# Cytotoxicity, Antimicrobial, Antioxidant, Anthelmintic, and Anti-Inflammatory Activities and FTIR Analysis ofCombretum nioroense Stem Bark

Authors: Clement Osei Akoto; Matilda Enyonam Adzimah; Anthony Yeboah Forkuoh; Patrick Obedi; Jacob Oguns; Felix Owusu Boahen
Journal: Journal of Chemistry
 (2023)

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## Abstract

Combretum nioroense is widely used in the folkloric treatment of a variety of bacterial and helminthic infections. The decoction of its leaves is traditionally fed to newborn babies in some rural parts of Ghana. The study focused on identifying the prime components of petroleum ether and ethanolic extracts of the stem bark using standard phytochemical screening protocols and chromatographic and spectroscopic techniques. The activities (cytotoxicity, antimicrobial, antioxidant, anthelmintic, and anti-inflammatory) of the extracts of C. nioroense were also investigated. Preliminary phytochemical analysis of the extracts revealed the presence of glycosides, saponins, phenols, coumarins, alkaloids, flavonoids, tannins, steroids, phytosterols, flavanols, terpenoids, and cardiac glycosides. The action of the extracts of C. nioroense on Milsonia ghanensis worms was concentration-dependent, with the least concentration (0.75 mg/mL) paralyzing and killing M. ghanensis after the maximal exposure time. The IC50 values for petroleum ether and ethanol extracts in the DPPH assay were >100.0 and 27.940 ± 1.005 μg/mL and those of the H2O2 assay were 400.900 ± 3.400 and 322.500 ± 1.005 μg/mL, respectively. The total antioxidant capacities (TACs) for petroleum ether and ethanol extracts were 47.197 ± 0.533 and 57.968 ± 0.560 gAAE/100 g, respectively. The IC50 value for ethanol extract in the cytotoxicity studies was 115.4 ± 1.332 μg/mL. The MICs of the extracts against the test organisms were within the range of 0.0122–25.0 mg/mL. The extracts (petroleum ether and ethanol) showed a concentration-dependent increase in anti-inflammatory activity with IC50 values of 31.254 ± 0.359 and 24.402 ± 0.569 μg/mL, respectively. Chromatographic separations of the ethanol extract gave three fractions. FTIR analysis of the extracts and purified fractions revealed the presence of functional groups, confirming the presence of the phytochemicals identified in the screening test. The results indicate that both extracts of C. nioroense exhibit cytotoxicity, antimicrobial, antioxidant, anthelmintic, and anti-inflammatory activities, thereby proving the folkloric use to treat ailments caused by worms and microorganisms.


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## Body

## 1. Introduction

Since ancient times and according to mythology and folklore, natural products (NPs) have been used to heal a variety of illnesses. Traditional natural product (NP) medicine methodologies have facilitated the discovery of a large spectrum of bioactive secondary metabolites from both terrestrial and marine sources [1, 2]. The cardiac glycoside digitoxin, the anti-inflammatory compound acetylsalicylic acid, and alkaloids like morphine, quinine, pilocarpine, and penicillin, among others, are well-known and commonly used natural products of plant origin [3, 4]. Despite the challenges for effective protocols in the development of drugs made from plant natural products (NP), as well as the difficulty in cultivating starting material, standardizing isolation methods, and determining medicinal effects, the percentage of NP-derived drugs was 40% in 2000 and remained approximately constant at 24% in 2001 and 26% in 2002 [5, 6]. By the end of 2013, the FDA had approved 547 NPs and derivatives [7]. In the area of cancer from 1981 to 2019, 62 (33.5%) out of the 185 small molecules approved by the FDA were NP-derived drugs, though that could be around 64.9%, including synthetic drugs with an NP pharmacophore or a mimic of NP [8]. However, the main setbacks are the developmental cost, lack of adequate supply, and multistep synthesis of their complex structural architecture. About ten tons of stem barks or leaves can be harvested to obtain a marginally small quantity of NP. Given that over 80% of the world’s plant varieties have yet to be tested for any potential biological activity and that each molecule could hypothetically have multiple targets, NPs will play an increasingly important role in drug development in the future. New discoveries of NPs are assisting in the opening of new horizons [9].The Combretaceae family comprises 20 genera and about 475 species globally, of which approximately 370 belong to the genus Combretum, and 264 in Tropical Africa, including Ghana, Burkina Faso, Zimbabwe, and South Africa [10, 11]. Plant species of the Combretaceae family, such as Combretum micranthum and Combretum erythrophyllum, have been shown to possess strong antioxidant and antimicrobial activities [12, 13]. Combretastatins are a class of chemical compounds first identified from a deciduous tree found in South African bushwillow (Combretum caffrum) [14]. Krajewska and Jarzab reported that fosbretabulin (combretastatin A4 phosphate), isolated from the bark of C. caffrum, as a vascular disrupting agent, targets tumor neovasculature, leading to an acute, reversible reduction in tumor blood flow and tumor central necrosis [15]. Infusions prepared with the aerial parts of Combretum leprosum are utilized in folk medicine as a sedative and for the treatment of uterine hemorrhages, gastric pains, and flu [16]. Combretum molle has also been shown to contain antioxidants such as punicalagin, ellagitannin [17], 1α-hydroxycycloartenoid saponins, mollic acid glucoside [18], and mollic acid 3β-D-xyloside [19]. Combretum nioroense, a species of the Combretaceae family, is less known in the literature, with less information regarding its chemical constituents and biological activity. It is used to treat a variety of ailments caused by worms and microorganisms (diarrhoea, dysentery), and the decoction of its leaves is traditionally given to newborns as a beverage in Burkina Faso and some rural parts of Ghana [10]. However, there is a dearth of information in scientific reports on the biological activities of the stem bark of C. nioroense, except for anecdotal reports from local herbalists. This study focuses on identifying some prime components (using standard phytochemical screening protocols, chromatographic, and spectroscopic techniques) and researching the activities (anthelmintic, antimicrobial, cytotoxic, antioxidant, and anti-inflammatory) of ethanol and petroleum ether stem bark extracts of C. nioroense.


## 2. Materials and Methods

### 2.1. Plant Collection, Identification, and Authentication

The collection of the stem barks ofC. nioroense was conducted at Nkawkaw in the Kwahu South District in the eastern region of Ghana (Latitude: 6°33′4.36″N, Longitude: 0°45′58.32″E) in October 2020. The plant material was authenticated by Mr. Clifford Osafo Asare, a botanist at the Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences (FPPS), KNUST, Kumasi, Ghana. A voucher specimen sample (specimen number: KNUST/HMI/2021/SB005) was deposited at the department’s herbarium.


### 2.2. Chemicals and Reagents

All chemicals (except for standard drugs) were purchased from Sigma Aldrich Co. Ltd., Irvine, U.K. All organic solvents (analytical grade) used in the experimental work were purchased from BDH Laboratory Supplies (England).


### 2.3. Preparation of Plant Material

The stem barks ofC. nioroense were cut into pieces, washed with water, air-dried at room temperature (28 to 32°C) for two weeks, then milled to powder and stored in a desiccator [1].


#### 2.3.1. Extraction of Ethanol and Petroleum Ether Soluble Plant Materials

A mass of 100 g of the pulverized sample ofC. nioroense was packed into a cellulose thimble and extracted via Soxhlet extraction following a method previously described [1]. The extraction was done successively, starting with 500 mL of petroleum ether and then ethanol. Each crude extract was filtered and evaporated using a rotary evaporator (Buchi Rotavapor R-114) at 50°C. The crude extracts were dried in an oven at 40°C, and their respective percentage yields of 10.416 and 0.590% for ethanol and petroleum ether extracts with respect to the mass of powdered plant samples were determined. The crude extracts were stored in a refrigerator at 4°C for further experimental analyses.


### 2.4. Phytochemical Screening

The phytochemical screening of the pulverized sample and the extracts (petroleum ether and ethanol) ofC. nioroense were carried out using standard protocols [20].


### 2.5. In Vitro Cytotoxicity Assay Using the MTT Method

The MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity [21]. Roswell Park Memorial Institute (RPMI) culture medium supplemented with 10% fetal bovine serum (FBS), containing penicillin, streptomycin, and L-glutamine, was maintained in culture at 37°C in the presence of a humidified 5% CO2 atmosphere in an incubator prior to the assay. The tetrazolium-based colorimetric MTT assay was used to determine the cytotoxicity of the ethanol extract on the JURKAT cell line. A 10 mL stock solution of MTT was prepared. A 50 mg MTT powder was dissolved in 10 mL phosphate-buffered saline (PBS), pH = 7.4, to make 0.5 mg/mL. The MTT solution was filter-sterilized through a 0.2 μM filter and transferred into a sterile, light protected container. The MTT solution was stored and protected from light at 4°C for frequent use. Cells were seeded into the 96-well plates at a concentration of 1 × 104 cells/well, treated with varying concentrations of the ethanol extract (0.0, 62.5, 125.0, 250.0, 500.0, and 1000.0 μg/mL), and incubated for 72 h. The concentration at 0 μg/mL had only seeded cells with no ethanol extract added (factor control), the one at 1000 μg/mL was used as the positive control with the highest amount of ethanol extract added, and lastly, acidified isopropanol was used in the last wells as a negative control. A colour control plate was also set up with varying concentrations of the drug standard, curcumin (0.0, 2.302, 4.604, 9.208, 18.417, and 36.834 μg/mL), and incubated for 72 h. MTT solution (0.5 mg/mL) was added to each well on the plate and incubation continued for a further 4 h. The reaction was stopped with an acidified isopropanol solution, and the plate was incubated in the darkness overnight at room temperature before the absorbance was read at 570 nm using a microplate reader (Tecan Infinite M200, Grodig, Austria). The assay was done in triplicates. The percentage of cell viability was determined as follows:(1)%cellviability=A0−A1A−A1×100,where Ao = mean absorbance of test wells, A1 = mean absorbance in negative control wells, and A = mean absorbance of factor control wells.The IC50 values were determined from the plot of percentage cell viability against the extract concentrations.


### 2.6. Determination of In Vitro Antimicrobial Activity

#### 2.6.1. Sources of Microorganisms

Pure cultures ofStaphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 29212), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 4853), and the clinical strain fungus, Candida albicans, were obtained from the Microbiology Section, Department of Pharmaceutics, College of Health Sciences, KNUST, Kumasi. These microbial strains were subcultured aseptically on freshly prepared nutrient agar slants and incubated at 37°C for 24 h. The test organisms were standardized at 0.5 M McFarland for use in each experiment [2, 22].


#### 2.6.2. Agar Well Diffusion Method

The antimicrobial activities of the extracts ofC. nioroense were determined using the agar well diffusion method previously described [23, 24]. The nutrient agar (Oxoid, United Kingdom) plates were inoculated with 100 μL of the test organisms using a micropipette and sterile pipette tips, as demonstrated by the pour plate method. By means of a sterile cork borer, 4 wells (10 mm in diameter) were made in the agar and filled with 200 μL of the extracts (6.25, 12.5, and 25 mg/mL) and reference antimicrobial drugs ciprofloxacin (for bacterial strains) and clotrimazole (for fungal strains) at 0.05 mg/mL concentrations. The experiment was done in triplicates for the extracts and reference drugs, and the plates were incubated. The diameter zones of growth inhibition produced were measured using a millimetre rule after 24 h of incubation at 37°C for bacteria and 72 h at 28°C for fungus, and the mean values were calculated.


#### 2.6.3. Broth Microdilution

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts ofC. nioroense were determined using the microwell broth dilution method [23, 24]. The inoculums of test microorganisms were prepared from 24-hour broth cultures by serial dilution of 1 mL culture into 9 mL sterile water and then compared to the 0.5 McFarland turbidity standard (1.5 × 105 CFU/mL). The polar plant extract (ethanol) was solubilized in dimethyl sulfoxide (DMSO), and the nonpolar plant extract (petroleum ether) was solubilized in polysorbate 20. A 100 μL single strength nutrient broth was transferred into each well of the 96-well sterile plates followed by 125 μL of two-fold serial dilutions of the extract concentrations (25.00, 12.50, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953, 0.09765, 0.048828, 0.02441, and 0.0122 mg/mL), as well as 25 μL of the inoculum was added. Ciprofloxacin and clotrimazole were the positive control drugs used in the analysis with prepared concentrations (0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.001563, 0.000781, and 0.000390 mg/mL). DMSO and polysorbate 20 were used as negative controls. The microplates were incubated at 37°C for 24 h and observed for inhibition of microbial growth by the addition of 20 μL (0.2 mg/mL) solution of MTT (Sigma-Aldrich, St. Louis, M.O., USA) for 30 min. After 30 min of addition of the dye solution, the wells that developed dark purple colour indicate the presence of microbial growth as the dehydrogenase enzymes in the live bacteria react with the tetrazolium salt to form a dark complex pink colour. However, wells that do not develop any colour change but retain the yellow colour of the dye solution are indicative of the inhibition of microbial growth by the extracts. For MBC determination, 100 μL cultures were transferred from all wells that showed clear inhibition into another 100 μL sterile Nutrient Broth in 96-well plates to a total volume of 200 μL and incubated for a further 24 h. Afterward, the plate was observed to ascertain which concentrations of the extracts caused 99.9% of the microorganisms’ deaths. All the experiments were conducted in triplicate.


