CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

Prostate cancer (PCa) is the most commonly diagnosed male malignancy and the

fourth leading cause of cancer-related male mortalities worldwide (Mattiuzzi et al.,

2019). Although PCa-related fatalities have been declining, incidence rates have

been on the rise with an increasing number of patients living with the disease

(Jemal et al., 2015). A significant number of PCa patients have used or are

interested in using natural products to supplement their therapy, a move that is

supported by a growing number of epidemiological, clinical, and pre-clinical

studies (Cicero et al., 2019), indicating lower toxicities, easier usage and greater

availability in many cases, compared to pharmaceuticals (Cragg et al., 2018).

Multiple pre-clinical studies have reported the anti-cancer properties of Neem

(Azadirachta indica) and Neem products in PCa cell lines and animal studies

(Singh et al., 2016). Key pathways known to drive PCa progression include the

androgen receptor (AR) and the phosphatidylinositol 3-kinase (PI3K)/protein

kinase B (PKB/Akt) pathway, both of which can be modulated by treatment with

Neem (Azadirachta indica) and its derivatives. Pathways which are linked to

chemoresistance (e.g., Bcl-2) are also targets of Neem (Azadirachta indica) and its

derivatives. Several clinical studies have tested the medicinal properties of Neem
1
(Azadirachta indica). For example, in a pilot study with 14 patients,

Bandyopadhyay et al. (2014) have demonstrated Neem (Azadirachta indica) bark

extract to have therapeutic potential in controlling gastric hypersecretion and

gastroduodenal ulcers. In this study, 30 mg of lyophilised aqueous extract of air-

dried Neem (Azadirachta indica) bark was encapsulated in the gelatine capsule that

was used for oral administration to the selected patients. Treatment with

lyophilized Neem (Azadirachta indica) bark extract orally for 10 days at 30 mg

twice daily resulted in a 77% decrease in gastric acid secretion in 9 patients, while

3 others showed a decrease of 10–13% while 2 exhibited no effect. In addition, the

bark extract at the dose of 30–60 mg twice daily for 10 weeks in 6 patients

displayed significant reduction of duodenal ulcers (Bandyopadhyay et al. 2014). A

larger study in 80 patients with type 2 diabetes mellitus described how the leaves

and twigs of Neem (Azadirachta indica) have the potential to significantly reduce

glycosylated hemoglobin (HbA1c) levels, insulin resistance (IR)/fasting blood

sugar (FBS) and systemic inflammation (Pingali et al., 2020). In this study,

subjects received capsules of 125, 250 or 500 mg of Neem (Azadirachta indica)

[aqueous extract of Azadirachta indica leaves (AIE) and twigs or placebo twice

daily for 12 weeks. At all doses, Neem (Azadirachta indica) not only significantly

reduced postprandial blood sugar (PPBS) level, HbA1c, FBS and IR but also

improved endothelial function, reduced oxidative stress and systemic inflammation

2
when compared to the placebo. The bioactives in these capsules were found to be

flavonoids and myo-inositol monophosphate was found to be the predominant

bioactive. Neem (Azadirachta indica) leaves, seed oil and the bark have also been

used against malaria in India, Nigeria, and some other parts of Asia (Pingali et al.,

2020). The larvicidal properties of Neem (Azadirachta indica) seed oil (0.03%

azadirachtin) and Neem (Azadirachta indica) leaf slurry (over heated Neem

(Azadirachta indica) leaves dried and minced, extracted using water and

water/acetonitrile) was reported to be successful in controlling malaria (Abiy et al.,

2015). No clinical studies have formally been conducted to evaluate the effects of

Neem (Azadirachta indica) in cancer patients; however, pre-clinical studies

support the usage of Neem (Azadirachta indica) and its various components in the

treatment of cancer.

1.2 Problem Statement

Azadirachta indica (neem) and its constituents have therapeutic significance that

has been traditionally used worldwide especially in the subcontinent since ancient

times. The clinical studies provided that A. indica plays a central role in the

prevention of several diseases. A. indica natural extracts have been shown to work

as a supplement to control several alignments such as inflammation and diabetics.

The role of A.indica active constituents with chemopreventive effect has been

noticed in several tumors by modulating various signaling pathways. The exact

3
molecular mechanism of A. indica extracts in deactivating the activity of several

genes in cancer progression and development in this regard is not understood

completely and yet needs to be explored

1.3 Aim and Objectives of the Study

The study focused on evaluation of neem as a component in the production of

prostate cancer drugs. To achieve this aim, the following specific objectives are to

be carried out:

i. To evaluate the efficacy of Azadirachta Indica as a component in the

production of prostate cancer drugs

ii. To identify the active compound responsible for the inhibition of prostate

cancer growth in Azadirachta Indica using the simulation approach

iii. To investigate the phytochemical composition of the root extract of

Azadirachta Indica

iv. To identify the bioactive compounds responsible for its use in prostate

cancer treatment using GC-MS analysis

1.4 Justification of the Study

Lack of diversity in the modes of treatment for cancer coupled with the robust

economic input required for the treatment of cancer has prompted the shift in focus

from conventional medicine to traditional medicine. Surgery, chemotherapy, and

radiotherapy are still the major conventional cancer therapies. Serious drawbacks
4
however can be pegged to these strategies despite the obvious milestones gained in

the fight against cancer. Therefore, there is an urgent need for the development of

novel combination therapies and treatment regimens with higher efficacies

Medicinal plants have great potential to be used as sources for anti-cancer

therapeutics. Recently, there has been a shift in the world of research and drug

development to concentrate more on the potential of medicinal plants as sources of

anticancer agents. This shift has been driven by the successful potency of plant

derived molecules such as the vinca alkaloids, vinblastine, vincristine and

cytotoxic podophyllotoxins as anticancer agents. There is a need to bridge the gaps

in such knowledge with regards to the use of Azadirachta indica, in cancer

management. With the investigation of in vitro properties of the plant extracts

against cell lines for both cervical cancer and prostate cancer, a deeper

understanding can be attained of their bioactivity leading to their validation and

more informed usage.

