Online Submissions: http://www.journaltcm.
com J Tradit Chin Med 2019 August 15; 39(4): 482-491
info@journaltcm.com
Microbiostatic, antioxidative and cytotoxic potentiation of some
grasses of Bahawalpur, Pakistan
Iram Fatima, Sobia Kanwal, Tariq Mahmood
aa
Iram Fatima, Tariq Mahmood, Department of Plant Scienc-
es, Faculty of Biological Sciences, Quaid-i-Azam University,
Islamabad 45320, Pakistan
Sobia Kanwal, Department of Zoology, University of Gujrat,
Sub-campus Rawalpindi, 46000, Pakistan.
Correspondence to: Tariq Mahmood, Department of Plant
Sciences, Faculty of Biological Sciences, Quaid-i-Azam Uni-
versity, Islamabad 45320, Pakistan. tmahmood.qau@gmail.
com; tmahmood@qau.edu.pk
Telephone: +92-51-9064 3050
Accepted: October 15, 2018
Abstract
OBJECTIVE: To evaluate biological potential of
methanol and n-hexane extracts of aerial parts in
seven species of family Poaceae.
METHODS: Qualitative phytochemical tests were
done by using standard protocols. In vitro antioxi-
dant activity was performed via different assays
and antimicrobial potential was observed via disc
diffusion method. Cytotoxic activity was carried out
using brine shrimps' assay.
RESULTS: Phytochemical studies revealed the pres-
ence of alkaloids, flavonoids, glycosides, phenols,
steroids, saponins, tannins, anthocyanins and cou-
marins in most of the plant extracts. Maximum anti-
oxidant and antimicrobial potential were observed
in Cymbopogon citratus methanol extract and
Cymbopogon citratus n-hexane extract along with
significant number of total flavonoids and phenols
contents. However, Polypogon monspeliensis
methanol extract and Polypogon monspeliensis
n-hexane extract showed minimum antioxidant as
well as antimicrobial potential. Moreover, methanol
extracts showed a cytotoxic effect with their effec-
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ISSN 0255-2922
© 2019 JTCM. All rights reserved.
RESEARCH ARTICLE
TOPIC
tiveness ranked Polypogon monspeliensis metha-
nol extract > Cymbopogon citratus methanol ex-
tract > Phalaris minor n-hexane extract > Aristida
funiculata methanol extract > Stipagrostis plumosa
methanol extract > Cenchrus ciliaris methanol ex-
tract > Panicum antidotale methanol extract. Simi-
larly, n-hexane plant extracts revealed cytotoxic ac-
tivity in decreasing order Cenchrus ciliaris n-hex-
ane extract > Stipagrostis plumosa n-hexane ex-
tract > Phalaris minor n-hexane extract > Aristida
funiculata n-hexane extract > Polypogon monspe-
liensis n-hexane extract > Panicum antidotale
n-hexane extract > Cymbopogon citratus n-hexane
extract respectively.
CONCLUSION: Methanol extracts exhibit signifi-
cant antioxidant and antimicrobial potential which
can be correlated to their medicinal values. The ob-
served brine shrimp's lethality of the plant extracts
revealed the presence of potent cytotoxic compo-
nents in these plants.
© 2019 JTCM. All rights reserved.
Keywords: Poaceae; Methanol; n-Hexane; Anti-in-
fective agents; Antioxidants; Cytotoxins
INTRODUCTION
Pakistan is a well-develop ecological zone, in which
plants are utilized as a medicine for human as well as
animals.1 Punjab is the second largest Province of Paki-
stan and Bahawalpur region is in one of the nine divi-
sions of the Punjab province, constructed on the south-
ern bank of Sutlej River.2 During summers, there is ex-
tremely harsh environment due to high temperature,
strong winds, low humidity and a high rate of evapora-
tion. However, it is very rich in biodiversity and the pe-
rennial grasses, by and large, serve as a significant
482 August 15, 2019 | Volume 39 | Issue 4 |
 I Fatima et al. / Research Article
source of fodder. Due to absence of health facilities,
people prefer indigenous plants as medicines. Poaceae
or the grass family is the fifth largest family of flower-
ing plants containing 660 genera and 10000 species.3
Some plants of family Poaceae are used as a medication
for hypertension, anti-inflammatory, anthelmintic, di-
uretic and antioxidant.4,5
Non-nutritive plant chemicals which provides protec-
tion or aids in preventing diseases are known as 'phyto-
chemicals'. Usually such chemicals are produced by
plants to protect themselves but recent studies prove
that these chemicals can also protect animals and hu-
mans against various diseases.6 Phytochemicals general-
ly include primary metabolites (carbohydrates, pro-
teins, amino acids and chlorophyll) and secondary me-
tabolites (alkaloids, steroids, tannins, saponins and fla-
vonoids).7 Oxygen is an essential component of life but
oxidative stress occurs when ROS production exceeds
antioxidant capability of the target cell. Naturally pres-
ent antioxidants in human body act by prevention, re-
moval and repair of oxidative damage.8 Recently, thera-
peutic potentials of medicinal plants have gained a lot
of interest because of less side effects. The preclude role
of plant phytochemicals as natural antioxidants has en-
