DR. B.

LAL INSTITUTE OF BIOTECHNOLOGY

DISSERTATION PRESENTATION

SESSION : 2018-19

ANTIBACTERIAL POTENTIAL OF BIOACTIVE

COMPOUNDS EXTRACTED FROM MORINGA OLEIFERA
BARK AND LEAVES AGAINST METHICILLIN RESISTANT
STAPHYLOCOCCUS AUREUS (MRSA) ISOLATED FROM
CLINICAL SAMPLES OF PATIENTS FROM RAJASTHAN

PRESENTED BY
SUPERVISOR Prachi Jodha
Ms. Apoorva Rana M.Sc. Biotechnology
Final Semester
 CONTENTS
• INTRODUCTION

• OBJECTIVES

• METHODOLOGY

• RESULTS

• CONCLUSION

• REFERENCES
 INTRODUCTION
•Antibiotics have revolutionized medicine in many respects, and countless lives have been
saved; their discovery was a turning point in human history. Penicillin was the first
antibiotic to be discovered by Alexander Fleming in 1928 from the fungus Penicillium
notatum.

•Despite the existence of potent antibiotic and antifungal agents, resistant or multi-resistant
strains are continuously appearing, imposing the need for a permanent search and
development of new drugs (Silver & Bostian 1993).

•Antibiotic resistance can be defined as the ability of an organism to survive and reproduce
in the presence of the antibiotic that was previously thought effective against them.

•The reason of resistance development is the indiscriminate use of commercial

antimicrobial drugs commonly used in the treatment of infectious disease. In addition to
this problem, antibiotics are sometimes associated with adverse effects on the host
including hypersensitivity, immune-suppression and allergic reactions.
•Methicillin Resistant Staphylococcus aureus is the most common antibiotic resistant
microorganism and a dangerous pathogen for both community-acquired as well as
hospital-associated infections.

•Methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health

issue because of worldwide spread of several clones.

• More than 20 years later, the specific genetic mechanism of its resistance has been
identified as a mobile genetic element (Staphylococcal cassette chromosome mec)
integrated into the Staphylococcus aureus chromosome, within which the mecA gene
encodes a specific methicillin-resistant transpeptidase (penicillin-binding protein 2a)
(PBP2a). This protein has a low affinity for β-lactam antimicrobial drugs. Thus, bacteria
expressing this protein are resistant to all types of these drugs.

•Methicillin resistant Staphylococcus aureus (MRSA) is now endemic in India. The

incidence of MRSA varies from 25 per cent in western part of India to 50 per cent in South
India. Community acquired MRSA (CA-MRSA) has been increasingly reported from
India.
• Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of
infectious diseases from medicinal plants.
• Several screening studies have been carried out in different parts of the world. There are
several reports on the antimicrobial activity of different herbal extracts in different
regions of the world.
• Because of the side effects and the resistance that pathogenic microorganisms build
against antibiotics, recently much attention has been paid to extracts and biologically
active compounds isolated from plant species used in herbal medicine.
• According to World Health Organization (WHO), more than 80% of the world’s
population relies on traditional medicines for their primary health care needs. The most
important bioactive compounds of plants are alkaloids, flavonoids, tannins and phenolic
compounds.
•The phytochemical research based on ethnopharmacological information is generally
considered an effective approach in the discovery of new anti-infective agents from
higher plants.
•The present study has been carried out to focus on screening the antibacterial potentials
of bioactive compounds extracted from the leaves and bark of Moringa oleifera. The
findings of our study can be further extended and the information obtained can be very
useful to frame the contaminant of MRSA in society.
• Staphylococcus aureus
 S. aureus cells are Gram-positive cocci. The name ‘Staphylococcus’ was derived from
Greek, meaning bunch of grapes (staphyle) and berry (kokkos) .The diameter of the cells
ranges from 0.5 to 1.0 µm
 S. aureus colonizes the nasal passage, axillae and can be found on skin as well.
 S. aureus forms fairly large yellow or white colonies on nutrient rich agar media.
 Nearly all isolates of S. aureus produce coagulase enzyme, a virulence factor that also
helps in identification of the organism. The organism is catalase and coagulase positive .

• Methicillin-Resistant Staphylococcus aureus

 The species of Staphylococcus aureus showing resistance to Methicillin are considered as
Methicillin Resistant Staphylococcus aureus(MRSA)
 The mecA gene (the gene responsible for methicillin resistance) is part of a mobile genetic
element found in all MRSA strains. Katayama et al. demonstrated that mecA is part of a
genomic island designated staphylococcal cassette chromosome mec (SCCmec).
• Medicinal plants
• India is one of the richest country with vast resource of medicinal and aromatic
plants. It constitutes of 11% of the total known world flora having medical
property (Sati et al., 2010).
• The bioactive substances in plants are their secondary metabolites and have
multiple mechanisms of action responsible for the antimicrobial property.
Moringa oleifera
Moringa oleifera is a native plant of India and is the most widely cultivated
species in the genus Moringa, the only member of Moringaceae family. It is
commonly known as ‘drumstick tree’.

