Screening of mentha cordifolia opiz (yerba buena) buffer crude extract for aspartyl protease pepsin inhibitory activity

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ISSN Print: 2278 – 2648 IJRPP |Vol 3 | Issue 1 | Jan - April-2014
ISSN Online: 2278-2656 Journal Home page: www.ijrpp.com
Research article Open Access
Screening of Mentha cordifolia Opiz (Yerba Buena) buffer crude
extract for aspartyl protease pepsin inhibitory activity
Alfredo A. Hinay Jr, Lilen Dorothy C.Sarol
College of Public Health, Department of Medical Microbiology, University of the Philippines,
Manila, Philippines.
* Corresponding author: Alfredo A. Hinay
E-mail id: redhinajr@yahoo.com
Abstract
To date, several hundred bioactive compounds have been isolated from plant sources. Among them, protease
inhibitors have drawn the attention recently owing to their pivotal role in pharmaceutical industry. With the
anticipation that commonly and widely available plant could provide pharmacologically important protease
inhibitor, an attempt will be made to screen Mentha cordifolia Opiz (Yerba buena) for aspartyl protease pepsin
inhibitor to select a potential candidate for possible HIV-1 protease inhibitor. This research is an experimental type
of study. It specifies the inhibitory activity of Mentha cordifolia Opiz (Yerba buena) against aspartyl protease
pepsin. Plant extracts preparation, pepsin kinetic assay, pepsin inhibitory activity assay, qualitative phytochemical
screening and Total Flavonoid Content were employed in the methodology. The percent inhibition of various
concentration of Pepstatin A 12.5, 25, 50, 100 and 1,000ug/ml obtained 63.43%, 72.26%, 78.62%, 82.86% and
96.47% respectively. All negative control concentrations were above 60% inhibitory activity. Mentha cordifolia
Opiz lyophilized buffer crude extract concentrations 12.5, 25, 50, 100 and 1,000ug/mL obtained 57.95%, 62.46%,
69.32%, 74.37% and 94.96% respectively with an IC60 value of 17.30ug/mL. The study aims to determine the
inhibitory activity of Mentha cordifolia Opiz (Yerba buena) on aspartyl protease pepsin. Relating to the objectives
of the study and based on the results of the experimentations, the proponent now concludes: Mentha codifolia Opiz
(Yerba buena) have Concentration 60% (IC60) value of 17.30ug/mL against pepsin and the effect of lyophilized
buffer crude extract of Mentha codifolia Opiz (Yerba buena) shows increasing response as the concentration of the
plant lyophilized buffer extract increases.
Keywords: Pre-screening of HIV-1 Protease, Pepsin inhibitor, Mentha codifolia Opiz.
INTRODUCTION
Proteolytic processing is a major form of post
translational modification which occurs when a
protease cleaves one or more bonds in a target protein
to modify its activity. This processing may lead to
activation, inhibition or destruction of the protein's
activity. Many cellular processes are triggered by
proteolytic processing. The attacking protease may
remove a peptide segment from either end of the
target protein, but it may also cleave internal bonds in
the protein that lead to major changes in the structure
and function of the protein (Pettit et al 2005;
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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Zhongbin et al 2007). Various RNA viruses express
their genomic RNA by synthesis of a high molecular
weight polyprotein which is post-translationally
cleaved by proteases into functional viral gene
products. The proteases catalysing the specific
cleavages of a polyprotein can either be encoded by
the virus or occurs in the host prior to infection
(Carter J. and Saunders V. 2007; Fields et al. 2007).
HIV-1 protease which is encoded by HIV-1, is one of
the viruses that encode protease that is small around
100-125 amino acids, active as homodimers and bear
a striking resemblance to pepsin both in their tertiary
fold and catalytic mechanism. As both HIV-protease
and Pepsin share the signature sequence Asp-Thr-
Gly, an overall similarity of primary structure,
inhibition by Pepstatin A and are inactivated by
mutation of the putative active site aspartates
therefore these all suggest that pepsin may be a
representative for the HIV protease in the aspartic
group (Singh et al. 2013; Abad et al. 2000). Thus,
aspartyl protease pepsin could be used as pre-
screening enzyme of HIV-1 protease inhibitors.
