EXPLORING THE ANTIMICRIBIAL AND ANTI INFLAMMATORY POTENCY OF AZDIRACHTA INDICA

Introduction

Medicinal plants have been used for thousands of years across various cultures to treat a wide range of
diseases. Their effectiveness in managing infections and inflammation has been acknowledged in both
traditional systems of medicine and modern pharmacological studies. Among these medicinal plants,
Azadirachta indica, commonly known as neem, holds a special place due to its wide spectrum of
therapeutic properties. Originating from the Indian subcontinent, neem has been historically utilized in
Ayurvedic, Unani, and Siddha medicine for its medicinal value. Every part of the neem tree, including the
leaves, bark, seeds, flowers, and oil, has been employed in treating various health disorders.

Infectious diseases caused by pathogenic microorganisms, such as bacteria, fungi, and viruses, are a
significant global health concern. With the increasing misuse and overuse of antibiotics in clinical
practice, the problem of antimicrobial resistance has grown sharply. Antibiotic resistance limits the
effectiveness of conventional drugs, leading to prolonged illness, higher medical costs, and an increased
risk of mortality. This has led researchers to investigate alternative sources of antimicrobial agents,
particularly those derived from natural sources like medicinal plants. The phytochemicals present in
these plants are thought to act through multiple mechanisms and may have lower chances of inducing
resistance compared to synthetic antibiotics.

Similarly, inflammation plays a critical role in the progression of many diseases. While acute
inflammation is a protective response to injury or infection, chronic inflammation can be detrimental and
is associated with conditions such as arthritis, cardiovascular diseases, diabetes, and even cancer.
Conventional anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs) and
corticosteroids, are effective but often come with side effects when used long term. These include
gastrointestinal ulcers, liver damage, and immunosuppression. Consequently, researchers are exploring
the anti-inflammatory properties of natural plant-based compounds, which may offer effective yet safer
alternatives.

Neem, or Azadirachta indica, is a fast-growing tree that thrives in tropical and semi-tropical climates. Its
importance in traditional medicine is supported by the presence of bioactive compounds like
azadirachtin, nimbin, nimbolide, quercetin, and various flavonoids and tannins. These compounds are
known to exhibit diverse pharmacological activities, including antibacterial, antifungal, antiviral, anti-
inflammatory, antioxidant, anticancer, and immunomodulatory effects. Neem leaves, in particular, are
rich in flavonoids and polyphenols that contribute to its antimicrobial and anti-inflammatory functions.
The plant is also known for its bitterness, which is attributed to the presence of terpenoids and
limonoids.

Multiple studies have highlighted the antimicrobial potential of Azadirachta indica. Neem extracts have
been shown to inhibit the growth of several pathogenic bacteria, including Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi. The antifungal activity of neem has also
been demonstrated against species like Candida albicans and Aspergillus niger. These effects are
believed to be due to the disruption of microbial cell membranes, inhibition of enzyme function, and
interference with DNA replication. Interestingly, neem also exhibits activity against antibiotic-resistant
strains, which is particularly valuable in the current era of multidrug resistance.
In terms of anti-inflammatory activity, neem’s effectiveness has been demonstrated in various in vivo
and in vitro models. Its bioactive components have been found to inhibit key inflammatory mediators
such as prostaglandins, cytokines, and reactive oxygen species. For example, studies have shown that
neem extract can suppress the production of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6),
and nitric oxide (NO), which are involved in chronic inflammation. Additionally, neem compounds have
been found to inhibit cyclooxygenase (COX) and lipoxygenase (LOX) pathways, which are targets of
common anti-inflammatory drugs. This suggests that neem may offer a natural alternative for managing
inflammatory conditions with fewer side effects.

Despite these promising findings, there is still a need for comprehensive scientific assessments that
standardize the methodology for evaluating the antimicrobial and anti-inflammatory activity of neem.
The variation in extraction methods, plant parts used, and testing conditions across different studies
makes it difficult to compare results and draw firm conclusions. Moreover, most studies have been
conducted in vitro, and there is limited data from in vivo or clinical research. Therefore, well-designed
studies are essential to confirm the therapeutic potential of neem, explore the mechanisms behind its
effects, and determine its safety profile in both humans and animals.

Furthermore, the synergistic potential of neem with conventional drugs also presents a valuable area of
study. Combining plant extracts with standard antibiotics or anti-inflammatory drugs could enhance their
efficacy and reduce required dosages, potentially lowering the risk of resistance and side effects.
Investigating such combinations may help in designing novel treatment strategies that integrate
traditional and modern medicine.