5
 CHAPTER TWO

LITERATURE REVIEW

2.1 Neem (Azadirachta indica) plant

Neem (Azadirachta indica) plant is a native of India, where it is known as divine

tree; “life giving tree”. It belongs to maliceae family. Away from India, it is

commonly found in Africa and America (Kumar et al., 2015). It occurs naturally in

tropical region and sub-tropical zones. However, it can still be planted or

cultivated. Neem (Azadirachta indica) tree is an incredible therapeutic plant that

has been declared the tree of the 21st century by the United Nations (Dhama et al.,

2013). The plant kingdom represents a rich store house of organic compounds,

many of which have been used for medicinal purposes and could serve as a lead for

the development of novel agents having good efficacy in various pathological

disorders in the coming years. Neem (Azadirachta indica) plant is considered to be

the richest sources of drugs for traditional medicine, modern medicine,

nutraceuticals, food supplements, folk medicine, pharmaceutical intermediates and

chemical entities for synthetic drugs (Mahima et al., 2018). Some of the

phytochemicals contained in Neem (Azadirachta indica) plant have been isolated,

quantified and identified through Intensive studies. These bioactive chemicals have

provided leads in the development of several lifesaving drugs, which are in use

today (Padal et al., 2018). Extract from Azadiracta indica, which is referred to as
6
dogonyaro in some parts of Nigeria are mostly recommended in ancient medical

texts. The leaves can be used as drug for diabetes, eczema and fever. Thus, the

objective of this research was to ascertain the phytochemical constituents of Neem

(Azadirachta indica) plant and relate it to some of its traditional use.

Fig 2.1: Neem (Azadirachta indica) plant (Padal et al., 2018)

2.2 Botanical description

It is a tree 40-50 feet or higher, with a straight trunk and long spreading branches

forming a broad round crown; it has rough dark brown bark with wide longitudinal

7
fissures separated by flat ridges. The leaves are compound, imparipinnate, each

comprising 5-15 leaflets. The compound leaves are themselves alternating with one

another. It bears many flowered panicles, mostly in the leaf axils. The selel are

ovate and about one cm long with sweet scented white oblanciolate petals. It

produces yellow drupes that are ellipsoid and glabrous, 12-20 mm long. Fruits are

green, turning yellow on ripening, aromatic with garlic like odour. Fresh leaves

and flowers come in March-April. Fruits mature between April and August

depending upon locality (Ross, 2021).

2.3 Phytochemistry

Biologically active principles isolated from different parts of the plant include:

Azadirachtin, meliacin, gedunin, nimbidin, nimbolides, salanin, nimbin, valassin,

meliacin forms the bitter principles of Neem (Azadirachta indica) oil, the seed also

contain tignic acid responsible for the distinctive odour of the oil (Sharma et al.,

2021). Neem (Azadirachta indica) kernels contain 30-50 % of oil mainly used by

the soap, pesticide and pharmaceutical industries and contain many active

ingredients which are together called triterpene or limnoids (Djenontin Tindo et al.,

2022). The four best limnoids compounds are: Azadirachtin Salannin, Meliantriol,

and Nimbin. Limonoids contain insecticidal and pesticidal activity (Mondal et al.,

2022).

8
2.4 Pharmacological actions

Abortifacient, analgesic, anthelminthic, antibacterial, antiyeast, antiulcer,

antifertility, antifilarial, antifungal, antihyperglycemic, anti-inflammatory,

antiviral, antimalarial, diuretic, antinematodal, antipyretic, antispasmodic,

insecticidal, antispermatogenic, antitumor, hypercholesteremic, hypoglycaemic,

immunomodulator locality (Ross, 2021).

2.5 Medicinal use

All parts of the tree have been used medicinally for centuries. It has been used in

Ayurvedic medicine for more than 4000 years due to its medicinal properties. The

earliest Sanskrit medical writings refer to the benefits of Neem (Azadirachta

indica)’s fruits, seeds, oil, leaves, roots and bark. Each has been used in the Indian

Ayurvedic and Unani medicine, and is now being used in pharmaceutical and

cosmetics industries (Brototi et al., 2021)

2.6 Application of Neem (Azadirachta indica)

Neem (Azadirachta indica) oil is extracted from the seeds of the Neem

(Azadirachta indica) tree and has insecticidal and medicinal properties due to

which it has been used in pest control in rice cultivation.

2.6.1 Neem (Azadirachta indica) used as Fertilizer

The material left after oil is squeezed out from seeds and is popularly known as the

seed cake; it acts as a bio fertilizer and helps in providing the required nutrients to
9
plants. It is widely used to ensure a high yield of crops. Neem (Azadirachta indica)

is used as a fertilizer both for food crops and cash crops, particularly rice and

sugarcane crop. Benefits: Neem (Azadirachta indica) seed cake performs the dual

function of both fertilizer and pesticide, acts as a soil enricher, reduces the growth

of soil pest and bacteria, provides macro nutrients essential for all plant growth,

and helps to increase the yield of plants in the long run, bio degradable and Eco

friendly and excellent soil conditioner (Vethanayagam et al., 2019).

2.6.2 Neem (Azadirachta indica) used as Manure

Manure is any animal or plant material used to fertilize land especially animal

excreta for improving the soil fertility and thus promoting plant growth. Neem

(Azadirachta indica) manure is gaining popularity because it is environmental

friendly and also the compounds found in it help to increase the nitrogen and

phosphorous content in the soil. It is rich in sulphur, potassium, calcium, nitrogen,

etc. Neem (Azadirachta indica) cake is used to manufacture high quality organic or

natural manure, which does not have any aftermaths on plants, soil and other living

organisms. It can be obtained by using high technology extraction methods like

cold pressing or other solvent extraction. It can be used directly by mixing with the

soil or it can be blended with urea and other organic manure like farm yard manure

and sea weed for best results (Vethanayagam et al., 2019).

