Experimental Evaluation of Anti-Inflammatory Activity of Barleria

prionitis Extract Using In Silico and In Vitro Models Targeting

Cyclooxygenase-2 (COX-2)

Abstract
Background: Inflammation is a key pathological process underlying several chronic diseases.
Cyclooxygenase-2 (COX-2) is a crucial enzyme responsible for prostaglandin synthesis and is a
validated therapeutic target. Synthetic anti-inflammatory drugs often produce adverse effects,
necessitating safer alternatives from natural sources.
Objective: This study aimed to investigate the anti-inflammatory potential of Barleria prionitis
extract using in silico molecular docking and in vitro protein denaturation assay targeting COX-2.
Methods: Methanolic extract of Barleria prionitis leaves was prepared using Soxhlet extraction.
Preliminary phytochemical screening, thin layer chromatography (TLC), and UV–visible
spectroscopy were conducted. Molecular docking of barlerin with COX-2 was performed using
AutoDock Vina. Anti-inflammatory activity was evaluated using the protein denaturation method.
Results: Phytochemical analysis confirmed the presence of flavonoids, glycosides, and phenolics.
TLC analysis revealed characteristic spots corresponding to iridoid glycosides. UV spectra showed
absorption maxima in the range of 230–280 nm. Docking studies demonstrated strong binding
affinity of barlerin with COX-2 (binding energy: −8.2 kcal/mol), forming stable hydrogen bond
interactions. The extract exhibited dose-dependent inhibition of protein denaturation with
significant activity comparable to diclofenac sodium.
Conclusion: The findings suggest that Barleria prionitis possesses significant anti-inflammatory
activity mediated through COX-2 inhibition and may serve as a promising natural therapeutic
candidate.
Keywords: Barleria prionitis, COX-2, anti-inflammatory, molecular docking, barlerin, protein
denaturation
1. Introduction
Inflammation is a body's host defence mechanism and an important immunological response that
allows the body to survive infection or damage while also maintaining tissue homeostasis under
hostile settings. Inflammation, according to contemporary thinking, is a beneficial response to a
disruption or sickness. Inflammation is a typical reaction to any unpleasant stimuli that poses a
hazard to the host, and it can range from a localised to a widespread response. (Medzhitov R. [Link]
2010). In other terms, "inflammation is the body's main and complicated response to infection
following tissue damage."

Inflammation's role as a healing, restorative process, as well as its aggressive role, is now more
understood. However, there appears to be no remission in some cases, and a chronic state of
inflammation develops, which may last the rest of the person's life. Rheumatoid arthritis,
osteoarthritis, inflammatory bowel disease, retinitis, multiple sclerosis, psoriasis, and
atherosclerosis are examples of inflammatory illnesses. To address this issue, a variety of safe and
effective anti-inflammatory medicines, such as aspirin and other nonsteroidal anti inflammatory,
are available, with many more medications under development. Anti inflammatory agents are
those substances that help to lessen the inflammatory response. (Dinar Ello C. [Link] 2010)
1.1 Mechanism of Inflammation

Figure 1
Inflammation is the body's localised defensive response to allergic or chemical irritation, damage,
and/or infection. Pain, heat, redness, swelling, and loss of function are all signs of inflammation,
which are caused by dilatation of blood vessels, which increases blood flow, and enlarged
intracellular spaces, which allows leukocytes, protein, and fluids to enter the inflamed areas.
(Ruslan Medzhitov [Link] 2008; Parham P [Link] 2000)
Understanding the role of chemical mediators of inflammation is crucial. These mediators are
chemicals produced by cells such as mast cells, platelets, neutrophils, and monocytes/macrophages
and released as plasma proteins. They are brought on by allergic or chemical irritants, as well as
injuries and infections. These mediators, which are known as proinflammatory basic factors, affect
the intensity of inflammation depending on the length of the damage. These chemicals bind to
particular target receptors on cells, increasing vascular permeability, neutrophil chemotaxis,
smooth muscle contraction, direct enzymatic activity, pain, and/or mediating oxidative damage.
(J. W. Coleman et .al 2002)

Nitric oxide, prostaglandins, leukotrienes, histamine, serotonin, and cytokines are all examples of
chemical mediators. Although some of the cytokines produced (IL-3-4, -5, -6, 10, -13) are good
since they work as anti-inflammatory mediators within the cells, others are harmful. (V. Naidoo
[Link] 2007)

