BIODIVERSITY OF CITRUS PEEL ESSENTIAL...

BIODIVERSITY OF CITRUS PEEL ESSENTIAL OILS

By

Rizwan Mahmood

2003-ag-1842

M.Sc (Hons.)

A Thesis Submitted in Partial Fulfillment of Requirements for the

degree of

DOCTOR OF PHILOSOPHY

IN

HORTICULTURE

INSTITUTE OF HORTICULTURAL SCIENCES

FACULTY OF AGRICULTURE

UNIVERSITY OF AGRICULTURE

FAISALABAD, PAKISTAN

2016

i

DECLARATION

I hereby declare that contents of the thesis, “Biodiversity of Citrus Peel Essential

Oils” are product of my own research and no part has been copied from any published

source (except the references some standard mathematical or genetic

models/equations/protocols etc). I further declare that this work has not been submitted

for award of any other diploma /degree. The university may take action if the above

statement is found inaccurate at any stage.

Signature of the Student

Rizwan Mahmood

2003-ag-1842

iii

DEDICATION

TO

MY LOVING

and

CARING PARENTS

I DEDICATE THIS HUMBLE EFFORT TO

WHO HAVE DONE GREAT FOR ME

WHO HAVE BELIEF IN ME

AND WHOSE PRAYERS ARE MY REAL ASSET

v

ACKNOWLEDGEMENTS

All praises and thanks to Almighty ALLAH, Who alone is the primary source of

knowledge and wisdom to mankind, He gave me the strength and capability to make this

humble contribution to the existing treasure of literature. Secondly all credits / devotions go to

The Holy Prophet Muhammad (S.A.W), who delivered the message of ALLAH.

I extend my deepest sense of gratitude to my Supervisor, Dr. Saeed Ahmed Associate

Professor Institute of Horticultural Sciences University of Agriculture, Faisalabad for his

scholarly guidance, encouragement, and sincere help in completing this work by correcting and

re-correcting the text with patience in that way enabling me to complete the research work. His

professional attitude became the constant source of inspiration throughout my research work. His

advice strengthened me to overcome all the obstacles that came in my way.

It is matter of great honor and pleasure for me to express my ineffable gratitude and

profound indebtedness to my committee member Prof. Dr. Muhammad Jafar Jaskani

Professor Institute of Horticultural Sciences, University of Agriculture, Faisalabad for his kind

supervision, valuable suggestions and intellectual activities, inexhaustible energy to steer for

the students.

I also appreciate efforts and Supervisory committee Member Prof. Dr. Rashid Ahmad

Dept. of Agronomy, University of Agriculture, and Faisalabad, for his sincere cooperation and

encouragement who helped me whenever needed.

Thanks are also payable to my all family members for their constant support and

encouragement in the completion of this research work .I really thankful to the love and

kindness of her Mother & Father (Mahmood Ahmad Naseem), Sisters, WIFE and

Daughters whose wished and prayed for me and remained a constant source of moral support

and encouragement to improve my education and research work. It can never be completed

without the moral support and encouragement of Prof. H. Muhammad Saeed, Sheikh Fayyaz

Ahmad, Dr. Ahmed Zeeshan and Muzammil Iqbal Hashmi.

Rizwan Mahmood

Dated: 10-10-2017

vi

CONTENTS

Sr. No. Title Page

1

INTRODUCTION

01-05

2 REVIEW OF LITERATURE 06-19 2.1 Importance of citrus 06

2.2 Origin of Citrus 06

2.3 Classification of citrus 07

2.4 Distribution of Citrus 07

2.5 Citrus by products 07

2.6 Citrus essential oils 08

2.7 Economic and medicinal value of essential oil 08

2.8 Composition of citrus peel essential oil 11

2.9 Methods for essential oils extraction 15

2.10 Diversity in essential oils 18

2.11 GC-MS Analysis 19

2.12 Conclusion 19

3 MATERIALS AND METHODS 21-33

3.1 Experiment # 1 Effects of different temperatures on citrus peel oil

extracted in steam distillation: 22

3.1(a) Location 22

3.1(b) Sample collection 22

3.1(c) Handling 22

3.1(d) Experimental detail 22

3.1(e) Treatments 23

3.1(f) Replications 23

3.1(g) Storage of Essential oils: 23

3.1.1 Yield attributes 23

3.1.1(a) Physical Attributes 23

3.1.1(b) Density (mg/cm3) of essential oils 23

3.1.1(c) Refractive Index 24

3.1.1 (d) Chemical characterization 25

3.1.2 Gas Chromatography-Mass Spectrometry (GC-MS) 25

3.1.3 Statistical analysis 25

3.2 Experiment # 2 26

Comparative study of essential oils of Grapefruit peels extracted

by different methods 26

3.2.1 (a) Location 26

3.2.1 (b) Sample collection 26

3.2.1(c) Handling 27

3.2.1(d) Experimental detail 27

3.2.2 Supercritical Fluid Extraction System 27

vii

3.2.3 Steam Distillation 28

3.2.4 Storage of oils 29

3.2.5 Comparison of SCFE and Steam Distillation techniques 29

3.2.5(a) Physical Attributes 29

3.2.5(b) Chemical Characterization 30

3.2.5(c) Statistical analysis 30

3.3 Experiment # 3 Effect of different climatic regions on citrus peel

essential oils 31

3.3.1 Location 31

3.3.2 Cultivars selected 31

3.3.3 Collection of fruits 31

3.3.4 Meteorological data for the regions 31

3.3.5 Detailed Experiment 32

3.3.6 Essential oils collection 32

3.3.7 Physical Attributes 32

3.3.8 Chemical Characterization 33

3.3.9 Statistical Analysis 33

4 RESULTS 34-131 4.1 (a) Physical attributes 34

4.1.1 Density of essential oils extracted at various temperatures in

steam distillation 34

4.1.2 Essential oil percentage extracted at various temperatures in

steam distillation 38

4.1.3 Refractive index of essential oils extracted at various

temperatures in steam distillation 42

4.1.4 Chemical Characterization of essential oils extracted at various

temperatures in steam distillation 46

4.1.5 Ward’s hierarchical method for compounds diversity in essential

oils extracted at various temperatures in steam distillation 61

4.1.6 Principal components on the basis of diversity of different

compounds 63

4.1.7 Principal Components on the basis of percentage of compounds 69

Experiment # 2 73

4.2 (a) Physical attributes 73

4.2.1 Density of essential oils extracted by two methods of extraction 73

4.2.2 Oil percentage extracted by two methods of extraction 73

4.2.3 Refractive index of essential oils extracted by two methods of

extraction 73

4.2.4 Chemical Characterization of Grape fruit essential oil extracted by

two methods 78

4.2.5 Principal components of the grapefruit essential oils of extracted by two methods

82

4.2.6 Principal Component Analysis for compounds percentages in grapefruit peel oil extracted by two methods

89

Experiment # 3 95

viii

4.3 (a) Physical attributes 95

4.3.1 Refractive index of essential oils of citrus cultivars collected from

different locations 95

4.3.2 Oil percentage of essential oils of citrus cultivars collected from


4.3.3 Density (mg/cm3) 101

4.3.4 Chemical Characterization 103

4.3.5 Ward’s hierarchical method to explain the diversity among the

different compounds found in essential oils extracted from three

cultivars from different localities

123

4.3.6 Principal Component Analysis for compounds percentages in the citrus cultivars essential oils of different locations

125

4.3.7 Correlation of environmental factors with compounds in citrus

peel essential oils 133

4.3.8 Correlation for environmental condition with compounds of essential oils extracted from Grapefruit

135

4.3.9 Correlation for environmental condition with compounds of essential oils extracted from Kinnow(C.nobilis Loureiro×C. deliciosa Tenore)

137

4.3.10 Correlation for environmental condition with compounds of essential oils extracted from Musambi

139

5 DISCUSSION 141-146

6 SUMMARY 147-149

LITERATURE CITED 150

ix

LIST OF TABLES

Table

No. Title Page #

3.1 Instruments used with their model and company 21

3.2 Meteorological data for the regions according to Pakistan

Meteorological Department 33

4.1.1 Effect of temperature and cultivar on density (mg/cm3) of

essential oils 38

4.1. 2 Cultivar- cultivar essential oil density (mg/cm3) interactions 38

4.1.3 Effect of temperature on essential oil percentage in SD 42

4.1.4 Tukey HSD table for cultivars 42

4.1.5 Effect of temperature on essential refractive index in SD 44

4.1.6 Percent composition for different compounds in citrus cultivars

essential oils at different temperature levels in steam distillation 50

4.1.7

Principal components of different compounds found in

essentials oils extracted from citrus cultivars at different

temperatures

67

4.1.8

Eigen values of correlation matrix, and related statistics of

different compounds found in essentials oils extracted from

three citrus cultivars at different temperatures

67

4.1.9

Principal components of percentage of different compounds found in

essentials oils extracted from three citrus cultivars at different

temperatures

70

4.1.10

Eigen values of correlation matrix, and related statistics of

percentage of different compounds found in essentials oils

extracted from three citrus cultivars at different temperatures

70

4.2.1 Effect of methods of extraction on density of essential oils

extracted by two methods 77

4.2.2 Effect of method of extraction on percentage of essential oils

extracted by two methods 77

x

4.2.3 Effect of method of extraction on Refractive Index of essential

oils extracted by two methods 77

4.2.4 List of compounds found grapefruit peel essential oil extracted

by two methods 80

4.2.5

Eigen values of correlation matrix, and related statistics on the basis

of presence and absence of different compounds inessential oil

extracted from grapefruit

85

4.2.6

Principal components of different compounds on the basis of presence

and absence of different compounds inessential oil extracted from

grapefruit

85

4.2.7 Factor coordinates of cases on the basis of presence and absence of

different compounds extracted from oil of grapefruit 86

4.2.8

Eigen values of correlation matrix, and related statistics on the basis

of percentage of different compounds inessential oil extracted from

grapefruit

92

4.2.9

Principal components of different compounds on the basis of

percentage of different compounds inessential oil extracted from

grapefruit

92

4.2.10 Factor coordinates of cases on the basis of percentage of different compounds

inessential oil extracted from grapefruit 93

4.3.1 ANOVA table for refractive index of EOs of Citrus cultivars

from different locations 98

4.3.2 ANOVA table for percentage of EOs of Citrus cultivars from


4.3.3 Tukey HSD table for locations 101

4.3.4 ANOVA table for Density (mg/cm3) of EOs of Citrus cultivars

from different locations. 102

4.3.5 List of compounds with percentage in peel essential oils of three

citrus cultivars from different locations 106

4.3.6 Eigen values of correlation matrix, and related statistics on the basis

of percentage of different compounds found in essentials oils 127

xi

extracted from three citrus cultivars collected from different

localities

4.3.7

Principal components of different compounds on the basis of

percentage found in essentials oils extracted from three citrus

cultivars collected from different localities

128

4.3.8

Factor coordinates of cases on the basis of percentage of different

compounds found in essentials oils extracted from three citrus

cultivars collected from different localities

129

4.3.9 Correlation for environmental condition with compounds of

essential oils of three citrus cultivars 134

4.3.10 Correlation for environmental condition with compounds of essential

oils extracted from Grapefruit. 136

4.3.11

Correlation for environmental condition with compounds of essential

oils extracted from Kinnow (C.nobilis Loureiro×C. deliciosa

Tenore).

138

4.3.12 Correlation for environmental condition with compounds of essential

oils extracted from Musambi. 140

xii

LIST OF FIGURES

Fig. No. Title Page No

3.1 Essential oils samples before storage 24

3.2 Flow diagram of SCFE apparatus 28

3.3 Schematic sketch of steam distillation apparatus 29

3.4 Comparison of essential oil extracted by SCFE (A) and Steam

distillation (B) 31

4.1.1 Density (mg/cm3) of essential oil recovered at various

temperatures in SD Interaction Plot 36

4.1.2 interaction plot for the density (mg/cm3) of essential oils at

various temperature in SD 37

4.1.3 Effect of temperature on essential oil percentage in SD 40

4.1.4 Interaction plot for percentage of essential oil extracted by SD

at various temperatures 41

4.1.5 Effect of temperature on Refractive index of essential oils in

SD 44

4.1.6 Interaction plot Refractive index of the essential oils in SD 45

4.1.7 Typical chromatogram of Grapefruit peel essential oil extracted

by Steam Distillation at 105oC 53





4.1.10

Typical chromatogram of Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore)peel essential oil extracted by Steam

Distillation at 105oC

57

4.1.11


deliciosa Tenore) peel essential oil extracted by Steam


57

xiii

4.1.12


deliciosa Tenore) peel essential oil extracted by Steam


58

4.1.13 Typical chromatogram of Musambi peel essential oil extracted






4.1.16 Diversity of different compounds found in essentials oils extracted

from citrus cultivars 63

4.1.17

Scree plot between eigen values and number of principal

components of different compounds found in essentials oils

extracted from citrus cultivars

66

4.1.18 Two dimensional ordination of different temperatures used to

extract essentials oils from 3 citrus cultivars on PC1 and PC2 68

4.1.19 Two dimensional ordination of different compounds in

essentials oils extracted from 3 citrus cultivars on PC1 and PC2 69

4.1.20


components of different compounds percentage found in

essentials oils extracted from three citrus cultivars

71

4.1.21 Two dimensional ordination of percentage of different compounds in

essentials oils extracted from 3 citrus cultivars on PC1 and PC2 73

4.2.1 Density of essential oils of Grapefruit by two methods of

extraction 75

4.2.2 Essential oil percentage in two methods of extraction 76

4.2.3 Refractive index of esential oils extracted by two methods 78


by Steam Distillation method 82


by Supercritical Fluid Extraction methods 82

xiv

4.2.6


components of different compoundson the basis of presence

and absence of different compounds inessential oil extracted

from grapefruit

84

4.2.7 Two dimensional ordination of two methods used to extract

essentials oils from grapefruit on PC1 and PC2 88

4.2.8 Two dimensional ordination of different compounds in essentials oils

extracted from grapefruit by two methods on PC1 and PC2 89

4.2.9

Scree plot between eigen values and number of principal components

of percentage of different compounds inessential oil extracted from

grapefruitby two methods

91

4.2.10 Two dimensional ordination of two methods used to extract

essentials oils percentage from grapefruit on PC1 and PC2 94

4.2.11

Two dimensional ordination of different compounds in essentials oils

extracted from grapefruit on the basis of percentage by two methods

on PC1 and PC2

95

4.3.1 Interaction plot of refractive index of essential oils of citrus

cultivars from different locations 97

4.3.2 Interaction plot of percentage of essential oils of citrus cultivars

from different locations 98

4.3.3 Interaction plot of Density (mg/cm3) of essential oils of citrus

cultivars from different locations 101

4.3.4 Typical chromatogram of peel essential oil of Grapefruit

collected from Abbotabad 108

4.3.5

Typical chromatogram of peel essential oil of Kinnow

(C.nobilis Loureiro×C. deliciosa Tenore)collected from

Abbotabad

109

4.3.6 Typical chromatogram of peel essential oil of Musambi

collected from Abbotabad 110


collected from Faisalabad 111

xv

4.3.8



Faisalabad

112


collected from Faisalabad 113


collected from Layyah 114

4.3.11



Layyah

115


collected from Layyah 116


collected from Rahim Yar Khan 117

4.3.14


(C.nobilis Loureiro×C. deliciosa Tenore)collected from Rahim

Yar Khan

118


collected from Rahim Yar Khan 119


collected from Sargodha 120

4.3.17



Sargodha

121


collected from Sargodha 122

4.3.19

Diversity of different compounds found in essentials oils


localities

124

xvi

4.3.20


components of different compounds found in essentials oils


localities

126

4.3.21 Two dimensional ordination of three citrus cultivars collected

from different localities on PC1 and PC2 131

4.3.22

Two dimensional ordination of different compounds found in

essentials oils extracted from three citrus cultivars collected

from different localities on the basis of percentage PC1 and PC2

132

ABSTRACT

Citrus is a major fruit crop of Pakistan. Its production is near about 2150 thousand tons in

Pakistan. Its peel is being wasted but citrus peel essential oils can be used in industries such

as cosmetics, perfumery, baking and medicines. Present study was to identify the best cultivar

for its essential oil yield percentage and chemical constituents. Three citrus cultivars namely

Citrus paradisi (grapefruit), Kinnow (C.nobilis Loureiro×C. deliciosa Tenore)) and Citrus

sinensis (musambi) were used in this study. Steam distillation is an easiest and cheapest method

for essential oils production. In steam distillation extraction temperature is an important

parameter to be effected on the basic quality of essential oils. Three temperature levels such as

105oC, 110oC and 120oC were used in steam distillation to extract the essential oils. It was

observed that at higher temperature yield, oil density and refractive index were minimum. With

the increase in temperature the chemical constituent’s number and percent composition were

also decreased. Chemical characterization of the essential oils was done by GC-MS. Maximum

total compounds in all the samples were 57 but maximum number of compounds in one sample

were 29 at 105oC. Essential oils extracted at this temperature showed the maximum oil yield

percentage 0.311% in grapefruit followed by musambi (0.310%) and Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore)(0.302 %). The numbers of chemical compounds were also

affected by heat and it was found that percentages of α-caryophyllene, α-pinene,

caryophyllene, citronellal, decanal and nootkatone were decreased at higher temperature of

120oC. Maximum chemical compounds 34 noted in grapefruit peel essential oil in SCFE

(Super Critical Fluid Extraction) method and 29 were observed in steam distillation method.

Yield of essential oil was found maximum (0.311%) in steam distillation as compared to SCFE

method (0.243%). The fruits harvested from different districts (climate) showed significant

variation in their chemical compounds in the essential oils. The district of cool climate

(Abbotabad) showed the maximum compounds (30) in their fruit peel oil, while minimum were

compounds (13) in hot climate district (Rahim Yar Khan). It is concluded that steam distillation

method is best being easiest and cheaper and the citrus fruits collected from different climatic

zones showed big variation in their peel essential oil composition. Moreover comparison of

three cultivars showed that the peel of grapefruit have maximum essential oil components as

compared to other both cultivars.

1

Chapter 1

INTRODUCTION

Citrus belongs to family Rutaceae and has a very important place in plant kingdom.

Although citrus is grown worldwide in more than 140 countries, mostly growing on both sides

of the belt around the equator 35o north and 35o south latitude covering tropical and subtropical

regions of the world. These countries with the cultivation and production are concentrated in

key areas in the northern Hemisphere (Ramana et al., 1981). Citrus fruits, such as oranges,

grapefruit and limes can be eaten fresh, but citrus fruits are in the global processing and

utilization. Orange juice comprises of 85% of the total processed citrus consumption.

Nutritional value of citrus fruits is established beyond the provision of vitamin C

(Nagy, 1980). The fruit is rich with different concentrations of minerals in the form of simple

reducing sugars and fiber contents as well as nutrients. Citrus possesses many micronutrients

such as phosphorus, calcium, copper, potassium, magnesium, vitamin B6, folic acid, thiamine,

pantothenic acid, niacin, , riboflavin, , which are the source for health and normal necessary

growth of the body (Economos and Clay 1999 , Rouseff and Nagy, 1994). Citrus fruits have a

low energy density, are sodium and cholesterol-free (Guthrie and Carroll, 1995; Whitney et

al., 2009). Othercompounds found in citrus fruit include limonoides, flavonoids and

carotenoids. Recent epidemiological studies and other researches has clearly shown that these

active compounds have a wide variety of physiological effects can contribute the prevention

of chronic diseases ( Silalahi 2002; Steinmetz and Potter; 1991 :Liu 2003; Yao. et al 2004), so

cardiac diseases (Clinton, 1998; Ford and Giles 2000), cancer (Nishino 1997; Steinmetz and

Potter 1996), nervous defects (Youdim et al., 2002), cataract (Taylor et al., 2002) related aging

effect on muscles (Gale et al., 2003; Zhou et al., 2011), and bones deterioration (Yang et al.,

2008). It is reported that the risk of lung cancer can be reduced by using grapefruit juice,

(Feskanich et al., 2000). In some other studies, there was a significantly negative correlation

between the risk of lung cancer and the uptake of orange or tangerine fruit juice or orange fruit

(Smith-Warner et al., 2003). Several other research reports have shown the importance about

the consumption of citrus may lower the possible risks of cancers up to 40-50% (Baghurst,

2003). LDL oxidation can be reduced with the intake of fresh orange juice (Harats et al., 1998).

http://onlinelibrary.wiley.com/doi/10.1111/j.1541-4337.2012.00201.x/full#b144

2

The improvement signs by the use of citrus were also observed in number of patients with

hypercholesterolemia at small clinical study (Liu et al., 2012).