10
2.6.3 Neem (Azadirachta indica) as urea coating agent

Neem (Azadirachta indica) and its parts are being used to manufacture urea

coating agent to improve and maintain the fertility of soil. The fertility of the soil

can be measured by the amount of Nitrogen, Potassium and Phosphorous it has;

there are certain bacteria found in soil, which denitrify it. Use of Neem

(Azadirachta indica) urea coating agent helps to retard the activity and growth of

the bacteria responsible for denitrification. It prevents the loss of urea in the soil. It

can also be used to control a large number of pests such as caterpillars, beetles,

leafhoppers, borer, mites etc. Urea coating is generally available either in liquid

form or powdered form. Properties of Neem (Azadirachta indica) Urea Coating are

Antifeedant, anti fertility and pest growth regulator (Vethanayagam et al., 2019).

2.6.4 Neem (Azadirachta indica) as Soil Conditioner

Neem (Azadirachta indica) seed granules or powdered seeds are used to

manufacture the soil conditioner. It can be applied during sowing of plants or can

be sprinkled and raked into the soil (Leos et al., 2022). The process of sprinkling

should be followed by proper irrigation so that the product reaches the roots. It is a

natural soil conditioner that helps improve the quality of soil, thereby enhancing

the growth of plants and fruits. Organic soil conditioner is gaining popularity in

agricultural industry, not only in Asian countries like India but also in western

counterparts such as USA, UK and Australia (Leos et al., 2022).

11
2.6.5 Neem (Azadirachta indica) as fumigant

Neem (Azadirachta indica) tree has been used against household, storage pests and

crop pests. Neem (Azadirachta indica) pest fumigant is available in gaseous state

and is used as a pesticide and disinfectant. It is being used by a large number of

countries on a commercial basis by farmers and agriculturists. This 100% natural

product is being exported as it is non toxic and does not affect the environment. It

assumes more importance in developing countries where millions of deaths are

reported every year due to the accidental intake of synthetic pest fumigants (Leos

et al., 2022).

2.6.6 Neem (Azadirachta indica) as pesticide

Neem (Azadirachta indica) pesticides play a vital role in pest management and

hence have been widely used in agriculture. There has been an evident shift all

over the world from synthetic pesticides to non synthetic ones; this is largely

because of the wide spread awareness of the side effects of these synthetic

pesticides not only on plants and soil but also on other living organisms. This is a

great opportunity for Neem (Azadirachta indica) pesticides manufacturers to cash

in on the growing popularity of natural or herbal pesticides. Neem (Azadirachta

indica) pesticides are being manufactured and exported to various countries as a lot

of research has been conducted to test the safety and efficacy of Neem

(Azadirachta indica) for use as a pesticide (Anis Joseph et al., 2020).

12
2.7 Prostate cancer

Prostate cancer is cancer that occurs in the prostate. The prostate is a small walnut-

shaped gland in males that produces the seminal fluid that nourishes and transports

sperm. Prostate cancer is one of the most common types of cancer. Many prostate

cancers grow slowly and are confined to the prostate gland, where they may not

cause serious harm. However, while some types of prostate cancer grow slowly

and may need minimal or even no treatment, other types are aggressive and can

spread quickly. Prostate cancer that's detected early — when it's still confined to

the prostate gland — has the best chance for successful treatment.

2.7.1 Symptoms

Prostate cancer may cause no signs or symptoms in its early stages.

Prostate cancer that's more advanced may cause signs and symptoms such as:

 Trouble urinating

 Decreased force in the stream of urine

 Blood in the urine

 Blood in the semen

 Bone pain

 Losing weight without trying

 Erectile dysfunction

13
2.7.2 Risk factors

Factors that can increase your risk of prostate cancer include:

 Older age. Your risk of prostate cancer increases as you age. It's most

common after age 50.

 Race. For reasons not yet determined, Black people have a greater risk of

prostate cancer than do people of other races. In Black people, prostate

cancer is also more likely to be aggressive or advanced.

 Family history. If a blood relative, such as a parent, sibling or child, has

been diagnosed with prostate cancer, your risk may be increased. Also, if you

have a family history of genes that increase the risk of breast cancer (BRCA1

or BRCA2) or a very strong family history of breast cancer, your risk of

prostate cancer may be higher.

 Obesity. People who are obese may have a higher risk of prostate cancer

compared with people considered to have a healthy weight, though studies

have had mixed results. In obese people, the cancer is more likely to be more

aggressive and more likely to return after initial treatment.

2.7.3 Complications

Complications of prostate cancer and its treatments include:

 Cancer that spreads (metastasizes). Prostate cancer can spread to nearby

organs, such as your bladder, or travel through your bloodstream or

14
 lymphatic system to your bones or other organs. Prostate cancer that spreads

to the bones can cause pain and broken bones. Once prostate cancer has

spread to other areas of the body, it may still respond to treatment and may

be controlled, but it's unlikely to be cured.

 Incontinence. Both prostate cancer and its treatment can cause urinary

incontinence. Treatment for incontinence depends on the type you have, how

severe it is and the likelihood it will improve over time. Treatment options

may include medications, catheters and surgery.

 Erectile dysfunction. Erectile dysfunction can result from prostate cancer or

its treatment, including surgery, radiation or hormone treatments.

Medications, vacuum devices that assist in achieving erection and surgery are

available to treat erectile dysfunction.

2.7.4 Prevention

 Choose a healthy diet full of fruits and vegetables. Eat a variety of fruits,

vegetables and whole grains. Fruits and vegetables contain many vitamins

and nutrients that can contribute to your health.

Whether you can prevent prostate cancer through diet has yet to be

conclusively proved. But eating a healthy diet with a variety of fruits and

vegetables can improve your overall health.

15
 Choose healthy foods over supplements. No studies have shown that

supplements play a role in reducing your risk of prostate cancer. Instead,

choose foods that are rich in vitamins and minerals so that you can maintain

healthy levels of vitamins in your body.

 Exercise most days of the week. Exercise improves your overall health,

helps you maintain your weight and improves your mood. Try to exercise

most days of the week. If you're new to exercise, start slow and work your

way up to more exercise time each day.

