Kuma Endale Thesis

ADDIS ABABA SCIENCE AND TECHNOLOGY UNIVERSITY
COLLEGE OF BIOLOGICAL AND CHEMICAL ENGINEERING
DEPARTMENT OF CHEMICAL ENGINEERING
Thesis Title: Extraction and Characterization of Essential oil

from basil leaves
Group members
NAME ID N.O
1. Aklilu Hailemariam……..………….…..ETS0092/08
2. Nurye Degu…………………………….ETS0912/08
3. Tsegaw G/Michael……………………..ETS1119/08
4. Walbeka Wako………………………..ETS1140/08
Advisor: Mr.Yoseph Asrat

Submission date 21/01/2021
Addis Ababa, Ethiopia
AASTU College of biological and chemical engineering department of chemical engineering
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ACKNOWLEDGMENT
First of all we would like to praise Almighty GOD for giving us the ability and strength to finalize
this thesis work. Secondly we would like to give great respect for AASTU collage of biological
and chemical engineering for giving us the opportunity to conduct this project work.
Next, special acknowledgement and great appreciation should be deserved for our mentor Mr.
YOSEF for his continuous guidance, good treatment and relevant consultation up on complement
of this project work. We also express our profound gratitude to the laboratory Technicians of
Chemical Engineering who assists us ideally and technically until the end of this project.
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ABSTRACT
Essential oils are highly concentrated substances extracted from flowers, leaves stems, roots,
seeds, barks, resins or fruit rinds. Basil (Ocimum basillicum L.) is an erect herbaceous annual
aromatic, spice and medicinal plant that belong to the Lamiaceae family. The application of basil
leaves in antioxidant, aromatherapy, food, pharmaceutical, cosmetic and other related industries
are limited due to lack of adequate research on their chemical and biological potential. Basil is a
useful plants which are not well known about its valuable uses and so far its many uses are not
discovered. Currently Ethiopia imports phenolic compounds from abroad for chemical and
pharmaceutical uses. This is a serious challenge which takes millions of dollars annually to meet
its sufficient essential oil demand. Principally, the project aims to extract essential oil from basil
leaves by solvent extraction method using Soxhlet apparatus. To do this 1200g of sample basil
leaves was taken and treated by passing through washing, drying, grinding, extraction, and
separation. The dried sample basil leaves was grinded to less than 1 mm particle size to facilitate
the extraction process. Then the yield of the oil for solute to solvent ratio of (0.05, 0.075 and
0.1), extraction time of (2hr, 3hr and 4hr) and at temperature of 69 oc were studied. Meanwhile
we have got the optimum yield which is 6.4 % at 0.075 solvent ratio and at 4 hours extraction
time. The characterization on physical properties such as specific gravity, density, pH value,
refractive index and color were analyzed using standard procedures and chemical properties like
the acid value, saponification value and peroxide value of basil essential oil were performed and
compared with literatures.
LIST OF FIGURE
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Figure 2.1 Basil plant…………………………………………………………………………….10
Figure 2.2 Soxhlet apparatus……………………………………………………………………..21 Figure
3.1 fresh basil leaves……………………………………………………………………...25
Figure 3.2 a) Grinded basil powder and b) sieved basil powder………………………………...26
Figure 3.3 Experimental set up of Soxhlet apparatus……………………………………………27
LIST OF TABLE
Table 2.1 Nutritional value of basil per 100 grams (3.5 ounces)……………………………….11
Table 3.1 The main materials / equipment’s required to conduct our research…………………24
Table: 3.4 Factors and respective ranges of the experiments……………………………………31
Table 4.1 Moisture content of fresh basil leaves………………………………………………...32
Table 4.2 Yield of basil essential oil by solvent extraction……………………………………...33
Table 4.3 Yield of essential oil for variable solute to solvent ratio……………………………...33
Table 4.4 Average density and specific gravity of essential oil…………………………………34
Table 4.5 Refractive index……………………………………………………………………….35 LIST OF

ABRIVATION
EDA Electro-dermal activity
EEG Electroencephalogram
EO Essential oil
EY Extracted yield
O.Basilicum L Ocimum basilicum Leaves
SG specific gravity
BPC British Pharmaceutical Conference
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Table of Contents
ACKNOWLEDGMENT................................................................................................................. ii
ABSTRACT ................................................................................................................................... iii
LIST OF FIGURE.......................................................................................................................... iv
LIST OF TABLE ............................................................................................................................ v
LIST OF ABRIVATION ............................................................................................................... vi
1. INTRODUCTION ...................................................................................................................... 1
1.1 Background ........................................................................................................................... 1
1.2 Statement of the Problem ...................................................................................................... 2
1.3 Objective ............................................................................................................................... 3
1.3.1 General objective ............................................................................................................ 3
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1.3.2 Specific objective ........................................................................................................... 3

1.4 Significance of the Study ...................................................................................................... 3
1.5 Scope of the Study................................................................................................................. 4
2. LITERATURE REVIEW ........................................................................................................... 5
2.1 Essential oil .......................................................................................................................... 5
2.1.1 The unique characteristics of essential oil ...................................................................... 6
2.1.2 Essential oils in tropical countries .................................................................................. 7
2.1.3 Chemical Constituents of Essential Oils ........................................................................ 8
2.1.4 Biosynthesis of Essential Oils ........................................................................................ 8
2.1.5 Major uses of Essential Oil ............................................................................................. 8
2.2 Basil Plant ............................................................................................................................. 9
2.2.1 Growth of Basil .............................................................................................................. 9
2.2.2 Basil production in Ethiopia ......................................................................................... 10
2.3 Uses of Basil........................................................................................................................ 10
2.4 Basil Essential Oils.............................................................................................................. 13
2.4.1 Physical properties of basil essential oils ..................................................................... 13
2.3.2 Application of Basil Essential Oil ................................................................................ 14
2.4 Essential oil extraction technologies ................................................................................... 16
2.4.1Turbo-Extractor ............................................................................................................. 16
2.4.2 Microwave Assisted Techniques .................................................................................. 16
2.4.3 Mechanical Expression ................................................................................................. 17
2.4.4 Steam and water distillation ......................................................................................... 17
2.4.5 Water /Hydro distillation .............................................................................................. 17
2.4.5 Steam Distillation ......................................................................................................... 18
2.4.6 Solvent Extraction ........................................................................................................ 19
2.5 Solvent Extractor (Soxhlet Apparatus) ............................................................................... 19
2.6 Factors Affecting Solvent Extraction of Basil Essential Oil ............................................... 21
2.6.1 Particle Size .................................................................................................................. 22
2.6.2 Solvent .......................................................................................................................... 22
2.6.3 Extraction time ............................................................................................................. 22
2.6.4 Solute to solvent ratio ................................................................................................... 22
2.6.5 Agitation of the Fluid ................................................................................................... 22
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3. MATERIALS AND METHODS .............................................................................................. 24

