Anti-MRSA Flavonoids from Iraqi Citrus sinensis (L.)

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Shaymaa Al-Majmaie, Lutfun Nahar, M. Mukhlesur Rahman, George P. Sharples, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7037612/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Methicillin-resistant Staphylococcus aureus (MRSA) infections represent a major global health challenge, contributing to millions of deaths annually due to antibiotic resistance. The development of new and effective antimicrobial agents is crucial to address this issue. In this study, the leaves and peels of Citrus sinensis (Rutaceae) were investigated as potential sources of anti-MRSA compounds. A bioassay-guided isolation protocol led to the identification of two polymethoxy flavones, 3-methoxynobiletin ( 4 ), sinensetin ( 7 ), and rutin 3’-methyl ether ( 12 ), which exhibited anti-MRSA activity against various strains with minimum inhibitory concentration (MIC) values ranging from 128–256 mg/mL. In addition, ten known flavonoids ( 1–3, 5, 6, 8, 9, 11–14 ) and a new flavanone glycoside, named sinensidin ( 10 ), which were not active against S. aureus . The chemical structures of all isolated compounds were determined using spectroscopic techniques, including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESIMS). Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Chemical biology Physical sciences/Chemistry Biological sciences/Drug discovery Biological sciences/Microbiology Biological sciences/Plant sciences Citrus sinensis Rutaceae MRSA polymethoxyflavone antimicrobial bioassay-guided protocol Figures Figure 1 1. Introduction Antibiotic resistance, also known as antimicrobial drug resistance (AMR), represents one of the most significant global public health challenges today.[ 1 ] The improper and excessive use of antibiotics has exacerbated this issue, leading to a worsening situation.[ 2 , 3 ] According to a report published by the World Health Organization (WHO), antimicrobial resistance was responsible for approximately 5 million deaths globally in 2019 alone.[ 4 , 5 ] Projections suggest that by 2050, the annual death toll due to AMR could rise to 10 million.[ 6 ] Given the severity of this public health crisis, the systematic search for novel antimicrobial agents from natural sources has become essential. These agents are necessary to combat various drug-resistant microbial strains, including methicillin-resistant Staphylococcus aureus (MRSA).[ 7 ] Citrus sinensis (L.) Osbeck, commonly known as the ‘sweet orange’, is an evergreen flowering tree that belongs to the Rutaceae family. This species typically grows to a height of 7–10 m and accounts for approximately 70% of the global annual production of Citrus species.[ 8 ] Native to Asia, particularly in regions such as China, India, Iraq, and Pakistan, C. sinensis is widely distributed in warm and tropical areas. The fruits of C. sinensis are widely consumed as a rich source of vitamin C, while various parts of the plant have been traditionally used in medicinal systems to treat a range of ailments. These include angina, anxiety, colds, constipation, coughs, cramps, colic, depression, diarrhoea, hypertension, menstrual disorders, obesity, stress, and microbial infections such as bronchitis, flu, and tuberculosis.[ 8 ] Previous phytochemical studies on C. sinensis have identified several bioactive secondary metabolites, including carotenoids, cinnamic acid derivatives, flavonoids (primarily polymethoxyflavones), alkaloids (mainly acridone types), limonoids, sterols, terpenoids, and vitamins B and C. These compounds have demonstrated a wide range of bioactivities, including anticancer, antihyperlipidemic, anti-inflammatory, antimalarial, antimicrobial, anti-obesity, antioxidant, anxiolytic, and sedative properties.[ 8 ] However, the anti-MRSA activity of C. sinensis has not been previously evaluated, and bioassay-guided isolation has never been conducted to identify anti-MRSA compounds from this plant. Some preliminary studies have revealed the antimicrobial activity of essential oils derived from the peels of C. sinensis against multidrug-resistant bacteria.[ 9 ] In addition, hesperitin, coumaric acid, and ferulic acid, present in the peel of Newhall navel orange, have shown anti-MRSA activity against a single MRSA strain.[ 10 ] As part of ongoing research aimed at discovering phytochemicals with anti-MRSA activity,[ 7 , 11 – 13 ] this study focuses on the bioassay-guided isolation of 3-methoxynobiletin ( 4 ), rutin 3’-methyl ether ( 12 ) and sinensetin ( 7 ) as potential anti-MRSA agents. These compounds were isolated from the leaves and peels of C. sinensis collected from Iraq, where the plant has been traditionally used to treat infections. Additionally, ten other known flavonoids ( 1 – 3 , 5 , 6 , 8 , 9 , 11 – 14 ) and a new flavanone glycoside, named sinensidin ( 10 ), were identified. The objective of this study was to isolate and identify anti-MRSA compounds from C. sinensis using a bioassay-guided protocol. 2. Experimental Section General Chromatographic solvents were from Fisher Scientific, UK, and used without further purification. The NMR experiments were performed on a Bruker AMX600 NMR spectrometer (600 MHz for 1 H, and 150 MHz for 13 C). MS was conducted using a high-resolution mass spectroscopy facility (HR-MS) at the National Mass Spectrometry Facility (NMSF) (Swansea, UK) on Xevo G2-S ASAP or LTQ Orbitrap XL1 spectrometers. Low- and high-resolution MS analyses were also performed at Liverpool John Moores University; HR-MS using an Agilent 6200 Series Accurate-Mass Time-of-Flight (TOF) LC/MS system with electrospray ionization (ESI) in positive ion mode, connected to an Agilent auto-sampler injection system [ 26 ]. The vacuum liquid chromatographic (VLC) fractions, prepared using silica gel (Sigma-Aldrich, UK), were analyzed on a Dionex Ultimate 3000 UHPLC system coupled with a photodiode array (PDA) detector. A Phenomenex Gemini-NX 5 U C18 column (150 x 4.6 mm, 5 µm, Phenomenex, USA), and gradient solvent systems comprising acetonitrile (ACN, solvent B) and water (solvent A) (both contained 0.1% TFA, flow rate: 1 mL/min) were employed for method development for preparative-scale separation and isolation of compounds. The reversed-phase preparative HPLC purification was performed on an Agilent Technologies 1260 Infinity Series prep-HPLC coupled with a photo-diode-array detector (Germany) using a Phenomenex LC-18 C18 stainless steel column (150 x 21.2 mm, 5 µm, Phenomenex, USA) with the same solvent system as mentioned above, but with a 10 mL/min flow rate. The column temperature was set at 25°C [ 26 ]. Plant material The leaves and peels of Citrus sinensis (L.) Osbeck were collected from Diyala, Central Iraq (N 33.79684, E 44.623337) in September 2015. The plant materials were air-dried at room temperature and ground into a fine powder using a coffee grinder. Dr. Shaymaa Al-Majmaie (Diyala University, Iraq) identified the plant by comparing its morphological features with herbarium specimens. A voucher specimen (No. 6534) was deposited at the National Herbarium of Iraq. Extraction The air-dried ground leaves (194.6 g) and peels (394.4 g) of C. sinensis were extracted separately. Sequential extraction was performed using solvents of increasing polarity: n -hexane, dichloromethane (DCM), and methanol (MeOH), using a Soxhlet apparatus (900 mL, ten cycles each). The crude extracts were concentrated to dryness using a rotary evaporator and stored at 4°C for further analysis. The DCM extracts of the leaves and peels, along with the MeOH extract of the peels, demonstrated the highest antimicrobial activity in initial in vitro screening using resazurin as an indicator of cell growth.[ 40 ] These active extracts were subjected to further fractionation to isolate the bioactive compounds. Antimicrobial assays Resazurin assay The modified resazurin test, as described by Sarker et al. [ 40 ], was used to determine the minimum inhibitory concentration (MIC) using a microtitre plate. The assay was performed under aseptic conditions. Two Gram-positive bacterial strains (Micrococcus luteus NCTC 7508 and Staphylococcus aureus NCTC 12981), two Gram-negative bacterial strains (Escherichia coli NCTC 12241 and Pseudomonas aeruginosa NCTC 12903), and a fungal strain (Candida albicans ATCC 90028) were used in this study. Preparation of standard microbial colonies Microbial strains were cultured in 20 mL of nutrient agar on Petri dishes and incubated for 12–48 h at 35°C. Single colonies from the incubated plates were transferred to sterilized tubes containing 100 mL of nutrient broth and incubated at 35°C for 24–48 h. After incubation, the tubes were centrifuged at 4000 rpm for 5 min. The supernatant was discarded, and 20 mL of sterile normal saline was added to the tubes, followed by centrifugation under the same conditions. This process was repeated until the supernatant became clear. The optical density of the bacterial suspension was measured using a spectrophotometer at 500 nm, and the concentration was adjusted to 5 × 10 6 CFU/mL through dilution and calculations [ 26 ]. Preparation of resazurin solution The resazurin solution was prepared by dissolving 1 mg of resazurin sodium salt (purchased from Aldrich, USA) in 5 mL of sterile distilled water. A vortex mixer was used to ensure complete dissolution and homogeneity. Resazurin served as an indicator of cell growth in this assay [ 26 ]. Preparation of tested materials The stock concentration of the tested materials was prepared by dissolving them in 10% (v/v) dimethyl sulfoxide (DMSO) or sterilized water. The stock concentration was 10 mg/mL for crude extracts and 1 mg/mL for fractions and pure compounds [ 26 ]. Preparation of 96-well plates All wells on 96-well plates were filled with 50 µL of sterilized normal saline. The test material (100 µL) was added to the first row of the plate, and serial dilutions were made using multichannel pipettes by transferring 50 µL. Resazurin (10 µL) was added to all wells, followed by 10 µL of bacterial suspension (5 × 10 6 CFU/mL). To prevent bacterial dehydration, each plate was loosely wrapped with cling film. Each plate included the antibiotic ciprofloxacin as a positive control for bacterial strains and nystatin for the fungal strain C. albicans [ 26 ]. Interpretation of results The normal colour of resazurin is blue. During incubation, if the test materials inhibited the microorganisms, the wells retained the blue colour of resazurin or turned purple or colourless, indicating a positive result. The development of a pink colour indicated no effect of the test materials on the microbes. The lowest concentration at which the colour change occurred was recorded as the MIC (minimum inhibitory concentration) value. The mean of three values was calculated for each sample [ 26 ]. Anti-MRSA screening The anti-MRSA screening was conducted against five methicillin-resistant Staphylococcus aureus strains: SA1199B, XU212, MRSA340702, EMRSA-15MRSA274819, and the standard strain ATCC25923. All bacterial strains were obtained from the UCL School of Pharmacy, and the experiments were performed at the University of East London. Preparation of culture medium Mueller-Hinton broth (MHB) was prepared according to the supplier’s instructions. The MHB was adjusted to contain cations at concentrations of 20 mg/L Ca 2+ and 10 mg/L Mg 2+ . Preparation of tested compounds The compounds and antibiotics were dissolved in predetermined amounts of dimethyl sulfoxide (DMSO), ensuring that the final concentration of DMSO in the well was less than 1%. The solutions were further diluted with MHB to achieve the targeted starting concentration of 128 µg/mL. Preparation of MTT solution The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was prepared by dissolving the required amount of MTT in methanol (MeOH) to obtain a concentration of 5 µg/mL. Suspension of the bacterial subculture All bacterial strains were subcultured one day before the experiment. The strains were streaked onto nutrient agar slopes using a loop and incubated at 37°C for 12–18 hours. Anti-MRSA assay The assay was performed using 96-well plates. The first step involved adding 100 µL of MHB to all wells except those in column 12. In the first row of the plate, 100 µL of the test compounds or antibiotics was added. Using a multi-channel pipette, the contents of the first well were mixed thoroughly, and 100 µL was transferred to the wells of the second column. This process was repeated until column 10. Finally, 100 µL of the contents from column 10 was transferred to the wells of column 12. An inoculum density of 5 × 10 5 colony-forming units (cfu) of each test organism was prepared in normal saline (9 g/L) by comparison with a 0.5 McFarland standard. MHB (125 µL) was dispensed into 10 wells of a 96-well microtitre plate (Nunc, 0.3 mL volume per well). The plates were incubated at 37°C for 18 hours. To determine the minimum inhibitory concentrations (MICs), 20 µL of MTT solution was added to the microtitre plate, followed by incubation for 20 min. This colorimetric method indicated bacterial growth by a colour change from yellow to dark blue. The MIC was recorded as the lowest concentration at which no growth was observed. Norfloxacin, a well-known antibiotic, was used as a positive control. The method was adapted from the broth microdilution technique according to the National Committee for Clinical Laboratory Standards, with modifications using nutrient broth as the medium. [ 13 , 41 – 45 ] Isolation of compounds The most active dichloromethane (DCM) extracts from the leaves and peels of Citrus sinensis were subjected to vacuum liquid chromatography (VLC) separation [ 46 ] on silica gel. A stepwise mobile phase of increasing polarity was used, consisting of chloroform (CHCl3) and MeOH as follows 100% CHCl 3 , 2% MeOH in 100% CHCl 3 , 4% MeOH in 100% CHCl 3 , 6% MeOH in 100% CHCl 3 , 8% MeOH in 100% CHCl 3 , 10% MeOH in 100% CHCl 3 , 20% MeOH in 100% CHCl 3 and 30% MeOH in 100% CHCl 3 (each fraction was 200 mL). The active methanol (MeOH) extract of the peels was further fractionated by solid-phase extraction (SPE) on a Strata C 18 20 g cartridge [ 46 ] using 30%, 50%, 80%, and 100% MeOH in water as eluents (200 mL each). The VLC and SPE fractions were assessed for antimicrobial activity. The most active VLC fraction F7 (20% MeOH in 100% CHCl 3 ) of the DCM extract of the leaves, and VLC fractions F6 (10% MeOH in CHCl 3 ) and F7 (20% MeOH in CHCl 3 ) from the peel DCM extract, were selected for further analysis. Additionally, the active SPE fractions SPE2 (60% MeOH in water) and SPE3 (80% MeOH in water) of the MeOH extract of the peels were analysed by reversed-phase analytical HPLC to develop the optimum separation conditions for preparative HPLC separation. Reversed-phase preparative HPLC analysis using various gradient elution methods, as described in the results section, yielded fourteen flavonoids ( 1 – 14 ). The structures of all compounds were confirmed through extensive 1D and 2D NMR data analysis, MS spectroscopic data interpretation, and comparison with literature data for known compounds 1 – 9 and 11 – 14 [ 26 ]. 