Crude Alkaloids form Phyllanthus fraternus, Webster: Antibacterial, Time-Kill Kinetics and Resistance Modulation Studies

Crude Alkaloids form Phyllanthus fraternus, Webster: Antibacterial, Time-Kill Kinetics and Resistance Modulation Studies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Crude Alkaloids form Phyllanthus fraternus, Webster: Antibacterial, Time-Kill Kinetics and Resistance Modulation Studies Samuel Asiamah Obiri, Denzel Opoku-Kwabi, Yaw Opoku-Boahen, Francis Ackah Armah, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5914968/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Antimicrobial resistance (AMR) continues to rise, making a growing number of infectious diseases difficult to cure. Millions of people worldwide die from infections with medication resistance. According to the World Health Organization (WHO), a resistant variant has a 64% higher chance of killing an infected victim than a non-resistant variant. As a result, scientists continue to focus research attention on finding novel chemotypes that could have different modes of action. Combination therapy has the potential to overcome AMR since the therapeutic components work together to suppress the etiological microorganism. In the current study, we investigated the antibacterial properties of crude alkaloidal extract of Phyllanthus fraternus (AEPF) using high-throughput spot culture growth inhibition (HT-SPOTi) assay. We performed time-kill kinetic assays to assess the interactions between the crude alkaloids and test microbial strains. The ability of the crude alkaloids to alter the antimicrobial action of standard tetracycline was evaluated by modulation study. Our findings indicate that P. fraternus alkaloids effectively suppress majority of clinically significant pathogenic strains in vitro. Bactericidal effect was shown by time-kill kinetics against S. aureus , E. coli (ATCC 43888), and E. coli (ATCC 10455). Tetracycline was successfully potentiated against Shigella sp. by the alkaloidal extract. The crude alkaloid extract of P. fraternus included two known alkaloids, epibubbialine and ent-norsecurinine, according to LC-ESI-MS analysis. Taken together, the antibiotic activity of P. fraternus is primarily due to its alkaloids and that the potential exists to develop isolated alkaloids as drug candidates for use in combination therapies against antimicrobial resistance. Natural Product Chemistry Antimicrobial Resistance Time Kill-Kinetic Modulation Studies Phyllanthus fraternus Alkaloidal Extract Liquid Chromatography-Mass Spectrometry (LC-MS) Figures Figure 1 Figure 2 Figure 3 Introduction Antimicrobial resistance remains a major public health concern globally with an increasing number of antibiotics and antifungal agents losing their efficacy. As microbes develop resistance and adapt to treatments designed to eliminate them, clinical treatment of infections become impaired and expensive, which poses a major threat to public health. Through a variety of defense mechanisms, bacteria render antibacterial agents ineffective. These include limiting access to antibiotics, expelling antibiotics from the body via active efflux strategies, inactivating the antibiotic or its target, and evading the drug's effects through mutations [ 1 – 2 ]. When bacteria have the right combination of resistance mechanisms, the effectiveness of antibacterial treatments is significantly diminished. Although antimicrobial resistance is a natural phenomenon, it impairs the effectiveness of current treatments because resistant strains can disseminate their resistance mechanisms [ 3 ]. Research continues to focus on the need to discover new antibacterial agents with a preferably novel mode of action. Natural products remain crucial in the search for novel molecules for various therapeutic applications. Among rural communities in developing countries, traditional treatments employing medicinal plants continue to play a key role in treating several ailments primarily due to the lack of an efficient primary healthcare system [ 4 ], [ 5 ]. Phyllanthus fraternus is a herbaceous weed widely distributed in Asia, the West Indies, and in Africa where the plant has been used traditionally in treating various diseases. In Ghana, aqueous decoction from the leaves of P. fraternus is used to treat a wide variety of ailments including jaundice, malaria, kidney disease, high blood pressure, diabetes, genital-urinary tract infections, stroke, liver disease, intestinal infections, anaemia, hepatocellular cancer, severe abdominal pains, and diarrhea [ 6 ]. A broad range of pharmacological activities have been reported in extracts from P. fraternus including antiviral, anti-inflammatory, antioxidant, anti-diabetic, antinociceptive, hepatoprotective and antifibromyalgic [ 7 – 9 ] which may have implications for their traditional uses. P. fraternus is known to contain different secondary metabolites including alkaloids, tannins, saponins, terpenoids and steroids. Alkaloids are nitrogen-containing natural products with significant pharmacological activities, including unique antibacterial capabilities [ 10 – 13 ]. Alkaloids served as the backbone for the design and development of various antibacterial agents. Notably, quinolones emerged inadvertently during quinine synthesis, metronidazole was derived through chemical modification of azomycin, and bedaquiline is based on the quinoline structure [ 10 ]. Other antibacterials such as trimethoprim and linezolid have alkaloids as foundational substructures [ 14 ]. Extensive investigations of the antibacterial activity of alkaloids have revealed their ability to alter DNA function [ 15 ], interfere with protein synthesis [ 16 ] and disrupt bacterial cell membrane [ 17 ]. In the present study, we selectively extracted the alkaloidal components from P. fraternus and evaluated the antimicrobial properties against a number of WHO priority pathogens. We assessed the potential of the alkaloidal extract to alter the activity of standard tetracycline. To assess the nature of interactions between the alkaloidal extract and the chosen pathogens, we conducted time-kill kinetic assays. We employed LC-MS methods to characterize the various alkaloids present in the alkaloidal extract after column chromatographic purification. We hereby report the antibacterial abilities of P. fraternus and point the way for future work on separating the active alkaloidal components for possible optimization into potential drug candidates. Materials and Methods Materials and Reagents The reagents used included the following: Wagner’s reagent, Mayer’s reagent, Dragendorff’s reagent, McFarland standard, Liebermann-Burchard Reagent, Crystal violet and Salkowski reagent. Materials used included the following: 96-well microtiter plate, Silica gel (70–230 mesh size) [ASTM, Merck, Germany], aluminium pre-coated silica gel plates 60 F 254 0.25 mm thick (Merck, Germany), Mueller-Hinton broth (MHB) (Merck, Germany), Mueller-Hinton agar (MHA) (Oxford Ltd, England), Methanol, Dichloromethane, Ethyl acetate and Hexane. All organic solvents used were of analytical grade and obtained from BDH Laboratory Supplies (Merck Ltd., Lutterw North, UK). Test Organisms Pure cultures of four Gram-positive: Staphylococcus aureus, MRSA, Streptococcus pyogenes, Staphylococcus lentus and six Gram-negative: Escherichia coli (ATCC 10455 & ATCC 43888), Klebsiella pneumoniae, Salmonella poona, Shigella sp., Salmonella typhi were obtained from the Microbiology laboratory of the Department of Biomedical Sciences, School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana. To establish that the bacteria were viable and actively proliferating, sub-culturing of the bacteria was conducted by taking a loop full of test organisms from falcon tubes and streaking on sterile nutrient agar. Collection of Plant Materials Phyllanthus fraternus (whole plant) was collected from Akotokyire (5° 8’3”N/ 1° 17’28”W) in Cape Coast, Ghana on March 31st, 2022. The plant sample was authenticated by Mr. Felix Fynn, a taxonomist at the Department of Botany, University of Cape Coast, Ghana. A specimen sample was deposited at the University’s herbarium with voucher number Eup. CC 5152. Crude Extraction The plant samples were washed to remove soil and other debris and air-dried. 800 grams of ground plant material was macerated with 1.5 L methanol-dichloromethane (1:1) mixture for 72 h. Following filtration, the “marc” was further extracted with 1.5 L methanol for 72 h. The combined extract was evaporated in vacuo at 45°C to afford the crude extract (58.14 g, 7.23%). The crude extract was stored in a desiccator until use. The crude extract was screened for the presence of secondary metabolites according to standard procedures [ 18 – 22 ]. Dragendorff’s, Mayer’s, and Wagner’s tests were used to confirm the presence of alkaloids. Extraction of Alkaloidal Components The alkaloidal extraction was carried out according to the reported literature procedure [ 23 ]. The crude extract (8.1 g) was dissolved in 30 ml 10% acetic acid solution. The resulting solution was defatted with hexane (7×50 ml). The combined aqueous layer was made alkaline with aq. NH 3 (70 ml) to a pH 8–9. The alkaline solution was extracted with chloroform (5×30 ml) and the combined chloroform extracts were concentrated at 40°C in vacuo to afford the alkaloidal extract of P. fraternus (AEPF, 244.2 mg, 0.64%). The presence of alkaloids was confirmed by standard tests [ 24 ]. The absence of other secondary metabolites was also confirmed using standard procedures [ 18 , 25 – 26 ]. Determination of Minimum Inhibition Concentration (MIC) of Crude Extract and Crude Alkaloidal Extracts of P. fraternus The MIC of the crude and alkaloidal extracts were determined using the high-throughput spot culture growth inhibition assay (HT-SPOTi) by visual inspection after 18–24 h of the incubation period as described by Danquah [ 27 ]. Tetracycline and ciprofloxacin were used as the positive controls. Antibiotic Resistance Modulation Study We used checkerboard assay [ 28 ] to evaluate the potential of the crude alkaloidal extract to alter the antimicrobial activity of standard tetracycline. Bacteria strains whose activity was inhibited at MICs ≤ 250 µg/ml by the alkaloid extract and, those with tetracycline MICs ≥ 15.6 µg/mL, were selected for the study. The following organisms (with their tetracycline MICs) were involved; Shigella sp , (125 µg/ml); MRSA, (125 µg/ml); Escherichia coli ATCC 43888, (15.6 µg/ml); Staphylococcus aureus , (15.6 µg/ml); Klebsiella pneumoniae , (15.6 µg/ml); Salmonella poona , (15.6 µg/ml); and Staphylococcus lentus , (15.6 µg/ml). The checkerboard assay was performed according to the standard protocols described in the literature [ 28 ] with minor modifications. Instead of broth, we used nutrient agar and bacterial growth was determined by visual inspection. For each test bacteria, stock solutions of tetracycline and the AEPF were prepared based on observed MICs. The concentration of each stock solution was 8 \(\:\times\:\) MICs observed for the different bacteria. Two-fold serial dilution afforded concentrations in ranges of 8×MIC, 4×MIC, 2×MIC, MIC, 1/2×MIC, 1/4×MIC, 1/8×MIC and 1/16×MIC on each row and column of a 96-well microtiter plates for tetracycline and the crude alkaloid extract. Equal volumes (10 µl) from the respective columns and rows were mixed to obtain the tetracycline-AEPF combination. These tetracycline-AEPF mixtures (2 µl) were transferred into corresponding wells on the second 96-well microtiter plate. Molten agar (196 µl) was dispensed into wells