{"id":49610,"date":"2023-06-30T10:34:28","date_gmt":"2023-06-30T10:34:28","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=49610"},"modified":"2023-07-18T11:44:09","modified_gmt":"2023-07-18T11:44:09","slug":"gas-chromatography-mass-spectrometry-analysis-phytochemical-screening-antioxidant-and-antibacterial-activity-of-methanol-root-extract-of-rhaphiostylis-beninensis-hook-f-planch-family-icacinacea","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no2\/gas-chromatography-mass-spectrometry-analysis-phytochemical-screening-antioxidant-and-antibacterial-activity-of-methanol-root-extract-of-rhaphiostylis-beninensis-hook-f-planch-family-icacinacea\/","title":{"rendered":"Gas Chromatography-Mass Spectrometry Analysis, Phytochemical Screening, Antioxidant and Antibacterial Activity of Methanol Root Extract of Rhaphiostylis beninensis (Hook.f.) Planch. [family ICACINACEAE] against Uropathogens"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

One of the most ubiquitous bacterial illnesses worldwide is urinary\ntract infections (UTIs). About 50\u201360% of women may experience UTIs at some\npoint in their lives, making it a predominant bacterial infection responsible for\nclose to 25% of all infections in women. About 150 million cases per year, as\nan estimate, was indicated in 2016. Although both men and women can become\nseriously ill from these diseases, women are more likely to contract them1<\/sup>.\nWomen who are sexually active, especially those who use spermicidal products\nand diaphragms for contraception, are more likely to develop UTIs due to immune\nsuppression in such females, even though the spread of these infections to the\nkidney is more proliferating in pregnant women owing to their enfeebled immune\nsystems in gestation. While UTIs in pregnant women may result in early labor\nand high blood pressure2<\/sup>, post-menopausal women experience higher\nrates of UTIs because of the pelvic prolapse, deficiency of estrogen, amplified\nperi-urethral colonization by Escherichia coli<\/em>, diabetes mellitus, and reduction\nof Lactobacilli<\/em> spp. amongst the vaginal flora3<\/sup>. <\/p>\n\n\n\n

Although Staphylococcus<\/em> spp., Proteus vulgaris<\/em> and Pseudomonas<\/em> spp. are infrequent bacteria responsible for UTIs4<\/sup>,\nProteus mirabilis, Enterobacter cloacae, Enterobacter\naerogenes, Pseudomonas aeruginosa, Proteus vulgaris, Staphylococcus aureus, Escherichia\ncoli <\/em>and Klebsiella pneumoniae<\/em> have been\nidentified as the causative agents of UTIs5,6<\/sup>. While E. coli<\/em> and K. pneumoniae<\/em> are the most common uropathogens and UTI indicator\norganisms, respectively, according to the World Health Organization’s Global\nAntimicrobial Surveillance System (GLASS)7,8<\/sup>, over and above 80% of\nall unsophisticated community acquired urinary tract infections (CA-UTIs) are triggered\nby E. coli<\/em> alone9<\/sup>. In\ngeneral, E. coli<\/em> in the intestine are\nusually normal flora and not detrimental, but if it gets to the vaginal hole\nduring washing or after sexual activity, it could cause infections on entering\nand colonizing the bladder. For instance, , some E. coli<\/em> pathotypes such as uropathogenic E. coli <\/em>(UPEC) in immunocompromised individuals have developed enabling\ncapabilities to breach the characteristically sterile urinary system and cause\nboth symptomatic and asymptomatic infections9<\/sup>. Numerous virulence\nfactors, including polysaccharide capsule, lipopolysaccharide (LPS), flagella, pili,\nnon-pilus adhesins, outer-membrane proteins, outer-membrane vesicles, secreted\ntoxins and TonB-dependent iron-uptake receptors such as siderophore receptors\nare used to colonize the bladder by this organism. All of these elements offer\ngreat potential for the establishment of treatments and vaccines10,11<\/sup>.\nAmphipathic molecules called LPS are composed of lipid A or endotoxin, core\nphosphorylated oligosaccharide and a lengthy side chain known as O antigen12<\/sup>.\nLPS structural components, rapid bladder colonization, reservoir formation, and\ninduction of innate and adaptive immune responses have roles to play in facilitating\nvarious phases of the UPEC life cycle13<\/sup>. When there is a decrease in\nthe quantity of LPS at the surface of the cell, LPS confers resistance to\nhydrophobic antibiotics and hypersensitivsity to hydrophobic harmful molecules such\nas bile salts and certain antibiotics14<\/sup>.<\/p>\n\n\n\n

Since many of the agents causing UTIs have developed resistance to majority\nof the antibiotics used to treat them15,16<\/sup>, diverse guidelines for\ntreating UTIs have been adopted in innumerable regions of the world10<\/sup>\nbased on noteworthy scientific evidence. According to Magyar et al.<\/em>15<\/sup>, resistance\nof E. coli<\/em> to ciprofloxacin increased\nremarkably from 19% to 25%, resistance of K.\npneumoniae<\/em> to cephalosporins was 60%, and the percentage of\ncarbapenem-resistant P. aeruginosa<\/em>\nalso increased noticeably.  While\nmultidrug-resistant pathogens are increasingly responsible for UTIsworldwide17<\/sup>,\nantibacterial resistance has led to higher rates of morbidity and mortality, higher\nresource use, increased costs, decreased hospital activity, and antibiotic\ntreatment guidelines that favor broad-spectrum empiric therapy18,19<\/sup>.\nA great impediment to using these empirical therapy options is provided by the\nrise in antibiotic resistance20<\/sup>. Due to the fact that herbal\nmedications are secure, affordable, and simple to use, concentrated attention\nhas been directed to the therapeutic potential of medicinal plants2<\/sup>.\nConsequently, medicinal plants have been widely investigated21<\/sup>, and\nvarious essential chemical constituents with tremendous therapeutic potential\nhave been identified to indicate their pharmacological properties22-24<\/sup>.\nWhile antimicrobial, anti-inflammatory, and antioxidant activities of plant extracts\nhave been documented25,26<\/sup>, a wide variety of their chemical\ncomponents have therapeutic potentials in\nvitro<\/em>27,28<\/sup>. Since there are no credible scientific data\nindicating that bacteria have evolved a resistance to therapeutic plants, these\nphytochemicals could serve as the basis for the manufacture of secure ground-breaking\nmedications for treating infections and diseases. Thus, by using these natural\nmedications, the issue of resistance resulting from the usage of conventional\ntreatment might be resolved.29<\/sup>. <\/p>\n\n\n\n

