CHAPTER TWO

INTRODUCTION

Moringa oleifera belonging to the family of moringaceae is an effective remedy

for malnutrition. Moringa is rich in nutrition owing to the presence of a variety
of essential phytochemicals present in its leaves, pods and seeds in fact,
moringa is said to provide 7 times more vitamin C than oranges, 10 times more
vitamin A than carrot, 17 times more calcium than milk 9 times more protein
than yoghurt, 15 times more potassium than bananas and 25 times more iron
than spinach (J.L. Rockwood et al, 2013). Children deprived of breast milk
tends to show symptoms of malnutrition. Laetogogus are generally prescribed to
lactating mother to augment milk production. Moringa is rich in phytosterols
like sigmasterol, sitosterol and rampesterol which are precursors for harmones.
These compound increase the estrogen production, which in turn stimulates the
proliferation of the mammary gland ducts to produce milk. It is used to treat
malnutrition in children younger than 3 years (J. Mutiana Titi, 2013). About 6
spoonful of leaf powder can meet a woman’s daily iron and calcium
requirements during pregnancy. This study provides an overview on the
cultivation nutritional values, medial properties for commercial use and
pharmacological properties of moringa.

Psidium guajava L. (guava), a fruit plant belonging to the family myntaceae, is

found all over the world. Guava leaves, roots and fruits have been used for the
prevention and treatment of diarrhea (Lutterodt, G.D et al, 2002). In several
studies, guava showed significant antibacterial activity against common food
borne diarrhea – causing bacteria such as staphylococcus Spp. Shigella Spp.
Salmonella Spp. Bacillus Spp. E. Coli clostridium Spp and food spoilage
bacteria such as Pseudomonas Spp. (Abdemahirm S.I et al, 2002) guava is also
used medically in many parts of the world as an anti-inflammatory and
antiseptic as well as in the treatment of diabetes, hypertension, pain fever,
respiratory disorders gastroenteritis, diarrhea and dyscentary (Gutierrez, R .M.P
et al, 2008). Guava also had antioxidant properties which were attributed to the
polyphenols found in the leaves.

WHAT IS MORINGA OLEIFERA

Moringa oleifera is a fast growing drought resistant tree of the family

moringaceae, native to the Indian subcontinent common names include
moringa, drumstic tree, horseradish tree, and ben oil tree on benzolive tree. A
preparation of the leaves, fruits, or other parts of the drumstick tree, used chiefly
as herb on dietary supplement.

WHAT IS GUAVA

Guava is a common tropical fruit cultivated in many tropical and subtropical

regions. Psidium guajava (common guava, lemon guava) is a small tree in the
myntle family (myretaceae), native to Mexico, cental America, the Caribbean
and Northern South America.

HISTORY OF GUAVA

Guava is of numerous trees and shrubs of the genus psidium (family myrtaceae)
native to tropical America. The term “guava” appears to derive from Arawak
yauyabo “guava tree” via the Spanish guayaba. It has been adapted in many
European and Asian languages, having a similar form. The common types of
guava include apple guava, yellow fruited cherry guava, strawberry guava, and
red apple guava. It is mostly eaten raw (ripe or semi-ripe) or consumed in the
form of juice, jams, and jellies. The common guava has a fruit with a yellow
skin and white, yellow or pink flesh. Guava are known for their sweet and
tangy flavor and many uses, but there’s much more to this fruit than meets the
eye guava’s believed to have originated from Mexico or central America. It is
now very population in Asian countries and is also increasingly available in
American countries, particularly after its health benefits have been revealed.
The most guava producing countries are Indian, China, Thailand, Pakistan,
Mexico, Indonesia, Brazil, Bangladesh, Philippines and Nigeria.

HISTORY OF MORINGA OLEIFERA

Moringa oleifera is a type of local medical Indian herb which has turn out to be
familiar in the tropical and subtropical countries. The other terms used for
Moringa are Horseradish tree, Sajna, rielor, Saijihan and moringa. Monga
oleifera is a small native tree of the sub-Himalayan region of North West
Indian, which is now indigenous to many regions in Africa. Arabia, South East
Asia, the pacific and Caribbean Island and South America. Traditionally,
besides being a daily used vegetable among people of these regions the Moringa
is also widely knowned and used for its health benefit. It has earned it name as
the “miracle tree” due to its amazing healing abilities for various ailments and
even some chronic diseases several investigations were carried out to isolate
bioactive compounds from various parts of the plant due to its various
application (Guava et al, 1999). Therefore herbal plants in medicine or renown
as phytomedicine are still trustworthy and widely applied as one of the
alternative affordable cot (Abalaka et al, 2009). For centuries and in many
cultures around the world, the medical usage of the moringa has been used to
treat problems such as grain infection, anaemia, anxiety, asthma, blackhead ,
blood impurities and cholera (Khawaja et al, 2010, Hamza, 2010; Singh et al,
2012).

