15

Chapter II

LITERATURE REVIEW

Related literature and studies are indicated in this chapter subsequent to

the conduct of research study done by the researcher. Associated concepts and

definition of terms will also be presented to provide better understanding of the

study. This chapter presents essential literature that is linked to the present study

entitled: “Phytochemical Analysis and Anti-angiogenic Activities of Patola (Luffa

acutangula (L.) Roxb.) Peel Extracts on Philippine Duck (Anas luzonica) Eggs

Chorioallantoic Membrane Assay”.

Cancer begins when a cell breaks free from the normal restraints on cell

division and begins to follow its own agenda for proliferation. All of the cells

produced by division of this first ancestral cell and its progeny also display

inappropriate proliferation. A tumor, or mass of cells, formed of these abnormal

cells may remain within the tissue in which it originated (a condition called in situ

cancer), or it may begin to invade nearby tissues (a condition called invasive

cancer). An invasive tumor is said to be malignant, and cells shed into the blood

or lymph from a malignant tumor are likely to establish new tumors (metastases)

throughout the body. Tumors threaten an individual's life when their growth

disrupts the tissues and organs needed for survival (Bethesda, 2007).

Angiogenesis, the formation of new blood vessels, is a complex process

that involves the migration, growth, and differentiation of endothelial cells lining the

inner walls of blood vessels (Thakur, 2018). Angiogenesis plays a critical role in

the growth and spread of cancer. A blood supply is necessary for tumors to grow
 16

beyond a few millimeters in size. Tumors can cause this blood supply to form by

giving off chemical signals that stimulate angiogenesis. Tumors can also stimulate

nearby normal cells to produce angiogenesis signaling molecules. The resulting

new blood vessels “feed” growing tumors with oxygen and nutrients, allowing the

cancer cells to invade nearby tissue, to move throughout the body, and to form

new colonies of cancer cells, called metastases. Because tumors cannot grow

beyond a certain size or spread without a blood supply, scientists are trying to find

ways to block tumor angiogenesis. The scientists are studying natural and

synthetic angiogenesis inhibitors, also called anti-angiogenic agents, with the idea

that these molecules will prevent or slow the growth of cancer. Angiogenesis

inhibitors are unique cancer-fighting agents because they tend to inhibit the growth

of blood vessels rather than tumor cells. In some cancers, angiogenesis inhibitors

are most effective when combined with additional therapies, especially

chemotherapy. It has been hypothesized that these drugs help normalize the blood

vessels that supply the tumor facilitating the delivery of other anticancer agents,

but this possibility is still being investigated. Angiogenesis inhibitor therapy does

not necessarily kill tumors but instead may prevent tumors from growing. Therefore,

this type of therapy may need to be administered over a long period (Oncology

Nurse Advisor, 2014).

When blood flow is initiated, physiological processes such as embryonic

development, wound healing, and immune reactions are then allowed to start and

develop (Steagall et al., 2014; Shibuya, 2013). On the other hand, the creation of

new blood vessels increases the supply of nutrients, oxygen, and growth factors
 17

to normal and tumor cells. If tumor cells can induce angiogenesis, subsequent

tumor expansion and transition from a benign state to a malignant one is started

(Yang, F. et al., 2013). Endothelial cells can migrate in order to initiate or progress

to angiogenesis, with the aid of a wide range of regulators and signaling molecules

such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF),

vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and

transforming growth factor (TGF). Thus, these angiogenic peptides play a critical

role in the creation of new blood vessels (Prevete et al., 2015; Thom et al., 2014;

Alasvand et al., 2015; as cited by Alasvand et al., 2019).

Since angiogenesis is the main requisite for cancer growth and invasion, its

inhibition is considered the mainstay in cancer treatment strategies (Abdalla et al.,

2018a). Angiogenesis inhibitors are classified into either direct inhibitors that target

endothelial cells in the growing vasculature or indirect inhibitors that prevent the

expression or block the activity of angiogenesis inducers (El-Kenawi & El-Remessy,

2013). Numerous bioactive chemical compounds of plant origin may influence the

angiogenic activity of various cell types and may affect the formation of blood

vessels (Loboda, Zarebski, Cisowski & Jazwa, 2005). Scientific and research

interest is drawing its attention towards naturally-derived compounds as they are

considered to have less toxic side effects compared to current treatments such as

chemotherapy. The Plant Kingdom produces naturally occurring secondary

metabolites which are being investigated for their anticancer activities leading to

the development of new clinical drugs. With the success of these compounds that

have been developed into staple drugs for cancer treatment, new technologies are
 18

emerging to develop the area further. New technologies include nanoparticles for

nanomedicines which aim to enhance anticancer activities of plant-derived drugs

by controlling the release of the compound and investigating new methods for

administration (Greenwell & Rahman, 2015a).

