ANTIOXIDANT, INHIBITION OF ADVANCED GLYCATION END-PRODUCT FORMATION AND ANTIMICROBIAL ACTIVITIES OF OCIMUM GRATISSIMUM (SCENT LEAF) METHANOLIC LEAF EXTRACT AGAINST Escherichia coli AND Bacillus SPP

The study investigates the antioxidant, inhibition of advanced glycation end-product formation and antimicrobial activities of methanolic leaf extract of Ocimum gratissimum . GC-MS, qualitative, antioxidant scavenging and antiglycation activities of the extract of Ocimum gratissimum were determined using standard procedures. The antimicrobial activity was evaluated by agar well diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using standard methods. The GC-MS analysis of Ocimun gratissimum leaf revealed the presence of 49 compounds with P –cymene been the most abundant. The phytochemical screening of the extract shows the presence of alkaloids, tannins, phenolic, flavonoids, saponins etc. The in-vitro antioxidant assay of the extract was found to have β-carotene, lycopene, total phenolic compounds, total flavonoid compounds, reducing power and DPPH scavenging activities. The extract showed significant inhibitory effects on the formation of compounds containing two carbonyl groups and Advanced Glycated End products formation with IC 50 of 75.85 ±4.87 µg/mL. Agar well diffusion assay was characterized by inhibition zones of 21.0 ± 0.30, 19.66 ± 0.20, 33.78± 0.30 and 33.25 ± 0.40 mm for Escherichia coli and Bacillus spp respectively at 250 mg/ml for Ocimun gratissimum and 25 mg/ml for tetracycline solution. The MIC values for E.coli and Bacillus spp were 33.33, 66.67, 0.78 and 0.78 for both extract and tetracycline while their MBC values were 66.67, 133.00, 1.56 and 1.56 respectively. MBC/MIC values showed that Ocimun gratissimum extract and tetracycline had bactericidal effects. These results indicated that Ocimum gratissimum possesses antioxidant, prevents glycation and has antimicrobial activities.


INTRODUCTION
The use of synthetic drugs in the treatment of various viral and bacterial diseases has leads to complication due to some of the chemical composition of these drugs can be cytotoxic and carcinogenic when administered in large doses [1].The use of natural or ethnomedicinal herbs as an alternative for orthodox drugs is very important to prevent the negative side effects and toxicity of synthetic drugs.Herbal medicines are used in place of synthetic drugs and may look primitive when compared to orthodox drugs, which are believed to be better than those made from herbal plants.In developing countries, 75-80% of their population depend on herbal medicines as their primary health care.Ocimun gratissimum also called scent leaf is widely distributed in the tropics of Asia and Africa.It Ocimum gratissimum is well known for its antioxidant property and its medicinal values may be due to its rich polyphenolic compounds [2].It is commonly used by traditional medicine practitioners and has also been reported to show certain biological activities such as: antimicrobial, phytotherapeutic properties, as well as insect repellant and antioxidant activities [3][4][5][6].Ocimum gratissimum has been used locally for the treatment of dysentery, diarrhea, candidiasis, and gastrointestinal disorders caused by various gastrointestinal organisms.
Glycation process occurs when carbonyl group of reducing carbohydrates reacts with amino group of proteins, nucleic acids and other molecules non-enzymatically to initiate the process glycation (fructosamine products) [7].The Amadori products formed will then undergo numerous irreversible reactions to form highly reactive carbonyl species like 3-deoxyglucosone, glyoxal and methylglyoxal.These

Collection and Identification of Ocimum gratissimum
The leaf of Ocimum gratissimum was obtained

Detection of components
The interpretation of mass spectrum gas chromatography-mass spectrometry was conducted by the database of the National Institute Standard and Technique (NIST) which contain more than sixty-two thousand patterns.
The spectrum of the unidentified compound was compared with the spectrum of the identified compounds stored in the NIST library.The names, molecular weight, structure of the compounds in the test material were ascertained.

Qualitative phytochemical analysis of Ocimum gratissimum methanolic leaf extract
Phytochemical analyses for secondary metabolite constituents were carried out on the Ocimum gratissimum methanolic leaf extract using standard phytochemical procedures [8,9].