3. Results and Discussion The Soxhlet extraction of the dried ground leaves of Citrus sinensis (194.6 g) yielded three extracts: n -hexane (6.2 g, 3.19%), dichloromethane (DCM) (10.2 g, 5.24%), and methanol (MeOH) (14.3 g, 7.35%). Similarly, the extraction of the peels (394.4 g) produced three additional extracts: n -hexane (7.3 g, 1.9%), DCM (12.7 g, 3.2%), and MeOH (25.2 g, 6.39%). Initial antimicrobial screening of these extracts revealed that the DCM extracts exhibited the highest activity, followed by the MeOH extracts of the leaves and peels (Table 1 ). These extracts were active against all tested microorganisms, with minimum inhibitory concentration (MIC) values ranging from 4.8–625 mg/mL. Table 1 Antimicrobial activity of the extracts of the leaves and peels of C. sinensis , the VLC fractions of the DCM extracts, SPE fraction of the MeOH extract of peels and the isolated compounds (1–14) Tested samples MIC values in mg/mL Gram-positive bacteria Gram-negative bacteria Fungus Micrococcus luteus Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Candida albicans Leaves n -Hexane extract 5000 5000 2500 NA NA DCM extract 312.5 625 625 5000 312.5 MeOH extract 1250 2500 2500 5000 5000 Peels n -Hexane extract 156 NA 5000 5000 2500 DCM extract 4.8 625 312.5 625 156 MeOH extract 312.5 5000 1250 1250 625 VLC fractions of the DCM extract of the leaves F1 NA NA NA NA NA F2 NA NA NA NA NA F3 NA NA NA NA NA F4 NA NA NA NA NA F5 NA NA NA NA NA F6 NA NA NA 1000 NA F7 625 625 NA 1000 625 F8 NA 1000 NA NA 1000 VLC fractions of the DCM extract of the peels F1 NA NA NA NA NA F2 NA NA NA NA NA F3 NA NA NA NA NA F4 500 NA NA NA 500 F5 500 NA NA 1000 0.50 F6 7.81 1000 500 500 7.81 F7 15.6 500 NA 1000 15.6 F8 NA NA NA NA NA SPE fractions of the MeOH extract of the peels SPE1 500 NA NA NA 500 SPE2 625 1000 1000 NA 500 SPE3 31.2 125 250 500 15.6 SPE4 500 NA AN NA 500 Isolated compounds ( 1 – 14 ) 1 500 NA NA NA NA 2 250 NA NA 500 250 3 250 NA NA 500 500 4 500 250 NA 500 250 5 500 NA NA 250 250 6 250 NA NA NA NA 7 250 250 500 250 62.5 8 NA NA NA NA NA 9 NA NA NA NA NA 10 125 NA NA NA 250 11 NA NA NA NA NA 12 250 250 250 250 250 13 125 NA NA 500 250 14 500 NA NA NA 500 Positive controls Ciprofloxacin 97.6 97.6 15.5 19.5 Not applicable Nystatin Not applicable Not applicable Not applicable Not applicable 97.6 NA = No activity at the tested concentration The n -hexane extract of the leaves was the least active among the six extracts, showing activity against E. coli , Micrococcus luteus , and S. aureus only at high concentrations (MIC = 2500–5000 mg/mL). The n -hexane extract of the peels was also active at high concentrations against all tested organisms except S. aureus , with its most potent activity observed against M. luteus (MIC = 156 mg/mL). The DCM extracts of both the leaves and peels were the most active among all extracts, showing activity against all strains. However, the DCM extract of the peels was more potent than that of the leaves. The DCM extract of the peels was particularly effective against M. luteus , with an MIC value of 4.8 mg/mL. A similar difference in potency was observed between the MeOH extracts of the leaves and peels, with the latter being more active (Table 1 ). The highest antifungal activity against Candida albicans was observed with the DCM extract of the peels (MIC = 156 mg/mL), while the least active was the MeOH extract of the leaves (MIC = 5000 mg/mL). Although the MeOH extracts were active against all five microbial strains, their MIC values (312.5–5000 mg/mL) were significantly higher than those of the DCM extracts (Table 1 ). Consequently, the active DCM extracts of the leaves and peels were subjected to vacuum liquid chromatography (VLC)-assisted fractionation, resulting in eight fractions each. Since the MeOH extract of the peels demonstrated a better antimicrobial profile than the MeOH extract of the leaves, it was further fractionated using solid-phase extraction (SPE) on a Strata C 18 cartridge (20 g, Phenomenex, UK). This process yielded four SPE fractions (SPE1-SPE4) eluted with 30%, 50%, 80%, and 100% MeOH in water. All VLC and SPE fractions were tested for antimicrobial activity to identify the most active fractions for subsequent isolation of bioactive compounds. The first five VLC fractions (F1-F5: 100% CHCl 3 , 2% MeOH in CHCl 3 , 4% MeOH in CHCl 3 , 6% MeOH in CHCl 3 , and 8% MeOH in CHCl 3 ) of the DCM extract of the leaves did not exhibit any antimicrobial activity against the tested strains at the concentrations used. VLC fraction 6 (F6, 10% MeOH in CHCl 3 ) was active only against Pseudomonas aeruginosa (MIC = 1000 mg/mL), while fraction 8 (F8, 30% MeOH in CHCl 3 ) showed activity against S. aureus and C. albicans (MIC = 1000 mg/mL). VLC fraction 7 (F7, 20% MeOH in CHCl 3 ) of the DCM extract of the leaves was the most active among the fractions, showing activity against all tested organisms (MIC = 625–1000 mg/mL) (Table 1 ). The first three VLC fractions (F1-F3: 100% CHCl 3 , 2% MeOH in CHCl 3 , and 4% MeOH in CHCl 3 ) and fraction 8 (F8, 30% MeOH in CHCl 3 ) of the DCM extract of the peels did not show any antimicrobial activity against the tested strains. VLC fractions 4 (F4, 6% MeOH in CHCl 3 ) and 5 (F5, 8% MeOH in CHCl 3 ) were active against M. luteus (MIC = 500 mg/mL) and C. albicans (MIC = 500 mg/mL), with F5 also active against P. aeruginosa (MIC = 1000 mg/mL). VLC fraction 6 (F6, 10% MeOH in CHCl 3 ) was active against all tested organisms (MIC = 7.81–1000 mg/mL), while fraction 7 (F7, 20% MeOH in CHCl 3 ) was active against all microorganisms (MIC = 156–1000 mg/mL) except E. coli (Table 1 ). Among the four SPE fractions, fraction 3 (SPE3, 80% MeOH in water) was the most active, showing activity against all microbial strains tested in this study (MIC = 156–500 mg/mL). Fraction 2 (SPE2, 60% MeOH in water) was also active against all organisms (MIC = 500–625 mg/mL) except P. aeruginosa (Table 1 ). The first fraction (SPE1, 30% MeOH in water) was only active against M. luteus and C. albicans (MIC = 500 mg/mL). Considering the overall antimicrobial activity profile, particularly against S. aureus , VLC fraction 7 (F7) of the DCM extract of the leaves, VLC fractions F6 and F7 of the DCM extract of the peels, and SPE fractions SPE2 and SPE3 were selected for further HPLC analysis to purify the active compounds. Reversed-phase preparative HPLC analysis of the most active VLC fraction F7 of the DCM extract of the leaves of C. sinensis (linear gradient 30–100% acetonitrile (ACN) in water over 30 min, isocratic 100% ACN for the next 5 min, linear gradient 100 − 30% ACN in water for 5 min, and isocratic 30% MeOH in water for 5 min, 10 mL/min) yielded five compounds (5, 3, 6, 1, and 8) (Fig. 1 ) with retention times ( t R ) of 20.39, 21.36, 22.32, 31.61, and 33.45 min, respectively. A similar preparative HPLC analysis (linear gradient 20–50% ACN in water over 30 min, isocratic 50% ACN for the next 3 min, linear gradient 50 − 20% ACN in water for 5 min, and isocratic 20% ACN in water for 5 min, 10 mL/min) of VLC fraction F6 of the DCM extract of the peels of C. sinensis yielded six compounds: 7 ( t R = 28.28 min), 14 (tR = 33.21 min), 4 ( t R = 34.35 min), 13 ( t R = 37.03 min), 2 ( t R = 39.84 min), and additional amounts of 5 ( t R = 32.0 min). The analysis of VLC fraction 7 of the peels (linear gradient 20–80% ACN in water over 30 min, isocratic 80% ACN for the next 5 min, linear gradient 80 − 20% ACN in water for 5 min, and isocratic 20% ACN in water for 5 min, 10 mL/min) resulted in the isolation of seven more compounds: 11 ( t R = 14.01 min), 9 ( t R = 17.7 min), 14 ( t R = 22.42 min), and additional amounts of 7 ( t R = 19.74 min), 5 ( t R = 21.75 min), 4 ( t R = 23.03 min), and 13 ( t R = 24.33 min). Reversed-phase preparative HPLC analysis of the SPE fractions SPE2 and SPE3 of the MeOH extract of the peels (linear gradient 20–80% MeOH in water over 30 min, isocratic 80% MeOH for the next 2 min, linear gradient 80 − 20% MeOH in water for 2 min, and isocratic 100% MeOH in water for 2 min, and isocratic 20% MeOH in water for 5 min) yielded compounds 11 ( t R = 13.78 min), 10 ( t R = 15.03 min), 12 ( t R = 15.5 min), 9 ( t R = 17.7 min), 8 ( t R = 19.18 min), 13 ( t R = 26.13 min), 11 ( t R = 28.33 min), and 5 ( t R = 28.82 min). Among the isolated compounds, compounds 1 – 9 and 11 – 14 were identified as known natural products: cirsilineol (1, 3.3 mg),[ 14 ] demethylnobiletin (2, 3.1 mg),[ 15 ] 5-desmethylsinensetin ( 3 , 2.8 mg),[ 16 ] 3-methoxynobiletin ( 4 , 4.4 mg),[ 17 ] nobiletin ( 5 , 2.9 mg),[ 18 ] 6,7,8,3',4'-pentamethoxyflavone ( 6 , 3.6 mg),[ 19 ] sinensetin ( 7 , 4.8 mg),[ 19 ] hesperidin ( 8 , 6.1 mg),[ 20 ] narirutin ( 9 , 1.9 mg),[ 21 ] rutin ( 11 , 9.1 mg),[ 22 ] rutin 3’-methyl ether ( 12 , 4.1 mg),[ 7 ] tangeritin ( 13 , 6.7 mg),[ 23 ] and tetramethylscutellarein ( 14 , 3.7 mg).[ 18 ] The structures of these compounds were confirmed through 1D and 2D NMR and high-resolution electrospray ionization mass spectrometry (HRESIMS) data analyses. The experimental ¹H and ¹³C NMR data were consistent with published literature for the respective compounds. Most of these isolated flavonoids are polymethoxyflavones, which are characteristic of the Citrus genus and are known to possess various bioactivities.[ 24 ] Compound 10 , isolated as a golden yellow oily substance, was identified as a new natural product. Its structure was elucidated using spectroscopic techniques. The high-resolution electrospray ionization mass spectrometry (HRESIMS) data suggested the empirical formula C 28 H 32 O 16 , with a pseudomolecular ion [M + H] + peak at m/z 625.1761 (calculated 625.1768). The 1 H NMR spectrum (Table 2 ) revealed aromatic proton signals at δH 7.47 (2H, d, J = 8.8 Hz) and 7.16 (2H, d, J = 8.8 Hz), indicative of a para-disubstituted benzene ring, typical of the B ring in flavonoids such as apigenin and kaempferol. A singlet at δH 6.22 (2H) was assigned to a methylenedioxy group attached to a benzene ring. Signals at δH 5.53 (1H, dd, J = 3.1, 12.4 Hz), 2.83 (1H, dd, J = 3.1, 17.1 Hz), and 3.29 (1H, dd, J = 17.1, 12.4 Hz) were characteristic of H-1 and H 2 -3 of a flavanone skeleton. Table 2 1H (600 MHz) and 13C (150 MHz) NMR data, and 1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC key correlations of sinensidin (10) Number Chemical shift d in ppm 1 H- 1 H COSY 1 H- 13 C HSQC 1 H- 13 C HMBC d H , coupling constant J in Hz d C 1 J 2 J 3 J Flavanone skeleton 2 5.53, dd, 1H, J = 3.1, 12.4 81.5 H-3 C-2 C-1’ C-4 3 2.83, dd, 1H, J = 3.1, 17.1 3.29, dd, 1H, J = 17.1, 12.4 43.1 H-2 C-3 C-2, C-4 C-1’ 4 - 203.6 - - - - 5 - 147.7** - - - - 6 - 135.5 - - - - 7 - 151.6 - - - - 8 - 129.4 - - - - 9 - 147.6** - - - - 10 - 111.6 - - - - 1’ - 132.7 - - - - 2’, 6’ 7.47, d, 2H, J = 8.8 129.1 H-3’. H-5’ C-3’, C-5’ C-1’ C-2, C-4’ 3’, 5’ 7.16, d, 2H, J = 8.8 118.0 H-2’, H-6’ C-2’, C-6’ C-4’ C-1’ 4’ - 160.4 - - - - 5,6-O-CH 2 -O- 6.22, s, 2H 101.3 - -O-CH 2 -O- - C-5, C-6 Glucose unit 1’’ 4.96, d, 1H, J = 7.0 102.3 H-2’’ C-1’’ - C-4’ 2’’ 3.88* 75.5 H-1’’, H-3’’ C-2’’ - C-4’’ 3’’ 3.48* 78.5 H-2’’, H-4’’ C-3’’ - C-5’’ 4’’ 3.39* 71.6 H-3’’, H-5’’ C-4’’ - C-2’’, C-6’’ 5’’ 3.34* 78.4 H-4’’, H-6’’ C-5’’ - C-3’’ 6’’ 3.92, 3.70* 69.9 H-5’’ C-6’’ - C-1’’’ Rhamnose unit 1’’’ 4.71, d, 1H, J = 1.4 103.4 H-2’’’ C-1’’’ - C-6’’ 2’’’ 3.48* 74.2 H-1’’’, H-3’’’ C-2’’’ - C-4’’’ 3’’’ 3.29* 71.8 H-2’’’, H-4’’’ C-3’’’ - C-5’’’ 4’’’ 3.69* 72.5 H-3’’’, H-5’’’ C-4’’’ - C-2’’’, C-6’’’ 5’’’ 3.60* 77.3 H-4’’’, H-6’’’ C-5’’’ - C-3’’’ 6’’’ 1.21, d, 3H, J = 6.2 18.1 H-5’’’ C-6’’’ C-5’’’ C-4’’’ Spectra obtained in CD 3 OD *Unresolved overlapped peaks, determined by 2D COSY, HSQC, and HMBC experiments **Interchangeable; could be either carbon Two anomeric proton signals at δH 4.96 (1H, d, J = 7 Hz) and 4.71 (1H, d, J = 1.4 Hz) were attributed to glucose and rhamnose moieties, forming a rutinosyl unit. Overlapping signals in the δH 3.33–4.20 range were assigned to the remaining sugar protons. The absence of additional aromatic proton signals indicated full substitution of the A ring of the flavanone skeleton. The 1 H- 1 H COSY spectrum (Table 2 ) confirmed scalar couplings, particularly aiding in the assignment of sugar protons. The 13 C NMR spectrum (Table 2 ) revealed 28 carbons, including a carbonyl signal at δC 203.6 (C-4 of the flavanone nucleus), 12 carbons for the rutinosyl unit (including anomeric carbons at δC 102.3 and 103.4, a rhamnose methyl signal at δC 18.1, and oxymethine carbons at δC 71.6–78.5), a methylenedioxy group at δC 101.3, and 12 aromatic carbons (four methine, six oxygenated quaternary, and two quaternary carbons) (Table 5). The 1 H- 13 C HSQC experiment (Table 2 ) confirmed the assignment of all protonated carbons. Key 3 J correlations in the ¹H-¹³C HMBC spectrum (Table 2 ) included H-1’’ (glucose anomeric) to C-4’, H-6’’ to C-1’’’ (rhamnose anomeric), and methylenedioxy protons to C-5 (δC 147.7) and C-6 (δC 135.5) of the flavanone A ring. These correlations confirmed the placement of the rhamnosyl unit at C-4’, the 6→1 linkage between glucose and rhamnose, and the methylenedioxy group at C-5/C-6. Due to the full substitution of the A ring and the absence of aromatic proton signals, the 13 C NMR data of compound 10 were compared with similar compounds, particularly those with oxygenation at C-5, C-6, C-7, and C-8. This comparison allowed for the unambiguous assignment of all oxygenated carbons in the A ring. Compound 10 was identified as a new flavanone glycoside, named sinensidin. Notably, the methylenedioxy linkage at C-5/C-6 is less common in nature compared to C-6/C-7 linkages. All fourteen compounds ( 1 – 14 ) were subjected to the resazurin assay, as described for the extracts and fractions. Compounds 3-methoxynobiletin ( 4 ), sinensetin ( 7 ), and rutin 3’-methyl ether ( 12 ) exhibited activity against all tested microorganisms, with MIC values ranging from 500–625 mg/mL (Table 1 ). These compounds were the only ones active against S. aureus (MIC = 250 mg/mL). Sinensetin ( 7 ) showed potent antifungal activity against C. albicans (MIC = 62.5 mg/mL), consistent with recent findings where a combination of sinensetin (7) with other compounds was effective in treating vulvovaginal candidiasis .[ 25 ] Hesperidin ( 8 ), narirutin ( 9 ), and rutin ( 11 ) did not show antimicrobial activity against the tested strains. However, other flavonoids ( 1 – 3 , 5 , 6 , 10 , and 12 – 14 ) were active against M. luteus (MIC = 125–500 mg/mL). Cirsilineol ( 1 ) and 6,7,8,3',4'-pentamethoxyflavone ( 6 ) were the least active, showing no activity against other organisms. Demethylnobiletin ( 2 ), 5-desmethylsinensetin ( 3 ), nobiletin ( 5 ), and tangeritin ( 13 ) exhibited similar antimicrobial profiles, with activity against M. luteus, Pseudomonas aeruginosa, and C. albicans (MIC = 250–500 mg/mL). Sinensidin ( 10 ) and tetramethylscutellarein ( 14 ) also displayed antifungal activity against C. albicans (MIC = 250 and 500 µg/mL, respectively) (Table 1 ). The activity of 3-methoxynobiletin ( 4 ), sinensetin ( 7 ), and rutin 3’-methyl ether ( 12 ) against S. aureus (MIC = 250 mg/mL) led to their further testing against five clinical MRSA strains: XU212, SA1199B, EMRSA15, MRSA340702, and MRSA274819 (Table 3 ). 