Anti-angiogenic Activity Of Musa Sapientum (banana) Peel Induced Leukemia In 10-day Old Duck Broiler Embryo.docx

Anti-angiogenic Activity of Musa Sapientum (Banana) Peel induced leukemia in 10-day Old Duck Broiler Embryo

Daquigan, Venneth S. Dela Cerna, Edsil Liga, Fretziel Mae E. Lambayan, Rachel Ann G. Dela Torre, Ahrmina B. Baltazar, Madelyn B.

Chapter I A. Introduction Angiogenesis is the process of the formation of new blood vessels and preconditioning can promote cancer progression and metastasis to nutrient and oxygen supply including waste removal (Seow et al. 2010; Hazel 2003; El-Kinawi and ElRemessy 2013). Increasing welfares of Anti-Angiogenic Activity are considered auspicious approaches for cancer therapy (Adini et al. 2009 and Seow et al.2010). many in vivo models can be used to screen potential antagonists and agonists of angiogenesis which include rats, mice and rabbit corneal pocket assays (Chen et al. 1995),the human or mouse chimeric angiogenesis assay (Brooks et al. 1995), and the chorioallantoic membrane (CAM) aasay (Nguyen et al. 1994), or the vascularization of chorioallantoic membrane in chicken embryo chick embryo (Hazel 2003). Chorioallantoic membrane (CAM) of the chick (Gallus domesticus) embryo is fast economical, and a widely used tool for both angiogenesis and anti-angiogenic activity of various drugs including plant crude extracts (Peng et al. 2009; Seow et al. 2010). The important advantage of this systemis that it allows inhibitors of angiogenesis to interfere with the development of new blood vessels without interfering with the growth of preexisting blood vessels. The success of the anti-angiogenic agents for cancer research therapy (Mathur et al. 2006; He at al. 2009; Seow et al. 2010). Pharmacological industries believed that traditional herbal plants will be the focus of attraction in international scientific communities as a source of powerful of bio-active substances. It is inexpensive , safe, and readily available medicinal products that have long been documented with its useful pharmacological properties and understanding its importance enables scientists venture more on the medical services it could offer (Catherine and Pravabathi-Nagarajan 2011). Many plant extracts showed to have antiangiogenic properties e.g. Nelumbo nucifera (Lee et al. 2015), Benincasa hispida(Lee et al.,2005), Saururus chinensis (Yoo et al. 2008), Gastrodia elata Blume (Ahn et al. 2007), Tripterygium wilfordii (He et al. 2009), Acer mono, Adenophora racemose, Caryopteris Incana, Cinnamomum japonicum, Eurya japonica and Reynoutria sachalinensis (Sei et al. 2013) and one of which is Musa Sapientum peel (Jenie and Meiyanto 2006; Muo and Dash 2016; Tan et al. 2016).

According to Husain and William (2010) Banana is a tropical fruit grown in over 122 countries worldwide. Until 2004, the cultivated area of 3.8 million hectares and a total production of 56.4 million metric tones of the fruit were produced ranking it fourth behind rice, corn and milk (Chai et al., 2004; Arumugam and Manikandan, 2011). In recent times, Banana peel has been utilized for various industrial applications including bio-fuel production, bio-sorbents, pulp and paper, cosmetics, energy related activities, organic

fertilizer, environmental clean up and biotechnology related processes (Morton, 1987; Gunaseelan, 2004; Bori et al., 2007). Bananas are naturally slightly radioactive, more so than most other fruits, because of their potassium content and the small amounts of the isotope potassium-40 found in naturally occurring potassium (Amarnath and Balakrishnan, 2007).

Ripe bananas fluoresce when exposed to ultraviolet light. The study suggested that this allows animals which can see light in the ultraviolet spectrum (tetrachromats and pentachromats) to more easily detect ripened bananas (Ashraf et al., 2010). This property is attributed to the degradation of chlorophyll leading to the accumulation of a fluorescent product in the skin of the fruit. The chlorophyll breakdown product is stabilized by a propionate ester group (Anhwange et al., 2009). Banana-plant leaves also fluoresce in the same way (Ashraf et al., 2010). Green bananas do not fluoresce (Bhat et al., 2010a).

