample. Conidia were removed from the agar slant surfaces using sterile 0.85% saline containing 0.1% Tween 20 (vol/vol). In a final amount of 100 μL per well, the conidia suspension was adjusted with sterile 0.85% saline to a concentration of roughly 1.0 × 10.5. For later usage, the inocula were stored at -20°C. To ensure there was no contamination and to confirm the inocula’s validity, dilutions of the inocula were cultivated on the solid MEA. Using 96-well microtitre plates and a serial dilution approach, the MIC was determined. Fungal inoculums (10 μL) were added to varying volumes of the studied material that had been dissolved in malt extract broth (MEB). The microplates were kept at 28°C for 72 hours. The concentration that totally inhibited fungal growth (MIC) was the lowest concentration at which no growth was discernible. Serial subcultivation of 2 μL onto microtitre plates containing 100 μL of MEB allowed for the determination of the minimal fungicidal concentration (MFC). The MFC, which indicates 99.5% death of the origin inoculum, was the lowest concentration with no discernible growth. Results and Discussion Characterization of patchouli oil fraction To obtain the dominant α-guaiene, the patchouli oil was fractionated using a BR Instrument Spinning Band Distillation System Model 36-100 and divided into five fractions. Fraction 4 was suspected to contain α-guaiene as the pre-dominant content. However, based on the GC-MS test results, the compound was higher in fraction 1 than in the others. After fractionation, the fractions were tested for antifungal activity. As determined by GC-MS test conducted on fraction 1 and 4, fraction 1 contained a significantly higher α-guaiene content than fraction 4. The percentage of α-guaiene was detected at peak 2 with retention time at 21.837 min. whereas that of fraction 4 was detected at peak 1 with retention time at 21.293 min. The percentage of α-guaiene content was calculated by dividing the peak area by the total area of the compound formed. Based on these calculations, the percentages in fractions 1 and 4 were 38.8% and 2.1% respectively. Subsequently, fraction 1 patchouli oil was used as the antifungal activity test material. Figure 1 show fragmentation pattern of α-guaiene. Figure 1. Electron Impact Mass Spectra of α-guaiene. Fungus identification Fungi were macroscopically and microscopically identified. Macroscopic identification involved observation with the eyes in order to directly identify the physical appearance ( Figure 2 ). Figure 2 (Line 1) show the physical appearance of C. albicans and A. niger grown on PDA medium. C. albicans cultures were white and formed round colonies, corroborating Lee et al’s finding ( Le et al., 2022 ), that C. albicans has a round shape. Allen et al. (2018 ) reported that A. niger culture exhibited white mycelium and brown-black conidia heads. Despite its higher colony size, A. niger had less dense colonies than C. albicans , which tend to form a firm line based on the groove of the ose needle stroke. In addition, C. albicans colonies were spherical, and had a relatively uniform size without hyphae due to their unicellularity, whereas A. niger colonies were irregularly spherical, had a nonuniform size, and formed hyphae owing to their multicellularity. Figure 2. Physical appearance of four pathogenic fungi on PDA medium. The colony of the fungus suspected to be M. gypseum resembled a pile of fine white cotton on top with a little brown powder scattered on the hyphae, corroborating a previous finding ( Putriningsih & Arjentinia, 2018 ) that M. gypseum colonies grew rapidly, were slightly powdery with a blackish-red brown color, and were scattered with a flat surface containing macroconidia. Macroscopically, although M. gypseum and T. mentagrophytes did not differ significantly, having white hyphae, T. mentagrophytes colonies appeared denser and slightly whiter with a protruding rough or powdery surface corroborating Frías-De-León et al’s findings ( Frías-De-León et al., 2020 ) that T. mentagrophytes colonies were often white to slightly yellowish-white and could sometimes turn violet-red, brown, or pale yellowish with a surface resembling cotton, wax wovwn or granules. The four test fungi were also identified microscopically (1000x) to corroborate their characteristics. A. niger , C. albicans , M. gypseum and T. mentagrophytes shared similar characteristics with the same fungi theoretically. Figure 3 showed the identification results of the test. The A. niger shows that this fungus has an elongated shape with visible fungal part such as conidia, vesicles, and conidiophores that were obvious by the staining process. Meanwhile, Figure 3 depicting the C. albicans compared to A. niger. C. albicans has a variable round shape with a bluish color and nonuniform cell size. Each cell shows a different individual because this fungus is a unicellular microorganism. Figure 3. Macroscopic identification of four pathogenic fungi. MIC MIC test on A. niger The MIC test results for A. niger showed that well with a clear appearance only occurred at 60% concentration treatment, whereas those at 30%, 15%, 7.5% and 3.75% were cloudy with fungal colonies on the surface medium. These observations are relatively weak compared to other studies, such as Yanti et al. (2017) which examined the essential antifungal test on kaffir lime against five type of Aspergillus fungi, revealing an inhibitory mechanism as delayed spore germination and the formation of mycelia at a concentration of 0.05%. The first observation results showed that the mechanism for fungal growth inhibition occurred only at 60%, indicating that the MIC value was reached at this concentration. Because the range of values between treatments was large, the MIC value may be reach before 60%, specially between 30% and 60%. The second testing process was conducted to minimize concentration different between treatments. Consequently, treatments with new concentrations were made, namely 45%, 50%, 55%, 60% and 65%. A fraction above 60% was intended to predict when the MFC value was not achieved. As seen in Table 1 , the well had a clear appearance before reaching a concentration of 60%, specifically at 55%. This concentration inhibited the growth of A. niger , as evidenced by its clear appearance, indicating that fungal growth was inhibited. Because treatment above 55% showed similar results, the MIC value was determined at this concentration. Table 1. MIC observation results in four pathogenic fungi tested. A. niger C. albicans M. gypseum T. mentagrophytes Treatments Appearance Treatments Appearance Treatments Appearance Treatments Appearance 45% Cloudy 35% Cloudy 40% Cloudy 80% Cloudy 50% Cloudy 40% Cloudy 45% Cloudy 85% Cloudy 55% Clear 45% Clear 50% Clear 90% Cloudy 60% Clear 50% Clear 55% Clear 95% Clear 65% Clear 155% Clear 60% Clear 100% Clear Ketoconazole 2% Clear Ketoconazole 2% Clear Ketoconazole 2% Clear Ketoconazole 2% Clear Fluconazole Clear