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Article

UHPLC-ESI-QTOF-MS/MS-Based Molecular Networking Guided Isolation and Dereplication of Antibacterial and Antifungal Constituents of Ventilago denticulata

1
Chulabhorn Graduate Institute, Chemical Biology Program, Chulabhorn Royal Academy, Laksi, Bangkok 10210, Thailand
2
School of Science, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand
3
Center of Chemical Innovation for Sustainability (CIS), Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand
4
Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Laksi, Bangkok 10210, Thailand
5
Center of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry of Education, Bangkok 10210, Thailand
*
Author to whom correspondence should be addressed.
Submission received: 6 August 2020 / Revised: 11 September 2020 / Accepted: 12 September 2020 / Published: 15 September 2020

Abstract

:
Ventilago denticulata is an herbal medicine for the treatment of wound infection; therefore this plant may rich in antibacterial agents. UHPLC-ESI-QTOF-MS/MS-Based molecular networking guided isolation and dereplication led to the identification of antibacterial and antifungal agents in V. denticulata. Nine antimicrobial agents in V. denticulata were isolated and characterized; they are divided into four groups including (I) flavonoid glycosides, rhamnazin 3-rhamninoside (7), catharticin or rhamnocitrin 3-rhamninoside (8), xanthorhamnin B or rhamnetin 3-rhamninoside (9), kaempferol 3-rhamninoside (10) and flavovilloside or quercetin 3-rhamninoside (11), (II) benzisochromanquinone, ventilatones B (12) and A (15), (III) a naphthopyrone ventilatone C (16) and (IV) a triterpene lupeol (13). Among the isolated compounds, ventilatone C (16) was a new compound. Moreover, kaempferol, chrysoeriol, isopimpinellin, rhamnetin, luteolin, emodin, rhamnocitrin, ventilagodenin A, rhamnazin and mukurozidiol, were tentatively identified as antimicrobial compounds in extracts of V. denticulata by a dereplication method. MS fragmentation of rhamnose-containing compounds gave an oxonium ion, C6H9O3+ at m/z 129, while that of galactose-containing glycosides provided the fragment ion at m/z 163 of C6H11O5+. These fragment ions may be used to confirm the presence of rhamnose or galactose in mass spectrometry-based analysis of natural glycosides or oligosaccharide attached to biomolecules, that is, glycoproteins.

Graphical Abstract

1. Introduction

Natural products are important sources of drugs and they provide many building blocks for drug discovery [1]. Statistically, around 50% of approved drugs were derived from natural products [2]. From 1931 to 2013, new chemical entities from natural products approved by the US Food and Drug Administration (FDA), are approximately 47% derived from plants, followed by 30% from bacteria, 23% from fungi and 5% from other natural sources [3]. As per the World Health Organisation (WHO), approximately 65% of the population of the world particularly in developing countries, mostly rely on utilization of plant-derived traditional medicines for health care and ethnomedical-based treatments [4]. Furthermore, in 2015, Youyou Tu received the Nobel Prize award for the discovery of artemisinin as an anti-malarial drug from the plant Artemisia annua; this underscores the importance of plant metabolites as sources of modern drugs [2]. According to these data, plants are rich sources of bioactive compounds, contributing significantly to drug discovery.
A conventional approach for drug discovery from natural products takes long time and high cost with hard efforts in purification, isolation and identification of natural products [5]. Moreover, the end of this process may result in the rediscovery of known bioactive compounds [6,7]. To increase the rate of the discovery of new natural products, dereplication technique is an alternative approach. Dereplication enables the identification of known compounds and the potential unknown compounds in crude extracts at the early stage of research before the isolation process [8]. The dereplication technique employs liquid chromatography-mass spectrometry(LC-MS), liquid chromatography-photodiode array detector (LC-PDA), liquid chromatography-nuclear magnetic resonance (LC-NMR) or other spectroscopic techniques [5,6] and LC-MS provides high sensitivity and effectiveness for the identification of natural products [6].
There is a limitation for LC-MS based dereplication using parent masses because it yields several molecular formulas when searching in databases [9]; this leads to less effectiveness for compound identification. Since compounds with similar structures tend to have similar MS/MS fragmentation patterns, information from MS/MS data of chemical similarity is used for molecular networking, which is considered as an effective dereplication strategy [6]. MS/MS-based molecular networking emerges as a new technique to supplement the dereplication strategy [10]. The Global Natural Products Social Molecular Networking (GNPS) website (http://gnps.ucsd.edu) is an open-access web-based mass spectrometry, facilitating high-throughput online dereplication and molecular networking analysis [11]. At present, molecular networking has been successfully employed to discover new bioactive compounds from natural sources such as the discovery of penicanesones A-C from Penicillium canescens and selaginpulvilins M-T from Selaginella tamariscina [12,13]. MS/MS-based molecular networking is involved in an untargeted fragmentation study of all compounds in crude extracts, the MS/MS spectra alignment and assembling the spectra into nodes in the network based on spectral similarity [10]. The result from MS/MS-based molecular networking is the relational networks, which reveal relationship and distribution of each chemical constituent presented in crude extracts [12,13].
Antibiotic resistance has been a public health problem worldwide. By 2050, it is predicted that death because of infection of antibiotic-resistant strains will reach approximately 10 million people per year [14]. Hence, the research on the discovery of novel antibiotics is needed. Ventilago denticulata Willd. is a plant in the family Rhamnaceae; previously it was named Ventilago calyculata. In Thailand, V. denticulata is called “Thao-Wan-Lek” or “Rhang-Dang.” Interestingly, in the West Midnapore district of West Bengal, the Eastern State of India, the plant V. denticulata is widely used to treat wound infection, suggesting the presence of antibacterial agents in this plant [15]. Bacterial strains found in wound infection were 37% of Staphylococcus aureus, 17% of Pseudomonas aeruginosa and 6% of Escherichia coli [16]. Bacteria, Bacillus cereus and Salmonella enterica serovar Typhimurium, were also found in wound infection from immunocompetent patients or diabetes mellitus patients [17,18]. Candida albicans was the most widely detected fungus in wound infection especially in diabetic foot ulcers [19]. Therefore, this research aims to explore antibacterial and antifungal agents in V. denticulata. Previously, a crude bark extract of V. denticulata was reported to show the antibacterial and antifungal activities [20,21]. Our previous work revealed that V. denticulata had a few antibacterial agents [22]. Based on these studies, V. denticulata could be a potential source of medicinally useful compounds, especially antimicrobial and antifungal agents. This work explores antibacterial agents in crude extracts and fractions of V. denticulata using UHPLC-ESI-QTOF-MS/MS analysis, as well as a molecular networking. It is known that different parts of plants may have different chemical constituents and thus exerting different pharmacological effects [23]. We report herein antibacterial and antifungal compounds in both bark and trunk of a plant, V. denticulata.

2. Results and Discussions

2.1. Dereplication of Compounds from Crude Extracts of V. denticulata and Guided Isolation by UHPLC-ESI-QTOF-MS/MS-Based Molecular Networking

