Science laboratory technology department, federal polytechnic, mubi, adamawa state, nigeria



Дата17.04.2016
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PHYTOCHEMICAL AND ANTIMICROBIAL STUDIES ON CITRULUS COLOCYNTHIS (WATER MELON) SEEDS EXTRACTS FROM NORTH EASTERN NIGERIA
*UZOH , RAYMOND D., AKANDE E. K. AND MANU M. A.

SCIENCE LABORATORY TECHNOLOGY DEPARTMENT, FEDERAL POLYTECHNIC, MUBI,

ADAMAWA STATE, NIGERIA
ABSTRACT

Although the extraction of bioactive agents from plants has been one of the most intensive areas of natural products research, a number of medicinal plants remain unexplored. The pulverized seeds of citrulus colocynthis (Water Melon) was extracted with n-hexane, petroleum ether and water. The oil extract tested positive for alkaloid, tannin, phlobatannin, phenol, flavannoid, and cardiac glycoside. Water extract tested positive for alkaloid, tannin, phlobatannin, phenol, saponins, and negative for flavanoids, and glycoside indicating absence of flavanoid and glycosides in the water extract. Antimicrobial assay with staphylococcus aureus and Escherichia coli at 0.25cm3, 0.5cm3, 1cm3 and 1.5cm3 concentrations produced moderate inhibition of the growth of the test organisms, while in the antimicrobial disc method, only the water extract produced significant zone of inhibition of 0.01mm and 0.02mm respectively. The zone of inhibition of the seeds extracts under study was below 10mm, the recommended value of inhibition for an antibiotic pending further research.


Keywords: Bioactive agents, Phytochemicals, Antibiotic, Antimicrobial, Natural Products.


Introduction

Citrullus Colocytnthis (watermelon) of the family cuccurbitaceac is a popular American summer fruit. It is known to have been cultivated by the Egyptians, early Christians and imperial Chinese, where the fruit was known as Si-kwa or “melon from the East”. The origin of this plant has been traced to the desert regions of the Kalahari Desert in Africa, other authorities claim it came from southern Italy or Asia. There are several types of watermelon, the most popular are honeydew, charentais, cantaloup, ogen etc. Ronald (1981).

Watermelon is used as desert fruit and a thirst quencher and in the very dry areas of Africa, it is relished by both humans and his animal as a source of water. The fruits are generally eaten raw and the seeds are sometimes grinded to form a flour. The young leaves are also cooked or added to soup. Water melon seed oil is an edible oil quite similar to sunflower oil, Margared, (1979) .

Citrullus Colocynthis (Watermelon) is a rich source of phytochemicals especially lycopene, a carotenoid according to United States Dietetic Association in Agricultural Research magazine of 2002. On average, water melon has about 40% more lycopene than raw tomatoes. Studies indicate that lycopene protects against cardiovascular disease and certain types of cancer most notably prostate. Other studies revealed protective properties against breast, endometrium and lung cancers as well, Craig (1997), Marvic, (1995). Watermelon is fat free and is a source of vitamins A, B6, C and thiamin. According to Folk Medicine, roots of citrullus Colocynthis may be used to treat urinary diseases and other infections. It is based on this fact that Staphylococcus aureus and Escherichia coli were used as target micro organism to test for bioactivity of the seed extracts.



Phytochemicals: Phytochemicals are substances found in plants and by the strictest definition, they are chemicals that are produced by plants. Currently the term is being used only for those plant chemicals that may have health-related effects but are not considered essential nutrients like proteins, carbohydrates, facts, minerals and vitamins. Craig (1997), Marvic (1995). Phytochemicals are thought to protect the cells from damage, stimulate anticancer enzymes and help remove cancer-causing substances from the body. They have been found in large quantities in fruits, vegetables, whole grains and even Soya beans. They affect the colour, flavour and odour of plants.

During the 1980s and 1990s, numerous carbohydrates began studying phytochemicals to “Mine” plants for bioactive agents that might be used as medicines (nutriceuticals). More than 600 phytochemical substances have been identified and there are probably hundreds more, Craig (1997).

Generally, phytochemicals are secondary metabolites and are often found in disposable parts of plants, these include stem and root barks, leaves, flowers, fruits and seeds. Common phytochemicals found in plants include tannins, phlobatannins, quinones, alkaloids, saponins, sugars etc. Kumar and Singh (1976).