Fluconazole Clear n-hexane Cloudy n-hexane Cloudy n-hexane Cloudy n-hexane Cloudy Growth control Cloudy Growth control Cloudy Growth control Cloudy Growth control Cloudy Medium control Clear Medium control Clear Medium control Clear Medium control Clear Observation on each control in the second MIC test showed similar results to the first. The positive control of 2% ketoconazole had a clear appearance, indicating that this compound had antifungal activity. The control medium had a clear appearance, showing an absence of fungal or microbial growth, whereas the growth control had a cloudy appearance, indicating that the fungus grew in this treatment. The negative control n-hexane also had a cloudy appearance, suggesting that this compound lacked antifungal activity against A. niger. MIC test on C. albicans Observation data revealed that none of the treatments produced perfectly transparent wells in C. albicans. In general, a higher concentration implies a clearer well, but at the highest value of 40%, fungal growth was still visible, warranting a re-test to determine perfectly transparent wells. The control treatment on C. albicans yielded similar result to the MIC test on A. niger. The second test was conducted with treatment concentrations of 35%, 40%, 45%, 50% and 55%. The results showed that a clear appearance began to develop at a concentration of 45%, whereas the treatment at 35% and 45% had a cloudy appearance, suggesting fungal growth. The difference in concentration appeared to have affected the presence of fungi, corroborating a previous study ( Górski et al., 2021 ) that tested the antifungal activity of patchouli oil at a concentration of 12.5%, 25%, 50% and 100% against C. albicans. Based on the observations, the MIC value was determined at a concentration of 45%, the result obtained was better than that of Kamoda ( Kamoda et al., 2020 ), stated that the MIC of the red galangal ethanolic extract against the fungus was achieved at 200 mg/mL, but the inhibition only reached at 60%. Differences in antifungal activity for each treatment with varying concentrations also reported by Ningtias et al. (2020) , where the MIC value of black garlic extract against C. albicans was reached at a concentration of 50%, but active inhibition was fully achieved at 75%. MIC test on M. gypseum The MIC observation test on M. gypseum fungus with concentration used in twofolds, notably 40%, 20%, 10%, 5% and 2.5% revealed fungal growth in all treatments. Positive and medium controls lacked fungal development, but negative and growth controls demonstrated the opposite. Furthermore, the first observation showed that the highest concentration exhibited no inhibition, although the technique was correctly executed, as indicated by the control treatment, which showed appropriate results. The second test was conducted by increasing the upper limit of concentration and decreasing it with a value range of 5%, hence the new concentration used were 60%, 55%, 50%, 45%, and 40%. The second MIC test revealed no fugal growth in the wells containing 50%, 55% and 60% of the teat compound, indicating maximum inhibition at these concentrations. The treatments wells overgrown as the number of fungal colonies that grew on the surface increased. Fungal growth was observed at 45% but was not as cloudy as that of 40%, suggesting that a 45% concentration inhibited growth insignificantly. The MIC value is often indicated by the smallest concentration that can inhibit total fungal growth based on visualization, it was determined at 50%. Positive controls with 2% ketoconazole and fluconazole inhibited fungal growth, as evidence by a clear appearance with a small amount of nonhomogeneous antibiotic precipitate. However, the negative control of n-hexane showed fungal growth, indicating that this compound lacked antifungal activity. The growth control also exhibited fungal growth, suggesting that growth on PDB media was not inhibited. Notably, the control media used was clear, indicating that no undesirable microorganism were present. MIC test on T. mentagrophyhes The MIC test results for T. mentagrophytes at concentration of 100%, 50%, 25%, 12.5%, and 6.25% of the test compounds showed that only 100% concentration treatment inhibited fungal growth. At 50%, 25%, 12.5% and 6.25% fungal growth remained on the surface, although in varying quantities. The inhibition activity of the test substance at a certain concentration was reflected in the appearance of the media, suggesting the presence or absence of microbes. In the first test, the MIC value was determined at a concentration of 100%. Because the range of concentrations capable of inhibiting fungal growth was extremely large, the MIC value could be obtained between 50% and 100%. The second test was conducted by minimizing the concentration difference, resulting in values of 100%, 95%, 90%, 85% and 80%. The second MIC observation test results showed that the wells with 95% and 100% concentration exhibited no fungal or other microbial growth, as indicated by their clear color surface. However, concentration of 90%, 85% and 80% showed varying fungal growth for each treatment. The MIC value is often indicated by the smallest concentration that can inhibit the total growth of the fungi or the appearance of clear media on visualization. Based on the results, the value was determined at 95%. Because the control treatment for T. mentagrophytes yielded identical results to those for M. gypseum , the technique used was appropriate. The difference in MIC values demonstrated that fungus resistance to fraction 1 as an antifungal agent differed for each fungal species. T. mentagrophytes value exceeded that of M. gypseum. These results are directly proportional to the previous inhibition zone test which showed that T. mentagrophytes was more resistant than M. gypseum to the fraction 1 antifungal agent containing 38.8% α-guaiene MFC The MFC test results for A. niger , C. albicans , M. gypseum and T. mentagrophytes revealed fungal growth in wells with a clear medium. Observations were made by creating a new inoculum from only the clear-appearing MIC test results. In order to reduce material requirements and minimize the possibility of errors as wells with cloudy appearance can be ascertained to have fungal growth. MFC test on A. niger The MFC test results for A. niger shown in Table 2 show that only the 55% concentration of the tree treatments indicated the present of fungal growth. Five of six inoculums created were colonized by fungus. Moreover, the number of fungal colonies in one petri disc was less that 5, this growth was relatively small compared to the original culture without treatment, indicating that the test substance inhibited A. niger growth at 55% but did not eradicate this fungus completely. Treatment concentration of 60% and 65% showed no fungal growth in each created inoculum. Based on these results, the MFC value was determined at a concentration of 60%. Table 2. MFC observation