All parts of the banana plant have medicinal applications (Amit and Shailandra, 2006): the flowers in bronchitis and dysentery and on ulcers; cooked flowers are given to diabetics; the astringent plant sap in cases of hysteria, epilepsy, leprosy, fevers, hemorrhages, acute dysentery and diarrhea, and it is applied on hemorrhoids, insect and other stings and bites; young leaves are placed as poultices on burns and other skin afflictions; the astringent ashes of the unripe peel and of the leaves are taken in dysentery and diarrhea and used for treating malignant ulcers (Girish and Satish, 2008); the roots are administered in digestive disorders, dysentery and other ailments; banana seed mucilage is given in cases of diarrhea in India (Bhat et al., 2010a). Antifungal and antibiotic principles are found in the peel and pulp of fully ripe bananas (Brooks, 2008). The antibiotic acts against Mycobacteria (Omojasola and Jilani, 2009). A fungicide in the peel and pulp of green fruits is active against a fungus disease of tomato plants (Ponnuswamy et al., 2011). Norepinephrine, dopamine, and serotonin are also present in the ripe peel and pulp (Ratule et al., 2007). The first two elevate blood pressure; serotonin inhibits gastric secretion and stimulates the smooth muscle of the intestines (Anhwang et al., 2009).

Some of the specific diseases known to be cured by banana are Anaemia: High in iron, bananas are believed to stimulate the production of haemoglobin in the blood and so helps in cases of anaemia (Amit and Shailandra, 2006). Blood Pressure: Banana is extremely high in potassium yet low in salt, making it the perfect food for helping to beat blood pressure (Debabandya et al., 2010). Depression: This is because bananas contain tryptophan, a type of protein that the body converts into serotonin known to make you relax, improve your mood and generally make you feel happier (Girish and Satish, 2008). Heartburn: Bananas have a natural antacid effect in the body so if you suffer from heartburn, try eating a banana for soothing relief (Mokbel et al., 2005). Morning Sickness: Snacking on bananas between meals helps to keep blood sugar

levels up and avoid morning sickness (Amit and Shailandra, 2006). Mosquito bites: Before reaching for the insect bite cream, try rubbing the affected area with the inside of a banana skin. Many people find it amazingly successful at reducing swelling and irritation (Odebiyi and Sofowora, 1978). Nerves: Bananas are high in B vitamins that help calm the nervous system (Singh and Bhat, 2003). Smoking: Bananas can also help people trying to give up smoking, as the high levels of Vitamin C, A1, B6, B12 they contain, as well as the potassium and magnesium found in them, help the body recover from the effects of nicotine withdrawal (Mokbel et al., 2005). Stress: Potassium is a vital mineral, which helps normalize the heartbeat, sends oxygen to the brain and regulates the body’s water-balance (Girish and Satish, 2008).

Strokes: eating bananas as part of a regular diet can cut the risk of death by strokes by as much as 40% (Amit and Shailandra, 2006). Temperature control: Many other cultures see bananas as a cooling fruit that can lower both the physical and emotional temperature of expectant mothers (Mokbel et al., 2005). Ulcers: The banana is used as the dietary food against intestinal disorders because of its soft texture and smoothness (Girish and Satish, 2008). And Warts: Those keen on natural alternatives swear that, if you want to kill off a wart, take a piece of banana skin and place it on the wart, with the yellow side out (Amit and Shailandra, 2006). Alleged hallucinogenic effects of the smoke of burning banana peel have been investigated scientifically and have not been confirmed (Anhwange et al., 2009). It has been observed that antimicrobial activity of the plants is associated with the presence of some chemical components such as phenols, tannis, saponins, alkaloids, steroids, flavonoids and carbohydrates (Singh and Bhat, 2003).

