Phytochemical Profiling, Spectroscopic Characterization, and Antimicrobial Activity of Solvent Extracts of Imperata Cylindrica

in the departmental herbarium. The collected plant samples were washed thoroughly with distilled water to remove soil particles and debris. The samples were air-dried at room temperature (25–28 °C) for seven days and pulverized into fine powder using an electric laboratory grinder (Model MX-AC400, Panasonic Corporation, Japan). The powdered samples were stored in airtight containers prior to analysis. 2.3 Chemicals and Reagents All chemicals used in this study were of analytical grade. Methanol, ethanol, chloroform, ethyl acetate, n-hexane, sulfuric acid (H2SO4), and ammonium hydroxide (NH4OH) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Silica gel (60–120 mesh) used for column chromatography was obtained from Merck KGaA (Darmstadt, Germany). Microbiological media including Mueller–Hinton agar and Sabouraud dextrose agar were procured from Oxoid Ltd (Basingstoke, UK). 2.4 Extraction Procedure Ten grams (10 g) of powdered plant material were homogenized in methanol–water (80:20 v/v) using a laboratory homogenizer (Model T25 Digital Ultra-Turrax, IKA Works GmbH & Co. KG, Germany) for five minutes. The mixture was filtered using Whatman No.1 filter paper and concentrated using a rotary evaporator (Model R-210, Büchi Labortechnik AG, Switzerland) at 40 °C under reduced pressure until one-tenth of the original volume remained. The concentrate was acidified with 2 mL of dilute H2SO4 and extracted three times with chloroform using a separatory funnel. The chloroform layer containing moderately polar compounds was evaporated to dryness. The aqueous acidic layer was subsequently basified to pH 10 using ammonium hydroxide and extracted with chloroform: methanol (3:1 v/v). The organic phase was evaporated to obtain alkaloid-rich fractions. Extracts obtained (aqueous, 80% methanol, and ethyl acetate) were stored at 4 °C for further analysis. 2.5 Phytochemical Secreening Qualitative phytochemical analysis was carried out to detect secondary metabolites including alkaloids, flavonoids, tannins, saponins, steroids, terpenoids, and glycosides using standard phytochemical procedures. Alkaloids were detected using Wagner’s and Mayer’s reagents, while tannins were identified using ferric chloride and lead acetate tests. Saponins were confirmed using frothing and emulsion tests, flavonoids by alkaline reagent tests, and terpenoids using the Salkowski reaction Harborne, (1998): Doughari, (2012). , 2.6 Quantitative Determination of phytochemicals Quantitative analysis of phytochemicals was carried out using established methods: Tannins: Van-Burden and Robinson method. Saponins: Obadoni and Ochuko method. Alkaloids: Harborne method. Flavonoids: Bohm and Kocipal-Abyazan method. Terpenoids: Gravimetric method Harborne, (1998) All determinations were carried out in triplicate and expressed as percentage composition (% w/w) relative to 4 g of crude plant sample. 2.7 Antibacterial Activity Assay Antibacterial activity of the plant extracts was evaluated using the agar well diffusion technique against the following bacteria: Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumonia, Pseudomonas aeruginosa, Bacillus licheniformis. Mueller–Hinton agar plates were inoculated with standardized bacterial suspensions (0.5 McFarland standard). Wells of 6 mm diameter were bored into the agar and filled with extract solutions. Plates were incubated at 37 °C for 24 h in an incubator (Model BD 115, Binder GmbH, Germany). Zones of inhibition were measured in millimeters. Minimum inhibitory concentration and minimum bactericidal concentration were determined using broth dilution techniques Cushnie, et (2011). 2.8 Antifungal Activity Assay Antifungal activity was assessed using Sabouraud dextrose agar against fungal strains including:Candida albicans, Candida parapsilosis, Aspergillus niger, Aspergillus flavus, Fusarium oxysporum. Inoculated plates were incubated at 28 °C for 48–72 h. Zones of inhibition were measured and minimum inhibitory concentration and minimum fungicidal concentration were determined. 