Investigation on the Phenolic Content in Moringa oleifera and Its Antimicrobial Activity

Moringa oleifera commonly known as drumstick in India belongs to family Moringaceae is widely well-known for its excellent nutritional and medicinal properties (Kumar et al., 2014). M. oleifera is known to possess anti-microbial, hypoglycemic, antioxidant immunomodulatory, antitumor activities and this is due to the presence of various secondary metabolites. It has also been used to treat gastro intestinal problems like gastritis, coltis and constipation. Some of the secondary plant metabolites present in M. oleifera are carotenoids, vitamins, minerals, amino acids, sterols, glycosides, alkaloids, flavonoids and phenolics that attribute to the biological activity of M. oleifera (Patel, 2017; Selmi et al., 2019). Moringa is used as a vegetable and is also used for water purification (Delelegn et al., 2018). It is a fast-growing tree commonly cultivated in tropical and sub-tropical areas. It is cultivated for its leaves, pods and seeds which are used for variety of purposes. All of its parts contain nutritional value including the oil pressed from seed. The seeds contain all vitamins mostly Vitamin B and C and minerals. The seeds can be used as fertilizers, water purifying agents and also as biofuel.
       
M. oleifera is used to treat malnutrition and iron deficiency (Ravani et al., 2017; Saini et al., 2016). M. oleifera is rich in phenolic acids and flavonoids, it’s extract exhibits significant antioxidant activity both in vitro and in vivo, especially  seeds has the highest abundance of identified phenolic compound and antioxidant activity (Adebayo et al., 2018). At present, the M. oleifera extract has been widely used in fields of medicine, functional food and cosmetics (Yadav and Ghimire, 2019; Zhao et al., 2019). Moringa, native to parts of Africa and Asia, is the sole genus in the flowering plant family Moringaceae. The name is derived from murungai. The species of M. oleifera is a little tree with wide, spreading branches and fragrant, cream-shaded or white blossoms that sprout on long, hanging panicles. Depending on the species and atmosphere, Moringa trees might be evergreen or semi-deciduous. Also, there are 13 known types of Moringa, few are developed outside their local natural surroundings. Moringa arborea, M. borziana, M. longituba, M. pygmaea, M. rivae, M. ruspoliana, M. drouhardii and M. hildebrandtii are the species.
       
M. oleifera seed flour was found to be a store house of phenolic compound produced from plant source act as free radical terminator (Shahidi et al., 1992). Most of the workers have reported the use of leaf and or bark extracts elsewhere, while the scientific and effective use of M. oleifera dried seed powder for total phenolic content (TPC) and antibacterial potential against gram positive and negative around the location of Vellore at Tamil Nadu is not reported so far. In view of the above, the present study, we have described the FTIR, HPLC and antibacterial characteristics of crude seed extract Moringa oleifera, the seed extract was further explored the analysis of Total phenolic contents.
       
Therefore, the present study was designed to investigate TPC and characterization crude seed extract specifically FTIR and HPLC and the role of seed extracts against bacterial growth namely Pseudomonas aeruginosa (MTCC 1688), Bacillus subtilis (MTCC 8561), Staphylococcus aureus (MTCC 3160) and Proteus mirabilis (MTCC 3310).

Preparation of M. oleifera seed extract
 
The experiment was conducted during 2020 at the School of BioSciences and Technology, VIT University. Mature seeds of M. oleifera were chosen from dry cracked fruits. The plucked fruits were cracked to obtain the seeds which were air-dried for 2 days. The shells surrounding the seed kernels were removed using a knife and the kernels were powdered using a laboratory mortar and pestle and sieved using a strainer with a pore size of 2.5 mm to obtain a fine powder. The powder was stored in a sterile bottle at room temperature in a dark place. The powdered sample was successively extracted with ethanol in increasing polarity. In this procedure, 50 g of M. oleifera powdered seeds were soaked in 250 mL of ethanol solvent (1: 5). Solvent mixture was left shaking on a horizontal shaker for 3 days. Then, the extracts were filtered separately through sterile Whatman no.1 filter paper. The filtrates were then centrifuged at 5000 rpm for 15 min. The supernatant of each extract was evaporated by using Rota vapor (Laboratory 4000-efficient, Heidolph, Germany). The crude extracts were stored at 4°C.
 
Estimation of total phenolic content (TPC)
 
Colorimetric method using Folin-Ciocalteau reagent was carried out for the estimation of total phenolic content as described by Siddiqui et al., 2017. A blank test tube was prepared using distilled water and gallic acid solution was used as standard. The working standard was prepared to contain a concentration of 0.1 mg/mL and a standard curve was obtained. A calibration curve of absorbance values against varying concentrations of gallic acid standard was plotted. A regression equation of the curve, absorbance value=0.0248 (gallic acid concentration)+0.0003 with R² value, 0.9865 was obtained. Total phenolic contents of the samples in mg gallic acid equivalence (GAE)/g of sample were calculated using the equation.
 