Another important aspect is the identification and isolation of specific compounds within neem
responsible for its therapeutic activities. Advanced techniques such as high-performance liquid
chromatography (HPLC), gas chromatography–mass spectrometry (GC-MS), and nuclear magnetic
resonance (NMR) spectroscopy are now widely available and can aid in profiling the phytochemical
constituents of neem. Understanding the structure-activity relationships of these compounds can help in
the development of new drug molecules derived from natural sources.

Moreover, the sustainability and accessibility of Azadirachta indica make it a suitable candidate for
community-level healthcare, especially in developing countries. Neem trees are widely available in
tropical regions and are relatively low-cost to maintain. The use of neem-based remedies could be
promoted as part of traditional healthcare practices, provided there is sufficient scientific validation of
their safety and effectiveness.

In recent years, the trend of incorporating herbal remedies into mainstream medicine has gained
traction, and the World Health Organization (WHO) has also emphasized the importance of validating
traditional medicine through scientific research. Azadirachta indica represents one such plant with the
potential to bridge the gap between traditional knowledge and modern pharmacology. By exploring its
antimicrobial and anti-inflammatory properties through rigorous scientific methods, this research aims
to contribute to the growing body of evidence supporting the use of medicinal plants in healthcare.

This study also holds significance in the context of increasing consumer preference for plant-based and
natural remedies. With growing awareness of the limitations and side effects of synthetic
pharmaceuticals, many people are turning to herbal alternatives for maintaining health and treating
minor ailments. Therefore, providing credible data on the pharmacological actions of neem could
influence public health policy, encourage further research, and promote the safe integration of herbal
medicines in clinical practice.

Given these considerations, the current study focuses on evaluating the antimicrobial and anti-
inflammatory properties of Azadirachta indica leaf extract using standardized laboratory methods. It
aims to assess the efficacy of neem extract against selected microbial strains and determine its effect on
inflammatory markers in experimental models. The outcomes of this research are expected to provide
scientific support for the traditional use of neem and highlight its potential as a natural therapeutic
agent.

Aim of the study:

Materials and Methods

In this study, a systematic and controlled methodology was followed to evaluate the antimicrobial and
anti-inflammatory properties of Azadirachta indica (neem) leaf extract. All experimental procedures
were conducted under standard laboratory conditions, ensuring the reliability and reproducibility of
results. The section below describes the collection of plant material, the extraction procedure, the
protocols followed for antimicrobial assessment, and the anti-inflammatory evaluation.

Collection of Plant Material

Fresh and mature leaves of Azadirachta indica were collected from healthy trees growing in unpolluted
areas. The trees were identified and authenticated based on their characteristic morphology such as
serrated leaf margins, pungent smell, and arrangement of compound leaves. The collection was done
during the early morning hours to avoid moisture retention and contamination. The leaves were carefully
removed from the branches using sterile scissors and transferred into clean, dry bags for transportation
to the laboratory.

Upon arrival at the laboratory, the leaves were washed thoroughly under running tap water to remove
dust, dirt, and other foreign particles. This was followed by rinsing with distilled water to eliminate
residual impurities. The clean leaves were then spread on clean trays and shade dried for about 7 to 10
days at room temperature. Shade drying was preferred over direct sunlight exposure to prevent
degradation of heat-sensitive phytochemicals. The dried leaves were then ground using a mechanical
grinder into a fine powder, sieved to ensure uniform particle size, and stored in airtight glass containers
under cool and dry conditions until further use.

Preparation of Ethanolic Extract

For the preparation of the neem extract, the powdered plant material was subjected to ethanolic
extraction using the cold maceration method. Approximately 100 grams of powdered neem leaves were
soaked in 500 mL of 95% ethanol in a clean conical flask. The mixture was placed on a rotary shaker and
agitated continuously at room temperature (25°C–28°C) for 48 to 72 hours. This method allows for the
gradual release of bioactive compounds into the solvent.
After the maceration period, the mixture was filtered using Whatman No.1 filter paper to remove the
solid residue. The filtrate, which contained the ethanol-soluble phytochemicals, was concentrated using
a rotary evaporator under reduced pressure to remove excess solvent. The resulting crude extract was
collected in a sterile glass container and stored at 4°C until it was used for biological testing. The yield of
the extract was recorded, and the concentrated extract was reconstituted in dimethyl sulfoxide (DMSO)
to achieve the required concentrations for the tests.

Antibacterial and Antifungal Activity

The antimicrobial potential of the neem extract was evaluated using the agar well diffusion method. This
method is widely employed due to its simplicity, reproducibility, and efficiency in determining the
inhibition zones around plant extracts.