 Maintain a healthy weight. If you’re current weight is healthy, work to

maintain it by choosing a healthy diet and exercising most days of the week.

If you need to lose weight, add more exercise and reduce the number of

calories you eat each day. Ask your doctor for help creating a plan for

healthy weight loss.

 Talk to your doctor about increased risk of prostate cancer. If you have

a very high risk of prostate cancer, you and your doctor may consider

medications or other treatments to reduce the risk. Some studies suggest that

taking 5-alpha reductase inhibitors, including finasteride (Propecia, Proscar)

and dutasteride (Avodart), may reduce the overall risk of developing prostate

16
 cancer. These drugs are used to control prostate gland enlargement and hair

loss.

However, some evidence indicates that people taking these medications may

have an increased risk of getting a more serious form of prostate cancer

(high-grade prostate cancer). If you're concerned about your risk of

developing prostate cancer, talk with your doctor.

2.7.5 Mechanism of Action of Active Compounds

Neem (Azadirachta indica), a member of the Meliaceae family, has therapeutics

implication in the diseases prevention and treatment. But the exact molecular

mechanism in the prevention of pathogenesis is not understood entirely. It is

considered that Azadirachta indica shows therapeutic role due to the rich source of

antioxidant and other valuable active compounds such as azadirachtin, nimbolinin,

nimbin, nimbidin, nimbidol, salannin, and quercetin (Sarmiento et al., 2021).

Possible mechanism of action of Azadirachta indica is presented as follows.

Neem (Azadirachta indica) plants parts shows antimicrobial role through

inhibitory effect on microbial growth/potentiality of cell wall breakdown.

Azadirachtin, a complex tetranortriterpenoid limonoid present in seeds, is the key

constituent responsible for both antifeedant and toxic effects in insects (Hossain et

al., 2021). Results suggest that the ethanol extract of Neem (Azadirachta indica)

leaves showed in vitro antibacterial activity against both Staphylococcus

17
aureus and MRSA with greatest zones of inhibition noted at 100% concentration

(Sarmiento et al., 2021).

i. Neem (Azadirachta indica) plays role as free radical scavenging properties

due to rich source of antioxidant. Azadirachtin and nimbolide showed

concentration-dependent antiradical scavenging activity and reductive

potential in the following order: nimbolide > azadirachtin > ascorbate

(Hossain et al., 2021).

ii. Neem (Azadirachta indica) ingredient shows effective role in the

management of cancer through the regulation of cell signaling pathways.

Neem (Azadirachta indica) modulates the activity of various tumour

suppressor genes (e.g., p53, pTEN), angiogenesis (VEGF), transcription

factors (e.g., NF-κB), and apoptosis (e.g., bcl2, bax).

iii. Neem (Azadirachta indica) also plays role as anti-inflammatory via

regulation of proinflammatory enzyme activities including cyclooxygenase

(COX), and lipoxygenase (LOX) enzyme (Hossain et al., 2021).

2.8 Neem (Azadirachta indica) tree extracts and phytochemicals: an overview

of general properties and clinical uses

The Neem (Azadirachta indica) tree is native to the Indian subcontinent and

Burma. For hundreds of years, its various components and derivatives have been

widely used in alternate medicine approaches, including Ayurveda, throughout

18
South Asia and beyond (Rupani et al., 2018). The Neem (Azadirachta indica)

leaves, bark, seeds, flowers, and twigs have been reported to have anti-microbial,

antiinflammatory, anti-arthritic, hepatoprotective, anti-diabetic, anti-ulcer, and

anti-cancer activities (Rupani et al., 2018). Several clinical studies have tested the

medicinal properties of Neem (Azadirachta indica). For example, in a pilot study

with 14 patients, Bandyopadhyay et al. (2014) have demonstrated Neem

(Azadirachta indica) bark extract to have therapeutic potential in controlling

gastric hypersecretion and gastroduodenal ulcers. In this study, 30 mg of

lyophilised aqueous extract of air-dried Neem (Azadirachta indica) bark was

encapsulated in the gelatine capsule that was used for oral administration to the

selected patients. Treatment with lyophilized Neem (Azadirachta indica) bark

extract orally for 10 days at 30 mg twice daily resulted in a 77% decrease in gastric

acid secretion in 9 patients, while 3 others showed a decrease of 10–13% while 2

exhibited no effect. In addition, the bark extract at the dose of 30–60 mg twice

daily for 10 weeks in 6 patients displayed significant reduction of duodenal ulcers

(Bandyopadhyay et al. 2014). A larger study in 80 patients with type 2 diabetes

mellitus described how the leaves and twigs of Neem (Azadirachta indica) have

the potential to significantly reduce glycosylated hemoglobin (HbA1c) levels,

insulin resistance (IR)/fasting blood sugar (FBS) and systemic inflammation

(Pingali et al., 2020). In this study, subjects received capsules of 125, 250 or 500
19
mg of Neem (Azadirachta indica), aqueous extract of Azadirachta indica leaves

(AIE) and twigs] or placebo twice daily for 12 weeks. At all doses, Neem

(Azadirachta indica) not only significantly reduced postprandial blood sugar

(PPBS) level, HbA1c, FBS and IR but also improved endothelial function, reduced

oxidative stress and systemic inflammation when compared to the placebo. The

bioactives in these capsules were found to be flavonoids and myo-inositol

monophosphate was found to be the predominant bioactive. Neem (Azadirachta

indica) leaves, seed oil and the bark have also been used against malaria in India,

Nigeria, and some other parts of Asia (Pingali et al., 2020). The larvicidal

properties of Neem (Azadirachta indica) seed oil (0.03% azadirachtin) and Neem

(Azadirachta indica) leaf slurry (over heated Neem (Azadirachta indica) leaves

dried and minced, extracted using water and water/acetonitrile) was reported to be

successful in controlling malaria (Abiy et al., 2015). The Neem (Azadirachta

indica) seed oil/leaf slurry has been shown to induce sterility in insects by

interrupting sperm production in males and hormone control of oogenesis thereby

exerting a cytotoxic effect on both follicular cells and oocytes of the malaria

vector.

