PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITY OF

RIND OF CITRULLUS LANATUS

Hussaini Abdullahi Dangani, Ibrahim Sada, Yusuf Hassan*

Department of Chemistry, Umaru Musa Yar’adua University, Katsina, Nigeria

*
Corresponding author: yusuf.hassan@umyu.edu.ng

ABSTRACT

The crude methanolic extract of citrullus lanatus rind was fractionated with n-hexane,

chloroform, ethylacetate and acetone to afford four soluble fractions, respectively. The fractions

were tested for antibacterial activity using agar well diffusion method. While ethylacetate

fraction was found to be the most active of the fractions against the tested bacteria, n-hexane

fraction do not inhibit the growth of the tested bacteria. Phytochemical analysis showed the

presence of alkaloids, saponins, reducing sugar and steroid.

Keywords: citrullus lanatus, escherichia coli, staphylococcus epidemidis, phytochemicals,

antibacterial activity

INTRODUCTION

The history of medicinal plants dated back to 4000 – 5000 B.C [2], presenting ability of plants to

serve as alternative medicine to control disease widely accepted by ethnic and religious groups.
[4]
The Chinese for instance uses plants as medicine for over 6000 years . Natural products from

various sources including terrestrial plants, terrestrial microorganisms, marine organisms and

1
vertebrate and invertebrate, have played an important role throughout the world in treating and
[2]
preventing diseases . The use of medicinal plants as a fundamental component of the African

traditional healthcare system is perhaps the oldest and the most assorted of all therapeutic

systems. In many parts of rural Africa, traditional healers prescribing medicinal plants are the

most easily accessible and affordable health resource available to the local community and at
[3]
times the only therapy that subsists . Citrullus lanatus (Watermelon) is a type of melon that is

cultivated extensively for its pleasant-tasting fruit; it is a member of the gourd family and one of

the most economically important fruit in the Cucurbitaceae family. It has many nutritional and

medicinal values including hydration of the body because of its high water content which

contained sugar and energy booster. The plant contains large amount of beta-carotene and it is a
[5]
natural source of lycopene. It is also rich in citrulline, an effective precursor of L-arginine .

Phenolic compounds are constituents of both edible and non-edible parts of the plant. The seeds

are sources of protein, tannins and minerals [1].

MATERIALS AND METHODS

Collection and Preparation of Rind Material

The rind was obtained by peeling off of the water melon fruit. The rind obtained was dried under

shade for five days, and then ground to powder using a blender. Powdered rind material was

weighed and kept in air-tight container for further usage.

Extraction

The ground samples of Acalypha wilkesiana and Maerua angolensis (200g each) were extracted

by percolation with 800ml of re-distilled ethanol for a period of two weeks. Each extract was

concentrated and evaporated to dryness on a rotary evaporator at 40°C to afford ethanol extract

[9].

2
Extraction and Fractionation

Air dried sample (200g) was percolated using methanol (500ml) at room temperature for 4 days.

The extract obtained was concentrated using a rotary evaporator weighed and stored. The crude

methanol extract was macerated using various amounts of solvents, n-hexane (80ml), chloroform

(60ml) ethylacetate (60ml) and acetone (60ml). The extracts were labeled as F1 (methanol), F2

(n-hexane), F3 (chloroform), F4 (ethylacetate) and F5 (acetone).

Phytochemical Screening

Test for Alkaloids

Each extract (0.5g) was stirred with 5ml of 1 per cent aqueous hydrochloric acid on a steam bath;

1ml of the filtrate was treated with a few drops of Mayer’s reagent and a second 1ml portion was

treated similarly with Dragendorff’s reagent. Turbidity or precipitation with either of these

reagents was taken as evidence for the presence of alkaloids in the extract being evaluated [6].

Test for Saponins

Each extract (0.5g) was shaken with water in a test tube. Frothing which persists on warming

was taken as evidence for the presence of saponins [7].

