CHAPTER IINTRODUCTION

Annona squamosa locally known as the Atis which is a member of the Annonaceae family that bears edible fruits called sugar apples. Studies have showed analgesic, anti-inflammatory, anti-microbial, anti-diabetic, cytotoxic, antioxidant, antilipimic, anti-ulcer, molluscicidal, genotoxic, vasorelaxant, hepatoprotective, larvicidal, anthelmintic properties. Ficus septica locally known as the labnog tree which is a member of the Moraceae family is known to have diuretic, sudorific, antiherpetic, antirheumatic properties. Studies have shown that its leaves yielded phenanthroindolizidine N-oxide, ficuseptine-A together with 18 known compounds. Some of the compounds showed possible cytotoxic activity against two human cancer cell lines.Medicinal plants play an important role in terms of healthcare system in some portion of the world. There are several medicinal plants which are widely used in the traditional systems of medicine for the prevention and cure of diseases like cancer. Several plant derived compounds have been found to play significant role in the development of clinically useful anti-cancer agents. According to the World Health Organization, there were an estimated 4 million new cancer cases and 8.2 million cancer-related deaths in 2012 worldwide. Cancer is the name given to a collection of related diseases. In all types of cancer, some of the bodys cells begin to divide without stopping and spread into surrounding tissues. Cancer can start almost everywhere in the human body, which is made up of trillion cells. Many cancers form solid tumors, which are masses of tissue. (NCI, 2015). Tumors can grow and interfere with the digestive, nervous, and circulatory systems and they can release hormones that alter body function. When a tumor successfully spreads to other parts of the body and grows, invading and destroying other healthy tissues, it is said to have metastasized. The process itself is called metastasis, and the result is a serious condition that is very difficult to treat. (Peter Crosta, 2008)

Background of the StudyStudy showed that cancer is the fifth leading cause of morbidity and mortality worldwide. There are a lot of therapies that cancer patients can undergo but not all can acquire it due to its expensiveness. People know that medicinal herbs and plants have been used in folk medicine for millennia. For years, Ficus septica and Annona squamosa are claimed to have properties that can cure common diseases. The said information aroused the researchers interest to investigate and to conduct a further study to confirm its feasibility as an alternative cure for cancer and tumors.

Statement of the ProblemThis study aimed to determine and identify the mitotic index and toxicity of the ethanolic leaf extracts Annona squamosa and Ficus septica . In particular, the researcher conducted a phytochemical screening to determine the active compounds present in each leaves, the brine shrimp lethality assay also being performed to further manifest its cytotoxic activity, the antioxidant potency of the ethanolic leaf extracts using DPPH Radical Scavenging Assay and to test the Allium cepa through distinguishing and discerning chromosomal aberration. Specifically, the study sought to answer the following questions:1. What are the phytochemical substances found in Annona squamosa and Ficus septica?2. Are the ethanolic extracts of Annona squamosa and Ficus septica exhibited cytotoxic activities when tested using the brine shrimp lethality test?3. Are the ethanolic extracts of Annona squamosa and Ficus septica has the antioxidant potential when tested using the DPPH Radical Scavenging Assay?4. Is there significant mean difference of the mitotic phases of the Allium cepa roots treated with the Annona squamosa and Ficus septica ethanolic extracts as compared to the control?

Significance of the Study

The results of this study may provide strong evidence about the uniqueness of the chemical characteristics of Annona squamosa and Ficus septica ethanolic leaf extracts. Positive results may stimulate the use of natural medication particularly in cancers and tumors. It may also equip baseline information to the locality in their way of finding possible alternative source of medicine for nursing common illness.

Scope and Limitations of the Study

This study only focuses on the determination of the following components: phytochemicals, cytotoxic activity, antioxidant activity and the chromosomal aberration in the ethanolic leaf extracts of Annona squamosa and Ficus septic leaves. These components are exhibited in terms of phytochemical analysis, brine shrimp lethality test, Free Radical Scavenging Activity and Allium cepa chromosomal aberration assay.

Operational Definition of Terms

Annona squamosa is a small, well-branched tree or shrub from the family Annonaceae that bears edible fruits called sugar apples.Brine shrimp lethality assay is a simple, high throughput cytotoxicity test of bioactive chemicals. It is based on the killing ability of test compounds on a simple zoological organism-brine shrimp.Cancer - A group of diseases involving abnormalcell growthwith the potential to invade or spread to other parts of the body. A potentially deadly form of uncontrolled cell division. Chromosomal Aberration A missing, extra, or irregular portion of chromosomal DNA. It can be atypical number of chromosomes or a structural abnormality in one or more chromosomes.Cytotoxic is the quality of being toxic to cell.DPPH is a common abbreviation for an organic 1-picrylhydrazyl. It is a dark colored crystanine powder composed of stable free-radical molecules.Ficus septica is a shrub or tree of the family moraceae living at low altitudes. Mitotic Index Is defined as the ratio between the numbers of cells in a population undergoing mitosis to the number of cells not undergoing mitosis.Phytochemical are chemical compounds that occur naturally in plants. Tumor - An abnormal growth of body tissue. Tumors can be cancerous (malignant) or noncancerous (benign).

CHAPTER IIREVIEW OF RELATED LITERATURE

This chapter represents the review of related literature, which is all directed towards the accomplishments of the aims and purposes of this research. These literature and research works are reviewed to serve as bases of the data gathered for this study.

Geographical location of Sta. Filomena

Figure 1. Map of Sta. Filomena (https://www.google.com.ph/maps/place)

Sta. Filomena is a barangay located in Iligan City, Philippines. It is where the researcher collected the samples of Annona squamosa and Ficus septica leaves.Annona squamosa is a small, well-branched tree or shrub from the family Annonaceae that bears edible fruits called sugar apples. It tolerates a tropical lowland climate better than its relatives helping make it the most widely cultivated of these species. (Morton, 1987)Ficus septica is a shrub or tree of the Moraceae family living at low altitudes. It lives on the edge of vegetation, often in degraded environments. (Shanahan et al., 2001)

