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Journal of Microbial & Biochemical Technology

Research - (2020) Volume 12, Issue 1

Reduce the Risk of Oxidation and Pathogenic Bacteria Activity by Moringa oleifera Different Leaf Extract Grown in Sudan
Rasha Khalid Abbas1,2, Amina AM Al-Mushhin3, Fatma S Elsharbasy4 and Kother Osman Ashiry5
 
1Department of Chemistry, Faculty of Science and Arts in Mukhwa, 65931, Saudi Arabia
2Department of Biochemistry, Faculty of Applied and Industrial Science, University of Bahri, Sudan
3Department of Biology, Science and Humanity College in Al –kharj, Prince Sattam bin Abdul-Aziz University Al-Khaarj 11942, Saudi Arabia
4Department of Chemistry, Faculty of Science and Humanity Studies, Prince Sattam Bin Abdul Aziz University Alkarj, Alkharj City 11942, Saudi Arabia
5Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki 12622, Egypt
 
*Correspondence: Rasha Khalid Abbas, Department of Chemistry, Faculty of Science and Arts in Mukhwa, 65931, Saudi Arabia, Tel: + 00966534556026, Email:

Received: 26-Oct-2019 Published: 07-Feb-2020, DOI: 10.35248/1948-5948.20.12.427

Abstract

High Performance Liquid Chromatography (HPLC) used in this study to identified Polyphenol constituents of Moringa oleifera leaf extract by different methods (aqueous, ethanol, ethyl acetate and chloroform), it contain gallic acid, Chlorogenic acid, Catechin, Coffeic acid, Rutin, Pyro catechol, Coumaric acid, Vanillin, Ferulic acid1, Naringenin, Propyl Gallate, 4`,7-Dihydroxyisoflavone, and Cinnamic Acid at conc. (µg/15 mg) in all extracts. Ellagic acid gave the highest concentration when extracted by ethyl acetate Caffeine gave the lowest concentration. in all different extract, The effect of moringa (aqueous, ethanol, ethyl acetate and chloroform) leaf extracts against four different pathogenic bacteria Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli, and Bacillus cereus, were examined using Mueller Hinton Agar and measuring inhibition zone (diameter mm), were found that, there were a significant different of all moringa leaf extracts against bacteria. The study was conducted to determine the polyphenol constituents of Moringa oleifera aqueous, ethanol, ethyl acetate and chloroform leaf extract. The effect of Moringa oleifera (aqueous, ethanol, ethyl acetate and chloroform) leaf extracts against four different pathogenic bacteria.

Keywords

Moringa oleifera; Polyphenol constituent; Microorganism; Antimicrobial

Introduction

Moringa oleifera species is widely cultivated around the world, in, East, West and Sudan, the origin is India. Moringa belong to family Moringaceae [1]. Flowers, leaves, bark, seeds and roots it used as medicinal purposes and food. Moringa leaves contain important constituent, including carbohydrates, protein, vitamin such as riboflavin, ascorbic acid, thiamine, niacin, mineral such as calcium, phosphorus and iron [2]. Moringa leaves extract reduce the free radicals and the oxidation of blood because it contain polyphenol, the leaves are used in medicinal, against AIDS, fever, respiratory diseases and antimicrobial [3,4]. Moringa contain poly phenol that prevents body against many diseases such as pathogenic bacteria, hypertensive, and cancer. It include carotenoids (including β-carotene or provitamin A) prevent body from free radicals, act as antioxidant are more commonly recognized as phytochemicals [5]. Many studies reported that moringa have chemo preventive properties and has potent cytotoxicity in human cancer cells, the leaves have best used to reduce oxidation, prevent from cancer and degenerative diseases [6]. Moringa leaves extract contain several compounds, including glucopyranoside, and niazimicinreduce reduce the risk of lymphoblastic anemia [7]. Moringa contain flavonoids which are an essential in diet prevent lipid peroxidation that lead to cancer, and thrombosis, flavonoids prevent body from free radical, inflammatory inhibition of oxidative and hydrolytic enzymes (cyclooxygenase, phospholipase A2 and lipoxygenase). In general Phenolic acid classified into hydro benzoic acid contain protocatechuic, gallic, vanillic, syringic acid and p-hydroxybenzoic, the other class is hydroxycinnamic acids contain sinapic, coffeic, coumeric and ferulic acids most of these compound found in moringa [8].

Materials and Methods

Moringa oleifera leaves were purchased in the super market and identified in the Department of Plant Botany, Faculty of Agriculture, Khartoum University, Sudan.

Microorganisms

The bacteria used in this work were isolated from Stak Laboratory (Khartoum), Sudan and identified by conventional biochemical methods [9]. These methods for identification were carried out on all isolates bacteria in the three separate laboratories. The results from the three separate laboratories were from routine clinical microbiology service. According to standard microbiology techniques, these microbes were Pseudomonas aeruginosa, Escherichia coli, Bacillus cereus and Salmonella typhimurium.