A great number of protease inhibitors have been
recovered and identified from plants (Bode and
Huber, 2000). Plant protease inhibitors endure to
attract the attention of researchers because of their
increasing use in medicine and biotechnology (Singh
et al. 2013). With the preliminary study done by
Victoriano showing the effect of Philippine local
mint Mentha cordifolia Opiz on HIV-1 replication in
latently infected cells, the findings provided the
evidence that an anti-HIV activity is contained in the
plant extract. However, which of the viral replication
step is inhibited was not elucidated. The chemical
constituent of the plant extract – Apigenin, Luteolin,
Oleanolic acid, Pomolic acid and Ursolic acid may be
the putative active compounds that inhibit a pepsin-
like viral protease (Rummel DJ. 2009; Villasenor JM.
1995; Dr. Duke’s Phytochemical and Ethnobotanical
Databases). An attempt will be made to screen local
plant – Mentha cordifolia Opiz (Yerba buena) for
aspartyl protease pepsin inhibitor to select a potential
candidate for possible HIV-1 protease inhibitor.
MATERIALS AND METHODS
RESEARCH DESIGN
This research is an experimental type of study. It
specifies the inhibitory activity of Mentha cordifolia
Opiz (Yerba buena) against aspartyl protease pepsin.
The >60% pepsin inhibitory activity of phosphate
buffer crude extract was main subject to be studied.
Plant extracts preparation, pepsin kinetic assay,
pepsin inhibitory activity assay, qualitative
phytochemical screening and Total Flavonoid
Content were employed in the methodology. Plant
extract preparation, pepsin kinetic assay and pepsin
inhibitory activity assay were done in the Department
of Medical Microbiology Laboratory, College of
Public Health and two grams of lyophilized buffer
crude extract was brought to the College of
Pharmacy, University of the Philippines for
phytochemical screening and Total Flavonoid
Content (TFT) determination.
PLANT MATERIAL
The leaves of Mentha cordifolia Opiz were purchased
at Manila Seedling Bank Environmental Center and
verified by the National Museum, Manila.
BUFFER CRUDE EXTRACT
PREPARATION
Plant materials for the study were washed thoroughly
in distilled water and air-dried. A buffer extract was
prepared in a 500 ml conical flask by homogenizing
25 g of plant materials in 100 ml of 0.1M phosphate
buffer with pH 7.0 in an electrical blender. The
homogenate was filtered through gauze and the
filtrate was centrifuged at 3,400 RPM for 30 minutes
to remove any cell debris that remains in the
preparation. The clear supernatant obtained
represents the crude extract and was lyophilized to
prepare a serial dilution of 100ug/mL to 50, 25 and
12.5ug/mL concentrations for use in the pepsin
inhibitory activity assay.
BUFFER EXTRACTS LYOPHILIZATION
A 200 mL of buffer extract was collected and
submitted for lyophilisation at the Spine Unit,
Norberto R. Agcaoili Memorial Tissue Bank
(NRAMTB), Philippine General Hospital. 3.83 grams
were obtained after lyophilisation; the dried extract
was prepared in various concentrations for pepsin
inhibitor activity assay.
PEPSIN KINETIC ASSAY

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Haemoglobin substrate concentration were prepared
in 10 tubes containing each 400ug, 600ug, 800ug,
1,000ug, 1,200ug, 1,400ug, 1600ug, 1,800ug,
2,000ug and 2,200ug and diluted with 250 uL of 0.3
N HCl. The tubes were then incubated with 50 ug of
pepsin / 250 uL of 0.01 N HCl at 370
C for 20
minutes. After 20 minutes of incubation 700 μl of 5%
TCA was added to stop the reaction. The material
was centrifuged at high speed for 10 minutes and the
supernatant collected. Optical Density (OD) was
recorded using ELISA Reader at 280 nm.
