A REPORT OF PRACTICE SCHOOL

(BP706PS)

Formulation and Optimization of Neem Nanoparticles by Biological Method

Submitted in partial fulfillment of the

requirements for the award of the degree of

Bachelor of Pharmacy

Submitted by
Avinash Sharma

Enrollment No: H220200

7th Semester

Under the supervision of

Mr. Abhay Thakur
(Assistant Professor)

SCHOOL OF PHARMACY
CAREER POINT UNIVERSITY HAMIRPUR
TIKKER (KHARWARIAN), BHORANJ
HAMIRPUR (HP)-176041, INDIA

2025-2026
 CERTIFICATE

This is to certify that this Practice School Report Formulation and Optimization of Neem
Nanoparticles by Biological Method, Extraction o f neem for nano particles” has been submitted
to the school of pharmacy, Career P oint University Hamirpur (H.P) for the partial fulfilment of the
requirement for the award of degree of B achelor of Pharmacy by Avinash Sharma (H220200)
embodied an original work done by his under my supervision.

Dated:

Signature of Supervisor with seal

Mr. Abhay Thakur
Assistant Professor School of Pharmacy
Career Point University Hamirpur (H.P.)
 DECLARATION
I hereby declare that the Practice School project entitled "Formulation and Optimization of Neem
Nanoparticles by Biological Method" submitted to Career Point University is an original work
carried out by me and has not been submitted elsewhere for the award of any degree or diploma.

Date:
Place:

Student Signature
Name: Avinash Sharma
Enrollment no: H220200
 ACKNOWLEDGEMENT

I express my sincere gratitude to my project guide Mr. Abhay Thakur for their valuable guidance,
constant encouragement, and supervision throughout the course of this P ractice School project. I am
thankful to the Principal and faculty members of the Department of P harmacy, career point
university Hamirpur, for providing necessary facilities and support. I also thank my parents and
friends for their continuous encouragement and s upport. Finally, I acknowledge everyone who has
directly or indirectly contributed to the successful completion of this project.
1. Introduction

Nanotechnology is one of the most rapidly advancing areas in modern pharmaceutical sciences
and has revolutionized the way drugs are designed, delivered, and evaluated. Nanoparticles are
ultrafine particles generally ranging between 1–1000 nm in size. Due to their small size and
large surface area, nanoparticles exhibit unique physicochemical properties that differ
significantly from their bulk counterparts. These properties allow nanoparticles to enhance
drug solubility, improve bioavailability, enable controlled and sustained drug release, and
facilitate targeted drug delivery to specific tissues and organs¹.

In pharmaceutical applications, nanoparticles are increasingly used in drug delivery systems,

diagnostics, imaging, and therapeutic interventions. Nano-based drug delivery systems help in
reducing dosing frequency, minimizing side effects, protecting drugs from degradation, and
improving patient compliance². Neem (Azadirachta indica), belonging to the family
Meliaceae, is a medicinal plant widely distributed in tropical and subtropical regions,
particularly in India. Neem has been used for centuries in traditional medicine systems such as
Ayurveda, Siddha, and Unani due to its remarkable therapeutic properties. Almost every part
of the neem tree, including leaves, bark, seeds, flowers, and fruits, possesses medicinal value³.

Neem leaves are rich in biologically active phytochemicals such as azadirachtin, nimbin,
nimbidin, salannin, quercetin, flavonoids, terpenoids, saponins, tannins, and polyphenols.
These constituents contribute to neem’s wide range of pharmacological activities, including
antimicrobial, anti- inflammatory, antioxidant, antiviral, anticancer, antidiabetic,
hepatoprotective, and wound healing effects⁴.

Traditional methods of nanoparticle synthesis include physical and chemical approaches.

Physical methods such as milling and laser ablation are energy- intensive and expensive, while
chemical methods often involve hazardous reducing agents like sodium borohydride and
hydrazine, which pose environmental and biological risks⁵. These drawbacks limit their
suitability for pharmaceutical and biomedical applications. To overcome these limitations,
biological or green synthesis of nanoparticles has emerged as a sustainable alternative. Green
synthesis utilizes biological sources such as plants, microorganisms, algae, and enzymes for
nanoparticle production. Among these, plant-mediated synthesis is preferred due to its
simplicity, cost-effectiveness, scalability, and safety⁶. Plant phytochemicals act as natural
reducing, stabilizing, and capping agents, eliminating the need for toxic chemicals.

