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 INTRODUCTION

Medicinal plants have been used for centuries as remedies for human
diseases because they contain components of therapeutic value. The plants
have always been vital for mankind irrespective of the era and area all over the
globe since the beginning of life. These were and will remain ever beneficial
from nutritional, social, cultural, religious, environmental and human’s etc.

However, a very important step in the screening of the sanitizing and

preservative activity of a plant material is to evaluate its antimicrobial
properties. It is important to evaluate the antimicrobial properties of
Moringa oleifera leaves on some selected microorganisms and also to verify its
Phytochemical constituent.

Medicinal plants contain numerous biologically active compounds,

many of which have been shown to have antimicrobial properties. Plant-
derived medicines have been part of traditional healthcare in most parts of the
world for thousands of years and there is increasing interest in plants as sources
of agents to fight microbial diseases.

Medicinal plants are a source of great economic value in the Indian

subcontinent. Nature has bestowed on us a very rich potancial wealth and a
large number of diverse types of plants grow in different parts of the country.
India is rich in all the three levels of biodiversity they are namely species
diversity, genetic diversity and habitat diversity. In India thousands of species
are known to have medicinal value and the use of different parts of several
medicinal plants to cure specific ailments has been in vogue since ancient
times.

1
 Today the pathogenic bacteria have developed resistances against
existing antibiotics due to indiscriminate use of antimicrobial drugs to treat
infectious disease. The antimicrobial resistant bacteria are the causes of
numerous clinical problems worldwide. The development and increase of
resistance among pathogens causing nosocominal and community acquired
infection are known to be associated with the widespread utilization of
antibiotics.

Moringa oleifera is a well-documented world removed plant herb known

for is extraordinary nutritional and medicinal properties. It is a known
antihelminth; antibiotic detoxifier and an outstanding immune builder used in
many countries tit ret malnutrition and malaria. The plant Moringa oleifera is a
highly valued plant, distributed in many countries of the tropics and subtropics.

Phytochemicals are chemical compounds that are naturally found in

plant. They are responsible for the colour and organoleptic properties of the
plant. It is also referred to as those chemicals that may have biological
significance but are not established as essential nutrients in plant.
Phytochemicals could be available as dietary supplements, but the potential
health benefits of Phytochemicals are derived from consumption of the whole
plant.

Moringa oleifera is considered to be effective in the treatment of many

diseases. It is an exceptionally nutritious vegetable tree with a variety of
potential uses. The tree itself is rather slender, with dropping branches. It is
often cut back annually to one meter or less and allowed to re-grow so that its
pods and leaves remain within arm’s reach. Moringa tree is mainly grow in the
semi-arid tropical and subtropical areas. It grows best in dry sandy soil and can
tolerate any other type oil soil. It is fast growing drought resistant tree that is
2
native to the Southern foothills of Himalayans in Northern India. It is
considered as one of the world’s most useful tree.

In traditional medicine Moringa oleifera tree is known is known as a

miracle tree as almost every part of this tree possesses product useful for
humans. The leaves and pods are eaten. The plant is also reported to be
medicinally important and almost all parts of the Moringa oleifera tree
considered to possess medicinal properties and are used in the treatment of as
cites, rheumatism venomous bites cardiac and circulatory stimulant.

Moringa leaves are known to have a high content of protein, minerals

and vitamin, hence an ideal nutritional supplement. Moringa oleifera leaves
have been used to combat malnutrition, especially among infants and nursing
mothers and hasten uterine contract ion during child birth in pregnant women.
It’s antihypertensive, diuretic, antispasmodic, antiulcer, and anticancer and
cholesterol lowering activities have been reported. The leaves are helpful in
increasing breast milk in nursing mothers during breast feeding and leaf
decoction has been found useful in the treatment of asthma, back pain and
rheumatism. Moringa oleifera tree has in recent times been advocated as an
outstanding indigenous source of highly digestible protein, carotenoids and
vitamin C suitable for utilization in many of the so called developing regions of
the world where under nourishment is a major concern. In some parts of the
world for example Senegal and Haiti, health workers have been treating
malnutrition in small children, pregnant and nursing women with Moringa leaf
powder.
The Moringa tree is considered one of the world’s most useful trees, as
almost every part of the Moringa tree can be used for food or has some other
beneficial properties. In the tropics, it is used as forage for livestock, and in

3
many countries, it is used as a micronutrient powder to treat various ailments.
The fruit of the tree is quite popular as a vegetable in Asia and African
countries. In India and other parts of the country the fruit called as drumstick.
The leaves contain more Vitamin A compared to carrots, more calcium than
milk, more iron than spinach, more Vitamin C than oranges, and more
potassium than bananas, and that the protein quality of Moringa leaves rivals
that of milk and eggs. The Moringa trees have been used to combat
malnutrition, especially among infants and nursing mothers. The non govern
mental organizations in particular-Trees for Life, Church World Service and
Educational Concerns for Hunger Organization—have advocated Moringa as
natural nutrition for the tropics. Its leaves as food supplement, recommended
for children with moderate malnutrition between the ages of 6 months to 5
years. It has been claimed to have an unusually high content of calcium, iron,
and vitamin A and high quality protein, and a low content of anti nutrients
(Natural or synthetic compounds that interfere with the absorption of nutrients)
such as tannins and oxalates. The leaves have therefore been promoted as a
potential low cost high quality food.