For many years, herbal medicines have been used and are still used in

developing countries as the primary source of medical treatment. Plants have been

used in medicine for their natural antiseptic properties. Thus, research has

developed into investigating the potential properties and uses of terrestrial plants

extracts for the preparation of potential nanomaterial based drugs for diseases

including cancer (Sivaraj, Rahman, Rajiv, Vanathi & Venckatesh, 2014). Many

plant species are already being used to treat or prevent development of cancer.

Multiple researchers have identified species of plants that have demonstrated

anticancer properties with a lot of focus on those that have been used in herbal

medicine in developing countries (Ochwang’I et al., 2014; Freiburghaus et al.,

1996; Kamatou et al., 2008; as cited by Greenwell & Rahman, 2015b).

Luffa acutnagula Linn. var. amara Roxberg of Cucurbitaceae family is a

perennial climber native to southern and western India (Kalaskar & Surana, 2010).

The plant possesses various medicinal properties such as treatment of jaundice,

splenic enlargement and laxative. The plant is also reported to have potent α-

glucosidase inhibitory effect. α-glucosidase is an enzyme that is responsible for

breakdown of carbohydrates in intestine (Andrade-Cetto, Becerra-Jimenez &

Cardenas-Vazquez, 2008; as cited by Pimple, Kadam & Patil, 2011).

 19

Luffa acutangula, a perennial plant grows mainly in India, Southeast Asia,

China, Japan, Egypt, and other parts of Africa, it is widely used in the traditional

Indian medicinal system to treat various health conditions. The plant has been

used in diabetes, hemorrhoids, dysentery, headache, ringworm infection, and

leprosy (Shendge & Belemkar, 2018c). The plant is loaded with nutrients such as

dietary fiber, vitamin C, vitamin A, riboflavin, zinc, thiamine, iron and magnesium.

The fruit has been found to have antioxidant, antibacterial and antifungal properties.

It is also considered demulcent, diuretic, nutritive (Stuart, 2019b).

Anticancer potential of a methanolic and aqueous extract of fruit was

studied in Dalton’s Lymphoma Ascites (DLA) cell induced solid tumor model. In the

study, Swiss albino mice received two doses (200 and 400 mg/kg, oral) of each

extract along with DLA cells. Development of solid tumor in mice was significantly

diminished on treatment with both extracts (Dashora & Chauhan, 2015b).

Furthermore, growth inhibitory effect of ethanolic extract of leaf was investigated

on human lung cancer cell line (NCI-H460). The IC50 value was found to be at 20

μg/ml in MTT assay while cell lines showed high DCF fluorescence and

significantly increased mitochondrial depolarization indicating anticancer activity of

the extract (Vanajothi et al., 2012a). However, not sufficient studies were

undertaken to prove anticancer activity of the plant, due to which it is quite early to

come to any conclusion. In vitro and in vivo anticancer studies are recommended

to prove anticancer efficacy of plant (Shendge & Belemkar, 2018d).

Plant derived phytochemicals are defined as bioactive non-nutrient

compounds which have been connected to reduction of major chronic diseases

 20

risk (Wiseman, 1999; Sreelatha & Padma, 2009; Doughari et al., 2009). The Greek

word ‘phyto’ in phytochemicals means plant (Liu R.H., 2004; Block et al., 2001). In

other words, phytochemicals are plant chemicals. It is predicted that more than

5000 particular phytochemicals have been recognized in grains, fruits and

vegetables but a large percentage are still unknown and must be identified before

understanding their health benefits in whole foods (Liu R.H., 2003; 2013; as cited

by Mollakhalili, Mortazavian, Monfared & Sohrabvandi, 2017).