PEPSIN INHIBITORY ACTIVITY ASSAY
For this assay, 50μg pepsin/250 uL 0.01 N HCl and
250 uL of 12.5ug/mL, 25μg/mL, 50μg/mL,
100μg/mL and 1,000ug/mL (1mg/mL) lyophilized
buffer crude extract were pre-incubated at 370
C for
15 minutes. The pre-incubated mixture was
transferred to a tube containing 1,800ug hemoglobin.
The 500 uL reaction mixtue was allowed to incubate
at 370
C. After 20 minutes of incubation 700 μl of 5%
TCA was added to stop the reaction. The material
was centrifuged at high speed for 10 minutes and the
supernatant collected. Optical Density (OD) was
recorded using ELISA Reader at 280 nm. Separate
blanks were used for both positive and negative
controls as well as for the samples. For positive
control, enzyme and substrate was utilized while for
negative control pepstatin A was used as a well-
known inhibitor of both pepsin and HIV-protease.
Each sample concentration was tested in duplicate, to
provide reproducible results.
The protease inhibitory activity was expressed in terms of percent inhibition and it will be calculated as:
% inhibition = Optical density of Positive control – Optical densty of Negative control
Optical density of Positive Control X 100
PHYTO CHEMICAL SUBSTANCES
SCREENING OF PLANT EXTRACT WITH
PEPSIN INHIBITORY ACTIVITY
Lyophilized extract of Mentha cordifolia Opiz (Yerba
Buena) was brought to the College of Pharmacy, UP
Manila for phytochemical substances screening. This
was done to elucidate what plant substances are
present in the extracts, as additional data. Test
parameters were listed in Table 3.1.
Table 3.1 Test parameters for qualitative phytochemical substances screening.
TESTS SPECIFIC TESTS
pH determination pH paper
Test for Tannins Ferric chloride test
Test for Glycosides Lead subacetate test
Test for Saponins Froth test
Test for plant acids Sodium carbonate test
Test for reducing substances Fehling’s test
Test for alkaloids Mayer’s test
Valser’s test
Wagner’s test
Dragendorff’s test
Test for Flavonoids Cyanidin Test
Test for Triterpenes Salkowski test
Test for Diterpenes Copper acetate test

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TOTAL FLAVONOID CONTENT OF
MENTHA CORDIFOLIA OPIZ
LYOPHILIZED BUFFER CRUDE
EXTRACT
The total flavonoid content was estimated using the
method described by Koncic´et al. (2010) and
Adedapo et al. (2008) with quercitin as standard. All
tests were performed in triplicate. To 0.5 mL of
extract solution, 0.5 mL of 2% AlCl3 ethanol solution
was added. Absorbance was measured at 420 nm
after an hour of incubation at room temperature. The
flavonoid content was calculated according to the
logarithmic regression equation that was obtained
from the quercetin standard curve.
DATA ANALYSIS
The absorbance generated from the pepsin inhibitory
activity assay at 280 nm were treated with
mathematical equation to obtain the percent
inhibition. The calculated percent inhibition of
Mentha cordifolia Opiz lyophilized buffer crude
extract must be above 60% to be considered as a
significant inhibitory activity.
RESULTS
PEPSIN KINETIC ASSAY
The optimum activity of pepsin to different
concentration of hemoglobin substrate were
determine based on the results obtained after
controlling the following variables shown in table
4.1. These variables are crucial points to ensure the
catalytic activity of pepsin to hemoglobin. The
increasing optical density (OD) shown in Table 4.2
shows the concentration of by-products released by
the substrate that is proportional to the concentration
of hemoglobin. The plateau point of hemoglobin
concentration which is 1,800ug shows the optimum
activity of pepsin; this indicates that above the
plateau point concentration of substrate the catalytic
activity of pepsin is constant.
Table 4.1 Variables controlled to get the optimum activity of enzyme.
Parameter Optimum value
pH 2-4
Incubation temperature 37 °C
Incubation period 20 minutes
Table 4.2 Optical density of pepsin-hemoglobin activity in 280 nm.