2.9 Neem (Azadirachta indica) Components and Their Effects on Cancer

Carcinogenesis represents a complex multifactorial process, characterized by

multiple steps from precancerous lesions to neoplasia, which modifies a normal

20
cell into a cancer cell with increased invasive and metastatic potential. Cancer cells

are characterized by proliferation that cannot be controlled, angiogenesis,

resistance to apoptosis, and a marked suppression of the immune reaction against

tumor cells (Hanahan et al., 2021). Tumor cells have a special ability to modulate

their environment, which facilitates cell invasion, increases inflammation, and

induces angiogenesis (Spano et al., 2022).

In order to rationalize the complexity of cancer, Hanahan et al. 2021 described the

biological capabilities, or hallmarks, of a neoplasia. These hallmarks of cancer

include resisting apoptosis, sustaining proliferative signaling, avoiding growth

suppressor molecules, allowing uncontrolled replicative immortality, inducing

vascular formation, activating invasion, and the metastasis process. All these

biological capabilities of neoplasms are sustained by instable genomes. Tumors are

insular masses constituted by apparently normal cells that possess the capacity of

creating a tumor microenvironment, characterized by all these hallmarks.

The important role of Neem (Azadirachta indica) components in cancer prevention

consists of the capacity to modulate the tumor environment through several

actions, including decreasing angiogenesis and increasing the toxicity of the cell.

A study by Mahapatra et al. 2022 assessed the antiangiogenic actions of the

ethanol extract of Neem (Azadirachta indica) leaves in endothelial cells originating

from the human umbilical vein. Their results suggest that the Neem (Azadirachta
21
indica) leaf extract is capable of regulating the genes involved in cellular

development and functions and decreasing the stimulatory effect of vascular

endothelial growth factor (VEGF), exerting strong antiangiogenic effects (Ricci et

al., 2018).

Neem (Azadirachta indica) extracts can be prepared in several ways, with a series

of various solvents consisting in diluted alcohol, ether, petrol ether, and ethyl

acetate. The majority of the studies based on the efficiency of Neem (Azadirachta

indica) extracts on cancer used different mixtures of Neem (Azadirachta indica)

compounds, and for this reason, the efficiency of individual components is still

insufficiently known (Ricci et al., 2018).

22
Fig 2.2: Neem (Azadirachta indica) Components and Their Effects on Cancer

(Ricci et al., 2018)

Gedunin is a tetranortriterpenoid isolated from Neem (Azadirachta indica)

seed oil with a D lactone ring. In Indian medicine, the active product gedunin has

been administered for infectious diseases such as malaria, but recent studies have

shown the potential anticancer efficiency of this product against tumor cells in the

ovaries, colon, and prostate through regulation of important signaling pathways

(Uddin et al., 2017).

Gedunin also demonstrated its anticancer activity as a preventive and

therapeutic agent in breast cancer. This bioactive compound exerts its function

23
through inhibition of tumoral cells, modulating several heat shock proteins (Uddin

et al., 2017).

Meliatetraolenone, also known as 24,25,26,27-tetranor-apotirucalla-

(apoeupha)-6alpha-O-methyl,7alpha-senecioyl(7-deacetyl)-11alpha,12alpha,21,23-

tetrahydroxy-21,23-epoxy-2,14,20(22)-trien-1,16-dione, is a new

tetranortriterpenoid compound that has been isolated from the fresh leaves of

Neem (Azadirachta indica), but its anticancer activity needs further investigation.

For the moment, only the insecticidal activity of this Neem (Azadirachta indica)

component has been reported (Uddin et al., 2017).

Presently, 35 limonoids isolated from Neem (Azadirachta indica) seed extracts

have proved their cytotoxic activity. The Neem (Azadirachta indica) limonoids

also include:

 Azadiradione type 1–15

 Gedunin type 16–20

 Azadirachtin type 21–24

 Nimbin type 25–33

 Degraded limonoids 34, 35.

Quercetin is a flavonoid from vegetables and fruits that can also be isolated from a

50% ethanolic extract of Neem (Azadirachta indica) leave (Uddin et al., 2017).

The structure of quercetin is represented by a basic diphenylpropane C6-C3-C6

24
skeleton. Even if this flavonoid is consumed daily by people through alimentation,

it is now available in dietary supplements known as functional food. After one

month of dietary supplementation, the quercetin level in plasma could be increased

with 1 g/day and the plasmatic concentration could reach 1.5 µM of quercetin

(Uddin et al., 2017).

25
 CHAPTER THREE

3.0 MATERIALS AND METHODS

The following materials and reagents were used for each of the steps taken in the

course of this work as follows:

3.1 Reagents

 N-Hexane (C6H14)

 Ethanol (C2H5OH)

 Distilled water

 Ferric chloride (feCl3)

 Magnesium dust

 Sulphuric Acid(H2So4)

 Petroleum ether (CHCOCH3)

 Chloroform (CHCL3)

 Methanol (CH3OH)

 Ethyl acetate (C4H8O2)

 Acetic acid (CH3COOH)

 Formaldehyde (CH20)

 Potassium iodine

 Iodine
26
3.1.2 Apparatus

 Beaker

 Conical flask

 Test tube

 Storage container

 Whitman filter paper

 Glass stirrer

 Separating funnel

 Electrical heating mantle

 Electric grinder

 Electric oven

 Electric weighing balance

 Bath column

 Measuring cylinder

 Gas Chromatography Mass Spectrophotometer.

27
3.2. Methodology

3.2.1 Sample collection

Fresh plant leaves of Azadirachta Indica sample were cut down from its tree in

ihiagwa village community from immaculate compound close to Federal

University Owerri L.G.A of Imo state in the eastern part of Nigeria.

3.2.2. Sample Preparation

Azadirachta indica leaves were properly cut down from the tree and the leaves

were air dried at room temperature for 60 days. The dried sample was crushed into

powdery form using an electrical blender and then stored in an air tight container.

