PSIDIUM GUAJAVA AND PREMMA CORDIFLOR AS ANTIOXIDANT

PSIDIUM GUAJAVA AND PREMMA CORDIFLORA LEAVES AS ANTIOXIDANT, ANTI ULCER AND ANTISEPTIC SCIENTIFIC INVESTIGATION GROUP: 46 GROUP MEMBERS: 1. MOHD FADZLI BIN MUSTAPA 084010 2. TAN SHEY LE 084193 3. ARVINDAN A/L GOPALAKRISHNAN 084131 4. NAVINTHARARAO A/L VICKMASWARAN 084125 THESIS ADVISOR: MR. ROZAKI B. PAIJAN CONFESSION “We admit that our thesis is done by ourselves except all the summary and the idea each one that we had written the source in our thesis. …………………….. (MOHD FADZLI BIN MUSTAPA) College no:- 084010 Date :- …………………………. (ARVINDAN S/O GOPALAKRISHNAN) College no:- 084131 Date :- . …………………………. (NAVINTHARARAO S/O VICMASWARAN) College no:- 084125 Date :- ……………………... (TAN SHEY LE) College no:- 084193 Date :- Checked by, …………………………. (EN ROZAKI BIN PAIJAN) Advisor Date :- GROUP MEMBER’S PERSONAL DETAILS NAME I/C NUMBER H/ROOM CLASS : TAN SHEY LE : 920905-04-5230 : IBNU AFLAH 5 : 5 INTAN COLLEGE NUMBER : 084193 ADDRESS : 7298-C,SPRING GARDEN, TANJUNG KLING, 76400 MELAKA. NAME I/C NUMBER H/ROOM CLASS : ARVINDAN A/L GOPALAKRISHNAN : 920892-01-6787 : IBNU JUBAYR 5 : 5 ZAMRUD COLLEGE NUMBER : 084131 ADDRESS : NO B-7,BLOK 11, RUMAH PANGSA L.P.J, 81700 PASIR GUDANG, JOHOR. NAME I/C NUMBER H/ROOM CLASS : NAVINTHARARAO A/L VICKMASWARAN : 920613-08-6479 : IBNU SINA 5 : 5 BAIDURI COLLEGE NUMBER : 084125 ADDRESS : 11, JALAN BUNGA CEMPAKA 2A, TAMAN EHSAN IBU, KAMPUNG KOSKAN, 48000 RAWANG, SELANGOR. NAME I/C NUMBER H/ROOM CLASS : MOHD FADZLI BIN MUSTAPA : 920521-06-5363 : IBNU AFLAH 5 : 5 NILAM COLLEGE NUMBER : 084010 ADDRESS : NO 45, FELDA CHINI TIMUR 1, JALAN PERDANA, 26690 PEKAN, PAHANG DARUL MAKMUR ACKNOWLEDGEMENT Praise be to God as we had succeed to complete the thesis within the period given around one month. We would like to take this opportunity to address our appreciation and millions of thank you to our adviser En. Rozaki Bin Paijan who had helped us a lot to find the information about our thesis. Thanks to him for his encouragement which had boosted our confidence to finish up this thesis. Beside that, billions of thank you to Mr. Kamarussolihin Bin Nordin for all his guidance, advice and attention throughout our thesis. Our experiment was held in University of Technology of Malaysia (UTM). We would also like to express our gratitude to both our parents for their never-ending support in completing our project. They had spent money just to make sure that our thesis went well. They also gave us encouragement and will let us go out to have our thesis completed. We would like to thank our friends who had given us a lot of help to complete our project. We would also like to thank the school management for the facilities and help given. Thank you very much. ABSTRAK Jambu batu merupakan buah tempatan yang biasa yang boleh dijumpai di seluruh Malaysia. Buah ini digunakan secara meluas oleh orang-orang trdahulu sebagai ubat tradisional untuk sakit perut. Buahnya boleh dimakan dan sesetengah buahnya tidak berbiji dan ada juga yang berbiji bebuas pula merupakan tumbuhan yang semakin sukar untuk ditemui di Negara Malaysia kini. Sesetengah orang percaya bahawa daun bebuas boleh digunakan sebagai awet muda untuk kulit yang cantik dan licin. Orang-orang dahulu menggunakan daunnya sebagai herba tradisional. Objektif utama kajian ini ialah untuk mengekstrak daun jambu batu dan bebuas. Kajian ini juga tertumpu kepada analisis komponen bahan kimia yang terdapat dalam kedua-dua daun tumbuhan ini. Ekstrak bahan kimia yang diperoleh dikapsulkan sebagai anti ulser,antiseptic dan daripada kedua-dua pokok ini akan diuji menggunakan kolum antioksidan.sampel kromatografi.komponen bahan kimia yang diperoleh akan dianalisis untuk memastikan bahan kimia tersebut berfungsi sebagai anti ulser, antiseptik, dan antioksidan. Bahan kimia yang dipilih akan dimampatkan dalam kapsul untuk digunakan sebagai ubat. Konklusinya, daun jambu batu dan bebuas mengandungi bahan kimia yang boleh digunakan sebagai antiulser, antiseptik, dan antioksidan. ABSTRACT Psidium guajava is a common local fruit that can be found in Malaysia. It has been used extensively by the elderly people as a traditional remedy for stomach ache. This fruit also can be eaten and some of the fruits are seedless and some of them have seed. Premma cordiflora is a local plant that can rarely be found in Malaysia nowadays. Some people believe that premma cordiflora leaves can be used to make the skin more beautiful and smooth. Some of the elderly people also used the leaves as traditional herbs. The main objective of this research is to extract psidium guajava and premma cordiflora leaves to investigate the chemical component consist in both leaves. Actually this research is more to the analysis of chemical components consist in both leaves of the psidium guajava and premma cordiflora. Extracts of the chemical