Substrate concentration Optical density Mean±SD
400 ug 0.1270 ± 0.0014
600 ug 0.1390 ± 0.0014
800 ug 0.1430 ± 0.0014
1,000 ug 0.1560 ± 0.0000
1,200 ug 0.1780 ± 0.0000
1,400 ug 0.2150 ± 0.0035
1,600 ug 0.2500 ± 0.0000
1,800 ug 0.2850 ± 0.0000
2,000 ug 0.2870 ± 0.0000
2,200 ug 0.2870 ± 0.0000
Assay done in duplicate

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0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0 2 2 0 0
0 .0 0
0 .0 2
0 .0 4
0 .0 6
0 .0 8
0 .1 0
0 .1 2
0 .1 4
0 .1 6
0 .1 8
0 .2 0
0 .2 2
0 .2 4
0 .2 6
0 .2 8
0 .3 0
0 .3 2
H e m o g lo b in c o n c e n t r a t io n ( u g )
PepsinActivity(Opticaldensityat280nm)
The concentration of haemoglobin by-products –
tyrosine and tryptophan after the catalytic activity of
pepsin was measured using ELISA reader absorbance
at 280 nm illustrated in Figure 4.1. The plateau point
concentration of substrate is 1,800 ug with a 95%
Confidence Interval optical density of 0.2572 to
0.9193; the positive control of both negative control
and plant test material optical density value must fall
under this range.
Figure 4.1 Michaelis-Menten pepsin kinetics
PEPSIN INHIBITORY ACTIVITY ASSAY
Inhibition of pepsin was tested using Pepstatin A as a
negative control – a known inhibitor of aspartyl
protease pepsin. The variables shown in Table 4.3
were controlled to obtain the optimum activity of
both the enzyme and the inhibitor (Pepstatin A and
Mentha cordifolia Opiz lyophilized buffer crude
extract). The pre-incubation period allows the
inhibitor to bind to the active site of pepsin.
Table 4.3 Variables controlled to obtain the optimum activity of the enzyme and inhibitor.
Parameter Optimum value
pH 2-4
Pre-incubation period (enzyme + inhibitor) 15 minutes
Incubation temperature 37 °C
Incubation period (enzyme+inhibitor+haemoglobin) 20 minutes
The optical density value is equivalent to the
concentration of haemoglobin by-products – Tyrosine
and Tryptophan. The descending optical density and
standard deviation error bar of the different
concentration of negative control was illustrated in
Figure 4.2. The positive control optical density
obtained was 0.2830 ±0.0000 and concentration of
Pepstatin A 12.5, 25, 50, 100 and 1,000ug/mL
obtained an optical density value of 0.1035±0.0092,
0.0785±0.0035, 0.0605±0.0078, 0.0485±0.0064 and
0.0100±0.0099 respectively. The result shows the
optical density value directly proportional to the
catalytic activity of pepsin and inversely proportional
to the activity of inhibitor to the enzyme.

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Figure 4.2 Optical density of different concentrations of Pepstatin A in 280 nm
Table 4.4 shows the shows the percent inhibition of each Pepstatin A concentrations were treated using the
mathematical formula:
% Inhibition = Optical density of Positive control – Optical densty of Negative control
Optical density of Positive Control X 100
Table 4.4. Percent inhibition of various concentrations of Negative Control.
Negative control concentration (Pepstatin A) % Inhibition±SD
12.5ug/mL 63.43 ± 3.25
25ug/mL 72.26 ± 1.24
50ug/mL 78.62 ± 2.74
100ug/mL 82.86 ± 2.25
1000ug/mL 96.47 ± 3.59
The pepsin inhibitory activity tested using the
negative control Pepstatin A established a baseline to
compare with the percent inhibition of the Mentha
cordifolia Opiz lyophilized buffer crude extract. The
positive control optical density obtained was
0.2770±0.0000 and different concentrations of
Mentha cordifolia Opiz lyophilized buffer crude
extract 12.5, 25, 50 and 100 and 1,000ug/mL
obtained an optical density value ± standard deviation
of 0.1165±0.0021, 0.1040±0.0155, 0.0850±0.0311,
0.0710±0.0113 and 0.0140±0.0142 respectively. The
descending optical density of the plant extract were
also noted and illustrated in Figure 4.3.
