Asian Pac J Trop Biomed 2014; 4(3):189-193 189

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Asian Pacific Journal of Tropical Biomedicine

journal homepage: www.elsevier.com/locate/apjtb

Document heading doi:10.1016/S2221-1691(14)60230-6 襃 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.

Phytochemical constituents and antibacterial activity of some green leafy

vegetables
Ramesa Shafi Bhat*, Sooad Al-Daihan
Biochemistry Department, College of Science, King Saud University, P. O. Box 22452, Riyadh 11495, Saudi Arabia

PEER REVIEW ABSTRACT

Peer reviewer Objective: To investigate the antibacterial activity and photochemicals of five green leafy
D r. A faf K . E l- A nsary, P rofessor, vegetables against a panel of five bacteria strains.
B iochemistry D epartment, S cience Methods: Disc diffusion method was used to determine the antibacterial activity, while
College, King Saud University, Saudi kanamycin was used as a reference antibiotic. The phytochemical screening of the extracts was
Arabia. performed using standard methods.
Tel: 009661-4769137 ext. 1396 Results: All methanol extracts were found active against all the test bacterial strains. Overall
Fax: 009661-4769137 maximum extracts shows antibacterial activity which range from 6 to 15 mm. Proteins and
E-mail: elansary@ksu.edu.sa carbohydrates was found in all the green leaves, whereas alkaloid, steroids, saponins, flavonoids,
tannins were found in most of the test samples.
Comments Conclusions: The obtain result suggests that green leafy vegetables have moderate antibacterial
This is a good and applicable study in activity and contain various pharmacologically active compounds and thus provide the scientific
which the author tested the efficacy basis for the traditional uses of the studied vegetables in the treatment of bacterial infections.
of 5 different green leafy plants as
antibacterial extracts. The obtained
results are promising showing the
possibility of using these plants to treat
different bacterial infections through
their antioxidant, detoxification and
antimicrobial effects. KEYWORDS
Details on Page 192 Green leafy vegetables, Antibacterial activity, Phytochemical screening.

1. Introduction source of carotene, ascorbic acid, riboflavin, folic acids

and minerals like calcium, iron and phosphorus[7]. Being a
Green leafy vegetables have been used as medicine since photosynthetic tissue, leafy vegetables have higher levels of
ancient times and have been playing a very important vitamin K when compared with other fruits and vegetables
role in our diet and nutrition. They are the most readily due to direct involvement of vitamin K (phylloquinone) in
available sources of carbohydrates, fats, important proteins, photosynthesis process. Vegetables as medicinal plants
vitamins, minerals, essential amino acids, and fibers[1]. contain none or less toxic effects[8,9], and have the ability
Their bioactive substances have a wide range of biological to synthesize several secondary metabolites of relatively
functions, including antioxidant and antimicrobial complex structures possessing antimicrobial activities[10-12].
activities[2-5] and can be helpful in management of oxidative Green leafy vegetables are also rich in compounds having
stress and age related human aliments[6]. They are rich antidiabetic[13], anti-histaminic[14], anti-carcinogenic[15]
*Corresponding author: Ramesa Shafi Bhat, Biochemistry Department, College of Article history:
Science, King Saud University, P. O. Box 22452, Riyadh 11495, Saudi Arabia. Received 25 Dec 2013
Tel: +96614785968 ext 1204 Received in revised form 3 Jan, 2nd revised form 11 Jan, 3rd revised form 20 Jan 2014
E-mail: ramesa.aftab@gmail.com, rbhat@ksu.edu.sa Accepted 29 Feb 2014
Foundation Project: Supported by a grant from the Research Center of the Center for Available online 28 Mar 2014
Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud
University.
190 Ramesa Shafi Bhat and Sooad Al-Daihan /Asian Pac J Trop Biomed 2014; 4(3): 189-193

