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Phytochemistry, cytotoxicity and antiviral activity of Eleusine indica (sambau)

Conference Paper · September 2015

DOI: 10.1063/1.4931234

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Rashidah Iberahim Ahmad Yaacob

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 Phytochemistry, Cytotoxicity and Antiviral Activity of
Eleusine indica (Sambau)

Rashidah Iberahim1,a), Wan Ahmad Yaacob2,b) and Nazlina Ibrahim1,c)

1
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan
Malaysia, 43600 UKM, Bangi, Selangor, Malaysia
2
School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti
Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia
a)
rshiey@yahoo.com
b)
wanyaa@ukm.edu.my
c)
Corresponding author: nazlina@ukm.edu.my

Abstract. Goose grass also known as Eleusine indica (EI) is a local medicinal plant that displays antioxidant,
antimicrobial and anticancer activities. Whole dried plants were macerated with methanol for crude extract
preparation and hexane used for fractionation. The present study is aimed at determining phytochemical
constituents, cytotoxicity and antiviral activities for both crude extract and fraction obtained from the plant.
The crude extract contained more secondary metabolites compared to the hexane fraction as gauged using
standard phytochemical tests. Cytotoxicity screening against Vero cells using MTT assay showed that the CC50
values for crude extract and the hexane fraction were 2.07 and 5.62 mg/ml respectively. The antiviral activity
towards Herpes Simplex Virus type 1 (HSV-1) was determined using plaque reduction assay. The selective
indices (SI = CC50 / EC50) for both methanol extract and hexane fraction were 12.2 and 6.2 respectively. These
results demonstrate that the extract prepared from E. indica possesses phytochemical compound that was non
cytotoxic to the cell with potential antiviral activity.

INTRODUCTION
Herpes viruses (subfamily of alphaherpesvirus) are large, double-stranded DNA viruses that contain
124-295 kb genome length. Commonly HSV-1 can cause mucocutaneous lesion in mouth and genital area
or sometimes encephalitis of immunocompromised patient [1]. HSV-1 strains are now resistant towards
acyclovir among immunocompromised patients. The resistant strain identified as thymidine kinase-
deficient reported to be less infectious in an animal model but more dangerous in latency reactivation as
evidenced in human clinical cases [2]. Eleusine indica is an annual turf grass found in tropical countries
[3]. This medicinal plant is used for traditionally for treatment of hypertension, oligouria, influenza [4],
hemorrhagic cough [5], liver, kidney and urinary bladder infections [6]. Methanol extract of E. indica was
reported to display antioxidant activity is worthy of further investigation [4]. However, the methanol
extract is not effective in antibacterial reaction as the hexane extract [4]. The aqueous extract of E. indica
was showed that the plant was not cytotoxic and has potential as antiviral agent towards HSV-1 [7]. On the
other hand, ethanol extracts of E. indica also showed antiviral effects in inhibiting HSV-1 infection that
might be due to its hydrocyanic acid content [4]. Thus, this study was carried out to further evaluate the
antiviral activity of the methanol extract and hexane fraction in search for active antiviral activity.
 MATERIALS AND METHODS

Plant Material
E. indica plants were collected from the vicinity of the Plant House, UKM, Bangi, and oven-dried
(60oC). The dried whole plants consisting of leaf, stem and root were cut into smaller pieces and ground
into powder using a grinder (CM-1). The extraction procedure was followed according to [8] with soaking
of powder (200 g) in 500 ml of methanol for three days instead of 24 hours. The extracts were filtered, and
solvent was evaporated under reduced pressure using rotary vacuum evaporator (Heidolph Laborota 4000).
The dried crude extracts were stored in small bottles at 4oC until used.

For hexane fraction, the procedure described by [9] was followed with slight modification by soaking
the powder (200 g) in distilled water and methanol (1:2) for 48 hours instead of 15 hours. The methanol
was evaporated using rotary evaporation and the liquid part successively partitioned with (3×50 ml) hexane
to obtain the fraction. The solvent was evaporated using rotary evaporation.

Cells and Viruses

The HSV-1 clinical strain and Vero cells used in this study were obtained from stocks available at the
Virology Laboratory, School of Biosciences and Biotechnology, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia. Vero cells were maintained in Dulbecco’s Modified Essential Medium
(DMEM) supplemented with 5% Fetal Bovine Serum (FBS, Jr. Scientific), penicillin/streptomycin (100
U/L), MEM non-essential amino acid and amphostate B (20 U/L). The cell culture was maintained in an
incubator at 37oC and humidified 5% CO2 atmosphere. HSV-1 was propagated in Vero cells and incubated
until cytophatic effects developed. The titer of the virus was estimated and stored at -80oC until used.