  1. Quinones: Quinone is a generic for a class of cyclic organic compounds containing two carboxyl groups either adjacent or separated by a vinylene group, - CH = CH -, in a usually six membered ring. Hydroguinone is known principally as a photographic developing agent. Quinones are found in bacteria, certain plants and in only a few animals and may be used in various dyes used to colour certain types of fabrics.

ii. Alkaloids:- Akaloids comprise the largest single class of secondary substances that are known to number over 2000, Godwin and Mercer, (1983). Alkaloids are basic substances, which contain one or more nitrogen atoms in a ring and because of their toxic effect and physiological activities, they are widely employed in medicine. They have marked effect on the central nervous system. Examples include cocaine caffeine, nicotine bellodna and oceanite. The anti-malaria drug, quinine obtained from the bark of cinchona plant is example of an alkaloid.

Other phytochemicals found in plants include tannins, phlobatannins, saponnins, lycopene, etc.

The aim of this research was to bring to limelight the inherent phytochemicals found in watermelon seed extracts with a view of establishing its potential to be used as an antibiotic or as a biocide.

Materials and Method: The Citrullus colocynthis (watermelon) used for the study was purchased from fruit vendors at Mubi North local government of Adamawa State which they bought from Maluri area of Yobe State, Nigeria, an area known to support the growth watermelon.

Test organisms used are staphylococcus aureus and Escherichia coli while the solvents used are n-hexane, petroleum ether and water.



Solvent Extraction:- The watermelon seeds were pulverized using pestle and mortar and 10g of the ground sample was place inside the soxhlet extractor with 90cm3 of the n-hexane as the extracting solvent. The temperature of the heating mantle was adjusted to cover the range of 40 – 60oC to keep the solvent volatile enough.

The same procedure was done for petroleum ether as well. The extraction was done for 48 hours.



Water Extract: The water melon seeds was ground using mortar and pestle. 30g of the ground seeds was placed into 500cm3 conical flask and 300cm3 distilled water was added and shaked vigoursly. The sample was allowed to stand for four days after which it was filtered and dried accordingly.

Phytochemical Analysis: Phytochemical analysis was done according to the method of Evans and Sofowora, (1974). The phytochemicals tested for include the following; alkaloids, tannin, phytobatannins, phenols, flavanoids and glycosides.

Bioassay on the solvent extract and water extract was done using Staphylococcus aureus and Escherichia coli. Culture media was prepared according to Baker and Silverton, 2001. Macconkey agar and blood agar was used.

Plates inoculation method was also used Baker and Silverton, 2001. antimicrobial disc method was also used in the in the bioassay.

Procedure: Filter paper was punched using punching machine and the filter paper was deepened into water and solvent extract for some minutes and it was dried.

Calculation:- For 1 Petri dish 0.7g of nutrient agar powder to 25cm3 of distilled water, therefore for 2 Petri dishes (0.7g x 2) of nutrient agar to (25cm3 x 2) of distilled water equal to 1.4.g of nutrient agar to 5cm3 of distilled water.

Procedure: 1.4g of nutrient agar was weighed and transferred into 200cm3 conical flask and 50cm3 of distilled water added and the mixture was shaked vigorously. Then it was sterilized using autoclave at 121oC for 15 minutes and then allowed to cool to 50oC.

It was poured aseptically into two sterile Petri dishes and was allowed to solidify. The plates were dried in the oven at 37oC.



Plate Inoculation: The wire loop was sterilized and a loopful of Staphylococcus aureus and Escherichia coli colony was smeared over the area. The loop was sterilized in the Bunsen flame and when cooled was streaked all over the plate. The sensitivity disc of water and solvents extract was placed in position on top of the plates. Then the plates were incubated at 37oC for 24 hours.
Results and Discussion

Table 1: Phytochemical analysis of solvent extract



Test

Present(+)/Absent(-)

Alkaloid

Tannin


Phlobatannins

Phenol


Flavanoids

Glycosides



Present (+)

Present (+)

Present (+)

Suspected (+)

Present (+)

Present (+)


Table 2: Phytochemical analysis of water extract



Test

Present/Absent

Alkaloid

Tannin


Phlobatanins

Phenol


Flavanoids

Saponins

Glycosides


Present (+)

Present (+)

Present (+)

Suspected (+)

Absent (-)

Present (+)

Absent (-)