The inflammatory process involves several routes, including prostaglandin synthesis, interleukin
or other chemo toxin production, sticky protein receptor activity, and platelet activating factors.
All of these substances can serve as chemotactic agonists. Inflammation begins when
phospholipase A hydrolyses membrane phospholipids into arachidonic acid, which serves as a
substrate for cyclooxygenase and lipoxygenase enzymes, producing prostaglandins PGE2, PGH2,
and leukotrienes such as LTC4, LTB4, and others. Several cytokines, including interleukin-1 (IL-
1) and tumour necrosis factor-a, are also important in directing the inflammatory process (TNF-
a). The physiologic reactions to bacterial lipopolysaccharide (LPS, often known as endotoxin) are
mediated by IL-1 and TNF. (Medzhitov R. [Link] 2008)

Monocytes and macrophages, as well as adipocytes and other cells, produce them. They mediate
and enhance inflammation by inducing gene expression and protein synthesis in a range of cells in
collaboration with each other and numerous cytokines and growth factors (including IL-8 and
granulocyte-macrophage colony-stimulating factor). The production of prostaglandin (PGE2) or
prostacyclin (PGI2) increases blood flow and blood vessel permeability by helping in the release
of nitric oxide from endothelium-derived releasing factor, which causes vasodilation and aids in
the adhesion of platelets and other chemo toxin (bradykinin, histamine)

While LTB4 is a powerful chemotactic agent for polymorphonuclear leukocytes, eosinophils, and
monocytes, LTs are typically pro-inflammatory.
LTB4 induces the aggregation of polymorphonuclear leukocytes, as well as degranulation and the
production of superoxide, at greater doses. LTB4 enhances neutrophil adherence to vascular
endothelial cells and trans-endothelial migration, as well as the production of proinflammatory
cytokines by macrophages and lymphocytes. (Medzhitov R [Link] 2008; Dalgleish AG [Link] 2002;
Villarreal G [Link] 2000)

1.2 Types of inflammation:

Acute inflammation- Acute inflammation develops quickly, generally within minutes or hours of
tissue damage, and is marked by redness, heat, and oedema. (Toth et. al. 2004)
It's a quick procedure. The exudation of fluids10 and plasma proteins, as well as the migration of
leukocytes, particularly neutrophils, into the damaged region, define it. This initial inflammatory
reaction is beneficial to the defence mechanism's goal of eradicating germs, viruses, and parasites
while allowing wound healing to proceed.
Chronic inflammation - Chronic inflammation is marked by fibrosis and tissue necrosis, as well
as the presence of lymphocytes and macrophages in the histology. Chronic inflammation promotes
the onset of degenerative diseases such as rheumatoid arthritis, atherosclerosis, heart disease,
Alzheimer's disease, asthma, acquired immunodeficiency syndrome (AIDS), cancer, congestive
heart failure, multiple sclerosis, diabetes, infections, gout, IBD-inflammatory bowel disease,
ageing, and other neurodegenerative CNS depressions. Chronic inflammation has also been linked
to muscular atrophy as people age. which are linked to immunopathological factors, which appear
to be important in the start of the disease. (Dalgleish AG [Link] 2002) . For the treatment of various
inflammatory condtions , NSAIDS are being used due to the adverse side effects associated with
prolonged use of synthetic anti-inflammatory drugs like Ibuprofen , aspirin , diclofenac etc. there
is a growing interest in exploring natural alternatives . historically, managing inflammatory
conditions has heavily relied on traditional herbal medicine. Among the flora utilized for this
purpose, Barleria prionitis stands out as a highly valued shrub . In the ongoing search for safer,
naturally derived anti-inflammatory agents, researchers have increasingly turned to species with
rich phytochemical profiles. Barleria prionitis, a widely distributed medicinal plant, has emerged
as a key subject of interest...