results in four pathogenic fungi tested. A. niger C. albicans M. gypseum T. mentagrophytes Treatments Appearance Treatments Appearance Treatments Appearance Treatments Appearance 55% Cloudy 45% Cloudy 45% Cloudy 95% Cloudy 60% Clear 50% Clear 50% Cloudy 100% Clear 65% Clear 55% Clear 55% Clear Ketoconazole 2% Clear Ketoconazole 2% Clear Ketoconazole 2% Clear Ketoconazole 2% Clear Fluconazole Clear Fluconazole Clear n-hexane Cloudy n-hexane Cloudy n-hexane Cloudy n-hexane Cloudy Growth control Cloudy Growth control Cloudy Growth control Cloudy Growth control Cloudy Medium control Clear Medium control Clear Medium control Clear Medium control Clear The inoculation wells with the control treatment yielded the expected outcomes under the expected conditions. The positive and medium controls lacked fungal growth, whereas the negative and growth controls indicated opposite results. Moreover, the growth control treatment showed the strongest fungal growth due to the absence of effective antifungal effects. MFC test on C. albicans The MFC test results for C. albicans presented in Table 2 indicated that the 45% treatment exhibited fungal growth compared to the 50% and 55% treatments. Compared to the control treatment, the number of colonies was much lower for the 45% treatment. Based on these observations, the MFC value was determined at a concentration of 50%. C. albicans ’s control treatment yielded similar results to those of A. niger. However, the positive control with 2% ketoconazole exhibited no fungal growth. Ketoconazole, a commonly used conventional antifungal agent exhibited antifungal activity that was significantly more potent than fraction 1 of patchouli oil. The results indicate that a concentration of at most 2% ketoconazole can eradicate fungi. MFC test on M. gypseum MFC test was conducted at three concentrations, notably 50%, 55% and 60% which were found to inhibit fungal growth in the previous MIC test. The results showed that, at 50% fungal growth remained on the PDA media in all petri dishes. At 55%, four petri dishes were still observed to be overgrown with fungus, whereas the other two were clear. The number of colonies form in each petri dish at 50% and 55% did not exceed three, this growth was relatively small, indicating that the antifungal agent only inhibited fungal growth but did not eradicate M gypseum. In addition, at 60%, fungal growth was unobserved in all petri dishes; hence, the MFC value was determined at this concentration. The MFC test results for the control treatment were consistent with the prior MIC test. The positive and medium controls exhibited no fungal growth compared to the negative and growth controls. MIC test on T. mentagrophytes MFC testing on T. mentagrophytes was conducted at two concentrations, notably 95% and 100%, which were found to inhibit fungal growth in the previous MIC test. The results showed that, at 95%, five petri dishes were still overgrown with fungus, whereas one was not. The number of colonies formed was not significantly high; hence, the antifungal agent appeared to only inhibit but not eradicate fungi. At 100%, fungal growth was unobserved in all Petri dishes; hence, the MIC value was determined at this concentration. The control treatment for the MFC test on T. mentagrophytes yielded similar results to those of M. gypseum. The positive control at 2% ketoconazole and fluconazole did not support fungal growth. This suggests that 2% ketoconazole and fluconazole as conventional antifungal agents, have significantly more potent effects than fraction 1 of patchouli oil. Conclusions Fraction 1 of patchouli oil containing 38.8% α-guaiene exhibited antifungal activity against A. niger , C. albicans , M. gypseum and T. mentagrophytes. The MIC values for each fungus were reached at concentrations of 55%, 45%, 50% and 95%, whereas the MFC value were achieved at 60%, 50%, 60% and 100%, respectively. This study demonstrates that α-guaiene is a prospective agent effective against the investigated pathogenic fungus. Data availability Underlying data Figshare: Data of MIC and MFC of alpha-guaiene againts four pathogenic fungi, Doi: 10.6084/m9.figshare.27925854.v1 ( Nurjanah et al., 2024 ). This project contains the following underlying data: • MIC of four pathogenic fungi.xlsx • MFC of four pathogenic fungi.xlsx Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). References Alen Y, Melati A, Sarina G, et al. : Isolation of Secondary Metabolite A. niger “In-Habiting” Queen M. gilvus Hagen.’s Nest. Indones. J. Pharm. Sci. Technol. 2018; 5 (2): 61. Publisher Full Text Almeida RN, Soares R d P, Cassel E: FRACTIONATION PROCESS OF ESSENTIAL OILS BY BATCH DISTILLATION. 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Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 02 Jan 2025 ADD YOUR COMMENT Comment Author details Author details 1 Department of Agricultural and Biosystem Engineering, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, West Java, 40600, Indonesia 2 Department of Food Industry Technology, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, West Java, 40600, Indonesia Sarifah Nurjanah Roles: Conceptualization, Data Curation, Funding Acquisition, Investigation, Resources, Writing – Original Draft Preparation, Writing – Review & Editing Zhaqqu Alhafidz Roles: Data Curation, Investigation, Methodology, Resources, Writing – Original Draft Preparation Maghfira Maulani Roles: Investigation, Methodology, Resources, Writing – Original Draft Preparation, Writing – Review & Editing Tita Rialita Roles: Conceptualization, Formal Analysis, Methodology, Validation Elazmanawati Lembong Roles: Data Curation, Methodology, Project Administration, Software, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information Universitas Padjadjaran-RKDU (Riset Kompetensi Dosen UNPAD) grant - 1427/UN6.3.1/LT/2020 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 13 Oct 2025, 14:4 https://doi.org/10.12688/f1000research.154021.2 version 1 Published: 02 Jan 2025, 14:4 https://doi.org/10.12688/f1000research.154021.1 Copyright © 2025 Nurjanah S et al . This is an open access article 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. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Nurjanah S, Alhafidz Z, Maulani M et al. Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.12688/f1000research.154021.