4
 AIM AND OBJECTIVES

 To validate the potential of the selected medicinal plant for its anti
microbial properties.

 Phytochemicals screening of the selected plant extract.

5
 REVIEW OF LITERATURE

Yogeshkumar et al., (2007) the methanol and acetone extracts of 14

plants belonging to different families were evaluated for antimicrobial activity
against certain Gram positive and Gram negative bacteria and some of fungi.
For the detection of antimicrobial activity, they use agar disc diffusion
methods. In their study they concluded that the methanol extracts showed more
antibacterial activity then acetone extracts.

Memnune Sengul et al., (2009) according to their Antimicrobial activity

and antioxidant activity of medicinal plant. β-carotene bleaching assay and
Folin-Ciocalteu reagent were used to determine total antioxidant activity and
total phenols of plant extract. Antimicrobial activity was determined by using
disk diffusion assay. The results provided evidence that the studied plant
might indeed be potential source of natural antioxidant and antimicrobial
agents.

Bukar et al., (2009) worked on the anti-pseudomonal activities

aqueous, chloroform and ethanol extracts of leaf of Sienna siamea (Lam) were
evaluated by agar disc diffusion method. This was aim of substantiating the
Ethanomedicinal use of the plant as anti-bacterial. Ciprofloxacin (oxide) as
positive control had zone diameter of inhibition of 35mm in all test plates.
However aqueous extract proved to be a more suitable candidate for use in the
treatment of disease cause by Pseudomonas aeruginosa.

Khawaja Tahir Mahmood et al., (2010) in their work on the

therapeutic uses of selected plants. Moringa has been used since 150B.C. by
ancient kings and queens in their diet for mental alertness and healthy skin.
6
Moringa are used in more than 80 countries. It is also improves eyesight,
mental alertness and bone strength and provide excellent support of the
body’s anti-inflammatory mechanisms, enrich anemic blood and support
immune system. We need to explore therapeutic, nutritional and benefit of this
gift of nature reported to be one of the world’s most useful trees.

Thilza et al., (2010) says about the in vitro antibacterial activity of the
water extract of Moringa oleifera. The highest activity was produced by
Escherichia coli at 1000mg/ml which comparably is less than of the standard
drug Tetracycline (250mg/ml),this study has shown that the water extract of
Moringa oleifera posses some degree of antimicrobial activity especially at
high dose.

Gustavo Hitzschky Fernandes et al., (2010) reported the antibacterial

effect against gram positive and gram negative bacteria. Using solvent aqueous
and ethanol extract. The aqueous extracts of soursop showed an antibacter ial
against Staphylococcus aureus, Vibrio cholera but the antibacterial activity by
the ethanol extracts of this plant was not demonstrated.

Bukar et al., (2010) the chloroform and ethanol extracts of seeds and
leaf Moringa oleifera were investigated for antimicrobial activity against some
selected food-borne microorganisms. The preliminary phytochemical screening
and antimicrobial assay were carried out using standard procedures. Saponins
were detected in all the extracts while tannin’s was only detected in Moringa
oleifera leaf chloroform extract. The MIC values ranged between 1.0
and >4.0mg/ml for the tested organisms respectively.

Josephine et al., (2010) it established the medicinal uses of Moringa

oleifera leaves in Uganda and identified Phytochemicals preset in Moringa

7
oleifera leaves extracts. Phytochemicals present included tannins, steroids,
triterpenoids, flavanoids, saponins, anthraquinones, alkaloids, and reducing
sugars. There is need to standardize Moringa oleifera leaves use for nutrition
and herbal medicine.

Renu Solanki et al., (2010) reported about the antimicrobial

properties of Cassia fistula, Acacia aroma, Azadirachta indica, Ziziphora
clinopodiodies, and Argemone mexicane etc. a review of some patents on
herbal products for the treatment of bacterial disease is provided that highlights
the recent technical advancements that have taken in this area.

Devendra et al., (2011) in folklore, and ayurvedic all parts of the tree
used in different healing procedures for different diseases. This plant leaves are
very good nutrient supplement for mall nutrition and also used antibiotic. So
this plant extracts having good healing properties without side effects when
compared with synthetic antibiotics.

Mohammed Abu Sayeed et al., (2012) the present study was designed
to investigate antibacterial and antifungal activity of Methanol extract from the
fruits of Moringa oleifera. The extracts were screened for antibacterial and anti
fungal by Disk diffusion method. The highest zone of inhibition was found at
the concentration of 200µg/ disk for Pseudomonas aeruginosa (22 mm) and
Collectitrichum Sp (14 mm).