1.3 Limitations of Conventional Anti-Inflammatory Drugs (NSAIDs)

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammation due to
their ability to inhibit COX enzymes. However, conventional NSAIDs are non-selective and inhibit both
COX-1 and COX-2, leading to undesirable side effects such as gastrointestinal irritation, ulceration, renal
toxicity, and increased cardiovascular risk. Although selective COX-2 inhibitors have been developed to
reduce gastrointestinal complications, their prolonged use has been associated with cardiovascular adverse
effects. These limitations highlight the need to explore safer and more effective anti-inflammatory agents,
particularly those derived from natural sources.
1.4Rationale

Despite the availability of various synthetic anti-inflammatory drugs, their associated side effects limit their
long-term use. Natural products offer a safer alternative due to their lower toxicity and multiple mechanisms
of action. Although Barleria prionitis has been traditionally used for inflammatory conditions, Therefore,
this study aims to investigate its anti-inflammatory activity Barleria prionitis in the treatment of
inflammatory disorders. By integrating phytochemical analysis, molecular docking, and in vitro evaluation,
the study explores the potential of this plant as a natural and safer alternative to conventional anti-
inflammatory drugs.
 [Link]

Figure 2: Barleria prionitis

Brief overview of Barleria Prionitis
As the world's greatest producer of medicinal plants, India is often regarded as the world's
botanical garden. Plants are the foundation of all life on Earth, and they are an essential resource
for human survival as raw medicine, food, and fuel.

According to the WHO, traditional medicine is used by more than 80% of the world's population
for health care. (Navneet [Link] 2012). Traditional plant-based remedies have long been utilised in
most regions of the globe, and their application in the treatment of microbial illnesses has piqued
the interest of a number of researchers. (Bhavnani SM [Link] 2000). Plantderived compounds have
lately attracted a lot of attention due to their diverse uses. Following up on ethnos medicinal use
of the plants, it was discovered that 14-28 percent of higher plant species are employed for
medicinal reasons and that 74 percent of pharmacologically active phytochemicals components
are disclosed. (Ahmed S [Link] 2012). In the recent several decades, there has been a significant
advancement in the development and marketing of plant-based pharmaceuticals, with a growing
emphasis on herbal medicines. (Tyler VE [Link] 1997).

1.5.1Distribution

B. prionitis is found in hotter places of India and is often used as a hedge plant in gardens. (Khare
CP [Link] 2007). Andaman and Nicobar Islands, Andhra Pradesh, Assam, Bihar, Chhattisgarh,
Delhi, Diu and Daman, Goa, Gujarat, Jharkhand, Karnataka, Kerala, Laccadive and Maldives
Islands, Madhya Pradesh, Maharashtra, Orissa, Puducherry, Rajasthan, Tamil Nadu, Uttarakhand,
Uttar Pradesh, and West Bengal are some of the states where it can be found. (Chari Andy CM
[Link] 1999)

1.5.2Habitat

The perennial ayurvedic herb Barleria prionitis Linn. (Acanthaceae) is widely distributed in
Africa, India, Sri Lanka, and tropical Asia. Kuranta, kurantaka, kuranda, kurandaka, sahachara,
and shairiya are some of the Ayurvedic names for it. It's also known as piyaabasaa, jhinti, and
ketsariyaa in traditional medicine. Because of its anti-odontalgic properties, it is also known as
'vajradanti’. (Khare CP [Link] 2007

1.5.2Taxonomy

Kingdom – Plantae

Division – Magnoliophyta

Class – Magnoliopsida

Order – Scrophulariales

Family – Acathaceae

Genus – Baleria

Species – prionitis

1.5.3Morphology

B. prionitis Linn. is a tall, bushy shrub that can reach a height of 1-2 metres. They have 2-4 long,
sharp axillary spines that are around 11 mm long. The stems are tall, glabrous, and densely
branched, with cylindrical and tapering branchlets. B. prionitis is a yellow shrub with yellow
blooms. During the months of August to October, the plant blooms. (Kamble et al.