B. Statement of the Problem This study seeks to determine the blood vessels of the 10-day old broiler duck embryo using anti-angiogenic activity of banana peel. The study are going to test to results on banana peel to lessen the growth of blood vessels. Its distinctively pursue to answer the following: General Problem Does Musa balbisiana peels extract are capable to be an anti-angiogenic agent? Specific Problems 1. What are the health benefits of banana peel in terms of curing cancer? 2. Is the banana peel recommended by the experts? 3. Does the banana peel accepted by the respondents?

C. Significance of the Study If proven, this investigation will benefit people because of the anti-angiogenic capabilities of saba. Considering saba can be found commonly in the provinces, this will be beneficial to the people who lives in the rural areas where some people do not have the access to pharmacies. This treatment can easily be made because people will only need to dehydrate the banana peels under the sun and pound it to turn it into powder. It will be a cheaper option to cure leukemia because they would only have to cultivate a plant that is easily found in their area. Thus, this will also be a way to reduce throwing away banana peels as a waste. There will be no presence of harmful chemicals in this traditional medicine because it is natural. Therefore, possible health damage because of wrong usage will not occur. Whereas this research is only for a probable cause and is not definitive, this will be a basis for future studies on the plant which may continue. This study also benefit the following: STUDENTS – Students may use this research as their related study in their future study. It may also use as an article in their school works. TEACHERS – Teachers may use this study as their basis in teaching medical or biology subject. FUTURE RESEARCHERS – This study may use by the future researchers by means of citation and basis in their medical research.

D. Scope and Delimitation The study aimed to test the capability of Musa balbisiana as anti-angiogenic agent. It aims to find out if banana peels can be used as a medicine, and not just a waste in the area. However, it cannot be tested on human subjects. The plant sample is limited due to its scarcity in the City of San Pedro, Province of Laguna. It would also be required to have a large amount of ripe banana peels to have an ample supply for the experiment. E. Hyphothesis Ho: Ha: F. Rationale This study focuses on the belief that banana peel can induced leukemia in 10day old broiler duck embryo. The researchers wanted to give cognizance to the future readers to switch in a better healthy living and researchers also want to change the abstraction of the banana peel.

G. Definition of Terms Antagonist- A substance that acts against and blocks an action. Are key players in the chemistry of the human body and in pharmacology. (MedicineNet 2017) Agonist- A substance that acts like another substance and therefore stimulates an action. . Are key players in the chemistry of the human body and in pharmacology. (MedicineNet 2017) Auspicious- Being just the same as the apertures of the human body. (Hsu 1970) Metastasis- The process by which cancer spreads from the place at which it first arose as a primary tumor to distant locations in the body. (MedicineNet 2017) Hallucinogens- work by altering serotonin levels in the brain. Serotonin, a primary neurotransmitter chemical, regulates Mood states, Arousal, Cognition, Stress responses. (National Institute on Drug Abuse) Hemorrhoids- also known as piles, are swollen veins in the lower part of the anus and rectum. When the walls of these vessels are stretched, they become irritated. (UIC 2017)

Chapter II: Review of Related Literature This Chapter presents the related literature and studies after the thorough and indepth search done by the researchers. This will also present the synthesis of the study to fully understand the research to be done. Related Literature Banana (Musa x paradisiaca L., Musaceae) represents one of the most important fruit crops, with a global annual production of more than 50 million tons. Peels constitute up to 30% of the ripe fruit. About 1000 banana plants are estimated to yield 20–25 tons of pseudostems providing about 5% edible starch (Anand and Maini, 1997). Attempts at utilization of banana waste include the biotechnological production of protein (Chung and Meyers, 1979), ethanol (Tewari et al., 1986), α-amylase (Krishna and Chandrasekaran, 1996), hemicellulases (Medeiros et al., 2000) and cellulases (Krishna, 1999). Very recently, anthocyanin pigments in banana bracts were evaluated for their potential application as natural food colorants and were found to be a good source of anthocyanins (Pazmino-Duran et al., 2001). Most of the carotenoids found in banana peels were demonstrated to be xanthophylls.