Physical DescriptionAnnona squamosa is a small, semi-deciduous, much branched shrub or small tree (9.8 to 26 ft tall) with a broad, open crown or irregularly spreading branches and a short trunk short, not buttressed at base. Its fruit has delicious whitish pulp, and is popular in tropical markets. Branches have light brown bark and visible leaf scars; inner bark light yellow and slightly bitter; twigs become brown with light brown dots.Thin, simple, alternate leaves occur singly, 5 centimeters to 17 centimeters long and 2 centimeters to 6 centimeters wide; rounded at the base and pointed at the tip (oblong-lanceolate). Pale green on both surfaces and mostly hairless with slight hairs on the underside when young. The sides sometimes are slightly unequal and the leaf edges are without teeth, inconspicuously hairy when young. Leaf stalks are 0.4 centimeters to 2.2 centimeters long, green, sparsely pubescent.Flowers are in solitary or in short lateral clusters of 2-4 about 2.5 centimeters long, greenish-yellow flowers on a hairy, slender 2 centimeters long stalk. Three green outer petals, purplish at the base, oblong and three inner petals reduced to minute scales or absent. Very numerous stamens; crowded, white, less than 1.6 centimeters long; ovary light green. Styles white, crowded on the raised axis. Each pistil forms a separate tubercle which matures into the aggregate fruit. Flowering occurs in spring-early summer and flowers are pollinated by nitidulid beetles.Aggregate and soft fruits form from the numerous and loosely united pistils of a flower which become enlarged and mature into fruits which are distinct from fruits of other species of genus. The round or heart-shaped greenish yellow, ripened aggregate fruit is pendulous on a thickened stalk with many round protuberances and covered with a powdery bloom. Fruits are formed of loosely cohering or almost free carpets.The pulp is white tinged yellow, edible and sweetly aromatic. Each carpel containing an oblong, shiny and smooth, dark brown to black, long seeds. (Wikipedia, 2015)

Ficus septica is an erect, small tree, growing 3 to 8 meters high, smooth, with more or less hairy young shoots. Leaves are smooth and shining, not all roughened, oblong-ovate to elliptic-ovate, 10-20 centimeters long, with tip tapering to a rather sharp point, and the base pointed. Receptacles are axillary, solitary, depressed-globose or turbinate, obscurely ridged or angled, 1.5 to 2 centimeters in diameter, and shortly peduncled. (Stuartxchange, n.d.)Figs grow often in pairs but can be solitary or in groups of up to four. Figs are depressed-globose to ellipsoid, the apex is flat or concave. Seven to twelve ribs toward to ostiole. At maturity, whitish to yellowish dots appear on the fig. The individuals from Philippines have their stems covered by short hairs while those found in Taiwan are glabrous. (Wikipedia, 2015)

DistributionAnnona squamosa is cultured throughout the Philippines; occasionally instinctive. It is also native to the tropical Americas and West Indies, but the spot-on origin is unknown. It is now the most widely cultured of all the species of Annona, being grown for its fruit throughout the tropics and warmer subtropics, such as Indonesia, Thailand, and Taiwan; it was acquainted to southern Asia before 1590. It is naturalized as far north as southern Florida in the United States and as south as Bahia in Brazil, and is an invasive species in some areas.

Ficus septica is distributed in thickets at low and medium altitudes throughout the Philippines. Also occurs in China, from Northeast India to Australia and throughout Malesia.

DevelopmentUsing histological techniques and microscopy, Annona squamosa seeds were studied with regard to their anatomical characteristics. In general, they presented the typical anatomical patterns of seeds of the Annonaceae family. The multilayered testa was comprised of exo-, meso- and endotestas, with the presence of longitudinal and oblique fibers. The embryo was small and straight with moderately developed embryonic axis, rudimentary plumule and flat and thin cotyledons, wherein one could observe the procambium. In particular, the perichalaza was observed in the medium longitudinal plane of the endotesta with more than two rows of vascular tissue. The tegmen or inner integument was collapsed. The micropylar plug was tapered, woody and composed of transverse fibers; a fracture line was also observed. The ruminate endosperm interfered with both the inner and external integuments. Oleiferous idioblasts were observed at the outer edges of the ruminations. (Martinez et al., 2013)Ficus septica Cotyledons are almost orbicular, about 3 mm diam. First pair of leaves toothed. At the tenth leaf stage: leaf blade broadly elliptic, apex acute, base obtuse, margins sinuate on the upper half of the leaf blade, teeth obscure, upper surface glabrous; oil dots not visible; flat glands sometimes visible on the underside of the leaf blade at the junction of the main lateral veins and the midrib; petiole with a few scattered hairs; stipules sheathing the terminal bud, glabrous, about 10-15 mm long. (Burman, 1768)

Life CycleSugar apple seeds have a relatively long life, having kept well for 3 to 4 years. They germinate better a week after removal from the fruit than when perfectly fresh. Germination may take 30 days or more but can be hastened by soaking for 3 days or by scarifying. The percentage of germination is said to be better in unsoaked seeds. While the tree is generally grown from seed, vegetative propagation is practiced where the crop is important and early fruiting is a distinct advantage.Sugar apple trees are spaced at 10 x 10 ft (3x3 m) in order to elevate atmospheric humidity and improve pollination. Commercial fertilizer containing 3% N, 10 % P and 10% K significantly increases flowering, fruit set and yield. Judicious pruning to improve shape and strength of tree must be done only in spring when the sap is rising; otherwise pruning may kill the tree. Irrigation during the dry season and once during ripening will increase fruit size. (Morton, 1987)Figure 2. Annona squamosa (Atis) Seedlings

Figure 3. Annona squamosa (Atis) Full grown tree

Ficus septicas obligate pollination mutualism is similarly specialized. Life cycles of pollinating fig wasps and their Ficus host plants are completely interdependent. Volatile signals attract female fig wasps to a receptive fig of a host where they gain access to the unisexual flowers of the enclosed inflorenscence through a narrow bract-covered opening, called the ostiole. Females oviposit in pistillate flowers and deposit pollen in the process. In most cases, pollinators do not have a second chance at reproduction as they cannot leave the fig upon entering (Moore et al. 2003). In monoecious figs, flowers either set seed, form galls that nourish fig wasp offspring, or produce pollen that eclosing wasps later transport to other receptive figs. In dioecious species, reproductive functions are separated between plants bearing either seed-producing inflorescences (female figs) or inflorescences producing wasps and pollen (male figs).Cotyledonsalmostorbicular, about 3 mmdiameter.First pair of leaves toothed. At the tenth leaf stage: leafbladebroadlyelliptic,apexacute, baseobtuse, marginssinuateon the upper half of the leafblade, teeth obscure, upper surfaceglabrous;oil dotsnot visible; flat glands sometimes visible on the underside of the leafbladeat the junction of the main lateral veins and themidrib;petiole with a few scattered hairs;stipulessheathingtheterminalbud,glabrous, about 10-15 mm long. (Moe and Weiblen, 2012)