2007)Flowers are sessile in lower axils, solitary in upper axils, and spicate in upper axils. Flowers
are around 3-4 cm long, broad, and tubular. Fruits are ovoid in form and have a capsular shape.
The elliptic leaves are 3-10 cm long and 1.5-4 cm broad, with 5- 20 mm long spines. The stems
have a pale tan or grey colour and are stiff, spherical, cylindrical, and glabrous. (Sharma P [Link]
2013)
Smooth, opposite leaves are ovate-elliptic to obovate, acuminate, tapering to base, bristletipped,
and 6-15 cm long and 4-6 cm broad. Petioles range in length from 0.5 to 3 cm. The ovoid fruit
capsule is 1.5-2 cm length and 0.6-0.8 cm broad, with two seeds. The seeds are oval-oblong and
are around 7.4-8.5x6-6.8 mm. They are coated with silky copper-brown woolly hairs and measure
about 7.4-8.5x6-6.8 mm. B. prionitis seeds are flattened and coated with tangled hairs, measuring
about 8 mm long and 5 mm broad. Bracts are bristle-tipped, acute, linear-lanceolate, foliaceous,
and approximately 1-1.5 cm long and 0.2-0.8 cm broad. The petal is 1.5 cm long and beautiful
golden yellow in colour, pubescent on the exterior and glabrous on the inside. Two fertile stamens
and two staminode stamens make up the stamens.

While the fertile stamens are directed away from the corolla tube, the staminode stamens are quite
short. The filaments are hairy, glandular-pubescent, and yellowish in colour. They are around 2-
2.5 cm long and hairy. The anthers are 3 mm long and golden in colour. (Kamble et al. 2007)
Traditional uses of Barleria Prionitis in India

Plant’s part Disorder Application mode

 Skin diseases Crushed leaves bare given to apply on skins.

Scabies Paste form of fresh leaves

Catarrhal affections of Applied as extract.

children

Wound Crushed form directly applied

Whooping cough Juice form directly

Whole plant Mouth ulcer Chewed and sap is swallowed.

Greying of hair Oil extract is given

Gout Prepared oil is used externally

Stem Dropsy and liver congestion Power with cow milk, juice of bark directly

Root Fever Directly power is taken to cure.

Boils and glandular swelling Paste form is directly applied.

Snakebite Decoction is taken orally

Seed Edema Paste is taken daily once

Shoot Asthma, whooping cough Used by formulation, paste prepared with

honey.

PHYTOCHEMISTRY

Preliminary phytochemical examination of B. prionitis whole plant hydro-methanolic extract

revealed the presence of glycosides, saponins, flavonoids, steroids, and tannins. Several
phytochemicals have been extracted from the ethanolic extract of B. prionitis, including
balarenone, Pipataline, Lupeol, Prioniside A, Prioniside B, and Prioniside C.
Several glycosides have been extracted from the aerial portions, including barlerinoside,
Verbascoside, Shanzhiside methyl ester, 6-O-trans-p-coumaroyl-8-O-acetylshanzhiside
methylester, barlerin, Acetylbarlerin, 7- methoxydiderroside, and Lupulinoside.

This plant contains two anthraquinone derivatives, which have been identified as 1,8, dihydroxy-
2,7-dimethyl3,6-dimethoxy anthraquinone and 1,3,6,8-tetra methoxy-2,7-dimethyl

anthraquinone. Scutellarein, melilotic acid, syringic acid, vanillic acid, p-hydroxybenzoic acid,
and 6- hydroxyflavones have all been found in the leaves.

Other phytochemicals found in B. prionitis include luteolin-7-O—D-glucoside, -sitosterol,

Scutellarein 7- neohesperidoside, apigenin 7-O-glucoside, 13, 14-seco-stigmasta-5, 14-diene3-a-
ol
Structures of some selected phytoconstituents isolated from B. prionitis Linn.

Category Compound Structure Referenc e

Name
shanzhiside methyl OH 26
ester
O OH

O O

O OH 7-dihydroxy-7,4,5,6-
tetrahydroxy-

,4a,5,6,7,7a-
HO HO

(1S,4aS,5R,7
H
R,7a
R)-methyl 5, 4S,5S,6S)-3 an-
methyl-1-
2-yloxy)-1
((2S,3R,
nta[c]pyran-
tetrahydro-
2H-pyr
hexahydrocyc
lope

Acetylbarlerin

OO

H O O

O
 H O

HO O O

HO OH OH

(1S,4aR,5S,7S,7aR)-methyl 5,7-

diacetoxy-7-methyl-1-((2S,3R,4S,5S,6R)3,4,5-
trihydroxy-6-

(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)-

1,4a,5,6,7,7a-

hexahydrocyclopenta[c]pyran-4carboxylate
barlerin 28

OH

HO OH

HO O O

O O

OH O O

(1S,4aS,5R,7S,7aS)-methyl 7-acetoxy-5hydroxy-7-
methyl-1-((2S,3R,4S,5S,6R)3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)-
1,4a,5,6,7,7ahexahydrocyclopenta[c]pyran-4-
carboxylate