### 2.7. Antioxidant Activity

#### 2.7.1. DPPH Radical Scavenging Assay

The antioxidant activity of the extracts ofC. nioroense was determined using the standard methods previously described for the DPPH assay [25, 26]. Ascorbic acid was used as the reference standard. The absorbance of each mixture was measured at 517 nm after incubation [26]. The experimental procedure was repeated in triplicate for data analysis. DPPH radical (%) scavenging was calculated using the following formula:(2)%scavenging=Ao−AAo×100,where AO = absorbance of the control and A = absorbance of the test solution.


#### 2.7.2. Hydrogen Peroxide (H2O2) Scavenging Assay

The H2O2 scavenging activity of the extracts of C. nioroense was assessed using the standard methods previously described for the H2O2 assay [27, 28]. Ferrous ammonium sulphate (0.5 mL, 1 mM), distilled water (3 mL), and 1, 10-phenanthroline (3 mL, 1 mM) were used as the negative control, and gallic acid as the reference standard. The absorbance was read at 510 nm using a UV-VIS spectrophotometer. The procedure was repeated in triplicate for data analysis. H2O2 percentage scavenging activity of the extracts was calculated using the following formula:(3)%scavenging=AtestAcontrol×100,where Atest is the absorbance of the test samples and Acontrol is the absorbance of the negative control. The results were further reported in IC50.


#### 2.7.3. Total Antioxidant Capacity (TAC) Assay

The total antioxidant capacity of the extracts ofC. nioroense was assessed using the standard protocol previously reported for the assay [27, 29]. Distilled water was used as the blank and ascorbic acid as the reference standard. The absorbance of the solutions was measured in triplicates using the UV-vis spectrophotometer at 695 nm. The procedure was repeated in triplicate for data analysis [27].


### 2.8.In Vitro Anthelmintic Activity (Adult Worm Mortality Assay)

#### 2.8.1. Collection ofMilsonia ghanensis for Anthelminthic Activity

Milsonia ghanensis (common name: adult Indian earthworms; order: Haplotaxida) were used for this analysis because they have an anatomical and physiological resemblance to the human intestinal roundworm [30, 31]. Adult M. ghanensis earthworms (measuring 6.3–7.2 cm in length and 0.20–0.30 cm in width) were collected from the soil on the banks of the Wiwi River behind the Department of Theoretical and Applied Biology Building within KNUST, Kumasi, Ghana, and authenticated by Mr. Lawrence Yeboah in the Zoology Unit, Department of Theoretical and Applied Biology, KNUST, Kumasi, Ghana. The earthworms were thoroughly washed in sterile distilled water to get rid of all soil debris prior to the experiment [27].


#### 2.8.2. Adult Worm Mortality Assay

The anthelmintic assay of the extracts ofC. nioroense was assessed using a modification of the methods previously reported [1, 31]. Concentrations of 6.00, 3.00, 1.50, and 0.75 mg/mL of petroleum ether and ethanol extracts of C. nioroense and the standard drug (albendazole) were prepared from a stock solution of 12.00 mg/mL using sterile distilled water as solvent. Double distilled water was used as a negative control. The time of death of the worms was only recorded after ascertaining that the worms neither moved nor responded to stimuli when they were vigorously shaken nor when they were dipped in hot water (50°C), followed by the fading away of their body colours. This experiment was run in triplicates for data analysis.


### 2.9. Anti-Inflammatory Assay

The assay was carried out on the extracts ofC. nioroense according to the standard protocol previously described [1, 32]. The egg albumin denaturation method was employed for this assay since anti-inflammatory agents have been reported to inhibit the thermal denaturation of proteins. Test samples containing 0.2 mL of the egg albumin sol (freshly laid egg of a hen), 2.8 mL of phosphate-buffered saline (PBS, pH of 6.4), and 2.0 mL of each of the crude extracts and standard drugs at various concentrations (100.0, 50.0, 25.0, and 12.5 μg/mL) were measured out into different test tubes. A volume of 2 mL of doubled distilled water solution served as a negative control. The mixture was then incubated at 37°C in a BOD incubator for about 15 min and heated at 70°C for 5 min. The absorbance of each reaction mixture was taken after cooling at a wavelength of 660 nm. Standard drugs (aspirin and diclofenac) were used as reference drugs and treated as such for the determination of absorbance. The experimental procedure was repeated in triplicates. The percentage inhibition of protein denaturation by the extracts and standard drugs was calculated using the following formula:(4)Percentageinhibition=Abscontrol−AbstreatedAbscontrol×100,where Abs(control) = absorbance of the negative control and Abs(treated) = absorbance of the test solution.


### 2.10. Thin Layer Chromatography (TLC)

TLC was employed in determining the number of components present in the extracts and obtaining an appropriate solvent system for column chromatography. The basis of separation with respect to TLC is dependent on polarity differences in compounds. The ratio of 1 : 4 (ethyl acetate-hexane) gave the best separation of the least polar components for both extracts [26]. The retardation factor (Rf) of the eluted spots was calculated as follows:(5)Rf=DistancetravelledbythespotDistancetravelledbythesolventfront.The ethyl acetate-hexane (1 : 4) solvent system was then adapted and used in column chromatographic separation.


### 2.11. Column Chromatographic Separation

Flash column chromatography was done to isolate the first three components (A, B, and C) of the ethanol extract using 40–63μm silica gel (200 × 400 mesh) [26]. A 1.0 g dry powdered ethanol extract was column chromatographed and eluted using solvent ratios of hexane-ethyl acetate (45.0 : 5.0, 47.5 : 2.5, and 50.0 mL each) to give fractions A–C. TLC (eluent, hexane-ethyl acetate 9 : 1 and 4 : 1) was used to ascertain the purity of the fractions. Phytochemical screening was further carried out on the three fractions to determine their accompanied class of secondary metabolite. Fractions A and C tested positive only for the presence of steroids, and fraction B gave a positive test only for terpenoids.


### 2.12. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

FTIR (UATR Two, PerkinElmer) was used to determine the functional groups present in the separated fractions (A-C). The wavenumbers between 4000 cm−1 and 400 cm−1 were scanned, followed by baseline correction.


### 2.13. Statistical Analysis of Data

Data were analyzed with GraphPad Prism Version 5.0 for Windows (GraphPad Software Inc, San Diego, CA, USA) and Microsoft Excel 2019 (Microsoft Office Suite, 2019). The data from the experiment were reported as mean ± standard error of the mean (SEM).


## 2.1. Plant Collection, Identification, and Authentication

The collection of the stem barks ofC. nioroense was conducted at Nkawkaw in the Kwahu South District in the eastern region of Ghana (Latitude: 6°33′4.36″N, Longitude: 0°45′58.32″E) in October 2020. The plant material was authenticated by Mr. Clifford Osafo Asare, a botanist at the Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences (FPPS), KNUST, Kumasi, Ghana. A voucher specimen sample (specimen number: KNUST/HMI/2021/SB005) was deposited at the department’s herbarium.


## 2.2. Chemicals and Reagents

All chemicals (except for standard drugs) were purchased from Sigma Aldrich Co. Ltd., Irvine, U.K. All organic solvents (analytical grade) used in the experimental work were purchased from BDH Laboratory Supplies (England).


## 2.3. Preparation of Plant Material

The stem barks ofC. nioroense were cut into pieces, washed with water, air-dried at room temperature (28 to 32°C) for two weeks, then milled to powder and stored in a desiccator [1].


### 2.3.1. Extraction of Ethanol and Petroleum Ether Soluble Plant Materials

A mass of 100 g of the pulverized sample ofC. nioroense was packed into a cellulose thimble and extracted via Soxhlet extraction following a method previously described [1]. The extraction was done successively, starting with 500 mL of petroleum ether and then ethanol. Each crude extract was filtered and evaporated using a rotary evaporator (Buchi Rotavapor R-114) at 50°C. The crude extracts were dried in an oven at 40°C, and their respective percentage yields of 10.416 and 0.590% for ethanol and petroleum ether extracts with respect to the mass of powdered plant samples were determined. The crude extracts were stored in a refrigerator at 4°C for further experimental analyses.


## 2.3.1. Extraction of Ethanol and Petroleum Ether Soluble Plant Materials

A mass of 100 g of the pulverized sample ofC. nioroense was packed into a cellulose thimble and extracted via Soxhlet extraction following a method previously described [1]. The extraction was done successively, starting with 500 mL of petroleum ether and then ethanol. Each crude extract was filtered and evaporated using a rotary evaporator (Buchi Rotavapor R-114) at 50°C. The crude extracts were dried in an oven at 40°C, and their respective percentage yields of 10.416 and 0.590% for ethanol and petroleum ether extracts with respect to the mass of powdered plant samples were determined. The crude extracts were stored in a refrigerator at 4°C for further experimental analyses.


## 2.4. Phytochemical Screening

The phytochemical screening of the pulverized sample and the extracts (petroleum ether and ethanol) ofC. nioroense were carried out using standard protocols [20].


## 2.5. In Vitro Cytotoxicity Assay Using the MTT Method

The MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity [21]. Roswell Park Memorial Institute (RPMI) culture medium supplemented with 10% fetal bovine serum (FBS), containing penicillin, streptomycin, and L-glutamine, was maintained in culture at 37°C in the presence of a humidified 5% CO2 atmosphere in an incubator prior to the assay. The tetrazolium-based colorimetric MTT assay was used to determine the cytotoxicity of the ethanol extract on the JURKAT cell line. A 10 mL stock solution of MTT was prepared. A 50 mg MTT powder was dissolved in 10 mL phosphate-buffered saline (PBS), pH = 7.4, to make 0.5 mg/mL. The MTT solution was filter-sterilized through a 0.2 μM filter and transferred into a sterile, light protected container. The MTT solution was stored and protected from light at 4°C for frequent use. Cells were seeded into the 96-well plates at a concentration of 1 × 104 cells/well, treated with varying concentrations of the ethanol extract (0.0, 62.5, 125.0, 250.0, 500.0, and 1000.0 μg/mL), and incubated for 72 h. The concentration at 0 μg/mL had only seeded cells with no ethanol extract added (factor control), the one at 1000 μg/mL was used as the positive control with the highest amount of ethanol extract added, and lastly, acidified isopropanol was used in the last wells as a negative control. A colour control plate was also set up with varying concentrations of the drug standard, curcumin (0.0, 2.302, 4.604, 9.208, 18.417, and 36.834 μg/mL), and incubated for 72 h. MTT solution (0.5 mg/mL) was added to each well on the plate and incubation continued for a further 4 h. The reaction was stopped with an acidified isopropanol solution, and the plate was incubated in the darkness overnight at room temperature before the absorbance was read at 570 nm using a microplate reader (Tecan Infinite M200, Grodig, Austria). The assay was done in triplicates. The percentage of cell viability was determined as follows:(1)%cellviability=A0−A1A−A1×100,where Ao = mean absorbance of test wells, A1 = mean absorbance in negative control wells, and A = mean absorbance of factor control wells.The IC50 values were determined from the plot of percentage cell viability against the extract concentrations.


## 2.6. Determination of In Vitro Antimicrobial Activity

### 2.6.1. Sources of Microorganisms

Pure cultures ofStaphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 29212), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 4853), and the clinical strain fungus, Candida albicans, were obtained from the Microbiology Section, Department of Pharmaceutics, College of Health Sciences, KNUST, Kumasi. These microbial strains were subcultured aseptically on freshly prepared nutrient agar slants and incubated at 37°C for 24 h. The test organisms were standardized at 0.5 M McFarland for use in each experiment [2, 22].


### 2.6.2. Agar Well Diffusion Method

The antimicrobial activities of the extracts ofC. nioroense were determined using the agar well diffusion method previously described [23, 24]. The nutrient agar (Oxoid, United Kingdom) plates were inoculated with 100 μL of the test organisms using a micropipette and sterile pipette tips, as demonstrated by the pour plate method. By means of a sterile cork borer, 4 wells (10 mm in diameter) were made in the agar and filled with 200 μL of the extracts (6.25, 12.5, and 25 mg/mL) and reference antimicrobial drugs ciprofloxacin (for bacterial strains) and clotrimazole (for fungal strains) at 0.05 mg/mL concentrations. The experiment was done in triplicates for the extracts and reference drugs, and the plates were incubated. The diameter zones of growth inhibition produced were measured using a millimetre rule after 24 h of incubation at 37°C for bacteria and 72 h at 28°C for fungus, and the mean values were calculated.