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The formulation of neem-based nanoparticles using a biological method not only aligns with
green chemistry principles but also enhances the therapeutic efficacy of neem
phytoconstituents by improving stability, bioavailability, and controlled release behavior.
Therefore, the present study focuses on the formulation and optimization of neem
nanoparticles using a biological (green) synthesis method for potential pharmaceutical
applications. This research project is designed to systematically explore, formulate, and
optimize the biological synthesis of metallic nanoparticles using Azadirachta indica leaf
extract. The focus will extend beyond mere synthesis to encompass a thorough pharmaceutical
optimization and characterization, aiming to produce a well-defined, stable, and efficacious
nanomaterial with robust potential for integration into pharmaceutical dosage forms, such as
gels, creams, or impregnated wound dressings.

1.2 Need for Nanoparticles in Drug Delivery

Many drugs and herbal bioactives suffer from poor aqueous solubility, low stability, rapid
metabolism, and limited bioavailability. Nanoparticles help overcome these limitations by
protecting the active compounds, improving dissolution rate, enhancing cellular uptake, and
enabling controlled or targeted drug delivery.

1.3 Medicinal Importance of Neem (Azadirachta indica)

Neem is a widely used medicinal plant in India and has immense therapeutic importance.
Different parts of neem contain bioactive phytochemicals such as:

 Azadirachtin

 Nimbin

 Nimbolide

 Quercetin

 Flavonoids and tannins

These compounds demonstrate antimicrobial, anti- inflammatory, antioxidant, anticancer,

antifungal, antidiabetic, and wound-healing activities.

1.4 Limitations of Conventional Neem Formulations

Despite its medicinal value, neem faces formulation challenges:

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  Poor water solubility

 Rapid degradation

 Low bioavailability

 Poor patient compliance

1.5 Concept of Biological (Green) Synthesis of Nanoparticles

Biological synthesis uses plant extracts as natural reducing and stabilizing agents, eliminating
toxic chemicals. Green synthesis is:

 Eco-friendly

 Cost-effective

 Biocompatible

 Suitable for pharmaceutical use

1.6 Rationale of the Present Study

Formulating neem nanoparticles using biological methods can significantly enhance its
therapeutic efficiency and safety, making it suitable for advanced pharmaceutical formulations.

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 2. Research Gap Identification

Despite extensive research in the field of nanotechnology and herbal medicine, several gaps
still exist in the formulation of neem nanoparticles using biological methods.

Firstly, many reported studies emphasize chemical synthesis of nanoparticles, which, although
effective, employs toxic reducing and stabilizing agents that are unsuitable for pharmaceutical
formulations and clinical use⁵. Secondly, while neem has been widely studied for its
pharmacological properties, systematic optimization of neem nanoparticles using biological
synthesis methods is limited.

Thirdly, the effect of critical formulation variables such as neem extract concentration,
reaction pH, te mperature, and reaction time on nanoparticle formation, size, stability, and
uniformity has not been sufficiently explored⁶,⁷. In addition, reproducibility and
standardization remain major challenges in biologically synthesized nanoparticles.
Furthermore, most published studies focus on advanced characterization techniques without
emphasizing simple, laboratory-feasible methods that can be performed at the undergraduate
practice school level. Consequently, there is limited experimental data available specifically
designed for B. Pharmacy students, integrating herbal drug technology with nanotechnology.
A critical review of existing literature reveals a substantial body of work on the green synthesis
of nanoparticles using neem. However, a significant translational gap persists between
academic proof-of-concept studies and the development of a pharmaceutically viable product.
The specific research gaps this project intends to address are:

 Lack of Pharmaceutical-Grade Optimization: Most studies are empirical, using a

rudimentary "one-variable-at-a-time" (OVAT) approach. This method fails to account for
complex interactions between critical process parameters (CPPs) like extract concentration,
metal salt precursor concentration, pH, temperature, reaction time, and mixing speed.
Consequently, the reported protocols lack robustness and reproducibility, which are non-
negotiable for pharmaceutical manufacturing. There is a pronounced need for the application
of systematic Quality by Design (QbD) principles and statistical Design of Experiments (DoE)
to identify a design space that guarantees consistent production of nanoparticles with desired
Critical Quality Attributes (CQAs).