3.1 Materials .............................................................................................................................. 24
3.2 Methodology ....................................................................................................................... 25
3.2.1 Collection and preparation of sample .......................................................................... 25
3.2.2 Treatment of the Basil leaves ....................................................................................... 25
3.2.3 Size reduction .............................................................................................................. 25
3.2.4 Determination of moisture content of the Basil leaves................................................ 26
3.2.5 Experimental set up ...................................................................................................... 26
3.2.6 Experimental Procedures .............................................................................................. 27
3.3 Characterization of Extracted Basil Essential Oil ............................................................... 28
3.3.1 Determination of physical properties .......................................................................... 28
3.3.2 Characterization of the chemical property of Basil essential oil .................................. 29
3.4 Experimental design ............................................................................................................ 31
4. RESULT AND DISCUSSION ................................................................................................. 32
4.1 Determination of moisture content of basil leaves .............................................................. 32
4.2 Percentage yield oil ............................................................................................................. 32
4.2.1 Effect of Extraction Time on Essential Oil Yield ......................................................... 33
4.2.2 Yield of essential oil for variable solute to solvent ratio .............................................. 33
4.3 Characterization of oil ......................................................................................................... 34
4.3.1 Density and Specific gravity of oil ............................................................................... 34
4.3.2 Color ............................................................................................................................. 34
4.3.3 PH ................................................................................................................................. 35
4.3.4 Refractive index of the oil ............................................................................................ 35
4.3.5 Acid Value or Acid Number ......................................................................................... 35
4.3.6 Peroxide value .............................................................................................................. 35
4.3.7 Saponification value ..................................................................................................... 36
5.1 CONCLUSION AND RECUMENDATION .........................................................................
37
5.2 Conclusion ........................................................................................................................... 37
5.3 Recommendation ................................................................................................................. 38
REFERENCESS ........................................................................................................................... 39
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1. INTRODUCTION
1.1 Background
Essential oils are very interesting natural plant products and among other qualities they possess various
biological properties. The term “biological” comprises all activities that these mixtures of volatile
compounds (mainly mono- and sesquiterpenoids, benzenoids, phenylpropanoids, etc.) exert on humans,
animals, and other plants. Essential oils are complex mixtures of volatile compounds produced by living
organisms and isolated by physical means only (pressing and distillation and/or extraction) from a whole
plant or plant parts of known taxonomic origin (Husnu and Gerhard, 2010). An essential oil is a
concentrated hydrophobic liquid containing volatile (easily evaporated at normal temperatures) chemical
compounds from plants. Essential oils are also known as volatile oils, ethereal oils, aetherolea, or simply
as the oil of the plant from which they were extracted, such as oil of clove. An essential oil is "essential"
in the sense that it contains the "essence of" the plant's fragrance, the characteristic fragrance of the plant
from which it is derived. It can be derived from one or more plant parts, such as flowers, leaves, leaves
and stems, bark, wood, roots, seeds, fruits, rhizomes or gums or oleoresin exudations.
Basil (Ocimum basillicum L.) is an erect herbaceous annual aromatic, spice and medicinal plant that
belong to the Lamiaceae family (Darrah, 1980). The name basil is derived from the Greek word basilus
which means “king” (Chang, 2004). Basil (Ocimum basilicum) is among the most important herbs across
the globe and it has the acronym of ‘King of herbs.’ In Ethiopia, the subspecies goes under the name
sacred basil and the local name bessobela. It has grown in the wild for generations, with locals harvesting
it by hand before sun-drying it for spicing meals like pasta and leguminous dishes. The original home of
the typical basil is India, where it has grown under cultivation since 3000 B.C. Basil was used also a
symbol of mourning in Greece where it was known as basilikon photon, meaning magnificent, royal, or
kingly herb. Ancient records from 907 AD indicate sweet basil in the Hunan region of China. Many
beliefs and rituals are accompanied with basil. In Italy it is a symbol of love, in France it is named as an
herb of royalty. Jewish people used it to get strength during fasting while an African legend claims that
basil protects against scorpions. However, a European group has considered it to be a symbol of Satan
(Marwat et al., 2011).
Basil comes with a magnesium content of 16 percent worth of the daily nutritional value, while that of
iron is 17 percent. Calcium represents 17 percent of the daily needs while vitamin C, for boosting
immunity, stands at 30 percent of the daily requirements. Each serving of the herb has zero percent
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saturated fat, and zero cholesterol, which keeps the heart healthy. With a dietary fiber content of 1.6
grams, basil provides 6% of the daily needs for this fiber that assists in digestion.
In this thesis we are going deal import information about extracting essential oils from local and
indigenous plant Ocimum basillicum L and determine the optimum operating conditions of process
parameters in small-scale solvent extraction method.
1.2 Statement of the Problem

According to National Program Coordinator of Aromatic, Medicinal and Bio-energy Crops Research
Ethiopia Imports more than 700 tons of essential oils annually (minimum 350 Million birr) (Addis Ababa
Development Study Associates (2008)) ≈ 18.42million USD. In 2012 it reached more than 1800 tones
(Custom Authority) ≈ 900 million birr ≈ 6.15 Million USD annually this Indicates Ethiopia is spending
huge foreign currency for importation of essential oil. Again in Ethiopia, 80-90% of the animals and
7080% of the human beings are reported to be treated with plant based traditional medicines (WHO,
2006; Endashaw, 2007). In general the essential oil market of our country increase in its size from time to
time, due to the increasing importance and usage of essential oil in pharmaceutical and aromatherapy
aids. The increasing demand for flavored food and beverages products among consumers is also enhance
this market dramatically. The application of plant in antioxidant, aromatherapy, food, pharmaceutical,
cosmetic and other related industries are limited due to lack of adequate research on their chemical and
biological potential. Basil is a useful plants which are not well known about its valuable uses and so far
its many uses are not discovered. As it is so, currently Ethiopia is importing phenolic compounds from
abroad for chemical and pharmaceutical uses. This is a serious challenge which takes millions of dollars
annually to meet its sufficient essential oil demand. And basil leaves are not used for any production in
our country. Therefore, extract the essential oil from basil leave to be used for different cosmetics
medicine, flavors etc. rather than using it in raw forms, detect the composition of the essential oil
extracted and investigate the optimum operating condition for the extraction process were needed.
1.3 Objective
1.3.1 General objective

The general objective of this study is extraction and characterization of essential oil from basil leaves
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(Ocimum basillicum L.)
1.3.2 Specific objective

 To characterize some physical and chemical properties of basil leaves essential oil extracted 
To determine the effect of Extraction time on the yield of the essential oil.
 To determine the effect of solvent ratio on the yield of the essential oil.
1.4 Significance of the Study

As we have stated in the statement of the problem above, Ethiopia Currently imports essential oils for
different pharmaceutical and cosmetics industries. It expense considerable amount of foreign currency to
import this essential oil. Thus, this research is aimed to provide import substitution by producing this oil
in our country using local raw material or indigenous plants.
1.5 Scope of the Study

This work involves extraction of essential oil from basil leaves (locally available biomass) by hexane
based solvent extraction method. The study covers starting from raw material preparation, extraction of
oil and characterization of some of the physical and chemical properties of the oil extracted. The
extraction process was carried out by varying parameters such as solute to solvent ratio, extraction time
and by making temperature constant which is at the boiling point of the solvent (hexane) to see the
response of the parameter on the yield of the oil product.
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2. LITERATURE REVIEW
2.1. Essential oil

Essential oils have been used in folk medicine throughout history. The earliest recorded mention of the
techniques and methods used to produce essential oils is believed to be that of Ibn al-Baitar (1188–
1248), an Al-Andalusian (Muslim Spain) physician, pharmacist and chemist. Essential oils are aromatic
substances present in the specialized cells or glands of certain plants used by them to protect themselves
from predators and pests, but also to attract polinators. In other words, essential oils are part of the
immune system of the plant.
Essential oils are subtle, concentrated, hydrophobic, aromatic liquids containing volatile compounds
which is extracted from the flowers, seeds, leaves, stems, bark &/or roots of trees, herbs, bushes &
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shrubbery through different extraction method. They were recovered in Egyptians, Romans, and Greeks
between
1500 B.C to 377B.C. The Bible story also refers to essential or anointing oils over 150 time, which means
"to smear with oil”, to make a person sacred, to set them apart & to dedicate them to serve a higher
spiritual purpose. Essential oils are used in the manufacture of high quality perfumes and lotions, food
flavorings, cooking, cleaning, skincare, hair care, massage, aromatherapy, cosmetics, homemade beauty
and cleaning products, healing and medicinal purposes and as fragrant seed and antiseptic additives in
many common products. Besides essential oils promote hormonal balance, improve digestion and cure
respiratory problems and infections (Suryawanshi & Kumbhar, 2016).
Each the volatile oil is unique, being different even within the same plant, and has wonderful
applications and as varied as Nature itself. The complex combination of organic substances that form a
volatile oil gives its specific note, which depends on the species, the harvest period, the climate, as well
as the part of the plant from which it is extracted. Numerous compounds that make up essential oils have
been identified. Only the mint oil has no more than 200 different components. Many the compounds are
structural isomers.
Essential oils contain the true essence of the plant it was derived from and are highly concentrated.
Essential oil is volatile oil, ethereal oils or aetherolea, or simply as the "oil of" the plant from which they
were extracted, usually having the characteristic odor or flavor of the plant from which it is obtained,
used to make perfumes and flavorings. Oil is essential in the sense that it carries a distinct scent, or
essence of the plant. Essential oils are not the same as perfume or fragrance oils. Where essential oils are
derived from the true plants, perfume oils are artificially created fragrances or contain artificial
substances and do not offer the therapeutic benefits that essential oils offer. Formerly, essential oils are
produced by tedious hand pressing and sponge pressing. They are now produced by high-speed
machines. The yield of essential oils varies widely from species to species (Vanhaelen et al., 2002).
The synthesis and accumulation of essential oils occur either outside the plant, in the glandular brushes
(Asteraceae, Geraniaceae, Laminaceae, etc.) and in the papillae, either inside the plant, in the secretory
cells, in the intercellular spaces (secretory channels) secretory bags (Anacardiaceae, Rutaceae,
Myrtaceae). Essential oils can accumulate in all plant organs, but in varying amounts. Thus we can meet
them in: roots, leaves, flowers, fruits, wood of the stems or in the bark. The content in essential oils of
plants is often below 1 %, rarely reaching 15 % or even more, in the dry product of some plants.
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2.1.1 The unique characteristics of essential oil