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0
0 .0 0
0 .0 1
0 .0 2
0 .0 3
0 .0 4
0 .0 5
0 .0 6
0 .0 7
0 .0 8
0 .0 9
0 .1 0
0 .1 1
0 .1 2
0 .1 3
0 .1 4
0 .1 5
0 .1 6
0 .1 7
0 .1 8
0 .1 9
0 .2 0
0 .2 1
0 .2 2
0 .2 3
0 .2 4
0 .2 5
0 .2 6
0 .2 7
0 .2 8
0 .2 9
0 .3 0
P e p s ta tin A (N e g a tiv e C o n tro l)
C o n c e n tr a tio n (u g /m L ) V S A b s o r b a n c e 2 8 0 n m
C o n c e n tra tio n (u g /m L )
Opticaldensity(Mean±SD)
P e p sta tin A P o sitive C o n tro l

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0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0
0 .0 0
0 .0 1
0 .0 2
0 .0 3
0 .0 4
0 .0 5
0 .0 6
0 .0 7
0 .0 8
0 .0 9
0 .1 0
0 .1 1
0 .1 2
0 .1 3
0 .1 4
0 .1 5
0 .1 6
0 .1 7
0 .1 8
0 .1 9
0 .2 0
0 .2 1
0 .2 2
0 .2 3
0 .2 4
0 .2 5
0 .2 6
0 .2 7
0 .2 8
0 .2 9
0 .3 0
M e n th a c o rd ifo lia O p iz ly o p h iliz e d b u ffe r c ru d e e x tra c t
C o n c e n t r a t io n ( u g /m L ) V S A b s o r b a n c e 2 8 0 n m
c o n c e n tra tio n (u g /m L )
Opticaldensity(Mean±SD)
M e n th a c o rd ifo lia O p iz P o s itiv e c o n tro l
Figure 4.3 Optical density of different concentration of Mentha cordifolia Opiz
lyophilized buffer crude extract in 280 nm.
The percent inhibition of the different concentrations
of Mentha cordifolia Opiz lyophilized buffer crude
extract 12.5, 25, 50 and 100 and 1,000ug/mL were
shown in Table 4.5. The results obtained shows that
the higher the concentration of the plant lyophilized
extract the higher the affinity to the enzyme.
Table 4.5. Percent inhibition of Mentha cordifolia Opiz lyophilized buffer crude
extract in different concentrations.
Mentha cordifolia Opiz lyophilized buffer
crude extract concentration
% Inhibition±SD
12.5 ug/mL 57.95 ± 0.76
25 ug/mL 62.46 ± 5.62
50 ug/mL 69.32 ± 11.23
100 ug/mL 74.37 ± 4.09
1,000 ug/mL 94.96 ± 5.09
The percent inhibition of various concentration of
Pepstatin A 12.5, 25, 50, 100 and 1,000ug/ml
obtained 63.43%, 72.26%, 78.62%, 82.86% and
96.47% respectively. All negative control
concentrations were above 60% inhibitory activity.
Mentha cordifolia Opiz lyophilized buffer crude
extract concentrations 12.5, 25, 50, 100 and
1,000ug/mL obtained 57.95%, 62.46%, 69.32%,
74.37% and 94.96% respectively.
Figure 4.4 illustrates the comparison of both negative
control and the plant lyophilized extract optical
density ± standard deviation error bars with the mean
of positive control optical density 0.2800±0.0000.
Limitations such as Mentha cordifolia Opiz plant
material used was crude extract that is not pure and
may introduce error that contributes to the varied
standard deviation.

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Figure 4.4 Comparison of Pepstatin A and Mentha cordifolia Opiz percent inhibition.
