Antibacterial Properties and Phytochemicals Screening of Tectona grandis (Teak) Leaf Extracts against Bacteria Implicated from Upper Respiratory Tract Infections

Screening of Tectona grandis (Teak) Leaf Extracts against Some


Introduction
Upper respiratory tract infections (URTIs)are caused by mainly viruses (human rhinovirus (hRV), coronavirus, parainfluenza viruses (PIVs), adenovirus (ADV), human metapneumovirus (hMPV), influenza, enterovirus, bocavirus and respiratory syncytial virus (RSV)) and some bacteria, though present with almost indistinguishable clinical symptoms (Heikkinen andJarvinen, 2003, Allander, 2008;Caliendo, 2011;Harris et al., 2016;Pokorski, 2016).In most cases, it spreads from person-to-person, when touching the secretions by hand or directly inhaling the respiratory droplets.Bacterial infections could be a prime cause of URTIs, but they may also be due to superinfection of a primarily viral infection (Turner, 2007).Risk factors for the development of upper respiratory tract infections are close contact with infected person, most likely close contact of children who attend the kindergarten or school, travelers with exposure to numerous individuals, smoking which may alter mucosal resistance, anatomic changes of respiratory tract, and nasal polyposis (Heymann,2014).URTIs causes variety of patient diseases such as acute bronchitis, the How to cite this article: O. O. Daramola, S. God'swealth U., Unwana E.O., O.M. Olugbenga1, (2022).Antibacterial Properties and Phytochemicals Screening of Tectona grandis (Teak) Leaf Extracts against Some Clinical Isolates Causative Agents of Upper Respiratory Tract Infections.Journal of Microbes and Research. 1 (1).DOI: 10.58489/2836-2187/004 Page 2 of 9 common cold, influenza, and respiratory distress syndromes (Thomas and Bomar, 2022).It has a high cost to society, being responsible for absenteeism from school and work, unnecessary medical care and is occasionally associated with serious sequelae (Cotton et al., 2008).It affects the nose, nasal cavity, pharynx, and larynx with subglottic area of trachea (Green, 2006)

Collection, Identification and Extraction of Plant Materials
Fresh leaves of Tectona grandis were collected from the vicinity of D. S. Adegbenro ICT Polytechnic, Itori, Ewekoro Local Government Area, Ogun State, Nigeria.The identification and authentication of the plant materials was done at Forest Research of Nigeria, JerichoHill, Ibadan, Oyo State, with the voucher number of FHI 113357.The collected sample was air dried at room temperature for twenty-one (21) days.The samples were ground into powdery state using an electric blender.200 g of the ground sample of the leaf was weighed and dissolved in 500 mL of ethanol, again 200 g of the ground sample of the leaf was weighed and dissolved in 500 mL of ethyl acetate.They were kept in the refrigerator for 72 hours.The extract was filtered using a chess cloth and Whatman filter paper No. 1 (125 cm), to obtain filtrates of the respective solvents of ethanol and ethyl acetate, which were then used for the study.

Collections and Maintenance of Test Organisms
The test organisms used for this study were all clinical isolates from the Department of Medical Microbiology and Parasitology, Sacred Heart Hospital, Lantoro, Abeokuta, Ogun State.The isolates include; Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus.The organisms were collected on a sterile agar slant and incubated at 37oCfor 24 hours.

Preparation of Agar
Agar was prepared according to manufacturer instruction and the agar used was nutrient agar for the analysis.Seven (7) grams of nutrient agar was weighed and dissolved in 250 ml of distilled water, mixed and was autoclaved at 121oC for 15 minutes.Antimicrobial Assay (Agar Well Diffusion Method) Antimicrobial activities of ethyl acetate and ethanol extracts of Tectona grandis L. leaf were evaluated by the agar well diffusion method (Bauer et al., 1996) using ciprofloxacin as positive control.The microbial culture was adjusted to 0.5 McFarland turbidity standards.The plate was flooded with 1 ml each of the standardized test organism, swirled and excess inoculums was carefully decanted.A sterile cork borer was used to make wells (6 mm in diameter) on the agar plates.Aliquots 0.2 ml of dilution were applied on each well in the culture plates previously inoculated with the test organisms.The holes were filled with the plant extract.Each well was labeled approximately; control experiment was also carried out where the hole was filled with ciprofloxacin as positive control for bacteria.However, each extract was tested in triplicates.These were then left on the bench for 1 h for proper diffusion of the nanoparticles (NCCLS, 1990).Thereafter the plates were incubated at 37oC for 24 h for bacteria.Antimicrobial activities were determined by measuring the zone of inhibition around each well (Excluding the diameter of the well) for the extract.Triplicate tests were conducted against each organism and diameter of zone of inhibition (mm) as expressed as Mean and Standard derivation.

Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) of the ethyl acetate and ethanol extracts of Tectona grandis L. leaf for the bacterial isolates were evaluated according to the method of Ochei and Kolhatkar (2008), using microtubes dilution method described by National Committee for Clinical Laboratory standards (NCCLS,2000).

Determination of Minimum Bactericidal Concentration (MBC)
The minimum bactericidal concentration (MBC) was determined by first selecting the tubes that showed no growth during the MIC determination.A loopful from each of the tube was sub-cultured on the sterile nutrient agar and incubated for 24 h at 37oC.The bactericidal effect was demonstrated when no growth occurred on the medium.

Quantitative
Phytochemical Screening Phytochemical screening was carried out on the plant extracts to ascertain the presence of secondary metabolites.Presence of saponin, tannin, alkaloid and flavonoid were determined using the procedure as described by Sofowora (2008).

Determination of the Mineral Composition
The mineral contents of the samples were determined by the procedure of AOAC (2000).Calcium, potassium, magnesium, phosphorus, iron and other elements were measured with Atomic Absorption Spectrophotometer (Thermo scientific S Series Model GE 712354) after digesting with perchloric-nitric acid mixture (AOAC, 2000).Prior to digestion, 5 ml of the samples were measured into a 125 mL Erlenmeyer flask with the addition of perchloric acid (4 mL), concentrated HNO3 (25 mL) and concentrated sulphuric acid (2 mL) under a fume hood.The contents were mixed and heated gently in a digester at low to medium heat on a hot plate under perchloric acid fume hood and heating was continued until dense white fume appeared.Heating was continued strongly for half a minute and then allowed to cool followed by the addition of 50 mL distilled water.The solution was allowed to cool and filtered completely with a wash bottle into a Pyrex volumetric flask and then made up with distilled water.The solution was then read on Atomic Absorption Spectrophotometer.

Quantitative Phytochemical study
In the study, tannins, saponins, alkaloids, flavonoids, glycoside and anthraquinone were observed in the quantitative analysis of the phytochemical study.Flavonoids were most abundant followed by alkaloids and tannins respectively (Tables 1).

Mineral compositions
The mineral compositions of the plant sample are presented in Table 2. Sodium (Na), potassium (K) and calcium (Ca) and iron (Fe) were presented in appreciable quantities in both solvents.But Sodium (Na), Potassium (K), and Calcium (Ca) were highly present in ethyl acetate extract than in the ethanol extract excluding Iron (Fe), which is more present in the ethanol extract than the ethyl acetate.Moreso, the heavy metals compositions of the plant sample were presented in Table 3. Lead (Pb), Cadmium (Cd) and Arsenic (As) were presented in significant quantities in both extracts of T. grandis leaf.However, high amount of the heavy metal's composition was observed in the ethanol extract.The Mean with difference along the same row is significant at (p<0> Tukey Duncan's Multiple range test.Values are mean of three replicates ± Standard Deviation.

Minimum Inhibitory Concentration (MIC)
MIC activity of Tectona grandis on the microorganisms is presented in Table 5.The same mean zone diameter of 7.29± 4.774 mm was observed for all the organisms with both extracts.However, a mean zone of inhibition of 8.33±3.608mm was obtained for E. coli and Staphylococcus aureus, whereas the least mean zone of inhibition of 5.21±1.804was recorded for Klebsiella pneumonia with the control antibiotics.The Mean with difference along the same row is significant at (p<0> Tukey Duncan's Multiple range test.Values are mean of three replicates ± Standard Deviation.The Mean with difference along the same row is significant at (p<0> Tukey Duncan's Multiple range test.Values are mean of three replicates ± Standard Deviation.