and hypolipidemic[16] properties and possess preventive or in 30 mL of 0.01 mol/L HCl containing 0.15 mol/L NaCl.
curative properties against cardiovascular disease, ageing, (sample:extract solution, 1:3 w/v). The residue was then
obesity, hypertension, insomnia and ageing[17-19]. Leafy removed by filtering through cheese cloth. The filtrate was
vegetables are natural source of antioxidants and rich then centrifuged at 8 100伊g, for 5 min. These leaves and
in phytochemicals[20,21]. The present work was therefore extract were subjected to antibacterial activity experiments
designed to investigate the antibacterial effects of five leafy and protein determination[22].
vegetables namely Coriandrum sativum (C. sativum), Lactuca
sativa (L. sativa), Mentha piperita (M. piperita), Portulaca 2.3.3. Methanol extraction
oleracea (P. oleracea) and Raphanus sativus (R. sativus) Ten grams of powdered sample was dissolved in 100 mL of
against some bacteria strains and their phytochemical methanol in a conical flask, plugged with cotton wool and
screening. then kept on a rotary shaker at 190-220 r/min for 24 h. The
supernatant was collected slowly and evaporated in wide
mouthed evaporating bowls at room temperature for 2-3 d
2. Materials and methods till the final volume was reduced to one fourth of the original
volume of the solvent used, giving the concentration of 400
2.1. Collection of plant material mg/mL[23] and stored at 4 °C in airtight bottles.

F resh leaves of C. sativum, L. sativa, M. piperita, 2.4. Media preparation

P.oleracea and R. sativus free from disease were purchased
from local farms in Al-Qassim. Samples were labeled and Twenty three grams of nutrient agar (Sigma-Aldrich,
stored at 4 °C in polythene bags till they were processed. Germany) were dissolved in 1 000 mL of distilled water and
Collected materials were washed thoroughly in running bring to boil. Agar was then autoclaved for 15 min at 121 °C
tap water, rinsed in distilled water and shade dried for and left to cool at room temperature. Once the LB medium
one week in open air, and then crushed using mortar and was cooled (about 45 °C), it was poured into Petri dishes.
pestle, reduced to powder using Waring laboratory blender Each Petri dish was left on the flat surface for 30-40 min
( MX - 7011 G ) for 5 min at high speed and then stored in until completely set.
airtight closed bottles for two days before used for analysis.
Fifty grams of all the fresh samples were stored for juice 2.5. Antibacterial activity
preparation.
A ntibacterial activity was assayed by disc diffusion
2.2. Microorganisms method. For all bacteria strains, overnight culture grown
in broth was adjusted to an inoculum’s density of 100 µL:
Bacteria cultures of Staphylococcus aureus, Streptococcus 0 . 1 A 600 culture containing 3 . 2 伊10 colony forming unit.
8

pyogenes, Escherichia coli, Bacilllus subtillis, and Further, 20 µL was spread onto 20 mL of sterile agar plates
Pseudomonas aeruginosa (clinical isolates) were obtained by using a sterile cotton swab. The surface of the medium
from Botany Department of King Saud University. The strains was allowed to dry for about 3 min. Sterile filter paper discs
were maintained on agar slant at 4 °C and activated at 37 °C (5 mm in diameter) impregnated with different test extracts
for 24 h on nutrient agar (Sigma-Aldrich, Germany) before (100 µL disc) were then placed on the surface of inoculated
any susceptibility test. agar plates. Kanamycin (30 µg/disc) was used as positive
control. The plates were then incubated at 37 °C for 24 h after
2.3. Preparation of leaf extract which microbial growth was determined by measuring the
diameter of the inhibition zone (mm) using a transparent
2.3.1. Juice preparation scale. Each extract was analyzed in triplicate, the mean
Fifty grams of raw leave samples after washing with water values are presented. Kanamycin disc (30 µg/disc) was used
were crushed by grinder without adding any solvent. The for comparing the bioassay.
residue was removed by filtering through 8 layers of muslin
cloth. The filtrate was collected in clean airtight bottle and 2.6. Phytochemical analysis
stored at 4 °C until use for antibacterial activity test.
2.6.1. Molisch’s test for Carbohydrates
2.3.2. Aqueous extraction At first 0.5 g of each powder was dissolved separately in
T en grams of dry powder of samples were dissolved 5 mL of distilled water and filtered. Few drops of Molisch’s
 Ramesa Shafi Bhat and Sooad Al-Daihan /Asian Pac J Trop Biomed 2014; 4(3): 189-193
191