Figure 4. Ficus septica (Labnog) Seedlings and full grown tree

Tumors and CancersThe terms tumor and cancer are sometimes used interchangeably which can be misleading. A tumor is not necessarily a cancer. The word tumor simply refers to a mass. For example, a collection of fluid would meet the definition of a tumor. A cancer is a particularly threatening type of tumor. It is helpful to keep these distinctions clear when discussing a possible cancer diagnosis. (Johns Hopkins University, n.d.)According to Canadian Cancer Society, malignant tumors are cancerous. Cancer can start in any one of the millions of cells in our bodies. Cancer cells have a largernucleus that looks different from a normal cells nucleus, and cancer cells behave, grow and function quite differently from normal cells. Malignant tumors vary in size and shape. They grow in an uncontrolled, abnormal way and can grow into (invade) nearby tissues, blood vessels or lymphatic vessels. They can interfere with body functions and become life-threatening. Cancer cells can break off and spread to distant locations in the body (metastasize). Cancer that spreads from its original location (the primary tumor) to a new part of the body is calledmetastatic cancer. Malignant tumors can also come back (recur) after they are removed.The most common way to name cancers is to do so according to the place in the body they start, such as breast cancer or prostate cancer. Cancers of the blood are called leukemia, while cancer of the plasma cells is called multiple myeloma and cancers of the lymphatic system are called lymphoma. Some cancers have been named after the person who first described them (for example, Hodgkin lymphoma or Wilms tumor). Another way to name some benign or malignant tumors is after the type of cell or tissue it develops from (tissue of origin). Most benign tumors and some malignant tumors have the suffix "oma" at the end of their name. When a malignant tumor has the same name as a benign tumor, the word "carcinoma" or "sarcoma" is added to the end to identify it as cancer. For example, a benign tumor of fatty tissue is called a lipoma, whereas a malignant tumor of fatty tissue is called a liposarcoma.

Bioactive compounds in Annona squamosaAnnona squamosa leaves yield an alkaloid, chloroplatinate. Anonaine, an alkaloid, is found in the bark, leaves and seeds. Seed yields an alkaloid, neutral resin, fixed oil, contains yellow, non-drying oil and an irritant which kills lice. Fruit peel extracts yielded alkaloids, proteins, carbohydrates, flavonoids, glycosides, saponins, and tannins. Roots yielded two oxoaporphine alkaloids: liriodenine and oxoanalobine. (Stuartxchange, n.d.)According to Vijayalaksham et al., (2011) Forty-four compounds were identified in the ethyl acetate fraction of Annona squamosa leaf extract by GC-MS analysis. The prevailing compounds were sodium benzoate (27.50%), 4, 4- Tert- Butylcalix(4)arene (12.34 %),4, 4- Dimethylcholesterol (10.30%), Butyloctylpthalate (9.67%), stigmasterol acetate (2.92%), isoamylacetyate (2.29%). The present investigation clearly indicates the highest percentage of sodium benzoate in ethylacetate fraction of Annona squamosa leaf extract. Sodium benzoate was used as an antifungal agent, antibacterial agent, as CNS stimulant and as a diagnostic aid in liver function tests.

Bioactive compounds in Ficus septicaPhytochemical study isolated isoflavones: ficusin A and ficusin B from the root bark. Two indolizidine alkaloids: a novel ficuseptine and antofine. Phytochemical screening of an ethanol extract yielded alkaloids, quarternary base, tannins, 2-deoxysugars and benzopyrone nucleus. Methanol extract of stems yielded eight new alkaloids, namely, ficuseptines B-D (1-3), 10R, 13aR-tylophorine N-oxide, 10R, 13aR-tylocrebrine N-oxide, 10S, 13aR-tylocrebrine N-oxide, 10S, 13aR-isotylocrebrine N-oxide, and 10S, 13aS-isotylocrebrine N-oxide, together with six known phenanthroindolizidine alkaloids.

Annona squamosa Health BenefitsAnnona squamosa leaves, fruit and seeds are vermicidal and insecticidal. The unripe fruit is astringent, used for diarrhea and dysentery and dyspepsia. The bark is astringent and tonic. Roots make a drastic purgative. Leaves are emmenagogue, febrifuge and tonic. Studies have showed analgesic, anti-inflammatory, anti-microbial, anti-diabetic, cytotoxic, antioxidant, antilipimic, anti-ulcer, molluscicidal, genotoxic, vasorelaxant, hepatoprotective, larvicidal, anthelmintic, insecticidal properties. .(Stuartxchange, n.d.)

High in Vitamin CA medium-size sweetsop fruit weighs about 5.5 ounces and contains 56.3 milligrams of vitamin C -- this is roughly 75 percent of the daily recommendation of 75 milligrams for women and 62 percent of the recommended daily intake of 90 milligrams for men. Vitamin C helps maintain healthy bones and muscles, as well as helps blood vessels stay supple and strong. Although the value of vitamin C to fight colds remains unconfirmed, the July 2012 issue of "American Family Physician" points out that vitamin C does seem to shorten the duration of a cold in adults and children.

Rich in FiberSweetsop is rich in fiber and carbohydrates. One 5.5-ounce serving, or roughly 3-inch fruit, provides you with 6.8 grams of fiber, which is about 18 percent of the daily recommendation for an adult. With 36.6 grams of carbohydrates per piece of fruit, sweetsop also is an excellent source of energy. In comparison, a medium-size apple, or about 6.4 ounces has only 25 grams of carbs and, even with the skin, just 4.4 grams of fiber. Fiber is an essential nutrient that helps lower cholesterol, normalizes bowel movements and helps control blood sugar levels. (Dray, 2015)

Antioxidant PropertiesIn a 2011 study published in the "Journal of Pharmacy Research," researchers studied Annona squamosa to see if it contains any compounds that could be useful for medical treatments or nutritional support. Results from test-tube research shows that sweetsop has free radical scavenging activity, making it an effective antioxidant.

Anecdotal UseSweetsop is used extensively in India and tropical American countries to treat a number of conditions. Most of its uses have been passed down through generations, though studies are needed to confirm whether sweetsop really works in treating these conditions. For example, sweetsop is used in India to treat wounds and ulcers, as well as an anti-diarrheal tonic. In tropical American countries, sweetsop has different medicinal uses, such as being a treatment for colds, digestive problems and high fever.

AntidiabeticBeneficial effects of Annona squamosa extract in streptozotocin-induced diabetic rats: Study results showed that Annona squamosa extract has an antihyperglycemic effect and alleviated liver and renal damage associated with STZ-induced diabetes mellitus in rats. Study of aqueous leaf extracts were investigated on STZ-nicotinamide induced diabetic rats. The diabetic groups treated with aqueous leaf extract were compared with standard glibenclamide.Study evaluated the antidiabetic activity of a hydroalcoholic extract of Annona squamosa in experimentally induced diabetic rat model. Extract of leaves showed significant reduction in blood glucose after glucose loading, with activity comparable to glibenclamide.

Anti-Inflammatory / Cyclic PeptidesStudy yielded two new cyclic peptides, cyclosquamosin H and I, together with six known cyclic peptides, squamin A, squamin B, cyclosquamosin A, D E and cherimolacyclopeptide B from the seeds. Compound 7 showed an inhibitory effect on the production of pro-inflammatory cytokines.