grossed their screening for therapeutic components. Re-
cently, many antioxidants have been isolated from
plants.9
In many countries bacterial infections are the main
cause of deaths. Fungi are also ubiquitous in the envi-
ronment leading to different fungal infections. Fungal
species of the genus Aspergillus and other species have
been considered to be major plant pathogens world-
wide.10 Antimicrobial is an agent that either kill or pre-
vent the growth of microbes. Serious infections caused
by bacteria and fungi have become a major problem in
21st century. Synthetic antibiotics as well as fungicides
have many side-effects. Hence, now-a-days antimicrobi-
als of plant origin are generally recommended as they
are cheap, locally available and more effective in treat-
ment of infectious.11 Several plant species are reported
to possess antifungal and antibacterial properties.12-14
Brine shrimps cytotoxic test are preferably used to
check plant lethality before conducting antitumor and
cytotoxic assays. Brine shrimp assay is also used to ex-
amine plant extracts for activities against fungi, bacte-
ria as well as anticancer potential and to identify com-
pounds present in these plants that are involved in
these activities.15 Unfortunately, many plants of Baha-
walpur region are more often used by the local inhabit-
ants, but their chemical constituents and medicinal im-
portance is still not documented. Hence, present stud-
ies were carried out to determine the efficacy of com-
monly occurring fodder grasses. Different biological
tests were performed to check whether the selected spe-
cies can control the growth of pathogenic microorgan-
isms or may act as natural antioxidants. Furthermore,
difference between the use of methanol and n-hexane
plant extracts was also evaluated.
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MATERIALS AND METHODS
Preparation of plant extracts
Cenchrus ciliaris L. (Acc. No. 129852), Phalaris minor
Retz. (Acc. No. 129856), Polypogon monspeliensis L.
(Acc. No. 129849), Cymbopogon citratus Stapf.
(Acc. No. 129848), Panicum antidotale Retz. (Acc.
No. 129845), Stipagrostis plumosa (Tausch) Bor
(Acc. No. 129843) and Aristida funiculata Trin. &
Rupr. (Acc. No. 129847) were collected from Baha-
walpur, Pakistan during the month of March, 2016
and accession numbers were allotted by Herbarium
of Pakistan, Quaid-e-Azam University, Islamabad. Ex-
tracts of aerial plant parts were prepared with two
different solvents viz. methanol and n-hexane (50 gm/
500 mL each).16
Preliminary phytochemical tests
Initially some phytochemical tests were performed to
check the presence of flavonoids, saponins, terpenoids,
steroids, alkaloids, glycosides, coumarins and phenolic
compounds.16, 17
Total phenols and flavonoids contents
For total phenols, 20 μL of plant extracts was mixed
with 90 μL of Folin-Ciocalteu reagent and 90 μL of
sodium carbonate (NaCO3) solution and absorbance
was measured. Standard curve was made by mixing
methanol solution of gallic acid with same reagent.18
To determine flavonoids, 20 μL of plant extract was
mixed with 10 μL AlCl3, 10 μL of potassium acetate
and 160 μL dH2O and absorbance was measured.
Standard curve was made by quercetin and total fla-
vonoids were expressed as mg quercetin equivalent
(QE)/g.19
Antioxidant activity determination
Antioxidant activity was evaluated via following differ-
ent assays and then antioxidant activity index was cal-
culated.20
(a) Free radicals scavenging capacity: 2,2-diphe-
nyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethyl-
benzothiazoline-6-sulphonic acid) (ABTS) and SOR
scavenging activity was performed. In DPPH assay,
DPPH solution was prepared in methanol (9.6 mg/
1000 mL). 180 μL of this solution was added to a 20 μL
of test solution and absorbance was taken at 515 nm.
In ABTS assay, 7 mM ABTS solution was added in
2.45 mM Potassium persulphate. 160 μL of ABTS + so-
lution was added in 10 μL of plant extracts and absor-
bance was taken at 734 nm. In SOR assay, 1 mL of
50 mM NaCO3 and 0.4 mL of 24 mM nitro-blue tetra-
zolium were added in 1 mL of each extract solution.
Then 0.2 mL of ethylenediaminetetraacetic acid solu-
tion and 0.4 mL of hydroxylamine HCl were added
and absorbance was taken at 540 nm. Then, scaveng-
ing activity percentage and half maximal inhibitory
483 August 15, 2019 | Volume 39 | Issue 4 |
 I Fatima et al. / Research Article
concentration (IC50) values were calculated:21-23
% scavenging activity = Abscontrol - Abssample × 100
Abscontrol
(b) Reducing Power Assay and CUPRAC assay: Add
plant extract (200 μL) in phosphate buffer (500 μL)
and potassium ferricyanide (500 μL) and incubate at
50 ℃ . Then add 10% trichloroacetic acid (500 μL)
and centrifuge for 10 min. Mix supernatant with FeCl3
(100 μL) and then read the absorbance.24 In CUPRAC
assay, 10 μL of 7.5 mM ethanol neocuproine solution,