Rhaphiostylis beninensis <\/em>Planch ex Benth (Icacinaceae) is a woody climber with a\nwide variety of curative and culinary uses. It grows in the\nWest African subcontinent and South-Western Nigeria. It goes by many different\nnames in Nigeria depending on one’s location and its usage. Atapata\n(Yoruba), osumadin (Benin), kpolokoto (Ibos), umeni (Urhobos), and kumeni\n(Itsekiris) are all names for the same plant. R. beninensis<\/em> belongs to the Icacinaceae family, which contains a\nvariety of bioactive phytochemicals30<\/sup>. Innumerable pharmacological\nand biological properties of the plant’s root extract have also been\nreported. Antibacterial, analgesic, and anti-inflammatory properties have been\nreported for the plant31<\/sup>. The leaf and root are used in folkloric treatment\nof arthritis, rheumatism, skin illnesses, mental disorders, convulsions, and\neye problems, while the leaf decoction is used as a mouthwash and a sore wash32,33<\/sup>.\nAdjanohoun et al.34<\/sup> reported mosquito repellent activities of its\naqueous leaf extract. Gram-positive and Gram-negative bacteria as well as\nfungus were found to be resistant to the antibacterial activities of the oil\nextracted from the root, stem bark extracts, and fruit35<\/sup>. While\nanthraquinones, flavonoids, and triterpenes were discovered in the plant36<\/sup>\nand a thiourea derivative, N, N-di (4-methyoxybenzyl) thiourea, with\nanti-inflammatory activity has been isolated from the root of the plant37<\/sup>,\ninformation on the\nchemis try, pharmacology, and antibacterial potential of oils produced from the\nmethanol extract of R. beninensis<\/em>\nroots is minimal. So, in response to its folkloric\nuse, this study investigated the antioxidant, and antibacterial activities of\nthe methanol extract of R. beninensis<\/em>\nagainst uropathogenic bacteria as well as identify bioactive phytochemicals\ncrucial in developing novel drugs from natural products. <\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Collection of Plant materials<\/strong><\/p>\n\n\n\n

The roots of Rhaphiostylis\nbeninensis<\/em> were harvested from its natural habitat in Ijebu-Ode, Ogun State,\nNigeria, rinsed with sterile distilled water and allowed to air-dry. The roots\nwere identified and deposited at the Nigerian Forestry Herbarium in Ibadan\n(FHI) under voucher number FHI 113354.<\/p>\n\n\n\n

Plant Extraction<\/strong><\/p>\n\n\n\n

The extraction was done using the method of Adeshina et al.<\/em>38<\/sup>. The roots were pulverized before\nbeing sealed up in an airtight container free of grease. Four hundred grams of\nthe sample was soaked in 1000 mL of methanol, left to stand for 72 h while\nbeing stirred frequently, sieved with three folds sterile muslin cloth and then\nfiltered with Whatman No. 1 filter paper29<\/sup>. The extracting solvent was effectively evaporated\nfrom samples with a rotary evaporator at 68\u00b0C. The extract was properly stored\npending additional analyses39<\/sup>.<\/p>\n\n\n\n

Qualitative Phytochemical analysis<\/strong><\/p>\n\n\n\n

Using standard methods outlined by Sofowora40<\/sup> and Trease & Evans41<\/sup>,\nthe methanol extract was examined for the occurrence of several phytochemicals.<\/p>\n\n\n\n

Test for Saponins<\/strong><\/p>\n\n\n\n

The extract was mixed briskly with 20 mL of water in a graduated\nmeasuring cylinder to dilute it, and the concoction was allowed to stand for 15\nmin to test for the presence of saponin. It was clear that saponin was present\nbecause foam was formed. The foam height (H2<\/sub>-H1<\/sub>) was then\ndetermined.<\/p>\n\n\n\n

Test for Alkaloids<\/strong><\/p>\n\n\n\n

A percentage of the extract was dispensed in a test tube, along with\nethanol and diluted hydrochloric (HCl) acid (50:50). The concoction was heated\nin boiling water for 10 min before filtering to check for alkaloids. Mayer\u2019s\nTest: When Mayer’s reagent (1.36 g of mercuric chloride and 5.00 g of potassium\niodide were mixed in 100 mL of water) was applied to the filtrate, a buff-white\nyellow precipitate formed, indicating the occurrence of alkaloids. <\/p>\n\n\n\n

Test for Tannins<\/strong><\/p>\n\n\n\n

A beaker containing 0.30 g of the methanol extract was boiled in a water\nbath for 10 min to test for tannins. Boiling was followed by filtering with Whatman\nnumber 42 (125 mm) filter paper. Then, 3 drops of 1% ferric chloride were added\nto 5 mL of the filtrate. The presence of tannins was suggested by the development\nof brownish-green or blue-black coloring.<\/p>\n\n\n\n

Test for Steroids<\/strong><\/p>\n\n\n\n

To test for steroids, chloroform (3 mL) was added to methanol extract (5\nmg) before filtering into test-tube. The test-tube was tilted and  concentrated Sulphuric acid (H2<\/sub>SO4<\/sub>)(2 mL) was carefully added through the side of the test-tube. The\npresence of brown and reddish color at chloroform phase showed existence of\nsteroid.<\/p>\n\n\n\n

Test for Terpenoids<\/strong><\/p>\n\n\n\n

For the terpenoids test, 0.30 g of the methanol extract was extracted\nfor 2 h in a beaker with sterile distilled water (30 mL). To partition the\naliquot, the extract (5 mL) was added to chloroform (2 mL) combined with concentrated\nH2<\/sub>SO4 <\/sub>(3 mL). Reddish brown coloring at the border line confirmed\nthe existence of terpenoids.<\/p>\n\n\n\n

Test for Phenols<\/strong><\/p>\n\n\n\n

For phenols test,<\/strong> 2 mL of1% FeCl3<\/sub> was mixed with the\nextract.The presence of blue-black (violet) or blue green\ncoloration confirmed the availability of phenol.<\/p>\n\n\n\n

Test for Phlobatannins<\/strong><\/p>\n\n\n\n

To test for phlobatannins, sterile distilled water (30 mL) was added to 0.30\ng of powdered sample in a beaker. After extracting for 24 h, the aqueous\nextract (10 mL) was boiled with 1% aqueous hydrochloric acid (5 mL). Red\nprecipitate indicated the presence of phlobatamins.<\/p>\n\n\n\n