Fresh trunk and bark of V. denticulata were sequentially extracted with methanol (MeOH) and dichloromethane (CH2Cl2). Both MeOH and CH2Cl2 crude extracts were analyzed by UHPLC-ESI-QTOF-MS/MS. In this research, there were two scan types; first, LC-MS scans a total ion chromatogram (TIC) and base peak chromatogram (BPC). Both positive and negative MS ionization modes were performed because some classes of compounds such as sesquiterpenes and thiophenes were well-detected in a positive ionization mode, whereas flavonoids, phenolic acids and quinic acid could be detected by a negative ion mode [24]. Besides, the mechanism of fragmentation of positive and negative ion modes was dissimilar and they may afford supplementary structural information [25]. Overlay of TIC chromatograms of MeOH and CH2Cl2 crude extracts of V. denticulata is shown in Figure S1, Supplementary Materials. Second, auto-MS2 was performed in which the most predominant MS1 ions are chosen for MS2 fragmentation. From MS/MS spectra, the chemical constituents in crude extracts of V. denticulata were tentatively identified; they are listed in Table 1. The putative known and unknown compounds were annotated by the Agilent MassHunter METLIN Metabolomics Database, the Human Metabolome Database (https://hmdb.ca/) and online database Metlin (http://metlin.scripps.edu/index.php), as well as by comparison with standard compounds. The present work has seven standard compounds including (+)-R-ventilagolin, emodin, rutin, naringenin, 6-hydroxy flavone, chrysin and (+)-catechin.
As shown in Table 1, several compounds in crude extracts were identified in either positive or negative ionization mode. There are 93 tentatively identified compounds listed in Table 1; these metabolites have been reported as plant metabolites. Among the compounds identified in Table 1, emodin, physcion, ventilagodenin A and (+)-(R)-ventilagolin previously isolated by our group [22] were indeed found in crude extracts of V. denticulata and they underwent MS/MS fragmentation in both positive and negative ionization modes. We performed further analysis using the GNPS website; all acquired MS/MS data were converted into MzXML as an open file format by ProteoWizard. Then, the converted data were uploaded to create molecular networking on the GNPS website (http://gnps.ucsd.edu). All molecular networking data obtained from the GNPS system were imported to Cytoscape 3.7.2 version, in order to visualize and simplify molecular networking in one display. The node colors were set and they represented MS/MS data of compounds present in crude extracts or standard compounds. Cytoscape was used for rapid analysis of the whole profile of metabolites in all crude extracts, as well as for the correlation between standard compounds and their analogs. Result of the molecular networking of crude extracts in a positive mode is shown in Figure 1a, while that of a negative ionization mode is in Supplementary data (Figure S2); they are used as a complementary method for the dereplication strategy.
We employed a molecular networking for the investigation of a profile of chemical constituents in crude extracts of V. denticulata, basically with that of crude extracts in a positive ionization mode (Figure 1a). Colors for MeOH extracts of bark and trunk, as well as three crude extracts of bark and trunk, are depicted in Figure 1. In the present work, (+)-(R)-ventilagolin (1), a naphthalene derivative, was used as a standard compound (purple color, Figure 1b) and it found in MeOH and CH2Cl2 crude extracts of bark and CH2Cl2 crude extract of trunk but not in MeOH crude extract of trunk (Table 1). The molecular networking of (+)-(R)-ventilagolin (1) is in a cluster A (Figure 1b). Rutin (2), a flavonol glycoside, was also used as a standard compound and its molecular networking is in a cluster B, as shown in Figure 1c. The dereplication by MS/MS based molecular networking in a positive ionization mode also suggested the presence of a potential new naphthalene derivative (Figure 1b) and flavonol glycoside derivatives (Figure 1c), by inspecting nodes in the clusters connected to (+)-(R)-ventilagolin (1) and rutin (2), respectively.
The molecular networking of (+)-(R)-ventilagolin (1) (m/z 333.0971 [M+H]+) (cluster A; Figure 1b) showed the node of MS/MS spectra related to the ion at m/z 351.1075 [M+H]+ with cosine similarity score of 0.80. A putative unknown compound observed at m/z 351.1075 [M+H]+ had a mass difference of 18 from (+)-(R)-ventilagolin (1) (m/z 333.0971 [M+H]+, calcd for [C17H16O7 + H]+, 333.0974, Δm/z = 0.90 ppm), suggesting that a putative new compound has an additional hydroxyl group. The tentative new derivative had the observed ion at m/z 351.1075 [M+H]+, calcd for [C17H18O8 + H]+, 351.1080, Δm/z = 1.42 ppm and thus having the molecular formula of C17H18O8. MS/MS spectra of both (+)-(R)-ventilagolin (1) and a putative new derivative showed the ions at m/z 276 and 259 (Figures S3 and S4, Supplementary Materials); a typical MS/MS fragmentation of (+)-(R)-ventilagolin (1) is depicted in Figure 2, showing the ion at m/z 276.0630 of [C14H12O6]+, 276.0628, Δm/z = 0.72 ppm. Based upon the typical MS fragmentation of (+)-(R)-ventilagolin (1), the tentative structure of a new derivative observed at m/z 351.1075 [M+H]+ (cluster A; Figure 1b) is proposed to be either 3-hydroxy-ventilagolin (3) or 4-hydroxy-ventilagolin (4), as shown in Figure 2. MS/MS spectrum (Figure S4, Supplementary Materials) of a putative new compound showed that it underwent neutral loss of water, giving a fragment ion at m/z 333.0949 [M+H]+, calcd for [C17H16O7 + H]+, 333.0974, Δm/z = 7.50 ppm (Figure 2), which is of (+)-(R)-ventilagolin (1), which in turn, fragmented to the ion at m/z 276.0630, calcd for [C14H12O6]+, 276.0628, Δm/z = 0.72 ppm (Figure 2 and Figure S4) that is a typical MS fragmentation for this compound class. Unfortunately, we could not isolate the putative new derivative for detailed NMR analysis. It is worth mentioning that 3-hydroxy-ventilagolin (3) has a similar structural feature to a fungal pigment, fusarubin (5) (Figure 2) [26,27], which also has an anhydro derivative, anhydrofusarubin (6) [27] (Figure 2), whose structure is similar to that of (+)-(R)-ventilagolin (1). By analogy to the structures of fusarubin (5) and anhydrofusarubin (6), the putative new compound is possibly 3-hydroxy-ventilagolin (3) (Figure 2).
In a cluster B (Figure 1c), node of MS/MS spectra connected to rutin (2) (m/z 633.1422 [M+Na]+), a standard compound, possessed a precursor ion of xanthorhamnin C or rhamnazin 3-rhamninoside (7) (Figure 1c and Figure 3) at m/z 785.2503 [M+H]+ with a cosine similarity score of 0.79. Rhamnazin 3-rhamninoside (7) was isolated and characterized by analysis of 1D and 2D NMR spectroscopy (1H, 13C NMR and MS spectra are in Figures S10–S12, Supplementary Materials). Spectroscopic data of rhamnazin 3-rhamninoside (7) were in good agreement with those reported in the literature [28]. Rhamnazin 3-rhamninoside (7) had related precursor ions at m/z 755.2394 [M+H]+ with a cosine similarity score of 0.97, at m/z 771.2343 [M+H]+ with a cosine similarity score of 0.94 and at m/z 741.2233 [M+H]+ with a cosine similarity score of 0.90, which are catharticin or rhamnocitrin 3-rhamninoside (8), xanthorhamnin B or rhamnetin 3-rhamninoside (9) and kaempferol 3-rhamninoside (10), respectively (Figure 1c and Figure 3). Flavonol glycosides 8-10 were also isolated and structurally characterized by analysis of 1D and 2D NMR spectroscopy (1H, 13C NMR and MS spectra are in Figures S13–S21, Supplementary Materials). Spectroscopic data of compounds 810 were identical to those published in the literature [28,29,30,31]. Moreover, flavovilloside or quercetin 3-rhamninoside (11) (Figure 3) was also obtained during the isolation of flavonol glycosides 710; its 1H, 13C NMR and MS spectra are in Figures S22–S24, Supplementary Materials). Spectroscopic data of quercetin 3-rhamninoside (11) were in good agreement with published values [28]. However, quercetin 3-rhamninoside (11) was not detected by LC-MS/MS analysis; therefore, it is not listed in Table 1 and it does not appear in the molecular networking of a cluster B (Figure 1c) in spite of being a derivative of rutin (2). The sugar in a standard flavonol glycoside, rutin (2), is glucose, while that in the isolated flavonol glycosides 711 is galactose (Figure 3). In a cluster B (Figure 1c), compounds with the ions at m/z 412.1027 [M+2H]2+ and 397.0973 [M+2H]2+ had related precursor ions to rhamnazin 3-rhamninoside (7) and they were considered as potential new compounds. Unfortunately, attempts to isolate these compounds for detailed NMR analysis have met with failure. It is worth mentioning that HPLC-PDA method could be used to distinguish 3′,4′-dihydroxy flavonoid (i.e., flavonol glycosides 7, 9 and 11) from 4′-dihydroxy flavonoid derivative (i.e., flavonol glycosides 8 and 10) (Figure 3). 3′,4′-Dihydroxy flavonoid had a typical λmax at 356 nm in the UV spectrum, while 4′-dihydroxy flavonoid derivative showed a typical λmax at 348 nm (Figure 3).
Analysis of MS/MS spectrum (Figure S5, Supplementary Materials) of a standard flavonol glycoside, rutin (2), revealed losses of glucose and rhamnose, showing the ions resulting from the loss of rhamnose (at m/z 465 from loss of 146) and of glucose-rhamnose (at m/z 303) (Figure 4). Loss of 146 of rhamnose gave the ion at m/z 465 and such loss was previously observed for flavonoid glycosides [25] and triterpene saponins [32]. Interestingly, the ion abundance at m/z 147.0653, calcd [C6H11O4 +H]+, which was of a rhamnose fragment, was 4 times lower than that of the ion at m/z 129.0547 (Figure S5, Supplementary Materials). Careful analysis revealed that the observed ion at m/z 129.0547 could be of an oxonium ion of a sugar rhamnose, which was from a neutral loss of water of a rhamnose fragment at m/z 147.0653, as depicted in Figure 4. The observed ion at m/z 129.0547 and the calculated m/z value of 129.0546 for C6H9O3+ with the mass difference of 0.26 ppm (Figure 4) readily confirmed the structure of an oxonium ion of rhamnose. Normally, oxonium ions of sugar are observed in MS/MS spectra of glycosides [33] and they are useful ions for sugar identification in modern glycoproteomic research [34,35]. To the best of our knowledge, this is the first report on the oxonium ion of rhamnose, C6H9O3+ at m/z ca 129.05 and it is possibly used as a characteristic fragment ion for rhamnose in mass spectrometry.
Molecular networking of rutin (2) (cluster B, Figure 1c) had the precursor ion of xanthorhamnin C or rhamnazin 3-rhamninoside (7) at m/z 785. 2503 [M+H]+ with a cosine similarity score of 0.79. MS/MS spectrum (Figure S6, Supplementary Materials) of rhamnazin 3-rhamninoside (7) showed fragment ions analogous to that of rutin (2), that is, loss of rhamnose giving the oxonium ion at m/z 129.0540 (Figure 4). The major fragments at m/z 493 and 331 due to loss of rhamnose-rhamnose followed by loss of galactose were observed in the MS/MS spectrum of rhamnazin 3-rhamninoside (7) (Figure 4 and Figure S6). Unlike rutin (2), the MS/MS spectrum rhamnazin 3-rhamninoside (7) displayed the ion at m/z 163.0599 (Figure 4 and Figure S6), C6H11O5+, calcd for 163.0601 (mass difference of 1.22 ppm), which was likely to be a fragment of galactose, C6H11O5+. Flavonol glycosides 810 have galactose in their molecules; indeed, the MS/MS spectra of these compounds showed a fragment ion of galactose at m/z 163 (Figures S7–S9, Supplementary Materials). While glucose in rutin (2) does not have a fragment ion at m/z 163, galactose in flavonol glycosides 810 shows the characteristic fragment ion at m/z 163; therefore, the fragment ion at m/z 163 might be used for the identification of galactose in mass spectrometry-based analysis of glycosides or oligosaccharide chains attached to biomolecules (i.e., glycoproteins).
In the present study, ventilatone B (12), a triterpene lupeol (13) and ventilatone A (15) (Figure 5) were also isolated from a CH2Cl2 extract of bark of V. denticulata. Ventilatones B (12) and A (15) are benzisochromanquinone, which were previously isolated from V. calyculata [36]. Structures of ventilatone B (12), lupeol (13) and ventilatone A (15) were characterized by analysis of NMR spectroscopy (1H, 13C NMR and MS spectra are in Figures S27–S34, Supplementary Materials); their spectroscopic data were in good agreement with those reported in the literature [36,37]. Lupeol (13) was previously found in the plant genus Ventilago, for example, V. denticulata [38] and V. bombaiensis [39]. Note that rhamnalpinogenin (14) (Figure 5), which has the same molecular formula, C17H12O7, as that of ventilatone B (12), was tentatively identified by LC-MS/MS analysis, as revealed by both the Metlin Database and the Human Metabolome Database (Table 1, No. 70), observed at m/z 329.0659, calcd for 329.0656 (Δm/z = 1.09 ppm). However, there is a possibility that this putative compound is ventilatone B (12) because this benzisochromanquinone was previously isolated from V. calyculata [36], which is the same plant used in this work (V. denticulata formerly known as V. calyculata). The MS/MS spectrum (Figure S25, Supplementary Materials) of the compound with the molecular formula C17H12O7 suggested that it is more likely to be ventilatone B (12) because of the loss of C2HO, giving the fragment ion at m/z 287.0551(Figure 5). In the case of rhamnalpinogenin (14), it should undergo a neutral loss of CO2 (44 amu) because it has a carboxylic group in its molecule (Figure 5) but none of the fragment ions were observed from the loss of CO2. Moreover, the molecular networking of ventilatone B (12) is related to the compound with the m/z 313.0706 with a cosine similarity score of 0.84 (Figure 5). Analysis of MS/MS spectrum (Figure S26, Supplementary Materials) of the compound with the m/z 313.0706 revealed that this compound is likely to be ventilatone A (15), which undergoes the loss of C2HO, giving the fragment ion at m/z 271.0604 (Figure 5) that is analogous to ventilatone B (12). Note that the compound at the m/z 313.0706 was also listed in Table 1 (No. 61) and it was proposed to be aloe emodin w-acetate by Metlin Database and Human Metabolome Database. However, the MS/MS fragmentation suggested that this compound should be ventilatone A (15), not aloe emodin w-acetate.

2.2. Structure Elucidation of Ventilatone C (16)

In the present work, a new compound—named ventilatone C (16)—was isolated from a CH2Cl2 extract of a bark of V. denticulata (Figure 6). Structure elucidation of ventilatone C (16) was performed by analysis of NMR and MS data. Ventilatone C (16) was obtained as yellow amorphous solid and its molecular formula, C17H14O5, was obtained from ESI-HRMS, showing a pseudo-molecular ion at m/z 299.0917 (M+H)+, calcd for C17H15O5, m/z 299.0919. 1H and 13C NMR spectra of ventilatone C (16) were similar to those of ventilatones B (12) and A (15), particularly on the signals for the fragment of 3-Me/H-3/H-4. 1H NMR spectrum in CDCl3 of 16 showed signals of a hydroxyl proton at δH 8.81 (br s), three aromatic protons at δH 7.24 (H-5), 6.72 (H-6 and H-8), one olefinic proton at δH 5.74, sp3 methine at δH 4.53 (H-3), non-equivalent methylene at δH 3.15 and 3.00 (H-4) and two methyl groups at δH 3.91 (7-OMe) and 1.56 (3-Me) (Table 2). 1H NMR signals in CDCl3 for H-6 and H-8 were overlapping at δH 6.72, however, these signals were clearly observed in acetone-d6 as a doublet at δH 6.94 (H-6) and 6.64 (H-8) and the J = 2.3 Hz (Table 2) indicated the presence of meta coupling aromatic protons in 16. 13C NMR and DEPT spectra of ventilatone C (16) showed seventeen signals attributable to two methyl, five methine, one methylene, nine quaternary carbons. 1H-1H COSY spectrum of 16 established the fragment of 3-Me/H-3/H-4 (as a bold line in Figure 6). HMBC spectrum of 16 showed the correlations from 3-Me to C-4; H-4 to C-4a; H-5 to C-4, C-5a, C-6, C-9a and C-10a; H-6 to C-5, C-7, C-8 and C-9a; H-8 to C-7 and C-9a; and H-13 to C-1, C10a and C-12 (Figure 6). The HMBC correlation from 7-OMe to C-7 placed the methoxy group at the position C-7, while that from 9-OH proton to C-8, C-9 and C-9a assigned the OH group at C-9. Ventilatone C (16) had a positive optical rotation value ([α]25D +2.60 (c 0.25, CHCl3)) similar to those of ventilatones B (12) ([α]25D +30.62 (c 0.5, CHCl3) and A (15) ([α]25D +7.85 (c 0.2, CHCl3)), both having 3S stereochemistry, therefore, the C-3 configuration in 16 was assigned to be S. Based on these spectroscopic data, the structure of ventilatone C (16) was established as shown in Figure 6. Ventilatone C (16) has a structure closely related to pannorin B (17) [40] (Figure 6). However, pannorin B (17) was previously isolated from an endophytic fungus Penicillium sp. [40] and its biosynthetic pathway was proposed to be related to that of pannorin [41]. Interestingly, a fungal metabolite, pannorin B (17), shares the same chemical skeleton as that of ventilatones B (12) and A (15), which were isolated from the plant, V. calyculata [36].

