Medicinal plants have played a central role in human healthcare for centuries, forming the backbone of traditional medicine and serving as critical sources for modern drug discovery. Their therapeutic relevance is largely attributed to diverse secondary metabolites such as flavonoids, terpenoids, alkaloids, and phenolic compounds, which exhibit antimicrobial, antioxidant, anti-inflammatory, and anticancer activities [1].

One such plant is Bauhinia purpurea L., commonly known as the Purple Orchid Tree, a member of the Fabaceae family widely distributed across tropical regions. It is extensively used in ethnomedicine for the treatment of wounds, ulcers, diabetes, infections, liver disorders, and other ailments. Phytochemicals isolated from B. purpurea have demonstrated antimalarial, antimycobacterial, antifungal, anti-inflammatory, and cytotoxic effects, highlighting its broad therapeutic potential [2].

The integration of plant science with nanotechnology has opened new research pathways, particularly through green synthesis techniques. In this approach, plant extracts act as both reducing and stabilizing agents in the production of metal nanoparticles, eliminating the need for toxic chemicals. These eco-friendly nanoparticles often display enhanced biological activities and biocompatibility compared to crude plant extracts [1,3]. For example, silver and gold nanoparticles synthesized using B. purpurea leaf extracts have shown remarkable antimicrobial, antioxidant, and anticancer properties in vitro [3].

Despite the extensive traditional use of B. purpurea and isolated pharmacological reports, few studies have combined phytochemical profiling, green nanoparticle synthesis, and bioactivity evaluation in a single framework. An integrated approach is therefore essential to advance this plant from traditional application to evidence-based biomedical development. This study was designed to provide a comprehensive evaluation of Bauhinia purpurea, combining phytochemical analysis, environmentally friendly nanoparticle synthesis, and biological activity assessment to explore its potential therapeutic relevance.

RESEARCH METHODOLOGY

Plant Material Collection and Taxonomic Authentication

Fresh leaves of Bauhinia purpurea (Orchid plant) were collected on March 10, 2025, in the early morning from naturally growing stands in Ado-Ekiti, Ekiti State, Nigeria. Early morning collection was chosen to minimize moisture loss and preserve heat-sensitive phytochemicals that may degrade under strong sunlight or elevated temperatures. Proper handling of the samples during collection ensured that primary bioactive constituents (e.g., flavonoids, phenolics, alkaloids, and saponins) remained intact for subsequent analysis. The species identity was authenticated at the Herbarium of the Department of Plant Science, Faculty of Science, Ekiti State University, Ado-Ekiti, Nigeria, where a voucher specimen was prepared and deposited for future reference.

Preparation of Aqueous Leaf Extract

Leaves were thoroughly rinsed under running tap water to remove surface dust and debris, then air-dried at ambient temperature until constant weight was achieved. The dried leaves were pulverized into fine powder using a sterile mortar and pestle to increase extraction efficiency. One hundred grams (100 g) of the powdered leaves were weighed and transferred into a clean extraction beaker. One litre (1 L) of distilled water was added to the powder, and the mixture was stirred to ensure uniform dispersion. The suspension was heated on a heating mantle at 70–80 °C for 2 h to facilitate the release of soluble phytochemicals into the solvent, a strategy commonly employed in plant extraction protocols to improve the yield of antioxidant constituents (e.g., phenolics and flavonoids) known to contribute to bioactivity in aqueous extracts [4]. After cooling to room temperature, the mixture was filtered through a fine cloth, and the resulting filtrate was collected into clean, labelled containers. The aqueous extract was stored at 4 °C until further analysis to maintain stability.

Qualitative Phytochemical Screening

The aqueous leaf extract was subjected to qualitative screening to detect major classes of secondary metabolites, including flavonoids, phenolic compounds, alkaloids, saponins, and steroids. Standard chemical tests, which involve reagent reactions producing characteristic colour changes or precipitates, were used to confirm the presence of these phytochemicals, as previously demonstrated in aqueous extracts of Bauhinia purpurea where multiple phytochemicals were identified in qualitative analyses [5,6].

In Vitro Antioxidant Assays

DPPH Free Radical Scavenging Assay

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was used to evaluate the antioxidant activity of the extract. Different extract concentrations (e.g., 20, 40, and 80 mg/mL) were prepared in clean test tubes. One millilitre (1 mL) of each concentration was mixed with 1 mL of 0.1 mM methanolic DPPH solution. The mixtures were vortexed and incubated in the dark at room temperature for 30 min to allow interaction between antioxidants in the extract and the DPPH radical. After incubation, absorbance was measured at ~516 nm (the characteristic absorption wavelength of DPPH radical) using a UV-visible spectrophotometer. A significant reduction in absorbance relative to a control indicates effective free radical scavenging, as previously reported for aqueous leaf extracts of Bauhinia purpurea with notable DPPH scavenging activity in published studies [7,8].

Green Synthesis of Metal Nanoparticles

Green synthesis of metal nanoparticles (e.g., silver, copper, zinc) was conducted using the aqueous leaf extract as a reducing and stabilizing agent, following plant-mediated synthesis protocols widely described in nanobiotechnology literature. In this approach, aqueous solutions of metal salts (such as AgNO₃ for silver nanoparticles) are mixed with plant extract under controlled stirring and temperature conditions, allowing bioactive phytochemicals to reduce metal ions and cap the resulting nanoparticles. Such eco-friendly synthesis methods are known to yield nanoparticles with bioactive surfaces suitable for downstream biological evaluation, including antioxidant and antimicrobial assays [3].