QUALITATIVE PHYTO CHEMICAL
SCREENING
Two (2) grams of lyophilized buffer crude extract of
Mentha cordifolia Opiz (Yerba Buena) were screened
for the presence of phytochemical groups. Table 4.6
shows the qualitative screening of phytochemical
groups with its result done at the College of
Pharmacy, University of the Philippines Manila.
The qualitative result of Cyanidin test shown in Table
4.6 indicates the presence of Flavonoids most
probably flavones due to the light green to light pink
color change. Based on the qualitative result the
lyophilized buffer plant extract was submitted to
quantify the amount of flavonoids present in the
sample.
Figure 4.5 illustrates Total Flavonoid Content (TFC)
using Quercetin as a standard curve. Total Flavonoid
Content allows quantifying the total amount of
flavonoid in lyophilized buffer crude extract of
Mentha cordifolia Opiz and identifies putative
flavonoid compounds that are the same in chemical
structure with Quercetin.
Table 4.6 Qualitative screening for phytochemical groups.
TESTS RESULT
pH determination pH of 6, slightly acidic
Test for Tannins Absence of Tannins
Test for Glycosides Presence of glycosides
Test for Saponins Absence of Saponins
Test for plant acids Absence of plant acids
Test for reducing substances Absence of reducing substances
Test for alkaloids Absence of Alkaloids
Test for Flavonoids Presence of Flavonoids (Light green to light pink)
Test for Triterpenes Presence of Triterpenes
Test for Diterpenes Presence of Diterpenes

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Figure 4.5 Mentha cordifolia Opiz lyophilized buffer crude extract Total Flavonoid …………….
Content using Quercetin as a standard curve.
The Total Flavonoid Content (TFC) of Mentha
cordifolia Opiz lyophilized buffer crude extract was
computed based on the standard curve of Quercetin.
Using the logarithmic regression equation the result
was expressed in mg/grams shown in Table 4.6.
Table 4.7. Total Flavonoid Content of Mentha cordifolia Opiz lyophilized buffer
crude extract in mg/grams.
DISCUSSION AND CONCLUSSION
Results obtained in determining the pepsin kinetic
assay showed that the optimum substrate
concentration for 50ug pepsin was 1,800ug with
700uL stop solution using trichloroacetic acid.
Several variables were controlled to obtain the
optimum activity of pepsin such as pH, incubation
temperature and incubation period. Based on the
related literatures, pepsin will obtain its maximal
activity if the environment has a pH of 2-4 and
temperature of 37 °C. Incubation period was also
crucial in the process to obtain the equilibrium of
pepsin and hemoglobin (Anson ML 1938; Singh KP
et.al 2010 and 2013; Rege AA and Chowdhary AS
2013). The haemoglobin concentration of 1,800ug
presented the point of plateau of tyrosine and
tryptophan liberation measured using ELISA reader
at 280 nm illustrated in Figure 4.1. The absorbance of
1,800ug concentration of haemoglobin was used as a
point reference for positive control in pepsin
inhibitory activity assay.
Pepsin inhibitory activity assay was done first using
the negative control – Pepstatin A which is a known
inhibitor for aspartyl protease pepsin. Additional
variable were controlled to obtain the maximal
activity of both the enzyme and the inhibitor. To
assure the environment of pepsin falls under pH 2-4
the diluent used was 0.01 N HCl (Anson ML 1938;
Singh KP et.al 2010 and 2013; Rege AA and
Chowdhary AS 2013). Pre-incubation period of
pepsin and inhibitor was also added in the controlled
Mentha cordifolia Opiz lyophilized buffer crude extract
TOTAL FLAVONOID CONTENT 353.44±2.1 mg/g of Quercetin equivalent flavonoid content
Assay done in triplicate

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variables before incubating the enzyme to the
substrate. This pre-incubation allows the inhibitor to
inhibit pepsin prior to hemoglobin interaction (Anson
ML 1938).