Hepatoprotective / DEN-induced HepatotoxicityStudy on diethylnitrosamine (DEN)-induced liver injury in Swiss albino mice showed hepatoprotective effect, with improvement in biochemical parameters and confirmation by histopathological studies.Study showed the extracts of Annona squamosa were not able to completely revert the hepatic injury induced by isoniazid + rifampin, but it could limit the effect of the drugs on the liver. The effect compared with standard drug silymarin.

AntibacterialStudy screened the ethanol crude extract of the fruit of Annona squamosa for antimicrobial activity against some pathogenic microorganisms. It showed inhibitory activity against S aureus and S pneumoniae. Results conclude the plant extract may serve as a valuable source of compounds with therapeutic antibiotic potentials.

Antithyroid Activity / QuercetinExtract of the seeds of Annona squamosa was evaluated for it ameliorative effect in the regulation of hyperthyroidism in a mouse model. Phytochemical study revealed the presence of quercetin in the seed extract and the results of the effects of quercetin suggest an involvement of this phytochemical in the mediation of antithyroidal activity of A squamosa seed extract.

Antigenotoxic ActivityStudy showed both aqueous and ethanolic bark extracts of Annona squamosa showed antigenotoxic effect. The bark extract demonstrated more prominent antigenotoxic effect in DBMA induced genotoxicity in Syrian hamsters.

Molluscicidal ActivityStudy on molluscicidal activity of leaves, bark and seed of Annona squamosa against snail Lymnaea acuminata was studied. Highest activity was observed in the seed extracts. The acetogenins from the seed were more toxic than synthetic pesticides.

Anti-Head Lice ActivityStudy identified the active compounds against head lice from the hexane extract of Annona squamosa seeds. The two major compounds were oleic acid and triglyceride with one oleate ester. The triglyceride with one oleate ester and the crude hexane extract diluted with coconut oil 1:1 were found to kill all tested head lice.

Antimicrobial / PhytochemicalsPhytochemical screening yielded phenols, tannins, alkaloids, saponins, flavanoids, reducing sugars and oil. The methanol extract showed maximum antibacterial activity against E. coli. Seed extract showed maximum antifungal activity against T. rubrum.

Anti-UlcerPhytochemical investigation of twigs isolated twelve known compounds. Three of the compounds, (+)-O-methylarmepavine (2), N- methylcorydaldine (3), isocorydine (6), showed promising anti-secretory activity, comparable to standard drug omeprazole. An ethanol extract and its chloroform and hexane fractions exhibited gastroprotection via inhibition of H+K+-ATPase (proton pump) activity and simultaneously strengthening mucosal defense mechanisms.

ToxicityThe seeds are acrid and poisonous. Bark, leaves and seeds contain the alkaloid, anonaine. Six other aporphine alkaloids have been isolated from the leaves and stems: corydine, roemerine, norcorydine, norisocarydine, isocorydine and glaucine. Aporphine, norlaureline and dienone may be present also. Powdered seeds, also pounded dried fruits serve as fish poison and insecticides in India. A paste of the seed powder has been applied to the head to kill lice but must be kept away from the eyes as it is highly irritant and can cause blindness. If applied to the uterus, it induces abortion. Heat-extracted oil from the seeds has been employed against agricultural pests. Studies have shown the ether extract of the seeds to have no residual toxicity after 2 days. High concentrations are potent for 2 days and weaken steadily, all activity being lost after 8 days. In Mexico, the leaves are rubbed on floors and put in hen's nests to repel lice. (Stuartxchange, n.d.)

Ficus septica Health BenefitsFicus septica have diuretic, sudorific, antiherpetic, antirheumatic properties. In the Philippines, Ficus septica are used for diarrhea, cough, malaria and stomach problems.

Mucarinic Receptor ActivityMalaysian study of 224 plant extracts from 50 plant families for muscarinic receptor binding activity showed the greatest inhibition, and with other extracts that exhibited significant muscarinic properties were suggested to be worthy of further investigation.

Anti-inflammatoryStudy examined the molecular mechanisms for the anti-inflammatory activity of phenanthroindolizidine alkaloids isolated from the leaves of Ficus septica. Study suggests that it exerts its anti-inflammatory effects by inhibiting expression of the proinflammatory factors and related signaling pathways.

Antimicrobial / Antifungal / AntiprotozoalStudy of ethanol extracts of Ficus septica and S foetida showed antibacterial activities, inhibiting the growth of S aureus and E coli. Antifungal assay showed inhibition of Candia albicans. Antiprotozoal assay showed activity against T vaginalis and Entamoeba histolytica. Results suggest the extracts can be used to produce alternative forms of antimicrobials.

Antioxidant / HepatoprotectiveStudy evaluated the antioxidant properties of leaf extract of Ficus septica by measuring malondialdehyde (MDA) levels, as by-products of lipid peroxidation, in the liver of ICR mice. Extract treated mice had lower MDA levels with various signs of histological cellular repair. Results suggested hepatoprotection.

Antiangiogenic ActivityStudy evaluating the potential of leaves of various plants, including Ficus septica for angiosuppressive activity. All the extracts tested except for P. laevigata can reduce CAM (chorio-allantoic membrane) vascular density pointing to an antiangiogenic activity. (Stuartxchange, n.d.)

Free RadicalsFree radicals are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain molecules. Once formed these highly reactive radicals can start a chain reaction, like dominoes. Their chief danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane. Cells may function poorly or die if this occurs. To prevent free radical damage the body has a defense system of antioxidants. (Antioxidants and Free Radicals, n.d.)A notable example of a free radical is the hydroxyl radical (HO), a molecule that has one unpaired electron on the oxygen atom. Two other examples are triplet oxygen and triplet carbine (:CH2) which have two unpaired electrons. In contrast, the hydroxyl anion (HO-) is not a radical, since the unpaired electron is resolved by the addition of an electron; singlet oxygen and singlet carbine are not radicals as the two electrons are paired. Free radicals may be created in a number of ways, including synthesis with very dilute or rarefied reagents, reactions at very low temperature, or breakup of larger molecules. The latter can be affected by any process that puts enough energy into the parent molecule, such as ionizing radiation, heat, electrical discharges, electrolysis, and chemical reactions. Indeed, radicals are intermediate stages in many chemical reactions. (Wikipedia, 2015)

Some conditions cause by free radicals include, deterioration of the eye lens, which contributes to blindness, inflammation of the joints (arthritis), damage to nerve cells in the brain, which contributes to the conditions such as Parkinsons disease, acceleration of the aging process, increased risk of coronary heart disease, since free radicals encourage low-density lipoprotein (LDL) cholesterol to stick to artery walls, certain cancers, triggered by damage cellDNA. (Anioxidants, n.d.))A diet in antioxidants may reduce the risk of many diseases, including heart disease and certain cancers. Antioxidants scavenge free radicals from the body cells, and prevent to reduce the damage caused by oxidation.