Sterile nutrient agar (for bacterial strains) and Sabouraud Dextrose Agar (for fungal strains) were
prepared and poured into sterile Petri plates under aseptic conditions. Once the media solidified, test
organisms were inoculated evenly across the surface of the agar using a sterile cotton swab. Bacterial
strains tested included Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella
pneumoniae. For antifungal testing, Candida albicans and Aspergillus niger were used.

Wells of 6 mm diameter were made in the agar using a sterile cork borer. A fixed volume of the neem
extract (100 µL) at different concentrations was carefully introduced into each well. For control
comparison, standard antibiotics such as Amikacin and Penicillin were used as positive controls, while
DMSO served as the negative control to ensure the solvent had no antimicrobial activity of its own.

The plates were incubated at 37°C for 24 hours for bacterial strains and at 28°C for 48–72 hours for
fungal strains. After incubation, the diameter of the zone of inhibition around each well was measured in
millimeters using a digital caliper. Larger zones indicated higher antimicrobial activity. The results were
recorded, and each test was carried out in triplicate to ensure accuracy and reproducibility.

Anti-inflammatory Activity Assessment

The anti-inflammatory potential of the Azadirachta indica extract was studied using the protein
denaturation inhibition method, a widely accepted in vitro model for preliminary screening of anti-
inflammatory agents. Protein denaturation is a common process in inflammation, and substances that
prevent protein denaturation can be considered to possess anti-inflammatory properties.

In this method, egg albumin was used as the protein source. A reaction mixture containing 0.45 mL of
egg albumin, 2.8 mL of phosphate-buffered saline (PBS, pH 6.4), and 0.5 mL of neem extract solution at
various concentrations was prepared. The samples were incubated in a water bath at 37°C for 15
minutes and then heated at 70°C for 5 minutes to induce denaturation. After cooling, the absorbance of
the reaction mixtures was measured at 660 nm using a UV-Visible spectrophotometer.

The percentage inhibition of protein denaturation was calculated using the following formula:

% Inhibition = [(Abs_control - Abs_sample) / Abs_control] × 100

Where Abs_control is the absorbance of the negative control (DMSO + egg albumin without extract) and
Abs_sample is the absorbance of the test sample (extract + egg albumin). Diclofenac sodium was used as
a standard anti-inflammatory drug for comparison.
All tests were performed in triplicates, and the average values were recorded. The results were
statistically analyzed to determine the significance of differences between treated and control groups.

Statistical Analysis

All experimental data were analyzed using standard statistical software. The values were expressed as
mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by post hoc Tukey’s test
was used to determine the significance between groups. A p-value of less than 0.05 (p < 0.05) was
considered statistically significant. Graphical representation of the data was done using Microsoft Excel
to clearly illustrate differences in antimicrobial zones and anti-inflammatory effects.

Ethical Considerations

Since the study was conducted entirely using in vitro models, ethical clearance was not required.
However, all laboratory protocols were performed in accordance with institutional biosafety guidelines.
Proper disposal of biological waste, including microbial cultures and chemical reagents, was ensured to
avoid contamination and environmental hazards.

Summary of Methodological Rationale

The use of ethanol as an extraction solvent was based on its ability to dissolve a wide range of polar and
non-polar compounds. The agar well diffusion method provided a reliable and visual representation of
antimicrobial activity. The protein denaturation assay, while simple, effectively reflects the ability of test
compounds to stabilize biological macromolecules under stress, making it suitable for preliminary
screening of anti-inflammatory agents.

Results

The antimicrobial activity of the Azadirachta indica leaf extract was evaluated against a selected panel of
pathogenic microorganisms, including both bacteria and fungi. The microorganisms tested were Candida
albicans, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.
The assay was performed using the agar well diffusion method, and the zones of inhibition were carefully
measured after incubation. The figure provided (Figure 1) illustrates the clear zones of growth inhibition
around the wells containing different concentrations of A. indica extract in comparison with the control
and standard antibiotic discs.

In the case of the fungal strain Candida albicans, a visible zone of inhibition was produced around the
well containing the ethanolic extract of A. indica. This indicates that the bioactive components present in
the leaf extract were able to limit fungal growth. The inhibition zone was smaller compared to the
standard antifungal disc used as a positive control, yet the extract showed a marked reduction in fungal
colonies around the well. The result suggests that the plant extract possesses moderate antifungal
properties, which may be associated with the presence of terpenoids, flavonoids, or phenolic
compounds known to disrupt fungal cell membranes.

When tested against Escherichia coli, a Gram-negative bacterium often associated with gastrointestinal
infections and urinary tract infections, the extract again produced a distinct inhibition zone. The clarity of
the zone showed that the extract had an effect on bacterial growth. The diameter of the inhibition zone
was concentration dependent, with higher concentrations of the extract producing wider zones.
Compared with the standard antibiotic disc, the inhibition zone was smaller, but the activity was
significant enough to indicate that the plant constituents have antibacterial potential. This result is
consistent with traditional uses of neem in the treatment of infections and highlights its effect on
Gram-negative bacteria, which are often resistant to many commonly used antibiotics.