containing tetracycline-AEPF mixture and the plate shaken for about 10 seconds to obtain a uniform distribution and allowed to solidify. Standardized microbial suspension (2 µl) was then added. The plate was sealed with parafilm, covered with aluminum foil, and incubated at 37°C for 24 hours. After 24 hours, the plate was visually observed for growth, or no growth. Fractional inhibition concentration (FIC) indices determined using the equation [ 29 ]: $$\:\frac{A}{MIC\:\left(A\right)}\:+\frac{B}{MIC\:\left(B\right)}\:=FIC\:\left(A\right)+FIC\:\left(B\right)=FIC\:Index$$ where A and B are the MIC of each antibiotic in combination (in a single well), and MIC (A) and MIC (B) are the individual MIC of each antibiotic. FIC indices were interpreted as follows: S, synergy (FIC 4). Time-kill Kinetic Study of Crude Alkaloidal of P. fraternus and Extract-Tetracycline Combinations Time-kill kinetics of AEPF and AEPF-tetracycline combinations were carried out in 24 hours following the procedure described by Mojsoska [ 30 ], with slight modifications. Briefly, in the presence or absence of the crude alkaloid extract, first and last six (6) hours constant MIC time-kill curves were performed in duplicates for activities at MICs less or equal to 250 µg/ml against the test strains. AEPF (2 µl) was dispensed into appropriate wells and Mueller-Hinton nutrient broth (196 µl) added. An inoculum equal to a 0.5 McFarland turbidity standard (2 µl) was dispensed into each well containing AEPF-media mixture. The optical density (OD) at 620 nm at time zero (t = 0) was read using the Elisa micro-plate reader. The plates were then sealed with parafilm, covered with aluminum foil, and incubated at 37°C. For the first 6 hours, the optical density at 30 minutes intervals was taken. For the last 6 hours (18–24 hours) the optical density at one hour (1h) interval was taken. A blank and a negative control experiment were also run simultaneously. Chromatographic Isolations and Purifications The alkaloidal extract (AEPF) was subjected to flash column chromatography. AEPF (0.3 g) was loaded on glass column packed with silica gel (40–63 µm) mesh (ASTM, Merck Germany) and eluted with hexane (100%), hexane-ethyl acetate (50:10 v/v and 50:30 v/v). Fractions were monitored by TLC to obtain eight (8) fractions (F1 – F8). The fractions were concentrated at 40 o C in vacuo , dried and stored in a desiccator until LC-MS analysis. LC-ESI-MS Analysis The profile of the alkaloids present in the fractions was ascertained using reverse-phase high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The triple quadruple mass spectrometer (AB Sciex, USA) was used in conjunction with an Agilent 1200 HPLC system (Agilent Technologies, USA) integrated with a binary gradient solvent pump, degasser, autosampler, and column oven. Separations were carried out at 25°C on a Zorbax SB-C18 column (2.1 × 50 mm, 1.8-µm particles; Agilent Technologies, USA) with a mobile phase of 0.1% formic acid in water (Solvent A) and methanol-acetonitrile (1:1 v/v, Solvent B), using 2 µl injections. The method was slightly modified from Nowacka [ 31 ]. The gradient time was 16 minutes, and the flow rate was 100 µl min − 1 , with a capillary temperature of 300°C, curtain gas pressure of 30 psi, and nebulizer gas pressure of 60 psi, the MS was run in the positive ion mode. The collision and curtain gas were nitrogen. Results and Discussion Extraction and Crude Alkaloidal Extract Crude extraction with 50% methanol in dichloromethane followed by 100% methanol afforded the phytoconstituents from the plant (58.14g, 7.23%). Phytochemical screening revealed the presence of secondary metabolites including alkaloids, steroids, tannins, and glycosides (Table 1 ) [ 24 ]. The alkaloids in the total crude extract (TCE) were converted to their water-soluble salts in the presence of acetic acid to enable their selective partitioning into the aqueous phase. To enable their extraction into chloroform, the alkaloids were further treated with a base [ 23 ]. P. fraternus contains 0.64% alkaloids, which agrees with the generally low levels of alkaloids usually reported in plants (Murphy, 2017). Using widely accepted standard reagents, we confirmed the presence of alkaloids in the extract when addition of Dragendorff’s reagent (1ml) gave orange red precipitate. The formation of yellowish precipitate with Meyer’s reagent gave further indication of alkaloids [ 24 ]. The formation of large brown amorphous and flocculent precipitate after treating the hydrochloric acid solution (5 ml) of the extract with some drops of Wagner’s reagent further confirmed the presence of alkaloids [ 25 ]. In order to ensure that the crude alkaloidal extract of P. fraternus (AEPF) was devoid of any other secondary metabolites, we repeated the phytochemical screening assays as shown in Table 1 . The presence of only alkaloids in the AEPF demonstrated that the acid-base extraction method for selective alkaloidal extraction was successful. Table 1 Phytoconstituents of the total crude extract (TCE) and the crude alkaloidal extract of P. fraternus (AEPF) Phytochemical P. fraternus a (b) Alkaloid Triterpenoid Steroids Flavonoid Tannin Saponins Glycosides + (+) + (-) + (-) - (-) + (-) + (-) + (-) Key: a = TCE; b = AEPF; + = detected; - = not detected Minimum Inhibition Concentration (MIC) We predicted that antibacterial activities of P. fraternus were primarily attributable to its alkaloidal phytoconstituents. We evaluated the antibacterial properties of both the total crude extract and the selectively extracted alkaloidal components from the plant. For most of the studied bacteria, the AEPF demonstrated superior growth inhibition over the TCE (Table 2 ). The high MICs (mostly > 500 mg/mL) recorded for the TCE are consistent with previous reports using crude extract against various pathogenic bacteria [ 32 – 34 ]. In some cases where the TCE showed no growth inhibitory activity, the AEPF demonstrated some activity. Our findings suggest that the alkaloidal components of P. fraternus contribute substantially to its antibacterial activity. Importantly, both the TCE and the AEPF demonstrated antibacterial activities on at least two of the test strains. In a previous study, the crude extract of the aerial parts of P. fraternus showed low inhibitory activity with MICs ranging from 50–250 mg/mL [ 35 ]. This finding was corroborated by [ 36 ] when crude extract from the leaves of the plant showed no antibacterial activity against E. coli. Ibrahim [ 34 ] verified antibacterial activity using n-hexane and ethyl acetate fractions from the leaves of P. fraternus . The authors reported MICs ranging from 80–120 mg/mL and 40–80 mg/mL for n-hexane and the ethyl acetate fractions respectively. Results from our investigations showed that the AEPF performed better than the TCE against E. coli (ATCC 104550, E. coli (ATCC 43888), S. poona , and S. aureus with MICs ranging from 12.5 to 25.0 mg/mL in comparison to ≥ 50 mg/mL for the TCE (Table 2 ). All positive controls showed growth, indicating that the nutrient agar sustained bacterial growth and the bacteria were actively proliferating. Absence of growth in the negative controls indicated that no microorganisms were introduced by the equipment used and the workspace into the nutrient agar. In the present study, E. coli (ATCC 43888), S. aureus, S. lentus and Shigella sp. were the most susceptible microorganisms to the AEPF. However, the AEPF was inactive against S. pyogenes, K. pneumoniae and MRSA . Based on MIC values, antimicrobial activity of plant extracts is classified as significant (MIC 625 µg/mL) (Kuete, 2010). As suspected, the AEPF demonstrated moderate antibacterial activity, whilst the TCE acted as a weak antibacterial agent. The AEPF gave MICs of 125 µg/mL for E. coli (ATCC 43888), S. aureus, Shigella sp. and S. lentus. Against S. typhi and E. coli (ATCC 10455), more AEPF was required to inhibit the growth (MICs of 250 µg/ml) of the bacteria with S. poona , showing even lower susceptibility to the AEPF (MIC 500 µg/ml). Gram-negative bacteria possess a cell wall surrounded by an outer membrane. Antibiotics must pass through the outer membrane to access their targets, and Gram-negative bacteria can alter this outer layer’s hydrophobic properties or develop mutations in porins and other factors to create resistance [ 37 ]. Due to the absence of this crucial layer in Gram-positive bacteria, Gram-negative bacteria are generally more resistant to antibiotics than Gram-positive bacteria [ 37 – 39 ]. Impressively, the AEPF showed excellent inhibitory activities against five (5) out of the six (6) Gram-negative bacteria used in this study; E. coli (ATCC 10455 & ATCC 43888), S. poona, Shigella sp. and S. typhi. The good inhibitory activity of the AEPF against E. coli (ATCC 43888), especially, is noteworthy, as the WHO categorizes this bacterium as a critical priority pathogen for research and development of new antibiotics [ 40 ]. S. aureus and S. lentus are also classified as high priority pathogens. The positive control, ciprofloxacin, showed better antibacterial activity (MIC < 3.9 µg/ml) than both the TCE and AEPF against all ten (10) bacteria strains. Standard tetracycline was less active in comparison to ciprofloxacin but more active than the plant extracts against all tested bacteria (Table 2 ). Our findings support the traditional use of this plant in the treatment of infectious diseases. Table 2 Minimum inhibition concentration (MIC) of total crude and alkaloidal crude extracts of P. fraternus against some pathogenic bacteria Drug/ Extract Minimum Inhibition Concentration (MIC) (µg/ml) E. coli (10) E. coli (43) S. aureus MRSA Kleb Shigella S. typhi S. poona S. lentus S. pyogenes TCE AEPF CP TT 500 250 < 3.9 500 125 < 3.9 15.6 500 125 500 500 > 500 < 3.9 15.6 N/A 125 500 250 500 500 < 3.9 15.6 500 125 500 < 3.9 < 3.9 Key; TCE = total crude extract; AEPF = alkaloidal extract of P. fraternus ; CP = Ciprofloxacin; TT = Tetracycline; E. coli (10) = E. coli (ATCC 10455); E. coli (43) = E. coli (ATCC 43888); ND = Not determined. Antimicrobial Resistance Modulation Study In Ghana and many other developing countries, patients on antibiotics might also use herbal preparations without the knowledge of their doctors. Although herb-drug interactions can be detrimental [ 41 – 42 ], the potential also exists for beneficial outcomes. This motivated us to investigate whether the alkaloidal extract from P. fraternus can modulate the antibacterial activity of tetracycline. Standard tetracycline was less active than ciprofloxacin against all bacteria tested and was therefore selected for this study. We selected bacteria for this study based on MICs when the AEPF and tetracycline were used alone. The AEPF (MIC; 125 µg/ml) in combination with tetracycline (MIC; 125 µg/ml) against Shigella sp. gave additive effects with fractional inhibitory concentration (FIC) indices of 1.25 and 1.0 for AEPF-tetracycline combinations in the ratios 1MIC:1/4MIC and 1/2MIC:1/2MIC respectively. Other AEPF-tetracycline combinations resulted in antagonistic effects with FIC indices ranging from 16–4.25. We did not observe synergistic effects for any of the combinations. Table 3 presents the FIC indices for different AEPF-tetracycline combinations and their interactions as antagonistic, additive, or synergistic. Inhibitors which target multi-drug resistant pathogens exhibit additive or synergistic effects when combined with other phytochemicals possessing antibacterial activity [ 43 ]. Findings from this study aligns with this evidence, as demonstrated by the enhanced activity of