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Title of Project

Solvent extraction of neem oil from Neem kernel by using Microwave

digester and Ultrasonic Wave (Azadirachta Indica kernel)

Students Name: Jeevan Kumar s/o Paramasivan

ID Number: 55201113079
Section: 6 BCP

Supervisors Name
Aiza Syuhaniz Bte Salleh

Malaysian Institute of Chemical And

Bioengineering Technology
University Kuala Lumpur

07 October 2015


1.1 Introduction

Continent of Asia will be a standout amongst those biggest biodiversity areas

on those world, holding exactly of the wealthiest nations on plant assets. One
of the highly value plant assets are neem tree. The neem tree (Azadirachta
indica A.Juss.) is a tropical evergreen tree (deciduous in drier areas) native to
Indian sub-continent. It has been used in Ayurvedic medicine for more than
4000 years due to its medicinal properties. Neem is called arista in Sanskrit
a word that means perfect, complete and imperishable (Roxburgh, 1874).

The Neem tree (Azadirachta indica A. Juss.) has been considered a

wonder tree for centuries in the Indian subcontinent. Nowadays it is useful for
health and food production all over the world. The history of the Neem tree is
inextricably linked to the traditional Indian way of life and has longly been
cherished as a symbol of health in India. Indian people have used the
Azadirachta indica from the very beginning of history.

Today, rural Indians call this tree their "village pharmacy" because of
claims it "cures" diseases and disorders ranging from teeth caves and bedbugs
to ulcers and malaria. The seeds, bark and leaves contain limonoids with

proven antiseptic, antiviral, antipyretic, anti-inflammatory, anti-ulcer and
antifungal uses (Nix, 2007). The insecticidal properties of neem, always known
in India, have attracted the attention of scientists not only for their
effectiveness, but also because non-polluting (Nix, 2007).

1.2 Background Studies

Neem is the most versatile, multifarious tree of tropics, with immense potential.
It possesses maximum useful non-wood products (leaves, bark, flowers, fruits,
seed, gum, oil and neem cake) than any other tree species. Known to have
antiallergenic, antidermatic, antifeedant, antifungal, anti-inflammatory,
antipyorrhoeic, antiscabic, cardiac, diuretic, insecticidal, larvicidal, nematicidal,
spermicidal and other biological activities. Due to these activities neem has
found enormous applications making it a green treasure (Biswas et al., 2002)

Neem has become important in the global context today because it

offers answers to the major concerns facing mankind. Neem (Azadirachta
indica) is considered harmless to humans, animals, birds, beneficial insects
and earthworms, and has been approved by the US Enviromental Protection
Agency for use on food crops (Chiranjib et al., 2010)

Biologically active principles isolated from different parts of the plant

inlcude: azadirachtin, meliacin, gedunin, salanin, nimbin, valassin and many
other derivatives of these principles (Kabeh & Jalingo, 2007). Azadirachtin has
been identified as A. indicas principal active compound. Extracts can be made
from leaves and other tissues, but the seeds contain the highest
concentrations of the compound (Perry, 1980). The active principles are
slightly hydrophilic, but freely lipophilic and highly soluble in organic solvents
like hydrophilic, alcohols, ketones and esters (Kabeh & Jalingo, 2007).

The extraction process of neem kernels is solvent extraction. Solvent

extraction is a common form of chemical extraction using organic solvent as

the extractant (EPA, 1988). In this project, solvent extraction will be conducted
together with microwave digester and ultrasonic waves.

The fundamentals of the microwave extraction (MAE) process are

different from those of conventional methods (solidliquid or simply extraction)
because the extraction occurs as the result of changes in the cell structure
caused by electromagnetic waves. In MAE, the process acceleration and high
extraction yield may be the result of a synergistic combination of two transport
phenomena: heat and mass gradients working in the same direction (Chemat,
Abert-Vian & Zill, 2009).

On the other hand, another method of extraction will be used is

ultrasonic waves. In general, ultrasound can lead to a permeabilization of cell
membranes to ions and it can reduce the selectivity of the cell membranes
significantly. The mechanical activity of the ultrasound supports the diffusion
of solvents into the tissue (Mummery 1978).

1.3 Problem Statement

The first problem identified is the indiscriminate use of synthetic pesticides has
resulted in ecological and health hazards along with development of resistance
in insect pests. This has led to the popularity of pest control agents derived
from plants as they are biodegradable, environmentally compatible and less
toxic to non-target organisms. Neem (Azadirachta indica A. Juss), belonging
to Meliaceae family, leads the list of plants with the highest potential for this
purpose (Kumar, 2003).

Extraction of a desired component and to obtain higher yield from a

solute or plant material requires a proper extraction method. In this project, two
methods will be used which is microwave assisted extraction and ultrasonic

extraction. Thus, comparative study will be made on which extraction will give
out a better quality as well as high yield of the essential oil extracted.