2.9 Isolation and Purification of Bioactive Compounds Eight grams (8 g) of ethyl acetate crude extract were subjected to silica gel column chromatography (70 cm × 3.5 cm) packed with silica gel (60–120 mesh; Merck, Germany). Elution was performed using gradient solvent systems of n-hexane and ethyl acetate (100:0, 90:10, and 85:15 v/v). Fractions were collected and monitored using thin layer chromatography on precoated silica gel plates (Merck TLC plates, F254, Germany). Fractions with similar Rf values were pooled, yielding seven combined fractions (IMP1–IMP7). Further purification by repeated column chromatography produced three pure isolates (IMP1, IMP2, and IMP3) Okwuego et (2025). 2.10 Structural Elucidation Structural elucidation of purified compounds was performed using spectroscopic techniques. FTIR Analysis: Infrared spectra were recorded using an FTIR spectrophotometer (Model Spectrum Two, PerkinElmer, USA) in the range of 4000–400 cm−1. Okwuego et al (2021):Ochie et (2025): . UV-Visible Spectroscopy: UV–Visible spectra were recorded using a UV–Vis spectrophotometer (Model UV-1800, Shimadzu Corporation, Japan). The absorption maxima (λmax) were recorded between 200–800 nm Mmuo et (2024): Okwuego et (2025). GC-MS Analysis: Gas chromatography–mass spectrometry analysis was performed using a GC–MS system (Model QP2010 Plus, Shimadzu Corporation, Kyoto, Japan). Compounds were identified by comparing mass spectra with the National Institute of Standards and Technology spectral library database Okwuego et (2025). , 2.11 Statistical Analysis All experiments were conducted in triplicate and results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using statistical software (Version 22.0, IBM SPSS Statistics). Statistical significance was considered at p < 0.05. 3.0 Results and Discussions 3.1 Phytochemical Composition of Imperata cylindrica Preliminary phytochemical screening of the leaf and root extracts of Imperata cylindrica revealed the presence of several important classes of secondary metabolites including alkaloids, flavonoids, tannins, saponins, terpenoids, steroids, and glycosides. The occurrence of these phytoconstituents confirms the plant's broad ethnopharmacological applications reported in traditional medicine. Alkaloids detected using Wagner’s and Mayer’s reagents produced characteristic precipitates, indicating the presence of nitrogen-containing heterocyclic compounds known for their antimicrobial and pharmacological activities. Flavonoids were confirmed through alkaline reagent tests that produced distinct color changes, suggesting the presence of polyphenolic compounds capable of modulating oxidative stress and inflammatory pathways. Tannins were identified through ferric chloride and lead acetate reactions, producing greenish-black and cream precipitates, respectively Harborne, (1998): . Doughari, (2012): Okwuego et (2025). Tannins are well known for their protein-binding properties and antimicrobial activity through enzyme inhibition and disruption of microbial membranes. Saponins showed positive frothing and emulsion tests, indicating amphiphilic glycosides capable of increasing membrane permeability. Similarly, terpenoids and steroids were confirmed using the Salkowski reaction, producing a reddish-brown interface, indicative of triterpenoid or steroidal skeletons. The presence of these metabolites provides a biochemical basis for the therapeutic applications of Imperata cylindrica, particularly its antimicrobial, anti-inflammatory, and diuretic effects reported in traditional medicine Harborne, (1998): Doughari, (2012). Table 1 Qualitative Phytochemical Screening of Imperata cylindrica Phytochemical Class Test Method Observation Inference Alkaloids Wagner’s reagent Reddish-brown precipitate Present (++) Mayer’s reagent White precipitate Present (++) Saponins Frothing test Persistent frothing Present (++) Emulsion