TAXONOMICAL CLASSIFICATION OF GUAVA

Kingdom - Plantae

Sub-kingdom – tracheobionta
Division – Magnoliophyta

Class - Magnoliopsida

Sub-class – Rosidae

Order – Myrtales

Family – myrtaceae

Genus – Psidium

Species – Psidium guajava

TAXONOMICAL CLASSIFICATION OF MORINGA

Kingdom - Plantae

Division – Magnoliophyta

Class - Magnoliopsida

Order – Brassicles

Family – Moringaceae

Genus – Moringa

Species – M. Oleifera (Fatiey, 2005).

EXTRACTION AND PREPARATION OF GUAVA

Preparation of plant extracts the leaf samples were collected from the guava tree
growing at the specialty plant. Random leaf samples were collected into plastic
zip lack bags with appropriate labeling and stored in an ice cooler until being
transported to the laboratory for extraction.
EXTRACTION METHODS USED ON GUAVA

The leaf samples were washed in tap water, dried and place into a blender to be
grounded into powder. Four solvent were arranged in increasing polarity; n-
hexane (>95%), methanol (>95%), ethanol (>19.5%) and boiling distilled water
were used for the creation extraction procedure. The leaf powder was added to
each of the solvent to make a 20% concentration. The mixture were made in
sterile 125ml tzrlenmeyer flask wrapped in aluminum foil to avoid evaporation
and exposure to light for 3 days at room temperature. The flasks were placed on
a platform shaker at 70rpm. After 3 days of soaking in solvent, the mixtures
were transformed to 50ml tubes and centrifuged for 10mins at 4,000 rpm 20 0c.
The supernatant was collected and stored at 40c until use.

PHYTOCHEMICAL ANALYSIS

Chemical tests for the screening and identification of bioactive chemicals

constituents in the guava were carried out with the extracts using the standard
procedure as described (J.B. Harbone, et al, 1973 J. Tanaka et al, 1992) for each
test 1ml of each solvent extract was used for analysis, in exception for the
saponin test in which 3ml solvent extract was used.

TEST FOR SAPONINS

Extracts was placed in a test tube and shaken vigorously. The formation of
stable foam was taken as an indication for the presence of saponins.

TEST FOR TANNINS

Extract was mixed with 2ml of 2% solution of FeCl 3 A blue-green or black

coloration indicated the presence of phenols and tannins.
EXTRACTION AND PREPRATION OF MORINGA

Preparation of plant & Extraction method moringa dry leaf powder extract in
20, 30 and 40 mins times water diluted concentration was obtained by drying
leaves under shade and then ground to form powder using pastle and mortar
which later was dissolved in water, overnight moringa fresh chopped leaf
extract was prepared by crusing fresh chopped moringa leaves under shade
pestle and mortar which later was dissolved in water.

USE OF GUAVA

Psidium guajava (Guava) is an ethnomedicance. It has special importance in the

importance in the traditional system of medicine. It is considered as an
important herbal medicine for dysentery and diarrhea in traditional chines
medicine system, it is used since ages to improve the wealth of humans.it Is use
for the treatment of gastrointestinal disorder.

The compounds, isoflavonoids, gallic acid, catercluin, epicathechin, rutin,

naringmin, caempferoal.

These compounds are used for hepatoprotection, antioxidant, anti-inflammatory,

anti-spansmodic anti-cancer, antimicrobial anti hyperglycemic, amalgesic
activity.