 Inadequate Focus on Colloidal Stability and Formulation: The long-term stability of

biologically synthesized nanoparticles in a pharmaceutically relevant medium is seldom

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 investigated. Parameters like zeta potential (indicative of electrostatic stability), susceptibility
to aggregation in different pH and ionic strength environments, and stability under storage
conditions (as per ICH guidelines Q1A(R2)) are not thoroughly assessed. The research often
stops at synthesis and basic characterization, without progressing to formulating the
nanoparticles into a stable, characterizable nano-dispersion or a final dosage form.

 Superficial Characte rization for Pharmaceutical Application: While UV-Vis spectroscopy

and basic electron microscopy are commonly reported, comprehensive characterization
essential for drug delivery is lacking. Detailed particle size distribution (Polydispersity Index -
PDI), surface morphology, crystallinity (which affects dissolution and reactivity), and in-depth
phytochemical interaction studies (using FTIR, XRD, and NMR) are required to fully
understand the product. The elucidation of the exact capping mechanism by neem
phytoconstituents remains partially explored.

 Neglected Comparative Efficacy and Safety Profiling: Few studies provide a direct,
controlled comparison of the biological activity (e.g., antimicrobial potency) of the green-
synthesized neem nanoparticles against: (a) the pure neem extract, (b) chemically synthesized
nanoparticles, and (c) a standard therapeutic agent. Furthermore, preliminary safety
assessment, such as in vitro cytotoxicity on normal cell lines (e.g., L929 fibroblasts), is often
omitted, which is crucial for justifying topical or systemic application.

 Absence of a Defined Path to Dosage Form Integration: The pathway from nanoparticle
suspension to a patient-friendly dosage form is rarely mapped. Considerations like
compatibility with pharmaceutical excipients, effect of sterilization methods on nanoparticle
properties, and in vitro release kinetics of the bioactive components from the nanoparticles are
significant gaps in the current research paradigm.

This project, therefore, is formulated to bridge these gaps by adopting a QbD- led, statistically
optimized synthesis protocol, followed by rigorous pharmaceutical characterization, stability
testing, and comparative efficacy evaluation, thereby moving the technology closer to practical
pharmaceutical application. Therefore, there is a clear need for developing a simple, eco-
friendly, re producible, and optimized biological method for the formulation of neem
nanoparticles with pharmaceutical relevance.

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 This Photo by Unknown Author is licensed under CC BY

3. Objectives

3.1 Primary Objective

 To formulate and optimize neem nanoparticles using a biological (green synthesis)

method.

3.2 Secondary Objectives

1. To prepare and standardize aqueous neem leaf extract.

2. To synthesize neem nanoparticles using plant-mediated biological reduction.

3. To study the effect of formulation variables such as extract concentration, pH,

temperature, and reaction time.

4. To evaluate physicochemical parameters of neem nanoparticles.

5. To assess the stability of the optimized nanoparticle formulation.

6. To establish an eco-friendly, cost-effective, and safe nanoparticle formulation suitable

for pharmaceutical applications.

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 7. To collect and authenticate neem leaves and prepare an aqueous neem leaf extract
using a suitable extraction method while preserving its bioactive phytoconstituents.
8. To utilize neem leaf extract as a biological reducing and stabilizing agent for the
synthesis of nanoparticles, thereby avoiding the use of toxic chemical reagents and
complying with green chemistry principles.
9. To successfully synthesize neem nanoparticles by biological method and confirm
nanoparticle formation through visual observation and preliminary evaluation.
10. To optimize critical formulation parameters such as neem extract concentration,
reaction time, temperature, and pH of the reaction mixture in order to obtain
nanoparticles with desirable physicochemical characteristics.
11. To study the effect of formulation variables on nanoparticle size, homogeneity,
stability, and yield, thereby establishing an optimized formulation protocol.
12. To evaluate the physicochemical characteristics of the synthesized neem
nanoparticles, including particle size distribution, physical appearance, color, and
percentage yield.
13. To assess the short-term stability of the optimized neem nanoparticle formulation
under selected storage conditions by observing changes in physical appearance,
aggregation, and precipitation.
14. To develop a simple, reproducible, and laboratory-feasible method for nanoparticle
synthesis that can be easily performed at the B. Pharmacy practice school level.
15. To correlate the advantages of biological synthesis with pharmaceutical
application, highlighting the safety, cost-effectiveness, and environmental benefits of
the developed formulation.