Vinoth et al., (2012) worked on the preliminary photochemical

screening indicated the presence of phenolics, flavanoids, tannins, glycosides
etc. We believe that the higher reducing power of the aqueous extract could be
due to the better solubility of the antioxidant components in water whereas the
predominant antibacterial activity in organic solvent extracts as compared to

8
aqueous extracts, indicates that the active components responsible for the
bactericidal activity are more soluble in organic solvent. These studies provid e
an evidence to support traditional medicinal uses of the plant.

Mudasser Zaffer et al., (2012) studied about the Chloroform, Ethyl

acetate, Methanol and aqueous extracts of Moringa oleifera bark traditionally
used in folkloric medicine for the treatment of various infections were
investigated for their antifungal activity against two fungal strains. The results
provide justification for the use of the plant in folk medicine to treat various
infectious diseases.

Abalaka et al., (2012) studied about the evaluation of the antibacterial

activity of medicinal plant. The chloroform extracts of Moringa oleifera
(leaves) and using the standard disc diffusion method and photochemical
analysis against four strains of bacterial species.

Kaniz Fatima Urmi et al., (2012) done the project aim of the present
study was to determine the antimicrobial and cytotoxic activity of different
parts of the plant Moringa oleifera Lam. Disc diffusion method and brine
shrimp lethality bioassay were used for antimicrobial activity and cytotoxic
activity respectively. Chloroform fractions of leaf and fruit part have shown
moderate antimicrobial activity within zone of inhibition ranging from 9-28mm
against all the experimental microbes.

Gruvinder Pal Singh et al., (2012) reported about the antimicrobial

evaluation of Moringa oleifera Lam. The root and bark are useful in treatment
of heart complaints, eye diseases, inflammation, dyspepsia and enlargement of
spleen. In the present study the antimicrobial activity was investigated by
employing main model Kirby-Bauer disk diffusion method.

9
 Rockwood et al., (2013) worked on the Moringa oleifera seeds and
leaves were extracted using three different solvents (de-ionized water,
inorganic ethanol, and organic ethyl acetate) and two different extraction
methods (crude, sophisticated). Sensitivity disks impregnated with the various
extracts were used for antibiotic susceptibility testing of fourteen bacterial
species: seven representatives Gram-negative and seven representatives Gram-
positive. De-ionized water was the only solvent capable of extracting plant
constituents which conferred bacterial inhibition. Seed extracts were found to
inhibit a broader range of organisms (4) than leaf extracts (1). 75% of the
organisms inhibited by seed extracts were Gram-positive bacteria. A single
parameter, the zone of inhibition, was used to compare antibacterial efficacy
between extraction methods, trials, and controls. No difference was observed
between the zone of inhibition of crude and sophisticated extracts. Seed
extracts demonstrated a zone of inhibition comparable to that of penicillin and
tetracycline.

Makanjuola et al., (2013) worked on the Antibacterial activities of

Moringa oleifera leaf extracts were carried out on coli forms isolated from
surface waters using growth inhibition indices based on agar plate technique.
The minimum inhibitory concentration ranged from 4.0 mg/ml to 35.0 mg/ml,
while the minimum bactericidal concentration ranged from 5.0 mg/ml to 45.0
mg/ml. The antibacterial activities examined in this study showed that Moringa
oleifera leaves are capable of exerting inhibitory effect on the target organisms.

Theophilus et al., (2013) studied about the Phytochemical screening

and antibacterial assays on Moringa oleifera leaves extract have been
documented but none of the reports showed the fractionation and separation of
the phytoconstituents by solvent extraction and thin layer chromatographic

10
procedures, respectively. The antibacterial assay results portrayed broad
activity spectrum against the test microbes with comparable inhibitory zones
by standard antibiotics. The MIC ranged between 10mg/ml and 90mg/ml for all
the organisms. The results from this research have shown the antibacterial
potentials of Moringa oleifera implying that the extract could help as a
chemotherapeutic agent or might be a lead compound for the development of
new potent antibacterial agents.

Ugwu Okechukwu et al., (2013) acute toxicity and phytochemical

studies of ethanol leaf extract of Moringa oleifera were analysed. The
Phytochemical analyses of the ethanol extract of Moringa oleifera leaf showed
the presence of tannins, carbohydrates, saponins, glycosides, reducing sugars,
terpenoids, steroids, flavanoids and alkaloids. Phytochemicals such as resins,
proteins and fat oils were not detected. The LD50of the ethanol leaf extract in
mice was found to be less than 5000mg/kg body weight.

Philip et al., (2013) Moringa oleifera defatted seed and seed coat was
conducted. Paper disc diffusion method was used to assess the effect of the
extracts on Pseudomonas aeruginosa, Escherichia coli, Klebsiella sp,
Salmonella typhi, Proteus mirabilis, all the examined extract showed varying
degrees of antibacterial activities against the selected pathogens. Highest
inhibitory effect was observed in Proteus mirabilis (19mm) following
Salmonella typhi (16mm) with the Moringa oleifera seed extract, these strains
Salmonella typhi and Proteus mirabilis were resistant to ethanol extract of
Moringa oleifera seed coat along with their respective minimum inhibitory
concentration (MIC) values ranging from extract on the test isolates ranges
from a 0.1mg/ml- 100mg/ml concentration.

