Phytochemical Screening and Anti-bacterial Potential of Watermelon Seed Extract (Citrullus lanatus) on P.

acnes

Juwan Howard Barlomento1, Joey Lopena1, Joshua Marcial1, Alexis Ane Pa-a1, Kristi Patrisha Sapungan1, Patricia Anne Yray1 and Dean Theresita V. Atienza2
1

Biology Student, Department of Biology, College of Science, Polytechnic University of the Philippines 2 Dean, College of Science, Polytechnic University of the Philippines

OVERVIEW Watermelon (Citrullus lanatus) is a scrambler and trailer flowering plant which originated from southern Africa and is also cultivated in the Philippines. The fruit it bears is called a watermelon, classified by botanists as a pepo a berry with thick rind and fleshy center. Though the leaves, roots, flowers, whole plants and stems were examined for useful phytochemicals in many research projects, few researches refer to seeds from the fruit as sources of biological properties, specifically anti-bacterial properties. Also, we found a number of claims in the internet, as well as in Filipino folklore, about the anti-acne effects of watermelon seeds. The researchers came up with the following questions with regards to the properties of watermelon seed extract: Which phytochemical constituents are present in the watermelon seed extract? How will these phytochemicals affect the growth of Propionibacterium acnes? Will the 4 extracts (HWE, CWE, ethanol, methanol) yield the same intensity of antibacterial property? Thus, the researchers hypotheses are: The seeds contain secondary metabolites such as Saponins, Alkaloids, Tannins, etc. The phytochemical constituents in the seeds could be helpful to inhibit the growth of P. acnes bacteria. The four extracts will have different levels of efficacy against Propionibacterium acnes.

OBJECTIVES The objective of this study is to determine the presence of phytochemicals and secondary metabolites on the 4 extracts of Citrullus lanatus, and to test the extracts have antibacterial properties against P. acnes. We also aim to find out if which of the HWE, CWE, ethanol, and methanol extracts are effective in inhibiting the growth of P. acnes SIGNIFICANCE OF THE STUDY We are living in this modernized society where commercially available anti-acne products sold in the market were considered expensive and the worst case brought some adverse effects to the consumers.

This study would like to unveil whether the phytochemicals in watermelon (Citrullus lanatus) seed extract have the anti-bacterial potential. Instead of wasting the seeds, it could be offered as one of the many possible sources of a cheap and accessible supply of readily available anti-acne remedy. This study may also be of significance to those who have acnes due to some disadvantageous effects of commercial products (e.g. those who suffered from acnes due to incompatibility or adverse effects of chemical-based product to their skin), since this extract is natural and organic. Additionally, considering the types phytochemicals we can screen, this study can be used for further researches on the possible uses of watermelon seed extract.

RELATED LITERATURE Watermelon leaves, roots, flowers, whole plants and stems were examined for useful phytochemicals in many research projects. The fruit has chemical compounds such as: phytonutrient lycopene (Collins, 2003) an amino acid citrulline (Collins, 2007), -carotene (Edwards,2003), and alphatocopherol (Charoensiri, 2009). The seeds are a source of protein (Akpapunam, 1981), B vitamins, minerals (such as magnesium, potassium, phosphorous, sodium, iron, zinc, manganese and copper) and fat (Vandermark, 2011; Collins et al., 2007). The oil extracted from the seeds are rich in Omega 6 and 9 EFAs, as reported by Natural Sourcing, LLC.* Propionibacterium acnes is a gram-positive human skin commensal that prefers anaerobic growth conditions and is involved in the pathogenesis of acne (Kirschbaum and Kligman, 1963). Acne is one of the most common skin diseases, affecting more than 45 million individuals in the United States. Also, the researchers found a number of claims in the internet, about the anti-acne effects of watermelon seeds. [1] Watermelon seed oils and extracts are being sold as facial serum, [2][3], facial tonic [4] , and facial oil [5][6][7], though there are only few studies about the anti-bacterial potential/activity of watermelon seeds, (Braide et al., 2012), and the researchers was unable to find researches about it having anti-bacterial activity against P.acnes. The results that we will have from this study will give us knowledge on the phytochemical constituents of watermelon seeds and its anti-bacterial properties, specifically against Propionibacterium acnes. The efficacy of each of extract will be compared to each other, to see which extract is more effective.

METHODOLOGY Collection and preparation of the seeds of watermelon

Watermelons that will be used for this study will be bought at Divisoria market. The seeds that will be obtained from the fruits will be washed and dried in laboratory oven maintained at 40C overnight, and will be stored in a dry container to avoid fungal growth. The seeds will be ground using laboratory blender, all methods as done by Braide (2012). The ground seeds will be subjected to extracting methods adopted by Cheesbrough (2000). Filtrate of the extract was obtained by separation the suspension in a glass funnel and filter paper. Hot and cold water extracts were neatly separated and also stored. Ethanol and methanol were allowed to evaporate and stored in an airtight conical flask (Braide 2012). Phytochemical Screening The analyses for phytochemical constituents will be performed using generally adapting the methodologies described by Martinez, Miranda, and Doughari (Martinez, et.al, 2003 and Miranda, 2000; Doughari 2012). The methods were accepted as qualitative methods in determining the presence of phytochemicals and secondary metabolites. Preparation of P. acnes Pure culture of Propionibacterium acnes will be ordered from Biotech Philippine National Collection Of Microorganisms, National Institute of Molecular Biology and Biotechnology, UPLB, College, Laguna. Preparation of paper discs We will prepare small circular high potency discs with diameter 6.25mm made from Whatman No.1 grade filter paper with the aid of a mechanical perforator, if available. (Braide et al.,2012) The circular discs will be sterilized in a glass Petri dish using the hot air oven at a temperature maintained at 160C for 1h (Cheesbrough, 2000; Harrigan and McCance, 1990).

