In-vitro studies of membrane stabilizing and lipoxygenase inhibitory

activities of ​Cordia dichotoma​ G. forst leaves

A Thesis Proposal
Presented to the Faculty
Department of Pharmacy
College of Allied Medical Professions
Lyceum of the Philippines University – Batangas

In Partial Fulfillment
of the Requirements of the Degree
Bachelor of Science in Pharmacy

Diane Antoinette A. Balitaan, Mar Donna Dei B. Cabrera, Jethro C. Hernandez,

Frances Lyn G. Mangubat, Leigh Danica D. Mendoza,
Aaron Dell A. Cobeng & Oliver Shane R. Dumaoal

INTRODUCTION to react with nonspecific stimuli like

exercise and cold air. Other symptoms
According to the World Health
include excessive mucus production,
Organization, asthma is one of the most
airway narrowing and airway wall
important non- communicable
remodeling, chest tightness, shortness
conditions. It is a chronic disease that
of breath and wheezing (Lambrecht &
inflames and narrows the air passages
Hammad, 2015). The cause of asthma
of the lungs. According to the most
is unknown, but there are risk factors
recent WHO estimates released in
that have been identified. One factor
December 2016, 383 000 asthma-
includes genetics and heritability ranges
related deaths occurred in 2015.
from 35% to 95%. Environmental factors
Meanwhile, the Philippines is ranked 9​th
such as exposure to pollutants, ozone,
in asthma mortality across the globe
smoke and tobacco increase the risks of
with approximately 315 million Filipinos
developing asthma. The underlying
suffering from this condition. Back in
inflammation can be asymptomatic and
2002, the prevalence of asthma was
inflammation control is central in asthma
reported at 26.7 percent according to
management. The disconnection
the National Epidemiology Survey
between inflammation and symptoms
(Jambora, 2018). Asthma is defined as
can lead to poor awareness of asthma,
a chronic inflammatory disease of the
which can lead to poor recognition and
bronchioles. Symptoms include
non-compliance treatment (Mims, 2015).
bronchial hyper-reactivity, which is
According to numerous studies, airway
described as the tendency of the cells of
inflammation is a central key to the
the smooth muscle in asthmatic patients
pathophysiology of asthma (Kudo,
Ishigatsubo, & Aoki, 2013). One of the
major causes of airway inflammation is
the production of leukotrienes via
lipoxygenase (LOX) pathway upon
exposure to certain allergens (Di
Gennaro & Haeggström, 2013). The
liberation of potent chemical mediators
from inflammatory cells resulting to
chronic inflammation of the airway,
airway remodeling, which is
characterized by thickening of airway
wall compartments that may lead to the
progression and development of the
disease (Kudo et al., 2013).
Membrane destabilization of the
immune system cells is a physiologic
indicator in the pathogenesis of several
inflammatory diseases. The secretion of
the lysosomal constituents (bacterial
enzymes and proteases) of the immune
system cell system (mast cells,
neutrophils, basophils, eosinophils and
leukocytes) in response to a stimuli may
lead to progression of the damage thus
resulting in several physiologic
response which are prominent in the
usual signs of inflammation (Sinbad,
Samuel, Adewale, & Adedoyin, 2017).
Lipoxygenases belong to a class
of non-heme iron containing
dioxygenases which catalyze the
addition of oxygen molecule to fatty
acids containing a cis,
cis-1,4-pentadiene system to yield an
unsaturated fatty acid hydroperoxides.
This enzyme is found to play a
significant role in numerous disorders
such as bronchial asthma, inflammation
and tumor angiogenesis. LOX is
responsible for the conversion of
arachidonic acid to 5-hydroxy-6, 8, 11,
14- eicosatetraenoic acid (5-HPETE).
5-HPETE when furthered converted
would result to a series of leukotrienes,
which are mediators of inflammation in
asthma (Sacan & Turhan, 2014).
Corticosteroids, B2- agonist,
lipoxygenase and leukotriene inhibitors
have been effective in suppressing
inflammatory responses like asthma but
prolonged treatment can result to
notable adverse effects and drug
resistance. (Sullivan, Ghushchyan,
Globe, & Schatz, 2018; Cazzola, Page,
Rogliani, & Matera, 2013 & Rossi et al.,
2010). Hence, discovery of natural
product alternative therapies that do not
produce or lessen significant adverse
effects has become a trend. Sustaining
and combating diseases through the
use of plant derived medicines from
their specific plant source is considered
therapeutic and the first line of defense
(Jamkhande, Barde, Patwekar, & Tidke,
2013). However, scientific investigation
or studies and proper screening from
different plant kingdoms specifically
higher plants, which can act as both
potential crude drug and a source of
developing chemotherapeutic agents
should be performed. (Ali, Kala , & Khan
, 2018).
Cordia dichotoma G. Forst
commonly known as ​anonang,​ is
claimed to originate in South and
Southeast Asia. It grows in tropical and
subtropical regions and belongs to the
Boraginaceae family. Anonang is a
medium sized tree found widespread in
the thickets and secondary forests at
low and medium altitudes in the
Philippines. (Jamkhande, Barde,
Patwekar, & Tidke , 2013; Ragasa, et
al., 2015)
FIGURE 1. ​Cordia dichotoma t​ ree
Retrieved from:
https://sylviatramos.blog/
Phytochemical analysis on
Cordia dichotoma ​proves that its leaves
contains Alkaloid, carbohydrate,
flavonoids, proteins, amino acids,
phenols, tannins, glycosides and
steroids are chemical constituents found
in the ​Cordia dichotoma plant (Ali, Kala ,
& Khan , 2018).Several scientific studies
have been made on ​Cordia dichotoma,
which proves different pharmacologic
activities of its leaves like analgesic,
antipyretic (Gupta & Kaur, 2014),
antifertility, juvenomimetic biliary
obstruction, nutritional food (Jamkhande
et., al, 2013) and contraceptives
(Bhattacharya & Saha, 2013).
The need for novel compounds
which can acts as an alternative for the
current anti-asthma drugs is evident