3.2.3 Extraction and Isolation

20g of sample (Azadirachta indica) powder was macerated in ethanol for 48hours

in 1liter of absolute ethanol, and left to stand for 48hours: after which the extract

was filtered out with the help of a cotton wool and a white filter cloth which is

referred toas cold extraction. The resulting ethanol extract was concentrated and

evaporated to dryness using a rotary evaporator at an optimum temperature of

between 40℃ to 45℃ to avoid denaturation of the active ingredients.

Hot extraction was performed with Soxhlet apparatus using n-Hexane as solvent.

The extract was re-extracted in chloroform to remove all the insoluble components

in ethanol which is dissolved in chloroform before they are placed into samples

28
which is sent for GC-MS analysis for their structural clarification and antimicrobial

analysis to determine their properties against positive and negative bacteria.

3.2.4 Phytochemical screening

3.2.4.1 Frothing Test for Saponins

This test is based on the ability of the saponin to produce froth in aqueous solution.

A small amount of the leaf sample was placed into a test tube and 50cm 3 of water

was added and extracted after 4hours.The water extract was shaken vigorously in a

conical flask. The production of a stable froth indicates the presence of saponins.

3.2.4.2 Test for Flavonoids (Shinoda’s test)

20cm3 of the water extract was measured into 25ocm 3 flask and few pieces of

magnesium chips was added to it, followed by the addition of drops of

concentrated hydrochloric acid (HCL). A precipitate was formed with a reddish

coloration which indicates the presence of flavonoids.

3.3.4.3. Test for Alkaloids

20cm3 of 5% acetic acid extract was measured into 25ocm 3 flask and the sample

was treated with the Wagner’s test (iodine crystals and potassium iodine) to give a

yellow coloration. Similartest was carried out with Meyers reagent to confirm the

presence of alkaloids.

29
3.3.4.4 Test for tannins

20cm3 of the water extract was measured into a 20cm 3 beaker and a few drops of

iron (iii) chloride solution was added to it. Theblue-black coloration formed is

evidence of the presence of tannins.

3.2.4.5 Test for steroids (Salkowski test)

Formaldehyde and concentrated Sulphuric acid (H 2SO4) was added to the water

extract which gives a reddish coloration. This indicates the presence id steroids.

3.2.4.6. Test for cardiac glycosides

A mixture of 20cm3 of Fehling solution A and B was added to the water extract

which appears blue in color, then it was heated and it turns to reddish brown

forming a precipitate. This indicates the presence of cardic glycosides.

3.2.5. GC-MS Experimental Procedure

GC-MS analysis was carried out with Gas Chromatography 5975 7890 with a fixed

GC column OV1011 coated with polymethyl silicon (o.25mmX50mm) and the

conditions are as follows: Temperature programming from 80 ℃-200 ℃ held at

80℃ for one minute, the rate is 5℃/min and 200℃ for twenty minutes. FID

temperature of 300℃, injection temperature of 250℃, carrier gas is Nitrogen at a

flow rate of 1cm3/ Mn and split ratio 1:75. GC-MS Gas Chromatography Mass

spectrum analysis was conducted using GC-MS QP 2010 with injector temperature

at 230℃ and carrier gas pressure on 100kpa. The column length was 30m with a
30
diameter of 0.25mm and flow rate of 50ml/min. the elements were automatically

passed into the Mass spectrometer with a detector voltage set at 1.5kv and

sampling weight 0.2 seconds. The Mass spectrometer was equipped with a

computer fed Mass Spectra data bank. Reagent and solvents such as Ethanol,

Chloroform, Diethyl ether, Hexane all of analytical grade were obtained.

3.2.6 Antimicrobial Analysis

The microorganism; staphylococcus aureus, streptococcus spp Pseudomonas

aeruginosa, candida albicans, E. coli, salmonella typhi, were used for the analysis.

They are clinical isolate of human pathogens obtained from the Federal medical

center Owerri and were brought to the laboratory and resuscitated in buffered

peptone broth (secharian chemie) and therefore into nutrient agar medium and

incubated at 37℃ for 24hrs.

Materials and methods

A. Micrological Neat: nutrient agar, Mueller-Tinton agar, MacConkey agar,

potato dextrose.

B. Reagents used are: hydrogen chloride, barium chloride, dihydrate

[Bacl2.2H20], Sulphuric acid, Kovac’s reagent, oxidase reagent, distilled

water.

C. Other include: petri-dishes, wire loop, test tubes, pipettes, autoclave, cotton

wool, conical flask, Bunsen burner, sterile scalpel, weighing balance.

31
Collection of Bacteria and Fungi Isolates

The isolates were obtained from clinical and environmental source using

streaking and pour plate method. Isolate obtained include staphylococcus

aureus, streptococcus sp, pseudomonas acraginosa, Serratia marcense and

penicillium sp.

 The organism wassubculture unto plates of nutrient agar, MacConkey

and potatoes dextrose agar to obtain pure culture of the organisms.

 Smear of the bacterial isolates were made unto clean grease-free slides,

air dried and heat fixed

 Gram staining was done for each of the bacterial isolate.

 Catalase test, coagulase test, indole test and citrate utilization test were

carried out to further identify the bacterial isolates using the standard

methods outlined by Chesbrough, 2004.

 Organisms were properly identified.

Gram Staining

 Neat fixed smears of each of the bacterial isolates were made unto clean

grease free slides.

 The smear was stained with crystal violet for 1 minute.

 They were washed in water.

32
  The smear was covered with lugoi’s iodine and allowed in minute.

 They were washed in water.

 The smears were decolorized with acetone until no more color appears to

ooze out.

 They were washed in water.

 They were counterstained with safranin for 1 minute.

 They were washed with water.

 The slides were blot dried with filter paper and allowed to dry.

 They were examined microscopically using x100 objectives of the

microscope.

Biochemical test for Identification of Bacterial Isolates

A. Catalase test

 Few colonies of the organisms were emulsified in drops of distilled

water on slides and placed in a petri-dishes.

 2 drops of H2O2[ hydrogen peroxide] was added and the dishes were

covered.

 The plates were observed for gas bubbles which indicate a positive

result.