10 μL of 0.01M CuCl2 solution, and 10 μL of 1.0 M
ammonium acetate buffer solution were added to 20 μL
of gallic acid and plant extracts and absorbance was
measured at 515 nm.25
(c) Phosphomolybdate assay: 50 μL of extract was incu-
bated with 500 μL of reaction mixture [28 mM sodium
phosphate, 0.6 M Sulphuric acid (H2SO4) and 4 mM
ammonium molybdate] for 90 min and then absor-
bance was taken at 630 nm.26
Antimicrobial assay
Eleven microbial strains were collected from Depart-
ment of Microbiology, Quaid-i-Azam University, Is-
lamabad and propagated in Plant Biochemistry and
Molecular Biology Laboratory, Quaid-i-Azam Univer-
sity, Islamabad, Pakistan. These include Escherichia
coli, Staphylococcus aureus, Listeria monocytogenes,
Bacillus spizizenii, Salmonella typhi, Wickerhamomy-
ces anomalus, Fusarium oxysporum, Mucor sp., As-
pergillus flavus, Aspergillus niger and Saccharomyces
cerevisae. These isolates were first sub-cultured in a
respective media (nutrient broth and SDA) and incu-
bated for 18-24 h. Agar disc diffusion method was
used and oxytetracycline and chloramphenicol were
used as a positive control.27, 28 The zones < 8 mm
were not considered significant. Minimal inhibitory
concentration (MIC) and activity index for each ex-
tract was evaluated.
Cytotoxic brine shrimp assay
Plant extracts were prepared in methanol (20 mg/ 5 mL)
and different plant concentrations (6, 12, 25, 50, 100
and 250 mg/mL) were added to each vial and final vol-
ume was made up to 5 mL with saline solution. After
24 h, ten shrimps were transferred to each vial and in-
cubated at 32° C for 24 h, after which the survivors
were counted.29
Statistical analysis
Each experiment was done three times, and then
mean and standard deviation ( xˉ ± s) was recorded.
Analysis of variance and by least significant difference
performed to check significant differences at P <
0.05 between groups for phytochemicals and antioxi-
dant activity.30 Graphpad prism was used to calculate
IC50 values. Fifty/median lethal concentration (LC50)
values were calculated via probit analysis program
and were assessed at 95% confidence interval.31
JTCM | www. journaltcm. com
RESULTS
Qualitative analysis of phytochemicals and
determination of total phenols and flavonoids
contents
The qualitative analysis revealed that flavonoids and
phenols are present in all selected methanol and n-hex-
ane plant extracts. Glycosides, steroids, saponins, alka-
loids, terpenoids and coumarins were detected in most
of the plant extracts. However, tannins and anthocya-
nins were weakly present or absent in selected plant ex-
tracts. Overall, phytochemicals were strongly present in
methanol plant extracts as compared to the n-hexane
plant extracts (Table 1).
Among methanol plant extracts, Cenchrus ciliaris
methanol extract (CeCME) and Aristida funiculata
methanol extract (ArFME) [(219.9 ± 28.1) and (195.2
± 17.0) mg gallic acid equivalent (GAE)/g] exhibited
maximum phenolic contents while Cymbopogon citra-
tus methanol extract (CyCME) and Panicum antidot-
ale methanol extract (PaAME) [(38.9 ± 2.2) and (28.4
± 1.6) mg QE/g] revealed maximum flavonoids con-
tents. However, among n-hexane plant extracts, Cen-
chrus ciliaris n-hexane extract (CeCHE) and Cymbo-
pogon citratus n-hexane extract (CyCHE) [(189.5 ±
13.9) and (180.6 ± 25.6) mg GAE/g] possess maxi-
mum phenolic contents while Panicum antidotale
n-hexane extract (PaAHE) and Stipagrostis plumosa
n-hexane extract (StPHE) [(29.5 ± 1.3) and (29.2 ±
3.9) mg QE/g] exhibited maximum flavonoid contents
(Figure 1).
Antioxidant activities
The free radicals including DPPH, ABTS and SOR
scavenging activity of respective plant extracts was de-
termined and compared with standard antioxidant
(ascorbic acid). Most of the plant extracts were found
to be effective in scavenging radicals and the IC50 val-
ues was also determined (Table 2). In present studies,
among methanol plant extracts CyCME showed stron-
ger DPPH radical scavenging activity [IC50 (18.5 ±
1.0) μg/mL] while P. monspeliensis methanol extract
(PoMME) showed minimum DPPH scavenging activi-
ty [IC50 (132.6 ± 5.9) μg/mL]. In case of n-hexane
plant extracts, CeCHE showed stronger DPPH radical
scavenging activity [IC50 (52.1 ± 2.9) μg/mL] while P.
monspeliensis n-hexane extract (PoMHE) showed
minimum DPPH scavenging activity [IC50 (400.9 ±
3.9) μg/mL].
Similarly, highly significant (P < 0.05) variable ABTS
radical scavenging activity of selected species was ob-
served. In case of methanol plant extracts, maximum
activity was detected in Aristida funiculata methanol
extract (ArFME) [IC50 (5.6 ± 2.7) μg/mL] and mini-
mum activity was observed in PaAME [IC50 (15.8 ±
3.8) μg/mL]. However, among n-hexane plant extracts,
maximum radical scavenging activity was revealed in
CeCHE [IC50 (18.5 ± 1.3) μg/mL] and minimum ac-
484 August 15, 2019 | Volume 39 | Issue 4 |
 I Fatima et al. / Research Article

