Biochemistry & Analytical Biochemistry

Phytochemical Screening, Antioxidant and Antimicrobial Activities of Methanol Extract from Baphia longipedicellata (De Wild.) leaf

Samuel Friday Ibitoye^1*, Semiu Olasoju Lawal¹, Arhmiyahu Ayodeji Alonigbeja² and Olayinka A. Adebayo^{3 1}Department of Pharmacy, Olabisi Onabanjo University, Ago Iwoye, Ogun State, Nigeria
²Department of Pharmaceutical Technology, Olabisi Onabanjo University, Ogun State, Nigeria
³Department of Biochemistry, Olabisi Onabanjo University, Oyo State, Nigeria
^*Correspondence: Samuel Friday Ibitoye, Department of Pharmacy, Olabisi Onabanjo University, Ago Iwoye, Ogun State, Nigeria, Email:

Received: 04-Apr-2023, Manuscript No. BABCR-23-20714; Editor assigned: 07-Apr-2023, Pre QC No. BABCR-23-20714 (PQ); Reviewed: 24-Apr-2023, QC No. BABCR-23-20714; Revised: 01-May-2023, Manuscript No. BABCR-23-20714 (R); Published: 09-May-2023, DOI: 10.35248/2161-1009.23.12.482

Introduction

Although the use of medicinal plant for medicine predate human history but recently, attention has been drifted toward the use of medicinal plants as alternative to drug in treatment and management of one disease or the other. The sudden attention may be due to the realization of the use of some of these plants in ancient and folklore medicine ranging from the acupuncture and Chinese herbal remedies as far back as 2,200 years (Huangdi neijing) and Ayurveda of ancient India up to the current traditional medicine which serves as the basis. Many studies have shown that plants contain uncountable phytoconstituents and this makes them to be effective antioxidant and antimicrobial agents [1-5].

Currently, the advancement in the awareness about free radicals and Reactive Oxygen Species (ROS) is yielding a medical turning point that guarantee a new breakthrough in health and disease management. Oxygen is a fundamental element for life but ironically, under certain circumstances possesses impairing effects on the body of human beings. Formation and actions of number of chemical compounds, ROS, which have a propensity to bequeath oxygen to other substances is responsible for most of the potentially catastrophic effects of oxygen. Free radicals and antioxidants have been incriminated in the causes and mechanisms of many diseases. An antioxidant is a molecule steady enough to grant an electron to a flagrant free radical and get it neutralized, thus overcoming destructive capacity by lagging or inhibiting cellular impairment mainly through their free radical scavenging property. Some of such antioxidants are glutathione, ubiquinol, and uric acid produced endogenously by humans; neutraceuticals and other plants’ secondary metabolites gotten exogenously [6-10].

Baphia longipedicellata is leguminous plant belonging to the family Fabaceae. It is found in Kenya and it is distributed across Congo Republic, Nigeria and some West African countries although it is vulnerable to habitat loss. B. longipedicellata leaf has history of folklore use in the treatment of dry coughs, whooping cough, bronchial catarrh, asthma, indigestion, gastritis, diarrhea, malaria and infertility in male.

Objective of the study

• Investigate the phytochemical constituents of Methanol Extract of Baphia longipedicellata leaf (MEBLL)

• Investigate the in vitro antioxidant activities MEBLL using various models

• Investigate the antimicrobial activities of MEBLL using different strains of gram positive and gram negative bacteria and fungi

Materials and Methods

The study was executed at the Department of Pharmaceutical Technology, School of Science and Technology, Mosshood Abiola Polytechnic, Ojere, Abeokuta, and Faculty of Pharmacy, Olabisi Onabanjo University, Sagamu Campus, Ogun State, Nigeria.

Collection and extraction of plant materials

Fresh leaves of B. longipedicellata were procured from Ibadan around the neighborhood of Federal Research Institute of Nigeria (FRIN), Jericho, Ibadan, Oyo State. The leaves were cleaned, air-dried (25°C-28°C) for 30 days, pulverized and extracted in methanol in reference to the method of Gomathi et al., 2015 by soaking 10 g of the leaves in 1.5 Litres methanol for 3 days. Thereafter, it was filtered and concentrated in a rotary evaporator at reduced temperature of 40°C and preserved for further studies [11-15].

Phytochemical screening

Phytochemical analysis was performed on the crude methanol extract of Baphia longipedicellata leaf to exemplify the presence of secondary metabolites like alkaloids, saponins, anthraquinones, tannins, flavonoids, terpenoids, etc. using standard procedure.