DPPH Radical Scavenging Assay DPPH is a common abbreviation for an organic chemical compound 2, 2-diphenyl-1picrylhydrazyl. It is a dark-colored crystalline powder composed of stabl free-radical molecules. DPPH has two major applications, both in laboratory research: one is a monitor of chemical reactions involving radicals, most notably it is a common antioxidant assay, and another is a standard of the position and intensity of electron paramagnetic resonance signals. (Sharma and Bhat, 2009)

Allium cepa Is known as garden onion or bulb onion. Its family is a large and diverse one containing over 500 species (Simoons, et al. 1998). Alliun cepa is a biological material or great importance for mutagenicity screening of many substances. The Allium cepa Test or Allium Test is widely employed because of its simplicity, the fast growth of its roots, and the response of its genetic material to the presence of substances with cytotoxic and genotoxic potential (Smacka-kincl et al., 1996; Evadri et al., 2000). The test is routinely used around the world, at laboratories working with toxicological genetics. It provides the assessment of the degree of pollution of environments and of the toxicity caused by industrial, agricultural and domestic effluents, by determining the reduction in the mitotic index (MI) and the formation of chromosomal aberrations. (Fisjek, 1993)

Mitotic PhasesMitosis simply refers to a type of cell division in which one cell (the mother) divides to produce two new cells (the daughters) that are genetically identical to itself. However, in the context of the cell cycle, mitosis also has a narrower definition: it refers to just one part of the overall division process, the part in which the DNA of the nucleus is split into two equal sets of chromosomes.The great majority of the cell divisions that happen in your body, or in the bodies of plants, animals, fungi, and other eukaryotic organisms, involve mitosis. During development and growth, mitosis populates an organisms body with cells, and throughout an organisms life, it replaces old, worn-out cells with new ones. For single-celled eukaryotes like yeast, mitotic divisions are actually a form of reproduction, adding new individuals to the population. (Khan Academy, 2015)Mitosis consists of four basic phases: prophase, metaphase, anaphase, and telophase. Some textbooks list five, breaking prophase into an early phase (simply called prophase) and a late phase (called prometaphase). These phases occur in strict sequential order, and cytokinesis - the process of dividing the cell contents to make two new cells - starts in anaphase or telophase.

Figure 5. Cell Cycle of Mitosis (http://biology.tutorvista.com/cell/cell-cycle.html)Mitosis begins with prophase, during which chromosomes recruit condensin (large protein complexes that play a central role in chromosome assembly and segregation during mitosis and meiosis) and begin to undergo a condensation process that will continue until metaphase. In most species, cohesin (a protein complex that regulates the separation of sister chromatids during cell division, either mitosis or meiosis) is largely removed from the arms of the sister chromatids during prophase, allowing the individual sister chromatids to be resolved. Cohesin is retained, however, at the most constricted part of the chromosome, the centromere. During prophase, the spindle also begins to form as the two pairs of centrioles move to opposite poles and microtubules begin to polymerize from the duplicated centrosomes.Prometaphase begins with the abrupt fragmentation of the nuclear envelope into many small vesicles that will eventually be divided between the future daughter cells. The breakdown of the nuclear membrane is an essential step for spindle assembly. Because the centrosomes are located outside the nucleus in animal cells, the microtubules of the developing spindle do not have access to the chromosomes until the nuclear membrane breaks apart.Prometaphase is an extremely dynamic part of the cell cycle. Microtubules rapidly assemble and disassemble as they grow out of the centrosomes, seeking out attachment sites at chromosome kinetochores, which are complex platelike structures that assemble during prometaphase on one face of each sister chromatid at its centromere. As prometaphase ensues, chromosomes are pulled and tugged in opposite directions by microtubules growing out from both poles of the spindle, until the pole-directed forces are finally balanced. Sister chromatids do not break apart during this tug-of-war because they are firmly attached to each other by the cohesin remaining at their centromeres. At the end of prometaphase, chromosomes have a bi-orientation, meaning that the kinetochores on sister chromatids are connected by microtubules to opposite poles of the spindle.After, chromosomes assume their most compacted state during metaphase, when the centromeres of all the cell's chromosomes line up at the equator of the spindle. Metaphase is particularly useful in cytogenetics, because chromosomes can be most easily visualized at this stage. Furthermore, cells can be experimentally arrested at metaphase with mitotic poisons such as colchicine. Video microscopy shows that chromosomes temporarily stop moving during metaphase. A complex checkpoint mechanism determines whether the spindle is properly assembled, and for the most part, only cells with correctly assembled spindles enter anaphase.The progression of cells from metaphase into anaphase is marked by the abrupt separation of sister chromatids. A major reason for chromatid separation is the precipitous degradation of the cohesin molecules joining the sister chromatids by the protease separase. Two separate classes of movements occur during anaphase. During the first part of anaphase, the kinetochore microtubules shorten, and the chromosomes move toward the spindle poles. During the second part of anaphase, the spindle poles separate as the non-kinetochore microtubules move past each other. These latter movements are currently thought to be catalyzed by motor proteins that connect microtubules with opposite polarity and then "walk" toward the end of the microtubules.Mitosis ends with telophase, or the stage at which the chromosomes reach the poles. The nuclear membrane then reforms, and the chromosomes begin to decondense into their interphase conformations. Telophase is followed by cytokinesis, or the division of the cytoplasm into two daughter cells. The daughter cells that result from this process have identical genetic compositions. (Nature Education, 2014)

CHAPTER IIIMETHODOLOGY

Materials

50

BeakerTest tubesTest tube holderWater bathSpatulaDropper

Chemicals Used

HClMayers reagent Wagners reagent Dragendroffs reagentHagers reagentSodium HydroxideLead Acetate 1% Gelatin Solution containing NaClAcetic AnhydrideConcentrated Sulphuric AcidChloroformAmmoniaFerric ChlorideH2SO4-naphtholPhenylhydrazine HydrochlorideSodium acetateGlacial acetic acid Potassium HydroxideAqueous copper(II) sulfateHNO3CaseinEgg albuminTyrosineAcetic Acid

Collection of SamplesHandpicked leaf samples of Annona squamosa and Ficus septica was collected at Barangay Sta. Filomena, Iligan City.

Preparation of SamplesLeaves of A. squamosa and F. septica were washed, air-dried until crisp, powdered prior to extraction with 95% ethanol at room temperature for 72 hours. The process was repeated 3 times and the combined ethanol fractions were evaporated under reduced pressure using a rotary evaporator at 55C.Phytochemical ScreeningPhytochemical examinations were carried out for all the extracts as per the standard methods.