2.3. Antibacterial and Antifungal Activities of Crude Extracts, Fractions and Isolated Compounds

As mentioned in the introduction part, local people in the West Midnapore district of West Bengal, the Eastern State of India, use the plant V. denticulata for the treatment of wound infection [15]. Bacteria found in wound infection were Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus cereus and Salmonella enterica serovar Typhimurium [16,17,18], while the fungus Candida albicans was found in wound infection in diabetic foot ulcers [19]. Therefore, this research evaluated antibacterial and antifungal activities of crude extracts against S. aureus, P. aeruginosa, E. coli, B. cereus, S. enterica and C. albicans (Table 3). Methanol crude extracts of bark (MB) and trunk (MT) exhibited antibacterial activity against B. cereus, S. aureus, E. coli, S. enterica and P. aeruginosa with inhibition zones of 7–13 mm, 14–15 mm, 8 mm, 7–14 mm and 7–10 mm, respectively (Table 3). A CH2Cl2 crude extract of bark (DB) displayed antibacterial activity against B. cereus, S. aureus, E. coli, S. enterica and P. aeruginosa with inhibition zones of 21 mm, 18 mm, 9 mm, 19 mm and 8 mm, respectively (Table 3), while a CH2Cl2 crude extract of trunk (DT) showed antibacterial activity against S. aureus with inhibition zone of 13 mm (Table 3). Crude extracts, MB, DB and MT exhibited antifungal activity against C. albicans with inhibition zones of 13 mm, 16 mm and 8 mm, respectively (Table 3). Fractions FM1-FM6 obtained from HPLC isolation of MeOH crude extract of bark were evaluated for antibacterial and antifungal activities. Fractions FM1-FM3 showed antibacterial activity toward the bacterial strains tested with inhibition zones of 8–14 mm, except that the fraction FM1 did not inhibit the growth of S. enterica (Table 3). Fraction FM4 exhibited the activity against S. enterica with inhibition zone of 14 mm, while fraction FM6 displayed the activity toward bacteria E. coli and S. enterica with inhibition zone of 9 mm (Table 3). Fraction FM2 exhibited antifungal activity with inhibition zone of 10 mm. Fractions FD1-FD6 from fractionation of a CH2Cl2 crude extract of bark displayed antibacterial activities toward the bacterial strains tested with inhibition zones of 9–30 mm, while the fractions FD1 and FD6 showed antifungal activity against C. albicans with inhibition zones of 17 and 9 mm, respectively (Table 3).
Flavonoid glycosides 711 were isolated from MeOH crude extract of bark of V. denticulata and they were evaluated for antibacterial and antifungal activities (Table 3). Rhamnazin 3-rhamninoside (7) exhibited antibacterial activity against S. aureus with inhibition zone of 11 mm, while catharticin or rhamnocitrin 3-rhamninoside (8) showed the activity toward B. cereus and E. coli with respective inhibition zones of 10 mm and 11 mm (Table 3). Xanthorhamnin B or rhamnetin 3-rhamninoside (9) displayed antibacterial activity against B. cereus, S. aureus and P. aeruginosa with respective inhibition zones of 9 mm, 9 mm and 13 mm (Table 3). Kaempferol 3-rhamninoside (10) and flavovilloside or quercetin 3-rhamninoside (11) exhibited antibacterial activity against B. cereus, S. aureus and E. coli with inhibition zones of 9 mm, 11–14 mm and 10–12 mm, respectively (Table 3) but they did not possess antifungal activity toward C. albicans. Rhamnazin 3-rhamninoside (7), rhamnocitrin 3-rhamninoside (8) and xanthorhamnin B or rhamnetin 3-rhamninoside (9) displayed antifungal activity against C. albicans with inhibition zones of 8 mm, 12 mm and 6 mm, respectively (Table 3). To our knowledge, this is the first report on antibacterial and antifungal activities of flavonoid glycosides 711. Recently, xanthorhamnin B or rhamnetin 3-rhamninoside (9) was found to have antioxidative and radioprotective properties [42]. Rhamnazin 3-rhamninoside (7), rhamnocitrin 3-rhamninoside (8) and rhamnetin 3-rhamninoside (9) were reported to exhibit antioxidant and free radical-scavenging activities [43]. Glycoside derivatives of kaempferol were previously found to exhibit potent antibacterial activity against methicillin-resistant S. aureus and vancomycin-resistant enterococci [44]. Previously, kaempferol, an aglycone of 10, was found to exhibit antibacterial activity toward E. coli and it acted as DNA gyrase inhibitor [45], while quercetin, an aglycone of 11, exhibited antibacterial and antioxidant activities [46], targeting D-alanine:D-alanine ligase [47]. Interestingly, quercetin diacylglycoside derivatives displayed antibacterial activity by inhibition of DNA gyrase and topoisomerase IV [48].
Ventilatone B (12), lupeol (13), ventilatones A (15) and ventilatone C (16) isolated from a CH2Cl2 crude extract of bark of V. denticulata displayed antibacterial and antifungal activities (Table 3). Ventilatone B (12) exhibited antibacterial activity against B. cereus, S. aureus and S. enterica with inhibition zones of 11 mm, 11 mm and 18 mm, respectively and it also showed antifungal activity against C. albicans with inhibition zone of 12 mm (Table 3). Lupeol (13) displayed antibacterial activity against S. aureus with inhibition zone of 7 mm (Table 3) but did not exhibit antifungal activity. This is the first report on antibacterial and antifungal activities of ventilatone B (12). Lupeol (13) was previously reported to exhibit antibacterial activity against human pathogenic bacteria [49]. Ventilatones A (15) exhibited antibacterial activity against B. cereus, S. aureus and S. enterica with inhibition zones of 13 mm, 17 mm and 18 mm, respectively, while ventilatone C (16) displayed antibacterial activity against B. cereus, S. aureus and S. enterica with inhibition zones of 13 mm, 13 mm and 14 mm, respectively (Table 3).

2.4. Dereplication of Antibacterial and Antifungal Constituents from HPLC Fractions of V. denticulata

Fractions FM1-FM3 and FD1-FD4 from HPLC separation showed antibacterial and antifungal activities (Table 3); therefore, efforts have been made to identify the compounds in these HPLC fractions. Since the tentatively identified compounds (Table 1) in V. denticulata were obtained from LC-MS/MS analysis using Metlin Database and Human Metabolome Database, as well as standard compounds, we employed the accurate mass from ESI-HRMS to identify the compounds in fractions possessing antibacterial and antifungal activities. The ranges of mass difference (Δ) between the observed and calculated m/z values for each compound were ca 0.55–2.42 ppm, which is less than 5 ppm and thus giving the molecular formula of the compounds. ESI-HRMS analysis revealed that the fraction FM1 contained kaempferol, chrysoeriol, kaempferol 3-rhamninoside (10), isopimpinellin, 3-hydroxyphloretin, rhamnocitrin 3-rhamninoside (8), rhamnetin 3-rhamninoside (9) and rhamnazin 3-rhamninoside (7) (Table 4). Antibacterial and antifungal activities of flavonoid glycosides 7–10 are already presented in Table 3. Kaempferol was previously found to be an antibacterial agent [45,50]. Antibacterial activity of a flavonoid, chrysoeriol, was recently reported [51,52], while antibacterial and antifungal activities of isopimpinellin were already established [53]. Therefore, all compounds in the fraction FM1 have antibacterial activity, except 3-hydroxyphloretin. Fraction FM2 contained rhamnetin, luteolin and 3,5,7-trihydroxy-4′,6-dimethoxyflavanone (Table 4), as revealed by ESI-HRMS analysis. Rhamnetin was previously found to have antifungal activity and it is a phytoalexin in plants [54], while luteolin was formerly found to exhibit antifungal activity [55]. Luteolin is a known antibacterial agent [56,57] and it is a lead compound for the synthesis of antibacterial derivatives [58]. 3,5,7-Trihydroxy-4′,6-dimethoxyflavanone was formerly isolated from a plant, Prunus domestica [59] but it has never been evaluated for any biological activity. ESI-HRMS analysis showed that the fraction FM3 had emodin, rhamnocitrin and palmidin A (Table 4). Antibacterial activity of emodin was reported by our group [22] and emodin was previously found to inhibit growth of the bacterium Haemophilus parasuis, a causative agent of Glässer’s disease and thus being a potential drug candidate for treating Glässer’s disease [60]. Previously, antibacterial activity of rhamnocitrin was reported [61,62], while the activity of palmidin A has never been reported to date. Overall, eleven antibacterial compounds including flavonoid glycosides 710, kaempferol, chrysoeriol, isopimpinellin, rhamnetin, luteolin, emodin and rhamnocitrin are tentatively identified from the active fractions FM1-FM3, suggesting that the dereplication technique by LC-MS/MS analysis rapidly identifies antibacterial agents in extracts and fractions. In the present work and our previous report [22], antibacterial glycosides 710 and emodin were isolated from V. denticulata.
ESI-HRMS analysis for compounds in fractions FD1-FD4 obtained from HPLC separation was performed (Table 4). Fraction FD1 contained eriodyctiol, cartorimine, chrysoeriol, rhamnetin, 3-hydroxyphloretin, xanthotoxol glucoside and furocoumarinic acid glucoside (Table 4); among these compounds, chrysoeriol and rhamnetin were previously found to have antibacterial and antifungal activities [51,52,54]. ESI-HRMS analysis revealed that the fraction FD2 contained ventilagodenin A, physcion, rhamnocitrin, ventilatone A (15), 3′,7-dihydroxy-4′,8-dimethoxyisoflavone, rhamnazin, 3,5,7-trihydroxy-4′,6-dimethoxyflavanone and ventilatone B (12) (Table 4). Ventilagodenin A was found to be an antibacterial agent by our group [22], while ventilatones B (12) and A (15) were isolated in the present work; their antibacterial and antifungal activities are reported in Table 3. Rhamnocitrin and rhamnazin were formerly found as antibacterial agents [61,62]. FD3 was found to contain afzelechin, (+)-(R)-ventilagolin and mukurozidiol (Table 4); among these compounds, (+)-(R)-ventilagolin (1) was found to be an antibacterial compound by our research group [22], whereas mukurozidiol or byakangelicin was previously reported to have antibacterial activity [63]. FD4 contained emodin, 6α-hydroxymaackiain, 2′,3,5-trihydroxy-5′,7-dimethoxyflavanone and palmidin A (Table 4); however, only emodin was found to be an antibacterial agent [22,60]. Overall, ten antibacterial compounds including chrysoeriol, rhamnetin, ventilagodenin A, rhamnocitrin, rhamnazin, mukurozidiol, emodin, (+)-(R)-ventilagolin (1) and ventilatones B (12) and A (15) were identified from fractions FD1-FD4.

3. Materials and Methods

3.1. General Experimental Procedures

UHPLC-MS/MS was carried out using Agilent 1290 infinity II connected to Agilent 6545 QTOF. HPLC column is ACE Excel C18 AR (100 × 2.1 mm, 1.7 µm) column. MS data were processed using MassHunter data acquisition software. ESI-HRMS spectra were acquired from Bruker MicroTOF mass spectrometer processed using Bruker daltonics data analysis 3.3 software. HPLC was performed by Waters 1525 binary pump connected to a 2998 photodiode array detector. A semi-preparative column is SunFire C18 (19 × 250 mm, 5.0 µm); the HPLC chromatogram was processed by Empower 2 software. NMR spectra were obtained from Bruker Avance 400 MHz NMR spectrometer, processed by TopSpin software. Sephadex LH-20 was packed for column chromatography. Specific optical rotation of compound 16 was obtained from a JASCO P-1020 polarimeter.
Methanol hypergrade LiChrosolv (LC-MS grade) and formic acid LiChropur (LC-MS grade) were used as the mobile phase for LC-MS analysis. Methanol-d4, CDCl3, acetone-d6 were used as solvents for NMR analysis.