In-vitro plant antioxidant assay
Determination of β-Carotene and lycopene determination β-carotene and lycopene were determined in the dried plant of Ocimum gratissimum by the method described by Barros et al., (2007) [10].

Total phenolic contents determination
The methanolic leaf extract of Ocimum gratissimum was used for the determination of total phenolic content (TPC) using Folin-Ciocalteu reagent with standard procedure [11].

In vitro antiglycation inhibition assay
In vitro antiglycation activity of the methanolic leaf extract of Ocimum gratissimum was examined by bovine serum albumin (BSA)glucose model.Antiglycation activity of the plant extract was determined using standard reported method [15], with slight modification.In

Determination of diameter of zone of inhibition using agar well diffusion method
The procedure of agar well-diffusion method was employed to determine the antimicrobial activity of Ocimum gratissimum.The susceptibility of the two different organisms (Escherichia coli and Bacillus spp) to Ocimum gratissimum methanolic leaf extract were assayed using standard method [16].The experiment was repeated thrice, for each replicate, the readings were taken in three different fixed directions and the average values were recorded [16].

Ocimum gratissimum extract
Minimum inhibition concentration is the lowest extract concentration that inhibited the growth of the test organism as indicated by the absence of visible turbidity in the tube compared with the control tubes.The MIC of Ocimum gratissimum methanolic leaf extract was determined according to standard method [16].The MIC of the Ocimun gratissimum extract was assayed using serial dilution method.The lowest concentration of the dilution of the methanolic leaf extract in mg/ml without bacterial growth was taken as the minimum inhibition concentration.

Minimum Bactericidal Concentration (MBC) of Ocimum gratissimum extract
The minimum bactericidal concentration of the Ocimum gratissimum methanolic leaf extract was carried out by standard method [16].

Statistical Analysis
The in vitro BSA-AGE assay for the Ocimum gratissimum methanolic leaf extracts and IC50 assays for the extract were represented as mean ± standard deviation.The data analysis was done using Graph Pad prism computer software version 5.01.Unpaired t-test suggested significant differences at p < 0.05.
The preliminary qualitative analysis of the active components (secondary metabolites) of Ocimun gratissimum leaf using methanolic extraction showed the presence of different constituents such as alkaloids, flavonoids, terpenoids, phenolic, cardiac glycosides, tannins which have been reported to have curative effects on pathogens by serving as defense mechanism against micro-organisms, in addition to their various biological activities such as, antidiabetes, anti-inflammatory, antimicrobial and anticancer [23].
The values obtained for lycopene and β-Carotene were 0.035±0.03μg/g and 0.136 ± 0.01 μg respectively.The reducing power activity assay shows that the Ocimun gratissimum extracts exhibited reducing capacity (0.873±0.05OD at 700 nm) less than ascorbic acid (0.924±0.012OD at 700 nm).The total flavonoids and total phenolic compounds obtained (Table 3) shows Ocimum gratissimum extracts was assessed for their antioxidant activity using 1, 1-diphenyl-2picrylhydrazyl radical method and expressed as percent DPPH inhibition as shown in Table 3 above.The strong antioxidant activities of Ocimun gratissimum extracts are possible as a result of flavonoids and phenolics which have been shown to have high antioxidant properties [24,25].
There are few studies reported on the antiglycation activity of Ocimun gratissimum, however, it is well known that active ingredients in this plant extracts are responsible for this activity [26,27].The mechanism of glycation process is also well understood, as oxygen free radicals such as, ROS (reactive oxygen species) plays a crucial role in protein glycation via lipid peroxidation during AGEs formation.
Incorporation of these free radicals into the glycation process leads to the formation of AGEs belongs to the family Labiatae and Ocimun gratissimum is the most abundant of the genus Oscimum.In Nigeria, scent leaf is called Nchonwu by Igbo, effinrin-nia by the people of the Yoruba´s speaking tribe, while in the northern region of Nigeria; the Hausas call it Daidoya.
reactive carbonyls species react with sulfhydryl, amino and functional groups found in guanidine of extracellular and intracellular proteins to produce stable advanced glycation end products (AGEs).The AGE products can cross-link with proteins like lens crystallin, collagen, low density lipoprotein and hemoglobin which lead to the alteration of the structures and functions of these proteins in vivo.The formation of protein glycation is a key molecular basis of diabetic complications which results from chronic increase in blood sugar.Synthetic AGEs inhibitor drug like aminoguanidine (AG), plant extracts and their purified constituents have been shown to possess antiglycation properties and suppress AGE formation.Antiglycation properties of plant extracts have been attributed to the present of substance containing phenolic compounds and correlated to their free radical scavenging properties.Therefore, glycation is an essential important target for the treatment of complication arising from diabetic.Despite the popular use of this medicinal plant, there are few data available on their antiglycation and antimicrobial potentials.For these reasons, this study aims to determine the antioxidant, antiglycation and antimicrobial activities of methanolic leaf extract of Ocimum gratissimum.