Table 3: Results of Bioassay with solvent extract



Concentration

Media

Inoculated organism

Control

Observation on sample media

0.25cm3

Blood agar

Saphylococcus aureus

Heavy growth

Moderate growth

0.25cm3

Macconkey agar

E. coli

Heavy growth

Moderate growth

0.5cm3

Blood agar

S. aureus

Heavy growth

Scanty growth

0.5cm3

Macconkey agar

E.Coli

Heavy growth

Scanty growth

Table: 4 Bioassay with water extract



Concentration

Media

Inoculated organism

Control

Observation on sample media

1cm3

Macconkey agar

E.coli

Heavy growth

Scanty growth

1cm3

Blood agar

S. aureus

Heavy growth

Scanty growth

1.5cm3

Macconkey agar

E.Coli

Heavy growth

No significant growth

1.5cm3

Blood agar

S. aureus

Heavy growth

No significant growth


Table: 5 Antimicrobial Disc Method results

Plate

Media

Inoculated organism

Extract

Zone of inhibition

Plate 1

Nutrient age

S. aureus

Water extract

0.01mm

Plate 2

Nutrient agar

E. coli

Water extract

0.02mm

Plate 3

Nutrient agar

S. aureus

Solvent extract

No zone of inhibition

Plate 4

Nutrient agar

S. aureus

Solvent extract

No zone of inhibition


Discussion: According to table 1, phytochemical screening tested positive for alkaloid, tannin, phlobatannin, flavanoids, phenol and glycosides. Similarly from table 2, screening for phytochemicals tested positive for alkaloid, tannin phlobatannins, phenol and saponins while flavanoids and glycosides are absent in the water extract.

According to table 3, bioassay with the solvents extract at 0.25cm3 concentration and with S. aureus and E. coli produced moderate growth. With 0.5cm3 concentration, the agar plates showed scanty growth similarly, with water extract at 1cm3, using E.Coli and S.aureus, scanty growth was produced while at 1-5cm3 , no significant growth was recorded. Table 4 result showed considerable and significant zone of inhabitation for the water extract with a value of 0.01mm and 0.02mm for S. aureus and E. Coli. The solvent extract with the cost organism yielded no zone of inhibition.



Conclusion: There is an established presence of most phytochemicals as discovered from the results. From antimicrobial assay, a pattern emerged here that at lower concentrations, the extract has little activity while at higher concentrations for both target organisms, the level of activity increases.

For antimicrobial disc method, the solvent extract produced no zone of inhibition while water extract produced significant zone of inhibition for both organisms, with values 0.01mm and 0.02mm respectively.

Finally these values are far below the recommended zone of inhibition value for strong antibiotic which is 10mm.

Oil extracted from the seeds is edible and of high nutritive value according to literature and watermelon is highly recommended for its high phytochemical content.



References

Blood D.C. and Students (1990). Baillure’s Comprehensive Veterinary Dictionary, 2 edition. Clays ltd. England. P59.


Craig, W.J. (1997), Phytochemicals: Guardians of our Health. Journal of the American Dietetic Association. Pp199-204.
Denniston K.J., Topping JJ. and Caret R.L. (2004) General, Organic and Biochemistry, Fourth Edition. McGraw Hill Publishers ltd, pp884
Duke, J.A. (1978). The Quest for Tolerance Germplasm. http://www.hort.purdue.edulnewcorp/duke_energy/citrullus colocythu.html
Frank A. (1995). An introduction to Veterinary Pharmacology, 4th edition. Long publishers, Londoa Pp290-340
George, M.S. (1981) Antimicrobial Agents, Encyclopedia of Science and Technology, 6th edition. McGraw-Hill, New York. Pp644.
Kumar. HD. and Singh, H.N. (1976) Plant Metabolism. Affiliated East West Press Private ltd. hxlia, pp246-27 I
Marvic. C. (1995). Learning How Phytochemicals Help fight Disease. Journal of American Medical Association. Pp 274.
Moore, W.R. and Winston, A. (1996). Laboratory Manual for Organic Chemistry. A Microscale Approach. The McGraw-Hill Company, New York. Pp 53-68,197.
Nester E. W. (2004). Microbiology; A Perspective, 4th edition. The McGraw-Hill Companies, Inc. New York. New York, pp537-54O, 694-695, 614-616.
Ronald, K. (1981). The Theory of Catering, 7th edition. 7122.
Sofowora, A (1982), Medicinal Plants and Traditional Medicine in Africa. John Willey and sons ltd, New York. Pp 128-129, 142-159.
Uzoh, R.D. and Agho, M.O. (2005) Investigation of the Medicinal Property of Khaya Senegalensis Seed oil and Application in the Formulation of

Antidandruff and Haircream (M.Sc Thesis) pp 19-22, 27-31.


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