Results obtained in pepsin inhibitory activity assay
using Pepstatin A showed a descending pattern of
absorbance from 12.5ug/mL, 25ug/mL, 50ug/mL,
100ug/mL and 1,000ug/mL Pepstatin A
concentration. This is expected because as the
concentration of inhibitor increases the greater
amount of pepsin is inhibited thus the lower
liberation of tyrosine and tryptophan. The tyrosine
and tryptophan are by-products of the substrate
hemoglobin when catalyzed by the enzyme pepsin
measured at 280 nm. In contrast to the descending
absorbance of the different concentration of Pepstatin
A the percent inhibition in its corresponding
concentrations has ascending pattern. This indicates a
dose response reaction; when concentration of
inhibitor increases the pepsin inhibition increases.
The negative control absorbance at 280 nm and the
percent inhibition shows the purity of Pepstatin A and
the effectiveness in controlling the variables to obtain
the optimum activity of both inhibitor and enzyme.
The negative control which is a known inhibitor of
aspartyl protease pepsin established the effectiveness
of the pepsin inhibitory activity assay. The controlled
variables such as pH, temperature, pre-incubation and
incubation period allows to obtain the maximal
activity of both inhibitor and enzyme.
The same procedure was done using Mentha
cordifolia Opiz lyophilized buffer crude extract.
Different concentrations were prepared using serial
dilutions from 100ug/mL, 50ug/mL,
25ug/mL,12.5ug/uL and 1,000ug/mL (1 mg/mL) was
also prepared as the highest concentration of the
lyophilized plant extract. Results obtained shows
57.95% of inhibition using the lowest concentration
of 12.5 ug/mL and 94.96% of inhibition using the
highest concentration of 1,000ug/mL. The results
were close to the value obtained using the negative
control which concentration of Pepstatin A were also
prepared in serial dilution from 100, 50, 25 and
12.5ug/mL. 1,000ug/mL was also used as the
standard concentration to inhibit aspartyl protease
pepsin.
The results obtained using the negative control shows
63.43% inhibition with the lowest concentration of
12.5 ug/mL and yield 96.47% inhibition using the
highest concentration of 1,000ug/mL Pepstatin A. As
to compare with the result to Mentha cordifolia Opiz
lyophilized buffer crude extract the lowest
concentration of 12.5ug/mL yields 57.95% and
1,000ug/mL yields 93.24%. The IC60 (Inhibitory
Concentration 60%) value was calculated using the
Microsoft Excel programme at which the logarithmic
regression equation was obtained. The concentration
needed for Pepstatin A and Yerba Buena to inhibit
60% of pepsin activity were 5.13ug/mL and
17.30ug/mL respectively.
The qualitative screening for phytochemical group
was done after determining >60% inhibition of
Mentha cordifolia Opiz lyophilized buffer crude
extract. Two (2) grams were submitted to the College
of Pharmacy, University of the Philippines for
phytochemical screening. The results obtained shows
that the plant lyophilized buffer crude extract
contains only glycosides, flavonoids, triterpenes and
diterpenes. Other phytochemical groups were absent
like tannins and alkaloids which are theoretically
present in Mentha cordifolia Opiz but because of the
sample preparation like using 0.01 M Phosphate
buffer as diluent to extract protease inhibitors from
the plant and the process of lyophilisation, the said
phytochemical groups were not qualified.
The qualitative screening for phytochemical group
results obtained shows the presence of Flavonoid. To
identify putative flavonoid compounds present in
Mentha cordifolia Opiz lyophilized buffer crude
extract a procedure was done to quantify the amount
of flavonoids using the standard curve of Quercetin, a
flavonoid compound. The result obtained in Total
Flavonoid Content (TFC) shows 353.44±2.1 mg/g of
Quercetin equivalent flavonoid content. The standard
curve of Quercetin used in the Total Flavonoid
Content of the lyophilized buffer crude extract
identified putative other flavonoid compounds that
have similar chemical structure to Quercetin such as
Luteolin and Apigenin.