### 2.6.3. Broth Microdilution

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts ofC. nioroense were determined using the microwell broth dilution method [23, 24]. The inoculums of test microorganisms were prepared from 24-hour broth cultures by serial dilution of 1 mL culture into 9 mL sterile water and then compared to the 0.5 McFarland turbidity standard (1.5 × 105 CFU/mL). The polar plant extract (ethanol) was solubilized in dimethyl sulfoxide (DMSO), and the nonpolar plant extract (petroleum ether) was solubilized in polysorbate 20. A 100 μL single strength nutrient broth was transferred into each well of the 96-well sterile plates followed by 125 μL of two-fold serial dilutions of the extract concentrations (25.00, 12.50, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953, 0.09765, 0.048828, 0.02441, and 0.0122 mg/mL), as well as 25 μL of the inoculum was added. Ciprofloxacin and clotrimazole were the positive control drugs used in the analysis with prepared concentrations (0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.001563, 0.000781, and 0.000390 mg/mL). DMSO and polysorbate 20 were used as negative controls. The microplates were incubated at 37°C for 24 h and observed for inhibition of microbial growth by the addition of 20 μL (0.2 mg/mL) solution of MTT (Sigma-Aldrich, St. Louis, M.O., USA) for 30 min. After 30 min of addition of the dye solution, the wells that developed dark purple colour indicate the presence of microbial growth as the dehydrogenase enzymes in the live bacteria react with the tetrazolium salt to form a dark complex pink colour. However, wells that do not develop any colour change but retain the yellow colour of the dye solution are indicative of the inhibition of microbial growth by the extracts. For MBC determination, 100 μL cultures were transferred from all wells that showed clear inhibition into another 100 μL sterile Nutrient Broth in 96-well plates to a total volume of 200 μL and incubated for a further 24 h. Afterward, the plate was observed to ascertain which concentrations of the extracts caused 99.9% of the microorganisms’ deaths. All the experiments were conducted in triplicate.


## 2.6.1. Sources of Microorganisms

Pure cultures ofStaphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 29212), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 4853), and the clinical strain fungus, Candida albicans, were obtained from the Microbiology Section, Department of Pharmaceutics, College of Health Sciences, KNUST, Kumasi. These microbial strains were subcultured aseptically on freshly prepared nutrient agar slants and incubated at 37°C for 24 h. The test organisms were standardized at 0.5 M McFarland for use in each experiment [2, 22].


## 2.6.2. Agar Well Diffusion Method

The antimicrobial activities of the extracts ofC. nioroense were determined using the agar well diffusion method previously described [23, 24]. The nutrient agar (Oxoid, United Kingdom) plates were inoculated with 100 μL of the test organisms using a micropipette and sterile pipette tips, as demonstrated by the pour plate method. By means of a sterile cork borer, 4 wells (10 mm in diameter) were made in the agar and filled with 200 μL of the extracts (6.25, 12.5, and 25 mg/mL) and reference antimicrobial drugs ciprofloxacin (for bacterial strains) and clotrimazole (for fungal strains) at 0.05 mg/mL concentrations. The experiment was done in triplicates for the extracts and reference drugs, and the plates were incubated. The diameter zones of growth inhibition produced were measured using a millimetre rule after 24 h of incubation at 37°C for bacteria and 72 h at 28°C for fungus, and the mean values were calculated.


## 2.6.3. Broth Microdilution

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts ofC. nioroense were determined using the microwell broth dilution method [23, 24]. The inoculums of test microorganisms were prepared from 24-hour broth cultures by serial dilution of 1 mL culture into 9 mL sterile water and then compared to the 0.5 McFarland turbidity standard (1.5 × 105 CFU/mL). The polar plant extract (ethanol) was solubilized in dimethyl sulfoxide (DMSO), and the nonpolar plant extract (petroleum ether) was solubilized in polysorbate 20. A 100 μL single strength nutrient broth was transferred into each well of the 96-well sterile plates followed by 125 μL of two-fold serial dilutions of the extract concentrations (25.00, 12.50, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953, 0.09765, 0.048828, 0.02441, and 0.0122 mg/mL), as well as 25 μL of the inoculum was added. Ciprofloxacin and clotrimazole were the positive control drugs used in the analysis with prepared concentrations (0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.001563, 0.000781, and 0.000390 mg/mL). DMSO and polysorbate 20 were used as negative controls. The microplates were incubated at 37°C for 24 h and observed for inhibition of microbial growth by the addition of 20 μL (0.2 mg/mL) solution of MTT (Sigma-Aldrich, St. Louis, M.O., USA) for 30 min. After 30 min of addition of the dye solution, the wells that developed dark purple colour indicate the presence of microbial growth as the dehydrogenase enzymes in the live bacteria react with the tetrazolium salt to form a dark complex pink colour. However, wells that do not develop any colour change but retain the yellow colour of the dye solution are indicative of the inhibition of microbial growth by the extracts. For MBC determination, 100 μL cultures were transferred from all wells that showed clear inhibition into another 100 μL sterile Nutrient Broth in 96-well plates to a total volume of 200 μL and incubated for a further 24 h. Afterward, the plate was observed to ascertain which concentrations of the extracts caused 99.9% of the microorganisms’ deaths. All the experiments were conducted in triplicate.


## 2.7. Antioxidant Activity

### 2.7.1. DPPH Radical Scavenging Assay

The antioxidant activity of the extracts ofC. nioroense was determined using the standard methods previously described for the DPPH assay [25, 26]. Ascorbic acid was used as the reference standard. The absorbance of each mixture was measured at 517 nm after incubation [26]. The experimental procedure was repeated in triplicate for data analysis. DPPH radical (%) scavenging was calculated using the following formula:(2)%scavenging=Ao−AAo×100,where AO = absorbance of the control and A = absorbance of the test solution.


### 2.7.2. Hydrogen Peroxide (H2O2) Scavenging Assay

The H2O2 scavenging activity of the extracts of C. nioroense was assessed using the standard methods previously described for the H2O2 assay [27, 28]. Ferrous ammonium sulphate (0.5 mL, 1 mM), distilled water (3 mL), and 1, 10-phenanthroline (3 mL, 1 mM) were used as the negative control, and gallic acid as the reference standard. The absorbance was read at 510 nm using a UV-VIS spectrophotometer. The procedure was repeated in triplicate for data analysis. H2O2 percentage scavenging activity of the extracts was calculated using the following formula:(3)%scavenging=AtestAcontrol×100,where Atest is the absorbance of the test samples and Acontrol is the absorbance of the negative control. The results were further reported in IC50.


### 2.7.3. Total Antioxidant Capacity (TAC) Assay

The total antioxidant capacity of the extracts ofC. nioroense was assessed using the standard protocol previously reported for the assay [27, 29]. Distilled water was used as the blank and ascorbic acid as the reference standard. The absorbance of the solutions was measured in triplicates using the UV-vis spectrophotometer at 695 nm. The procedure was repeated in triplicate for data analysis [27].


## 2.7.1. DPPH Radical Scavenging Assay

The antioxidant activity of the extracts ofC. nioroense was determined using the standard methods previously described for the DPPH assay [25, 26]. Ascorbic acid was used as the reference standard. The absorbance of each mixture was measured at 517 nm after incubation [26]. The experimental procedure was repeated in triplicate for data analysis. DPPH radical (%) scavenging was calculated using the following formula:(2)%scavenging=Ao−AAo×100,where AO = absorbance of the control and A = absorbance of the test solution.


## 2.7.2. Hydrogen Peroxide (H2O2) Scavenging Assay

The H2O2 scavenging activity of the extracts of C. nioroense was assessed using the standard methods previously described for the H2O2 assay [27, 28]. Ferrous ammonium sulphate (0.5 mL, 1 mM), distilled water (3 mL), and 1, 10-phenanthroline (3 mL, 1 mM) were used as the negative control, and gallic acid as the reference standard. The absorbance was read at 510 nm using a UV-VIS spectrophotometer. The procedure was repeated in triplicate for data analysis. H2O2 percentage scavenging activity of the extracts was calculated using the following formula:(3)%scavenging=AtestAcontrol×100,where Atest is the absorbance of the test samples and Acontrol is the absorbance of the negative control. The results were further reported in IC50.


## 2.7.3. Total Antioxidant Capacity (TAC) Assay

The total antioxidant capacity of the extracts ofC. nioroense was assessed using the standard protocol previously reported for the assay [27, 29]. Distilled water was used as the blank and ascorbic acid as the reference standard. The absorbance of the solutions was measured in triplicates using the UV-vis spectrophotometer at 695 nm. The procedure was repeated in triplicate for data analysis [27].


## 2.8.In Vitro Anthelmintic Activity (Adult Worm Mortality Assay)

### 2.8.1. Collection ofMilsonia ghanensis for Anthelminthic Activity

Milsonia ghanensis (common name: adult Indian earthworms; order: Haplotaxida) were used for this analysis because they have an anatomical and physiological resemblance to the human intestinal roundworm [30, 31]. Adult M. ghanensis earthworms (measuring 6.3–7.2 cm in length and 0.20–0.30 cm in width) were collected from the soil on the banks of the Wiwi River behind the Department of Theoretical and Applied Biology Building within KNUST, Kumasi, Ghana, and authenticated by Mr. Lawrence Yeboah in the Zoology Unit, Department of Theoretical and Applied Biology, KNUST, Kumasi, Ghana. The earthworms were thoroughly washed in sterile distilled water to get rid of all soil debris prior to the experiment [27].


### 2.8.2. Adult Worm Mortality Assay

The anthelmintic assay of the extracts ofC. nioroense was assessed using a modification of the methods previously reported [1, 31]. Concentrations of 6.00, 3.00, 1.50, and 0.75 mg/mL of petroleum ether and ethanol extracts of C. nioroense and the standard drug (albendazole) were prepared from a stock solution of 12.00 mg/mL using sterile distilled water as solvent. Double distilled water was used as a negative control. The time of death of the worms was only recorded after ascertaining that the worms neither moved nor responded to stimuli when they were vigorously shaken nor when they were dipped in hot water (50°C), followed by the fading away of their body colours. This experiment was run in triplicates for data analysis.


## 2.8.1. Collection ofMilsonia ghanensis for Anthelminthic Activity

Milsonia ghanensis (common name: adult Indian earthworms; order: Haplotaxida) were used for this analysis because they have an anatomical and physiological resemblance to the human intestinal roundworm [30, 31]. Adult M. ghanensis earthworms (measuring 6.3–7.2 cm in length and 0.20–0.30 cm in width) were collected from the soil on the banks of the Wiwi River behind the Department of Theoretical and Applied Biology Building within KNUST, Kumasi, Ghana, and authenticated by Mr. Lawrence Yeboah in the Zoology Unit, Department of Theoretical and Applied Biology, KNUST, Kumasi, Ghana. The earthworms were thoroughly washed in sterile distilled water to get rid of all soil debris prior to the experiment [27].


## 2.8.2. Adult Worm Mortality Assay

The anthelmintic assay of the extracts ofC. nioroense was assessed using a modification of the methods previously reported [1, 31]. Concentrations of 6.00, 3.00, 1.50, and 0.75 mg/mL of petroleum ether and ethanol extracts of C. nioroense and the standard drug (albendazole) were prepared from a stock solution of 12.00 mg/mL using sterile distilled water as solvent. Double distilled water was used as a negative control. The time of death of the worms was only recorded after ascertaining that the worms neither moved nor responded to stimuli when they were vigorously shaken nor when they were dipped in hot water (50°C), followed by the fading away of their body colours. This experiment was run in triplicates for data analysis.


## 2.9. Anti-Inflammatory Assay

The assay was carried out on the extracts ofC. nioroense according to the standard protocol previously described [1, 32]. The egg albumin denaturation method was employed for this assay since anti-inflammatory agents have been reported to inhibit the thermal denaturation of proteins. Test samples containing 0.2 mL of the egg albumin sol (freshly laid egg of a hen), 2.8 mL of phosphate-buffered saline (PBS, pH of 6.4), and 2.0 mL of each of the crude extracts and standard drugs at various concentrations (100.0, 50.0, 25.0, and 12.5 μg/mL) were measured out into different test tubes. A volume of 2 mL of doubled distilled water solution served as a negative control. The mixture was then incubated at 37°C in a BOD incubator for about 15 min and heated at 70°C for 5 min. The absorbance of each reaction mixture was taken after cooling at a wavelength of 660 nm. Standard drugs (aspirin and diclofenac) were used as reference drugs and treated as such for the determination of absorbance. The experimental procedure was repeated in triplicates. The percentage inhibition of protein denaturation by the extracts and standard drugs was calculated using the following formula:(4)Percentageinhibition=Abscontrol−AbstreatedAbscontrol×100,where Abs(control) = absorbance of the negative control and Abs(treated) = absorbance of the test solution.