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 02 Jan 2025 Views 0 Cite How to cite this report: Kuspradini H. Reviewer Report For: Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.5256/f1000research.168997.r356464 ) The direct URL for this report is: https://f1000research.com/articles/14-4/v1#referee-response-356464 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 16 Jan 2025 Harlinda Kuspradini , Mulawarman University Jl. Ki Hajar Dewantara, East Kalimantan, Indonesia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.168997.r356464 The manuscript is well organized but needs improvement. The discussion lacks information on the antifungal mechanism of α-guaiene and does not compare it with patchouli alcohol (PA) or patchouli oil, which would strengthen the argument. ... Continue reading READ ALL The manuscript is well organized but needs improvement. The discussion lacks information on the antifungal mechanism of α-guaiene and does not compare it with patchouli alcohol (PA) or patchouli oil, which would strengthen the argument. The effect of α-guaiene concentration on its antifungal activity is unclear. Include the number of replicates and the statistical analysis method used to validate the results. The method and results sections are inconsistent, particularly in presenting the GC-MS data. The results show that α-guaiene has antifungal properties. It should specify which fungi, α-guaiene is most effective against and discuss its limitations for broader use. Fix some minor inconsistencies in the use of italics for scientific names. 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? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Medicinal and Aromatic Plants 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. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Kuspradini H. Reviewer Report For: Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.5256/f1000research.168997.r356464 ) The direct URL for this report is: https://f1000research.com/articles/14-4/v1#referee-response-356464 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 13 Oct 2025 Sarifah Nurjanah , Department of Agricultural and Biosystem Engineering, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, 40600, Indonesia 13 Oct 2025 Author Response 1.Thank you for your valuable feedback. We appreciate your suggestion to improve the discussion section. In response, we have added a brief explanation of the antifungal mechanism of α-guaiene , highlighting ... Continue reading 1.Thank you for your valuable feedback. We appreciate your suggestion to improve the discussion section. In response, we have added a brief explanation of the antifungal mechanism of α-guaiene , highlighting its potential interaction with the fungal cell membrane and its role in disrupting ergosterol biosynthesis, similar to other sesquiterpenes. Furthermore, we have included a comparative discussion between α-guaiene, patchouli alcohol (PA) , and patchouli oil based on available antifungal activity data from recent studies. This comparison is intended to strengthen the argument regarding the potential of α-guaiene as an active antifungal component. 2.Thank you for your constructive comment. We agree that clarification on the concentration-dependent antifungal activity of α-guaiene is important. To address this, we have added supporting information in the Results and Discussion section regarding the correlation between α-guaiene concentration and its inhibitory effect against the tested fungal strain(s). Where available, we also refer to relevant quantitative data from previous studies that demonstrate dose-dependent responses. These additions aim to provide a clearer understanding of the antifungal behavior of α-guaiene across different concentrations. 3.Thank you for your valuable suggestion. We have now included the number of replicates for each experiment and specified the statistical analysis methods used to validate our results. All experiments were performed in triplicate (n = 3) , and the data are expressed as mean ± standard deviation . Statistical significance was analyzed using one-way ANOVA , followed by Tukey’s post-hoc test for multiple comparisons, with a significance level set at p < 0.05. 4.Thank you for your insightful comment. We acknowledge the inconsistency between the Methods and Results sections regarding the presentation of GC-MS data. In response, we have carefully revised the Methods section to provide a clearer description of the GC-MS operating conditions, including the type of column used, temperature program, injection volume, and identification criteria (comparison with NIST library and retention indices). Additionally, we have ensured that the compounds reported in the Results section match the analytical parameters described, and we have clarified how the major and minor components were selected and quantified. 5.Thank you for your thoughtful and constructive feedback. In response, we have clarified in the Results and Discussion section that α-guaiene exhibited the highest antifungal activity against Fusarium oxysporum , as indicated by the largest inhibition zone and lowest MIC value among the tested fungal strains. To address the limitations, we have added a brief discussion noting that while α-guaiene shows strong activity against certain fungi, its effectiveness may vary depending on the fungal species, concentration applied, and formulation conditions. Additionally, we highlighted the need for further studies on its stability, toxicity, and synergistic interactions with other components to support its broader application in commercial antifungal formulations. 6.Thank you for pointing this out. We have carefully reviewed the manuscript and corrected all inconsistencies in the formatting of scientific names. All genus and species names have now been italicized in accordance with scientific writing conventions throughout the text, including in the abstract, tables, and figure captions. 1.Thank you for your valuable feedback. We appreciate your suggestion to improve the discussion section. In response, we have added a brief explanation of the antifungal mechanism of α-guaiene , highlighting its potential interaction with the fungal cell membrane and its role in disrupting ergosterol biosynthesis, similar to other sesquiterpenes. Furthermore, we have included a comparative discussion between α-guaiene, patchouli alcohol (PA) , and patchouli oil based on available antifungal activity data from recent studies. This comparison is intended to strengthen the argument regarding the potential of α-guaiene as an active antifungal component. 2.Thank you for your constructive comment. We agree that clarification on the concentration-dependent antifungal activity of α-guaiene is important. To address this, we have added supporting information in the Results and Discussion section regarding the correlation between α-guaiene concentration and its inhibitory effect against the tested fungal strain(s). Where available, we also refer to relevant quantitative data from previous studies that demonstrate dose-dependent responses. These additions aim to provide a clearer understanding of the antifungal behavior of α-guaiene across different concentrations. 3.Thank you for your valuable suggestion. We have now included the number of replicates for each experiment and specified the statistical analysis methods used to validate our results. All experiments were performed in triplicate (n = 3) , and the data are expressed as mean ± standard deviation . Statistical significance was analyzed using one-way ANOVA , followed by Tukey’s post-hoc test for multiple comparisons, with a significance level set at p < 0.05. 