Are considered among the first fruits to be harvested by primitive agricultures and have been present in diverse cultures and civilizations for centuries. Bananas originated in Southeast Asia, including northern India, Cambodia, Sumatra, Java, the Philippines,

and Taiwan. In the sixteenth century, the banana cultivar was introduced in the islands of Santo Domingo and Cuba. At the end of the nineteenth century, the first commercial plantations were established in Jamaica, and thereafter in diverse countries of Central America and Mexico. Banana is a climateric fruit cultivated in many countries, primarily those located in the tropical and subtropical regions (approximately 120–130 countries), and it represents a major staple. Annual world production of banana is approximately 104 millions tons. The main producers of banana are Brazil, China, Ecuador, the Philippines, and India. The leading exporters of banana are Ecuador, Colombia, Costa Rica, and the Philippines. Edible Musa plants are classified into AA, AB, BB, AAA, AAB, ABB, AAAA, AAAB, and ABBB genomic groups. In general, dessert banana cultivars are AA or AAA, with this last group including almost all bananas sold. Cooking bananas, also named

plantains, are predominantly AAB, ABB, or BBB. The great biodiversity of banana plants provides potential for diverse uses and applications (Aurore et al., 2009). Unripe banana contains large amounts of starch, cellulose, hemicellulose, and lignin in the pulp. The nutritional/nutraceutical potential of unripe banana starch and fiber has been reported (Langkilde et al., 2002). Bananas and plantains belong to the section Eumusa of the genus Musa. Many authors use the Linnaean classification, e.g. Musa paradisiaca, M. x paradisiaca and M. sapientum and many others. However, Cheesman (1947–1949) defined the two species, M. balbisiana and M. acuminata as the basis for almost all cultivated bananas and plantains. Simmonds and Shepherd (1955) and Stover and Simmonds (1987) confirmed this, and reported that many edible varieties are derived from M. acuminata, some being diploid and a few being tetraploid but most being triploid. M. balbisiana has also contributed to the origin of edible bananas and plantains by hybridization with M. acuminate. They recommended that in place of the species name, an A genome or a B genome should be used showing the origin and contribution of the two species. So a triploid variety whose origin is M. acuminate, e.g. the Giant Cavendish and Gros Michel varieties, would be referred to as Musa AAA. Where the triploid has one-third M. bulbisiana and two-thirds M. acuminate, as in most plantain varieties, it would be referred to as Musa AAB. The chemistry of these phytoconstituents suggests that the functional groups such as amide, methyl, and hydroxyl adjacent to different other heteroatoms enhance the medicinal potency. Similarly, conjugated double bonds also play active role to make these constituents biologically active. In addition to tumor progression, uncontrolled angiogenesis is a major contributor to a number of diseases such as, inflammatory disorders, obesity, asthma, diabetes,

proliferativeretinopathies, multiple sclerosis, psoriasis, endometriosis, AIDS, bacterial infections, atherosclerosis, rheumatoid arthritis and autoimmune disease, due to its ability to supply oxygen and nutrients to the pathological ischemic area. Therefore, modulation of angiogenesis is considered to be a therapeutic strategy of great importance for human health. VEGFR-2 is believed to be the most important regulator of angiogenesis, mediating the majority of angiogenic effects of VEGF-A, including enhanced vascular permeability, endothelial cell proliferation, invasion, migration and survival. As several literature reports indicated VEGFR-2 as a promising target for antiangiogenesis, it was selected as the target for the study.

Chalcone was selected as the backbone structure for the present study. All of the features of the active site of VEGFR -2 were taken into consideration during design of the chalcone analogues as angiogenesis inhibitors. Literature reports were referred for design of VEGFR-2 inhibitor using molecular modeling techniques, synthesis of 1,3diarylpropenone analogues and pharmacological evaluation of synthesized analogues by CAM assay for anti-angiogenic activity.