Notes: CeCME: Cenchrus ciliaris methanol extract; CeCHE: Cenchrus ciliaris n-hexane extract; PhMME: Phalaris minor methanol extract; PhMHE: Phalaris minor n-hexane extract; PoMME: Polypogon
monspeliensis methanol extract; PoMHE: Polypogon monspeliensis n-hexane extract; CyCME: Cymbopogon citratus methanol extract; CyCHE: Cymbopogon citratus n-hexane extract; PaAME: Panicum anti-
dotale methanol extract; PaAHE: Panicum antidotale n-hexane extract; StPME: Stipagrostis plumosa methanol extract; StPHE: Stipagrostis plumosa n-hexane extract; ArFME: Aristida funiculata methanol ex-
tivity was found in PoMHE [IC50 (96.3 ± 7.2) μg/mL].
ArFHE
In SOR assay, among methanol extracts maximum ac-

+++
+++

+++
++
+

+
+
-

-
-
tivity was present in PaAME [IC50 (46.3 ± 6.0) μg/mL]
and minimum activity was observed in Stipagrostis plu-
mosa methanol extract (StPME) [IC50 (186.4 ± 5.6) μg/
ArFME

+++
+++

+++

+++
mL]. However, in case of n-hexane plant extracts, maxi-
++

++
+

+
-
mum scavenging activity was detected in CyCHE [IC50
(34.6 ± 3.2) μg/mL] and minimum activity in Phalaris
minor n-hexane extract (PhMHE) [IC50 (68.6 ± 4.2)
StPHE

+++
+++

+++
++ μg/mL]. Overall, methanol extracts showed remarkably
+
-

-
-
-
-
stronger free radicals scavenging activity as compared
to the n-hexane extracts. Moreover, analysis of variance
StPME

also revealed significant variations among all plant spe-

+++
+++

+++

+++
++

++
++
+

+
-
cies.
In reducing power assay, the higher absorbance indi-
cates greater reducing power. In present studies, among
PaAHE
+++
++

++

++

methanol plant extracts, maximum reducing power

+

+
-

-
-

ability was found in CeCME [(30.8 ± 3.8) mg/g] and

minimum in StPME [(25.1 ± 1.1) mg/g]. However,
PaAME

among n-hexane plant extracts, maximum reducing

+++

+++

+++

+++
++
++

++

++
+
+

power ability was found in PaAHE [(35.4 ± 1.9) mg/g]

and minimum in StPHE [(15.0 ± 1.3) mg/g] respec-
tively (Figure 2).
CyCHE

Similarly, CUPRAC assay was performed in which

tract; ArFHE: Aristida funiculata n-hexane extract; +++: strongly present; ++: moderately present; +: weakly present; -: absent.
+++

+++

+++

+++
++

++

++

++
+

complex is formed between neocuproine and copper

(I). Results revealed that cupric ion reducing power of
the methanol plant extracts ranked in order: CyCME >
CyCME

CeCME > PaAME > ArFME > PhMME > StPME >
+++
+++

+++
+++

+++
+++

+++
++

++

PoMME [(119.3 ± 4.5), (104.6 ± 2.5), (95.7 ± 3.4),

(85.0 ± 2.5), (84.8 ± 1.7), (72.7 ± 5.7), (58.9 ± 1.8)
mg/g]. Similarly, cupric ion reducing power of the
PoMHE

n-hexane plant extracts was found in order: CeCHE >

++
++

++

++
+
+
+

+
-

CyCHE > PaAHE > PhMHE > ArFHE > StPHE >
PoMHE [(97.6 ± 2.4), (96.5 ± 1.5), (94.4 ± 3.1),
(84.6 ± 2.0), (80.1 ± 2.8), (54.5 ± 0.8), (53.5 ± 1.8)
PoMME

mg/g] respectively (Figure 2).

+++

+++

+++
++

++

++
+

+
+

The results obtained from phosphomolybdate assay

Table 1 Qualitative analysis of phytochemicals of selected plant extracts

showed that methanol extract has strong reducing abili-

ty as compared to the n-hexane extracts. In comparing
PhMHE
+++
+++

+++

methanol plant extracts, total antioxidant capacity

++

++
++

++
+
+
+

(TAC) was observed maximum in ArFME [(42.8 ±

3.6) mg/g] and minimum in CeCME [(33.3 ± 0.9) mg/
g] while among n-hexane plant extracts TAC was
PhMME
+++

+++
+++

+++

+++
++

++

++

found maximum in PaAHE [(48.6 ± 2.5) mg/g] and

+

minimum in ArFHE [(33.4 ± 2.1) mg/g]. Analysis of

variance also showed significant variations among se-
CeCHE

lected plant species (Figure 2).

+++

+++
++
++

++
+

+
-

Antioxidant activity index (AAI)

The AAI was used to grade the selected plant species
CeCME
+++
+++
+++

+++
+++

+++

on the basis of their antioxidant potential. Some plant

++
++
++

species showed different levels of activities in different

assays. For example, methanol extract of CyCME was
revealed as a good antioxidant by DPPH, ABTS and
Phytochemi-cal

Anthocyanin

CUPRAC assay but moderate or weak antioxidant by

Terpenoids
Flavonoids
Glycosides

Coumarin
Alkaloids

SOR scavenging assay, total reducing power assay and

Saponins
Tannins
Steroids
Phenols

total antioxidant assay. However, in case of n-hexane

extract, CeCHE was shown to be a good antioxidant

JTCM | www. journaltcm. com 485 August 15, 2019 | Volume 39 | Issue 4 |
 I Fatima et al. / Research Article

300 TPC (Methanol) TPC (n-hexane)

TFC (Methanol) TFC (n-hexane)
Concentration

250
200
(mg/g)

150
100
50
0 is r s
liar ino nsi tus tale osa lata
ci P. m lie citra ido lum icu
C. spe C. nt S. p . fu
n
on A P. a
P. m TPC and TFC of selected plant extracts
Figure 1 Total phenolic and flavonoids contents in the plant extracts
TPC: total phenolic contents; TFC: total flavonoids contents; C. ciliaris: Cenchrus ciliaris; P. minor: Phalaris minor; P. monspeliensis:
Polypogon monspeliensis; C. citratus: Cymbopogon citratus; P. antidotale: Panicum antidotale; S. plumosa: Stipagrostis plumosa;
A. funiculata: Aristida funiculata.