Detection of Alkaloids - Extracts were dissolved individually in dilute Hydrochloric acid and filtered.a) Mayers Test Filtrates were treated with Mayers reagent (Potassium Mercuric Iodide). Formation of a yellow colored precipitate indicates the presence of alkaloids.b) Wagners Test: Filtrates were treated with Wagners reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids. c) Dragendroffs Test Filtrates were treated with Dragendroffs reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids. d) Hagers Test Filtrates were treated with Hagers reagent (saturated picric acid solution). Presence of alkaloids confirmed by the formation of yellow colored precipitate.

Detection of Flavonoidsa) Alkaline Reagent Test Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow color, which becomes colorless on addition of dilute acid, indicates the presence of flavonoids.b) Lead acetate Test Extracts were treated with few drops of lead acetate solution. Formation of yellow color precipitate indicates the presence of flavonoids.

Detection of Tannins Gelatin Test To the extract, 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicates the presence of tannins.

Detection of Saponinsa) Froth Test Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.Detection of Steroidsa) Libermann Burchard Reaction: 2 ml extract was mixed with chloroform. To this 1-2 ml acetic anhydride and 2 drops concentrated sulphuric acid were added from the side of test tube. First red, then blue and finally green color appears.

Detection of Anthraquinonea) Borntragers Test To 3 ml extract dilute sulphuric acid was added, boiled and filtered. To the cold filtrate equal volume benzene or chloroform was added. The organic layer was separated and ammonia was added. Ammonical layer turns pink or red.

Detection of Glycosides: Extracts were hydrolysed with dil. HCl, and then subjected to test for glycosides.a) Modified Borntragers Test Extracts were treated with Ferric Chloride solution and immersed in boiling water for about 5 minutes. The mixture was cooled and extracted with equal volumes of benzene. The benzene layer was separated and treated with ammonia solution. Formation of rose-pink color in the ammonical layer indicates the presence of anthranol glycosides.b) Foam Test 0.5 gm of extract was shaken with 2 ml of water. If foam produced persists for ten minutes it indicates the presence of saponins.

Brine Shrimp Lethality Test/ Cytotoxic Activity TestPlant test extract preparation50 mg of the plant extract was dissolved in 5 mL of methanol. 0.5 mL of the above Solution A was diluted to 10 mL with methanol (Solution B). Pipette 100 L of Solution B, 50 L of Solution A and 500 L of Solution A were separated into vials, respectively labelled 1, 2 and 3. A control vial was prepared containing 1 mL of methanol. The solutions were dried in each of the above vials under nitrogen and then prepared five replicates for each dose level.

Hatching the shrimpA shallow rectangular dish (22 cm x 32 cm; a soap dish will do) was filled with the artificial sea water. A plastic divider was placed, punched with several 2 mm holes, in the dish to divide into two unequal compartments. The minute brown brine shrimp eggs were sprinkled (ca. 50 mg) into the larger compartment. The larger compartment was covered to keep away from light; the smaller compartment was left open. The smaller open compartmentwas illuminated then after 48 hours, pipette the hatched brownish orange nauplii from the illuminated compartment of the dish.Note: The brine shrimp egg shells are left in the darkened side of the dish.

BioassayCounting the naupliiPipette nauplii and was counted macroscopically in the stem of the pipette, and was held against a well lighted background.

Concentration of sample vials 1, 2 and 3Each sample vials diluted to 5 mL with artificial seawater made a final concentration of 10,100 and 1000 g/mL respectively.

With a 9 in. pipette, ten nauplii was transfered into each sample vial labelled 1, 2 and 3 and control vial. Artificial sea water was added to each vial, samples and control, to make a total volume of 5 mL. A drop of yeast suspension (3 mg/ 5 mL of sea water) was added as food in each vial. The vials were kept under illumination. The survivors were counted first after 6 hours then after 24 hours; The 24 hour count was used to record the number of deaths then determined the percent deaths for each dose level and for the control vials.In cases where control deaths occurred, the data was corrected using Abbotts formula:% deaths = (death in test vial-death in control vial) x 100 death in control vial

Free Radical Scavenging ActivitySpray reagents:Solution A 1 % iron(III) chloride aqueous solutionSolution B 1 % potassium ferricyanide aqueous solutionEqual volumes of Solution A and b were mixed before use. This spray reagent can only detect phenolic antioxidants.

Thin-layer plates were prepared and then take an equivalent of 10 g plant material from the stock plant extract prepared and were evaporated to incipient dryness over a steam bath; and were cooled to room temperature. The residue was extracted with just enough diethyl ether and drops of methanol were added so as to break down any emulsified material. Spots of 2 L were applied of the appropriate plant sample solutions and 2 L each of the standard solutions of BHA and BHT (0.05% in ethyl alcohol) to a thin layer of silica gel chromatographic plate. The chromatogram was developed in the dark in a developing chamber that has been equilibrated with n-hexane:2-butanone:n-butyl ether (34:7:6) for 30 minutes. When the solvent reaches the solvent front, the plate was removed from the tank and allow the chromatogram to stand in a stream of cold air until most of the solvent has evaporated then immediately spray the chromatogram with the spray reagent. And the sizes were compared and intensities of the sample blue spots with that of the standard.

Alternative spray reagents for chromatograms C1 and C2:Solution C consists of a 2% solution of -ocimene in hexane.Solution D1 consists of a 2% solution of iron (II) thiocyanate in 30% perchloric acid.Solution D2 consists of a 2% solution of 2.4-dinitrophenylhydrazine in 30% perchloric acid.

Alternatively develop two chromatograms, C1 and C2, containing the same plant sample and standards. The developed chromatograms was sprayed, C1 and C2, with Solution C, and then were allowed standing at room temperature for about 1 hour or heat at 50C for 10-15 minutes. Chromatogram C1 was sprayed with Solution D1. White spots in a pink background indicate positive results. The results were immediately documented because the color is very unstable. The chromatogram C2 was sprayed with Solution D2. The antioxidants are revealed as white spots on a yellow to brown background. The spots remained visible for days on inert supports.

Determination of the Eukaryotic Cell Inhibition: Antimitotic AssayOnion Collection16 pieces of the same size Onion Bulbs were bought from the market and were then separated into two variables: Four onion bulbs for the negative control (distilled water) and to the respective treatments with the concentrations of 20%, 40% and 60%.

Preparation of TreatmentsA 20%, 40%, and 60% concentration of Annona squamosa and Ficus septica extracts already mixed with distilled water was prepared separately and a 100% concentration of Annona squamosa and Ficus septica extracts and was placed in a 15 ml glass.