since drugs for this disease such as
corticosteroids, B2-agonist and
5-Lipoxygenases and leukotrienes
inhibitors are predominantly used but
these agents contains various drug
interactions and adverse effects. Herbal
medicine greatly depends on herbs for
curing numerous illnesses. Even though
plants are accepted as a product used
in developing new drugs, empirical data
from scientific studies should strongly
validate the therapeutic effects of
natural products to strengthen and
continue the development of natural
resources and medicinal compounds.
Because of these reasons, the
researchers decided to conduct a study
on the leaves of ​Cordia dichotoma
G.forst a ​ nd evaluate its anti-asthma
activity through lipooxygenase inhibition
and membrane stabilization as
parameters. In addition, discovery of
new source of drug molecules for the
treatment of asthma with the main goal
of identifying safer and more tolerable
substitute is also one of the aims of this
study.
METHODOLOGY
Plant Collection​ ​and Authentication
 The ​Cordia dichotoma ​leaves will
be obtained from Barangay Ticub,
Laurel, Batangas and will be brought to
the UST Herbarium located in España,
Manila for authentication and
certification purposes.
Preparation of Plant
The leaves of ​Cordia dichotoma
will be properly clean with water and
shade dry under room temperature.
Complete drying should be observed.
Dried leaves will be coarsely powdered
through a mechanical mixer (Hatware et
al., 2018).
Extraction of Plant
A sample of 100g from the aerial
part will be taken in a soxhlet and
addition of 80% methanol to the 2
siphons which is up to 500ml shall be
filled. The temperature is set to 70 °C
and the extraction will be performed in a
span of 5 hours. After that, the extract
that will be obtain will be filtered and
concentrated at 70 °C.(Linn, 2013).
Determination of Flavonoid Content
By using the aluminum chloride
colorimetric method, the total flavoinoid
content would be estimated. The
principle in this colorimetric method is
that AlCl3 will form stable complex with
keto groups of C-4 and either the C-3 or
C-5 hydroxyl group of flavonols and
flavones. Other than that, formation of
acid labile complexes with
ortho-dihydroxyl groups in the rings A or
B of flavonoids. According to studies,
quercetin is the suitable reference for
total flavonoid determination in the plant
sample extract. Various concentrations
of quercetin will be prepared to make
the standard calibration curve. In 100 ml
of methanol, 10 mg of quercetin will be
dissolved and will be diluted to 6.25,
12.5, 25, 50, 80, and 100 µg/ml with
methanol. To prepare the plant extract
stock solution, dissolve 100 mg
methanolic extract in 5ml methanol and
transfer it to a 10ml volumetric flask. 1M
potassium citrate and 10% aluminum
chloride will be prepared using distilled
water. The assay will be determined by
0.5 ml of each dilution of standard
quercetin and each of the plant extract
stock solution in separate test tubes. In
a test tube create a mixture of 1.5ml
methanol, 0.1ml potassium acetate
solution, 0.1 ml aluminum chloride
solution and 2.8ml of distilled water.
Prepare a sample blank for the three
methanolic extract and all dilution of
standard quercetin. But instead of using
aluminum chloride replace it with
distilled water. All solutions will be
filtered through a Whatmann filter paper
before absorbance is measured (Bag &
Devi, 2015).
Isolation and purification of bioactive
compound
Pack a column chromatography
(CC) (50 x 9 cm) starting off with 1.5 cm
fine sand layer and silica gel (particle
size 63-200 mm, 430 g) at a depth of 20
cm. Cover again with 1.5 cm sand layer
to protect the adsorbent surface. ​A
volume of hexane (4 x 1 L) will pass
through the CC and would result to
rising of bubbles and settling of silica
gels allowing them to pack tightly into
the column. Organic fraction must be
dissolved in 40ml of CH​2​Cl​2 and mix it
with silica gel forming a slurry. Use the
rotary evaporator to remove solvent and
obtaining dry powder. Slowly and evenly
apply the dried powder extract on the
top portion of the packed column and
perform a gradient elution with a mixture
of hexane/EtOAc. The mixture of the
solvents and eluents that will be
collected are of increasing polarity
varying in concentration. Starting off
with 100% hexane and will be followed
by hexane/EtOAc mixtures of 8:2, 6:4,
4:6, 2:8, 1:9 and lastly with 100% ethyl
acetate. A ratio of 10:0 to 0:10 will be
observed. In round-bottom flasks (1 L)
collect the resulting fractions and
remove the mobile phase in each
fraction by using a rotary evaporator (at
≤ 4°C). (Martín-Rodríguez et al., 2015)
Thin-Layer Chromatography
According to their TLC profile
similarities, incorporate the 15 fractions
into 10 sub-fractions. Add the hexane /
ethyl acetate solvent mixtures with
increasing polarity from 8:2 (8 ml
hexane plus 2 ml ethyl acetate) to 6:4 in
the TLC chamber at a depth of just
under 0.5 cm to run the TLC plates. Use
a micro capillary to apply an aliquot of
each fraction to the baseline (measuring
0.7 cm from one end of the plate) of the
TLC (about 7 x 5 cm for TLC) and place
it in the TLC chamber. Develop TLC,
leaving the solvent with capillary action
to raise the TLC plate, until the solvent
reaches almost the top (0.5 cm from the
plate). Then view the developed TLC
with 254 nm wavelengths under a UV
lamp and stain the plates with the oleum
system and heat them to 80 - 100 ° C in
a hot plate up until to the appearance of
the spots (Martín-Rodríguez et al.,
2015).
Membrane Stabilization Assay
Preparation of Human Red Blood Cells
(HRBC) Suspension
Collect fresh whole human blood
and mix it with equal volume of sterilized
Alsever solution (0.05% citric acid,
0.42% sodium chloride, 0.8% sodium
citrate and 2% dextrose in water). Blood
will be centrifuged at 3000 rpm for 10
minutes. Pack cells will be washed
thrice with isosaline (0.85%, pH 7.2).
Volume of blood will be measured and
reconstituted as a 10% v/v suspension