## 2.10. Thin Layer Chromatography (TLC)

TLC was employed in determining the number of components present in the extracts and obtaining an appropriate solvent system for column chromatography. The basis of separation with respect to TLC is dependent on polarity differences in compounds. The ratio of 1 : 4 (ethyl acetate-hexane) gave the best separation of the least polar components for both extracts [26]. The retardation factor (Rf) of the eluted spots was calculated as follows:(5)Rf=DistancetravelledbythespotDistancetravelledbythesolventfront.The ethyl acetate-hexane (1 : 4) solvent system was then adapted and used in column chromatographic separation.


## 2.11. Column Chromatographic Separation

Flash column chromatography was done to isolate the first three components (A, B, and C) of the ethanol extract using 40–63μm silica gel (200 × 400 mesh) [26]. A 1.0 g dry powdered ethanol extract was column chromatographed and eluted using solvent ratios of hexane-ethyl acetate (45.0 : 5.0, 47.5 : 2.5, and 50.0 mL each) to give fractions A–C. TLC (eluent, hexane-ethyl acetate 9 : 1 and 4 : 1) was used to ascertain the purity of the fractions. Phytochemical screening was further carried out on the three fractions to determine their accompanied class of secondary metabolite. Fractions A and C tested positive only for the presence of steroids, and fraction B gave a positive test only for terpenoids.


## 2.12. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

FTIR (UATR Two, PerkinElmer) was used to determine the functional groups present in the separated fractions (A-C). The wavenumbers between 4000 cm−1 and 400 cm−1 were scanned, followed by baseline correction.


## 2.13. Statistical Analysis of Data

Data were analyzed with GraphPad Prism Version 5.0 for Windows (GraphPad Software Inc, San Diego, CA, USA) and Microsoft Excel 2019 (Microsoft Office Suite, 2019). The data from the experiment were reported as mean ± standard error of the mean (SEM).


## 3. Results and Discussion

### 3.1. Phytochemical Screening

The phytochemical screening ofC. nioroense revealed the presence of twelve out of the seventeen phytoconstituents tested in both the pulverized sample and the ethanol extract, except anthocyanins, anthraquinones, carotenoids, proteins and amino acids, and phlobatannins, which were absent. However, in the petroleum ether extract, saponins, phenols, alkaloids, flavonoids, tannins, anthocyanins, anthraquinones, steroids, carotenoids, phytosterols, phlobatannins, proteins and amino acids, and cardiac glycosides were absent (Table 1).Table 1
Phytoconstituents present in the pulverized sample and extracts ofC. nioroense.
PhytoconstituentsPulverized sampleEthanol extractPetroleum ether extractGlycosides+++Saponins++−Phenols++−Coumarins+++Alkaloids++−Flavonoids++−Tannins++−Anthocyanins−−−Anthraquinones−−−Steroids++−Carotenoids−−−Phytosterols++−Phlobatannins−−−Flavanol++−Proteins and amino acids−−−Terpenoids+++Cardiac glycosides++−(+) = presence of the secondary metabolite; (−) = absence of the secondary metabolite.The ethanol and petroleum ether extracts had three phytochemicals in common, namely, glycosides, coumarins, and terpenoids. The phytochemicals present could be responsible for some of the biological activities ofC. nioroense. The phytochemicals such as flavonoids, tannins, steroids, saponins, terpenoids, triterpenes, and alkaloids identified in many species of Combretum are known to exhibit a wide array of pharmacological activities such as antinociceptive, antimicrobial, antioxidant, haematological, anti-inflammatory, anthelminthic, cicatrizing, cytotoxic, mutagenic, neuroprotective activity, and many others [10, 11, 16, 33–38]. Viau et al. reported that Combretum leprosum is rich in cycloartanes, triterpenes (arjunolic acid, mollic acid, and 3β, 6β, 16β-trihydroxy-lup-20(29)-ene), and flavonoids (3-O-methylquercetin, 5, 3′-dihydroxy-3, 7, 4′-trimethoxyflavone, 5, 3′, 4′-trihydroxy-3, 7-dimethoxyflavone, and quercetin) [39]. The phytochemicals identified in C. nioroense could contribute to cytotoxicity, antimicrobial, antioxidant, anthelmintic, and anti-inflammatory activities.


### 3.2. In Vitro Cytotoxicity Assay

This assay has been widely used to measure thein vitro cytotoxic effects of extracts and standard drugs in cell lines. The assay is based on the reduction of a yellow tetrazolium salt MTT to purple formazan crystals by metabolically active living cells, which determines mitochondrial activity. This is because for most cell populations, the total mitochondrial activity is related to the number of viable cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes, which reduce the MTT to formazan [21, 40]. The insoluble formazan crystals were solubilized for homogenous measurement, and the resulting-coloured solution was quantified by measuring absorbance at 570 nm using a multiwell spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells [40]. The ethanol extract was found to be cytotoxic, with an IC50 value of 115.4 ± 1.332 μg/mL (Figures 1(a) and 1(b), Table 2).Table 2
Cytotoxicity of the ethanol extract ofC. nioroense and curcumin expressed as IC50.
SamplesIC50 (μg/mL)Standard (curcumin)6.562 ± 1.007Ethanolic extract115.4 ± 1.332Figure 1
(a) Cytotoxicity of curcumin at various concentrations. (b) Cytotoxicity of ethanol extract ofC. nioroense at various concentrations.
(a)(b)The results showed cytotoxic activity in a concentration-dependent manner (Figure1). Pettit et al. reported that Combretum caffrum was found to be significantly active against murine P-388 lymphocytic leukemia [41]. Similar reports indicated that fosbretabulin (combretastatin A4 phosphate), isolated from the bark of C. caffrum, showed leads in the treatment of thyroid cancer [15]. The cytotoxicity results show that extracts of the stem bark of C. nioroense may be beneficial to humans in minimizing diseases related to cancer.


### 3.3. In Vitro Antimicrobial Activity

The extracts ofC. nioroense stem bark were assessed using the agar well diffusion and broth microdilution methods (MIC and MBC) to analyse the efficacy of the extracts as antimicrobial agents.


#### 3.3.1. Susceptibility Testing Using the Agar Well Diffusion Method

The agar well diffusion method’s simplicity and reproducibility were used to determine the antimicrobial activities of the extracts in this assay at concentrations of 6.25, 12.5, and 25.0 mg/mL, with the reference drugs at 0.05 mg/mL [27]. The dose-dependent antimicrobial activity exhibited by the reference drugs and the extracts is shown in Table 3.Table 3
Mean zones of growth inhibition (ZI) of ethanol and petroleum ether extracts ofC. nioroense against test organisms.
Sample/drugConcentration (mg/mL)Mean zones of growth inhibition (mm) (mean ± SEM)E. coliS. aureusB. subtilisP. aeruginosaC. albicansCiprofloxacin0.0535.5 ± 0.12134.5 ± 0.21735.5 ± 0.08145.0 ± 0.164NDClotrimazole0.05NDNDNDND18.5 ± 0.171Ethanol extract25.0020.5 ± 0.01921.0 ± 0.06121.0 ± 0.09924.0 ± 0.07724.5 ± 0.18412.5016.0 ± 0.02018.0 ± 0.09017.0 ± 0.09716.5 ± 0.06817.5 ± 0.0886.2514.5 ± 0.21314.5 ± 0.18612.0 ± 0.18611.0 ± 0.12812.5 ± 0.089Petroleum ether extract25.0017.0 ± 0.14613.0 ± 0.11910.5 ± 0.210——12.5014.5 ± 0.02411.5 ± 0.030———6.25—————ND = not determined, the diameter of well (10 mm).From the results obtained, the antimicrobial activity of the extracts ofC. nioroense was concentration-dependent (Table 1). The petroleum ether extract showed no activity against P. aeruginosa and C. albicans at the tested concentrations. The lowest concentration at which the ethanol extract recorded a zone of inhibition was 6.25 mg/mL, whilst the petroleum ether extract was 12.5 mg/mL. The most susceptible organism to the ethanol extract was C. albicans, with a mean diameter zone of inhibition of 24.5 ± 0.184 mm, with E. coli (20.5 ± 0.019 mm) being the least susceptible organism at the highest concentration of 25.0 mg/mL. S. aureus was the most susceptible organism to the ethanol extract at a concentration of 12.5 mg/mL, whereas S. aureus and E. coli were the most susceptible organisms at the lowest concentration of 6.25 mg/mL. The most susceptible organism to the petroleum ether extract was E. coli, with a mean diameter zone of inhibition of 17.0 ± 0.146 mm, with B. subtilis (10.5 ± 0.210 mm) being the least susceptible organism at the highest concentration of 25.0 mg/mL. E. coli was the most susceptible organism, with a mean diameter zone of inhibition of 14.5 ± 0.024 mm at a lower concentration of 12.5 mg/mL. P. aeruginosa was the most susceptible organism to ciprofloxacin (standard drug). Clotrimazole (standard drug) showed activity against the fungus C. albicans. The results showed that the extracts of C. nioroense contained broad-spectrum antimicrobial agents that were effective and capable of inhibiting the growth of the selected microorganisms employed for the assay with variable activity.


#### 3.3.2. Minimum Inhibitory Concentration (MIC) of the Extracts ofC. nioroense

The MICs of the extracts of the stem bark ofC. nioroense against the test organisms are shown in Table 4.Table 4
MICs values ofC. nioroense extracts and reference drugs concentrations against test organisms.
Test organismsMinimum inhibitory concentration, MIC (mg/mL)Ethanol extractPetroleum ether extractCiprofloxacinClotrimazoleE. coli0.048812.501.5625 × 10−3NDS. aureus0.048812.501.5625 × 10−3NDP. aeruginosa0.096525.006.25 × 10−3NDB. subtilis0.048825.001.5625 × 10−3NDC. albicans0.012225.00ND3.125 × 10−3ND = not determined.The MICs of the extracts ofC. nioroense against the test organisms were in the range of 0.0122–25.0 mg/mL. The ethanol extract showed the highest inhibitory antimicrobial activity against C. albicans and the least against P. aeruginosa, with MICs of 0.0122 and 0.0965 mg/mL, respectively. The highest inhibitory antimicrobial activity of the petroleum ether extract was observed against E. coli and S. aureus at 12.5 mg/mL, whilst the lowest was observed against P. aeruginosa, B. subtilis, and C. albicans at 25.0 mg/mL. The broad spectrum antimicrobial activity exhibited by the extracts of C. nioroense could be attributed to the presence of the actions of individual phytoconstituents present in the stem bark or synergistic activities [17, 20–23].


#### 3.3.3. Minimum Bactericidal Concentration (MBC) of the Extracts ofC. nioroense

The ethanol and petroleum ether extracts of the stem bark ofC. nioroense exhibited varying antimicrobial activity against the test organisms, as shown in Table 5.Table 5
MBCs values ofC. nioroense extracts and reference drugs concentrations against test organisms.
Test organismsMinimum bactericidal concentration, MBC (mg/mL)Ethanol extractPetroleum ether extractCiprofloxacinClotrimazoleE. coli6.25>25.006.25 × 10−3NDS. aureus6.25>25.006.25 × 10−3NDP. aeruginosa12.5>25.0012.5 × 10−3NDB. subtilis6.25>25.003.125 × 10−3NDC. albicans3.125>25.00ND25.0 × 10−3ND = not determined.The MBCs of the ethanol extract ofC. nioroense against the test organisms at various concentrations ranging from 3.125 to 12.5 mg/mL do not only inhibit the growth of microorganisms but also kill them entirely. The ethanol extract of C. nioroense exhibited the most antimicrobial activity against C. albicans and the least against P. aeruginosa, with MBCs of 3.135 and 12.5 mg/mL, respectively. The MBCs of the ethanol extract of C. nioroense for E. coli, S. aureus, and B. subtilis were 6.25 mg/mL, whilst the petroleum ether extract did not register any activity against the test organisms. The antimicrobial properties displayed by the ethanol extract of C. nioroense may be associated with the synergistic activities or the actions of individual secondary metabolites present in the stem bark [42]. Therefore, this confirms the traditional usage of boiling both the leaves and stem bark as medication for the treatment of bacterial and helminthic infections and the broad spectrum antimicrobial properties [10].