Applications of Moringa oleifera

 OBJECTIVES
Isolation and identification of Methicillin Resistant
Staphylococcus aureus (MRSA) from clinical samples

To evaluate the antibacterial potential of the crude extracts of bark and

leaves of Moringa oleifera against MRSA isolated from clinical samples

To screen the phytochemicals of the plant extracts responsible for showing

the antibacterial activity against MRSA isolates

Extraction of the pure compounds

To perform Thin Layer Chromatography of pure bioactive compounds

extracted from Moringa leaves and bark.
 METHODOLOGY
Isolation and identification of Methicillin Resistant Staphylococcus aureus (MRSA)
from clinical samples

Isolation of bacterial isolates from

Identification of Staphylococcus Identification of MRSA by
clinical samples of infected
aureus by Biochemical Testing Double Disk Diffusion Test
patients by Streaking Method

* The MRSA isolates were procured from MCRD, CIRD

Collection and Processing of Moringa oleifera leaves and bark

Collection of bark and Grinding of dried bark and

leaves of Moringa Processing of the plant (Washing, leaves in the form of fine
oleifera shade drying) powder
Evaluation of the antibacterial potentials of the crude extracts of bark and leaves of Moringa oleifera
against Methicillin Resistant Staphylococcus aureus (MRSA) isolated from clinical samples

Plant extraction using different solvents by Antimicrobial activity of crude extracts against
Soxhlet extraction method MRSA isolates from clinical samples by Kirby
Baeur Method

Qualitative and Quantitative screening of the phytochemicals, extraction of the pure compound
and the screening of the phytoconstituent responsible for the antibacterial potential of the
medicinal plants against MRSA isolates

Qualitative screening of phytochemicals from the Quantitative estimation of the present

plant extracts showing antibacterial potentials phytochemicals

Extraction of the phytochemical found in the To perform Thin layer Chromatography of

detectable limit bioactive compounds extracted from
Moringa leaves and bark
RESULTS
Figure 1:- Isolation of bacterial isolates from clinical samples of infected
patients by streaking method
 Gram’s Staining for Microscopic Identification

Catalase Test for Biochemical Identification Coagulase Test for Biochemical Identification

Figure 2:- Microscopic and Biochemical Identification for Staphylococcus aureus

 Cefoxitin Cefoxitin

Penicillin Penicillin

MNC-3202 C - 191

Figure 3:- Antibiotic susceptibility pattern by Kirby Bauer Method (Disk

Diffusion Test) for MRSA Identification
(For MRSA – Penicillin G ≤ 28 mm and Cefoxitin ≤ 21 mm)
 LEAVES BARK LEAVES

BARK LEAVES

Figure 4:- Washing & Shade drying of Moringa oleifera Leaves and Bark
 Methanolic Methanolic
Extract of Extract of
Moringa leaves Moringa bark

Ethanolic Ethanolic
Extract of Extract of
Moringa leaves Moringa bark

Figure 5:- Plant extraction of different solvents using Soxhlet Extraction Method
 Table 1:- Antibacterial Activity of Ethanolic Extracts of Moringa oleifera Leaves
Isolate PC 100 mg/ml 200 mg/ml 400 mg/ml NC

S. aureus 32 10 10 11 0

MRSA 27 11 12 13 0

C - 191 35 13 14 15 0

85/30 36 12 13 14 0

C - 2156 34 11 12 13 0

CDC - 519 33 11 13 15 0

MNC - 3202 30 12 13 14 0

CDC - 553 30 10 11 12 0

C - 1954 30 7 9 11 0

CDC - 404 29 7 9 13 0

MSC - 308 20 6 7 8 0

CDC - 361 28 8 10 12 0

71/746 32 7 10 13 0

Mean 30.6±4.4 9.5±2.5 11±2.2 12.7±2 0

 40
400 mg/ml 200 mg/ml 100 mg/ml

35 Activity
0.42 0.36 0.31
Index
30
30.6
Mean Zone of Inhibition (mm)

25

20

15

12.7
10 11
9.5

0
0
Positive Control 400.0 200.0 100.0 Negative Control
Concentration (mg/ml)

Graph 1:- Antibacterial Activity of Ethanolic Extracts of Moringa

oleifera Leaves
 Table 2:- Antibacterial Activity of Ethanolic Extracts of Moringa oleifera Bark
Isolate PC 100 mg/ml 200 mg/ml 400 mg/ml NC