Table 2 Evaluation of IC50 values of free radical scavenging activities (n = 3, xˉ ± s)

DPPH radical scavenging activity ABTS radical scavenging activity SOR scavenging activity
Plant material
Methanol n-hexane Methanol n-hexane Methanol n-hexane
Cenchrus ciliaris 23.35±5.48 52.08±2.95 8.71±3.76 18.49±1.27 59.04±5.81 36.09±4.48
Phalaris minor 77.43±8.11 200.63±6.86 6.56±3.24 52.08±8.40 75.88±7.44 68.61±4.20
Polypogon monspeliensis 132.60±5.90 400.90±3.93 12.06±2.31 96.32±7.18 182.83±2.4 61.48±1.36
Cymbopogon citratus 18.48±1.03 80.03±9.18 9.42±2.78 19.53±5.06 87.32±8.24 34.63±3.24
Panicum antidotale 75.64±4.76 302.93±6.71 15.85±3.80 49.53±7.05 46.34±6.04 60.92±4.90
Stipagrostis plumosa 33.40±2.99 150.20±6.85 12.95±2.32 42.10±5.47 186.37±5.60 61.52±5.44
Aristida funiculata 34.80±7.34 259.76±7.84 5.63±2.69 26.41±8.31 58.68±7.80 50.25±9.73
Ascorbic acid 16.91±2.57 16.91±2.57 2.80±0.29 2.80±0.29 32.25±4.67 32.25±4.67
Notes: DPPH: 2,2-diphenyl-1picrylhydrazyl; ABTS: 2, 2'-azino-bis (3- ethylbenzthiazoline-6-sulphonic acid); SOR: superoxide radical.

TRP (Methanol) TRP (n-hexane) CUPRAC (Methanol)

140 CUPRAC (n-hexane) TAC (Methanol) TAP (n-hexane)
120
Concentration

100
80
(mg/g)

60
40
20
0
C.ciliaris P.monspeliensis
P.minor C.citratus P.antidotale S.plumosa A.funiculata
TPC, CUPRAC and TAC of selected plant extracts
Figure 2 Total reducing power (TRP), cupric reducing antioxidant capacity (CUPRAC) and total antioxidant capacity (TAC) of the se-
lected plant extracts
TRP and TAC expressed as ascorbic acid equivalent (mg AE/g extract); cupric ions reducing assay expressed as gallic acid equiva-
lent (mg GA/g extract). C. ciliaris: Cenchrus ciliaris; P. minor: Phalaris minor; P. monspeliensis: Polypogon monspeliensis; C. citratus:
Cymbopogon citratus; P. antidotale: Panicum antidotale; S. plumosa: Stipagrostis plumosa; A. funiculata: Aristida funiculata.

by DPPH radical scavenging assay, ABTS radical scav- Antimicrobial assay

enging assay and cupric ions reducing assay but moder- Totally 14 plant extracts were tested for their bioactivi-
ate or weak antioxidant by SOR radical scavenging as- ty. Most of the plant extracts showed significant or lit-
say, total reducing power assay and total antioxidant ac- tle antimicrobial potential against nine microbes. How-
tivity. Owing to such difficulty in comparing results, ever, no activity was observed against two microorgan-
AAI was calculated to combine the average results of isms (Fusarium oxysporum and Aspergillus niger) in
six assays. both methanol and n-hexane plant extracts. Most sus-
Present studies revealed that CyCME, CyCHE ceptible organisms among various strains were Staphy-
(53.96% and 47.28%) and CeCME, CeCHE (51.66% lococcus aureus and Bacillus spizizenii followed by Lis-
and 48.50% ) showed highest relative antioxidant po- teria monocytogenes and Wickerhamomyces anoma-
tential while PoMME and PoMHE (38.21% and lous against which, most of the plant extracts formed
29.83% ) showed poor antioxidant activity. However, inhibition zone. The range of MIC of extracts recorded
other plant species showed relatively moderate antioxi- was 50-75 μg/mL (Table 3).
dant potential. Overall, methanol plant extracts exhibit In present studies, little or non-significant antibacterial
maximum antioxidant activity as compared to the activity was observed against Salmonella typhi and
n-hexane plant extracts (Figure 3). Escherichia coli. Among methanol plant extracts,

JTCM | www. journaltcm. com 486 August 15, 2019 | Volume 39 | Issue 4 |
 I Fatima et al. / Research Article