Its peel is also used in many ways, such as for activated carbon, fodder at fisheries,

paper making bio-ethanol and cosmetics. However, this peel is being wasted in huge amounts

every year (Song et al., 2002; Sharma et al., 2007b; Kim et al., 2008). Citrus by-products can

be used as the feed for the; ruminants this may include fresh citrus pulp, dried citrus pulp, citrus

silage citrus, citrus molasses, citrus peel (Bampidis and Robinson 2006).The usage of orange

peel and pulp in biscuits formula increased dietary fiber and ash in a reasonable amount and

unwanted proteins and fat in the backery products were decreased (Nassar et al., 2008)

Citrus peel has the oil sacs in the flavedo. Citrus has been extensively studied for their

essential oils but their biological activity is still under study. Some authors reported these

essential oils are very effective (Fisher and Phillips, 2008), while others say the effect is

variable (Burt, 2004). Oils extracted from plants have a number of applications like in

pharmaceuticals, food additives and preservatives because they have antioxidant, antimicrobial

and anti-inflammatory effects (Baik et al., 2008; Kim et al., 2008c; Oh et al., 2009; Imelouane

et al., 2009a and b; Yoon et al., 2009a, b, and c).Countries such as Turkey, Southern France,

China, Italy and Brazil are producing the major citrus peel essential oils

(Mitsuri,1997).According to Pakistan Statistical Year book (2010) the total cost of essential

oils, perfumes and flavours was PKR 10158.35 million. This is the mix figure for all types of

essential oils and fragrances, etc. Export data is not available. A very large amount of money

is being spent on these essential oils. Enhancing value of small scale production units elevates

the income and potential (Younis et al., 2009). Most plant phenolic compounds are classified

as generally recognized as safe (GRAS) substances. These substances are used in foods to

prevent growth of pathogenic organisms and potential spoilage (Burt, 2004; Nedorostova et

al., 2011). Among polyphenols, flavonoids are secondary metabolites very efficiently reported

for their anticancer, antimutagenic, anti-inflammatory and, antiviral biological effects and

activities (Benavente et al 1997, Vuorela et al. 2005). Citrus peel essential oils resulted in

antifungal activity against the moulds. These oils could be considered as alternatives to

chemical additives for use in the food industry (Viuda-Martos et al., 2008). Essential oil

extracted from peel of different citrus genotypes exhibited the antibacterial activity against a

large number of strains of microbes obtained from the different food matrices. (Randazzo et

3

al., 2016).A recent report shows that in Sicily (Italy) citrus extracted essential oils showed

good potential for prevention of food spoilage due to effective antimicrobial compounds

(Settanni et al.,2012). Mansour et al., (2004) mixed the citrus peel essential oils with

Malathion, a well established insecticide and resulted in marvelous effects against the mosquito

larvae.

Citrus peel essential oil contains pleasant sensory characteristics components that are

popular in food, pharmaceuticals and cosmetics industries.Forty-six compounds were found

in the essence of lime, and the highest concentration of these compounds were limonene,

linalool, sabinene and bergamol (Maria et al., 2012). Monoterpene hydrocarbons constituted

the majority (88.96%, w/w) of the total oil. (Tao et al., 2014). The compounds particularly

responsible for aroma of yuzu were limonene, α-pinene, α- and β-phellandrene, myrcene, c-

terpinene, (E)-b-farnesene and linalool (Lan-Phi et al., 2009). Sour lime peel essential oil

contains constituents such as o-cymene (16.62%) alpha-cedrene (10.57%), decadienal

(8.043%), bisabolene (5.066%), β-humelene (4.135%), Citronellyl acetate (2.371%), linalool

acetate (2.371%), carvone (1.806%), decanone (1.474%), isopulegol acetate (1.296%),farnesol

(1.254%), 40-methoxyacetophenone (1.207%), D-carene (1.070%) , α-terpineole (0.607%),

dihydroxylinalool acetate (0.650%), cis-nerone (0.574%), caryophyllene oxide (0.433%), and

2,2-dimethyl-3,4-octadienal (0.375%) (Mahmud et al., 2009).

Steam distillation, hydro distillation, and solvent extraction are the different methods

of essential oil extraction (Heath, 1981). Steam distillation and organic solvent extraction are

the traditional methods being used for centuries.Steam distillation was found to be the best

extraction method (Maria et al., 2012). In steam distillation method of essential oil extraction,

different heat levels may result in different chemical and physical achievements. High heat

results in the decay of the sensitive compounds in steam distillation method of essential oil

extraction. A controlled temperature in steam chamber is very important for the purpose

(Zakiah et al., 2013). Alternative methods for essential oils extraction are being used for

research purpose today. These methods use less time and energy for the purpose. Microwave

dry-diffusion and gravity method performed in the similar to steam distillation but in much

less time and energy consumption (Farhat et al., 2010). Supercritical fluid Extraction System

(SCFE) is a rapid, selective and easy method for sample preparation for the volatile compounds

of plant origin (Eikani et al., 1999; Paroul et al., 2002). Supercritical fluid extraction system

4

(SCFE) is a new technique for essential oil extraction that is environmentaly friendly. Carbon

Dioxide is usually used for the oil extraction as extracting material so this inert gas creates no

serious hazards (Pourmortazai and Hajimirsadeghi, 2007).This low temperature process

prevents the chemical constituents from degradation that occurs in other higher temperature

methods (Gamiz-Gracia and Luque, 2000; Raeissi and Peters, 2005). Water distillation method

of essential oil extraction was found to have the lowest environmental impact and carbon

footprint with full energy integration (Moncada et al., 2016).

Citrus peel essential oils have diversity in their chemical constituents. This diversity is

dependent upon the different cultivars, locations, genetic variations and even in seasonal

changes (Hosni et al 2010). Not only the chemical constituents are different but also the oil

percentage is different among the cultivars (Ahmad et al., 2006).Climatic conditions also affect

the taste of citrus fruit quality and yield. In addition to altitude, agronomic management and

environmental conditions can influence essential oil composition (Ghasemi Pirbalouti et al.,

2013). It is a general phenomenon that the chemical pathways, and chemical composition of

the oil bearing plant species are effecte by the environmental factors such as wind, average

temperature, and rainfall and relative humidity radiation while the factor such as soil properties

and time of harvest also effect. (Celiktas et al., 2007; Djouahri et al., 2015; Formisano et al.,

2015)

Essential oil constituents are identified by different techniques such as HPLC or GC-

MS.GC-MS is a combination of two different analytical tools, gas chromatography (GC) and

mass spectrometry (MS). This technique is used to analyze organic and complex biochemical

compounds. GC can separate volatile and semi-volatile compounds with high resolution, but

it can not recognize them. MS gives the detailed structural information of the compounds in

manner that they can be accurately be recognised and measured (Hussain and Khushnuma,

2014).

References show that efforts have been made to identify the constituents of essential

oil, their antimicrobial activity and relative percentages of different cultivars. Exact answers

about the variation in the citrus peel essential oil components extracted in steam distillation at

various temperature levels, heat effects on the essential oil constituency, oil yield, climatic

effects on the essential oils in citrus and an easiest method of citrus peel essential oil extraction

with a good composition are still lacking.

5

Objectives of this research were

• To compare the conventional oil extraction methods for citrus peel essential oils.

• To recognise the variation in volatile components of citrus peel essential oils collecting

from different climatic regions.

• To identify the best citrus cultivar for essential oil components.

6

Chapter 2

REVIEW OF LITERATURE

2.1. Importance of citrus

The genus Citrus, is from the Rutaceae family, it contains one hundred forty genera

and thirteen hundred species. Citrus sinensis, Citrus medica, Citrus paradise, Citrus limon,

Citrus reticulata, Citrus aurantium, Citrus grandis and Citrus aurantifolia are some important

fruits of the Citrus (Singh et al., 1983; Anwar et al., 2008). Citrus is at the top of the fruits in

many countries such as Brazil, the United States, Japan, China, Spain, Mexico, Pakistan,India

and Mediterranean region. Citrus production is up to 105 million metric tons (MMT) per year

worldwide. Pakistan's annual citrus production is about 21500 thosand tons. (FAO Stat 2013)

2.2 Origin of Citrus crop

The original center of citrus fruits has been the subject of speculation and discussion.

Citrus taxonomist Tanaka believes that modern citrus species originate in northeastern India

and neighboring northern Myanmar as quoted by Gmitter and Hu (1990). There are a variety

of citrus germplasm resources in the northeastern region of India, including 68 varieties as

described by Sharma et al., (2004). Climate and soil factors are particularly suitable for citrus

plants for plant growth and fruit quality. However, Gmitter and Hu (1990) suggested that

according to the recent Chinese survey, most of the citrus genebanks in the citrus genome, as

well as available natural dispersal mechanisms, provide strong evidence that the origin and

distribution of modern citrus varieties in Yunnan and its surrounding areas plays a vital role.

Mesopotamia found citrus seeds dating back to 4000B.C. (Mabberley, 2008; Scora, 1988;

Webber et al., 1967).

Citrus is placed in the major fruit crops in all over the world and is being sown in more than

125 countries with suitable weathers and optimum temperature range. Citrus fruit appeared on

globe 30 million years ago.

2.3 Citrus classification

According to Fang et al., (1998), "Taxonomy and phylogeny of citrus family is

complicated by cross compatibility and apomixis, in some taxa. While the citrus classification

7

is a complicated, to the selection and propagation of a number of natural and man-made hybrids

and many mutants during a long history of cultivation makes citrus classification even more

difficult. The most widely accepted citrus classification system is Swingle (1943) and Tanaka

(1977), which identify 16 genera and 162 species, respectively, C. maxima (Burm.) Merril, C.

medica L. and C. reticulata Blanco are ancestral species in subgenus citrus, and other "forms"

in subgenus may originate from the hybridization between other species.

2.4 Citrus Production

The total production and consumption of citrus fruits has been growing strongly since

the last decades of 20th century. The current annual output of citrus is estimated at more than

105 million tonnes, of which more than half are oranges. According to the United Nations

Conference on Trade and Development (UNCTAD 2004), the rise in citrus production is

directly propotional to the rise in growing aeas, development in technology, and increased

demand by users. However this was elevated to 2000 to 2010 million tones by the growing

trends (Naseer, 2010).

2.5 Citrus by products

Citrus fruits are mainly consumed as desserts, extracting juice and making jams. The

food and agro-food producing industry yields a huge amount of debris or by-products such as

peels, seeds and pulps which prevails the 50% of the raw fruit (Anwar 2008). These by-

products are considered to be valuable sources of efficient constituents, namely flavonoids,

dietary fiber and essential oils (Senevirathne, 2009). Citrus fruits and their by-products are of

high economic value. Because of their relatively safe status, widely accepted by consumers.

These factors have made citrus fruits and their by-products of high economic and medicinal

value (Ormanceyet al., 2001; Sawamura, 2000). Essential oil is one of the citrus by-products

attracting important vision of people (Njoroge, 2005). Usually the fruit pulp or seeds are used

by the consumers. (Morales et al., 2009). Peels, seeds and pulps of Citrus processing industry

left after juice extraction can be used as a potential source of valuable by-products (Silalahi,

2002; Saidan et al., 2004).

In recent years, the essential oils have been identified in different parts of fruits as in

leaves, showing that its major components are the major aromatic compounds of many citrus

species (Stashenko et al., 1996; Caccioni, 1998; Lota et al., 2001 and Minh et al., 2002). These

8

aromatic compounds are relatively cheap and plentiful raw materials used in flavour and food

industries (Reische et al., 1998). They can also serve as the initiaters in the synthesis of fine

chemicals and of new colognes for the beautifying products (Lis-balchin and Hart 1999).

2.6 Citrus essential oils

After processing citrus fruits, the peel and film residues are about 40-50% of the "wet

fruit pieces". Citrus fruit peel and leaves are a potential source of essential oils (Baddock, 1999)

and essential oil extraction ranges to 0.5- 3.0 kg/ton (Sattar and Mahmud, 1986a). Essential

oils sometimes called volatile oil are concentrated aromatic compounds (Arce, 2007; Lucchesi

et al., 2004) that show a very little portion of the plant part in the leaves , flowers and fruits as

well (Roldan-Gutierrez et al., 2008; Bousbia et al., 2009). These oils have natural antioxidant

and antimicrobial properties (Tepe et al., 2005; Jayaprakasha et al., 2007; Viuda-Martos et al.,

2008). The biological properties of plant material are closely related to its specific chemical

composition, mainly secondary metabolites usualy known as plant phenols and flavonoids

(Jayaprakasha et al., 2007; Viuda-Martos et al., 2008). Drying of plant materials under

different conditions has a significant impact on the chemical and biological properties of

essential oils (Asekun et al., 2007a; Masotti et al., 2003; Angioni et al., 2006).

2.7 Economic and medicinal value of essential oil

Citrus peels which are considered as agro industrial waste are a potential source of plant

secondary metabolites in the form of essential oils (Andrea et al., 2003). These essential oils

have a wide range of potential activities in food, perfumery, sanitary, cosmetics and

pharmaceutics (Mondello et al., 2005). The important application of citrus peel essential oils

is due to some bioactive compounds in them which serve as alternatives to the synthetic

antioxidants (Tepe et al., 2006; Viuda- Martos et al., 2008; Choi et al., 2000).Essential oils are

used in food industry for flavoring foods, drinks and other goods, cosmetics and medicines.

Citrus essential oils act as natural antioxidants because flavanone glycosides namely

naringin, narirutin, hesperidin and neohesperidin are valuable phenolic compounds found in

citrus peel oil which make them liable to avert rancidity of food (Anagnostopoulou et al.,

2006). Essential oils of Citrus peels are medicinally very important and show variety of

biological effects because they are rich in flavonoids (flavone, flavonol and flavanone),

terpenes, carotenes and coumarines which are responsible for antimicrobial activity (Tepe et

9

al., 2005). Citrus essential oils are extensively used in pharmaceutics as an antimicrobial, anti-

diabetic, antioxidant, insect repellent, carminative, larvicidal, antiviral, antihepatotoxic and

antimutagenic agent. In pharmaceutical industries they are employed as flavoring agents to

mask unpleasant tastes of drugs (Kanaze et al., 2008).These natural properties are in line with

the increasing demand from consumers to limit the use of synthetic additives, as these artificial

chemicals have been established as potential health hazards in some instances, due to toxic

impurities deriving from the synthetic pathways. Essential oils are much more acceptable to

the end consumers than are synthetic substances and they do not cause bacterial resistance

mainly, because they are comprised of a wide spectrum of compounds (Maggi et al., 2009).

The infusion of the peel have been widely used as tonic, diuretic, antipyretic, laxative

and anti-inflammatory in Iranian traditional medicine (Zargari, 1990).

For a long period of time, plants have been a valuable source of natural products for

maintaining human health. The use of plant extracts and phytochemicals, both with known

antimicrobial properties, can be of great significance in therapeutic treatments (Seenivasan et

al., 2006). Many plants have been used because of their antimicrobial traits, which are due to

compounds synthesized in the secondary metabolism of the plant. These products are known

by their active substances e.g. the phenolic compounds which are part of the essential oils, as

well as tannin (Tyagi and Malik, 2010). Essential oils are more effective in controlling biofilm

cultures due to their better diffusibility and mode of contact (Al-Shuneigatet al., 2005). They

have been screened for their potential uses as alternative remedies for the treatment of many

infectious diseases (Tepe et al., 2004; Dorman and Deans, 2000).

The food and pharmaceutical industries in Pakistan are using citrus oils as flavouring

and masking agents in abundant quantities. Citrus essential oils are widely used for aroma and

flavor of many food products, including alcoholic and non-alcoholic beverages, candy and

gelatins. In perfumery and cosmetic, they are used in many preparations (Guenther, 1948;

Dugo and Giacomo, 2002).

Citrus essential oils have been used as flavouring agents in foods, beverages, liquors

and confectionaries and as aromatic agents in perfumery, soaps and other household products

(Matsura et al., 2006). In some cases, the composition of the flavouring agents can play an

active role in the microbiological stability of the products. Moreover, various products made

from essential oils have been used in aroma therapy and may relax some physical and

10

psychological conditions (Susan, 1996). Essential oils repell insects and animals and have

inhibitory effects against microorganisms. Moreover, citrus essential oils possess

physiological activities such as antioxidative action against linoleic acid oxidation (Song et al.,

2001; Fahad et al., 2013), DPPH radical scavenging activity and tyrosinase inhibitory activity

(Choi et al., 2000).

The antimicrobial activity of the peel extract is directly dependent upon the components

that they contain. The studies showed that essential oils, protopine and corydaline alkaloids,

lactons, polyacetylene, acyclic sesquiterpenes, hypericin and pseudohypericin compounds are

effective against various bacteria. Nevertheless, other active terpenes, as well as alcohols,

aldehydes, and esters, can contribute to the overall antimicrobial effects of the essential oils

(Keles et al., 2001).The lemon peel extracts in different solvents such as ethanol, methanol and

acetone were subjected to antibacterial assay. Lemon extract in solvent shows higher

antimicrobial activity against tested microorganisms in comparison with the extracts of lemon

peel in other solvents like methanol and acetone. Moreover, citrus essential oils have been

recognized as safe due to their wide spectrum of biological activities such as antimicrobial,

antioxidant anti-inflammatory and anxiolytic (Fisher and Phillips 2008; Chutia et al., 2009 and

Rehman 2006). Due to their great nutraceutical and economic importance, numerous

investigations have been performed aimed at identifying the chemical composition,

antimicrobial activities of the essential oils from peel of different citrus species.

Bacterial resistance has often been a topic of discussion amongmedical and research

groups since the 1960s and 1970s, where it was observed that the indiscriminant use of semi-

synthetic penicillins resistant to penicillinases and cephalosporins favored the emergence of

strains of S. aureus resistant to methicillin, and atthe same time, the extensive use of ampicillin

favored the emergence of strains of ampicillin-resistant E. coli. When we analyze the1970s

and 1980s, gram-negative bacteria were the major therapeutic obstacle, but in the new

millennium, the Gram-positive alsocame to occupy a prominent place (Rossi and Andreazzi,

2005). The antifungal activity in terms of zone of inhibition and minimum inhibitory

concentration of essential oils of Citrus limettioides was tested against ten fungal strains viz.

Alternaria alternata, Rhizoctonia solani, Curvularia lunata, Fusarium oxysporum,

Helminthosporium oryzae, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus,

Cladosporium herbarum and Trichoderma viride. The essential oils of Citrus limettioides

11

exhibited varying antifungal activity against the various test strains (Vasudeva and Tanu,

2012).

Essential oils are mainly used to reduce high blood pressure, mental health, respiratory

problems, arthritis and rheumatism (Saidan et al., 2004; Silalahi, 2002). It is also used to

prevent kidney stones. In addition, lemon fruit and juice are used to wash for oral health to

freshen your breath and to treat flaky dandruff, headaches and reduce asthma symptoms

(Saidan et al., 2004; Silalahi, 2002; Reichling et al., 2009).

Synthesized antioxidants, such as butylated hydroxyanisole, butylated hydroxytoluene,

and tertiary butylhydroquinone, are very commonly used in food items to cut short the lipid

peroxidation. However, the foods supplemented with these synthetic antioxidants are not

promoted due to their toxic effects (Buxiang and Fukuhara, 1997) and carcinogenicity (Hirose

et al., 1998). Therefore, some essential oils and their components are of importance due to

their antioxidant potentials as alternatives to the artificially synthesized antioxidants without

showing any secondary effects (Carson et al., 2002).

