A plant's therapeutic value is attributed to the presence of various phytoconstituents present in the plant. Flavonoids, alkaloids, phenols, tannins, carboxylic acids, terpenes, amino acids, inorganic acids, etc. are a few of these plants’ chemical compounds. These phytoconstituents provide plants with their characteristics and originality [1]. Considering these facts, the current study is intended to assess the phytochemicals of Pterocarpus osun, a plant that has been known to contain a variety of therapeutic effects in traditional medicine. Its leaves and stem bark have been found to contain alkaloids, tannins, saponins, flavonoids, terpenoids, steroids, cardiac glycosides, and phenols [2]. The separation of plant extracts into its constituting chemical compounds remains an important subject for the identification and characterization processes because they are made up of several types of bioactive chemicals compounds with distinct polarities. It is common practice to use a variety of separation techniques, including thin-layer chromatography (TLC), column chromatography, to isolate these bioactive chemicals. To acquire pure substances, high-performance liquid chromatography (HPLC) can be utilized [3]. High-performance liquid chromatography, or HPLC [4], or planar chromatography [5], is typically used for the separation and separation of chemical compounds in multicomponent mixtures, such as plant extracts. Numerous instrumentation approaches, including UV-VIS, FTIR, and GC-MS analysis, have been used to identify bioactive compounds from medicinal plants [6]. The best method for detecting the bioactive components of substances like long-chain hydrocarbons, alcohols, acids, esters, alkaloids, steroids, amino and nitro compounds, etc. is gas chromatography and mass spectrophotometry (GC-MS). As a result, sophisticated methods for analyzing different chemicals have been developed using gas chromatography (GC) and mass spectroscopy (MS) in conjunction with specific detection techniques [7]. Because advances in biotechnology have made it possible to investigate natural compounds faster and more precisely than ever before, which has led to the isolation of bioactive compounds with health benefits, this research is focused on elucidating potential sources of ethnomedicinal plants using cutting-edge scientific analysis like gas chromatography-mass spectrometry. GC-MS has solidified its position as a major technology platform for secondary metabolite profiling in both plant and non-plant species over the past few years [8]. The best method for finding new phytochemicals with potential as medicines for various ailments is still GC-MS analysis of bioactive components in plants [9,10]. There is growing interest in phytomedicine, which will eventually contribute to the standardization of plant components for therapeutic effects. The study of plant-derived organic molecules and their actions has garnered more interest in recent years. The study of plant active compounds has led to the development of numerous extraction techniques as well as analytical techniques like spectrophotometry, high-performance liquid chromatography, capillary electrophoresis, gas chromatography (GC) with flame ionization detection (FID), and gas chromatography-mass spectrometry (GC-MS). The best qualitative and quantitative technique for volatile and semi-volatile chemical compounds was created by combining the best separation technique (GC) with the best identification technique (MS). Additionally, the adoption of an appropriate extraction technique is required [11].

Extraction and Isolation

The stembark of P. osun was harvested, air dried and blended to powder. The powdered sample was macerated in succession with n-hexane, ethyl acetate and methanol. The ethyl acetate crude extract was chromatographed using column chromatographic technique. A viscous dark brown oily substance which is soluble in chloroform was obtained as a fraction.  

Gas Chromatography-Mass Spectrometry (GC-MS)

Gas chromatography - mass spectrometry (GC-MS) was used to evaluate the oily fraction component isolated from the ethyl acetate extract of the stembark of P. osun. To determine and identify the bioactive compound in the plant. By comparing the resulting mass spectra of unknown peaks with those kept in the Wiley and NIST (National Institute of Standards and Technology) mass spectral electronic libraries, the compounds were identified [12,13].

Fourier Transform Infrared (FTIR) Analysis

FTIR spectroscopy analysis of the oily samples was scanned between 4000–400cm^-1 wavelengths on a Nicolet IS 5 Thermo Fisher Scientific, USA FTIR spectrophotometer. FTIR spectra give information about the characteristic functional groups identified in the samples.

Antibacterial Studies: Preparation of Different Concentration of Extracts

To make the stock solution 100mg of the different concentrated extracts from the shell, pulp, and seed of the plants were weighed into sterile tubes. 2ml of 10% Dimethyl sulfoxide (DMSO) was used to dissolve the extracts and mixed thoroughly using vortex machine. From the stock solution different concentration was made through serial dilution to get a concentration of 10mg/mL, 5mg/mL, and 2.5mg/mL.