with isosaline.
Heat Induced Hemolysis
The membrane lysis caused by
hypotonicity through the stabilization of
human red blood cell is the concept
tangled here. 1ml of phosphate buffer
with 7.4 pH and 0.15M, 2 ml hyposaline
(0.36%), 10% v/v of 0.5 ml HRBC
suspension with extracts of plant 0.5ml
and diclofenac sodium as a standard
with different concentrations (50, 100,
250, 500, 1000, 2000 µg/ml) and control
(distilled water to produce 100 percent
hemolysis instead of hyposaline) are the
mixtures that comprises the assay and
will be incubated at 37°c for 30min and
will be centrifuged correspondingly. The
spectrophotometer at 560nm will be
used to the project the content of
hemoglobin suspension (Chippada,
Volluri, Bammidi, & Vangalapati, 2011).
Calculation of percentage hemolysis of
HRBC membrane:​
OD of test sample
%Hemolysis= OD of control
x 100
Lipoxygenase Inhibition Assay
Cayman Chemical’s
Lipoxygenase Inhibitor Screening Assay
Kit will be purchased at Sapphire
Bioscience and manufacturer’s protocol
will be followed. Cayman’s
Lipoxygenase Inhibitor Screening Assay
Kit detects and measures the
hydroperoxides produced in the
lipoxygenation reaction using a purified
LO. The detection reaction is equally
sensitive to hydroperoxides at various
positions within the fatty acid, and will
work with fatty acids of any carbon
length. It is thus a general detection
method for LO, and can be used to
screen libraries of compounds for those
which inhibit LO enzymes.
REVIEW OF RELATED LITERATURE
DESCRIPTION
Asthma is defined as a chronic
inflammatory disease of the bronchioles.
Symptoms include bronchial
hyper-reactivity, which is described as
the the tendency of the cells of the
smooth muscle in asthmatic patients to
react with nonspecific stimuli like
exercise and cold air. Other symptoms
include excessive mucus production,
airway narrowing and airway wall
remodeling, chest tightness, shortness
of breath and wheezing (Lambrecht &
Hammad, 2015).
PREVALENCE
According to the World Health
Organization, asthma is one of the most
important non- communicable
conditions. It is a chronic disease that
inflames and narrows the air passages
of the lungs. Asthma is a common
childhood disease and approximately
235 million people suffer from it. The
majority of deaths associated with
asthma occur in countries with low
medium incomes. According to the most
recent WHO estimates released in
December 2016, 383 000 asthma-
related deaths occurred in 2015.
On the other hand, the
Philippines rank No. 9 in asthma
mortality with approximately 315 million
suffering from the ailment globally. The
prevalence of asthma in the Philippines
was reported at 26.7 percent back in
2002 based on the National
Epidemiology Survey (Jambora, 2018).
ETIOLOGY
The cause of asthma is unknown,
but risk factors have been identified and
interactions between gene and
environment are important. Genetics is
a factor in asthma and heritability
ranging between 35% and 95%.
Hundreds of genetic variants is related
to an increased risk of asthma have
been identified in large genetic studies.
Epigenetic variations in the transition of
the genetic code have also been
demonstrated to play a role in the
development of asthma. Respiratory
infections particularly early life viral
infections increase the risk of
developing asthma, especially when
symptoms are severe. Airborne
exposures to the environment increase
the risk of asthma tobacco, smoke,
ozone and pollutants. The development
of asthma is also associated with atopic
conditions and sensitization to inhalant
allergens. Other factors are also
involved in developing asthma like
microbiome effects, vitamin D, chemical
exposure, dietary changes, stress and
metabolites. Current understanding of
asthma involves a wide range of genetic
diversity, which is variable and
environmentally influenced by
epigenetic and transcriptional factors,
leading to less diverse histopathological
characteristics with resulting cardinal
asthmatic symptoms (Mims, 2015).
Several possible risk factors
related to the development of asthma
have been studied. Atopy is often
identified as a strong risk factor for the
development of asthma. Some studies
have shown that early sensitization of
mites and maternal asthma are
important predictors of asthma. Parental
smoking is a major risk factor for acute,
lower respiratory infections in infants
and wheezing and asthma development
in children. However, children who are
not exposed to tobacco smoke can also
have a high incidence of asthma. Active
smoking in adults is linked to the later
development of asthma. Air pollutions
and viral infections are well known
irritant for exacerbations of asthma, but
there are conflicting data as to whether
these factors contribute to the
development of asthma. Microbial
exposure is inversely linked to the
development of asthma and atopy and
may account for the disparate
prevalence of asthma in urban versus
rural environments. Ultimately, asthma
is likely to develop in genetically
sensitive people by combining complex
environment exposures (Maslan &
Mims, 2014).
In most cases, the inflammation
underlying asthma is chronically
present, but asthma often occurs
clinically in attacks or episodes. The
underlying inflammation can be present
with a lack of symptoms and
inflammation control is central in the
asthma management. The
disconnection between inflammation
and symptoms can lead to a poor
awareness of asthma that can cause
poor recognition and non-compliance
treatment. The inflammation and
associated changes in pathological
tissue leading to a constellation of
symptoms like shortness of breath,
coughing, and tightness of the chest.
The bronchial obstruction is mainly due
to the production of mucus, tissue
edema and smooth muscle constriction.
Smooth muscle constriction in the
bronchi usually responds to inhaled β2
agonists and creates a reversible
component for episodes of asthma
(Mims, 2015).
PATHOPHYSIOLOGY OF ASTHMA
Dendritic cells, like airway