test Stable emulsion formed Present (++) Fehling’s test Light reddish precipitate Weakly present (+) Flavonoids Ammonium test Green/light-green layer formation Present (++) NaOH/Acetic acid test Green/light-green layer formation Present (++) Tannins Ferric chloride test Greenish-black precipitate Present (++) Lead acetate test Cream precipitate Present (++) Steroids/Terpenoids Ethanol + chloroform + H2SO4 test Reddish-brown interface Present (++) Glycosides Fehling’s test Brick-red precipitate Present (++) , Note: (++ strongly present; + weakly present) Quantitative Phytochemical Analysis of Imperata cylindrica Quantitative evaluation of phytochemical constituents demonstrated varying concentrations of bioactive compounds within the crude plant material. Terpenoids were the most abundant constituent, accounting for approximately 43.3% of the phytochemical composition. Flavonoids were the second most abundant at 10.3%, followed by tannins (5.7%), alkaloids (5.4%), and saponins (2.25%) Nkachukwu. et al (2025): Okwuego et (2025): Okonkwo et (2025). The high terpenoid content suggests that these compounds may play a dominant role in the biological activities of the plant. Terpenoids are known to possess diverse pharmacological properties, including antimicrobial, antioxidant, and anti-inflammatory activities. Their ability to disrupt microbial membranes and interfere with metabolic processes may explain the strong antimicrobial activities observed in this study. Flavonoids, which were present in appreciable amounts, are widely recognized for their antioxidant potential. They can neutralize free radicals, chelate metal ions, and inhibit lipid peroxidation. Furthermore, flavonoids have been reported to inhibit microbial nucleic acid synthesis and cytoplasmic membrane function Harborne, (1998): Nkachukwu. et al (2025): Okwuego et (2025). The presence of tannins and alkaloids further strengthens the pharmacological potential of the plant. Tannins exert antimicrobial effects through precipitation of microbial proteins and inhibition of extracellular enzymes, whereas alkaloids often interact with DNA and essential enzymes within microbial cells. The phytochemical profile observed in this study is consistent with previous reports on medicinal grasses and supports the therapeutic use of Imperata cylindrica in ethnomedicine Harborne, (1998): Nkachukwu. et al (2025). Table 2. Quantitative Phytochemical Composition of Imperata cylindrica Notes Values reported as Mean ± SD (n = 3) Statistical analysis performed using ANOVA 3.3 Antifungal Activity The antimicrobial screening revealed that extracts of Imperata cylindrica exhibited inhibitory activity against several pathogenic bacteria including Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Bacillus licheniformis Cowan, (1999): Cushnie, et (2011).. Among the extracts tested, the 80% methanol extract exhibited the highest antibacterial activity with inhibition zones reaching up to 20 mm against Escherichia coli. This result suggests that methanol effectively extracted bioactive compounds with antibacterial properties. Polar solvents such as methanol are known to efficiently extract phenolic compounds and flavonoids, which are often responsible for antimicrobial activity. Phytochemical Mean Concentration (mg/g) Percentage (%) p-value Tannins 0.057 ± 0.002 5.7 <0.05 Flavonoids 0.103 ± 0.004 10.3 <0.01 Alkaloids 0.054 ± 0.003 5.4 <0.05 Saponins 0.023 ± 0.001 2.25 <0.05 Terpenoids 0.433 ± 0.008 43.3 <0.001 , The ethyl acetate extract also demonstrated moderate antibacterial activity, indicating that moderately polar compounds such as terpenoids and fatty acid derivatives may contribute to bacterial inhibition. In contrast, aqueous extracts showed relatively lower activity, which may be attributed to the limited solubility of certain bioactive compounds in water. Minimum inhibitory concentration values ranged between 0.2 and 0.8 mg/mL, indicating strong antimicrobial potency of the extracts. The bactericidal effect may be associated