Addis Ababa University

Addis Ababa institute of technology

School of chemical and bioengineering

Chemical Product design and development second report

Tittle: production of weevils protection from V.amygdalina essential oil and

D.limonene extracted from orange peel.

Section.A Group 4

Done by: Name id no

1. Gelana Mideksa ETR/2922/08

2. Abdela Chilot ATR/0609/08
3. Asres Yityaw ATR/8780/08
4. Melese Yemer ATR/ 5879/08
5. Ebbisa Tolasa ATR/7177/08
6. Nafiriya Hinsarmu ATR/7749/08
7. Azeb Belay ATR/8759/08
8. Efriam Tsegay ATR/1570/08
9. Tsega Alena ATR/2212/08

Submission date: January 31/2020

Submitted to :mr.Solomon and mr. Getiye

 1. Introduction

The essential oil is the product obtained from a vegetable raw material, either by steam
distillation or by mechanical processes from the epicarp of Citrus, or “dry”” distillation. The
essential oil is then separated from the aqueous phase by physical means. Essential oils are
soluble in alcohol, ether, and fixed oils, but insoluble in water. These volatile oils are generally
liquid and colorless at room temperature. They have a characteristic odor, are usually liquid at
room temperature and have a density less than unity, with the exception of a few cases
(cinnamon, sassafras, and vetiver). They have a refractive index and a very high optical activity.
These volatile oils contained in herbs are responsible for different scents that plants emit (Wissal
Dhifi, 2016).

Pesticidal or Insecticidal activity of v.amygdalina essential oil

V.amygdalina powder possessed pesticidal activity against Sitophilus zeamais and

Callosobruchus maculates. The principle of this effect may be due to the presence of
hydrocyanic acid and oxalic acid. However, concentrated extract was recommended to give
better response. One to five grams of V. amygdalina dry leaf powder was able to control the
growth of S. zeamais and Sitophilus oryzae on stored maize grains and rice(Swee Keong Yeap,
et al; 2010).

The essential oils extracted from v.amygdalina plant are a mixture of monoterpenes and
sesquiterpenes. 1, 8 Cineole occurred in the largest quantity (25.11%). This is followed by Beta
pinene (14.54%), Myrtenal (6.52%), Transpinocarveol (6.24%), Alpha pinene (4.93%) and
Linalool (4.28%). 1,8-Cineole showed stronger toxicity, and anisole as well as eugenol exhibited
the same level of fumigant toxicity as the essential oil against maize weevils (Asawalam, 2006;
Meirong Quan et al, 2018).

D-Limonene Toxicity

D-Limonene has very low toxicity in mammalian species. A number of studies have addressed
citrus oil and d-limonene toxicity toward insects. In their undistilled form, citrus oils have
exhibited topical toxicity against the cowpea weevil (Callosobruchus maculatus), rice weevil
(SiCophilus oryzae), house fly (Musca domestica), and red imported fire ant (Solenopsis
LnvLcta). Smith (1965) found that, of several ponderosa pines monoterpenoid vapors tested,
limonene vapors were the most toxic to western pine beetles, Dendroctonus brevicomis. In
similar studies, Coyne and Lott found topical applications of limonene to be toxic to the southern
pine beetle, Dendroctonus frontalis. Taylor and Vickery (1974) noted that limonene exhibited
contact toxicity in flies (Musca spp.), black flies (Simulium damanosum), mosquitoes (Culex and
Anopheles spp.), and cowpea weevils (Laura L. Karr Iowa State University, 1989).

2. Production of the active ingredients

The main active ingredients that we must have to develop our product are essential oil extracted
from v. amygdalina plant and d-limonene extracted from orange peel. The v.amygdalina leaves
are just collected from the plant by the help of scissors. They are dried before introduced into
steam distillation. Drying plant’s leave before distillation could affect essential oil yield and
composition of the oil considerably. The essential oil components are however, evaporated or
some oxidation products can also appears during drying, therefore the method of drying is very
important. For our process the leaves are air dried in well-ventilated area for five days to
increase the concentration of essential oil by removing the more volatile components. The
components of this essential oil that we need for weevil protection are less volatile and do not
removed from the leaves at room temperature. 1,8cineole is the main component of this essential
oil and its greater yield obtained when the leave is dried in air. The dried leaves are grinded by
using 1mm sieve size, measured and then introduced into steam distillation.