Related Studies (Tribological evaluation MBS oil was conducted using pin on disc tribometer which is a bio-oil extracted from banana peel waste of Musa Aluminata Balbisiana.) This review critically assessed the phytochemical properties and biological activities of Musa spp fruit pulp and peel. Banana is a fruit with nutritional properties and also with acclaimed therapeutic uses, cultivated widely throughout the tropics as source of food and income for people. Banana peel is known by its local and traditional use to promote wound healing mainly from burns and to help overcome or prevent a substantial number of illnesses, as depression. A survey on the literature on banana (Musa spp, Musaceae) covering its botanical classification and nomenclature, as well as the local and

traditional use of its pulp and peel was performed. Besides, the current state of art on banana fruit pulp and peel as interesting complex matrices sources of high-value compounds from secondary metabolism was also approached. The biological potential of those biomasses is directly related to their chemical composition, particularly as provitamin A supplementation, as potential antioxidants attributed to their phenolic constituents, as well as in the treatment of Parkinson’s disease considering their contents in l-dopa and dopamine. Banana’s pulp and peel can be used as natural sources of antioxidants and pro-vitamin A due to their contents in carotenoids, phenolics, and amine compounds, for instance. For the development of a phytomedicine or even an allopathic medicine, e.g., banana fruit pulp and peel could be of interest as raw materials riches in beneficial bioactive compounds

The invention mentions Chalcone and Chalcone derivatives as angiogenesis inhibitors. The patent includes inexpensive synthesis of propenone, 3,6-diphenyl-2cyclohexenone, indandione, tetralone, propynone, benzylideneacetone, 1cycloalkylpropenone, camphor, stilbene, benzoyldihydronaphthylene, etc derivatives and determination of anti-angiogenic activity by SVR cell growth inhibition assay

(Bowen et al, 2001). These four patents (US 6,462,075, US 6,906,105 B2, US 7,432,303 and WO 01046110) were reviewed to ensure patent non infringing approach in synthesis of chalcone analogues Further the angiogenesis inhibitors of natural origin are reported to inhibit the key events of the angiogenic process such as proliferation, migration, and tube formation of endothelial cells; expression of vascular endothelial growth factor (VEGF) (Ferrara et al, 2004); and activation of MMPs, PI3K/Akt signaling pathway (Mojzisa et al, 2008), etc. Chalcones, the derivatives of phenylpropanoids are precursors of the flavonoids in higher plants and display a wide variety of pharmacological effects, including anti-proliferative and anticancer activities. One of the mechanisms of the anticancer activity of chalcone is suppression of angiogenesis. In a study, chalcone inhibited the proliferation of human breast cancer cell lines by inducing apoptosis and blocking cell cycle progression in the G2/M phase (Hsu et al, 2006). Potential anti-angiogenic effects of xanthohumol and isoxanthohumol, chalcones isolated from hop plant (Humulus lupulus L.) were studied under in vitro conditions (Albini et al, 2005). A licochalcone E, a new retrochalcone isolated from the roots of Glycyrrhiza inflata, was found to be an inducer of apoptosis in endothelial cells (Kim et al, 2010). Anti-angiogenic effect of 2-Hydroxy-4-methoxychalcone was observed under in vitro as well as in vivo conditions using chick embryosin the CAM assay and basic

fibroblast growth factor (bFGF) induced vessel formation in the mouse Matrigel plug assay (Lee et al, 2006). Hydroxychalcones were tested for melanoma cell line (B16F10) where they induced apoptosis in B16-F10 melanoma cells via GSH and ATP depletion (Lilian et al, 2009) Chalcones and their analogues extracted from the twigs of Dorstenia barteri were investigated for their capacity to inhibit matrix metalloproteinase (MMP)-2 secretion from brain tumour derived U87 glioblastoma cells (Ngameni et al, 2007). Methoxy chalcones inhibited certain steps of angiogenesis including cell migration, micro capillary tube formation, and MMP expression (Mojzisa et al, 2008). 1Furan-2-yl-3-pyridin-2-ylpropenone was found to inhibit the invasion and migration of HT1080 human fibrosarcoma cells by inhibiting MMP-2 and MMP-9 modulating the process of angiogenesis (Park et al, 2009). Therefore, Chalcone (1,3-diarylpropenone) was selected as the backbone for this study. Also, this extensive literature survey revealed that no work has been done on the molecules which are under consideration in the study