In vitro examination of the crude leaf extract for antioxidant activity

2, 2-diphenyl-1-picrylhydrazyl (DPPH•) scavenging activity: The DPPH• radical tramping action of crude methanol extract of Baphia longipedicellata leaf was estimated according to the method of Elizabeth and Olarte, 2010. The preparation of DPPH solution was made by mixing 100 mg of DPPH powder in 0.1 L of methanol. The plant crude extract and catechin (standard antioxidant) were also prepared at concentrations ranging from 50 uL to 1500 uL into properly labelled and arranged test tubes. Thereafter, 3000 uL of DPPH solution was added to the extract was incubated for 30 mins in the dark at 25°C. The absorbance was read at 570 nm using methanol as the blank [15-20].

Lipid Per-Oxidation (LPO) inhibition: The reticence of LPO was estimated in reference to the method of El-Mahmood and Doughari, 2018 where fresh liver sample of goat was purchased from Kuto market in Abeokuta, Ogun state and was weighed and homogenized in chilled phosphate buffer (pH 7.4) to get a clear homogenate with a homogenizer. 1000 uL of the liver homogenate was combined with the different concentrations of the plant extract and catechin (100 µg/mL-500 µg/mL). Initiation of Lipid peroxidation followed by adding 100 µL of the mixture to reaction mixture and incubated in a water bath at 80°C for 1800 seconds. Pink coloured complex was produced whose intensity was measured in a spectrophotometer at 535 nm.

Inhibition of 2, 2–azinobis-3–ethylbenzothiazoline–6–sulfonic acid (ABTS) radical: The reticence of ABTS radical was assayed following the method reported by Eseyin et al., 2005.3 g/L of ABTS and 132 g/mol NH₄SO₄ were prepared separately, mixed together at ratio 1:1 and was allowed to stand in the dark at 25°C for half a day prior to use. 600 uL of the mixture of ABTS and NH₄SO₄ was added to the different concentrations (100 µg/mL-1500 µg/mL) of the extract and then followed by the addition of 400 uL ethanol. Lastly, the absorbance was taken at 745 nm [21-28].

Iron II reducing capability: Ability of the extract to reduce iron II to iron III was analyzed according to the method revealed by Gupta et al., 2013. 2000 uL of the plant crude extract was introduced into 2500 uL of phosphate buffer (0.2 M, pH 6.6) and 2500 uL of 1% potassium ferricyanide. The resulting mixture was incubated at 50ºC for 1200 seconds. Subsequently, it was briskly cooled, amalgamated with 2500 uL of 10% Trichloroacetic Acid (TCA) and centrifuged at 6500 rpm for 600 seconds. 2500 uL of distilled water was use to dilute 2500 uL of supernatant, afterwards 500 uL of 0.1% ferric chloride was added, left on the bench for 600 seconds and the absorbance was taken spectrophotometrically at 700 nm.

Nitric oxide radical scavenging activity: The scavenging action of the plant extract against Nitric Oxide (NO) radical was determined by the method related by Haslam, 2013. The different concentrations of the crude plant extract (100 µg/mL-1500 µg/ mL) dissolved in methanol was mixed with 2000 uL of sodium nitroprusside (1.5 mM) in phosphate buffered saline and allowed to stand on the bench for 300 minutes at 25°C. Afterwards, 1500 uL of the incubated solution was taken, amalgamated with 500 uL of Griess reagent and the absorbance was read at 546 nm.

Superoxide (SO) radical scavenging activity: Superoxide scavenging activity of the extract was analyzed following the method related by Gamathi et al., 2015. 500 uL of the different concentrations (100 µg/mL-1500 µg/mL) of the crude plant extract was added to 1 mL of Dimethyl Sulphoxide (DMSO) in different test tubes and then 200 uL Nitroblue Tetrazolium (NBT) was added. The solutions were prepared in 0.1 M sodium phosphate buffer (pH 7.4). The absorbance was read at 540 nm and then 560 nm.

In vitro examination of the crude leaf extract for antimicrobial activity

Preparation of microorganisms: The pure cultures of the microorganisms were gotten from the Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Olabisi Onabanjo University, Sagamu, Ogun State. The bacteria isolate included Gram-positive Staphylococcus aureus and Streptococcus pneumoniae, and Gram-negative Escherichia coli and Pseudomonas aeruginosa. The fungi used for testing were Candida albicans and Aspergilus niger. These organisms were collected based on their clinical and pharmacological importance.