component which is obtained will be compacted in a capsule form to act as an anti ulcer, antiseptic and antioxidant..a sample from both plant leaves will be tested using the column chromatography. The chemical components that are collected from the result will be analysed to make sure that the chemical components has the properties of an anti ulcer, antiseptic, and antioxidant. The selected chemical components will be compacted into a capsule form to be used as a medicine. In conclusion psidium guajava and premma cordiflora leaves contain the suitable chemical components to be used as an anti ulcer, antiseptic, and antioxidant. CONTENTS Confession Group member’s Personality List of figure Acknowledgement Abstract Contents Chapter 1 – Introduction 1.1 – Introduction 1.2 - Research Background 1.3 - Problem Statement 1.4 - Objective 1.5 - Importance Of This Research 1.6 - Definition Chapter 2 – Literature Review (Past year research) 2.1- Introduction 2.2 – Relevant literature 2.3 – Relevant Research Chapter 3 – Research Methodology/Experiments 3.1 – Introduction 3.2 – Procedure 3.3 – Apparatus and Materials 3.4 – Obtaining Data 3.5 – Analysing Data Chapter 4 – Research Result – Introduction Results Chapter 5 – Conclusion and Suggestion 5.1 – Discussion 5.2 – Conclusion 5.3 – Implication 5.4 – Suggestion 5.5 – Summary References Attachment PAGE CHAPTER 1 INTRODUCTION 1.1 INTRODUCTION We have been given a task to complete a thesis report as one the graduation criteria. After much consideration and discussion, we have decided to do a research on the capability of extracts of Psidium guajava Linnaeus and Premma Cordiflora to act as an antioxidant, antiseptic, and anti ulcer. 1.2 RESEARCH BACKGROUND An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols or polyphenols. Antioxidants are also widely used as ingredients in dietary supplements in the hope of maintaining health and preventing diseases such as cancer and coronary heart disease. Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction. They should generally be distinguished from antibiotics that destroy bacteria within the body, and from disinfectants, which destroy microorganisms found on non-living objects. Some antiseptics are true germicides, capable of destroying microbes (bacteriocidal), whilst others are bacteriostatic and only prevent or inhibit their growth. Antibacterials are antiseptics that only act against bacteria. Microbicides which kill virus particles are called viricides. Anti ulcer is used to treat various kind of ulcers. An ulcer is a sore or lesion that forms in the lining of the stomach or duodenum where acid and pepsin are present. Ulcers in the stomach are called gastric or stomach ulcers. Those in the duodenum are called duodenal ulcers. In general, ulcers in the stomach and duodenum are referred to as peptic ulcers. Ulcers rarely occur in the esophagus or in the first portion of the duodenum, the duodenal bulb. Although ulcers may cause discomfort, rarely are they life threatening. With an understanding of the causes and proper treatment, most people find relief. Eradication of H.pylori infection is a major medical advance that can permanently cure most peptic ulcer diseases. 1.3 PROBLEM STATEMENT While doing the thesis we faced a lot of problems. One of them is the permission to conduct this experiment in UTM Skudai, Johor. At first we decided to do this experiment during our school holidays last year, but we could not do because the professor who was incharge of our research went outstation. So we have to wait until this year to conduct the experiment and obtain the result. This delayed us in completing the folio regarding our thesis. Next is the financial problem. During our stay in UTM, Skudai, we faced a financial problem as we had to stay there for a longer period than that we had expected because the column chromatography didn’t show the accurate picks. Therefore we had to redo the experiment once again to obtain the accurate picks to prove that the extracts of psidium guajava and premma cordiflora can show the properties of antioxidant, anti ulcer, and antiseptic. We also faced the problem to gain the sample leaf of psidium guajava and premma cordiflora as all of us are living in cities. So we requested the help of uor group adviser Mr. Rozaki to get the sample leaves to be taken to the UTM. 1.4 AIM OF EXPERIMENT  To find more nature friendly method to produce antioxidant, antiseptic, and anti ulcer.  To determine the suitable leave that can act as antioxidant, antiseptic, and anti ulcer.  To analyse the chemical components in psidium guajava and premma cordiflora leaves and extract them to be used as antioxidant, anti ulcer and antiseptic.  To determine whether psidium guajava and premma cordiflora can replace the function of commercial substances that is being used as antioxidant, antiseptic and anti ulcer.  