In early work, the term "essential oils" was defined as the volatile oils obtained though different
extraction method from plants. This definition was clearly intended to make a distinction between "fatty
oils" and the oils, which are easily volatile. Gradually with the advance of science came improvements
in the methods of preparing the oils, and parallel with this development a better knowledge of the
constituents of the oils was gained. It was found that the oils contain many classes of organic substances
with varying volatility. Although a list of all the known oil components would include a variety of
chemically unrelated compounds (Becker, 2005).
 Aromatic
Currently fragrant of essential oils are mainly used by the cosmetic industry. Smell has quite an effect on
human body and nervous responses. Essential oils are highly aromatic and therefore, many of the
benefits can be obtain by simply inhaling them. This can be done by breathing in the fragrance from the
bottle, or they can be diffused into the room. Essential oils, when diffused, can be the best air filtration
system in the world. They will purify the air by removing metallic particles and toxins from the air,
increase atmospheric oxygen, increase ozone and negative ions in the house, which inhibits bacterial
growth, destroy mold, cigarettes and animal odors, fill the air with a fresh, herbal aromatic scent
(Becker, 2005).
 Penetrating Ability
The penetrating characteristic of essential oils greatly enhances their ability to be effective. Essential oils
will penetrate into the body when applied to the skin. Essential oils rubbed into the feet will be
distributed to every cell in the body in minutes. They will even penetrate a finger or toe nail to treat
fungal infection underneath (Becker, 2005).
 High frequency
The effectiveness of essential oils is sometimes also described in terms of frequency. It has been
reported that the human body has an electrical frequency and that much about a person's health can be
determined by frequency. In 1992, Bruce Tainio of Tanio Technology, an independent division of
Eastern State University in Cheny, Washington, built the first frequency monitor in the world. Tainio
has determined that the average frequency of the human body during the day time is 62-68 MHz. (a
healthy body frequency is 62-72). When the frequency drops, the immune system is compromised. If the
frequency drops to 58 MHz, cold and flu symptoms appear, at 55 MHz, diseases like Candida take hold,
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at 52 MHz, Epstein bar and at 42 MHz, cancer. According to Dr. Royal R. Rife, every disease has a
frequency. He found that certain frequencies can prevent the development of disease and that others
would destroy disease. Substances with higher frequency will destroy diseases of a lower frequency.
The study of frequencies raises important questions, concerning the frequencies of substances we eat
breath and absorb. Many pollutants lower healthy frequency. Processed canned food has a frequency of
zero. Fresh produce has up to 27 MHz Essential oil start at 52 MHz and go as high as 320 MHz, which
is the frequency of Rose oil. Clinical research shows that essential oils have the highest frequency of any
natural substance known to man, creating an environment in which disease, bacteria, virus, fungus, etc.,
cannot live (Balami, 2007).
2.1.2 Essential oils in tropical countries

Developing countries are endowed with vast resources of aromatic plants which have been used by their
people for centuries as food, health care products, flavour and fragrances. Apart from the traditional
ways of using these plants, many are exported to industrialized countries as raw materials for drugs,
fragrances and flavour. The value-added products are then imported back costing the countries several
times more than the original revenue gained from exporting the raw materials. The actual value of these
resources has not been retained in many countries for want of know-how and trained manpower.
Today the promotion and development of plant based products have gained momentum due to certain
ground realities:
 Green consumerism and the current resurgence on the use of “Naturals” in developed countries
which has given a fresh impetus to the development of plant based products.
 Free market economy bringing in more openness and expanding markets and demand for new
resources, materials and products.
 A growing acceptability of the social responsibility of minimizing socio-economic inequalities in
favour of rural people resulting in opening up of additional job and income opportunities for the
poor people.
 Poor economic conditions in developing countries restricting imports thereby placing increasing
reliance on substitutes using indigenous plant resources.
 Increasing awareness regarding biodiversity conservation and therefore sustainable and protective
use of plant resources.
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2.1.3 Chemical Constituents of Essential Oils

Essential oils are complex mixtures (5000–7000 chemical constituents) in which mono– and
sesquitrpene constituents predominate, but also contain aromatic compounds, often phenylpropane
derivatives, and rarely meet diterpenes. The terpenic compounds may be hydrocarbons or oxygenated
derivatives (oxides, alcohols, aldehydes, ketones, acids) or reaction products thereof (esters, ethers).
Terpenic compounds are substances of vegetable origin that enter into the natural composition of
molecular mixtures that lead to the formation of volatile (essential, etheric) oils.
2.1.4 Biosynthesis of Essential Oils

The biosynthesis of odorous substances takes place in the leaves, where most of them are found and
remains until flowering. Flowering, essential oils migrate into flowers, and part is consumed in the
fertilization process. After fertilization, it accumulates in fruits and seeds or there is a migration to
leaves, bark and root.
2.1.5 Major uses of Essential Oil

Essential oils are valuable natural products used as raw materials in many fields, including perfumes,
cosmetics, aromatherapy, phytotherapy, spices and nutrition, insecticides (Buchbauer 2000).
Aromatherapy is the therapeutic use of fragrances or at least mere volatiles to cure or mitigate or prevent
diseases, infection and indisposition by means of in halation. Inhalation of essential oils or their
individual volatile terpenes has a significant role in controlling the central nervous s ystem. For instance,
aroma inhibit of storax pill essential oil and pre inhalation of Aconus gramineus rhizome essential oils
are used in Chinese folk medicine in the treatment ofepilepsy (Boukhatem, M. N. 2014).
2.2 Basil Plant

Ocimum basilicum is an erect, almost glabrous herb, which grows to between 30 and 90 cm high. The
leaves are ovate, lanceolate, cucuminate, toothed or entire, glabrous on both surfaces and glandular.
When mature, they reach approximately 5 cm in length, excluding the petiole, which is approximately 2
cm long. The upper surface is smooth and lustrous; on the lower surface along the midrib and on the
petiole short, stiff hairs occur sparingly (Prakash, 1990).
Basil (Ocimumbasilicum L.) is an annual herb, up to 40cm tall which cultivated as a spice 1 000-2600m
altitude. It is widely cultivated in tropical to temperate regions. The plants are more herbaceous and
probably shorter lived, but might despite that be taller and stouter, and have always a strong fragrance
(Ryding, 2006).
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2.2.1 Growth of Basil

Basil requires well-drained soils, with a pH ranging from 6.0-7.5 and sown seed is easily germinate with
optimum temperature 20°C, but the seeds will germinate well between 1 5 to 30 °C in about 7 days after
sowing of seed. Basil requires 500 - 800 mm annual precipitation for optimum growth and development.
Although, basil is cultivated in different climatic and ecological conditions, the most favorable conditions
are found in countries with a warm climate. Light, temperature and moisture are the basic ecological
requirements for sweet basil cultivation. Sweet basil develops best under long days in sunny conditions.
Planting density/spacing high plant populations are recommended for essential oil and dried basil. Basil
grows quickly, and harvests of stems may begin approximately 40-60 days after seedling emergence.
Stems may be clipped off, leaving several sets of leaves on the below the cut. Stems are bunched and sold
individually at farm markets, or placed in 1 5 bunch crates and sold wholesale. Some producers of
essential oils harvest basil only once and then during the full flowering stage. Other producers harvest the
crop just as flowering commences and allows re-growth to have additional harvests during the same
season. 4 up to 14 cuttings are possible that regular harvesting of stems encourages lateral growth and
limits the production of flowers, which are not marketable (Agriculture, Forestry and Fisheries, 201 2).
Figure 2.1 Basil plant
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2.2.2 Basil production in Ethiopia

Ethiopia is a mother of wide agro ecology and suitable for cultivation of many aromatic and medicinal
plant which are traditionally used as medicine and food flavoring spice. For these reason, a number of
basil accessions are cultivated and widely used throughout the country. Even though research work and
information is limited on this crop, farmers of Ethiopia conventionally cultivate and use basil for house
consumption and provide for local market. Demand of basil is high for international market due to its
multipurpose and some cultivated accession of Ethiopia also exported to different countries to fetch
foreign currency. According to Mesresha Yimer, (201 0), export of Basil is mainly destined to Sudan
with 91.4% share of total export value of basil from Ethiopia, and the rest of export goes to Israel (7.4%),
and USA (1 .2%).
2.3 Uses of Basil

 Direct uses of Basil
Basil (Ocimum basilicum L.) is a popular herb grown for the fresh market or for its aromatic leaves, in
which its aromatic leaves are used in fresh or dried forms as drug in traditional medicine and as flavoring
agent in food and confectionary products as well as beverages (Prakash, 1 990; Marottiet al., 1996).
Mainly in Ethiopia fresh and dried and leaves, fresh and dried seeds are using for centuries. For instance
low level families traditionally use basil addition for spices purpose, directly add in common daily food
of Ethiopian Shirowot for flavoring purpose by simply putting fresh matured basil inflorescence with
stalk without any processing which used for replacement of butter and also they believe it serve them as
appetizer in many country of the country.
Table 2.1 Nutritional value of basil per 100 grams (3.5 ounces)
Energy - 94 kilojoules (22 kilocalorie) Carbohydrates - 2.65 grams
Dietary fiber - 1.6 grams Fat - 0.64 grams

Protein - 3.15 grams Water - 92.06 grams
Vitamin A - 264 micrograms Thiamine - 0.034 micrograms

Riboflavin - 0.076 milligrams Niacin - 0.902 milligrams
Vitamin B6 - 0.155 micrograms Folate - 68 micrograms