USES OF MORINGA

Moringa can be used as a potent neuroprotectant, cevebral ischemia is cused

due to obstruction of blood flow to the brain this leads to reperfusion and lipid
peroxidation, which in turn results in reactive oxygen species, moringa with its
antioxidant can reduces the brain (W. Kirisattay a Kul et al, 2013) its used to
treat demeatial as it has been shown to be a promoter of spatial memory. The
leaf extracts have shown to decrease the acetylcholine esterase activity, thereby
improving cholinergic function and memory (C.sutalangria et al) 2013).
Morninga decrease acidity in gastric ulcers by a percentage of 86.15% and
85.13% at does of 500mg and 350mg respectively and therefore can be used as
an antiulcer agent (M.K Choudhary et al., 2013). Moringa is prescribed by
herbal practitioners for patients with AIDS, moringa is suggested to be included
in the diet, with the view of boosting the immune system of HIV positive
individuals.

Moringa leaves can be used to treat asthma, hyperglycemia, Dyslipidemia, flu

heart burn, syplilis, malaria, pheumonia, diarrhea, headaches, scurvy, srain
disease, bronchitis eye and ear infections. Also reduces blood pressure and
cholesterol and acts as an anticancer antimicrobial antioxidant, antidibetic and
anti-atherosclerotic agents, Nero protectant.

BENEFITS OF MORINGA

 Anti-fibrotic/ulcer
Major contributors to the treatment of liver constant efforts and studies on
these natural drugs to treat liver fibrosis discovered to date are natural
drugs. Constant efforts and studies on these natural drugs to treat liver
fibrosis are being carried out in search for effective anti-fibrotic agent.
 Anti-inflammatory effect.
Moringa has been practically used in medicinal filed, throughout the
decades to heal a huge amount of acute and chromic conditions in vitro
and in vivo studies with the plant have recommended its effectiveness in
treating inflammation, hyperlipidemia, and hyperglycemia (Bennett et al.,
2003; family, 2005, mbiriay, 2002).
 Antimicrobial effect
 The assorted extracts of morniga’s morphological parts such as seeds
cotyledon, seeds coat, stem bark, leaves, root bark are repointed to posses
antimicrobial potential (arora et al., 2013)

 Anti-hyperglycemic
Diabetes mellitus (DM) is a chronic metabolic disorder. Diabetic patients
exhibit stage of chronic hyperglycemia and glucose tolerance impairment
(tiwari & Roa, 2002). It is well know for its pharmacology actions and is
used for he traditional treatment of diabetes mellitus (Bhishagrata, 1991;
Babu and chauduri, 2005).

 Antioxidant properties
Naturally occurring antioxidants, particularly polyphenols, are the main
plant compounds that ar able to decrease oxidative damage in tissues by
indirect enhancement of a cell or by free radical scavenging (Du et al.,
2010).the moringa leaves have been reported to demonstrate antioxidant
activity dues to its high amount of polyphenols (Sneelatha and Padma
2009; verma et al., 20090.

 Anti-tumor properties
Inhibitor against the two stage mouse tumourigenesis from in vitro
screening suggested that several of test compounds, particularly 4(x-2—
hamonsyloxy) benzyl isothiocyanate, niazimicin and B-Sitosterol -3-0-B-
D-glucopyranoside were strong anti-tumor promoters whilst in the invivo
two-stage. Carcinogenesis test on mouse skin mazimicn exhibited 50%
delay in their promotion of tumours and decreased the incidence of
papilloma bearing mice by 30% at 100 weeks and 17% at 20 weeks of
promotion (Guevera et al., 1999).
  Anti-cancer-properties
Moringa is revealed to possess potential therapeutic effects to fight
cancer, rheumatoid arthritis, diabetes, and some other ailments,
particularly in south Asia, it work as treatment for disserent disease in the
indigenous system of medicine (Mehta et al, 2007; roy et al., 2007).

 Anti-Clastogenic properties
The moringa oleifera demonstrated free radical scavenging properties
that directly indicate anti-clastogenic effects which was found to be due
to its rich vitamin C content. The anti-clastogeniticity test in this stud
showed activity against both direct mitomycin C (MMC) indirect-acting
DMBA clastogens it was finally concluded that bMO at 2,1,4,3 and 8.5
g/kg BW does did not show clastogenic potential I modulated by the
direct acting carcinogene process.