Test for Flavonoids<\/strong><\/p>\n\n\n\n

To test for flavonoids, 0.30 g of powdered plant material was extracted for\n2 h with 30 mL of distilled water before filtering with Whatman filter paper\nnumber 42 (125 mm). Five milliliters of 1.0 M diluted ammonia solution were added\nto 10 mL of the aqueous filtrate of extract. This was followed by adding 5 mL\nof H2<\/sub>SO4<\/sub>. The presence of yellow colour that vanished\nupon standing indicated the occurrence of flavonoids.<\/p>\n\n\n\n

Test for Reducing Sugars<\/strong><\/p>\n\n\n\n

A test tube containing 2 mL of aqueous extract and 1 mL of each of\nFehling solutions A and Solution B was boiled for 10 min to check for reducing\nsugars. The yellow or brick-red precipitate produced indicated occurrence of\nreducing sugar.<\/p>\n\n\n\n

Test for Glycosides<\/strong><\/p>\n\n\n\n

In this test, 20 mL of water and 2 g of the extract were combined,\nheated for 5 min in a water bath and then filtered through Whatman number 42\nfilter paper (125 mm). To detect glycosides, two tests were run with the\nfiltrate. First, each of Fehling’s solution A and solution B (0.2 mL) were\nmixed with 5 mL of the filtrate until an alkaline mixture was obtained.\nPositive results were seen when brick-red coloring was present on heating. Second,\nthe aforesaid test was repeated using 15 mL of 1.0 M H2<\/sub>SO4<\/sub>\ninstead of water, and the quantity of precipitate obtained was compared to those\nobtained from the initial test. The presence of glycosides was confirmed by\nhigh precipitate content and low precipitate content confirmed the nonexistence\nof glycosides.<\/p>\n\n\n\n

Phytochemical analysis<\/strong><\/p>\n\n\n\n

Quantitative determination of flavonoid concentration<\/strong><\/p>\n\n\n\n

The total flavonoid content of the methanol extract of R. beninensis <\/em>was determines as described by Ejikeme et al.42<\/sup>. Fifty milliliters of 80% aliquot were added to 2.50 g of the plant material in a 250 mL beaker. The mixture was covered and allowed to stand for 24 h at room temperature. The extraction was repeated three consecutive times by adding the same volume of methanol after removing the supernatant. The mixture was filtered before transferring to a crucible and dried in a water bath. The crucible and its content were weighed after being cooled down in a desiccator.  <\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Quantitative determination of alkaloid concentrations<\/strong><\/p>\n\n\n\n

The alkaloid was determined quantitatively as described by Ezeonu & Ejikeme43<\/sup>. The extract (2.50 g) was liquefied in 200 mL of 10% acetic acid in methanol, and permitted to stand for 4 h. The filtrate was concentrated in a water bath to a quarter of its initial volume before 15 drops of concentrated ammonium hydroxide (NH4<\/sub>OH) was added until a precipitate was formed. After 3 h of sedimentation, the supernatant was removed by filtration, and the precipitate was washed with 20 mL of 0.1 M ammonium hydroxide before filtering.  After drying the residue in the oven, the percentage of alkaloid was estimated.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Quantitative determination of total saponin<\/strong><\/p>\n\n\n\n

The saponin was determined quantitatively as described by Ejikeme et al.42 <\/sup>and Obadoni & Ochuko44<\/sup>. Here, 5 g of powdered sample  was added to 100 mL of aqueous ethanol (20%) in a conical flask (250 mL)before being heated with constant stirring at a temperature of 55o<\/sup>C in a water bath for 4 h. After filtration, the procedure was repeated two more times. The extract was vaporized to 40 mL at 90o<\/sup>C in a water bath. In a separating funnel, diethyl ether (20 mL) was added to the concentrate before shaken strongly. The aqueous layer was recovered and the ether layer was discarded. This purification process was repeated twice. n-butanol (60 mL) was added to the aqueous layer recovered before extracting twice with 10 mL of 5% sodium chloride. The residual solution was heated in a water bath for 30 min after discarding the sodium chloride layer, transferred to a crucible and dried in an oven to a constant weight.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Quantitative determination of total tannins<\/strong><\/p>\n\n\n\n

The tannin\ncontent of the methanol extract of R.\nbeninensis<\/em> was determined as described\nby Ejikeme et al.42<\/sup> and Amadi et al.45<\/sup>. Folin-Denis reagent was prepared by liquefying\nsodium tungstate (Na2<\/sub>WO4<\/sub>) (50 g) in distilled water (37 mL)\nwhile phosphomolybdic acid (H3<\/sub>PMo12<\/sub>O40<\/sub>) (10 g)\nand orthophosphoric acid (H3<\/sub>PO4<\/sub>) (25 mL) were added to\nthis reagent. This mixture was refluxed for 2 h, cooled, and made up to 500 mL with\ndistilled water. The extract (1 g) was added to distilled water (100 mL) in a\nconical flask, boiled for 1 h on an electric hot plate and filtered using Whatman\nnumber 42 (125 mm) filter paper in a volumetric flask (100 mL). Folin-Denis\nreagent (5 mL), saturated Na2<\/sub>CO3<\/sub> (10 mL) solution,\ndiluted extract (10 mL) and distilled water (50 mL) were added together in a conical\nflask (100 mL) for a change in colour. After constant stirring, the aliquot stood\nfor 30 min in a water bath at 25\u00b0C before determining the optical density at\n700 nm using a UV\/VIS spectrophotometer, and its results were compared to a\ntypical tannic standard curve obtained by dissolving 0.20 g of tannic acid in\ndistilled water before being diluted up to 200 mL mark (1 mg\/mL). Five\ndifferent test tubes were filled with varied amounts of the standard tannic\nacid solution (0.2-1.0 mg\/mL), followed by adding Folin-Denis reagent (5 mL),\nsaturated Na2<\/sub>CO3<\/sub> (10 mL), and distilled water to bring\nthe total volume to 100 mL. The solution was allowed to stand for 30 min in a\nwater bath at 25o<\/sup>C. Optical density (absorbance) versus tannic acid\nconcentration was plotted on a graph. <\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

where \ud835\udc36 is concentration of tannic acid read off the graph.<\/p>\n\n\n\n

Quantitative determination of total phenolic\nconcentration<\/strong><\/p>\n\n\n\n