FTIR analysis
 
FTIR relies on the fact that molecules absorb light in the infra-red region of the electromagnetic spectrum. The absorption corresponds specifically to the bands present in the molecule. The frequency ranges are measured as wave numbers typically over the range 4000-400 cm^-1. FTIR was done using Shimadzu IRAffinity-1.
 
HPLC analysis
 
High performance liquid chromatography, help us to get an accurate concentration of phenolic component in compare to the standard which is Gallic acid solution. The mobile phase was prepared using acetonitrile and 20 mM phosphoric acid in the ratio 9:1. It was degassed and filtered before using it for analytical purpose. The flow rate was set to 50 µl/ minute and the injection volume was 20 µl. The run time was 25 minutes. Gallic acid was used as standard with the concentration of 0.1 mg/mL. The wavelength was set to 280 nm and the chromatogram peaks were compared with the standard peaks and the results were interpreted (Gaafar et al., 2016).
 
Antibacterial assay
 
The agar well diffusion method was used in assessing the antibacterial activity of the M. oleifera seed extract. Nutrient agar medium was poured into sterile petri dishes and allowed to solidify. The test organisms were poured on into the solidified agar and was swab all over the surface of the agar. Three different organisms were tested namely Pseudomonas aeruginosa (MTCC 1688), Bacillus subtilis (MTCC 8561), Staphylococcus aureus (MTCC 3160) and Proteus mirabilis (MTCC 3310). After this the plate was kept for incubation at 37°C for 24 hrs. After 24 hrs the anti-bacterial activity was checked by measuring the diameter of the zone of inhibition. Bacterial broth culture was prepared to a density of 10⁸ cells ml of 0.5 McFarland standards. The aliquot was spread evenly onto Mueller-Hinton agar using a sterilized cotton swab. Then, the plated medium was allowed to dry at room temperature for 30 min. On each plate, equidistant wells were prepared with a 9 mm diameter sterilized, cork borer, which were 5 mm from the edge of the plate. Fifty microliters of seed extract (50 mg/mL) was aseptically introduced into a respective agar well. Tetracycline (25 μg/mL) was used as standard (positive) control and sample free solutions as blank control. This was followed by allowing the agar plate on the bench for 40 min pre-diffusion followed by incubation at 37°C for 24-48 h. The formation of clear inhibition zone around the well was regarded as significant susceptibility of the organisms to the extract. The experiment was performed in triplicates (Delelegn et al., 2018).

Sampling and Pre-treatment
 
Pre-treatment makes the phenolic compound active, which was further undergone ftir, tpc and hplc analysis. M. oleifera seeds contain phenolic compounds that are useful as natural antimicrobial agent. The ethanol extract for 50 gms of powdered Moringa oleifera seed yielded 0.6 gms of crude extract.
 
Total phenolic content
 
Phytochemical screening of the ethanolic extracts of M. oleifera seed was carried out by UV-Spectrometer. The graph (Fig 1) shows the presence of phenolic compounds. The total phenolic in terms of the gallic acid equivalent (GAE) in mg/g of the extract. The total phenolic content was calculated with the help of the graph shown in Fig 1 and the standard curve equation was:
 
Where
R²= 0.9599.
       
The average phenolic contents in the ethanolic extracts was calculated to be 11.85 GAE mg/g.
 

 
FTIR
 
The moringa seed extract was characterized by FTIR. The FTIR spectra (Fig 2 and Table 1) revealed that moringa seed  contains phenolic compounds confirmed by specific peaks such as 3280.92 cm^-1 (O-H groups of phenols), 2922.16 cm^-1 (C-H groups of phenols), 1544.98 cm^-1 (C=O carbonyl group of phenols) and 1230.58 (C-O group of phenol) and 1743.65 cm^-1 (C=C group of aromatic compounds) as shown in Table 1.
 

 

 
HPLC
 
HPLC chromatogram of the phenolic compounds in crude extract in the seeds of M. oleifera and standard res are shown in Fig 3a and b respectively. Identified phenolic compounds are listed in Table 2b. The major constituents of the extract are phenolic acids such as, gallic acid, ellagic acid, ascorbic acid, acetyl salicylic acid. These phenolics were identified by comparing with the retention times of the standards. The major phenolic constituents are listed with their RT (Retention Time) and Peak area for each standard with peak area of seed sample in Table 2a and b.
 

 

 

 

 
Antibacterial activity
 
From the agar well diffusion result, it was found that P. aeruginosa, B. subtilis, S. aureus and P. mirabilis was significantly susceptible to ethanol extracts. The ethanol extract had the maximum (17 mm) antibacterial activity against S. aureus, while minimum (7 mm) antibacterial activity against P. mirabilis. The M. oleifera seed extract of ethanol was found to be effective against all the four pathogenic organisms showing a zone of inhibition ranging from 10-17 mm. It was found to be highly effective against S. aureus and least against P. aeruginosa.
       