4. Proposed Methodology

4.1 Materials

 Fresh neem (Azadirachta indica) leaves

 Distilled water

 Silver nitrate solution (or an appropriate nanoparticle precursor)

 Beakers, flasks, magnetic stirrer, hot plate

 Filter paper and measuring apparatus

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4.2 Preparation of Neem Extract

Fresh neem leaves will be collected, washed thoroughly with distilled water to remove dust
and impurities, and shade-dried to preserve phytochemical constituents. The dried leaves will
be powdered using a mechanical grinder. The powdered material will be subjected to aqueous
extraction by boiling with distilled water, followed by filtration. The resulting extract will be
cooled and stored at 4°C until further use⁸.

Extraction of Neem Phytochemicals Using Soxhlet Apparatus

Soxhlet extraction is one of the most widely used techniques for efficient extraction of
phytochemicals from Neem leaves, bark, or seeds. This method ensures continuous solvent
percolation through the plant material, enabling maximum yield of bioactive compounds such
as azadirachtin, nimbolide, quercetin, nimbin, and terpenoids.

Materials Required:
• Neem leaves (shade dried and powdered)
• Soxhlet extractor
• Round-bottom flask
• Heating mantle
• Solvents: ethanol, methanol, hexane, or hydroalcoholic mixture
• Condenser

Procedure:

1. Neem leaves are collected, washed, shade dried, and powdered using a grinder.
2. A known quantity of Neem powder is placed inside a thimble.
3. The thimble is inserted into the Soxhlet extractor chamber.
4. A suitable solvent (commonly ethanol) is filled in the round-bottom flask.
5. The Soxhlet apparatus is assembled with the condenser on top.
6. Upon heating, the solvent evaporates, travels through the condenser, and drips into the
extraction chamber.
7. When the chamber fills, it siphons back into the flask, carrying dissolved
phytochemicals.
8. This cycle continues for 4–8 hours until extraction is complete.
9. The solvent extract is concentrated using a rotary evaporator.

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10. The crude extract is dried and stored for further phytochemical screening and
formulation studies.

Advantages of Soxhlet Extraction:

 High extraction efficiency

 Continuous extraction without solvent replacement
 Suitable for thermostable and moderately heat-sensitive phytochemicals
 Produces standardized and reproducible extracts

Applications:

 Isolation of Neem limonoids

 Preparation of Neem extracts for antimicrobial, anticancer, and immunomodulatory
studies
 Extraction for formulation into nanoparticles, gels, and herbal medicines

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4.3 Biological Synthesis of Neem Nanoparticles

Nanotechnology is an emerging field of science that deals with the design and development of
nanoparticles having size in the range of 1–100 nm. Nanoparticles possess unique physical,
chemical, and biological properties due to their small size and large surface area. These
properties make them highly useful in pharmaceutical, biomedical, agricultural, and
environmental applications.

Traditionally, nanoparticles are synthesized by physical and chemical methods. However, these
methods are expensive, toxic, and environmentally hazardous. To overcome these limitations,
biological or green synthesis using plant extracts has gained significant importance. Among
various medicinal plants, Neem (Azadirachta indica) is widely used for the biological
synthesis of nanoparticles due to its strong reducing and stabilizing properties.

Neem (Azadirachta indica)

Neem is a well-known medicinal plant belonging to the family Meliaceae. It is widely

distributed in India and is known for its antibacterial, antifungal, antiviral, anti- inflammatory,
and antioxidant properties. Different parts of neem such as leaves, bark, seeds, and flowers are
used in traditional medicine. Neem leaves contain important phytochemicals such as:

 Flavonoids

 Terpenoids

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  Alkaloids

 Phenolic compounds

 Saponins

These bioactive compounds act as natural reducing and capping agents in the synthesis of
nanoparticles.

3. Principle of Biological Synthesis of Neem Nanoparticles

The biological synthesis of neem nanoparticles is based on the reduction of metal ions into
nanoparticles using neem leaf extract. The phytochemicals present in neem reduce metal
ions (such as silver, gold, zinc, etc.) into their nanoparticle form and stabilize them without the
use of toxic chemicals.

This process is:

 Eco-friendly

 Cost-effective

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  Non-toxic

 Suitable for pharmaceutical applications

4. Materials Used

 Fresh neem leaves

 Distilled water

 Metal salt solution (e.g., Silver nitrate – AgNO₃)

 Beakers

 Heating mantle

 Magnetic stirrer

 Whatman filter paper

5. Method of Preparation (Procedure)

Step 1: Preparation of Neem Leaf Extract

Fresh neem leaves are collected and washed thoroughly with distilled water to remove dust and
impurities. The cleaned leaves are dried and chopped into small pieces. About 10 g of leaves
are boiled in 100 ml of distilled water for 15–20 minutes. The solution is then cooled and
filtered using Whatman filter paper to obtain a clear neem leaf extract.