tetracycline when combined with alkaloids from P. fraternus . The findings also underscore the potential for combining existing antibiotics with antibacterial phytoconstituents to boost treatment outcomes especially in cases where drug resistance is eminent. Antimicrobial resistance is known to arise from six mechanisms including extrusion of the antimicrobial agent by active efflux pumps and biofilm formation. Although the mechanisms of antibacterial action of the crude alkaloids cannot be inferred from the available data, inhibitors of bacterial efflux systems might potentially reverse antimicrobial resistance and allow reuse of existing antimicrobial agents rendered ineffective via efflux-mediated mechanism. The inhibition of Bmr efflux pump by the alkaloid, reserpine, prevented extrusion of tetracycline in Bacillus subtilis [ 44 ]. In another study, the inhibition of Tet(K) efflux protein by reserpine in two clinical isolates of MRSA potentiated the activity of tetracycline [ 45 ]. The ability of plant extracts to exhibit antibacterial activity via biofilm inhibition has been reported [ 46 ]. The observed antibacterial activity of the crude alkaloids from P. fraternus could result from efflux pump or biofilm inhibition mechanisms although further studies are required. Table 3 Fractional Inhibitory Concentration (FIC) Indices for AEPF in Combination with Tetracycline against Shigella sp., S. lentus and S. aureus AEPF:Tetracycline AEPF:Tetracycline AEPF:Tetracycline MIC Combinations (µg/ml:µg/ml) Shigella sp. MIC Combinations (µg/ml:µg/ml) S. lentus MIC Combinations (µg/ml:µg/ml) S. aureus FIC Activity FIC Activity FIC Activity 1000:15.6 500:62.5 125:31.25 62.5:62.5 31.25:500 8.0625 4.5 1.25 1.0 4.25 A A I I A 1000:62.4 250:124.8 62.5:62.5 15.6:124.8 12 10 4.5 8.0625 A A A A 1000:3.9 500:3.9 250:31.2 125:124.8 8.25 4.25 4 9 A A A A A = Antagonism; I = Additive; AEPF: alkaloidal extracts of Phyllantus fraternus Time-Kill Kinetic Study Time-kill curves offer a dynamic data for assessing the relationship between an antibacterial concentration and its potency over time. This allows for direct comparison of MICs and antibacterial effects of a potential lead to those of existing conventional antibiotic [ 47 – 48 ]. A graph of optical density at 620 nm (OD 620 ) against time (h) was generated to study the bactericidal or bacteriostatic rate of the extracts compared to two positive control drugs in 24-hours. The exponential (last 6 h) and lag (first 6 h) phases of time-kill curves are the most informative parts of bacteria time-kill activity graphs [ 49 – 50 ]. As expected, the negative control showed an increasing regrowth in bacteria concentration for both the exponential and lag phases for all test bacteria, suggesting an increasing number of viable cells (Fig. 1 ). The curves for AEPF and positive controls deviate from typical negative control curves which indicate the effects of the alkaloidal extracts or standard drug on the bacteria. When the maximum optical density is reached at a lower optical density than the negative control, the effect is bacteriostatic. However, a straight line with no curves gives indication of bactericidal action [ 51 – 52 ]. The average optical density (OD ave ) during the last 6 h for the test strains were as follows; E. coli (ATCC 10455), 0.7481; E. coli (ATCC 43888), 0.7600; S. aureus , 1.0040; S. typhi , 0.8214; S. poona , 0.8286; S. lentus , 0.7581; K. pneumoniae , 1.8390; Shigella sp. , 0.6816; MRSA , 0.8095. The test strain, K. pneumoniae , showed the highest OD ave , followed by S. aureus and S. poona indicating their rapid growth rate in 24 h. The Shigella sp. showed the slowest growth rate. Against all test strains, ciprofloxacin showed a constant OD 620 during the first and last six hours, ranging from 0.0063–0.0255, indicating a constant reduction in viable cells. Again, the rate of activity of ciprofloxacin against all test strains over the course of the entire 24 h period demonstrated a bactericidal effect with zero tolerance for bacteria proliferation, indicating that it is more efficient at killing the test organisms as soon as they were introduced into the wells. Tetracycline, however, was bactericidal to E. coli ATCC 43888 and S. poona , but bacteriostatic agent against Shigella sp , S. lentus and S. aureus . The time-kill kinetic profile of AEPF against six (6) test strains was studied. The results show a better reduction (83%) in the number of viable cells against E. coli (ATCC 10455) compared to standard tetracycline (68%) (Fig. 1 b) and 94% reduction against E. coli (ATCC 43888) compared to standard tetracycline (90%) (Fig. 1 a). Against both bacteria, AEPF showed bactericidal activity with OD ave of 0.0898 and 0.1333 for the first and last six (6) hours respectively against E. coli (ATCC 10455) and OD ave of 0.0418 and 0.0517 for the first and last six (6) hours respectively against E. coli (ATCC 43888). The standard tetracycline also showed reduction in bacteria growth against these two bacteria although it was less effective in comparison to AEPF. Against E. coli (ATCC 43888), the OD ave for tetracycline was 0.0337 and 0.0740 for the lag and exponential phases respectively. Against E. coli (ATCC 10455) the OD ave were 0.1863 and 0.2412 for the lag and exponential phases respectively. AEPF and tetracycline showed similar activity from 18–24 h against S. aureus (Fig. 1 c) and S. poona (Fig. 1 d). In fact, both AEPF and tetracycline acted as bactericides against these two bacteria. Against S. aureus , an OD ave of 0.2242 for AEPF and 0.2000 for tetracycline whilst for S. poona , OD ave of 0.1320 and 0.1405 were observed for AEPF and tetracycline respectively. The AEPF showed better reduction (2 fold) in the number of viable cells with OD ave of 0.1798 compared to tetracycline with OD ave of 0.3470 from 18–24 h against Shigella sp. (Fig. 1 e). Tetracycline, however, was bactericidal whilst AEPF acted bacteriostatically. Tetracycline was three (3) fold better as antibacterial agent against S. lentus compared to AEPF (Fig. 1 f) with OD ave of 0.1005 compared to 0.2855 for AEPF from 18–24 h. The AEPF, however, showed better antibacterial activity against S. typhi with OD ave of 0.1622 compared to 0.2707 for tetracycline at the exponential phase. Time-Kill Kinetics of Modulated Tetracycline with Additive Interactions When combined with standard tetracycline, the AEPF-tetracycline in the ratio 1MIC:1/4MIC and 1/2MIC:1/2MIC demonstrated additive effect against Shigella with FIC indices 1.25 and 1.0 respectively. We investigated these AEPF-tetracycline combinations using time-kill kinetic to evaluate their rate of action and their interactions as bacteriostatic or bactericidal. We observed a bactericidal activity similar to standard ciprofloxacin for tetracycline in combination with AEPF in 24 hours (Fig. 2 ). The presence of the alkaloidal extract in the ratio 1/4MIC:1MIC tetracycline:AEPF, enhanced the antimicrobial activity of tetracycline and modified its action from bacteriostatic to bactericidal against Shigella (Fig. 2 ). The observed OD ave of 0.2259 and 0.3470 for the lag and exponential phases respectively for tetracycline improved significantly to 0.0226 and 0.0304 when combined with the AEPF in a 1/4MIC:MIC ratio, representing 10-fold increase in activity. We observed a similar trend when tetracycline was combined with the AEPF in 1/2MIC:1/2MIC ratio. Again, the time-kill kinetics profile showed 10-fold improvement in activity compared to tetracycline alone. It was gratifying to observe that the AEPF modulated the activity of tetracycline to a level comparable to ciprofloxacin, demonstrating the potential antibacterial combination therapy using alkaloids from P fraternus. Identification of Alkaloids Based on LC-ESI-MS Fragmentation Following chromatographic purification, the fractions were subjected to LC-MS analysis. Flash chromatography afforded 7 fractions, 5 of which contained a single spot by TLC. The molecular weights determined from the mass spectra were compared with known alkaloids from literature. LC-ESI-MS analysis of the crude alkaloid of P. fraternus revealed two known alkaloids from literature: epibubbialine ( a) and ent-norsecurinine ( b ) (Fig. 3 ) [ 53 – 55 ]. For epibubbialine, the spectrum showed ion peaks at m/z 240.2 (weak), 170.2 (weak), 204.3, 124.1, 222.3 and 111.7 (strong). The molecular ion peak [M + H] + was attributed to the peak at m/z 222.3, in agreement with molecular weight 221.3 amu [ 54 ]. The doubly charged pseudomolecular ion [M + 2H] 2+ corresponded to the peak at m/z 111.7. The weak peak at m/z 240.2 was attributed to the adduct [M + H 2 O + H] + ( m/z 221.3 → m/z 240.2). No adduct was assigned for the peaks at m/z 170.2 and 124.1. The spectrum for ent-norsecurinine showed ion peaks at m/z 244.3 (weak), 154.1 (weak), 204.3 and 102.6 (strong). The molecular ion peak [M + H] + was attributed to the peak at m/z 204.3 consistent with molecular weight 203.3 amu [ 53 ], [ 55 ]. The peak m/z 203.3 → m/z 102.6 corresponded to the doubly charged adduct [M + 2H] 2+ . The weak peak at m/z 244.3 corresponded to an [M + Na + H 2 O] + adduct. Conclusion The low content of alkaloids observed for Phyllanthus fraternus (0.64%)agrees with the generally lowlevels of alkaloids reported in most plants. The crude alkaloids demonstrated superior antibacterial activity in comparison to the total crude extract. The crude alkaloids of P. fraternus effectively suppress majority of clinically significant pathogenic strains in vitro . Significantly, the crude alkaloids successfully potentiated the biological activity of tetracycline against drug resistant Shigella resulting in ten-fold increase in antimicrobial activity. Time-kill kinetic assay revealed bactericidal effect of the crude alkaloids against S. aureus , E. coli (ATCC 43888), and E. coli (ATCC 10455). Interestingly, the antibacterial activity of tetracycline was modified by the crude alkaloids from bacteriostatic to bactericidal against Shigella and the level of activity became comparable to ciprofloxacin in the presence of the crude alkaloids. LC-ESI-MS experiments revealed the presence of two known alkaloids, epibubbialine and ent-norsecurinine, as components of P. fraternus. Taken together, the antibiotic activity of P. fraternus is primarily due to its alkaloids. Efforts are underway to isolate the individual alkaloids which could potentially be developed as drug candidates for use in combination therapies against antimicrobial resistance. Further investigations into the synergistic nature of these alkaloids with other conventional antimicrobial agents along with in-vivo study are required. Additionally, anti-biofilm activity, efflux pump inhibition potential, among others, should be examined to shed more light on the mechanisms of the action of the crude alkaloids present in the plant. Declarations Availability of data and materials: The data which support the current study are available from the corresponding author on reasonable request. Competing interests : The authors have no competing interests to declare. Funding: This study received no financial support. The authors, however, received our regular salaries from University of of Cape Coast or Kwame Nkrumah University of Science and Technology. Authors’ contributions SAO: Experimental methods design and implementation, methods validation, data curation, results analysis and interpretation and drafting original manuscript. DOK: Experimental methods design and implementation, result analysis and interpretation. YOB, FAA, PMF and LSB: Conceptualization, research design, manuscript review and editing. IA: Conceptualization, research design and supervision, project administration, data analysis, manuscript writing, review and editing. All the listed authors contributed to this study, read and approved the final manuscript Acknowledgements The authors wish to acknowledge the support of our colleagues in the medicinal chemistry and drug discovery research group at the department of chemistry, universities of cape coast (UCC), for their constructive feedback during group presentation. We are also thankful to the department of biomedical science, UCC, providing access to their microbiology laboratory. References L. S. Redgrave, S. B. Sutton, M. A. Webber, and L. J. V. 