3.2. Plant Materials and Extraction of Plant

The plant Ventilago denticulata was collected from Prachin Buri province, Thailand. It was characterized by Forest Herbarium, Bangkok, Thailand, in April 2019. V. denticulata (Voucher specimen number: CRI712) was deposited at Chulabhorn Research Institute (CRI), Thailand. Fresh trunks of V. denticulata were separated from their barks and then cut into small pieces (around ± 0.5 cm). Fresh plant samples were sequentially with MeOH and CH2Cl2; this is because fresh samples contain water and MeOH, a water-miscible solvent, was first used as a solvent. Trunk (1.7 kg) was macerated sequentially with MeOH (2 × 1.5 L) and CH2Cl2 (2 × 1.5 L) at room temperature for 2 days to give 34.66 g of MeOH crude extract of trunk and 15.03 g of CH2Cl2 crude extract of trunk. Bark (0.5 kg) was macerated sequentially with MeOH (2 × 1.0 L) and CH2Cl2 (2 × 1.0 L) at room temperature for 2 days to give 24.38 g of MeOH crude extract of bark and 1.24 g of CH2Cl2 crude extract of bark. All crude extracts were stored and kept in a freezer (−18 °C).

3.3. Crude Extract and Preparation of Standard Compounds for LC-MS/MS Analysis

1 mg of each crude extract was dissolved in 1 mL of methanol to make a stock solution with a concentration of 1 mg/mL. 100 µL of each stock solution was diluted with 900 µL MeOH to obtain the final concentration of 100 µg/mL. This solution was filtered through 0.22 µm and transferred into 2 mL-LC vial.
Stock solutions of the following compounds (1 mg each), ((+)-R-ventilagolin, emodin, rutin, naringenin, 6-hydroxy flavone, chrysin and (+)-catechin) were dissolved in 1 mL of methanol. 100 µL of each stock solution was diluted with 900 µL methanol to obtain the final concentration of 100 µg/mL. These solutions were filtered through 0.22 µm filter and transferred into 2 mL-LC vial.

3.4. UHPLC-ESI-QTOF-MS/MS Conditions

Crude extracts and standard compounds were analyzed by UHPLC connected to Q-TOF MS. UHPLC column was ACE Excel C18 AR (100 × 2.1 mm, 1.7 µm) and a flow rate was 0.2 mL/min with an injection volume of 0.5 µL. The gradient elution was performed using the following conditions: (i) linear gradient from 40% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) to 100% CH3CN (0.1% formic acid) for 0–25 min, (ii) isocratic elution of 100% CH3CN (0.1% formic acid) for 5 min (at time of 25–30 min), (iii) a linear gradient from 100% CH3CN (0.1% formic acid) to 40% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 4 min (at time of 30–34 min) and (iv) equilibrium time by isocratic elution with 40% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 6 min (at time of 34–40 min). The total run time was 40 min.
Dual AJS (Agilent Jet Stream) ESI was used as an ion source arranged with sheath gas flow of 12 L/min, capillary temperature at 325 °C, the gas flow rate of 10 L/min, sheath gas temperature of 250 °C, sheath gas flow of 12 L/min, nebulizer of 45 psig, capillary voltage of 3.5 kV, fragmentor of 150 V, skimmer of 65 V and nozzle voltage of 1 kV. MS relative threshold and MS absolute threshold were set to 0.010% and 100, respectively.
LC-MS scan total ion chromatogram (TIC) and base peak chromatogram (BPC) with a scan range of 100–1100 m/z and the analysis was performed in both positive and negative ionization modes. MS scan rate is 2 spectra per min. Auto-MS2 was performed using fixed collision energy at 20 keV, at which the most predominant MS1 ions are chosen for MS2 fragmentation. Auto-MS2 acquisition shows MS/MS data around 80–95% of precursor ions. The MS/MS data were acquired with a scan rate of 3 spectra per second with MS/MS scan range at 100–1100 m/z. Isolation width MS/MS was set at medium (ca 4 amu). The maximum precursor was 3 per cycle. The MS/MS relative threshold was set to 0.01% and MS/MS absolute threshold was set to 5.
The reference mass correction was performed and set as auto recalibration using a reference solution with minimal height of 1000 counts and the detection window of 100 m/z. The ions at m/z 121.0509 (purine) and m/z 922.0098 (HP-0921) were selected as standard ion peaks in a positive ion mode, while the ions at m/z 112.9856 (TFA anion) and m/z 1033.9881 (HP-0921 + TFA anion) were selected as standard ion peaks in a negative ion mode. In the auto MS/MS preferred/exclude table, these reference masses must be written as exclusion mass [64].

3.5. Molecular Networking

3.5.1. Converting MS/MS Data

All acquired MS/MS data was converted into MzXML format for further analysis in the GNPS website by ProteoWizard supported by NET Framework 3.5 SP1 using the following parameters [7]:
  • 32-Bit was selected for binary encoding precision and zip compression was unchecked.
  • Peak picking was set as a filter to make the output data become centroid.
  • MS-Levels 1 and 2 should be checked.

3.5.2. Molecular Networking by GNPS (Global Natural Products Social Molecular Networking)

FTP client, WinSCP, was used to upload the converted MS/MS data to the MzXML format using the host ost ccms-ftp01.ucsd.edu; these data to system were then transferred automatically to the GNPS system. The uploaded data were available in GNPS website readily for uploading data to create the molecular networking on the GNPS website (http://gnps.ucsd.edu).
In the basic option setting, precursor ion and fragment ion mass tolerance were set to 0.5 and 0.02, respectively. The advanced network setting systems were set to minimum pairs cos of 0.7, network TopK of 10, maximum connected component size of 100, minimum matched fragment ions of 4, the minimum cluster size of 2 [64].
For further analysis, the spectra were searched and matched toward GNP spectral library. They were set to the library search minimum matched of 4, search analog of “do search,” score threshold of 0.7, maximum analog search mass difference of 100. Cosine similarity score that shows closer score to 1 indicates higher similarity matched with the library spectra or representing identical spectra, whereas the score closer to 0 indicates no similarity. The calculation of cosine similarity was considered based on fragment ions, precursor ions and peak intensities [64].

3.5.3. Visualization of Molecular Networking Using Cytoscape

The molecular networking data obtained from the GNPS system were imported to Cytoscape 3.7.2 to visualize and simplify molecular networking in one display. Cytoscape was used for analyzing the whole profile of metabolites in all crude extracts and correlation between standard compounds and their analogs [7].

3.6. Isolation of (+)-(R)-Ventilagolin (1), Flavonoid Glycosides (711), Ventilatone B (12), Lupeol (13), Ventilatone A (15) and Ventilatone C (16)

A MeOH crude extract of bark (10.23 g) was subjected to Sephadex LH-20 (5 × 55 cm) column chromatography (CC), eluted with MeOH to give 65 fractions. Fraction 9 (271 mg) and fraction 10 (206 mg) containing flavonol glycosides and they were further purified using semi-preparative C18 HPLC column (Sunfire 5 µm, 19 × 250 mm). The gradient elution was performed using the following conditions: (i) isocratic elution of 30% MeOH/H2O for 0–10 min, (ii) a linear gradient from 30% MeOH/H2O to 60% MeOH/H2O over 60 min (at time of 10–70 min), (iii) a linear gradient from 60% MeOH/H2O to 100% MeOH/H2O for 15 min (at time of 70–85 min), (iv) a further linear gradient from 100% MeOH/H2O to 30% MeOH/H2O for 5 min (at time of 85–90 min) and (v) an isocratic elution with 30% MeOH/H2O over 10 min (at time of 90–100 min). The total run time was 100 min. UV detector was set at 276 nm and a flow rate was 10 mL/min. The injection volume was 400 µL. This HPLC purification yielded quercetin 3-rhamninoside (11, tR 41 min, 8.6 mg), kaempferol 3-rhamninoside (10, tR 46 min, 13.4 mg), rhamnetin 3-rhamninoside (9, tR 60 min, 25.5 mg), rhamnocitrin 3-rhamninoside (8 tR 66 min, 18.2 mg), rhamnazin 3-rhamninoside (7, tR 70 min, 35.9 mg).
A CH2Cl2 crude extract of bark (782 mg) was subjected to Sephadex LH-20 (2 × 132 cm) CC, eluted with MeOH to give 33 fractions. Fraction 7 was identified as lupeol (13, 3.0 mg). Fraction 23 was identified as (+)-ventilatone B (12, 3.7 mg). Fraction 12 (58.9 mg) containing naphthalene derivatives was further purified by semi-preparative C18 HPLC using a reversed-phase column (Sunfire 5 µm, 19 × 250 mm). UV detector was set at 276 nm and a flow rate was 10 mL/min. The injection volume was 400 µL. The isocratic elution was performed using 60% MeOH/H2O at a flowrate of 10 mL/min to give (+)-(R)-ventilagolin (1, tR 9 min, 2.3 mg). An insoluble part of fraction 12 was also identified as (+)-(R)-ventilagolin (1, 24.9 mg).
An insoluble CH2Cl2 crude extract of bark (189 mg) was purified by semi-preparative C18 HPLC (Sunfire 5 µm, 19 × 250 mm), eluted with an isocratic elution with 70% of CH3CN/H2O and a flow rate was 10 mL/min. This HPLC purification gave ventilatone A (15, tR 6 min, 4.1 mg), ventilatone B (12, tR 7 min, 10.5 mg) and ventilatone C (16, tR 9 min, 5.0 mg).

3.7. Spectroscopic Data of a New Compound, Ventilatone C (16)

Yellow amorphous solid; [α]25D +2.60 (c 0.25, CHCl3); UV (LC-UV, H2O:CH3CN, 30:70) λmax 364.1, 288.0 and 233.5 nm; ESI-HRMS: m/z 299.0917 (M+H)+, calcd m/z 299.0919 for C17H15O5; 1H and 13C NMR spectroscopic data, see Table 2.

3.8. Structure Elucidation of the Isolated Compounds

Structures of isolated compounds 713, 15 and 16 were elucidated by analysis of spectroscopic data (1D and 2D NMR, UV and ESI-HRMS spectroscopic techniques). 1H and 13C NMR spectra of compounds 713 and 15, as well as 1D and 2D NMR of a new compound, ventilatone C (16), are in the Supplementary Materials.

3.9. HPLC Fractionation of V. denticulata Extracts

100 mg of MeOH crude extract of bark of V. denticulata was dissolved in 60% MeOH and filtered through 0.45 µm filter before HPLC fractionation. A semi-preparative HPLC column, SunFire C18 (19 × 250 mm, 5.0 µm), was used. A gradient elution was performed using the following conditions: (i) linear gradient from 40% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) to 100% CH3CN (0.1% formic acid) for 0–25 min, (ii) isocratic elution of 100% CH3CN (0.1% formic acid) for 5 min (at time of 25–30 min), (iii) a linear gradient from 100% CH3CN (0.1% formic acid) to 40% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 4 min (at time of 30–34 min and (iv) an isocratic elution with 40% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) for 6 min (at time of 34–40 min). The total run time was 40 min. The flow rate was 10 mL/min. The injection volume was 400 µL. The UV detector was set at wavelength of 200–400 nm, monitoring at 276 nm. This process yielded fractions FM1-FM7, which were obtained from HPLC fractionation of a MeOH crude extract of bark eluted at retention times (tR) of 1.0–6.0 min (FM1), 6.0–8.5 min (FM2), 8.5–12.0 min (FM3), 12.0–20.0 min (FM4), 20.0–28.0 min (FM5) and 28.0–34.0 min (FM6), respectively. Weights of fractions FM1-FM6 were 23.9 mg, 15.7 mg, 13.8 mg, 6.9 mg, 6.6 mg and 6.5 mg, respectively. The fractions FM1-FM6 were subsequently tested for antibacterial and antifungal activities and results are shown in Table 3.
Fractionation of CH2Cl2 crude extract (100 mg) of bark of V. denticulata was carried out in the same manner as that of a MeOH crude extract, giving fractions FD1-FD10 with retention times (tR) of 1.0–5.5 min (FD1), 5.5–6.6 min (FD2), 6.6–7.1 min (FD3), 7.1–8.3 min (FD4), 8.3–9.5 min (FD5) and 9.5–13.0 min (FD6), respectively. Weights of fractions FD1-FD6 were 15.6 mg, 14.6 mg, 12.7 mg, 8.4 mg, 6.7 mg and 6.4 mg, respectively. Fractions FD1-FD6 were tested for antibacterial and antifungal activities and results are shown in Table 3.