reagent were added to each solution, this was then followed 3. Results
by addition of 1 mL of concentrated H2SO4 by the side of the
test tube. The mixture was then allowed to stand for 2 min 3.1. Antibacterial activity of the vegetable extracts
and then diluted with 5 mL of distilled water. Formation of
a red or dull violet colour at the interphase of the two layers The antibacterial activity of five green vegetables extract was
was taken as positive test[24]. assayed in vitro by agar disc diffusion against five bacterial
species. The data in Table 1 show the antibacterial activities
2.6.2. Test for alkaloids of the tested extracts on a panel of five Gram positive or Gram
A given weight of 0.1 g of each powder was dissolved in 5 negative bacteria. All methanol extracts were found active
mL of methanol separately and then filtered. A volume of 2 against all the test bacterial strains. All the extracts of C.
mL of each filtrate from each sample were stirred with 5 mL sativum showed maximum inhibitory activity against the entire
of 1% aqueous HCl on water bath and then filtered. Of the test bacterial strains while all extracts from M. piperita were
filtrate, 1 mL was taken individually into 2 test tubes. To the less active against test organism under study. The best activity
first portion (1 mL), few drops of Dragendorff’s reagent were was obtained with methanol extract from C. sativum against E.
added. Occurrence of orange-red precipitate was taken as coli.
positive. To the second 1 mL, Mayer’s reagent was added and
Table 1
appearance of buff-colored precipitate was taken as positive Antibacterial activity of various extracts of test samples against bacterial
test for the presence of alkaloids[24]. species tested by disc diffusion assay.
Plant extract Zone of inhibition (mm)
S. aureus S. pyogenes B. subtillis E. coli P. aeruginosa
2.6.3. Liebermann-Burchard test for steroids C. sativum Fresh juice 8.50依0.57 7.00依0.10 8.00依0.33 10.00依0.77 8.50依0.00
At the beginning 0.2 g of crude powder of each sample Aqueous 11.00依0.20 12.00依0.00 11.00依0.42 10.00依0.43 11.00依0.15

was dissolved in 2 m L of acetic acid separately. T he Methanol 14.50依0.00 13.00依0.60 12.00依0.45 15.00依0.56 11.00依0.11
L. sativa Fresh juice - - 6.00依0.44 9.00依0.45 -
solutions were cooled well in ice followed by the addition of Aqueous 10.00依0.55 9.00依0.20 - 11.00依0.23 -
concentrated H2SO4 carefully. Color development from violet Methanol 13.00依0.10 14.00依0.66 11.50依0.55 12.50依0.44 11.00依0.50

to blue or bluish-green indicated the presence of a steroidal M. piperita Fresh juice 7.00依0.50 8.00依0.56 6.50依0.00 7.50依0.76 -
Aqueous 8.50依0.40 11.00依0.68 - 9.00依0.23 -
ring[24]. Methanol 12.00依0.00 12.50依0.33 10.00依0.42 12.00依0.56 12.00依0.00
P. oleracea Fresh juice 9.00依0.15 8.50依0.57 - 7.00依0.63 6.50依0.15
Aqueous 12.00依0.33 12.00依0.34 - 12.00依0.71 -
2.6.4. Test for saponins
Methanol 14.00依0.66 13.00依0.40 11.50依0.33 14.00依0.55 10.00依0.33
One gram of crude powder of each sample was boiled with R. sativus Fresh juice 10.00依0.80 8.00依0.00 - 8.00依0.00 7.00依0.15