33
B. Coagulase test

 A drop of saline was placed on a clean slide

 One or two colonies of the organism was emulsified unto the drop of

saline.

 A straight wire loop dipped

 Clumping of the mixture was observed for as it indicates a positive

result.

C. Indole test

 The organism was grown overnight in peptone water

 A few drops of Kovac’s reagent were added to the overnight peptone

water culture.

 A red ring was observed for above the peptone water as this indicates

a positive result.

D. Citrate utilization test

 A light suspension of the organism was made in saline.

 It was stab inoculated into simmon’s citrate agar.

 A growth of blue color in Simmons’s agar indicates a positive result.

E. Oxidase test

 A loopful of oxidase reagent wad added to a filter paper in a petri-dish

34
  With the aid of a platinum loop, a colony of the organism was smeared

unto the wetted filter paper.

 A purple color was observed for as it indicates a positive result.

Fungai Identification Using Potassium Hydroxide Preparation

 A drop of potassium hydroxide was placed on a slide

 The fungi isolated was placed unto the drop

 It was teased using two dissecting needles.

 A coverslip was placed over it.

 It was allowed to stand at room temperature.

 It was then observed microscopically.

Evaluation of Antimicrobial Activity

A. Dilution of the Organism

 Serial dilution of the organism was made to get a concentration the

corresponds to 0.5 MacFarland’s turbidity standard.

 0.5 MacFarland’s turbidity standard was prepared by missing 0.05ml of

1.175% barium chloride dihydrate [ Bacl2].2H2O] with 9.95ml of 1%

Sulphuric acid [H2So4].

 0.9ml of sterile nutrient broth was added to four clean test tube set up on a

rack.

35
 0.1ml of the broth culture of one of the organisms was added to the first tube

containing 0.9ml of nutrient broth.

 It was well mixed and 0.1ml of the mixture was pipettes and transferred to

the second tube.

 Content of the tube 2 was well mixed and 0.1ml of the mixture was pipetted

out and transferred to tube 3

 Content of tube 4 was mixed well, after which 0.1ml of the mixture was

pipetted out and discarded.

 The diluted bacterial suspension was compared usually against the 0.5

MacFarland’s turbidity standard by placing against a while background

 This procedure was repeated for each of the bacterial isolates.

A. Dilution of Extract

 Varying concentration of each of the extract was obtained using doubling

dilution.

 The dilution was achieve using four test tube rack. The tube was labelled as

follows: tube 1[Neat], tube 2 [1/10], tube 3 [1/20], tube 4 [1/40]

 1ml of extract A was added to the first tube

 1ml of ethanol was added to each tube from test tube 2-test tube 4.

36
  Another 1ml of extract A was added to test tube 2. A homogenate of the

mixture was achieved by gentle and careful shaking of the tube.

 1ml of the homogenate was taken using a sterile pipette and transferred to

tube 3.

 The content of tube 3 was also mixed properly and then 1ml of the

homogenate was aspirated using a sterile pipette and transferred to tube 4

The content of tube 4 was carefully mixed and the 1ml of the homogenate as

aspirated and discarded.

Evaluation of Antimicrobial Activity Using Well in Agar, Diffusion Method

 Standard concentration of the test bacteria [s. aureus, streptococcus sp, p.

aeruginosa and Serratia marcense and fungi [Penicillium sp] were streaked

on the surface of the freshly prepared Mueller-Tinton Agar plates and

potatoes dextrose agar plates with a sterile wire loop.

 These were allowed for 30mins to diffuse and a no 4 cork borer was used to

bore hole of 40mm diameter on each of the agar plates containing the five

isolates.

 A volume of 0.1ml [ 10µL] of each three extracts was used to fill the agar

wells made in the Mueller-Tinton plates were allowed to stand for 1 hour to

allow the extract diffusion into the agar and were incubated at 37% for

37
 24hours while the potato dextrose plates were incubated at room temperature

for 5days.After incubation the zones of inhibition around the extract was

measured using a ruler. zones ≥8mm were regarded sensitive while zone ≤

8mm were regarded as resistant.

3.2.7 Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration of the extract was determined by

incorporating constant volume 0.2cm3 of each dilutes of the extract into the

perforated disc on a seed nutrient agar plate as described in the anti-microbial

susceptibility test section. 0.1g of each extract was dissolved in 1cm3 of DMSO to

obtain 100mg/cm3. This concentration of DMSO was then doubled to obtain

50mg/cm3 then doubled again to obtain 12.5mg/cm3 and again to obtain

6.25mg/cm3. Each concentration was then used in the method earlier described to

obtain zone inhibition. The least concentration that showed inhibitory zones was

taken as the Mic.

38
 CHAPTER FOUR

RESULTS AND DISCUSSION

4.1. RESULTS

Table 4.1: Phytochemical analysis results of Azadirachta Indica leaf:

PHYTOCHEMICALS CONSTITUENT TEST INFERENCE

Tannins FeCl3 +

Alkaloids Mayer's test +

Wagner's test

Saponin Foam test +

Cardiac glycoside Fehling's solution +

Flavonoids Shinoda's test +

Steroids Salkowski's test +

Key: + presence of secondary metabolites

- Absence of secondary metabolites

39
The phytochemical analysis of Azadirachta Indica ethanol extract were found to

show the presence alkaloids, saponin, cardiac glycoside, flavonoids, steroids and

tannins. (Tables 4.1)

4.2. GC-MS Analysis Result

Fig. 4.1: GC-MS Spectra of Plant Extract.

4.2.1 GC-MS RESULTS DISCUSSION

The GC-MS spectra of AZADIRACHTA INDICA are shown in Fig. 2 which

shows Ten (10) absorption peaks. These peaks revealed twenty-three

phytochemical constituents and were interpreted with mass spectra.

40
Peak 1 appeared at molecular weight 236 with molecular formula C 15H24O2 and is

identified as -Limonenen-6-ol, pivalate.

Peak 2 appeared at molecular weight 228 with molecular formula C 15H230 and is

identified as -1 dodecanol, 3, 7,11-trimethyl-.