2.8 Composition of citrus peel essential oil

Essential oils are a composite blend of terpene hydrocarbons and oxidized derivatives

such as aldehydes, alcohols, ketones, organic acids and esters that can be derived from peel

from different extraction approaches (Clarke, 2002; Merle et al., 2004). Monoterpene

hydrocarbons and sesquiterpene hydrocarbons give characteristic flavor to citrus oils.

Aldehydes contribute to the overall matter of oxidized compounds and help to establish the

quality and price of essential oils (Braddock, 1995; Dugo, 1994; Diaz et al., 2005). Citrus peel

essential oils are rich source of compounds i.e. flavonoids, coumarins, terpenes, carotenes and

linalool etc. (Mondello et al., 2005).

The citrus peel is a rich source of flavonoid glycosides, coumarins, sitosterol,

glycosides and volatile oils (Shahnah et al., 2007). Many poly methoxylated flavones have

important functions, which are very uncommon in other plants (Ahmad et al., 2006). In

addition the fiber of citrus fruit also contains bioactive compounds, such as polyphenols, the

most important having vitamin C (or ascorbic acid), and they can certainly prevent and cure

the disease of scurvy (Aronson, 2001).

Citrus oils are mixtures of very volatile components as terpenes and oxygenated

compounds (Sato et al., 1996). Limonene, a monoterpene, is the major component of lime and

12

other related citrus essential oils (Lanças and Cavicchioli, 1990). These oils are used in the

edicinal industry, perfumery and food industries (Huet, 1991). The quality of the oils is related

to the value of total aldehydes, basically citral content which is between 4-5% (Shaw, 1979).

The chemical compositions of the essential oils is affected by different factors such as

humidity, soil conditions, temperature and weather, leading them to be considered the best time

to choose the oil and the amount of material of interest (Evergetis et al., 2016; Kiazolu et al.,

2016; Almeida et al., 2016; Sarrazin et al., 2015).

Three main chemical constituents types viz: limonene, γ-terpinene and linalyl

acetate/limonene were differentiated for peel oils while sabinene linalool, γ-terpinenelinalool

and methyl N-methyl anthranilate for leaf oils. Kaffir lime from the Rutaceae family contains

two types of essential oils, leaf oil and fruit peel oil (Srisukh et al., 2012).

From orange peel essential oils (92.42%) limonene and (3.89%) β-myrcene were

recorded in Iran (Yaghoub et al., 2006) while in Italy limonene and β-myrcene were (93.67%)

(2:09%), respectively (Verzera et al., 2004). Other organics available in citrus essential oils

are aliphatic hydrocarbons, alcohols, aldehydes, acids, esters and some aromatic compounds

(Sharma andTripathi, 2006). Svoboda and Greenaway (2003) reported the ajor chemical part

of citrus essential oils as limonene with a range of 32 to 98%.

The The essential oil extracted from citrus has a monocyclic haploid whose main

component is d-limonene (-mentha-1,8-dene) with insecticidal action (Karr and Coats, 1988).

Mansour et al., 2004 already found that limonene was the main compound of different citrus

species, the incidence is 51.97% ~ 95.32%. Many researchers have further concluded that

individual limonene has a basic biological properties (Su and Horvat, 1987; Lota et al., 2002),

but its effectiveness may vary widely depending on the citrus and insect species being studied.

According to Choi and Sawamura (2000) this change in limonene content in citrus peel

may be related to harvest time, freshness and fruit size. In addition, consideration should be

given to geographical locations, fruit varieties and extraction methods. Essential oils are multi-

component chemicals. Mixtures of oily compounds that make up essential oils include polar

and non-polar compounds Fleisher and Fliesher, 1991 and Bohra et al. 1994. Some of the

compounds in the composite oil are lost in the wastewater. In the case where the plant material

and water are mixed in the stationary material, the oil is lost in the water in the distilled water

and in the aqueous phase of the distillate.

13

Salamon et al., (2010) analyzed the composition of essential oils and sesquiterpene for

environmental and genetic variation in natural chamomile population of Iran and compared

those with Egyptian cultivars. The α-bisaboloxide, A and α-bisabololoxide B contents were

high in chamomile plants, the flower anthodia of whom were collected from several places of

Egypt.

Baik et al., (2008) investigated fourteen kinds of citrus oils for the chemical

composition and to study their biological activities. From Jeju island immature fruits were

collected and the oil was extracted using the steam distillation method. Gas chromatograph

(GC) with FID and with mass spectrometer (MS) were used to analyze these oils. 55.4% to

91.7% of Limonene, 2.1% to 32.1% myrcene, 0.6% to 1.6 % α-pinene and 0.4% to 6.9%

linalool.

Yang et al., (2009) examined the chemical proportion of essential oils obtained from

citrus peel using the method of distillation. The efficiency of these oils against different types

of microorganism, including drug resistant and drug susceptible skin pathogens was also

investigated. The GC-MS was used for chemical analysis. This revealed that 94.5% of total oil

was comprised by only six compounds. The oil contained limonene, γ-terpinene, cymene ,β-

myrcene, α-pinene and α-terpinolene (80.51%), (6.80%), (4.02%), (1.59%), (1.02%) and

(0.56%) respectively.

Kamal et al., (2011) isolated essential oils from healthy, fully ripened and hot dried

peel of Citrusreticulata, Citrus sinensis and Citrus paradisii for comparison of variation in

chemical composition and yield. Oven dried samples were rich in oil contents as compared to

fresh peel samples. The chemical constituents of C. reticulate, C. sinensis and C. paradisii

were 16-27, 17-24 and 18-40 respectively using GC and GC/MS analysis. The most abundant

chemical component, limonene, was 64.1-71.1% (C. reticulata), then 66.8-80.9% (C. sinensis)

and 50.8-65.5% (C. paradisii).

Viuda-Martos et al., (2008) observed the effect of the essential oils of (Citrus lemon

L.) lemon, (Citrus reticulata L.) mandarin, (Citrus paradisi L.) grapefruit and orange (Citrus

sinensis L.) on moulds growth. These essential oils could be used as the best chemical additives

in food industry.

Gong et al., (2014) investigated Lamiaceae and Origanum vulgare L. collected from

six different locations in Pakistan and China. The samples were analyzed by GC-FID and the

volatile constituents were examined by GC-MS. About 11 to 46 components could be

identified through this method among six locations which represent 98.5% to 99.9% of the

14

total oil extract. The production of the extracted oil of O. vulgare from the six locations ranged

from 0.1 to 0.7% oxygenated monoterpenes were highest in all the essential oils extracted.

However, sesquiterpene hydrocarbons were in abundance in two ares. This study provided

comprehensive evaluation of essential oil contents of O. vulgare. According to this study of

the essential oil components the cluster analysis of O. vulgare was classified into three subsets.

Furthermore, researches resulted thatchemical and biological profile of the essential

oils are changed due to drying of the material (Asekun et al., 2007a; Masotti et al., 2003;

Angioni et al., 2006).

The percentage of primary and secondary chemical components is considered to be the

chemical composition of each essential oil. In addition, the chemical composition of essential

oils depends on the content of the main chemical composition. The biological activities such

as antimicrobial activity are not only depend on the chemical component but also depend on

the structure activity relationship that might occur for the other chemical components (Faleiro

et al., 2003).

Essential oils from natural sources are more effective than the various antimicrobial

agents used for air disinfection because essential oils have low levels of toxicity and high

volatility (Inouye et al., 2003). In addition, essential oil as natural food preservatives are widely

used and accepted by consumers around the world (Militello et al., 2011).

The chemical composition of lemon juice apart from water contains certain acidic

substances called citric acid and carboxylic acid helpful for health benefits (Saidan et al., 2004;

Silalahi, 2002).

2.9 Methods for essential oils extraction

Essential oils are derived by many of the the methods around the globe but most of

them are extracted by steam distillation (Reverchon and Senatore, 1992). The major part of

different essential oils extracted is by steam distillation which is 93% and only 7 % is derived

by other methods (Masango, 2001). Major compounds in the essential oils decide the quality

of the essential oil (Kasuan et al., 2009). Extraction methods and parameters i.e. temperature,

pressure, flow rate, extraction time, particle size of extract material and raw state material (dry

or natural) also influence the quality of oils (Grosso et al., 2008; Kristiawan et al., 2008).

Pressure and flow rate have less effect on result yield as compared to the temperature,

extraction time and size of material (Eikani et al., 2007; Rezzoug, 2009; Louli et al., 2004)

Monitoring the temperature at the time of extraction is vital as a minor alteration of a few

degrees may result in big losses of oil with very different chemical characteristics, making

15

them either efficient or ineffeicient, for the purpose. Presently Pakistan is importing citrus peel

essential oils worth Rs.300 million (Saeed, 1989) which suggests that there is There is an

urgent need to develop a viable technology for the production of essential oils from very own

resources.

Common commercial methods for producing oil from citrus fruits and peels are

machine cold pressing and steam distillation. However, the oil obtained by distillation is easily

deteriorated due to the instability of the terpene hydrocarbons present and produces an odor,

particularly d limonene (Yamauchi and Sato, 1990). Supercritical fluid extraction is an

advantageous alternative to the refining of citrus oil due to its low operating temperature and

the absence of solvent residues (Iwai et al., 1994).

Qadir and Wajahat (2014) carried out a research in order to observe the chemical

onstituents, anticancer, antioxidant and antibacterial penetration of Pinus roxburghii. Hydro-

distillation method was used to extract the oil. Agar well diffusion method was applied to

evaluate the antibacterial activity whereas antioxidant activity was checked through DPPH

assay. MTT method was adopted to evaluate the anticancer activity. It was found that the major

constituents of the oil were α -pinene and β -pinene.

Lota et al., (2001) used 58 mandarin cultivars from15 different species for peel and leaf

oil extraction. The chemical compositions of these oils were studied by capillary GC-MS and

13C NMR.

Many of the research studies have been resulted that the quality of essential oils in

reference to the presence of chemical components may change due to drying methods (Asekun

et al., 2007a; Asekun et al., 2007b). However, as far as we know, it has not been reported that

any comparative study investigated the effects of dry pretreatment on the yield and chemical

composition of essential oils peeled from different citrus fruits. In order to optimize the

recovery of essential oils, the loss of some oil components, such as the moisture content of the

distillate and the polar component of the moisture in the stationary material, must be re-

distilled, known as the joint action. Recycling wastewater to recover dissolved oil components

results in increased utility costs, mainly for heating or energy costs (Holman, 1997; Brennan,

1998).

Typically, essential oils are separated from the plant by solvent extraction, steam

distillation or hydrogenation distillation. When the essential oil is extracted with an organic

16

solvent, it is usually necessary to evaporate the solvent, and the operating conditions for the

evaporation may cause the product to degrade (Morales et al., 1998). Steam distillation and

hydrogenation distillation as alternatives to organic solvents; however, these methods have

some degradation. In recent years, the application of supercritical fluid technology in the food

processing industry has been paid more and more attention. Supercritical fluid extraction has

become a meaningful alternative to traditional extraction methods (Assis et al., 2000; Khajeh

et al., 2005).

Ahmad et al., (2006) adopted cold pressing method to extract oil from the peels of

mousami (C. sinensis), malta (C. sinensis), eureka lemon (C. limon) and grapefruit (C.

paradise). Malta was richest in the oil contents and it was followed by eureka lemon and

mousami, whereas, grapefruit was lowest in oil contents. The extracted oils were investigated

for their constituents by GC-FID on carbowax 20 M packed glass column. The peel of malta

was richest in limonene.

Waheed et al., (2011) extracted the essential oil of Zanthoxylum armatum through

hydro distillation method and analyzed by GC-MS. The oil contained high portion of

oxygenated compounds (39.21%) and the portion of hydrocarbons was fairly low (17.35%).

The sesquiterpenes and monoterpenes were 10.83% and 47.33% respectively. Zanthoxylum

armatum contained high percentage of chromatogram of essential oil i.e. 37.23%, whereas, the

monoterpene hydrocarhons were found 10.09%. The percentage of alcohol was higher i.e.

26.76%. The only cyclic ester, 15-Hexadecanoloide was found in high percentage i.e. 6.58%.

Rozzi et al., (2002) used supercritical extraction method to extract oil from four

varieties of lemon scented plants. Lemon eucalyptus (Eucalyptus citriodora), lemongrass

(Cymbopogoncitratus), lemon bergamot (Monarda citriodora) and lemon balm (Melissa

officinalis) were used in this study. Three samples of each species from different plants were

used. The samples were analyzed through gas chromatography and further confirmed with GC-

MS. Lemon balm contained; citronellal, geranial, caryophyllene oxide, neral acetate, neral and

caryophyllene. Lemon bergamont; thymol, α –terpine and thymolmethylester, lemon

eucalyptus; citronellal, caryophyllene oxide, caryophyllene, and neral whereas, lemongrass

contained neral, caryophyllene, and geranial. Three pressure levels (13,790, 27,580, and

41,370 kPa) of supercritical fluid were applied with a range of temperature of 40o and 60°C.

17

The results indicated that as the temperature and pressure were increased the mass percent

extract was also increased.

Chanthaphon et al., (2008) extracted citrus oils through steam distillation. The major

components kaffir lime from SFC with ethyl acetate as extractant were limonene (31.64 %),

citronellal (25.96 %) and β-pinene (6.83 %) while β-pinene (30.48 %), sabinene (22.75 %) and

citronellal (15.66 %) were obtained by the steam distillation method of oil extraction.

Lan-Phi et al., (2009) used cold-pressing method for extraction of essential oils and

found 69 compounds from different yuzu cultivars viz, kumon ,nagano , yasu, jimoto ,

komatsusadao and komatsukoichi . GC-olfactometry and aroma extraction dilution analysis

techniques were used in stepwise dilution of the neat oil. This resulted eight odorants with the

maximum flavour dilution values. Those were limonene, α-pinene, α- and β-phellandrene,

myrcene, γ-terpinene, (E) β-farnesene and linalool. ‘KOS’ showed maximum number of

constituents.

Moncada et al., (2016) extracted the essential oils from oregano and rosemary and

samples were analyzed to determine the composition of the oil and use the data as a starting

point in technical analysis. Three extraction techniques (i.e., supercritical fluid, solvent, and

water distillation) were used for modeling and assessment. Efficient use of energy was the

subject to be determined. Also the impact of essential oil production on environment was

minimum in the distillation method.

2.10 Diversity in essential oils

Melito et al., (2016) examined the chemical profile of essential oils of 146 H. italicum

species in Sardinia. They were discussed from two distinct habitats. Significant differences

were found between the two groups in the volatile components, displays of secondary

metabolites production and habitat types. Multivariate analysis of variance showed that

monoterpenes, sesquiterpenes, alcohols, esters were in different quantities result in plants

grown under different environmental conditions. Patel et al., (2016) revealed the difference

between the essential oil contents and oil yield percentage of the essential oil extracted from

five Ocimum species from two different locations of India. Environment factors diversified the

oil components.

18

2.11 GC-MS Analysis

After the extraction of essential oils from some plants or plant parts it is necessary to

investigate the oil components. GC-MS (Gas Chromatography- Mass Spectrometry) analysis

is done to separate and identify them. GC-MS today is an important part of the essential oil

research. This analysis gives detail about column, carrier gas, sample injection, temperature

programming and detection (Adams, 1991). Terada et al., (2010) studied the effect of

temperature and pressure on the extraction of yuzu essential oil and analyzed it by GC-MS.

They revealed by help of GC-MS that yuzu oil is potential source of elemene. Boukhatem et

al.,(2014) examined the quality of the essential oil from Eucalyptus globulus Labill of Blida

(Algeria) origin. Chemical compounds of the essential oil were analysed by GC-MS.

Identification of compounds based of retention time and comparing with mass spectral

database of standard compounds. Zakiah et al.,(2013) analyzed the chemical compounds

profile of kaffir lime peel essential oils with the help of GC-MS. Peak area percentages of all

the compounds was compared to identify the compounds. Boutekedjiret et al., (2003) extracted

the essential oil of rosemary by steam and hydro distillation and analyzed by the help of GC-

MS. They found that these oils were characterized by monoterpene hydrocarbons, oxygenated

mono and sesquiterpenes.

2.12 Conclusion

From the above literature review it is clear that many scientists have done their research

on different methods of essential oil extractions. Their physical and chemical attributes were

examined. Cumulatively it is known that steam distillation method is the preferred method for

citrus peel essential oil extraction. It is an easy method to be adopted. Worldwide production

of essential oils mainly depends upon this method. Comparatively it is an economic method

having least factors on the environment. However it is also important to be noted that the

controlled temperature in steam distillation is more preferred than the uncontrolled.

Supercritical fluid extraction system is also being used for essential oil extraction but it is not

adopted commercially for essential oil production because it is an expensive and complex

technique to be applied. Different cultivars have different ratio of chemical compounds in their

essential oil. This is why their aroma are different. Climate affects the quality and quantity of

the essential oils. Low or high altitudes with different temperature ranges in their climate result

in biodiversity of the chemical constituents. Moreover, the genetic variation may also be the

19

responsible for the variation in the chemical components of these essential oils. These essential

oil is a good source of antibacterial and antifungal substances can be used for the edible film

making for fruits and vegetable storage. These oils can be used for medicinal purposes and

aromatherapy against diseases such as cancer, high cholesterol, memory loss, stress, etc. Citrus

peel essential oils can also be used in the synthesis of bio-insectcides and bio pesticides. These

essential oils may deliver a reasonable capital amount for the country.

20

Chapter 3

MATERIALS AND METHODS

The present studies were carried out in Rosa Oil extraction Lab and Tissue Culture Lab

Institute of Horticultural Science, University of Agriculture, Faisalabad. Some parts of the

experiments were completed in the Chemistry Labs, Forman Christian College University

Lahore. The present research work was done to compare the oil extraction methods, to check

the variation in volatile compounds of citrus peel essential oils from different climatic regions

and to identify the best citrus cultivar for essential oil components. Steam distillation method

of extraction was mainly used for the research. Physical and chemical attributes were examined

for the essential oils extracted. The chemical constituents were separated and identified by the

use of GC-MS.

Table 3.1 Instruments used with their model and company

Name of instrument Manufacturing company

Steam Distillation NIAB, Pakistan

Supercritical Fluid Extraction System DEVEN, Supercritical Pvt.Ltd, India

Ultra Low Freezer Sanyo, Germany

Electric Balance MP-300 Ohyo, Japan

Refractometer ATAGO RX-5000 Atago Co.,Ltd Japan

GC-MS 6890N-5975B Agilent-Technologies,California,USA

21

3.1 Experiment # 1Effects of different temperatures on citrus peel oil extracted in steam

distillation:

3.1(a) Location:

Rosa project Lab, Institute of Horticultural Sciences, University of Agriculture,

Faisalabad

3.1(b) Sample collection:

The fruits for the oil extraction were collected from experimental orchard Square no 9

of Horticultural Sciences, University of Agriculture Faisalabad for this experiment. Following

cultivars were used for the experiment.

Grapefruit

Musambi

Kinnow(C.nobilis Loureiro×C. deliciosa Tenore)

All the fruits were peeled off for manually, only the flavedo was used for the experiments.

3.1(c) Handling

The peels were cleaned by washing them with soft water so that no inert matter or the

leaves may not present in the samples. All the unwanted materials from the samples were

removed.

3.1(d) Experimental detail:

Fruit peel was collected and weighed carefully. Steam distillation method for essential

oil extraction was used in this experiment. For each sample to be run in steam distillation

12.5kg peel was used. The sample peel was placed in the steam chamber. It was closed tightly

so that no steam may be lost during the process. In the steam distillation method The mixture

of two substantially immiscible liquids is heated with stirring to expose the surface of each

liquid to the gas phase, each of which independently exerts its own vapor pressure as a function

of temperature as if the other components were not present same. As a result, the vapor pressure

of the entire system increases. When the sum of the vapor pressures of the two immiscible

liquids just exceeds the atmospheric pressure (sea level of about 101 kPa), boiling begins. In

this way, many water-insoluble organic compounds are purified at temperatures well below

the point of decomposition. The steam chamber temperature was the variable in this

experiment. As 100oC is the boiling temperature of water so above the boiling point or steam

production temperature was kept and modified for the results.