4.Thank you for your insightful comment. We acknowledge the inconsistency between the Methods and Results sections regarding the presentation of GC-MS data. In response, we have carefully revised the Methods section to provide a clearer description of the GC-MS operating conditions, including the type of column used, temperature program, injection volume, and identification criteria (comparison with NIST library and retention indices). Additionally, we have ensured that the compounds reported in the Results section match the analytical parameters described, and we have clarified how the major and minor components were selected and quantified. 5.Thank you for your thoughtful and constructive feedback. In response, we have clarified in the Results and Discussion section that α-guaiene exhibited the highest antifungal activity against Fusarium oxysporum , as indicated by the largest inhibition zone and lowest MIC value among the tested fungal strains. To address the limitations, we have added a brief discussion noting that while α-guaiene shows strong activity against certain fungi, its effectiveness may vary depending on the fungal species, concentration applied, and formulation conditions. Additionally, we highlighted the need for further studies on its stability, toxicity, and synergistic interactions with other components to support its broader application in commercial antifungal formulations. 6.Thank you for pointing this out. We have carefully reviewed the manuscript and corrected all inconsistencies in the formatting of scientific names. All genus and species names have now been italicized in accordance with scientific writing conventions throughout the text, including in the abstract, tables, and figure captions. Competing Interests: The authors declare no competing interests. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 13 Oct 2025 Sarifah Nurjanah , Department of Agricultural and Biosystem Engineering, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, 40600, Indonesia 13 Oct 2025 Author Response 1.Thank you for your valuable feedback. We appreciate your suggestion to improve the discussion section. In response, we have added a brief explanation of the antifungal mechanism of α-guaiene , highlighting ... Continue reading 1.Thank you for your valuable feedback. We appreciate your suggestion to improve the discussion section. In response, we have added a brief explanation of the antifungal mechanism of α-guaiene , highlighting its potential interaction with the fungal cell membrane and its role in disrupting ergosterol biosynthesis, similar to other sesquiterpenes. Furthermore, we have included a comparative discussion between α-guaiene, patchouli alcohol (PA) , and patchouli oil based on available antifungal activity data from recent studies. This comparison is intended to strengthen the argument regarding the potential of α-guaiene as an active antifungal component. 2.Thank you for your constructive comment. We agree that clarification on the concentration-dependent antifungal activity of α-guaiene is important. To address this, we have added supporting information in the Results and Discussion section regarding the correlation between α-guaiene concentration and its inhibitory effect against the tested fungal strain(s). Where available, we also refer to relevant quantitative data from previous studies that demonstrate dose-dependent responses. These additions aim to provide a clearer understanding of the antifungal behavior of α-guaiene across different concentrations. 3.Thank you for your valuable suggestion. We have now included the number of replicates for each experiment and specified the statistical analysis methods used to validate our results. All experiments were performed in triplicate (n = 3) , and the data are expressed as mean ± standard deviation . Statistical significance was analyzed using one-way ANOVA , followed by Tukey’s post-hoc test for multiple comparisons, with a significance level set at p < 0.05. 4.Thank you for your insightful comment. We acknowledge the inconsistency between the Methods and Results sections regarding the presentation of GC-MS data. In response, we have carefully revised the Methods section to provide a clearer description of the GC-MS operating conditions, including the type of column used, temperature program, injection volume, and identification criteria (comparison with NIST library and retention indices). Additionally, we have ensured that the compounds reported in the Results section match the analytical parameters described, and we have clarified how the major and minor components were selected and quantified. 5.Thank you for your thoughtful and constructive feedback. In response, we have clarified in the Results and Discussion section that α-guaiene exhibited the highest antifungal activity against Fusarium oxysporum , as indicated by the largest inhibition zone and lowest MIC value among the tested fungal strains. To address the limitations, we have added a brief discussion noting that while α-guaiene shows strong activity against certain fungi, its effectiveness may vary depending on the fungal species, concentration applied, and formulation conditions. Additionally, we highlighted the need for further studies on its stability, toxicity, and synergistic interactions with other components to support its broader application in commercial antifungal formulations. 6.Thank you for pointing this out. We have carefully reviewed the manuscript and corrected all inconsistencies in the formatting of scientific names. All genus and species names have now been italicized in accordance with scientific writing conventions throughout the text, including in the abstract, tables, and figure captions. 1.Thank you for your valuable feedback. We appreciate your suggestion to improve the discussion section. In response, we have added a brief explanation of the antifungal mechanism of α-guaiene , highlighting its potential interaction with the fungal cell membrane and its role in disrupting ergosterol biosynthesis, similar to other sesquiterpenes. Furthermore, we have included a comparative discussion between α-guaiene, patchouli alcohol (PA) , and patchouli oil based on available antifungal activity data from recent studies. This comparison is intended to strengthen the argument regarding the potential of α-guaiene as an active antifungal component. 2.Thank you for your constructive comment. We agree that clarification on the concentration-dependent antifungal activity of α-guaiene is important. To address this, we have added supporting information in the Results and Discussion section regarding the correlation between α-guaiene concentration and its inhibitory effect against the tested fungal strain(s). Where available, we also refer to relevant quantitative data from previous studies that demonstrate dose-dependent responses. These additions aim to provide a clearer understanding of the antifungal behavior of α-guaiene across different concentrations. 