screening of mentha cordifolia opiz (yerba buena) buffer crude extract for aspartyl protease pepsin

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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: [email protected]

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

Alfredo A. Hinay et al / Int. J. of Res. in Pharmacology & Pharmacotherapeutics Vol-3(1) 2014 [28-39]

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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 370C 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 370C 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 370C. 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

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Pe

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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


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


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

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P e p s ta tin A (N e g a tiv e C o n tro l)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 )

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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

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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 )

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tic

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M e n th a c o rd ifo l ia 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.


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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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

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P e p s ta tin A v e rs u s M e n th a c o r d ifo l ia O p iz

P e rc e n t in h ib it io n ± S D

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Per

cent

inhi

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P e p s ta tin A M e n th a c o rd ifo l ia O p izP o s itv e c o n tro l

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


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


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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screening of mentha cordifolia opiz (yerba buena) buffer crude extract for aspartyl protease pepsin

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