epithelial cells, are also directly exposed
to the external environment. Pulmonary
dendritic cells act as antigens
presenting cells and express on their
cell surface a variety of pattern
recognition receptors. In response to
pathogens and allergens, dendritic cells
can also be recruited to the airway. The
dendritic cells can be directly stimulated
by binding of allergens or infectious
agents on the surface or stimulated
indirectly by epithelial cells in the airway
(by mediators like IL-25, IL-33, GM-CSF
and TSLP). Locally, eosinophils can be
recruited by dendritic cells. Multiple
factors such as C-C chemokine receptor
types 7 (CCR7), CCR8, and CCRL2
mediate the migration of dendritic cells
from the lymphatics to the regional
lymph nodes. T cell differentiation is
affected by dendritic cells and under
certain circumstances produce T-helper
type 2 (TH2) response seen in atopic
asthma (Olin & Wechsler, 2014).
In allergic sensitization, the
dendritic cell (DCs), which processes
the antigenic molecules, absorbs
allergen. Once the allergen is taken up
by the dendritic cell, it is presented to
the naïve T helper cells. These inhaled
allergens stimulate cell proliferation of
T- helper type 2( Th2), subsequently
Th2 cytokines, interleukin( IL)-4, IL-5
and IL-13 production and release (Kudo
et al., 2013). Through the signaling of
IL-4 and IL-13 to eosinophils and B
cells, it promotes inflammation.
Remodeling by signaling of the
interleukins to fibroblasts, airway
smooth muscle, dendritic cells and
epithelial cells (Olin & Wechsler, 2014).
The leading candidates in conferring
susceptibility to asthma are the genes of
the IL-4-IL-13-IL-4R cytokine pathways.
Th2-dependent responses like
modulation of lymphocyte and
antigen-presenting cell function,
stimulation of IgE synthesis and
induction of allergic inflammation are
stimulated by these functionally
overlapping cytokines. Upon exposure
to antigen, allergic airway inflammation
will develop in the presence of IL-4R Arachidonic acid (AA) is a
chain expression in the resident airway precursor to two different metabolic
tissues. Events such as induction of pathways, cyclooxygenase (COX) and
airway hyperresponsiveness (AHR), lipoxygenase (LOX), for several families
mucin overproduction, and goblet cell of lipid mediators such as
metaplasia are reliant on the signaling of prostaglandins, thromboxane,
IL-4R signaling in the airway epithelial leukotrienes and lipoxins. These
cells. Moreover, atopy and asthma compounds, commonly referred to as
includes coding genetic polymorphisms eicosanoids, are important lipid
in the human IL-4R chain gene mediators involved in maintaining
(Tachdjian et al., 2009). normal homeostasis, blood pressure
regulation, reproduction and host
B cells play an important role in
defense. Of all the eicosanoids, the
atopic asthma since they produce IgE.
leukotrienes (LTs) play a significant role
Activating factors such as IL-5 and B
in the inflammatory process.
cell promotes the survival of B cell. B
Leukocytes, which are myeloid cells,
cells must bind to T cells under the
particularly the granulocytes,
influence of IL-4 or IL-13 (through CD40
macrophages/monocytes, and mast
and CD40 ligand respectively) to
cells are responsible for the synthesis of
produce IgE, usually within regional
the leukotrienes. Leukotriene is a
lymph nodes. High affinity FC receptors
product of AA metabolism through the
on basophils and mast cells are
5-lipoxygenase (5-LO) pathway. In a
primarily bound with the secreted IgE,
paracrine signaling, they exert their
and these cells will undergo
activity at nanomolar concentrations in
degranulation when crosslinked by
the local cellular milieu targeting cells
aeroallergens thereby causing the
implicated with receptors that transduce
release of their mediators. Mast cells
the intracellular signaling and
play an important role in the
specifically recognize LTs. Leukotrienes
pathogenesis of asthma. These cells are
can be classified into two major classes,
maintained on distinct mucosal surfaces
namely the dihydroxy acid leukotriene
by IL-9 and can be stimulated by binding
B4 (LTB4) and the
of the surface receptor c-kit to the stem
cysteinyl-leukotrienes (cys-LT). LTB4 is
cell factor, binding of tyrosine kinase or
a potent chemoattractant which has an
by crosslinking of IgE. Cysteinyl
activity on immune cells such as
leukotrienes, prostaglandins and
macrophages eosinophils and
histamine are released when the mast
neutrophils. And the cys-LT possesses
cells are activated (Olin & Wechsler,
a powerful spasmogenic effect,
2014).
especially in the respiratory tract. There
are two enzymes involved in the
committed step in the LTB4 and cys-LTs Air-way smooth muscle cell hyperplasia,
biosynthesis. Leukotriene A4 hydrolase eosinophilic inflammation, mucus
(LTA4H) catalyzes LTB4 biosynthesis hypersecretion, mucus gland
and leukotriene C4 synthase (LTC4S) hyperplasia, and collagen accumulation
catalyzes cys-LTs biosynthesis, both underneath the epithelial layer and in
from LTA4 which is the common the lung interstitium at leucocyte
substrate. The AA has both infiltration sites, are involved in the
pro-inflammatory and anti-inflammatory process of airway remodeling where
mediators. Examples of those included cysLTs may participate in. LTB4 is a
in the biosynthesis of anti-inflammatory factor that may contribute to the
and pro-resolving mediators are lipoxins narrowing of the airway by increasing
(LXs) such as LXA4, LXB4 and aspirin mucus secretion and production of local
triggered lipoxins. With LTA4 as an edema. But it has no bronchoconstrictor
intermediate and through the effect in asthmatic patients and in
consecutive actions of 12-LOX, healthy subjects. In neutrophilic
acetylated COX-2 or 5-LO via phenotype of asthma, LTB4 plays and
transcellular routes, lipoxins are formed important role because of its highly
(Di Gennaro & Haeggström, 2013). potent chemoattractant activity for
neutrophils that may lead to asthma
Leukotrienes such as cys-LTs