S. aureus 31 16 20 22 0

MRSA 27 13 14 15 0

C - 191 34 6 8 10 0

85/30 35 16 20 22 0

C - 2156 34 12 14 16 0

CDC - 519 33 5 7 10 0

MNC - 3202 31 13 15 22 0

CDC - 553 31 8 11 14 0

C - 1954 31 6 7 8 0

CDC - 404 30 10 13 16 0

MSC - 308 20 6 7 8 0

CDC - 361 28 5 6 7 0

71/746 31 6 7 8 0

Mean 30.7±4.1 8.5±3.8 10.5±4.5 12.8±5.6 0

 40
400 mg/ml 200 mg/ml 100 mg/ml
35 Activity
0.42 0.34 0.28
Index
30
Mean Zone of Inhibition (mm)

30.7

25

20

15

12.8
10
10.5
8.5
5

0
0
Positive Control 400.0 200.0 100.0 Negative Control

Concentration (mg/ml)

Graph 2:- Antibacterial Activity of Ethanolic Extracts of Moringa oleifera Bark

Anti-MRSA Activity of Moringa oleifera Anti-MRSA Activity of Moringa oleifera
Leaves – Ethanolic extract Bark – Ethanolic extract

PC PC

NC 100 mg
NC 100 mg
400 mg 400 mg

200 mg
200 mg

Figure 6:- Antibacterial activity of Ethanolic Extracts of Moringa oleifera

 Table 3:- Antibacterial Activity of Methanolic Extracts of Moringa oleifera Leaves

Isolate PC 100 mg/ml 200 mg/ml 400 mg/ml NC

S. aureus 32 9 9 10 0

MRSA 27 10 11 12 0

C - 191 35 7 9 11 0

85/30 36 7 10 13 0

C - 2156 34 7 9 11 0

CDC - 519 32 10 11 12 0

MNC - 3202 30 9 11 12 0

CDC - 553 30 10 12 12 0

C - 1954 31 6 7 8 0

CDC - 404 29 7 8 9 0

MSC - 308 19 6 7 8 0

CDC - 361 28 11 12 13 0

71/746 31 7 8 9 0

Mean 30.5±4.5 7.9±1.8 9.5±1.9 10.7±1.9 0

 40
400 mg/ml 200 mg/ml 100 mg/ml

35 Activity
0.35 0.31 0.26
Index
30
Mean Zone of Inhibition (mm)

30.5

25

20

15

10
10.7
9.5
7.9
5

0
0
Positive Control 400.0 200.0 100.0 Negative Control

Concentration (mg/ml)

Graph 3:- Antibacterial Activity of Methanolic Extracts of Moringa oleifera

Leaves
 Table 4:- Antibacterial Activity of Methanolic Extracts of Moringa oleifera Bark

Isolate PC 100 mg/ml 200 mg/ml 400 mg/ml NC

S. aureus 32 9 12 13 0

MRSA 27 5 7 9 0

C - 191 35 9 10 11 0

85/30 34 11 12 13 0

C - 2156 33 9 11 14 0

CDC - 519 33 6 9 12 0

MNC - 3202 31 8 10 12 0

CDC - 553 31 10 12 13 0

C - 1954 30 7 9 11 0

CDC - 404 29 4 6 8 0

MSC - 308 19 6 7 8 0

CDC - 361 28 6 7 8 0

71/746 32 6 8 10 0

Mean 30.5±4.3 7.5±2.1 9.2±2 10.9±2.2 0

 40
400 mg/ml 200 mg/ml 100 mg/ml
35 Activity
0.36 0.3 0.25
Index
30
Mean Zone of Inhibition (mm)

30.5

25

20

15

10
10.9
9.2
5 7.5

0
0
Positive Control 400.0 200.0 100.0 Negative Control

Concentration (mg/ml)

Graph 4:- Antibacterial Activity of Methanolic Extracts of Moringa oleifera

Bark
Anti-MRSA Activity of Moringa oleifera Anti-MRSA Activity of Moringa oleifera
Leaves – Methanolic extract Bark – Methanolic extract

PC PC

NC NC
100 mg 400 mg 100 mg
400 mg

200 mg
200 mg

Figure 7:- Antibacterial activity of Methanolic Extracts of Moringa oleifera

Table 5:- Qualitative Estimation of Phytochemicals in Moringa oleifera Leaves and Bark

Moringa leaves Moringa bark Moringa leaves Moringa bark

(Ethanolic Extract) (Ethanolic Extract) (Methanolic Extract) (Methanolic Extract)