11
 Mona et al., (2013) accordingly, rocket (Eruca vesicaria sub sp.
Sativa) plants were foliar sprayed with the aqueous extracts of leaves and twigs
of Moringa oleifera at rates of 1, 2 and 3%. Among these concentrations,
fertilization of rocket plants with 2% leaf and 3% twig extracts potentially
increased all measured growth criteria (plant height, fresh and dry herb
weight), photosynthetic rates, stomatal conductance, the amounts of each of
chlorophyll a and b, carotenoids, total sugars, total protein, phenols, ascorbic
acid, N, P, K, Ca, Mg, Fe as well as growth promoting hormones (auxins,
gibberellins and cytokinins).

Kalpana et al., (2013) worked on the antimicrobial activity of

petroleum ether, chloroform, ethanol and aqueous extracts of Moringa oleifera
leaf against four microorganism’s viz., Escherichia coli, Staphylococcus
aureus, Klebsiella pneumoniae and Streptococcus pneumoniae. The
antimicrobial activity was performed by Kirby-Bauer disc diffusion method.
Ethanolic extract showed maximum zone of inhibition against Staphylococcus
aureus.

Patil et al., (2013) ) studied about, antioxidant activity and in vitro

antibacterial activity of acetone, methanol and chloroform extracts of Moringa
oleifera roots were investigated., The antibacterial effects of Moringa oleifera
alone as well as in combination with Cleome viscosa against all tested
pathogens suggest their potential use as alternative tools for controlling otitis
media.
Ashok et al., (2014) The antibacterial activity of leaf extracts of
Moringa oleifera and Saraca indica was evaluated in vitro, using disc diffusion
method. Six different extracts each for both the plants were used which
included aqueous, acetone, chloroform, ethanol, methanol and petroleum ether.

12
The study showed that aqueous and ethanol extract of Moringa oleifera was
inhibitory against Escherichia coli (12mm each), Proteus vulgaris (10mm
each) and Salmonella typhi (12mm and 10mm respectively). Moringa oleifera
leaf extract showed that Bacillus subtilis and Klebsiella pneumoniae were
resistant to all the extracts.

Eman et al., (2014) Moringa oleifera leaves can be used as a medicine

for many diseases, as antibacterial, antioxidant, anticancer. In addition it can be
used as a protein supplement for human being and animals due to high protein
content. Moringa oleifera leaves are unique because of their tremendous
amounts of minerals but lower amounts of harmful compounds and it can be
considered as safe supplement because besides that it is not toxic

Riad et al., (2014) describe the first record on resistance of

Moringa oleifera plants against root rot and wilt diseases caused by Fusarium
solani , Fusarium oxysporum , Rhizoctonia solani , Sclerotium rolfsii and
Macrophomina phaseolina .There is no clearly effect of the tested pathogens
on vegetative growth parameter such as plant height , fresh and dry weight of
shoots and roots of Moringa . It could be suggested that seeds and media used
for Moringa ought to be sterilized with hypochlorite or another material or
procedure to ensure good seed emergence and control seed and soil borne
pathogenic fungi.

Mudasser Zaffer et al., (2014) Moringa oleifera were evaluated for

their antibacterial activity against four bacteria viz. Staphylococcus aureus,
Citrobacter freundii, Bacillus megaterium and Pseudomonas fluorescens using
erythromycin as positive control. The activity was analyzed using paper disc
diffusion method at different concentration of the extract. The study revealed
that all the bark extracts irrespective of their types, in different concentrations
13
inhibited growth of the test pathogens to varying degrees. Ethyl acetate extract
showed maximum activity against all the bacterial strains followed in
descending order by chloroform, methanol and aqueous extracts.

Tona et al., (2014) Sixty one days feeding trial was conducted
to determine the total feed intake, live weight gain and digestibility coefficients
of WAD goats (bucks) fed basal diet of Panicum maximum (guinea grass) and
concentrate diet containing Moringa oleifera leaf meal (MOL) at 0%, 5%, 10%
and 15% levels. The results showed that the WAD goats fed the concentrate
diet with 15% level of inclusion of Moringa oleifera leaf meal had significantly
higher (P<0.05) rate of growth and digestibility coefficients than those on the
5% and 10% inclusion levels.

Kodia et al., (2014) This study shows that we can overcome Buruli
ulcer with medicinal plants that is abundantly available in Africa. It also shows
that it is necessary to conduct research on a wider range of fract ions of
Moringa oleifera to improve the activity of anti mycobacterienes molecules in
the aqueous extract of Moringa oleifera.