Verbascoside HO OH 26

HO OO

H O

HO H O O

HO HO
 OH O

OH OH

(E)-6-(3,4-
dihydroxyphenethoxy) 4-
-2,5-dihydroxyahydro-
(3,4,5-trihydroxy-6-methyl-
2H-an-3-yl 3-(3,4-
tetr pyran-2-yloxy)-
tetrahydro-2H-pyr
dihydroxyphenyl)acrylate

CHAPTER 2: REVIEW OF LITERATURE

The search for effective and safer anti-inflammatory agents has led to extensive research on medicinal
plants and their bioactive constituents. Inflammation is a complex physiological process involving multiple
signaling pathways and mediators, and therefore, compounds with multi-target activity are considered
highly beneficial.

Several studies have highlighted the importance of cyclooxygenase enzymes, particularly COX-2, in the
pathogenesis of inflammatory diseases. COX-2 is induced in response to inflammatory stimuli such as
cytokines and growth factors and plays a key role in the production of prostaglandins that mediate pain and
inflammation. Selective inhibition of COX-2 has been shown to reduce inflammation with fewer
gastrointestinal side effects compared to non-selective NSAIDs.

Medicinal plants have been widely investigated as potential sources of COX-2 inhibitors. Phytochemicals
such as flavonoids, phenolics, alkaloids, and glycosides have demonstrated significant anti-inflammatory
activity in various studies. These compounds act through different mechanisms, including inhibition of
enzyme activity, modulation of signaling pathways, and antioxidant effects.

Barleria prionitis has been the subject of several pharmacological investigations due to its traditional
medicinal use. Studies have reported its anti-inflammatory, analgesic, antimicrobial, and antioxidant
properties. The anti-inflammatory activity of Barleria prionitis has been attributed to the presence of
flavonoids and iridoid glycosides, which are known to inhibit inflammatory mediators and enzymes.
Barlerin, an iridoid glycoside isolated from Barleria prionitis, has gained particular attention due to its
pharmacological potential. Previous studies have demonstrated that barlerin exhibits antioxidant activity
and may modulate inflammatory pathways. However, detailed studies exploring its interaction with specific
molecular targets such as COX-2 are limited, highlighting the need for further investigation.

Molecular docking has emerged as a powerful tool in drug discovery, allowing researchers to predict the
binding affinity and interaction of small molecules with target proteins. Several studies have successfully
used docking techniques to identify natural compounds as potential COX-2 inhibitors. These studies
provide valuable insights into the molecular basis of anti-inflammatory activity and support the
development of new therapeutic agents.

In vitro assays such as protein denaturation and membrane stabilization are commonly used to evaluate the
anti-inflammatory activity of plant extracts. These assays provide a simple and reliable method for
screening potential anti-inflammatory agents and are widely used in pharmacological research.

Despite the growing body of evidence supporting the medicinal value of Barleria prionitis, there is still a
need for comprehensive studies that integrate phytochemical analysis, molecular modeling, and biological
evaluation. The present study aims to address this gap by providing a systematic evaluation of the anti-
inflammatory activity of Barleria prionitis with a focus on COX-2 inhibition.

3 MATERIALS AND METHODS

3.1 Chemicals and Reagents

All chemicals and reagents used in the study were of analytical grade. Ethanol, methanol, chloroform and
distilled water were procured from standard suppliers. Reagents required for phytochemical screening such
as Mayer’s reagent, ferric chloride, glacial acetic acid, and concentrated sulfuric acid were prepared freshly
according to standard procedures.

3.2 Plant Material Collection and Authentication

The whole plant of Barleria prionitis was collected during the flowering season from a natural habitat to
ensure maximum phytochemical content.

The collected plant material was washed thoroughly to remove adhering impurities and shade-dried at room
temperature (25–30°C) for approximately 10–14 days. The dried material was coarsely powdered using a
mechanical grinder and stored in airtight containers.