22

3.1(e) Treatments:

Following were the temperature ranges used for the essential oil extraction in steam distillation

method of oil extraction

T1=105°C

T2=110°C

T3=120°C

3.1(f) Replications: 3

3.1(g) Storage of Essential oils:

Falcon tubes were used for storing the essential oils recovered directly from the steam

distillation unit. Then the extracted essential oils were shifted to the glass vials. These vials

prevent the essential oils from the decay or the air contact. These vials also prevent the essential

oils from volatilization of the compounds. The essential oil samples were kept at 4oC in the

calibrated freezer. At this temperature there are minimum chances of loss of the volatile

compounds from these oils

3.1.1 Yield attributes:

Oil percentage was measured by weighing the extracted oil just after the oil recovery.

It was done by the weight balance.

Oil percentage = wt of oil

wt of peels × 100

3.1.1(a) Physical Attributes:

To evaluate the quality and determine the composition of essential oils, used the

available facilities to analyze certain physical properties. In conducting these analyzes,

maximum attention was paid to the achievement of satisfactory results.

3.1.1(b) Density of essential oils

A density tube is a glass tube numerated an accurate volume. It was used to determine

the density ρ or specific gravity, which measures the volume, use the balance to determine the

quality. Manual glassware is generally used to determine the density or specific gravity of the

23

liquid. This is an important criterion for determining the quality and purity of essential oils. In

order to determine the physical properties of the essential oil, the specific tube or density tube

is filled one by one, leaving no bubbles in the vessel and then weighed.Density was calculated

by

ρ = m/V where

ρ means the density

m means the weight of the essential oil

V is the volume of the essential oils

3.1.1(c) Refractive Index

It is the ratio of the speed of light in space to the speed of light in a particular medium.

The refractive index is the basic physical property of the substance, which is usually used to

identify a particular substance, to confirm its purity or to measure its concentration. The

refractive index is used to measure solids, liquids, and gases. The most commonly used to

measure the concentration of solute in aqueous solution. It can also be used as a useful tool to

distinguish between different types of substances In this research work a refractometer was

used available in Tissue culture Lab Institute of Horticultural Sciences UAF.

Figure: 3.1 Essential oils samples before storage

24

3.1.1 (d) Chemical characterization

The extracted essential oils were subjected to the chemical analysis by GC-MS. Gas

Chromatography separated the chemical constituents of the essential oils and the Mass

Spectrometer identified these compounds by their structures.

3.1.2Gas Chromatography-Mass Spectrometry (GC-MS)

The qualitative exploration of citrus peel essential oils was performed by the GC-MS

using Agilent technologies 6890N-5975B system. Capillary column was at 30m×0.25µm.

temperature was programmed at 50oC to 280oC. Helium was used as the carrier. It was set a

constant scan mode wit electron energy of 70eV.Mass range was 35-400 Da while the

quadruple temperature was 150oC and source temperature was 230oC. Acquired data were

analyzed by Agilent Chem Station along with a paired NIST MS search software and AMIDIS

(Automated Mass Spectral Deconvolution and Identification System).The finally the

identification was made possible by the comparison of their relative retention indices and

retention times with recorded values (Adams, 1995; Vagionas et al., 2007; Grbovic, et al.,

2010)

Adams (1991) suggested that although a large number of spectra could be obtained

from reliable sources such as the National Bureau of Statistics (NBS), only the main

components could be easily identified. However, since then, the mass spectrometry library has

been greatly improved. In this regard, the relevant literature is also available online.

3.1.3 Statistical analysis:

The data obtained was subjected to the statistical analysis using the Analysis Of

Variance (ANOVA) technique at 5% probability (Steel et al., 1997). Then this was subjected

to the PCA. Principal component analysis (PCA) and cluster analysis techniques were used for

statistical analysis of the resulting data. PCA is one of the most important results of applying

linear algebra, and perhaps the most common use is to try the first step in analyzing large data

sets. After the PCA, the data were clustered. Clustering analysis has developed tools and

methods for data matrices containing multivariate measurements of a large number of

individuals (or objects), with the aim of building some natural subgroups or individual clusters.

25

This is done by grouping "similar" individuals by some appropriate criteria. Once clustered, it

is often useful to use some descriptive tools to describe each group (Hardle and Leopold 2007).

3.2 Experiment # 2 Comparative study of essential oils of Grapefruit peels extracted by

different methods

In this experiment two methods of essential oil extraction were used and compared with each

other. In this experiment grapefruit peel was used. Grapefruit peel had the maximum oil

contents and this experiment was only to compare the methods of essential oil extraction.

3.2.1 (a) Location:

This experiment was done at Rosa Oil Extraction Lab, Institute of Horticultural Sciences.

University of Agriculture, Faisalabad.

3.2.1 (b) Sample collection:

The fruits for the oil extraction were collected from same Sq# 9 Institute of

Horticultural Sciences, University of Agriculture, Faisalabad for this experiment. Grapefruit

was used for this experiment. All the fruits were peeled off for the peel oil extraction.

3.2.1(c) Handling

The peels were cleaned so that no inert matter or the leaves may not present in the

samples. All the unwanted materials from the samples were removed.

3.2.1(d) Experimental detail:

The present study was done in the Rosa oil extraction Lab, Institute of Horticultural

Sciences, University of Agriculture, Faisalabad. Fruit peel was collected and weighed

carefully. For each sample to be run in steam distillation the fruit peel was 12.5 Kg. Steam

distillation method of extraction was adopted as one of the two methods. It was maintained at

105oC as in the first experiment it was observed that at this temperature maximum number of

compounds and maximum oil percentage was obtained.

3.2.2 Supercritical Fluid Extraction System

Any compound above its critical temperature and pressure is called supercritical. At

supercritical stage, neither liquid nor gaseous from exists. An intermediate of both liquid and

26

gas known as fluid can be found with properties of liquid and gas molecules. The extraction

with SCFE was performed in a pilot unit in Rose Project, Institute of Horticultural Sciences,

University of Agriculture, Faisalabad. The SCFE pilot plant has one CO2 uphold tank, heat

exchanger, gas booster, one extractor and one separator vessel.

The SCFE include following steps

CO2 conditioning

Extraction process

Extract recovery

A gas booster received liquid CO2 (100 % pure) from a cylinder and pressurized a jacketed

surge tank which in turn provided gas to the jacketed extraction vessel. The jacketed surge tank

was placed between the gas booster and the extraction vessel in order to avoid potential

pressure overshoots allowing a better pressure control. The temperature of the surge tank and

extraction vessel was controlled by a thermostatic water bath. The grapefruit peels were filled

in the extraction vessel after weighing 12.5 kg weight. The extraction pressure was maintained

by the gas booster, monitored by a pressure conductor and controlled by a pneumatic control

valve. The temperature and pressure were maintained through connected computer having

SCFE run software. Extraction is carried out typically at temperature between 35-60oC (Paroul

et al., 2002). The supercritical solvent was passed into a vessel at lower pressure than the

extraction vessel. The density, and thus, dissolving power, of supercritical fluids varied sharply

with pressure, and hence, the solubility in the lower density CO2 was much lower, and the

material precipitated for collection. It was possible to fractionate the dissolved material using

a series of vessels at reducing pressure. The CO2 was recycled or depressurized to atmospheric

pressure and vented. Essential oil of the grapefruit peel was collected in flask.

27

Fig.3.3 Flow diagram of SCFE apparatus

3.2.3 Steam Distillation

In steam distillation process the compounds of a mixture of liquid are separated by

creating difference in their vapor pressure. In the present study the fruit peels were used and

weighed 12.5kg. The sample peels were placed in the steam chamber containing the sufficient

quantity of water. A sieve plate kept the water and peels separated from each other. A

continuous flame beneath the unit produced the steam in the chamber. The steam vapors and

the essential oil vapors raised and passed through the condenser. The vapors and oil drops

cooled down. These water vapors and oil drops were collected in a flask. The water was shed

by opening the beneath valve and then the oil was collected in the falcon tubes.

28

Fig.3.4 Schematic sketch of steam distillation apparatus

3.2.4 Storage of oils

The essential oil samples were kept at 4oC in the calibrated freezer. At this temperature there

are minimum chances of loss of the volatile compounds from these oils.

3.2.5 Comparison of SCFE and Steam Distillation techniques

From the experiment # 1 the results of grapefruit peel oil at its best result was used as the steam

distillation method’s result. The essential oil extracted by SCFE was compared. This

comparison was made on the basis of following attributes and efficiency of the technique.

3.2.5(a) Physical Attributes

Oil percentage

Refractive index

Density of oils

29

3.2.5 (b) Chemical Characterization



Spectrometer identified these compounds by their structures. The conditions for GC-MS were

same as in experiment # 1.

A B

Fig3.5 comparison of essential oil extracted by SCFE (A) and Steam distillation (B)

3.2.5(c) Statistical Analysis

The collected data was subjected to statistical analysis using Analysis Of Variance (ANOVA)

technique at 5% probability (Steel et al., 1997)

30

3.3 Experiment # 3 Effect of different climatic regions on citrus peel essential oils

Climate affects on the yield, quality and taste of the fruit so this affects on the essential oils as

well. With the change in altitude, latitude, rainfall and average temperature changes the

chemical characterization of the essential oils.

3.3.1 Location

The experiment was done at Rose Project Lab, Institute of Horticultural Sciences, University

of Agriculture, Faisalabad.

3.3.2 Cultivars selected

Citrus cultivars selected for the essential oil extraction were as under

Grapefruit


Musambi

3.3.3 Collection of fruits

Following districts were selected for the collection of fruits. One orchard from each district

was selected and fruits were obtained at full maturity stages.

Rahim Yar Khan

Layyah

Faisalabad

Sargodha

Abbotabad

These regions have different climatic and environmental conditions. The purpose of the

selection of the regions was the diversity in the climatic conditions and their correlation with

the production and quality of the essential oils.

3.3.4 Meteorological data for the regions

Here is the meteorological data for the selected region. This data reveals the variation in the

climatic conditions of the regions. This variation has different effects on the quality of the fruits

as well.

31

Table 3.2 Meteorological data for the regions according to Pakistan Meteorological

Department

Region Elevation (m) Average

Temperature (oC)

Annual Rainfall

(mm)

Rahim Yar Khan 82.93 26.2 101

Layyah 143 25.2 195

Faisalabad 185.6 23.2 346

Sargodha 187 22.8 410

Abbotabad 1308 16.7 1145

3.3.5 Detailed Experiment

Methodology for the experiment was adopted as in case of the first experiment. The

method adopted for the essential oil extraction was steam distillation because of eay access of

the equipment and to meet the energy effecient solutions. In this method of essential oil

extraction the temperature of the steam chamber was maintained at 105oC. At this ambient

temperature the yield percentage of the citrus peel essential oils was measured at highest level.

The chemical constituents were also maximum in number and percentage according to the

Experiment #1.

3.3.6 Essential oils collection

Essential oils from the peels of the citrus cultivars discussed above were collected in

the falcon tubes at the time of extraction. They were weighed and stored in the HPLC vial at

4oC in the freezers for further use or analysis

3.3.7 Physical Attributes

Oil percentage

Refractive index

Density of oils

32

3.3.8 Chemical Characterization



Spectrometer identified these compounds by their structures. NIST library and online

chemistry libraries helped much in the identification of the common names of the chemical

constituents of the essential oils.

3.3.9 Statistical Analysis

The data obtained was subjected to the statistical analysis using the Analysis Of

Variance (ANOVA) technique at 5% probability (Steel et al., 1997). Then this was subjected

to the Principal Component Analysis (PCA) and cluster analysis. Meteorological data of the

selected districts was correlated with components of the essential oils.

33

Chapter 4

RESULTS 4.1 Experiment # 1Effects of different temperatures on citrus peel oil extracted in steam

distillation:

4.1 (a) Physical attributes

4.1.1 Density of essential oils extracted at various temperatures in steam distillation

Statistical analysis showed that the mean density of oil was significantly different in three

cultivars i.e., grapefruit, Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and musambi

(Fig.4.1.1). Maximum oil density was recorded in grapefruit (0.881 mg/cm3) followed by

Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) (0.846 mg/cm3) and Musambi (0.833

mg/cm3) at 105°C. The density of oil gradually reduced in grapefruit peel extracted at 110°C

(0.872 mg/cm3) and 120°C (0.864 mg/cm3), respectively. Oil density was recorded inversely

proportional to the temperature used in the steam distillation method of extraction. The

density of Kinnow (C.nobilis Loureiro×C. deliciosa Tenore)peel oil extracted at 105°C

(0.846 mg/cm3) was more than oil extracted at 110°C (0.845mg/cm3) and 120°C (0.830

mg/cm3) respectively. The Musambi peel oil density extracted at 105°C was maximum

(0.833 mg/cm3) which gradually reduced in extracted oils at 110°C (0.831 mg/cm3) and

120°C (0.822 mg/cm3), respectively. The Musambi oil density (0.831 mg/cm3) at 110°C was

more than the Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) oil extracted at 120°C

(0.830).

Interaction plot (Fig. 4.1.2) showed the decreasing trend in oil density with the increase in

temperature in all cultivars i.e. Grapefruit, Kinnow (C.nobilis Loureiro×C. deliciosa Tenore)

and Musambi. Oil density of grapefruit was significantly greater from the Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) and Musambi which have close interaction. The individual

effect of temperature and cultivar for density of essential oils was significant. Two way

interaction of temperature and fruit was also significant (Table. 4.1.1). Tukey test (Table:

4.1.2) revealed the differences between all the cultivars for their densities.

34

Figure: 4.1.1 Density (mg/cm3) of essential oil recovered at various temperatures in Steam

distillation

35

Fig. 4.1.2 Interaction plot for the density of essential oils at various temperatures in steam

distillation

36

Table 4.1.1 Effect of temperature and cultivar on density (mg/cm3) of essential oils

SOV df SS MS F value Pr(>F)

Temperatur

e 1 1.167 1.1675 10.213 0.00435**

Cultivar 2 1.427 0.7136 6.243 0.00745**

Temp×Cult. 2 1.657 0.8284 7.247 0.00404**

Residuals 21 2.401 0.1143

Table: 4.1.2Cultivar- cultivar essential oil density (mg/cm3) interactions

Tukey HSD Table for Fruit

Difference Lower Upper P-value

Kinnow-Grapefruit -0.033 -0.036 -0.029 0.000

Musambi-Grapefruit -0.043 -0.047 -0.041 0.000

Musambi-Kinnow -0.010 -0.014 -0.007 0.000

37

4.1.2 Essential oil percentage extracted at various temperatures in steam distillation

The oil percentage showed significant difference in three cultivars i.e., grapefruit,

Kinnow (C.nobilis Loureiro×C. deliciosa Tenore)and Musambi (Fig.4.1.3). Maximum oil

percentage was recorded in grapefruit (0.311 %) followed by Musambi (0.310 %) and

Kinnow (C.nobilis Loureiro×C. deliciosa Tenore)(0.302 %). The maximum oil percentage

was recorded in grapefruit peel extracted at 105°C. The percentage of oil gradually reduced

in grapefruit peel extracted at 110°C (0.304 %) and 120°C (0.294 %), respectively. The

percentage of Kinnow (C.nobilis Loureiro×C. deliciosa Tenore)peel oil extracted at 105°C

(0.302 %) was more than oil extracted at 110°C (0.302 %) and 120°C (0.287 %)

respectively.The Musambi peel oil percentage extracted at 105°C was maximum (0.310 %)

which gradually reduced in extracted oils at 110°C (0.308 %) and 120°C (0.302 %),

respectively. The Musambi oil percentage at 105°C and 110°C was more than the Kinnow

(C.nobilis Loureiro×C. deliciosa Tenore) oil extracted at 105°C, 110°C and 120°C. The

Musambi oil percentage at 110°C and 120°C was more than the Grapefruit oil percentage

extracted at 110°C and 120°C.

Interaction plot (Fig: 4.1.4) showed the decreasing trend in oil percentage with increase

in temperature in all the cultivars. Oil percentage of Musambi was significantly different from

the Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and grapefruite which have close

interaction. At 105°C the interaction of oil percentage of Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore) and grapefruit was very close.

The individual effect of temperature and citrus cultivars for percentage of essential oils

was highly significant. Two way interaction of temperature and cultivars was insignificant for

percentage of essential oils (Table. 4.1.3). Tukey HSD test (Table 4.1.4) revealed the difference

between oil percentage in all the cultivars for their essential oil percentages.

38

Figure 4.1.3 Effect of temperature on essential oil percentage in Steam distillation

39

Figure 4.1.4 Interaction plot for percentage of essential oil extracted by Steam distillation

at various temperatures

40

Table 4.1.3 Effect of temperature on essential oil percentage in Steam distillation

SOV DF SS MS F value Pr(>F)

Temperature 1 0.0008932 0.0008932 84.769 0.000**

Cultivar 2 0.0003117 0.0001558 14.789 0.000**

Temperature cultivar 2 0.0000545 0.0000273 2.588 0.098*

Residuals 21 0.0002213 0.0000105

Table 4.1.4 Tukey HSD table for cultivars

Diff lower upper P value

Kinnow)-Grape -0.0045859 -0.0084430 -.0007288 0.018077

Musambi-Grape 0.0036667 -0.0001904 0.0075238 0.06435

Musambi-Kinnow 0.008252604 0.0043955 0.0121097 0.0000683

41

4.1.3 Refractive index of essential oils extracted at various temperatures in steam

distillation

The refractive index of essential oils showed significant difference in three cultivars

i.e., Grapefruit, Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and Musambi (Fig. 4.1.5).

Maximum oil refractive index (1.472) was recorded in Grapefruit followed by Musambi

(1.471) and Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) (1.470). The maximum oil

refractive index was recorded in Grapefruit peel extracted at 105°C i.e. 1.472. The refractive

index of essential oil gradually decreased in grapefruit peel extracted at 110°C (1.470) and

120°C (1.468), respectively. The refractive index of Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore) peel oil extracted at 105°C (1.470) was more than oil extracted at 110°C

(1.469) and 120°C (1.467), respectively. The musambi peel oil refractive index extracted at

105°C was maximum (1.4713) which gradually reduced in extracted oils at 110°C (1.471)

and 120°C (1.466), respectively. The musambi peel oil refractive index at 105°C and 110°C

was more than the Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) oil extracted at 105°C,

110°C and 120°C. The musambi peel oil refractive index at 110°C and 120°C was more than

the Grapefruit oil refractive index extracted at 110°C and 120°C. The refractive index of

Musambi oil extracted at 120°C was less than Kinnow (C.nobilis Loureiro×C. deliciosa

Tenore) and Grapefruit essential oil refractive index extracted at 120°C.

Interaction plot (Figure 4.1.6) showed the decreasing trend oil refractive index with

increase in temperature in all cultivars i.e. Grapefruit, Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore) and Musambi. Oil refractive index of Musambi was very close to Grapefruit

extracted at 105°C and 110°C. The decreasing trend in refractive index of Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) was significantly different from Musambi and Grapefruit.The

individual effect (Table. 4.1.5) of temperature and fruit for refractive index of essential oils

was significant. Two way interactions of temperature and fruit was also significant for

refractive index of essential oils.

42

Figure 4.1.5 Effect of temperature on Refractive index of essential oils in Steam

distillation

43

Figure 4.1.6 Interaction plot Refractive index of the essential oils in Steam distillation

44

Table 4.1.5 Effect of temperature on citrus peel essential oil refractive index in Steam

Distillation

SOV Df SS MS F value Pr(>F)

Temperature 1 0.0000703 0.0000703 73.319 0.0000000*

Cultivar 2 0.0000051 0.0000026 2.671 0.0926000*

Temp×Cultivar 2 0.0000092 0.0000046 4.769 0.0196000*

Residuals 21 0.0000201 0.0000010

45

4.1.4 Chemical Characterization of essential oils extracted at various temperatures in

steam distillation

The chemical characterization of essential oils was analyzed by the help of GC-MS. Gas

chromatography isolated the compounds and Mass Spectrometry identified the compounds

making their peaks in the chromatogram sketches. These compounds were matched with NIST

libraries for their nomenclature.

