When the fresh leaves were collected, the immediately were washed in running water to remove all dirt and bugs that may be on the leaves. The motive of conducting the collection and drying process in one day is to attain the compounds found in the leaves. The dried leaves were blended and stored in tightly closed scotch bottles to avoid moisture.
5.1 Extraction Process
The drying process used was oven drying and according to Olabode et al., 2015, Temperature had a significant effect on the mineral composition of moringa leaves, except magnesium. No significant difference occurred in the magnesium, sodium, and phosphorus contents, along with the color, aroma, and flavor of water extract of moringa leaves at drying temperatures of 60 and 80ºC. Moringa leaves contain essential nutrients that are essential to the well-being of man. Furthermore, the temperature of drying had varying effects on the components of the leaves. Over-dying the leaves will drastically change the green colour of the leaves in turn affect the phytochemical compounds too.
The sonication extraction method had more yield as compared to the Soxhlet extraction this can be due to the simplicity of the sonication extraction and the effectiveness ultrasound frequencies. To improve the yield of the soxhlet sample, the heating mantel temperature should not be placed at a high temperature as this will minimize the solvent in the round bottom flask and alter the extraction process.
Soxhlet method proved best in terms of high extraction efficiency, and extraction of phenolic compounds. Microwave assisted extraction with intermittent cooling (MAE), room temperature extraction by shaking (ERT), and ultra-sonication assisted extraction (UAE) proved good at extracting antibacterial compounds from plant seeds. Latter also proved effective at extracting antioxidant compounds. Methanol extraction proved most suitable solvent for of flavonoids
5.1.1 Soxhlet extraction
In this extraction process 15 grams of sample was placed in the extractor, prior to this, cotton wool was used to close the thimble to allow filtration in the siphon arm. From the 300ml of ethanol, 150 ml was placed the extractor and the excess in the round bottom flask set in a heating mantel.
For the soxhlet to function effectively, the condenser must have continuous water supply .In this case, the water inlet was at the top and outlet at the bottom. The water pressure ought to be at a moderate flow to avoid improper condensation which could crack the condenser.
5.1.2 Sonication process
The same amount of sample and solvent was used, except they were placed in a beaker and covered with aluminium foil. The process was 6 hours at a frequency of 20Hz, temperature of 70ºC
Unlike the soxhlet method, the sonication process had no condenser instead the components are broken down at a particular frequency .It assures full extraction as compaired to soxhlet.
The similarity in the two extraction methods is the use of high temperature which solely correspond with the solvent used. This intention is to allow the solvent to evaporate causing the extraction of compounds from the leaves. They both require rotary evaporation process or fume hood drying in order to obtain a crude extract.
5.1.3 Rotary Evaporator
This equipment was used to distill the solvent and separate the solvent from the sample .The parameters as follows; the boiling point of the solvent (ethanol) is 78 ºC hence the water bath temperature was set at 80ºC, the round bottom flask rotated at a 150-160 rpm and the water pressure was at a medium flow.
To have substantial results, the round bottom flask should be rinsed with the solvent of use before the process and the water bath should only be filled with distilled water to avoid rusting.
5.1.4 McFarland solution
A McFarland Standard is a chemical solution of barium chloride and sulfuric acid; the reaction between these two chemicals results in the production of a fine precipitate, barium sulfate. The standard most commonly used in the clinical microbiology laboratory is the 0.5 McFarland Standard, which is prescribed for antimicrobial susceptibility testing and culture media performance testing.
5.2 Subculture microorganisms
The microorganisms where subculture in an aseptic manner. With a metal hook, the bacteria was streaked in 4 quadrants with the motive of obtaining a single colony in the 4th quadrant. The plates were then placed in an incubator at 37º C for 24hours in an inverted position.
Contamination of bacteria was avoided not leaving the plate exposed for a long time, thoughly heating the hook before and after streaking a bacteria ,insuring that the hook is not too hot resulting in killing the microorganism and placing the plates in an inverted position to avoid water vapour from interfering bacterial growth.
The antimicrobial action of plant extract compounds was related to inactivation of cellular enzymes, which depended on the rate of penetration of the substance into the cell or caused by membrane permeability changes. Increased membrane permeability is a major factor in the mechanism of antimicrobial action, where compounds may disrupt membranes and cause a loss of cellular integrity and eventual cell death
5.3 Thin layer chromatography
According to Figure 4.3.1 both soxhlet and sonication extraction samples had similar phytochemicals that appeared in various colours ranging from light yellow, yellow, orange ,green to dark green.