with membrane disruption, inhibition of protein synthesis, or interference with bacterial metabolic pathways. These findings are consistent with previous reports indicating that plant-derived secondary metabolites such as flavonoids, tannins, and terpenoids can exert antimicrobial effects through multiple mechanisms including membrane permeabilization and enzyme inhibition Cowan, (1999): Cushnie, et (2011).. Table 3. Antibacterial Activity of Imperata cylindrical extract Test Organism Methanol Extract (mm) Mean ± SD Aqueous Extract (mm) Ethyl Acetate Extract (mm) Ciprofloxacin Control (mm) MIC (mg/mL) MBC (mg/mL) p- value Pseudomonas aeruginosa 11.88 ± 0.42 5.64 ± 0.21 9.14 ± 0.33 26.00 ± 0.45 0.4 0.2 <0.01 Bacillus cereus 6.89 ± 0.31 7.00 ± 0.28 0.82 ± 0.05 11.11 ± 0.29 0.6 0.3 <0.05 Staphylococcus epidermidis 13.24 ± 0.38 10.69 ± 0.41 8.86 ± 0.34 21.23 ± 0.39 0.4 0.2 <0.01 Klebsiella pneumonia 3.05 ± 0.12 3.12 ± 0.15 4.00 ± 0.18 12.36 ± 0.22 0.2 0.1 <0.05 Escherichia coli 20.00 ± 0.55 12.30 ± 0.39 7.37 ± 0.27 23.00 ± 0.41 0.8 0.3 <0.001 Notes Zone of inhibition expressed in mm Values represent Mean ± SD (n = 3) Ciprofloxacin used as standard antibacterial control Nkachukwu. et al (2025) 3.4 Antifungal Activity The antifungal evaluation demonstrated inhibitory activity against several fungal pathogens including Candida albicans, Candida parapsilosis, Aspergillus niger, Aspergillus flavus, and Fusarium oxysporum. The methanol extract showed the most pronounced antifungal activity, particularly against Candida albicans and Aspergillus parasiticus, with inhibition zones exceeding 7 mm. The observed antifungal effects may be attributed to the presence of phenolic compounds and terpenoids that can interfere with fungal cell membrane integrity and inhibit ergosterol biosynthesis Cowan, (1999): Cushnie, et (2011).. The moderate antifungal activity observed against filamentous fungi suggests that the plant extracts possess broad-spectrum antimicrobial potential. Minimum fungicidal concentration values ranged from 0.2 to 1.0 mg/mL, indicating fungistatic or fungicidal effects depending on concentration. , The antifungal activity may also be associated with fatty acid derivatives detected in the extracts, which have been reported to disrupt fungal cell membranes and inhibit spore germination Cowan, (1999): Cushnie, et (2011). Table 4. Antifungal Activity of Imperata cylindrical extract Test Organism Methanol Extract (mm) Mean ± SD Water Extract (mm) Ethyl Acetate Extract (mm) Nystatin Control (mm) MIC (mg/mL) MFC (mg/mL) p- value Candida albicans 7.33 ± 0.28 7.00 ± 0.32 5.44 ± 0.21 22.00 ± 0.40 0.8 0.3 <0.01 Candida parapsilosis 2.87 ± 0.14 2.00 ± 0.11 2.00 ± 0.09 8.77 ± 0.22 1.0 0.4 <0.05 Aspergillus niger 4.22 ± 0.17 2.81 ± 0.13 2.29 ± 0.12 12.85 ± 0.31 1.0 0.3 <0.05 Aspergillus parasiticus 10.01 ± 0.33 7.36 ± 0.29 8.23 ± 0.25 18.91 ± 0.37 0.6 0.2 <0.01 Microsporum gypseum 0.97 ± 0.06 1.08 ± 0.08 1.00 ± 0.07 5.34 ± 0.18 1.0 0.4 <0.05 Notes Nystatin used as standard antifungal drug Nkachukwu. et al (2025) Chromatographic Separation and Isolation of bioactive compounds Column chromatography of the ethyl acetate extract resulted in the separation of 56 fractions, which were further combined into seven pooled fractions based on similar TLC profiles. Further purification yielded three major isolates designated IMP1, IMP2, and IMP3. Thin-layer chromatography analysis revealed distinct Rf values for each isolate, indicating successful separation of individual constituents. The use of gradient solvent systems consisting of n-hexane and ethyl acetate allowed efficient separation of compounds based on polarity differences. The isolation of discrete fractions enabled subsequent structural characterization using spectroscopic techniques. 