### 3.4. In Vitro Antioxidant Activity

Reports indicate that plants with good antimicrobial activity are likely to exhibit antioxidant activity as well [43]. Due to the different mechanisms of antioxidant actions, the DPPH and H2O2 radical scavenging assays and the TAC assays were used to evaluate the antioxidant properties of the extracts of C. nioroense [1].


#### 3.4.1. DPPH Radical Scavenging Capacity

The ethanol and petroleum ether extracts ofC. nioroense and ascorbic acid (reference standard) scavenged DPPH at concentrations of 6.25 μg/mL to 100 μg/mL, as presented in Figure 2.Figure 2
DPPH free radical scavenging activity of the extracts ofC. nioroense and ascorbic acid.The extracts ofC. nioroense showed a dose-dependent increase in antioxidant activity. The IC50 values of the ethanol and petroleum ether extracts of C. nioroense and ascorbic acid ranged from <6.25 to >100.0 μg/mL, as shown in Table 6.Table 6
IC50 of DPPH/H2O2 scavenging activity for the ethanol and petroleum ether extracts of C. nioroense and ascorbic acid/gallic acid.
DPPH radical scavenging activityH2O2 scavenging activitySamplesIC50 (μg/mL)SamplesIC50 (μg/mL)Ascorbic acid<6.25Gallic acid211.50 ± 2.985Ethanolic extract27.94 ± 1.005Ethanol extract322.50 ± 1.005Petroleum ether extract>100.0Petroleum ether extract400.90 ± 3.400The results demonstrated significant antioxidant activity in a concentration-dependent manner, with the ethanol extract showing higher effectiveness than the petroleum ether extract (Figure2 and Table 6). The antioxidant potential of the ethanol extract could be due to the presence of additional phytochemicals like flavonoids, tannins, and phenols [44, 45]. Therefore, C. nioroense may be beneficial to humans in preventing oxidative damage from free radicals.


#### 3.4.2. H2O2 Radical Scavenging Assay

Figure3 indicates the H2O2 radical scavenging capacity of the ethanol and petroleum ether extracts of C. nioroense and gallic acid as a reference standard at different concentrations.Figure 3
H2O2 radical scavenging capacity of the extracts of C. nioroense and gallic acid.The ethanol and petroleum ether extracts ofC. nioroense demonstrated a concentration-dependent increase in antioxidant activity, with IC50 values ranging from 211.50 ± 2.985 to 400.90 ± 3.400 μg/mL, as shown in Table 6.The ethanol extract showed better antioxidant activity than petroleum ether extract, which might be due to the presence of extra phytochemicals like flavonoids, tannins, and phenols, although they are all good antioxidants that could be exploited in minimizing oxidative stress [14].


#### 3.4.3. Total Antioxidant Capacity (TAC)

Ascorbic acid concentrations of 6.25–100μg/mL showed antioxidant activity and mean absorbance values between 0.313667 ± 0.002082 and 0.909667 ± 0.000208 at a wavelength of 695 nm, as presented in Figure 4.Figure 4
Mean absorbance ofPMo4VMo8VIO407− against the concentration of ascorbic acid.The TAC was established to be proportional to the concentration of the extracts. The TAC of the extract results was expressed as grams of ascorbic acid equivalent per 100 grams of extract (gAAE/100 g) [1]. The ethanol and petroleum ether extracts of C. nioroense had 57.968 ± 0.560 and 47.197 ± 0.533 gAAE/100 g, respectively (Table 7). This result implies that 57.968 g of ethanol extract and 47.197 g of petroleum ether extract of stem bark extracts of C. nioroense out of 100 g of the sample will function as ascorbic acid.Table 7
TAC of ethanol and petroleum ether extracts expressed as gAAE.
SampleTAC (gAAE/100 g)Ethanolic extract57.968 ± 0.560Petroleum ether extract47.197 ± 0.533TAC: total antioxidant capacity; AAE: ascorbic acid equivalent.Generally, the TAC showed a concentration-dependent increase; thus, the higher the TAC, the better the activity of the extract. The extracts displayed appreciable antioxidant activities associated with the secondary metabolites present in the stem bark [1].


### 3.5. In Vitro Anthelminthic Activity

The ethanol and petroleum ether extracts ofC. nioroense were screened against Milsonia ghanensis earthworms in vitro because M. ghanensis has been documented to possess physical and anatomical resemblance to intestinal roundworm parasites of human beings, as well as their ease of availability [1, 16]. The action of the extracts of C. nioroense on the worms was concentration-dependent, with all concentrations (12.0, 6.0, 3.0, 1.5, and 0.75 mg/mL) capable of paralyzing and killing the worms after the maximal exposure time. At the highest concentration of 12 mg/mL, ethanol extract paralyzed and killed all worms within 30.0 ± 0.881 and 54.0 ± 0.207 min of exposure, respectively. Similarly, the petroleum ether extract paralyzed and killed all worms within 39.0 ± 0.171 and 69.0 ± 0.121 min of exposure, respectively. However, the standard drug albendazole paralyzed and killed all worms within 70.0 ± 0.221 and 121.0 ± 0.200 min of exposure, respectively. At the least concentration of 0.75 mg/mL, ethanol extract paralyzed and killed all worms within 88.0 ± 0.714 and 139.0 ± 0.011 min of exposure, respectively. Similarly, the petroleum ether extract paralyzed and killed all worms within 94.0 ± 0.011 and 182.0 ± 0.712 min of exposure, respectively, whilst albendazole (the standard drug) paralyzed and killed all worms within 121 ± 0.070 and 261 ± 0.032 min of exposure, respectively (Figures 5 and 6). The anthelmintic activities of ethanol and petroleum ether extracts at test concentrations were observed to be significantly p<0.001 higher compared to albendazole-treated  worms.Figure 5
Anthelmintic activity of the extracts ofC. nioroense and albendazole (for paralysis) [1].Figure 6
Anthelmintic activity of the extracts ofC. nioroense and albendazole (for death) [1].The anthelmintic activity of the extracts ofC. nioroense (Figures 5 and 6) may be due to the presence of phytochemicals such as alkaloids, saponins, tannins, flavonoids, and glycosides. This is because the aforementioned secondary metabolites in the extracts have been reported to possess anthelmintic activity [46–49]. The mode of action of some anthelmintics like imidazothiazoles, derquantel, and piperazine, which are all alkaloids, is to cause paralysis of worms, such that they can be expelled in the faeces of humans and animals [1, 14]. The extracts not only exhibited such potency but also caused both paralysis and death of the M. ghanensis earthworms at a rate much higher than albendazole. The higher anthelminthic potency exhibited by the ethanol extract than the petroleum ether extract could probably be due to the presence of alkaloids, saponins, and tannins and may lead to the death of helminths.


### 3.6. Anti-Inflammatory Activity

The anti-inflammatory activity of a plant extract was determined by its ability to inhibit the thermal denaturation of protein (egg albumin) [1, 50]. The extracts of C. nioroense and standard drugs (aspirin and diclofenac) displayed concentration-dependentanti-inflammatory activity in preventing thermally induced protein denaturation at test concentrations of 500 to 100 μg/mL. The diclofenac anti-inflammatory activity was significantly p<0.01 higher than that of the extracts. However, the anti-inflammatory activity of the ethanol extracts was observed to be significantly higher p<0.01 than that of aspirin, as presented in Figure 7. The ethanol and petroleum ether extracts showed good activities at all test concentrations (Figure 7) with IC50 values of 24.402 ± 0.569 and 31.254 ± 0.359 μg/mL, respectively (Table 8).Figure 7
Anti-inflammatory action of ethanol and petroleum ether extracts ofC. nioroense [27].Table 8
IC50 of anti-inflammatory activity of extracts of C. nioroense.
SampleIC50 (μg/mL)Diclofenac19.256 ± 0.362Aspirin26.061 ± 0.087Ethanol extract24.402 ± 0.569Petroleum ether extract31.254 ± 0.359The significant anti-inflammatory activity exhibited by the extracts ofC. nioroense could be attributed to the presence of phytoconstituents such as phenols, flavonoids, tannins, saponins, glycosides, terpenoids, alkaloids, and steroids [1, 51–56]. Polyphenols such as flavonoids, tannins, and phenols are reported to inhibit enzymes and serve as mediators of the inflammation process such as C-reactive protein or adhesion molecules [1, 52, 57]. Hence, the anti-inflammatory activity of the extracts shown by preventing thermally induced protein denaturation may be attributed to the actions of individual secondary metabolites or synergistic activities present in the stem bark of C. nioroense.


### 3.7. Thin Layer Chromatography (TLC)

The analytical TLC method using a solvent system of ethyl acetate-hexane (1 : 4) was used to determine the number of less polar components present in the extracts. The various separated TLC spots, representing the less polar compounds in the extracts and theirRf values, were observed and determined (Table 9).Table 9
TLCs of the less polar constituents ofC. nioroense extracts.
ComponentsRetardation factor,RfEthanol extractPetroleum ether extractA0.086NDB0.1710.171C0.2290.229D0.3140.314E0.4000.400F0.5710.571G0.7570.757ND = not detected.The TLC results of the petroleum ether and ethanol extracts ofC. nioroense showed seven and six distinct spots, respectively, with Rf values ranging from 0.171 to 0.757 and 0.086 to 0.757, respectively, using a solvent system of EtOAc-hexane (1 : 4). A flash column chromatography purification system was used to separate the spots in the ethanol extract.


### 3.8. Column Chromatographic Separation

The less polar components of the ethanol extracts ofC. nioroense were separated employing flash column chromatography. After elution with a solvent system of hexane-EtOAc (50.0 : 0.0–45.0 : 5.0), three fractions, namely, A, B, and C, were separated (Table 10).Table 10
Chromatographic fractions separated, their respectiveRf values and confirmed phytochemical screening results.
FractionsRetardation factor,RfPhytochemical presentA0.757SteroidB0.571TerpenoidC0.400SteroidTheRf values from the column chromatography results confirm the analytical TLC results. The TLC (eluent, EtOAc-hexane (1 : 4)) was used to monitor the separated fractions.


### 3.9. Phytochemical Screening of Separated Fractions

The separated fractions A, B, and C obtained from column chromatography were screened qualitatively to determine the type of secondary metabolite present in the ethanol extract ofC. nioroense (Table 10). The phytochemical screening results identified fractions A and C as steroids and fraction B as a terpenoid. Ongoing research is focused on separating all the components present in the extracts and determining their biological activity. The identified secondary metabolites give credence to their folkloric use.


### 3.10. FTIR Analysis

FTIR analysis was performed on the petroleum ether and ethanol crude extracts as well as the separated fractions (A, B, and C) obtained from the ethanol extract after separation with flash column chromatography (Figures8–12 and Tables 11 and 12).Figure 8
FTIR spectrum of ethanol crude extract ofC. nioroense.Figure 9
FTIR spectrum of petroleum ether crude extract ofC. nioroense.Figure 10
FTIR spectrum of fraction A from the ethanol extract ofC. nioroense.Figure 11
FTIR spectrum of fraction B from the ethanol extract ofC. nioroense.Figure 12
FTIR spectrum of fraction C from the ethanol extract ofC. nioroense.Table 11
FTIR interpretation of the extracts ofC. nioroense.
Ethanolic crude extractPetroleum ether crude extractFunctional groupPeak value (cm−1)Functional groupPeak value (cm−1)O-H/N-H stretch3358Csp2-H stretch3027Csp3-H stretch2922, 2852Csp3-H stretch2935, 2862C=O stretch1690C=O stretch1738, 1713C=C stretch1638, 1513C=C stretch1530C-H bend1452C-H bend1444, 1379C-O stretch1034C-O stretchAr-ring C-H bend1175730Table 12
FTIR interpretation of fractions A, B, and C from the ethanol extract ofC. nioroense.
Fraction AFraction BFraction CFunctional groupPeak value (cm−1)Functional groupPeak value (cm−1)Functional groupPeak value (cm−1)Csp3-H stretch2957, 2924, 2873Csp3-H stretch2924, 2853Csp3-H stretch2956, 2925, 2873C=O stretch1750C=O stretch1737C-H bend1460, 1378C-H bend1460, 1378C-H bend1464, 1365C-O stretch1214C-O stretch1237, 1049C-O stretch1216The wave numbers (cm−1) around 3358 (O-H or N-H), 2922 to 28512 (Csp3-H), 1690 (C=O), 1638, 1513 (C=C), 1452 (C-H), and 1034 (C-O) were observed for the stretching and bending vibrations for the spectrum of the ethanolic crude extract (Table 11). However, the spectrum of the petroleum ether crude extract registered wave numbers (cm−1) around 3027 (Csp2-H), 2935 to 2862 (Csp3-H), 1738, 1713 (C=O), 1530 (C=C), 1444, 1379 (C-H), 1175 (C-O), and 730 (Ar-C-H), representing the identified phytochemicals in the extracts of C. nioroense (Table 1).