exacerbations (Montuschi &
and LTB4, which are derived from
Peters-Golden, 2010).
arachidonic acid via the 5-lipoxygenase
(5-LO) pathway, is a potent lipid According to numerous studies,
mediator. Different types of airway inflammation is a central key to
inflammatory cells implicate the the pathophysiology of asthma.
complete synthesis of CysLTs pathway Existence of chronic inflammation of the
and become stimulated during airway has been acknowledged for a
inflammation of the allergic airway. long time. The liberation of potent
Several cell types that lack the complete chemical mediators from inflammatory
synthetic pathway like endothelial cells cells resulting to chronic inflammation of
and platelets induce CysLTs through the airway, airway remodeling, which is
intercellular transfer mechanism from characterized by thickening of airway
the reactive mediator LTA4. LTs play a wall compartments that may lead to the
central role in asthma, but their progression and development of the
importance may vary in patients with disease (Kudo et al., 2013).
asthma. CysLTs causes diverse
ENZYME ROLE
pathophysiological responses that
contribute to asthma, while LTB4’s role Lipoxygenases belong to a class
in disease expression is more restricted. of non-heme iron containing
dioxygenases which catalyze the
addition of oxygen molecule to fatty
acids containing a cis,
cis-1,4-pentadiene system to yield an
unsaturated fatty acid hydroperoxides.
These LOX products have been found
to play an important role in various
disorders, including inflammation,
bronchial asthma, and tumor
angiogenesis. LOX is expressed in
platelet, neutrophils, eosinophils,
synovial fluid, colonic tissues,
monocytes, lungs, and bone marrow
cells in human tissues. 5-LOX found in
the neutrophils, converts the arachidonic
acid to to 5-hydroxy-6, 8, 11, 14-
eicosatetraenoic acid 5-HPETE.
5-HPETE is converted into a series of
leukotrienes and the tissue varies
depending on the nature of the final
product. Leukotrienes are allergic
reactions and inflammation mediators.
Leukotriene receptor antagonists and
5-LOX inhibitors are used in asthma
treatment (Sacan & Turhan, 2014).
MEMBRANE DESTABILIZATION
ROLE IN INFLAMMATION
Membrane destabilization of the
immune system cells is a physiologic
indicator in the pathogenesis of several
inflammatory diseases. The secretion of
the lysosomal constituents (bacterial
enzymes and proteases) of the immune
system cell system (mast cells,
neutrophils, basophils, eosinophils and
leukocytes) in response to a stimuli may
lead to progression of the damage thus
resulting in several physiologic
response which are prominent in the
usual signs of inflammation.(Sinbad et
al., 2017) Glucocorticoid receptor can
be inactivated by the released
lysosomal enzymes through
antagonizing the interaction between
glucocorticoids and GR and by altering
the size of the receptor complex.
Moreover, the release of lysosomes can
regulate this immune response by
degradation or secretion of inflammatory
cytokines. On a case-to-case basis,
lysosomes can either have
anti-inflammatory or pro-inflammatory
mechanisms. This negative and positive
regulation of inflammation by lysosomes
has been defined as “flexible” and is
significant in maintaining this balance.
However, lysosomes contain a
substantial number of pro-inflammatory
membrane protein which acts as targets
for the discovery of new drugs.
Lysosomes contribute to the
progression of autoimmune diseases
through the increase production of
autoantigens by the lysosomal
enzymes. Cathepsins (also known as
lysosomal proteases) were recognized
to have an important role in the
pathogenesis of some autoimmune
diseases namely rheumatoid arthritis
(RA), psoriasis, systemic lupus
erythematosus (SLE) and multiple
sclerosis (MS) (Ge, Li, Gao, & Cao,
2015). In preventing initiation of
inflammation, limiting the release of
lysosomal constituents by stabilizing the
lysosomal membrane is significantly
evident. HRBC membrane stabilization
has been introduced as a technique to
observe anti-inflammatory for it has
been analogous to the lysosomal
membrane in which the component can
modify the stabilization of the said
membrane. NSAIDS stabilize lysosomal
membranes or may inhibit the effects of
these lysosomal enzymes.
(Leelaprakash & Mohan Dass, 2011).
TREATMENT
CORTICOSTEROIDS
The most effective
anti-inflammatory therapy for asthma
are inhaled corticosteroids, these drugs
works in almost every patient. But then,
inhaled corticosteroids have the
potential systemic side effects because
of its lungs absorption which has placed
an effort for the researchers to find safer
ICs with reduced absorption from the
lungs and oral bioavailability or
circulation inactivation (P. Barnes,
2012). Down regulation arises by the
pro-inflammatory proteins and the
asthma-induced structural changes
(airway remodeling) are reversed with
the increased vascularity of the
bronchial wall (Hossny et al., 2016).
Adverse effects associated with
corticosteroid are reportedly Metabolic
syndrome, osteoporosis Hypertension,
Obesity, Type 2 Diabetes Mellitus,
Cataracts, Avascular Necrosis,
Dyslipidemia, Gastrointestinal
Ulcers/Bleeds, fractures, Tuberculosis
and Glaucoma in relation to the
published studies about asthma and
other conditions. These adverse effects
are considered chronic conditions
because of its development over time.
Acute adverse effects (e.g.,
gastrointestinal ulcers/bleed) may also
be present with its long-term use
(Sullivan et al., 2018).
BETA AGONIST
β2-Agonists are effective
bronchodilators mainly because of their
ability to relax airway smooth muscles
(ASM). They initiate a signaling effect
that arises to a several events leading to
the relaxation of ASM by binding to the
active site of β2-adrenoceptors on ASM.
Some differences exist between
β2-agonists. The inhaled traditional
short-acting β2-agonists, albuterol,
terbutaline and fenoterol afford rapid
symptom relief and short- term
prophylactic protection against