Quantitative determination of phenol was carried out as described by Ezeonu & Ejikeme43<\/sup>. Two grams of powdered sample were defatted\nfor 2 h in ether (100 mL) using a soxhlet apparatus. The defatted sample (0.50\ng) was boiled in ether (50 mL) for 15 min to extract the phenolic compounds.\nFor colour development, distilled water (10 mL), 0.1 N ammonium hydroxide (2 mL)\nsolution and concentrated amyl alcohol (5 ml) were added to  the extract (5 mL) and left to react for 30 min.\nThe optical density was measured at 505 nm while tannic acid (0.20 g) was\ndissolved in distilled water (200 mL) mark (1 mg\/mL) to prepare the phenol\nstandard curve. Varied concentrations (0.2\u20131.0 mg\/mL) of the standard tannic\nacid solution were pipetted into five different test tubes to which NH4<\/sub>OH\n(2 mL), amyl alcohol (5 mL), and water (10 mL) were added for colour\ndevelopment after reacting for 30 min. The optical density was measured with a\nUV\/VIS TG 50 spectrophotometer at 505 nm.<\/p>\n\n\n\n

GC-MS Quantification<\/strong><\/p>\n\n\n\n

GC\u2013MS is one of the best, fast and most accurate approach to detect\nvarious compounds including alcohols, nitro compounds, alkaloids, organic\nacids, steroids, long chain hydrocarbons, esters and amino acids, and requires little\namount of plant extracts. In this study, the GC\u2013MS analysis detected and\nidentified the chemical compounds present in R. beninensis<\/em> as described by Fagbemi et al.24<\/sup>.<\/em> <\/p>\n\n\n\n

Determination of antioxidant Activity by DPPH Radical Scavenging Activity Assay<\/strong><\/p>\n\n\n\n

The DPPH radical scavenging assay, as described by Chen et al.46<\/sup>, was used to examine the extract’s capacity to scavenge\nfree radicals. Briefly, 0.1 mM DPPH (1 mL) in methanol was thoroughly mixed with\nextract (1 mL) in methanol at different concentrations (25\u2013100 \u03bcg\/mL),\nallowed to stand for 30 min at room temperature before measuring the absorbance\nwith a UV-Vis Spectrophotometer at 517 nm. The decreased absorbance in the\nreaction mixture indicated a higher free radical scavenging capacity. DPPH\nscavenging effect\u2019s percentage inhibition was calculated using the equation: <\/p>\n\n\n\n

DPPH Scavenging effect (%) = [(A0<\/sub>-A1<\/sub>)\/A0<\/sub>]\nx 100<\/p>\n\n\n\n

Where A0 <\/sub>was\nthe absorbance of the control and A1<\/sub> was the absorbance in the\npresence of the standard sample or extract. The IC50<\/sub> value\nrepresented the concentration of the extract that inhibited production of DPPH activities\nby 50%. The experiment was repeated three times at different concentrations.<\/p>\n\n\n\n

Determination of Antioxidant Activity by Ferric Reducing Antioxidant Power Assay (FRAP)<\/strong><\/p>\n\n\n\n

Using Otang et al.47<\/sup> approach, the ferrous reducing\nantioxidant capacity of the extract was assessed. One milliliter of the extract (25-100 \u00b5g\/mL) was mixed\nwith 0.1 M sodium phosphate buffer (2.5 mL) (pH 6.6) and 1% w\/v potassium\nferrocyanate (2.5 mL) [K3<\/sub>Fe(CN)6<\/sub>] in a 250 ml conical\nflask before incubating at 50\u00b0C for 20 min. Following this, trichloroacetic\nacid (2.5 mL) (10% w\/v) was added to the mixture before centrifuging at 5000 rpm\nfor 10 min. The upper layer (5 ml) was mixed with of fresh FeCl3<\/sub>\n(0.1%, w\/; 0.5 mL), and the absorbance was measured at 700 nm in comparison to\na blank. Gallic acid was used as the control. The percentage inhibition in FRAP\nwas calculated using the following equation: <\/p>\n\n\n\n

FRAP percentage inhibition (%) = [(A0<\/sub>-A1<\/sub>)\/A0<\/sub>]\nx 100<\/p>\n\n\n\n

where A0 <\/sub>was\nthe absorbance of the control and A1<\/sub> was the absorbance in the\npresence of the standard sample or extract. All tests were performed in\ntriplicate. Higher absorbance of the reaction mixture indicated great reducing\npower.<\/p>\n\n\n\n

Determination of Antioxidant Activity by Nitric Oxide Radical Scavenging Assay<\/strong><\/p>\n\n\n\n

The nitric oxide radical scavenging activity was evaluated as previously described by Silva and Soysa48<\/sup>. Different concentrations (25\u2013100 \u00b5g\/mL) of the extract and the standard ascorbic acid were prepared by serially diluting them with distilled water. These were kept for subsequent use and stored at 4o<\/sup>C. Griess reagent was prepared by mixing equal volumes of sulphanilamide (1%) in phosphoric acid (2.5%) and naphthylethylene diamine dihydrochloride (0.1%) in phosphoric acid (2.5%) proximately before use. Following this, sodium nitroprusside (10 mM) in phosphate buffered saline (0.5 mL) was mixed with 1 mL of the different concentrations of the methanol extract (25\u2212100 \u00b5g\/mL) and incubated at 25o<\/sup>C for 180 min. The extract and the ascorbic acid used as control were mixed with an equivalent volume of recently prepared Griess reagent. The tubes used as control contained the same concentrations of methanol extract without sodium nitroprusside while 150 \u00b5L of the reaction mixture was transferred to a 96-well plate. Using a UV\/VIS TG 50 Plus UV-VIS microplate reader, the absorbance at 546 nm was determined (Molecular Devices, GA, USA). The percentage inhibition of the extract and the standard was calculated and recorded. The inhibition percentage of the nitrite radical scavenging activity of both methanol extract and ascorbic acid were calculated using the following formula: <\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Where \ud835\udc34control<\/sub> = absorbance of the control sample and \ud835\udc34test<\/sub> = absorbance in the presence of the\nextract or standard. The experiment was conducted three times at different\nconcentrations.<\/p>\n\n\n\n

Determination of Antioxidant Activity by Lipid Peroxidation Assay<\/strong><\/p>\n\n\n\n