Antimicrobial susceptibility testing: The agar well diffusion method was used to determine the sensitivity to the plant extract, as defined by the International Commission on Microbiological Specifications for Foods (ICMSF, 1998). Firstly, the pure strains of bacteria were developed in nutritional broth for 18 hours and sub- cultured in Mueller-Hinton agar prior to use. The susceptibility test was carried out using agar diffusion method where the plates with solidified agar were bored. The degree of inhibition was depicted in terms of diameter of the inhibition zone as measured with a transparent ruler. The effect of the extract on bacteria and fungi was compared with that of a standard antibiotic; ciprofloxacin which is a broad spectrum antibiotic against bacteria and griseofulvin for fungi at a maximum concentration of 100 mg/ml.

Determination of Minimum Inhibitory Concentrations (MIC’s) of the plants extract: MIC is the slightest quantity of the antimicrobial material that impedes the microbial growth post 24 hours incubation. The plant extracts which display a tenacious bacteria inhibition at as low as 10 mg/ml was employed in the determination of MIC using disk diffusion method as described by Pimmada et al., 2022. Different concentrations of the crude extract (1.25, 2.5, 5.0, 10.0, 12.5 and 15.0 mg/ml) were made separately. Mueller-Hilton agar was seeded with bacteria suspension of interest and was poured into sterile Petri dishes. The loaded filter paper discs with different concentrations of the effective plant extract were placed on the top of the Mueller-Hilton agar plates and were then incubated at 35°C for 24 hours. The inhibition zones were measured and recorded against the concentrations of the crude extracts.

Determination of Minimum Bactericidal Concentrations (MBC’s): The culture plates of two lowest concentrations with non-apparent growth were streaked and sub cultured. The plates were incubated at 35°C for 24 hours and then studied for bacterial growth relative to the plant extract concentration. MBC was taken as the concentration of plant extract that did not exhibiting any bacterial growth on the freshly inoculated agar plates.

Statistical analysis: All data were expressed using means ± standard deviation and were statistically analyzed using one-way ANOVA followed by student t-test analysis to test the significance difference of the data. The differences were considered statistically at P<0.05 while graph Pad prism program (v.5) was used to analyse and present the data.

Results and Discussion

Most pharmaceutical industries rely on in vitro methods for large scale production due to the ease of culture and the ability to monitor stages of production when compared to the use of animals. Natural compounds of plant origin have continued to gain prominence as an essential substitute for modern synthetic drug agents due to low toxicity and effect treatment or/ and management of diseases and infections by the former. Phytochemicals are highly complex and widely distributed in natural plants. The antioxidant activities inherent in various plant extracts can therefore not be assessed by just one technique, and the capacity of these antioxidants to mop of free radicals solely depends on the number of free radicals present. Result from this study showed that out of the eight phytochemicals screened from the Methanol extract of Baphia longipedicellata leaf (MEBLL), only five of them were present in significant amount, namely alkaloids, tannins, cardiac glycosides, flavonoids and terpenoids while anthraquinone, saponins and Phlobatanins were conspicuously absent in the plants (Figure 1). It is of importance to know that alkaloids are well documented to be performing vital functions in the body such as anti-inflammatory and cardioprotective agents. The presence of tannins is a good indicator that MEBLL could be used for the treatment of management of cancer because study has shown that tannins have the ability to play synergistic role alongside cisplatin to prevent progression to liver cancer (Figure 2).

Figure 1: Inhibition of malonaldehyde (index of Lipid peroxidation) by the methanolic leaf extract of Baphia longipedicellata. Note: MEBLL CATECHIN

Figure 2: Fe³⁺ Reducing ability of the methanolic leaf extract of Baphia longipedicellata. Note: MEBLL CATECHIN