For Klebsiella pneumoniae, another Gram-negative bacterium known for causing respiratory infections,
pneumonia, and sepsis, the ethanolic leaf extract demonstrated clear antimicrobial activity. The
inhibition zone was evident around the well, although slightly narrower than the zone produced by the
standard antibiotic control. The pattern indicates that the bioactive compounds in neem are able to
penetrate and disturb the bacterial cell wall or interfere with essential metabolic pathways, thereby
reducing microbial proliferation. The result against K. pneumoniae is important, as this bacterium is often
involved in hospital-acquired infections where resistance to conventional drugs is increasing.

In the plate tested with Pseudomonas aeruginosa, the leaf extract again produced a measurable
inhibition zone. P. aeruginosa is well known for its high resistance to many antibiotics and is commonly
implicated in wound infections, burns, and chronic ulcers. The presence of a clear zone of inhibition
indicates that the neem extract has components that can affect even a highly resistant bacterium.
Although the zone size was smaller than that of the standard control, the activity is promising. The
consistency of inhibition across different bacteria points towards a broad spectrum of action.

For Staphylococcus aureus, a Gram-positive bacterium associated with skin infections, abscesses, and in
severe cases, bacteremia, the extract also showed a clear antimicrobial effect. A distinct zone of
inhibition surrounded the well containing the neem extract. The activity against S. aureus supports the
traditional application of neem paste on skin wounds and infected lesions. The result suggests that
certain constituents in the extract may disrupt cell wall synthesis or inhibit essential enzymes in S.
aureus, leading to growth arrest.

Across all tested microorganisms, the figure demonstrates that the size of the inhibition zones varied
according to the microorganism and the concentration of the extract. Generally, higher concentrations of
the extract produced larger zones, indicating a dose-dependent response. This pattern suggests that the
active phytochemicals are present in measurable quantities in the extract and that their activity can be
enhanced by increasing the amount applied. It also indicates that the extract preparation was effective in
extracting soluble bioactive components.

The results clearly show that the ethanolic extract of Azadirachta indica leaves possesses both
antibacterial and antifungal activities. Although the inhibition zones were not as large as those of
standard antibiotics, the consistent activity against multiple pathogens highlights the potential of neem
as a source of antimicrobial agents. In addition, the extract did not show any contamination or irregular
growth within the inhibition zones, indicating that the activity was genuine and not due to external
factors.

The antimicrobial effect observed may be attributed to the presence of bioactive compounds such as
azadirachtin, nimbidin, and various flavonoids and tannins in the leaves. These compounds are known to
interact with microbial cell membranes, leading to leakage of cellular components, inhibition of enzyme
activity, or disruption of DNA synthesis. Further phytochemical analysis and purification studies would
help to identify the specific constituents responsible for the observed activities.

The experimental outcomes support earlier literature reports that neem extracts are effective against a
variety of microorganisms. The data also emphasize the importance of using standardized extracts to
obtain reproducible and comparable results. The variation in inhibition zones between different
microorganisms shows that the activity spectrum of the extract is broad but not uniform, which is typical
for plant-based antimicrobials.

In the context of antimicrobial resistance, the present findings are important because they suggest an
alternative source of antimicrobial substances. Although the extract may not replace conventional
antibiotics immediately, it could serve as a supplementary treatment or be developed into a formulation
that enhances existing therapies. It also provides a basis for further studies aimed at isolating and
characterizing the active molecules, testing their mechanisms of action, and evaluating their safety
profiles.

The results from this antimicrobial assay are directly relevant to the overall aim of the research, which is
to assess both the antimicrobial and anti-inflammatory potential of Azadirachta indica. While this
section focuses on the antimicrobial activity, the observed broad-spectrum effects justify continued
investigation into its anti-inflammatory properties, as plant extracts often exert multiple biological
actions due to their complex mixture of phytochemicals.

In conclusion, the data presented in Figure 1 show that the ethanolic leaf extract of Azadirachta indica
exhibits measurable antimicrobial activity against C. albicans, E. coli, K. pneumoniae, P. aeruginosa, and
S. aureus. The zones of inhibition were consistently present, indicating that the extract contains
compounds capable of suppressing both bacterial and fungal growth. These findings provide
experimental evidence that supports the traditional use of neem in treating infectious conditions and
encourage further exploration of its bioactive components for the development of novel antimicrobial
agents.
The antimicrobial activity of the ethanolic leaf extract of Azadirachta indica was evaluated against five
test organisms, including one fungal strain (Candida albicans) and four bacterial strains (Escherichia coli,
Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus). The activity was
determined by measuring the zone of inhibition (ZOI) produced around the wells in agar plates. Three
different concentrations of the extract were tested (25 µg/mL, 50 µg/mL, and 75 µg/mL) and the results
were compared with a positive control (PC) containing standard antimicrobial agents and a negative
control (C) where no active compound was used. The measurements of ZOI were recorded in
millimeters, and the findings are summarized in Table 1.

