Effect of Different Fractions of Eleusine indica (Paragis) on the

Renin-Angiotensin System, An In-Vitro Study

Abstract
Hypertension has an alarming increase in cases that it is now considered a global public health crisis.
Drugs used in the management of hypertension are acting on the renin- angiotensin system (RAS), but
synthetic drugs are associated with a variety of side effects that even lead to life threatening events.
Additionally, despite the strategic location of renin in the system the potential of the substances that can
inhibit it, is still not fully studied. Eleusine indica (Paragis) an abundant grass in the Philippines is said to
contain abundant flavonoids that exhibit different pharmacologic properties including the capacity to act
on the RAS. This study assesses the effect of the different fractions of E. indica in the RAS. The
methanolic extract of E. indica was fractionated using column chromatography. Fractions were then
subjected to renin and ACE assay kits using the principle of fluorometry and spectrophotometry
respectively. HPLC and FTIR analysis were conducted to further isolate the compound present in the
highest-inhibiting fractions. In ACE inhibition assay, fraction 4 showed an inhibiting activity of 71.23%
that is comparable to captopril with 70.55% inhibition using a post hoc analysis. Renin inhibition assay
revealed fraction 5 (0.05050271) having a comparable activity with the positive control (0.02640686) via
post hoc analysis. FTIR analysis revealed the presence of hydroxyl and alkene groups in fractions 4 and 5
but both revealed absence of quercetin via HPLC analysis. Fractions of E. indica have renin and ACE
inhibiting activity that is comparable with their respective positive controls. Absence of quercetin in those
fractions reveals the potential of other flavonoids that is present in E. indica capable of acting in the RAS.
Key words: Hypertension, Eleusine indica, Paragis, Renin inhibitor, ACE inhibitor

INTRODUCTION

Hypertension, is a condition in which the blood vessels have persistently raised blood
pressure. It has an alarming increase in the number of cases and, once detected, warrants
significant lifestyle changes. One factor in maintaining the arterial blood pressure is by using
medications acting on the renin-angiotensin system (RAS). Two of the main enzyme components
in this system are renin and angiotensin-converting enzyme (ACE), in which antihypertensive
drugs act upon. Different studies regarding the flavonoid content of E. indica (Paragis), a weed
found abundantly in the Philippines, exhibit different pharmacologic properties including the
capacity to act on the renin-angiotensin system. In a study conducted by Guerrero et al,
flavonoids, one of which is quercetin, demonstrated an inhibitory effect on ACE. Additionally,
Motaal et al concluded that the ethyl acetate with bioactive markers for quercetin and apigenin
showed renin and ACE inhibition mechanisms. Eleusine indica (Paragis) is noted to contain the
flavonoids quercetin and apigenin. In a study conducted by Tutor et al. certain fractions of the
methanol crude extract of E. indica showed an inhibitory activity to ACE but the specific
constituent responsible for this activity remains unidentified. Up to date there is an obvious lack
of literature characterizing the constituents of E. indica and its potential in treating
diseases. With the need to explore the potential of E. indica and anchoring with the established
theories and literature of hypertension and flavonoids we conceptualized the study, “Effect of
Different Fractions of Eleusine indica (Paragis) on Renin-Angiotensin System, An In-Vitro
Study.”

METHODS

Plant Collection and Extraction

The fresh plant of Eleusine indica (Paragis) was collected from New Corella, Davao del
Norte, and was validated by Fe Magpayo-Bagajos a taxonomist from Ateneo de Davao
University.After verification, whole plant of E. indica was washed and air-dried for at least 72
hours. The roots were separated from the leaves and powdered through an electric blender. It was
macerated in distilled methanol for three days and then filtered. The filtrate was then
concentrated in vacuo utilizing a rotary evaporator at 85-95 revolutions per minute (rpm) at
40°C. The concentrated filtrate was then collected, weighed and stored in sterile bottles prior to
use. Iodoform test was performed to ensure the absence of alcohol. Phytochemical screening for
alkaloids, flavonoids and tannins using 25mL of the methanolic extract was done.