The DPPH radical test has been explored significantly to serve as an essential test for free radical in order to evaluate the ability of various extracts to act as scavengers of free radicals or reactive oxygen species and to also assess their antioxidant activities. The reaction mechanism between antioxidants and DPPH is via the donating or sharing electron (hydrogen), thereby reducing DPPH to 1,1 diphenyl- 2 -hydrazine (DPPH-H). This reaction product gives up a strong violet coloration which readily changes to colourless, and which absorbs maximally at 512 nm. Our study was able to show that MEBLL demonstrated substantial scavenging effect in a dose dependent manner, though has less activity when compared to catechin. The DPPH scavenging effect of most extract has been attributed to the phenolic content of the extract. Also, ABTS was significantly inhibited by MEBLL where its inhibition ability was almost similar to standard catechin. The probable mechanism by which MEBLL scavenges ABTS+ radical might be through formation of peroxodisulphate via transfer of electron. It could therefore be inferred that MEBLL contains phenols flavonoids containing hydroxyl side chain capable of stabilizing radicals or scavenging them. Nitric oxide is an important component of the body due to the fact that it plays important role the vascular smooth muscle where it helps to maintain vascular tone and aid easy blood flow in the smooth muscles of most blood capillaries. Alongside the importance of nitric oxide, superoxide serves essential function especially in cellular signalling and via the activation of membrane receptors and physiological amount of superoxide can induce apoptosis by altering the permeability of the mitochondrial membrane (Tables 1-6).

Table 1: Phytochemical constituents of the methanol extract of Baphia longipedicellata leaf (MEBLL)

Table 2: Antioxidant activity of methanol extract of Baphia longipedicellata leaf (MEBLL) measured as % inhibition of DPPH and ABTS relative to Catechin

Table 3: Antioxidant activity of methanol extract of Baphia longipedicellata leaf (MEBLL) measured as % inhibition of nitric oxide and superoxide radical relative to Catechin

Table 4: Antibacterial activities of methanol extract of Baphia longipedicellata leaf as a measure of zone of Inhibition

Table 5: Antifungal activities of methanol extract of Baphia longipedicellata leaf as a measure of zone of inhibition

Table 6: Minimum inhibitory concentration and minimum bacteriocidal concentration of methanol extract of Baphia longipedicellata leaf

However, during electron transport across the electron transport chain, electrons can leak prematurely and react with oxygen to form superoxide radical. In this experiment, MEBLL was reacted with nitric oxide and the result showed that MEBLL significantly inhibited in it a dose dependent manner, an action that is more than catechin. Also, nitric oxide was inhibited, though it has less action when compared to standard catechin. This further gives credence to the ability of MEBLL to serve as a potent free radical scavenging agent. One of the resultant effects of free radical attack is peroxidation of the membrane bilayer. This study utilized the method of El-Mahmood and Doughari, 2018 to induce lipid peroxidation and it was observed that MEBLL inhibited lipid peroxidation when compared with the control. It is of note that MEBLL demonstrated greater inhibition than standard catechin even at the same concentration. The decreased lipid peroxidation product, Malondialdehyde (MDA) further supports earlier results obtained in the study.

This study also monitored the reducing ability of MEBLL from ferricyanide to ferrous, and result showed a dose dependent increase in the reduction of Fe³⁺ to Fe²⁺ when compared to the standard catechin. This is an indication that the reducing ability of MEBLL is greater than catechin. Some studies have also reported that most plants have powerful reducing ability. The ability of MEBLL can also be attributed to the presence of flavonoids.in substantial amount. Concerning the anti-bacterial properties of MEBLL, the extract was evaluated against four different strains of bacteria and findings from this study showed that MEBLL displayed strong antibacterial activity against Staphylococus aureus, Streptococcus pneumonae, Escherichia coli and Pseudomonas aeruginosa respectively in the order of inhibition when compared with ciprofloxacin control. It is therefore obvious that MEBLL has strong antibacterial properties which can be harnessed to treat bacterial diseases and infections. Many studies have shown that most plant extracts rich in bioactive metabolites like alkaloids, saponins and terpenoids have characteristic anti-fungal properties. In this study, we evaluated the anti-fungal properties of MEBLL against two potent fungi namely Candida albicans and Aspergillus niger and result showed that MEBLL strongly inhibited these two fungi when compared to griseofulvin control. This is an indication that MEBLL possesses the ability to treat fungi diseases and infections, leveraging on its anti-fungi properties.

Conclusion

Plant possesses many thousands of secondary metabolites that could found useful in diseases management and intervention if its potential is maximally harnessed. Findings from this study revealed that Methanol Extract of Baphia longipedicellata leaf (MEBLL) exhibited excellent free radical scavenging ability, bacteriostatic and bacteriocidal actions against the various microorganisms accommodated in this study. The activities of this plant extract may be due to the presence of a number of phytochemicals in it. Therefore, this plant has demonstrated a promising intervention and leads as an antioxidant and antimicrobial agent.

Recommendation

Further studies should be carried out to isolate, characterize and elucidate the active ingredients in the plant and also test the in vivo mitigating power against some diseases using rat model.

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