“Screening of aerial parts of Ziziphus nummularia for biological activities”

1. Introduction:

Nature has been a source of medicinal agents for thousands of years and an impressive

number of modern drugs have been isolated from natural sources; many of these isolations were

based on the uses of the agents in traditional medicine. This plant-based, traditional medicine

system continues to play an essential role in health care, with about 80% of the world’s

inhabitants relying mainly on traditional medicines for their primary health care [1]. Natural

products of plants may provide a new source of antimicrobial agents with possibly novel

mechanism of action [2]. The search of biologically active compounds from plants has always

been of great interest to scientists looking for new sources of useful drugs against infectious

diseases. In the recent years, infections have increased to a great extent and antibiotic resistance

becomes an ever increasing therapeutic problem [3].

Zizyphus nummularia commonly called jujube, belongs to family Rhamnaceae [4]. This

family consists of 50 genera and more than 900 species. It is almost cosmopolitan and found

mainly in subtropical to tropical areas [5]. It is a shrub plant up to 2 meters high and a

multipurpose species valued for edible fruits, leaves as forage, branches for fencing and as folk

medicine. This plant grows in warm and dry climate and on sandy and silicic soil. A much

branched shrub, flexuous, tomentose, young branches puberulous, grey, spines in unequal pairs,

smaller recurved, bark light colour. The roots, leaves, seeds of zizyphus species have been used

against many diseases such as malaria, jaundice, anxiety, insomnia, sore throat, liver diseases

and many yields edible fruits [4].

2. Aims and Objectives:

The aim and objective of this research is to screen the aerial parts of Ziziphus nummularia specie

for antibacterial, phytotoxic and haemagglutination activities.

3. Plan of Work and Methodology:

Phase 1:

Plant selection:

The aerial parts of Z. nummularia will be collected from the native Northern region of Khyber

Pukhtunkhawa, Pakistan. The sample identification would be done.

Phase 2:

Extract preparation:

The plant material will be shade dried, chopped into small pieces and ground to powder using an

electric grinder. The powdered material of Z. nummularia will be soaked in methanol for 15

days, twice, at room temperature, with occasional shaking. Each time, the material will be

filtered and the filtrate will be concentrated at 40°C under vacuum, by rotary evaporator and

extract of Z. nummularia will be obtained.

Phase 3

Fractionation:

The crude methanolic extract of Z. nummularia will be suspended in distill water and partitioned

with n-hexane, chloroform and ethyl acetate respectively, to yield their fractions. Some of the

crude methanolic extract of Z. nummularia will be left for biological activities. All the fractions

will only contain their particular compounds based on the solubility from the crude extract.

Phase 4

Biological activities:

Biological activities will be done for crude and fractionated extract samples.

1. Antibacterial activity

Determining percent inhibition

Antibacterial activity of Z. nummularia will be determined against different strains of bacteria.

Eighteen hours old culture of the test organism from the nutrient broth will be transferred to

sterile nutrient agar plates to make bacterial lawn. Using a sterile borer, wells will be dug in

plates. Stock solutions of the test samples will be prepared in sterile DMSO. 100 µl of crude

methanolic extract and fractions will be loaded to their respective wells. Antibiotic drug and

DMSO will be used as positive and negative controls, respectively. Zone of inhibition will be

measured in comparison with positive control.

Determination of minimum inhibitory concentration (MIC)

After determining the percent inhibition, the MIC50 of the test samples at different

concentrations will be measured against the test organisms . To sterile nutrient broth in the test

tubes, test samples and test organisms will be inoculated and then incubated. Results will be

recorded after 24 hours based on the percent clarity.

2. Phytotoxic activity:

Phytotoxic activity of the crude methanolic extract and various fractions of Z. nummularia will

be determined against Lemna minor. Stock solutions will be prepared and will be introduced in

sterilized flasks, inoculated with test samples and will be left overnight. E-media will be then

added to the flasks and healthy L. minor plants will be introduced into each flask. A standard

growth inhibitor will be added. All the flasks will be then incubated in growth chamber for 7

days. The results will be noted by counting the number of fronds damaged and growth inhibition

will be calculated in reference with the negative control.

3. Haemagglutination activity:

The crude methanolic extract and various fractions of Z. nummularia fractions will be screened

for possible haemagglutination activity against human erythrocytes of all blood groups. Fresh

blood will be collected from healthy volunteers, centrifuged and the erythrocytes will be

separated. Erythrocytes suspension will be prepared in phosphate buffer. For activity, stock

solution of the test samples will be prepared in DMSO and different dilutions will be made from

it. From each dilution, small amount will be added to erythrocytes suspension and incubated at

37°C. Positive and negative results will be indicated by rough granules and smooth button

formation. Extent of deposition will be determined the intensity of positive result.

4. References:

1. Owolabi, O.J., E.K. Omogbai, and O. Obasuyi, Antifungal and antibacterial activities of the

ethanolic and aqueous extract of Kigelia africana (Bignoniaceae) stem bark. African Journal of

Biotechnology, 2007. 6(14).

2. Barbour, E.K., et al., Screening of selected indigenous plants of Lebanon for antimicrobial

activity. Journal of Ethnopharmacology, 2004. 93(1): p. 1-7.

3. Shahwar, D. and M.A. Raza, In vitro antibacterial activity of extracts of Mimusops elengi against

gram positive and gram negative bacteria. African Journal of microbiology research, 2009. 3(8):

p. 458-462.

4. Verma, S., A REVIEW ON ZIZIPHUS NUMMULARIA: VALUABLE MEDICINAL PLANT OF DESERT.

2016.

5. Jan, G., M.A. Khan, and F. Gul, Ethnomedicinal plants used against jaundice in Dir Kohistan

valleys (NWFP), Pakistan. Ethnobotanical Leaflets, 2009. 2009(8): p. 7.