Choline - 11.4 milligrams Vitamin C - 18.0 milligrams
Vitamin E - 0.80 milligrams Vitamin K - 414.8 micrograms
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Calcium - 177 milligrams Iron - 3.17 milligrams
Magnesium - 64 milligrams Manganese - 1.148 milligrams

Phosphorus - 56 milligrams Potassium - 295 milligrams
Sodium - 4 milligrams Zinc - 0.81 milligrams
Source: USDA Nutrient Database

 Indirect uses of Basil
Basil (Ocimum basillicum L.), the oldest spices belonging to the Lamiaceae family, is commercially and
extensively cultivated for essential oil production in many continents of the world. Oil and oleoresin of
basil are widely utilized for flavor and fragrance in the food, pharmaceutical, cosmetic, and aromatherapy
industries. Basil is one of the most popular aromatic plants and it has been used extensively in
pharmaceutical, cosmetics, aromatherapy and food industry. Medicinally used in the treatment of
malaria, colic vomiting, common cold, cough, headaches, diarrhea, inflammation, pain, skin diseases and
others (Vanacioet al., 2011).
A number of authors have mentioned the biological activity of basil plant on microbial, fungal and insect
repelling properties (Ottaiet al., 201 2). These plants are economically important due to the large quantity
of diverse essential oil compounds that are derived from them. For centuries, basil leaves have been a
very popular spice and also today, basil essential oils are used hygiene and cleaning products, perfumes,
cosmetics and local aesthetics and antiseptics. Most commercial basil cultivars available belong to the
common basil (Ocimumbasilicum L.). Because of the constant human demand for new flavors, many
Ocimumbasilicum cultivars have been bred during the long cultivation of the herb (Carovic-Stankoet al.,
2011).
 Other importance of basil
Research indicates that there may be several health benefits associated with basil. Studies have shown
that basil "contains a wide range of essential oils, rich in phenolic compounds, and a wide array of other
natural products including polyphenols such as flavonoids and anthocyanins. " According to research
conducted at the Swiss Federal Institute of Technology, basil contains (E)- beta-caryophyllene (BCP),
which may be useful in treating arthritis and inflammatory bowel diseases.
Fighting cancer: A review, published in 2013, investigated the potential for holy basil to prevent
cancer. They concluded that, thanks to the phytochemicals it contains, including eugenol, rosmarinic
acid, apigenin, myretenal, luteolin, β-sitosterol, and carnosic acid, it may help prevent certain types of
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skin, liver, oral, and lung cancers. It appears to do this by increasing antioxidant activity, changing gene
expression, triggering cell death, and slowing cell division. Although more research needs to be done,
there is potential for basil extract to be used alongside current cancer treatments.
Reducing the effects of oxidative stress: An adaptogen is a herbal medicine that helps the body adapt to
stress; ginseng is perhaps the most well-known. Basil may also fall into this category.
In one study, rabbits were exposed to oxidative stress (an increase in damaging free radicals). The
rabbits were given 2 grams of fresh basil leaves each day for 30 days, and cardiovascular and respiratory
adaptation was monitored. The researchers measured a significant decrease in blood sugar levels and an
increase in antioxidant activity. The basil appeared to help the rabbits deal better with oxidative stress.
Anti-aging properties: According to research presented at the British Pharmaceutical Conference

(BPC) in Manchester, basil also have properties that might help prevent some of the harmful effects of
aging. Holy basil extract was effective at killing off harmful molecules and preventing damage caused
by some free radicals in the liver, brain, and heart.
Reduce inflammation and swelling: Basil extract may help reduce inflammation. One study, presented
at the Royal Pharmaceutical Society's annual event, found: "Extracts of O. tenuiflorum (Holy basil) were
shown to reduce swelling by up to 73%, 24 hours after treatment.
These effects on swelling were similar in extent to those seen with the drug diclofenac, an anti-
inflammatory medication that is widely used in the treatment of arthritis.
Rich in antioxidants: Results of a study published in the Journal of Advanced Pharmacy Education &
Research showed that ethanol extract – Ocimum basilicum had more antioxidant activity than standard
antioxidants.
Antibacterial properties: Basil may have an antibacterial effect. Lab studies have demonstrated that
basil has antibacterial properties; this may be because of the volatile oils it contains, which include
estragole, linalool, cineole, eugenol, sabinene, myrcene, and limonene. Basil restricts the growth of
numerous bacteria, including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli
O157:H7, Yersinia enterocolitica, and Pseudomonas aeruginosa. This could mean that adding fresh basil
to a salad not only adds flavor, it also helps reduce the number of harmful bacteria on the plate.
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2.4 Basil Essential Oils

2.4.1 Physical properties of basil essential oils
There are several physical property standards which must be fulfilled to maintain the quality of Ocimum
basilicum essential oils (http://www.fao.org//docrep/nutmeg and derivatives). Some of them are:
 Appearance, color, fragrance: Basil essential oil is a transparent fluid with pale-yellow color. It
has a distinctive flavor and fragrance.
 Specific gravity: Specific gravity is an important criterion of the quality and purity of an essential
oil. At 25oC, the specific gravity value of basil essential oil varies between the limits of 0.95200-
0.97300. In general, the specific gravity of essential oil is less than 1 .0 (Guenther, 1 960). Thus,
essential oil can be collected over (floating on) water.
 Refractive index: Refractive index is the ratio of the speed of light at a definite wave length in a
vacuum to its speed in the medium and this varies with the wave length of light. When a ray of
light passes from a less dense to a more dense medium, it bents or “refracted” toward the normal.
The refractive index of Ocimum basillicum oil is between 1.51200-1 .51900 at 20oC.
 Solubility: Ocimum basillicum essential oil is insoluble in water and it is well miscible with hydro
citronellal. However, it can be dissolved in paraffin oil.
2.3.2 Application of Basil Essential Oil

Exhibits Antimicrobial Properties: Basil essential oil is a good safeguard against disease causing
bacteria. It exhibits potential in inhibiting bacterial strains that are immune to antibiotic treatments
according to studies. In fact, a group of researchers from the Medical University of Lodz in Poland can
attest to its potency. They conducted a test involving sick patients and showed that basil essential oil is
highly effective in inhibiting the bacterial activity of E.coli and other strong types of bacteria.
Provides a Number of Skincare Benefits: Basil essential oil’s skincare properties make the oil an
effective remedy for a number of chronic skin conditions such as acne. Acne can result from overactive
sebaceous glands. Accumulation of excess sebum and dirt manifests the growth of bacteria and thereby
produce painful red swellings on your face. Applying a face pack using basil oil on a daily basis is
beneficial for acne-inflamed skin due to its anti-inflammatory and antibacterial properties. You may also
add other ingredients such as lemon juice or rose hip oil to amplify its effects.
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Likewise, basil essential oil can help unclog blackheads and whiteheads to give you a clearer and
smoother complexion. Moreover, basil essential oil’s potent antibacterial properties can remedy
ringworm infections. You can also relieve itchy skin with the assistance of basil essential oil.
Eases Leg Cramps and Sore Muscles: Leg cramps or muscle spasms occur when leg muscles are under
severe stress brought by strenuous activity or due to a prolonged fixed position. Leg cramps are harmless
but it can be very painful and frustrating, especially if it disrupts your good night’s sleep. The pain can
last anywhere from a few minutes to a quarter of an hour. Fortunately, basil essential oil is a natural
analgesic and provides fast relief from pain. You can always keep a bottle of basil essential oil on your
bedside table so you can massage your legs with the oil when needed. Likewise, basil essential oil can
ease tired and sore muscles. It is also helpful in the treatment of arthritis, sprains, and sports injuries.
Aids in Stress Management and Prevent Adrenal Fatigue: Basil is among the best essential oils for
the healing of the mind. In fact, basil has been traditionally used in royal families to strengthen resolve,
provide clarity, and to raise the spirit. These traits of basil are especially beneficial to those who have
debilitating illnesses or are prone to mental fatigue brought by workplace stress. Work stress can be
chronic and takes a toll on your job productivity as well as your general physical and emotional health. A
basil steam can help keep your stress in check. Its aromatic vapors can eliminate brain fog and improve
your concentration so you can perform any task at hand.
Promotes Proper Digestion: Poor digestion can hamper the proper absorption of vitamins and minerals
that our body needs to stay well nourished. Fortunately, basil essential oil promotes proper digestion by
restoring the body’s pH level. By doing so, this helps the good bacteria within the gut to flourish and
improve digestive function.
Before prescription drugs, people use basil oil to treat acid reflux, bloating, stomach cramps, loss of
appetite, as well as kills intestinal parasites. Such theories have already been supported by modern
studies. It shows that basil essential oil exhibits carminative properties that can provide immediate relief
from constipation, indigestion, and flatulence.
Assists in the Treatment of Urinary Tract Infections: Basil essential oil alone has a broad spectrum
antimicrobial property that helps speed up the healing process of such infections. Moreover, basil
essential oil is a diuretic that increase urine production and helps expel excess salt and water from the
body.
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Helps Treat Earache and Mouth Ulcers: Basil essential oil can help cure minor ear infections and
alleviate pain associated with the infection. All you need is a few drops to ear canal. You can also rub
basil essential oil around your ear to reduce inflammation.
Likewise, adding a few drops of basil essential oil can prevent halitosis as it eliminates odor- causing
bacteria. It also helps protect your mouth from ulcers and sores.
Treats Respiratory Ailments: Breathing in the enticing aroma of the basil essential oil can help you get
through cold wintry weathers. As temperatures drop, the air begins to dry and burdens your respiratory
system. Fortunately, basil essential oil exhibits favorable respiratory effects due to its antispasmodic
properties. It helps decongest clogged sinuses, treats asthma, coughs, and bronchitis. Moreover, basil
essential oil also has properties that induce sweat and antipyretic properties that relieve fever.
Basil Essential Oil Fights Allergies: The majority of illnesses are rooted to inflammation. When your
body deals with high inflammation, allergens amplify an increased reaction. Basil essential oil can
diminish the inflammatory response of allergens and lessen your body’s vulnerability to outside sources
to prevent the overreaction of your immune system. Likewise, basil essential oil helps detoxify your body
and flushes out toxins and harmful microorganisms that act as allergens.
Nourishes Hair: Massaging your scalp with basil essential oil stimulates hair growth. It is also very
effective in eliminating excess oil in the scalp, which can lead to dandruff. Likewise, its anti-fungal
properties can treat existing dandruff and prevent it from reoccurring. Basil essential oil also adds luster
while it strips away grease buildup on your hair.
Natural Insect Repellent and Air Freshener: Basil essential oil is the best substitute to harmful
aerosols and expensive air filtration systems. Diffusing basil essential oil is a natural, low-cost, effective,
and therapeutic way to purify and scent your home. Its antibacterial and antifungal properties eliminate
foul odors caused by bacteria and mold; thus, preventing contamination. Likewise, you can effectively
repel mosquitoes using basil essential oil. You can also massage basil essential oil to relieve itch caused
by insect bites.
Generally though basil essential oil is notable for its culinary benefits, it also features a variety of uses for
both mind and body. Its uses extend far beyond the kitchen and can help treat several ailments.
Furthermore, its warm and uplifting aroma can promote a sense of focus and eliminate stress
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2.4 Essential oil extraction technologies