#### 3.10.1. Fraction A

The wave numbers (cm−1) around 2873–2957 (Csp3-H), 1750 (C=O), 1460, 1378 (C-H), and 1237, 1049 (C-O) were observed for the stretching and bending vibrations typical of saturated steroidal structural backbones (Table 12) [58, 59]. The functional groups therefore confirm fraction A as a steroid, as identified from the phytochemical screening (Table 1).


#### 3.10.2. Fraction B

The wave numbers (cm−1) around 2924, 2853 (Csp3-H), 1737 (C=O), 1464, 1365 (C-H), and 1216, 978 (C-O) were identified for the stretching and bending vibrations typical of saturated steroidal or terpenoid structural backbones (Table 12) [58, 59]. The observed functional groups, therefore, confirm fraction B as a saturated terpenoid, as identified from the phytochemical screening (Table 1).


#### 3.10.3. Fraction C

The wave numbers (cm−1) around 2873–2957 (Csp3-H), 1460, 1378 (C-H), and 1214 (C-O) were registered for the stretching and bending vibrations for fraction C, thus confirming the presence of a saturated steroid identified from the phytochemical screening (Table 1).The FTIR analysis confirms the identified steroids (fractions A and C) and terpenoids (fraction B) from the screening test (Table1).


## 3.1. Phytochemical Screening

The phytochemical screening ofC. nioroense revealed the presence of twelve out of the seventeen phytoconstituents tested in both the pulverized sample and the ethanol extract, except anthocyanins, anthraquinones, carotenoids, proteins and amino acids, and phlobatannins, which were absent. However, in the petroleum ether extract, saponins, phenols, alkaloids, flavonoids, tannins, anthocyanins, anthraquinones, steroids, carotenoids, phytosterols, phlobatannins, proteins and amino acids, and cardiac glycosides were absent (Table 1).Table 1
Phytoconstituents present in the pulverized sample and extracts ofC. nioroense.
PhytoconstituentsPulverized sampleEthanol extractPetroleum ether extractGlycosides+++Saponins++−Phenols++−Coumarins+++Alkaloids++−Flavonoids++−Tannins++−Anthocyanins−−−Anthraquinones−−−Steroids++−Carotenoids−−−Phytosterols++−Phlobatannins−−−Flavanol++−Proteins and amino acids−−−Terpenoids+++Cardiac glycosides++−(+) = presence of the secondary metabolite; (−) = absence of the secondary metabolite.The ethanol and petroleum ether extracts had three phytochemicals in common, namely, glycosides, coumarins, and terpenoids. The phytochemicals present could be responsible for some of the biological activities ofC. nioroense. The phytochemicals such as flavonoids, tannins, steroids, saponins, terpenoids, triterpenes, and alkaloids identified in many species of Combretum are known to exhibit a wide array of pharmacological activities such as antinociceptive, antimicrobial, antioxidant, haematological, anti-inflammatory, anthelminthic, cicatrizing, cytotoxic, mutagenic, neuroprotective activity, and many others [10, 11, 16, 33–38]. Viau et al. reported that Combretum leprosum is rich in cycloartanes, triterpenes (arjunolic acid, mollic acid, and 3β, 6β, 16β-trihydroxy-lup-20(29)-ene), and flavonoids (3-O-methylquercetin, 5, 3′-dihydroxy-3, 7, 4′-trimethoxyflavone, 5, 3′, 4′-trihydroxy-3, 7-dimethoxyflavone, and quercetin) [39]. The phytochemicals identified in C. nioroense could contribute to cytotoxicity, antimicrobial, antioxidant, anthelmintic, and anti-inflammatory activities.


## 3.2. In Vitro Cytotoxicity Assay

This assay has been widely used to measure thein vitro cytotoxic effects of extracts and standard drugs in cell lines. The assay is based on the reduction of a yellow tetrazolium salt MTT to purple formazan crystals by metabolically active living cells, which determines mitochondrial activity. This is because for most cell populations, the total mitochondrial activity is related to the number of viable cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes, which reduce the MTT to formazan [21, 40]. The insoluble formazan crystals were solubilized for homogenous measurement, and the resulting-coloured solution was quantified by measuring absorbance at 570 nm using a multiwell spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells [40]. The ethanol extract was found to be cytotoxic, with an IC50 value of 115.4 ± 1.332 μg/mL (Figures 1(a) and 1(b), Table 2).Table 2
Cytotoxicity of the ethanol extract ofC. nioroense and curcumin expressed as IC50.
SamplesIC50 (μg/mL)Standard (curcumin)6.562 ± 1.007Ethanolic extract115.4 ± 1.332Figure 1
(a) Cytotoxicity of curcumin at various concentrations. (b) Cytotoxicity of ethanol extract ofC. nioroense at various concentrations.
(a)(b)The results showed cytotoxic activity in a concentration-dependent manner (Figure1). Pettit et al. reported that Combretum caffrum was found to be significantly active against murine P-388 lymphocytic leukemia [41]. Similar reports indicated that fosbretabulin (combretastatin A4 phosphate), isolated from the bark of C. caffrum, showed leads in the treatment of thyroid cancer [15]. The cytotoxicity results show that extracts of the stem bark of C. nioroense may be beneficial to humans in minimizing diseases related to cancer.


## 3.3. In Vitro Antimicrobial Activity

The extracts ofC. nioroense stem bark were assessed using the agar well diffusion and broth microdilution methods (MIC and MBC) to analyse the efficacy of the extracts as antimicrobial agents.


### 3.3.1. Susceptibility Testing Using the Agar Well Diffusion Method

The agar well diffusion method’s simplicity and reproducibility were used to determine the antimicrobial activities of the extracts in this assay at concentrations of 6.25, 12.5, and 25.0 mg/mL, with the reference drugs at 0.05 mg/mL [27]. The dose-dependent antimicrobial activity exhibited by the reference drugs and the extracts is shown in Table 3.Table 3
Mean zones of growth inhibition (ZI) of ethanol and petroleum ether extracts ofC. nioroense against test organisms.
Sample/drugConcentration (mg/mL)Mean zones of growth inhibition (mm) (mean ± SEM)E. coliS. aureusB. subtilisP. aeruginosaC. albicansCiprofloxacin0.0535.5 ± 0.12134.5 ± 0.21735.5 ± 0.08145.0 ± 0.164NDClotrimazole0.05NDNDNDND18.5 ± 0.171Ethanol extract25.0020.5 ± 0.01921.0 ± 0.06121.0 ± 0.09924.0 ± 0.07724.5 ± 0.18412.5016.0 ± 0.02018.0 ± 0.09017.0 ± 0.09716.5 ± 0.06817.5 ± 0.0886.2514.5 ± 0.21314.5 ± 0.18612.0 ± 0.18611.0 ± 0.12812.5 ± 0.089Petroleum ether extract25.0017.0 ± 0.14613.0 ± 0.11910.5 ± 0.210——12.5014.5 ± 0.02411.5 ± 0.030———6.25—————ND = not determined, the diameter of well (10 mm).From the results obtained, the antimicrobial activity of the extracts ofC. nioroense was concentration-dependent (Table 1). The petroleum ether extract showed no activity against P. aeruginosa and C. albicans at the tested concentrations. The lowest concentration at which the ethanol extract recorded a zone of inhibition was 6.25 mg/mL, whilst the petroleum ether extract was 12.5 mg/mL. The most susceptible organism to the ethanol extract was C. albicans, with a mean diameter zone of inhibition of 24.5 ± 0.184 mm, with E. coli (20.5 ± 0.019 mm) being the least susceptible organism at the highest concentration of 25.0 mg/mL. S. aureus was the most susceptible organism to the ethanol extract at a concentration of 12.5 mg/mL, whereas S. aureus and E. coli were the most susceptible organisms at the lowest concentration of 6.25 mg/mL. The most susceptible organism to the petroleum ether extract was E. coli, with a mean diameter zone of inhibition of 17.0 ± 0.146 mm, with B. subtilis (10.5 ± 0.210 mm) being the least susceptible organism at the highest concentration of 25.0 mg/mL. E. coli was the most susceptible organism, with a mean diameter zone of inhibition of 14.5 ± 0.024 mm at a lower concentration of 12.5 mg/mL. P. aeruginosa was the most susceptible organism to ciprofloxacin (standard drug). Clotrimazole (standard drug) showed activity against the fungus C. albicans. The results showed that the extracts of C. nioroense contained broad-spectrum antimicrobial agents that were effective and capable of inhibiting the growth of the selected microorganisms employed for the assay with variable activity.


### 3.3.2. Minimum Inhibitory Concentration (MIC) of the Extracts ofC. nioroense

The MICs of the extracts of the stem bark ofC. nioroense against the test organisms are shown in Table 4.Table 4
MICs values ofC. nioroense extracts and reference drugs concentrations against test organisms.
Test organismsMinimum inhibitory concentration, MIC (mg/mL)Ethanol extractPetroleum ether extractCiprofloxacinClotrimazoleE. coli0.048812.501.5625 × 10−3NDS. aureus0.048812.501.5625 × 10−3NDP. aeruginosa0.096525.006.25 × 10−3NDB. subtilis0.048825.001.5625 × 10−3NDC. albicans0.012225.00ND3.125 × 10−3ND = not determined.The MICs of the extracts ofC. nioroense against the test organisms were in the range of 0.0122–25.0 mg/mL. The ethanol extract showed the highest inhibitory antimicrobial activity against C. albicans and the least against P. aeruginosa, with MICs of 0.0122 and 0.0965 mg/mL, respectively. The highest inhibitory antimicrobial activity of the petroleum ether extract was observed against E. coli and S. aureus at 12.5 mg/mL, whilst the lowest was observed against P. aeruginosa, B. subtilis, and C. albicans at 25.0 mg/mL. The broad spectrum antimicrobial activity exhibited by the extracts of C. nioroense could be attributed to the presence of the actions of individual phytoconstituents present in the stem bark or synergistic activities [17, 20–23].


### 3.3.3. Minimum Bactericidal Concentration (MBC) of the Extracts ofC. nioroense

The ethanol and petroleum ether extracts of the stem bark ofC. nioroense exhibited varying antimicrobial activity against the test organisms, as shown in Table 5.Table 5
MBCs values ofC. nioroense extracts and reference drugs concentrations against test organisms.
Test organismsMinimum bactericidal concentration, MBC (mg/mL)Ethanol extractPetroleum ether extractCiprofloxacinClotrimazoleE. coli6.25>25.006.25 × 10−3NDS. aureus6.25>25.006.25 × 10−3NDP. aeruginosa12.5>25.0012.5 × 10−3NDB. subtilis6.25>25.003.125 × 10−3NDC. albicans3.125>25.00ND25.0 × 10−3ND = not determined.The MBCs of the ethanol extract ofC. nioroense against the test organisms at various concentrations ranging from 3.125 to 12.5 mg/mL do not only inhibit the growth of microorganisms but also kill them entirely. The ethanol extract of C. nioroense exhibited the most antimicrobial activity against C. albicans and the least against P. aeruginosa, with MBCs of 3.135 and 12.5 mg/mL, respectively. The MBCs of the ethanol extract of C. nioroense for E. coli, S. aureus, and B. subtilis were 6.25 mg/mL, whilst the petroleum ether extract did not register any activity against the test organisms. The antimicrobial properties displayed by the ethanol extract of C. nioroense may be associated with the synergistic activities or the actions of individual secondary metabolites present in the stem bark [42]. Therefore, this confirms the traditional usage of boiling both the leaves and stem bark as medication for the treatment of bacterial and helminthic infections and the broad spectrum antimicrobial properties [10].