bronchoconstriction induced by exercise
or other stimuli. The β2-agonists,
formoterol and salmeterol comprise an
important advance due to its twice-daily
administration. Significant clinical
benefits are offered because of their
bronchodilating effect and long-term
improvement in lung functions to
patients with asthma (Cazzola et al.,
2013).
β2-agonists have an acceptable
safety profile. But it possesses adverse
effects such as palpitations, increased
heart rate, transient decrease in PaO2
and tremor. β2-adrenoceptors
desensitization during the first few days
of treatment is responsible for the
observed tenacity most of these adverse
effects. Nonetheless β2-agonists
bronchoprotective effects can also
induce tolerance and reduce the
bronchodilator sensitivity to them
(Cazzola et al., 2013).
Short-acting beta agonists has a short
half-life confines them as maintenance
treatment. They are currently used as
rescue medications. In contrast,
sustained bronchodilation provided by
Long-acting beta agonists and
ultra-long-acting beta agonists are
incapable to influence to decline the
accelerated lung function characteristics
in chronic obstructive pulmonary
disease (COPD) (Cazzola et al., 2013).
The complementary actions of
corticosteroids with long-acting beta
agonists like salmeterol and formoterol
are now the most effective available
therapy for asthma. LABAs are
bronchodilators and important for
negating and relieving
bronchoconstriction which last for over
12 hours (P. J. Barnes, 2010).
5-LIPOXYGENASE INHIBITORS/
LEUKOTRIENE INHIBITOR
Zileuton the only approved
5-lipoxygenase inhibitor for the
prevention and treatment of asthma in
adults and children 12 years old and
above (Montuschi & Peters-Golden,
2010). Zileuton has various
disadvantages like hepatic toxicity and
its opposing pharmacokinetic profile
from its short half-life (Rossi et al.,
2010).
Zileuton is a benzothiophene
N-hydroxyurea and is known to interfere
with inflammation and allergic diseases
via inhibition of leukotriene (LT)
biosynthesis. Zileuton chelates the
active site of iron of the enzyme with its
weak reducing properties asserting that
it belongs to 5-lipoxygenase iron
ligand-type inhibitors. Knowing the
importance of leukotrienes in the airway
inflammation, this drug has been added
to provide a therapeutic option in the
therapy of persistent and chronic
asthma. However, the clinical use of
Zileuton was limited because of its liver
toxicity thus leading to the need of
monitoring the hepatic enzyme levels.
Nonetheless this hepatic injury caused
by Zileuton is not related to the inhibition
of 5-LOX direct toxic effect (Rossi et al.,
2010).
Leukotrienes (LTs), including
LTB4, cysteinyl LTs (CysLTs) has a role
in pathophysiology of asthma that is a
potent mediator of lipids. Increased
airway smooth muscle activity, airway
secretion and microvascular
permeability are the pathophysiological
effect of the CysLTs activation in
asthma. Montelukast, Zafirlukast and
Pranlukast are selective CysLTs1
receptor antagonist approved for
treatment of asthma (Montuschi &
Peters-Golden, 2010).
Leukotrienes are 5-lipoxygenase
products synthesized by mast cells,
eosinophils and other inflammatory cells
of arachidonic acid. LTB4 and cysteinyl
leukotrienes (cysLT: LTC4, LTD4, LTE4)
were both involved in asthma
pathogenesis. Potent
bronchoconstrictors like cysLT also
have a blood vessel, mucociliary
clearance and eosinophilic effect in
inflammation. LTB4 is a potent
chemoattractant for neutrophils and
eosinophils and a cell activator.
Leukotriene synthesis is inhibited by
5-lipoxygenase. CysLT1 receptors are
block by leukotriene receptor
antagonists on smooth muscle in the
airway and other cells. The
anti-inflammatory and bronchodilator
effects of these drugs are modest
(Marinho & Custovic, 2008).
The prominent hepatic enzyme
elevation incidence in Zileuton was not
seen in Montelukast and its shorter
half-life that requires an administration
of four times daily (Montuschi &
Peters-Golden, 2010).
OTHER PLANTS WITH ANTI-LOX ACT
Cordia Africana ​(Boraginaceae) a​
folkloric plant medicine is used in
inflammation and other related
infectious diseases and conditions​. The
stem and bark of ​Cordia Africana
anti-inflammatory activity was evaluated
using LOX-inhibitor screening assay kit
(Catalog No. ab133087, abcam, UK).
The extracts presented a
15-lipoxygenase inhibitory effect at a
certain level. 88.81% of ethyl acetate
leaf extract yield the inhibitory activity of
15-LOX inhibitor and the bark extract of
acetone with IC50 value of 41± 1.3
µg/mL was the highest (Isa, A. I., Saleh,
M. I. A., Abubakar, A., Dzoyem, J. P.,
Adebayo, S. A., Musa, I. Sani, U. F.,
Alexandra, 2016).
Fresh leaves of ​Bambusa
spinosa, Amaranthus viridis ,
Commelina diffusa, Eclipta alba,
Crataeva religiosa, Euphorbia hirta,
Isotoma longiflora, Monochoria
vaginalis, Plumeria rubra, , Pistia
stratiotes a ​ nd Premna odorata was
assayed with 15-lipoxygensae inhibitor
activity. 51.3% of inhibition ​from C.
diffusa and 48.5% from ​E. hirta of LOX
inhibition activity showed the highest
effects at a concentration of 167µg/ml.
On the other hand, not all samples
exhibited higher activity compared with
quercetin the positive control at 62.4%
at 17µg/ml concentration. The traditional
use of these plant extracts as
respiratory remedy in inflammation was
validated via the spectrophotometric
assay related to oxygen and
polyunsaturated fatty acid
1,4-diene-type structure of 15-LOX
reaction. Flavonoids and terpenoids
contents were both positive in ​C. diffusa
and ​E. hirta.​ (D. Paguigan &
Chichioco-Hernandez, 2014)
Studied natural compounds comprises low and medium altitudes in thickets and
isolated flavonoids from orange peels, secondary forests.(Gupta & Kaur,
and from the leaves of ​Orthosiphon 2015)(Jamkhande et al., 2013)
spicatus​. The leaves and seeds extract
of ​Coriandrum sativum correlatedly
showed its effect because of its phenolic
content (D. Paguigan &
Chichioco-Hernandez, 2014)