DPPH Radical scavenging activity assay of methanolic leaf extract of Ocimum gratissimum 1 , 1 -
Total flavonoids content was determined in the extract by using the protocol ofSakanaka et al., (2005) [12].The ferric reducing power capacities of the methanolic leaf extract of Ocimum gratissimum was determined using method described byWang et al., (2008) [13], with ascorbic acid being used as the positive controls.The experiments were carried out in triplicate.Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity was measured in the plant according to the procedure described byGyamfi et al., (1999) [14].The DPPH radical scavenging activity was calculated by using the following equation: Radical scavening activity (%) = Acontrol -Asample x 100 Acontrol The IC50 was defined as the concentration (in μg/mL) of the Ocimum gratissimum leaf extract required to deplete the amount of DPPH radical by 50%.Ascorbic acid (AA) was used as the standard.
different tubes assays, each test tube contain 60 µL reaction mixtures containing 20 µL of BSA, 20µL glucose anhydrous and 20 µL of methanolic leaf extract of Ocimun gratissimum sample of different concentration.Glycated control test tube contain 20 µL BSA, 20 µL glucose, and 20 µL sodium tetraoxophosphate (vi) buffer (pH 7.4, 67mM), while blank control test tube contains 20 µL of BSA and 40 µL sodium tetraoxophosphate (vi) buffer.The reaction mixture was incubated at 37ºC for 7 days.After incubation, 6 µL (100%) of trichloroacetic acid (TCA) was added to each test tube and centrifuge at 1500 rpm for 20 minutes at 4 o C.After centrifugation, the pellets were rewashed with 60 µL (10%) of TCA.The supernatant containing glucose inhibitor and interfering substance was removed and pellet contains AGE-BSA were dissolved in 60 µL sodium tetraoxophosphate (vi) buffer (PBS).The assessment of fluorescence spectrum (ex.370 nm), and change in fluorescence intensity (ex.370 nm to 440 nm) based on AGEs were monitored by using a spectrofluorimeter.Percentage (%) inhibition was calculated using the following formula: % inhibition = [1 -[fluorescence of sample/fluorescence of glycated)] × 100.Test organisms To study the antimicrobial activity of Ocimum gratissimum methanolic leaf extract, we use two bacterial strains (Escherichia coli and Bacillus spp) obtained from Chemical Sciences Department (Biochemistry unit), Lagos State University of Science and Technology, Lagos, Nigeria.The two microorganisms were maintained at 4°C on Nutrient Agar slant in the Department of Chemical Sciences and fresh subcultures were made before use.

Figure 2 :
Figure 2: Zone of inhibition at 250 mg/ml concentration of the Ocimum gratissimum extract against E.coli

Figure 3 :
Figure 3: Zone of inhibition at 250 mg/ml concentration of the Ocimum gratissimum extract against Bacillus spp.