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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-5914968","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":407883207,"identity":"c0e14078-52af-49f5-b88b-ec4a42ccbab5","order_by":0,"name":"Samuel Asiamah Obiri","email":"","orcid":"","institution":"University of Cape Coast","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"Asiamah","lastName":"Obiri","suffix":""},{"id":407883208,"identity":"3887e829-c791-4662-ae88-2ead3298d913","order_by":1,"name":"Denzel Opoku-Kwabi","email":"","orcid":"","institution":"University of Cape Coast","correspondingAuthor":false,"prefix":"","firstName":"Denzel","middleName":"","lastName":"Opoku-Kwabi","suffix":""},{"id":407883209,"identity":"f0cfd797-eb4e-4e58-84b5-15da1fdb4824","order_by":2,"name":"Yaw Opoku-Boahen","email":"","orcid":"","institution":"University of Cape Coast","correspondingAuthor":false,"prefix":"","firstName":"Yaw","middleName":"","lastName":"Opoku-Boahen","suffix":""},{"id":407883210,"identity":"59a655a9-bd04-4697-93a1-67c399f8d78f","order_by":3,"name":"Francis Ackah Armah","email":"","orcid":"","institution":"University of Cape Coast","correspondingAuthor":false,"prefix":"","firstName":"Francis","middleName":"Ackah","lastName":"Armah","suffix":""},{"id":407883211,"identity":"5743059c-8725-4c17-9487-7ddeafb7184f","order_by":4,"name":"Malcolm Patrick Fynn","email":"","orcid":"","institution":"University of Cape Coast","correspondingAuthor":false,"prefix":"","firstName":"Malcolm","middleName":"Patrick","lastName":"Fynn","suffix":""},{"id":407883212,"identity":"76ea451c-5b02-4467-836d-6d085e46f442","order_by":5,"name":"Lawrence Sheringham Borquaye","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACdh4Yi/kAA2MDMVqY4VrYEkjWwmNAnBb5Zt6DjyvKDssbHO/5JvFzh40cA/vhoxvwaTE4zJdseObcYcMNZ85uk+w9k2bMwJOWdgOvFmYeM8nGtsOMG27kbpPgbTuc2CDBY4ZXi3wzRIv9hvtvnkn+JUYLw2GIlsQNN3jYpImyBeyXhnPpyTOB3rCWbUszZiPkF/n23oMPG8qsbfuOH354822bjRw/++Fj+B0GBmwMDAoHGFgkoGxiAFCZfAMD8wfiVI+CUTAKRsFIAwCpeUyLJI2N0QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-5037-0777","institution":"Kwame Nkrumah University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Lawrence","middleName":"Sheringham","lastName":"Borquaye","suffix":""},{"id":407883213,"identity":"2978375e-05ce-4688-814f-e3fa4be077e8","order_by":6,"name":"Isaac Asiamah","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYBACNiA+AMQJbOwNYAHGBoggEVr4eA4QqQUGEuQkEojUwsd+/OGBnzl2eWySbww//GCwkd1wgP3aA7wO48kxONi7LbmYTTrHWLKHIc14wwGecgP8fslhOMC7jTmxTTrHjJmB4XAiUEuaBF4t/M8fHPy7rT6xTfIMSMt/IrRIJBgc5t12OLFNggek5QBQC/sxAlreGByW3XY8sY0nrViyxyDZeOZhHja8WuT70x9/fLutOnF+++GNH35U2Mn2HW9/hlcLGgAFFTMP3gDDCtgfkKxlFIyCUTAKhjUAAGXmSP0rjxjwAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3451-4379","institution":"University of Cape Coast","correspondingAuthor":true,"prefix":"","firstName":"Isaac","middleName":"","lastName":"Asiamah","suffix":""}],"badges":[],"createdAt":"2025-01-27 20:58:34","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5914968/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5914968/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74999705,"identity":"97dd0e18-ab45-40bc-a9a0-64a07947af55","added_by":"auto","created_at":"2025-01-29 09:37:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93538,"visible":true,"origin":"","legend":"\u003cp\u003eTime-kill kinetics curve for E. coli ATCC 43888 (a), E. coli ATCC 10455 (b), S. aureus (c), S. poona (d), Shigella sp. (e) and S. lentus (f) subjected to the minimum inhibition concentrations of AEPF (blue), Tetracycline (orange) and Ciprofloxacin (grey) in 24 hours. Optical density readings were taken for the first and last 6 hours in 24 hours and compared with a negative control curve (yellow).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5914968/v1/a044448e7a1fb3ee9295583a.png"},{"id":75001065,"identity":"687e1d66-3ae0-4032-929e-35370fa1d1a4","added_by":"auto","created_at":"2025-01-29 09:45:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33511,"visible":true,"origin":"","legend":"\u003cp\u003eTetracycline (TT) in combination with crude alkaloidal constituents of P. fraternus, AEPF (AEPF: TT) in the ratios 1/2MIC:1/2MIC (red) and 1MIC:1/4MIC (green) with additive interactions showing bactericidal action against Shigella similar to ciprofloxacin (CP) (blue). Both AEPF (grey) and Tetracycline (orange) only inhibited bacterial growth. Bacteriostatic actions of tetracycline when used alone (orange) was successfully optimized in the presence of AEPF, acting in a bactericidal fashion similar to CP\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5914968/v1/05f945e5db3fc80dff179cda.png"},{"id":74999706,"identity":"ef16def8-b7f1-449a-bac8-8f544c9c8cd5","added_by":"auto","created_at":"2025-01-29 09:37:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12470,"visible":true,"origin":"","legend":"\u003cp\u003eThe two bioactive alkaloids Epibubbialine (a) and Ent-norsecurinine (b) identified from the crude alkaloidal extract of \u003cem\u003eP. fraternus\u003c/em\u003e (AEPF) by LC-MS/MS analysis of chromatographic fractions\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5914968/v1/9de5b2d84788d8abb323e49c.png"},{"id":75003435,"identity":"4c98aab8-734d-45f1-b4f9-6fb0914cbd79","added_by":"auto","created_at":"2025-01-29 10:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1138915,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5914968/v1/5adcb964-239b-4d00-9ef1-2f6ae2f9f76c.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eCrude Alkaloids form \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePhyllanthus fraternus, \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eWebster: Antibacterial, Time-Kill Kinetics and Resistance Modulation Studies\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntimicrobial resistance remains a major public health concern globally with an increasing number of antibiotics and antifungal agents losing their efficacy. As microbes develop resistance and adapt to treatments designed to eliminate them, clinical treatment of infections become impaired and expensive, which poses a major threat to public health. Through a variety of defense mechanisms, bacteria render antibacterial agents ineffective. These include limiting access to antibiotics, expelling antibiotics from the body via active efflux strategies, inactivating the antibiotic or its target, and evading the drug's effects through mutations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. When bacteria have the right combination of resistance mechanisms, the effectiveness of antibacterial treatments is significantly diminished. Although antimicrobial resistance is a natural phenomenon, it impairs the effectiveness of current treatments because resistant strains can disseminate their resistance mechanisms [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Research continues to focus on the need to discover new antibacterial agents with a preferably novel mode of action. Natural products remain crucial in the search for novel molecules for various therapeutic applications.\u003c/p\u003e \u003cp\u003eAmong rural communities in developing countries, traditional treatments employing medicinal plants continue to play a key role in treating several ailments primarily due to the lack of an efficient primary healthcare system [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003ePhyllanthus fraternus\u003c/em\u003e is a herbaceous weed widely distributed in Asia, the West Indies, and in Africa where the plant has been used traditionally in treating various diseases. In Ghana, aqueous decoction from the leaves of \u003cem\u003eP. fraternus\u003c/em\u003e is used to treat a wide variety of ailments including jaundice, malaria, kidney disease, high blood pressure, diabetes, genital-urinary tract infections, stroke, liver disease, intestinal infections, anaemia, hepatocellular cancer, severe abdominal pains, and diarrhea [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A broad range of pharmacological activities have been reported in extracts from \u003cem\u003eP. fraternus\u003c/em\u003e including antiviral, anti-inflammatory, antioxidant, anti-diabetic, antinociceptive, hepatoprotective and antifibromyalgic [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] which may have implications for their traditional uses.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. fraternus\u003c/em\u003e is known to contain different secondary metabolites including alkaloids, tannins, saponins, terpenoids and steroids. Alkaloids are nitrogen-containing natural products with significant pharmacological activities, including unique antibacterial capabilities [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Alkaloids served as the backbone for the design and development of various antibacterial agents. Notably, quinolones emerged inadvertently during quinine synthesis, metronidazole was derived through chemical modification of azomycin, and bedaquiline is based on the quinoline structure [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Other antibacterials such as trimethoprim and linezolid have alkaloids as foundational substructures [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Extensive investigations of the antibacterial activity of alkaloids have revealed their ability to alter DNA function [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], interfere with protein synthesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and disrupt bacterial cell membrane [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, we selectively extracted the alkaloidal components from \u003cem\u003eP. fraternus\u003c/em\u003e and evaluated the antimicrobial properties against a number of WHO priority pathogens. We assessed the potential of the alkaloidal extract to alter the activity of standard tetracycline. To assess the nature of interactions between the alkaloidal extract and the chosen pathogens, we conducted time-kill kinetic assays. We employed LC-MS methods to characterize the various alkaloids present in the alkaloidal extract after column chromatographic purification. We hereby report the antibacterial abilities of \u003cem\u003eP. fraternus\u003c/em\u003e and point the way for future work on separating the active alkaloidal components for possible optimization into potential drug candidates.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and Reagents\u003c/h2\u003e \u003cp\u003eThe reagents used included the following: Wagner\u0026rsquo;s reagent, Mayer\u0026rsquo;s reagent, Dragendorff\u0026rsquo;s reagent, McFarland standard, Liebermann-Burchard Reagent, Crystal violet and Salkowski reagent. Materials used included the following: 96-well microtiter plate, Silica gel (70\u0026ndash;230 mesh size) [ASTM, Merck, Germany], aluminium pre-coated silica gel plates 60 F\u003csub\u003e254\u003c/sub\u003e 0.25 mm thick (Merck, Germany), Mueller-Hinton broth (MHB) (Merck, Germany), Mueller-Hinton agar (MHA) (Oxford Ltd, England), Methanol, Dichloromethane, Ethyl acetate and Hexane. All organic solvents used were of analytical grade and obtained from BDH Laboratory Supplies (Merck Ltd., Lutterw North, UK).