Namrata Prasad et al., (2014) In this paper leaves of Moringa

oleifera, Dalbergia sissoo and Alstonia scholaris in different solvent were
subjected to antibacterial analysis against selected bacterial pathogens and
Phytochemical analysis was done. The dry crude sample extracts were tested
for its antibacterial activities using agar well diffusion technique. These
samples were further taken to determine the MIC value. The MIC value was
determined using broth dilution method. Acetone and ethyl acetate extracts of
Moringa oleifera were subjected to get MIC against Staphylococcus aureus
and it was found to be 0.003mg/ml and 0.096mg/ml respectively. MIC values
for methanolic extract of Dalbergia sissoo were 0.386 mg/ml for
14
Staphylococcus aureus and 0.005 mg/ml for Pseudomonas aeruginosa.
Important sources of Phytochemicals of immense medicinal and
pharmaceutical potential were present.

15
 MATERIALS AND METHODS

Antimicrobial properties of Moringa oleifera were screened by Disc

diffusion method.

COLLECTION OF PLANT MATERIALS

The material used in the present study was the plant Moringa oleifera
collected from Pokkam Palayam, Namakkal District, and Tamilnadu, India.

PLANT (MORINGA OLEIFERA): Plate 1

The plant MORINGA OLEIFERA is also known as horseradish tree or
benzoil tree. It is a species of the genus Moringa in the Moringaceae family.

GENERAL DESCRIPTION

Moringa oleifera is a fast-growing tree. It can reach a height of 10–12 m

(32-40 ft) and the trunk can reach a diameter of 45 cm (1.5 ft). The bark has a
whitish-grey colour and is surrounded by thick cork. Young shoots have
purplish or greenish-white, hairy bark. The tree has an open crown of drooping,
fragile branches and the leaves build up feathery foliage of leaves.

The flowers are about 1.0-1.5 cm long and 2.0 cm broad. They grow on
slender, hairy stalks in spreading or drooping later flower clusters which have a
length of 10–25 cm.

The fruit is a hanging, three-sided brown capsule of 20–45 cm size

which holds dark brown, globular seeds with a diameter around 1 cm. The
seeds have three whitish papery wings and are dispersed by wind and water.

16
PREPARATION OF EXTRCTS

The dried plant material was crushed in to fine particles (powder) using
a mixer. About 25 grams of each plant powdered material was separately
extracted with 100 ml of solvents (Acetone, Chloroform, Methanol and
Petroleum ether) respectively. All the solvents were kept at room temperature,
for 7days to allow the extraction of compounds from plants. Each mixture was
stirred every 24 hours using sterile glass rod. The greenish extracts were
obtained and passed through the whattman filter paper No: 1 and the respective
solvents were evaporated (at 40°C) with the help of heating mantel. The sticky
black substances were obtained and stored in refrigerator and these are
suspended/ dissolved in DMSO (Dimethyl Sulphoxide) prior to use.

MICROBIAL INOCULUM PREPARATION

The young microbial inoculums / culture was prepared and used in the
entire research period. The nutrient broth was prepared and poured into several
tubes and sterilized. The pure microbial cultures were collected from the
Microbiology Department of the institute (either solid or liquid medium) and
inoculated in the tubes using inoculation needles or loops, after that all the tubes
were incubated at different temperatures and time duration (at 37° C for 24 to
48 hours).

SCREENING FOR ANTIBACTERIAL ASSAY

The antibacterial activities of the plant were tested against the selected
bacterial strains. The sterilized Mueller Hinton agar medium was poured in to
each sterile petriplate and allowed to solidify. Using sterile cotton swabs the test
bacterial cultures were evenly spread over the appropriate media. The sterile
discs were individually loaded with different concentrations of organic solvents
17
extracts (Acetone, Chloroform, Methanol, and Petroleum Ether) of the plant.
These discs were kept in undisturbed place for the evaporation of the solvents.
Then the discs were placed on the top layer of the Petri dishes pertaining to the
test cultures. All the plates were incubated at 37°C for 24-48 hours. After the
incubation period the results were observed and measured the diameter to
demarcate inhibition zone around the each disc/ organism.

MICROBIAL STRAINS USED FOR ANTIBACTERIAL ASSAY

Ten different bacterial strains are used for the antibacterial assay they
are, Escherichia coli, Staphylococcus aureus, Bacillus sp, Streptococcus
pyogens, Proteus sp, Salmonella sp, Shigella sp, Pseudomonas aeruginosa,
Klebsiella sp, Streptococcus mutants.

PRELIMINARY PHYTOCHEMICAL SCREENING

Various extracts collected from the shade-dried powdered stem bark of
Moringa oleifera was tested for identification of its active chemical
constituents.

TEST FOR ALKALOIDS

To the small quantity of test solution, a few drops of diluted
Hydrochloric acid was added and filtered. The filtrate may be tested carefully
with various alkaloidal reagents such as,

a) Mayor’s reagent – Creamy precipitation

b) Dragondroff’s reagent – Orange brown precipitate

c) Hager’s reagent – Yellow precipitate

d) Wager’s reagent – Reddish brown precipitation

18
TEST FOR FLAVONOIDS

a) To the small quantity of test solution, add aqueous sodium hydroxide

solution, appearance of blue to violet colour indicates the presence of
anthocyanins, yellow colour indicates the presence of flavones, yellow

b) To the small quantity of test solution, add concentrated sulphuric acid

indicates the presents of anthocyanins, yellow to orange colour indicates the
presence of flavones and orange to crimson colour indicates the presence of
flavonones.