Column Chromatography

Column chromatography of the methanol extract was performed utilizing successive

gradient elution with a series of solvents consisting of hexane, ethyl acetate and methanol. The
different fractions obtained was then subjected to rotatory evaporation and was stored in sterile
bottles and labeled with numbers to be used for ACE and renin activity assay.

ACE Assay
The cleavage of the substrate hippuryl-histidyl-leucine (HHL) by ACE was mixed with pyridine
and benzene sulfonyl chloride to produce hippuric acid (HA) and histidyl-leucine (HL) followed
the method developed by Jimsheena and Gowda. The therapeutic drug captopril was used as the
positive control. The assay mixture was composed of 35 μL of 0.05 M sodium borate buffer (pH
8.2) containing 0.3 M NaCl, 35 μL 500 μg/mL of plant extracts, 20 μL of 5 mM HHL, and 10 μL
of ACE enzyme extract from rabbit lung.

The first step of the method was a 10-minute pre-incubation at 37°C to establish contact between
the inhibitor and ACE. After the pre-incubation step, 20 μL of 5 mM HHL was added to each, to
initiate ACE reaction. The mixtures were then incubated at 37°C for 30 min. Adding 50μL of 1
M hydrochloric acid (HCl) arrested the reaction. This was immediately followed by the addition
of 100 μL pyridine and 50 μL benzenesulfonyl chloride (BSC) to develop the yellow color. The
absorbance was then measured at 410 nm using a spectrophotmeter.

The following formula was used to determine percent inhibition:

Where A1 is the ACE without inhibitor; A2 is the inhibitor (plant extract/positive

control). The fraction that was considered active or have a 50% inhibition of ACE was subjected
to HPLC for qualitative and quantitative determination for specific flavonoids.

Renin Assay

The renin inhibition assay method that was used in this study was based on the method
described by Merck International for screening renin inhibitors. The first step was to make the
renin assay mixture using a 50 uL of the renin substrate and 50 uL of the assay buffer. 50 μL of
the assay mixture is required for each reaction (well). The 100× renin substrate was aliquoted
and stored at –20 °C to avoid repeated freeze-thaws. The plate and the inhibitor were pre-
incubated for 10–15 minutes at the desired temperature (25 °Cor 37 °C) for the enzyme reaction.
Addition of 50 μL of the renin assay mixture into each of the sample, standard, and blank wells
was done. The plate was incubated at the desired temperature for 30–60 minutes taking
measurements (λex = 540/λem = 590 nm, cut off = 570 nm) every 5 minutes.

High Performance Liquid Chromatography (HPLC)

 HPLC analysis was performed for the qualitative determination of the flavonoid
quercetin of the fractions that showed the highest inhibiting activity to renin and ACE.

Fourier Transform Infrared Spectroscopy (FTIR)

Active fractions of E. indica were subjected to FTIR through the methods employed by
the chemist of DMSFI for the determination of functional groups that are present in the
compound exhibiting the inhibiting activity.

RESULTS

Phytochemical analysis of Eleusine indica (Paragis) methanolic extract revealed the

presence of flavonoids, alkaloids, and tannins.

ACE

Figure 1. Percentage inhibition of ACE assay

Percentage inhibitions for the ACE inhibiting activity between the different fractions E.
indica (Paragis) methanolic extract and the positive control when compared showed that fraction
4 with a 71.23% enzyme inhibition compared to the positive control that yielded a 70.55%
enzyme inhibition. A One-way ANOVA was conducted and the positive control revealed a p-
value is <0.05. It was further analyzed with Tukey’s post hoc analysis, wherein fraction 4 has the
most comparable activity to the positive control, thus it was subjected to HPLC. HPLC analysis
showed no peak observed at 12.283 minutes that would indicate the absence of quercetin.
However, a peak in the chromatogram was seen at 5 minutes. FTIR analysis was then conducted
on fraction 4 of E. indica (Paragis) methanolic extract the functional groups were identified,
which are alkene, conjugated acid, isothiocyanate, carboxylic acid, alkane, alcohol and primary
aliphatic amine.