3.6 Spectroscopic Characterization of Isolation compounds 3.6.1 FTIR Analysis: Fourier transform infrared spectra of the isolated fractions revealed several characteristic absorption bands corresponding to functional groups commonly found in natural products. Strong absorption bands around 3200–3400 cm−1 indicated the presence of hydroxyl (–OH) groups associated with alcohols and phenolic compounds. Peaks observed around 1600–1700 cm−1 correspond to carbonyl (C=O) stretching vibrations, suggesting the presence of ketones, carboxylic acids, or amide groups Okwuego et al (2021): Okwuego (2023): Ochie et (2025): Okwuego et (2025): Okonkwo et (2025) , Table 5 FTIR Functional Group Analysis of Extracts Extract Major FTIR Peaks (cm−1) Functional Groups IMP1 (Ethyl acetate) 3344, 3125 O–H, N–H stretching 2786, 2660 C–H stretching 1629 C=O stretching 1516 C=C stretching 1185 C–O stretching IMP2 (Methanol) 3521–3249 O–H stretching 3093 C–H alkenes 1612 C=O 1251 C–O IMP3 3572–3341 O–H stretching 1617 C=O 1410 C=C 1043 C–O Additional bands between 1000–1200 cm−1 were attributed to C–O stretching vibrations typical of alcohols and esters. These spectral features indicate that the isolated compounds belong to oxygenated organic molecules such as fatty acids, esters, or phenolic derivatives Okwuego et al (2021); Ochie et (2025): Okwuego et (2025). 3.6.2 UV-Visible Spectroscopy The UV–Visible spectra showed absorption maxima (λmax) within the range of 368–664 nm. Absorption bands observed in the region of 350–450 nm are typically associated with π→π* transitions in conjugated systems such as flavonoids and phenolic compounds. Meanwhile, absorption peaks near 600 nm suggest the presence of extended conjugated chromophores or carbonyl-containing compounds Mmuo et (2024): Okwuego et (2025): Okonkwo et (2025). Table 6 UV-Visible Spectroscopic Analysis Extract λmax (nm) Electronic Transition Chromophore Ethyl acetate (IMP1) 664 n → π* Carbonyl 532 n → π* Conjugated C=C 439 π → π* Alkene Methanol (IMP2) 652 n → π* Carbonyl 413 π → π* Aromatic Water (IMP3) 662 n → π* Carbonyl 515 n → π* Conjugated double bonds These spectral characteristics further support the presence of conjugated bioactive molecules capable of exhibiting antioxidant and antimicrobial activities. 3.6.3 GC-MS Analysis Gas chromatography–mass spectrometry analysis revealed the presence of several bioactive compounds including: 1,6-Anhydro-heptanoic acid, 1,6-Dichloro-cyclohexaneacetic acid, 1,6- Anhydro-5-hexenoic acid, 5-Hexenoic acid and Cyclohexaneacetic acid Okwuego et (2025): Okonkwo et (2025). , Table 6 GC-MS Identification of Major of bioactive compounds Peak No. Retention Time Identified Compound Molecular Formula Molecular Weight Peak Area (%) Biological Activity 1 5.32 Hexanoic acid derivative C6H12O2 116 8.45 Antimicrobial 2 7.11 1,6- Anhydroheptanoic acid C7H12O3 144 12.23 Anti- inflammatory 3 8.56 5-Hexenoic acid C6H10O2 114 16.11 Antimicrobial 4 10.73 Cyclohexaneacetic acid derivative C8H14O2 142 9.84 Antioxidant 5 12.61 Phenolic compound C9H10O3 166 18.92 Antioxidant 6 14.82 Terpenoid derivative C15H24 204 21.45 Antimicrobial 7 16.10 Fatty acid ester C18H34O2 282 13.00 These compounds are structurally related to fatty acid derivatives known to possess antimicrobial, anti-inflammatory, and antioxidant properties. Fatty acid esters are known to exert antimicrobial activity through disruption of lipid membranes and inhibition of enzyme systems within microbial cells Okwuego et (2025): Okonkwo et (2025). The identification of these compounds suggests that fatty acid derivatives may be responsible for the antimicrobial activities observed in the crude extracts. 3.7 Implication for Ethanomedicinal Application The results obtained in this study provide scientific validation for the traditional medicinal use of Imperata cylindrica in the treatment of infections and inflammatory conditions. The presence of diverse bioactive phytochemicals and the demonstrated antimicrobial activity support the plant's ethnopharmacological relevance Okwuego et (2026). Furthermore, the isolation and structural characterization of bioactive compounds indicate that this plant may serve as a promising source of lead molecules for the development of novel antimicrobial agents. The increasing prevalence of antimicrobial resistance highlights the need for new therapeutic compounds derived from natural sources. 