C) Shimoda’s test: The small quantity of test solution is dissolved in

alcohol, to that piece of magnesium followed by concentrated hydrochloric acid
drop was added and heated. Appearance of magneta shows the presence of
flavanoids.

TEST FOR TANNIN

To the small quantity of test solution was taken separately in water and
tested for the presence of tannin.

1) Dil. Ferric chloride solution (5%) – Violet colour

2) 1% Solution of gelatin containing 10% Nacl – White precipitate

3) 10% Lead acetate solution – White precipitate

TEST FOR CARBOHYDRATES

To the small quantity of test solution was dissolved in 5ml of distilled

water and filtered was subjected to test for carbohydrates.

19
 a) Molish test: The filtrate was treated with 2-3 drops of 1% alcoholic
alpha napthol and 2ml of concentrated sulphuric acid was added along the sides
of the test tubes.

b) Fehling’s test: The filtrate was treated with 1 ml of fehling’s solution

and heated. Orange precipitate was obtained shows the presence of
carbohydrates.

TEST FOR SAPONINS

Small quantity of test solution was diluted with 20 ml of distilled water
and it was agitated on a graduated cylinder for 15 minutes, the presence of
saponins was indicated by the formation of 1 cm layer of foam.

TEST FOR TRITERPENOIDS

1) Salkowski Test: Approximately 2 mg of dry extract was shaken with 1
ml of chloroform and a few drops of concentrated sulfuric acid were added
along the side of the test tube. A red brown color formed at the interface
indicated the test as positive for triterpenoids.
TEST FOR PHYTOSTEROLS
Liebermann Burchard’s Test
The small quantity of test solution was dissolved in few drops of dry
acetic, 3ml of acetic anhydride was added followed by few drops of conc.
Sulphuric acid. Appearance of bluish green colour indicates the presence of
phytosteroles.

20
 RESULTS

SCREENING OF ANTIMICROBIAL ACTIVITY

Four different organic solvents (Acetone, Chloroform, Methanol,
Petroleum Ether) at various concentrations (25µl, 50 µl, 75 µl, 100 µl) of
Moringa oleifera was evaluated for its antimicrobial activity. Both Gram
positive and Gram negative organisms were screened in this study. The results
were tabulated in Table 1, Plate 1-2

The results of the present study shoes that most of the organic solvent
extracts of Moringa oleifera Leaf showed significant activity against the tested
bacterial strains.

The disc containing lest amount of solvent extract, 25µl/ disc had similar
antibacterial activity of the tested microorganisms, Escherichia coli (8 mm),
Staphylococcus aureus (11 mm), Klebsiella sp (11-17 mm), Pseudomonas
aeruginosa (9-15 mm), Streptococcus pyogens (11-15 mm), Proteus sp (15
mm), Salmonella sp (8-12mm), Shigella sp (8-12mm), Streptococcus mutants
(11 mm), Bacillus sp (10-20 mm)

In case of 50 µl shows a fair antibacterial activity against the bacterial

tested, Escherichia coli (9 mm), Staphylococcus aureus (11-14 mm),
Klebsiella sp (9-17 mm), Pseudomonas aeruginosa (9-10 mm),
Streptococcus pyogens (7-18 mm), Proteus sp (10-17mm), Salmonella sp (8-
17mm), Shigella sp (8-17mm), Streptococcus mutants (12mm), Bacillus sp
(12-21mm).

21
 The disc containing 75 µl/ disc extracts showed a better result,
Escherichia coli (9-13 mm), Staphylococcus aureus (10-17 mm), Klebsiella sp
(11-15 mm), Pseudomonas aeruginosa (11-13 mm), Streptococcus pyogens (8-
20 mm), Proteus sp (11-21mm), Salmonella sp (9-17mm), Shigella sp (9-
17mm), Streptococcus mutants (9-12mm), Bacillus sp (12-24mm).

The higher concentration of the extract 100 µl/ disc shows a good
activity the tested organisms, Escherichia coli (10-18 mm),
Staphylococcus aureus (12-20 mm), Klebsiella sp (10-17 mm),
Pseudomonas aeruginosa (12-16 mm), Streptococcus pyogens (8-28mm),
Proteus sp (11-31mm), Salmonella sp (10-20mm), Shigella sp (13-27mm),
Streptococcus mutants (10-15mm), Bacillus sp (12-28mm). (Table.1)

PHYTOCHEMICAL ANALYSIS OF MORINGA OLEIFERA

The presence of Phytochemicals in the plant under study was evaluated.
It is reported to predominantly contain a wide range of chemicals in different
extracts. The acetone extract contains the following constituents, Terpenoids,
Tannins, Saponins, Flavanoids and Alkaloids. The chloroform extract contains
Carbohydrates, Terpenoids, Tannins, Saponins, Flavanoids and Alkaloids. The
methanol extract contains Terpenoids and Phyto Steroids. The petroleum ether
extract contains Alkaloids, Flavanoids, Saponins, Terpenoids, Phyto Steroids
and Tannins. (Table.2)