There are several types of phytochemicals. Flavonoids, a common

phytochemical, specifically, inhibit tumor growth, reduce inflammation and boost

immunity. Other phytochemicals like polyphenols can also prevent cancer

formation and inflammation (Bramlet, 2017b). Flavonoids, alkaloids, saponins, and

triterpenes altogether show anti-cancer properties (Racadio, 2016; Sharma D.,

2006).

In 2017, a phytochemical screening performed on ethanol extract of Patola

fruit yielded alkaloids, saponins, carotenoids, and terpenoids with the absence of

flavonoids, tannins, and anthraquinones (Suryanti, Marliyan & Astuti, 2017a).

However, in the following year, phytochemical components of the plant was

evaluated again and the researchers have isolated and identified about 50

compounds, such as flavonoids, anthraquinones, proteins, fatty acids, saponin

triterpene, volatile compounds, among others. Nutritional evaluation of the seed

showed the presence of fats, proteins, and minerals. Protein and fat from the

kernel were 39% and 44% of total weight, respectively (Shendge & Belemkar,
 21

2018e; as cited by Stuart, 2019c). With this, further analysis of the plant

components is suggested to prove the presence of phytochemicals in it.

In phytochemical screening methods, the extracts of medicinal plants are

analyzed for the presence of alkaloids, saponins, tannins, cardiac glycoside,

anthraquinones, steroid, coumarins, carbohydrates and flavonoids, according to

standard methods (Chinedu, David, and Ameh, 2015).

Studies have consistently linked abundant consumption of plant-based food

to a substantial reduction in risk of developing various cancers (Béliveau &

Gingras, 2007). Furthermore, there are some bioactive compounds in foods that

have potential health benefits, such as flavonoids, carotenoids, stilbenes, lignans,

and phenolic acids (Xu et al., 2017; Shashirekha et al., 2015). Flavonoids are large

group of phenolic compounds and are usually involved in protection against harsh

environmental conditions, UV radiation, and microorganism attacks in plants

(Nabavi et al., 2018; Liu J. et al., 2018; as cited by Rodríguez-García, Sánchez-

Quesada & Gaforio, 2019). Major molecular mechanisms of action of flavonoids

are given as follows:

(1) down regulation of mutant p53 protein,

(2) cell cycle arrest,

(3) tyrosine kinase inhibition,

(4) inhibition of heat shock proteins,

(5) estrogen receptor binding capacity,

(6) inhibition of expression of Ras proteins (Kumar & Pandey, 2013).

 22

In a study to determine effects of isoflavones genistein, daidzein, and

biochanin A on mammary carcinogenesis, genistein (a flavonoid) was found to

suppress the development of chemically induced mammary cancer without

reproductive or endocrinological toxicities (Barnes, 1995b).

Tannins are plant polyphenolic compounds that are contained in large

quantities in food and beverages consumed by humans daily. It has been shown

that various tannins exert broad cancer chemoprotective activity in a number of

animal models (Nepka et al., 1999b).

Ellagic acid, which is produced by ellagitannin (a tannin) hydrolysis, has

been shown to exert anticarcinogenic activities in a number of systems and

experimental animal models. The vast majority of the studies have focused on the

effects of ellagic acid on the initiation phase of the carcinogenic process. The

mechanisms involved can be summarized as:

(1) changing the phase l/phase 2 enzymes ratio, either by lowering, or by

inhibiting the cytochrome P450 enzymes responsible for carcinogen

metabolism activation; and

(2) inhibiting the binding of carcinogens to DNA either by direct binding of

ellagic acid to the carcinogen, or by blocking the carcinogen-DNA

interaction (Boukharta, Jalabert & Castonquay, 1992).

Saponins have long been known to be plant- originated, but they can also

be found in marine organisms. Saponins, from a structural viewpoint, are

composed of one or more hydrophilic glycoside moieties along with a lipophilic and

derived aglycone and finally one or more sugar chains. Therefore, it is not a
 23

surprise that they exhibit pharmacological effects. Anticancer activity is one of

these important properties. Saponins interfere with the replication of cellular DNA

and they prevent the proliferation of cancer cells (Yildirim & Kutlu 2015b). They

have been ascribed to a number of pharmacological actions. In fact, saponins

isolated from B. aegyptiaca (Zygophyllaceae) had anti-proliferative property. This

saponin exhibited potent anti-proliferative activity against MCF-7 human breast,

cancer cells and HT-29 human colon cancer cell (Beit-Yannai et al., 2011).