Grapefruit peel essential oil extracted 105oC according to (Fig:4.1.7), have the compounds (-

) α-Neoclovene , ϒ-Muurolene , 1-Decene , 28 Menthadien-1-ol , α-caryophyllene , α-pinene

, Aromadendrene , Caryophyllene , Caryophyllene oxide , Citronellal , Citronellol , Copaene ,

Corane , D-Cadinene , Decanal , E-carvool , Elemol , Epiglobulol , Limonene-4-ol , Limonene

, Limonine oxide , Linalool , Linalool oxide , Nootkatone , Perillyl acetate , Sabinene , ß-

Cubebene , ß-Pinene , Terpinene-4-ol and Valencene.

Grapefruit peel essential oil extracted at 110oC showed the following compounds (Figure

4.1.8) α-caryophyllene , α-pinene , Aromadendrene, Carvene , Carveol , Carvone ,

Caryophyllene , Citronellal , Copaene , D-Cadinene , Decanal , Limonene , Linalool ,

Nootkatone , Terpinene-4-ol , Trans-Carved and Valencene. Grapefruit peel essential oil

extracted at 120oC showed the following compounds (Figure 4.1.9) α-pinene , Copaene ,

Farnesol , Heneicosane , Heptacosane , Levoverbenone , Limonene , Linalool , Nerolidol ,

Nootkatone , Octacosane , ß-elemene , ß-Myrcene , Terpinene-4-ol , Trans-2-Menthenol and

Valencene.

Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel essential oil extracted in steam

distillation at 105oC contained the compounds (Fig:4.1.10) (-) α-Neoclovene , 1-Decene , 22-

Tritetracontanone , 4-terpinene , α-caryophyllene , α-pinene , α-Sinensal , Anethol ,

Caryophyllene , Citronellal , Citronellol , Copaene , D-Cadinene , Decanal , Heneicosane ,

Levoverbenone , Limonene , Linalool , Nootkatone , ß-elemene , ß-Pinene , ß-Terpenyl Acetate

, Terpinene-4-ol and Valencene. Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel

essential oil extracted in steam distillation at 110oC contained the compounds (Fig:4.1.11) α-

caryophyllene , Anethol , Caryophyllene , Citronellal , Citronellol , Copaene , D-Cadinene ,

Decanal , E-Nerolidol , Elemol , Fernesyl acetate , Levoverbenone , Limonene , Linalool ,

Nootkatone , ß-Terpenyl Acetate and Valencene. Kinnow (C.nobilis Loureiro×C. deliciosa

Tenore) peel essential oil extracted in steam distillation at 120oC contained the compounds

46

(Fig:4.1.12) α-Fernesene , Aromadendrene , Caryophyllene , Citronellal , Citronellol , Decanal

, Estragole , Limonene , Linalool , Nootkatone , Octacosane , ß-Myrcene and Valencene.

Musambi peel essential oil extracted by steam distillation at 105oC (Fig:4.1.13) showed the

compounds α-citral , α-pinene , Aromadendrene , Caryophyllene , Citronellol , D-Cadinene ,

Decanal , Levoverbenone , Limonene , Linalool , Nootkatone , Perillyl acetate , ß -Citral , ß-

Pinene and Valencene. Musambi peel essential oil extracted by steam distillation at 110oC

(Fig:4.1.14) showed the compounds 4-terpinene , Aromadendrene , Calarene , Citronellol , D-

Cadinene , Decanal , Juniper Camphor , Levoverbenone , Limonene , Limonene Oxide-trans ,

Linalool , Nootkatone , ß-Pinene and Valencene. Musambi peel essential oil extracted by

steam distillation at 120oC (Fig:4.1.15) showed the compounds (-) α-Neoclovene , Citronellal

, Copaene , D-Cadinene , Decanal , Farnesol , Levoverbenone , Limonene , Linalool ,

Nootkatone , Sabinene , ß-Pinene and Valencene.

47

Table: 4.1.6 Percent composition for different compounds in citrus cultivars essential oils

at different temperature levels in steam distillation

Compound

Grapefruit

Kinnow (C.nobilis

Loureiro×C. deliciosa

Tenore)

Musambi

105°C 110°C 120°C 105°C 110°C 120°C 105°C 110°C 120°C

(-) α-Neoclovene 0.26 0 0 0.52 0 0 0 0 0.11

ϒ-Muurolene 0.29 0 0 0 0 0 0 0 0

1-Decene 0.2 0 0 0.14 0 0 0 0 0

22-Tritetracontanone 0 0 0 0.26 0 0 0 0 0

28 Menthadien-1-ol 0.97 0 0 0 0 0 0 0 0

4-terpinene 0 0 0 0.23 0 0 0 0.2 0

α-caryophyllene 0.24 0.15 0 0.28 0.25 0 0 0 0

α-citral 0 0 0 0 0 0 0.15 0 0

α-Fernesene 0 0 0 0 0 0.27 0 0 0

α-pinene 2.01 0.81 0.24 0.29 0 0 0.6 0 0

α-Sinensal 0 0 0 0.16 0 0 0 0 0

Anethol 0 0 0 0.33 0.29 0 0 0 0

Aromadendrene 0.23 0.34 0 0 0 0.16 0.11 0.12 0

Calarene 0 0 0 0 0 0 0 0.2 0

Carvene 0 0.57 0 0 0 0 0 0 0

Carveol 0 3.14 0 0 0 0 0 0 0

Carvone 0 2.1 0 0 0 0 0 0 0

Caryophyllene 2.3 1.19 0 1.94 1.85 0.14 0.24 0 0

Caryophyllene oxide 1.18 0 0 0 0 0 0 0 0

Citronellal 0.29 0.27 0 0.34 0.26 0.2 0 0 0.17

Citronellol 0.51 0 0 0.39 0.21 0.18 0.48 0.44 0

48

Compound Grapefruit

Kinnow (C.nobilis


Tenore) Musambi

105°C 110°C 120°C 105°C 110°C 120°C 105°C 110°C 120°C

Corane

0.36

0

0

0

0

0

0

0

0

D-Cadinene 1.35 0.95 0 1.73 0.41 0 0.2 0.19 0.19

Decanal 1.87 0.67 0 1.31 1.09 1.04 0.38 0.29 0.29

E-carvool 0.69 0 0 0 0 0 0 0 0

E-Nerolidol 0 0 0 0 0.63 0 0 0 0

Elemol 0.38 0 0 0 0.23 0 0 0 0

Epiglobulol 0.22 0 0 0 0 0 0 0 0

Estragole 0 0 0 0 0 0.23 0 0 0

Farnesol 0 0 0.44 0 0 0 0 0 0.22

Fernesyl acetate 0 - 0 0 0.9 0 0 0 0

Heneicosane 0 0 0.65 0.27 0 0 0 0 0

Heptacosane 0 0 0.43 0 0 0 0 0 0

Juniper Camphor 0 0 0 0 0 0 0 0.17 0

Levoverbenone 0 0 0.48 0.54 0.24 0 0.17 0.34 0.6

Limonene-4-ol 0.47 0 0 0 0 0 0 0 0

Limonene 82.86 84.33 85.44 84.45 87.23 89.56 91.97 93.16 94.19

Limonene Oxide-trans 0 0 0 0 0 0 0 0.3 0

Limonine oxide 0.19 0 0 0 0 0 0 0 0

Linalool 0.4 0.29 0.16 0.55 0.46 0.29 1.5 0.58 0.56

Linalool oxide 0.16 0 0 0 0 0 0 0 0

Nerolidol 0 0 0.3 0 0 0 0 0 0

Nootkatone 1.92 1.79 0.53 3.14 2.12 0.15 0.17 0.15 0.15

Octacosane 0 0 0.37 0 0 0.34 0 0 0

Perillyl acetate 0.43 0 0 0 0 0 0.11 0 0

Sabinene 0.29 0 0 0 0 0 0 0 0.1

ß-Citral 0 0 0 0 0 0 0.17 0 0

49

Compound Grapefruit

Kinnow (C.nobilis


Tenore) Musambi

105°C 110°C 120°C 105°C 110°C 120°C 105°C 110°C 120°C

ß-elemene 0 0 0.18 0.41 0 0 0 0 0

ß-Myrcene 0 0 0.58 0 0 0.59 0 0 0

ß-Pinene 0.94 0 0 0.63 0 0 0.74 0.84 1.41

ß-Terpenyl Acetate 0 0 0 0.25 0.25 0 0 0 0

Terpinene-4-ol 0.31 0.24 0.12 0.9 0 0 0 0 0

Trans-2-Menthenol 0 0 0.94 0 0 0 0 0 0

Trans-Carved 0 1.83 0 0 0 0 0 0 0

Valencene 0.65 0.95 1.02 0.23 2.24 2.65 1.81 2.21 2.34

50

Figure: 4.1.7 Typical chromatogram of grapefruit peel essential oil extracted by steam

distillation at 105oC

51

Figure: 4.1. 8 Typical chromatogram of grapefruit peel essential oil extracted by steam


52

Figure: 4.1.9 Typical chromatogram of grapefruit peel essential oil extracted by steam


53

Figure: 4.1.10. Typical chromatogram of Kinnow (C.nobilis Loureiro×C. deliciosa

Tenore) peel essential oil extracted by steam distillation at 105oC

54

Figure: 4.1.11. Typical chromatogram of Kinnow (C.nobilis Loureiro×C. deliciosa

Tenore) peel essential oil extracted by steam distillation at 110oC

55

Figure: 4.1. 12 Typical chromatogram of Kinnow (C.nobilis Loureiro×C. deliciosa Tenore)

peel essential oil extracted by steam distillation at 120oC

56

Figure: 4.1.13 Typical chromatogram of Musambi peel essential oil extracted by steam


57

Figure: 4.1.14Typical chromatogram of Musambi peel essential oil extracted by steam


58

Figure: 4.1.15. Typical chromatogram of Musambi peel essential oil extracted by steam


59

4.1.5 Ward’s hierarchical method for compounds diversity in essential oils extracted at

various temperatures in steam distillation

Hierarchical clustering by using Ward’s hierarchical method (Ward, 1963) grouped the

compounds in three main clusters is represented on the hierarchical tree using Euclidean

distance and showed a fairly clear picture (Figure 4.1.16). Cluster I included these compounds:

E-carveol, Epiglobulol, Limonene-4-ol, Limonene oxide, α-caryophyllene, Caryophyllene,

Citronellal, Copaene, α-pinene, Terpinene-4-ol, Aromadendrene, Citronellol, D-Cadinene,

Decanal, Levoverbenone, β-Pinene, Limonene, Linalool, Nootkatone andValencene. Cluster

II contained Limonene oxide-trans, Carvene, Carveol, Carvone, Trans-carved, α-Fernesene,

Estragole, Octacosane, β-Myrecene, Fernesol, Heptacosane, Nerollidol, Trans-2-Menthenol,

ϒ-Muurorene, 2 8 Menthadien-1-ol, Caryophyllene oxide, Corane, Linalool oxide and β-

Cubebene. Cluster IIIincluded these compounds(-)α-Neoclovene, 1-Decene, Sabinene, 22-

Tritetracontanone, α-Sinesal, 4-terpinene, Heneicosane, β-elemene, Anethol, β-Terpenyl

Acetate, E-Nerolidol, Elemol, Fernesyl acetate, α-citral, β-citral, Perillyl acetate, Calarene and

Juniper camphor (Figure 4.1.16).

In each cluster, the number of compounds are different and decide the number of sub-clusters

and groups on hierarchical tree. Cluster I is subdivided into two sub-clusters. Sub-cluster II of

cluster I was further divided into two groups. Each group included different compounds. Group

I of sub-cluster II included following compounds namely Aromadendrene, Citronellol, D-

Cadinene, Decanal, Levoverbenone and β-Pinene. Group II of sub-cluster II included

following compounds namelyα-caryophyllene, Caryophyllene, Citronellal, Copaene, α-pinene

and Terpinene-4-ol (Figure 4.1.16). Cluster III has subdivided into two sub-clusters. In sub-

cluster I of cluster III following compounds included namely β-citral, Perillyl acetate, Calarene

and Juniper camphor. Sub-cluster II of cluster III has further divided into two groups. Group I

of sub-cluster II of cluster III included compounds viz., β-Terpenyl Acetate, E-Nerolidol,

Elemol andFernesyl acetate. Group II of sub-cluster II of cluster III included compounds

namely (-)α-Neoclovene, 1-Decene, Sabinene, 22-Tritetracontanone, α-Sinesal, 4-terpinene

and Heneicosane (Figure 4.1.16).

60

Fig 4.1.16: Diversity of different compounds found in essentials oils extracted from citrus

cultivars

Ward`s method

Euclidean distances

0 5 10 15 20

Linkage Distance

ValenceneNootkatone

LinaloolLimoneneß-Pinene

LevoverbenoneDecanal

D-CadineneCitronellol

AromadendreneTerpinene-4-ol

a-pineneCopaene

CitronellalCaryophyllene

a-caryophylleneLimonine oxideLimonene-4-ol

EpiglobulolE-carvool

ß-CubebeneLinalool oxide

CoraneCaryophyllene oxide28 Menthadien-1-ol

?-MuuroleneTrans-2-Menthenol

NerolidolHeptacosane

Farnesolß-Myrcene

OctacosaneEstragole

a-FerneseneTrans-Carved

CarvoneCarveol

CarveneLimonene Oxide-trans

Juniper CamphorCalarene

Perillyl acetateß-Citrala-citral

Fernesyl acetateElemol

E-Nerolidolß-Terpenyl Acetate

Anetholß-elemene

Heneicosane4-terpinenea-Sinensal

22-TritetracontanoneSabinene1-Decene

(-) a-Neoclovene

61

4.1.6 Principal components on the basis of diversity of different compounds

The principal component analysis based on the correlation matrix was performed to

evaluate diversity and grouping pattern of the compounds in oils extracted from three citrus

cultivars and that of the temperatures used to extract the oils from the cultivars. The criterion

of the significance of the Eigen values, established by Kaiser (1960), was used to select the

statistically significant principal components (PCs). Only those principal components that

exhibited the eigenvalues greater than one were considered as significant. Scree plot explained

the percentage variance associated with each principal component, obtained by drawing graph

between Eigen values and principal component numbers.

Out of nine principal components, the first two showed eigenvalues greater than one

(significant) in different compounds found in essentials oils extracted from citrus cultivars at

different temperatures (Fig 4.1.17). The other PCs exhibited non-significant variation and were

not worth interpreting. The first two PCs showed cumulative variability of 40.09% and 12.62%

respectively in different compounds found in essentials oils extracted from citrus cultivars at

different temperatures (Fig 4.1.17).

The method established by Johnson and Wichern (1988) was used to estimate the

importance of a trait coefficient for each significant principal component. The first PC was

highly related to temperature (110oC) used to extract essential oil from grapefruit, temperatures

(105oC, 110oC and 120oC) used to extract essential oils from Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore) and to temperatures (105oC, 110oC and 120oC) used to extract essential oils

from Musambi (Table 4.1.7). The second PC was related to temperature (105oC) used to extract

essential oil from grapefruit (Table 4.1.7).

A principal component scatter plot of different compounds found in essential oils

extracted from citrus cultivars at different temperatures depicted that compounds and

temperatures that are close together are being similar when related with other compounds and

temperatures. The projection of compounds and temperatures on PC1 and PC2 showed

thediverse nature of both factors. To identify the better transgressive pattern of compounds and

temperatures in dissimilar groups of pattern, the projection of compounds and temperatures on

first two principal components was useful. The projection of traits on PC1 and PC2 revealed

that the temperature (105oC) used to extract essential oil from grapefruit were opposite in

62

ordination with the temperature (120oC) used to extract essential oil from grapefruit and

temperature (105oC and 110oC) used to extract essential oil from Musambi, are positively

related to temperature (1100C) used to extract essential oil from grapefruit and to temperature

(110oC) used to extract essential oil from Kinnow (C.nobilis Loureiro×C. deliciosa Tenore)

but these all temperatures which are positively correlated to each other are negatively

correlated to temperature (120oC) used to extract essential oil from Musambi and to

temperatures (105oC and 120oC) used to extract essential oil from Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) (Fig. 4.1.18).

The projection of compounds on PC1 and PC2 showed population structure on the basis

of presence of compounds (Fig. 4.1.19). From the different compounds in essential oils

extracted from three cultivars of citrus, the following diverse groups were identified. The

compounds (D-Cadinene, Decanal,caryophyllene, citronellol, β-pinene, Perillyl acetate,

Sabinene,1- Decene, (-)α-Neoclovene, Aromadendrene, Fernyl acetate, β-Citral, α-

caryophyllene and Elemol) were opposite to the compounds (α-pinene, Copaene, Terpinene-

4-ol, levoverbenone, Fernesol, β-terpenyl acetate, Nootakatone, Trans-2-menthenol, 4-

terpinene, Heptacosane, Limonene oxide-trans).