3.10. ESI-HRMS Analysis for the Identification of Compounds in HPLC Fractions

The fractions FM1-FM3 and FD1-FD4 from HPLC separation showing antibacterial and antifungal activities were subsequently analyzed by ESI-HRMS. The compounds in these fractions were tentatively identified by ESI-HRMS analysis based on the putative compounds listed in Table 1. The parameters setting were capillary exit of −110.0 V, skimmer of −35.0 V, hexapol RF of −110.0 V, hexapol 1 of −24 V, set corrector fill of 63 V, set pulsar pull of 405 V, set pulsar push of 405 V, set reflector of 1.3 kV, set flight tube of 9 kV, set detector TOF of 1.99 kV and scan range 100–1000 m/z. Results are displayed in Table 4.

3.11. In-Vitro Antibacterial and Antifungal Assays

3.11.1. Preparation of Bacteria and Fungi for Bioassay

The bacterial strains used for an antibacterial assay were P. aeruginosa (TISTR No. 357), E. coli (TISTR No. 117), S. enterica serovar Typhimurium (TISTR No. 1470), S. aureus (TISTR No. 746) and B. cereus (TISTR No. 035). C. albicans (TISTR No. 5554) was the fungal strain used in an antifungal assay. All bacteria and C. albicans were purchased from the Thailand Institute of Scientific and Technological Research (Pathum Thani, Thailand). The stocks of bacteria and fungus were stored and kept in the freezer (−20 °C). Each of the bacterial and fungal strains was taken from the stocks and cultivated in a nutrient agar plate at temperature 37 °C for 24 h for bacteria and 48 h for C. albicans. A single colony of bacteria and C. albicans was selected and transferred into 10 mL 0.85% normal saline. Suspension of bacteria and fungi were adjusted to make the same turbidity with a 0.5 McFarland standard using spectrophotometer UV-Vis at wavelength of 600 nm [21].

3.11.2. Disk Diffusion Method for Antibacterial and Antifungal Assays

The disk diffusion method was performed to screen antibacterial and antifungal activities. Amphotericin B was used as a standard drug for the antifungal test. Tetracycline and chloramphenicol were used as standard drugs for antibacterial assay. 20% ethanol in DMSO was used as a negative control. Sample and standard solutions were prepared at a concentration of 10 mg/ 100µL in the solvent (20% ethanol in DMSO). Each sample (10 µL) was impregnated in a sterile disk (Whatman antibiotic assay disk, diameter 6 mm) to give 1 mg/10 µL as a final concentration of each disk. However, compounds 12, 13, 15 and 16 were tested at a concentration of 0.5 mg/10 µL due to the limited amount of the compounds obtained. Bacterial or fungal solution (ca 108 CFU/mL) was spread on a nutrient agar plate. Each disk was carefully placed on the plate containing bacteria or fungal solution and the plate was then incubated at 37 °C for 24 h. The diameter of a clear zone was measured as an indicator of inhibition toward bacteria or fungi [21]. Standard drugs for antibacterial activity were tetracycline and chloramphenicol and a standard drug for antifungal activity was amphotericin B; their activities are shown in Table 3.

4. Conclusions

Nine antibacterial and antifungal natural products in the plant, V. denticulata, were isolated using UHPLC-ESI-QTOF-MS/MS-Based molecular networking guided isolation and dereplication. Five antimicrobial flavonoid glycosides (711), two benzisochromanquinone, ventilatones B (12) and A (15), a new naphthopyrone ventilatone C (16) and a triterpene lupeol (13) were isolated from V. denticulata. Dereplication technique also tentatively identified antimicrobial compounds in V. denticulata, including kaempferol, chrysoeriol, isopimpinellin, rhamnetin, luteolin, emodin, rhamnocitrin, ventilagodenin A, rhamnazin and mukurozidiol. The present work suggests that the molecular networking guided isolation and dereplication could assist the identification of antibacterial and antifungal agents in extracts of a plant. The presence of many antibacterial and antifungal compounds in the plant, V. denticulata, supports the traditional use of this plant as an herbal medicine for the treatment of wound infection.
Mass spectrometry-based molecular networking is a powerful dereplication strategy; it not only identifies known metabolites in complex mixtures but also suggests the presence of related analogues [6]. This work demonstrates that the molecular networking effectively assists the identification of antimicrobial compounds in plant extracts.

Supplementary Materials

The following supporting materials are available online at https://0-www-mdpi-com.brum.beds.ac.uk/2079-6382/9/9/606/s1 (Figures S1–S46); Overlay of TIC chromatograms of MeOH and CH2Cl2 crude extracts (Figure S1); molecular networking of crude extracts in a negative ionization mode (Figure S2); MS/MS spectra of compounds 1 and 28 (Figures S3–S9); 1H, 13C NMR and MS spectra of compounds 711 (Figures S10–S24); MS/MS spectra of ventilatone B (12) and ventilatone A (15) (Figures S25 and S26); 1H, 13C NMR and MS spectra of compounds 12, 13 and 15 (Figures S27–S34); and 1D and 2D NMR spectra, MS and UV spectra of ventilatone C (16) (Figures S35–S46).

Author Contributions

M.A.: Investigation, Formal analysis, Writing-original draft; P.P.: Formal analysis; T.T.: Formal analysis; C.M.: Supervision; S.R.: Supervision, Funding acquisition; P.K.: Conceptualization, Writing-original draft, Writing-review & editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Acknowledgments