Phytochemicals are the chemical constituents in plants with distinct physiological action on the human body. Alkaloids, flavonoids, phenolics, terpenoids, and essential oils are some of the important bioactive phytochemicals. It is observed that the geographic location of the plant and the solvent system used in the extraction process may act as determining a factor for the distribution of these phytochemical (Sankhalkar and Vernekar., 2016)
The yellow colour phytochemical observed on the TLC plate shows a consistency in table 4.3.2 of having the highest Rf value in both samples ranging from (0.71-0.98). The lowest Rf value is 1 recorded from the movement of the light orange colour phytochemical. According to Sharma and Paliwal .,2013 TLC of ethanol extract of Moringa oleifera pods revealed the presence of 3 compounds having Rf values of 0.75, 0.87 and 0.91 respectively when a solvent phase of Chloroform.
All the plates observed have a similarity is the separation of the phytochemicals, the green colour always appears in between the orange and the yellow colour. Flavonoids like quercetin and kaempferol are reported in ethanolic leaf extracts of M. oleifera (Shakhalkar and Vernekar., 2016) from this statement the yellow coloured phytochemical can be identified as kaempferol.
In order to obtain accurate results, the chamber was rinsed with the stationary phase (methanol and chloroform) before the process began. This is create an atmosphere in the chamber and eliminate previous solvents used. The plates were touched at the edges or corners because the oils found in our hands can interfere with the final results. A pencil, instead of a pen or marker was used because ink is prone to be separated on the TLC plate.
In comparision with the Rf values from the two different extraction process, sonication sample G had the highest value and the lowest value was from a soxhlet sample D at 0.29 according to table 4.3.2.
Various phytochemicals gave different Rf values in different solvent system. This variation in Rf values of the phytochemicals provided a very important clue in understanding of their polarity and also helps in selection of appropriate solvent system for separation of pure compounds by column chromatography. Mixture of solvents with variable polarity in different ratio was used for separation of pure compound from plant extract. The selection of appropriate solvent system for a particular plant extract can only be achieved by analyzing the Rf values of compounds in different solvent system .Different Rf values of the compound also reflect an idea about their polarity. This information will help in selection of appropriate solvent system for further separation of compound from these plant extracts.
5.4 Antimicrobial test
The currently available screening methods for the detection of antimicrobial activity of natural products fall into three groups, including bio autographic, diffusion, and dilution methods. The bio autographic and diffusion methods are known as qualitative techniques since these methods will only give an idea of the presence or absence of substances with antimicrobial activity. On the other hand, dilution methods are considered quantitative assays once they determine the minimal inhibitory concentration (Valgus et al., 2006).
Plants and other natural sources can provide a huge range of complex and structurally diverse compounds. Recently, many researchers have focused on the investigation of plant and microbial extracts, essential oils, pure secondary metabolites and new synthetized molecules as potential antimicrobial agents as stressed by Balouiri et al .,2015.
According to Figure 4.4 In all the bacteria used, inhibition occurred at a range of (1-12.5mm) .This activity against both gram negative and gram positive bacteria may be attributed to presence of some broad-spectrum antibacterial compounds. These compounds are claimed to be from bioactive secondary metabolites such as alkaloids, flavonoids, saponins and tannins (Abdallah, 2016).
Flavonoids enhance the effects of Vitamin C and function as antioxidants. They are also known to be biologically active against liver toxins, tumors, viruses and other microbes. Tannins have shown potential Antiviral, Antibacterial and Antiparasitic effects. Saponins cause haemolysis of red blood cells (Patel et al., 2014).
As stressed by Idris and Abubakar., 2016, It is important also to note that phytochemical constituent is an important factor that determines the antimicrobial properties of the leave extract. This explain why medicinal plants are used as antimicrobial drugs, several authors have linked the presence of bioactive compound to the antimicrobial properties of the plant extract.
5.4.1 Disc diffusion
The principle of disc diffusion according to Balouiri et al., 2015, a filter paper discs (about 6 mm in diameter) is used, containing the test compound at a desired concentration, which are then placed on the agar surface. The Petri dishes are incubated under suitable conditions. Generally, antimicrobial agent diffuses into the agar and inhibits germination and growth of the test microorganism and then the diameters of inhibition growth zones are measured.
Given that a duplicate had to be performed, 2 plates for each bacteria and 1 plate was used for disc diffusion .In the sample plates 6 discs were used for the different concentrations, and the control plate had 6 discs ( 2 each for antibiotic, solvent and distilled water).
The purpose of having a control plate is to have guidance in the experiment being conducted, the positive control shows the expected results whereas the negative control shows the unexpected results.