Test for Tannins

Each extract (0.5g) was stirred with 10ml of distilled water. This was filtered and ferric chloride

reagent was added to the filtrate, a blue-black precipitate was taken as evidence for the presence

of tannin [8].

3
Test for Flavonoids

A portion of each extract was heated with 10ml of ethylacetate over a steam bath for 3min. The

mixture was filtered and 4ml of the filtrate was shaken with 1ml of dilute ammonia solution. A

yellow coloration was taken as evidence for the presence of flavonoids [9].

Test for Steroid

The dried extract (2mg) was shaken with chloroform. Sulphuric acid was added slowly by the

sides of test tube to the chloroform layer. Formation of red colour indicates the presence of

steroids.

Reducing Sugars

A portion of the extract was dissolved in distilled water followed by the addition of a mixture of

Fehling’s solution A & B and warmed. A brick brown precipitate appears which indicates the

presence of reducing sugar.

Preparation of Test Solution

Each of the extracts (0.1g) F1 F2, F3, F4, and F5 was dissolved in dimethylsulfoxide (DMSO)

(1ml) to yield a concentration of 0.1g/ml (100,000 μg/ml) solution which was labeled as stock

solution from which other concentrations were formed (serial dilution).

From the stock solution, 1ml was transferred into a test tube containing DMSO (9ml) to effect 10

times dilution, this gives a concentration of 10,000 μg/ml. subsequently 1ml was transferred into

another test tube containing DMSO (9ml) to give a concentration of 1000 μg/ml and further

diluted to 100 μg/ml, 10 μg/ml, and 1.0 μg/ml respectively.

Culturing and Sensitivity Testing

The test organisms used were clinical bacterial isolates obtained from Microbiology laboratory

of Umaru Musa Yar’adua University Katsina. The isolated bacteria were cultured on nutrient

4
agar plates prepared by dissolving nutrient agar (28g) in one liter (1L) of distilled water and

heated to boiling before autoclaving at 121mpa for 15min. The media (10ml) was poured in each

plate and allowed to solidify before inoculation. With the aid of sterile cock borer, wells of

about 6mm in diameter were bored on the plates and different concentrations of the extracts

(0.5ml) was dispensed into the wells and then allowed to stand for about 15mins. These were

then incubated at 370C for 24hrs. At the end of the period, sensitivity of different bacterial strains

to various extracts was measured in terms of zone of inhibition as described by Bauer et al. [10].

RESULTS AND DISCUSSION

Table 1. Result of Phytochemical Screening
Metabolite Methanol n-hexane Chloroform Ethylacetate Acetone

Alkaloid + + - - +

Flavonoid - - - - -

Reducing sugar + - + - -

Saponin + + - + -

Steroid + - - + -

Tannin - - - - -

+ = Present, - = Absent

The results of phytochemical screening showed the presence of secondary metabolites such as

alkaloids, flavonoid, reducing sugar, saponins and tannins in the crude methanolic extract, n-

hexane, chloroform, and ethylacetate fractions of the rind of citrullus lanatus. While alkaloids

were found present in the crude methanolic extract, n-hexane and acetone fractions, flavonoid

and tannins were absent in all the fractions. The reducing sugar was found to be present in the

crude extract and chloroform extract and the presence of saponin was detected in the crude, n-

hexane and ethylacetate extracts but not detected in the chloroform and acetone extracts. The

5
presence of steroid was also detected in crude and ethylacetate extracts but absent in n-hexane,

chloroform and acetone fractions.

Table 2. Result for Antibacterial activity

Diameter Zones of inhibition (mm)

Disc potency (mg/disc)

Bacterial specie Fraction 10,000 1000 100 10 1

Escherichia coli Methanol (F1) 13 - - - -

n-Hexane(F2) - - - - -
Chloroform (F3) 10 - - - -
Ethylacetate (F4) - 18 13 12 -
Acetone (F5) - - - - -
Staphylococcus. Methanol (F1) 27 13 10 - -
Epidemidis n-Hexane(F2) - - - - -
Chloroform (F3) - - - - -
Ethylacetate (F4) 20 13 15 16 -
Acetone (F5) 16 13 - 14 -

Antibacterial susceptibility test (Table 2) showed the zones of inhibition measured in millimetre

(mm) on the bacteria susceptible to the plant extract. When considering the macerated fractions,

the ethylacetate soluble fraction (F4) was observed to be the most active of all the fractions with

highest zone of inhibition of 20mm at 10,000μgml-1 recorded against staphylococcus epidemidis.