Phytochemical Screening
Phytochemical qualitative screening was carried out for crude extract and hexane fraction according to
standard procedures used to test for the presence of tannins, alkaloids, flavanoids, saponins, steroids,
triterpenoids as described in [10]. Anthra-quinones and quinones were determined using the method
described by [11]. Phenol and C-glycoside content follows the method by [12].

Cytotoxicity Test
Cytotoxicity was tested using the method described by [7] using reagent. After addition of DMSO to
MTT treated cells, in incubator at 37oC for 5-10 minutes. The absorbance readings were recorded using
microplate reader (Chromate 4300). The 50% cytotoxicity concentration (CC50) was defined as the sample
concentration that able to reduce 50% of cell viability compared to the untreated cells.

Plaque Reduction Assay

The procedures used were follows the method described by [9]. Firstly, 80% confluent Vero cell with
concentration of 2×108 cells/ml were prepared in 24-well plates. Then, the media were replaced with 50 pfu
of HSV-1, DMEM and 5% FBS with the total volume of 300 µL for each well. The plates were incubated
for an hour in 37oC. The inoculum were removed and replaced with 500 µL of extract with 1%
Methylcellulose (5% FBS). The tested plates were then further incubated for 48 hours and stained with
crystal violet. The wells were overlaid with medium without test sample as the control. The percentage of
plaque inhibition was calculated as follows:

EC50= [(mean number of plaques in control)− (mean number of plaques in sample)] × 100
Mean number of plaques in control
 The EC50 was calculated as the concentration of extracts that inhibit 50% of virus plaque compared to
control. The mean number of plaques for all replicates used to determine the EC50. The experiment was
done in triplicate. Selective index (SI) value was calculated as (CC50/EC50) to indicate the safety of drug to
be used as medicine.

RESULTS AND DISCUSSION

The percentage of crude extract and hexane fractions yields’ were 2.5% and 2.23% respectively. This is
considered as low compared to the aqueous extract yield of previously produced by [7]. Methanol is a less
polar solvent compared to water thus lesser ability for the phytochemical to be extracted. Phytochemical
analysis shown in TABLE 1 indicates the crude extract does not contain saponin, anthrax-quinones and C-
Glycosides. As for hexane fraction, a few secondary metabolites such as tannin, alkaloids and steroid were
extracted. The crude extract contained more secondary metabolites due to high polar compounds contain in
E. indica [13].

TABLE 1 Phytochemical analysis of methanol extract and hexane fraction of E. indica.

Phytochemical substances Methanol extract Hexane fraction
Tannin + +
Saponin - -
Flavonoid + -
Triterpenoids + -
Alkaloids + +
Steroid + +
Anthra-quinones - -
C-Glycosides - -
Quinones + +
Phenols + -
+: Present; -: Absent.

TABLE 2 shows the crude extract inhibited 50% of the cell viability at concentration of 2.07 mg/ml
while hexane fraction inhibited the cell growth at greater concentration of 5.62 mg/ml. Both the crude
extract and fraction are consider as not cytotoxic to the cell as the CC50 value are more than 0.02 mg/ml
[14].

In this study, for crude extract, the EC50 was 0.17 mg/ml and hexane extract EC50 0.9 mg/ml. The
absence of flavonoid, triterpenoids and phenols reduces the antiviral activity in hexane fraction as indicated
in the reduction of SI value in TABLE 2 compared to the crude extract. As reported in the previous study
done on E. indica towards HSV-1 showed aqueous extract EC50 vale of 0.725 mg/ml on Vero cells [7] and
the ethanol extract’s concentration of 100% plaque inhibition (IC100) on HeLa cell was 0.1 mg/ml [4].

TABLE 2. CC50, EC50 and selective index values of plant extracts.

Eleusine indica CC50(mg/ml) EC50(mg/ml) SI

Methanol extract 2.07±0.17 0.17±0.12 12.2

Hexane fraction 5.62±0.13 0.9±0.15 6.2

The SI value is shown in TABLE 2 were between the safe range for the plant to be used as antiviral
agent for human which was between 5 to10 [9]. Referring to the previous study on aqueous extract
described by [7] (SI value = 4.3) was also safe to be used as an antiviral agent. The methanol extract
showed better antiviral activity (SI value = 12.2) might be due to the presence of phenolic groups
compound in the extract.
 CONCLUSION
As a conclusion, our findings suggest that crude extract and hexane fraction prepared from Eleusine
indica contains antiviral active compounds and could be potential antiviral agent.

ACKNOWLEDGMENTS
The authors are grateful to Universiti Kebangsaan Malaysia (BKBP-FST-K006401 and DPP-2014-021)
and Ministry of Education Malaysia for supporting the financial, plant source and laboratory facilities to
conduct the experimental work.

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