1.0 Introduction
The project seeks to extract phytochemicals from Crombetum molle and evaluate their antibacterial activity against methicillin-resistant Staphylococcus aureus specie. Indigenous plants possess the ability to produce phytochemicals which they use to protect themselves against insect infection and environmental harsh conditions. Currently, the phenomenon of antibiotic resistance is the greatest challenge on the treatment of bacterial infection in Zimbabwe (Weinstein R.A, 1998). In Zimbabwe vanocomycim, meropenem, imipenem are antibiotics which are being used against methicillin resistant S.aureus specie and are considered very expensive because they are not manufactured in Zimbabwe. Combretum molle is an indigenous plant which produces phytochemicals which possess the ability to inhibit the growth of bacteria and have several biological activities like antioxidant, cytotoxic, anti-inflammatory, antiviral, antibacterial activities. These phytochemicals can be used in rotation with the conventional drugs since they possess a different structure which is new to the bacteria. Using phytochemicals as antibacterial agents from Combretum molle plant can be of great advantage since it is readily available in Zimbabwe thereby cutting cost of importing and they do not cause undesirable toxic effects because they are natural.

1.1 Problem statement

Currently, the phenomenon of antibiotic resistance is the greatest challenge on the treatment of bacterial infection in Zimbabwe especially the methicillin-resistant Staphylococcus aureus specie. The number of bacteria that are resistant toward these synthetic antibacterial agents has increased each year where 70% of the bacteria that cause infections in hospitals are resistant to at least one of the most commonly antibiotic agents. These conventional antibiotics have been reported to cause undesirable toxic effects in humans and the exorbitant rising cost of antibiotics has been a major problem to this nation.

1.2 Justification

In Zimbabwe there is no production of conventional drugs and the start material of manufacturing antibiotics is costly, so there is need to use phytochemicals from plants as antibacterial agents since the source material is readily available compared to that of conventional drugs. The phytochemicals do not cause undesirable toxic effects and can be used in rotation with conventional drugs.

2.0 Goal
To determine and obtain the antibacterial activity of phytochemicals in Crombretum molle (mupembere) against methicillin-resistant Staphylococcus aureus specie
2.1 Objectives * To determine the antibacterial activity of the phytochemicals against the methicillin-resistant Staphylococcus aureus specie * To identify the phytochemicals present in Combretum molle

2.2 Literature view

Infectious diseases are transmissible diseases that comprise clinically evident illnesses which result from infection of pathogenic biological agents in an individual host organism. Infectious diseases represent a leading cause of morbidity and mortality worldwide, accounting for more than 26% of all death with developing countries carrying the major burden. Pathogenic biological agents include viruses, bacteria, fungi, protozoa and multicellular parasites that have the ability to interact with human being by creating a community and cause an infection which reflected in possession of certain pathogenic factors. Nosocomial infection is known to be a major cause of death and increased morbidity among hospitalized patients. (Weinstein R.A,1998). According to the World Health Report, 1.8 million childhood death annually due to acute diarrheal illness which is also a very common illness in developed countries. Antimicrobial agents or antibiotics are used to prevent, control and reduce the occurrence of the infection. Antibiotics are semi-synthetic or synthetic substances produced by the natural metabolic processes of some microorganisms that can inhibit or destroy other microorganism.( Vroumsia T et al,2009), The greatest number of antibiotics is derived from bacteria from the genera Streptomyces and Bacillus and molds from genera Penicillium and Cephalosporium whereas only 7% of antimicrobial metabolites are plant-derived compounds (Weinstein R.A,1998) . Currently, the phenomenon of antibiotic resistance is the greatest challenge on the treatment of bacterial infection.

Plants are found to be an enormous source for variety of bioactive compounds with diverse molecular structure and function. These molecules are primarily derived from the secondary metabolism of plants and are used to protect it against predation by microorganisms, insects and herbivorous (Todar, K et al. 2002) Phytochemicals have two categories primary and secondary constituents. Primary constituents have chlorophyll, proteins sugar and amino acids. Secondary constituents contain terpenoids and alkaloids (Wadood A et al, 2013). Phytochemicals are naturally occurring in the medicinal plants, leaves, vegetables and roots that have defence mechanism from various diseases. Phytochemicals are primary and secondary compounds. Chlorophyll, proteins and common sugars are included in primary constituents and secondary compounds have terpenoids, alkaloids and phenolic compounds.

Terpenoids exhibit various important pharmacological activities that are anti-inflammatory, anticancer, anti-malarial, inhibition of cholesterol synthesis, anti-viral and anti-bacterial activities. Terpenoids are very important in attracting useful mites and consume the herbivorous insects.( Cowan M, 1999) Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties. They are non-essential nutrients, meaning that they are not required by the human body for sustaining life. It is well-known that plants produce these chemicals to protect themselves but recent research demonstrates that they can also protect humans against diseases. There are many phytochemicals and each works differently. (Doughari, J.H. et al,2009) These are some possible actions:
Antioxidant - Most phytochemicals have antioxidant activity and protect cells against oxidative damage and reduce the risk of developing certain types of cancer. Phytochemicals with antioxidant activity: allyl sulphides (onions, leeks, garlic), carotenoids (fruits, carrots), flavonoids (fruits, vegetables), polyphenols (tea, grapes).