To compact the chemical components found in the psidium guajava and premma cordiflora leaves into capsule form or liquid form to ease the consumers. 1.5 IMPORTANCE OF THIS RESEARCH This experiment is important to find an alternative product which can be used to treat peptic ulcer. This is because the ulcer drugs that is being used now is costly and most of the peptic ulcer patients are suffering and having financial problems in paying for their treatment. Furthermore the commonly used ulcer drugs such as H2-blockers, acid pump inhibitors, and mucosal protective agents take a longer period to give an effect to the disease. So if we managed to find the alternative product which can treat this disease efficiently and more quickly, we can reduce the peptic ulcer patients suffering. Moreover this experiment is important because some of the ulcer drugs used today has an adverse effect on the user. Therefore this experiment is necessary to find out whether we can find a cure for ulcer which has no side effect. 1.6 DEFINITION OF TERMINALOGY  Psidium guajava: The apple guava or common guava is an evergreen shrub or small tree native to Mexico, the Caribbean, and Central and South America.[1] It is easily pollinated by insects; in culture, mainly by the common honey bee, Apis mellifera  Premma Cordiflora: Local Name is Bebuas Bebuas is commonly used as ulam by the Malay community. Its crunchy stem and slightly bitter taste increases ones appetite. When eaten raw as ulam, this plant is said to help rid the "wind" in ones body quickly. The leaves of this plant can be boiled and the water can be drunk to help get rid of worms in the body and also helps lactating mothers.  Antioxidant: An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols or polyphenols.  Antiseptic: Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction. They should generally be distinguished from antibiotics that destroy bacteria within the body, and from disinfectants, which destroy microorganisms found on non-living objects.  Ulcer: Ulcers are crater-like sores (generally 1/4 inch to 3/4 inch in diameter, but sometimes 1 to 2 inches in diameter) which form in the lining of the stomach (called gastric ulcers), just below the stomach at the beginning of the small intestine in the duodenum (called duodenal ulcers) or less commonly in the esophagus (called esophageal ulcers).  Anti ulcer: Anti ulcer is a drug that is used to treat ulcers.  Column chromatography: Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms. The classical preparative chromatography column is a glass tube with a diameter from 5 to 50 mm and a height of 50 cm to 1 m with a tap at the bottom. A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles. A solution of the organic material is pipetted on top of the stationary phase. This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluant. Eluant is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the column. The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluant. At the end of the column they elute one at a time. During the entire chromatography process the eluant is collected in a series of fractions. The composition of the eluant flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV absorption, or fluorescence. Colored compounds (or fluorescent compounds with the aid of an UV lamp) can be seen through the glass wall as moving bands. FIGURE 1 FIGURE 2 CHAPTER 2 LITERATURE SURVEY 2.1 Introduction Our group had done a research on leaves of Psidium guajava and Premma Cordiflora to find out whether their extract can be use as antioxidant, antiulcer and antiseptic. FIGURE 3 Common name: Guava, Amroo (Hindi), Pera (Malayalam), Segappu koyyaa (Tamil), Pungton (Manipuri), Pairr (Konkani), Peru (Marathi), Goyyapandu(Telugu), Peyaaraa (Bengali) Botanical name: Psidium guajava- [ (SIGH-dee-um) from the Greek for pomegranate; (gwahJAV-vuh) from the Spanish guayaba ] Synonyms: Guaiava pyriformis Gaertn. • Guajava pyrifera (L) Kuntze • Psidium fragrans Macfadyen Psidium pyriferum L. • Psidium pomiferum L. • Psidium sapidissimum Jacq. • Psidium aromaticum Blanco Family: Myrtaceae (myrtle family) Origin: Tropical America Guava (from Spanish Guayaba; Goiaba in Portuguese; Perakkya in Malayalam) is a genus of about 100 species of tropical shrubs and small trees in the myrtle family Myrtaceae, native to the Caribbean, Central America and northern South America. The leaves are opposite, simple, elliptic to ovate, 5-15 cm long. The flowers are white, with five petals and numerous stamens. In Asia, a tea is made from guava fruits and leaves. In Egypt, guava juice is popular. The whole guava is edible, from seeds to rind, but many people choose to cut out the seeds and the middle of the guava. The guava flesh is sweet (the middle part with the seeds is sweetest), and the rind is slightly