Screening of plant kingdom for natural products with anticancer properties

contributed to the discovery of anticancer alkaloids and recent progress in cancer

chemotherapy through drugs development (Wall et al., 1966; Wani et al., 1971;

Cragg & Newman, 2009; as cited by Isah, 2016b). The alkaloids are secondary

metabolites that are biosynthesized by the plants for a defensive role, and in many

cases, no biological function is attributed to the molecules (Levinson, 1976; as

cited by Isah, 2016c). The alkaloids and their congeners target DNA replication or

protein synthesis in the mechanism of action on tumor cells, resulting in apoptosis

of the neoplastic cells (Mollov et al., 1968; Parness & Horwitz, 1981; Hsiang et al.,

1985; Faddeeva & Beliaeva, 1997; as cited by Isah, 2016d).

Plant sterols (PS) are C28 and C29 carbon steroid alcohols (Otaegui-

Arrazola, Menendez-Carreno, Ansorena & Astiasaran, 2010) that are integral

components of plant cell membranes, have been shown to be key components of

plant plasma membrane microdomains (Roche et al., 2008), and may exert similar

functions in human cells (as cited by, Grattan, 2013b). In animal models of colon

cancer, PS have been shown to exert beneficial effects (Baskar et al., 2012). In a
 24

case-control study by Mendilaharsu a 50% reduction (95% CI 0.31–0.70) in the

risk of lung cancer was seen among those with the highest quartile intake of

phytosterols (Mendilaharsu, de Stefani, Deneo-Pellegrini, Carzoglio & Ronco,

1998; as cited by, Grattan, 2013c). Similarly, case-control work by De Stefani (de

Stefani et al., 2000; as cited by, Grattan, 2013d) assessing the effects of PS on

stomach cancer risk showed an odds ratio of 0.33 (95% CI 0.17–0.65) among

those with the highest PS intake. While limited, epidemiological data suggests a

correlation between plant sterol intake and a reduction in cancer risk. It has been

estimated that phytochemical intake may be related to a reduction in cancer risk

upwards of 20% (The American Institute for Cancer Research, 2006; as cited by,

Grattan, 2013e).

Cardiac glycosides comprise a large family of naturally derived compounds,

the core structures of which contain a steroid nucleus with a five-membered

lactone ring (cardenolides) or a six-membered lactone ring (bufadienolides) and

sugar moieties (Mijatovic, Ingrassia, Facchini & Kiss, 2008; as cited by

Pongrakhananon, 2012b). Cardiac glycosides have been shown to have

anticancer activities during various stages of carcinogenesis. These activities

include antiproliferative, pro-apoptotic, and chemotherapy sensitization effects

(Pongrakhananon, 2012c). They have been demonstrated to have antiproliferative

activities via their regulation of the cell cycle. The extract from the skin glands of

Bufo bufo gargarizans which contains bufalin (a cardiac glycoside), is able to

induce arrest in human malignant melanoma cells in the G2/M phase of the cell

cycle (Yang, P. et al., 2006; as cited by Pongrakhananon, 2012d). Likewise,

 25

digitoxin (a cardiac glycoside) causes cell cycle arrest in G2/M in a dose-

dependent manner, resulting in a large increase in the number of cells in the sub-

G0 phase (Elbaz et al., 2012; as cited by Pongrakhananon, 2012e).

Terpenes are a wide-spread group of natural compounds with considerable

practical significance which are produced by arrangement of squalene epoxide in

a chair-chair-chair-boat arrangement followed by condensation (Patočka, 2003).

Triterpene glycosides have hemolytic, cytotoxic, antifungal, and other biological

activities caused by membranotropic action. These natural products suppress the

proliferation of various human tumor cell lines in vitro and, more importantly,

intraperitoneal administration in rodents of solutions of some sea cucumber

triterpene glycosides significantly reduces both tumor burden and metastasis. The

anticancer molecular mechanisms include the induction of tumor cell apoptosis

through the activation of intracellular caspase cell death pathways, arrest of the

cell cycle at S or G2/M phases, influence on nuclear factors, NF-κB, and up-down

regulation of certain cellular receptors and enzymes participating in

cancerogenesis, such as EGFR (epidermal growth factor receptor), Akt (protein

kinase B), ERK (extracellular signal-regulated kinases), FAK (focal adhesion

kinase), MMP-9 (matrix metalloproteinase-9) and others (Aminin et al., 2015b).