The study has been aimed to evaluate and compare phytochemical content and the antioxidant activity in peel extracts of nine local varieties of banana, i.e. Musa sapientum species. Ethanolic extract of peels of these varieties were subjected to in

vitro free radical scavenging assays like DPPH, ABTS and lipid peroxidation inhibition assay. Total antioxidant capacity assay to confirm the antioxidant potential and phytochemical content such as total phenols, flavonoids were also determined. The results obtained were analyzed statistically by ANOVA and DMRT analysis. The peel extracts of all the nine varieties of banana exhibited significant antioxidant and phytochemical activities with Musa spp – Blueggoe (Monthan) - AAB and Musa spp – Rasthali – AAB showing highest free radical scavenging activity and Musa spp – Karpooravalli – ABB, Musa spp – Rasthali – AAB, Musa spp - Ney Poovan (Kadali) – AB and Musa spp – Mysore (Poovan) – AAB having highest phytochemical content. The study suggests that peel extracts of these banana varieties could be useful to combat free radical mediated diseases.

Definition of Banana Banana is a long curve with a thick peel that comes in a variety of sizes and colors when ripe, including yellow, purple and red. Health Benefits of Banana Bananas are good for your heart. They are packed with potassium, a mineral electrolyte that keeps electricity flowing throughout the body, a requirement to keep your heart beating. Bananas’ high potassium and low sodium content may also help to protect your cardiovascular system against high blood pressure, according to FDA. Chemical Content of Banana Musa sapientum peels were analysed for minerals, nutritional and anti – nutritional contents. The result of mineral content indicate the concentrations (mg/g) of potassium, calcium, sodium, iron, manganese, bromine, rubidium, strontium, zirconium and niobium to be 78.10, 19.20, 24.30, 0.61, 76.20, 0.04, 0.21, 0.03, 0.02 and 0.02 respectively. The percentage concentrations of protein, crude lipid, carbohydrate and crude fibre were 0.90, 1.70, 59.00 and 31.70 respectively. The results indicate that if the peels are properly exploited and process, they could be a high-quality and cheap source of carbohydrates and minerals for livestock. (Electronic Journal of Environmental, Agricultural and Food Chemistry 2009)

Traditional Uses of Banana with

Bananas contain three natural sugars - sucrose, fructose and glucose combined fiber.A banana gives an instant, sustained and substantial boost of

energy.Research has proven that just two bananasprovide enough energy for a strenuous 90-minute workout. No wonder the banana is the number one fruit with the world's leading athletes. But energy isn't the only way a banana can help us keep fit. It can also help overcome or prevent a substantial number of illnesses and conditions, making it a must to add to our daily diet. • Depression: According to a recent survey undertaken by MIND amongst people suffering from depression, many felt much better after eating a banana. •This is because bananas contain tryptophan, a type of protein that the body converts into serotonin, known to make you relax, improve your mood and generally make you feel happier. • PMS: Forget the pills - eat a banana. The vitamin B6 it contains regulates blood glucose levels, which can affect your mood. • Anemia: High in iron, bananas can stimulate the production of hemoglobin in the blood and so helps in cases of anemia. • Blood Pressure: This unique tropical fruit is extremely high in potassium yet low in salt, making it perfect to beat blood pressure. So much so, the US Food and Drug Administration has just allowed the banana industry to make official claims for the fruit's ability to reduce the risk of blood pressure and stroke. • Brain Power: 200 students at a Twickenham (Middlesex) school were helped through their exams this year by eating bananas at breakfast, break, and lunch in a bid to boost their brain power. • Research has shown that the potassiumpacked fruit can assist learning by making pupils more alert. • Constipation: High in fiber, including bananas in the diet can help restore normal bowel action, helping to overcome the problem without resorting to laxatives. • Hangovers: One of the quickest ways of curing a hangover is to make a banana milkshake, sweetened with honey. The banana calms the stomach and, with the help of the honey, builds up depleted blood sugar levels, while the milk soothes and rehydrates your system. • Heartburn: Bananas have a natural antacid effect in the body, so if you suffer from heartburn, try eating a banana for soothing relief. • Morning Sickness: Snacking on bananas between meals helps to keep blood sugar