bitter tasting. Guava leaves are used for medicinal purposes, as a remedy for diarrhea, and for their supposed antimicrobial properties. The same anti-diarrheal substances which are useful in folk medicine may also cause constipation in the case of consumption of large amounts of guava fruits. In Cuba their leaves are also used in barbecues providing a nice smoked flavor and scent to the meat. FIGURE 4: Psidium Guajava FIGURE 5: Premma Cordiflora Medicinal purposes: Among the medicinal uses of Premma Cordiflora when eaten raw is enabling us to get rid of the air in the body at a very fast rate. Water boiled with the stem of Premma Cordiflora can increase the production of mother’s milk. Antioxidant An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols or polyphenols. Although oxidation reactions are crucial for life, they can also be damaging; hence, plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases. Low levels of antioxidants, or inhibition of the antioxidant enzymes, causes oxidative stress and may damage or kill cells. As oxidative stress might be an important part of many human diseases, the use of antioxidants in pharmacology is intensively studied, particularly as treatments for stroke and neurodegenerative diseases. However, it is unknown whether oxidative stress is the cause or the consequence of disease. Antioxidants are also widely used as ingredients in dietary supplements in the hope of maintaining health and preventing diseases such as cancer and coronary heart disease. Although initial studies suggested that antioxidant supplements might promote health, later large clinical trials did not detect any benefit and suggested instead that excess supplementation may be harmful.[1] In addition to these uses of natural antioxidants in medicine, these compounds have many industrial uses, such as preservatives in food and cosmetics and preventing the degradation of rubber and gasoline. Antiulcer Recurrent gastric and duodenal ulcers are caused by Helicobacter pylori infections, and are treated with combination treatments that incorporate antibiotic therapy with gastric acid suppression. Additionally, bismuth compounds have been used. The primary classes of drugs used for gastric acid suppression are the proton pump inhibitors, omeprazole, lansoprazole, pantoprazole and rabeprazole. The H-2 receptor blocking agents, cimetidine, famotidine, nizatidine, and ranitidine have been used for this purpose, but are now more widely used for maintenance therapy after treatment with the proton pump inhibitors. Sucralfate, which acts by forming a protective coating over the ulcerate lesion, is also used in ulcer treatment and may be appropriate for patients in whom other classes of drugs are not indicated, or those whose gastric ulcers are caused by non-steroidal anti-inflammatory drugs (NSAIDs) rather than H. pylori infections. Antiseptics Antiseptics (from Greek αντί - anti, '"against" + σηπτικός - septikos, "putrefactive") are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction. They should generally be distinguished from antibiotics that destroy bacteria within the body, and from disinfectants, which destroy microorganisms found on non-living objects. Some antiseptics are true germicides, capable of destroying microbes (bacteriocidal), whilst others are bacteriostatic and only prevent or inhibit their growth. Antibacterials are antiseptics that only act against bacteria. Microbicides which kill virus particles are called viricides 2.2 Connected experiment There had been research done with the objective nearly like what our group had chosen, however, all those research were done either using different organic substances or using the same organic substances but for different purposes. One of the experiment done with objective nearly the same as our group is “Asiatica as reagent antioxidant” Centella asiatica FIGURE 6 Scientific classification Kingdom: (unranked): (unranked): (unranked): Order: Family: Genus: Species: Plantae Angiosperms Eudicots Asterids Apiales Mackinlayaceae Centella C. asiatica Centella asiatica is a small herbaceous annual plant of the family Mackinlayaceae or subfamily Mackinlayoideae of family Apiaceae, and is native to Sri Lanka, northern Australia, Indonesia, Iran, Malaysia, Melanesia, New Guinea, and other parts of Asia. Common names include Gotu Kola, Asiatic Pennywort, Luei Gong Gen, Takip-kohol, Antanan, Pegagan, Pegaga, vallaarai, Kula kud, Bai Bua Bok, Brahmi (although this last name is shared with Bacopa monnieri and other herbs) and rau má (Vietnamese). In Assamese it is known as Manimuni. It is used as a medicinal herb in Ayurvedic medicine and traditional Chinese medicine. Botanical synonyms include Hydrocotyle asiatica L. and Trisanthus cochinchinensis (Lour.) In sinhalese (Sri Lanka) Gotu = conical shape and Kola= leaf The stems are