Antibacterial Activity

Three pathogenic bacteria such as Staphylococcus auerus, Echerichia coli, and Salmonella Typhi were collected from the biotechnology advanced research center, Sheda Science and Technology Complex, confirmed using appropriate media and were then used for these studies. The antibacterial activity was done using Agar Well diffusion methods. All the bacteria were grown on tryptic soy agar for 24h. Three colonies were inoculated in to muller Hinton broth and incubated for 4h and the turbidity was compared with 0.5 MacFarland and adjusted accordingly.  A sterile swab was used to spread each of the inoculum on the Muller Hinton Agar plates. Four wells were bored on the media using cup borer of 6mm, the wells were sealed using MHA, the plates were allowed to stay for 15min. 100µl of different concentration of the extracts was dispensed into the different well and was allowed to stay for 30mins. The fourth well was used as control which contained 100µl of 10mg/ml of chloramphenicol. The plates were incubated for 18-24h the zone of inhibition was measured in mm [24].

The antimicrobial screening of the viscous oily substance obtained from the chromatography separation of the ethyl acetate extract of P. osun stembark at various concentration (Table 1) showed positive activities comparable to the standard drug, chloramphenicol which was used as controlled experiment. The test organisms Escherichia coli, Salmonella typhi and Staphylococcus aureus are all susceptible to the oily sample.  Staphylococcus aureus is a gram-positive bacterium which causes inflammation diseases such as skin infections, pneumonia, endocarditis, septic arthritis, osteomyelitis, and abscesses. Escherichia coli is a gram-negative organism which is responsible for ill health conditions such as intestinal and extraintestinal infections, gastrointestinal, pneumonia, bacteremia, urinary tract infection, abdominal and pelvic infection, also, meningitis. Salmonella typhi, a gram-negative pathogen that is responsible typhoid fever. At the highest concentration of test i.e., 10 mg/mL, all the test organisms recorded zones of inhibitions which compared favourably with the chloramphenicol used as standard control drug (Table 1). 

Table 1: Antibacterial activity of ethyl acetate oily fractions from leave and stembark of P. osun.

C = Control (Chloramphenicol 10mg/ml)

The FTIR analysis was run by scanning the sample through the wavelength of 4000–400cm^-1. The spectrum is shown in Figures 5 and 6 and the absorption bands, and the functional groups present are displayed in Tables 2 and 3 for both fractions of leaf and stembark respectively. From the spectra and the tables, the functional groups noticeably present include C-H alkane stretch at 2914, 2847cm^-1, C=O (aldehyde functional group) at 1728cm^-1. At 2359 cm^-1, we have the Si-H group and sulphonyl at 1175cm^-1 for the leaf sample and in addition to these functional groups, the stembark bark sample shows carboxylic acid C-O stretch at 1165 and 1032cm^-1, also ester at 720cm^-1.

Figure 1: FTIR spectrum of stembark oily fraction of P. osun.

Table 2: FTIR analysis of oily fraction from the stembark of P.osun.

Figure 2: GCMS chromatogram of oil fraction of the stembark of P. osun.

The sample was dissolved in chloroform and the GC-MS analysis was carried out, the result is presented in Table 3 and Figure 1. Some of the chemical compounds identified by the GC-MS analysis are shown, with their chemical formula, molecular weight, and pharmacological functions. The pharmacological uses of these compounds confirmed the earlier work [2] on the extracts of P. osun, which showed the plant as a good antioxidant and antimicrobial agents. The GCMS analysis confirmed the presence of important chemical compounds which have been pharmacologically confirmed as therapeutic agents.  Some of these chemical compounds are responsible for the antimicrobial properties demonstrated by the plant’s extract in our previous studies, and the basis for the traditional medicinal usage of the plants in treatment of various health related issues.

Table 3:  Main Phytochemical compounds identified in P. osun stembark oily fraction.

The results of this research work give insight into the chemical composition of the isolated oily fraction obtained from the ethyl acetate extract of the stembark of P. osun, its antimicrobial prowess and the pharmacological uses of the chemical composition of the isolate. This suggest that plant is loaded with many chemical compounds which could be a lead to the development of many therapeutic drugs.

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