Peak 3 appeared at molecular weight 228 with molecular formula C 15H230 and is

identified as -1 dodecanol,3,7,11-trimethyl-.

Peak 4 appeared at molecular weight 236 with molecular formula C 15H24O2 and is

identified as -Limonenen-6-ol, pivalate.

Peak 5 appeared at molecular weight 204 with molecular formula C 15H24 and is

identified as β-longipinene.

Peak 6 appeared at molecular weight 204 with molecular formula C 15H24 and is

identified as -γ-Elemene.

Peak 7 appeared at molecular weight 206 with molecular formula C 15H24 and is

identified as 7-1,1,4a-Trimethyl-5,6-dimethylenedecahydronaphthalene.

Peak 8 appeared at molecular weight 206 with molecular formula C 14H24O and is

identified as -2,4-Di-tert-butylphenol

Peak 9 appeared at molecular weight 284 with molecular formula C 14H21Bro and is

identified as -2,4-Di-tert-butylPhenol.

Peak 10 appeared at molecular weight 592 with molecular formula C 16H48O8Si8 and

is identified as -Cyclooclasiloxane, hexadecamethyl-

41
 Table 4.2: GC-MS identified in the Ethanoic Extract of Azadirachta Indica

Leaf

Chromatographic Chemical name Molecular Molecular Structure

Formula weight

1. Limonenen-6-ol, pivalate C15H24O2 236

2. -1dodecanol,3,7,11 trimethyl-. C15H230 228

3. -1dodecanol,3,7,11-trimethyl-. C15H230 228

4. -Limonenen-6-ol, pivalate. C15H24O2 236

5. β-longipinene. C15H24 204

42
6. -γ-Elemene. C15H24 204

7. 7-1,1,4a-Trimethyl-5,6- C15H24 206

dimethylenedecahydronaphthalene

8. -2,4-Di-tert- C14H24O 206

butylphenol

9. -2,4-Di-tert- C14H21Bro 284

butylPhenol

10. - C16H48O8Si8 592

Cyclooclasiloxane,

hexadecamethyl-

43
4.3. ANTIMICROBIAL RESULT

Table 4.3: Antimicrobial table result of Occimum Gratissimum Leaf

Microorganism Diameter of Mic mg/g

inhibition mm

Staphylococcus aureus 8 6.25

Escherichia. Coli 10 3.125

P. aeruginosa 14 12.5

Klebsiella 13 3.125

Candida albicans 18 6.25

 STAPHYLOCOCCUS AUREUS:

The extract inhibited the growth of Staphylococcus aureus by 20mm with MIC

50mg/g. Staphylococcus aureus is the most dangerous of all of the many common

staphylococcal bacteria. These gram-positive, sphere-shaped (coccal) bacteria

often cause skin infections but can cause pneumonia, heart valve infections, and

bone infections. It has been found to be normal flora of upper respiratory tract and

vagina. S. aureus has been known to produce heat stable toxins which are

44
implicated in illnesses and stomach upset. From literatures, S. aureus had shown to

be very resistant to a wide variety of antibiotics.

 STREPTOCOCCUS SPP:

The extract also inhibited the growth Streptococcus spp by 15mm with MIC

50mg/g. Streptococcus spp, is a group of bacteria responsible for streptococcal

infections caused by any one of several species of Streptococcus. These gram-

positive, sphere-shaped (coccal) bacteria cause many disorders, including strep

throat, pneumonia, and wound, skin, heart valve, and bloodstream infections.

 ESCHERICHIA COLI:

The extract also inhibited the growth of Escherichia Coli by 18mm with MIC

25mg/g. The gram-negative bacterium, Escherichia Coli is the most numerous

aerobic commensal inhabitants of the large intestine. Certain of their strains cause

diarrhea and all can infection when they invade sterile (e.g., the urinary tract).

Diagnosis is by standard culture techniques. Toxin assays may help identify the

cause of diarrhea. Treatment with antibiotics is guided by susceptibility testing.

 SALMONELLA TYPHI:

The extract showed activity against Salmonella typhi by 16mm with MIC 50mg/g.

Salmonella typhi is a gram-negative, rod-shaped, flagellated bacterium that is

45
responsible for typhoid fever and has been a burden on developing nations for

generations. Salmonella typhi is usually contracted by ingestion of food or water

that is contaminated with the excrements of those that carry the organism and must

survive the gastric pH barrier in the stomach prior to adherence in the small

intestine.

 KLEBSIELLA:

The extract showed activity against Klebsiella by 18mm with MIC 50mg/g.

Klebsiella is a genus of gram-negative oxidase-negative, rod-shaped bacteria with

a prominent polysaccharide-based capsule that is responsible for urinary tract

infection, pneumonia, bloodstream infection, and meningitis.

CANDIDA ALBICANS:

The extract also inhibited the growth of Candida Albicans by mm with mg/g.

Candida Albicans is an opportunistic pathogenic yeast that is a common member

of the human gut flora. It can also survive outside the human body.

CHAPTER FIVE

5.1 Conclusion

In conclusion, Azadirachta Indica, commonly known as neem, possesses

46
remarkable phytochemical diversity and valuable medicinal properties. Through

various scientific studies, it has been established that these leaves are rich in

compounds such as Nimbolide, Quercetin, Salannin, Nimbidin, Azadirachtin and

many others, which contribute to their anti-inflammatory, antipyretic, anti-gastric

and antimicrobial effects.

This plant (Neem) has demonstrated potential in combating various health

conditions, including cancer, diabetes, ulcer, and hypertension, as well as boosting

immune system support. The neem plant (Azadirachta Indica) can inhibit the

growth of cancer cells, as well as induce cell death and prevent the spread of

cancer.

There are a number of studies that have shown the anti-cancer potential of the

neem plant, However, further research is required to fully understand its safety,

efficacy and harness the therapeutic potential of these leaves. The findings suggest

that Azadirachta Indica leaves hold promise as a natural source of health benefits

and may be valuable addition to traditional medicine and pharmaceutical research.

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