slender, creeping stolons, green to reddish green in color, interconnecting one plant to another. It has long-stalked, green, reniform leaves with rounded apices which have smooth texture with palmately netted veins. The leaves are borne on pericladial petioles, around 20 cm. The rootstock consists of rhizomes, growing vertically down. They are creamish in color and covered with root hairs. The flowers are pinkish to red in color, born in small, rounded bunches (umbels) near the surface of the soil. Each flower is partly enclosed in two green bracts. The hermaphrodite flowers are minute in size (less than 3 mm), with 5-6 corolla lobes per flower. Each flower bears five stamens and two styles. The fruit are densely reticulate, distinguishing it from species of Hydrocotyle which have smooth, ribbed or warty fruit. The crop matures in three months and the whole plant, including the roots, is harvested manually. Centella grows along ditches and in low wet areas. In Indian and Southeast Asian centella, the plant frequently suffers from high levels of bacterial contamination, possibly from having been harvested from sewage ditches. Because the plant is aquatic, it is especially sensitive to pollutants in the water, which easily are incorporated into the plant. Centella is used as a leafy green in Sri Lankan cuisine. It is most often prepared as mallung; a traditional accompaniment to rice and curry, and goes especially well with vegetarian dishes such as parippu' (dhal), and jackfruit or pumpkin curry. It is considered quite nutritious. In addition to finely chopped gotu kola, mallung almost always contains grated coconut and may also contain finely chopped green chilis, chili powder (1/4 teaspoon), turmeric powder (1/8 teaspoon) and lime (or lemon) juice. A variation of the extremely nutritious porridge known as Kola Kenda is also made with Gotu Kola by the Sinhalese people of Sri Lanka. Kola Kenda is made with very well boiled red rice (with extra liquid), coconut milk and Gotu Kola which is liquidized. The porridge is accompanied with Jaggery for sweetness. Centella leaves are also used in the sweet "pennywort drink. When eaten raw as a salad leaf, pegaga is thought to help maintain youthfulness. In Thailand cups with gotu kola leaves are used as an afternoon pick me up. A decoction of juice from the leaves is thought to relieve hypertension. This juice is also used as a general tonic for good health. A poultice of the leaves is also used to treat open sores. Interestingly, chewing on the plant for several hours induces entheogenic meditation, similar to the effects of salvia divinorum, although this practice is widely considered dangerous, as it can cause temporomandibular joint pains. Richard Lucas claimed in a book published in 1979 that a subspecies "Hydrocotyle asiatica minor" allegedly from Sri Lanka also called "Fo ti tieng", contained a longevity factor called 'youth Vitamin X' said to be 'a tonic for the brain and endocrine glands' and maintained that extracts of the plant help circulation and skin problems. However according to medicinal herbalist Michael Moore, it appears that there is no such subspecies and no Vitamin X is known to exist. Nonetheless some of the cerebral circulatory and dermatological actions claimed from centella (as hydrocotyle) have a solid basis. Several scientific reports have documented Centella asiatica's ability to aid wound healing, which is responsible for its traditional use in leprosy. Upon treatment with Centella asiatica, maturation of the scar is stimulated by the production of type I collagen. The treatment also results in a marked decrease in inflammatory reaction and myofibroblast production. The isolated steroids from the plant have been used to treat leprosy. In addition, preliminary evidence suggests that it may have nootropic effects. Centella asiatica is used to re-vitalize the brain and nervous system, increase attention span and concentration, and combat aging. Centella asiatica also has anti-oxidant properties. It works for venous insufficiency. It is used in Thailand for opium detoxification. 1. Introduction. Lipid peroxidation is considered a principal mean of deterioration in the quality of food stuffs. It not only imparts rancid and undesireable flavours to fat products, but also it generates reactive oxygen species which are linked to carcinogenesis, inflammation, aging and cardiovascular disorders. (Pezzuto and Park, 2002). Lipids containing polyunsaturated fatty acids are readily oxidized by molecular oxygen, and such oxidation proceeds by a free radical chain mechanism (Aruoma, 1998).It also decreases organoleptic value of foods and imparts rancid and unpleasant flavors to the raw and end-use oil and fat products, thus making them unacceptable to consumers.