Philippine Journal of Science

151 (6B): 2365-2384, December 2022

ISSN 0031 - 7683
Date Received: 06 Jun 2022

Isolates from Eleusine indica (Poaceae)

Aerial Shoot Fraction Dually Inhibits 5-LOX
and COX Enzyme Systems

Adrien Kyle M. Jacinto1*, Francisco M. Heralde3, Patrick Gabriel G. Moreno3,

Joshua H. Santos4, Joanna V. Toralba2, and Noel S. Quiming2

1Department of Pharmacy, Faculty of Pharmacy, University of Santo Tomas,

España Blvd., Manila 1015 Philippines
2Pharmaceutical Chemistry Department, College of Pharmacy,
University of the Philippines Manila, Metro Manila 1000 Philippines
3Department of Biochemistry and Molecular Biology, College of Medicine,
University of the Philippines Manila, Metro Manila 1000 Philippines
4Industrial Technological Development Institute, Department of Science and Technology,
General Santos Avenue, Upper Bicutan, Taguig City 1631 Philippines

Recent studies supported the use of Eleusine indica in inflammatory processes as evidenced
by the reduction of lipopolysaccharide-induced inflammation in vitro and in vivo. The aim of
this research was to evaluate the anti-inflammatory activity of E. indica in the context of dual
inhibition of 5-lipoxygenase (5-LOX)/ cyclooxygenase (COX). Drugs able to block the two
main arachidonic acid pathways are of pharmacological interest due to the wide range of anti-
inflammatory activity with no ulcerogenic risk. In addition, cytotoxicity screening using the
HepG2 and HK-2 cells and analysis of the most active subfraction via UPLC-QTOF-MSE were
performed. Preliminary screening of the crude methanolic extract and its fractions revealed
significant dual 5-LOX/COX inhibition, with no significant difference among the fractions (p
< 0.05). Ethyl acetate fraction, the most active among the four fractions and found to be rich
in phenolic compounds after TLC derivatization, was considered for further purification and
screening at 50 μg mL–1. Among its nine subfractions, subfraction 6 was regarded as the most
promising dual inhibitor with a moderately hepatotoxic and nephrotoxic profile. Its median
inhibitory concentration (IC50) was found to be 16.47 μg mL–1 for 5-LOX, 19.64 μg mL–1 for
COX-1, and 22.26 μg mL–1 for COX-2. Two putative compounds namely Naringenin-7-O-β-D-
glucuronide and Tricin-7-O-β-D-glucopyranoside were identified after the mass spectral analysis
of subfraction 6. These results confirm that E. indica elicits its anti-inflammatory activity by
targeting the arachidonic acid metabolic pathways.

Keywords: anti-inflammatory, arachidonic acid pathway, dual 5-LOX/COX inhibitors, goose grass,
herbal medicine, inflammation