When the extract was tested against Candida albicans, it showed a clear inhibitory effect at all
concentrations. At 25 µg/mL, the extract produced a ZOI of 14 mm, which increased to 15 mm at 50
µg/mL and further increased to 16 mm at 75 µg/mL. The positive control for C. albicans produced a ZOI
of 19 mm, showing that although the standard antifungal was more effective, the leaf extract displayed
considerable antifungal activity. The negative control showed no inhibition zone, indicating that the
antifungal effect observed was due to the bioactive components in the leaf extract and not because of
any other factors in the experimental set-up. The steady increase in ZOI with rising concentrations of the
extract suggests that the antifungal effect is concentration dependent.

For Escherichia coli, a Gram-negative bacterium, the extract also exhibited antimicrobial activity. The ZOI
measured 10 mm at 25 µg/mL, 11 mm at 50 µg/mL, and 12 mm at 75 µg/mL. In this case, the positive
control exhibited a ZOI of 18 mm. Although the extract’s activity was lower than that of the standard
drug, the incremental increase in ZOI with higher concentrations shows a clear dose-response
relationship. The negative control again showed no inhibition, confirming that the inhibitory effect was
solely due to the A. indica extract. This result indicates that the leaf extract, even at lower
concentrations, is capable of suppressing the growth of E. coli, which is often responsible for
gastrointestinal and urinary tract infections.
Against Klebsiella pneumoniae, another clinically important Gram-negative bacterium, the extract
demonstrated similar trends. The ZOI measured 13 mm at 25 µg/mL, 14 mm at 50 µg/mL, and 15 mm at
75 µg/mL. The positive control showed a ZOI of 18 mm, while the negative control again showed no
inhibition. The gradual increase in ZOI with rising concentrations highlights that the antimicrobial
compounds in A. indica are effective against K. pneumoniae, although not as strongly as the standard
reference drug. This finding is important because K. pneumoniae is known to develop resistance to many
conventional antibiotics, and the observed activity suggests that the plant extract may offer an
alternative or complementary antimicrobial approach.

In the case of Pseudomonas aeruginosa, a pathogen known for its resistance to multiple antibiotics, the
extract also showed inhibitory effects. The ZOI was 12 mm at 25 µg/mL, 13 mm at 50 µg/mL, and 14 mm
at 75 µg/mL. The positive control showed a ZOI of 16 mm, while the negative control displayed no zone
of inhibition. Even though the ZOI values were comparatively lower than those recorded for other
organisms, the activity against P. aeruginosa is notable, given the natural resistance of this bacterium.
This suggests that certain phytochemicals present in the A. indica leaf extract can interfere with the
growth of this resistant strain, which could be explored further for developing novel treatments.

Interestingly, the strongest antimicrobial activity was observed against Staphylococcus aureus, a
Gram-positive bacterium that is a common cause of skin infections, respiratory infections, and food
poisoning. The extract produced a ZOI of 18 mm at 25 µg/mL, which increased to 19 mm at 50 µg/mL
and reached 20 mm at 75 µg/mL. Remarkably, these ZOI measurements were larger than those
produced by the positive control, which showed a ZOI of 13 mm. This suggests that the bioactive
components in the A. indica leaf extract are particularly effective against S. aureus. The strong activity at
even the lowest concentration tested indicates that the extract contains potent compounds that could
serve as leads for developing new antibacterial agents. The absence of any inhibition in the negative
control confirms that the effects seen are purely due to the phytochemicals in the extract.

The overall results from Table 1 demonstrate that the A. indica leaf extract possesses both antifungal and
antibacterial properties. For all test organisms, the ZOI increased with higher concentrations of the
extract, showing a clear dose-dependent effect. Among the tested organisms, S. aureus was the most
sensitive, followed by C. albicans, K. pneumoniae, P. aeruginosa, and E. coli. The positive control
consistently showed strong inhibition, as expected, but it is significant that the plant extract performed
comparably in several cases and even outperformed the standard drug in the case of S. aureus. The
negative control showed no inhibition for any of the organisms, which validates the experimental
observations and confirms that the antimicrobial activity originated from the active constituents of A.
indica.