The lipid peroxidation assay was valued as described by Murugan & Parimelazhagan49<\/sup>. Ten microliters of extract and a standard solution containing 1,1,3,3-tetramethoxypropane (TEP) at varying concentrations of 25, 50, 75 and 100 \u03bcg\/mL and phosphate buffer (20 mM; 40 \u03bcL) (pH 7.0) were added to test tubes in an ice bath. In a tightly sealed test tube, sodium dodecyl sulfate (SDS) (3%; 50 \u03bcL), HCl (0.1 N;  200 \u03bcL), phosphotungstic acid (10%; 30 \u03bcL) and 2-thiobarbituric acid (TBA) (0.7%;100 \u03bcL) were combined and boiled at 100\u00b0C for 30 min in a water bath. The reaction mixture was mixed with n-butanol (400 \u03bcL) and then centrifuged at 3000 rpm for 10 min. Supernatants were collected and pass through a UV\/VIS spectrophotometer at a wavelengths of 515\/555 nm.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Where \ud835\udc34control<\/sub> = absorbance of the control sample and \ud835\udc34test<\/sub> = absorbance in the presence of the\nextract or standard. The experiment was conducted three times at each of the\nconcentrations.<\/p>\n\n\n\n

Determination of Total Antioxidant Capacity<\/strong><\/p>\n\n\n\n

Total antioxidant capacity (TAC) of the sample was evaluated as indicated by Kattamis et al.50<\/sup>.  Aliquots (100 \u00b5L) of KMnO4<\/sub> solution (5 mmol\/L) made by dissolving 79 mg of KMnO4<\/sub> in distilled water (100 mL) were added to the extract and evenly mixed by shaking. After warming for 30 min in a water bath at 37\u00b0C, the mixture’s absorbance was measured using a UV\/VIS spectrophotometer (optical density, OD, of 570 nm).<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Where \ud835\udc34control<\/sub> = absorbance of the control sample and \ud835\udc34test<\/sub> = absorbance in the presence of the\nsamples of extracts or standards. The experiment was conducted three times at\neach of the concentrations.<\/p>\n\n\n\n

Antimicrobial activity<\/strong><\/p>\n\n\n\n

Bacterial isolates used in this study included\nPseudomonas putida<\/em> PA25T, Pseudomonas aeruginosa<\/em> PA129T, Pseudomonas aeruginosa<\/em> PA29T, Morganella morganii<\/em> PA17T, Serratia marcescens<\/em> PA18T, Klebsiella pneumoniae<\/em> KB24AT, Proteus mirabilis<\/em> EC28T, Escherichia coli<\/em> EC3AT, Acinetobacter indicus<\/em> KB45AT and Acinetobacter indicus<\/em> EC41TT isolated\nfrom the urinary tract infection of patients at Olabisi Onabanjo University Teaching\nHospital, Sagamu, Ogun State, Nigeria, were used for sample collections between\nJune and October, 2017. The ethical approval\nto carry out the study was granted with a certificate indicating National code\nfor Health Research Ethics NHREC\/24\/01\/2020 and Babcock University Health\nResearch Ethics Committee BUHREC 651\/21 number from Babcock University, Ilisan\nRemo, Ogun State, Nigeria. Susceptibility of the\nuropathogens to the methanol extract of R.\nbeninensis<\/em> was evaluated by determining the minimum inhibitory\nconcentrations (MICs) and minimum bactericidal concentrations (MBCs) of the extract\nwith macrobroth dilution methods26<\/sup>.<\/p>\n\n\n\n

Determination of minimum inhibitory concentration (MIC)\nof the extracts<\/strong><\/p>\n\n\n\n

Minimum inhibitory concentrations (MICs) of the extract, defined as the\nlowest concentrations maintaining or reducing of inoculums viability51<\/sup>,\nwas determined by serial tube dilution technique26<\/sup> against the\nbacterial isolates. For antibacterial assay, concentrations of extract that ranged\nbetween 20 \u00b5g\/mL and 10,000 \u00b5g\/mL and those of erythromycin used as positive\ncontrol ranged between 0.0122 \u00b5g\/mL and 50 \u00b5g\/mL were prepared by serial\ndilutions in tubes containing double strength Mueller Hinton broth and\ninoculated with 100 \u00b5L of each of the bacterial isolates. Blank broth of\nMueller Hinton was used as negative control. \n<\/p>\n\n\n\n

Determination of minimum bactericidal concentration (MBC)\nof the extracts<\/strong><\/p>\n\n\n\n

For the MBC assay, one loopful of culture from each of the first five\nbroth cultures without bacterial growth in the MIC tubes was inoculated into\nfresh nutrient agar plates26<\/sup> and incubated at 37\u00b0C for 24 h. After\nthe incubation periods, the lowest concentration of the extract that showed no sign\nof bacterial growth on the solid medium was regarded as the MBC value for this extract.\nThis finding was supported by the MIC test tube, which showed no growth after\n48 h of the incubation period.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

The qualitative phytochemical analysis of the methanol extract of R. beninensis<\/em> indicated alkaloids,\nsaponin, tannins, cardiac glycosides, flavonoids and reducing sugar which are\nof pharmacological importance (Table 1). <\/p>\n\n\n\n

Table 1: Qualitatively determined phytochemicals in the methanol extract of R. beninensis<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Phytochemical compounds<\/strong><\/p>\n<\/td>\n

\n

Methanolic extract<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Saponins<\/p>\n<\/td>\n

\n

+<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Tannins<\/p>\n<\/td>\n

\n

+<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Phlobatannins<\/p>\n<\/td>\n

\n

–<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Flavonoids<\/p>\n<\/td>\n

\n

+<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Cardiac Glycosides<\/p>\n<\/td>\n

\n

+<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Alkaloid<\/p>\n<\/td>\n

\n

+<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Reducing Sugar<\/p>\n<\/td>\n

\n

+<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Terpenoid<\/p>\n<\/td>\n

\n

+<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Phenol<\/p>\n<\/td>\n

\n

+<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Steroids<\/p>\n<\/td>\n

\n

–<\/strong><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Quantitative phytochemical analysis<\/strong><\/p>\n\n\n\n

The quantitative phytochemical analysis of the methanol extract of R. beninensis<\/em> in Table 2 showed that\nthe total reducing sugar content was 31.27\u00b10.08 mg\/g, alkaloid content was\n28.99\u00b10.03 mg\/g, terpenoid content was 25.96\u00b10.06 mg\/g and saponin content\nwas 22.73\u00b10.19 mg\/g while those of tannins and phlobatannins in the\nextract were 12.39\u00b10.04 and 6.73\u00b10.07 mg\/g, respectively. However, the cardiac\nglycoside, and steroid content were absent. <\/p>\n\n\n\n