The orange peel is also collected and air dried in well-ventilated area for 5 days for extraction
of D-limonene. D-Limonene is a colorless liquid aliphatic hydrocarbon classified as a cyclic
monoterpene, and is the major component in the oil of citrus fruit peels. It is less a volatile
compound that can be obtained in higher quantity when the orange peel is dried in air. The
reason why we dry the leaves is to remove the more volatile components that may affect the
efficiency of separation by reducing the rate of heat transfer. These more volatile components
may need additional separation techniques and reduce the quantity of needed product. The dried
peel is then grinded at 1mm size and introduced into the steam distillation.
2.1 Extraction of essential oil from v.amygdalina plant by steam distillation

Steam distillation method is used for the extraction of essential oil from v.amygdalina plant. The
essential oil is extracted by this steam distillation using Clavenger apparatus. The condensing oil
is collected in n-hexane solvent. It is allowed to condense in n-hexane solvent because of the
density of essential oil is close to the density of steam (water). The first higher component of this
essential oil is 1,8 cineole which has density 922kg/m3 and the third higher component, Myrtenal
has 987kg/m3 of density. These components are soluble in n-hexane. The density of n-hexane
(655kg/m3) is much less than density of water (1000kg/m3). Therefore the essential oil dissolved
in n-hexane is taken to the top of the cyclone and separated by density difference.

The solution will be filtered with a filter paper containing anhydrous sodium sulphate in a funnel
to remove any remaining traces of water. Hexane will be then removed by distillation at 60 ºC
from 'Contes' Short Path distillation apparatus. When condensation stopped, the oil was collected
and weighed into small amber- coloured vials.

Figure 1. Flow diagram for extraction of essential oil from v.amygdalina

2.2 Extraction of D-limonene from orange peel plant by steam distillation

In the same way to v.amygdalina essential oil D-limonene is also extracted by steam distillation
from orange peel. Since the density difference between the D-limonene and water is higher it is
not necessary to add n-hexane on cyclone. The condensate is stored in cyclone (oil separator) and
essential oil is easily separated by density difference. Then trace of water is removed by using
filter paper containing anhydrous sodium sulphate and the solution is weighed into small amber-
coloured vials.

Figure 2. Flow diagram for extraction of D-limonene from orange peel

The active ingredients of our product are the v.amygdalina essential oil and D-limonene
extracted by above techniques. They have different density. But since both of them are
hydrophobic no need of adding any emulsifier to mix them.

3. Additives

The essential oil by itself has its own preservative component. Therefore it is not necessary to
add any preservative. The carriers that must be added into the mixture to ensure the uniform
distribution over the grain are hexane and ethanol. These are available at free market.
 4. Product (weevil protection) development

The two active-ingredients are added on the tanker in 1:1 ratio. Carrier (n-hexane and ethanol)
are also added on the tanker in quantity of essential oil 1.3% to ensure the uniform distribution
over the grain. Equal amount of n-hexane and ethanol are added. Axial and radial flow impellers
are effective for fine emulsion, therefore they are used.

Figure 3. Flow diagram for weevil protection development

5. Analysis of essential oil

Characterization, identification and determination of relative amounts of components of the

essential oil will be done through Gas-Liquid Chromatography (GC), Gas Chromatography -
Mass Spectrometry (GC-MS), and GC Co injection of the essential oil with authentic standards.

6. Test for toxicity of the developed product

Mortality assessment assays

 Essential oil will be dissolved in carrier (n-hexane, ethanol) and applied to the grains at the
different rate. Treated grains will be kept for 24 h to allow the hexane and ethanol to evaporate
completely before bioassays will be conducted.

Essential oil extracts will be mixed separately grains in glass jars at the three different dosages.
Two blank controls will run periodically consisting of hexane treated grain and the untreated
grain. A pairs of 5-7 day old weevil adults will be introduced into jars containing the different
treated and untreated grains. Jars will be covered with nylon mesh held with rubber bands, and
shaken gently for proper admixture. Each treatment was replicated four times. The experiment
will be arranged in completely randomized design in the laboratory. Number of dead insects in
each vial will be counted after 1, 2, 3, 4, 5, 6, and 7 days to estimate maize weevil mortality.

Progeny assessment assays

In a similar experiment to mortality assessment, a pairs of weevils will be introduced into treated
and untreated grains. After 30 days oviposition period, parent adults will be removed. Insects
subsequently emerging will be counted to estimate FI progeny production.

Repellency

Repellent action of the essential oil extracts against weevils will be assessed in a choice bioassay
system, consisting of two 1-L glass jars connected together at their rims by means of a nylon
mesh tube. A circular hole will be cut at the middle of the mesh for the introduction of test
insects. A grain will be put into the glass jars. Grains in one jar will be treated with essential oil
extracts while untreated grains in the other jar acted as control. Adults of weevils will be
introduced into the nylon mesh tube through the circular hole. Number of insects present at the
control (NC) and treated (NT) jars will be recorded after 1 hour exposure.