CHAPTER ONE

INTRODUCTION

Generally, plant parts (seeds, leaves, bark, fruits and stems) contain bioactive agents. These

confer such plants with nutritive and antimicrobial properties which could contribute to the

management of diseases. However, proper assessment on the possible dietary and therapeutic

potentials of such plant parts are required for informed use in animals, including human.

There is growing awareness worldwide, for using medicinal plants, over the conventional

antibiotics, as an alternative for the treatment, management and prevention of infectious

diseases. This is because the conventional antibiotics are fast becoming less effective due to

the emergence of multidrug resistance amongst microbial strains (Ali, et al., 2015). This

renewed interest for alternative therapy may also have been prompted by an increase in

incidence of disease and opportunistic infections, especially in immune- compromised

individuals which have become difficult to manage with orthodox antibiotics (Badria and

Zidan, 2004). One approach to circumvent this challenge is to use plant’s bioactive

phytochemicals (Singh et al., 2009).

The use of plants as phytotherapeutic agents are comparatively, less expensive than

conventional drugs and if administered at higher concentration seldom have such drastic side

effects on the host(Saraf,2010).Plants are known to have a broad spectrum of antimicrobial

activity and constitute a source of many of such compounds (Ayodele and Banuso, 2004).

The most important bioactive constituents of plants are flavonoids , tannins, phenolic

compounds, alkaloids, and their antimicrobial activity may reside in a variety of these

phytochemicals(Gonclaves et al., 2005). These phytochemicals are secondary metabolites

present in plants which enable them combat many disease-causing pathogens hence possess
pharmacological potentials (Hussain et al., 2011). Persea Americana (avocado pear) is one of

the most important fruits used as fruits used as food in most topical and subtropical countries.

Different parts of the plant are used in herbal preparations. The peel, leaves and fruits are

used in the treatment of haemorrhagia, hypertension. The leaf extract effectively inhibited

herpes simple virus types I and II (Sturluson,2017). The seeds of P.americana have been

applied for the treatment of diverse diseases such as dysentery, skin infections, toothache

(Pamplora and Roger,1999). The seeds are equally rich in tannin and carotenoids (Lu et al.,

2009); Enterococcus faecalis, Pseudomonas aeruginosa (Chia and Dykes, 2010). The seed

oil and leaves have diverse application in ethnomedicine for the treatment of diarrhea,

toothache, dysentery, intestinal parasites, skin treatment and beautification (Roger, 1999;

Adeyemi et al., 2002).

Despite these potential uses of the seeds, they are usually discarded hence underutilized. In

view of the current trend of antibiotics resistant microorganisms, there is a need to look into

the potential of P.americana seed extracts as an antimicrobial agent. Research into the

possible therapeutic/antimicrobial activity of this underutilized agro-food waste, will also

reduce the environmental waste burden it may constitute. Therefore, this study is a

comparative study of the phytochemical composition and the antimicrobial activities of

ethanolic extracts from the leaves and seeds of P. Americana

1.1 JUSTIFICATION OF STUDY

The study related to antimicrobial activity of medicinal plant is growing due to increasing

cases of antibiotic resistance occurring in almost parts of the world. Traditional healing

system around the world utilize herbal medicine have already produced compounds that are

effective against antibiotics antibiotic-resistant strain of bacteria. Medicinal plants have

immunomodulatory and antioxidant properties, leading to antimicrobial activity. This study

was carried out to investigate the phytochemical, proximate, minerals, antioxidant and

antimicrobial properties of avocado pear (Persea americana) seed and fruit peel.

AIM AND OBJECTIVES OF THE STUDY

The aim of the study is to investigate the proximate, phytochemical, mineral, antioxidant and

antimicrobial properties of the ethanolic extract of avocado pear (Persea americana) seeds

and fruit peel.

The objectives of the study include:

 Collection and identification of Avocado pear (Persea americana) fruits.

 Extraction of avocado pear (Persea americana) seed and fruit peel by cold maceration

using ethanol and water as different solvents.

 Phytochemical, proximate, mineral and antioxidant screening of the plant extracts.

 To carry out antimicrobial susceptibility tests of the plant extracts on clinical bacteria

isolates ( )

 Determination of minimum inhibitory concentration [MIC] of the plant extracts on

susceptible organism.

 Comparison of the various parameters of the various plant extracts analyzed to

ascertain the one with better activity.

Disposal of clinical isolates after use will be done using standard microbiological methods.

Isolates would be treated with chemicals before disposal.

 CHAPTER TWO

LITERATURE REVIEW

Avocado

Avocado (Persea americana Mill.) is one of the oldest known flowering plants belong to the

family Lauraceae and order Laurales, mainly cultivated for its edible fruit. It is believed to

have been originated from Mexico and Central America (Rohwer, 1999), having a diploid

chromosome number of 2n =24. Botanically, the fruit is a large berry containing a single

large seed. The avocado was introduced to india from Sri Lanka in the twentieth century

(Ghosh et al.2000).

