Study of mechanism of inhibition of HIV-1 infection and cytotoxicity

assessment of Mentha cordifolia Opiz (Yerba Buena) buffer crude

extract
Alfredo A. Hinay Jr.,* Dr. Lilen Dorothy C. Sarol. University of the Philippines Manila

Apart from the significance placed by man on plants as source of food, their other
boundless use has been in the area of medicine. The World Health Organization
(WHO) has projected that 80% of the inhabitants of the world depend on traditional
medicine; hence plants and plant products have been utilized in the management of
infections many centuries before the active principles in the plant products could be
elucidated through the improvements in science and technology (Abonyi et. al.,
2006). Anti-HIV inhibitory activity is extensively distributed in nature in the form of
medicinal plants (Chinsembu KC and Hedimbi M, 2009, 2010). People with HIV/AIDS
are very likely to seek herbal drugs particularly in the area of the world where
Antiretroviral Therapy (ART) is not freely available or affordable. Therefore, plants
and their products can be utilized as a source of new anti-HIV drugs (Singh et. al.,
2010, Govindappa M et.al 2011, Rege AA and Chowdhary AS, 2013).
Yamasaki et.al, 1998 demonstrated aqueous extracts from leaf, flower, roots, stem
and aerial parts of Labiateae plant origin showing potent anti-HIV activity. Crude
extract of Mentha x peperita grapefruit mint was one of the 46 species of
Labiateae tested in the same study and one of those which showed a strong antiHIV activity. In the Philippines, Vicroriano conducted a preliminary study 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 viral replication step was not
elucidated (Victoriano F, 2003). To specify what post-infection interaction was
inhibited by the plant and to elucidate the mechanism of inhibition of the Philippine
mint leaf crude extract a preliminary study was done for aspartly protease pepsin
inhibition; where pepsin structure is similar to HIV-1 Protease (Hinay AJR, 2014). The
result showed 93.27% inhibition of aspartyl protease pepsin that supports a putative
action of the plant extract impedes the post-translational HIV-1 viral protease.
The qualitative result of Cyanidin test shown in the study of Hinay AJR indicated the
presence of flavonoids, most probably flavones, in the amount of 353.442.1 mg/g
Quercitin equivalent flavonoid. This phytochemical in the mint buffer extract may be
the putative active group that is responsible in the inhibition of HIV-1 viral protease
(Rummel DJ. 2009; Villasenor JM. 1995; Dr. Dukes Phytochemical and
Ethnobotanical Databases, Hinay AJR, 2014). However, with the anticipation that the
phytochemical Flavonoids were also active in the inhibition activity of viral entry to
host cells, other HIV-1 viral enzymes (Integrase, Reverse Transcriptase) and
regulation of NF-kB that is a major host transcription factor of HIV replication at the
transcriptional step, in this study and based on the 93.27% inhibition of the plant
extract against pepsin, an attempt will be made to test local plant Mentha
cordifolia Opiz (Yerba buena) for HIV-1 viral enzyme Protease inhibition using
fluorescence resonance energy transfer technique.

General Objective:

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 Mentha
cordifolia Opiz buffer crude extract could provide pharmacologically important
protease inhibitor, an attempt will be made to test for HIV-1 protease inhibition.
Specific objectives:
1. To determine the inhibitory activity of Mentha cordifolia Opiz (Yerba buena)
lyophilized buffer leaf extract against HIV-1 protease.
2. To determine the dose response effect of Mentha cordifolia Opiz lyophilized
buffer leaf extract (1,500, 1,250, 1,000ug/mL, 500ug/ml, 250ug/mL,
125ug/mL, 62.5ug/mL) against HIV-1 protease.
3. To determine the inhibition kinetics of Mentha cordifolia Opiz lyophilized
buffer leaf extract against HIV-1 protease.
Significance of the study:
Many
major
physiological
processes
depend
on
regulation
of proteolytic enzyme activity and there can be dramatic consequences when
equilibrium between an enzyme and its substrates is disturbed. In this prospective,
the discovery of small-molecule ligands, like protease inhibitors, that can modulate
catalytic activities has an enormous therapeutic effect (Cuccioloni, M. et al. 2009).
Hence, inhibition of the HIV protease is one of the most important approaches for
the therapeutic intervention in HIV infection and their development is regarded as
major success of structure-based drug design (Chen X, et al. 2003; Adachi M,et al.
2009). They are highly effective against HIV and have, since the 1990s, been a key
component of anti-retroviral therapies for HIV/AIDS (Yanchunas, J. et al. 2005).
The effects of Mentha cordifolia Opiz (Yerba buena) on the in vitro study of HIV-1
protease activity as observed in this study would lead to additional knowledge and
understanding of the plants biochemical property. The results would have has far
reaching implications on its current use in traditional medicine as an antiinflammatory and anti-microbial agent. This study may give impetus to further
research targeting the beneficial properties of the plant useful in developing
alternative natural-based anti-viral compounds. If proven that the local Philippine
mint Mentha cordifolia Opiz (Yerba buena) has inhibitory activity against HIV-1 posttranslational protease this could be used as a potential drug component against
HIV/AIDS infection.
The outcome of this study will be of significance to the following:
Individuals with HIV/AIDS. A formulation of local-based drug using the inhibitory
property of Philippine mint Mentha cordifolia Opiz (Yerba buena) against HIV-1
protease that will be available in a cheaper but reliable, guaranteed, accessible and
safe medication product to the individuals with HIV/AIDS.
Research Agencies & Institutions. The inhibitory activity of Mentha cordifolia
Opiz (Yerba Buena) Leaf Extract against HIV-1 protease will serve as a platform in
therapeutic virology and pharmacology in the Philippines that will encourage the
researchers and research institutions to formulate a local-based drug component
against HIV-1 replication cycle.

Proposed Methods:
Virus generation
HIV-1NL4.3 (CXCR4 tropic virus) will prepared by transfecting HEK-293 T cells with
proviral plasmid DNA clone pNL4.3 (AIDS Research and Reference Reagent Program
[ARRRP], Division of AIDS, National Institute of Allergy and Infectious Diseases, USA)
using CaPO4. The medium will be changed 24 h post-transfection and the
supernatant will be harvested after 48 h of incubation and frozen at 80C. The
concentration of virus stock will be determined by the HIV-1 p24 Antigen Capture
Assay ELISA (SAIC-Frederick Inc., NCI-Frederick, USA) and by determining the
infectious titer using TZM-bl cells.
Anti-HIV activity using TZM-bl cells
In TZM-bl cells-based assay, HIV-1NL4.3 viral strain at a multiplicity of infection
(MOI) of 0.05 will be treated with varying concentrations (250 g/ml) of Mentha
cordifolia Opiz lyophilized buffer extract for 1 h at 37C. Subsequently, HIV-1 pretreated with lyophilized buffer extract will be added to TZM-bl cells (4 104/well;
seeded on the previous day in 24-well plate) and incubated for 4 h. Subsequently,
cells will be washed with cold 50 mM PBS, fresh culture medium with extracts added
and further incubated for 48 h in humidified atmosphere of 5% CO2 at 37C.
Azidothymidine (AZT; Sigma-Aldrich Inc.) will be used as positive reference control.
After incubation, cells will be washed twice with PBS and lysed with 1X lysis buffer
(Promega Corporation, Madison, WI, USA). Supernatant will be collected and
luciferase activity will be measured using white optiplate in the Fluorimeter (BMG
Labtech GmbH, Offenberg, Germany) at a spectral range of 240-740 nm. The results
will be expressed as percentage inhibition, calculated by taking the luminescence in
experimental group divided by the luminescence in infected cells in absence of test
extracts/AZT multiplied by hundred. Percent inhibition will be calculated by
subtracting the above value from hundred.
Luciferase activity assay (BrightGlo Luciferase Assay Kit)
After 48 h incubation at 37C, 5% CO2 the medium will be aspirated and 35 ul of
cell culture lysis reagent (Promega, Wisconsin, USA) will be added to each well. 5 ul
of each sample will be mixed with 25 ul of Luciferase Assay Substrate (Promega)
and luminescence will be measured using the FLUOstar OPTIMA multi-detector
reader (BMG Labtech) at a spectral range of 240-740 nm.
Cytotoxicity assay using MTT
The cytotoxicity of Mentha cordifolia Opiz lyophilized buffer extract on TZM-bl cell
lines will be assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium
bromide; Sigma-Aldrich Inc.] assay. In brief, cells will be seeded (6 103 adherent
cells/ well; 5 104 suspension cells/well) in 96-well cell culture plates (Greiner BioOne, GmbH, Frickenhausen, Germany) and grown overnight at 37C in humidified
atmosphere of 5% CO2. After 24 h, cells will be treated with varying concentrations
of the lyophilized buffer extract ranging from 10400 g/ml, for the duration as used
to determine the anti-HIV activity [TZM-bl cells - 48 h]. Negative control included
cells treated with solvent/medium. After incubation, cell viability will be assessed by
adding 20 l MTT (5 mg/ml in 50 mM PBS) per well and incubated at 37C for 3 h
followed by addition of MTT solvent (100 l/well; 20% SDS and 50% dimethyl