*Corresponding author: amjacinto1@alum.up.edu.ph

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INTRODUCTION
Inflammation is the immune response of the body tissues
against infection and injury, but it also contributes to the
pathophysiology of many chronic diseases – including
diabetes mellitus, cardiovascular disease, certain types
of cancers, bowel diseases, arthritis, osteoporosis, and
chronic obstructive pulmonary disease (Libby 2007;
Ricciotti and Fitzgerald 2011; Wouters et al. 2007).
Interactions of cells in the innate immune system, adaptive
immune system, and inflammatory mediators orchestrate
aspects of acute and chronic inflammation that underlie
diseases of many organs (Libby 2007).
The arachidonic acid pathway constitutes one of the
main mechanisms for the production of inflammation.
Enzymes such as cyclooxygenases (constitutive COX-1
and inducible COX-2) and lipoxygenase (5-LOX) are
responsible for catalyzing various structural modifications
of free arachidonic acid into the potent biologically
active lipid mediators that are intimately involved in
inflammation (Patrono 2011). Although different anti-
inflammatory drugs are already available on the market,
certain anti-inflammatory agents are not advisable to
be taken on a longer basis mainly due to their adverse
effects, whereas only very few are free from toxicity.
Non-selective COX-inhibitors such as aspirin are
associated with gastrointestinal (GI) ulceration, as
well as bleeding and platelet dysfunction due to the
inhibition of COX-1 (Patrono 2011; Sagnia et al. 2014;
Xue et al. 2011). Selective COX-2 inhibitors seem to
be effective and safe non-steroidal anti-inflammatory
drugs (NSAIDs); however, such compounds also pose
some adverse effects (Leval et al. 2002; Xue et al.
2011). In particular, COX-2 selective inhibitor rofecoxib
(Vioxx®) was withdrawn from the market due to the
associated increased risk of renal and cardiovascular
events (Rao and Knaus 2008). In addition, blocking the
COX pathway results in the upstream process on the
5-LOX pathway forming high levels of 5-leukotrienes,
which are potent pro-inflammatory compounds. Based
on emerging studies, 5-LOX-derived leukotrienes may
also contribute to colorectal cancer development and
the risk of thrombotic events (Rao et al. 2012). On the
other hand, LOX inhibitors, although included among the
effective therapies for asthma, appear to be an insufficient
single therapeutic approach to inflammation (Leval et al.
2002). Hence, drugs able to block both 5-LOX and COX
metabolic pathways with less or no side effects have
gained interest among researchers.
The interest in developing such compounds is supported
by a large number of pharmacological studies. Compared
to COX or LOX single inhibitors, dual inhibitors present
three major advantages: [1] it possess a wide range of
anti-inflammatory activity by acting on the two major
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Jacinto et al.: Eleusine indica Inhibits 5-LOX and COX Enzymes
arachidonic acid pathways; [2] appear to be almost
exempted from gastric toxicity mainly because of the
inhibition of leukotriene, which plays also a role in gastric
damage due to tissue ischemia and inflammation; and
[3] could be a promising approach for cancer treatment
since both leukotrienes and prostanoids are involved in
other pathologies such as cancer proliferation (Becker
et al. 2004; Leval et al. 2002). Hence, it is imperative to
determine other alternative therapies in targeting both the
COX and LOX pathway (Attiq et al. 2018). The abundance
of natural compounds provides a great advantage in drug
discovery and development. Natural compounds eliciting
unrivaled activity are then isolated and purified to serve as
a template for the structural modification and synthesis of
new generation anti-inflammatory drugs with low toxicity
and higher therapeutic values (Anilkumar 2010).
Eleusine indica Linn Gaertner (Poaceae) – commonly
known as goose grass, wire grass, and “paragis” in Tagalog
– is a strictly xerophytic grass abundant in settled areas
throughout the Philippines, China, Japan, Malaysia, India,
Australia, America, Africa, and other warm countries. It
is an annual, tufted, erect, glabrous plant that is 10 cm–1
m in height. Its flower spikes mostly have 2–7 spikelets,
producing a characteristic of a windmill-like appearance
(Waterhouse 1994). The genus Eleusine can either be an
annual or perennial species, where hybridization of the two
groups of Eleusine is possible resulting in intermediates
(Philips 1972).
In different parts of the world, the grass of E. indica is
traditionally used as sudorific, diuretic, anthelminthic,
and for treating hemoptysis, fevers, dysentery, and liver
complaints. It is also applied as a poultice for sprains
and dislocations (Quisumbing 1978). Further, E. indica
has been noted to possess antioxidant, hepatoprotective,
antibacterial, cytotoxic, antiviral, antihyperlipidemic,
and antiurolithiatic activities (Ong et al. 2017; Amoah et
al. 2017; Iberahim et al. 2015; Abdul et al. 2011; Iqbal
and Gnanaraj 2012; Sagnia et al. 2014). Studies in Brazil
supported the popular use of aerial parts of E. indica
against airway inflammatory processes like influenza and
pneumonia after investigation of the flavonoid-enriched
fraction and the C-glycosylflavone isolates (e.g. vitexin
and schaftoside) in inhibiting the lipopolysaccharide
(LPS) induced mouse lung inflammation (de Melo et
al. 2005). Furthermore, the ethanolic leaf extract of E.
indica exhibited an anti-inflammatory effect on γδ T-cells
and immature dendritic cells, as evidenced by the dose-
dependent reduction in TNF-α production (Sagnia et al.
2014). In the study of Ettebong and Nwafor (2014), the
ethanolic extract of E. indica elicited its anti-inflammatory
activity by inhibiting the paw edema caused by carrageenin
and egg albumin, and xylene-induced ear edema. Akah
and Ezeugo (2020) supported the result through their
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Vol. 151 No. 6B, December 2022
study where the ethanolic extract and ethyl acetate fraction
of the said plant resulted in 48–54% edema inhibition
against xylene-induce topical edema. Interestingly, gastric
mucosa damage was not observed. In addition, leukocyte
migration and membrane stabilization were noted upon
pre-treatment of the plant. Nevertheless, previous studies
have already reported E. indica use in other inflammatory
processes as a nasal decongestant and as a therapeutic
agent for osteoporosis and plantar fasciitis (de Oliveira