ACTIVITIES OF FLAVONOIDS

Flavonoids are plant extracted PLANT TAXONOMY

products and belong to an essential
classification of natural products; they
belong in a particular class of secondary
plant metabolites with a polyphenolic
structure commonly found in certain
drinks, fruits, and vegetables. There are
several subgroups of flavonoids,
including flavones, chalcones,
isoflavones, and flavonols. Flavonoids
possess anti-inflammatory, antioxidant,
anti-carcinogenic and anti-mutagenic
properties coupled with their ability to
regulate key cellular enzyme functions
such as cyclooxygenase (COX),
xanthine oxidase (XO), lipoxygenase
and phosphoinositide 3-kinase. FIGURE 1. ​Cordia dichotoma t​ ree
(Panche, Diwan, & Chandra, 2016) Retrieved from:
FAMILY OF CORDIA DICHOTOMA https://sylviatramos.blog/

The genus ​Cordia b​ elongs to the Kingdom: Plantae

Boraginaceae family, with around 300 Division: Magnoliophyta
species worldwide, mainly in the warmer
regions of the world. The species is Class: Dicotyledons
common in the Philippines and found in
 Subclass: Astaridae
Order: Lamiales
Family: Boraginaceae
Genus: Cordia
Fragrant: Manjack
Species: C. dichotoma
DESCRIPTION OF CORDIA
DICHOTOMA
Boraginaceae is a family of a
small to moderate deciduous tree with a
short bole, short crooked trunk and
spreading crown. The stem bark is
smooth and longitudinally wrinkled in
grayish brown. Leaves are simple,
completely and slightly dented,
elliptical-lanceolate to wide ovate with a
round and cordate base. The flowers
are short, bisexual and white to pink and
appear in loose cymes of corymbose.
Fruits with sticky flesh mass are edible.
It is a yellow or pinkish-yellow glistening
globose or ovoid drupe sitting in an
enlarged calyx like a saucer. It becomes
black on maturation and the pulp
becomes visible (Hussain, & Kakoti,
2013).
PHYTOCHEMICAL PRESENT AND
PHARMACOLOGIC STUDIES
The presence of pyrrolizidine
alkaloids, coumarins, flavonoids,
terpenes and sterols was demonstrated
by chemical screening of both the
leaves and fruit. Four flavonoid
glycosides (robinin, rutin, rutoside,
datiscoside and hesperidine) and two
phenolic derivatives (chlorogenic acid
and caffeic acid) have been isolated
from fruits and leaves. The fruits have
astringent, anthelminthic, diuretic,
demulcent and expectorant activity. The
fruit’s mucilage treats cough and other
chest complaints. It is also used to treat
uterine disorders and urethra. The
powder form the fruit kernels are mixed
with oil to heal tinea. The plant is also
used as laxative and diuretic. The
leaves are used as tonic, stimulant, and
febrifuge. In cough and cold, leaf
decoction is used. The leaves are also
used for headache relief and ulcer
treatment. The traditional use of ​C.
dichotoma ​G. Forst leaves methanol
extract as analgesic, antipyretic, and
anti-inflammatory drug is also evident.
(Gupta & Kaur, 2015).
BIBLIOGRAPHY
Bag, G. C., & Devi, P. G. (2015).
Full-Text, ​30(​ 28), 154–159.
Barnes, P. (2012). New Drugs for
Asthma. ​Seminars in Respiratory
and Critical Care Medicine​, ​33​(06),
685–694.
https://doi.org/10.1055/s-0032-1326
965
Barnes, P. J. (2010). New therapies for
asthma: Is there any progress?
 Trends in Pharmacological
Sciences,​ ​31(​ 7), 335–343.
https://doi.org/10.1016/j.tips.2010.0
4.009
Cazzola, M., Page, C. P., Rogliani, P., &
Matera, M. G. (2013). β 2 -Agonist
therapy in lung disease, (C), 1–21.
https://doi.org/10.1164/rccm.20120
9-1739PP
Chippada, S. C., Volluri, S. S., Bammidi,
S. R., & Vangalapati, M. (2011). In
vitro anti inflammatory activity of
methanolic extract of centella
asiatica by HRBC membrane
stabilisation. ​Rasayan Journal of
Chemistry​, ​4(​ 2), 457–460.
https://doi.org/10.13005/bbra/867
D. Paguigan, N., &
Chichioco-Hernandez, C. L. (2014).
15-Lipoxygenase inhibition of
selected Philippine medicinal
plants. ​Pharmacognosy Journal,​
6​(1), 43–46.
https://doi.org/10.5530/pj.2014.1.7
Di Gennaro, A., & Haeggström, J. Z.
(2013). Targeting leukotriene B4 in
inflammation. ​Expert Opinion on
Therapeutic Targets,​ ​4,​ 1–15.
https://doi.org/10.1517/14728222.2
013.843671
Ge, W., Li, D., Gao, Y., & Cao, X.
(2015). The Roles of Lysosomes in
Inflammation and Autoimmune
Diseases. ​International Reviews of
Immunology​, ​34​(5), 415–431.
https://doi.org/10.3109/08830185.2
014.936587
Gupta, R., & Kaur, J. (2015). Evaluation
of analgesic, antipyretic and
anti-inflammatory activity on Cordia
dichotoma G. Forst. Leaf.
Pharmacognosy Research.​
https://doi.org/10.4103/0974-8490.1
47227
Hatware, K. V., Sharma, S., Patil, K.,
Shete, M., Karri, S., & Gupta, G.
(2018). Evidence for
gastroprotective, anti-inflammatory
and antioxidant potential of
methanolic extract of Cordia
dichotoma leaves on indomethacin
and stress induced gastric lesions
in Wistar rats. ​Biomedicine and
Pharmacotherapy.​
https://doi.org/10.1016/j.biopha.201
8.04.007
Hossny, E., Rosario, N., Lee, B. W.,
Singh, M., El-Ghoneimy, D., Soh, J.
Y., & Le Souef, P. (2016). The use
of inhaled corticosteroids in
pediatric asthma: Update. ​World
Allergy Organization Journal,​ ​9(​ 1),
1–24.
https://doi.org/10.1186/s40413-016-
0117-0
Isa, A. I., Saleh, M. I. A., Abubakar, A.,
Dzoyem, J. P., Adebayo, S. A.,
Musa, I. Sani, U. F., Alexandra, D.
P. (2016). Evaluation of
anti-inflammatory, antibacterial and
cytotoxic activities of Cordia
Africana leaf and stem bark
extracts. ​Bayero J Pure Applied
Scienc​, ​9(​ 1), 228–235.
Jambora, A. (2018).
Prevalence-By-WHO. ​Philippine
Daily Inquirer​. Retrieved from
https://lifestyle.inquirer.net/293087/
ph-among-top-10-countries-asthma
-mortality/
Jamkhande, P. G., Barde, S. R.,
Patwekar, S. L., & Tidke, P. S.
 (2013). Plant profile,
phytochemistry and pharmacology
of Cordia dichotoma (Indian cherry):
A review. ​Asian Pacific Journal of
Tropical Biomedicine​.
https://doi.org/10.1016/S2221-1691
(13)60194-X
Kudo, M., Ishigatsubo, Y., & Aoki, I.
(2013). Pathology of asthma.
Frontiers in Microbiology,​ ​4​(SEP),
1–16.
https://doi.org/10.3389/fmicb.2013.0
0263
Lambrecht, B. N., & Hammad, H.
(2015). The immunology of asthma.
Nature Immunology,​ ​16(​ 1), 45–56.
https://doi.org/10.1038/ni.3049
Leelaprakash, G., & Mohan Dass, S.
(2011). Invitro anti-inflammatory
activity of methanol extract of
enicostemma axillare. ​International
Journal of Drug Development and
Research,​ ​3​(3), 189–196.
Linn, S. (2013). Research Article
Estimation of Total Flavonoids
Content ( Tfc ) and Anti Oxidant
Activities of Methanolic Whole Plant
Extract of Biophytum, ​3(​ 4), 33–37.
https://doi.org/10.22270/jddt.v3i4.54
6
Marinho, S., & Custovic, A. (2008). ​40 –
Management of the asthmatic
patient.​ ​Clinical Immunology​ (Third
Edit). Elsevier.
https://doi.org/10.1016/B978-0-323-
04404-2.10040-5
Martín-Rodríguez, A. J., Ticona, J. C.,
Jiménez, I. A., Flores, N.,
Fernández, J. J., & Bazzocchi, I. L.
(2015). Flavonoids from Piper
delineatum modulate
quorum-sensing-regulated
phenotypes in Vibrio harveyi.
Phytochemistry,​ ​117​, 98–106.
https://doi.org/10.1016/j.phytochem.
2015.06.006
Maslan, J., & Mims, J. W. (2014). What
is asthma? Pathophysiology,
demographics, and health care
costs. ​Otolaryngologic Clinics of
North America​, ​47​(1), 13–22.
https://doi.org/10.1016/j.otc.2013.09
.010
Mims, J. W. (2015). Asthma: definitions
and pathophysiology. ​International
Forum of Allergy & Rhinology​,
5​(S1), S2–S6.
https://doi.org/10.1002/alr.21609
Montuschi, P., & Peters-Golden, M. L.
(2010). Leukotriene modifiers for
asthma treatment. ​Clinical and
Experimental Allergy,​ ​40(​ 12),
1732–1741.
https://doi.org/10.1111/j.1365-2222.
2010.03630.x
Olin, J. T., & Wechsler, M. E. (2014).
Asthma: pathogenesis and novel
drugs for treatment. ​BMJ,​
349​(nov24 8), g5517–g5517.
https://doi.org/10.1136/bmj.g5517
Panche, A. N., Diwan, A. D., & Chandra,
S. R. (2016). Flavonoids: An
overview. ​Journal of Nutritional
Science,​ ​5​.
https://doi.org/10.1017/jns.2016.41
Rossi, A., Pergola, C., Koeberle, A.,
Hoffmann, M., Dehm, F., Bramanti,
P., … Sautebin, L. (2010). The
5-lipoxygenase inhibitor, zileuton,
suppresses prostaglandin
biosynthesis by inhibition of
arachidonic acid release in
 macrophages. ​British Journal of
Pharmacology​, ​161(​ 3), 555–570.
https://doi.org/10.1111/j.1476-5381.
2010.00930.x