3-Methoxynobiletin ( 4 ) was active only against EMRSA15 (MIC = 256 mg/mL), while sinensetin ( 7 ) and rutin 3’-methyl ether ( 12 ) showed activity against three MRSA strains. Sinensetin ( 7 ) was most potent against MRSA340702 (MIC = 128 mg/mL), while rutin 3’-methyl ether ( 12 ) was active against XU212, MRSA340702, and MRSA274819 (MIC = 256 mg/mL). Table 3 Anti-MRSA activity of 3-methoxynobiletin (4), sinensetin (7) and rutin 3’-methyl ether (12) Tested compounds MIC values in mg/mL MRSA strains Standard S. aureus strain XU212 SA1199B EMRSA15 MRSA340702 MRSA274819 ATCC25923 3-Methoxynobiletin ( 4 ) NA NA 256 NA NA 256 Sinensetin ( 7 ) NA NA 256 128 256 128 Rutin 3’-methyl ether ( 12 ) 256 NA NA 256 256 128 Norfloxacin 16 32 1 64 64 2 NA = No activity at the tested concentrations 3-Methoxynobiletin ( 4 ), sinensetin ( 7 ), and rutin 3’-methyl ether ( 12 ) also exhibited antibacterial activity against the standard S. aureus strain ATCC25923, with MIC values of 256, 128, and 128 mg/mL, respectively. The anti-MRSA activity of sinensetin ( 7 ) was two-fold and four-fold less potent against MRSA340702 and MRSA274819, respectively, compared to the positive control norfloxacin (MIC = 64 mg/mL). 3-Methoxynobiletin ( 4 ) has been previously reported from the peels of Citrus aurantium var. sinensis [ 27 ] and C. unshiu Markob.[ 28 ] Although its antimicrobial potential was described, no activity against MRSA strains was reported. This polymethoxyflavone was also isolated from Ageratum conyzoides [ 29 ] and Ageratina colestinum L.[ 30 ] of the Asteraceae family, where its antifungal properties were noted.[ 30 ] Sinensetin ( 7 ), widely found in the Citrus genus (e.g., C. aurantium , C. grandis , C. reticulata , and C. sinensis ),[ 26 , 31 , 32 ] has been reported to exhibit antibacterial activity,[ 26 , 33 – 35 ] particularly against S. aureus . This activity is believed to be mediated by targeting staphylocoagulase and inhibiting biofilm formation.[ 36 ] Rutin 3’-methyl ether ( 12 ), also known as isorhamnetin 3- O -rutinoside, was previously isolated as an antimicrobial compound with anti-MRSA properties from Ruta chalepensis (Rutaceae).[ 7 ] This compound has also been found in Campanula rapunculoides L. (Campanulaceae),[ 37 ] Caragana sinica (Buc’hoz) Rehder (Fabaceae),[ 38 ] and Coriandrum sativum L. (Apiaceae).[ 39 ] 4. Conclusions A bioassay-guided isolation study led to the identification of two polymethoxy flavones, 3-methoxynobiletin ( 4 ) and sinensetin ( 7 ), along with rutin 3’-methyl ether ( 12 ), as potential anti-MRSA agents from the leaves and peels of Citrus sinensis of Iraqi origin. These three flavonoids exhibited activity against various strains of methicillin-resistant S. aureus (MRSA), with minimum inhibitory concentration (MIC) values ranging from 128 to 256 mg/mL. In addition to the anti-MRSA compounds, ten other known flavonoids ( 1–3 , 5 , 6 , 8 , 9 , 11 – 14 ) and a new flavanone glycoside, named sinensidin ( 10 ), were isolated from the active fractions. However, none of these compounds showed activity against S. aureus . Among the isolated flavonoids, 3-methoxynobiletin ( 4 ), sinensetin ( 7 ), and rutin 3’-methyl ether ( 12 ) demonstrated the most potent antibacterial activity against all tested microorganisms. The anti-MRSA potential of 3-methoxynobiletin ( 4 ) is reported here for the first time. Based on the findings of this study, these flavonoids ( 4 , 7 , and 12 ) could serve as valuable templates for the development of new structural analogues with enhanced anti-MRSA properties. However, further physicochemical, preclinical, and clinical studies are necessary to fully evaluate the therapeutic potential of these polymethoxyflavones and their analogues in the treatment of MRSA infections. Declarations Acknowledgements The authors would like to thank the Iraqi Ministry of Higher Education and Scientific Research, as well as the College of Science, University of Diyala, Iraq, for providing a PhD Scholarship to Shaymaa Al-Majmaie to conduct this study. Additionally, the authors appreciate the support of the EPSRC National Mass Spectrometry Service, Swansea, UK, for the HRMS analyses. Lutfun Nahar gratefully acknowledges the support from the European Regional Development Fund (Project ENOCH #CZ.02.1.01/0.0/0.0/ 16_019/0000868), and the Czech Science Foundation (Project #23-05474S). Author Contribution Statement SA: carried out all aspects of the research work, including data analysis; LN: supervised the project, prepared the first draft of the manuscript and carried out the final editing. MMR: MRSA work; GPS: supervised the project, data analysis and editing; HA: data analysis; SDS: concept, supervised the project, prepared the first draft of the manuscript and carried out the final editing. Data Availability Statement The data that support the findings of this study are available from the corresponding authors upon reasonable request. Conflicts of Interest The authors declare no conflicts of interest. Funding Declaration No funding was received for this work. References Livermore, D. M. Bacterial resistance: Origins, epidemiology, and impact. Clin. Infect. Dis. 36 , S11–S23 (2003). Sweileh, W. M. Global research publications on irrational use of antimicrobials: call for more research to contain antimicrobial resistance. Globalization Health . 17 , 94 (2021). Hossain, M. J. et al. Irrational use of antibiotics and factors associated with antibiotic resistance: Findings from a cross-sectional study in Bangladesh. Health Sci. Rep. 6 , e1465 (2023). Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis. Lancet 399 , 629–655 (2022). WHO (The World Health Organization. ), Antimicrobial resistance. Available online: (2024). https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance (accessed on 27 October 2024). GBD 2021 Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019–2021: A systematic analysis with forecast to 2050. Lancet 404 , 1199–1226 (2024). Al-Majmaie, S. et al. Anti-MRSA constituents from Ruta chalepensis (Rutaceae) grown in Iraq, and in silico studies on two of most active compounds, chalepensin and 6-hydroxy-rutin 3’, 7-dimethyld ether. Molecules 26 , 1114 (2021). Favela-Hernandez, J. M. J., Gonzalez-Santiago, O. & Ramirez-Cabrera, M. A. Esquivel-Ferrino, M. del Rayo Camacho-Corona, Chemistry and pharmacology of Citrus sinensis . Molecules 21 , 247 (2016). Azghar, A. et al. Chemical composition and antibacterial activity of Citrus peels essential oils against multidrug-resistant bacteria: A comparative study. J. Herb. Med. 42 , 100799 (2023). Liang, Y., Zhang, L. Y., Lai, C. L., Ouyang, Z. G. & Zhang, J. Evaluation of antibacterial activity of compounds isolated from the peel of Newhall navel orange. Nat. Prod. Res. 37 , 2060–2064 (2023). Datta, B. K. et al. Polygosumic acid, a new cadinane sesquiterpene, from Polygonum viscosum inhibits the growth of drug-resistant Escherichia coli and Staphylococcus aureus (MRSA) in vitro . J. Nat. Med. 61 , 391–396 (2007). Alloush, M., Mallion, C., Sarker, S. D. & Rahman, M. M. Coumarins from the roots of Angelica archangelica and their antibacterial activity against methicillin-resistant Staphylococcus aureus . Dhaka Univ. J. Pharm. Sci. 20 , 275–281 (2022). Nurunnabi, T. R. et al. D. Anti‐MRSA activity of oxysporone and xylitol from the endophytic fungus Pestalotia sp. growing on the Sundarbans mangrove plant Heritiera fomes . Phytother. Res. 32 , 348–354 (2018). Hammoud, L. et al. A new flavonoid and other constituents from Centaurea nicaeensis All. var. walliana M. Nat. Prod. Res. 26 , 203–208 (2012). Wang, X. et al. Preparative isolation and purification of polymethoxylated flavones from Tangerine peel using high-speed counter-current chromatography. J. Chromatogr. A . 1090 , 188–192 (2005). Alarif, W. M. et al. Selective cytotoxic effects on human breast carcinoma of new methoxylated flavonoids from Euryops arabicus grown in Saudi Arabia. Eur. J. Med. Chem. 66 , 204 (2013). Li, S. L., Lo, C. Y. & Ho, C. T. Hydroxylated polymethoxyflavones and methylated flavonoids in sweet orange ( Citrus sinensis ) peel. J. Agric. Food Chem. 54 , 4176–4185 (2006). Li, Z., Zhao, Z. & Zhou, Z. Simultaneous Separation and Purification of Five polymethoxylated flavones from Dahongpao Tangerine ( Citrus tangerina Tanaka) using macroporous adsorptive resins combined with prep-HPLC. Molecules 23 , 2660 (2018). Han, S., Kim, H. M., Lee, J. M., Mok, S. Y. & Lee, S. Isolation and identification of polymethoxyflavones from the hybrid Citrus , Hallabong. J. Agric. Food Chem. 58 , 9488–9491 (2010). Nieto, J. Gutierrez, 1 H NMR spectra at 360 MHz of diosmin and hesperidin in DMSO solution. Spectrosc. Lett. 19 , 427–434 (1986). Abu-Gharbieh, E. & Shehab, N. G. Therapeutic potentials of Crataegus azarolus var. eu azarolus Maire leaves and its isolated compounds. BMC Complement. Altern. Med. 17 , 218 (2017). Kamel, K. M., Abd El-Raouf, O. M., Metwally, S. A., Abd El‐Latif, H. A. & El‐Sayed, M. E. Hesperidin and rutin, antioxidant Citrus flavonoids, attenuate cisplatin‐induced nephrotoxicity in rats. J. Biochem. Mol. Toxicol. 28 , 312–319 (2014). Hamdan, D. et al. Chemical composition and biological activity of Citrus jambhiri Lush. Food Chem. 127 , 394–403 (2011). Toledo, R., Tomas-Navaro, M., Yuste, J. E., Crupi, P. & Vallejo, F. An update on citrus polymethoxyflavones: chemistry, metabolic fate and relevant bioactivities. Eur. Food Res. Technol. 250 , 2179–2192 (2024). Zeng, Z. et al. A non-antibiotic antimicrobial drug, a biological bacteriostatic agent, is useful for treating aerobic vaginitis, bacterial vaginosis and vulvovaginal candidiasis. Frontierts Microbiol. 15 , 1341878 (2024). Al-Majmaie, S., Nahar, L., Rahman, M. M., Sharples, G. P. & Sarker, S. D. Antimicrobial potential of the leaves of Citrus grandis (L.) Osbeck collected from Iraq: Bioassay-guided isolation of sinensetin as the anti-MRSA compound. Fitoterapia 106393. (2025). Chakroun, I. et al. Phytochemicals constituents and potential applications of Thomson Navel orange Citrus x aurantium var. sinensis L.) peel extracts: Antioxidant, antimicrobial and antiproliferative properties. Ind. Crops Prod. 206 , 117597 (2023). Vu, T. O. et al. Flavonoids from the peels of Citrus unshiu Markov. And their inhibitory effects on RANKL-induced osteoclastogenesis through the downregulation of c-Fos signaling. vitro Bioorg. Chem. 107 , 104613 (2021). Vyas, A. V. & Mulchandani, N. B. Polyoxygenated flavones from Ageratum conyzoides . Phytochemistry 25 , 2625–2627 (1986). Boner, P. L. Isolation and structural studies of natural products of Solidago ohioensis Riddell, Amphiachyris dracunculoides (DC.) Nutt., and Ageratina colestinum L. PhD Thesis , The Ohio State University, USA. (1997). Cai, W., Zhang, S., Wang, Y., Liu, C. & Luo, R. Differential distribution of characteristic constituents in peel and pulp of Auruntium Fructus Immaturus ( Citrus aurantium L.) using MALDI mass spectrometry imaging. Fitoterapia 177 , 106067 (2024). Shi, J. J. et al. Evaluation of chemical components and quality in Xinhui Chenpi ( Citrus reticulata ‘Chach’) with two different storage times by GC-MS. Food Sci. Nutr. 12 , 5036–5051 (2024). Jie, L. H., Jantan, I., Yusoff, S. D., Jalil, J. & Husain, K. Sinensetin: An insight on its pharmacological activities, mechanisms of action and toxicity. Front. Pharmacol. 11 , 553404 (2021). Patel, K. & Patel, D. K. Therapeutic effectiveness of sinensetin against cancer and other human complications: A review of biological potential and pharmacological activities. Cardiovasc. Henatological Disorders Drug Targets . 22 , 144–154 (2022). Permadi, N., Nurzaman, M., Doni, F. & Julaeha, E. Elucidation of the composition, antioxidant and antimicrobial properties of essential oil and extract from Citrus aurantifolia (Christm.) Swingle peel. Saudi J. Biol. Sci. 31 , 103987 (2024). Ge, B. et al. Sinensetin interferes with Staphylococcus aureus infections by targeting staphylocoagulase and improves infections survival rates in mouse model pneumonia. J. Appl. Microbiol. 135 , lxae235 (2024). Teslov, L. S. Phenolic compounds of the above-ground part of Campanula rapunculoides L. Rastitel’nye Resursy . 32 , 87–92 (1996). Tai, Z. G. et al. Antioxidant activities of Caragana sinica flower extracts and their main chemical constituents. Molecules 15 , 6722–6732 (2010). Hussain, F., Jahan, N., Khalil-ur-Rahman, B., Sultana, S. & Jamil Identification of hypotensive biofunctional compounds of Coriandrum sativum and evaluation of their angiotensin-converting enzyme (ACE) inhibition potential. Oxidative Medicine and Cellular Longevity 2018 , 4643736. (2018). Sarker, S. D., Nahar, L. & Kumarasamy, Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 42 , 321–324 (2007). Rahman, M. M., Garvey, M., Piddock, L. J. & Gibbons, S. Antibacterial terpenes from the oleoresin of Commiphora molmol (Engl). Phytother. Res. 22 , 1356–1360 (2008). Rahman, M. M., Shiu, W. K., Gibbons, S. & Malkinson, J. P. Total synthesis of acylphloroglucinols and their antibacterial activities against clinical isolates of multi-drug resistant (MDR) and methicillin-resistant strains of Staphylococcus aureus . Eur. J. Med. Chem. 155 , 255–262 (2018). Ioannou, E. et al. Structures and antibacterial activities of minor dolabellanes from the brown alga Dilophus spiralis . European Journal of Organic Chemistry 2012 , 5177–5186. (2012). Shiu, W. K. et al. Antibacterial acylphloroglucinols from Hypericum olympicum . J. Nat. Prod. 75 , 336–343 (2011). Tareq, F. S. et al. Anti- Staphylococcal calopins from fruiting bodies of Caloboletus radicans . J. Nat. Prod. 81 , 400–404 (2018). Reid, R. & Sarker, S. Isolation of natural products by low-pressure column chromatography, in Natural Products Isolation, 3rd edition, Humana Press – Springer-Verlag, USA, (2012). Additional Declarations No competing interests reported. Supplementary Files SupplementarymaterialsREV2.