and Romão 2015). Despite its therapeutic potential,
only little is known about the chemical composition and
bioactive compounds of E. indica. Hence, the study aims
to investigate the anti-inflammatory activity of E. Indica
via inhibition of 5-LOX, COX-1, and COX-2 enzymes,
which can contribute to an understanding of their possible
mechanism in the treatment of inflammatory diseases.
MATERIALS AND METHODS
Chemicals
5-LOX inhibition assay screening kit (human recombinant,
BioVision, Cat. No. K980-100) and 96-well white
plate with a flat bottom (Cayman, Cat. No. 700029)
were purchased from Sapphire Bioscience Pty. Ltd.
Indomethacin secondary standard (PHR1247, Sigma
Aldrich), and 2-aminoethyl diphenylborinate (≥ 97%,
Supelco 42810) were purchased from Chemline Scientific.
Merck TLC Silica Gel 60 F254 was purchased from
Interlab. All other solvents (HPLC and analytical grade),
reagents, and standards were obtained from the University
of the Philippines (UP) College of Pharmacy, Belman
Laboratories, and Chemline Scientific.
Plant Material and Extraction
E. indica aerial shoots were collected in January 2018
(between 09:00–11:00 AM) from a farm vicinity of Brgy.
Masagana, Pandi, Bulacan, Philippines (14.883938,
120.932902). The voucher specimen was authenticated by
Manuel D. Ching (Chief, CIPGR Section) of the Bureau of
Plant Industry, Quirino, Manila. The genetic identity of the
plant was also validated via DNA barcoding using matK
and ITS primers at Thomas Aquinas Research Center,
España, Manila. About 50 g of air-dried and pulverized
material was exhaustively macerated with 800-mL 80%
methanol (MeOH) for 3 d. The extract was concentrated
using a rotary evaporator at 40 ºC and then lyophilized.
The crude MeOH extract underwent liquid-liquid
partitioning using the procedure below. The extract was
dissolved with 50 mL 80% MeOH using sonication and
then partitioned eight times with 50 mL hexane (Hex).
The methanolic layer was concentrated and suspended
Jacinto et al.: Eleusine indica Inhibits 5-LOX and COX Enzymes
in 50-mL distilled water (Aq.) and then partitioned eight
times with 50-mL dichloromethane (DCM) and ethyl
acetate (EtOAc), successively. The resulting fractions
were concentrated using a rotary evaporator at 40 °C until
one-fourth of the solvent was removed. Thereafter, it was
placed in the evaporating dish and then oven-dried at 40
ºC for 8 h to further remove the excess solvent.
Phytochemical Screening
The phytochemical tests were conducted by derivatizing
the TLC plates of samples developed using the following
optimized solvent systems: ethyl acetate-methanol-water-
toluene (100:15.5:13.5:5.2) for crude MeOH extract and
aq. fraction, benzene-ethyl acetate-formic acid (40:10:5)
for hex. fraction, chloroform-methanol (6:1) for DCM
fraction, and ethyl acetate-acetic acid-formic acid-water
(100:11:11:27) for EtOAc fraction.
The spray reagents used were the following: AlCl3 in 95%
ethanol, ammonia vapors, Wilson’s reagent (3% boric
acid – 10% oxalic acid, 3:1), and Naturstoff’s reagent
(ethanolamine diphenylborinate – PEG 4000) for flavonoids
(reference: quercetin 1 mg mL–1); Potassium ferricyanide-
ferric chloride for phenolics (reference: quercetin 1 mg
mL–1); Borntraeger’s reagent (potassium hydroxide in
MeOH) for anthraquinones, anthrones, and coumarins;
vanillin-sulfuric acid reagent for triterpenes, sterols, and
essential oils; Dragendorff’s reagent for alkaloids (reference
material: quinine HCl 1 mg mL–1); Hemolysis reagent
(blood gelatin suspension) for saponins (reference: “gugo”
bark extract 1 mg mL–1), ninhydrin’s reagent for amino
acids (reference: phenylalanine 1 mg mL-1), and Molisch’s
reagent (α-naphthol-sulfuric acid) for sugars (reference:
glucose 1 mg mL–1). These phytochemical tests were
conducted based on the methods from Cai et al. (2011),
Jork et al. (1990), and Wagner et al. (1984). Additionally,
for flavonoid tests, the Shinoda test (aka Willstatter cyanidin
test) was done using the test tube method. The color
intensity of the reaction was the basis of relative abundance.
In Vitro 5-LOX and COX Inhibition Assay
The ability of the test samples to elicit anti-inflammatory
activity by inhibiting the cyclooxygenase (COX-1 and
COX-2) and lipoxygenase (5-LOX) enzymes at 50 and/
or 100 µg mL–1 concentration was determined using the
protocols outlined below. The percent of inhibition was
calculated as follows:
Slope uninhibited − Slope inhibited
% inhibition = x 100 (1)
Slope uninhibited
5-lipoxygenase inhibition assay. The 5-LOX inhibition
assay (fluorometric) was performed using the optimized
protocol provided by BioVision (Milpitas, CA, USA).
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Vol. 151 No. 6B, December 2022
About 1 µL of the test samples at their respective final
well concentrations were mixed with 19 µL LOX assay
buffer in the 96-well white plate (flat bottom). Zileuton
(the provided 5-LOX inhibitor) was used as the positive
control, whereas DMSO (the solvent used to dissolve the
samples and controls) was used as the negative control.
Indomethacin and quercetin were also used as additional
controls for the assay. The working LOX substrate solution
was obtained by diluting the provided LOX substrate
(12500 X) in LOX assay buffer to get 5 X concentration.
For each well, 20 µL reaction mix containing 18 µL LOX
assay buffer, 1 µL LOX probe, and 1 µL LOX enzyme
were added. After incubating the plate at 25 ºC for 10
min, 10 µL of working LOX substrate solution was added
to each well. The fluorescence at excitation/emission
wavelength of 500/536 nm was recorded on the second
minute after the addition of substrate at 30-s intervals for
10–20 min.
Cyclooxygenase-1 and cyclooxygenase-2 inhibition
assay. Using the protocol of Bonner and Fry (2012)
for COX inhibition assay (fluorometric), the following
were added to 150 µL of 100 mM Tris-HCl pH 8.0, in
a sequential manner: 10 µL of test compounds at their
respective concentrations, 10 µL of 1,000 µM Hemin, and
10 µL of 250 U mL–1 COX-1 or COX-2 (ovine) enzyme.
The positive controls were 50 µg mL–1 indomethacin,
diclofenac, and quercetin in 100% DMSO (final well
concentration), and the negative control was 10 µL 100%
DMSO. The mixture was incubated at 25 °C for 15 min.
After incubation, 10 µL of 200 µM Amplex® Red probe
(ADHP; 10-acetyl-3,7-dihydroxyphenoxazine) was added
to the mixture. 10 µL of 2,000 µM of arachidonic acid was
then added and the reaction was monitored for 2 min using
the Fluorescence mode of the Varioskan Flash (Thermo
Scientific) with the following parameters; excitation/
emission wavelength at 535/590 nm.