Sacan, O., & Turhan, E. Y. (2014).

Lipoxygenase Inhibitory Activities of
Some Plant Extracts and Chemical
Compounds Bazı Bitki Ekstreleri ve
Kimyasal Bileşiklerin. ​Journal of
Biology,​ ​73(​ 2), 47–52.

Sinbad, O., Samuel, F., Adewale, A., &

Adedoyin, O. (2017). Evaluation of
Membrane Stabilizing, Proteinase
and Lipoxygenase Inhibitory
Activities of Ethanol Extract of Root
and Stem of Sphenocentrum
jollyanum Pierre. ​Journal of
Advances in Biology &
Biotechnology​, ​13​(1), 1–8.
https://doi.org/10.9734/JABB/2017/
34121

Sullivan, P. W., Ghushchyan, V. H.,

Globe, G., & Schatz, M. (2018).
Oral corticosteroid exposure and
adverse effects in asthmatic
patients. ​Journal of Allergy and
Clinical Immunology,​ ​141​(1),
110–116.e7.
https://doi.org/10.1016/j.jaci.2017.0
4.009

Tachdjian, R., Mathias, C., Al Khatib, S.,

Bryce, P. J., Kim, H. S., Blaeser, F.,
… Chatila, T. A. (2009).
Pathogenicity of a
disease-associated human IL-4
receptor allele in experimental
asthma. ​The Journal of
Experimental Medicine,​ ​206​(10),
2191–2204.
https://doi.org/10.1084/jem.200914
80