Cell Counting and Determination of Mitotic IndexThe assay was carried out with four sample concentrations. A 100 ml of distilled water as the negative control followed by a 20%, 40% and 60% extracts of Annona squamosa and Ficus septica with distilled water (treatment). 4 onions were exposed to each concentration. In each beaker containing the solution, an onion bulb was placed on the top of it and the roots were allowed to grow for 4 days. The test was carried out at a room temperature and the onions were kept away from direct sunlight. 5 root tips at a length of 10 mm was cut off for the cell counting of different mitotic phases and was placed in a test tube with 2 ml acetic acid and hydrochloric acid solution and heated for 3 minutes at 50C. Then the roots were placed on a slide and the terminal root tips which measure 1-2 mm are cut off for further preparation. Two drops of Benedicts Reagent was also added and mixed with the root tips. Then the cover slip was placed on the root cells and absorbed stain for 4 minutes. The cells were squashed by placing the clean absorbent fabric on the cover glass and press it slightly with the hand. Then, cell counts of different mitotic phases were counted under a compound microscope.

CHAPTER IVRESULTS AND DISCUSSIONS

Annona squamosa (Atis)

Kingdom: Plantae Phylum: Spermatophyta Class: Dicotyledonae Order: Magnoliales Family: Annonaceae Genus: Annona Species: A. squamosa

Ficus septica (Labnog)

Kingdom: Plantae Phylum: Tracheophyta Class: Magnoliopsida Order: Rosales Family: Moraceae Genus: Ficus Species: F. septica

Qualitative Determination of PhytochemicalsPhytochemical Components TestedPhytochemical ComponentsAnnona squamosaFicus septica

Flavanoids++++++

Alkaloids++++++

Tannins++++++

Saponins++++++

Steroids+++-

Anthraquinone--

Cyanogenic glucoside--

Legend:(+) slight turbidity(++) definite turbidity(+++) heavy precipitation(-) no detection

Findings exhibited that Annona squamosa contains Flavanoids, Alkaloids, Tannins, Saponins and Steroids while Ficus septica contains Flavanoids, Alkaloids, Tannins and Saponins only.Alkaloids are an amazing group of phytoprinciples. Some alkaloids, such as nicotine, are used in pesticides, and others are used as chemical reagents. The primary use of alkaloids, however, is in medicine, because they can act quickly on specific areas of the nervous system. Alkaloids have potent anticancer activity against various cancers (Mohan et al., 2011).Saponins are glucosides with foaming characteristics. Studies have shown that saponins haveantitumorandanti-mutagenicactivities and canlower the risk of human cancers, by preventing cancer cells from growing. Saponins seem to react with the cholesterol rich membranes of cancer cells, thereby limiting their growth and viability. Studies have also shown that saponins can cause apoptosis ofleukemiacells by inducing mitotic arrest (Top Cultures, N.D.).Flavonoids are plant-based compounds with powerful antioxidant properties found in many fruits and vegetables like blueberries and grapes. They serve a variety of functions such as protecting blood vessel walls in people who have heart disease or diabetes, alleviating allergies, protecting brain health against dementia and even preventing some cancers. Flavonoids or bioflavonoids another word for the same compounds havemedicinalproperties that include the ability to defend against cancer, viruses, not to mention anti-microbial, antihistamine and anti-inflammatory characteristics (Mother Nature Network, 2015).Steroids on the other hand were developed as medical treatments and they come in two varieties. Anabolic steroids are the kind you hear about the most. They behave like male sex hormones, and doctors prescribe them for treating problems like late puberty as well as significant muscle loss in patients with cancer and AIDS (Kristen Philipkoski, 2012).Table 1. Cytotoxic Activities of A. squamosa and F. septica leaf extracts within 6 and 24 hoursSampleLC50 (6 hours)LC50 (24 hours)Interpretation

Annona squamosa137.08 ppm192.34 ppmActive (Toxic)

Ficus septica105.76 ppm126.59 ppmActive (Toxic)

As presented in Table 1, the leaf extracts of both Annona squamosa and Ficus septica exhibited cytotoxic activity. This implies that both were found to be toxic.

Table 2. Mitotic activities in Allium cepa root cells treated with F. septicaFormul-ationProphaseCellsMetaphaseCellsAnaphaseCellsTelophase cellsTotal dividing cellsTotal cells countedMitotic Index

Control128102116174520127640.75

T1 (20%)301242115332886238.05

T2 (40%)36294412423374631.23

T3 (60%)30863711526895428.09

T4 (100%)1126116411262118.03

Table 3. Mitotic activities in Allium cepa root cells treated with A. squamosaFormul-ationProphaseCellsMetaphaseCellsAnaphaseCellsTelophase cellsTotal dividing cellsTotal cells countedMitotic Index

Control1439398152486121639.96

T1 (20%)25682210221791523.71

T2 (40%)937135611560119.13

T3 (60%)1513527415484518.22

T 4 (100%)172512399371113.08

Graph 1. Mitotic Activities of A. squamosa and F. septica leaf extracts

FORMULATIONMITOTICINDEX

Tables 2 and 3 exhibited the data on the mitotic activities observed in onion root cells treated with Annona squamosa and Ficus septica leaf extracts. The mitotic index values of each leaf extracts was obtained by dividing the total dividing cells to the total cells counted and was multiplied to 100. The mitotic index values were lowered in the treated onion root cells when compared with the control, and the mitotic index values were observed to be decreasing with increasing concentrations of the Annona squamosa and Ficus septica leaf extract.The decreased mitotic index values in the treated onion roots is an indication of the presence of cytotoxic substances in the Annona squamosa and Ficus septica leaf extracts, which caused inhibition of mitotic activities.Table 4. Chromosomal aberrations an Allium cepa root cells treated with F. septica leaf extract.Formul-ationTotalcellscountedTotal dividingCellsSmall and big frag-mentsBridge and two frag-mentsLaggard Chromo-someVagrant Chromo-someTotal aber-rant cellsAber-ration inci-dence

Control1276520000000

T1 (20%)862328000000

T2 (40%)746233012031.28

T3 (60%)954268213282.98

T4 (100%)6211122521108.92

Table 5. Chromosomal aberrations an Allium cepa root cells treated with A. squamosa leaf extract.Formul-ationTotalcellscountedTotal dividingCellsSmall and big frag-mentsBridge and two frag-mentsLaggard Chromo-someVagrant Chromo-someTotal aber-rant cellsAber-ration inci-dence

Control1216486000000

T1 (20%)915217000000

T2 (40%)601115000000

T3 (60%)845154112153.24

T4 (100%)71193231177.52

Tables 4 and 5 exhibited that chromosome aberrations observed includes small and big fragments, bridge and two fragments, laggard chromosome, vagrant chromosome and chromosome breakages. The aberration incidence of each leaf extracts was obtained by dividing the total aberrant cells to the total dividing cells and was multiplied to 100. The number of aberrant cells was also observed to be increasing with the concentration of the Annona squamosa and Ficus septica leaf extracts. The observation of aberrant cells in the treated onion root tip indicates genotoxic effects of the leaf extracts.