Isolation of Phytochemicals
The phytochemicals from the most active fraction (60 mg
mL–1) were isolated via a micro-preparative technique
using the optimized solvent system and pre-washed and
pre-activated silica gel G F254 TLC plates (20x20, 0.25
mm thick) at a distance of 18 cm (Francis and Andersen
1996). After subsequent elution-drying of the plates, the
Rf values of the target bands were recorded. Using the
modified method of isolation studied by Gwatidzo et
al. (2018), the target bands were scraped off, suspended
with 10 mL HPLC-grade methanol, vortexed, sonicated,
and then centrifuged. The supernatant liquid was filtered
using a 0.2-micron nylon filter and then collected in
a pre-weighed vial for the removal of excess solvent.
The method was repeated until the collected weight of
subfractions was sufficient for further study. The Rf value
is expressed mathematically as:
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Jacinto et al.: Eleusine indica Inhibits 5-LOX and COX Enzymes
Distance of solute from the point of application
Rf value = (2)
Distance of solvent from the point of application
Cell-based Hepatotoxicity and Nephrotoxicity Assay
of the Most Active Subfraction
Sample preparation. About 5 mg of the subfractions
were sent for assay at the Disease Molecular Biology
and Epigenetics Laboratory, UP Diliman. Upon receipt,
they were stored at 4 °C and protected from light. Prior
to the assay, they were dissolved in DMSO to achieve
a stock solution of 20,000 µg mL–1. From this starting
concentration, the 50 µg mL–1 concentration was obtained
through serial dilution.
Cell culture preparation. Liver cancer (HepG2) and
normal kidney (HK-2) cell lines were cultured using the
appropriate media and grown to 80% confluency. They
were then seeded with a seeding density of 5,000 cells per
well into 96-well plates for subsequent assay.
Cytotoxicity assay. Cytotoxicity of the subfraction was
determined using the protocol provided by CytoTox
96® NonRadioactive Cytotoxicity Assay (Promega).
Absorbance values were measured using a 96-well plate
reader at 490 nm. The percentage of cytotoxicity of the
samples was computed based on the absorbance values.
The test samples were assayed in two independent trials,
each having two replicates.
The in vitro cytotoxicity assay was based on the cytotoxic
percentage versus compound concentration computed
through a logarithmic model. The cytotoxicity of the
samples was classified as < 0%, non-cytotoxic; < 1%,
mildly cytotoxic; 1–10%, moderately cytotoxic; and >
10%, highly cytotoxic (unsafe). The 10% cytotoxicity was
arbitrarily used as the threshold for flagging the sample for
testing discretion. This would imply a margin of safety as
increasing the sample concentration will also increase the
bioactivity but may reach the 50% cytotoxicity boundary,
which is deemed toxic to humans. Based on the two
trials performed, the higher cytotoxic concentration was
used to designate the cytotoxicity profile, as previously
mentioned.
Analysis of the Most Active Subfraction Using
UPLC-QTOF-MSE
The sample was dissolved in 80% MeOH to make a 1 mg
mL–1 final concentration and then filtered using a 0.2-µm
PTFE syringe prior to injection. 2 µL of the sample was
injected to ACQUITY HSS T3 C18 (100 mm x 2.1 mm
x 1.8 mm) column (Waters Co.) at 30 ºC. The mobile
phases were composed of eluent A (water with 0.1%
v/ formic acid) and eluent B (acetonitrile with 0.1% v/
v v
formic acid) at a flow rate of 0.4 mL min–1. The applied
elution conditions were based on Lin et al. (2019) with
Philippine Journal of Science
Vol. 151 No. 6B, December 2022
modifications: 0-2 min, 10% B; 2-26 min, 10-90% B;
26-28 min, 90% B; 28-28.1 min, 90-10% B; and 28.1-30
min, 10% B. The mixtures of water/acetonitrile at the ratio
of 10/90 and 90/10 were the strong wash and weak wash
solvent, respectively.
To determine the molecular weight as well as the
fragmentations associated with the most active subfraction,
UPLC-QTOF-MSE analysis was performed on a Waters
Xevo G2-XS QTOF mass spectrometer (Waters Co.,
Milford, MA, USA) equipped with a UPLC system through
an electrospray ionization (ESI) interface in positive mode
using Leucine-enkephalin (m/z 556.2771) as the external
reference. The instrumental parameters in this mode were
based on Lin et al. (2019) with modifications: capillary
voltage, 2.6 kV; source temperature, 120 ºC; desolvation
temperature, 300 ºC; cone voltage, 40 V; desolvation gas
flow, 800 L/h; cone gas flow, 50 L/h; and collision energy,
6 V for low energy function and 20–40 V for high energy
function. The mass spectrum was acquired from 50–1,200
m/z in MSE mode.
For the identification of compounds, the UPLC-QTOF-
MSE raw files were subjected to mass screening using
the UNIFI data analysis software. The base peak ions of
distinct peaks were subjected to library matching using
Traditional Chinese Medicine (TCM) and ChemSpider.
The criteria used for the identification of a good match
were as follows: mass accuracy error of ≤ 5 mDa (or ≥
–5 mDa), response for precursor ion ≥ 2,000, isotope
Figure 1. Dual 5-LOX/COX inhibition by E. indica crude methanolic extract.
Jacinto et al.: Eleusine indica Inhibits 5-LOX and COX Enzymes
match intensity RMS percent ≤ 20, and isotope match Mz
RMS PPM < 15. The most active subfraction was sent
for analysis to the Biochemistry Department, College of
Medicine, UP Manila.
Data Processing and Analysis
All statistical analyses were performed using SPSS.
Results from at least three individual experiments (n = 3)
were expressed as mean ± SD. Statistical methods, e.g.
one-sample Kolmogorov-Smirnov test, Levene’s test, and
one-way ANOVA followed by Tukey’s multi-comparisons
test or Welch and Brown-Forsythe followed by Games-
Howell test were employed using a 95% confidence
interval (α = 0.05).
RESULTS AND DISCUSSION
DNA barcoding of the plant sample confirmed the
identity of Eleusine indica as supported by the BLAST
result in Table A1. Initial screening of the crude MeOH
extract at 50 and 100 µg mL–1 inhibited 5-LOX by 73.84
± 1.07% and 85.49 ± 9.74%, COX-1 by 74.72 ± 0.85%
and 81.40 ± 1.46%, and COX-2 by 77.26 ± 3.13% and
84.33 ± 0.89%, respectively, as shown in Figure 1. This
revealed that the aerial shoots of E. indica can prevent
and/or treat inflammatory conditions by potentially acting
as a dual 5-LOX/COX inhibitor. The methanolic extract
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Vol. 151 No. 6B, December 2022
of E. indica posed a greater advantage compared to the
classical NSAIDs which only inhibit COX-1 and COX-2