Essential oils can be extracted using a variety of methods, although some are not commonly used today.
The specific extraction method employed is dependent upon the plant material to be distilled and the
desired end product. The essential oils from aromatic plants are for the most part volatile and thus, lend
themselves to several methods of extraction such as steam distillation, solvent extraction, supercritical
fluid extraction, etc.
2.4.1Turbo-Extractor
This method is used for solvent or water-extraction of numerous solid raw materials, mainly vegetal, used
in the perfume, flavor, cosmetic, pharmaceutical, and spirit and food industries. It is energy and time
saving production of natural extracts. The equipment is equipped with a high blade turbine. It is used for
crushing materials within the solvent and fluidizing the comminuted pieces. Extraction is accelerated by
the intimate and turbulent contacts between the solid and the liquid phase. Nevertheless, this type of
equipment is very expensive and not economical.
2.4.2 Microwave Assisted Techniques
Microwave energy is a superior alternative to several thermal applications owing to its efficient
volumetric heat production. The volumetric heating or heating of the bulk as opposed to transferring heat
from the surface, inwards, is more efficient, uniform and less prone to overkill or supererogation.
Controllability is by far the greatest advantage of microwaves over conventional thermal technologies. In
processing applications, the ability to instantaneously shut the heat source makes enormous difference to
the product quality and hence the production economics. The raw material is heated directly by
microwaves and this brings about quality consistency and minimizes the impact on the environment as
opposed to using fossil fuels or less efficient, indirect electrical heating systems. Specifically in the
essential oil extraction, microwave mediated processes are highly desirable due to their small equipment
size (portability) and controllability through mild increments of heating. However, so far the microwave
technology has found application in very few industrial bio-processing installations due to the lack of
available data on microwave interaction with heterogeneous natural raw materials. The sensing and close
control of microwave process is a challenging science and there seems to be insufficient literature in this
regard.
2.4.3 Mechanical Expression

Prior to the discovery of distillation most essential oils were expressed mechanically or cold pressed
(Sellar, 2001). It is the simple process of heating the plant material to low temperatures and then
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physically pressing the essential oil out. Today mechanical expression is used mainly for citrus peels and
is unpopular due to the low extraction yield.
2.4.4 Steam and water distillation
To overcome the drawbacks of the water/ hydro distillation, the technique was modified and wet steam
distillation was developed. The plant material is supported on a cage / perforated grid below which water
is boiled. Direct contact of plant material with hot furnace bottom is thus avoided. The water below the
grid is heated by open fire which produces saturated and wet steam which rises through the plant material
vaporizing the contained essential oil.
Disadvantages:
 Unsafe, time consuming due to low pressure steam, poor quality oil.
 Improper condensation, oil separation incomplete, less recovery.
 Poor material of construction and excessive pollution (Guenther, 1 960).
2.4.5 Water /Hydro distillation

In this method, the charge (which is usually comminuted) is immersed totally in boiled water. The stills
are of the simplest type and are used extensively by smallholder producers of essential oils (Guenther, 1
972). Often they are heated over an open fire.
Disadvantages of the Hydro Distillation

 The process is slow and the distillation time is much longer thereby consuming more fuel
making process uneconomical.
 Variable rate of distillation due to difficult control of heating.

 Extraction of the herb is not always complete.
 As the bottom walls of the still comes in direct contact with the fire from furnace there is a
possibility of adjacent plant material getting charred and thus imparting an objectionable odor
to the essential oil.
 Prolonged action of hot water can cause hydrolysis of some constituents of the essential oils
such as esters, which reacts with the water at high temperatures to form acids and alcohols.
 Not suitable for large capacity / commercial scale distillations and not suitable for high Boiling
hardy roots / woody plant materials (Guenther, 1 960). However, it is necessary for the efficient
distillation of certain woody materials e.g. sandalwood and cinnamon barks (Noor Azian, 2001).
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2.4.5 Steam Distillation

The vast majority of essential oils are produced by steam distillation. There are, however, different
processes that are used. In all of them, water is heated to produce steam, which is used to extract the most
volatile aromatic chemicals. The steam is then cooled (in a condenser) and the resulting distillate is
collected. The essential oils will normally float on top of the hydrosol (the distilled water component) and
may be separated off. Steam distillation is the most commonly used method for extracting essential oils.
Many traditional distillers favor this method for distilling most oils as they claim that none of the newer
methods produces better quality oils (Boucard et al., 2005). Steam distillation, as described by Boucard et
al. (2005), is carried out in a still in which fresh or sometimes dried plant material is placed in a chamber
of the still. Pressurized steam, generated in a separate chamber, is then circulated through the plant
material. The heat of the steam forces open the tiny intercellular pockets in which the essential oils are
contained releasing the oils. During steam distillation, the temperature of the steam must be moderated so
that it is high enough to open the oil pouches without destroying the plants, fracturing or burning the
essential oils as has been recommended in the literature (Sheridan, 2000). Some or most essential oils
have been found to be heat sensitive and hence thermo degradable. As the tiny droplets of essential oils
are released, they evaporate and mingle with the steam, travelling through a pipe into a condenser. The
steam and oil vapor are then condensed to a liquid mixture. As the oil-water mixture has been found to be
nearly immiscible at a temperature lower than about 65 (Sheridan, 2005). The mixture can be separated
using various gravity related techniques. Due to the immiscibility of the oil and water at low temperature,
the essential oil can be separated from the water by either decanting off the water or skimming of the oil
from the top, as the oil is less dense than water at these conditions. The density of some essential oils
such as lavender oil has been reported to average 0.89g /L, as opposed to lg/L (Ndou, 1986) for water at
room temperature and atmospheric pressure conditions. The water obtained as a byproduct of distillation
is referred to as floral water or distillate and retains many of the therapeutic properties of the plant. For
this very reason, floral waters are valuable in skin care for making facial mists and toners and are also
preferred to essential oils when treating a sensitive individual or child or when a more diluted treatment is
required (Sheridan, 2000).
2.4.6 Solvent Extraction