## 3.3.1. Susceptibility Testing Using the Agar Well Diffusion Method

The agar well diffusion method’s simplicity and reproducibility were used to determine the antimicrobial activities of the extracts in this assay at concentrations of 6.25, 12.5, and 25.0 mg/mL, with the reference drugs at 0.05 mg/mL [27]. The dose-dependent antimicrobial activity exhibited by the reference drugs and the extracts is shown in Table 3.Table 3
Mean zones of growth inhibition (ZI) of ethanol and petroleum ether extracts ofC. nioroense against test organisms.
Sample/drugConcentration (mg/mL)Mean zones of growth inhibition (mm) (mean ± SEM)E. coliS. aureusB. subtilisP. aeruginosaC. albicansCiprofloxacin0.0535.5 ± 0.12134.5 ± 0.21735.5 ± 0.08145.0 ± 0.164NDClotrimazole0.05NDNDNDND18.5 ± 0.171Ethanol extract25.0020.5 ± 0.01921.0 ± 0.06121.0 ± 0.09924.0 ± 0.07724.5 ± 0.18412.5016.0 ± 0.02018.0 ± 0.09017.0 ± 0.09716.5 ± 0.06817.5 ± 0.0886.2514.5 ± 0.21314.5 ± 0.18612.0 ± 0.18611.0 ± 0.12812.5 ± 0.089Petroleum ether extract25.0017.0 ± 0.14613.0 ± 0.11910.5 ± 0.210——12.5014.5 ± 0.02411.5 ± 0.030———6.25—————ND = not determined, the diameter of well (10 mm).From the results obtained, the antimicrobial activity of the extracts ofC. nioroense was concentration-dependent (Table 1). The petroleum ether extract showed no activity against P. aeruginosa and C. albicans at the tested concentrations. The lowest concentration at which the ethanol extract recorded a zone of inhibition was 6.25 mg/mL, whilst the petroleum ether extract was 12.5 mg/mL. The most susceptible organism to the ethanol extract was C. albicans, with a mean diameter zone of inhibition of 24.5 ± 0.184 mm, with E. coli (20.5 ± 0.019 mm) being the least susceptible organism at the highest concentration of 25.0 mg/mL. S. aureus was the most susceptible organism to the ethanol extract at a concentration of 12.5 mg/mL, whereas S. aureus and E. coli were the most susceptible organisms at the lowest concentration of 6.25 mg/mL. The most susceptible organism to the petroleum ether extract was E. coli, with a mean diameter zone of inhibition of 17.0 ± 0.146 mm, with B. subtilis (10.5 ± 0.210 mm) being the least susceptible organism at the highest concentration of 25.0 mg/mL. E. coli was the most susceptible organism, with a mean diameter zone of inhibition of 14.5 ± 0.024 mm at a lower concentration of 12.5 mg/mL. P. aeruginosa was the most susceptible organism to ciprofloxacin (standard drug). Clotrimazole (standard drug) showed activity against the fungus C. albicans. The results showed that the extracts of C. nioroense contained broad-spectrum antimicrobial agents that were effective and capable of inhibiting the growth of the selected microorganisms employed for the assay with variable activity.


## 3.3.2. Minimum Inhibitory Concentration (MIC) of the Extracts ofC. nioroense

The MICs of the extracts of the stem bark ofC. nioroense against the test organisms are shown in Table 4.Table 4
MICs values ofC. nioroense extracts and reference drugs concentrations against test organisms.
Test organismsMinimum inhibitory concentration, MIC (mg/mL)Ethanol extractPetroleum ether extractCiprofloxacinClotrimazoleE. coli0.048812.501.5625 × 10−3NDS. aureus0.048812.501.5625 × 10−3NDP. aeruginosa0.096525.006.25 × 10−3NDB. subtilis0.048825.001.5625 × 10−3NDC. albicans0.012225.00ND3.125 × 10−3ND = not determined.The MICs of the extracts ofC. nioroense against the test organisms were in the range of 0.0122–25.0 mg/mL. The ethanol extract showed the highest inhibitory antimicrobial activity against C. albicans and the least against P. aeruginosa, with MICs of 0.0122 and 0.0965 mg/mL, respectively. The highest inhibitory antimicrobial activity of the petroleum ether extract was observed against E. coli and S. aureus at 12.5 mg/mL, whilst the lowest was observed against P. aeruginosa, B. subtilis, and C. albicans at 25.0 mg/mL. The broad spectrum antimicrobial activity exhibited by the extracts of C. nioroense could be attributed to the presence of the actions of individual phytoconstituents present in the stem bark or synergistic activities [17, 20–23].


## 3.3.3. Minimum Bactericidal Concentration (MBC) of the Extracts ofC. nioroense

The ethanol and petroleum ether extracts of the stem bark ofC. nioroense exhibited varying antimicrobial activity against the test organisms, as shown in Table 5.Table 5
MBCs values ofC. nioroense extracts and reference drugs concentrations against test organisms.
Test organismsMinimum bactericidal concentration, MBC (mg/mL)Ethanol extractPetroleum ether extractCiprofloxacinClotrimazoleE. coli6.25>25.006.25 × 10−3NDS. aureus6.25>25.006.25 × 10−3NDP. aeruginosa12.5>25.0012.5 × 10−3NDB. subtilis6.25>25.003.125 × 10−3NDC. albicans3.125>25.00ND25.0 × 10−3ND = not determined.The MBCs of the ethanol extract ofC. nioroense against the test organisms at various concentrations ranging from 3.125 to 12.5 mg/mL do not only inhibit the growth of microorganisms but also kill them entirely. The ethanol extract of C. nioroense exhibited the most antimicrobial activity against C. albicans and the least against P. aeruginosa, with MBCs of 3.135 and 12.5 mg/mL, respectively. The MBCs of the ethanol extract of C. nioroense for E. coli, S. aureus, and B. subtilis were 6.25 mg/mL, whilst the petroleum ether extract did not register any activity against the test organisms. The antimicrobial properties displayed by the ethanol extract of C. nioroense may be associated with the synergistic activities or the actions of individual secondary metabolites present in the stem bark [42]. Therefore, this confirms the traditional usage of boiling both the leaves and stem bark as medication for the treatment of bacterial and helminthic infections and the broad spectrum antimicrobial properties [10].


## 3.4. In Vitro Antioxidant Activity

Reports indicate that plants with good antimicrobial activity are likely to exhibit antioxidant activity as well [43]. Due to the different mechanisms of antioxidant actions, the DPPH and H2O2 radical scavenging assays and the TAC assays were used to evaluate the antioxidant properties of the extracts of C. nioroense [1].


### 3.4.1. DPPH Radical Scavenging Capacity

The ethanol and petroleum ether extracts ofC. nioroense and ascorbic acid (reference standard) scavenged DPPH at concentrations of 6.25 μg/mL to 100 μg/mL, as presented in Figure 2.Figure 2
DPPH free radical scavenging activity of the extracts ofC. nioroense and ascorbic acid.The extracts ofC. nioroense showed a dose-dependent increase in antioxidant activity. The IC50 values of the ethanol and petroleum ether extracts of C. nioroense and ascorbic acid ranged from <6.25 to >100.0 μg/mL, as shown in Table 6.Table 6
IC50 of DPPH/H2O2 scavenging activity for the ethanol and petroleum ether extracts of C. nioroense and ascorbic acid/gallic acid.
DPPH radical scavenging activityH2O2 scavenging activitySamplesIC50 (μg/mL)SamplesIC50 (μg/mL)Ascorbic acid<6.25Gallic acid211.50 ± 2.985Ethanolic extract27.94 ± 1.005Ethanol extract322.50 ± 1.005Petroleum ether extract>100.0Petroleum ether extract400.90 ± 3.400The results demonstrated significant antioxidant activity in a concentration-dependent manner, with the ethanol extract showing higher effectiveness than the petroleum ether extract (Figure2 and Table 6). The antioxidant potential of the ethanol extract could be due to the presence of additional phytochemicals like flavonoids, tannins, and phenols [44, 45]. Therefore, C. nioroense may be beneficial to humans in preventing oxidative damage from free radicals.


### 3.4.2. H2O2 Radical Scavenging Assay

Figure3 indicates the H2O2 radical scavenging capacity of the ethanol and petroleum ether extracts of C. nioroense and gallic acid as a reference standard at different concentrations.Figure 3
H2O2 radical scavenging capacity of the extracts of C. nioroense and gallic acid.The ethanol and petroleum ether extracts ofC. nioroense demonstrated a concentration-dependent increase in antioxidant activity, with IC50 values ranging from 211.50 ± 2.985 to 400.90 ± 3.400 μg/mL, as shown in Table 6.The ethanol extract showed better antioxidant activity than petroleum ether extract, which might be due to the presence of extra phytochemicals like flavonoids, tannins, and phenols, although they are all good antioxidants that could be exploited in minimizing oxidative stress [14].


### 3.4.3. Total Antioxidant Capacity (TAC)

Ascorbic acid concentrations of 6.25–100μg/mL showed antioxidant activity and mean absorbance values between 0.313667 ± 0.002082 and 0.909667 ± 0.000208 at a wavelength of 695 nm, as presented in Figure 4.Figure 4
Mean absorbance ofPMo4VMo8VIO407− against the concentration of ascorbic acid.The TAC was established to be proportional to the concentration of the extracts. The TAC of the extract results was expressed as grams of ascorbic acid equivalent per 100 grams of extract (gAAE/100 g) [1]. The ethanol and petroleum ether extracts of C. nioroense had 57.968 ± 0.560 and 47.197 ± 0.533 gAAE/100 g, respectively (Table 7). This result implies that 57.968 g of ethanol extract and 47.197 g of petroleum ether extract of stem bark extracts of C. nioroense out of 100 g of the sample will function as ascorbic acid.Table 7
TAC of ethanol and petroleum ether extracts expressed as gAAE.
SampleTAC (gAAE/100 g)Ethanolic extract57.968 ± 0.560Petroleum ether extract47.197 ± 0.533TAC: total antioxidant capacity; AAE: ascorbic acid equivalent.Generally, the TAC showed a concentration-dependent increase; thus, the higher the TAC, the better the activity of the extract. The extracts displayed appreciable antioxidant activities associated with the secondary metabolites present in the stem bark [1].


## 3.4.1. DPPH Radical Scavenging Capacity

The ethanol and petroleum ether extracts ofC. nioroense and ascorbic acid (reference standard) scavenged DPPH at concentrations of 6.25 μg/mL to 100 μg/mL, as presented in Figure 2.Figure 2
DPPH free radical scavenging activity of the extracts ofC. nioroense and ascorbic acid.The extracts ofC. nioroense showed a dose-dependent increase in antioxidant activity. The IC50 values of the ethanol and petroleum ether extracts of C. nioroense and ascorbic acid ranged from <6.25 to >100.0 μg/mL, as shown in Table 6.Table 6
IC50 of DPPH/H2O2 scavenging activity for the ethanol and petroleum ether extracts of C. nioroense and ascorbic acid/gallic acid.
DPPH radical scavenging activityH2O2 scavenging activitySamplesIC50 (μg/mL)SamplesIC50 (μg/mL)Ascorbic acid<6.25Gallic acid211.50 ± 2.985Ethanolic extract27.94 ± 1.005Ethanol extract322.50 ± 1.005Petroleum ether extract>100.0Petroleum ether extract400.90 ± 3.400The results demonstrated significant antioxidant activity in a concentration-dependent manner, with the ethanol extract showing higher effectiveness than the petroleum ether extract (Figure2 and Table 6). The antioxidant potential of the ethanol extract could be due to the presence of additional phytochemicals like flavonoids, tannins, and phenols [44, 45]. Therefore, C. nioroense may be beneficial to humans in preventing oxidative damage from free radicals.


## 3.4.2. H2O2 Radical Scavenging Assay

Figure3 indicates the H2O2 radical scavenging capacity of the ethanol and petroleum ether extracts of C. nioroense and gallic acid as a reference standard at different concentrations.Figure 3
H2O2 radical scavenging capacity of the extracts of C. nioroense and gallic acid.The ethanol and petroleum ether extracts ofC. nioroense demonstrated a concentration-dependent increase in antioxidant activity, with IC50 values ranging from 211.50 ± 2.985 to 400.90 ± 3.400 μg/mL, as shown in Table 6.The ethanol extract showed better antioxidant activity than petroleum ether extract, which might be due to the presence of extra phytochemicals like flavonoids, tannins, and phenols, although they are all good antioxidants that could be exploited in minimizing oxidative stress [14].


## 3.4.3. Total Antioxidant Capacity (TAC)

Ascorbic acid concentrations of 6.25–100μg/mL showed antioxidant activity and mean absorbance values between 0.313667 ± 0.002082 and 0.909667 ± 0.000208 at a wavelength of 695 nm, as presented in Figure 4.Figure 4
Mean absorbance ofPMo4VMo8VIO407− against the concentration of ascorbic acid.The TAC was established to be proportional to the concentration of the extracts. The TAC of the extract results was expressed as grams of ascorbic acid equivalent per 100 grams of extract (gAAE/100 g) [1]. The ethanol and petroleum ether extracts of C. nioroense had 57.968 ± 0.560 and 47.197 ± 0.533 gAAE/100 g, respectively (Table 7). This result implies that 57.968 g of ethanol extract and 47.197 g of petroleum ether extract of stem bark extracts of C. nioroense out of 100 g of the sample will function as ascorbic acid.Table 7
TAC of ethanol and petroleum ether extracts expressed as gAAE.
SampleTAC (gAAE/100 g)Ethanolic extract57.968 ± 0.560Petroleum ether extract47.197 ± 0.533TAC: total antioxidant capacity; AAE: ascorbic acid equivalent.Generally, the TAC showed a concentration-dependent increase; thus, the higher the TAC, the better the activity of the extract. The extracts displayed appreciable antioxidant activities associated with the secondary metabolites present in the stem bark [1].