enzymes. It has been established that inhibition of the
COX pathways can lead to the upstream synthesis of
leukotrienes (Rao et al. 2012).
Dual inhibitors had been widely investigated in animal
models, and the results demonstrated that these drugs
possessed a broad spectrum of anti-inflammatory activity
without presenting marked ulcerogenic risk (Leval et al.
2002). This unique property can be attributed in part to
the suppression of leukotriene production under the LOX
pathway (Jacob and Kumar 2015; Leval et al. 2002).
Like prostanoids, leukotrienes are pro-inflammatory
mediators that, according to Leval et al. (2002),
significantly contribute to gastric epithelial injury. In
addition, cysteinyl leukotriene (LTC4, LTD4, and LTE4)
promotes local vasoconstriction – which leads to less
blood flow, induces mucosal microvascular injury and
mucosal barrier degradation, and stimulates gastric acid
interleukin-1 secretion (Peskar 1991; Buccellati et al.
1997). This cascade will make the gastric mucosa more
prone to injury. Leukotriene B4 enhances leukocyte
infiltration, which synergizes with cysteinyl leukotriene on
damaging the gastric mucosa (Wallace and Keenan 1990;
Wallace et al. 1990). Moreover, dual inhibitors could also
prevent seizure-induced neurovascular leakage and cancer
Figure 2. Dual 5-LOX/COX inhibition by E. indica fractions; *significantly higher activity (p < 0.05) than
the crude methanol extract; #significantly lower activity (p < 0.05) than the crude methanol extract.
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Jacinto et al.: Eleusine indica Inhibits 5-LOX and COX Enzymes
proliferation, as these conditions were also mediated
by COX and LOX (Leval et al. 2002; Sokola 2018).
This marked activity can be attributed to the following
phytochemical constituents: phenolics, flavonoids,
anthraquinones, anthrones, coumarins, sterols, amino
acids, and sugars – as summarized in Table B1. The result
of this analysis was close to the literature report of Morah
and Otuk (2015), excluding the presence of alkaloids.
Partitioning of the crude MeOH extract resulted in four
fractions with varying degrees of activity for both 5-LOX
and COXs, as illustrated in Figure 2. Particularly, mid-polar
fractions such as the ethyl acetate and dichloromethane
fractions produced significantly higher dual inhibitory
activity than the crude MeOH extract. This can be attributed
to the presence of abundant amounts of phenolics, such
as flavonoids, as determined through TLC derivatization
tests. Conversely, the dual inhibitory activity of the aqueous
fraction significantly declined. Based on the phytochemical
analysis, the aqueous fraction was rich in amino acids and
sugars. On the other hand, the hexane fraction, although
it has comparable 5-LOX inhibitory activity to the crude
MeOH extract, also suffered a significant decline in the
activity against COX enzymes. It was determined that this
fraction contains sterols, as well as sugars. Nonetheless, it
was determined that there is no significant difference among
the fractions themselves (p < 0.05).
Philippine Journal of Science
Vol. 151 No. 6B, December 2022
In line with the previous result from the 5-LOX and COX
inhibitory assay, the ethyl acetate fraction was subjected
to a micro-preparative technique using the optimized
solvent system of ethyl acetate-acetic acid-formic acid-
water (100:11:11:27) to obtain the compounds. Nine
subfractions were afforded in this procedure. Interestingly,
flavonoids were detected in five subfractions and anthrone
in one subfraction using Naturstoff’s and Borntraeger’s
reagents, respectively (as summarized in Table C1).
Screening of subfractions for dual inhibition had
resulted in a significant decline in the activity except for
subfractions 6, 8, and 9 in inhibiting 5-LOX and COX-1
enzymes (as illustrated in Figure 3 and summarized in
Tables D1 and D2). No significant increase in the activity
of these three subfractions was observed, but the results
were still comparable with the parent fraction. Increasing
evidence suggests that the previous classification of COX-
1 as a homeostatic enzyme and COX-2 as an inflammatory
enzyme was uncertain, as more recent studies showed
that the prostaglandins generated by COX-2 are involved
in both inflammation and homeostatic processes with
cardioprotective effects. Conversely, the thromboxane
formed by COX-1 had been associated with an increased
risk of coronary heart disease (Armstrong et al. 2011).
Thus, COX-1 inhibition had been found to be effective
in the prevention of cardiovascular diseases, as revealed
by the application of low doses of aspirin, which is a
preferential COX-1 inhibitor. There was also increasing
evidence that suggested COX-1 is a potent mediator of
Figure 3. Dual 5-LOX/COX inhibition of E. indica subfractions at 50 µg mL–1; *no significant difference in
activity (p > 0.05) as compared to the ethyl acetate fraction.
Jacinto et al.: Eleusine indica Inhibits 5-LOX and COX Enzymes
neuroinflammation; hence, selective inhibitors of COX-1
can be effective in the prevention of neurodegenerative
diseases, such as Alzheimer’s disease, Parkinson’s disease,
and traumatic brain injury (Langhansova et al. 2017).
Table D3 summarizes the selectivity ratios of crude MeOH
extract, fractions, and subfractions from E. indica for
5-LOX, COX-1, and COX-2 enzymes at 50 µg mL–1.
Samples with a COX-2/COX-1 selectivity ratio of ≥ 1
were as follows: crude MeOH extract; fractions hexane,
dichloromethane, and ethyl acetate; and subfractions 1,
5, 6, 7, and 9. This data implies that the aforementioned
samples can potentially act as non-selective COX
inhibitors. On the other hand, the hexane fraction and
subfractions 1, 2, 3, 4, 6, and 9 resulted in a selectivity
ratio of ≥ 1 for 5-LOX/COX-1 and 5-LOX/COX-2.
Meanwhile, aqueous fraction and subfraction 8 also have
5-LOX/COX-2 selectivity ratio of ≥ 1 only. In relation to
5-LOX, the data suggests that most of the samples from
E. indica act as dual inhibitors of the arachidonic acid
pathway and not just non-selective COX inhibitors, as
seen in the case of indomethacin.
The cytotoxicity of the subfractions in liver cancer
(HepG2) and normal kidney (HK-2) cell lines at 50 µg
mL–1 were listed in Table E1. Using the 10% cytotoxicity
threshold, the initial cytotoxicity assessment revealed
that all the subfractions were moderately hepatotoxic and
nephrotoxic except for subfraction 8 (which is deemed to
be highly hepatotoxic) and subfractions 1, 2, and 9 (which
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Vol. 151 No. 6B, December 2022