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 20 Aug, 2025 Reviews received at journal 19 Aug, 2025 Reviews received at journal 17 Aug, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers agreed at journal 29 Jul, 2025 Reviews received at journal 25 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers invited by journal 15 Jul, 2025 Editor assigned by journal 15 Jul, 2025 Editor invited by journal 15 Jul, 2025 Submission checks completed at journal 08 Jul, 2025 First submitted to journal 08 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7037612","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":486191059,"identity":"96be4bd8-f054-41e9-956b-6b6f122f60b7","order_by":0,"name":"Shaymaa Al-Majmaie","email":"","orcid":"","institution":"University of Diyala","correspondingAuthor":false,"prefix":"","firstName":"Shaymaa","middleName":"","lastName":"Al-Majmaie","suffix":""},{"id":486191060,"identity":"3f8d7df5-ca08-4eed-b509-53db9b97eb69","order_by":1,"name":"Lutfun Nahar","email":"","orcid":"","institution":"Palacký University, The Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lutfun","middleName":"","lastName":"Nahar","suffix":""},{"id":486191061,"identity":"ea3f4c4e-4472-4084-bfc0-481d7dfa8a79","order_by":2,"name":"M. Mukhlesur Rahman","email":"","orcid":"","institution":"University of East London","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"Mukhlesur","lastName":"Rahman","suffix":""},{"id":486191068,"identity":"0d80de0b-cf1e-4970-8fd2-dd3fb4b2128a","order_by":3,"name":"George P. Sharples","email":"","orcid":"","institution":"Liverpool John Moores University","correspondingAuthor":false,"prefix":"","firstName":"George","middleName":"P.","lastName":"Sharples","suffix":""},{"id":486191071,"identity":"86b114eb-6cab-493a-a148-e3b325bfd0a8","order_by":4,"name":"Hesamoddin Arabnozari","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBACAzSGhBwDAw+JWoxJ1sKQ2EBIizl7+8NPN2ruyZtLNx/+8HOPRfqG42cPPvjAYCen24Bdi2XPGWPpnGPFhjvnHEsw7HkmkbvhTF6y4QyGZGOzAzgcdiOHQTqHLYFxw40cgwSeA0AtB3LMpHkYDiRuw6Xl/vPHv3P+JdiDtBz8c0Ai3eD8GwJabjCYSee2JSQCtRg2A21JANpLQMuZHDPr3L6E5J0z0pKZZQ5IGM688cbYcIYBHr8cP/74ds63BNvtEsmHP745UCfPdz7H8MGHCjs5XFowgQJYpQEBVShAvoEU1aNgFIyCUTASAAAapmLj38WHFQAAAABJRU5ErkJggg==","orcid":"","institution":"Mazandaran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Hesamoddin","middleName":"","lastName":"Arabnozari","suffix":""},{"id":486191075,"identity":"51606c7b-c854-4648-8f36-3e8a6d594ce3","order_by":5,"name":"Satyajit D. Sarker","email":"","orcid":"","institution":"Liverpool John Moores University","correspondingAuthor":false,"prefix":"","firstName":"Satyajit","middleName":"D.","lastName":"Sarker","suffix":""}],"badges":[],"createdAt":"2025-07-03 11:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7037612/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7037612/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87025106,"identity":"6640f5b4-07df-40b9-a95e-0b5365f3851e","added_by":"auto","created_at":"2025-07-18 11:55:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62601,"visible":true,"origin":"","legend":"\u003cp\u003eStructures of flavonoids isolated from the active fractions of the extracts of the leaves and peels of C. sinensis\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7037612/v1/05c8727f81fc507a86c0d040.png"},{"id":87025799,"identity":"08601e87-3ffe-47e0-a604-3275818e7cd9","added_by":"auto","created_at":"2025-07-18 12:03:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1657998,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7037612/v1/b75a142f-7287-4106-b011-fe6118e6c9a2.pdf"},{"id":87024211,"identity":"a864973a-ff09-492c-aa4e-0103734181fa","added_by":"auto","created_at":"2025-07-18 11:47:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":206994,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialsREV2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7037612/v1/b142223c2c89fdd36f656883.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-MRSA Flavonoids from Iraqi Citrus sinensis (L.)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntibiotic resistance, also known as antimicrobial drug resistance (AMR), represents one of the most significant global public health challenges today.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] The improper and excessive use of antibiotics has exacerbated this issue, leading to a worsening situation.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] According to a report published by the World Health Organization (WHO), antimicrobial resistance was responsible for approximately 5\u0026nbsp;million deaths globally in 2019 alone.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Projections suggest that by 2050, the annual death toll due to AMR could rise to 10\u0026nbsp;million.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Given the severity of this public health crisis, the systematic search for novel antimicrobial agents from natural sources has become essential. These agents are necessary to combat various drug-resistant microbial strains, including methicillin-resistant Staphylococcus aureus (MRSA).[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e (L.) Osbeck, commonly known as the \u0026lsquo;sweet orange\u0026rsquo;, is an evergreen flowering tree that belongs to the Rutaceae family. This species typically grows to a height of 7\u0026ndash;10 m and accounts for approximately 70% of the global annual production of \u003cem\u003eCitrus\u003c/em\u003e species.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Native to Asia, particularly in regions such as China, India, Iraq, and Pakistan, C. sinensis is widely distributed in warm and tropical areas. The fruits of \u003cem\u003eC. sinensis\u003c/em\u003e are widely consumed as a rich source of vitamin C, while various parts of the plant have been traditionally used in medicinal systems to treat a range of ailments. These include angina, anxiety, colds, constipation, coughs, cramps, colic, depression, diarrhoea, hypertension, menstrual disorders, obesity, stress, and microbial infections such as bronchitis, flu, and tuberculosis.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e\u003cp\u003ePrevious phytochemical studies on \u003cem\u003eC. sinensis\u003c/em\u003e have identified several bioactive secondary metabolites, including carotenoids, cinnamic acid derivatives, flavonoids (primarily polymethoxyflavones), alkaloids (mainly acridone types), limonoids, sterols, terpenoids, and vitamins B and C. These compounds have demonstrated a wide range of bioactivities, including anticancer, antihyperlipidemic, anti-inflammatory, antimalarial, antimicrobial, anti-obesity, antioxidant, anxiolytic, and sedative properties.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] However, the anti-MRSA activity of C. sinensis has not been previously evaluated, and bioassay-guided isolation has never been conducted to identify anti-MRSA compounds from this plant.\u003c/p\u003e\u003cp\u003eSome preliminary studies have revealed the antimicrobial activity of essential oils derived from the peels of \u003cem\u003eC. sinensis\u003c/em\u003e against multidrug-resistant bacteria.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] In addition, hesperitin, coumaric acid, and ferulic acid, present in the peel of Newhall navel orange, have shown anti-MRSA activity against a single MRSA strain.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] As part of ongoing research aimed at discovering phytochemicals with anti-MRSA activity,[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] this study focuses on the bioassay-guided isolation of 3-methoxynobiletin (\u003cb\u003e4\u003c/b\u003e), rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e) and sinensetin (\u003cb\u003e7\u003c/b\u003e) as potential anti-MRSA agents. These compounds were isolated from the leaves and peels of C. sinensis collected from Iraq, where the plant has been traditionally used to treat infections. Additionally, ten other known flavonoids (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e3\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, \u003cb\u003e6\u003c/b\u003e, \u003cb\u003e8\u003c/b\u003e, \u003cb\u003e9\u003c/b\u003e, \u003cb\u003e11\u003c/b\u003e\u0026ndash;\u003cb\u003e14\u003c/b\u003e) and a new flavanone glycoside, named sinensidin (\u003cb\u003e10\u003c/b\u003e), were identified. The objective of this study was to isolate and identify anti-MRSA compounds from C. sinensis using a bioassay-guided protocol.\u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cp\u003e\u003cb\u003eGeneral\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChromatographic solvents were from Fisher Scientific, UK, and used without further purification. The NMR experiments were performed on a Bruker AMX600 NMR spectrometer (600 MHz for \u003csup\u003e1\u003c/sup\u003eH, and 150 MHz for \u003csup\u003e13\u003c/sup\u003eC). MS was conducted using a high-resolution mass spectroscopy facility (HR-MS) at the National Mass Spectrometry Facility (NMSF) (Swansea, UK) on Xevo G2-S ASAP or LTQ Orbitrap XL1 spectrometers. Low- and high-resolution MS analyses were also performed at Liverpool John Moores University; HR-MS using an Agilent 6200 Series Accurate-Mass Time-of-Flight (TOF) LC/MS system with electrospray ionization (ESI) in positive ion mode, connected to an Agilent auto-sampler injection system [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe vacuum liquid chromatographic (VLC) fractions, prepared using silica gel (Sigma-Aldrich, UK), were analyzed on a Dionex Ultimate 3000 UHPLC system coupled with a photodiode array (PDA) detector. A Phenomenex Gemini-NX 5 U C18 column (150 x 4.6 mm, 5 \u0026micro;m, Phenomenex, USA), and gradient solvent systems comprising acetonitrile (ACN, solvent B) and water (solvent A) (both contained 0.1% TFA, flow rate: 1 mL/min) were employed for method development for preparative-scale separation and isolation of compounds. The reversed-phase preparative HPLC purification was performed on an Agilent Technologies 1260 Infinity Series prep-HPLC coupled with a photo-diode-array detector (Germany) using a Phenomenex LC-18 C18 stainless steel column (150 x 21.2 mm, 5 \u0026micro;m, Phenomenex, USA) with the same solvent system as mentioned above, but with a 10 mL/min flow rate. The column temperature was set at 25\u0026deg;C [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlant material\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe leaves and peels of \u003cem\u003eCitrus sinensis\u003c/em\u003e (L.) Osbeck were collected from Diyala, Central Iraq (N 33.79684, E 44.623337) in September 2015. The plant materials were air-dried at room temperature and ground into a fine powder using a coffee grinder. Dr. Shaymaa Al-Majmaie (Diyala University, Iraq) identified the plant by comparing its morphological features with herbarium specimens. A voucher specimen (No. 6534) was deposited at the National Herbarium of Iraq.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe air-dried ground leaves (194.6 g) and peels (394.4 g) of \u003cem\u003eC. sinensis\u003c/em\u003e were extracted separately. Sequential extraction was performed using solvents of increasing polarity: \u003cem\u003en\u003c/em\u003e-hexane, dichloromethane (DCM), and methanol (MeOH), using a Soxhlet apparatus (900 mL, ten cycles each). The crude extracts were concentrated to dryness using a rotary evaporator and stored at 4\u0026deg;C for further analysis. The DCM extracts of the leaves and peels, along with the MeOH extract of the peels, demonstrated the highest antimicrobial activity in initial in vitro screening using resazurin as an indicator of cell growth.[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] These active extracts were subjected to further fractionation to isolate the bioactive compounds.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eResazurin assay\u003c/p\u003e\u003cp\u003eThe modified resazurin test, as described by Sarker et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], was used to determine the minimum inhibitory concentration (MIC) using a microtitre plate. The assay was performed under aseptic conditions. Two Gram-positive bacterial strains (Micrococcus luteus NCTC 7508 and Staphylococcus aureus NCTC 12981), two Gram-negative bacterial strains (Escherichia coli NCTC 12241 and Pseudomonas aeruginosa NCTC 12903), and a fungal strain (Candida albicans ATCC 90028) were used in this study.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePreparation of standard microbial colonies\u003c/strong\u003e\u003cp\u003eMicrobial strains were cultured in 20 mL of nutrient agar on Petri dishes and incubated for 12\u0026ndash;48 h at 35\u0026deg;C. Single colonies from the incubated plates were transferred to sterilized tubes containing 100 mL of nutrient broth and incubated at 35\u0026deg;C for 24\u0026ndash;48 h. After incubation, the tubes were centrifuged at 4000 rpm for 5 min. The supernatant was discarded, and 20 mL of sterile normal saline was added to the tubes, followed by centrifugation under the same conditions. This process was repeated until the supernatant became clear. The optical density of the bacterial suspension was measured using a spectrophotometer at 500 nm, and the concentration was adjusted to 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL through dilution and calculations [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePreparation of resazurin solution\u003c/strong\u003e\u003cp\u003eThe resazurin solution was prepared by dissolving 1 mg of resazurin sodium salt (purchased from Aldrich, USA) in 5 mL of sterile distilled water. A vortex mixer was used to ensure complete dissolution and homogeneity. Resazurin served as an indicator of cell growth in this assay [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePreparation of tested materials\u003c/strong\u003e\u003cp\u003eThe stock concentration of the tested materials was prepared by dissolving them in 10% (v/v) dimethyl sulfoxide (DMSO) or sterilized water. The stock concentration was 10 mg/mL for crude extracts and 1 mg/mL for fractions and pure compounds [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePreparation of 96-well plates\u003c/strong\u003e\u003cp\u003eAll wells on 96-well plates were filled with 50 \u0026micro;L of sterilized normal saline. The test material (100 \u0026micro;L) was added to the first row of the plate, and serial dilutions were made using multichannel pipettes by transferring 50 \u0026micro;L. Resazurin (10 \u0026micro;L) was added to all wells, followed by 10 \u0026micro;L of bacterial suspension (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL). To prevent bacterial dehydration, each plate was loosely wrapped with cling film. Each plate included the antibiotic ciprofloxacin as a positive control for bacterial strains and nystatin for the fungal strain \u003cem\u003eC. albicans\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eInterpretation of results\u003c/strong\u003e\u003cp\u003eThe normal colour of resazurin is blue. During incubation, if the test materials inhibited the microorganisms, the wells retained the blue colour of resazurin or turned purple or colourless, indicating a positive result. The development of a pink colour indicated no effect of the test materials on the microbes. The lowest concentration at which the colour change occurred was recorded as the MIC (minimum inhibitory concentration) value. The mean of three values was calculated for each sample [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnti-MRSA screening\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe anti-MRSA screening was conducted against five methicillin-resistant Staphylococcus aureus strains: SA1199B, XU212, MRSA340702, EMRSA-15MRSA274819, and the standard strain ATCC25923. All bacterial strains were obtained from the UCL School of Pharmacy, and the experiments were performed at the University of East London.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePreparation of culture medium\u003c/strong\u003e\u003cp\u003eMueller-Hinton broth (MHB) was prepared according to the supplier\u0026rsquo;s instructions. The MHB was adjusted to contain cations at concentrations of 20 mg/L Ca\u003csup\u003e2+\u003c/sup\u003e and 10 mg/L Mg\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePreparation of tested compounds\u003c/strong\u003e\u003cp\u003eThe compounds and antibiotics were dissolved in predetermined amounts of dimethyl sulfoxide (DMSO), ensuring that the final concentration of DMSO in the well was less than 1%. The solutions were further diluted with MHB to achieve the targeted starting concentration of 128 \u0026micro;g/mL.