SCOPE AND LIMITATION The researchers will only use fresh C. lanatus seeds. The C. lanatus specimens will be acquired straight from a farm to ensure freshness and availability. Furthermore, the research will only require the use of the specimen C. lanatus. The specimens will be sent to the Bureau of Plant Industry for identification and authentication. For the extraction, only polar solvents will be used, thus only the ethanol, ethyl acetate and maceration methods are to be performed. For the phytochemical analysis, the presence of only the following phytochemicals will be tested: flavonoids, saponins, terpenes, sterols, anthocyanins, alkaloids and tannins. Furthermore, the researchers will only use qualitative tests in determining the presence of the said phytochemicals. For the bacterial specimens, both gram-positive and negative bacteria are to be used. During bacterial testing, the zones of inhibition will be the determinants of the antibacterial potential. Additionally, only the antibacterial potential is to be analyzed.

BUDGET ALLOCATION Things needed Watermelon Laboratory Expenses Transportation Other expenses TOTAL Allocation Php 150.00 Php 1500.00 Php 250.00 Php 100.00 Php 2000.00

SCHEDULE OF ACTIVITIES

Nov 20

Nov 27

Dec 4

Dec 11

Dec 18

Dec 25

Jan 1

Jan 8

Jan 15

Jan 22

Jan 29

Research and Additional Info Gathering Watermelon Preparation of seeds Storage Extraction Place Order Phytochemical Screening Antibacterial test Finalizing Paper Analyzation of Results Final Writing

REFERENCES

*Natural Sourcing, LLC is and FDA registered specialist in cosmeceutical ingredients that publish reports on ingredients (here, watermelon seeds) for evaluation purposes Akpapunam, M., Markakis, P. (1981) Protein Supplementation of Cowpeas with Sesame and Watermelon Seeds, Journal of Food Science Volume 46, Issue 3, pages 960961, May 1981 Braide, W, Odiong IJ, and Oranusi S (2012) Phytochemical and Antibacterial properties of the seed of watermelon (Citrullus lanatus), Journal of Microbiology Research (PJMR) Vol. 2(3), pp. 99-104, March 2012 Collins, JK, Ph.D., Wu, G, Ph.D., Perkins-Veazie, P, Ph.D., Spears, K, Ph. D., Claypool, LP, Ph.D., Baker, RA, Ph.D., Clevidence, BA, Ph.D. (2007) Watermelon Consumption Increases Plasma Arginine Concentrations In Adults, 11 January 2007. Collins, JK,Ph.D., Perkins-Veazie, P, Ph.D. (2003) Flesh quality and lycopene stability of freshcut watermelon Charoensiri R, Kongkachuichai R, Suknicom S et al. (2009) Beta-carotene, lycopene, and alphatocopherol contents of selected Thai fruits. Food Chemistry, Volume 113, Issue 1, 1 March 2009, Pages 202-207. Cheesbrough M (2000). District Laboratory Practice in Tropical Countries Part 2. Cambridge University Press, UK. Harrigan WF, McCance M (1990). Laboratory Methods in Food and Diary Microbiology. Academic Press Inc., London, pp. 25-28. Doughari, J. H. Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents , Phytochemicals -A Global Perspective of Their Role in Nutrition and Health, 21 March, 2012, pages 1-32 Edwards AJ, Vinyard BT, Wiley ER, Brown ED, Collins JK, Perkins-Veazie P, Baker RA, and Clevidence BA Consumption of Watermelon Juice Increases Plasma Concentrations of Lycopene and -Carotene in Humans Vandermark T (2011). The Health Benefits of Watermelon Seeds. Retrieved Dec. 09, 2011 [1] http://healthmedicalblog.blogspot.com/2010/01/benefits-of-watermelon-seeds.html [2] http://www.asterandbay.com/product/purify-facial-serum-watermelon-seed-myrtle [3] https://oneloveorganics.com/shop/love-springs-eternal-youth [4] http://www.starkskincare.com/products/white-willow-bark-perfecting-tonic [5] http://www.sheaterraorganics.com/Kalahari-Ootanga-Oil-Prehistoric-Watermelon-SeedOil_p_51.html [6] http://www.aromantic.co.uk/buy-watermelon-seed-oil-uk.htm

[7] http://www.fromnaturewithlove.com/soap/product.asp?product_id=OILWATERMELON