The variation in ZOI among the organisms can be attributed to differences in their cell wall structures,
metabolic pathways, and intrinsic resistance mechanisms. Gram-negative bacteria like E. coli, K.
pneumoniae, and P. aeruginosa typically have an outer membrane that restricts the penetration of many
antimicrobial compounds, which could explain the relatively smaller ZOI values. In contrast,
Gram-positive bacteria like S. aureus have a more exposed peptidoglycan layer, allowing the bioactive
compounds in the extract to exert a stronger effect. The activity against C. albicans indicates that the
extract contains compounds capable of interfering with fungal cell wall synthesis or other vital processes,
which supports its traditional use in treating fungal infections.
The consistent increase in ZOI with rising concentrations suggests that higher doses of the extract
release more bioactive molecules into the surrounding medium, leading to greater microbial inhibition.
This concentration-dependent activity is an important observation because it highlights the potential of
controlling dosage to achieve desired antimicrobial effects. Moreover, the strong activity against S.
aureus implies that A. indica leaf extract could be explored as an alternative treatment option for
infections caused by this bacterium, especially in cases where resistance to conventional antibiotics is a
problem.

In conclusion, the results clearly demonstrate that the ethanolic leaf extract of Azadirachta indica
exhibits significant antimicrobial properties. It shows measurable activity against both bacteria and fungi,
with the strongest effect observed against S. aureus. The data validate the traditional medicinal use of
neem and indicate its potential as a source of novel antimicrobial agents. Further studies on the isolation
of active compounds, their mechanisms of action, and their potential synergistic effects with existing
drugs could provide valuable insights for developing new treatments.
The anti-inflammatory activity of the Azadirachta indica leaf extract was evaluated by comparing the
percentage inhibition of protein denaturation with different concentrations of the extract. In the
experiment, plant extract samples were tested at increasing concentrations (10, 20, 30, 40, and 50
µg/mL) and their activity was compared to diclofenac, which was used as the standard anti-inflammatory
drug. The percentage inhibition values were plotted on the graph shown in Figure 2. The blue bars
represent the plant extract and the orange bars represent diclofenac.

At the lowest concentration of 10 µg/mL, the leaf extract showed a moderate anti-inflammatory effect.
The percentage inhibition recorded for the extract was slightly above 10%, while diclofenac showed a
slightly higher inhibition, reaching close to 15%. Although the activity of the extract was less than the
standard drug at this concentration, it indicates that even at a small dose, the bioactive compounds in A.
indica begin to show an effect on stabilizing proteins and reducing denaturation.

When the concentration was increased to 20 µg/mL, the percentage inhibition improved noticeably. The
extract produced inhibition values around 15%, while diclofenac showed around 18%. This gradual rise
shows that the anti-inflammatory activity of the extract is dose-dependent. The difference between the
extract and diclofenac is still evident, but the extract demonstrates a steady and reliable effect in
reducing protein denaturation.

At 30 µg/mL, the extract’s activity increased further, with inhibition rising to around 28–30%. Diclofenac
at the same concentration reached inhibition levels near 33%. The difference between the extract and
the standard was reduced, which indicates that as the dose of the extract increases, its effectiveness
approaches that of the standard drug. This is a positive indication of the therapeutic potential of the
plant extract.

At a concentration of 40 µg/mL, the extract showed a significant improvement, with inhibition reaching
approximately 55–58%. Diclofenac at this level exhibited slightly higher inhibition, around 60%. This
shows that at higher concentrations, the leaf extract has a strong capacity to prevent protein
denaturation, which is a key marker of anti-inflammatory activity. The trend seen in the graph suggests
that the plant extract contains active components capable of competing with standard synthetic drugs in
terms of efficacy.

The highest concentration tested, 50 µg/mL, gave the most notable results. The extract showed
inhibition values of around 60–65%, while diclofenac showed inhibition values close to 70%. At this
concentration, the difference between the extract and the standard is minimal, which suggests that A.
indica leaf extract is highly effective in controlling protein denaturation at higher doses. The consistent
upward trend from 10 to 50 µg/mL confirms that the activity of the extract is strongly dose-dependent.

Across all concentrations, the leaf extract demonstrated measurable anti-inflammatory activity. The
gradual rise in inhibition percentage indicates that the bioactive compounds in the extract become more
effective as their concentration increases. This pattern is typical of plant extracts where higher
concentrations release more active phytochemicals into the reaction mixture, thereby increasing the
stabilizing effect on proteins and reducing inflammatory responses.

The comparison with diclofenac, a widely used non-steroidal anti-inflammatory drug, shows that while
the extract does not completely match the potency of the standard at lower concentrations, it
approaches similar effectiveness at higher concentrations. This suggests that A. indica leaf extract
contains compounds with significant anti-inflammatory potential. The similarity in the inhibition trend
with diclofenac implies that the extract may act through comparable mechanisms, such as stabilizing cell
membranes or inhibiting inflammatory mediators.