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePreparation of MTT solution\u003c/strong\u003e\u003cp\u003eThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was prepared by dissolving the required amount of MTT in methanol (MeOH) to obtain a concentration of 5 \u0026micro;g/mL.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSuspension of the bacterial subculture\u003c/strong\u003e\u003cp\u003eAll bacterial strains were subcultured one day before the experiment. The strains were streaked onto nutrient agar slopes using a loop and incubated at 37\u0026deg;C for 12\u0026ndash;18 hours.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAnti-MRSA assay\u003c/strong\u003e\u003cp\u003eThe assay was performed using 96-well plates. The first step involved adding 100 \u0026micro;L of MHB to all wells except those in column 12. In the first row of the plate, 100 \u0026micro;L of the test compounds or antibiotics was added. Using a multi-channel pipette, the contents of the first well were mixed thoroughly, and 100 \u0026micro;L was transferred to the wells of the second column. This process was repeated until column 10. Finally, 100 \u0026micro;L of the contents from column 10 was transferred to the wells of column 12.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eAn inoculum density of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e colony-forming units (cfu) of each test organism was prepared in normal saline (9 g/L) by comparison with a 0.5 McFarland standard. MHB (125 \u0026micro;L) was dispensed into 10 wells of a 96-well microtitre plate (Nunc, 0.3 mL volume per well). The plates were incubated at 37\u0026deg;C for 18 hours. To determine the minimum inhibitory concentrations (MICs), 20 \u0026micro;L of MTT solution was added to the microtitre plate, followed by incubation for 20 min.\u003c/p\u003e\u003cp\u003eThis colorimetric method indicated bacterial growth by a colour change from yellow to dark blue. The MIC was recorded as the lowest concentration at which no growth was observed. Norfloxacin, a well-known antibiotic, was used as a positive control. The method was adapted from the broth microdilution technique according to the National Committee for Clinical Laboratory Standards, with modifications using nutrient broth as the medium. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42 CR43 CR44\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation of compounds\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe most active dichloromethane (DCM) extracts from the leaves and peels of \u003cem\u003eCitrus sinensis\u003c/em\u003e were subjected to vacuum liquid chromatography (VLC) separation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] on silica gel. A stepwise mobile phase of increasing polarity was used, consisting of chloroform (CHCl3) and MeOH as follows 100% CHCl\u003csub\u003e3\u003c/sub\u003e, 2% MeOH in 100% CHCl\u003csub\u003e3\u003c/sub\u003e, 4% MeOH in 100% CHCl\u003csub\u003e3\u003c/sub\u003e, 6% MeOH in 100% CHCl\u003csub\u003e3\u003c/sub\u003e, 8% MeOH in 100% CHCl\u003csub\u003e3\u003c/sub\u003e, 10% MeOH in 100% CHCl\u003csub\u003e3\u003c/sub\u003e, 20% MeOH in 100% CHCl\u003csub\u003e3\u003c/sub\u003e and 30% MeOH in 100% CHCl\u003csub\u003e3\u003c/sub\u003e (each fraction was 200 mL). The active methanol (MeOH) extract of the peels was further fractionated by solid-phase extraction (SPE) on a Strata C\u003csub\u003e18\u003c/sub\u003e 20 g cartridge [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] using 30%, 50%, 80%, and 100% MeOH in water as eluents (200 mL each). The VLC and SPE fractions were assessed for antimicrobial activity.\u003c/p\u003e\u003cp\u003eThe most active VLC fraction F7 (20% MeOH in 100% CHCl\u003csub\u003e3\u003c/sub\u003e) of the DCM extract of the leaves, and VLC fractions F6 (10% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) and F7 (20% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) from the peel DCM extract, were selected for further analysis. Additionally, the active SPE fractions SPE2 (60% MeOH in water) and SPE3 (80% MeOH in water) of the MeOH extract of the peels were analysed by reversed-phase analytical HPLC to develop the optimum separation conditions for preparative HPLC separation.\u003c/p\u003e\u003cp\u003eReversed-phase preparative HPLC analysis using various gradient elution methods, as described in the results section, yielded fourteen flavonoids (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e14\u003c/b\u003e). The structures of all compounds were confirmed through extensive 1D and 2D NMR data analysis, MS spectroscopic data interpretation, and comparison with literature data for known compounds \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e9\u003c/b\u003e and \u003cb\u003e11\u003c/b\u003e\u0026ndash;\u003cb\u003e14\u003c/b\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThe Soxhlet extraction of the dried ground leaves of \u003cem\u003eCitrus sinensis\u003c/em\u003e (194.6 g) yielded three extracts: \u003cem\u003en\u003c/em\u003e-hexane (6.2 g, 3.19%), dichloromethane (DCM) (10.2 g, 5.24%), and methanol (MeOH) (14.3 g, 7.35%). Similarly, the extraction of the peels (394.4 g) produced three additional extracts: \u003cem\u003en\u003c/em\u003e-hexane (7.3 g, 1.9%), DCM (12.7 g, 3.2%), and MeOH (25.2 g, 6.39%). Initial antimicrobial screening of these extracts revealed that the DCM extracts exhibited the highest activity, followed by the MeOH extracts of the leaves and peels (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These extracts were active against all tested microorganisms, with minimum inhibitory concentration (MIC) values ranging from 4.8\u0026ndash;625 mg/mL.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntimicrobial activity of the extracts of the leaves and peels of \u003cem\u003eC. sinensis\u003c/em\u003e, the VLC fractions of the DCM extracts, SPE fraction of the MeOH extract of peels and the isolated compounds (1\u0026ndash;14)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eTested samples\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eMIC values in mg/mL\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eGram-positive bacteria\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eGram-negative bacteria\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFungus\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMicrococcus luteus\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eCandida albicans\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eLeaves\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003en\u003c/em\u003e-Hexane extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDCM extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e312.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e312.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeOH extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003ePeels\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003en\u003c/em\u003e-Hexane extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e156\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDCM extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e312.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e156\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeOH extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e312.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e625\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eVLC fractions of the DCM extract of the leaves\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e625\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eVLC fractions of the DCM extract of the peels\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eSPE fractions of the MeOH extract of the peels\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSPE1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSPE2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e625\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSPE3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSPE4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eIsolated compounds (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e14\u003c/b\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e62.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003ePositive controls\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCiprofloxacin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e97.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e97.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNystatin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e97.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eNA\u0026thinsp;=\u0026thinsp;No activity at the\u0026nbsp;tested concentration\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003en\u003c/em\u003e-hexane extract of the leaves was the least active among the six extracts, showing activity against \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eMicrococcus luteus\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e only at high concentrations (MIC\u0026thinsp;=\u0026thinsp;2500\u0026ndash;5000 mg/mL). The \u003cem\u003en\u003c/em\u003e-hexane extract of the peels was also active at high concentrations against all tested organisms except \u003cem\u003eS. aureus\u003c/em\u003e, with its most potent activity observed against \u003cem\u003eM. luteus\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;156 mg/mL).\u003c/p\u003e\u003cp\u003eThe DCM extracts of both the leaves and peels were the most active among all extracts, showing activity against all strains. However, the DCM extract of the peels was more potent than that of the leaves. The DCM extract of the peels was particularly effective against \u003cem\u003eM. luteus\u003c/em\u003e, with an MIC value of 4.8 mg/mL. A similar difference in potency was observed between the MeOH extracts of the leaves and peels, with the latter being more active (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe highest antifungal activity against \u003cem\u003eCandida albicans\u003c/em\u003e was observed with the DCM extract of the peels (MIC\u0026thinsp;=\u0026thinsp;156 mg/mL), while the least active was the MeOH extract of the leaves (MIC\u0026thinsp;=\u0026thinsp;5000 mg/mL). Although the MeOH extracts were active against all five microbial strains, their MIC values (312.5\u0026ndash;5000 mg/mL) were significantly higher than those of the DCM extracts (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Consequently, the active DCM extracts of the leaves and peels were subjected to vacuum liquid chromatography (VLC)-assisted fractionation, resulting in eight fractions each.\u003c/p\u003e\u003cp\u003eSince the MeOH extract of the peels demonstrated a better antimicrobial profile than the MeOH extract of the leaves, it was further fractionated using solid-phase extraction (SPE) on a Strata C\u003csub\u003e18\u003c/sub\u003e cartridge (20 g, Phenomenex, UK). This process yielded four SPE fractions (SPE1-SPE4) eluted with 30%, 50%, 80%, and 100% MeOH in water. All VLC and SPE fractions were tested for antimicrobial activity to identify the most active fractions for subsequent isolation of bioactive compounds.\u003c/p\u003e\u003cp\u003eThe first five VLC fractions (F1-F5: 100% CHCl\u003csub\u003e3\u003c/sub\u003e, 2% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e, 4% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e, 6% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e, and 8% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) of the DCM extract of the leaves did not exhibit any antimicrobial activity against the tested strains at the concentrations used. VLC fraction 6 (F6, 10% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) was active only against Pseudomonas aeruginosa (MIC\u0026thinsp;=\u0026thinsp;1000 mg/mL), while fraction 8 (F8, 30% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) showed activity against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;1000 mg/mL). VLC fraction 7 (F7, 20% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) of the DCM extract of the leaves was the most active among the fractions, showing activity against all tested organisms (MIC\u0026thinsp;=\u0026thinsp;625\u0026ndash;1000 mg/mL) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe first three VLC fractions (F1-F3: 100% CHCl\u003csub\u003e3\u003c/sub\u003e, 2% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e, and 4% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) and fraction 8 (F8, 30% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) of the DCM extract of the peels did not show any antimicrobial activity against the tested strains. VLC fractions 4 (F4, 6% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) and 5 (F5, 8% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) were active against \u003cem\u003eM. luteus\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;500 mg/mL) and \u003cem\u003eC. albicans\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;500 mg/mL), with F5 also active against \u003cem\u003eP. aeruginosa\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;1000 mg/mL). VLC fraction 6 (F6, 10% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) was active against all tested organisms (MIC\u0026thinsp;=\u0026thinsp;7.81\u0026ndash;1000 mg/mL), while fraction 7 (F7, 20% MeOH in CHCl\u003csub\u003e3\u003c/sub\u003e) was active against all microorganisms (MIC\u0026thinsp;=\u0026thinsp;156\u0026ndash;1000 mg/mL) except \u003cem\u003eE. coli\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the four SPE fractions, fraction 3 (SPE3, 80% MeOH in water) was the most active, showing activity against all microbial strains tested in this study (MIC\u0026thinsp;=\u0026thinsp;156\u0026ndash;500 mg/mL). Fraction 2 (SPE2, 60% MeOH in water) was also active against all organisms (MIC\u0026thinsp;=\u0026thinsp;500\u0026ndash;625 mg/mL) except \u003cem\u003eP. aeruginosa\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The first fraction (SPE1, 30% MeOH in water) was only active against \u003cem\u003eM. luteus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;500 mg/mL).\u003c/p\u003e\u003cp\u003eConsidering the overall antimicrobial activity profile, particularly against \u003cem\u003eS. aureus\u003c/em\u003e, VLC fraction 7 (F7) of the DCM extract of the leaves, VLC fractions F6 and F7 of the DCM extract of the peels, and SPE fractions SPE2 and SPE3 were selected for further HPLC analysis to purify the active compounds.