M. oleifera is a versatile horticulture tree with important medicinal, nutritional and industrial applications, widely distributed and used in India (Pandey et al., 2019). M. oleifera is among  the  most common plants usually consumed by Indian medicine and Almost all the parts of the plant, roots, leaves, flowers and seeds have been used in one way or other in the treatment of various ailments in the indigenous system (Ramachandran and Gopalakrishnan, 2010). In this study, antimicrobial activity of M. oleifera seed extract are due to the total phenolic contents of the seed (Abdulkadir et al., 2015). Total phenolic contents of M. oleifera seeds of different polar fractions yields are displayed in Fig 1. Ethanol extract (11.85 mg GAE/g sample) has showed the highest amount of phenolics. The same scenario reported (Singh et al., 2013) found that ethanolic extract had TPC range from 7.6 to 11.5 g/100 g defatted M. oleifera seed. The values of phenolic content in this current study slightly varied compared to those in the literatures. This may be due to the presence of different amounts of sugars, carotenoids or ascorbic acid, or the duration, geographical variation or methods of extraction, which may alter the amount of phenolics (Burri et al., 2017).
       
According to our study, FTIR range of (4000-500) cm^-1 having many peaks recorded in the Table 1. For more confirmation, the FTIR was compared with the graph of other study, where M. oleifera seeds from Indonesia was used, which show similar peaks, like for -OH phenolic stretch at 3280.92 cm^-1, C-O stretch of phenol at 1097.50 cm^-1 and -OH bending of phenolics at 1057.65 cm^-1; these similarity in peak values confirm the presence of phenolic compounds (Izza et al., 2018). This mild variation in peak values, due to the variation in amount and type of phenolic compounds due to geographic, climatic or environmental changes, which varies from place to place. Phenolic compounds are an important group of active compounds in herbals since they act disrupting the bacterium cell wall, interfering with the ATP pool and altering its membrane potential, resulting in bacterium’s death (Aliyu et al., 2016).
       
The HPLC graph recorded for 280 nm (Fig 3a and 3b) was compared with a standard graph studies by (Mradu et al., 2012), where they analysed eight well known phenolic compounds which were collected from different compounds. Their recorded HPLC retention time were ellagic acid (11.86 min), catechol (9.08 min), gallic acid (3.50 min), resorcinol (7.15 min), vanillin (12.77 min), acetyle salicylic acid (17.46 min), benzoic acid (19.19 min) and ascorbic acid (2.56 min). This study is in accordance with Mradu et al., (2012) which showed a similar values for the seed extract. The similarity with their standard, represent the same phenolic compound in ethanol extract of M. oleifera seed powder. The slight variation in retention times, could be due to difference in calibration of HPLC, same solvent from different manufacturer and run time. Interestingly similar report was observed by Kadam et al., (2017) the retention time of ascorbic acid, galic acid and catechol were 2.56, 3.5 and 9.08 respectively.
       
The antibacterial activity of ethanolic extracts of dried M. oleifera seeds was determined using four bacterial species such as Bacillus subtilis, Staphylococcus aureus (Gram positive) and Pseudomonas aeruginosa, Proteus mirabilis (Gram negative). The ethanolic extract had appreciable antimicrobial activity proved against all the 4 tested pathogens. Similar report were recorded by (Anwar and Rashid, 2007) and (Bello and Jamiu, 2017). Which also had both bactericidal and bacteriostatic activity on gram positive and negative organisms. This indicates that the seed extracts could also be used in the treatment of some gastro intestinal or wound infections caused by the above tested Gram negative and positive bacteria. Other studies have also shown that the antibacterial activity of M. oleifera seeds ethanolic extract is due to the presence of phenolic compound (Abdulkadir et al., 2015; Gebregiorgis Amabye and Mekonen Tadesse, 2016; Ruttarattanamongkol and Petrasch, 2015). Phenolic acid has a beneficial role of forming both ester and ether linkage on reacting with carboxylic and hydroxyl group respectively, this bifunctional nature resulted into efficient anti-microbial activity by cross link with cell wall macromolecules (Yu et al., 2001).
 

 

The anti-microbial activity of the M. oleifera seed is due to the presence of various phytochemicals like phenols. The total value phenolic content was estimated in the M. oleifera seed. By using these natural extracts, the biggest emerging problem like anti-microbial resistance can be overcome and it is a promising alternative. The total phenolic content estimated 11.85 mg GAE/g of dried seed, by observing the phenolic content of -OH stretches and bindings along with C-O stretches by FTIR, further HPLC analysis proves the proved the presence of Ascorbic acid, Gallic acid and Catechol in 280 nm wavelength. This study could further analyse in-depth for the unknown peaks of HPLC, which could be some unique phenolic compounds. All these are quite beneficial in microbial susceptibility tested on species Pseudomonas aeruginosa (MTCC 1688), Bacillus subtilis (MTCC 8561), Staphylococcus aureus (MTCC 3160) and Proteus mirabilis (MTCC 3310), which may be a broad-spectrum activity from M. oleifera extract can be proved by further research which using active bioactive compounds over other pathogens.

There is no conflict of interest.