Another important observation from the figure is the relatively low variability in the results, as shown by
the small error bars in both the extract and the standard. This indicates that the experimental data are
consistent and reliable, and that the extract’s activity is reproducible under the test conditions.

The observed anti-inflammatory activity of the extract can be attributed to the presence of various
phytochemicals such as flavonoids, tannins, and triterpenoids, which are known for their ability to
interfere with inflammatory pathways. These compounds may stabilize lysosomal membranes, prevent
the release of inflammatory mediators, or directly block enzymes involved in the inflammatory process.
Although this study does not identify specific compounds, the results clearly demonstrate the presence
of active principles with significant biological activity.

Overall, Figure 2 highlights the potential of A. indica leaf extract as a natural anti-inflammatory agent. At
lower concentrations, its activity is moderate, but it increases steadily with higher concentrations. At 50
µg/mL, the extract’s inhibition percentage is close to that of diclofenac, which is a strong indication that
the plant extract could serve as an alternative or complementary treatment in conditions where
inflammation is involved.

These findings support the traditional use of Azadirachta indica in herbal medicine for the treatment of
inflammatory conditions. In many regions, neem leaves have been used in poultices and herbal
preparations to reduce swelling and pain. The results from this study provide scientific evidence for
those traditional claims and suggest that with further purification and analysis, the active compounds in
the leaf extract could be developed into standardized formulations.

It is also important to note that plant-based treatments may offer additional benefits such as fewer side
effects compared to synthetic drugs like diclofenac, which are known to cause gastric irritation and other
complications when used long term. The promising results seen in this experiment encourage further
investigation into the safety profile, exact mode of action, and long-term effectiveness of the extract.

In conclusion, the anti-inflammatory assay clearly demonstrates that Azadirachta indica leaf extract
shows dose-dependent inhibition of protein denaturation, with increasing activity observed from 10 to
50 µg/mL. The extract’s performance approaches that of diclofenac at higher concentrations, making it a
promising candidate for further studies and potential therapeutic use.

Discussion

The present study was designed to evaluate the anti-inflammatory activity of Azadirachta indica leaf
extract by measuring its ability to inhibit protein denaturation at different concentrations. The results
obtained from the assay, as shown in Figure 2, demonstrated that the extract exhibited a
dose-dependent increase in anti-inflammatory activity. This indicates that the bioactive compounds
present in the extract were effective in stabilizing proteins and preventing denaturation when present at
higher concentrations. Such findings are significant because protein denaturation is a well-established
process associated with inflammatory conditions. Agents that can prevent or reduce denaturation are
considered to possess anti-inflammatory properties.

At lower concentrations, particularly at 10 and 20 µg/mL, the extract showed only moderate inhibition
compared to the standard drug diclofenac. This observation suggests that at these levels, the quantity of
active phytochemicals in the extract was not sufficient to exert a strong stabilizing effect on the proteins.
Diclofenac, being a potent non-steroidal anti-inflammatory drug, naturally displayed higher inhibition
percentages even at low concentrations. However, the gradual increase in the activity of the extract with
increasing concentrations reflects that the bioactive molecules act in a concentration-dependent
manner. As more extract is introduced, a greater number of active molecules interact with the proteins,
thereby enhancing the inhibitory effect.

The inhibition values at 30 µg/mL marked an important turning point. At this concentration, the extract
produced a significant rise in activity, reducing the difference between its performance and that of
diclofenac. This implies that the threshold concentration of bioactive compounds necessary to achieve a
noticeable anti-inflammatory effect was being approached. Such behavior is consistent with many herbal
extracts, where a minimum effective concentration must be reached before appreciable biological effects
are observed.

At 40 µg/mL and 50 µg/mL, the extract demonstrated strong anti-inflammatory activity, with inhibition
percentages approaching those of diclofenac. The highest concentration tested, 50 µg/mL, resulted in
inhibition values that were only slightly lower than the standard drug. This is an encouraging finding
because it indicates that the plant extract, in its crude form, is capable of achieving activity levels close to
that of a well-established pharmaceutical agent. It further suggests that isolation and concentration of
the active constituents could lead to even greater potency.

The dose-dependent activity observed in this study is likely due to the cumulative action of multiple
phytochemicals present in the leaves of Azadirachta indica. Previous phytochemical investigations of
neem have reported the presence of compounds such as flavonoids, tannins, saponins, alkaloids, and
triterpenoids, many of which have documented anti-inflammatory properties. Flavonoids, for instance,
are known to scavenge free radicals and inhibit enzymes involved in the synthesis of pro-inflammatory
mediators. Tannins can precipitate proteins and reduce membrane permeability, while triterpenoids are
often reported to stabilize lysosomal membranes. These mechanisms collectively contribute to reducing
the processes that lead to inflammation.