\u003c/p\u003e\u003cp\u003eReversed-phase preparative HPLC analysis of the most active VLC fraction F7 of the DCM extract of the leaves of \u003cem\u003eC. sinensis\u003c/em\u003e (linear gradient 30\u0026ndash;100% acetonitrile (ACN) in water over 30 min, isocratic 100% ACN for the next 5 min, linear gradient 100\u0026thinsp;\u0026minus;\u0026thinsp;30% ACN in water for 5 min, and isocratic 30% MeOH in water for 5 min, 10 mL/min) yielded five compounds (5, 3, 6, 1, and 8) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) with retention times (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e) of 20.39, 21.36, 22.32, 31.61, and 33.45 min, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA similar preparative HPLC analysis (linear gradient 20\u0026ndash;50% ACN in water over 30 min, isocratic 50% ACN for the next 3 min, linear gradient 50\u0026thinsp;\u0026minus;\u0026thinsp;20% ACN in water for 5 min, and isocratic 20% ACN in water for 5 min, 10 mL/min) of VLC fraction F6 of the DCM extract of the peels of C. sinensis yielded six compounds: \u003cb\u003e7\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 28.28 min), 14 (tR\u0026thinsp;=\u0026thinsp;33.21 min), \u003cb\u003e4\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 34.35 min), \u003cb\u003e13\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 37.03 min), \u003cb\u003e2\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 39.84 min), and additional amounts of \u003cb\u003e5\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 32.0 min).\u003c/p\u003e\u003cp\u003eThe analysis of VLC fraction 7 of the peels (linear gradient 20\u0026ndash;80% ACN in water over 30 min, isocratic 80% ACN for the next 5 min, linear gradient 80\u0026thinsp;\u0026minus;\u0026thinsp;20% ACN in water for 5 min, and isocratic 20% ACN in water for 5 min, 10 mL/min) resulted in the isolation of seven more compounds: \u003cb\u003e11\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 14.01 min), \u003cb\u003e9\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 17.7 min), \u003cb\u003e14\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 22.42 min), and additional amounts of \u003cb\u003e7\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 19.74 min), \u003cb\u003e5\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 21.75 min), \u003cb\u003e4\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 23.03 min), and \u003cb\u003e13\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 24.33 min).\u003c/p\u003e\u003cp\u003eReversed-phase preparative HPLC analysis of the SPE fractions SPE2 and SPE3 of the MeOH extract of the peels (linear gradient 20\u0026ndash;80% MeOH in water over 30 min, isocratic 80% MeOH for the next 2 min, linear gradient 80\u0026thinsp;\u0026minus;\u0026thinsp;20% MeOH in water for 2 min, and isocratic 100% MeOH in water for 2 min, and isocratic 20% MeOH in water for 5 min) yielded compounds \u003cb\u003e11\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 13.78 min), \u003cb\u003e10\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 15.03 min), \u003cb\u003e12\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 15.5 min), \u003cb\u003e9\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 17.7 min), \u003cb\u003e8\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 19.18 min), \u003cb\u003e13\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 26.13 min), \u003cb\u003e11\u003c/b\u003e (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 28.33 min), and 5 (\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e = 28.82 min).\u003c/p\u003e\u003cp\u003eAmong the isolated compounds, compounds \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e9\u003c/b\u003e and \u003cb\u003e11\u003c/b\u003e\u0026ndash;\u003cb\u003e14\u003c/b\u003e were identified as known natural products: cirsilineol (1, 3.3 mg),[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] demethylnobiletin (2, 3.1 mg),[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] 5-desmethylsinensetin (\u003cb\u003e3\u003c/b\u003e, 2.8 mg),[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] 3-methoxynobiletin (\u003cb\u003e4\u003c/b\u003e, 4.4 mg),[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] nobiletin (\u003cb\u003e5\u003c/b\u003e, 2.9 mg),[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] 6,7,8,3',4'-pentamethoxyflavone (\u003cb\u003e6\u003c/b\u003e, 3.6 mg),[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] sinensetin (\u003cb\u003e7\u003c/b\u003e, 4.8 mg),[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] hesperidin (\u003cb\u003e8\u003c/b\u003e, 6.1 mg),[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] narirutin (\u003cb\u003e9\u003c/b\u003e, 1.9 mg),[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] rutin (\u003cb\u003e11\u003c/b\u003e, 9.1 mg),[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e, 4.1 mg),[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] tangeritin (\u003cb\u003e13\u003c/b\u003e, 6.7 mg),[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and tetramethylscutellarein (\u003cb\u003e14\u003c/b\u003e, 3.7 mg).[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] The structures of these compounds were confirmed through 1D and 2D NMR and high-resolution electrospray ionization mass spectrometry (HRESIMS) data analyses. The experimental \u0026sup1;H and \u0026sup1;\u0026sup3;C NMR data were consistent with published literature for the respective compounds. Most of these isolated flavonoids are polymethoxyflavones, which are characteristic of the \u003cem\u003eCitrus\u003c/em\u003e genus and are known to possess various bioactivities.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eCompound \u003cb\u003e10\u003c/b\u003e, isolated as a golden yellow oily substance, was identified as a new natural product. Its structure was elucidated using spectroscopic techniques. The high-resolution electrospray ionization mass spectrometry (HRESIMS) data suggested the empirical formula C\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e, with a pseudomolecular ion [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e peak at \u003cem\u003em/z\u003c/em\u003e 625.1761 (calculated 625.1768).\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectrum (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed aromatic proton signals at δH 7.47 (2H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz) and 7.16 (2H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz), indicative of a para-disubstituted benzene ring, typical of the B ring in flavonoids such as apigenin and kaempferol. A singlet at δH 6.22 (2H) was assigned to a methylenedioxy group attached to a benzene ring. Signals at δH 5.53 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.1, 12.4 Hz), 2.83 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.1, 17.1 Hz), and 3.29 (1H, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.1, 12.4 Hz) were characteristic of H-1 and H\u003csub\u003e2\u003c/sub\u003e-3 of a flavanone skeleton.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e1H (600 MHz) and 13C (150 MHz) NMR data, and 1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC key correlations of sinensidin (10)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNumber\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eChemical shift d in ppm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH COSY\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HMBC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ed\u003csub\u003eH\u003c/sub\u003e, coupling constant \u003cem\u003eJ\u003c/em\u003e in Hz\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ed\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eFlavanone skeleton\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.53, dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.1, 12.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e81.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC-1\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.83, dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.1, 17.1\u003c/p\u003e\u003cp\u003e3.29, dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.1, 12.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC-2, C-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-1\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e203.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e147.7**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e135.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e151.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e129.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e147.6**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e111.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e132.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u0026rsquo;, 6\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.47, d, 2H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e129.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-3\u0026rsquo;. H-5\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-3\u0026rsquo;, C-5\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC-1\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-2, C-4\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u0026rsquo;, 5\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.16, d, 2H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e118.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-2\u0026rsquo;, H-6\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-2\u0026rsquo;, C-6\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC-4\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-1\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e160.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5,6-O-CH\u003csub\u003e2\u003c/sub\u003e-O-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.22, s, 2H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e101.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-O-CH\u003csub\u003e2\u003c/sub\u003e-O-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-5, C-6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eGlucose unit\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.96, d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e102.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-2\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-1\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-4\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.88*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-1\u0026rsquo;\u0026rsquo;, H-3\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-2\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-4\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.48*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e78.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-2\u0026rsquo;\u0026rsquo;, H-4\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-3\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-5\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.39*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e71.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-3\u0026rsquo;\u0026rsquo;, H-5\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-4\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-2\u0026rsquo;\u0026rsquo;, C-6\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.34*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e78.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-4\u0026rsquo;\u0026rsquo;, H-6\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-5\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-3\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.92, 3.70*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e69.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-5\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-6\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-1\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eRhamnose unit\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.71, d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e103.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-2\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-1\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-6\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.48*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e74.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-1\u0026rsquo;\u0026rsquo;\u0026rsquo;, H-3\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-2\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-4\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.29*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e71.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-2\u0026rsquo;\u0026rsquo;\u0026rsquo;, H-4\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-3\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-5\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.69*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e72.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-3\u0026rsquo;\u0026rsquo;\u0026rsquo;, H-5\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-4\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-2\u0026rsquo;\u0026rsquo;\u0026rsquo;, C-6\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.60*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e77.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-4\u0026rsquo;\u0026rsquo;\u0026rsquo;, H-6\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-5\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-3\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.21, d, 3H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eH-5\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC-6\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eC-5\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC-4\u0026rsquo;\u0026rsquo;\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eSpectra obtained in CD\u003csub\u003e3\u003c/sub\u003eOD\u003c/p\u003e\u003cp\u003e*Unresolved overlapped peaks, determined by 2D COSY, HSQC, and HMBC experiments\u003c/p\u003e\u003cp\u003e**Interchangeable; could be either carbon\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTwo anomeric proton signals at δH 4.96 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 Hz) and 4.71 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.4 Hz) were attributed to glucose and rhamnose moieties, forming a rutinosyl unit. Overlapping signals in the δH 3.33\u0026ndash;4.20 range were assigned to the remaining sugar protons. The absence of additional aromatic proton signals indicated full substitution of the A ring of the flavanone skeleton.\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH COSY spectrum (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) confirmed scalar couplings, particularly aiding in the assignment of sugar protons. The \u003csup\u003e13\u003c/sup\u003eC NMR spectrum (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed 28 carbons, including a carbonyl signal at δC 203.6 (C-4 of the flavanone nucleus), 12 carbons for the rutinosyl unit (including anomeric carbons at δC 102.3 and 103.4, a rhamnose methyl signal at δC 18.1, and oxymethine carbons at δC 71.6\u0026ndash;78.5), a methylenedioxy group at δC 101.3, and 12 aromatic carbons (four methine, six oxygenated quaternary, and two quaternary carbons) (Table\u0026nbsp;5).