Steroids + + + _

Phenols _ + _ _

Phlobatannins _ _ _ +

Quinones _ _ _ _

Coumarins _ + _ _

Flavonoids + _ + +

Volatile oils + _ + -

Terpenoids _ + _ +

Reducing sugars _ _ _ _

Cardiac glycosides _ + _ +

Alkaloids + + _ +

Saponins _ + _ +

Tannins + _ _ _
 Alkaloids Flavonoids Phenols Tannins

Figure 8 :- Qualitative Screening of Phytochemicals of Moringa oleifera Leaves and Bark

 0.8
0.75
0.7 0.6995
0.65
0.6 0.6005 y = 0.0034x + 0.3817
R² = 0.9495
0.55
Absorbance(at 725nm)

0.5
0.476
0.45
0.4 0.4145
0.3795
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0 20 40 60 80 100 120
Concentration (mg/ml)

Graph 5:- Quantitative Estimation of Tannins in Ethanolic Extract of

Moringa oleifera Leaves
Standard – Gallic Acid (Concentration -100 mg/ml)
 0.085
0.08
0.075
0.073
0.07
0.065
y = 0.0008x - 0.0014
0.06
R² = 0.9778
Absorbance(at 510nm)

0.055
0.05
0.045 0.045
0.04
0.035
0.03
0.025
0.02 0.019
0.015
0.01
0.005 0.006
0 0.001
0 20 40 60 80 100 120
Concentration (mg/ml)

Graph 6:- Quantitative Estimation of Flavonoids in Methanolic Extract of

Moringa oleifera Leaves
Standard - Quercetin (Concentration -100 mg/ml)
 0.08
0.075
0.073
0.07
0.065
0.06 y = 0.0008x - 0.0028
0.055 R² = 0.9908
Absorbance(at 510 nm)

0.05
0.045
0.04 0.041
0.035
0.03
0.025
0.02
0.017
0.015
0.01
0.005 0.005
0 0.001
0 20 40 60 80 100 120

Concentration (mg/ml)

Graph 7:- Quantitative Estimation of Flavonoids in Methanolic Extract of

Moringa oleifera Bark
Standard – Quercetin (Concentration -100 mg/ml)
 1.1
1.05
1 0.999
0.95
0.9
0.85
0.8
0.75 y = 0.0101x + 0.0139
0.7 R² = 0.9942
Absorbance(at 765nm)

0.65
0.6
0.55 0.566
0.5
0.45
0.4
0.35
0.3
0.25 0.2735
0.2
0.15
0.1 0.1235
0.05 0.0605
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105

Concentration (mg/ml)

Graph 8:- Quantitative Estimation of Phenols in Ethanolic Extract of

Moringa oleifera Bark
Standard – Gallic Acid (Concentration -100 mg/ml)
 Flavonoid Flavonoid Flavonoid
Extraction Extraction Extraction
(Petroleum ether (Diethyl ether (Ethyl acetate
fraction) from fraction) from fraction) from
Moringa Bark Moringa Bark Moringa Bark

Flavonoid Flavonoid Flavonoid

Extraction Extraction Extraction
(Petroleum ether (Diethyl ether (Ethyl acetate
fraction) from fraction) from fraction) from
Moringa Leaves Moringa Moringa
Figure 9 :- Flavonoid Extraction Leaves Leaves
from Moringa oleifera Bark and
Leaves
 Standard – Quercetin
PE – Fraction 1 (Petroleum Ether)
0.66 DE – Fraction 2 (Diethyl Ether)
EA – Fraction 3 (Ethyl Acetate)
0.63

0.34 0.43
0.55

0.24

TLC of TLC of
Flavonoid in Flavonoid in
Moringa Moringa
Standard PE DE EA Leaves StandardPE DE EA Bark

Figure 11 :- TLC Plates

 CONCLUSION
 The antibacterial potentials of Ethanolic and Methanolic Extract of Moringa oleifera leaves
are 12.7±2 and 10.7±1.9, respectively and the antibacterial potentials of Ethanolic and
Methanolic Extract of bark are 12.8±5.6 and 10.9±2.2, respectively.
 The activity indexes shown by the Ethanolic and Methanolic Extract of Moringa oleifera
leaves are 0.42 and 0.35 and the activity indexes shown by the Ethanolic and Methanolic
Extract of bark are 0.42 and 0.36.
 Of the two solvents (Methanol & Ethanol) used for extract preparation, results have shown
that Ethanolic extracts show better antibacterial activity.
 As per the quantitative estimation, the total flavonoid content in methanolic extracts of
Moringa oleifera leaves and bark was 306.75 mg/ml and 127.25 mg/ml respectively.
 The TLC of the Extracts of M. oleifera leaves and bark provided flavonoids with the Rf
values of 0.63, 0.34, 0.43, 0.24 and 0.55, 0.66 respectively.
 The phytoconstituents responsible for the antibacterial potentials can be extracted from the
plants and can lead to the development of novel therapeutic agents capable of controlling
or curbing the problem of drug resistance.
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