(Min and Lee, 1998). The oil industry has to pay special attention in this context, as oils,fats and fatty foods suffer from stability problems (Wu and Nawar, 1986).Traditionally, chemically synthesized compounds, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), are used as antioxidants in oil products. However, some of these compounds have been questioned for their safety (Bran 1975; Whysner et al., 1994). The use of BHA and BTH is proved to be carcinogeneic. Therefore, there is an increasing interest in the antioxidative activity of natural compounds (Amakura et al., 2002; Orhan et al., 2003). Ter-butyl hydroquinone (TBHQ) is not allowed in Japan, Canada and Europe. Similarly, BHA has also been removed from the generally recognized as safe (GRAS) list of compounds. (Frag et al., 1998). Higher and aromatics plants have traditionally been used in folk medicine as well as to extend the shelf life of foods (Hulin et al., 2002). Most of their properties are due to essential oils produced by their secondary metabolism (Adam et al., 1998). Plant essential oils as antioxidants were researched in detail with the view to investigating their protective role for highly unsaturated lipids (Deans et al., 1993). Centella asiatica (L) Urb (Apiaceae), commonly called pennywort or gotu kola, is a perennial creeping weeds commonly found in moist places (Fosberg et al., 1979; Whistler, 1988; Van Wky et al., 1997). The composition of essential oil of centella asiatica and other phytochemicals reported by various scientists can lead us to an idea to exploit the essential oil of Centella sciatica as an antioxidant in case of lipid oxidation. (Schaneberg et al., 2003 and Oyedeji et al., 2005). In Pakistan Centella sciatica is used for wound treatment and to alleviate skin diseases. No earlier reports on antioxidant activity of Centella asiatica in vegetable oils are available in context of Pakistan. Present research work is designed to investigate the antioxidant activity of essential oil of Cantella asiatica or brahmi booti. It is very essential to develop natural antioxidants to meet up the challenges and demands especially in case of lipid oxidation. 2. Material and methods Refined, bleached and deodorized (RBD) sunflower oil samples were used to investigate the antioxidant activity of essential oil of Centella asiatica. Sunflower oil was selected due to its high use in food as it is a rich source of linoleic acid and it easily can undergo rancidity due to high degree of unsaturation. (Shahidi et al., 1992) 2.1 Collection of plant material. The plant material of Centella asiatica was collected from Botanical garden of GC University, Lahore, Pakistan in February 2007. 2.2. Extraction. Extraction of essential oil was carried out by using steam distillation.500 gm of whole plant material was used for 3 hours to obtain essential oil. Ten Kg of plant was used to collect oil. The oil was filtered through 1mm sieve and stored at 4 co till use. Percentage yield was 0.18%. 2.3. Initial Screening of Antioxidant Potential Essential Oil. 2.3.1. DPPH assay on TLC. DPPH assay with TLC was used to measure the antioxidant activity of essential oil. Method of Bektas was followed. (Bektas et al., 2005). 1:10 dilution of essential oil was made in methanol. Five microlitre of this dilution was applied on the TLC plate. Plate was developed by methanol and ethyl acetate in ratio of 1:1. Then the plate was sprayed with 0.2% of DPPH reagent in methanol and stayed for 30 min at room temperature. Purple color of DPPH reagent bleaching by yellow spots is the indication of positive antioxidant activity. 2.4. Determination of antioxidant activity in sunflower oils. 2.4.1. Storage of samples Seven refined, bleached and deodorized sunflower oil samples (SFO) were stored in triplicate in transparent polyethylene bottles of 250 ml capacity each. Out of total twenty one bottles, seven bottles contained 120 ml blank deodorized, refined and bleached SFO (blank). Other seven bottles contained 200 ppm of BHA per 120 ml deodorized, refined and bleached sunflower oil samples (SFO). Remaining seven bottles contained 200 ppm of essential oil per 120 ml of RBD sunflower oil. (Duh and Yen, 1997) All these samples were stored at ambient conditions. All the investigations in triplicate were made on weekly basis. Data was analyed by GraphPad Prism 3.0 (Graph Pad Software, Inc.San Diego, USA). 2.4.2. Analysis of rancidity parameters in Sunflower oil. Free fatty acid (FFA) values and Peroxide values (PV) and Iodine values (IV) were determined by following the recommended methods of AOCS (AOCS, 1989). Conjugated dienes (CD) and Conjugated trienes (CT) were determined according to recommended methods of IUPAC (IUPAC 1987). For the determination of Conjugated dienes (CD) and and Conjugated trienes (CT) sunflower oil samples were diluted with iso-octane to bring the absorbance with in the limits. The absorbance was measured at wavelength 232 nm and 268 nm for conjugated diene and triene values respectively (Hitachi, U-2001, Model 7400 spectrometer), Tokyo, Japan (IUPAC, 1987). All these parameters were good indicators of lipid oxidation. Many scientists monitored the phenomenon of lipid oxidation to judge the extent of oxidation and antioxidant potential of plant extracts (Anwar et al., 2006; Anwar et al., 2006; Freja, et al., 1999 and Gulcan, et al, 2007). 