Avocado is the most nutritive fruit and the pulp is rich in proteins(up to 4%),fat (up to

30%),copper and iron but low in carbohydrates. The fat is similar to olive oil in composition

and is widely used in the pharmaceutical and cosmetic industries. The fat content of avocado

had zero cholesterol level and found to decreases the LDL level in the body by 22 per cent; it

also increases the HDL level by 11 percent. Avocado is mainly used as fresh. The protein

content of an avocado is three times that of an apple, nearly equal to that of the local plantain

and less than olive fruit.

Avocado plant bears fruit known as avocado pear or alligator pear that contains the avocado

pear seed. Reported uses of avocado pear seed include use in the management of

hypertension, diabetes, cancer and inflammation. The fruit is known as ube oyibo (loosely

translated to ‘foreign pear’) in Ojoto and neighbouring Igbo speaking communities south east

Nigeria. Different parts of avocado pear were used in traditional medications for various

purposes including as an antimicrobial. That notwithstanding, the avocado pear seeds are

essentially discarded as agro-food wastes hence underutilized. Exploring the possible dietary
and therapeutic potentials of especially underutilized agro-food wastes will in addition reduce

the possible environmental waste burden.

Taxonomy

Kingdom: Plantae

Phylum: Magnoliophyta

Class: Magnoliopsida

Order: Laurales

Family: Lauraceae

Genus: Persea

Subject: Persea americana P. Mill.

Morphology:

Avocado belongs to the family lauraceae, It is a shiny evergreen tree which has height of 9-

20m for medium to large tree, much more branched, vigorous and round topped[1,2].

Avocado is cultivated in all tropical and subtropical regions in the world for its delicious and

nutritious fruit. The leaves are 6-30cm in length and 3.5-19cm in wide and narrow to broadly

elliptical in shape. They are constantly pubescent and reddish when young. When they

mature, becoming smooth, leathery and dark green. Its fruit is consumed as food. Essential

oils and flavonoids were reported to be present in Persea americana leaf. Also, avocado

leaves contain persin which is a toxin for lactating livestock.

DESCRIPTION: Avocado trees can be short or tall, and its elliptic to egg-shaped leaves

range in size from 10-30cm(4-12inches) in length. The tiny greenish flowers are produced in

dense inflorescences. The flowers feature a one-called ovary and nine stamens that are

grouped in three series. It’s interesting to note that different cultivars produce different sorts

of avocado blooms, A and B. The male and female sections of these flowers mature

separately, and each bloom only opens twice. Type A flowers bloom as functionally male in

the afternoon of the following day after closing as functionally female in the morning,

lunchtime, and evening. Type B flowers open the following morning as functionally female

after being functionally female in the afternoon, close that night and then reopen the

following morning as functionally male.

The fruit can range in size from being no bigger than an egg in some Mexican races to

occasionally weighing 1-2kg (2 -4 pounds) in others. The color ranges from green to dark

purple, and the shape might be spherical or pear shaped with a long, slender neck. The fruit is

a berry according to botany, and it has a single, enormously rounded seed with two

cotyledons. The fruit’s outer peel can vary in texture from being rough and woody to being

no thicker than an apple’s.

PHYTOCHEMICAL AND MINERAL CONSTITUENTS:

PHARMACOLOGICAL ACTIVITY:

Use of Avocado in traditional herbal medicine can be attributed to its pharmacological

activity.

1. Analgesic effect

It has been reported that administration of 1600mg/kg an aqueous leaf extract of

avocado was able to reduce the writhing in mice induced by administration of acetic

acid, by 57%. It could also increase the threshold of pain by 87.2% upon
 administration of 800mg/kg as noted by the hot plate test and inhibit both phases of

formalin induced pain in a dose-dependent manner. These results were similar to

those obtained after administration of drugs like acetylsalicylic acid and morphine for

the mouse writhing assay and hot plate test respectively. The results along with the

inhibition of formalin induced pain indicated the analgesic effect of the extract on

mice.

2. Anti-inflammatory activity

Anti-inflammatory activity of the extract was observed upon its use in mice with

carrageenan induced edema, as it resulted in reduction of swelling. Moreover, no

toxicity symptoms in mice were found even after administration of 10g/kg of extract.

3. Anti-Diabetic Activity

The anti-diabetic activity of hydroalcholic extract of Avocado leaves on

streptozotocin-induced diabetic rats has been reported. Upon administration of the

extract, reduction in blood glucose levels(hypoglycemia) has been observed. In

addition, treatment of rats with the persea americana resulted in an increase in the

phospho-PKB expression in the soleus muscle. The activation of this enzyme leads to

the translocation of the GLUT-2 molecule from the cytoplasm to the cell membrane in

the uptake of glucose.