are mildly nephrotoxic). This implies that the subfractions, subfraction 6 for 5-LOX, COX-1, and COX-2 is shown in
excluding subfraction 8, were regarded as safe to be used Figure 4. The median inhibitory concentration (IC50) for
at 50 µg mL–1. 5-LOX enzyme was interpolated from the graph and was
determined to be at 16.47 µg mL–1. On the other hand,
Despite being moderately toxic to both HepG2, and NK-2 for the COX-1 and COX-2 enzymes, the IC50 values were
cells, the biological activity of subfraction 6 against found to be at 19.64 and 22.26 µg mL–1.
5-LOX (80.17 ± 2.60%), COX-1 (65.24 ± 8.47%),
and COX-2 (66.23 ± 5.81%) was comparable with the A total of nine peaks were detected in the base peak
ethyl acetate fraction. In relation to this, the selectivity intensity chromatogram of subfraction 6. Their mass
ratios displayed by subfraction 6 was higher compared spectral profiles using the ESI+ mode were listed in Table
to the other subfractions (i.e. 1.02, 1.23, and 1.21 for 1 and illustrated in Figures F1–F7. Among the nine peaks,
COX-2/COX-1, 5-LOX/COX-1, and 5-LOX/COX-2, two putative flavonoid compounds were determined after
respectively). By taking all of this into consideration, it matching their mass spectral profiles with the TCM and
was revealed that subfraction 6 is the most promising dual ChemSpider databases. These were the naringenin-7-O-
inhibitor among the nine subfractions. Analysis revealed β-D-glucuronide (4) and tricin-7-O-β-D-glucopyranoside
that it is more selective in inhibiting 5-LOX than COX (5), which belong to the dihydroxyflavanone and
enzymes. In the study of de Melo et al. (2005), it was glycosyloxyflavone subclasses.
determined that the E. indica crude extract, flavonoid-
enriched fraction, and isolated vitexin and schaftoside
inhibited lung inflammation in the groups of mice exposed
to aerosols of LPS in a dose-dependent manner. The
LPS pathway produces leukotriene, a pro-inflammatory
mediator, catalyzed by 5-LOX enzyme from arachidonic
acid. Leukotriene (particularly the LT-D otherwise known
as the slow-reacting substance of anaphylaxis) binds
to the leukotriene D4 receptor thus triggering airway (4) Naringenin-7-O-β-D-glucuronide
constriction, and inflammation (Hedi and Norbert 2004).
The release of leukotriene will also trigger the activity of
other pro-inflammatory mediators such as NO, TNF-α, IL-
1β, and IL-6 (Huang et al. 2020). This result may justify
the popular use of E. indica against airway inflammatory
processes.
Based on the assay results, subfraction 6 also showed
a non-selective COX inhibition. Since dual inhibition
needs both COX-metabolic pathways to be blocked
along with the 5-LOX, the risk for GI ulceration is more
likely to be low. Although in vivo studies demonstrated
(5) Tricin-7-O-β-D-glucopyranoside
its effectiveness in the treatment of inflammation without
causing GI toxicity, the results of the clinical trials
Flavonoids were reported to have anti-inflammatory
performed on such agents were still insufficient (Leval
activity by targeting one or more of the following
et al. 2002). The concentration-response curve of the