Figure 6 :
Figure 6: Zone of inhibition at 12.5 mg/ml concentration of tetracycline against Bacillu spp Figure 7: Zone of inhibition at 25 mg/ml concentration of tetracycline against Bacillus spp.
that O. gratissimum may serve as good scavenger of free radicals.The free radicals produce by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) are normally used for the screening of medicinal plants to investigate their antioxidant properties.The principle of this antioxidant assay is the ability of 1, 1-diphenyl-2-picrylhydrazyl, a stable free radical, to diminish the deep purple color in the presence of an antioxidant.The color of 1, 1-diphenyl-2-picrylhydrazyl radical is as a result of the presence of an odd electron found in it.When an antioxidant compound donates an electron to 1, 1-diphenyl-2-picrylhydrazyl, the DPPH is decolorized; this can easily be quantified by noting the change in absorbance at 517 nm.

[
28], hence, it is possible that the reduction of these free radical formation contribute to AGEs inhibition.Several plants with secondary metabolites that act as antioxidants such as, polyphenols and flavonoids with free radicals scavenging and reducing capacity[29,30] have been reported for their high inhibitory activity against non-enzymatic glycation process[25,31].The antiglycation activity of Ocimun gratissimum methanolic leaf extracts was evaluated for the inhibition of advanced glycation endproducts formation based on the BSA/glucose system.The results showed that Ocimun gratissimum exhibited potential antiglycation activity (> 50% inhibition) in a dose dependent manner more than ascorbic acid used in the study (

Mass Spectrometry (GC- MS) Analysis of Ocimum gratissimum
from Lagos State University of Science and Technology Ikorodu in Lagos State, Nigeria.The leaves were identified using PictureThis-Plant identifier.Methyl Siloxane (30 m length × 0.32 mm diameter × 0.25 μm film thickness).The carrier gas used was helium at a constant flow rate of 1.573 ml/min, an initial nominal pressure of 1.9514 psi and at an average velocity of 46 cm/s.

Table 1 :
Phytocomponents identified in the leaf of Ocimum gratissimum using GC-MS analysis

Table 2 :
The qualitative phytochemical constituents of Ocimum gratissimum methanolic leaf extract

Table 3 :
Estimation of antioxidant and free radical scavenging activities of methanolic leaf extract of

Table 4 :
Result showing the percentage inhibition of glycation by methanolic leaf extract of Ocimum gratissimum Values are expressed as mean ± SD

Table 5 :
Zone of inhibition of Ocimum gratissimum methanol leaf extract and tetracycline solution against Escherichia coli and Bacillus spp

Table 6 :
Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) forOcimum gratissimum methanolic leaf extract and tetracycline solution against Escherichia coli and Bacillus spp

Table 4
[1]r et al., (2019)t the MIC values for E.coli and Bacillus spp were 33.33, 66.67, 0.78, and 0.78 for both Ocimun gratissimum extract and tetracycline while their MBC values were 66.67, 133.00, 1.56 and 1.56 respectively.Umar et al., (2019)[1]study shows that methanol and chloroform extracts of Ocimun gratissimum have an MIC of 50 mg/mL against S. typhi and E. coli and the same extracts recorded MBC of 50 mg/mL for B. subtilis and S. typhi.MBC/ MIC results showed that Ocimun gratissimum methanolic leaf extract and tetracycline had bactericidal effects.Ocimum gratissimum has strong potency against these microorganisms with E. coli being the most susceptible.Aderele et al., (2020) [16] study shows that calculated MBC/MIC ratio has bactericidal effect if the values of the ratio are less than or equal to 4 and bacteriostatic if the ratio is greater than 4. Ocimum gratissimum has potential as natural therapeutic agents against diabetes as shown from the antiglication activity.The methanolic leaf extract of Ocimum Gratissimum is effective on Escherichia coli and Bacillus spp.This is an indication that the extract has antimicrobial activity.The MIC values for E.coli and Bacillus spp were 33.33, 66.67 while their MBC values were 66.67, 133.00 respectively.MBC/MIC values showed that Ocimum gratissimum extract had bactericidal effects.
against Escherichia coli and Inhibition of 19.66 ± 0.20 mm compared to tetracycline (29.13 ± 0.30 a low concentration of 50 mg/ml.P-cymene been the most abundant.The in-vitro antioxidant assay of the extract was found to have β-carotene, lycopene, total phenolic compounds, total flavonoid compounds, reducing power and DPPH radical scavenging activities.