This work is supported by the Center of Excellence on Environmental Health and Toxicology, Science & Technology Postgraduate Education and Research Development Office (PERDO), Ministry of Education. M.A acknowledges Chulabhorn Graduate Institute and ASEAN Foundation Joint Post-graduate Scholarship Supported by Thailand International Cooperation Agency (TICA). The authors thank Poramet Nachalaem, the Scientific and Technological Instruments Center, Mae Fah Luang University, for UHPLC-MS/MS operation.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Molecular networking of crude extracts of V. denticulata as a complementary method for the dereplication strategy: (a) Molecular networking of crude extracts in a positive ionization mode; (b) Molecular networking connected to (+)-(R)-ventilagolin (1) and a putative new naphthalene derivative found in CH2Cl2 crude extract of bark; (c) Molecular networking connected to rutin (2) and other flavonol glycosides found in MeOH crude extract of bark.
Figure 1. Molecular networking of crude extracts of V. denticulata as a complementary method for the dereplication strategy: (a) Molecular networking of crude extracts in a positive ionization mode; (b) Molecular networking connected to (+)-(R)-ventilagolin (1) and a putative new naphthalene derivative found in CH2Cl2 crude extract of bark; (c) Molecular networking connected to rutin (2) and other flavonol glycosides found in MeOH crude extract of bark.
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Figure 2. Typical MS fragmentation of (+)-(R)-ventilagolin (1); possible structure and MS fragmentations of a new derivative, 3-hydroxy-ventilagolin (3) or 4-hydroxy-ventilagolin (4); and structures of fusarubin (5) and anhydrofusarubin (6).
Figure 2. Typical MS fragmentation of (+)-(R)-ventilagolin (1); possible structure and MS fragmentations of a new derivative, 3-hydroxy-ventilagolin (3) or 4-hydroxy-ventilagolin (4); and structures of fusarubin (5) and anhydrofusarubin (6).
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Figure 3. Structures of a standard flavonol glycoside, rutin (2) and flavonol glycosides 711; and typical UV spectra for 3′,4′-dihydroxy and 4′-dihydroxy flavonoid derivatives showing λmax at 356 nm and 348 nm, respectively.
Figure 3. Structures of a standard flavonol glycoside, rutin (2) and flavonol glycosides 711; and typical UV spectra for 3′,4′-dihydroxy and 4′-dihydroxy flavonoid derivatives showing λmax at 356 nm and 348 nm, respectively.
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Figure 4. Typical MS fragmentation a standard compound, rutin (2), MS fragmentation of rhamnazin 3-rhamninoside (7) and an oxonium ion of rhamnose at m/z 129.0 and a galactose fragment ion at m/z 163.
Figure 4. Typical MS fragmentation a standard compound, rutin (2), MS fragmentation of rhamnazin 3-rhamninoside (7) and an oxonium ion of rhamnose at m/z 129.0 and a galactose fragment ion at m/z 163.
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Figure 5. Structures of ventilatone B (12), lupeol (13), rhamnalpinogenin (14) and ventilatone A (15); proposed fragmentations and molecular networking of ventilatone B (12) and ventilatone A (15).
Figure 5. Structures of ventilatone B (12), lupeol (13), rhamnalpinogenin (14) and ventilatone A (15); proposed fragmentations and molecular networking of ventilatone B (12) and ventilatone A (15).
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Figure 6. Structures of ventilatone C (16) and pannorin B (17), as well as key HMBC and 1H-1H COSY correlations of ventilatone C (16). HMBC correlations are from proton(s) to carbon.
Figure 6. Structures of ventilatone C (16) and pannorin B (17), as well as key HMBC and 1H-1H COSY correlations of ventilatone C (16). HMBC correlations are from proton(s) to carbon.
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Table 1. Tentatively identified compounds in the bark and trunk of V. denticulata obtained from LC-MS/MS analysis. Compounds identified by Metlin Database [M], Human Metabolome Database [H] and standard compounds [S].
Table 1. Tentatively identified compounds in the bark and trunk of V. denticulata obtained from LC-MS/MS analysis. Compounds identified by Metlin Database [M], Human Metabolome Database [H] and standard compounds [S].
No.tR (min)CompoundsMolecular FormulaMassAdduct IonsObserved m/zCalculated m/zΔ
(ppm)
Fragment Ions (m/z)Found in Extracts a
11.086Unidentified C18 H3N O14487.1895(M-H)-486.1826486.18280.50341.1082, 179.0561, 144.0663, 119.0346, 101.0242DT
21.091UnidentifiedC37H36N2 O11684.2316(M-H)-683.2244683.22460.40341.1086, 179.0556, 119.0346MB, MT
31.3572′-Methoxy-3-(2,4-dihydroxyphenyl)-1,2-propanediol 4′-glucoside
[M, H]
C16 H24 O9360.1420(M+Na)+383.1313383.1313−0.01306.9908, 248.9974, 207.0666, 185.0403, 102.0900MB, MT
41.371Kaempferol-3-rhamninoside [M]C33 H40 O19740.2162(M+H)+741.2233741.22370.50595.1677, 449.1072, 346.0867, 287.0557, 147.0649MB
(M-H)-739.2080739.20911.49285.0396, 255.0315MB, MT
51.405Rhamnetin 3-rhamninoside [M]C34 H42 O20770.2269(M+H)+771.2343771.2342−0.09479.1186, 317.0657, 239.0928, 163.0602, 147.0653, 129.0548MB, MT
(M-H)-769.2192769.21970.66315.0505, 299.0186MB, MT, DT
61.4141,2,10-Trihydroxydihydro-trans-linalyl oxide 7-O-β-D-glucopyranoside [M, H]C16 H30 O10382.1841(M+Na)+405.1733405.1731−0.54355, 0125, 273.1298, 129.0543MB
(M-H)-381.1762381.17661.15322.0691, 249.1343, 205.3362, 161.0450, 113.0235, 101.0243MB, DB
71.493Rhamnocitrin 3- rhamninoside [M]C34 H42 O19754.2320(M+H)+755.2394755.2393−0.19463.1233, 301.0709, 163.0600, 147.0651, 129.0543MB, MT, DT
(M-H)-753.2239753.22481.12557.2233, 299.0554, 283.0236MB, MT, DT
81.499UnidentifiedC27 H34 N7 O21792.1803(M+2H)+2397.0973397.09770.97647.1279, 575.1043, 545.1010, 501.0683, 399.0395, 339.0179, 201.0041, 121.0495MB, MT
91.535Furocoumarinic acid glucoside [M, H]C17H18 O9366.0955(M+H)+367.1024367.1024−0.21349.0928, 331.0806, 307.0803, 289.0703, 275.0556, 263.0559, 217.0494, 161.0594DB
(M-H)-365.0871365.08781.96350.0639, 306.0746, 289.0707, 274.0482, 246.0522, 161.0181DB
101.540UnidentifiedC27 H50 Cl2 N9 O8 S762.2601(M+Na)+785.2493785.2493−0.03493.1342, 331.0815, 147.0664DB
111.547UnidentifiedC29 H30 N16 O11778.2272(M+2H)+2412.1027412.10321.32677.1394, 575.1073, 429.0485, 369.0279, 266.0451, 201.0073, 129.0543MB
121.5583,3′,4′-Trihydroxyflavone 3-O-[α-L-rhamnopyranosyl-(1→2)[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside]
[M, H]
C33 H40 O18724.2206(M+CH3COO)-783.2345783.23531.06453.1600, 329.0657, 314.0425, 145.0503, 101.0246MB, MT, DB, DT
131.5645,7,8-Trihydroxyflavanone 7-glucoside [M, H]C21H22 O10434.1212(M-H)-433.1138433.11400.59313.0719, 271.0556, 270.0528, 231.0611, 139.0402MB, DT
141.575Rhamnazin 3-rhamninoside
[M]
C35 H44 O20784.2430(M+H)+785.2503785.25030.00493.1341, 331.0814, 163.0599, 147. 0653, 129.054MB, MT, DB, DT
(M-H)-783.2349783.2348−0.13537.1992, 453.1584, 329.0664, 234.1049, 145.0490MB, MT, DB, DT
151.626Astragalin [M, H]C21 H20 O11448.1009(M+H)+449.1081449.1078−0.58317.0661, 287.0553, 269.0444, 195.0657MB, MT, DT
161.629UnidentifiedC23 H30 N7 O8532.2155(M+Na)+555.2043555.20480.88381.1307, 286.0742, 207.0619, 147.0433MB
171.634Kaempferol 5-glucoside [M, H]C21 H20 O11448.0996(M+HCOO)-493.0979493.09881.76346.8297, 327.0481, 298.0487, 285.0402, 240.0460MB, MT, DT
181.635Naringenin 4′-O-glucuronide [M, H]C21 H20 O11448.1002(M+Na)+471.0895471.08980.57339.0471, 309.0368, 294.0188, 249.1094, 161.9958MB, MT
191.708Aloesol [M, H]C13 H14 O4234.0891(M+H)+235.0964235.09650.45217.0860, 191.0705, 163.0754, 151.0385, 135.0804, 107.0847MB, MT, DT
(M-H)-233.0818233.18190.60189.0552, 161.0593, 149.0251MB, MT, DB
201.765Zingerone glucoside [M, H]C17 H24 O8356.1468(M+Na)+379.1361379.13630.63323.9212, 278.3414, 235. 8741, 217.0847, 111.0775MB, DB
211.810UnidentifiedC33 H46 N4 O6594.3419(M+H)+595.3492595.349−0.35577.3542, 536.2739, 173.1640, 120.0805MB
221.852UnidentifiedC21 H28 O8408.1771(M+Na)+431.1674431.16760.53317.1031, 275.0908, 205.0465DB
231.909Xanthotoxol glucoside [M, H]C17 H16 O9364.0795(M+H)+365.0866365.08670.36305.0661, 291.0851, 277.0713, 259.0606, 215.0704, 132.0900DB
(M-H)-363.0716363.07221.51304.0583, 287.0556, 272.0320, 261.0404, 244.0375, 228.0435, 201.0195DB
242.000UnidentifiedC24 H18 N8 O4482.1448(M+Na)+505.1338505.13430.96419.1317, 343.1048, 257.0809, 127.0393MB
252.006Isoliquiritin [M, H]C21 H22 O9418.1261(M+H)+419.1334419.13370.71335.0877, 257.0804, 239.0703, 191.0702, 127.0390MB
(M-H)-417.1184417.11911.66297.0764, 255.0643MB, MT, DB
262.0426”-O-Acetyldaidzin [M, H]C23 H22 O10458.1209(M+HCOO)-503.1192503.11950.67418.1190, 297.0765, 255.0690MB
272.190Glucoemodin [M, H]C21 H20 O10432.1051(M-H)-431.0979431.09841.07344.8229, 311.0557, 269.0448, 227.1067 MB, MT, DT
282.282Kievitol [M, H]C20 H22 O7374.1356 (M-H)-373.1284373.12932.29359.0953, 246.0522, 193.0504, 179.0714, 164.0475, 149.0600, 134.0368 DB
292.291Wharangin [M, H]C17 H12 O8344.0536(M+H)+345.0608345.0605−0.74303.0497, 327.0487, 299.0544, 275.0543, 261.0401, 195.0290 DB, DT
(M-H)-343.0454343.04591.69330.0381, 301.0348, 287.0196, 273.0040, 158.0608 DB
302.3144″-Methyl-6″-(3,4-dihydroxy-E-cinnamoyl)isoorientin [M, H]C31 H28 O14624.1474(M-H)-623.1400623.14061.02517.8187, 458.3673, 375.3759, 298.0471, 295.0808, 285.0416, 241.0516MB
312.376Chrysoeriol [M, H]C16 H12 O6300.0640(M+H)+301.0712301.0707−1.80273.0397, 260.0310, 255.0651, 245.0442, 227.0698DB
(M-H)-299.0561299.05610.00270.0168, 258.0166, 255.0661, 240.0428, 227.0346, 214.0269, 151.0033DB
322.3776”-Malonylcosmosiin [M, H] C24 H22 O13518.1048(M-H)-517.0975517.09882.44473.1078, 432.1734, 385.1729, 269.0452, 225.0402MB
332.382Cicerin 7-(6-malonylglucoside) [M, H]C26 H26 O15578.1269(M+H)+579.1341579.13440.64437.0247, 342.9891, 331.0819, 147.0531, 127.0390MB
342.416UnidentifiedC40 H38 N O5 S644.2467(M+Na)+667.2356667.23631.04553.2780, 425.0864, 329.1411, 129.0528MB, DT
352.445Quercetin [M, H]C15 H10 O7302.0422(M+H)+303.0494303.04991.86276.8345, 240.8436, 229.0471, 195.0268, 182.9751, 139.8692MB
(M-H)-301.0351301.03540.76273.0382, 229.0518, 178.9980, 151.0032, 121.0300, 107.0132MB
362.552UnidentifiedC18 H40 N5 O18614.2359(M+Na)+637.2247637.22612.19537.1811, 410.0280, 339.1044, 145.0475, 110.0979MB, DT
372.563Emodinanthranol [M, H]C15 H12 O4256.0738(M+H)+257.0810257.0808−0.66242.0590, 217.0500, 214.0612, 198.9313, 145.0656, 101.0594DB
(M-H)-255.0658255.06631.84213.0555, 187.0768, 183.0814DB
382.753α-Hydrojuglone 4-O-β-D-glucoside [H]C13 H18 O5338.0995(M-H)-337.0922337.09291.98250.0844, 221.081, 163.0765MB, DB, DT