According to Berkeley Wellness (2019), fruits and vegetable peels have the

following benefits:

(1) The pigments in produce are healthful, and the peels or skins of fruits

and vegetables are often the most colorful part. Colorful peels can be a

concentrated source of potentially beneficial phytochemicals.

 26

(2) Vegetable peels or skins are particularly good sources of insoluble fiber,

which helps prevent constipation. Some peels, notably apple, are rich in

pectin, a soluble fiber that helps lower blood cholesterol and control blood

sugar.

(3) Fruit peels may also help prevent cancer. Laboratory research has found

that whole apples have a much greater effect on cancer cells than peeled

ones, probably because of antioxidants in the apple skin.

In the observation of potential anti-angiogenic activity, the Patola peel

extracts was treated on Chorioallantoic Membrane (CAM) assay of duck embryos.

Among various animal model systems designed to study the mechanisms

underlying angiogenesis, chick embryo models have been useful tools in analyzing

the angiogenic potential of purified factors and intact cells. The CAM assay, a

specialized, highly vascularized tissue of the avian embryo, serves as an ideal

indicator of the anti- or pro-angiogenic properties of test compounds (Deryugina &

Quigley, 2008b). In the duck embryo, the chorioallantois is formed between days

4 and 5 of development, when the outer mesodermal layer of the allantois fuses

with the mesodermal lining of the chorion, and a network of blood vessels is

gradually formed between the two layers. The central portion of the CAM is fully

developed by day 8 to 10 at which time it becomes capable of sustaining tissue

grafts, while the outskirts of the CAM are still developing and expanding until the

CAM fully envelopes the embryo at day 12 of incubation. Histologically, the CAM

consists of three germ layers – ectoderm, mesoderm, and endoderm. The

ectoderm faces the shell membrane and is underlined by the respiratory capillary
 27

plexus, which starts to form between days 5 and 6 of embryonic development by

both angiogenesis and vasculogenesis (Melkonian et al., 2002; as cited by

Deryugina & Quigley, 2008c).

The Chorioallantoic Membrane model has many advantages, such as (a)

the highly vascularized nature of the CAM greatly promotes the efficiency of tumor

cell grafting; (b) high reproducibility; (c) simplicity and cost effectiveness, and

finally (d) as the CAM assay is a closed system, the half-life of many experimental

molecules such as small peptides tend to be much longer in comparison to animal

models, allowing experimental study of potential anti-metastatic compounds that

are only available in small quantities (Tufan & Satiroglu-Tufan, 2005b; Ossowski,

1988; Zhai et al., 2007b; Cimpean, Ribatti & Raica, 2008; as cited by Lokman et

al., 2012c).

In CAM assay, duck eggs were usually used to study angiogenesis because

their eggs are bigger than chicken eggs that makes it easier to evaluate. Also, they

are known to have thicker and stronger shell with higher calcium and

lower magnesium shell content than those of the chickens (Bordessa, 2019). With

that, higher survival rate of duck embryos can be observed.

Inclusively, in 2012b, Vanajothi et al. determined the potential of the Luffa

acutangula extracts in lung cancer treatment. Meanwhile, Dashora and Chauhan’s

study in 2015c demonstrated the anti-cancer activity of the extracts in Swiss albino

mice. The findings of these studies may serve as an evidence for the anticancer

property of the Patola peel extracts. Moreover, a phytochemical testing on the fruit

extracts was performed wherein the presence alkaloids, saponins, carotenoids,

 28

and terpenoids with the absence of flavonoids, tannins, and anthraquinones was

determined (Suryanti, Marliyan & Astuti, 2017b). On the other hand, presence of

50 compounds, such as flavonoids, anthraquinones, proteins, fatty acids, saponin

triterpene, volatile compounds were yielded in the following year (Shendge &

Belemkar, 2018f). Previous studies suggest that the results of phytochemical

testing of Luffa actutangula extracts have varying results. Because of this, further

investigation is needed to determine which compounds are present in the Luffa

acutangula (L.) Roxb. peel extracts.