Table 2: Quantitatively determined phytochemicals in the methanol extract of R. beninensis<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Phytochemical compounds<\/strong><\/p>\n<\/td>\n

\n

MRB (mg\/100g)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Saponins<\/p>\n<\/td>\n

\n

22.73 \u00b1 0.19<\/p>\n<\/td>\n<\/tr>\n

\n

Tannins<\/p>\n<\/td>\n

\n

12.39 \u00b1 0.04<\/p>\n<\/td>\n<\/tr>\n

\n

Phlobatannin<\/p>\n<\/td>\n

\n

6.73 \u00b1 0.07<\/p>\n<\/td>\n<\/tr>\n

\n

Cardiac Glycosides<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Alkaloid<\/p>\n<\/td>\n

\n

28.99 \u00b1 0.03<\/p>\n<\/td>\n<\/tr>\n

\n

Reducing Sugar<\/p>\n<\/td>\n

\n

31.27 \u00b1 0.08<\/p>\n<\/td>\n<\/tr>\n

\n

Terpenoid<\/p>\n<\/td>\n

\n

25.96 \u00b1 0.06<\/p>\n<\/td>\n<\/tr>\n

\n

Steroids<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Gas Chromatography Mass Spectrometry (GCMS) Analysis <\/strong><\/p>\n\n\n\n

Figure 1 shows the GCMS chromatogram of the chemical compounds in the\nextract of R. beninensis<\/em>. The GCMS\nanalysis showed 42 chemical compounds in the methanol (MRB) extract of R. beninensis<\/em>. The name, chemical\nstructure and pharmacological activities of each of the chemical compounds in\nthe methanol extract are presented in Table 3. The most notable compounds\nwith chemical composition\u2019s percentages \u2265 1.0% are (Z,Z)-9,12-Octadecadienoic\nacid (19.96%), Lupeol (18.96%), cis-13-Octadecenoic\nacid (9.96%), n-Hexadecanoic\nacid (9.87%),\n3′,5′-Dimethoxyacetophenone\n(6.67%), 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol (3.21%), Octadecanoic acid (2.98%), Capsaicin (2.58%), \u03b1-Amyrin\n(2.53%), 9-Octadecenoic\nacid (Z)-, 2,3-dihydroxypropyl ester (2.49%), Vitamin\nE (2.45%),\nChondrillasterol (1.98%), Gingerol (1.70%), Dihydrocapsaicin\n(1.53%), (Z,Z)-9,12-Octadecadienoic\nacid, 2,3-dihydroxypropyl ester (1.45%),\n2,6-dimethoxy-4-(2-propenyl)-phenol\n(1.23%), 4,7-Dimethoxy-2-methylindan-1-one (1.38%), Hexadecanoic acid, and 2-hydroxy-1-(hydroxymethyl)ethyl\nester 1.15%. From the literature search, most\nof these bioactive compounds exhibited varied pharmacological activities and\nother therapeutic potentials except 3′,5′-Dimethoxyacetophenone, 4-amino-2-ethyl-6-methyl-3-Pyridinol, Benzene, ethenylpentaethyl-, 2-Allyl-1,4-dimethoxy-3-vinyloxymethylbenzene\nBenzene, hexaethyl- and Benzene, hexaethyl-, having no biological activities reported in\nliteratures.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Chromatogram of the GC-MS analysis of methanol extract of Rhaphiostylis beninensis<\/em><\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Table 3: GC-MS Analysis of the bioactive compounds of methanol extract of <\/strong>R. beninensis<\/strong> (MRB)<\/strong><\/p>\n

<\/p>\n

Click here to view Table<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

DPPH radical scavenging activity<\/strong><\/p>\n\n\n\n

In this study, the radical scavenging activity of the methanol extract\nof R. beninensis<\/em> was concentration-dependent.\nThe capacity of the extract to mop up DPPH radicals increases as its concentration\nincreases as shown in Figure 2. At all the studied concentrations, the extract\nproduced a noticeably high DPPH radical scavenging action. Ascorbic acid,\nhowever,, used as control showed higher radical scavenging action and an IC50<\/sub>\nvalue of 28.42 \u00b5g\/mL compared with that of the extract having a higher IC50<\/sub>\nvalue of 42.19 \u00b5g\/mL.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: DPPH radical scavenging activity (%) (Mean <\/strong>\u00b1<\/strong> Standard deviation) of methanol extract of R. beninensis<\/em>. Key: MRB = Extract with Methanol, AA= Ascorbic acid used as control.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Ferric reducing power antioxidant assay (FRAP)<\/strong><\/p>\n\n\n\n

Figure\n3 illustrates the dependence of the reducing power of the methanol extract of R. beninensis<\/em> (25\u2013100 \u00b5g\/mL) on concentration. As concentrations increased,\nso did the extract’s FRAP activity. At all the studied\nconcentrations, the methanol extract of R.\nbeninensis<\/em> produced a considerably high FRAP activity. The\nascorbic acid was used as control and it showed\nFRAP activity and an IC50<\/sub> value of 77.29 \u00b5g\/mL\nin comparison with the methanol extract of R. beninensis<\/em> having an IC50<\/sub>\nvalue of 332 \u00b5g\/mL.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Ferric reducing antioxidant power (FRAP) activity of methanol extract of R. beninensis<\/em>. Key: MRB = Extract with Methanol, AA= Ascorbic acid.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Nitric oxide radical scavenging assay<\/strong><\/p>\n\n\n\n

The nitric oxide scavenging effect of the methanol extract of R. beninensis<\/em> (25\u2013100 \u00b5g\/mL) was concentration-dependent. The effects of the methanol extract’s nitric oxide scavenging activity at various doses were depicted in Figure 4. Nitric oxide scavenging effets for R. beninensis<\/em> had an IC50<\/sub> value of 41.29 \u00b5g\/mL, whereas ascorbic acid demonstrated potent nitric oxide inhibition with an IC50<\/sub> value of 34.13 \u00b5g\/mL.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 4: Nitric oxide radical scavenging activity of methanol extract of R. beninensis<\/em>. Key: MRB = Extract with Methanol, AA= Ascorbic acid used as control.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Lipid peroxidation assay<\/strong><\/p>\n\n\n\n