Having 2 gram positive bacteria and 2 gram negative bacteria, their to the extract varied, the difference in bacterial response to the Moringa extracts was possibly due to the nature of the bacterial species ( how old or fresh the bacteria is )
The advantages of using disc diffusion is that it is cheap, efficient, it can be used for many types of microorganisms and the results are easily interpreted.
5.4.3 Gram Positive bacteria
The bacteria used was Staphylococcus aureus and Bacillus cereus, according to table 4.4.1 the zone of inhibition in these two bacterias fell in a range of 1mm – 12.5mm. The highest inhibition can be observed in B.cereus was 8mm and in S.aureus was 12.5mm.
The control shows inhibition around the disc that contained the antibiotic with 11.5mm and 5mm inhibition of ethanol B.cereus .Streptococcus showed 11mm inhibition for antibiotic and 3mm for ethanol.
5.4.4 Gram Negative bacteria
The bacteria used was E.coli and Pseudomonas aeruginosa .according to table 4.4.1 the range of zone on inhibition was 1mm to 5mm. The highest inhibition can be observed in E.coli was 5mm and in P. aeruginosa was 5mm.
Between the two types of bacteria, the gram positive bacteria had the highest range and score of 12.5mm form S. aureus. The control results showed Pseudomonas had the overall highest inhibition at 18mm for antibiotic and 4.5mm for ethanol. E.coli had the least inhibition at 3.5mm for antibitotic and 4mm inhibition for ethanol
The use of MHA (Mueller Hinton Agar) instead of NA (Nutrient Agar) should have been used because MHA has a much firm surface area that NA and the inhition is clearly shown in MHA.
As stressed by Kilany, 2016, Gram-positive bacteria are more susceptible because of the presence of an outer peptidoglycan layer, which is not an efficient permeability barrier. The antimicrobial activity of the ethanol plant extract is less than that of antibiotics; this may be because antibiotics are in a p urified form, whereas the plant extracts are still in a crude state. Therefore, the antimicrobial activity of the plant extract might be higher after purification.
The antibiotic used was Rifampin, it inhibits DNA-dependent RNA polymerase activity in susceptible cells. Specifically, it interacts with bacterial RNA polymerase but does not inhibit the mammalian enzyme. It is bactericidal and has a very broad spectrum of activity against most gram-positive and gram-negative organisms (including Pseudomonas aeruginosa) and specifically Mycobacterium tuberculosis. Because of rapid emergence of resistant bacteria, use is restricted to treatment of mycobacterial infections and a few other indications
5.5 Minimum inhibition Concentration
Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism.
The MIC is the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in tubes or micro dilution wells as detected by the unaided eye.
Balouiri et al .,2015 elaborates that, the agar disk-diffusion method is not appropriate to determine the minimum inhibitory concentration (MIC), as it is impossible to quantify the amount of the antimicrobial agent diffused into the agar medium. Nevertheless, an approximate MIC can be calculated for some microorganisms and antibiotics by comparing the inhibition zones with stored algorithms.
The lane 1 to 8 was used for the dilution, therefore the sample concentration was higher in lane 1 as compared to lane 8 and the vice versa fir the bacteria ( lower concentration in 1 and highest in 8). The rows A1, C1 and D1 show inconsistency in the decrease of concentration from 1-8 where as in rows B1, E1, F1 and G1 there is a uniform decrease before incubation as shown in figure 4.5.1
After incubation, Overflow was observed in C1, E1 and H1, though a decrease was analysed from the lane 2-8. A1, B1, D1, F1and G1 shows consistent decrease in readings as shown in figure 4.5.2.
Various factors can contributed to the wrong interpretation of result such as not calibrating the ELISA plate reader before using it, not wiping any liquid that might be on the surface of the plate, not covering the plate with aluminium foil before incubation, failure to read the OD of the bacteria immediately after placing it in the well plate and minimal to excess incubation time.
For the determination of MIC endpoint, viewing devices can facilitate reading micro dilution tests and recording results with high ability to discern growth in the wells. Moreover, several colorimetric methods based on the use of dye reagents have been developed
To improve the results obtained, a dye should have been added to clearly visualise the growth inhibition of the bacteria such as Alamar blue dye (resazurin) and to determine the acturate end point Tetrazolium salts, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) can be used.
Prior to MIC, the OD of the bacteria was conducted by using the UV spectrophotometer at 625nm and the appropriate range was 0.08 -0.11. The OD readings were as follows; B. cereus 0.104, E.coli 0.100, P.aeruginosa 0.09 and S.aureus 0.103.
OD600 or above is preferable to UV spectroscopy when measuring the growth over time of a cell population because, at this wavelength, the cells will not be killed as they would under too much UV light. UV light has also been shown to cause small to medium-sized mutations in bacteria potential altering or destroying genes of interest.