A. funiculata Hex (against seven tested microbial strains) and minimum

S. plumosa Meth activity was recorded for PoMME (i.e against three
P. antidotale strains only) and PoMHE (i.e against five strains only)
C. citratus extracts respectively. However, remaining plant extracts
P. monspeliensis
P. minor
revealed some activity against few strains as shown in
C. ciliaris Figures 4 and 5.
010 20 30 40 50 60
Antioxidant activity index (% age) Cytotoxic activity
Figure 3 Antioxidant activity index (AAI) of methanol and All plants revealed good brine shrimp cytotoxic activity
n-hexane extracts of selected species when tested at six different concentrations (6, 12, 25,
A. funiculata: Aristida funiculata; S. plumosa: Stipagrostis 50, 100, 250 μg/mL). Maximum mortalities (100% )
plumosa; P. antidotale: Panicum antidotale; C. ciliaris: Cen-
were observed at a concentration of 250 parts per mil-
chrus ciliaris; P. monspeliensis: Polypogon monspeliensis; P.
minor: Phalaris minor; C. citratus: Cymbopogon citratus. lion in both methanol and n-hexane extracts. Metha-
nol extracts showed a cytotoxic effect with their effec-
PhMME and PaAME showed maximum activity tiveness ranked PoMME > CyCME > PhMME >
(ZOI: 9 mm, AI: 0.310) against Listeria monocyto- ArFME > StPME > CeCME > PaAME. Similarly,
genes and Bacillus spizizenii while among n-hexane n-hexane plant extracts revealed cytotoxic activity in
plant extracts PhMHE showed maximum antibacterial decreasing order CeCHE > StPHE > PhMHE > ArF-
activity (ZOI: 11 mm, AI: 0.379) against Listeria HE > PoMHE > PaAHE > CyCHE respectively (Ta-
monocytogenes. Among bacterial strains, lowest MIC ble 4).
values (50 μg/mL) were recorded for PaAME (against
Bacillus spizizenii) and PhMME (against Listeria
monocytogenes) and PhMHE, PoMHE, CyCHE, PaA-
DISCUSSION
HE (against Listeria monocytogenes). Similarly, in anti- In present study, the selected plant extracts contain fla-
fungal activity PhMME (ZOI: 13 mm, AI: 0.448) and vonoids, phenols, alkaloids, tannins, terpenoids, ste-
ArFHE (ZOI: 11 mm, AI: 0.379) showed maximum roids, anthocyanins and coumarins compounds. Quan-
activity against S. cerevisae as compared to the other titative analysis revealed that phenolic contents are pres-
plant extracts. However, no inhibition zones were ent in greater concentration than flavonoids in all spe-
observed against Fusarium oxysporum and Aspergillus cies and the maximum phenols and flavonoids are pres-
niger. ent in methanol plant extracts as compared to the
Overall, maximum antimicrobial activities were record- n-hexane plant extracts. Similarly, Abbas et al 40 and
ed for CyCME and CyCHE (i.e against eight tested Umar et al 41 reported the presence of various second-
microbial strains) followed by PhMME and PhMHE ary metabolites in Cenchrus ciliaris and Cymbopogon
Table 4 Percentage mortality of brine shrimps in probits at five different concentrations and respective LC50 values
Plant Mortality (%) in Probits at different doses
Slope Intercept R square LC50 95% CI
extract 6 12 25 50 100
CeCME 4.75 4.90 5.08 5.15 6.48 1.110 3.693 0.836 15.051 7.687-29.470
CeCHE 5.00 5.25 5.52 5.61 6.08 0.818 4.355 0.926 6.152 2.305-16.416
PhMME 4.75 5.00 5.41 5.61 6.75 1.437 3.480 0.928 11.418 6.401-20.367
PhMHE 4.82 5.15 5.33 5.61 6.08 0.966 4.054 0.945 9.520 4.152-21.828
PoMME 4.75 5.25 5.52 5.61 6.08 0.985 4.072 0.940 8.756 3.846-19.937
PoMHE 4.75 5.00 5.41 5.52 6.75 1.394 3.517 0.910 11.595 6.451-20.839
CyCME 4.82 5.15 5.33 5.52 6.75 1.286 3.692 0.891 10.406 5.568-19.446
CyCHE 3.87 4.75 5.15 5.61 6.28 1.845 2.569 0.981 20.770 12.968-33.266
PaAME 4.48 4.90 5.08 5.25 6.28 1.252 3.441 0.916 17.577 9.369-32.975
PaAHE 4.26 4.56 5.25 5.33 6.48 1.652 2.864 0.947 19.639 11.856-32.532
StPME 4.56 4.75 5.15 5.95 6.75 1.788 2.939 0.961 14.194 8.680-23.210
StPHE 5.00 5.33 5.61 6.08 - 1.140 4.093 0.968 6.241 2.915-13.363
ArFME 4.90 5.00 5.15 5.41 6.48 1.094 3.843 0.872 11.424 5.632-23.171
ArFHE 4.90 5.08 5.33 5.41 6.48 1.076 3.919 0.880 10.102 4.886-20.883
Notes: CeCME: Cenchrus ciliaris methanol extract; CeCHE: Cenchrus ciliaris n-hexane extract; PhMME: Phalaris minor methanol ex-
tract; PhMHE: Phalaris minor n-hexane extract; PoMME: Polypogon monspeliensis methanol extract; PoMHE: Polypogon monspeliensis
n-hexane extract; CyCME: Cymbopogon citratus methanol extract; CyCHE: Cymbopogon citratus n-hexane extract; PaAME: Panicum
antidotale methanol extract; PaAHE: Panicum antidotale n-hexane extract; StPME: Stipagrostis plumosa methanol extract; StPHE: Stipa-
grostis plumosa n-hexane extract; ArFME: Aristida funiculata methanol extract; ArFHE: Aristida funiculata n-hexane extract; LC50: fifty/
median lethal concentration; CI: confidence interval.