4.0 Conclusion This study provides comprehensive phytochemical, antimicrobial, and spectroscopic evaluation of extracts obtained from the leaves and roots of Imperata cylindrica. Qualitative and quantitative phytochemical analyses confirmed the presence of several important bioactive constituents including alkaloids, flavonoids, tannins, saponins, terpenoids, steroids, and glycosides. Among these metabolites, terpenoids and flavonoids were present in relatively high concentrations, suggesting their major contribution to the biological activity of the plant. The antimicrobial assays demonstrated significant inhibitory activity of the extracts against clinically relevant bacterial and fungal pathogens, including Escherichia coli, Staphylococcus epidermidis, and Candida albicans. Methanolic extracts exhibited the strongest antimicrobial activity, indicating that polar solvents are effective for extracting the active constituents responsible for microbial inhibition. Chromatographic separation and purification of the crude extract resulted in the isolation of three major fractions. Spectroscopic characterization using FTIR, UV–Vis, and GC–MS analysis revealed the presence of several fatty acid derivatives including 1,6-anhydro- heptanoic acid, 1,6-dichloro-cyclohexaneacetic acid, and 5-hexenoic acid. These compounds , are known to possess antimicrobial and anti-inflammatory properties and may be responsible for the biological activities observed. The results of this study provide scientific validation for the traditional medicinal use of Imperata cylindrica and highlight its potential as a natural source of bioactive compounds for antimicrobial drug development. , References Cowan, M.M., (1999). Plant products as antimicrobial agents. 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(2025) Phytochemical, Antioxidant and Antimicrobial Evaluation of Oil Palm Tree (Elaeis guineensis) Bark for Potential Nutraceutical and Pharmaceutical Applications International Journal of Health and Pharmaceutical Research , Okwuego. P. O., Offiah, V. O. Okey-Nzekwe .C. M. (2026) Synthesis and Characterization of Methanolic Root Extract of Imperata Cylindrica and Its Nanoencapsulation with Chitosan. International Journal of Latest Technology in Engineering, Management & Applied Science (Ijltemas) Issn 2278. Okwuego Peter Obinna, Mmaduakor Ebuka Chidiebere, Omoh Theodore Oshokha (2026) Green Engineering of Structured Lignocellulosic Sorbents from Pumpkin Stem Fibres for Oil Adsorption. International Journal of Engineering and Modern Technology E-ISSN 2504-8848 P-ISSN 2695-2149 Okwuego Peter Obinna , Nnaoma Ikenna Elvis , Orakwue Foster Chikeobi , Saater Mstushima Jennifer (2026) Comparative FTIR Characterization of Caffeine Functional Groups in Different Varieties of Kola Nuts (Cola Acuminata and Cola Nitida). International Journal Of Research And Innovation In Applied Science Issn No. 2454-6194 | Doi: 10.51584/Ijrias |Volume Xi Issue Ii. Mmuo V.E and Okwuego P.O (2024) Spectrophotometric Determination Of Proximate, Vitamin B Complex and Elemental Contents of Pigeon Peas (Cajanus Cajan) Anachem Journal, 15(1), 85-95, DOI:https://doi.org/10.5281/zenodo.14567423 Okonkwo S.1, Ughanze B.N, Okonkwo C.K, Ezigbo V.O, Okafor, E.C , Okwuego P.O, Okonkwo A.T, Okonkwo V. S.(2025) Determination of the Chemical and Morphological Structure of Liposomes Encapsulating Lycopene Derived from Tomatoes Using FTIR, SEM, TEM, and Ultrasound Imaging Techniques . International Journal of pharmaceutical Sciences Volume 03 | Issue 04 | Article Id IJPS/250304235 Okonkwo S. I., Mmuo V. E. , Okafor E. C. , Offiah V.O. , Okonkwo C. K. , Okwuego P. O. ,Okonkwo V. S.,Kene-Okonkwo A. T (2025) Extraction, Fatty Acid Profile and Elemental Analysis of Gongronema latifolium Leaf. International Journal Of Pharmaceutical Sciences [Issn: 0975-4725; Coden: Ijps00] Journal Homepage: https://www.ijpsjournal.com