It was also active at concentrations of 1000μgml-1, 100μgml-1 and 10μgml-1 against both bacterial

species. The acetone soluble fraction shows noticeable activity against staphylococcus

epidemidis only, at concentrations of 10,000μgml-1, 1000μgml-1 and 10μgml-1, as there was no

activity observed against escherichia coli at all concentrations. While chloroform soluble extract

shows the least activity against extract escherichia coli at 10,000μgml-1 concentrations, there was

6
no observed activity against staphylococcus epidemidis at the various concentrations. The n-

hexane soluble extract does not have any inhibitory effect against all the tested bacteria. It could

be noticed that the crude methanolic extract also shows most inhibitory action against

staphylococcus epidemidis at 10,000μgml-1, 1000μgml-1 and 100μgml-1compared to the activity

observed against escherichia coli at only 10,000μgml-1 concentration.

CONCLUSIONS
The results of this study showed that citrullus lanatus rind may be useful as potential

antibacterial agent and afford efficient and safe antimicrobials which will certainly contribute to

the ongoing search for new antimicrobial agents to fight against infectious diseases caused by

antibiotic resistant strains. The presence of phytochemicals as indicated by the phytochemical

analysis results may be responsible for the antibacterial activity of the plant part. It is

recommended that isolation of possible bioactive compound be carried out especially on the

most active fractions (ethylacetate).

ACKNOWLEDGEMENTS

The authors are grateful to Malam Munnir Kabir for the bioassay.

REFERENCES

1. Collin, J.K; Wu, Ji; Parker, R.A; Porkins, P. (2007). Watermelon Consumption Increase
Plasma Arginine Concentrations in Adults. Nutritional Journal, 23(3), 261-266.
2. Hosseinzadeh, S. J. (2015). The Application of Medicinal Plants in Traditional and
Modern Medicine: A Review of Thymus vulgaris. International Journal of Clinical
Medicine, 6, 635-642.
3. Mahomoodally, M. F. (2013). Traditional Medicine in Africa: An Appraisal of Ten
Potent African Medicinal Plants. Evidence Based Complementary and Alternative
Medicine, 1.

7
4. Patil, D. A. (2011). Ethnomedicine to Modern Medicine: Genesis through Ages. Journal
of Experimental Sciences Vol, 2(3), 25-29.
5. Taiwo A.A, Agbotaba M.O, Oyedepo J.A and Shobo M. (2008). Effects of Drying
Methods on Properties of Watermelon Oil. African Journal of Food and Development.,
8(4), 130-139.
6. Adegoke AA, Iberi PA, Akinpelu DA, Aiyegoro OA, Mboto CI. Studies on
phytochemical screening and antimicrobial potentials of Phyllanthus amarus against
multiple antibiotic resistant bacteria. International Journal of Applied Research in Natural
Products 2000; 3(3): 6-12.

7. Wall ME, Krider MM, Krewson CF, Eddy CR, Willaman JJ, Corell DS, Gentry HS.
Steroidal sapogenins VII: Survey of plants for steroidal sapogenins and other
constituents. Journal of American Pharmaceutical Association 1954; 43(1): 1-7.
8. Harborne JB. Phytochemical methods, a guide to modern techniques of plant analysis.
Chapman and Hall, New York; 1973, pp 88-185.

9. Edeoga HO, Okwu DE, Mbaebie BO. Phytochemical constituents of some Nigerian
medicinal plants. African Journal of Biotechnology 2005; 4: 685-688.

10. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a
standardized single disk method. American Journal of Clinical Pathology 1966; 45(4):
493-496.