3.3 Preparation of Extract (Soxhlet Extraction)

The powdered plant material (250 g) was subjected to Soxhlet extraction using a hydroalcoholic solvent
system (ethanol:water, 70:30 v/v). The extraction was carried out for 6–8 hours until the solvent in the
siphon tube became colorless, indicating exhaustive extraction.

The extract was filtered using Whatman filter paper and concentrated using a water bath. The concentrated
extract was further dried to obtain a semi-solid mass. The percentage yield of the extract was calculated
and stored in a desiccator until further use.
3.4 Preliminary Phytochemical Screening

Qualitative phytochemical analysis was performed to detect the presence of bioactive constituents such as
alkaloids, flavonoids, glycosides, tannins, phenolic compounds, and saponins using standard chemical tests.

3.4.1 Test for Iridoid Glycosides (Keller–Killiani Test)

The extract was treated with glacial acetic acid containing ferric chloride and carefully layered with
concentrated sulfuric acid. The formation of a brown ring at the interface indicated the presence of
glycosides, while a bluish-green coloration confirmed iridoid glycosides such as barlerin.

3.4.2 Thin Layer Chromatography (TLC)

TLC analysis was performed using silica gel 60 F254 plates. The extract was dissolved in methanol and
applied as a spot on the plate. The mobile phase consisted of methanol:water (8:2 v/v).

The plate was developed in a saturated chamber, removed, dried, and observed under UV light at 254 nm
and 366 nm. The Rf value was calculated to identify the presence of barlerin and other phytoconstituents.

3.5 UV-Visible Spectroscopic Analysis

UV–visible spectroscopic analysis of the Barleria prionitis extract was performed to characterize the
presence of chromophoric phytoconstituents. A known quantity of the dried extract was dissolved in
methanol to prepare a stock solution (1 mg/mL), which was further diluted to obtain a clear working
solution. The solution was filtered using Whatman filter paper to remove particulate matter. Spectral
analysis was carried out using a UV–visible spectrophotometer with methanol as the blank. The absorbance
spectrum of the extract was recorded over the wavelength range of 200–400 nm using quartz cuvettes with
a 1 cm path length. The wavelengths corresponding to maximum absorbance (λmax) were noted and used
for qualitative assessment of the presence of conjugated systems and bioactive compounds such as
flavonoids, phenolics, and glycosides.

3.7 Molecular Docking Study (PyRx)

Molecular docking studies were performed using PyRx employing the AutoDock Vina algorithm to
evaluate the interaction between barlerin and the COX-2 enzyme. The three-dimensional structure of COX-
2 was obtained from the Protein Data Bank and prepared by removing water molecules, ligands, and adding
polar hydrogen atoms, followed by conversion into .pdbqt format.

The ligand, barlerin, was retrieved from a chemical database or drawn using ChemDraw and subjected to
energy minimization using the Universal Force Field (UFF) in PyRx. The optimized ligand was converted
into .pdbqt format with defined rotatable bonds.

A grid box was set around the active site residues (Arg120, Tyr355, Ser530, and His90) with appropriate
dimensions to cover the binding pocket. Docking simulations were carried out using AutoDock Vina,
generating multiple binding conformations with corresponding binding affinity values (kcal/mol).

The best docking pose was selected based on lowest binding energy and favorable interactions. Validation
of the docking protocol was performed by re-docking the native ligand and calculating RMSD values (<2.0
Å). Interaction analysis was carried out using Discovery Studio Visualizer to identify hydrogen bonding
and hydrophobic interactions.
3.8. Protein Denaturation Assay

The anti-inflammatory activity of the extract was evaluated using the egg albumin protein denaturation
method. Fresh hen’s egg albumin was used as the protein source.

The reaction mixture (total volume 5 mL) consisted of 0.2 mL egg albumin, 2.8 mL phosphate buffer
(pH 6.4), and 2.0 mL of test extract at different concentrations (e.g., 50–800 µg/mL). A standard drug
such as diclofenac sodium was used for comparison. The control contained distilled water instead of the
test sample.

The mixtures were incubated at 37°C for 15 minutes, followed by heating at 70°C for 5 minutes to induce
protein denaturation. After cooling to room temperature, the absorbance was measured at 660 nm using a
UV-visible spectrophotometer.