formamide in 50 mM PBS). The absorbance (OD) will be read at 570 nm with

reference filter at 690 nm. Experiments will be performed in duplicates and percent
viability will be calculated by dividing the OD obtained in treatment group by OD of
untreated cell control multiplied by hundred.
HIV-1 entry assay
TZM-bl cells (4 104) will be pre-treated with or without lyophilized plant extract at
37C for 1 h before being infected at an MOI of 0.05. Bicyclam will be used as a
reference control. The infected cells will bet rypsinized and washed with PBS to
remove the unattached virus and cultured in fresh medium. After 48 h incubation,
percent inhibition in HIV-1 infection will be determined using p24 assay.
Tat-inhibitor assay
TZM-bl cells (4.0104/well) will be seeded in 24-well plate and cultured overnight.
The cells will be transfected with pTat (0.1 g/well; ARRRP, USA) using
Lipofectamine reagent (Invitrogen) according to the manufacturers instructions.
After 4 h of transfection, cells will be treated with the Mentha cordifolia Opiz
lyophilized buffer extract at 37C. Forty-eight hours post-treatment, luciferase
activity will be measured.
FRET-based inhibition of HIV-1 protease activity by Mentha cordifolia Opiz
The EnzoLyte 520 HIV-1 protease assay kit (AnaSpec Co.) will be used to measure
HIV protease enzyme activity and inhibition by Mentha cordifolia Opiz (Yerba buena)
leaf extract. This protease assay kit provides a convenient assay for measuring HIV
protease enzyme activity and screening HIV-1 Protease inhibitors using a HiLyte
Fluor488/QXL520 fluorescence resonance energy transfer (FRET) peptide. The
sequence of this FRET peptide is derived from the native p17/p24 cleavage site on
Prgag for HIV-1 protease. In the FRET peptide, the fluorescence of HiLyte Fluor488 is
quenched by QXL520 until this peptide is cleaved into two separate fragments by
HIV-1 protease. Upon cleavage, the fluorescence of HiLyte Fluor488 is recovered,
and can be monitored at excitation/emission (490 nm/520 nm). Recombinant HIV-1
protease will be used in this assay.
To validate the effect of Mentha cordifolia Opiz lyophilized buffer extract on HIV-1
protease activity, release of mature viral particles by HIV-1 infected and extract
treated PBLs will be investigated. The culture supernatant will be used to infect
TZM-bl cells to quantify the infectivity of the released virions and hence correlate it
with the HIV-1 protease activity.
Cell-associated virus based assay using peripheral blood lymphocytes
(PBLs)
Experiments using human blood cells will be carried out under informed consent
and following the clearance from the Institutional Bio-safety and Human Ethical
Committee. Blood (5 ml) will be taken from healthy HIV-1 sero-negative donors and
lymphocytes will be isolated using Ficoll density gradient method. Cells (2.0106
cells/ml) will be stimulated with phytohemagglutinin (PHA-P; Sigma-Aldrich Inc.) at
3g/ml for 3 days. After stimulation, cells will be washed twice with PBS. PBLs will be
infected with HIV-1NL4.3 at an MOI of 0.05, in presence of IL-2 (10 U/ml) for 4 h.
Cells will be washed twice with plain medium to remove the unbound virus and

seeded in 96-well plate (5.0104/well/200 l) in RPMI medium supplemented with

10% FBS and recombinant human IL-2 (10 U/ml). Plate will be further incubated at
37C, 5% CO2 after adding the Mentha cordifolia Opiz (Yerba buena) leaf extract at
varying concentrations to the wells. Cell culture supernatant will be collected on 5th
day for p24 analysis. The culture supernatant (100 l) will be further used for
infection of TZM-bl cells for 4 h, and luciferase expression will be estimated after 48
h as described above to validate the infectivity of virion particles released by PBLs
as an effect of treatment with Mentha cordifolia Opiz (Yerba buena) luyophlized
buffer extract.