Hormonal action - Isoflavones, found in soy, imitate human estrogens and help to reduce menopausal symptoms and osteoporosis. Stimulation of enzymes - Indoles, which are found in cabbages, stimulates enzymes that make the estrogen less effective and could reduce the risk for breast cancer. Other phytochemicals, which interfere with enzymes, are protease inhibitors (soy and beans), terpenes (citrus fruits and cherries).Interference with DNA replication - Saponins found in beans interfere with the replication of cell DNA, thereby preventing the multiplication of cancer cells. Capsaicin, found in hot peppers, protects DNA from carcinogens. (Babayi, H et al , 2004)

Anti-bacterial effect - The phytochemical allicin from garlic has anti-bacterial properties. Some phytochemicals bind physically to cell walls thereby preventing the adhesion of pathogens to human cell walls. Proanthocyanidins are responsible for the anti-adhesion properties of cranberry. Consumption of cranberries will reduce the risk of urinary tract infections and will improve dental health. ( Wadood A et al, 2013)

Combretum molle, the velvet bush willow, is a plant species in the genus Combretum found in Zimbabwe. It is recorded to contain antioxidants such as punicalagin, which is also found in the other Myrtale pomegranates (Punica granatum), a somewhat related plant. It also contains the 1alpha-hydroxycycloartenoid saponins mollic acid glucoside and mollic acid 3β-D-xyloside.Extracts from the bark or leaves of C. molle show antibacterial and antifungal as well as in vitro antiprotozoal activities.Mollic acid glucoside shows cardiovascular effects. Strains of Staphylococcus aureus are usually resistant to a variety of different antibiotics. Methicillin resistance is widespread and most methicillin-resistant strains are also multiply drug-resistant. Staphylococcus aureus is a gram-positive coccal bacterium that is a member of the Firmicutes, and is frequently found in the respiratory tract and on the skin. Pathogenic strains often promote infections by producing potent protein toxins, and expressing cell-surface proteins that bind and inactivate antibodies. The emergence of antibiotic-resistant forms of S. aureus such as MRSA is a worldwide problem in clinical medicine.( Vroumsia T et al ,2009)

3.0 Main Innovation of the proposed project
Currently in Zimbabwe the research on extracting and evaluating of phytochemicals from indigenous plant Combretum molle as antibacterial agents has not been carried out and use of phytochemicals as antibacterial agents on a large scale has not been done .

4.0 Research Methodology

1. Sources, identification, and processing of plant materials
Collection of the leaves of Combretum molle. Air dry the leaves to keep photocells intact for three days, pound in a mortar with pestle and store in sterile bottles for further study.

2. Test bacteria used
Bacteria used in this study is a pure culture of methicillin resistant Staphylococcus aureus specie.

3. Extraction Procedures
Soak twenty five grammes (25g) of the leaf powder in 100 ml of distilled water, 95% ethanol and methanol (BDH chemical, Poole, UK) in Erlenmeyer flasks and allow to soak for 72hours. The percolate obtained is passed through a Whatman filter paper No.1 and concentrate in vacuum using a rotary evaporator at 400C. Store the extracts in a sterile screw capped bottle and keep in the refrigerator at 40C.

4. Phytochemical screening of crude extracts of Combratum molle
Use the method of Harbone (1984) and Trease and Evans (1989) to test for the presence of phytochemicals in the plant leaves.

i) Test for anthracenes
One millilitre of the plant extract is shaken with equal volume of chloroform and 10% ammonia solution is added to the chloroform layer. Formation of red precipitate indicates the presence of anthracene.

ii) Test for tannins
Four millilitres (4.0 ml) of water is added to 0.5ml of the plant extract; add drops of ferric acid chloride. The immediate development of green precipitate indicates the presence of tannins. The presence of tannins is further confirmed by the development of red colouration on addition of lead acetate to the extract.

iii) Test for volatile oils
Mix one milliliter (1.0ml) of the plant extract and 0.1ml of dilute sodium hydroxide solution and dilute hydrochloric acid. The formation of a white precipitate indicates the presence of volatile oils.

iv) Test for saponins
Five grammes (5g) of the crude extract is added to 10ml of water and shaken for 2 minutes. Frothing which persists on warming is an evidence of the presence of saponins.

v) Test for alkaloids
Five drops of 100% tannic acid is added to 0.5ml of the extract. Formation of cream colour indicates the presence of alkaloids. Confirmatory test for alkaloids is done by adding drops of Dragendroffs reagent to the extract and the development of deep brown precipitate confirms the presence of alkaloids.

vi) Test for sesquiterpones
Aqueous extract (0.5 ml) of the plant is mixed with 0.1ml of methanol and shaken vigorously. To this mixture, 0.4ml of 5% sulphuric acid (H2SO4) plus 0.5% ferric chloride is added. The content is stirred using a small glass rod. The mixture is boiled in a water bath for one minute. A change in colour from colourless to pink indicates the presence of sesquiterpones.

vii) Test for phenols
Mix equal volumes (0.2ml) of the plant extracts and ferric chloride (FeCl3). The development of deep bluish green colour indicates the presence of phenols.