GENERAL SUMMARY OF THE STUDY
This study aims to determine the inhibitory activity of
Mentha cordifolia Opiz (Yerba buena) against
aspartyl protease pepsin. The study adopted the
methods of Govindappa M. and Singh KP for the
screening of plant extract against the catalytic activity

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of pepsin to haemoglobin substrate. Different
concentrations of Mentha cordifolia Opiz lyophilized
buffer crude extract were prepared in serial dilution
from 100ug/mL, 50ug/mL, 25ug/mL and 12.5ug/mL.
The highest concentration was also prepared
in1,000ug/mL (1 mg/mL) both negative control and
the plant lyophilized buffer crude extract.
Pepsin kinetic assay was performed to establish the
optimum activity of constant pepsin to various
concentrations of substrate haemoglobin. The assay
allows setting what concentration of haemoglobin
will be used as a positive control. The optimum
haemoglobin concentration obtained in the assay
result shows 1,800 ug that will be used as a standard
concentration in the study in pepsin inhibitory
activity assay.
pH, pre-incubation temperature and period,
incubation temperature and period, reaction mixture
and amount of stop solution were controlled to obtain
the optimum activity of both the pepsin and the
inhibitor. The various concentration of pepstatin A
and plant lyophilized buffer crude extract were tested
for pepsin inhibitory activity using ELISA reader at
280 nm. The optical density of each concentration
indicates the liberation of tyrosine and tryptophan
when haemoglobin is catalysed by pepsin. The
optical density value of each concentration reflects
the affinity of the inhibitor to pepsin; the lower the
optical density value the higher the affinity of the
inhibitor to pepsin.
The percent inhibition of Mentha cordifolia Opiz
lyophilized buffer crude extract shows a 94.96% with
2% difference of % inhibition with the known
inhibitor of pepsin which is 96.47%. The result of the
study allows the interpretation that the Mentha
cordifolia Opiz lyophilized buffer crude extract have
an inhibitory activity mechanism the same with the
negative control – Pepstatin A. The qualitative
screening for phytochemical groups supports that the
possible active group that inhibit pepsin are
flavonoids, triterpenes and diterpenes. The
quantitative flavonoid content test allows to identify
putative compounds that are active in the flavonoid
group which possibly contain Apigenin and Luteolin
that closely resemble in chemical structure to
Quercetin.
CONCLUSION
The study aims to determine the inhibitory activity of
Mentha cordifolia Opiz (Yerba buena) lyophilized
leaf buffer extract on aspartyl protease pepsin.
Relating to the initial statements of the problem of
the study and based on the results of the
experimentations, the proponent now concludes the
following:
 The lyophilized buffer crude extract of Mentha
codifolia Opiz (Yerba buena) have Concentration
60% (IC60) value of 17.30ug/mL against pepsin.
 The effect of lyophilized buffer crude extract of
Mentha codifolia Opiz (Yerba buena) shows
increasing response as the concentration of the
plant lyophilized buffer extract increases. The
data obtained in optical density value of each
plant lyophilized buffer crude extract
concentrations reflects the activity of the
inhibitor to pepsin; the lower the optical density
value the higher the activity of the inhibitor to
pepsin.
 Lyophilized buffer crude extract of Mentha
codifolia Opiz (Yerba buena) shows 353.44±2.1
mg/g of Quercetin equivalent flavonoid content.
The standard curve of Quercetin used in the
Total Flavonoid Content of the lyophilized buffer
crude extract identifies putative other flavonoid
compounds that have similar chemical structure
to Quercetin such as Luteolin and Apigenin.
Acknowledgement
I would like to thank Accelerated Science and
Technology Human Resource Development program
– Science Education Institute, Department of Science
and Technology (ASTHRDP-SEI, DOST) for the
graduate scholarship opportunity. To my advisor,
Dr.Lilen Dorothy C. Sarol for the guidance in
completing this research. And to professor Teresita S.
de Guzman and Dr.Alice Alma C.Bungay for the
comments and suggestions to improve this study.
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