Renin

The p-value of One-way ANOVA between the different fractions of E. indica (Paragis)
methanolic extract and the positive control shows that there is a significant difference between
the different fractions and the positive control, indicating that the null hypothesis should be
rejected. A Post hoc analysis was then conducted of the renin inhibiting activity of different
fractions of E. indica (Paragis) and the positive control showed that the ability of the fraction 5 to
inhibit renin is comparable to the positive control. HPLC analysis of fraction 5 was then
conducted wherein no peak is observed at 12.283 minutes that would indicate the absence of
quercetin. However, a peak in the chromatogram was seen in 5 minutes. FTIR analysis was then
conducted wherein the functional groups of fraction 5 of E. indica (Paragis) methanolic extract
were identified, which are alkene, cyclic alkene, amine, conjugated alkene, isothiocyanate,
alcohol and primary aliphatic amine.

DISCUSSION

ACE assay

Fraction 4, the fraction with the highest ACE inhibiting activity was then subjected to HPLC
analysis and showed the absence of such compound. This finding is significant for the fact that
despite certain fractions having the highest percentage of quercetin, it does not possess the
highest inhibiting activity and why pure samples of quercetin does not have the highest inhibiting
activity when compared with plants extracts.

In a study by Khalil et. al, the butanol fraction of the 70 % ethyl acetate extract of Hyphaene
thebaica showed a higher inhibiting activity at a same concentration than the pure quercetin
standard when tested for ACE inhibition noting that quercetin solely is not responsible for ACE
inhibiting activity. This further supports the study of Guerrero et. al, evaluating the ACE
inhibiting activity of 17 different flavonoids, in which quercetin does not have the highest
inhibiting activity and there are other flavonoids which possess a higher inhibiting activity.
Therefore, the higher inhibitory activity of fraction 4 might be attributed to another flavonoid
with higher inhibiting activity than quercetin.

FTIR analysis of fraction 4 of the methanolic extract indicated a significant presence of hydroxyl
groups, this is in line with the findings of Chen & Lin in his study “Inhibition of Angiotensin-I-
Converting Enzyme by Tetrahydroxyxanthones Isolated from Tripterospermum lanceolatum” in
which he emphasized the importance of free hydroxyl groups of phenolic compounds as
structural moieties capable of chelating the zinc ions, thus inactivating the ACE activity.

Desai et. al conducted an in vivo hypotensive study of E. indica in rats and points to iso-orientin
that possesses the hypotensive activity. Additionally, vitexin, another flavonoid identified in the
survey of E. indica is also said to have ACE inhibiting activity as revealed by the study of
Ridzuan et. al.

The structure of hydroxyl, carboxyl and alkene groups and as conjugated acids are consistent
with the structure of vitexin, an apigenin flavone glycoside, and iso-orientin, an 8-C glucoside of
luteolin. Thus, it highly recommended to test for the presence of these flavonoids in the
methanolic fraction of E. indica in future studies.