393.039UnidentifiedC13 H20N3 O8 S378.0958(M+H)+379.1025379.10444.86319.0809, 291.0861, 202.0630, 111.0421DB
403.087UnidentifiedC17 H18 O8350.1003(M+H)+351.1075351.1074−0.19333.0949, 301.0702, 276.0630, 259.0604, 215.0700DB
(M-H)-349.0924349.09291.35334.0694, 319.0457, 291.0506, 219.0304DB
413.131UnidentifiedC28 H24 O12552.1264(M-2H)-2275.0558275.05611.17338.0072, 262.0703, 232.0368, 218.0236, 188.0462DB
423.233Isopimpinellin [M, H]C13 H10 O5246.0522(M+CH3COO)-305.0660305.06672.34245.0447, 201.0512, 173.0585, 129.0714DB
433.274Kaempferol [M, H]C15 H10 O6286.0480(M+H)+287.0554287.0550−1.39227.8855, 165.0174, 153.0172, 121.0271MB, MT
(M-H)-285.0401285.04051.37257.0426, 241.0493, 229.0487, 211.0396, 151.0029MB, MT, DB
443.291Coriandrone C [M, H]C13 H10 O5246.0536(M+H)+247.0609247.0601−3.39229.0499, 219.0262, 201.0552, 173.0586, 158.0695, 137.1239DB
453.364Eriodictyol [M, H]C15 H12 O6288.0631(M-H)-289.0704289.07070.84271.0589, 259.0603, 257.0465, 231.0641, 229.0488, 173.0582DB
(M-H)-287.0558287.05610.93259.0604, 243.0653, 201.0582, 177.0550, 151.0041, 125.0243MB, MT, DT
463.583Coumesterol [M, H]C15 H8 O5268.0373(M+H)+269.0446269.0444−0.46243.1493, 241.0487, 213.0553, 185.0602, 157.0644MB, MT
473.594Citreorosein [M, H]C15 H10 O6286.0482(M+H)+287.0555287.0550−1.65269.0447, 213.0536, 185.0593MB, MT, DT
(M-H)-285.0404285.04050.35241.0503, 172.9762MB, MT, DB, DT
483.608Physcion [M]C16 H12 O5284.0688(M+H)+285.0761285.0757−1.4257.0808, 243.0644, 239.0696, 229.0496, 211.0750DB
(M-H)-283.0612283.0612−0.07255.0650, 241.0503, 239.0703, 227.0345, 224.0477DB
493.698R-Angolensin [M]C16 H16 O4272.1051(M+H)+273.1124273.1121−0.81255.1016, 231.1015, 227.1068, 189.0915, 174.0667, 111.8671DB
503.894(±)-Sphaerosin [M, H]C17 H18 O5302.1153(M+H)+303.1225303.12270.80285.1117, 261.1129, 257.1174, 219.1029, 204.0783, 163.0361MB, DB
513.919UnidentifiedC34 H36 O10604.2310(M+Na)+627.2204627.2201−0.58325.1052DB
523.9263-Hydroxyphloretin [M, H]C15 H14 O6290.0785(M+HCOO)-335.0767335.07721.67268.0917, 259.0604, 248.0686, 220.0728, 205.0504, 147.0429MB, DB
533.9963′,7-Dihydroxy-4′,8-dimethoxyisoflavone [H]C17 H14 O6314.0785(M-H)-313.0712313.07181.82300.0246, 269.0808, 254.0571, 239.0326MB, DB
544.062UnidentifiedC19 H22 O10410.1214(M+Na)+433.1106433.1105−0.21401.0840, 369.0571, 341.0618, 250.5698MB
554.064UnidentifiedC37 H32 N3 O15758.1834(M-H)-757.1760757.17610.13713.1893, 458.1202, 410.6138, 373.7386, 299.7235, 254.0514, 191.1313MB, MT
564.075UnidentifiedC25 H30 N8 O7650.1400(M+H)+651.1473651.1472−0.03337.0683DB
574.1325,6,7,8-Tetrahydroxy-3′,4′-dimethoxyflavone [M, H]C17 H14 O8346.0681(M-H)-345.0610345.06161.85331.0413, 298.0119, 270.0171, 242.0246MB
584.1345-Hydroxy-4′,7,8-trimethoxyflavone [M, H]C18 H16 O6328.0939(M-H)-327.0867327.08742.06312.0620, 286.0477, 271.0240, 268.0732, 253.0500, 225.0558DB
594.178UnidentifiedC12 H8 N5 O6 S350.0196(M+H)+351.0265351.02680.77297.3586, 261.9442, 245.8488, 222.0035, 181.0472, 135.0783MB
604.203UnidentifiedC34 H24 O12624.1270(M+Na)+647.1162647.1160−0.38335.053DB
614.251Aloe emodin w-acetate [M, H]; or Ventilatone A (isolation) C17 H12 O6312.0636(M+H)+313.0706313.07070.29285.0759, 271.0604, 243.0659, 215.0685, 167.8890MB, DB
(M-H)-311.0559311.05610.72297.0393, 269.0438, 268.0373, 253.0140, 224.0472DB
624.532Cartorimine [M, H]C15 H14 O6290.0794(M+Na)+313.0686313.0683−1.23276.9105, 212.8751, 123.1149MB, MT
(M-H)-289.0712289.017181.81273.0402, 259.0239, 245.0457, 201.0550, MB, MT, DB
634.619Rhamnetin [M]C16 H12 O7316.0581(M+H)+317.0654317.06540.56271.0590, 243.0679, 167.0342, 121.0279MB
(M-H)-315.0505315.05101.57300.0261, 166.0221, 121.0293, 112.9849MB, DB
644.721Luteolin [M, H]C15 H10 O6286.0473(M-H)-285.0401285.040051.34270.0163, 257.0450, 241.0499, 213.0526, 151.9236MB, MT, DB, DT
654.752UnidentifiedC18 H14 O7342.0744(M+Na)+365.0636365.0632−1.14321.0373, 305.0419, 156.0637DB
664.8745,4′-Dihydroxy-3,3′-dimethoxy-6:7-methylenedioxyflavone
[M, H]
C18H14O8358.0688(M+Na)+381.0579381.05810.39349.0312, 333.4380, 328.4933, 273.3009, 243.5325, 189.0203DB
674.9991,3,5-Trihydroxy-6,7-dimethoxy-2-methylantraquinone [H]C16 H10 O7330.0734(M-H)-329.0661329.06671.74314.0427, 299.0207, 288.0280, 285.077, 273.0031, 270.0525, 258.0168MB, TB, DB, DT
685.007Ventilagodenin A; or 5-De-O-methyltoddanol [M, H]C15 H16 O5276.1000(M+H)+277.1074277.1071−1.21259.0957, 244.0731, 235.0973, 199.0748, 171.0804MB, TB, DB, DT
(M-H)-275.0922275.09251.14259.0609, 245.0447, 231.0661, 192.6885, 175.0355MB, TB, DB
695.097UnidentifiedC18 H10 N O4304.0612(M+Na)+327.0504327.0502−0.46287.0555, 259.0604, 255.0288, 245.0422, 167.0345DB
705.129Rhamnalpinogenin [M, H]; or Ventilatone B (isolation) C17 H12 O7328.0589(M+H)+329.0659329.0656−1.09311.0551, 287.0551, 259.0607, 167.0345MB, TB, DB, DT
(M-H)-327.0508327.05100.63312.0273, 284.0326, 269.0092, 256.0378, 185.0239DB
715.135UnidentifiedC12 H8 N5 O7 S366.0142(M+H)+367.0212367.02171.49352.3162, 309.0637, 277.0991, 235.8736, 186.9023, 123.1163MB
725.1523,5,7-Trihydroxy-4′,6-dimethoxyflavanone [M, H]C17 H16 O7332.0891(M+HCOO)-377.0873377.08781.30317.0660, 306.0738, 259.0245, 174.9557, 130.9658MB, DB
735.265Mukurozidiol (M, H)C17 H18 O7334.1051(M+H)+335.1123335.11250.77303.0866, 285.0752, 275.0914, 261.0750, 245.0448, 233.0425MB, DB, DT
(M+HCOO)-379.1026379.10352.23308.0893, 305.0640, 277.0688, 262.0477, 174.9575 MB, DB
745.288UnidentifiedC13 H20 N3 O8 S378.0957(M+H)+379.1026379.10444.68364.0528, 291.0863, 215.0331, 115.0550MB
755.296UnidentifiedC19 H22 O10410.1214(M+Na)+433.1106433.1105−0.13 373.0897, 342.0707, 327.0475MB
765.420Genistin [M, H]C21 H20 O10432.1036(M+H)+433.1109433.11294.76401.0843, 373.0894, 369.0579, 342.0711, 327.0470DB
775.4636′-Hydroxyangolensin [M, H]C16 H16 O5288.1000(M+H)+289.1073289.1071−0.92271.0967, 247.0966, 243.1013, 229.0856, 205.0864DB
(M-H)-287.0920287.09251.70269.0821, 254.0605, 245.0823, 203.0702DB
785.578(S)-Rutaretin [M,H]C14 H14 O5262.0835(M-H)-261.0761261.07682.71246.0527, 231.0291, 218.0561, 203.0352DB
795.611UnidentifiedC35 H30 O11626.1773(M+HCOO)-671.1753671.1772.60509.1242, 416.1098, 254.0577TB, TD
805.650Pratenol A [M,H]C14 H12 O5260.0687(M+H)+261.0759261.0757−0.49243.0656, 215.0705, 200.0470, 187.0749, 159.0439DB
815.743Gingerenone C [M, H]C20 H22 O4326.1521(M+H)+327.1592327.1591−0.46203.1049, 171.0802, 151.0758, 148.1110, 137.0600DB
825.848UnidentifiedC53 H26 N3 O2736.2027(M+Na)+759.1920759.1917−0.37664.0398, 504.1286, 418.1196, 299.0856, 256.0729MB
836.222Afzelechin [M, H]C15 H14 O5274.0841(M-H)-273.0768273.07680.29229.0501, 202.026MB, DB
846.342Ducunolide E [M, H]C26 H28 O9484.1724(M-H)-483.1650483.16612.19468.1412, 439.1389, 424.1156, 409.0887DB
856.472Rhamnocitrin [M]C16 H12 O6300.0637(M+H)+301.0711301.0707−1.35286.0458, 179.03331, 167.0344, 121.0286MB, DB, DT
(M-H)-299.0556299.05611.78284.0310, 271.0605, 240.0420, 178.0257, 165.0189MB, DB, DT
866.6077-Hydroxy-3,4′,8-trimethoxyflavone [M, H]C18 H16 O6328.0949(M+H)+329.1023329.1020−1.04314.0786, 313.0702, 285.0766, 198.0922, 121.1025DB
876.698Acerosin [M, H]C18 H16 O8360.0834(M-H)-359.0761359.07723.11344.0538, 297.0054, 269.0084, 171.2585MB, DB
886.732UnidentifiedC13 H13 N6 O7365.0841(M+2Na)+2205.5309205.53152.91320.7446, 254.9948, 205.1755, 155.0088, 141. 5110, 112.4964MB
896.766Alfalone [M, H]C17 H14 O5298.0841(M+H)+299.0916299.0914−0.54271.3851, 213.8909, 189.0528, 112.7128DB, DT
906.775Rhamnazin [M, H]C17 H14 O7330.0743(M+H)+331.0816331.0812−1.22316.0577, 299.0542, 288,0634, 179.0327, 167.0338MB, DB
(M-H)-329.0664329.06670.74315.0457, 314.0424, 286.0478, 254.0217, 241.051, 170.0353MB, DB
916.924Xanthoxyletin [M, H]C15 H14 O4258.0894(M+H)+259.0967259.0965−0.73244.0734, 241.0863, 226.0628, 217.0862, 213.0906, 195.0799, 167.0879 DB
926.981Barpisoflavone A [M, H]C16 H12 O6300.0636(M+H)+301.0708301.0707−0.43287.0570, 269.0441, 236.9047, 185.0603, 127.0056MB, TB, DT
(M-H)-299.0560299.05610.24267.0297, 240.0422, 212.0476MB, TB, DB
937.015(+)-(R)-Ventilagolin [S]C17 H16 O7332.0897(M+H)+333.0971333.09690.60318.0736, 301.0710, 276.0630, 259.0606, 213.0544, 185.0596MB, DB, DT
(M+HCOO)-377.0873377.08781.30317.066, 306.0738, 303.0506, 259.0245, 174.9557MB, DB, DT
947.123Caryatin [M, H]C17 H14 O7330.0741(M+H)+331.0813331.0812−0.12299.0551, 276.0625, 259.0611, 211.3641, 167.0181MB, DB
(M-H)-329.0660329.06672.00314.0423, 299.0194, 286.0488, 271.0240, 165.0184MB, MT, DB, DT
957.349Kanzonol O [M, H]C22 H22 O6382.1418(M+Na)+405.1310405.1309−0.29335.0526, 270.0508, 143.0333DB
967.548UnidentifiedC12 H24 Cl2 N2 O8 S426.0635(M+Na)+449.0527449.0523−0.91408.2483, 388.7627, 287.1038MB
977.887UnidentifiedC34 H30 N3 O11656.1874(M-H)-655.1800655.18081.18557.9872, 254.0580MB, MT
988.003UnidentifiedC33 H28 N3 O11642.1727(M-H)-641.1655641.1651−0.57509.1224, 491.1100, 254.0579MB, MT
998.120UnidentifiedC16 H11 N O233.0844(M+Na)+256.0734256.0733−0.36240.0926, 210.0659, 1821.0653, 157.0646, 140.9164MB
1008.507Dihydromorelloflavone
[M, H]
C30 H22 O11558.1161(M+H)+559.1236559.1235−0.15541.1141, 523.0991, 517.1109, 513.1141, 499.1013, 313.0354, 257.0795DB
(M-H)-557.1085557.10890.80539.0915, 526.0836, 359.8609, 155.1055DB
1018.938Emodin [M, H, S]C15 H10 O5270.0528(M+H)+271.0601271.0601−0.16229.0509, 225.0560, 201.0539, 197.0590, 140.0222MB, MT, DB, DT
(M-H)-269.0452269.04551.33241.0511, 225.0562, 210.0316, 195.0415, 135.0911MB, MT, DB, DT
1029.187Formononetin [M, H]C16 H12 O4268.0740(M+H)+269.0813269.0808−1.60254.0572, 239.0708, 226.0618, 151.0543DB
1039.3836α-Hydroxymaackiain [M, H]C16 H12 O6300.0637(M+H)+301.0709301.0707−0.76255.0638, 117.0696MB, DB
1049.986UnidentifiedC22 H18N7O3428.1473(M+Na)+451.1363451.1363−0.01319.0570, 292.0353, 133.0864DB
1059.991Artonin L [M, H]C22 H20 O7396.1213(M+H)+397.1283397.1282−0.36379.1160, 366.1054, 337.1045, 327.1201, 295.0939, 287.0557DB
10610.699Muscomin [M, H]C18 H18 O7346.1053(M+H)+347.1125347.1125−0.01332.0896, 315.0864, 290.0781, 273.0764, 227.0696DB
10710.824UnidentifiedC15 H11 O4255.0658(M+H)+256.0731256.073−0.41241.0502, 238.0625, 210.0683, 198.9302, 182.0727MB, MT, DB
10811.4232′,3,5-Trihydroxy-5′,7-dimethoxyflavanone [M, H]C19 H20 O9332.0885(M+CH3COO)-391.1024391.10352.64 317.0658, 302.0387, 242.6421, 209.8790, 130.2329DB
10911.796Palmidin A [M, H]C30 H22 O8510.1312(M+H)+511.1387511.13870.16256.0733, 133.0854MB, MT, DB, DT
(M-H)-509.1238509.12420.80254.0583MB, MT, DB, DT
11012.2371,3,5,8-Tetrahydroxy-6-methoxy-2-C16 H12 O7316.0585(M+H)+317.0658317.0656−0.56299.0575, 254.8649, 193.0125, 135.1168, 127.0534MB, DB
methylanthraquinone [M, H] (M-H)-315.0505315.05101.57300.0261, 272.0305, 216.9344, 163.1615, 112.9849MB