As demonstrated in Figure 5, concentration has an effect on the lipid\nperoxidation assay of the methanol extract of R. beninensis<\/em> (25\u2013100 \u00b5g\/mL). In this investigation, the\nconcentration of the plant extract that can scavenge 50% of the lipid\nperoxidation radicals (IC50<\/sub>) was established. The results indicated that\nthe IC50<\/sub> value for the methanol extract of R. beninensis<\/em> was 59.18 \u00b5g\/mL as opposed to 33.44 \u00b5g\/mL for the ascorbic\nacid used as standard.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 5: Lipid peroxidation activity of methanol extract of R. beninensis<\/em>. Key: MRB = Extract with Methanol, AA= Ascorbic acid used as control.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Total antioxidant capacity<\/strong><\/p>\n\n\n\n

Table 4 showed methanol extract of R. beninensis <\/em>has a higher total\nantioxidant capacity (TAC) content (42.11 \u00b1 1.13 mg\/100 g) when compared with\nthe total flavonoid content of 28.42 \u00b1 0.44 mg\/100g and total phenolic content\nof 40.38 \u00b1 0.19 mg\/100 g.<\/p>\n\n\n\n

Table 4: Total antioxidant capacity of methanol extract of R. beninensis<\/strong><\/p>\n\n\n\n\n\n\n
\n

 <\/strong><\/p>\n<\/td>\n

\n

Total Antioxidant Capacity (mg\/100g)<\/strong><\/p>\n<\/td>\n

\n

Total Flavonoid (mg\/100g)<\/strong><\/p>\n<\/td>\n

\n

Total Phenol (mg\/100g)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

MRB<\/strong><\/p>\n<\/td>\n

\n

42.11 \u00b1 1.13<\/p>\n<\/td>\n

\n

28.42 \u00b1 0.44<\/p>\n<\/td>\n

\n

40.38 \u00b1 0.19<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Antimicrobial activity<\/strong><\/p>\n\n\n\n

The antibacterial activity of the extract was\npresented in Table 5. The result indicated that the extract inhibited the\nisolates at minimum inhibitory concentrations (MICs) and minimum bactericidal\nconcentrations (MBCs) ranging from 3.125 – 50 mg\/mL and 6.25 – 25 mg\/mL,\nrespectively.<\/p>\n\n\n\n

Table 5: Antimicrobial activity of methanol extract of R. beninensis<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Organism code<\/strong><\/p>\n<\/td>\n

\n

Antibacterial activity of R. beninensis<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

MIC (mg\/ml)<\/strong><\/p>\n<\/td>\n

\n

MBC (mg\/ml)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

E. coli<\/em> EC3AT<\/p>\n<\/td>\n

\n

12.5<\/p>\n<\/td>\n

\n

25<\/p>\n<\/td>\n<\/tr>\n

\n

P. mirabilis<\/em> EC28T<\/p>\n<\/td>\n

\n

6.25<\/p>\n<\/td>\n

\n

12.5<\/p>\n<\/td>\n<\/tr>\n

\n

A. indicus<\/em> KB45AT<\/p>\n<\/td>\n

\n

6.25<\/p>\n<\/td>\n

\n

12.5<\/p>\n<\/td>\n<\/tr>\n

\n

K. pneumoniae<\/em> KB24AT<\/p>\n<\/td>\n

\n

6.25<\/p>\n<\/td>\n

\n

12.5<\/p>\n<\/td>\n<\/tr>\n

\n

A. indicus<\/em> EC41TT<\/p>\n<\/td>\n

\n

6.25<\/p>\n<\/td>\n

\n

12.5<\/p>\n<\/td>\n<\/tr>\n

\n

P. aeruginosa<\/em> PA29T<\/p>\n<\/td>\n

\n

3.125<\/p>\n<\/td>\n

\n

6.25<\/p>\n<\/td>\n<\/tr>\n

\n

M. morganii<\/em> PA17T<\/p>\n<\/td>\n

\n

6.25<\/p>\n<\/td>\n

\n

12.5<\/p>\n<\/td>\n<\/tr>\n

\n

P. aeruginosa<\/em> PA129AT<\/p>\n<\/td>\n

\n

3.125<\/p>\n<\/td>\n

\n

6.25<\/p>\n<\/td>\n<\/tr>\n

\n

S. marcescens<\/em> PA18T<\/p>\n<\/td>\n

\n

12.5<\/p>\n<\/td>\n

\n

12.5<\/p>\n<\/td>\n<\/tr>\n

\n

P. putida<\/em> PA25T<\/p>\n<\/td>\n

\n

>50<\/p>\n<\/td>\n

\n

ND<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

KEYS: MICs = Minimum Inhibitory Concentrations, MBCs =\nMinimum Bactericidal Concentrations, ND = Not determined<\/p>\n\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

Using medicinal\nplants to treat a variety of infections, including UTIs, has received\nincreasing attention in recent years. Consequently, seventy percent of the population of the developing world use traditional systems of\nhealthcare39,90<\/sup>. Although various antibiotics have been administered\nto patients, it is necessary to seek for and use “non-antibiotic” approaches\nfor therapeutic purposes due to development of antibiotic resistance, which is\na somber concern in the worldwide healthcare arena91<\/sup>. In order to efficiently\ntreat bacterial infections, particularly urinary tract infections, it is essential\nto use bioactive compounds possessing therapeutic efficacy. Numerous studies\nhave demonstrated that plant bioactive compounds may meaningfully add to the\ndevelopment of new and potent medications that can modify bacterial resistance\nas an alternate and complementary method of addressing microbial resistance23,24,92<\/sup>.<\/p>\n\n\n\n