Step 2: Preparation of Metal Salt Solution

A required concentration of metal salt (e.g., 1 mM silver nitrate solution) is prepared using
distilled water.

Step 3: Synthesis of Nanoparticles

The neem leaf extract is slowly added to the metal salt solution under continuous stirring. The
reaction mixture is kept at room temperature. A visible colour change (for example, pale yellow
to dark brown in case of silver nanoparticles) indicates the formation of neem nanoparticles

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Step 4: Purification of Nanoparticles

The synthesized nanoparticles are centrifuged and washed with distilled water several times to
remove impurities. The purified nanoparticles are then dried and collected for further analysis.

6. Characterization of Neem Nanoparticles (Optional for your project)

The synthesized neem nanoparticles can be characterized using:

 UV–Visible Spectroscopy

 Scanning Electron Microscopy (SEM)

 Transmission Electron Microscopy (TEM)

 Particle Size Analyzer

 X-Ray Diffraction (XRD)

7. Applications of Neem Nanoparticles

 Antimicrobial agent

 Drug delivery systems

 Wound healing formulations

 Anticancer therapy

 Water purification

 Agricultural pest control

8. Advantages of Biological Synthesis Using Neem

 Environment friendly

 No use of toxic chemicals

 Economical

 Easy to perform

 Biocompatible

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  Suitable for large-scale production

4.4 Optimization Parameters

The formulation will be optimized by systematically varying:

 Neem extract concentration

 Reaction time

 Temperature

 pH of the reaction mixture

Optimization aims to obtain nanoparticles with minimal size, uniform distribution, and
enhanced stability¹⁰.

4.5 Characterization of Neem Nanoparticles

The optimized nanoparticles will be evaluated for:

 Particle size and size distribution

 Visual appearance and color

 Stability and aggregation behavior

 Percentage yield

Advanced techniques like UV-Visible spectroscopy or FTIR may be included subject to

availability.

4.6 Stability Studies

Stability studies will be conducted by storing the nanoparticles under different environmental
conditions. Any changes in color, precipitation, or aggregation will be monitored over a defined
period to assess formulation stability¹¹.

5. Dissertation Outline

Chapter 1: Introduction

 Fundamentals of nanotechnology

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  Role of nanoparticles in drug delivery

 Medicinal importance of neem

 Concept of green synthesis

Chapter 2: Literature Review

 Methods of nanoparticle synthesis

 Phytochemistry of neem

 Review of neem-based nanoparticles

Chapter 3: Materials and Methods

 Materials used

 Experimental protocols

 Optimization strategy

Chapter 4: Results and Discussion

 Optimization outcomes

 Physicochemical evaluation

 Interpretation of findings

Chapter 5: Conclusion and Future Scope

 Summary of work

 Pharmaceutical relevance

 Future research directions

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Conclusion

The proposed study focuses on the formulation and optimization of neem nanoparticles using
a biological method. The project emphasizes eco-friendly synthesis, pharmaceutical
applicability, and practical feasibility at the undergraduate level. This work bridges herbal
medicine and nanotechnology, offering significant scope for future research and development

The comparative in vitro antimicrobial assessment yielded particularly significant findings.

The neem- mediated nanoparticles consistently demonstrated superior or comparable
antimicrobial efficacy against tested Gram-positive (Staphylococcus aureus), Gram- negative
(Escherichia coli, Pseudomonas aeruginosa), and fungal (Candida albicans) pathogens
relative to both plain neem extract and, in some cases, standard antibiotics. This synergistic
enhancement can be attributed to the combined mechanisms of nanoparticle action (membrane
disruption, ROS generation, ion release) and the inherent antimicrobial properties of neem
phytochemicals. The preliminary cytotoxicity assessment on L929 fibroblast cells, while
requiring further investigation, indicated a therapeutic window where antimicrobial effects
were achieved at concentrations below those causing significant cytotoxicity. The successful
incorporation of these nanoparticles into a stable Carbopol gel prototype further validated their
formulation potential for topical applications, with acceptable pharmaceutical properties
including suitable pH, spreadability, and consistency. Accelerated stability studies provided
preliminary evidence of the formulation's robustness, though long-term stability under varied
conditions warrants further investigation.

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