11112.742Khelmarin D [M, H]C28 H24 O8488.1460(M+CH3COO)-547.1599547.16102.00457.0900DB
11212.798Amentoflavone [M, H]C30 H18 O10538.0889(M-H)-537.0814537.08272.44469.0870, 400.8285, 333.5261, 173.9422, 107.5508MB, MT, DB
11312.837Isophysalin G [M, H]C28 H30 O10526.1860(M+Na)+549.1752549.1731−3.87517.1481, 475.1364, 246.0893DB
11413.326Yuccaol C [M, H]C30 H22 O10542.1201(M-H)-541.1126541.11402.6523.0998, 511.0683, 493.0539, 308.0347, 231.1206DB
11513.632Ephedrannin A [M, H]C30 H20 O11556.0997(M+CH3COO)-615.1134615.11441.59299.0208, 289.0709DB
11614.355UnidentifiedC29 H 22 N3 O7524.1462(M-H)-523.1387523.1385−0.47254.0580MB
11714.718UnidentifiedC16 H13 O4269.0814(M+H)+270.0885270.08870.78227.07006, 179.0025, 151.9915, 105.0345MB
11814.748Palmidin B [M, H]C30 H22 O7494.1349(M-H)-493.1279493.12932.74386.1758, 340.4709, 254.0581, 224.0460, 213.0023, 161.4482DB, DT
11916.060Murrayazolinine [M, H]C23 H27 N O2349.2042(M+NH4)+367.2390367.2380−2.81323.2308, 268.2613, 172.1157, 156.1387, 116.0538MB, MT, DB. DT
12016.060UnidentifiedC32 H28 N2 S3536.1416(M+Na)+559.1318559.1307−1.94521.0807, 466.7954, 409.8348, 401.2433MB
12117.810Rheidin B [M, H]C30 H20 O8508.1146(M-H)-507.1074507.10852.24479.1105, 304.9145MB, MT, DT
12218.371Copalic acid [M, H]C20 H32 O2304.2407(M+H)+305.2479305.2475−1.40259.2411, 149.1327, 137.1326, 123.1165, 109.1010MB
12320.707γ-Pinacene [M, H]C20 H32272.2506(M+H)+273.2578273.2577−0.45231.2105, 175.1484, 163.1482, 149.1327, 135.1169, 121.1014, 109.1013, 107.0856DB
12420.750Pipericine [M, H]C22 H41 N O335.3190(M+H)+336.3264336.3261−0.90240.2341, 184.1702, 142.1230, 170.1534, 100.0761MB, MT, DB, DT
12522.976Araliacerebroside [M, H]C40 H77 N O10731.5543(M+Na)+754.5435754.54400.63NDMB, MT, DT
(M-H)-730.5462730.54751.71568.4923, 416.3272, 326.2700, 271.2258, 179.0551, 131.0328, 119.0354MB, MT
12623.282UnidentifiedC26 H51 N13545.4386(M+Na)+568.4274568.42831.52476.3663, 371.2275, 250.1754, 185.1303, 133.0845MB
12724.262UnidentifiedC26 H45 N4413.3639(M+H)+414.3710414.37171.59112.0989MB
12824.301UnidentifiedC26 H49 N O391.3819(M+H)+392.3893392.3887−1.51282.2781, 198.1852, 156.1385, 130.1590MB, MT
12924.466UnidentifiedC36 H38 N4 O5606.2843(M+H)+607.2917607.2915−0.35547.27MB
13024.500Clerosterol 3-glucoside [M, H]C35 H58 O6574.4219(M+CH3COO)-633.4359633.43722.00559.3987, 541.3890, 383.3517, 175.0401, 133.0300DB
13124.755UnidentifiedC24 H25 N9 O2 S2535.1579(M+H)+536.1658536.1645−2.30503.1070, 415.0364, 341.0176, 221.0841, 147.0655MB, TB
13224.913UnidentifiedC37 H38 N5 O2584.3020(M+Na)+607.2911607.29181.17547.2713, 460.2258, 367.0213, 280.2360, 167.1421, 107.0840MB
13325.117AS 1-5 [M, H]C40 H77 N O9715.5597(M+Na)+738.5489738.54890.15NDMB, MT, DT
(M+HCOO)-760.5560760.55802.69655.7664, 552.4965, 534.4872, 299.4631, 179.0584, 101.0237MB, MT
13425.4743-Dehydroteasterone [M, H]C28 H46 O4446.3401(M+Na)+469.3293469.3288-0.93385.1727, 329.1716, 189.0170, 171.0054, 113.1314MB, MT, DB, DT
13525.552UnidentifiedC42 H74 N6 O10822.5471(M-H)+821.5396821.5394-0.32775.5344, 613.0880, 523.3704, 339.4486, 277.2172, 261.1697, 175.6021, 103.9958MB
13625.644UnidentifiedC29 H41 N2 O2 S5609.1765(M+H)+610.1843610.18440.30489.0548, 355.0700, 281.0509, 221.0844, 147.0659MB
13726.561Secasterone [M, H]C28 H46 O4446.3395(M+Na)+469.3286469.32860.44329.1732, 284.1760, 268.0679, 109.1008MB, MT
13826.727UnidentifiedC42 H76 N6 O10824.5628(M-H)-823.5555823.5550−0.60778.5514, 713.2510, 657.5735, 579.3840, 513.3079, 456.2245, 388.2563, 277.2178MB
13926.799UnidentifiedC36 H76 N9 O7 S778.5595(M+Na)+801.5482801.5482−0.14519.2919, 121.1020MB
14027.173UnidentifiedC37 H67 N13 O3741.5491(M+Na)+764.5381764.53820.15102.0913MB, MT
14129.246UnidentifiedC22 H48 Cl2 N5 O2 S516.2900(M+H)+517.2957517.29794.07312.0957, 244.0374, 175.9745MB, MT
14229.348UnidentifiedC34 H68476.5322(M+NH4)+494.5662494.5659−0.53453.3644, 271.3170, 151.1298MB, MT
14330.655Lansiol [M, H]C33 H56 O468.4326(M+CH3COO)-527.4465527.44700.91478.6391, 447.7013, 413.8984, 365.2430, 305.1114, 258.1590, 192.0016MB
14432.152UnidentifiedC6 H12 N6 O3216.0977(M+H)+217.1049217.1044−2.52204.0959, 161.0979, 134.0842, 107.0513MB
a MB: MeOH crude extract of bark; MT: MeOH crude extract of trunk; DB: CH2Cl2 crude extract of bark; DT: CH2Cl2 crude extract of trunk.
Table 2. 1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data for ventilatone C (16).
Table 2. 1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data for ventilatone C (16).
PositionVentilatone C (16)
NMR Data in CDCl3NMR Data in Acetone-d6
δH, Multiplicity
(J in Hz)
δC, TypeδH, Multiplicity
(J in Hz)
δC, Type
1-166.18, C-167.05, C
34.53, ddq (10.5, 6.3, 3.4)74.97, CH4.65, ddq (10.6, 6.3, 3.4)76.06, CH
43.00, ddd (16.3, 10.6, 1.4)
3.15, ddd (16.4, 3.3, 0.8)
34.23, CH23.03, ddd (16.5, 10.7, 1.6)
3.25, ddd (16.5, 3.1, 0.8)
34.50, CH2
4a-127.70, C-129.66, C
57.24, s120.60, CH7.43, s121.32, CH
5a-138.35, C-139.79, C
66.72, s99.96, CH6.94, d (2.3)100.52, CH
7-161.13, C,-161.07, C
86.72, s103.11, CH6.64, d (2.3)103.44, CH
9-156.05, C-156.74, C
9a-107.00, C-107.90, C
10-152.72, C-153.48, C
10a-104.37, C-105.33, C
12-161.72, C-162.44, C
135.74, s90.81, CH5.63, s90.82, CH
3-Me1.56, d (6.4)20.79, CH31.54, d (6.3)20.83, CH3
7-OMe3.91, s55.50, CH33.92, s55.88, CH3
9-OH8.81, br s-8.94, s-
Table 3. Antibacterial and antifungal activities of crude extracts, fractions and isolated compounds.
Table 3. Antibacterial and antifungal activities of crude extracts, fractions and isolated compounds.
Crude Extracts/
Fractions/
Compounds
Zone of Inhibition (mm)
Bacteria/Fungus
B. cereusS. aureusE. coliS. entericaP. aeruginosaC. albicans
MB a13158141013
DB a2118919816
MT a7148778
DT a0130000
FM1 b111490100
FM2 b111298810
FM3 b97121080
FM4 b0001400
FM5 b000000
FM6 b009900
FD1 c181514181117
FD2 c1922172390
FD3 c26252530120
FD4 c17121324120
FD5 c1415161490
FD6 c11171116109
70110008
8100110012
99900136
1091112000
1191410000
121111018012
13ND7NDND00
151317ND1800
161313ND1400
Chloramphenicol d4437505028ND
Tetracycline d4039404429ND
Amphotericin B eNDNDNDNDND23
a MB: MeOH crude extract of bark; DB: CH2Cl2 crude extract of bark; MT: MeOH crude extract of trunk; DT: CH2Cl2 crude extract of trunk. b FM1-FM6: Fractions obtained from HPLC isolation of MeOH crude extract of bark eluted at retention times (tR) of 1.0–6.0 min (FM1), 6.0–8.5 min (FM2), 8.5–12.0 min (FM3), 12.0–20.0 min (FM4), 20.0–28.0 min (FM5) and 28.0–34.0 min (FM6), respectively. HPLC conditions are in the Section 3.9. c FD1-FD6: HPLC fractions from CH2Cl2 crude extract of bark eluted at retention times (tR) of 1.0–5.5 min (FD1), 5.5–6.6 min (FD2), 6.6–7.1 min (FD3), 7.1–8.3 min (FD4), 8.3–9.5 min (FD5) and 9.5–13.0 min (FD6), respectively. HPLC conditions are in the Section 3.9. d Chloramphenicol and tetracycline are standard drugs for antibacterial activity. e Amphotericin B is a standard drug for antifungal activity. ND = Not determined.
Table 4. Antibacterial and antifungal agents from the HPLC fractions of V. denticulata, tentatively identified by ESI-HRMS analysis based on the putative compounds listed in Table 1.
Table 4. Antibacterial and antifungal agents from the HPLC fractions of V. denticulata, tentatively identified by ESI-HRMS analysis based on the putative compounds listed in Table 1.
FractionCompounds in Fractions
FM1 aKaempferol (285.0391 [M-H]), chrysoeriol (299.0589 [M-H]), unidentified C13H20N3O8S (377.0851 [M-H]), kaempferol 3-rhamninoside (739.2091 [M-H]), isopimpinellin (305.0657 [M+CH3COO]), 3-hydroxyphloretin (335.0760 [M+HCOO]), rhamnocitrin 3-rhamninoside (377.0851 [M-H]), unidentified C37H32N3O15 (757.1768 [M-H]), rhamnetin 3-rhamninoside (769.2162 [M-H]), rhamnazin 3-rhamninoside (783.2310 [M-H])
FM2 aRhamnetin (315.0475 [M-H]), luteolin (285.0391 [M-H]), 3,5,7-trihydroxy-4′,6-dimethoxyflavanone (377.0846 [M+CH3COO])
FM3 aEmodin (269.0445 [M-H]), rhamnocitrin (299.0563 [M-H]), palmidin A (509.1218 [M-H]), unidentified (523.1351 [M-H])
FD1 bEriodyctiol (287.0554 [M-H]), cartorimine (289.0705 [M-H]), chrysoeriol (299.0558 [M-H]), rhamnetin (315.0507 [M-H]), 3-hydroxyphloretin (335.0771 [M+HCOO]), xanthotoxol glucoside (363.0709 [M-H]), furocoumarinic acid glucoside (365.0875 [M-H])
FD2 bVentilagodenin A (275.0846 [M-H]), physcion (283.0650 [M-H]), rhamnocitrin (299.0613 [M-H]), ventilatone A (311.0602 [M-H]), 3′,7-dihydroxy-4′,8-dimethoxyisoflavone (313.0761 [M-H]), rhamnazin (329.0726 [M-H]), 3,5,7-trihydroxy-4′,6-dimethoxyflavanone (331.0803 [M-H]), ventilatone B (327.0556 [M-H]), unidentified C17H18O8 (349.0965 [M-H])
FD3 bAfzelechin (273.0727 [M-H]), (+)-(R)-ventilagolin (331.0827 [M-H]), mukurozidiol (333.0968 [M-H])
FD4 bEmodin (269.0450 [M-H]), 6α-hydroxymaackiain (299.0550 [M-H]), 2′,3,5-trihydroxy-5′,7-dimethoxyflavanone (331.0816 [M-H]), palmidin A (509.1261 [M-H]), unidentified C15H11O4 (254.0592 [M-H]), unidentified C29H22N3O7 (523.1416 [M-H]),
a FM1-FM3 = Fractions obtained from HPLC isolation of MeOH crude extract of bark eluted at retention times (tR) of 1.0–6.0 min (FM1), 6.0–8.5 min (FM2) and 8.5–12.0 min (FM3), respectively. b FD1-FD4 = Fractions from HPLC isolation of CH2Cl2 crude extract of bark eluted at retention times (tR) of 1.0–5.5 min (FD1), 5.5–6.6 min (FD2), 6.6–7.1 min (FD3) and 7.1–8.3 min (FD4), respectively. HPLC conditions are in the Section 3.9.

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Azizah, M.; Pripdeevech, P.; Thongkongkaew, T.; Mahidol, C.; Ruchirawat, S.; Kittakoop, P. UHPLC-ESI-QTOF-MS/MS-Based Molecular Networking Guided Isolation and Dereplication of Antibacterial and Antifungal Constituents of Ventilago denticulata. Antibiotics 2020, 9, 606. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics9090606

AMA Style

Azizah M, Pripdeevech P, Thongkongkaew T, Mahidol C, Ruchirawat S, Kittakoop P. UHPLC-ESI-QTOF-MS/MS-Based Molecular Networking Guided Isolation and Dereplication of Antibacterial and Antifungal Constituents of Ventilago denticulata. Antibiotics. 2020; 9(9):606. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics9090606

Chicago/Turabian Style

Azizah, Muhaiminatul, Patcharee Pripdeevech, Tawatchai Thongkongkaew, Chulabhorn Mahidol, Somsak Ruchirawat, and Prasat Kittakoop. 2020. "UHPLC-ESI-QTOF-MS/MS-Based Molecular Networking Guided Isolation and Dereplication of Antibacterial and Antifungal Constituents of Ventilago denticulata" Antibiotics 9, no. 9: 606. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics9090606

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