In this study, the phytochemical analysis of\nthe root extract of R. beninensis<\/em>\nrevealed that reducing sugar, alkaloids, and terpenoids were found in higher\nconcentrations than phlobatannins, tannins, and saponins. These phytochemicals ensure diverse biological effects including\nantioxidant activity93<\/sup>. The relative conformation of phytochemicals obtained\ndiverges from phytochemicals in R.\nbeninensis<\/em> stem extract as reported by Lasisi et al.<\/em>31<\/sup>.\nThe differences could result from dissimilarities in the phytochemical\nconcentrations in different parts of the plant94,95<\/sup>. However, the\nexistence of these chemical constituents in methanol root extract of R. beninensis<\/em> suggests that, if judiciously\nscreened, the studied plant could provide pharmacologically significant\nplant-derived pharmaceuticals. This could further bolster the fact that\ndifferent parts of this plant have been employed in ethnomedicine in the\ntreatment of a number of diseases. Alkaloids are nitrogen-based compounds and\nare known to have antimalarial, antihypertensive, antiarrhythmic, and\nanticancer effects96<\/sup> and work as potent analgesics and stimulants\nfor the central nervous system97<\/sup>. While saponins have antimalarial\nproperties98<\/sup>, antioxidant, anti-inflammatory, anticancer, antiallergic,\nand antiplatelet activities have been reported for flavonoids99<\/sup>. The\navailability of these biologically important chemical compounds in R. beninensis<\/em>, therefore, underscores\nits medicinal values. Thus, the therapeutic potential of various extracts\nof R. beninensis<\/em> may be due to\nthese phytochemical compounds. <\/p>\n\n\n\n

The therapeutic\nproperties of this plant species are influenced by the different bioactive\ncompounds identified in the methanol extracts of R. beninensis<\/em>. While several prominent bioactive compounds from this\nextract included (Z,Z)-9,12-Octadecadienoic\nacid (19.96%), Lupeol (18.96%), cis-13-Octadecenoic acid (9.96%),\nn-Hexadecanoic acid (9.87%), 3′,5′-Dimethoxyacetophenone (6.67%), and\n4-((1E)-3-Hydroxy-1-propenyl)-2 -methoxyphenol (3.21%) have been known to have\nvarious inhibitory effects and therapeutic uses, the pharmacological activities\nof some chemical compounds identified in this study have been corroborated by Fagbemi et al.24<\/sup> who identified\n36 bioactive compounds and many of them possessed antioxidant, antibacterial,\nantiasthma, antifungal, antidiabetic, anti-cancer and anti-inflammatory activities.\nKuete100<\/sup> and Dzotam & Kuete101<\/sup> indicated that\nantimicrobial activities of studied Cameroonian plants depended on the occurrence\nof alkaloids, flavonoids, phenolics, steroids and triterpenes. While the antimicrobial\nactivity of methanol extracts of Artemisia\nvulgaris, Cinnamomum tamala, Oxalis corniculata, <\/em>and Ageratina adenophora<\/em> was against Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Citrobacter\nkoseri <\/em>and Salmonella typhi<\/em>102<\/sup>,\nhydromethanol extracts of Cistus\nmonspeliensis, Punica granatum <\/em>and Berberis\nvulgaris<\/em> indicated tremendous antibacterial activity against Enterococcus faecalis, Staphylococcus\naureus, <\/em>and Enterobacter cloacae<\/em>103<\/sup>.\nThese identified phytochemicals in the\nextract are believed to be a constituent of defense mechanisms in plant, and\nthey can be categorized as protective substances present in this plant as\n“phytoanticipins” and “phytoprotectants”104<\/sup>.  Despite the low percentages of some of the detected\nchemical compounds in this study, scientific reports indicated that each of the\ncompounds had considerable medicinal importance, and the presence of the\nidentified chemical compounds may have an impact on the antibacterial and\nantioxidant activities of R. beninensis<\/em>.\nThe chemical compounds identified underscore the veracity of this plant\u2019s\nusefulness in traditional medicine while identified compounds without\nbiological activities found in literature could also contribute individually or\nsynergistically to the pharmacological activities of the extract. <\/p>\n\n\n\n

Disproportionate free radical\nproduction damages biomolecules during cellular metabolism causing few\ndisorders including diabetes, cancer, chronic inflammation, and\nneurodegenerative illnesses105<\/sup>. The most practical method in managing\noxidative stress\u2013related diseases is antioxidant therapy106<\/sup>. Despite\nthe availability of various synthetic medications for managing oxidative\nstress, their high costs and unfavorable side effects have reduced their\neffectiveness107<\/sup>. As a result, alternative, low-cost, non-toxic\nantioxidants are required to fight oxidative stress and the diseases\nassociated with it106<\/sup>. Consequently, the high potency and\neffectiveness at low concentrations exhibited by the methanol extract of R.\nbeninensis<\/em> implied that its redox properties are dependent on its polyphenolic\ncontents satiating singlet and triplet oxygen and decomposing peroxides, adsorbing\nand neutralizing free radicals108<\/sup> and directly scavenging free\nradicals109<\/sup>.  <\/p>\n\n\n\n

The methanol extract of R. beninensis<\/em> roots was more effective\nagainst Pseudomonas spp.<\/em> PA29T. At\nlow concentrations, strong antibacterial activity was recorded against all the Gram-negative\nbacteria. The MBC values revealed that the extract could be more bactericidal\nagainst the Gram-negative bacteria.  This\nwas in consonance  with the findings of\nSemwal et al.<\/em> 110<\/sup>, Manivannan et al.<\/em>111<\/sup>,\nand Kouadri112<\/sup>. While antibacterial agents with lower MICs and MBCs\nare thought to be more effective, Tripathi113<\/sup> indicated that\nantibacterial agents are more bactericidal when the MIC is closer to the MBC. However, the variations in bacterial\nsusceptibility could be attributed to the differences in microorganisms’\nintrinsic tolerance or the physico-chemical properties of phytochemicals\ncontained in plant extracts112<\/sup> and phospholipid membranes that\ncontain structural lipopolysaccharide components making their cell walls impermeable\nto antimicrobials114<\/sup>. While the antibacterial activity of the\nmethanol extract could depend on the most prominent chemical compounds, the\ntherapeutic potential of the chemical compounds with lesser percentages and\nthose without pharmacological activities identified in literature could not be\nundermined. <\/p>\n\n\n\n

Conclusion <\/strong><\/p>\n\n\n\n

In conclusion, this study confirmed the presence of different\nphytochemical compounds and chemical compounds with diverse pharmacologically\nimportant principles or activities in the aqueous methanol extract of R. beninensis<\/em> roots. The extract’s\nantioxidant and antibacterial effects against the test bacterial isolates might\nbe accredited to the occurrence of the aforementioned chemicals found in the\nextract. This study demonstrates the medicinal value of R. beninensis<\/em>‘ root, identifies the plant as a significant source\nof innovative drug compounds, and suggests that this plant may be a viable\nalternative for treating urinary tract infections, supporting its use in the\nfolkloric treatment of these diseases. Further studies on isolation of\nbioactive compounds of therapeutic values to determine their biological\nactivities are ongoing in our research laboratory.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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