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTest Organisms\u003c/h3\u003e\n\u003cp\u003ePure cultures of four Gram-positive: \u003cem\u003eStaphylococcus aureus, MRSA, Streptococcus pyogenes, Staphylococcus lentus\u003c/em\u003e and six Gram-negative: \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 10455 \u0026amp; ATCC 43888), \u003cem\u003eKlebsiella pneumoniae, Salmonella poona, Shigella sp., Salmonella typhi\u003c/em\u003e were obtained from the Microbiology laboratory of the Department of Biomedical Sciences, School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana. To establish that the bacteria were viable and actively proliferating, sub-culturing of the bacteria was conducted by taking a loop full of test organisms from falcon tubes and streaking on sterile nutrient agar.\u003c/p\u003e\n\u003ch3\u003eCollection of Plant Materials\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003ePhyllanthus fraternus\u003c/em\u003e (whole plant) was collected from Akotokyire (5\u0026deg; 8\u0026rsquo;3\u0026rdquo;N/ 1\u0026deg; 17\u0026rsquo;28\u0026rdquo;W) in Cape Coast, Ghana on March 31st, 2022. The plant sample was authenticated by Mr. Felix Fynn, a taxonomist at the Department of Botany, University of Cape Coast, Ghana. A specimen sample was deposited at the University\u0026rsquo;s herbarium with voucher number Eup. CC 5152.\u003c/p\u003e\n\u003ch3\u003eCrude Extraction\u003c/h3\u003e\n\u003cp\u003eThe plant samples were washed to remove soil and other debris and air-dried. 800 grams of ground plant material was macerated with 1.5 L methanol-dichloromethane (1:1) mixture for 72 h. Following filtration, the \u0026ldquo;marc\u0026rdquo; was further extracted with 1.5 L methanol for 72 h. The combined extract was evaporated \u003cem\u003ein vacuo\u003c/em\u003e at 45\u0026deg;C to afford the crude extract (58.14 g, 7.23%). The crude extract was stored in a desiccator until use. The crude extract was screened for the presence of secondary metabolites according to standard procedures [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Dragendorff\u0026rsquo;s, Mayer\u0026rsquo;s, and Wagner\u0026rsquo;s tests were used to confirm the presence of alkaloids.\u003c/p\u003e\n\u003ch3\u003eExtraction of Alkaloidal Components\u003c/h3\u003e\n\u003cp\u003eThe alkaloidal extraction was carried out according to the reported literature procedure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The crude extract (8.1 g) was dissolved in 30 ml 10% acetic acid solution. The resulting solution was defatted with hexane (7\u0026times;50 ml). The combined aqueous layer was made alkaline with aq. NH\u003csub\u003e3\u003c/sub\u003e (70 ml) to a pH 8\u0026ndash;9. The alkaline solution was extracted with chloroform (5\u0026times;30 ml) and the combined chloroform extracts were concentrated at 40\u0026deg;C \u003cem\u003ein vacuo\u003c/em\u003e to afford the alkaloidal extract of \u003cem\u003eP. fraternus\u003c/em\u003e (AEPF, 244.2 mg, 0.64%). The presence of alkaloids was confirmed by standard tests [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The absence of other secondary metabolites was also confirmed using standard procedures [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of Minimum Inhibition Concentration (MIC) of Crude Extract and Crude Alkaloidal Extracts of\u003c/b\u003e \u003cb\u003eP. fraternus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe MIC of the crude and alkaloidal extracts were determined using the high-throughput spot culture growth inhibition assay (HT-SPOTi) by visual inspection after 18\u0026ndash;24 h of the incubation period as described by Danquah [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Tetracycline and ciprofloxacin were used as the positive controls.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic Resistance Modulation Study\u003c/h2\u003e \u003cp\u003eWe used checkerboard assay [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] to evaluate the potential of the crude alkaloidal extract to alter the antimicrobial activity of standard tetracycline. Bacteria strains whose activity was inhibited at MICs\u0026thinsp;\u0026le;\u0026thinsp;250 \u0026micro;g/ml by the alkaloid extract and, those with tetracycline MICs\u0026thinsp;\u0026ge;\u0026thinsp;15.6 \u0026micro;g/mL, were selected for the study. The following organisms (with their tetracycline MICs) were involved; \u003cem\u003eShigella sp\u003c/em\u003e, (125 \u0026micro;g/ml); MRSA, (125 \u0026micro;g/ml); \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 43888, (15.6 \u0026micro;g/ml); \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, (15.6 \u0026micro;g/ml); \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, (15.6 \u0026micro;g/ml); \u003cem\u003eSalmonella poona\u003c/em\u003e, (15.6 \u0026micro;g/ml); and \u003cem\u003eStaphylococcus lentus\u003c/em\u003e, (15.6 \u0026micro;g/ml). The checkerboard assay was performed according to the standard protocols described in the literature [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] with minor modifications. Instead of broth, we used nutrient agar and bacterial growth was determined by visual inspection. For each test bacteria, stock solutions of tetracycline and the AEPF were prepared based on observed MICs. The concentration of each stock solution was 8\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003eMICs observed for the different bacteria. Two-fold serial dilution afforded concentrations in ranges of 8\u0026times;MIC, 4\u0026times;MIC, 2\u0026times;MIC, MIC, 1/2\u0026times;MIC, 1/4\u0026times;MIC, 1/8\u0026times;MIC and 1/16\u0026times;MIC on each row and column of a 96-well microtiter plates for tetracycline and the crude alkaloid extract. Equal volumes (10 \u0026micro;l) from the respective columns and rows were mixed to obtain the tetracycline-AEPF combination. These tetracycline-AEPF mixtures (2 \u0026micro;l) were transferred into corresponding wells on the second 96-well microtiter plate. Molten agar (196 \u0026micro;l) was dispensed into wells containing tetracycline-AEPF mixture and the plate shaken for about 10 seconds to obtain a uniform distribution and allowed to solidify. Standardized microbial suspension (2 \u0026micro;l) was then added. The plate was sealed with parafilm, covered with aluminum foil, and incubated at 37\u0026deg;C for 24 hours. After 24 hours, the plate was visually observed for growth, or no growth. Fractional inhibition concentration (FIC) indices determined using the equation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\frac{A}{MIC\\:\\left(A\\right)}\\:+\\frac{B}{MIC\\:\\left(B\\right)}\\:=FIC\\:\\left(A\\right)+FIC\\:\\left(B\\right)=FIC\\:Index$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere A and B are the MIC of each antibiotic in combination (in a single well), and MIC (A) and MIC (B) are the individual MIC of each antibiotic. FIC indices were interpreted as follows: S, synergy (FIC\u0026thinsp;\u0026lt;\u0026thinsp;0.5); I, additive (FIC 0.5\u0026ndash;4); and A, Antagonistic (FIC\u0026thinsp;\u0026gt;\u0026thinsp;4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTime-kill Kinetic Study of Crude Alkaloidal of\u003c/b\u003e \u003cb\u003eP. fraternus\u003c/b\u003e \u003cb\u003eand Extract-Tetracycline Combinations\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTime-kill kinetics of AEPF and AEPF-tetracycline combinations were carried out in 24 hours following the procedure described by Mojsoska [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], with slight modifications. Briefly, in the presence or absence of the crude alkaloid extract, first and last six (6) hours constant MIC time-kill curves were performed in duplicates for activities at MICs less or equal to 250 \u0026micro;g/ml against the test strains. AEPF (2 \u0026micro;l) was dispensed into appropriate wells and Mueller-Hinton nutrient broth (196 \u0026micro;l) added. An inoculum equal to a 0.5 McFarland turbidity standard (2 \u0026micro;l) was dispensed into each well containing AEPF-media mixture. The optical density (OD) at 620 nm at time zero (t\u0026thinsp;=\u0026thinsp;0) was read using the Elisa micro-plate reader. The plates were then sealed with parafilm, covered with aluminum foil, and incubated at 37\u0026deg;C. For the first 6 hours, the optical density at 30 minutes intervals was taken. For the last 6 hours (18\u0026ndash;24 hours) the optical density at one hour (1h) interval was taken. A blank and a negative control experiment were also run simultaneously.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChromatographic Isolations and Purifications\u003c/h3\u003e\n\u003cp\u003eThe alkaloidal extract (AEPF) was subjected to flash column chromatography. AEPF (0.3 g) was loaded on glass column packed with silica gel (40\u0026ndash;63 \u0026micro;m) mesh (ASTM, Merck Germany) and eluted with hexane (100%), hexane-ethyl acetate (50:10 v/v and 50:30 v/v). Fractions were monitored by TLC to obtain eight (8) fractions (F1 \u0026ndash; F8). The fractions were concentrated at 40 \u003csup\u003eo\u003c/sup\u003eC in \u003cem\u003evacuo\u003c/em\u003e, dried and stored in a desiccator until LC-MS analysis.\u003c/p\u003e\n\u003ch3\u003eLC-ESI-MS Analysis\u003c/h3\u003e\n\u003cp\u003eThe profile of the alkaloids present in the fractions was ascertained using reverse-phase high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The triple quadruple mass spectrometer (AB Sciex, USA) was used in conjunction with an Agilent 1200 HPLC system (Agilent Technologies, USA) integrated with a binary gradient solvent pump, degasser, autosampler, and column oven. Separations were carried out at 25\u0026deg;C on a Zorbax SB-C18 column (2.1 \u0026times; 50 mm, 1.8-\u0026micro;m particles; Agilent Technologies, USA) with a mobile phase of 0.1% formic acid in water (Solvent A) and methanol-acetonitrile (1:1 v/v, Solvent B), using 2 \u0026micro;l injections. The method was slightly modified from Nowacka [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The gradient time was 16 minutes, and the flow rate was 100 \u0026micro;l min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with a capillary temperature of 300\u0026deg;C, curtain gas pressure of 30 psi, and nebulizer gas pressure of 60 psi, the MS was run in the positive ion mode. The collision and curtain gas were nitrogen.