5 mL of distilled water separately and then filtered. To each Aqueous 11.50依0.43 10.00依0.20 10.00依0.32 9.00依0.31 9.00依0.35
Methanol 15.00依0.12 12.00依0.33 13.00依0.10 11.00依0.45 13.00依0.25
filtrate, about 3 mL of distilled water was further added and Kanamycin (30 µg/disc) 26.50依0.33 28.00依0.57 21.00依0.15 20.00依0.33 25.00依0.10
shaken vigorously for about 5 min. Frothing which persisted Values are mean inhibition zone (mm)依SD of three replicates.
on warming was taken as an evidence for the presence of
saponins[24].
3.2. Chemical composition of the vegetable extracts
2.6.5. Shinoda’s test for flavonoids
About 0.5 g of each powder was dissolved in 5 mL of T he results of the qualitative analysis showed that
ethanol separately, warmed and then filtered. Three pieces carbohydrates and proteins were found in all the leaves under
of magnesium chips was then added to the filtrate followed study (Table 2). Flavonoids were present in all samples except
by few drops of concentrated HCl. A pink, orange, or red to in M. piperita. Only green leaves of M. piperita and R. sativus
purple colouration indicates the presence of flavonoids[25]. contain steroids while alkaloids were found in L. sativa, P.
oleracea and R. sativus. Saponins were found in M. piperita, P.
2.6.6. Test for tannins oleracea and R. sativus, and tannin was found in C. sativum, M.
About 0.5 g of each portion of crude powder was stirred piperita and P. oleracea.
with about 10 mL of distilled water separately and then
Table 2
filtered. Few drops of 1% ferric chloride solution were added Phytochemical composition of different salad leaves.
to 2 mL of each filtrate occurrence of a blue-black, green or Pleants Part used Carbohydrates Alkaloids Steroids Saponins Flavonoids Tannins Proteins

blue-green precipitate indicates the presence of tannins[25]. C. sativum Leaves + - - - + + +

L. sativa Leaves + + - - + - +
M. piperita Leaves + - + + - + +

2.7. Statistical analysis P. oleracea

R. sativus
Leaves
Leaves
+ + - + +
-
+ +
+ + + + + +

+: Present; -: Absent.
The results were analyzed by using standard deviation (SD)
statistical methods[26].
192 Ramesa Shafi Bhat and Sooad Al-Daihan /Asian Pac J Trop Biomed 2014; 4(3): 189-193

4. Discussion Comments

The various leave extracts showed varied antimicrobial Background

activity to the test organism which was species dependent. The authors gave a strong and well written background
Gram-positive bacterial strains were more susceptible to on the medicinal uses of green leafy vegetables, their
the extracts when compared to Gram negative bacteria. antioxidant and antimicrobial efficacy. The cited references
Gram negative bacteria are surrounded by the cell wall covered a period from 1998 until 2013 which reflect the
which restricts diffusion of hydrophobic compounds awareness of the author with the presented topic.
through its lipopolysaccharide covering. The absence of
this barrier in Gram positive bacteria allows the direct Research frontiers
contact of the essential oil’s hydrophobic constituents with Green leafy vegetables have phyto-protectants that are
the phospholipids bilayer of the cell membrane, causing important for cell growth, replacement, and body building.
either an increase of ion permeability and leakage of vital T heir detoxification and antimicrobial effects increase
intracellular constituents, or impairment of the bacterial their nutritional values.
enzyme systems[27]. Also two groups of bacteria differ in
their structure of cell wall. Ability of tannin to disintegrate Related reports
bacterial colonies is hinder with bacterial cell wall [28]. The antimicrobial effects of the five green leafy plants
Medicinal plants which are rich in tannins are used to treat were ascertained in this study.
inflamed or ulcerated tissues[29].
B ioactive compounds are normally accumulated Innovations and breakthroughs
as secondary metabolites in all plant cells but their The present study foucuses on the possibility of the
concentration varies according to the plant parts, seasons, traditional use of green leafy plant extracts in treating
climates and particular growth phases. Leaves are one bacterial infections.
of the highest sources of accumulation and are highly
beneficial[30,31]. Applications
Most of the secondary metabolite identified in the test The research results indicate that the five plants could
samples like flavonoids, saponins, tannin, steroids and be used in developing antimicrobials.
alkaloids are phytoprotectants and are important for cell
growth, replacement, and body building[32]. Their medicinal Peer review
value is due to presence of some chemical substances This is a good and applicable study in which the author
that can produce a defined physiological action on human tested the efficacy of 5 different green leafy plants as
body with antioxidant, antibacterial, anti-inflammatory, antibacterial extracts. The obtained results are promising,
antiviral, immune system stimulant and detoxification showing the possibility of using these plants to treat
activities[33]. different bacterial infections through their antioxidant,
Green leafy vegetables contain various pharmacologically detoxification and antimicrobial effects.
active compounds. On the whole the present investigation
confirmed the traditional uses of the studied vegetables in
the treatment of bacterial infections. References

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