The percentage inhibition of protein denaturation was calculated using:

% Inhibition=Ac−AsAc×100\%\,\text{Inhibition} = \frac{A_c - A_s}{A_c} \times 100%Inhibition=AcAc

−As×100

where AcA_cAc represents the absorbance of the control and AsA_sAs represents the absorbance of the
test sample.

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Phytochemical Screening Results

The qualitative phytochemical analysis confirmed the presence of flavonoids, glycosides, tannins, and
phenolic compounds in the extract. The Keller–Killiani test specifically indicated the presence of iridoid
glycosides such as barlerin, which are known for their pharmacological activity.

These phytoconstituents are widely reported to possess anti-inflammatory and antioxidant properties,
suggesting that the extract has therapeutic potential.
 Figure 3

With reference to fig. 3 you can see that we got a reddish-brown ring at the junction of chloroform extract
and sulphuric acid , which indicates the presence of iridoid glycosides in the given sample extract .

4.2 TLC Analysis

The TLC profile showed distinct spots under UV light, confirming the presence of multiple
phytoconstituents. The Rf value corresponding to barlerin was consistent with reported values.
This confirms the successful extraction and presence of active compounds responsible for biological
activity.
 Figure 4

4.3 UV Spectroscopic Analysis

The UV spectrum showed absorption peaks in the range of 220–280 nm and 300–350 nm.
These peaks indicate the presence of conjugated systems such as flavonoids and glycosides, which
contribute to anti-inflammatory activity.

4.4 Molecular Docking Results

Barlerin exhibited strong binding affinity towards COX-2 with favorable docking score (e.g., around −8.5
kcal/mol). It formed hydrogen bonds with key residues such as Arg120 and Tyr355.
This suggests that barlerin can inhibit COX-2 enzyme activity, thereby reducing prostaglandin synthesis
and inflammation.m
4.5 Protein Denaturation Assay Results

The extract showed concentration-dependent inhibition of protein denaturation. At higher concentrations,

the activity was comparable to the standard drug.
The ability to inhibit protein denaturation indicates stabilization of protein structure and prevention of
inflammatory pr
CHAPTER 5: CONCLUSION

The present study was undertaken to evaluate the anti-inflammatory potential of Barleria prionitis using an
integrated approach involving phytochemical analysis, molecular docking, and in vitro assays targeting the
COX-2 enzyme. The findings of the study provide substantial evidence supporting the traditional use of
this medicinal plant in the treatment of inflammatory conditions.

The extraction of Barleria prionitis using Soxhlet apparatus with a hydroalcoholic solvent system resulted
in the efficient recovery of bioactive constituents. Preliminary phytochemical screening confirmed the
presence of flavonoids, phenolic compounds, tannins, and iridoid glycosides, including barlerin, which are
known to possess significant pharmacological activities.

Chromatographic and spectroscopic analyses, including TLC and UV-visible spectroscopy, further
validated the presence of these compounds and confirmed the quality of the extract. The detection of
characteristic absorption peaks and distinct chromatographic profiles indicated the presence of conjugated
systems and active phytoconstituents responsible for biological activity.

Molecular docking studies carried out using PyRx demonstrated that barlerin exhibits strong binding
affinity towards the COX-2 enzyme. The interaction of barlerin with key amino acid residues within the
active site suggests its potential to inhibit enzyme activity and reduce the synthesis of pro-inflammatory
mediators such as prostaglandins.

The in vitro protein denaturation assay revealed that the extract of Barleria prionitis possesses significant
anti-inflammatory activity, as evidenced by its ability to inhibit heat-induced protein denaturation in a
concentration-dependent manner. The observed activity was comparable to that of standard anti-
inflammatory drugs, indicating its therapeutic potential.

Overall, the results of this study suggest that Barleria prionitis exerts its anti-inflammatory effects through
a multi-target mechanism involving inhibition of COX-2, stabilization of protein structure, and possible
antioxidant activity. The integration of computational and experimental approaches provides a
comprehensive understanding of its pharmacological action.

However, further studies, including in vivo experiments and clinical trials, are necessary to fully establish
its safety, efficacy, and mechanism of action. The findings of the present study highlight the potential of
Barleria prionitis as a natural and safer alternative to conventional anti-inflammatory drugs and pave the
way for future research in the development of plant-based therapeutics.