Figure 4. Concentration-response curve of the subfraction 6 for 5-LOX (A), COX-1 (B), and COX-2 enzymes (C).

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Vol. 151 No. 6B, December 2022

Table 1. Compounds identified from subfraction 6.

m/z Mass
Candidate mass/ com- tR Mol. (Da) MS2 fragments;
No. Response error
pound (min) formula MS3 fragments
Exp. Calc. (mDa)

150;
1 149.9533 0.53 103865 – – – –
221
2 – 0.58 – – – – – –
238;
3 238.1441 5.28 28850 – – – –
220
Naringen- 449;
4 5.66 199900 C21H20O11 448.1006 448.1014 0.8164
in-7-O-β-D-glucuronide 329, 299, 243
Tricin-7-O-β-D-gluco- 493;
5 7.57 173522 C23H24O12 492.1268 492.1273 0.5482
pyranoside 331, 315, 270
6 – 13.1 – – – – – –
318;
7 318.3004 13.27 39243 – – – –
256, 224
415;
8 415.2112 15.68 81285 – – – –
261
353;
9 353.2691 17.64 59053 – – – –
333, 261

mechanisms: inhibition of eicosanoid generating enzymes
(e.g. phospholipase A2, COX, and LOX), inducible nitric
oxide synthase, histamine release, phosphodiesterases,
protein kinases, and transcription factors NF-kb and
activator protein-1 (AP1); reduction of mitogen-activated
protein kinase (MAPK) activity; and activation of nuclear
factor-erythroid 2-related factor 2 (Nrf-2) (Rathee et al.
2009; Serafini et al. 2010). It was also suggested that
flavonoids can inhibit enzymes such as aldose reductase,
xanthine oxidase, and Ca2+-ATPase. In addition, they
have a regulatory role on different hormones like
estrogens, androgens, and the thyroid hormone. Their
anti-inflammatory activities have been found to act in
both proliferative and exudative phases of inflammation
(Rathee et al. 2009).
Naringenin activates the Nrf2 expression on macrophages,
which promotes HO-1 expression. In addition, the
compound inhibited LPS- and carrageenan-induced
NF-κB activation, and IL-33, TNFα, IL-1β, IL-6, and
COX-2 expression (Manchope et al. 2017). Aside from
the stated activities, Naringenin also exhibits anticancer,
antidiabetic, immunomodulatory, hepatoprotective,
and cardioprotective activity (Arafah et al. 2020). In
comparison, Tricin prevented the LPS-induced activation
of TLR4, MYD88, and TRIF proteins. Simultaneously,
p38MAPK, JNK1/2, and IRF3 kinases were also inhibited.
Moreover, Tricin inhibited the MYD88 dependent
and TRIF dependent pathways, which promoted the
modulation of NF-κB and IRF3. It also displayed a COX-
2 inhibitory effect through the prevention of cytosolic
phospholipase A2 activation. Lastly, tricin inhibited the
promotion of STAT1 and STAT3 via downregulating
JAK1 and JAK2 phosphorylating enzymes (Shalini et
al. 2015). Other activities noted in tricin includes anti-
angiogenic activity (Han et al. 2016), chemoprevention,
antihistamine, anti-ulcerogenic, antiviral, antidiabetic,
antibacterial, and antifungal (Jiang et al. 2020). The
reported activities of the two putative compounds found
in E. indica would confer the result of the test done in
this study.
CONCLUSION
The aerial shoots of E. indica was determined to
possess anti-inflammatory activity by blocking the two
main pathways of the arachidonic acid metabolism
simultaneously. Significantly higher dual inhibitory
activity was also noted for mid-polar fractions (most
especially the ethyl acetate fraction). Subfraction
6, the most promising dual inhibitor with moderate
hepatotoxicity and nephrotoxicity, revealed two putative
flavonoid compounds – namely, naringenin-7-O-β-
D-glucuronide and tricin-7-O-β-D-glucopyranoside,
along with seven other unknown compounds. Further
purification of the bioactive compounds, in vivo tests, and
cytotoxicity assays were recommended to further establish
the activity as well as safety of the E. indica as a dual
inhibitor of COX and LOX enzyme systems.

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Vol. 151 No. 6B, December 2022
ACKNOWLEDGMENTS
The researchers were supported by the Department of
Science and Technology–Science Education Institute
through the Accelerated Science and Technology Human
Resource Development Program scholarship, National
Institute of Health, and Graduate Assistance Program by
the National Graduate Office for the Health Sciences,
UP Manila.
STATEMENT ON CONFLICT OF
INTEREST
No potential conflict of interest was reported by the
authors.
NOTES ON APPENDICES
The complete appendices section of the study is accessible
at https://philjournsci.dost.gov.ph
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APPENDIX A

DNA Barcoding of E. indica

Table A1. BLAST result of E. indica DNA sequence.

BLAST results*
Primer
Description Accession Max score Total score E value Identity
MatK E. indica chloroplast, complete genome KU833246.1 1411 1411 93% 99%
Eleusine kigeziensis voucher DU/Bot/cyto/56 tnrK KF357742.1
gene, partial sequence; and maturase K (matK)
gene complete cds; chloroplast
E. indica voucher DU/Bot/cyto/53 tnrK gene, KF357739.1
partial sequence; and maturase K (matK) gene,
compete cds; chloroplast
Eleusine tristachya plastid plastid matK gene for FN908053.1
maturase k, specimen voucher BM<GBR-LON-
DON>:H. Schaefer 2008/602
ITS E. indica small subunit ribosomal RNA gene, MF029701.1 1186 1186 84% 99%
partial sequence; internal transcribed spacer 1 and
5.8S ribosomal RNA gene, complete sequence; and
internal transcribed spacer 2, partial sequence
*Highest sequence producing significant alignment

APPENDIX B

Phytochemical Screening of Eleusine indica Crude and Fractions

Table B1. Phytochemicals of E. indica.

Crude MeOH Fractions
Compound Test Std.
E. Hex. DCM EtOAc Aq.
Shinoda – – – + – +++
AlCl3 + – – ++ + +++
Flavonoids Ammonia – – ++ ++ – +++
Wilson’s + + + ++ + +++
Naturstoff’s + – + ++ + +++
FeCl3–potassium
Phenolics – – + ++ – +++
ferricyanide
Anthraquinones, Anthrones,
Borntraeger’s – – +a, c ++b, c –
Coumarins
Triterpenes, Sterols, Essen-
Vanillin–sulfuric acid – +++d ++d – –
tial oils
Alkaloids Dragendorff’s – – – – – +++
Saponins Hemolysis – – – – – +
Amino acids Ninhydrin ++ – + – ++ +++
Sugars Molisch’s +++ ++ + – +++ +++
[–] negative; [+] low intensity; [++] moderate intensity; [+++] high intensity
aanthraquinones; bantrones; ccoumarins; dsterols

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APPENDIX C

Summary of the Retention Factor Values of the Subfractions of E. indica Ethyl Acetate Fraction
Table C1. Rf values of subfractions from E. indica ethyl acetate fraction.
UV366
Sub-fraction Rf Visual UV254 Assigned substance
Pre-spray Post-spray
1 0.26 White Green Blue – Unknown
2 0.33 White Dark Blue – Unknown
3 0.47 – Green Light blue Yellowa Anthrone
4 0.50 Yellow Dark Dark Yellowb Flavonoid
5 0.53 – – Pale blue – Unknown
6 0.61 Yellow Dark Dark Yellowb Flavonoid
7 0.68 Yellow Dark Dark red Yellow greenb Flavonoid
8 0.81 Yellow Dark Dark Yellowb Flavonoid
9 0.87 Yellow Dark Dark Yellowb Flavonoid
awith Borntraeger’s spray reagent; bwith Naturstoff’s spray reagent

APPENDIX D

Dual 5-LOX/COX Inhibition of E. indica

Table D1. Dual 5-LOX/COX inhibition of E. indica crude extract and fractions.