1.4 Research Objectives

The objectives of this project are:

To extract neem oil from neem kernels using solvent extraction method
(EtoH + hexane)
To compare yield produced by using microwave digester and ultrasonic
To study the efficiency on the extraction based on solvent ratio and time
of extraction
To analyze azadirachtin by using High Performance Liquid
Chromatography (HPLC).


2.1 Neem Background

Azadirachta indica is the botanical name of the neem tree. In Farsi

(Persian), these words mean "The Free Tree of India." The tree is believed to
have originated in northeastern India and Myanmar and spread throughout the
subcontinent. Early 20th century Indian settlers in Australia, Africa and South
America brought neem trees with them to their new countries. There are
multiple references to neem and its medicinal uses in ancient medical
documents, namely the Charaka Samhita and the Susruta Samhita, dating
back to the second and fourth centuries respectively. They form the basis of
the Indian system of natural healing, Ayurveda. The Indian Institute of Science
in Bangalore conducted research into potential neem products as early as the

2.2 Introduction on Neem tree

The neem tree (Azadirachta indica) is native to tropical South East Asia.
It is fast growing, can survive drought and poor soil and keeps its leaves all

year round. It is a small to medium-sized tree, usually evergreen, up to 15 (30
max.) m tall, with a round, large crown up to 10 (20 max.) m in diameter
(Roshetko & Evans, 1997). Many white flowers which smell of honey appear
for the first time when the tree is 2 to 3 years old, and the tree bears fruit after
3 to 5 years. The ripe fruit are about 2 centimetres (cm) long and oval shaped.
Inside the fruit there is a lightcoloured seed about 1.5 cm long. Neem trees can
be grown in areas which have between 400 millimetres (mm) and 1500mm of
rain each year. It performs best at an altitude of less than 1,500 metres (HDRA,

2.3 Solvent Extraction

2.3.1 Ultrasonic Assisted Extraction

The intensification of extraction process using ultrasound has been
attributed to the cavitation phenomena. The effects caused by the
ultrasonic waves are compression and expansion cycles during the
passage through the fluid. The expansion can create bubbles or cavities in
a liquid. This is so when the negative pressure exerted exceeds the local
tensile strength of the liquid, which varies depending on its nature and
purity. The process by which vapor bubbles form, grow and undergo
implosive collapse is known as cavitation (Luque-Garcia & Luque de
Castro, 2003). The conditions within these imploding bubbles can be
dramatic, with temperatures of 4500C and pressures up to 100 MPa,
which in turn produces very high shear energy waves and turbulence in the
cavitation zone. The combination of these factors (pressure, heat and
turbulence) is used to accelerate mass transfer in the extraction process
(Patist & Bates, 2008).

Ultrasound also exerts a mechanical effect. In pure liquids, the bubble

retains its spherical shape during the collapse, as its surroundings are
uniform (Luque-Garcia & Luque de Castro, 2003). However, when the
bubble collapses near a solid surface it occurs asymmetrically and
produces high-speed jets of solvent toward the cell walls. These jets have
a strong impact on the solid surface, therefore, increasing the solvent

penetration into the cell and increasing the contact surface area between
solid and liquid phase (Rostagno et al., 2009). Another effect caused by
the ultrasound wave on the solid material is that the ultrasound waves can
facilitate the swelling and hydration and so cause an enlargement in the
pores of the cell wall. This will improve the diffusion process and therefore
enhancing mass transfer (Vinatoru, 2001)

2.3.2 Microwave Assisted Extraction

Microwave-assisted extraction (MAE) or simply microwave extraction is a
relatively new extraction technique that combines microwave and
traditional solvent extraction. Application of microwaves for heating the
solvents and plant tissues in extraction process, which increases the kinetic
of extraction, is called microwave-assisted extraction. MAE has a number
of advantages, e.g., shorter extraction time, less solvent, higher extraction
rate and lower cost, over traditional method of extraction of compounds
from various matrices, especially natural products (Delazar, Nahar, &
Sarker, 2012)

During the extraction process, the rate of recovery of the extract is not a
linear function of time: the concentration of solute inside the solid varies,
leading to a nonstationary or unsteady condition. A series of
phenomenological steps must occur during the period of interaction
between the solid-containing particle and the solvent effectuating the
separation, including (1) penetration of the solvent into the solid matrix; (2)
solubilization and/or breakdown of components; (3) transport of the solute
out of the solid matrix; (4) migration of the extracted solute from the external
surface of the solid into the bulk solution; (5) movement of theextract with
respect to the solid; and (6) separation and discharge of the extract and
solid (Aguilera, 2003). Therefore, the solvent penetrates into the solid
matrix by diffusion (effective), and the solute is dissolved until reaching a
concentration limited by the characteristics of the solid. The solution
containing the solute diffuses to the surface by effective diffusion. Finally,
by natural or forced convection, the solution is transferred from the surface
to the bulk solution (Hu, Cai, & Liang, 2008)