The purpose of distillation is to separate a mixture of several ingredients by taking advantage of their
different volatility, or to separate volatile ingredients of a raw material from its non-volatile parts. If the
final product is too sensitive to heat or humidity, solvent extraction could be used. Solvent extraction is
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adapted in producing essential oils generated by some flowers (Rose, Violetta, and Geranium), gums and
resins. The raw material is placed in a glass, vessel and soaked with a suitable solvent (petroleum, ether
or benzene). After the extraction, the solids are separated from the liquid mixture. The latter is heated so
that the more volatile essential oils can be evaporated to be subsequently condensed. Alternatively if the
solvent is more volatile, such as ethanol, it could then be vaporized leaving behind the essential oils
(Ndou, 1 986). As solvent extraction uses very little heat, it is found to be advantageous in producing
essential oils with whole fragrances that would otherwise be destroyed or altered during steam
distillation. Therefore, this extraction technique can be used to extract essential oils from very delicate
plants to produce higher amounts of essential oils at lower costs (Ndou, 1 986). There are, however, some
disadvantages associated with the solvent extraction technique. Solvent residues often contaminate the
product causing side effects which make the use of essential oil undesirable for skin applications but
could still be fine for fragrances or perfumes (Ndou, 1 986). Therefore, with solvent extraction effective
separation of the extracted oil from the solvent is necessary to remove any solvent which may
contaminate the essential oils.
2.5 Solvent Extractor (Soxhlet Apparatus)

Soxhlet extractor is a piece of laboratory apparatus invented in 1 879 by Franz von Soxhlet. It was
originally designed for the extraction of lipid from a solid material. Typically, soxhlet extraction is used
when the desired compound has limited solubility in a solvent, and the impurity is in soluble in that
solvent. It allows for unmonitored and unmanaged operation while efficiently recycling a small amount
of solvent to dissolve a large amount of material. A Soxhlet Extractor has three main sections: A
percolator (boiler and reflux) which circulates the solvent, a thimble (usually made of thick filter paper)
which retains the solid to be laved, and a siphon mechanism, which periodically empties the thimble.
The Soxhlet method is the most commonly used semi-continuous process for the extraction of lipids from
foods. According to Soxhlet procedure, oil and fat from solid material are extracted by repeated washing
(percolation) with an organic solvent, usually hexane or petroleum ether, ethanol. The hexane was used
for the purpose of solvent. The grounded basil leaves samples were placed in a filter paper. The filter
paper is then placed in an extraction chamber which is being suspended above a flask containing the
solvent and below a condenser.
Heat is being applied to the flask and the solvent evaporates and moves to the condenser where it is
converted into liquid that trickles in to the extraction chamber containing the sample. The extraction
chamber is made in such a way that when the solvent surrounding the sample exceeds a certain level it
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over flows and trickles back down in to the boiling flask. The flask containing solvent and lipid is
removed at the end of the extraction process. This cycle may be allowed to repeat many times, over hours
or days. During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many
cycles the desired compound is concentrated in the round bottom flask. The advantage of this system is
that instead of many portions of warm solvent being passed through the sample just one batch of solvent
is recycled.
After extraction, the non-soluble portion of the extracted solid remains in the thimble, and is usually
discarded. The mixture of solvent and essential oil has been separated by means of a rotary evaporator,
and then the essential oil was used for further characterization of analysis and other applications but
solvent has been used other extraction process (recycling) operation.
Assembly Soxhlet apparatus; (Source: http:// www.olroresins melbia.com.)

1 .The source material containing the compound to be extracted is placed inside the thimble
2. The thimble is loaded into the main chamber of the Soxhlet extractor.
3. The extraction solvent to be used is placed in a distillation flask.
4. The flask is placed on the heating element.
5. The Soxhlet extractor is placed atop the flask.
6. A reflux condenser is placed atop the extractor.
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Figure 2.2 Soxhlet apparatus
2.6 Factors Affecting Solvent Extraction of Basil Essential Oil

Essential oils have increasing importance in flavor and fragrance industries. In order to produce oil with
market potential its optimum production parameters have to be well known prior to its commercial
production. Essential oils are very complex products. Each is made up of many, sometimes hundreds, of
distinct molecules which come together to form the oil's aroma and therapeutic properties. Some of
these molecules are fairly delicate structures which can be altered or destroyed by adverse
environmental conditions. So, much like a fine meal is more flavorful when made with patience, most
oils benefit from a long, slow 'cooking' process (Satish, 2010). A number of factors determine the final
quality of a steam distilled essential oil. Apart from the plant material, most important factors are
extraction time, steam temperature, operating pressure, the steam flow rate, the quality of the distillation
equipment, the particle size and the condition of plant materials.
The selection of the equipment for an extraction process is influenced by the factors which are
responsible for limiting the extraction rate. Thus, if the diffusion of the solute through the porous
structure of the residual solids is the controlling factor, the material should be of small size so that the
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distance the solute has to travel is small. On the other hand, if diffusion of the solute from the surface of
the particles to the bulk of the solution is the controlling factor, a high degree of agitation of the fluid is
required (Coulson et al.,2002).
2.6.1 Particle Size

Particle size influences the extraction rate in a number of ways. The smaller the size, the greater is the
interfacial area between the solid and liquid, and therefore the higher is the rate of transfer of material
and the smaller is the distance the solute must diffuse within the solid as already indicated. On the other
hand, the surface may not be so effectively used with a very fine material if circulation of the liquid is
impeded, and separation of the particles from the liquid and drainage of the solid residue are made more
difficult. It is generally desirable that the range of particle size should be small so that each particle
requires approximately the same time for extraction and, in particular, the production of a large amount
of fine material should be avoided as this may wedge in the interstices of the larger particles and impede
the flow of the solvent.
2.6.2 Solvent
The liquid chosen should be a good selective solvent and its viscosity should be sufficiently low for it to
circulate freely. Generally, a relatively pure solvent will be used initially, although as the extraction
proceeds the concentration of solute will increase and the rate of extraction will progressively decrease,
first because the concentration gradient will be reduced, and secondly because the solution will generally
become more viscous.
2.6.3 Extraction time

Oil yield obtained (expressed in percent) is extraction time dependent. In general, the oil yield increased
with increase in extraction time.
2.6.4 Solute to solvent ratio

Solute to solvent ratio is another important parameter that affects oil extraction efficiency. The volume
of the solvent should not be more than an optimized volume because the cost of the solvent recovery
could be too high, thereby increasing the total operational cost.
2.6.5 Agitation of the Fluid

Agitation of the solvent is important because this increases the eddy diffusion and therefore the transfer
of material from the surface of the particles to the bulk of the solution. Further, agitation of suspensions
of fine particles prevents sedimentation and more effective use is made of the interfacial surface. In most
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cases, the solubility of the material which is being extracted will increase with rial which is being
extracted will increase with
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3. MATERIALS AND METHODS
3.1 Materials
 Raw materials
The main raw material used for our study is basil leaf (Ocimum basillicum L.)
 Equipment’s:
Table 3.1 the main materials / equipment’s required to conduct our research.
No. Equipment’s Purpose
1 Electronic weight balance Measuring the weight of the sample
2 Rotary evaporator Separation of essential oil from the solvent
3 Beaker Used to store, mix and heat liquids in
laboratories.
4 Sieve Separation of desired size of sample

5 PH meter Measuring the acidity and basicity
6 Oven For drying the sample
7 Spectrometer for measuring refractive Index
 Chemicals used
Hexane Phenolphthalein
Ethanol sodium theosulphate
Sodium hydroxide carbon tetra chloride
Hydrochloric acid potassium iodide solution
Starch
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3.2 Methodology
3.2.1. Collection and preparation of sample
A bulk sample of Basil leaves was collected from Akaki market (gebeya). Which is in Addis Ababa
Akaki kality sub city, on December 2020. Akaki is located at altitude 8.895831 and longitude
38.789161 in the southern hemisphere and having an elevation of 1500 to 2300 m above sea level.
(www.geodatos.net)
Figure 3.1 fresh basil leaves
3.2.2 Treatment of the Basil leaves

A sample of Basil leaves was collected from the market. Then it’s washed to remove dusts and other
un-necessary materials. Sample 1 is exposed for two full sunny day drying period and sample 2 is
dried by oven at 69 0c. After completion of drying, the leaves were re-weighed to determine any
potential mass loss as a result of the evaporation of moisture due to the drying effect.
3.2.3. Size reduction

Once the moisture of the leaves is removed by drying the basil leaves was crushed using grinder
which grinds the material into smaller size in order to increase the contact area of the leaves for
maximum extraction of the oils (Coulson J.M. and Richardson J.F 2002).
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a b
Figure 3.2 a) Grinded basil powder and b) sieved basil powder
3.2.4. Determination of moisture content of the Basil leaves

Four samples of the basil leaves were taken, weighted and dried in sun light and the weight was
measured after two days of full sunlight. The percentage moisture content of the basil leaves was
determined using the following formula (General Health Welfare, &Delhi, 2005).
Moisture content % = …………… eqn 3.1
Where: W1= original weight of the sample before drying
W2 = weight of the sample after drying
3.2.5 Experimental set up

In solvent extraction of the plant material was packed into the extraction chamber so that extraction
could commence. For each load, plant materials were placed into the extraction chamber. The first
load was conducted to set-up and establishes the procedure and determines processing parameters. The
experiments of this study were conducted in the laboratory of chemical engineering department in
Addis Ababa science and Technology University.
The experimental setup of the apparatus that was used for the extraction of the essential oil from basil
leaves by means of solvent extraction method for this specific study is shown in figure below.
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Figure 3.3 Experimental set up of Soxhlet apparatus
3.2.6 Experimental Procedures