4. Results and discussion. Trends of variations among Free fatty acid (FFA) values and Peroxide values (PV) and Iodine values (IV) are given below. 4.1. Free Fatty Acid value (FFA). Formation of free fatty acids might be an important measure of rancidity of foods. FFAs are formed due to hydrolysis of triglycerides and may get promoted by reaction of oil with moisture. (Freja et al., 1999). Figure number 1, showed the changes in Free Fatty Acid values. Free Fatty Acid (FFA) value of blank RBD sunflower oil was found to be 0.055 + 0.01. After one week the FFA values were promoted to 0.076+ 0.001 . While the FFA value for the BHA containing sample after one week of storage protocol was found to be 0.058 + 0.001. This value was same as essential oil containing sample. After the completion of seven week storage protocol FFA value for blank solution was increased to 0.298 + 0.002. This change in FFA contents was significant according to statistical analysis. FFA value for BHA containing sample was increased from 0.058 + 0.001 (1st week value) to 0.101+ 0.002 at the end of seven week experimental protocol. Changes in BHA containing RBD sunflower oil samples showed the significant blockage of oxidation phenomenon as compared to blank RBD sunflower oil sample. Similar findings were observed in case of essential oil containing RBD sunflower oil sample. In this case FFA value was jumped from 0.058+ 0.001 (1 st week value) to 0.102+ 0.001(7th week value). There was a strong co-relation (R2 = 0.91) between FFA values of BHA and essential oil containing sample. This relationship explored the strong antioxidant ability of essential oil of centella asiatica. 4.2. Peroxide Value (PV) Peroxide value is a widely used measure of the primary lipid oxidation indicating the amount of peroxides formed in fats and oils during oxidation. (Gulcan et al., 2007). Changes in Peroxide values are showed in Figure, 2. Peroxide value of blank RBD sunflower oil sample was 0.85+ 0.01. It was increased to 7.01± 0 . 0 1 at the end of seven week trial. These changes were significant indicating the noticeable phenomenon of lipid oxidation. Peroxide value of BHA containing RBD sunflower oil was found to be 0.87+ 0.01 after one week. It was subjected to 0.97± 0 . 0 1 at the completion of seven week analysis. Investigations in case of essential oil containing RBD sunflower oil samples expressed the peroxide value increase from 0.88+0.02 (1 st week value) to 0.99+0.01. Changes in case of BHA and essential oil containing RBD sunflower oil samples were very minor indicating the strong antioxidant activity of BHA and essential oil of centella asiatica. Like FFA values, Peroxide values also exhibited the strong co-relation. (R2 = 0.93) 4.3. Iodine Value (IV) Decrease in iodine value (IV) is an authentic tool to monitor lipid oxidation. (Naz et al., 2004). Magnitude of variation in iodine values was measured according to recommended methods of AOCS (AOCS 1989). Changes in Iodine values were presented in figure, 3. Iodine value for blank RBD sunflower oil sample was calculated as 141+ 1.20. it was subjected to 109+ 2.50. These variations were statistically significant. While variation in BHA and essential oil containing RBD sunflower oil samples was not huge indicating the presence of antioxidants in the form of BHA and essential oil. RBD sunflower oil samples having 200 ppm of BHA showed a change from first week value, 140+ 3.25 to 134 + 2.50 at the ending of experiments. The Iodine values of first and last week for essential oil containing RBD sunflower oil samples were 140 + 2.50 and 132 + 2.50 respectively. Iodine values for first two weeks in BHA containing RBD sunflower oil samples were same. (Figure,3). Excellent co-relation was observed during the comparison of BHT and essential oil containing SFO. (R 2 =0.98) These results were in accordance to the findings of many scientists worked on antioxidant activities of various essential oils. Conclusion Above mentioned analytical investigation reveals that essential oil of Centella asiatica is an excellent antioxidant for lipids and lipids containing foods. Its activity was quite comparable with the synthetic antioxidant BHA. Antioxidant activity of essential oil was probably due to presence of terpenes and phenolics mostly present in essential oils. There is need to identify and to isolate the antioxidant components of Centella asiatica. There is also a need to cultivate this plant in Pakistan. This can lead to great health and economic benefits for the nation in terms of vegetable oil industries in Pakistan. REFERENCE BOOKS 1) Clungston, M. & Flewming, R., 2000. Advanced Chemistry. New York: Oxford University Press. 