63

Fig: 4.1.17 Scree plot between eigen values and number of principal components of different

compounds found in essentials oils extracted from citrus cultivars

Eigenvalues of correlation matrix

40.09%

12.62%

10.52%9.19%

8.35%

6.18%5.56%

4.18%3.30%

-1 0 1 2 3 4 5 6 7 8 9 10 11

Eigenvalue number

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0E

igenvalu

e

40.09%

12.62%

10.52%9.19%

8.35%

6.18%5.56%

4.18%3.30%

64

Table: 4.1.7 Principal components of different compounds found in essentials oils extracted from

citrus cultivars at different temperatures

Cultivars Principal component 1 Principal component 2

Grapefruit (105°C) -0.447127 0.650540

Grapefruit(110°C) -0.627192 0.071089

Grapefruit(120°C) -0.327010 -0.815402

Kinnow (105°C) -0.680216 -0.107705

Kinnnow (110°C) -0.724606 0.065890

Kinnnow (120°C) -0.607715 -0.100372

Musambi(105°C) -0.745835 0.105604

Musambi (110°C) -0.659647 -0.007276

Musambi(120°C) -0.748035 -0.075216

Table: 4.1.8 Eigenvalues of correlation matrix, and related statistics of different

compounds found in essentials oils extracted from three citrus cultivars at different

temperatures

PCs Eigenvalue % Total – variance Cumulative – Eigenvalue Cumulative - %

1 3.608255 40.09172 3.608255 40.0917

2 1.136015 12.62239 4.744270 52.7141

3 0.946669 10.51855 5.690939 63.2327

4 0.827354 9.19282 6.518293 72.4255

5 0.751173 8.34637 7.269466 80.7718

6 0.556484 6.18316 7.825951 86.9550

7 0.500529 5.56143 8.326479 92.5164

8 0.376613 4.18459 8.703092 96.7010

9 0.296908 3.29898 9.000000 100.0000

65

Fig.4.1.18 Two dimensional ordination of different temperatures used to extract essentials oils

from 3 citrus cultivars on PC1 and PC2

Projection of the variables on the factor-plane ( 1 x 2)

GF_105°C

GF_110°C

GF_120°C

Kn_105°C

Kn_110°C

Kn_120°C

Ms_105°C

Ms_110°CMs_120°C

-1.0 -0.5 0.0 0.5 1.0

Factor 1 : 40.09%

-1.0

-0.5

0.0

0.5

1.0

Facto

r 2 :

12

.62

%

GF_105°C

GF_110°C

GF_120°C

Kn_105°C

Kn_110°C

Kn_120°C

Ms_105°C

Ms_110°CMs_120°C

66

Fig: 4.1.19. Two dimensional ordination of different compounds in essentials oils extracted from

3 citrus cultivars on PC1 and PC2

Projection of the cases on the factor-plane ( 1 x 2)

Cases with sum of cosine square >= 0.00

(-) a-Neoclovene

?-Muurolene

1-Decene

22-Tritetracontanone

28 Menthadien-1-ol

4-terpinene

a-caryophyllene

a-citral

a-Fernesenea-pinene

a-Sinensal

Anethol

Aromadendrene

Calarene

CarveneCarveolCarvone

CaryophylleneCaryophyllene oxide

Citronellal

Citronellol

Copaene

CoraneD-Cadinene

DecanalE-carvool

E-Nerolidol

Elemol

Epiglobulol

Estragole

Farnesol

Fernesyl acetate

Heneicosane

Heptacosane

Juniper Camphor

Levoverbenone

Limonene-4-ol

Limonene

Limonene Oxide-trans

Limonine oxide

Linalool

Linalool oxide

Nerolidol

Nootkatone

Octacosane

Perillyl acetate

Sabinene

ß-Citral

ß-Cubebene

ß-elemeneß-Myrcene

ß-Pinene

ß-Terpenyl Acetate

Terpinene-4-ol

Trans-2-Menthenol

Trans-Carved

Valencene

-6 -5 -4 -3 -2 -1 0 1 2 3

Factor 1: 40.09%

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5F

acto

r 2: 1

2.6

2%

(-) a-Neoclovene

?-Muurolene

1-Decene

22-Tritetracontanone

28 Menthadien-1-ol

4-terpinene

a-caryophyllene

a-citral

a-Fernesenea-pinene

a-Sinensal

Anethol

Aromadendrene

Calarene

CarveneCarveolCarvone


Citronellal

Citronellol

Copaene

CoraneD-Cadinene

DecanalE-carvool

E-Nerolidol

Elemol

Epiglobulol

Estragole

Farnesol

Fernesyl acetate

Heneicosane

Heptacosane

Juniper Camphor

Levoverbenone

Limonene-4-ol

Limonene

Limonene Oxide-trans

Limonine oxide

Linalool

Linalool oxide

Nerolidol

Nootkatone

Octacosane

Perillyl acetate

Sabinene

ß-Citral

ß-Cubebene

ß-elemeneß-Myrcene

ß-Pinene

ß-Terpenyl Acetate

Terpinene-4-ol

Trans-2-Menthenol

Trans-Carved

Valencene

67

4.1.7 Principal Components on the basis of percentage of compounds

In the case of percentage of different compounds in essential oils extracted from three cultivars

of citrus, only one PC showed eigen values greater than one (significant) at different

temperatures (Fig 4.1.20). The other PCs exhibited non-significant variation and were not

worth interpreting. The first PC showed cumulative variability of 99.91% in different

compounds percentages found in essentials oils extracted from citrus cultivars at different

temperatures (Fig 4.1.20).The method established by Johnson and Wichern (1988) was used

to estimate the importance of a trait coefficient for each significant principal component. The

first PC was negatively related to all temperature used to extract the essential oils from

Grapefruit, Musambi and Kinnow (C.nobilis Loureiro×C. deliciosa Tenore)(Table 4.1.9).

A principal component scatter plot of different compounds found in essentials oils extracted

from citrus cultivars at different temperatures depicted that percentages of different compounds

in essential oils extracted from citrus cultivars that are close together are being similar when

related with other percentages of compounds. The projection of percentages of different

compounds on PC1 and PC2 showed the diverse nature. To identify the better transgressive

pattern of percentages of compounds in dissimilar groups of pattern, the projection of

percentages of compounds on first two principal components was useful.

The projection of compounds on PC1 and PC2 showed population structureon the basis of

percentages of compounds (Fig. 4.1.21). From the different compounds in essential oils

extracted from three cultivars of citrus, the following diverse groups were identified. The

compounds (Nootkatone, Caryophyllene, D-Cadinene, Copaene, Decanal,Carveol, α-pinene,

Carvone,Trans-Carved, Terpinene-4-ol, Caryophyllene oxide and 28 Menthadien-1-ol) were

opposite to the compounds (Aromadendrene, juniper Camphor, Trans- 2 Menthenol, Limonene

oxide trans, Heniecosane, levoverbenone, β-Myrecene, limonene, valencene, 22

tritetracontanone) (Fig. 4.1.21).

68

Fig: 4.1.20 Scree plot between eigen values and number of principal components of different

compounds percentage found in essentials oils extracted from three citrus cultivars


99.91%

.04% .03% .01% .01% .00% .00% .00% .00%

-1 0 1 2 3 4 5 6 7 8 9 10 11

Eigenvalue number

-1

0

1

2

3

4

5

6

7

8

9

10

Eig

envalu

e

99.91%

.04% .03% .01% .01% .00% .00% .00% .00%

69

Table: 4.1.9 Principal components of percentage ofdifferent compounds found in essentials oils

extracted from three citrus cultivars at different temperatures

Cultivars Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Factor 7 Factor 8 Factor 9

Grapefruit (105°C) -0.999214 0.027753 0.018974 0.020450 0.001950 0.004092 0.000100 -0.001252 0.000239

Grapefruit(110°C) -0.998865 0.024099 -0.041002 0.002214 0.001069 -0.000655 -0.000036 -0.000163 -0.000014

Grapefruit(120°C) -0.999685 -0.013760 -0.001748 -0.003573 -0.015497 0.013277 0.002678 -0.000420 -0.000049

Kinnow (105°C) -0.999296 0.029000 0.014307 -0.014475 -0.009463 -0.006865 -0.003855 0.001009 -0.000160

Kinnnow (110°C) -0.999698 0.006628 0.007685 -0.013981 0.015818 0.004605 0.005805 -0.000572 0.000061

Kinnnow (120°C) -0.999713 -0.019361 -0.000464 -0.000425 0.008277 0.005464 -0.009710 0.002368 -0.000917

Musambi(105°C) -0.999790 -0.015427 0.001519 0.006138 -0.001328 -0.007046 0.004477 0.008340 -0.001325

Musambi (110°C) -0.999784 -0.019293 0.000030 0.001482 -0.000029 -0.005434 -0.000290 -0.001639 0.005023

Musambi(120°C) -0.999744 -0.019590 0.000680 0.002173 -0.000799 -0.007438 0.000829 -0.007673 -0.002858

Table: 4.1.10 Eigenvalues of correlation matrix, and related statistics of percentage ofdifferent

compounds found in essentials oils extracted from three citrus cultivars at different temperatures

Eigenvalue % Total - variance Cumulative – Eigenvalue Cumulative - %

1 8.991581 99.90645 8.991581 99.9065

2 0.003794 0.04216 8.995375 99.9486

3 0.002311 0.02568 8.997686 99.9743

4 0.000886 0.00984 8.998571 99.9841

5 0.000656 0.00729 8.999227 99.9914

6 0.000426 0.00474 8.999653 99.9961

7 0.000171 0.00190 8.999824 99.9980

8 0.000140 0.00155 8.999964 99.9996

9 0.000036 0.00040 9.000000 100.0000

70

Fig: 4.1.21 Two dimensional ordination of percentage of different compounds in essentials oils

extracted from 3 citrus cultivars on PC1 and PC2



(-) a-Neoclovene

?-Muurolene1-Decene22-Tritetracontanone

28 Menthadien-1-ol

4-terpinene

a-caryophyllene

a-citrala-Fernesene

a-pinene

a-SinensalAnetholAromadendrene

Calarene

Carvene

Carveol

Carvone

Caryophyllene

Caryophyllene oxide

CitronellalCitronellol

Copaene

Corane

D-Cadinene

Decanal

E-carvool

E-NerolidolElemol

EpiglobulolEstragoleFarnesol

Fernesyl acetateHeneicosaneHeptacosaneJuniper Camphor

Levoverbenone

Limonene-4-olLimonene

Limonene Oxide-transLimonine oxide

Linalool

Linalool oxideNerolidol

Nootkatone

Octacosane

Perillyl acetateSabineneß-Citral

ß-Cubebeneß-elemene

ß-Myrcene

ß-Pinene

ß-Terpenyl Acetate

Terpinene-4-ol

Trans-2-Menthenol

Trans-Carved

Valencene

-25 -20 -15 -10 -5 0 5

Factor 1: 99.91%

-0.2

-0.1

0.0

0.1

0.2

0.3

Facto

r 2:

.04%

(-) a-Neoclovene

?-Muurolene1-Decene22-Tritetracontanone

28 Menthadien-1-ol

4-terpinene

a-caryophyllene

a-citrala-Fernesene

a-pinene

a-SinensalAnetholAromadendrene

Calarene

Carvene

Carveol

Carvone

Caryophyllene

Caryophyllene oxide

CitronellalCitronellol

Copaene

Corane

D-Cadinene

Decanal

E-carvool

E-NerolidolElemol

EpiglobulolEstragoleFarnesol

Fernesyl acetateHeneicosaneHeptacosaneJuniper Camphor

Levoverbenone

Limonene-4-olLimonene

Limonene Oxide-transLimonine oxide

Linalool

Linalool oxideNerolidol

Nootkatone

Octacosane

Perillyl acetateSabineneß-Citral

ß-Cubebeneß-elemene

ß-Myrcene

ß-Pinene

ß-Terpenyl Acetate

Terpinene-4-ol

Trans-2-Menthenol

Trans-Carved

Valencene

71

Experiment # 2 Comparative study of essential oils of Grapefruit peels extracted by

different methods


4.2.1 Density of essential oils extracted by two methods of extraction

Density (mg/cm3) of essential oils recovered by Supercritical fluid extraction system and

Steam distillation was measured.The oil density of grapefruit was significantly different in the

peel oil extracted after Super Critical Fluid Extraction (SCFE) and Steam Distillation (SD)

method. The oil density was highest in case of SCFE i.e. 0.834mg/cm3and lower in Steam

distillation i.e., 0.829 mg/cm3 (Figure: 4.2.1). The individual effect of two methods

(Supercritical Fluid Extraction and Steam Distillation) was significant for oil density of

grapefruit (Table. 4.2.1).

4.2.2 Oil percentage extracted by two methods of extraction

The oil percentage of grapefruit was significantly different in the peel oil extracted after Super

Critical Fluid Extraction (SCFE) i.e.(0.243 %) and Steam Distillation (SD) method it was

(0.311%). The oil percentage was highest in case of SD and lowest in SCFE. The individual

effect of two methods (Super Critical Fluid Extraction and Steam Distillation) was significant

for oil percentage of Grapefruit (Table. 4.2.2).

4.2.3 Refractive index of essential oils extracted by two methods of extraction

The oil refractive index of grapefruit peel oil differs significantly in Super Critical Fluid

Extraction (SCFE) and Steam Distillation (SD) method. The oil refractive index was much

higher in SCFE than SD Figure 4.2.3. The individual effect of two methods (Super Critical

Fluid Extraction and Steam Distillation) was significant for oil refractive index of grapefruit

(Table. 4.7).

72

Figure 4.2.1 Density of essential oils of grapefruit by two methods of extraction

.

73

Figure: 4.2.2 Essential oil percentage in two methods of extraction

74

Table: 4.2.1 Effect of methods of extraction on density of essential oils extracted by two

methods

SOV DF SS MS F-value Pr(>F)

Methods 1 0.001 0.002 24.14 0.008

Residuals 4 0.004 0.001

Table: 4.2.2 Effect of method of extraction on percentage of essential oils extracted by

two methods

SOV Df SS MS F value Pr(>F)

Methods 1 0.007280000 0.007280000 1986 1.5E-06

Residuals 4 0.000015000 0.000004000

Table: 4.2.3 Effect of method of extraction on Refractive Index of essential oils extracted

by two methods

SOV DF SS MS F value Pr(>F)

Methods 1 0.000013500 0.000013500 40.5 0.00313

Residuals 4 0.000001300 0.000000300

75

Fig:4.2.3 Refractive index of esential oils extracted by two methods

76

4.2.4 Chemical Characterization of Grape fruit essential oil extracted by two methods

Chemical characterization was done by GC-MS.The grapefruit peel essential oil extracted by

steam distillation have the compounds i.e. 1-Decene , a-caryophyllene , α-pinene ,

Caryophyllen , Caryophyllene oxide , Citronellal , Citronellol , Copaene , Corane , d-cardinene

, Decanal , Elemol , Epiglobulol , g-Muurolene

Limonene , Limonine oxide , Linalool , Linalool oxide , Nootkatone , Perillyl acetate ,

Sabinene ß-Cubebene , ß-Pinene , Terpinene-4-oland Valencene.

The essential oil obtained by the supercritical fluid extraction system have the compounds with

the percentage a-bergamotene (1.37%), a-cadinol (0.39%) , a-caryophyllene (1.39%) , α-

pinene (0.27%), Ageratochromene (0.36%), Caryophyllen e(1.86%), Caryophyllene oxide

(1.14%) , Citronellol (0.84%) , D-corvone (0.41%) ,Estragole (0.24%) ,Ethyl

9,12Octadecedienoate (0.37%) ,Ethyl linolenate (0.83%) , g-Cardinene(0.25%), g-

Caryophyllene (3.29%) , ϒ-teripineol (0.65%), Globulol(0.38%) , Heneicosane(1.10%) ,

Heptacosane(0.58%), Hexa Tetra Contane(2.66%), Limonene(45.65%) , Linalool(5.77%),

Menthol(2.99%) , Nerollidol(0.24%), Nootkatone(0.77%) , Octacosane(9.94%), Palmintic

acid (5.42%), Octadocanal(0.52%) ,Pipri tone oxide(0.39%), Selina -3,7(11)dien (0.97%),

Squalene(0.28%), ß-famesene(0.62%) , ß-pinene(0.25%), Tetra TetraContane(3.56%) and

Valencene(7.48%)

77

Table: 4.2.4 List of compounds found grapefruit peel essential oil extracted by two

methods

Compound

Steam distillation Super critical fluid

extraction

1-Decene 0.2 0

α-bergamotene 0 1.37

α-cadinol 0 0.39

α-caryophyllene 0.24 1.39

α-pinene 2.01 0.27

Ageratochromene 0 0.36

Caryophyllene 2.3 1.86

Caryophyllene oxide 0.38 1.14

Citronellal 0.24 0

Citronellol 0.51 0.84

Copaene 1.22 0

Corane 0.36 0

d-cadinene 1.35 0.25

D-corvone 0 0.41

Decanal 1.87 0

Elemol 0.38 0

Epiglobulol 0.22 0

Estragole 0 0.24

Ethyl l-9,12-Octadecedienoate 0 0.37

Ethyl linolenate 0 0.83

ϒ-Caryophyllene 0 3.29

ϒ-Muurolene 0.29 0

α-teripineol 0 0.65

Globulol 0 0.38

Heneicosane 0 1.1

Heptacosane 0 0.58

Hexa Tetra Contane 0 2.66

Limonene 82.86 45.65

Limonine oxide 0.19 0

Linalool 0.4 5.77

Linalool oxide 0.16 0

Menthol 0 2.99

Nerollidol 0 0.24

Nootkatone 1.92 0.77

Octacosane 0 9.94

78

Compound

Steam distillation Super critical fluid

extraction

Octadacanal 0 5.42

Palmitic acid 0 0.52

Perillyl acetate 0.43 0

Pipri tone oxide 0 0.39

Sabinene 0.29 0

Selina -3,7(11)dien 0 0.97

Squalene 0 0.28

ß-Cubebene 0.3 0

ß-farnesene 0 0.62

ß-pinene 0.94 0.25

Terpinene-4-ol 0.31 0

Tetra TetraContane 0 3.56

Valencene 0.65 7.48

79

Figure: 4.2.4. Typical chromatogram of grapefruit peel essential oil

extracted by Steam Distillation method

Figure: 4.2.5Typical chromatogram of grapefruit peel essential oil

extracted by Supercritical Fluid Extraction methods

80

4.2.5 Principal components of the grapefruit essential oils of extracted by

two methods

In the case of presence and absence of different compounds in essential oil extracted from

grapefruit by different methods, only one PC showed eigenvalues greater than one

(significant)(Fig 4.2.6). The other PCs exhibited non-significant variation and were not worth

interpreting. The first PC showed cumulative variability of 80.77% in different compounds

found in essentials oils extracted fromgrapefruit by different methods (Table 4.2.5).The

method established by Johnson and Wichern (1988) was used to estimate the importance of a

trait coefficient for each significant principal component. The PC 1 was positively related to

steam distillation method used to extract the essential oils from grapefruit and was negatively

related to super critical fluid extraction systemused to extract the essential oils from grapefruit

(Table 4.2.6).


from grapefruit by two methods depicted that the two methods used for extraction of

compounds are opposite to each other when related to each other (Fig 4.2.7). The projection of

extraction methods on PC1 and PC2 showed the diverse nature of both methods (Fig 4.2.7).

The projection of compounds on PC1 and PC2 showed population structure on the basis of

presence of compounds in essential oil extracted from grapefruit by different methods (Fig.

4.1.8). From the different compoundsin essential oil extracted from grapefruit by different

methods, the following diverse groups were identified. The compounds (1-Decene, α-

caryophyllene, α-pinene, Caryophyllene, Caryophyllene oxide, Citronellal, Citronellol,

Copaene, Corane, d-cadinene, Decanal, Elemol, Epiglobulol, ϒ–Muurolene, Limonene,

Limonineoxid, Linalool, Linalool oxide, Nootkatone, Perillyl acetate, Sabinene, ß-Cubebene,

ß-pinene, Terpinene-4-ol andValencene) were opposite to the compounds (α–bergamotene, α–

cadinol, Ageratochromene, D-corvone, Estragole, Ethyl l-9,12-Octadecedienoate, Ethyl

linolenate, ϒ-Caryophyllene, α–teripineol, Globulol, Heneicosane, Heptacosane, Hexa Tetra

Contane, Menthol, Nerollidol, Octacosane, Octadacanal, Palmitic acid, Pipri tone oxide, Selina

-3,7(11)dien, Squalene, ß-farnesene, Tetra TetraContane) (Fig 4.1.8 and Table 4.2.7).