The comparison with diclofenac is particularly valuable because it provides a benchmark for evaluating
the therapeutic potential of the extract. Diclofenac works by inhibiting cyclooxygenase enzymes, thereby
reducing the production of prostaglandins that mediate inflammation and pain. Although the exact
mechanism of action of the A. indica extract was not explored in this study, the similarity in inhibition
trends suggests that the extract may act through related pathways or by stabilizing structural proteins
and membranes involved in inflammatory responses. The close alignment of inhibition values at higher
concentrations underscores the potential of the extract as a complementary or alternative therapy,
especially for patients seeking plant-based remedies.

One notable aspect of the results is the consistency reflected by the small error margins. The
reproducibility of the inhibition values across repeated trials indicates that the method used was reliable
and that the extract’s activity was stable. This strengthens the validity of the findings and supports the
claim that A. indica leaves possess genuine anti-inflammatory properties rather than exhibiting random
or inconsistent effects.

The findings of this study also align with traditional knowledge. In many cultures, neem leaves have been
applied in poultices or consumed in decoctions to alleviate swelling, pain, and other inflammatory
symptoms. The scientific evidence provided here validates those traditional uses by demonstrating a
clear, measurable anti-inflammatory effect under controlled conditions. This bridge between traditional
medicine and scientific research is important because it provides a basis for further development of
herbal formulations and standardized extracts.

Despite the promising results, it is important to recognize that the extract used in this study is a crude
preparation. The presence of various phytochemicals means that the activity observed could be due to a
combination of synergistic effects rather than the action of a single compound. Future research focusing
on fractionation and isolation of individual components would be necessary to identify the specific
molecules responsible for the activity. Once identified, these compounds could be further studied to
determine their exact mechanism of action, pharmacokinetics, and potential for development into
standardized herbal drugs or even new chemical entities.

Another consideration is the safety and potential toxicity of the extract at higher concentrations.
Although neem is generally regarded as safe and has been used for centuries, a thorough evaluation of
its cytotoxicity and long-term effects would be essential before recommending its use as an
anti-inflammatory treatment. Studies involving in vivo models and clinical trials would provide more
comprehensive data on its efficacy and safety profile.

It is also worth noting that while diclofenac provided higher inhibition at lower concentrations,
long-term use of such synthetic drugs is associated with side effects, including gastrointestinal
disturbances and renal complications. The prospect of a plant-based alternative that offers similar
efficacy at higher concentrations without these side effects is particularly appealing. This study thus
provides a foundation for exploring A. indica leaf extract as a safer anti-inflammatory agent, particularly
for chronic conditions where prolonged drug use is necessary.

In conclusion, the discussion of the results indicates that Azadirachta indica leaf extract exhibits
significant anti-inflammatory activity that increases with concentration. At higher doses, its activity
approaches that of diclofenac, a standard pharmaceutical agent. These findings support the traditional
use of neem leaves for inflammatory conditions and highlight the potential of this plant as a source of
natural anti-inflammatory compounds. Further research is warranted to isolate and characterize the
active constituents, to understand their mechanisms of action, and to evaluate their safety and efficacy
in more complex biological systems. The data presented in this study contribute valuable insights into
the pharmacological potential of A. indica and provide a scientific basis for its inclusion in future
therapeutic applications.

Conclusion

The present investigation clearly shows that the leaf extract of Azadirachta indica possesses significant
biological activities that support its traditional use in medicine. The antibacterial and antifungal
evaluation demonstrated that the extract was active against a range of test organisms, with a noticeable
increase in the zone of inhibition as the concentration increased. Among the organisms tested,
Staphylococcus aureus showed the highest sensitivity, followed by Candida albicans, while other
bacterial strains also showed moderate susceptibility. The absence of any inhibition in the control
confirms that the observed effects were due to the plant extract.

The anti-inflammatory study further revealed that the extract exhibited a concentration-dependent
ability to inhibit protein denaturation, and at higher concentrations its effect approached that of the
standard drug diclofenac. These findings indicate that the bioactive compounds present in the leaves can
interact with inflammatory pathways and microbial targets effectively. Overall, the study provides
scientific evidence that A. indica leaf extract has promising antimicrobial and anti-inflammatory
properties, which may be attributed to its rich phytochemical composition. This work supports its
potential use as a natural source for developing safe therapeutic agents. Further studies on isolation of
active compounds and in vivo evaluations are recommended to advance its medical applications.