2) Darrell D.Ebbing, 1984. General Chemistry. Boston: Houghton Mifflin Company 3) Fullick A. & Fullick P., 1994. Chemistry. Oxford: Heinemar 4) Jamilia Jamil. & Tan Qeuk Cheng., 2007. Smart Chemistry. Selangor: Petaling Jaya 5) Tan On Tin., 2007. Terminology Chemistry. Selangor: Penerbitan Daya Sdn. Bhd. 6) Matill HA , 1947. Antioxidants. Annu Rev Biochem 16: 177–192. 7) Wolf G, 2005. "The discovery of the antioxidant function of vitamin E: the contribution of Henry A. Mattill". J Nutr 135 (3): 363–6. 8) Agerberth B, Gudmundsson GH, 2006. "Host antimicrobial defence peptides in human disease". Curr. Top. Microbiol. Immunol. 306: 67–90. 9) Sies H, 1997. "Oxidative stress: oxidants and antioxidants" (PDF). Exp Physiol 82 (2): 291–5. 10) Miller RA, Britigan BE, January 1997. "Role of oxidants in microbial pathophysiology". Clin. Microiol. Rev. 10 (1): 1–18. 11) Sies H, 1993. "Strategies of antioxidant defense". Eur J Biochem 215 (2): 213 – 9. 12) Azzi A, 2007. "Molecular mechanism of alpha-tocopherol action". Free Radic. Biol. Med. 43 (1): 16–21. 13) Ho YS, Magnenat JL, Gargano M, Cao J, October 1998. "The nature of antioxidant defense mechanisms: a lesson from transgenic studies". Environ. Health Perspect. 106 (Suppl 5): 1219–28. 14) Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C, 2008. "Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases". Cochrane Database of Systematic Reviews (2): 15) Cullen DJ, Hawkey GM, Greenwood DC, 1997. "Peptic ulcer bleeding in the elderly: relative roles of Helicobacter pylori and non-steroidal anti-inflammatory drugs". Gut 41 (4): 459–62 16) Abdelrahim, SI; Almadboul, AZ; Omer, MEA; Elegami A, 2002. Antimicrobial activity of Psidium guajava L. Fitoterapia. 73(7-8):713–715. 17) Arima, H; Danno, G, 2002. Isolation of antimicrobial compounds from guava (Psidium guajava L.) and their structural elucidation. Bioscience Biotechnology and Biochemistry. 66(8):1727–1730. 18) Begum, S; Hassan, SI; Ali, SN; Siddiqui, BS, 2004. Chemical constituents from the leaves of Psidium guajava . Natural Product Research.18(2):135–140. 19) Chulasiri, M; Suthienkul, O; Pavaro, C; Wongkrajang, Y, 1986. Herbal extracts for diarrheal treatment: antibacterial activity in vitro. Journal of Public Health. 16:21–35. 20) Jaiarj, P; Khoohaswan, P; Wongkrajang, Y; Peungvicha, P; Suriyawong, P; Saraya, MLS; Ruangsomboon, O, 1999. Anticough and antimicrobial activities of Psidium guajava Linn. Leaf extract. Journal of Ethnopharmacology. 67(2):203–212. 21) Qian, H; Nihorimbere, V, 2004. Antioxidant power of phytochemicals from Psidium guajava leaf. Journal of Zhejiang University Science. 5(6):676–683. 22) Still, W. C.; Kahn, M.; Mitra, A. J, 1978. Org. Chem. 43(14), 2923-2925. 23) Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54. 24) Don R.; Johnson, Todd M. J. Chem. Educ. 2007. Isolation of Three Components from Spearmint Oil: An Exercise in Column and Thin-Layer Chromatography Davies, 84 318. 25) Harrison, 2003 . Bioseparations Science and Engineering. Oxford University Press. New York, New York. 26) Whysner L, Wang CX, Zang E, Iatropoulos MJ, Williams GM. 1994. Dose response of promotion by butylated hydroxyanisole in chemically initiated tumours of the rat forestomach. Food and Cheml Toxicol. 32 (30), 215–222. 27) Amakura Y, Umino Y, Tsuji S, Ito H, Hatano T, Yoshida T. 2002. Constituents and their antioxidative effects in eucalyptus leaf extract used as a natural food additive. Food Chem. 77, 47–56. 28) Deans SG, Svoboda KP, Gundidza M, Brechany EY, 1992. Essential oil profiles of several temperate and tropical aromatic plants: their antimicrobial and antioxidant activities. Acta Hortic. 306, 229 – 232. 29) Aruoma OI. 1998. Free radicals, oxidative stress, and antioxidants in human health and disease. J. Am. Oil. Chem. Soc. 75, 199–212 30) International union of pure and applied chemistry (IUPAC). 1987. Standard methods for the analysis of oils, fat and derivatives; 7th revised and enlarged ed., edited by C. Paquat and A.Hautfenne, Blackwell Scientific,London. INTERNET  http://folk.uio.no/toreben/a-monohalo-ethers/index.html  http://ull.chemistry.uakron.edu/erd/  http://www.ux1.eiu.edu/~cfthb/links/research/  http://en.wikipedia.org/wiki/antioxidants.  http://en.wikipedia.org/wiki/antiseptic  http://en.wikipedia.org/wiki/ulcer  http://en.wikipedia.org/wiki/psidium guajava  http://en.wikipedia.org/wiki/premma cordiflora  http://www.specialchem4adhesives.com/tc/antioxidants  http://www.innospecinc.com/americas/products/fuel_antitoxidants.cfm  http://www.pathologyatlas.ro/chronic-peptic-ulcer.php.