81


80.77%

19.23%

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Eigenvalue number

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Eig

en

va

lue

80.77%

19.23%

Fig: 4.2.6 Scree plot between eigen values and number of principal components of

different compounds on the basis of presence and absence of different compounds in

essential oil extracted from grapefruit

82

Table: 4.2.5 Eigenvalues of correlation matrix, and related statistics on the basis of presence and

absence of different compounds in essential oil extracted from grapefruit

PCs Eigenvalue % Total - variance Cumulative - Eigenvalue Cumulative - %

1 1.615486 80.77432 1.615486 80.7743

2 0.384514 19.22568 2.000000 100.0000

Table: 4.2.6 Principal components of different compounds on the basis of presence and absence

of different compounds in essential oil extracted from grapefruit

Methods of extraction Factor 1 Factor 2

Steam distillation method 0.898745 0.438471

Super critical fluid extraction system -0.898745 0.438471

83

Table: 4.2.7 Factor coordinates of cases on the basis of presence and absence of different

compounds extracted from oil of grapefruit

Compounds Factor 1 Factor 2

1-Decene 1.76154 -0.419276

α–bergamotene -1.17848 -0.280499 α–cadinol -1.17848 -0.280499

α-caryophyllene 0.22214 1.120122

α-pinene 0.22214 1.120122 Ageratochromene -1.17848 -0.280499

Caryophyllene 0.22214 1.120122 Caryophyllene oxide 0.22214 1.120122 Citronellal 1.76154 -0.419276 Citronellol 0.22214 1.120122

Copaene 1.76154 -0.419276

Corane 1.76154 -0.419276 d-cadinene 0.22214 1.120122

D-corvone -1.17848 -0.280499 Decanal 1.76154 -0.419276

Elemol 1.76154 -0.419276 Epiglobulol 1.76154 -0.419276

Estragole -1.17848 -0.280499

Ethyl l-9,12-Octadecedienoate -1.17848 -0.280499 Ethyl linolenate -1.17848 -0.280499 ϒ-Caryophyllene -1.17848 -0.280499

ϒ–Muurolene 1.76154 -0.419276

α–teripineol -1.17848 -0.280499 Globulol -1.17848 -0.280499

Heneicosane -1.17848 -0.280499 Heptacosane -1.17848 -0.280499 Hexa Tetra Contane -1.17848 -0.280499 Limonene 0.22214 1.120122

Limonine oxide 1.76154 -0.419276

Linalool 0.22214 1.120122 Linalool oxide 1.76154 -0.419276

Menthol -1.17848 -0.280499 Nerollidol -1.17848 -0.280499

Nootkatone 0.22214 1.120122 Octacosane -1.17848 -0.280499

Octadacanal -1.17848 -0.280499

Palmitic acid -1.17848 -0.280499 Perillyl acetate 1.76154 -0.419276 Pipri tone oxide -1.17848 -0.280499

Sabinene 1.76154 -0.419276

Selina -3,7(11)dien -1.17848 -0.280499

Squalene -1.17848 -0.280499

ß-Cubebene 1.76154 -0.419276

84

ß-farnesene -1.17848 -0.280499 ß-pinene 0.22214 1.120122

Terpinene-4-ol 1.76154 -0.419276

Tetra TetraContane -1.17848 -0.280499 Valencene 0.22214 1.120122

85

Fig.4.2.7 Two dimensional ordination of two methods used to extract essentials oils from

grapefruit on PC1 and PC2


SDSCFE

-1.0 -0.5 0.0 0.5 1.0

Factor 1 : 80.77%

-1.0

-0.5

0.0

0.5

1.0

Facto

r 2 :

19

.23

%

SDSCFE

86

Fig: 4.2.8 Two dimensional ordination of different compounds in essentials oils extracted

fromgrapefruit by two methods on PC1 and PC2



1-Decene

a-bergamotenea-cadinol

a-caryophyllenea-pinene

Ageratochromene


Citronellal

Citronellol

CopaeneCorane

d-cadinene

D-corvone

DecanalElemolEpiglobulol

EstragoleEthyl l-9,12-OctadecedienoateEthyl linolenate?-Caryophyllene

?-Muurolene

a-teripineolGlobulolHeneicosaneHeptacosaneHexa Tetra Contane

Limonene

Limonine oxide

Linalool

Linalool oxide

MentholNerollidol

Nootkatone

OctacosaneOctadacanalPalmitic acid

Perillyl acetate

Pipri tone oxide

Sabinene

Selina -3,7(11)dienSqualene

ß-Cubebene

ß-farnesene

ß-pinene

Terpinene-4-ol

Tetra Tetra Contane

Valencene

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5

Factor 1: 80.77%

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4F

acto

r 2: 1

9.2

3%

1-Decene

a-bergamotenea-cadinol

a-caryophyllenea-pinene

Ageratochromene


Citronellal

Citronellol

CopaeneCorane

d-cadinene

D-corvone

DecanalElemolEpiglobulol

EstragoleEthyl l-9,12-OctadecedienoateEthyl linolenate?-Caryophyllene

?-Muurolene

a-teripineolGlobulolHeneicosaneHeptacosaneHexa Tetra Contane

Limonene

Limonine oxide

Linalool

Linalool oxide

MentholNerollidol

Nootkatone

OctacosaneOctadacanalPalmitic acid

Perillyl acetate

Pipri tone oxide

Sabinene

Selina -3,7(11)dienSqualene

ß-Cubebene

ß-farnesene

ß-pinene

Terpinene-4-ol

Tetra Tetra Contane

Valencene

87

4.2.6 Principal Component Analysis for compounds percentages in grapefruit peel oil extracted

by two methods

In the case of percentage of different compounds in essential oils extracted from grapefruit by

different methods, only one PC showed eigenvalues greater than one (significant) (Table

4.2.8). The other PCs exhibited non-significant variation and were not worth interpreting. The

first PC showed cumulative variability of 97.47% in different compounds percentages found

in essentials oils extracted from grapefruit by different methods (Fig 4.2.9). The method

established by Johnson and Wichern (1988) was used to estimate the importance of a trait

coefficient for each significant principal component. The first PC was positively related to both

methods of extraction used to extract the essential oils from grapefruit (Table 4.2.9).


from grapefruit by different methods depicted that percentages of different compounds in

essential oils extracted from grapefruit by different methods that are close together are being

similar when related with other percentages of compounds. The projection of percentages of

different compounds on PC1 and PC2 showed the diverse nature. To identify the better

transgressive pattern of percentages of compounds in dissimilar groups of pattern, the

projection of percentages of compounds on first two principal components was useful.


from grapefruit by two methods depicted that the two methods used for extraction of

compounds are opposite to each other but are present on the same plane when related to each

other (Fig 4.2.10). The projection of extraction methods on PC1 and PC2 showed the diverse

nature of both methods (Fig 4.2.7). The projection of compounds on PC1 and PC2 showed

population structure on the basis of percentages of compounds (Fig 4.2.11). From the different

compounds in essential oils extracted from three cultivars of citrus, the following diverse

groups were identified. The compounds (1-Decene, α–cadinol, α-pinene, Ageratochromene,

Caryophyllene, Caryophyllene oxide, Citronellal, Citronellol, Copaene, Corane, d-cadinene,

D-corvone, Decanal, Elemol, Epiglobulol, Estragole, Ethyl l-9,12-Octadecedienoate, Ethyl

linolenate, ϒ–Muurolene, α–teripineol, Globulol, Heptacosane, Limonene, Limonine oxide,

Linalool oxide, Nerollidol, Nootkatone, Palmitic acid, Perillyl acetate, Pipri tone oxide,

Sabinene, Selina -3,7(11)dien, Squalene, ß-Cubebene, ß-farnesene, ß-pinene and Terpinene-4-

ol) were opposite to the compounds (α–bergamotene, α-caryophyllene, ϒ-Caryophyllene,

Heneicosane, Hexa Tetra Contane, Linalool, Menthol, Octacosane, Octadacanal, Tetra

TetraContane and Valencene) (Fig 4.2.11 and Table4.2.10).

88

Fig: 4.2.9 Scree plot between eigen values and number of principal components of percentage of

different compounds in essential oil extracted from grapefruit by two methods


97.44%

2.56%

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Eigenvalue number

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2E

ige

nva

lue

97.44%

2.56%

89

Table: 4.2.8 Eigen values of correlation matrix, and related statistics on the basis of percentage

of different compounds in essential oil extracted from grapefruit

PCs Eigenvalue % Total - variance Cumulative - Eigenvalue Cumulative - %

1 1.948843 97.44216 1.948843 97.4422

2 0.051157 2.55784 2.000000 100.0000

Table: 4.2.9 Principal components of different compounds on the basis of percentage of

different compounds in essential oil extracted from grapefruit

Methods of extraction Factor 1 Factor 2

Steam distillation method 0.987128 0.159932

Super critical fluid extraction system 0.987128 -0.159932

90

Table: 4.2.10 Factor coordinates of cases on the basis of percentage of different compounds in essential oil

extracted from grapefruit

Compounds Factor 1 Factor 2

1-Decene -0.337400 0.113943 α–bergamotene -0.205504 -0.041678 α–cadinol -0.308339 0.061157 α-caryophyllene -0.189171 -0.029542 α-pinene -0.201714 0.192965 Ageratochromene -0.311487 0.064305 Caryophyllene -0.017670 0.043322 Caryophyllene oxide -0.207100 0.004995 Citronellal -0.335028 0.116315 Citronellol -0.230870 0.044186 Copaene -0.276902 0.174441 Corane -0.327910 0.123433 d-cadinene -0.242958 0.155918 D-corvone -0.306240 0.059058 Decanal -0.238349 0.212993 Elemol -0.326724 0.124619 Epiglobulol -0.336214 0.115129 Estragole -0.324078 0.076896 Ethyl l-9,12-Octadecedienoate -0.310437 0.063255 Ethyl linolenate -0.262168 0.014986 ϒ-Caryophyllene -0.004033 -0.243149 ϒ–Muurolene -0.332062 0.119281 α–teripineol -0.281056 0.033874 Globulol -0.309388 0.062206 Heneicosane -0.233836 -0.013346 Heptacosane -0.288401 0.041219 Hexa Tetra Contane -0.070141 -0.177041 Limonene 9.355503 0.226470 Limonine oxide -0.337993 0.113350 Linalool 0.279925 -0.479658 Linalool oxide -0.339772 0.111570 Menthol -0.035513 -0.211669 Nerollidol -0.324078 0.076896 Nootkatone -0.154585 0.135161 Octacosane 0.693771 -0.940953 Octadacanal 0.219474 -0.466656 Palmitic acid -0.294697 0.047515 Perillyl acetate -0.323758 0.127584 Pipri tone oxide -0.308339 0.061157 Sabinene -0.332062 0.119281 Selina -3,7(11)dien -0.247477 0.000295 Squalene -0.319881 0.072699 ß-Cubebene -0.331469 0.119874 ß-farnesene -0.284204 0.037022 ß-pinene -0.267276 0.131600 Terpinene-4-ol -0.330876 0.120467

Tetra TetraContane 0.024299 -0.271481 Valencene 0.474189 -0.644265

91

Fig:4.2.10 Two dimensional ordination of two methods used to extract essentials oils percentage

from grapefruit on PC1 and PC2


SD

SCFE

-1.0 -0.5 0.0 0.5 1.0

Factor 1 : 97.44%

-1.0

-0.5

0.0

0.5

1.0

Facto

r 2 :

2.5

6% SD

SCFE

92

Fig: 4.2.11 Two dimensional ordination of different compounds in essentials oils extracted from

grapefruit on the basis of percentage by two methods on PC1 and PC2



1-Decene

a-bergamotene

a-cadinol

a-caryophyllene

a-pinene

AgeratochromeneCaryophylleneCaryophyllene oxide

Citronellal

Citronellol

CopaeneCorane

d-cadinene

D-corvone

Decanal

ElemolEpiglobulolEstragoleEthyl l-9,12-Octadecedienoate

Ethyl linolenate

?-Caryophyllene

?-Muurolene

a-teripineolGlobulol

Heneicosane

Heptacosane

Hexa Tetra Contane

Limonene

Limonine oxide

Linalool

Linalool oxide

Menthol

Nerollidol

Nootkatone

Octacosane

Octadacanal

Palmitic acid

Perillyl acetate

Pipri tone oxide

Sabinene

Selina -3,7(11)dien

Squaleneß-Cubebene

ß-farnesene

ß-pineneTerpinene-4-ol

Tetra Tetra Contane

Valencene

-4 -2 0 2 4 6 8 10

Factor 1: 97.44%

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4F

acto

r 2:

2.5

6%

1-Decene

a-bergamotene

a-cadinol

a-caryophyllene

a-pinene

AgeratochromeneCaryophylleneCaryophyllene oxide

Citronellal

Citronellol

CopaeneCorane

d-cadinene

D-corvone

Decanal

ElemolEpiglobulolEstragoleEthyl l-9,12-Octadecedienoate

Ethyl linolenate

?-Caryophyllene

?-Muurolene

a-teripineolGlobulol

Heneicosane

Heptacosane

Hexa Tetra Contane

Limonene

Limonine oxide

Linalool

Linalool oxide

Menthol

Nerollidol

Nootkatone

Octacosane

Octadacanal

Palmitic acid

Perillyl acetate

Pipri tone oxide

Sabinene

Selina -3,7(11)dien

Squaleneß-Cubebene

ß-farnesene

ß-pineneTerpinene-4-ol

Tetra Tetra Contane

Valencene

93

Experiment # 3 Effect of different climatic regions on citrus peel essential oils


4.3.1 Refractive index of essential oils of citrus cultivars collected from different locations

The refractive index of oil showed significant difference in three cultivars i.e., grapefruit,

Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and Musambi in all regions (Fig. 4.3.1).

Maximum oil refractive index was recorded in Musambi (1.472) and Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) (1.472) in Abbotabad while grapefruit showed less oil

refractive index (1.471). In Faisalabad, Musambi ranked 1st with maximum oil refractive

index (1.471) followed by grapefruit (1.470) and Kinnow (C.nobilis Loureiro×C. deliciosa

Tenore) (1.467). In Layyah region oil refractive index of Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore) (1.463) was maximum second to which was Musambi (1.462) and

grapefruit (1.460). The refractive index of all the fruits peels oils was significantly lower

from Abbotabad, Faisalabad and Sargodha. In Rahim Yar Khan, Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) oil showed more refractive index than Musambi and

grapefruit while latter two showed similar refractive index. In Sargodha, the refractive index

of Musambi, grapefruit and Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) was in

descending order i.e., 1.471, 1.470 and 1.468 respectively.

Interaction plot (Figure 4.3.1) showed that oil refractive index of Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) and Musambi was more than grapefruit. Oil refractive index of

Musambi and Grapefruit was very close in Faisalabad and Sargodha region while Kinnow

(C.nobilis Loureiro×C. deliciosa Tenore) oil refractive index was different in these two

regions. Similarly, oil refractive index of Musambi, Kinnow (C.nobilis Loureiro×C. deliciosa

Tenore) and Grapefruit was very close in Rahim Yar Khan and Layyah region.

The individual effect of region and fruit for refractive index of essential oils was significant.

Two way interactions of region and fruit was also significant for refractive index of essential

oils (Table. 4.3.1).

94

Figure 4.3.1 Interaction plot of refractive index of essential oils of citrus cultivars from

different locations

95

Table: 4.3.1 ANOVA table for refractive index of EOs of Citrus cultivars from different

locations

SOV df SS MS F Pr(>F)

Region 4 0.000796 0.000199 123.897 0.001

Fruit 2 8.4E-06 4.21E-06 2.622 0.089

Region:Fruit 8 3.58E-05 4.47E-06 2.781 0.019

Residuals 30 4.82E-05 1.61E-06

96

4.3.2 Oil percentage of essential oils of citrus cultivars collected from different locations

The percentage of oil showed significant difference in three cultivars i.e., grapefruit, Kinnow

(C.nobilis Loureiro×C. deliciosa Tenore) and Musambi in all regions (Fig. 4.3.2). Maximum

oil percentage was recorded in Grapefruit in Layyah region which was significantly different

from Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and Musambi oil percentage. The

grapefruit oil percentage gradually decreased in Rahim Yar Khan, Faisalabad, Sargodha and

Abbotabad, respectively.

The oil percentage of Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and Musambi was

maximum in Rahim Yar Khan followed by Layyah, Faisalabad, Sargodha and Abbotabad.

Interaction plot showed that oil percentage of grapefruit was higher than oil percentage of

Grapefruit in Rahim Yar Khan, Faisalabad, Sargodha and Abbotabad. The percentage of

Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) oil was significantly lower in Layyah than

grapefruit. Maximum oil percentage was in Rahim Yar Khan followed by Layyah, Faisalabad,

Sargodha and Abbotabad. Musambi oil percentage in Abbotabad was significantly less than

other four districts. Musambi oil percentage in Sargodha and Faisalabad was similar.

The individual effect of region and fruit for percentage of essential oils was significant. Two

way interactions of region and fruit was nonsignificant for percentage of essential oils (Table.

4.3.2). Tukey HSD table revealed the significant difference among the locations (Table: 4.3.3).

97

Figure: 4.3.2 Interaction plot of percentage of essential oils of citrus cultivars from

different locations

98

. Table: 4.3.2 ANOVA table for percentage of EOs of Citrus cultivars from different

locations

SOV DF SS MS F Pr(>F)

Region 4 0.003 0.009 9.772 0.003*

Cultivar 2 0.007 0.003 38.977 0.001*

Region:cultivar 8 0.001 0.002 1.327 0.269

Residuals 30 0.003 0.001

Table 4.3.3 Tukey HSD table for locations

TukeyHSD

diff Lower value Upper value p value

Faisalabad-Abbotabad -0.00233 -0.00407 -0.0006 4.20E-03

Layyah-Abbotabad -0.01011 -0.01184 -0.00838 4.25E-14

R Y Khan-Abbotabad -0.00956 -0.01129 -0.00782 4.52E-14

Sargodha-Abbotabad -0.00188 -0.00361 -0.00014 2.86E-02

Layyah-Faisalabad -0.00778 -0.00951 -0.00604 7.53E-13

R Y Khan-Faisalabad -0.00722 -0.00896 -0.00549 4.64E-12

Sargodha-Faisalabad 0.000456 -0.00128 0.002189 9.39E-01

R Y Khan-Layyah 0.000556 -0.00118 0.002289 8.83E-01

Sargodha-Layyah 0.008233 0.0065 0.009967 2.08E-13

Sargodha-R Y Khan 0.007678 0.005944 0.009411 1.03E-12

99

4.3.3 Density (mg/cm3)

The density (mg/cm3) of oil was different in Grapefruit, Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore) and Musambi in Abbotabad, Faisalabad, Layyah, Rahim Yar Khan and

Sargodha (Fig. 6). Minimum oil density (mg/cm3) was recorded in Grapefruit in Abbotabad

followed by Faisalabad, Layyah, RahimYar Khan and Sargodha region. After the grapefruit,

Musambi and Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) oil showed similar trend in

all the five districts for density (mg/cm3). In Sargodha region, Musambi oil density (mg/cm3)

was higher than grapefruit.Interaction plot showed that oil density (mg/cm3) of grapefruit in

Rahim Yar Khan was significantly higher than oil density (mg/cm3) of grapefruit in Layyah,

Faisalabad, Sargodha and Abbotabad. In all the five districts oil density (mg/cm3) of Musambi

was less than grapefruit and more than Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) was

recorded. The density (mg/cm3) of grapefruit and Kinnow (C.nobilis Loureiro×C. deliciosa

Tenore) oil was almost similar in case of Sargodha and Faisalabad regions while it showed a

slight different trend was observed in Musambi oil density (mg/cm3). Oil density (mg/cm3) for

Grapefruit, Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and Musambi was close in

Rahim Yar Khan and Layyah while fruits from Layyah were low in oil density (mg/cm3).

Interaction plot Figure 4.3.3 of density of essential oils of all the citrus cultivars selected from

different locations showed the individual effect of location and location for density (mg/cm3)

of essential oils was high. Two way interactions of region and fruit was also significant for

density (mg/cm3) of essential oils (Table. 4.3.4).

100

Figure: 4.3.3 Interaction plot of Density (mg/cm3) of essential oils of citrus cultivars from

different locations

101

Table: 4.3.4 ANOVA table for Density (mg/cm3) of EOs of Citrus cultivars from different

locations.

SOV DF SS MS F Pr(>F)

Location 4 0.0003456 0.00008641 155.54 0.005

Cultivar 2 0.0001115 0.00005576 100.36 0.094

Location:Cultivar 8 0.0000545 0.00000681 12.26 0.003

Residuals 30 0.0000167 0.00000056

102

4.3.4 Chemical Charecterization of citrus essential oils of different locations

Grapefruit peel oil fromAbbotabad that was extracted using steam distillation showed the

presence of the compounds (Fig:4.3.4) (-)α-Neoclovene α-caryophyllene , Elemol , 4-

terpinenol , α-pinene , Carvyol acetate (z) , Caryophyllene , Caryophyllene oxide , Citronellal

, Citronellol , Copaene , d-cadinene , D-Germacrene , Decanal , Farnesyl acetate , Globulol ,

Heneicosane , Heptacosane , Levoverbenone , Limonene , Linalool , Nerolidol , Nootkatone ,

ß-cubebene , ß-pinene and Valencene (Table: 4.3.5). Steam distilled extracted Kinnow

(C.nobilis Loureiro×C. deliciosa Tenore) peel oil from Abbotabad showed the presence of the

compounds viz β-myrcene , 4-terpinenol , α-citral , α-pinene , Anethol , Caryophyllene ,

Citronellal , Citronellol , d-cadinene , Decanal , Globulol , Limonene , Limonene oxide ,

Linalool , Perillaldehyde and Valencene (Table 4.3.5). Abbotabad originated Musambi peel

essential oil extracted by steam distillation showed the presence of the compounds (Fig: 4.3.6)

(-) -α-Panasinsene , β-Citronellol , Elemene , α -Selinene , 4,11-Selinadiene , α-pinene ,

Caryophyllene , Citronellol , E-Nerolidol , Farnesol , Juniper Camphor , Limonene , Linalool

, Menthol , Nootkatone , Sabinene , ß-pinene and Valencene. (Table 4.3.5)

Grapefruit peel oil from Faisalabad region showed the presence of the compounds (Fig:

4.3.7) (-)α-Neoclovene , Elemene , α-farnesol , α-pinene , Caryophyllene , Citronellal ,

Citronellol , d-cadinene , Decanal , Heneicosane , Heptacosane , Limonene , Linalool ,

Nerolidol , Nootkatone , Octacosane , Perillyl acetate and Valencene (Table 4.3.5). Faisalabad

originated Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel essential oil showed the

presence of the compounds (Fig: 4.3.8) β-myrcene , α -phellenderene , α-pinene ,

Caryophyllene , d-cadinene , Estragole , Globulol , Limonene , Linalool , Octanal , Terpinene-

4-ol and Valencene (Table 4.3.5). Faisalabad originated Musambi peel essential oil showed

the presence of the compounds (Fig: 4.3.9)β-myrcene , 4-terpinenol , α-citral , α-pinene ,

Aromadendrene , Citronellal , Citronellol , d-cadinene , Decanal , Iso-Caryophyllene , Juniper

Camphor , Levoverbenone , Limonene , Limonene oxide , Linalool , Nootkatone , Sabinene ,

ß-Citral and Valencene (Table 4.3.5).