BENEFIT OF GUAVA

Antimicrobial activity

Guava has a high antimicrobial activity guava leafs extract does can reduce the
amount of cough due to its anti-cough activity. Aqueous, chloroform and
methanol extract of leaves can reduce the growth of different bacteria due to its
anti-cough activity it is recommended in the condition of cough. (P rhoohaswan
et al., 1999)

 Anti-diarrhea is one of the most common and well recognized health

problems and a global issue. It is very common even in developed
countries. It is estimated that above 2.2 million people die annually by
diarrhea: most of them are children or infants (Venratesan N. et al., 2005)
Guava levels have quercetin -3- arabinose and quercetin which can be a
 compound which has morphine line action. It controls the muscular tone.
Quercetain repressed intestinal contraction encouraged by enhanced
absorption of calcium. It has a strong effect on ileum. It is thought that
quercetin in quava leaf are responsible for its spasmolytic activity. Guava
has high cytotoxicity (teixeria RDO, et al, 2003)
 Anti-Inflammatory activity extract of guava in ethyl acetate can stop the
germ infection and thymus production.it can act as anti-viral agent.it can
enhance the MRNA expression. Guava can alter the hence oxygenase-1
protein work. And due to this reason, it can be used as anti-inflammatory
agent for skin extract of guava in ethanol inhabit the lipopolysaccharide
from manufacturing of nitric oxide. It suppress the expression of Ez. In
this way it works as anti-inflammatory agent. (Jeongs, et al., 2014)
extract in ethyl acetate has the ability to minimize the antigen. It can stop
the release of the B-hexosaminidase with instamine into RBL-2H 3 cells.
In this way the antigen inhibits and 1kB-& become spoil. Benzophenone
and flavonoids are important compound found in guava. These
compounds are responsible for the instamine inhibition and nitric acid
production matsuzaki K, et al; 2010).

ANTIOXIDANT ACTIVITY

Antioxidants are molecules which retard the oxidation process. The oxidation
reaction may produce free radicals which damage the cells by starting various
chain reactions free radicals which damage the cells cause cancer and many
other disease. Antioxidant terminate the free radical and stop the chain reaction.
Examples of antioxidants include bête-carotene, lycopene, vitamin C, B, and A
and other substances. Oxidative reaction is one of the most important
destruction reaction free radicals damage is responsible for a lot of disorders in
human like nervous disorders, inflammation, debates and viral infections when
drugs are metabolized in body the free radicals are produced. Sometimes the
environmental changes and hormones become the reason for free radical
production. These free radicals are responsible for all the oxidation reaction
(Masuda T. et al; 2003).

MEDICAL IMPORTANCE OF GUAVA

Psidium guajava L. is consumed not only as food but also as folk medicine in
subtropical areas all over the world due to its pharmacologic activities (Deguchi
Y. et al, 2010). Medicinal plants find a very important place in medical system
all reflected from traditional knowledge. It is well known that guava is
frequently employed in numerous parts of the world for the cure of a lot of
sickness like diarrhea reducing fever, dysentery, gastroenteritis, hypertension,
diabetes, caries, pain relief and wounds. Guava contains high content of organic
and inorganic compounds like secondary metabolites e.g antioxidant,
polyphenols, antiviral compound and anti-inflammatory compounds. It has a
higher number of vitamins and minerals phenolic compounds like flavonoids
also find an important antioxidants. They help in the cure of cancerous cells
and help to prevent skin aging before time (anand V, et al, 2016). Guava skin
can control level of diabetes after 21days treatment (Rai pre, et al, 2010).

MEDICINAL PROPERTIES OF MORINGA

Moringa oleifera is often referred as a panacea and can be used to cure more
than 300 diseases. Moringa has long been used in herbal medicine by Indians
and Africans. The presence of phytochemicals makes it a good medicinal agent.

PRESERVATIVE METHOD OF MORINGA

Moringa can also be preserved for a long time without loss of nutrients. Drying
or freezing can be done to store the leaves. A report by (Yang et al; 2006)
shows that a low temperature oven used t to dehydrate the leaves retain more
nutrients except vitamin C than freeze dried leaves. Hence, drying can be done
using economical household appliance like stove to retain a continuous supply
of nutrients in the leaves. Preservation by dehydration improves the shelf life of
moringa without change in nutritional value.

EFFECT OF OVERDOS OF MORINGA

An overdose of moringa may cause high accumulation of iron. High iron can
cause gasterointestinal distress and hemochromatosis. Hence a daily dose of 70g
of moringa is suggested to be good and prevents over accumulation of nutrients
(I.J Asieda, et al, 2014).
 CHAPTER THREE

MATERIALS AND METHODS OF GUAVA LEAF

Preparation of plant Extract the leaf samples were collect from the guava trees
growing at the specialty plant House at fort valley state University. Randam leaf
samples were collocated into plastic zip lock bags with appropriate labeling and
stored in an ice cooler until being transported to the laboratory for extraction.

