Phytochemical Composition, Antioxidant and Antimicrobial Potentials of some Indigenous Plants in Umudike, Abia State, Nigeria

: Twenty four ethanol leaf and stem bark extracts of 17 indigenous plants were examined for their phytochemical composition, antimicrobial and antioxidant properties. Phytochemical compositions were analysed with GC-MS while antimicrobial activities on Staphylococcus aureus and Pseudomonas aeruginosa were investigated by the agar well diffusion method. The antioxidant activities were determined with Ferric reducing antioxidant power (FRAP), total phenolic content (TPC) and 2, 2,-dihenyl-1-picryhydazyl (DPPH) radical scavenging assays. The antibacterial activity was more towards the gram positive S. aureus than the gram negative P. aeruginosa for all the plant extracts. A wide range of phenolic concentrations among the aqueous plant extracts which varied from 28.04 to 500.26mg GAE per gram were observed. Inhibition percentages of DPPH ranged from 19.13 to 95.77% showing effectiveness in radical scavenging. GC-MS characterization of the plant extracts showed a total of 18 components including alkaloids, flavonoids, phenols, saponins, terpenoids, steroids and glycosides. Irvingia gabonensis leaf (IGL) extract and Tamarind stem bark (TSB) exhibited excellent ferric reducing abilities of 2.11 and 1.56 respectively while Voucanga Africana leaf (VCA) extract indicated the lowest ferric reducing power of 0.50. Extracts of IGL and TSB exhibited the highest antioxidant capacities and therefore could be the main sources of natural antioxidant. An important relationship between total phenolic content was observed showing that the major contributor to the antioxidant properties were phenolic compounds.

Antioxidants are secondary metabolities that fight against oxidative damage caused by free radicals (Shen et al., 2012;Subhasree et al., 2009). Free radicals are known to display essential activity in the development of tissue damage in many human diseases such as neurodegenerative disorders, cancer, cardiovascular diseases and pathological events in living organism. They rapidly inactivate enzymes, destroy membranes, and damage cell organelles by inducing degradation of nucleic acids and proteins lipids (Giweli et al., 2013;Tuo et al., 2015;Khalaf et al., 2008). Free radicals include reactive nitrogen species (RNS), reactive oxygen species (ROS), and reactive chlorine species (RCS). The human anatomy possesses innate defence mechanisms, such as uric acid, glutathione peroxides, superoxide dismutase, glutathione, catalase, and ubiquinone which counteract free radicals in the form of endogenous antioxidants (Spiegel et al., 2020;Fernandes et al., 2015;Udem et al., 2018). However, the quantities of these body generated defenders seem to be inadequate, most likely under oxidative stress conditions or inflammation during which the quantity of free radicals produced is increased (Ahn and Je, 2011;Gutteridge, 1994). Antioxidants plays important roles in preventing most of these diseases induced by free radicals by preventing or inhibiting the oxidation of oxidizable materials, decreasing oxidative stress and scavenging free radicals (Lim et al., 2009). Plants contain large numbers of biologically active compounds that can act as antioxidants. Under high environmental stress, plants contain non-enzymatic and enzymatic antioxidants. The enzymatic antioxidants are peroxidase (POX), superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) while non-enzymatic antioxidants include -tocopherol, anthocyanins, polyphenolic, ascorbic acid, catechins, lignans, -carotene, coumarins, and flavonoid compounds. Furthermore, the most synthetic antioxidants commonly used in cosmetic and food industries are propyl gallate (PG) butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and butylated hydroquinone (BHQ) (Duracková, 2010). However, these synthetic antioxidants are known to be promoters of carcinogenesis. This necessitates the search for natural antioxidants that have little or no side effects for use in the cosmetic and food industries and also as a material in medicine to displace the synthetic antioxidants. Plants that have curative uses are the main sources of antioxidants like phenolic compounds such as flavonoids, tannins, lignin, phenolic acids and stilbenes. They are also rich sources of vitamins such as E, C and A (Karuppanapandian et al., 2011;Erdemoglu et al., 2006). They also exhibit antibacterial, anticancer, immune stimulating, antiviral and anti-inflammatory activities (Reuter et al., 2010). Many studies have shown that plants exhibit important health benefits such as antimicrobial and antioxidants properties and this has led to the development of products for scavenging of free radicals (Kaur et al., 2009)

