The document summarizes a study that screened the aerial parts of Ziziphus nummularia for biological activities. Z. nummularia is a shrub plant found in subtropical and tropical regions that is used in traditional medicine. The aim of the study was to screen the aerial parts of Z. nummularia for antibacterial, phytotoxic, and haemagglutination activities. The methodology involved collecting plant material from Northern Pakistan, preparing extracts and fractions, and testing the crude extract and fractions for antibacterial activity against various bacteria strains, phytotoxic activity against Lemna minor, and haemagglutination activity against human erythrocytes.

1. “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:

2. 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.

3. 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., etal., Screening of selected indigenousplantsof Lebanon forantimicrobialactivity.
Journal of Ethnopharmacology, 2004. 93(1): p. 1-7.

4. 3. Shahwar, D. and M.A. Raza, In vitro antibacterial activity of extracts of Mimusopselengi against
gram positive and gram negative bacteria. African Journal of microbiology research, 2009. 3(8):
p. 458-462.
4. Verma,S., A REVIEWON ZIZIPHUSNUMMULARIA:VALUABLEMEDICINALPLANTOFDESERT. 2016.
5. Jan,G., M.A.Khan,and F.Gul, Ethnomedicinalplantsused againstjaundicein Dir Kohistan valleys
(NWFP), Pakistan. Ethnobotanical Leaflets, 2009. 2009(8): p. 7.