5. Determination of Antibacterial activity of plants extracts
Well diffusion method is used in this test. About 0.2g of each extract is dissolved in 5ml of distilled water, ethanol and methanol respectively. Three hour peptone water grown culture of the test organism is inoculated on nutrient agar plates. Create holes of 6mm diameter in nutrient plates using a cork borer and add 0.2ml of the extracts to each hole. Incubate the plates at 300C for 24hours.
The development of zones of inhibition around the holes containing the extracts indicates the antibacterial activity of the plant extracts against the test S.aureus.

6. Determination of Minimum Inhibitory
0.5mls of the reconstituted extracts (20mg/ml) of C. mole is placed in test tubes containing peptone water. The first tube should contain 1.5ml of peptone water while tubes 2-8 contain 1.0ml of peptone water each. One tube serves as the control experiment. The tubes are inoculated with a loopful of the test microorganisms and incubated at 370C for 24h. Compare the zone of inhibition with that of the conventional antibiotic to determine the efficacy of the phytochemicals.

5.0 Expected Results

* Obtaining the phytochemicals from Combretum molle * Comparable results of zone of inhibition of the phytochemicals with vancomycin antibiotic which is currently used in hospitals

6.0 Budget

Peptone water | $30 | Vancomycin | $30 | Ethanol | $25 | Methanol | $25 | Nutrient Agar | $25 | Ferric chloride | $30 | Sulphuric acid | $40 | Tannic acid | $40 | Sodium hydroxide | $30 | Ammonia | $40 | Transport | $15 | Total | $325 |

7.0 Environmental Impact

This project will not have a negative effect on the environment. Less effluent is produced and it is going to be disposed of accordingly.

8.0 WORK PLAN

Month | Activity | Time frame in week | Anticipated outcomes | December | Culturing of S.aureus in media | 2weeks | Growth of bacteria in media | January | Collection of the plant leaves | 2weeks | Access the C.molle plant | February | Obtaining the phytochemicals from the leaves | 1week | Crude extract obtained | February | Screening of the phytochemicals | 2weeks | Positive presence of phytochemicals | February | Determination of the antibacterial activity of phytochemicals against S.aureus | 1week | Large zone of inhibition of phytochemicals compared to that of antibiotics |

References 1) Wadood A, Ghufran M, Babar S Jamal, Naeem M, Khan A and Ghaffar R ,(2013), Phytochemical Analysis of Medicinal Plants Occurring in Local Area of Mardan, Research Institute of Chemistry, University of Karachi, Pakistan

2) Babayi, H., Kolo, I., Olokun, J.I and Ijah, U.J.J (2004).The antimicrobial activities of methanoic extracts of Eucalyptus camaldulensis and Terminalia catappa against pathogenic microorganisms. Biokemistri 16(2): 106-111.

3) Cowan M, (1999) Plant Products as Antimicrobial Agents,Department of Microbiology,Miami University, Oxford, Ohio 45056,Clinical Microbiology Reviews, American Microbiology

4) Doughari, J.H.; Human, I.S, Bennade, S. & Ndakidemi, P.A. (2009). Phytochemicals as chemotherapeutic agents and antioxidants: Possible solution to the control of antibiotic resistant verocytotoxin producing bacteria. Journal of Medicinal Plants
Research. 3(11): 839-848.

5) EL-mahmood, M.A. (2009). Efficacy of crude extracts of garlic (Allium sativum Linn.) against nosocomial Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniea and Pseudomonas aeruginosa. Journal of Medicinal Plants Research. 3(4): 179-185.

6) Weinstein, R.A. (1998) Nosocomial Infection Update. Emerging Infectious Diseases, , 416-420.http://dx.doi.org/10.3201/eid0403.980320

7) Todar, K. (2002) Antimicrobial Agents Used in the Treatment of Infectious Disease. Textbook of Bacteriology, University of Wisconsin-Madison, 1-11

8) Vroumsia T, Saotoing P,Dawé A, Djaouda M, Ekaney M and Abeng ,2009,The Sensitivity of Escherichia coli to Extracts of Combretum fragrans, Combretum micranthum and Combretum molle Locally Used in the Treatment of Diarrheal Diseases in,Department of Life and Earth Sciences, The University of Maroua Cameroon