Conc. % inhibition ± SD
Samples
(µg mL–1) 5-LOX COX-1 COX-2
Crude MeOH extract 73.84 ± 1.07 74.72 ± 0.85 77.26 ± 3.13
Hex. fraction 72.71 ± 0.70 68.24 ± 2.41b 70.43 ± 6.60
DCM fraction 50 79.61 ± 5.06 90.23 ± 1.60a 93.66 ± 1.40a
EtOAc fraction 77.97 ± 1.06a 93.58 ± 0.18a 93.25 ± 1.67a
Aq. fraction 66.54 ± 1.05b 68.07 ± 3.69b 66.24 ± 4.12b
Crude MeOH extract 85.49 ± 9.74 81.40 ± 1.46 84.33 ± 0.89
Hex. fraction 90.90 ± 2.31 76.39 ± 0.75b 79.97 ± 1.66b
DCM fraction 100 97.15 ± 5.88 95.90 ± 0.59a 97.51 ± 0.57a
EtOAc fraction 109.45 ± 3.61a 97.22 ± 0.42a 97.96 ± 0.37a
Aq. fraction 85.24 ± 3.12 71.64 ± 1.31b 77.56 ± 2.83b
Zileution std. 5 52.09 ± 0.54 – –
Indomethacin std. 0.76 ± 12.56 14.22 ± 9.10 19.02 ± 3.31
Diclofenac std. 50 – 19.69 ± 8.25 26.57 ± 4.18
Quercetin std. 97.69 ± 2.14 100.05 ± 0.23 99.81 ± 0.63
aIndicates significantly higher activity (p < 0.05) than the crude methanol extract
bIndicates significantly lower activity (p < 0.05) than the crude methanol extract

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Table D2. Dual 5-LOX/COX Inhibition of E. indica Subfractions at 50 µg mL–1.

Samples % inhibition ± SD (at 50 µg mL–1)
5-LOX COX-1 COX-2
Subfraction 1 65.50 ± 2.12b 52.54 ± 7.44b 54.85 ± 5.75b
Subfraction 2 57.50 ± 0.98b 57.14 ± 4.78b 48.05 ± 5.41b
Subfraction 3 65.51 ± 0.22b 58.06 ± 4.76b 54.30 ± 3.47b
Subfraction 4 57.12 ± 2.77b 51.30 ± 6.41b 49.77 ± 2.81b
Subfraction 5 50.46 ± 1.44b 52.34 ± 7.38b 54.58 ± 5.04b
Subfraction 6 80.17 ± 2.60a 65.24 ± 8.47a 66.23 ± 5.81b
Subfraction 7 46.65 ± 1.43b 52.93 ± 1.54b 55.86 ± 4.47b
Subfraction 8 67.86 ± 5.23a 72.23 ± 6.69a 56.82 ± 0.88b
Subfraction 9 74.57 ± 2.13a 61.91 ± 5.68b 66.61 ± 2.05b
aNo significant difference in activity (p > 0.05) as compared to the ethyl acetate fraction
bSignificantly lower activity (p < 0.05) as compared to the ethyl acetate fraction

Table D3. Selectivity ratios of E. indica samples at 50 µg mL–1

Selectivity ratio (at 50 µg mL-1)
Samples
COX-2/COX-1 5-LOX/ COX-1 5-LOX/ COX-2

Crude MeOH 1.03 0.99 0.96

Fractions
Hex.* 1.03 1.07 1.03
DCM 1.04 0.88 0.85
EtOAc 1.00 0.83 0.84
Aq. 0.97 0.98 1.00
Subfractions
1* 1.04 1.25 1.19
2 0.84 1.01 1.20
3 0.94 1.13 1.21
4 0.97 1.11 1.15
5 1.04 0.96 0.92
6* 1.02 1.23 1.21
7 1.06 0.88 0.84
8 0.79 0.94 1.19
9* 1.08 1.20 1.12
Controls
Quercetin 1.00 0.98 0.98
Indomethacin 1.34 0.05 0.04
Diclofenac 1.35 - -
Zileuton - - -
*Samples with a selectivity ratio of ≥ 1 for COX-2/COX-1, 5-LOX/COX-1, and 5-LOX/COX-2

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APPENDIX E

Summary of the Cytotoxicity Study of the Subfractions of E. indica

Table E1. Cytotoxicity of the subfractions in HepG2 and NK-2 cells at 50 µg mL–1.
% hepatotoxicity % nephrotoxicity
Subfractions
Trial 1 Trial 2 Trial 1 Trial 2
1 3.62c 1.63c 0.30b –0.87a
2 4.92c –1.43a 0.89b –3.15a
3 3.18c 2.61c 1.32c –4.77a
4 3.86c 4.80c 2.37c –5.52a
5 7.82c 4.17c 3.43c –3.48a
6 9.38c 6.20c 1.67c –4.88a
7 8.84c 3.50c 2.01c –0.72a
8 15.53d 4.45c 1.31c –4.72a
9 1.27c 6.08c 0.30b –0.87a
aNon-cytotoxic; bmildly cytotoxic; cmoderately cytotoxic; dhighly cytotoxic

APPENDIX F

Mass Spectra of Isolates from Subfraction 6

Figure F1. ESI+ mass spectrum of compound 1.

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Vol. 151 No. 6B, December 2022

Figure F2. ESI+ mass spectrum of compound 3.

Figure F3. ESI+ LC-MS/MS spectrum of compound 4.

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Vol. 151 No. 6B, December 2022

Figure F4. ESI+ mass spectrum of compound 5.

Figure F5. ESI+ mass spectrum of compound 7.

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Vol. 151 No. 6B, December 2022

Figure F6. ESI+ mass spectrum of compound 8.

Figure F7. ESI+ mass spectrum of compound 9.

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APPENDIX G

Graphical Abstract