Antimicrobial Evaluation of Synthesized Nanoparticles

The antimicrobial activities of the green-synthesized nanoparticles were assessed against selected pathogenic bacterial strains using standard microbiological assays such as the agar well diffusion method. Test organisms were cultured on appropriate media, and standardized inocula were spread onto agar plates. Wells loaded with nanoparticle suspensions were incubated, and zones of inhibition were measured to evaluate bacteriostatic and bactericidal effects relative to controls.

Additional Antioxidant Assessments

To complement the DPPH assay, total phenolic content and flavonoid content of the extract were estimated using spectrophotometric assays with relevant standards. These complementary assays provide quantitative insights into specific antioxidant components contributing to overall activity, which is essential in studies exploring extracts as both biological agents and reducing agents for green nanoparticle synthesis [4].

RESULTS

The proximate composition of Bauhinia purpurea leaf is presented in Table 1, showing moisture (7.40 ± 0.01%), ash (11.30 ± 0.03%), crude fat (4.79 ± 0.05%), crude fibre (10.20 ± 0.02%), crude protein (17.74 ± 0.05%), and carbohydrate (48.57 ± 0.03%). The antinutrient profile in Table 2 indicates oxalate (3.16 ± 0.01 mg/100 g), phytate (11.52 ± 0.02 mg/100 g), tannin (16.55 ± 0.00 mg/100 g), and trypsin inhibitor (21.00 ± 0.39%).

Mineral composition is summarized in Table 3, with potassium (4.52 ± 0.02%), sodium (2.53 ± 0.04%), calcium (2.13 ± 0.01%), copper (0.93 ± 0.00%), chromium (0.11 ± 0.00%), magnesium (0.98 ± 0.03%), iron (2.68 ± 0.00%), manganese (0.29 ± 0.00%), and lead (0.10 ± 0.00%).

Phytochemical screening of the aqueous extract is shown in Table 4, indicating the presence of saponins, phenolics, flavonoids, anthraquinones, terpenoids, and cardiac glycosides, while steroids were not detected.

Table 5 presents the flavonoid and phenolic contents of ethanol and aqueous extracts at 100 µg/mL. The ethanol extract showed flavonoids of 16.54 ± 0.47 mg AAE/g and phenolics of 13.01 ± 0.28 mg GAE/g, while the aqueous extract showed flavonoids of 21.13 ± 0.01 mg AAE/g and phenolics of 32.56 ± 0.11 mg GAE/g.

The antioxidant activities of the aqueous extract at 100 µg/mL are shown in Table 6, with DPPH (48.39 ± 0.04), FRAP (57.15 ± 0.57), nitric oxide scavenging (65.15 ± 0.85), and TBAR (22.23 ± 0.84). Table 7 summarizes the antioxidant activities of the ethanol extract at 100 µg/mL, showing DPPH (76.54 ± 0.47), FRAP (74.93 ± 0.13), nitric oxide scavenging (88.13 ± 1.56), and TBAR (26.30 ± 0.11).

The antimicrobial activity of the aqueous extract and silver nanoparticles is presented in Table 8. At 100 mg/mL, the aqueous extract produced inhibition zones of 3.7 mm against E. coli, 2.8 mm against S. typhi, and 1.3 mm against S. aureus, with no activity against P. aeruginosa. At 50 mg/mL, inhibition zones were 2.9 mm, 2.2 mm, and 1.0 mm against E. coli, S. typhi, and S. aureus, respectively. The silver nanoparticles at 100 mg/mL showed inhibition zones of 5.3 mm (E. coli), 4.1 mm (S. typhi), 3.0 mm (S. aureus), and 1.2 mm (P. aeruginosa).

Table 1: Proximate Composition of Bauhinia purpurea Leaf

Table 2: Antinutrient Composition of Bauhinia purpurea Leaf

Table 3: Mineral Composition of Bauhinia purpurea leaf

PARAMETERS    VALUE%

K                                                                                                           4.52±0.02  

Na                                                                                                         2.53±0.04

Ca                                                                                                          2.13±0.01

Cu                                                                                                          0.93±0.00

Cr                                                                                                           0.11±0.00

Mg                                                                                                          0.98±0.03

Fe                                                                                                            2.68±0.00

Mn                                                                                                          0.29±0.00

Pb                                                                                                           0.10±0.00

Table 4: Phytochemical screening of the Aqueous plant extracts (Bauhinia purpurea)

Parameters                                                                                  Value (%)

Saponin Test                                                                                            +        

Phenolic Test                                                                                           ++++

Wagner’s Test                                                                                          ++++

Anthraquinone Test                                                                                  +

Terpenoid Test                                                                                          ++++

Keller-killani Test                                                                                     +++

Lieberman’s Test                                                                                       -

Table 5: Antioxidant potentials of Aqueous Plant extracts of Bauhinia purpurea leaf

Table 6: Antioxidant potentials of aqueous plant’s extracts of Bauhinia purpurea leaf

Table 7: Antioxidant potentials of ethanol plant’s extracts of Bauhinia purpurea leaf

DPPH – 2,2‑diphenyl‑1‑picrylhydrazyl, FRAP – Ferric Reducing Antioxidant Power.  NO – Nitric Oxide scavenging assay, TBAR – Thiobarbituric Acid Reactive Substances,

Table 8: Antimicrobial activity of Aqueous extract and Silver Nanoparticle of Bauhinia purpurea leaf on some selected bacteria