Extraction Methods used on Guava. The leaf were washed in tap water, dried
and placed into a blender to be grounded into powder, four solvents were
arranged in increasing polarity; N-Hexane (>95%) methanol (>95%), ethanol
(>99.5%), and boiling distilled water were used for the maceration extraction
procedure. The leaf powder was added to each of solvents to make a 20%
concentration. The Mixture were made in sterile 125mL Erlenmeyer flask

Test for saponins. Extract was placed in a test tube and shaken vigorously. The
formation of stable foam was taken as an indication for the presence of saponins

Test for phenols and tannins. Extract was mixed with 2ml of 2% solution of
FeCL3. A blue-green or black coloration indicated the presence of phenols and
tannins
 MATERIALS AND METHODS OF MORINGA LEAF

The collected swab samples were streaked onto blood agar plates (nutrients agar
13g/l containing 5% citrated sheep blood) MacConkey agar plates (52g/l0. And
mannitol salt agar 7.5% plates (111g/l). All samples were streaked in duplicated
plates and were incubated aerobically and anaerobically at 370C for 24h and
370C for 48-72h, respectively.

Identification Of Bacteria Isolates

Bacteria isolates were microscopically identified according to Cruickshank et al.

(24) Biochemical identification of Gram-positive coccobacilli was carried out
according to funk et al. (25) Biochemical identification of gram-positive cocci
and Gram negative bacteria was carried out according to quinn et al (26) and
Cruickshank et al ., respectively (24).

Antibiotic Sensitive Test for Identification Strains

Fifteen standard antibiotic disks were used against the isolated bacteria after
preparation of the standardized bacterial inculums matching with 0.5 McFarland
tubes (108 colony-forming unit (UFU)/ml. then, 25 ul of the inculum was
distributed on Muller-Hinton agar plates and incubated at 370C for 24h. the
degree of sensitivity was determined by measuring the inhibition zone. The
result was interpreted according to Dzotam et al. 9 (16).

Plant Material of M.Oleifera

Moringa leaf powder was obtained from moringa Unit; NRC. It was collected
from a farm of Alexandria desert road. The plant material was collected in
January 2018 and presented in the study.
Cold Aqueous Extract Of M.Oleifera Leaves

100g of M. Oleifera leaf powder was weighed out and dissolved in 400 ml of
cold distilled water into a conical flask stooped with rubber corks and left for
mixture was filtered off using a sterile filter paper (whatman no.1) Into clean
conical flask and subjected to water bath evaporated where the aqueous solvent
was evaporated at its boiling temperature of 1000C the standard extracts
obtained were than stored in refrigerator at 40C for antibacterial activity test(27).

Hot aqueous extracts of m. Oleifera leaves

The same protocol as in cold water treatment was used with 30 min of boiling
while the plant material was dipped in distilled water.

Ethonal (95%) extracts of m. Oleifera leaves

Here, the same procedures as in cold water treatment was followed.

Test Microorganisms

All the isolated bacteria from camel abscesses were used to assess the
antibacterial effect of M. Oleifera. It included C. pseudotuberculosis, C.
ulcerans and S. aurens as gram –positive bacteria and E. coli, K. Pueumoniae,
Citrobacter spp., proteus vulgaris and p.aeruginosae as Gram-negative bacteria.

Antibacterial assey of M. Oleifera

The antibacterial activity of the three different samples, namely (1) cold water
extract (CWE) of leaves, (2) hot water extract of leaves and (3) ethanol extracts
(EEs) of leaves, was individually tested against the studied bacteria, in vitro
antibacterial test was then carried out by disk diffusion method (28,29) using
25 ul of the standardized bacterial suspension of the tested bacteria (10 8
CFU/ml) spread on plates. The disks (6 mm in diameter) were impregnated for
different samples with 10ul of 0.1g/ml (100 mg/disk), followed by air drying,
and placed on seeded agar plates. Negative controls were prepared using the
same solvents to dissolve the plant extracts. Tetracycline (TE) 30 ug/disk) was
used as a positive control to determine the sensitivity of bacterial strain. The
plates were incubated at 370C for 24h. the antimicrobial activity was evaluated
by measuring the zones of inhibition against the tested bacteria.