Renin assay

HPLC Analysis of fraction 5 revealed the absence of quercetin. This is congruent with the
studies conducted by Khalil et. al comparing different bioactive markers wherein quercetin is not
that superior in inhibiting renin. Fraction 5 having the comparable activity with the positive
control has the highest retention factor and is considered the most nonpolar among the fractions.
The hydrophobic nature of the fraction compared with the rest of the fractions could possibly
explain its higher inhibiting activity. In a study conducted by Ajibola et al. data showed that the
non-polar polyphenolic extracts of Vernonia amygdalina and Gongronema latitolium leaves had
a higher inhibiting activity compared to the polar fractions, in the same study he concluded that
hydrophobic nature of the chlorophyllic fraction may have contributed to the increased
interaction with enzyme protein and therefore the inhibition of the activities of renin. Khalil et al.
also studied the renin inhibiting activity of standardized bioactive fractions of Hyphaene thebaica
where he noted that despite the fact that the ethyl acetate fraction, a more polar fraction, had
higher percentages of the three bioactive markers chlorogenic acid, quercetin and apigenin, the
butanol fraction, the least polar among the fractions, still possessed the highest renin inhibiting
activity. From these, we can assume that a component of fraction 5 that is hydrophobic in
character is responsible for its activity. However, due to limited studies of renin inhibitor both
natural and synthetic products isolation of a specific phytochemical at this point will be difficult.
Various studies attribute the renin inhibiting activity to flavonoids and alkaloids, both of which
compounds are present in the preliminary phytochemical screening of the methanolic extract of
E. indica.

FTIR Analysis of Fraction 5 revealed significant functional groups of hydroxyl group, alkene,
cyclic alkene and amine possibly pointing to secondary phytochemical constituents of flavonoid
and alkaloid. Highlighting the further characterization of the compound in future studies.

Conclusion

There is a significant difference between the different fractions of E. indica methanolic

extract and the positive control, captopril. Fraction 4 has the closest percentage inhibiting
activity against ACE when compared to the positive control while fraction 5 is the most
comparable in terms of the renin inhibiting activity.

Results of both the HPLC analysis indicated the absence of quercetin in both fraction 4
and 5 and the FTIR analysis showed presence of functional groups uncharacteristic of Quercetin.
Thus it can be implied that other flavonoids may be accountable for the majority of the ACE and
renin inhibiting activity.
Acknowledgement
Davao Medical School Foundation Research Center, Philippine Genome Center and San Pedro
College of Davao
Funding
Funding of the study are done by the researchers themselves.
Conflicting interest
The authors are declared no conflict of interest

Authors
Aragon, Marjorie Kristine S. Affiliation: Davao Medical School
E-mail: [email protected] Foundatio
Affiliation: Davao Medical School Name : Sharifa Rasheda A. Baid
Foundation E-mail: [email protected]
Affiliation: Davao Medical School
Foundation
Name: Aranan, Asgari Nazel A.
E-mail: [email protected]
Affiliation:Davao Medical School
Name : Joshua Miles Balanza
Foundation
E-mail: [email protected]
Affiliation: Davao Medical School
Name : Arani, Fatima Raaysa M.
Foundation
E-mail: [email protected]
Affiliation: Davao Medical School
Foundation
Name : Bañez, Zaida Kamille B.
E-mail: [email protected]
Name : Arayan, Gene Carlo B. Affiliation: Davao Medical School
E-mail: [email protected] Foundation
Affiliation: Davao Medical School
Foundation Name : Basiga, Roven C.
E-mail: [email protected]
Name : Ascaño, Alyssa Louize B. Affiliation: Davao Medical School
E-mail: [email protected] Foundation
Affiliation: Davao Medical School
Foundation Name : Belandres, Chessa Louise L.
E-mail: [email protected]
Name : Aturdido, Patricia Ann G. Affiliation: Davao Medical School
E-mail: [email protected] Foundation
 Affiliation: Davao Medical School
Name : Beltran, Carlene Joy P. Foundation
E-mail: [email protected]
Affiliation: Davao Medical School Name : Julie Anne J. Bongalo
Foundation E-mail: [email protected]
Affiliation: Davao Medical School
Name : Boiser, Gaebrelle Anne E. Foundation
E-mail: [email protected]

Co-Author

Dr. Gladys O. Sermon

[email protected]
Davao Medical School Foundation

About the Authors

The authors are Bachelor Degree Holders of Science and Health related courses and currently
studying for a Degree in Medicine. They are very interested in exploring and revealing the
potential of Philippine Plants as natural sources of compounds that could potentially alleviate the
leading problems of the current time.

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