levels up and avoid morning sickness. • Mosquito bites: Before reaching for the insect bite cream, try rubbing the affected area with the inside of a banana skin. • Many people find it amazingly successful at reducing swelling and irritation. Description of Musa balbisiana Musa balbisiana is one of the wild ancestors of the cultivated plantain. Plantain, which is a hybrid between Musa acuminata and Musa balbisiana, is much starchier and less sweet than dessert bananas which are mainly bred from Musa acuminata. Chemical Content of Musa balbisiana Traditional Uses of Musa balbisiana Modern Research and Uses of Musa Balbisiana

Chapter III : Materials and Methods METHODOLOGY A. Research Design This experimental study determines the angiogenic effect of M. Sapientum to the chorioallantonic membrane (CAM) of a 10-day old broiler duck embryo. It utilized Randomized Complete Block Design (RCBD) consisting of subjects that were arranged in 8 treatments: To control group, T1 (100ppt), T2 (300ppt), and T3 (500ppt) for GP crude plant extract and T4 (100ppt + benzene solution (10%)), T5 (300ppt + benzene solution (10%)), and T6 (500ppt + benzene solution (10%)), T7 (benzene solution (10%)). Each treatment was replicated 3 times.

Research Procedure Test Eggs (Haddad and Vedar 2009) The collection and incubation of 72 pieces of 10-day old duck eggs were brought at Victoria located at Laguna near Calamba. Shoot Extraction of M. Sapientum (Gopez and Madrid 2012) The peels of MS were extracted separately using rotary evaporation. Approximately 200 grams of MS peels were washed and air dried, powdered by a homogenizer and was soaked in 95% ethanol for 48 hours then it was filtered using cheesecloth and further with the filter paper. The filtrate collected was subjected to the rotary evaporator to be able to collect the shoot crude extract.

Preparation of Concentration (Bunda and Octavio 2009) In preparing the desired concentration for the first treatment of the sample, 1 ml/L of each extract was diluted in separate test tubes including the additional water using the formula:

C1 x V1 = C2 x V2 The dilution factor was obtained by the difference between the final and the initial volume. The same procedure was used to prepare the 2 nd and the 3rd concventrations. Administrations of the crude shoot extract (Bunda and Octavio 2009) Incubated eggs (37oC and 70% humidity) were administered with the crude extracts through the use of sterile needle, the eggs were carefully punctured on the animal pole to make sure that the CAM was not damaged. 0.1 mL of benzene solution (10%) was first administered to the eggs following the 0.3 mL of the crude extract using a 1ml syringe with a 26-gauge needle. A drop of melted candle will be use to reseal the egg shell so as to avoid rotting and contamination. Each concentration used one different syringe. The eggs were incubated for two more days. Observation and examination of CAM (Gopez and Madrid 2012) After the 12th day of incubation the eggs were examined to determine the vascular activity on the CAM. The CAM was harvested by removing the shell of the egg. The embryos were transferred to a Petri dish and the soft membrane was extracted from the embryo and was observed using the microscope. The number of collaterals for each points of intersection of the blood vessels on the CAM of the eggs treated with MS and with benzene solution (10%) were counted and compared. B. C. D. E.

Sample of the Study Sampling technique Research Instrument Data Gathering Procedure F. Statistical treatment