4. Hypercholesterolemia

It has also been found that administration of aqueous leaf extracts to rats with

hypercholesterolemia caused a decrease in the levels of plasma glucose, total

cholesterol and LDL and a significant increase in the HDL levels.

5. Vasorelaxant properties

The vasorelaxant properties of aqueous leaf extract of avocado on isolated rat aorta

has been investigated. A significant vasorelaxation in aorta has been observed due the
 synthesis of endothelium derived relaxing factors (EDRF’S)and the release of

prostanoid. The treatment of the aorta with the extract also reduces vasoconstriction,

the probable reason for it being the inhibition of Ca 2+ influx through calcium

channels.

6. Anti-ulcer activity

Aqueous leaf extract of Avocado consisting of alkaloids, flavonoids, saponins and

tannins produced significant dose-dependent anti-ulcer activity when administered

orally to sick rats (rats pre-treated with ulcerogenic drugs-indomethacin and ethanol).

7. Anti-convulsant activity

Avocado was found to possess anticonvulsant activity as it was antagonized seizures

induced in mice by administration of the drugs, viz pentylenetetrazole (PTZ) and

picrotoxin (PCT). The anticonvulsant property of the extract can be attributed to its

ability to enhance the neurotransmission and or action of GABA in the brain.

8. Anti-platelet and Anti-thrombic activity.

Avocado has acetogenin compounds which are responsible for anti-platelet and anti-

thrombic activity. Platelet aggregation and thrombus formation occur during ischemic

diseases.

9. Lowers LDL

Avocado oil also reduces the very low density lipids (VLDL) levels without affecting

the high density lipids(HDL) levels in sick mice.

10. Anti-diarrheal property

The Anti-diarrheal property of bioactive compounds present in avocado seeds has

been reported. Administration of chloroform-methanolic extracts of these seeds to

castor oil induced diarrheal rats caused significant reduction in wetness of feaces in a

dose dependent manner. Also, the LD50 values of the extracts were found to be lower
 than 5000mg/kg body weight of the rats. This attested the safety of the extracts with

only a remote chance of acute toxicity.

11. Osteoarthritis

A mixture of unsaponifiables of soyabean and avocado oils, which constitute a drug

known as piascledine, has been used to effectively treat osteoarthritis. The drug

enhanced the synthesis of collagen and proteoglycan and decreased the synthesis of

fibronectin. The drug also inhibited the release and activity of metalloproteinases and

pro-inflammatory cytokines which play a major role in the development of

osteoarthritis. Within two years of drug administration, reduction in pain and

analgesic drug demands in patients revealed delayed joint destruction.

12.Anti-fungal property

A compound (E,Z,Z)-1-Acetoxy-2-hydroxy-4-oxoheneicosa-5,12,15-triene has been

isolated from avocado fruit. This compound was found to exhibit anti-fungal property

against the fungal pathogen Colletotrichumgloeo sporioides by inhibiting spore

germination. Seeds extracts of avocado have also shown to be toxic towards other

fungal pathogens like

Candida sp, Cryptococcus neoformans. Another study has shown that a topical cream

consisting of mixture of Avocado oil, tea tree oil ,emu oil and jojoba oil inhibited the

growth of the canine skin pathogens- Staphylococcus pseudintermedius and

Malasseziapachy dermatis and could be used as an alternative for the commonly used

antibiotic and antifungal agents.

13. Wound healing properties

The wound healing properties of the Avocado fruit extract has been confirmed. The

topical or oral administration of the fruit extract in wounded rats resulted in the
 complete epiyhelialization of the wound. Other parameters like rate of wound

contraction and hydroxyproline content of tissues along healing with histological

observations also indicated the wound healing property of persea Americana .

14. Platelet Aggregation

Avocado pulp contains bioactive compounds which inhibit platelet aggregation and

prevents thrombus formation. Thus a diet supplemented with Avocado pulp can be

beneficial for patients suffering with ischemic diseases. The anti-platelet and anti-

thrombic activity of Avocado has been confirmed.

15.Type 1 Diabetes

Administration of Avocado oil attenuates the alteration in electron transfer in rat

kidney mitochondria caused by type 1 diabetes.

16. Antioxidants

Antioxidants in the Avocado oil reduce ROS in the damaged mitochondria. The

beneficial effects of Avocado oil as a dietary supplement, over control of metabolic

illnesses, based on the evidence that the oil positively affected hepatic markers in

sucrose fed rats (which had altered hepatic markers) has been reported. It also results

in liver regeneration.

TOXICITY:

Although persin has not been quantified in any of those investigations, it has been suggested

that its presence is what causes avocado’s harmful effects. In a recent study, the persin
content of various avocado fruit sections (such as the peel, pulp, and pit),both raw and

cooked, was measured in the laboratory of Dr. Godoy(PI) and Dr. David Sarlah, Assistant

Professor of Chemistry. It was discovered that the peel and pulp of avocados contain the

highest amounts of the acetogenin persin, which is more prevalent in raw avocados.