Mueller Hinton Agar

Mueller Hinton Agar (MHA) (Becton Dicknson M.D USA), media was prepared according to the manufacturer’s instruction. Sterile Mueller Hinton agar plates were inoculated with the test culture by surface spreading using sterile wire loops and each bacterium evenly spread on the entire surface of the plate to obtain uniformity of the inoculum. Concentrations of 12.5, 25, 50 and 100 mg/ml prepared from the dry leaves powder were used for antibacterial analysis using agar well incorporation methods. Plates of Mueller Hinton agar were prepared and allowed to solidify on Petri dishes. Each plate was then seeded with a test bacterium. Four holes were made in each of the plate with a sterile 2.0 mm diameter cork borers. Each of the four holes was filled with a given concentration of the extract mixed with plane sterile agar. The plates were then incubated at 37°C for 24 hours. The diameters of zones of inhibition were measured using a meter rule and the mean value for each organism was recorded [10].

Preparation of plants extracts

The powdered sample (100 g, of leaf plant) was weighed, and were subjected to different extraction solvents separately extracted with ethyl acetate 80% at 50°C-60°C for 2 h, Chloroform 80% at 50°C-60°C for 2 h, ethanol 98% at 60°C for 2h in a Sox let apparatus. The all solvents extract were evaporated by a Buchi Rotary evaporator under reduced pressure.. The extract was similarly evaporated exhaustively; air dried for about 18 h and the yield was preserved in a covered flask, also the plant was extracted by distilled water over night at room temperature (25-30°C) filtered and dried [11].

Method of analysis by HPLC

In this analysis, High Performance Liquid Chromatography (HPLC) (Shimadzu corporation (Koyoto, Japan) an Agilent 1260 series was used. Temperature 35°C and C18 column (4.6 mm × 250 mm i.d., 5 μm) were used. The mobile phase contained water (A) and 0.02% tri-floro-acetic acid in acetonitrile (B), with a flow rate of 1 ml/ min. The mobile phase was programmed consecutively in a linear gradient as follows: 0 min (80% A); 0–5 min (80% A); 5-8 min (40% A); 8-12 min (50% A); 12-14 min (80% A) and 14-16 min (80% A). The wavelength was 280 nm and volume injected 10 μl. Authentic compound were obtained from the Central laboratory of The National Research Centre, Egypt.

Statistical analysis

It was done according to Duncan, Multiple Range Test [12].

Results and Discussion

The solvent of the extraction is the main factor in the prognosis of the qualitative and quantitative composition of the isolated phenolic compounds by HPLC, Table 1 and Figures 1-4 shows that the polyphenol constituents of Moringa oleiferan contain gallic acid, chlorogenic acid, catechin, coffeine, coffeic acid, syringic acid, rutin, pyro catechol, ellagic acid, coumaric acid, vanillin, ferulic acid1, naringenin, propyl gallate, 4’,7-Dihydroxyisoflavone, querectin and cinnamic acid in different extract methods (aqueous, ethanol, ethyl acetate and chloroform) at Conc. (μg/15 mg). Ellagic acid gave the highest concentration when extracted by ethyl acetate, Caffeine gave the lowest concentration in all different extracts, these result were confirm with the previous studies [8,13]. Antimicrobial activity of Moringa oleifer leaf extracts by (water, ethanol, ethyl acetate and chloroform) at different concentrations of (12.5, 25, 50 and 100%) against four pathogenic organisms (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium and Bacillus cereus) were examined by using Mueller Hinton Agar and measuring inhibition zone (diameter mm) (Table 1)( Figure 1-4).

microbial-biochemical-technology-polyphenol-constituents

Figure 1: The polyphenol constituents of Moringa oleifera aqueous leaf extract.

microbial-biochemical-technology-ethanol-leaf

Figure 2: The polyphenol constituents of Moringa oleifera s ethanol leaf extract.

microbial-biochemical-technology-moringa-oleifera

Figure 3: The polyphenol constituents of Moringa oleifera ethyl acetate leaf extract.

microbial-biochemical-technology-chloroform-leaf

Figure 4: The polyphenol constituents of Moringa oleifera chloroform leaf extract.

Amount of phenolic compounds of in different method of extract (µg/15mg)
Phenolic compound Aqueous Extract µg/ 15 mg Ethanol Extract µg/15 mg) Ethyl acetate Conc. (µg/15 mg) Chloroform Conc. (µg/15 mg) Mean Phenolic compound
Gallic acid 241.52a 233.97b 52.69c 0.67d 132.21b
Chlorogenic acid 149.12a 118.73b 24.38c 4.29d 74.13c
Catechin 144.56a 86.97b 37.44c 6.85d 68.96c
Caffeine 0.70b 5.78a 0.35d 0.45c 1.82d
Coffeic acid 16.16b 13.71c 24.45a 4.20d 14.63d
Syringic acid 0.65d 3.79c 23.88a 5.51b 8.46d
Rutin 58.36b 0.33d 130.00a 11.45c 50.04c
Pyro catechol 64.52a 41.48b 10.2c 9.3d 31.38d
Ellagic acid 0.81c 0.78d 1811.52a 3.57b 454.17a
Coumaric acid 3.95c 5.35b 78.80a 3.74d 22.96d
Vanillin 10.92b 5.58c 74.96a 0.50d 22.99d
Ferulic acid 11.51b 4.85d 31.13a 11.15c 14.66d
Naringenin 65.87b 62.45c 507.55a 8.81d 161.17b
Propyl Gallate 8.05b 8.78a 7.40c 6.7d 7.73d
4`,7-DihydroxyisoFlavone 74.67a 5.52b 0.55d 0.77c 0 20.38d
Querectin 0.91d 10.55c 512.45a 151.37b 168.82b
Cinnamic acid 1.38d 1.71c 2.97b 531.03a 134.27b
Mean Moringa oleifera leaf extract 50.22b 35.90d 195.92a 44.73c  

Table 1: Content of phenolic compounds of Moringa oleifera in different methods of extracts.