From the prepared sample, about 50g was weighed and placed in a carefully folded filter paper. The
filter paper containing the sample then inserted into the Soxhlet apparatus. The weight of the filter
paper and sample was recorded. A 660 ml of solvent (n-hexane) was measured using a measuring
cylinder and then poured into reboiler (bottom flask) of the Soxhlet apparatus and heated at a
temperature of about 690C. When the solvent is boiling, the vapor was raised through the vertical tube
into the condenser at the top. Then the liquid condensate was drips into the filter paper, which
contains the oil to be extracted. The extract seeps through the pores of the filter paper and was filled
the siphon tube, where it flows back down into the round bottom flask. It was then removed from the
tube. The experiment was repeated by varying parameters (extraction time at 2hr, 3hr, 4hr, at
temperature of 69 oc, particle size of bellow 1mm and mass to volume of solvent ratio of 0.1, 0.075
and 0.05. At the end of the extraction, the resulting mixture containing the oil was passing through a
rotary evaporator to recover solvent from the oil. The oil extracted was then stored in a plastic
container and characterization was performed. In our case we separate the oil from the solvent
mixture by direct evaporation of the hexane at its boiling temperature.
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Figure 3.4 Basil leaves oil inside filter paper
3.3 Characterization of Extracted Basil Essential Oil

The yield of essential oil, specific gravity at 25 0c, density, pH value, refractive index and color were
analyzed using standard procedures. The acid value, saponification value and peroxide value of basil
essential oil were performed and compared with literatures.
3.3.1. Determination of physical properties

3.3.1.1 Percentage of basil leaves essential oil extracted
The percentage yield of basil leave essential oil was calculated using two formulas, i.e. percentage of
oil yield and percentage of extraction yield.
Percentage of extraction Yield ……eqn 3.2
3.3.1.2. Specific gravity of essential oil

The density of the oil was determined by using density bottle. A clean and dry empty bottle of 25ml
capacity was weighed (W1) and then the bottle was filled with the oil, stopper inserted and reweighed
to give (W2). The oil was substituted with water after washing and drying the bottle and weighed to
give (W3) (General Health Welfare & Delhi, 2005). The expression for specific gravity (Sp.gr) is:
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Specific gravity (Sp.gr) ………eqn 3.3
3.3.1.3 PH value of essential oil

PH value of the extracted essential oil was measured directly using PH meter.
3.3.1.4. Refractive index

The refractive index of a substance is the ratio of the speed of light in a vacuum to the speed of light
in the substance. For practical measurements, including this method, the scales of standard
instruments indicate refractive indices with respect to air rather than vacuum. Refractive Index of the
sample was measured by refractometer. The index of refraction of oils is characteristic within certain
limits for each kind of oil. It is related to the degree of saturation particularly to the extent of
conjugation, but it is affected by other factors such as free fatty acid content, oxidation, and heat
treatment.
3.3.2 Characterization of the chemical property of Basil essential oil

3.3.2.1 Determination of saponification value
Saponification value or saponification number (SV or SN) represents the number of milligrams of
potassium hydroxide (KOH) required to saponify one gram of fat under the conditions specified. It is
a measure of the average molecular weight (or chain length) of all the fatty acids present in the
sample as triglyceride (shttps://en.wikipedia.org/wiki/Saponification_value).
0.5g of the oil will be placed in a conical flask to which 25ml of ethanoic potassium hydroxide (0.1M)
will be added and the mixture allowed boiling gently for about 1hr. With shaking, at regular intervals
of 5 min. Few drops of phenolphthalein indicator, as specified by International Standards
Organization (ISO 3657, 1988) will be added to the warm solution and then titrated with 0.5M HCl.
The end point will be reached when the pink color of the indicator just disappeared. The same
procedure will be followed for the blank. The saponification value (SV) is given by:
SV=56.1 × …….eqn 3.4
Where: VO = volume of HCl solution used for the blank test, VI = volume of HCl solution for the
determination, N = actual molarity of HCl used, and m = mass of sample
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3.3.2.2 Determination of Peroxide Value

Detection of peroxide gives the initial evidence of rancidity in unsaturated fats and oil. It gives the
measure to which an oil has undergone primary oxidation. Peroxide value, concentration of peroxide in
an oil or fat, is useful for assessing the extent to which spoilage has advanced. The peroxide value
points out the state of oxidation of a substance. If the oxidation proceeds for long, it makes the oil
rancid and gives the oil an unpleasant smell.
Procedure:
To prepare acetic acid and chloroform solution, mix in ratio of 2:3, 90 ml of acetic acid mix with 60
ml of chloroform. To prepare 0.01N of sodium thiosulfate, add 0.25 gram sodium thiosulfate in 80 ml
of distilled water and heat the solution to facilitate the solubility. To prepare saturated potassium
iodide, dissolve potassium iodide crystal in 2ml of distilled water until its maximum solubility limit.
Measure 0.5 gram of basil oil in Erlenmeyer. Then add 30 ml of acetic acid chloroform mixture the
sample. Again add 1 ml of saturated potassium iodide solution and shake to mix it well. Then add 30
ml of distilled water and shake again. Take 0.01 N sodium thiosulfate in the burette and record initial
reading. Titrate the sample mixture by 0.01 N sodium thiosulfate after adding starch indicator to the
sample solution.
End the titration when the black color change to white.
Peroxide Value ………..eqn 3.5
N = normality of sodium thiosulfate, Ws = weight of sample oil, V = volume of titrant used
3.3.2.3 Determination of Acid Value or Acid Number

Acid value is the mass of potassium hydroxide (KOH) in mg that is required to neutralize one gram of
chemical substance. The acid number is a measure of the amount of carboxylic acid groups in a
chemical compound. The acid number is used to quantify the amount of acid present, in basil
essential oil sample. Acid value was determined according to the method of European Pharmacopeia.
Basil essential oil (0.8g) was accurately weighted and dissolved in 10ml of 95% ethanol and 2-3
drops of phenolphthalein indicator was added. The mixture was then titrated with standard 0.1 N
aqueous sodium hydroxide solutions by adding the alkali drop-wise at a uniform rate. The content of
the flask was continuously agitated. The primary manifestation of the red coloration that did not fade
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within 10 seconds was considered the end point. Afterward, the acid value was determined using the
following equations:
Acid value = MWNaOH ……eqn 3.6

3.4 Experimental design
Table: 3.4 Factors and respective ranges of the experiments
Factors Name Unit Lower range Medium range Higher range
1 Extraction Hours 2 3 5
time
2 Solvent ratio g/ml 0.05 0.075 0.1
Number of experiments in 3 level full factorial with 2 factors, 1 replication,
N=K*Y n
Where N is total number of expected experiments
K is number of replica
Y is number of level and
n is number of factors
N=1*3 2 = 9 We
have designed to conduct 9 experiments.
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4. RESULT AND DISCUSSION
4.1 Determination of moisture content of basil leaves

Two samples of basil leaves were collected at different times for moisture content analysis. Then, the
moisture content was calculated using equation (3.1). Sample 1 was dried using oven and sample 2 was
dried by sun.
Table 4.1 Moisture content of fresh basil leaves.
Initial mass (g) Drying time (hr) Final mass (g) Moisture contents
Samples (%)
1 552.6 24 107.4 80.6
2 621.4 22 136 78.114
From the table the average moisture content is 79.357
From the above experimental result the moisture content of the basil leaves has some variation from
that of literature. The percent of moisture content of the basil leaves on literature is 82-83%. The
mean average moisture content of the two basil leaves sample is found 79.357%. The variation may
come from difference in drying time, drying temperature (since we have used oven drying in case of
sample 1) and even the variation may come due to different harvesting stage of the basil leaves. When
we buy the basil leaves, it was in the second or fruit stage which is not our desired sample.
4.2 Percentage yield oil

Designed experiments were carried out to map quantitative effects of the two parameters these are
extraction time and solute to solvent ratio. The yields of the extraction is calculated from the relation
between the essential oil mass obtained and the raw material mass used in the extraction
(Y=extract/raw material %). The temperature of the heater for consecutive tests was set at 69 oC. The
yield of oil extracts of basil leaves calculated using equation (3.2).
4.2.1 Effect of Extraction Time on Essential Oil Yield

Table 4.2 Yield of basil essential oil by solvent extraction.
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Weight of Extraction time Solute/ solvent Mass of oil (g) Yield (%)
Run Sample (hr) Ratio (g/ml)
(g)
1 50 4 0.075 3.2 6.4
2 50 3 0.075 2.65 5.3
3 50 2 0.075 2.4 4.8
From the above experiment we can see that as the extraction time increase the yield of the oil increase.
At time t= 2hrs the yield was 4.8 % but at time t= 4 hrs the yield become 6.4 %.
4.2.2 Yield of essential oil for variable solute to solvent ratio