22
 TABLE. 1 Antimicrobial activities of Moringa oleifera
Diameter Zone of inhibition
NAME OF THE (mm) at different concentration
SI.NO SOLVENT of the extracrs.
ORGANISMS
25µl 50µl 75µl 100 µl
Acetone - 9 10 11
Chloroform - 9 9 10
1. Escherichia coli Methanol - - - -
Petroleum ether 8 9 13 18
Acetone 11 14 17 20
Chloroform - - 10 12
2. Staphylococcus aureus
Methanol - 11 13 16
Petroleum ether - 11 15 19
Acetone - - 12 9
Chloroform 9 10 10 10
3. Klebsiella sp
Methanol - - 13 -
Petroleum ether 7 9 12 9
Acetone 10 - 12 10
Chloroform 13 - 12 -
4.. Pseudomonas aeruginosa Methanol - - - -
Petroleum ether 10 11 11 9
Acetone 11 12 17 20
Chloroform - 7 8 8
5. Streptococcus pyogens Methanol 14 16 20 20
Petroleum ether 15 18 20 30
Acetone 20 21 24 28
Chloroform 12 15 18 20
6. Bacillus sp
Methanol 13 15 17 18
Petroleum ether 10 10 12 12
Acetone - 10 10 13
Chloroform - 10 10 11
7. Proteus sp Methanol - 10 11 13
Petroleum ether 15 17 21 31
Acetone 12 17 17 20
Chloroform 8 8 9 10
8. Salmonella sp Methanol 10 11 12 16
Petroleum ether - 10 10 12

23
 Acetone 11 12 12 14
Chloroform 11 12 13 15
9. Streptococcus mutants Methanol - - 11 12
Petroleum ether - - 9 10
Acetone 12 17 17 20
Chloroform 8 8 9 10
10. Shigella sp Methanol 10 11 12 16
Petroleum ether - 10 10 12

24
 TABLE.2
Preliminary Phytochemical screening of various extracts of the
Moringa oleifera

SI.NO Constituents Acetone Chlorofom Methanol Petroleum

Ether

1 Alkaloids + + _ +

2 Flavonoids + + _ +

_
4 Saponins + + +

_
5 Tannins + + _

6 Terpenoids + + + +

7 Phyto Steroids _ _ + +

8 Carbohydrates _ + _ _

25
 DISCUSSION

Antibiotic resistance has been a global concern in recent years. This

problem is a great significance especially in developing countries, because
infectious diseases are one of the major cause of mortality in these countries.
Medicinal plants have been found useful in the cure of a number of diseases
including bacterial diseases. Medicinal plants are rich source of antimicrobial
agents. Due to the rapid increase in the rate of infections, antibiotic resistance in
microorganisms and due to side effects of synthetic antibiotics, medicinal plants
are gaining popularity over these drugs. Although medicinal plants produce slow
recovery, the therapeutic use of Medicinal plant is becoming popular because of
their lesser side effects and low resistance in microorganisms. Antimicrobial
activities of many plants have been reported by the researchers.

Plant based antimicrobial represents the vast untapped source for medicine.
Plant based antimicrobials have enormous therapeutic potential as they can solve
the purpose without any side effects , that are often associated with synthetic
antimicrobials, continued further research and exploration of plant derived
antimicrobials needed today.

Medicinal plants are important source for the development of potential new
chemotherapeutic drugs and the in vitro antibacterial test from basis. Many of the
studies were useful in identifying the active principle responsible for such
potentials and to develop clinically important therapeutic drugs for mankind.

The present study focused on the medicinal plant Moringa oleifera for its
antibacterial activity, Phytochemical analysis.

26
ANTIBACTERIAL ACTIVITY

In the present study, the plant Moringa oleifera was evaluated for its
antibacterial properties against both gram positive and gram negative organisms.
Various organic solvent extracts with different concentrations were analysed for
the current investigation. From the screening results it was observed that most of
the extracts were found to inhibit the growth of the organisms. The results
indicated that some of the secondary metabolites present in the plant part may be re
sponsible for this activity.
Ashok et al., (2014) Fresh leaves of Moringa oleifera and Saraca indica
were collected for the evaluation of their antimicrobial activity against test
bacterial pathogens. The antibacterial activity of the aqueous extracts of Moringa
oleifera was inhibitory against Staphylococcus aureus, Escherichia coli Proteus
vulgaris and Salmonella typhi. All the selected bacterial pathogens were resistant
to acetone and chloroform extract of M. oleifera. Ethanol extract was effective
against Escherichia coli while both ethanol and methanol extracts were inhibitory
against Proteus vulgaris and Salmonella typhi (10mm each zone of inhibition
respectively) zone of inhibition. Petroleum extract was not effective against all the
test pathogens except Peudomonas aeruginosa.
Vinoth et al., (2012) in their study about Antimicrobial activity on Moringa
oleifera leaves chloroform leaves chloroform extract. It is clearly noted that
aqueous extract of M. oleifera leaf posses significant antimicrobial activity against
most of the selected bacterial pathogens, thus signaling its broad spectrum of
antibacterial activity, thus rendering water as a good solvent of extraction for the
M. oleifera leaves, much so, that most people who use this leaf as a traditional
mean of treatment of various skin ailments and other diseases make use of water-