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExtraction and Crude Alkaloidal Extract\u003c/h2\u003e \u003cp\u003eCrude extraction with 50% methanol in dichloromethane followed by 100% methanol afforded the phytoconstituents from the plant (58.14g, 7.23%). Phytochemical screening revealed the presence of secondary metabolites including alkaloids, steroids, tannins, and glycosides (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The alkaloids in the total crude extract (TCE) were converted to their water-soluble salts in the presence of acetic acid to enable their selective partitioning into the aqueous phase. To enable their extraction into chloroform, the alkaloids were further treated with a base [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eP. fraternus\u003c/em\u003e contains 0.64% alkaloids, which agrees with the generally low levels of alkaloids usually reported in plants (Murphy, 2017). Using widely accepted standard reagents, we confirmed the presence of alkaloids in the extract when addition of Dragendorff\u0026rsquo;s reagent (1ml) gave orange red precipitate. The formation of yellowish precipitate with Meyer\u0026rsquo;s reagent gave further indication of alkaloids [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The formation of large brown amorphous and flocculent precipitate after treating the hydrochloric acid solution (5 ml) of the extract with some drops of Wagner\u0026rsquo;s reagent further confirmed the presence of alkaloids [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In order to ensure that the crude alkaloidal extract of \u003cem\u003eP. fraternus\u003c/em\u003e (AEPF) was devoid of any other secondary metabolites, we repeated the phytochemical screening assays as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The presence of only alkaloids in the AEPF demonstrated that the acid-base extraction method for selective alkaloidal extraction was successful.\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\u003ePhytoconstituents of the total crude extract (TCE) and the crude alkaloidal extract of \u003cem\u003eP. fraternus\u003c/em\u003e (AEPF)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhytochemical\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP. fraternus\u003c/em\u003e \u003csup\u003ea (b)\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlkaloid\u003c/p\u003e \u003cp\u003eTriterpenoid\u003c/p\u003e \u003cp\u003eSteroids\u003c/p\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003cp\u003eTannin\u003c/p\u003e \u003cp\u003eSaponins\u003c/p\u003e \u003cp\u003eGlycosides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ (+)\u003c/p\u003e \u003cp\u003e+ (-)\u003c/p\u003e \u003cp\u003e+ (-)\u003c/p\u003e \u003cp\u003e- (-)\u003c/p\u003e \u003cp\u003e+ (-)\u003c/p\u003e \u003cp\u003e+ (-)\u003c/p\u003e \u003cp\u003e+ (-)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eKey: \u003cem\u003ea\u003c/em\u003e\u0026thinsp;=\u0026thinsp;TCE; \u003cem\u003eb\u0026thinsp;=\u003c/em\u003e\u0026thinsp;AEPF; + = detected; - = not detected\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMinimum Inhibition Concentration (MIC)\u003c/h2\u003e \u003cp\u003eWe predicted that antibacterial activities of \u003cem\u003eP. fraternus\u003c/em\u003e were primarily attributable to its alkaloidal phytoconstituents. We evaluated the antibacterial properties of both the total crude extract and the selectively extracted alkaloidal components from the plant. For most of the studied bacteria, the AEPF demonstrated superior growth inhibition over the TCE (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The high MICs (mostly\u0026thinsp;\u0026gt;\u0026thinsp;500 mg/mL) recorded for the TCE are consistent with previous reports using crude extract against various pathogenic bacteria [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In some cases where the TCE showed no growth inhibitory activity, the AEPF demonstrated some activity. Our findings suggest that the alkaloidal components of \u003cem\u003eP. fraternus\u003c/em\u003e contribute substantially to its antibacterial activity. Importantly, both the TCE and the AEPF demonstrated antibacterial activities on at least two of the test strains. In a previous study, the crude extract of the aerial parts of \u003cem\u003eP. fraternus\u003c/em\u003e showed low inhibitory activity with MICs ranging from 50\u0026ndash;250 mg/mL [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This finding was corroborated by [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] when crude extract from the leaves of the plant showed no antibacterial activity against \u003cem\u003eE. coli.\u003c/em\u003e Ibrahim [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] verified antibacterial activity using n-hexane and ethyl acetate fractions from the leaves of \u003cem\u003eP. fraternus\u003c/em\u003e. The authors reported MICs ranging from 80\u0026ndash;120 mg/mL and 40\u0026ndash;80 mg/mL for n-hexane and the ethyl acetate fractions respectively. Results from our investigations showed that the AEPF performed better than the TCE against \u003cem\u003eE. coli\u003c/em\u003e (ATCC 104550, \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888), \u003cem\u003eS. poona\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e with MICs ranging from 12.5 to 25.0 mg/mL in comparison to \u0026ge;\u0026thinsp;50 mg/mL for the TCE (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All positive controls showed growth, indicating that the nutrient agar sustained bacterial growth and the bacteria were actively proliferating. Absence of growth in the negative controls indicated that no microorganisms were introduced by the equipment used and the workspace into the nutrient agar.\u003c/p\u003e \u003cp\u003eIn the present study, \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888), \u003cem\u003eS. aureus, S. lentus\u003c/em\u003e and \u003cem\u003eShigella sp.\u003c/em\u003e were the most susceptible microorganisms to the AEPF. However, the AEPF was inactive against \u003cem\u003eS. pyogenes, K. pneumoniae\u003c/em\u003e and \u003cem\u003eMRSA\u003c/em\u003e. Based on MIC values, antimicrobial activity of plant extracts is classified as significant (MIC\u0026thinsp;\u0026lt;\u0026thinsp;100 \u0026micro;g/mL), moderate (100\u0026thinsp;\u0026le;\u0026thinsp;MIC\u0026thinsp;\u0026le;\u0026thinsp;6.25 \u0026micro;g/mL) or weak (MIC\u0026thinsp;\u0026gt;\u0026thinsp;625 \u0026micro;g/mL) (Kuete, 2010). As suspected, the AEPF demonstrated moderate antibacterial activity, whilst the TCE acted as a weak antibacterial agent. The AEPF gave MICs of 125 \u0026micro;g/mL for \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888), \u003cem\u003eS. aureus, Shigella sp.\u003c/em\u003e and \u003cem\u003eS. lentus.\u003c/em\u003e Against \u003cem\u003eS. typhi\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e (ATCC 10455), more AEPF was required to inhibit the growth (MICs of 250 \u0026micro;g/ml) of the bacteria with S. \u003cem\u003epoona\u003c/em\u003e, showing even lower susceptibility to the AEPF (MIC 500 \u0026micro;g/ml). Gram-negative bacteria possess a cell wall surrounded by an outer membrane. Antibiotics must pass through the outer membrane to access their targets, and Gram-negative bacteria can alter this outer layer\u0026rsquo;s hydrophobic properties or develop mutations in porins and other factors to create resistance [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Due to the absence of this crucial layer in Gram-positive bacteria, Gram-negative bacteria are generally more resistant to antibiotics than Gram-positive bacteria [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Impressively, the AEPF showed excellent inhibitory activities against five (5) out of the six (6) Gram-negative bacteria used in this study; \u003cem\u003eE. coli\u003c/em\u003e (ATCC 10455 \u0026amp; ATCC 43888), \u003cem\u003eS. poona, Shigella sp.\u003c/em\u003e and \u003cem\u003eS. typhi.\u003c/em\u003e The good inhibitory activity of the AEPF against \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888), especially, is noteworthy, as the WHO categorizes this bacterium as a critical priority pathogen for research and development of new antibiotics [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. lentus\u003c/em\u003e are also classified as high priority pathogens. The positive control, ciprofloxacin, showed better antibacterial activity (MIC\u0026thinsp;\u0026lt;\u0026thinsp;3.9 \u0026micro;g/ml) than both the TCE and AEPF against all ten (10) bacteria strains. Standard tetracycline was less active in comparison to ciprofloxacin but more active than the plant extracts against all tested bacteria (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Our findings support the traditional use of this plant in the treatment of infectious diseases.\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\u003eMinimum inhibition concentration (MIC) of total crude and alkaloidal crude extracts of \u003cem\u003eP. fraternus\u003c/em\u003e against some pathogenic bacteria\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDrug/\u003c/p\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c9\" namest=\"c4\"\u003e \u003cp\u003eMinimum Inhibition Concentration (MIC) (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e(10)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e(43)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMRSA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eKleb\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eShigella\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eS. typhi\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eS. poona\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eS. lentus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003eS. pyogenes\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTCE\u003c/p\u003e \u003cp\u003eAEPF\u003c/p\u003e \u003cp\u003eCP\u003c/p\u003e \u003cp\u003eTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e250\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e \u003cp\u003e125\u003c/p\u003e\u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e125\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e125\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003cp\u003e125\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e125\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e \u003cp\u003e250\u003c/p\u003e\u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e7.8\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e \u003cp\u003e500\u003c/p\u003e\u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e125\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eND\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eKey; TCE\u0026thinsp;=\u0026thinsp;total crude extract; AEPF\u0026thinsp;=\u0026thinsp;alkaloidal extract of \u003cem\u003eP. fraternus\u003c/em\u003e; CP\u0026thinsp;=\u0026thinsp;Ciprofloxacin; TT\u0026thinsp;=\u0026thinsp;Tetracycline; \u003cem\u003eE. coli\u003c/em\u003e (10)\u0026thinsp;=\u0026thinsp;\u003cem\u003eE. coli\u003c/em\u003e (ATCC 10455);\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e (43)\u0026thinsp;=\u0026thinsp;\u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888); ND\u0026thinsp;=\u0026thinsp;Not determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial Resistance Modulation Study\u003c/h2\u003e \u003cp\u003eIn Ghana and many other developing countries, patients on antibiotics might also use herbal preparations without the knowledge of their doctors. Although herb-drug interactions can be detrimental [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], the potential also exists for beneficial outcomes. This motivated us to investigate whether the alkaloidal extract from \u003cem\u003eP. fraternus\u003c/em\u003e can modulate the antibacterial activity of tetracycline. Standard tetracycline was less active than ciprofloxacin against all bacteria tested and was therefore selected for this study. We selected bacteria for this study based on MICs when the AEPF and tetracycline were used alone.\u003c/p\u003e \u003cp\u003eThe AEPF (MIC; 125 \u0026micro;g/ml) in combination with tetracycline (MIC; 125 \u0026micro;g/ml) against \u003cem\u003eShigella sp.\u003c/em\u003e gave additive effects with fractional inhibitory concentration (FIC) indices of 1.25 and 1.0 for AEPF-tetracycline combinations in the ratios 1MIC:1/4MIC and 1/2MIC:1/2MIC respectively. Other AEPF-tetracycline combinations resulted in antagonistic effects with FIC indices ranging from 16\u0026ndash;4.25. We did not observe synergistic effects for any of the combinations. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the FIC indices for different AEPF-tetracycline combinations and their interactions as antagonistic, additive, or synergistic. Inhibitors which target multi-drug resistant pathogens exhibit additive or synergistic effects when combined with other phytochemicals possessing antibacterial activity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Findings from this study aligns with this evidence, as demonstrated by the enhanced activity of tetracycline when combined with alkaloids from \u003cem\u003eP. fraternus\u003c/em\u003e. The findings also underscore the potential for combining existing antibiotics with antibacterial phytoconstituents to boost treatment outcomes especially in cases where drug resistance is eminent.\u003c/p\u003e \u003cp\u003eAntimicrobial resistance is known to arise from six mechanisms including extrusion of the antimicrobial agent by active efflux pumps and biofilm formation. Although the mechanisms of antibacterial action of the crude alkaloids cannot be inferred from the available data, inhibitors of bacterial efflux systems might potentially reverse antimicrobial resistance and allow reuse of existing antimicrobial agents rendered ineffective via efflux-mediated mechanism. The inhibition of Bmr efflux pump by the alkaloid, reserpine, prevented extrusion of tetracycline in \u003cem\u003eBacillus subtilis\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In another study, the inhibition of Tet(K) efflux protein by reserpine in two clinical isolates of MRSA potentiated the activity of tetracycline [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The ability of plant extracts to exhibit antibacterial activity via biofilm inhibition has been reported [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The observed antibacterial activity of the crude alkaloids from \u003cem\u003eP. fraternus\u003c/em\u003e could result from efflux pump or biofilm inhibition mechanisms although further studies are required.\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\u003eFractional Inhibitory Concentration (FIC) Indices for AEPF in Combination with Tetracycline against \u003cem\u003eShigella sp., S. lentus\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAEPF:Tetracycline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eAEPF:Tetracycline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eAEPF:Tetracycline\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMIC Combinations\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml:\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eShigella sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMIC Combinations\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml:\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cem\u003eS. lentus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMIC Combinations\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml:\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eActivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eActivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eActivity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000:15.6\u003c/p\u003e \u003cp\u003e500:62.5\u003c/p\u003e \u003cp\u003e125:31.25\u003c/p\u003e \u003cp\u003e62.5:62.5\u003c/p\u003e \u003cp\u003e31.25:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.0625\u003c/p\u003e \u003cp\u003e4.5\u003c/p\u003e \u003cp\u003e1.25\u003c/p\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e4.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003eI\u003c/p\u003e \u003cp\u003eI\u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1000:62.4\u003c/p\u003e \u003cp\u003e250:124.8\u003c/p\u003e \u003cp\u003e62.5:62.5\u003c/p\u003e \u003cp\u003e15.6:124.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003cp\u003e10\u003c/p\u003e \u003cp\u003e4.5\u003c/p\u003e \u003cp\u003e8.0625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1000:3.9\u003c/p\u003e \u003cp\u003e500:3.9\u003c/p\u003e \u003cp\u003e250:31.2\u003c/p\u003e \u003cp\u003e125:124.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.25\u003c/p\u003e \u003cp\u003e4.25\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eA\u0026thinsp;=\u0026thinsp;Antagonism; I\u0026thinsp;=\u0026thinsp;Additive; AEPF: alkaloidal extracts of \u003cem\u003ePhyllantus fraternus\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTime-Kill Kinetic Study\u003c/h2\u003e \u003cp\u003eTime-kill curves offer a dynamic data for assessing the relationship between an antibacterial concentration and its potency over time. This allows for direct comparison of MICs and antibacterial effects of a potential lead to those of existing conventional antibiotic [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. A graph of optical density at 620 nm (OD\u003csub\u003e620\u003c/sub\u003e) against time (h) was generated to study the bactericidal or bacteriostatic rate of the extracts compared to two positive control drugs in 24-hours. The exponential (last 6 h) and lag (first 6 h) phases of time-kill curves are the most informative parts of bacteria time-kill activity graphs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. As expected, the negative control showed an increasing regrowth in bacteria concentration for both the exponential and lag phases for all test bacteria, suggesting an increasing number of viable cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The curves for AEPF and positive controls deviate from typical negative control curves which indicate the effects of the alkaloidal extracts or standard drug on the bacteria. When the maximum optical density is reached at a lower optical density than the negative control, the effect is bacteriostatic. However, a straight line with no curves gives indication of bactericidal action [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe average optical density (OD\u003csub\u003eave\u003c/sub\u003e) during the last 6 h for the test strains were as follows; \u003cem\u003eE. coli\u003c/em\u003e (ATCC 10455), 0.7481; \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888), 0.7600; \u003cem\u003eS. aureus\u003c/em\u003e, 1.0040; \u003cem\u003eS. typhi\u003c/em\u003e, 0.8214; \u003cem\u003eS. poona\u003c/em\u003e, 0.8286; \u003cem\u003eS. lentus\u003c/em\u003e, 0.7581; \u003cem\u003eK. pneumoniae\u003c/em\u003e, 1.8390; \u003cem\u003eShigella sp.\u003c/em\u003e, 0.6816; \u003cem\u003eMRSA\u003c/em\u003e, 0.8095. The test strain, \u003cem\u003eK. pneumoniae\u003c/em\u003e, showed the highest OD\u003csub\u003eave\u003c/sub\u003e, followed by \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. poona\u003c/em\u003e indicating their rapid growth rate in 24 h. The \u003cem\u003eShigella sp.\u003c/em\u003e showed the slowest growth rate. Against all test strains, ciprofloxacin showed a constant OD\u003csub\u003e620\u003c/sub\u003e during the first and last six hours, ranging from 0.0063\u0026ndash;0.0255, indicating a constant reduction in viable cells. Again, the rate of activity of ciprofloxacin against all test strains over the course of the entire 24 h period demonstrated a bactericidal effect with zero tolerance for bacteria proliferation, indicating that it is more efficient at killing the test organisms as soon as they were introduced into the wells. Tetracycline, however, was bactericidal to \u003cem\u003eE. coli\u003c/em\u003e ATCC 43888 and \u003cem\u003eS. poona\u003c/em\u003e, but bacteriostatic agent against \u003cem\u003eShigella sp\u003c/em\u003e, \u003cem\u003eS. lentus\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe time-kill kinetic profile of AEPF against six (6) test strains was studied. The results show a better reduction (83%) in the number of viable cells against \u003cem\u003eE. coli\u003c/em\u003e (ATCC 10455) compared to standard tetracycline (68%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and 94% reduction against \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888) compared to standard tetracycline (90%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Against both bacteria, AEPF showed bactericidal activity with OD\u003csub\u003eave\u003c/sub\u003e of 0.0898 and 0.1333 for the first and last six (6) hours respectively against \u003cem\u003eE. coli\u003c/em\u003e (ATCC 10455) and OD\u003csub\u003eave\u003c/sub\u003e of 0.0418 and 0.0517 for the first and last six (6) hours respectively against \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888). The standard tetracycline also showed reduction in bacteria growth against these two bacteria although it was less effective in comparison to AEPF. Against \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888), the OD\u003csub\u003eave\u003c/sub\u003e for tetracycline was 0.0337 and 0.0740 for the lag and exponential phases respectively. Against \u003cem\u003eE. coli\u003c/em\u003e (ATCC 10455) the OD\u003csub\u003eave\u003c/sub\u003e were 0.1863 and 0.2412 for the lag and exponential phases respectively. AEPF and tetracycline showed similar activity from 18\u0026ndash;24 h against \u003cem\u003eS. aureus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) and \u003cem\u003eS. poona\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In fact, both AEPF and tetracycline acted as bactericides against these two bacteria. Against \u003cem\u003eS. aureus\u003c/em\u003e, an OD\u003csub\u003eave\u003c/sub\u003e of 0.2242 for AEPF and 0.2000 for tetracycline whilst for \u003cem\u003eS. poona\u003c/em\u003e, OD\u003csub\u003eave\u003c/sub\u003e of 0.1320 and 0.1405 were observed for AEPF and tetracycline respectively. The AEPF showed better reduction (2 fold) in the number of viable cells with OD\u003csub\u003eave\u003c/sub\u003e of 0.1798 compared to tetracycline with OD\u003csub\u003eave\u003c/sub\u003e of 0.3470 from 18\u0026ndash;24 h against \u003cem\u003eShigella sp.\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Tetracycline, however, was bactericidal whilst AEPF acted bacteriostatically. Tetracycline was three (3) fold better as antibacterial agent against \u003cem\u003eS. lentus\u003c/em\u003e compared to AEPF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) with OD\u003csub\u003eave\u003c/sub\u003e of 0.1005 compared to 0.2855 for AEPF from 18\u0026ndash;24 h. The AEPF, however, showed better antibacterial activity against \u003cem\u003eS. typhi\u003c/em\u003e with OD\u003csub\u003eave\u003c/sub\u003e of 0.1622 compared to 0.2707 for tetracycline at the exponential phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTime-Kill Kinetics of Modulated Tetracycline with Additive Interactions\u003c/h2\u003e \u003cp\u003eWhen combined with standard tetracycline, the AEPF-tetracycline in the ratio 1MIC:1/4MIC and 1/2MIC:1/2MIC demonstrated additive effect against \u003cem\u003eShigella\u003c/em\u003e with FIC indices 1.25 and 1.0 respectively. We investigated these AEPF-tetracycline combinations using time-kill kinetic to evaluate their rate of action and their interactions as bacteriostatic or bactericidal. We observed a bactericidal activity similar to standard ciprofloxacin for tetracycline in combination with AEPF in 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The presence of the alkaloidal extract in the ratio 1/4MIC:1MIC tetracycline:AEPF, enhanced the antimicrobial activity of tetracycline and modified its action from bacteriostatic to bactericidal against \u003cem\u003eShigella\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The observed OD\u003csub\u003eave\u003c/sub\u003e of 0.2259 and 0.3470 for the lag and exponential phases respectively for tetracycline improved significantly to 0.0226 and 0.0304 when combined with the AEPF in a 1/4MIC:MIC ratio, representing 10-fold increase in activity. We observed a similar trend when tetracycline was combined with the AEPF in 1/2MIC:1/2MIC ratio. Again, the time-kill kinetics profile showed 10-fold improvement in activity compared to tetracycline alone. It was gratifying to observe that the AEPF modulated the activity of tetracycline to a level comparable to ciprofloxacin, demonstrating the potential antibacterial combination therapy using alkaloids from \u003cem\u003eP fraternus.