3.Thank you for your valuable suggestion. We have now included the number of replicates for each experiment and specified the statistical analysis methods used to validate our results. All experiments were performed in triplicate (n = 3) , and the data are expressed as mean ± standard deviation . Statistical significance was analyzed using one-way ANOVA , followed by Tukey’s post-hoc test for multiple comparisons, with a significance level set at p < 0.05. 4.Thank you for your insightful comment. We acknowledge the inconsistency between the Methods and Results sections regarding the presentation of GC-MS data. In response, we have carefully revised the Methods section to provide a clearer description of the GC-MS operating conditions, including the type of column used, temperature program, injection volume, and identification criteria (comparison with NIST library and retention indices). Additionally, we have ensured that the compounds reported in the Results section match the analytical parameters described, and we have clarified how the major and minor components were selected and quantified. 5.Thank you for your thoughtful and constructive feedback. In response, we have clarified in the Results and Discussion section that α-guaiene exhibited the highest antifungal activity against Fusarium oxysporum , as indicated by the largest inhibition zone and lowest MIC value among the tested fungal strains. To address the limitations, we have added a brief discussion noting that while α-guaiene shows strong activity against certain fungi, its effectiveness may vary depending on the fungal species, concentration applied, and formulation conditions. Additionally, we highlighted the need for further studies on its stability, toxicity, and synergistic interactions with other components to support its broader application in commercial antifungal formulations. 6.Thank you for pointing this out. We have carefully reviewed the manuscript and corrected all inconsistencies in the formatting of scientific names. All genus and species names have now been italicized in accordance with scientific writing conventions throughout the text, including in the abstract, tables, and figure captions. Competing Interests: The authors declare no competing interests. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Batubara I. Reviewer Report For: Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.5256/f1000research.168997.r356456 ) The direct URL for this report is: https://f1000research.com/articles/14-4/v1#referee-response-356456 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 07 Jan 2025 Irmanida Batubara , IPB University, Bogor, Indonesia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.168997.r356456 The manuscript need to add following: 1. Please add the background why the guaiene concentration only at 38.8%. What is the important, how if lower or higher concentration of guaiene? 2. Why the author did not compare the ... Continue reading READ ALL The manuscript need to add following: 1. Please add the background why the guaiene concentration only at 38.8%. What is the important, how if lower or higher concentration of guaiene? 2. Why the author did not compare the activity with only PA or with the patchouli oil before fractionation. 3. The author more to clarify the fungi name compare to discuss about the activity. 4. It is important to explain the mechanism of the sample as antifungi. 5. In the method for GCMS, the author compare the retention time, but in the result author used MS data. Please make it clear 6. Please add information how many repetition for each measurement 7. It is better to make table about MIC and MFC value in one table not prepare the observation result. 8. Please compare the result with positive control as well 9. in the abstract only MIC value but have two values? 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? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: analytical chemistry 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. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Batubara I. Reviewer Report For: Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.5256/f1000research.168997.r356456 ) The direct URL for this report is: https://f1000research.com/articles/14-4/v1#referee-response-356456 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 13 Oct 2025 Sarifah Nurjanah , Department of Agricultural and Biosystem Engineering, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, 40600, Indonesia 13 Oct 2025 Author Response We would like to sincerely thank the reviewer for the valuable comments and constructive suggestions that have significantly improved the quality and clarity of our manuscript. We carefully considered each ... Continue reading We would like to sincerely thank the reviewer for the valuable comments and constructive suggestions that have significantly improved the quality and clarity of our manuscript. We carefully considered each point and have made the following revisions: Several studies have shown that a-guaiene can be isolated using molecular distillation and spinning band vacuum distillation. However, to obtain pure a-guaiene is still very difficult. Molecular distillation can only isolate guaiene with a purity level of 18.80% while using spinning band distillation can produce a-guaiene with a higher purity of 31.05%. For this reason, this study used a-guaiene with a purity of 38.8%, which was obtained from modifying the previous process conditions using spinning bands vacuum distillation. Antimicrobial activity in patchouli oil with several concentrations of PA has been carried out by the author, so that in this paper the focus is on the antimicrobial component of a-guaiene. All other necessary corrections have been incorporated into the revised version of the manuscript (3-9) We would like to sincerely thank the reviewer for the valuable comments and constructive suggestions that have significantly improved the quality and clarity of our manuscript. We carefully considered each point and have made the following revisions: Several studies have shown that a-guaiene can be isolated using molecular distillation and spinning band vacuum distillation. However, to obtain pure a-guaiene is still very difficult. Molecular distillation can only isolate guaiene with a purity level of 18.80% while using spinning band distillation can produce a-guaiene with a higher purity of 31.05%. For this reason, this study used a-guaiene with a purity of 38.8%, which was obtained from modifying the previous process conditions using spinning bands vacuum distillation. Antimicrobial activity in patchouli oil with several concentrations of PA has been carried out by the author, so that in this paper the focus is on the antimicrobial component of a-guaiene. All other necessary corrections have been incorporated into the revised version of the manuscript (3-9) Competing Interests: The authors declare no competing interests. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 13 Oct 2025 Sarifah Nurjanah , Department of Agricultural and Biosystem Engineering, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, 40600, Indonesia 13 Oct 2025 Author Response We would like to sincerely thank the reviewer for the valuable comments and constructive suggestions that have significantly improved the quality and clarity of our manuscript. We carefully considered each ... Continue reading We would like to sincerely thank the reviewer for the valuable comments and constructive suggestions that have significantly improved the quality and clarity of our manuscript. We carefully considered each point and have made the following revisions: Several studies have shown that a-guaiene can be isolated using molecular distillation and spinning band vacuum distillation. However, to obtain pure a-guaiene is still very difficult. Molecular distillation can only isolate guaiene with a purity level of 18.80% while using spinning band distillation can produce a-guaiene with a higher purity of 31.05%. For this reason, this study used a-guaiene with a purity of 38.8%, which was obtained from modifying the previous process conditions using spinning bands vacuum distillation. Antimicrobial activity in patchouli oil with several concentrations of PA has been carried out by the author, so that in this paper the focus is on the antimicrobial component of a-guaiene. All other necessary corrections have been incorporated into the revised version of the manuscript (3-9) We would like to sincerely thank the reviewer for the valuable comments and constructive suggestions that have significantly improved the quality and clarity of our manuscript. We carefully considered each point and have made the following revisions: Several studies have shown that a-guaiene can be isolated using molecular distillation and spinning band vacuum distillation. However, to obtain pure a-guaiene is still very difficult. Molecular distillation can only isolate guaiene with a purity level of 18.80% while using spinning band distillation can produce a-guaiene with a higher purity of 31.05%. For this reason, this study used a-guaiene with a purity of 38.8%, which was obtained from modifying the previous process conditions using spinning bands vacuum distillation. Antimicrobial activity in patchouli oil with several concentrations of PA has been carried out by the author, so that in this paper the focus is on the antimicrobial component of a-guaiene. All other necessary corrections have been incorporated into the revised version of the manuscript (3-9) Competing Interests: The authors declare no competing interests. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 02 Jan 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 2 (revision) 13 Oct 25 read read Version 1 02 Jan 25 read read Irmanida Batubara , IPB University, Bogor, Indonesia Harlinda Kuspradini , Mulawarman University Jl. Ki Hajar Dewantara, East Kalimantan, Indonesia Lal R.K , CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Kuspradini H. 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. 08 Nov 2025 | for Version 2 Harlinda Kuspradini , Mulawarman University Jl. Ki Hajar Dewantara, East Kalimantan, Indonesia 0 Views copyright © 2025 Kuspradini H. 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. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The revised version shows clear improvements. The authors have addressed the previous comments satisfactorily. I have no further major comments at this stage. Competing Interests No competing interests were disclosed. Reviewer Expertise Medicinal and Aromatic Plants 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. reply Respond to this report Responses (0) Kuspradini H. Peer Review Report For: Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.5256/f1000research.189216.r423550) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-4/v2#referee-response-423550 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 R.K L. 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. 29 Oct 2025 | for Version 2 Lal R.K , CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India 0 Views copyright © 2025 R.K L. 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. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript's title renders as "Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi." I have precisely examined this revised research manuscript. The authors have now updated the revised manuscript to include all suggested revisions. The manuscript presents an interesting and relevant study focusing on the isolation and characterization of α-guaiene and its antifungal efficacy. The work contributes to the growing interest in natural bioactive compounds from essential oils as potential alternatives to synthetic fungicides. The study holds potential and addresses an important natural product research area. Finally, the manuscript may be accepted for indexing. 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 Plant breeding and Genetic Resource Conservation. 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. reply Respond to this report Responses (0) R.K L. Peer Review Report For: Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.5256/f1000research.189216.r424402) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-4/v2#referee-response-424402 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Kuspradini H. 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. 16 Jan 2025 | for Version 1 Harlinda Kuspradini , Mulawarman University Jl. Ki Hajar Dewantara, East Kalimantan, Indonesia 0 Views copyright © 2025 Kuspradini H. 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. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript is well organized but needs improvement. The discussion lacks information on the antifungal mechanism of α-guaiene and does not compare it with patchouli alcohol (PA) or patchouli oil, which would strengthen the argument. The effect of α-guaiene concentration on its antifungal activity is unclear. Include the number of replicates and the statistical analysis method used to validate the results. The method and results sections are inconsistent, particularly in presenting the GC-MS data. The results show that α-guaiene has antifungal properties. It should specify which fungi, α-guaiene is most effective against and discuss its limitations for broader use. Fix some minor inconsistencies in the use of italics for scientific names. 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? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Medicinal and Aromatic Plants 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. reply Respond to this report Responses (1) Author Response 13 Oct 2025 Sarifah Nurjanah, Department of Agricultural and Biosystem Engineering, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, 40600, Indonesia 1.Thank you for your valuable feedback. We appreciate your suggestion to improve the discussion section. In response, we have added a brief explanation of the antifungal mechanism of α-guaiene , highlighting its potential interaction with the fungal cell membrane and its role in disrupting ergosterol biosynthesis, similar to other sesquiterpenes. Furthermore, we have included a comparative discussion between α-guaiene, patchouli alcohol (PA) , and patchouli oil based on available antifungal activity data from recent studies. This comparison is intended to strengthen the argument regarding the potential of α-guaiene as an active antifungal component. 2.Thank you for your constructive comment. We agree that clarification on the concentration-dependent antifungal activity of α-guaiene is important. To address this, we have added supporting information in the Results and Discussion section regarding the correlation between α-guaiene concentration and its inhibitory effect against the tested fungal strain(s). Where available, we also refer to relevant quantitative data from previous studies that demonstrate dose-dependent responses. These additions aim to provide a clearer understanding of the antifungal behavior of α-guaiene across different concentrations. 3.Thank you for your valuable suggestion. We have now included the number of replicates for each experiment and specified the statistical analysis methods used to validate our results. All experiments were performed in triplicate (n = 3) , and the data are expressed as mean ± standard deviation . Statistical significance was analyzed using one-way ANOVA , followed by Tukey’s post-hoc test for multiple comparisons, with a significance level set at p < 0.05. 4.Thank you for your insightful comment. We acknowledge the inconsistency between the Methods and Results sections regarding the presentation of GC-MS data. In response, we have carefully revised the Methods section to provide a clearer description of the GC-MS operating conditions, including the type of column used, temperature program, injection volume, and identification criteria (comparison with NIST library and retention indices). Additionally, we have ensured that the compounds reported in the Results section match the analytical parameters described, and we have clarified how the major and minor components were selected and quantified. 5.Thank you for your thoughtful and constructive feedback. In response, we have clarified in the Results and Discussion section that α-guaiene exhibited the highest antifungal activity against Fusarium oxysporum , as indicated by the largest inhibition zone and lowest MIC value among the tested fungal strains. To address the limitations, we have added a brief discussion noting that while α-guaiene shows strong activity against certain fungi, its effectiveness may vary depending on the fungal species, concentration applied, and formulation conditions. Additionally, we highlighted the need for further studies on its stability, toxicity, and synergistic interactions with other components to support its broader application in commercial antifungal formulations. 6.Thank you for pointing this out. We have carefully reviewed the manuscript and corrected all inconsistencies in the formatting of scientific names. All genus and species names have now been italicized in accordance with scientific writing conventions throughout the text, including in the abstract, tables, and figure captions. View more View less Competing Interests The authors declare no competing interests. reply Respond Report a concern Kuspradini H. Peer Review Report For: Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.5256/f1000research.168997.r356464) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-4/v1#referee-response-356464 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Batubara I. 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. 07 Jan 2025 | for Version 1 Irmanida Batubara , IPB University, Bogor, Indonesia 0 Views copyright © 2025 Batubara I. 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. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript need to add following: 1. Please add the background why the guaiene concentration only at 38.8%. What is the important, how if lower or higher concentration of guaiene? 2. Why the author did not compare the activity with only PA or with the patchouli oil before fractionation. 3. The author more to clarify the fungi name compare to discuss about the activity. 4. It is important to explain the mechanism of the sample as antifungi. 5. In the method for GCMS, the author compare the retention time, but in the result author used MS data. Please make it clear 6. Please add information how many repetition for each measurement 7. It is better to make table about MIC and MFC value in one table not prepare the observation result. 8. Please compare the result with positive control as well 9. in the abstract only MIC value but have two values? 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? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise analytical chemistry 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. reply Respond to this report Responses (1) Author Response 13 Oct 2025 Sarifah Nurjanah, Department of Agricultural and Biosystem Engineering, Faculty of Agro-Industrial Technology, Universitas Padjadjaran, Jatinangor, 40600, Indonesia We would like to sincerely thank the reviewer for the valuable comments and constructive suggestions that have significantly improved the quality and clarity of our manuscript. We carefully considered each point and have made the following revisions: Several studies have shown that a-guaiene can be isolated using molecular distillation and spinning band vacuum distillation. However, to obtain pure a-guaiene is still very difficult. Molecular distillation can only isolate guaiene with a purity level of 18.80% while using spinning band distillation can produce a-guaiene with a higher purity of 31.05%. For this reason, this study used a-guaiene with a purity of 38.8%, which was obtained from modifying the previous process conditions using spinning bands vacuum distillation. Antimicrobial activity in patchouli oil with several concentrations of PA has been carried out by the author, so that in this paper the focus is on the antimicrobial component of a-guaiene. All other necessary corrections have been incorporated into the revised version of the manuscript (3-9) View more View less Competing Interests The authors declare no competing interests. reply Respond Report a concern Batubara I. Peer Review Report For: Alpha-guaiene isolated from patchouli oil exhibits antifungal activity against four pathogenic fungi [version 1; peer review: 2 approved with reservations] . F1000Research 2025, 14 :4 ( https://doi.org/10.5256/f1000research.168997.r356456) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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