27
based extract of the leaf. Another reason for M. oleifera aqueous extract might be
due to the presence of flavanoids, saponins, tannins, glycosides and terpenoids
Bukar et al., (2010) who reported in their study on the antibacterial
activity from the aqueous, acetone and ethanolic extracts of the leaves of Moringa
oleifera that ethanolic extract of the plant demonstrated the highest activity, while
the aqueous extract showed the least activity at 100 mg/ml.

PRILIMINARY PHYTOCHEMICAL ANALYSIS

The preliminary Phytochemical analysis of selected plant part displayed the
presence of various compounds. Phytochemicals are non nutritive plant chemicals
which have the protective activity, they can prevent the plant diseases. Many
researchers found that these compounds are also used against human diseases. The
present Phytochemical analysis results showed that the plant is credited with
various bioactive components like alkaloids, flavanoids, tannins, saponins,
triterpenoids, phyto steroids and carbohydrates.

Ugwu Okechukwu et al., (2013). Acute toxicity and phytochemical studies

of ethanol leaf extract of Moringa oleifera were analysed. The phytochemical
analyses of the ethanol extract of Moringa oleifera leaf showed the presence of
tannins, carbohydrates, saponins, glycosides, reducing sugars, terpenoids, steroids,
flavanoids and alkaloids.
Vinoth et al., (2012). In their investigations of the Phytochemical test was
done to find the presence of active chemical constituents such as glycosides,
alkaloids, tannins, flavonoids, terpenoids and saponins.

28
 Doughari et al., (2007) revealed the prominent presence of alkaloids,
phenolics, flavonoids and tannins in hydro-ethanolic extract of Moringa oleifera
pods. In present study alkaloids, tannins, saponins and flavonoids are detected in
Moringa oleifera.

29
 SUMMARY
The current investigations revealed that potentials of Moringa oleifera
against bacterial pathogens tested. Different organic solvents such as Acetone,
Chloroform, Methanol and Petroleum ether of this plant were evaluated for its
antibacterial activity using standardized procedure and methodologies.

The results showed that the different solvent extracts of varying

concentrations (25µl, 50 µl, 75 µl, 100 µl/ disc) showed concentration dependent
activity. All the organisms exhibited inhibitory activity in the higher concentration
of the extracts tested. The polarity of the solvent seems to play an important role in
exhibiting potential antimicrobial activity.

Phytochemical analysis helped to detect the presence of secondary

metabolites present in the plant extracts which may be responsible for the
inhibitory activity.

30
 CONCLUSION
Plants used in traditional are assumed to be safe due to the long-term use by
traditional healers. Herbal medicinal preparations and their proprietary products
are being used more and more widely throughout the world for treating varies
ailments. Hence evaluating and ensuring their quality becomes increasingly urgent.
Many medicinal plants have been found effective in the cure of bacterial diseases.
Due to increasing antibiotic resistance in microorganisms and side effects of
synthetic medicinal plants are now gaining popularity in the treatment of bacterial
infections. Medicinal plants are considered as clinically effective and safer
alternatives to the synthetic antibiotics.

In conclusion the results revealed that the organic solvent extracts of

Moringa oleifera have been found to be effective against wide range of
microorganisms.

The extract was more effective than traditional antibiotics to combat the
pathogenic microorganisms studied.

The phytochemical screening revealed the details about the bioactive

compounds present in the plant.

Moringa oleifera leaves to treat common medical conditions but a few use it
for preventing and treating malnutrition. Presence of Phytochemicals indicates
possible preventive and curative properties of Moringa oleifera leaves. There is
need to carry out more pharmacological studies to support the use of Moringa
oleifera as a medicinal plant

31
 FUTUROLOGY

 Development of herbal medicine is needed as an alternative theraphy.

 Further extensive work is needed to find out the active principles,

characterization of compounds and its pharmacological evalution.

32
 APPENDIX

MEDIA COMPOSITION
Nutrient Broth
Peptone - 5.0gm
Beef extract - 3.0gm
NaCl - 5.0gm
Yeast extract - 2.0gm
Distilled water - 1000ml
pH - 7.2 to 7.6

Nutrient Agar
Peptone - 5.0gm
Beef extract - 3.0gm
NaCl - 5.0gm
Yeast extract - 2.0gm
Agar - 20.gm
Distilled water - 1000ml
pH - 7.2

Mueller Hinton Agar

Beef infusion - 300.0gm
Casamino acid - 17.5gm
Starch - 1.5gm
Agar - 17.gm
Distilled water - 1000ml
pH - 7.4

33
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