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of Alkaloids Based on LC-ESI-MS Fragmentation\u003c/h2\u003e \u003cp\u003eFollowing chromatographic purification, the fractions were subjected to LC-MS analysis. Flash chromatography afforded 7 fractions, 5 of which contained a single spot by TLC. The molecular weights determined from the mass spectra were compared with known alkaloids from literature. LC-ESI-MS analysis of the crude alkaloid of \u003cem\u003eP. fraternus\u003c/em\u003e revealed two known alkaloids from literature: epibubbialine (\u003cb\u003ea)\u003c/b\u003e and ent-norsecurinine (\u003cb\u003eb\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. For epibubbialine, the spectrum showed ion peaks at \u003cem\u003em/z\u003c/em\u003e 240.2 (weak), 170.2 (weak), 204.3, 124.1, 222.3 and 111.7 (strong). The molecular ion peak [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e was attributed to the peak at m/z 222.3, in agreement with molecular weight 221.3 amu [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The doubly charged pseudomolecular ion [M\u0026thinsp;+\u0026thinsp;2H]\u003csup\u003e2+\u003c/sup\u003ecorresponded to the peak at m/z 111.7. The weak peak at \u003cem\u003em/z\u003c/em\u003e 240.2 was attributed to the adduct [M\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e (\u003cem\u003em/z\u003c/em\u003e 221.3 \u0026rarr; \u003cem\u003em/z\u003c/em\u003e 240.2). No adduct was assigned for the peaks at m/z 170.2 and 124.1. The spectrum for ent-norsecurinine showed ion peaks at \u003cem\u003em/z\u003c/em\u003e 244.3 (weak), 154.1 (weak), 204.3 and 102.6 (strong). The molecular ion peak [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e was attributed to the peak at \u003cem\u003em/z\u003c/em\u003e 204.3 consistent with molecular weight 203.3 amu [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The peak \u003cem\u003em/z\u003c/em\u003e 203.3 \u0026rarr; \u003cem\u003em/z\u003c/em\u003e 102.6 corresponded to the doubly charged adduct [M\u0026thinsp;+\u0026thinsp;2H]\u003csup\u003e2+\u003c/sup\u003e. The weak peak at \u003cem\u003em/z\u003c/em\u003e 244.3 corresponded to an [M\u0026thinsp;+\u0026thinsp;Na\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e+\u003c/sup\u003e adduct.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe low content of alkaloids observed for\u0026nbsp;\u003cem\u003ePhyllanthus fraternus\u0026nbsp;\u003c/em\u003e(0.64%)agrees with the generally lowlevels of alkaloids reported in most plants. The crude alkaloids demonstrated superior antibacterial activity in comparison to the total crude extract. The crude alkaloids of \u003cem\u003eP. fraternus\u003c/em\u003e effectively suppress majority of clinically significant pathogenic strains \u003cem\u003ein vitro\u003c/em\u003e. Significantly, the crude alkaloids successfully potentiated the biological activity of tetracycline against drug resistant \u003cem\u003eShigella\u003c/em\u003e resulting in ten-fold increase in antimicrobial activity. Time-kill kinetic assay revealed bactericidal effect of the crude alkaloids against \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888), and \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003e(ATCC 10455). Interestingly, the antibacterial activity of tetracycline was modified by the crude alkaloids from bacteriostatic to bactericidal against \u003cem\u003eShigella\u0026nbsp;\u003c/em\u003eand the level of activity became comparable to ciprofloxacin in the presence of the crude alkaloids. LC-ESI-MS experiments revealed the presence of two known alkaloids, epibubbialine and ent-norsecurinine, as components of \u003cem\u003eP. fraternus.\u0026nbsp;\u003c/em\u003eTaken together, the antibiotic activity of \u003cem\u003eP. fraternus\u0026nbsp;\u003c/em\u003eis primarily due to its alkaloids. Efforts are underway to isolate the individual alkaloids which could potentially be developed as drug candidates for use in combination therapies against antimicrobial resistance. Further investigations into the synergistic nature of these alkaloids with other conventional antimicrobial agents along with\u003cem\u003e\u0026nbsp;in-vivo\u003c/em\u003e study are required. Additionally, anti-biofilm activity, efflux pump inhibition potential, among others, should be examined to shed more light on the mechanisms of the action of the crude alkaloids present in the plant.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe data which support the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors have no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study received no financial support. The authors, however, received our regular salaries from University of of Cape Coast or Kwame Nkrumah University of Science and Technology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSAO: Experimental methods design and implementation, methods validation, data curation, results analysis and interpretation and drafting original manuscript. DOK: Experimental methods design and implementation, result analysis and interpretation. YOB, FAA, PMF and LSB: Conceptualization, research design, manuscript review and editing. IA: Conceptualization, research design and supervision, project administration, data analysis, manuscript writing, review and editing. All the listed authors contributed to this study, read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to acknowledge the support of our colleagues in the medicinal chemistry and drug discovery research group at the department of chemistry, universities of cape coast (UCC), for their constructive feedback during group presentation. We are also thankful to the department of biomedical science, UCC, providing access to their microbiology laboratory.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eL. S. Redgrave, S. B. Sutton, M. A. Webber, and L. J. V. Piddock, \u0026ldquo;Fluoroquinolone resistance: mechanisms, impact on bacteria, and role in evolutionary success,\u0026rdquo; \u003cem\u003eTrends Microbiol.\u003c/em\u003e, vol. 22, no. 8, pp. 438\u0026ndash;445, 2014, doi: 10.1016/J.TIM.2014.04.007.\u003c/li\u003e\n \u003cli\u003eM. Kvist, V. 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Methods\u003c/em\u003e, vol. 66, no. 3, pp. 381\u0026ndash;389, Sep. 2006, doi: 10.1016/J.MIMET.2006.01.002.\u003c/li\u003e\n \u003cli\u003eA. L. Koch, \u0026ldquo;Turbidity measurements of bacterial cultures in some available commercial instruments,\u0026rdquo; \u003cem\u003eAnal. Biochem.\u003c/em\u003e, vol. 38, no. 1, pp. 252\u0026ndash;259, 1970, doi: 10.1016/0003-2697(70)90174-0.\u003c/li\u003e\n \u003cli\u003eG. Komlaga \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Antiplasmodial Securinega alkaloids from Phyllanthus fraternus: Discovery of natural (+)-allonorsecurinine,\u0026rdquo; \u003cem\u003eTetrahedron Lett.\u003c/em\u003e, vol. 58, no. 38, pp. 3754\u0026ndash;3756, Sep. 2017, doi: 10.1016/J.TETLET.2017.08.045.\u003c/li\u003e\n \u003cli\u003eP. J. Houghton, T. Z. Woldemariam, S. O\u0026rsquo;Shea, and S. P. Thyagarajan, \u0026ldquo;Two securinega-type alkaloids from Phyllanthus amarus,\u0026rdquo; \u003cem\u003ePhytochemistry\u003c/em\u003e, vol. 43, no. 3, pp. 715\u0026ndash;717, 1996, doi: 10.1016/0031-9422(96)00345-7.\u003c/li\u003e\n \u003cli\u003eB. S. Joshi, D. H. Gawad, S. W. Pelletier, G. Kartha, and K. Bhandary, \u0026ldquo;Isolation and structure (X-ray analysis) of ent-norsecurinine, an alkaloid from Phyllanthus niruri,\u0026rdquo; \u003cem\u003eJ. Nat. Prod.\u003c/em\u003e, vol. 49, no. 4, pp. 614\u0026ndash;620, 1986, doi: 10.1021/NP50046A009.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"We received no funding for this study","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial Resistance, Time Kill-Kinetic, Modulation Studies, Phyllanthus fraternus, Alkaloidal Extract, Liquid Chromatography-Mass Spectrometry (LC-MS) ","lastPublishedDoi":"10.21203/rs.3.rs-5914968/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5914968/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial resistance (AMR) continues to rise, making a growing number of infectious diseases difficult to cure. Millions of people worldwide die from infections with medication resistance. According to the World Health Organization (WHO), a resistant variant has a 64% higher chance of killing an infected victim than a non-resistant variant. As a result, scientists continue to focus research attention on finding novel chemotypes that could have different modes of action. Combination therapy has the potential to overcome AMR since the therapeutic components work together to suppress the etiological microorganism. In the current study, we investigated the antibacterial properties of crude alkaloidal extract of \u003cem\u003ePhyllanthus\u003c/em\u003e \u003cem\u003efraternus \u003c/em\u003e(AEPF)\u003cem\u003e \u003c/em\u003eusing\u003cem\u003e \u003c/em\u003ehigh-throughput spot culture growth inhibition (HT-SPOTi) assay. We performed time-kill kinetic assays to assess the interactions between the crude alkaloids and test microbial strains. The ability of the crude alkaloids to alter the antimicrobial action of standard tetracycline was evaluated by modulation study. Our findings indicate that \u003cem\u003eP. fraternus\u003c/em\u003e alkaloids effectively suppress majority of clinically significant pathogenic strains \u003cem\u003ein vitro.\u003c/em\u003e Bactericidal effect was shown by time-kill kinetics against \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e (ATCC 43888), and \u003cem\u003eE. coli \u003c/em\u003e(ATCC 10455). Tetracycline was successfully potentiated against \u003cem\u003eShigella sp.\u003c/em\u003e by the alkaloidal extract. The crude alkaloid extract of \u003cem\u003eP. fraternus\u003c/em\u003e included two known alkaloids, epibubbialine and ent-norsecurinine, according to LC-ESI-MS analysis. Taken together, the antibiotic activity of \u003cem\u003eP. fraternus \u003c/em\u003eis primarily due to its alkaloids and that the potential exists to develop isolated alkaloids as drug candidates for use in combination therapies against antimicrobial resistance.\u003c/p\u003e","manuscriptTitle":"Crude Alkaloids form Phyllanthus fraternus, Webster: Antibacterial, Time-Kill Kinetics and Resistance Modulation Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-29 09:37:34","doi":"10.21203/rs.3.rs-5914968/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"35b7d983-21c8-4711-b1a0-9087f98dd233","owner":[],"postedDate":"January 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43561982,"name":"Natural Product Chemistry"}],"tags":[],"updatedAt":"2025-01-29T09:37:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-29 09:37:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5914968","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5914968","identity":"rs-5914968","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}