JTCM | www. journaltcm. com 487 August 15, 2019 | Volume 39 | Issue 4 |
 I Fatima et al. / Research Article

E.coli S.aureus L.monocytogenes B.spizizenii S.typhi

0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
E E E E
CM eCH MM hMH
E ME HE yCM CH
E
ME aAHE ME tPHE ME HE
Ce C Ph P PoM PoM C Cy PaA P StP S ArF ArF
Activity index of antibacterial activity
Figure 4 Activity Index of antibacterial activity of selected methanol and n-hexane plant extracts
CeCME: Cenchrus ciliaris methanol extract; CeCHE: Cenchrus ciliaris n-hexane extract; PhMME: Phalaris minor methanol extract;
PhMHE: Phalaris minor n-hexane extract; PoMME: Polypogon monspeliensis methanol extract; PoMHE: Polypogon monspeliensis
n-hexane extract; CyCME: Cymbopogon citratus methanol extract; CyCHE: Cymbopogon citratus n-hexane extract; PaAME: Pani-
cum antidotale methanol extract; PaAHE: Panicum antidotale n-hexane extract; StPME: Stipagrostis plumosa methanol extract;
StPHE: Stipagrostis plumosa n-hexane extract; ArFME: Aristida funiculata methanol extract; ArFHE: Aristida funiculata n-hexane
extract.

W. anomalus F. oxysporum Mucor A.f lavus A. niger S. cerevisae

0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
E
CME CH
E
MM hMH
E ME HE yCM
E
CH
E
ME aAHE ME HE ME HE
Ce Ce Ph P PoM PoM C Cy PaA P StP StP ArF ArF
Activity index of antifungal activity
Figure 5 Activity Index of antifungal activity of selected methanol and n-hexane plant extracts
CeCME: Cenchrus ciliaris methanol extract; CeCHE: Cenchrus ciliaris n-hexane extract; PhMME: Phalaris minor methanol extract;
PhMHE: Phalaris minor n-hexane extract; PoMME: Polypogon monspeliensis methanol extract; PoMHE: Polypogon monspeliensis
n-hexane extract; CyCME: Cymbopogon citratus methanol extract; CyCHE: Cymbopogon citratus n-hexane extract; PaAME: Pani-
cum antidotale methanol extract; PaAHE: Panicum antidotale n-hexane extract; StPME: Stipagrostis plumosa methanol extract;
StPHE: Stipagrostis plumosa n-hexane extract; ArFME: Aristida funiculata methanol extract; ArFHE: Aristida funiculata n-hexane
extract.

citratus species. StPHE. In CUPRAC assay, strong cupric ion reducing

In present study, methanol plant extracts showed signif-
icantly stronger inhibitory concentration ranging from
(18.5 ± 1.0) to (132.6 ± 5.9) μg/mL as compared to
the n-hexane plant extracts which exhibited IC50 (52.1
± 2.9) to (400.9 ± 3.9) μg/mL respectively. Overall, Cy-
CME, CyCHE, CeCME and CeCHE showed maxi-
mum DPPH radical scavenging activity and PoMME
and PoMHE showed least antioxidant potential where-
as other species showed moderate antioxidant potential.
In ABTS assay, ABTS + radical cation is produced by
the reaction of ABTS and potassium persulfate which
is then reduced in the presence of antioxidants. Similar-
ly, Superoxide radical is a major source of ROS.43 Al-
though it is a weak oxidant, but it produces powerful
radicals which causes oxidative stress.44 Results revealed
that selected methanol and n-hexane extracts possess ef-
fective antioxidant potential.
The antioxidant capacity of the plants was also evaluat-
ed via phosphomolybdenum method. All species
showed reducing power ability except StPME and
ability was observed in CyCME, CyCHE, CeCME
and CeCHE while weak cupric ion reducing potential
in StPME, StPHE, PaAME and PaAHE respectively.
Overall, present studies correlate with the studies of
Rathabai et al,45 who reported significant antioxidant
activity in Cymbopogon citratus and Cenchrus ciliaris
using DPPH, ABTS, SOR free radical assays, CU-
PRAC, TAC and total reducing power assays. Similar-
ly, Balakrishnan et al 46 and Nambiar et al 47 also re-
vealed strong antioxidant potential in the methanol ex-
tracts of Cymbopogon citratus.
Antibiotic-resistant bacteria continue to emerge rapid-
ly, causing a problem in the treatment of diseases
caused by them. Plant-based drugs have been proved ef-
fective in the treatment of many diseases. Antibacterial
activity of methanol and n-hexane plant extracts re-
vealed that most of the plant extracts were more active
against Staphylococcus aureus, Listeria monocyto-
genes, Bacillus spizizenii and Wickerhamomyces anom-
alous while Fusarium oxysporum and Aspergillus niger

JTCM | www. journaltcm. com 488 August 15, 2019 | Volume 39 | Issue 4 |
 Table 3 Antimicrobial activity determined as zone of inhibition (mm) and MIC (µg/mL) of selected species against tested microbial strains ( xˉ ± s)
Plant extract CeCME CeCHE PhMME PhMHE PoMME PoMHE CyCME CyCHE PaAME PaAHE StPME StPHE ArFME ArFHE Control
ZOI NI NI NI NI NI NI NI NI NI NI NI NI NI NI 30.0±1.5
Escherichia
coli MIC - - - - - - - - - - - - - - -
ZOI 7.0±0.5 4.0±3.7 4.0±3.7 NI NI NI NI 7.0±0.5 NI NI 7.0±1.7 NI NI NI 32.0±2.8
Staphylococ
cus aureus MIC - - - - - - - - - - - - - - -
Listeria ZOI NI 7.0±1.0 9.0±1.7 11.0±0.3 NI 9.0±1.0 8.0±2.0 9.0±2.0 8.0±1.5 9.0±1.0 NI 8.0±0.5 NI 7.0±1.1 29.0±1.5
monocytoge
MIC - - 50.0 50.0 - 50.0 75.0 50.0 75.0 50.0 - 75.0 - - -