Table 6. Antioxidant activity of A. squamosa using the DPPH methodppm extractMicrogramextractVol. used mLConc. Of DPPH, mMVol. of DPPH, mLFinal vol., mLNew conc., ppmAve. abs reading at 517 nmControl at 517 nm%FRSAC

103030.12560.0570.38185.04

206030.125120.0460.38187.93

309030.125180.0390.38189.76

4012030.125240.0330.38191.34

5015030.125300.0260.38193.18

Table 7. Antioxidant activity of F. septica using the DPPH methodppm extractMicrogramextractVol. used mLConc. Of DPPH, mMVol. of DPPH, mLFinal vol., mLNew conc., ppmAve. abs reading at 517 nmControl at 517 nm%FRSAC

103030.12560.3680.3813.41

206030.125120.3490.3818.40

309030.125180.340.38110.76

4012030.125240.3330.38112.60

5015030.125300.3290.38113.65

Table 8. Antioxidant activity of Vit. C using the DPPH method (Positive control)ppm extractMicrogramextractVol. used mLConc. Of DPPH, mMVol. of DPPH, mLFinal vol., mLNew conc., ppmAve. abs reading at 517 nmControl at 517 nm%FRSAC

103030.12560.3130.3346.29

206030.125120.2950.33411.68

309030.125180.2530.33424.25

4012030.125240.2090.33437.57

5015030.125300.0090.33497.31

The antioxidant activity was determined using the DPPH method that stands for Diphenyl Picrylhydrazyl. There are assorted methods in determining the antioxidant activity however; scavenging of DPPH free radical is the common basis for antioxidant assay. Antioxidants play an important role in human health, scientist have said that antioxidants can lessen the issue of chronic diseases such as cancer and heart disease. Plants sourced antioxidants like Vitamin C that has the potential to reduce disease risk. (Tailor Chandra, 2014)

The percentage of Free Radical Scavenging Activity was also observed to be increasing along with the concentration of the Annona squamosa and Ficus septica leaf extracts. The observation of the increased percentage of FRSAC indicates stronger antioxidant activity.

CHAPTER VCONCLUSION AND RECOMMENDATIONS

ConclusionPhytochemical Findings exhibited that Annona squamosa contains Flavanoids, Alkaloids, Tannins, Saponins and Steroids while Ficus septica contains Flavanoids, Alkaloids, Tannins and Saponins only which means that both leaf extracts have useful bioactive compounds. The brine shrimp lethality test results also implies that both were found to be toxic and could be preceded for further studies like anti-microbial and anti-viral. Both leaf extracts also exhibited decreased mitotic index by the use of Allium cepa chromosome aberration assay which is an indication of the presence of cytotoxic substances and which also caused inhibition of mitotic activities. The Free Radical Scavenging Activity exhibits that the Annona squamosa and Ficus septica have antioxidant activity using the DPPH method.Therefore, both Annona squamosa and Ficus septica leaf extracts exhibited an active substance and is toxic. But among the two leaf extracts, results showed that Annona squamosa leaf extract have stronger cytotoxic activity compared to the Ficus septica leaf extract. Thus, it could be a promising substance in the form of medicine for possible treatment for anti-tumor and anti-cancer.

RecommendationsThe researcher cited the following recommendations for further related studies:1. Investigate the possibility or potency to produce alternative form of antimicrobials and anti diabetic in the leaf extracts.2. The results suggest that more specific bioassays should be performed on those plant extracts in order to confirm the conclusions.

BIBLIOGRAPHY

Online JournalsBurman, N.L. (1768). Ficus septica. Australian Tropical Rainforest Plants. Retrieved from: http://keys.trin.org.au/key-server/data/0e0f0504-0103-430d-8004-060d07080d04/media/Html/taxon/Ficus_septica.htmDray, T. (2015). What are the Health Benefits of Sweetsop?. Livestrong.com. Retrieved from: http://www.livestrong.com/article/549365-what-are-the-health-benefits-of-sweetsop/Martinez, F. M., Miranda, D.L., Magnitskiy, S. (2013). Anatomy of sugar apple (Annona squamosa L.) seeds (Annonaceae). Agronomia Colombiana. Retrieved from: http://www.scielo.org.co/scielo.php?pid=S0120-99652013000300003&script=sci_arttextMorton, J. (1987). Sugar Apple Annona squamosa. Fruits of Warm Climate. Department of Horticulture & Landscape Architecture, Purdue University. p.69. Retrieved from https://hort.purdue.edu/newcrop/morton/sugar_apple.html

PDFsMoe, A.M., Weiblen G.D., (2012). Pollinator-Mediated Reproductive Isolation among Dioecious Fig Species (Ficus, Morecaeae). doi: 10.1111/j.1558-5646.2012.01727.xShanahan, M., Compton, S. G., So, S. & Corlett, R. (2001). Fig-eating by vertebrate frugivores: a global review. Biological Reviews 76 (4). 529-72. doi: 10.1017/S1464793101005760. PMID 11762492Sharma, O.P., Bhat, T.K. (2009). DPPH Antioxidant Assay Revisited. Food Chemistry. doi: 10.1016/j.foodchem.2008.08.008Vijayalakshmi, K., Umadevi, K. J., & Vanitha, V. (2011). Determination of Bioactive Components of Annona squamosal L Leaf by GC-MC Analysis. International Journal of Pharmaceutical Sciences and Drug Research. ISSN 0975-248X

WebsitesAntioxidants. (n.d.) Retrieved from: http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles.nsf/pages/antioxidantsAntioxidants and Free Radicals. (n.d.) Retrieved from: http://www.rice.edu./~jenky/sports/antiox.htmlCanadian Cancer Society. (n.d.). Retrieved from: http://www.cancer.ca/en/cancer-information/cancer-101/what-is-cancer/types-of-tumours/?region=onJohns Hopkins University. (n.d.). Retrieved from: http://pathology.jhu.edu/pc/BasicTypes1.php?area=ba

Khan Academy. (2015) Retrieved From: https://www.khanacademy.org/science/biology/cellular-molecular-biology/mitosis/a/phases-of-mitosisNature Education. (2014) Retrieved from: http://www.nature.com/scitable/topicpage/mitosis-and-cell-division-205Stuartxchange. (n.d.). Retrieved from: http://www.stuartxchange.com/Atis.htmlStuartxchange. (n.d.). Retrieved from: http://www.stuartxchange.com/Hauili.htmlWikipedia. (2015). Retrieved from: https://en.wikipedia.org/wiki/Annona_squamosa#cite_ref-FNA_8-6Wikipedia. (2015) Retrieved from: https://en.wikipedia.org/wiki/Ficus_septicaWikipedia. (2015). Retrieved From: https://en.wikipedia.org/wiki/Radical_(chemistry

APPENDIX

Phytochemical screening

Boiling of water

Chemicals used

Ficus septica (Labnog)

Annona squamosa (Atis)

Allium cepa test

Preparation of Onion root tip

Preparation of Onion root tip

Squashing of onion root tips

Visualization using the light microspe