\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC experiment (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) confirmed the assignment of all protonated carbons. Key \u003csup\u003e3\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e correlations in the \u0026sup1;H-\u0026sup1;\u0026sup3;C HMBC spectrum (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) included H-1\u0026rsquo;\u0026rsquo; (glucose anomeric) to C-4\u0026rsquo;, H-6\u0026rsquo;\u0026rsquo; to C-1\u0026rsquo;\u0026rsquo;\u0026rsquo; (rhamnose anomeric), and methylenedioxy protons to C-5 (δC 147.7) and C-6 (δC 135.5) of the flavanone A ring. These correlations confirmed the placement of the rhamnosyl unit at C-4\u0026rsquo;, the 6\u0026rarr;1 linkage between glucose and rhamnose, and the methylenedioxy group at C-5/C-6.\u003c/p\u003e\u003cp\u003eDue to the full substitution of the A ring and the absence of aromatic proton signals, the \u003csup\u003e13\u003c/sup\u003eC NMR data of compound \u003cb\u003e10\u003c/b\u003e were compared with similar compounds, particularly those with oxygenation at C-5, C-6, C-7, and C-8. This comparison allowed for the unambiguous assignment of all oxygenated carbons in the A ring. Compound \u003cb\u003e10\u003c/b\u003e was identified as a new flavanone glycoside, named sinensidin. Notably, the methylenedioxy linkage at C-5/C-6 is less common in nature compared to C-6/C-7 linkages.\u003c/p\u003e\u003cp\u003eAll fourteen compounds (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e14\u003c/b\u003e) were subjected to the resazurin assay, as described for the extracts and fractions. Compounds 3-methoxynobiletin (\u003cb\u003e4\u003c/b\u003e), sinensetin (\u003cb\u003e7\u003c/b\u003e), and rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e) exhibited activity against all tested microorganisms, with MIC values ranging from 500\u0026ndash;625 mg/mL (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These compounds were the only ones active against \u003cem\u003eS. aureus\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;250 mg/mL). Sinensetin (\u003cb\u003e7\u003c/b\u003e) showed potent antifungal activity against \u003cem\u003eC. albicans\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;62.5 mg/mL), consistent with recent findings where a combination of sinensetin (7) with other compounds was effective in treating \u003cem\u003evulvovaginal candidiasis\u003c/em\u003e.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eHesperidin (\u003cb\u003e8\u003c/b\u003e), narirutin (\u003cb\u003e9\u003c/b\u003e), and rutin (\u003cb\u003e11\u003c/b\u003e) did not show antimicrobial activity against the tested strains. However, other flavonoids (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e3\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, \u003cb\u003e6\u003c/b\u003e, \u003cb\u003e10\u003c/b\u003e, and \u003cb\u003e12\u003c/b\u003e\u0026ndash;\u003cb\u003e14\u003c/b\u003e) were active against \u003cem\u003eM. luteus\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;125\u0026ndash;500 mg/mL). Cirsilineol (\u003cb\u003e1\u003c/b\u003e) and 6,7,8,3',4'-pentamethoxyflavone (\u003cb\u003e6\u003c/b\u003e) were the least active, showing no activity against other organisms. Demethylnobiletin (\u003cb\u003e2\u003c/b\u003e), 5-desmethylsinensetin (\u003cb\u003e3\u003c/b\u003e), nobiletin (\u003cb\u003e5\u003c/b\u003e), and tangeritin (\u003cb\u003e13\u003c/b\u003e) exhibited similar antimicrobial profiles, with activity against M. luteus, Pseudomonas aeruginosa, and \u003cem\u003eC. albicans\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;250\u0026ndash;500 mg/mL). Sinensidin (\u003cb\u003e10\u003c/b\u003e) and tetramethylscutellarein (\u003cb\u003e14\u003c/b\u003e) also displayed antifungal activity against \u003cem\u003eC. albicans\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;250 and 500 \u0026micro;g/mL, respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe activity of 3-methoxynobiletin (\u003cb\u003e4\u003c/b\u003e), sinensetin (\u003cb\u003e7\u003c/b\u003e), and rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e) against \u003cem\u003eS. aureus\u003c/em\u003e (MIC\u0026thinsp;=\u0026thinsp;250 mg/mL) led to their further testing against five clinical MRSA strains: XU212, SA1199B, EMRSA15, MRSA340702, and MRSA274819 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). 3-Methoxynobiletin (\u003cb\u003e4\u003c/b\u003e) was active only against EMRSA15 (MIC\u0026thinsp;=\u0026thinsp;256 mg/mL), while sinensetin (\u003cb\u003e7\u003c/b\u003e) and rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e) showed activity against three MRSA strains. Sinensetin (\u003cb\u003e7\u003c/b\u003e) was most potent against MRSA340702 (MIC\u0026thinsp;=\u0026thinsp;128 mg/mL), while rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e) was active against XU212, MRSA340702, and MRSA274819 (MIC\u0026thinsp;=\u0026thinsp;256 mg/mL).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAnti-MRSA activity of 3-methoxynobiletin (4), sinensetin (7) and rutin 3\u0026rsquo;-methyl ether (12)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eTested compounds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eMIC values in mg/mL\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eMRSA strains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eStandard \u003cem\u003eS. aureus\u003c/em\u003e strain\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXU212\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSA1199B\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEMRSA15\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMRSA340702\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMRSA274819\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eATCC25923\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-Methoxynobiletin (\u003cb\u003e4\u003c/b\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSinensetin (\u003cb\u003e7\u003c/b\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e128\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNorfloxacin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eNA\u0026thinsp;=\u0026thinsp;No activity at the tested concentrations\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e3-Methoxynobiletin (\u003cb\u003e4\u003c/b\u003e), sinensetin (\u003cb\u003e7\u003c/b\u003e), and rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e) also exhibited antibacterial activity against the standard \u003cem\u003eS. aureus\u003c/em\u003e strain ATCC25923, with MIC values of 256, 128, and 128 mg/mL, respectively. The anti-MRSA activity of sinensetin (\u003cb\u003e7\u003c/b\u003e) was two-fold and four-fold less potent against MRSA340702 and MRSA274819, respectively, compared to the positive control norfloxacin (MIC\u0026thinsp;=\u0026thinsp;64 mg/mL).\u003c/p\u003e\u003cp\u003e3-Methoxynobiletin (\u003cb\u003e4\u003c/b\u003e) has been previously reported from the peels of \u003cem\u003eCitrus aurantium\u003c/em\u003e var. \u003cem\u003esinensis\u003c/em\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and \u003cem\u003eC. unshiu\u003c/em\u003e Markob.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Although its antimicrobial potential was described, no activity against MRSA strains was reported. This polymethoxyflavone was also isolated from \u003cem\u003eAgeratum conyzoides\u003c/em\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and \u003cem\u003eAgeratina colestinum\u003c/em\u003e L.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] of the Asteraceae family, where its antifungal properties were noted.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eSinensetin (\u003cb\u003e7\u003c/b\u003e), widely found in the \u003cem\u003eCitrus genus\u003c/em\u003e (e.g., \u003cem\u003eC. aurantium\u003c/em\u003e, \u003cem\u003eC. grandis\u003c/em\u003e, \u003cem\u003eC. reticulata\u003c/em\u003e, and \u003cem\u003eC. sinensis\u003c/em\u003e),[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] has been reported to exhibit antibacterial activity,[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] particularly against \u003cem\u003eS. aureus\u003c/em\u003e. This activity is believed to be mediated by targeting staphylocoagulase and inhibiting biofilm formation.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eRutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e), also known as isorhamnetin 3-\u003cem\u003eO\u003c/em\u003e-rutinoside, was previously isolated as an antimicrobial compound with anti-MRSA properties from \u003cem\u003eRuta chalepensis\u003c/em\u003e (Rutaceae).[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] This compound has also been found in \u003cem\u003eCampanula rapunculoides\u003c/em\u003e L. (Campanulaceae),[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] \u003cem\u003eCaragana sinica\u003c/em\u003e (Buc\u0026rsquo;hoz) \u003cem\u003eRehder\u003c/em\u003e (Fabaceae),[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and \u003cem\u003eCoriandrum sativum\u003c/em\u003e L. (Apiaceae).[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eA bioassay-guided isolation study led to the identification of two polymethoxy flavones, 3-methoxynobiletin (\u003cb\u003e4\u003c/b\u003e) and sinensetin (\u003cb\u003e7\u003c/b\u003e), along with rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e), as potential anti-MRSA agents from the leaves and peels of \u003cem\u003eCitrus sinensis\u003c/em\u003e of Iraqi origin. These three flavonoids exhibited activity against various strains of methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA), with minimum inhibitory concentration (MIC) values ranging from 128 to 256 mg/mL. In addition to the anti-MRSA compounds, ten other known flavonoids (\u003cb\u003e1\u0026ndash;3\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, \u003cb\u003e6\u003c/b\u003e, \u003cb\u003e8\u003c/b\u003e, \u003cb\u003e9\u003c/b\u003e, \u003cb\u003e11\u003c/b\u003e\u0026ndash;\u003cb\u003e14\u003c/b\u003e) and a new flavanone glycoside, named sinensidin (\u003cb\u003e10\u003c/b\u003e), were isolated from the active fractions. However, none of these compounds showed activity against \u003cem\u003eS. aureus\u003c/em\u003e. Among the isolated flavonoids, 3-methoxynobiletin (\u003cb\u003e4\u003c/b\u003e), sinensetin (\u003cb\u003e7\u003c/b\u003e), and rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e) demonstrated the most potent antibacterial activity against all tested microorganisms.\u003c/p\u003e\u003cp\u003eThe anti-MRSA potential of 3-methoxynobiletin (\u003cb\u003e4\u003c/b\u003e) is reported here for the first time. Based on the findings of this study, these flavonoids (\u003cb\u003e4\u003c/b\u003e, \u003cb\u003e7\u003c/b\u003e, and \u003cb\u003e12\u003c/b\u003e) could serve as valuable templates for the development of new structural analogues with enhanced anti-MRSA properties. However, further physicochemical, preclinical, and clinical studies are necessary to fully evaluate the therapeutic potential of these polymethoxyflavones and their analogues in the treatment of MRSA infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Iraqi Ministry of Higher Education and Scientific Research, as well as the College of Science, University of Diyala, Iraq, for providing a PhD Scholarship to Shaymaa Al-Majmaie to conduct this study. Additionally, the authors appreciate the support of the EPSRC National Mass Spectrometry Service, Swansea, UK, for the HRMS analyses.\u0026nbsp;Lutfun Nahar gratefully acknowledges the support from the European Regional Development Fund (Project ENOCH #CZ.02.1.01/0.0/0.0/ 16_019/0000868), and the Czech Science Foundation (Project #23-05474S).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSA: carried out all aspects of the research work, including data analysis; LN: supervised the project, prepared the first draft of the manuscript and carried out the final editing. MMR: MRSA work; GPS: supervised the project, data analysis and editing; HA: data analysis; SDS: concept, supervised the project, prepared the first draft of the manuscript and carried out the final editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLivermore, D. M. Bacterial resistance: Origins, epidemiology, and impact. \u003cem\u003eClin. Infect. Dis.\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, S11\u0026ndash;S23 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSweileh, W. M. Global research publications on irrational use of antimicrobials: call for more research to contain antimicrobial resistance. \u003cem\u003eGlobalization Health\u003c/em\u003e. \u003cb\u003e17\u003c/b\u003e, 94 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHossain, M. J. et al. Irrational use of antibiotics and factors associated with antibiotic resistance: Findings from a cross-sectional study in Bangladesh. \u003cem\u003eHealth Sci. Rep.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, e1465 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAntimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis. \u003cem\u003eLancet\u003c/em\u003e \u003cb\u003e399\u003c/b\u003e, 629\u0026ndash;655 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHO (The World Health Organization. ), Antimicrobial resistance. Available online: (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance\u003c/span\u003e\u003cspan address=\"https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed on 27 October 2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGBD 2021 Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019\u0026ndash;2021: A systematic analysis with forecast to 2050. \u003cem\u003eLancet\u003c/em\u003e \u003cb\u003e404\u003c/b\u003e, 1199\u0026ndash;1226 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Majmaie, S. et al. Anti-MRSA constituents from \u003cem\u003eRuta chalepensis\u003c/em\u003e (Rutaceae) grown in Iraq, and \u003cem\u003ein silico\u003c/em\u003e studies on two of most active compounds, chalepensin and 6-hydroxy-rutin 3\u0026rsquo;, 7-dimethyld ether. \u003cem\u003eMolecules\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 1114 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFavela-Hernandez, J. M. J., Gonzalez-Santiago, O. \u0026amp; Ramirez-Cabrera, M. A. Esquivel-Ferrino, M. del Rayo Camacho-Corona, Chemistry and pharmacology of \u003cem\u003eCitrus sinensis\u003c/em\u003e. \u003cem\u003eMolecules\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 247 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAzghar, A. et al. Chemical composition and antibacterial activity of \u003cem\u003eCitrus\u003c/em\u003e peels essential oils against multidrug-resistant bacteria: A comparative study. \u003cem\u003eJ. Herb. Med.\u003c/em\u003e \u003cb\u003e42\u003c/b\u003e, 100799 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiang, Y., Zhang, L. Y., Lai, C. L., Ouyang, Z. G. \u0026amp; Zhang, J. Evaluation of antibacterial activity of compounds isolated from the peel of Newhall navel orange. \u003cem\u003eNat. Prod. Res.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 2060\u0026ndash;2064 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDatta, B. K. et al. Polygosumic acid, a new cadinane sesquiterpene, from \u003cem\u003ePolygonum viscosum\u003c/em\u003e inhibits the growth of drug-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eJ. Nat. Med.