Minimum Bactericidal Concentration (MBC)
The resistance to antibiotics is becoming an increasingly important health and economic menace.However, different antimicrobial substances are elucidated by higher tropical plants (Ogunmefun et al., 2017).The antimicrobial activities of these plants are due to the presence of secondary metabolites present mainly in the leaves, as they are the site for the synthesis of bioactive compounds, hence the presence of high concentrations of active compounds and antibacterial activities in the leaves (Moses et al., 2019).These bioactive compounds, phytochemicals, are known for their interference with the proteins and enzymes of the microbial cell membrane, destroying its structure, leading to the inhibition of various cell functions and the eventual death of the microorganism (Mostafa et al., 2018;Muhammed et al., 2021).
From the results in Tables 4-6, all the microorganisms exhibited varying degrees of susceptibility to both extracts of Tectona grandis.The greater mean zone diameter indicated greater antimicrobial effect of the leaf extracts on the microorganisms.The variation could be attributed to the differences in the cell wall composition of the microorganisms.Furthermore, the genetic composition of plasmids which are easily distributed among the microbial strains might account for these differences (Karaman et al., 2003;Bitchagno et al., 2015).It is surprising to note that the zones of inhibition for the MIC (Table 5) was the same for both extracts of Tectona grandis on all the microorganisms, whereas the mean zone diameter of the MBC (

Materials And Methods Equipment, Chemicals and Reagents
(Iwu et al., 1999)4)e has become a global concern(Westh et al., 2004)as the clinical efficacy of many existing antibiotics is being threatened by the emergence of multi-drug resistant pathogens(Bandow et al., 2003).The majority of bacteria are resistant to many antibiotics therefore, the use of plant extracts against resistant bacteria leads to new choice for the treatment of infectious diseases (Purushotham et al., 2010).Antimicrobials of plant origin have enormous therapeutic potentials(Evans  and Turnbull,2004).Plants are rich source of phytochemicals which are responsible for the therapeutic potentials, therefore, plants are used for making different drugs having medicinal property.25% of the medicinal drugs prepared in the developed countries are based on the plants and their derivatives (Moses et al., 2019).Therefore, this has led to the screening of several medicinal plants for possible antimicrobial activity(Iwu et al., 1999).

Table 1 :
Phytochemical Analysis of Ethyl acetate and Ethanol Extracts of T. grandis leaf (%).

Table 2 :
Mineral compositions of Ethyl acetate and Ethanol Extracts of T. grandis leaf (mg/L).

Table 3 :
Heavy metals compositions of Ethyl acetate and Ethanol Extracts of T. grandis leaf.
Note: All results are represented as mg/L expect Arsenic (As) which is represented in µg/L.

Table 4 :
Antibacterial Activity of Tectona grandis Extracts on some Pathogens.

Table 5 :
Minimum Inhibitory Concentration Activity of Tectona grandis extracts on some Pathogens.

Table 6 :
Table 6 presents the highest mean zone diameter of 100± 0.000 mm obtained for E. coli with ethyl acetate and Staphylococcus aureus with ethanol extract.The control antibiotic had the least mean zone of diameter for all the microorganisms.Minimum Bactericidal Concentration Activity of Tectona grandis Extracts on some Pathogens.

Table 6
(Hashemi et al., 2017)vy metals are metallic chemical element with a relatively high density that are toxic, poisonous and harmful to human well-being at low concentrations(Sobha et al.,  2007).Lead (Pb), cadmium (Cd), and arsenic (As) are typical examples of these heavy metals(Tadesse et al., 2018).In this study, Lead (Pb), cadmium (Cd) and arsenic (As) were presented in significant quantities in both extracts of Tectona grandis leaves.However, high amount of the heavy metal's composition was observed in the ethanol extract.As, Cd, and Pb are the most common heavy metals potentially hazardous to human well-being(Lambert et al.,  2000).However, cadmium and lead have more significant side effects on human well-being(Hashemi et al., 2017).The trivial amounts of heavy metals exhibited, suggests that there are not toxic to the body.Staphylococcus aureus.The plants also contained various phytochemical, minerals and in sufficient quantities of heavy metals thereby validating the use of this plants for therapeutic tenacities.6.Bitchagno, G. T. M., Sama Fonkeng, L., Kopa, T. K., Tala, M. F., Kamdem Wabo, H., Tume, C. B., ... & Kuiate, J. R. (2015).Antibacterial activity of ethanolic extract and compounds from fruits of Cotton, M. F., Innes, S., Jaspan, H., Madide, A., How to cite this article: O. O. Daramola, S. God'swealth U., Unwana E.O., O.M. Olugbenga1, (2022).Antibacterial Properties and Phytochemicals Screening of Tectona grandis (Teak) Leaf Extracts against Some Clinical Isolates Causative Agents of Upper Respiratory Tract Infections.Journal of Microbes and Research.1(1).DOI: 10.& Rabie, H. (2008).Management of upper respiratory tract infections in children.South African Family Practice, 50(2), 6-12.