MATERIALS AND METHODS
Collection and identification of leaf and stem bark samples: The leaf and stem bark samples were collected within and around Michael Okpara University of Agriculture, Umudike. They were tightly packed into plastic bags and transferred to the laboratory..  Table 1 Pre-treatment of Samples: The samples were washed thoroughly thrice with double distilled water and were shade dried for 14 days.
Extraction: This was achieved based on the procedure reported by Azwanida (2015) with little modification. The dry samples were mechanically pulverized into powder with wooden mortar and pestle. The plant powder (40 g) was soaked in 200 mL of absolute ethanol for 20 h followed by filtration under applied vacuum through Whatman no 1 filter paper spread on a fitting Buchner funnel. The filtrate (extract) was then concentrated using a rotary evaporator to 2 ml. The extracts for DPPH, total phenolic and FRAP assays were left overnight for complete evaporation of the ethanol and the resulting solid residue was used for these analyses. The extract for GC-MS analysis was transferred into a labelled Teflon screw-cap vial and was cleaned up with 3 g of anhydrous sodium sulphate in a well packed column before analysis.

2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) photometric assay:
The free radical scavenging activity of the extract was investigated by the DPPH assay according to the method described by Mensor et al. (2001) using a Bio-base double beam scanning UV-VIS spectrophotometer (model BK-D 590). The crude extract at concentrations (25, 50, 100, 200 and 400) µg/mL each was mixed with 1 mL of 0.5 mM DPPH (in methanol) in a cuvette. The absorbance at 517 nm was taken after 30 min of incubation in the dark at room temperature. The experiment was done in triplicate. The percentage antioxidant activities were calculated as follows.

% = 100 -(ABS sample-ABS blank) × 100} ABS control
Where AA = antioxidant activity Methanol (1 mL) plus 2.0 mL of the test extract was used as the blank while 1.0 mL of the 0.5 mM DPPH solution plus 2.0 mL of methanol was used as the negative control. Ascorbic acid (vitamin C) was used as the reference standard (Iwalewa et al., 2008;Nurhaslina et al., 2019). The half maximal inhibitory concentrations (IC50) of the plant extracts were calculated from the plot of mean percentage DPPH inhibitory activity versus the equivalents of the tested samples concentrations in linear regression.
Total Phenolic Content Assay: Total phenol content (TPC) of each extract was determined using the Folin-Ciocalteau (FC) method described by Do et al. (2014) with minor modifications. The dried extract was dissolved in distilled water to a concentration of 50μg/mL. The calibration curve was established using gallic acid (0-60 μg/mL). The diluted extract or gallic acid (1.6 mL) was added to 0.2 mL FC reagent (5-fold diluted with distilled water) and mixed thoroughly for 3 min. Sodium carbonate (0.2 mL, 10% w/v) was added to the mixture and the mixture was allowed to stand for 30 min at room temperature. The absorbance of the mixture was measured at 760 nm using a Biobase double beam scanning UV-VIS spectrophotometer (model BK-D 590). TPC was expressed as milligram gallic acid equivalent per gram of extract (mg GAE/g extract).
Ferric Reducing Antioxidant Potential Assay: The ferric reducing antioxidant potential assay is a procedure for determining the reducing power of substances that are electron donors. This was determined according to the method described by Duh et al. (1999). Different concentrations (15-240 μg/mL) of the solvent fractions and the standard (gallic acid) were mixed with 2.5 mL of phosphate buffer (0.2 mol/L, pH 6.6) and 2.5 mL of 1% w/v potassium ferricyanide. The mixture was incubated for 20 min at 50°C. 2.5 mL of 10% trichloroacetic acid was added to acidify the mixture. Thereafter, 1 mL of the acidified mixture was mixed with 1 mL of distilled water and 0.5 ml of 0.1% FeCl3. The absorbance of the resulting solution was measured at 700 nm. The antioxidant power of the plant fractions was expressed as: The relative percentage amount of each component was calculated by comparing the average peak area to the total areas. The software adapted to handle mass spectra and chromatograms was chemstation.
Interpretation of the mass spectrum of GCMS was conducted using the database of National Instrument of Standard and Technology (NIST) having more than 63,000 patterns. Unknown components were compared to the known ones using the NIST library. Molecular weights and structures of the components of the test materials were ascertained. The spectrum profile of GC-MS confirms the presence of the main components with their retention times. The height of the peak aligned with the concentration of the components in the extracts.

Antioxidant activity (DPPH):
The antioxidant activities of various indigenous plant extracts were evaluated by DPPH radical scavenging mechanism which has been widely used to examine the free radical scavenging abilities of numerous plant extracts Durga et al., 2020). The results are shown in Table 2 and are expressed as the relative activities against standard ascorbic acid.