The temperature and the extraction time is kept constant which is 69 0c and 4 hrs respectively. The
following result was obtained based on eqn 3.2.
Table 4.3 Yield of essential oil for variable solute to solvent ratio
Weight Solute/solvent Extraction Mass of oil Yield (%)
of (g)
Run (g/ml) Time (hr)
Sample
(g)
1 50 0.05 4 2.9 5.8
2 50 0.075 4 3.2 6.4
3 50 0.1 4 3 6
The above experiments were conducted by varying the extraction time and solute to solvent
ratio. The maximum yield obtained was 6.4 % and we have selected it as the optimum value at 4
hours extraction time and medium range of solute to solvent ratio. The yield we have obtained is
high enough. But ones should understand us this oil may requires further purification to make it
clear.
So the yield may less from this value.
4.3 Characterization of oil
4.3.1 Density and Specific gravity of oil
An empty washed and dried sample holder was weighed on the top load weighing balance. The
weight of the sample holder was recorded. The volume of each of the oil sample were measured and
January 20, 2021 40

pour into the sample holder and weighed. Then the density of the oil can be obtained by taking the
ratio of the mass per unit volume of the oil. Based on eqn 3.3 the following results are obtained. Table
4.4 Average density and specific gravity of essential oil
Run Volume Mass Density Density of Specific
of oil (ml) of oil water
of oil (g/ml) gravity of
(g)
(g/ml)
oil
1 3.5 3.2 0.93 0.99 0.932
7
2 2.9 2.65 0.89 0.99 0.893
7
3 2.6 2.4 0.92 0.99 0.923
7
From this the average density and specific gravity that we have obtained is 0.913 and 0.916
respectively. The standard specific gravity of basil essential oil obtained from varies between the
limits of 0.95200- 0.97300 and from our experiment we have got 0.916 SG. This variation
indicates the presence of impurities in the oil. But as we see the variation is to small so the
standard value could be achieved by removing those impurities.
4.3.2 Color
Color of the extracted basil essential oil was greenish black. This is slightly different from the
standard color which is greenish yellow color. This is due to different factors. The use of
matured part of leaves, moisture content of powder and operating temperature may be the reason
for variation of oil color. Besides, the proportion of solute and solvent is a critical factor for oil
color. Thus, as it has been observed from the result at low solute and solvent proportion the color
becomes more greenish black.
4.3.3 PH
PH value of the extracted essential oil was measured directly using PH meter and it is recorded as
average of 6.18. The standard PH value of essential oil is ranging from 5.7 to 7.4. so the result
confirms with the literature value.
4.3.4 Refractive index of the oil

The following results are recorded from refract meter
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Table 4.5 Refractive index

Run Solute/solvent(g/ml) Extraction time Refractive index
1 0.005 2 1.33887
2 0.075 3 1.45818
3 0.1 4 1.45929
From this the average refractive index is 1.41878.
The refractive index of Ocimum basillicum oil obtained from literature is ranging between 1 .51 2001 .
51 900 at 20oC. But our result shows 1.41878. This variation also due to the presence of impurities.
4.3.5 Acid Value or Acid Number

Based on equation 3.6
Acid value = MWNaOH ……eqn 3.6

Acid value = (40 * 0.1 * 0.8)/0.8
Acid value = 4 Mg KOHJg
Acid value refers to the amount of "free" fatty acids in the oil. higher acid value, materials allow
faster appearing but less stable suds creation and Lower acid value materials allow slower to appear
but more stable suds formation. Our result shows the acid value is small and it confirm with the
literature value.
4.3.6 Peroxide value
Peroxide Value = ×𝐍 ∗𝐕∗𝟏𝟎𝟎𝟎

………..eqn 3.5 Peroxide Value
𝑾𝒔
= 8 MeqO2Ikg oil
There is no official maximum limit in peroxide value for essential oils but we can note that vegetable
oils (theoretically consumed in much larger quantities) must have a peroxide value under 10-15. The
peroxide value the degree of primary oxidation. The higher the peroxide mean the oil likeness of
becoming rancid. But our value shows it’s small and so have good shelf life.
4.3.7 Saponification value

After the 0.5M KOH and 0.5g of oil was titrated the result for the solvent hexane and the pink
color was disappeared when the titrate volume reaches at 14 ml from 25 ml and saponification
value of oil can be calculated based on eqn 3.4
January 20, 2021 40

SV=56.1 ×
SV = SV=56.1 ×0 .
SV
= 28.05 mgKOH/g
Saponification value refers to the amount of esters that can be hydrolyzed and turned into soap.
Lower saponification mean the oil has low proportion of fatty acids. So the above results show
that the basil essential oil has lower saponification value that mean this oil is not suitable for soap
making. This behavior has its own advantage for nutrition.
5.0 CONCLUSION AND RECUMENDATION
5.1 Conclusion
The work was intended to study on the possibility of oil extraction from the basil leave and the
influence of the extraction parameters such as extraction time and proportion of solute to solvent on
the yield of the oil. In this study, based on the analysis of experimental results. The maximum yield of
oil was achieved under the optimum conditions at extracting time of 4 hour and at medium solute to
solvent ratio which is 0.075. The yield at this point is about 6.4 %.
The observed some quantitative and qualitative variation of the oil from the standard value was due to
the operating parameters like extraction time, amount of solvent and sample size. Thus, determination
of the appropriate optimum parameters for the recommended particle size needs to have a
consideration to get the maximum amount of the required product.
January 20, 2021 40

5.2 Recommendation
Based on this research findings, the following recommendations are forwarded.
 Production technology is an essential element to improve the overall yield and quality of essential
oil. Therefore, using more efficient methods of extraction is recommended to extract more yields
with good quality.
 Once the required activities of the extraction process have been accomplished, further treatments
are recommended to be taken on the extracts before using the essential oil for cosmetics, food,
pharmaceutical and etc. the followings are also our recommendation.
 Comparing to exporting the raw basil leaves, extracting essential oils can save more the country’s
foreign currency and hence the production of essential oils could still be a good source of foreign
exchange revenue for our country.
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 The development of the essential oils industry is therefore important to our country which has rich
resources of raw materials (basil plant) and the climatic conditions for the initiation of crop wise
cultivation programs.
 Further study should be conducted on the quality of the basil leaves essential oil obtained at each
operating conditions using GC-MS. This will provide alternative to manufacturers on the
minimum conditions required to get a specific quality of basil leave essential oil.
 Further detail research on feasibility studies are required before implementing this research result.
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Kuma Endale Thesis

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ADDIS ABABA SCIENCE AND TECHNOLOGY UNIVERSITY

COLLEGE OF BIOLOGICAL AND CHEMICAL ENGINEERING

DEPARTMENT OF CHEMICAL ENGINEERING

Thesis Title: Extraction and Characterization of Essential oil

Advisor: Mr.Yoseph Asrat

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Figure 2.1 Basil plant…………………………………………………………………………….10

Figure 2.2 Soxhlet apparatus……………………………………………………………………..21 Figure

3.1 fresh basil leaves……………………………………………………………………...25

Figure 3.2 a) Grinded basil powder and b) sieved basil powder………………………………...26

Figure 3.3 Experimental set up of Soxhlet apparatus……………………………………………27

Table: 3.4 Factors and respective ranges of the experiments……………………………………31

Table 4.1 Moisture content of fresh basil leaves………………………………………………...32

Table 4.2 Yield of basil essential oil by solvent extraction……………………………………...33

Table 4.4 Average density and specific gravity of essential oil…………………………………34

Table 4.5 Refractive index……………………………………………………………………….35 LIST OF

EDA Electro-dermal activity

O.Basilicum L Ocimum basilicum Leaves

BPC British Pharmaceutical Conference

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1.3.2 Specific objective ........................................................................................................... 3

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3. MATERIALS AND METHODS .............................................................................................. 24

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1.2 Statement of the Problem

1.3.1 General objective

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(Ocimum basillicum L.)

1.3.2 Specific objective

1.4 Significance of the Study

1.5 Scope of the Study

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2.1. Essential oil

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2.1.1 The unique characteristics of essential oil

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2.1.2 Essential oils in tropical countries

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2.1.3 Chemical Constituents of Essential Oils

2.1.4 Biosynthesis of Essential Oils

2.1.5 Major uses of Essential Oil

2.2 Basil Plant

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2.2.1 Growth of Basil

Figure 2.1 Basil plant

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2.2.2 Basil production in Ethiopia

2.3 Uses of Basil

Dietary fiber - 1.6 grams Fat - 0.64 grams

Vitamin A - 264 micrograms Thiamine - 0.034 micrograms

Vitamin B6 - 0.155 micrograms Folate - 68 micrograms

Vitamin E - 0.80 milligrams Vitamin K - 414.8 micrograms

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Calcium - 177 milligrams Iron - 3.17 milligrams

Magnesium - 64 milligrams Manganese - 1.148 milligrams

Sodium - 4 milligrams Zinc - 0.81 milligrams