Table 2 shows that a significant differences among bacteria when Moringa oleifera aqueous leaf extract were added against microorganism, the highest inhibition zone were detected against Salmonella typhimurium (13 mm) and Escherichia coli (13 mm) while the lowest inhibition zone against Pseudomonas aeruginosa (5 mm) (Table 2).

Zone of inhibition by microorganism Concentration of the of Moringa oleifera leaf aqueous extract Mean microorganism
12.5 25 50 100
Salmonella typhimurium 10 12 14 16 13
Pseudomonas aeruginosa 0 0 10 10 5
Escherichia coli 10 13 14 15 13
Bacillus cereus 10 10 14 15 12.25
Mean Moringa oleifera leaf aqueous extract 7.5 8.75 13 14  

Table 2: Inhibition zone (in mm) for different concentrations of Moringa oleifera leaf aqueous extract.

Table 3 shows a significant differences among bacteria, when moringa ethanol leaf extract were added against microorganism, the highest activity against Pseudomonas aeruginos (13.25 mm) and lowest inhibition detected against Salmonella typhimurium (7.5 mm) (Table 3).

Zone of inhibition by microorganism Concentration of the of Moringa oleifera leaf ethanol extract Mean microorganism
  12.5 25 50 100  
Salmonella typhimurium 0 10 10 10 7.5
Pseudomonas aeruginosa 10 12 15 16 13.25
Escherichia coli 10 13 14 15 13
Bacillus cereus 10 12 14 15 12.75
Mean Moringa oleifera leaf ethanol extract 7.5 11.75 13.25 14  

Table 3: Inhibition zone (in mm) for different concentrations of Moringa oleifera leaf ethanol extract

Table 4 shows that a significant differences among bacteria when Moringa oleifera ethyl acetate leaf extract were added against microorganism, the highest inhibition zones against Salmonella typhimurium (17 mm) and lowest inhibition detected against Bacillus cereus (13.5 mm) (Table 4).

Zone of inhibition by microorganism Concentration of the of Moringa oleifera leaf extract by ethyl acetate Mean Microorganism
  12.5 25 50 100  
Salmonella typhimurium 17 17 17 17 17
Pseudomonas aeruginosa 14 15 15 16 15
Escherichia coli 13 13 15 15 14
Bacillus cereus 13 13 14 14 13.5
Mean Moringa oleifera leaf extract by chloroform 14.25 14.5 15.25 15.5  

Table 4: Inhibition zone (in mm) for different concentrations of Moringa oleifera leaf extract by ethyl acetate

Table 5 shows that a significant differences among bacteria when Moringa oleifera chloroform leaf extract were added against microorganism the highest inhibition zones against Salmonella typhimurium (15.25 mm) and lowest inhibition detected against Escherichia coli (13 mm) these results agree with the previous studies showed that the powder from the leaves of Moringa have potential antibacterial activity against the tested gram positive bacteria; Staphylococcus aurous and gram negative bacteria Escherichia coli and Pseudomonas aeruginosa [13-18] (Table 5).

Zone of inhibition by microorganism Concentration of the of Moringa oleifera leaf extract by chloroform Mean Microorganism
  12.5 25 50 100  
Salmonella typhimurium 14 14 16 17 15.25
Pseudomonas aeruginosa 13 13 14 16 14
Escherichia coli 12 12 13 15 13
Bacillus cereus 14 15 15 16 15
Mean Moringa oleifera leaf extract by chloroform 13.25 13.5 14.5 16  

Table 5: Inhibition zone (in mm) for different concentrations of Moringa oleifera leaf extract by chloroform

Conclusion

As a conclusion, Moringa oleifera contains polyphenol compounds in all extract (aqueous, ethanol, ethyl acetate and chloroform) have antioxidant, anticancer and anti-inflammatory activity, also all extracts of moringa have antibacterial activities.

Acknowledgement

The authors would like to thank the staff of the Laboratories of Chemistry and Microbiology, National Research Centre, Khartoum, Sudan.

REFERENCES

Citation: Citation: Abbas RK, Al-Mushhin AAM, Elsharbasy FS, Ashiry KO (2020) Reduce the Risk of Oxidation and Pathogenic Bacteria Activity by Moringa oleifera Different Leaf Extract Grown in Sudan. J Microb Biochem Technol. 12:427. doi: 10.35248/1948-5948.20.12.427

Copyright: Copyright: © 2020 Abbas RK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.