JTCM | www. journaltcm. com

nes
ZOI 8.0±1.1 NI 8.0±0.5 NI NI NI 8.0±1.5 8.0±2.3 9.0±2.5 8.0±3.4 7.0±1.1 NI 6.0±0.5 NI 29.0±3.5
Bacillus
spizizenii MIC 75.0 - 75.0 - - - 75.0 75.0 50.0 75.0 - - - - -
Salmonella ZOI NI NI 7.0±0.5 NI NI NI NI NI NI NI NI NI NI NI 28.0±2.0
typhi MIC - - - - - - - - - - - - - - -
Wickerhamo ZOI NI 7.0±1.7 7.0±1.7 7.0±1.5 8.0±1.7 NI NI 7.0±1.0 NI NI NI NI NI NI 31.0±2.0
myces
anomalus MIC - - - - 75.0 - - - - - - - - - -
Fusarium ZOI NI NI NI NI NI NI NI NI NI NI NI NI NI NI 30.0±1.8

489
oxysporum MIC - - - - - - - - - - - - - - -
Mucor sp. ZOI NI NI 7.0±1.1 6.0±0.5 NI NI 8.0±1.5 7.0±1.5 NI NI NI NI NI NI 27.0±2.0
MIC - - - - - - 75.0 - - - - - - - -
10.0±
I Fatima et al. / Research Article

Aspergillus ZOI NI 9.0±1.5 NI 8.0±2.0 NI NI 9.0±1.0 7.0±1.1 7.0±1.7 NI 8.0±2.0 NI 9.0±3.0 30.0±2.0
2.0
flavus
MIC - 75.0 - 75.0 - - 75.0 - - - 75.0 - 50.0 75.0 -
Aspergillus ZOI NI NI NI NI NI NI NI NI NI NI NI NI NI NI 31.0±1.8
niger MIC - - - - - - - - - - - - - - -

Saccharomy- ZOI NI NI 13.0±6.3 10.0±3.7 NI NI 11.0±3.6 9.0±1.5 NI NI 10.0±2.5 10.0±3.2 8.0±2.0 11.0±3.7 29.0±2.0
ces cerevisiae
MIC - - 50.0 50.0 - - 50.0 75.0 - - 50.0 50.0 50.0 50.0 -
Notes: CeCME: Cenchrus ciliaris methanol extract; CeCHE: Cenchrus ciliaris n-hexane extract; PhMME: Phalaris minor methanol extract; PhMHE: Phalaris minor n-hexane extract; PoMME: Polypogon
monspeliensis methanol extract; PoMHE: Polypogon monspeliensis n-hexane extract; CyCME: Cymbopogon citratus methanol extract; CyCHE: Cymbopogon citratus n-hexane extract; PaAME: Panicum anti-
dotale methanol extract; PaAHE: Panicum antidotale n-hexane extract; StPME: Stipagrostis plumosa methanol extract; StPHE: Stipagrostis plumosa n-hexane extract; ArFME: Aristida funiculata methanol ex-
tract; ArFHE: Aristida funiculata n-hexane extract; ZOI: zone of inhibition; MIC: minimum inhibitory concentration; NI: no inhibition; Meth: methanol extract; Hex: n-hexane extracts; Positive control: oxy-
tetracycline (Bacterial strains), Chloramphenicol (Fungal strains).

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 I Fatima et al. / Research Article
were known to be the most resistant strains. Our results
are in the line with the findings of Singariya et al,48 who
reported that Cenchrus ciliaris show no activity against
Aspergillus niger. Earlier findings of Gothandam et al 49
also proved that Panicum antidotale forms no signifi-
cant inhibition zone against Salmonella typhi and Esch-
erichia coli. Similarly, our results are also supported by
the findings of Al-Shamma et al 50 who documented
that Polypogon monspeliensis and Stipagrostis plu-
mosa species form no inhibition zone against Esche-
richia coli. Antifungal results are in agreement with the
findings of Ochanga et al,51 who reported significant
antifungal potential of Cymbopogon citratus specie.
The antimicrobial potential of grasses can be due to
the presence of phenols, flavonoids and other polyphe-
nolic compounds.52
Previous literature confirmed that plant extracts with
LC50 values below 20 μg/mL may yield anticancer com-
pounds.53, 54 In present studies, all plant extracts re-
vealed strong to moderate cytotoxic potential. These re-
sults on the lethality of Cymbopogon citratus on brine
shrimps is supported by the findings of Ochanga et
al.51 Our results are also in accord with those of
Hamidi et al 55 who revealed moderate cytotoxicity of
Cymbopogon citratus specie.
In conclusion, our findings reveal that the extracts of
these species can be used to combat various microbial
infections and also as a natural antioxidant. Further-
more, plant extracts made in methanol solvent showed
more significant results as it is a strongly polar mole-
cule having ability to dissolve all polar molecules as
compared to those extracts which were made in n-hex-
ane solvent as it is a non-polar substance. Hence, select-
ed crude extracts can be used in pharmaceutical indus-
try against various diseases. However, in future, tech-
niques such as high-performance liquid chromatogra-
phy and nuclear magnetic resonance spectroscopy
should be done to characterize principle active com-
pounds from these species which can be effectively uti-
lized in pharmaceutical industries.
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