2.4 Solvents

2.4.1 Ethanol

Ethanol commonly called ethyl alcohol, drinking alcohol, or

simply alcohol is the principal type of alcohol found in alcoholic beverages,
produced by the fermentation of sugars by yeasts. It is a neurotoxic
psychoactive drug and one of the oldest recreational drugs used by
humans. It can cause alcohol intoxication when consumed in sufficient

Ethanol is a volatile, flammable, colorless liquid with a slight chemical

odor. It is used as an antiseptic, a solvent, a fuel, and, due to its low
freezing point, the active fluid in post-mercury thermometers. Its structural
formula, CH3CH2OH, is often abbreviated as C2H5OH, C2H6O or EtOH.

2.4.2 Hexane

Hexane is an alkane of six carbon atoms, with the chemical

formula C6H14. Hexanes are significant constituents of gasoline. They are
all colorless liquids at room temperature, odorless when pure, with boiling
points between 50 and 70 C. They are widely used as cheap, relatively
safe, largely unreactive, and easily evaporated non-polar solvents.


3.1 Chemicals, Materials and Apparatus

Chemicals: ethanol, hexane

Raw Materials: neem seeds / kernels

Instruments: microwave digester, laboratory ultrasonic cleaner, HPLC, digital

weighing scale, rotary evaporator

3.2 Summarized Methodology

Depulping of neem fruits

Depulping is a process to remove seed coat and pulp from the neem seed. It
is done by hand and using mechanical depulper. Rub the ripe neem fruits
between palms in the bucket of water and wash the seed. Use clean water for
depulping. After depulping and cleaning, dry the neem seeds in the shade in a
thin layer. Keep the neem seeds in a cool and dry place.

Grinding neem kernels and sieving into desired particle sizes
Grinding the neem kernels into powder form using a suitable blender and
sieving it to particle size approximately below 0.71 mm

Preparation of sample solution

For extraction of neem oil by solvent process, Neem kernel powder were
extracted using the combination of two solvent (n-hexane and ethanol). The
amount of neem kernel powder used is kept constant and ratio of the two
solvents used is being manipulated. For example using solvent ratio of (50:50),
(60:40) and (40:60).

Microwave assisted extraction

Prepared sample will be introduced into microwave digester for about 30
minutes at the temperature of 55 degree Celsius.

Ultrasonic assisted extraction

At this stage, a comparative study have been made on the extraction kinetics
of azadirachtin with and without the ultrasonic waves. The setting of frequency
is to be determined.

Evaporation via rotary evaporator

Solution will be heated and evaporated to obtain solvent free oil at temperature
of 80 degree Celsius.

Compound solutions were prepared and injected to HPLC. Performed analysis
to calculate/identify the concentration bioactive component in the oil solution.



One of the main objectives of this research is to study the efficiency on the
extraction based on solvent ratio and time of extraction. Liauw et al. (2008)
investigated that ratio of 60% ethanol to 40% hexane produced higher oil yield
at optimum temperature of 55 degree Celsius over a long period of 6 hours.
Hexane performs better as extraction solvent at short period of operation while
at long period of extraction process, ethanol is preferred. And also, 60%
ethanol/40% hexane gave the maximum oil yield as shown in Figure 1.

Figure 1: Neem Oil Yield Percentage in Neem Oil Extraction at Different Time
using Ethanol, Hexane and Ethanol-Hexane Mixtures as Solvents (Source:
International Journal of Science and Technology)

Figure 2: Oil Yield obtained during Neem Oil Extraction at different

Temperature using Ethanol and Hexane (Source: International Journal of
Science and Technology)


Aug Sept Oct No Dec Jan Feb Mar Apr May Jun
Search related journal,
identify the method for the
Submission the first draft
of the proposal
Submission of the
Presentation of the
FYP Report
Writing the first chapter of
the report
Prepare all the raw
materials, and equipment
Run the experiment

FYP Report
Writing the second and
third chapter of the report
Submission of draft
chapter 1,2 and 3
Finish writing the report
Submission of the first
Presentation of slides
FYP Report
Submission of the final
draft and hard cover


As a conclusion, it is expected that 60% ethanol to 40% hexane will produce

higher oil yield at optimum temperature of 55 degree Celsius over a long period
of 6 hours. The proportions of ethanol-hexane mixtures (60/40, 50/50, and
40/60% respectively) served as efficient solvent alternatives to the use of
hexane only, in neem oil solvent extraction. These mixtures are more
environmentally friendly, have a reduced risk of flammability, less toxic and
give higher oil yields than both hexane and ethanol. And can be operated at
higher temperature. Another advantage is also the reduced cost associated
with the use of hexane only, as a solvent.


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