## 3.5. In Vitro Anthelminthic Activity

The ethanol and petroleum ether extracts ofC. nioroense were screened against Milsonia ghanensis earthworms in vitro because M. ghanensis has been documented to possess physical and anatomical resemblance to intestinal roundworm parasites of human beings, as well as their ease of availability [1, 16]. The action of the extracts of C. nioroense on the worms was concentration-dependent, with all concentrations (12.0, 6.0, 3.0, 1.5, and 0.75 mg/mL) capable of paralyzing and killing the worms after the maximal exposure time. At the highest concentration of 12 mg/mL, ethanol extract paralyzed and killed all worms within 30.0 ± 0.881 and 54.0 ± 0.207 min of exposure, respectively. Similarly, the petroleum ether extract paralyzed and killed all worms within 39.0 ± 0.171 and 69.0 ± 0.121 min of exposure, respectively. However, the standard drug albendazole paralyzed and killed all worms within 70.0 ± 0.221 and 121.0 ± 0.200 min of exposure, respectively. At the least concentration of 0.75 mg/mL, ethanol extract paralyzed and killed all worms within 88.0 ± 0.714 and 139.0 ± 0.011 min of exposure, respectively. Similarly, the petroleum ether extract paralyzed and killed all worms within 94.0 ± 0.011 and 182.0 ± 0.712 min of exposure, respectively, whilst albendazole (the standard drug) paralyzed and killed all worms within 121 ± 0.070 and 261 ± 0.032 min of exposure, respectively (Figures 5 and 6). The anthelmintic activities of ethanol and petroleum ether extracts at test concentrations were observed to be significantly p<0.001 higher compared to albendazole-treated  worms.Figure 5
Anthelmintic activity of the extracts ofC. nioroense and albendazole (for paralysis) [1].Figure 6
Anthelmintic activity of the extracts ofC. nioroense and albendazole (for death) [1].The anthelmintic activity of the extracts ofC. nioroense (Figures 5 and 6) may be due to the presence of phytochemicals such as alkaloids, saponins, tannins, flavonoids, and glycosides. This is because the aforementioned secondary metabolites in the extracts have been reported to possess anthelmintic activity [46–49]. The mode of action of some anthelmintics like imidazothiazoles, derquantel, and piperazine, which are all alkaloids, is to cause paralysis of worms, such that they can be expelled in the faeces of humans and animals [1, 14]. The extracts not only exhibited such potency but also caused both paralysis and death of the M. ghanensis earthworms at a rate much higher than albendazole. The higher anthelminthic potency exhibited by the ethanol extract than the petroleum ether extract could probably be due to the presence of alkaloids, saponins, and tannins and may lead to the death of helminths.


## 3.6. Anti-Inflammatory Activity

The anti-inflammatory activity of a plant extract was determined by its ability to inhibit the thermal denaturation of protein (egg albumin) [1, 50]. The extracts of C. nioroense and standard drugs (aspirin and diclofenac) displayed concentration-dependentanti-inflammatory activity in preventing thermally induced protein denaturation at test concentrations of 500 to 100 μg/mL. The diclofenac anti-inflammatory activity was significantly p<0.01 higher than that of the extracts. However, the anti-inflammatory activity of the ethanol extracts was observed to be significantly higher p<0.01 than that of aspirin, as presented in Figure 7. The ethanol and petroleum ether extracts showed good activities at all test concentrations (Figure 7) with IC50 values of 24.402 ± 0.569 and 31.254 ± 0.359 μg/mL, respectively (Table 8).Figure 7
Anti-inflammatory action of ethanol and petroleum ether extracts ofC. nioroense [27].Table 8
IC50 of anti-inflammatory activity of extracts of C. nioroense.
SampleIC50 (μg/mL)Diclofenac19.256 ± 0.362Aspirin26.061 ± 0.087Ethanol extract24.402 ± 0.569Petroleum ether extract31.254 ± 0.359The significant anti-inflammatory activity exhibited by the extracts ofC. nioroense could be attributed to the presence of phytoconstituents such as phenols, flavonoids, tannins, saponins, glycosides, terpenoids, alkaloids, and steroids [1, 51–56]. Polyphenols such as flavonoids, tannins, and phenols are reported to inhibit enzymes and serve as mediators of the inflammation process such as C-reactive protein or adhesion molecules [1, 52, 57]. Hence, the anti-inflammatory activity of the extracts shown by preventing thermally induced protein denaturation may be attributed to the actions of individual secondary metabolites or synergistic activities present in the stem bark of C. nioroense.


## 3.7. Thin Layer Chromatography (TLC)

The analytical TLC method using a solvent system of ethyl acetate-hexane (1 : 4) was used to determine the number of less polar components present in the extracts. The various separated TLC spots, representing the less polar compounds in the extracts and theirRf values, were observed and determined (Table 9).Table 9
TLCs of the less polar constituents ofC. nioroense extracts.
ComponentsRetardation factor,RfEthanol extractPetroleum ether extractA0.086NDB0.1710.171C0.2290.229D0.3140.314E0.4000.400F0.5710.571G0.7570.757ND = not detected.The TLC results of the petroleum ether and ethanol extracts ofC. nioroense showed seven and six distinct spots, respectively, with Rf values ranging from 0.171 to 0.757 and 0.086 to 0.757, respectively, using a solvent system of EtOAc-hexane (1 : 4). A flash column chromatography purification system was used to separate the spots in the ethanol extract.


## 3.8. Column Chromatographic Separation

The less polar components of the ethanol extracts ofC. nioroense were separated employing flash column chromatography. After elution with a solvent system of hexane-EtOAc (50.0 : 0.0–45.0 : 5.0), three fractions, namely, A, B, and C, were separated (Table 10).Table 10
Chromatographic fractions separated, their respectiveRf values and confirmed phytochemical screening results.
FractionsRetardation factor,RfPhytochemical presentA0.757SteroidB0.571TerpenoidC0.400SteroidTheRf values from the column chromatography results confirm the analytical TLC results. The TLC (eluent, EtOAc-hexane (1 : 4)) was used to monitor the separated fractions.


## 3.9. Phytochemical Screening of Separated Fractions

The separated fractions A, B, and C obtained from column chromatography were screened qualitatively to determine the type of secondary metabolite present in the ethanol extract ofC. nioroense (Table 10). The phytochemical screening results identified fractions A and C as steroids and fraction B as a terpenoid. Ongoing research is focused on separating all the components present in the extracts and determining their biological activity. The identified secondary metabolites give credence to their folkloric use.


## 3.10. FTIR Analysis

FTIR analysis was performed on the petroleum ether and ethanol crude extracts as well as the separated fractions (A, B, and C) obtained from the ethanol extract after separation with flash column chromatography (Figures8–12 and Tables 11 and 12).Figure 8
FTIR spectrum of ethanol crude extract ofC. nioroense.Figure 9
FTIR spectrum of petroleum ether crude extract ofC. nioroense.Figure 10
FTIR spectrum of fraction A from the ethanol extract ofC. nioroense.Figure 11
FTIR spectrum of fraction B from the ethanol extract ofC. nioroense.Figure 12
FTIR spectrum of fraction C from the ethanol extract ofC. nioroense.Table 11
FTIR interpretation of the extracts ofC. nioroense.
Ethanolic crude extractPetroleum ether crude extractFunctional groupPeak value (cm−1)Functional groupPeak value (cm−1)O-H/N-H stretch3358Csp2-H stretch3027Csp3-H stretch2922, 2852Csp3-H stretch2935, 2862C=O stretch1690C=O stretch1738, 1713C=C stretch1638, 1513C=C stretch1530C-H bend1452C-H bend1444, 1379C-O stretch1034C-O stretchAr-ring C-H bend1175730Table 12
FTIR interpretation of fractions A, B, and C from the ethanol extract ofC. nioroense.
Fraction AFraction BFraction CFunctional groupPeak value (cm−1)Functional groupPeak value (cm−1)Functional groupPeak value (cm−1)Csp3-H stretch2957, 2924, 2873Csp3-H stretch2924, 2853Csp3-H stretch2956, 2925, 2873C=O stretch1750C=O stretch1737C-H bend1460, 1378C-H bend1460, 1378C-H bend1464, 1365C-O stretch1214C-O stretch1237, 1049C-O stretch1216The wave numbers (cm−1) around 3358 (O-H or N-H), 2922 to 28512 (Csp3-H), 1690 (C=O), 1638, 1513 (C=C), 1452 (C-H), and 1034 (C-O) were observed for the stretching and bending vibrations for the spectrum of the ethanolic crude extract (Table 11). However, the spectrum of the petroleum ether crude extract registered wave numbers (cm−1) around 3027 (Csp2-H), 2935 to 2862 (Csp3-H), 1738, 1713 (C=O), 1530 (C=C), 1444, 1379 (C-H), 1175 (C-O), and 730 (Ar-C-H), representing the identified phytochemicals in the extracts of C. nioroense (Table 1).


### 3.10.1. Fraction A

The wave numbers (cm−1) around 2873–2957 (Csp3-H), 1750 (C=O), 1460, 1378 (C-H), and 1237, 1049 (C-O) were observed for the stretching and bending vibrations typical of saturated steroidal structural backbones (Table 12) [58, 59]. The functional groups therefore confirm fraction A as a steroid, as identified from the phytochemical screening (Table 1).


### 3.10.2. Fraction B

The wave numbers (cm−1) around 2924, 2853 (Csp3-H), 1737 (C=O), 1464, 1365 (C-H), and 1216, 978 (C-O) were identified for the stretching and bending vibrations typical of saturated steroidal or terpenoid structural backbones (Table 12) [58, 59]. The observed functional groups, therefore, confirm fraction B as a saturated terpenoid, as identified from the phytochemical screening (Table 1).


### 3.10.3. Fraction C

The wave numbers (cm−1) around 2873–2957 (Csp3-H), 1460, 1378 (C-H), and 1214 (C-O) were registered for the stretching and bending vibrations for fraction C, thus confirming the presence of a saturated steroid identified from the phytochemical screening (Table 1).The FTIR analysis confirms the identified steroids (fractions A and C) and terpenoids (fraction B) from the screening test (Table1).


## 3.10.1. Fraction A

The wave numbers (cm−1) around 2873–2957 (Csp3-H), 1750 (C=O), 1460, 1378 (C-H), and 1237, 1049 (C-O) were observed for the stretching and bending vibrations typical of saturated steroidal structural backbones (Table 12) [58, 59]. The functional groups therefore confirm fraction A as a steroid, as identified from the phytochemical screening (Table 1).


## 3.10.2. Fraction B

The wave numbers (cm−1) around 2924, 2853 (Csp3-H), 1737 (C=O), 1464, 1365 (C-H), and 1216, 978 (C-O) were identified for the stretching and bending vibrations typical of saturated steroidal or terpenoid structural backbones (Table 12) [58, 59]. The observed functional groups, therefore, confirm fraction B as a saturated terpenoid, as identified from the phytochemical screening (Table 1).


## 3.10.3. Fraction C

The wave numbers (cm−1) around 2873–2957 (Csp3-H), 1460, 1378 (C-H), and 1214 (C-O) were registered for the stretching and bending vibrations for fraction C, thus confirming the presence of a saturated steroid identified from the phytochemical screening (Table 1).The FTIR analysis confirms the identified steroids (fractions A and C) and terpenoids (fraction B) from the screening test (Table1).


## 4. Conclusion

The petroleum ether and ethanol extracts ofC. nioroense revealed the presence of varying phytoconstituents, including glycosides, saponins, phenols, coumarins, alkaloids, flavonoids, tannins, steroids, phytosterols, flavanols, terpenoids, and cardiac glycosides. This study has shown that extracts of the stem bark of C. nioroense contain promising bioactive phytocomponents that possess cytotoxicity, antimicrobial, antioxidant, anthelminthic, and anti-inflammatory activities. The ethanol extract exhibited higher biological activities compared to the petroleum ether extract. This study has provided some justification for the folkloric use of this medicinal plant for the treatment of bacterial and helminthic infections. Further studies towards the isolation and determination of biological activities of isolates from the stem bark of C. nioroense are in progress in our research laboratory.



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*Source: 5424386-2023-02-18.xml*