\u003c/em\u003e \u003cb\u003e61\u003c/b\u003e, 391\u0026ndash;396 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlloush, M., Mallion, C., Sarker, S. D. \u0026amp; Rahman, M. M. Coumarins from the roots of \u003cem\u003eAngelica archangelica\u003c/em\u003e and their antibacterial activity against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. \u003cem\u003eDhaka Univ. J. Pharm. Sci.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 275\u0026ndash;281 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNurunnabi, T. R. et al. D. Anti‐MRSA activity of oxysporone and xylitol from the endophytic fungus \u003cem\u003ePestalotia\u003c/em\u003e sp. growing on the Sundarbans mangrove plant \u003cem\u003eHeritiera fomes\u003c/em\u003e. \u003cem\u003ePhytother. Res.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 348\u0026ndash;354 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHammoud, L. et al. A new flavonoid and other constituents from \u003cem\u003eCentaurea nicaeensis\u003c/em\u003e All. var. \u003cem\u003ewalliana\u003c/em\u003e M. \u003cem\u003eNat. Prod. Res.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 203\u0026ndash;208 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, X. et al. Preparative isolation and purification of polymethoxylated flavones from Tangerine peel using high-speed counter-current chromatography. \u003cem\u003eJ. Chromatogr. A\u003c/em\u003e. \u003cb\u003e1090\u003c/b\u003e, 188\u0026ndash;192 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlarif, W. M. et al. Selective cytotoxic effects on human breast carcinoma of new methoxylated flavonoids from \u003cem\u003eEuryops arabicus\u003c/em\u003e grown in Saudi Arabia. \u003cem\u003eEur. J. Med. Chem.\u003c/em\u003e \u003cb\u003e66\u003c/b\u003e, 204 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, S. L., Lo, C. Y. \u0026amp; Ho, C. T. Hydroxylated polymethoxyflavones and methylated flavonoids in sweet orange (\u003cem\u003eCitrus sinensis\u003c/em\u003e) peel. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e54\u003c/b\u003e, 4176\u0026ndash;4185 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, Z., Zhao, Z. \u0026amp; Zhou, Z. Simultaneous Separation and Purification of Five polymethoxylated flavones from Dahongpao Tangerine (\u003cem\u003eCitrus tangerina\u003c/em\u003e Tanaka) using macroporous adsorptive resins combined with prep-HPLC. \u003cem\u003eMolecules\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 2660 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan, S., Kim, H. M., Lee, J. M., Mok, S. Y. \u0026amp; Lee, S. Isolation and identification of polymethoxyflavones from the hybrid \u003cem\u003eCitrus\u003c/em\u003e, Hallabong. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e58\u003c/b\u003e, 9488\u0026ndash;9491 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNieto, J. Gutierrez, \u003csup\u003e1\u003c/sup\u003eH NMR spectra at 360 MHz of diosmin and hesperidin in DMSO solution. \u003cem\u003eSpectrosc. Lett.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 427\u0026ndash;434 (1986).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbu-Gharbieh, E. \u0026amp; Shehab, N. G. Therapeutic potentials of \u003cem\u003eCrataegus azarolus\u003c/em\u003e var. \u003cem\u003eeu azarolus\u003c/em\u003e Maire leaves and its isolated compounds. \u003cem\u003eBMC Complement. Altern. Med.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 218 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKamel, K. M., Abd El-Raouf, O. M., Metwally, S. A., Abd El‐Latif, H. A. \u0026amp; El‐Sayed, M. E. Hesperidin and rutin, antioxidant \u003cem\u003eCitrus\u003c/em\u003e flavonoids, attenuate cisplatin‐induced nephrotoxicity in rats. \u003cem\u003eJ. Biochem. Mol. Toxicol.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 312\u0026ndash;319 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHamdan, D. et al. Chemical composition and biological activity of \u003cem\u003eCitrus jambhiri\u003c/em\u003e Lush. \u003cem\u003eFood Chem.\u003c/em\u003e \u003cb\u003e127\u003c/b\u003e, 394\u0026ndash;403 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eToledo, R., Tomas-Navaro, M., Yuste, J. E., Crupi, P. \u0026amp; Vallejo, F. An update on citrus polymethoxyflavones: chemistry, metabolic fate and relevant bioactivities. \u003cem\u003eEur. Food Res. Technol.\u003c/em\u003e \u003cb\u003e250\u003c/b\u003e, 2179\u0026ndash;2192 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeng, Z. et al. A non-antibiotic antimicrobial drug, a biological bacteriostatic agent, is useful for treating aerobic vaginitis, bacterial vaginosis and vulvovaginal candidiasis. \u003cem\u003eFrontierts Microbiol.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 1341878 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Majmaie, S., Nahar, L., Rahman, M. M., Sharples, G. P. \u0026amp; Sarker, S. D. Antimicrobial potential of the leaves of \u003cem\u003eCitrus grandis\u003c/em\u003e (L.) Osbeck collected from Iraq: Bioassay-guided isolation of sinensetin as the anti-MRSA compound. \u003cem\u003eFitoterapia\u003c/em\u003e 106393. (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChakroun, I. et al. Phytochemicals constituents and potential applications of Thomson Navel orange \u003cem\u003eCitrus x aurantium\u003c/em\u003e var. \u003cem\u003esinensis\u003c/em\u003e L.) peel extracts: Antioxidant, antimicrobial and antiproliferative properties. \u003cem\u003eInd. Crops Prod.\u003c/em\u003e \u003cb\u003e206\u003c/b\u003e, 117597 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVu, T. O. et al. Flavonoids from the peels of \u003cem\u003eCitrus unshiu\u003c/em\u003e Markov. And their inhibitory effects on RANKL-induced osteoclastogenesis through the downregulation of c-Fos signaling. \u003cem\u003evitro Bioorg. Chem.\u003c/em\u003e \u003cb\u003e107\u003c/b\u003e, 104613 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVyas, A. V. \u0026amp; Mulchandani, N. B. Polyoxygenated flavones from \u003cem\u003eAgeratum conyzoides\u003c/em\u003e. \u003cem\u003ePhytochemistry\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 2625\u0026ndash;2627 (1986).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoner, P. L. Isolation and structural studies of natural products of \u003cem\u003eSolidago ohioensis\u003c/em\u003e Riddell, \u003cem\u003eAmphiachyris dracunculoides\u003c/em\u003e (DC.) Nutt., and \u003cem\u003eAgeratina colestinum\u003c/em\u003e L. \u003cem\u003ePhD Thesis\u003c/em\u003e, The Ohio State University, USA. (1997).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai, W., Zhang, S., Wang, Y., Liu, C. \u0026amp; Luo, R. Differential distribution of characteristic constituents in peel and pulp of Auruntium Fructus Immaturus (\u003cem\u003eCitrus aurantium\u003c/em\u003e L.) using MALDI mass spectrometry imaging. \u003cem\u003eFitoterapia\u003c/em\u003e \u003cb\u003e177\u003c/b\u003e, 106067 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi, J. J. et al. Evaluation of chemical components and quality in Xinhui Chenpi (\u003cem\u003eCitrus reticulata\u003c/em\u003e \u0026lsquo;Chach\u0026rsquo;) with two different storage times by GC-MS. \u003cem\u003eFood Sci. Nutr.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 5036\u0026ndash;5051 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJie, L. H., Jantan, I., Yusoff, S. D., Jalil, J. \u0026amp; Husain, K. Sinensetin: An insight on its pharmacological activities, mechanisms of action and toxicity. \u003cem\u003eFront. Pharmacol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 553404 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatel, K. \u0026amp; Patel, D. K. Therapeutic effectiveness of sinensetin against cancer and other human complications: A review of biological potential and pharmacological activities. \u003cem\u003eCardiovasc. Henatological Disorders Drug Targets\u003c/em\u003e. \u003cb\u003e22\u003c/b\u003e, 144\u0026ndash;154 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePermadi, N., Nurzaman, M., Doni, F. \u0026amp; Julaeha, E. Elucidation of the composition, antioxidant and antimicrobial properties of essential oil and extract from \u003cem\u003eCitrus aurantifolia\u003c/em\u003e (Christm.) Swingle peel. \u003cem\u003eSaudi J. Biol. Sci.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 103987 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGe, B. et al. Sinensetin interferes with \u003cem\u003eStaphylococcus aureus\u003c/em\u003e infections by targeting staphylocoagulase and improves infections survival rates in mouse model pneumonia. \u003cem\u003eJ. Appl. Microbiol.\u003c/em\u003e \u003cb\u003e135\u003c/b\u003e, lxae235 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTeslov, L. S. Phenolic compounds of the above-ground part of \u003cem\u003eCampanula rapunculoides\u003c/em\u003e L. \u003cem\u003eRastitel\u0026rsquo;nye Resursy\u003c/em\u003e. \u003cb\u003e32\u003c/b\u003e, 87\u0026ndash;92 (1996).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTai, Z. G. et al. Antioxidant activities of \u003cem\u003eCaragana sinica\u003c/em\u003e flower extracts and their main chemical constituents. \u003cem\u003eMolecules\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 6722\u0026ndash;6732 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHussain, F., Jahan, N., Khalil-ur-Rahman, B., Sultana, S. \u0026amp; Jamil Identification of hypotensive biofunctional compounds of \u003cem\u003eCoriandrum sativum\u003c/em\u003e and evaluation of their angiotensin-converting enzyme (ACE) inhibition potential. \u003cem\u003eOxidative Medicine and Cellular Longevity 2018\u003c/em\u003e, 4643736. (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSarker, S. D., Nahar, L. \u0026amp; Kumarasamy, Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the \u003cem\u003ein vitro\u003c/em\u003e antibacterial screening of phytochemicals. \u003cem\u003eMethods\u003c/em\u003e \u003cb\u003e42\u003c/b\u003e, 321\u0026ndash;324 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRahman, M. M., Garvey, M., Piddock, L. J. \u0026amp; Gibbons, S. Antibacterial terpenes from the oleoresin of \u003cem\u003eCommiphora molmol\u003c/em\u003e (Engl). \u003cem\u003ePhytother. Res.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 1356\u0026ndash;1360 (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRahman, M. M., Shiu, W. K., Gibbons, S. \u0026amp; Malkinson, J. P. Total synthesis of acylphloroglucinols and their antibacterial activities against clinical isolates of multi-drug resistant (MDR) and methicillin-resistant strains of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. \u003cem\u003eEur. J. Med. Chem.\u003c/em\u003e \u003cb\u003e155\u003c/b\u003e, 255\u0026ndash;262 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIoannou, E. et al. Structures and antibacterial activities of minor dolabellanes from the brown alga \u003cem\u003eDilophus spiralis\u003c/em\u003e. \u003cem\u003eEuropean Journal of Organic Chemistry 2012\u003c/em\u003e, 5177\u0026ndash;5186. (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShiu, W. K. et al. Antibacterial acylphloroglucinols from \u003cem\u003eHypericum olympicum\u003c/em\u003e. \u003cem\u003eJ. Nat. Prod.\u003c/em\u003e \u003cb\u003e75\u003c/b\u003e, 336\u0026ndash;343 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTareq, F. S. et al. Anti-\u003cem\u003eStaphylococcal\u003c/em\u003e calopins from fruiting bodies of \u003cem\u003eCaloboletus radicans\u003c/em\u003e. \u003cem\u003eJ. Nat. Prod.\u003c/em\u003e \u003cb\u003e81\u003c/b\u003e, 400\u0026ndash;404 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReid, R. \u0026amp; Sarker, S. Isolation of natural products by low-pressure column chromatography, in Natural Products Isolation, 3rd edition, Humana Press \u0026ndash; Springer-Verlag, USA, (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Citrus sinensis, Rutaceae, MRSA, polymethoxyflavone, antimicrobial, bioassay-guided protocol","lastPublishedDoi":"10.21203/rs.3.rs-7037612/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7037612/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMethicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) infections represent a major global health challenge, contributing to millions of deaths annually due to antibiotic resistance. The development of new and effective antimicrobial agents is crucial to address this issue. In this study, the leaves and peels of \u003cem\u003eCitrus sinensis\u003c/em\u003e (Rutaceae) were investigated as potential sources of anti-MRSA compounds. A bioassay-guided isolation protocol led to the identification of two polymethoxy flavones, 3-methoxynobiletin (\u003cb\u003e4\u003c/b\u003e), sinensetin (\u003cb\u003e7\u003c/b\u003e), and rutin 3\u0026rsquo;-methyl ether (\u003cb\u003e12\u003c/b\u003e), which exhibited anti-MRSA activity against various strains with minimum inhibitory concentration (MIC) values ranging from 128\u0026ndash;256 mg/mL. In addition, ten known flavonoids (\u003cb\u003e1\u0026ndash;3, 5, 6, 8, 9, 11\u0026ndash;14\u003c/b\u003e) and a new flavanone glycoside, named sinensidin (\u003cb\u003e10\u003c/b\u003e), which were not active against \u003cem\u003eS. aureus\u003c/em\u003e. The chemical structures of all isolated compounds were determined using spectroscopic techniques, including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESIMS).\u003c/p\u003e","manuscriptTitle":"Anti-MRSA Flavonoids from Iraqi Citrus sinensis (L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 11:46:55","doi":"10.21203/rs.3.rs-7037612/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-20T05:29:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-19T10:56:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-17T23:07:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21973974138777573911562714157115985393","date":"2025-07-30T13:23:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280728586801877514161512660358493881437","date":"2025-07-29T08:10:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-25T14:06:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159411861643131995130493720813704487718","date":"2025-07-16T08:20:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-15T07:03:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-15T06:57:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-15T06:20:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-08T19:57:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-08T19:54:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"106c2ca8-686a-446b-85d5-4214bf87d012","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51611181,"name":"Biological sciences/Biochemistry"},{"id":51611182,"name":"Biological sciences/Biological techniques"},{"id":51611183,"name":"Biological sciences/Biotechnology"},{"id":51611184,"name":"Biological sciences/Chemical biology"},{"id":51611185,"name":"Physical sciences/Chemistry"},{"id":51611186,"name":"Biological sciences/Drug discovery"},{"id":51611187,"name":"Biological sciences/Microbiology"},{"id":51611188,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-09-27T21:08:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-18 11:46:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7037612","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7037612","identity":"rs-7037612","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}