Layyah originated Grapefruit peel essential oil showed the presence of the compounds

(Fig: 4.3.10)α-gurjenene , β-myrcene , α -phellenderene , α-pinene , Caryophyllene , Copaene

, d-cadinene , D-Germacrene , Decanal , Limonene , Nootkatone , ß-cubebene and Valencene

(Table 4.3.5). Layyah originated Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel

103

essential oil showed the presence of the compounds (Fig: 4.3.11) β-myrcene , α-pinene ,

Caryophyllene , Citronellal , Copaene , d-cadinene , Decanal , Heneicosane , Limonene ,

Linalool , Nootkatone , Octacosane , Trans-Anethol and Valencene (Table 4.3.5). Layyah

originated Musambi peel essential oil showed the presence of the compounds (Fig: 4.3.12) ϒ-

Muurolene , α-pinene , Caryophyllene , Citronellol , Copaene , d-cadinene , Decanal ,

Limonene , Linalool , Nootkatone , ß-cubebene , ß-pinene and Valencene (Table 4.3.5).

Rahim Yar Khan originated grapefruit peel essential oil showed the presence of the

compounds (Fig: 4.3.13) α-pinene , Caryophyllene , Citronellol , Copaene , d-cadinene ,

Decanal , Levoverbenone , Limonene , Linalool , Nootkatone , Octacosane , ß-cubebene ,

Trans-carane and Valencene (Table 4.3.5). Rahim Yar Khan originated Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) essential oil showed the presence of the compounds (Fig:

4.3.14) (z) – Carveol , 1-Decanol , Anethol , Carveol , Caryophyllene oxide , Cis-Carveol ,

Copaene , Levoverbenone , Limonene , Limoneneglycol , Linalool , Menthadien-1-ol , ß-

pinene , Trans-Carveol (Table 4.3.5). Rahim Yar Khan originated Musambi peel essential oil

showed the presence of the compounds (Fig: 4.3.15) (-) -α-Panasinsene , 4-terpinenol ,

Aromadendrene , Cis, Trans-Farnesol , Citronellal , Citronellol , Juniper Camphor ,

Levoverbenone , Limonene , Linalool , Nootkatone , ß-pinene , Trans-A-Terpineol , Trans-

nerolidol and Valencene (Table 4.3.5).

Sargodha originated grapefruit peel essential oil showed the presence of the compounds

(Fig: 4.3.16) β-myrcene , 2,6-Octadiene , Caryophyllene , Citronellal , Copaene , d-cadinene ,

Decanal , Heneicosane , Heptacosane , Hexahydrothymol , Levoverbenone , Limonene ,

Linalool , Nootkatone and Valencene (Table4.3.5). Sargodha originated Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) peel essential oil extracted by steam distillation showed the

presence of the compounds (Fig: 4.3.17) β-myrcene , α-pinene , Caryophyllene , Cis, Trans-

Farnesol , Citronellal , Copaene , d-cadinene , Decanal , Estragole , Heptacosane , Limonene ,

Linalool , Nootkatone , Octacosane and Valencene (Table4.3.5). Sargodha originated

Musambi peel essential oil showed the presence of the compounds (Fig: 4.3.18) β-myrcene, α-

pinene, Citronellal, d-cadinene , Decanal , Levoverbenone , Limonene , Linalool and

Valencene (Table4.3.5)

104

Table: 4.3.5 List of compounds with percentage in peel essential oils of three citrus cultivars

from different locations Compound Abbotabad Faisalabad Layyah R.Y.Khan Sargodha

C1 C2 C3 C1 C2 C3 C1 C2 C3 C1 C2 C3 C1 C2 C3

(-) -α-Panasinsene 0 0 0.23 0 0 0 0 0 0 0 0 0.13 0 0 0

(-)α-Neoclovene 0.75 0 0 0.36 0 0 0 0 0 0 0 0 0 0 0

(z) – Carveol 0 0 0 0 0 0 0 0 0 0 0.65 0 0 0 0

α-caryophyllene 0.55 0 0 0 0 0 0 0 0 0 0 0 0 0 0

β-Citronellol 0 0 1.99 0 0 0 0 0 0 0 0 0 0 0 0

Elemene 0 0 0.26 0.28 0 0 0 0 0 0 0 0 0 0 0

Elemol 0.44 0 0 0 0 0 0 0 0 0 0 0 0 0 0

α-farnesol 0 0 0 0.63 0 0 0 0 0 0 0 0 0 0 0

α-gurjenene 0 0 0 0 0 0 0.12 0 0 0 0 0 0 0 0

ϒ-Muurolene 0 0 0 0 0 0 0 0 0.15 0 0 0 0 0 0

β-myrcene 0 0.71 0 0 0.67 0.87 0.9 0.64 0 0 0 0 0.59 0.65 0.71

α -phellenderene 0 0 0 0 0.08 0 0.13 0 0 0 0 0 0 0 0

α -Selinene 0 0 1.07 0 0 0 0 0 0 0 0 0 0 0 0

1-Decanol 0 0 0 0 0 0 0 0 0 0 0.87 0 0 0 0

2,6-Octadiene 0 0 0 0 0 0 0 0 0 0 0 0 0.18 0 0

4-terpinenol 0.36 0.24 0 0 0 0.14 0 0 0 0 0 0.23 0 0 0

4,11-Selinadiene 0 0 0.22 0 0 0 0 0 0 0 0 0 0 0 0

α-citral 0 0.17 0 0 0 0.16 0 0 0 0 0 0 0 0 0

α-pinene 0.32 0.22 0.34 0.43 0.31 0.39 1.46 0.43 0.58 0.52 0 0 0 0.39 0.15

Anethol 0 0.44 0 0 0 0 0 0 0 0 0.53 0 0 0 0

Aromadendrene 0 0 0 0 0 0.44 0 0 0 0 0 0.51 0 0 0

Carveol 0 0 0 0 0 0 0 0 0 0 0.24 0 0 0 0

Carvyol acetate (z) 0.47 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Caryophyllene 0.66 0.14 0.16 1.7 1.23 0 2.68 0.51 0.47 5.73 0 0 1.5 0.34 0

Caryophyllene

oxide

1.33 0 0 0 0 0 0 0 0 0 0.54 0 0 0 0

Cis-Carveol 0 0 0 0 0 0 0 0 0 0 0.2 0 0 0 0

Cis, Trans-Farnesol 0 0 0 0 0 0 0 0 0 0 0 0.25 0 0.13 0

Citronellal 0.88 0.24 0 0.56 0 0.34 0 0.14 0 0 0 0.23 0.54 0.54 0.15

Citronellol 0.37 0.33 0.62 0.19 0 0.33 0 0 0.21 0.12 0 0.39 0 0 0

Copaene 0.31 0 0 0 0 0 0.65 0.24 0.27 1.82 0.47 0 0.41 0.16 0

d-cadinene 0.39 0.39 0 1.41 1.02 0.24 2.33 0.41 0.33 1.14 0 0 1.49 0.33 0.1

D-Germacrene 0.18 0 0 0 0 0 0.14 0 0 0 0 0 0 0 0

Decanal 0.79 1.71 0 0.9 1 0.3 0.17 0.15 0.22 0.17 0 0 0.73 0.85 0.12

105

Compound Abbotabad Faisalabad Layyah R.Y.Khan Sargodha

C1 C2 C3 C1 C2 C3 C1 C2 C3 C1 C2 C3 C1 C2 C3

Estragole 0 0 0 0 0.31 0 0 0 0 0 0 0 0 0.18 0

Farnesol 0 0 0.61 0 0 0 0 0 0 0 0 0 0 0 0

Farnesyl acetate 0.29 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Globulol 0.28 0.21 0 0 0.12 0 0 0 0 0 0 0 0 0 0

Heneicosane 1.51 0 0 0.17 0 0 0 0.44 0 0 0 0 0.37 0 0

Heptacosane 0.65 0 0 0.48 0 0 0 0 0 0 0 0 0.26 0.12 0

Hexahydrothymol 0 0 0 0 0 0 0 0 0 0 0 0 0.13 0 0

Hexyl Hexanoate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Iso-Caryophyllene 0 0 0 0 0 0.12 0 0 0 0 0 0 0 0 0

Juniper Camphor 0 0 0.27 0 0 0.14 0 0 0 0 0 0.63 0 0 0

Levoverbenone 0.41 0 0 0 0 0.16 0 0 0 2.13 0.41 0.22 0.39 0 0.4

Limonene 83.17 90.05 84.73 89.21 96.9

1

91.66 87.34 94.04 94.83 84.45 94.61 88.04 92.01 94.34 97.36

Limonene oxide 0 0.19 0 0 0 0.19 0 0 0 0 0 0 0 0 0

Limoneneglycol 0 0 0 0 0 0 0 0 0 0 0.24 0 0 0 0

Linalool 1.02 0.67 0.37 0.44 0.23 0.77 0 0.34 0.65 0.34 0.19 0.55 0.22 0.23 0.38

Menthadien-1-ol 0 0 0 0 0 0 0 0 0 0 0.25 0 0 0 0

Menthol 0 0 0.19 0 0 0 0 0 0 0 0 0 0 0 0

Nerolidol 0.7 0 0 0.48 0 0 0 0 0 0 0 0 0 0 0

Nootkatone 3 0 0.44 2.59 0 0.14 1.53 0.39 0.37 0.45 0 0.33 0.5 0.39 0

Octacosane 0 0 0 0.18 0 0 0 0.66 0 0.97 0 0 0 0.11 0

Octanal 0 0 0 0 0.09 0 0 0 0 0 0 0 0 0 0

Perillaldehyde 0 0.18 0 0 0 0 0 0 0 0 0 0 0 0 0

Perillyl acetate 0 0 0 0.22 0 0 0 0 0 0 0 0 0 0 0

Sabinene 0 0 0.12 0 0 0.13 0 0 0 0 0 0 0 0 0

ß-Citral 0 0 0 0 0 0.11 0 0 0 0 0 0 0 0 0

ß-cubebene 0.18 0 0 0 0 0 0.21 0 0.15 0.32 0 0 0 0 0

ß-pinene 1.21 0 0.49 0 0 0 0 0 0.54 0 0.49 0.39 0 0 0

Terpinene-4-ol 0 0 0 0 0.09 0 0 0 0 0 0 0 0 0 0

Trans-A-Terpineol 0 0 0 0 0 0 0 0 0 0 0 0.26 0 0 0

Trans-Anethol 0 0 0 0 0 0 0 0.24 0 0 0 0 0 0 0

Trans-carane 0 0 0 0 0 0 0 0 0 0.58 0 0 0 0 0

Trans-Carveol 0 0 0 0 0 0 0 0 0 0 0.31 0 0 0 0

Trans-nerolidol 0 0 0 0 0 0 0 0 0 0 0 0.27 0 0 0

Valencene 0.35 4.12 7.31 0.58 0.2 3.7 1.54 1.26 1.22 0.83 0 6.87 0.59 1.31 0.63

106

Figure: 4.3.4 Typical chromatogram of peel essential oil of Grapefruit

collected from Abbotabad

107

Figure: 4.3.5 Typical chromatogram of peel essential oil of Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore) collected from Abbotabad

108

Figure: 4.3.6 Typical chromatogram of peel essential oil of Musambi collected from

Abbotabad

109

Figure: 4.3.7 Typical chromatogram of peel essential oil of grapefruit collected from

Faisalabad

110

Figure: 4.3.8 Typical chromatogram of peel essential oil of Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore) collected from Faisalabad

111


Faisalabad

112

Figure: 4.3. 10Typical chromatogram of peel essential oil of grapefruit collected from

Layyah

113

Figure: 4.3.11 Typical chromatogram of peel essential oil of Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) collected from Layyah

114


Layyah

115

Figure: 4.3.13 Typical chromatogram of peel essential oil of grapefruit collected

from Rahim Yar Khan

116

Figure: 4.3.14 Typical chromatogram of peel essential oil of Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) collected from Rahim Yar Khan

117


Rahim Yar Khan

118

Figure: 4.3. 16Typical chromatogram of peel essential oil of Grapefruit collected from

Sargodha

119

Figure: 4.3. 17 Typical chromatogram of peel essential oil of Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) collected from Sargodha

120


Sargodha

121

4.3.5 Ward’s hierarchical method to explain the diversity among the different

compounds found in essential oils extracted from three cultivars from different localities

Hierarchical clustering by using Ward’s hierarchical method (Ward, 1963) grouped the

compounds in three main clusters is represented on the hierarchical tree using Euclidean

distance and showed a fairly clear picture (Figure 4.1.19). Cluster I included Limonene glycol,

Menthadiene-1-ol, Trans-carveol, β-myrecene, Citronellal, α-pinene, d-cadinene, Decanal,

Limonene, Linalool, Valencene, Caryophyllene, Nootkatone, Copaene, Citronellol, β -pinene

and Levoverbenone. Cluster I is subdivided into two sub-clusters. Sub-cluster I of cluster I

included these compounds viz., Caryophyllene, Nootkatone, Copaene, Citronellol, β-pinene

and Levoverbenone. Sub-cluster II of cluster I included the compounds β-myrecene,

Citronellal, α-pinene, d-cadinene, Decanal, Limonene, Linalool and Valencene.

Cluster II contained Farnesol, Menthol, (z)-Carveol, 1-Decanol, Carveol and Cis-

carveol. ClusterIII included these compounds(-)-α-Panasinsene, Juniper Camphor,

Aromadendrene, Cis,Trans-Farnesol, Trans-α-Terpineol, Trans-nerolidol,4-terpineol, α-citral,

Limonene oxide, Anethol, Perillaldehyde, Globulol, Iso-Caryophyllene, β-Citral, Sabinene, (-

)α-Neoclovene, Nerolidol, Heneicosane, Heptacosane, Elemene, α-farnesol, Perillal acetate, α-

caryophyllene, Trans-Anethol, Octacosane, α-gurjenene, D-Germcrene, α-phellenderene, ϒ-

Muurolene, Trans-carane, β-cubebene, 2,6-Octadiene, Hexahydrothymol, Estragole,

Octanal,Terpinene-4-ol, Elemol, Carvoyl acetate (z), Fernesyl acetate, Caryophyllene oxide,

β-Citronellol , α-Selinene, 4,11-Selinadiene and E-Nerolidol (Figure 4.1.19).

In each cluster, the number of compounds are different and decide the number of sub-

clusters and groups on hierarchical tree. Cluster III is subdivided into two sub-clusters. Sub-

cluster I of cluster III was further divided into two groups and sub-cluster II of cluster III also

has two groups. Each group included different compounds. Group I of sub-cluster I of cluster

III included following compounds namely α-caryophyllene, Trans-Anethol, Octacosane, α-

gurjenene, D-Germcrene, α-phellenderene, ϒ-Muurolene, Trans-carane and β-

cubebene.Group II of sub-cluster I of cluster III included following compounds

namelyNerolidol, Heneicosane, Heptacosane, Elemene, α-farnesol(Figure 4.1.19). Similarly

group I of sub-cluster II of cluster III has following compounds included namely 4-terpineol,

α-citral, Limonene oxide, Anethol, Perillaldehyde, Globulol, Iso-Caryophyllene, β-Citral and

Sabinene. Group II of sub-cluster II of cluster III compounds viz., (-)-α-Panasinsene, Juniper

Camphor, Aromadendrene, Cis,Trans-Farnesol and Trans-α-Terpineol(Figure 4.1.19).

122

Figure:4.3.19 : Diversity of different compounds found in essentials oils extracted from

three citrus cultivars collected from different localities

Ward`s method

Euclidean distances

0 5 10 15 20 25 30

Linkage Distance

Levoverbenoneß-pinene

CitronellolCopaene

NootkatoneCaryophyllene

ValenceneLinalool

LimoneneDecanal

d-cadinenea-pinene

Citronellalß-myrcene

Trans-CarveolMenthadien-1-olLimoneneglycol

Cis-CarveolCarveol

1-Decanol(z) – Carveol

MentholFarnesol

E-Nerolidol4,11-Selinadiene

a -Selineneß-Citronellol

Caryophyllene oxideFarnesyl acetate

Carvyol acetate (z)Elemol

Terpinene-4-olOctanal

EstragoleHexahydrothymol

2,6-Octadieneß-cubebene

Trans-carane?-Muurolene

a -phellendereneD-Germacrene

a-gurjeneneOctacosane

Trans-Anethola-caryophyllenePerillyl acetate

a-farnesolElemene

HeptacosaneHeneicosane

Nerolidol(-)a-Neoclovene

Sabineneß-Citral

Iso-CaryophylleneGlobulol

PerillaldehydeAnethol

Limonene oxidea-citral

4-terpinenolTrans-nerolidol

Trans-A-TerpineolCis, Trans-Farnesol

AromadendreneJuniper Camphor

(-) -a-Panasinsene

123

4.3.6 Principal Component Analysis for compounds percentages in the citrus cultivars essential

oils of different locations

In the case of percentage of different compounds in essential oils extracted from three citrus

cultivars collected from different localities, four PCs showed eigen values greater than one

(significant)(Table 4.3.6). The other PCs exhibited non-significant variation and were not

worth interpreting. The first PC showed cumulative variability of 42.67%, second PC showed

9.61 variability, third PC showed 8.10% variability and fourth PC showed 7.59% variability in

different compounds percentages found in essentials oils extracted from three citrus cultivars

collected from different localities (Fig 4.3.20). The method established by Johnson and

Wichern (1988) was used to estimate the importance of a trait coefficient for each significant

principal component. The first PC was negatively related to three citrus cultivars collected

from three localities except Musambi collected from districts Abottabad and Rahim Yar Khan

and Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) collected from district Rahim Yar Khan

which are not related to first PC (Table 4.3.7). The second PC is positively related to Musambi

collected from districts Abbotabad and Rahim Yar Khanand the fourth PC is positively related

to Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) collected from district Rshim Yar Khan

(Table 4.3.7)

A principal component scatter plot depicted that percentages of different compounds are close

together are being similar when related with other percentages of compounds. The projection

of percentages of different compounds on PC1 and PC2 showed the diverse nature. To identify

the better transgressive pattern of percentages of compounds in dissimilar groups of pattern,

the projection of percentages of compounds on first two principal components was useful.


from three citrus cultivars collected from different localities depicted that citrus cultivars

collected are diverse to each other when compared to each other (Fig 4.3.21). The projection

of three citrus cultivars from different localities on PC1 and PC2 showed the diverse nature

(Fig 4.3.21). The projection of compounds on PC1 and PC2 showed population structure on

the basis of percentages of compounds (Fig 4.3.22). From the different compounds in essential

oils extracted from three citrus cultivars collected from different localities also showed the

diverse nature because they were scattered in different ordinations on the factor-plane (Fig

4.3.21 and Table 4.3.22).

124

Figure:4.3.20 Scree plot between eigen values and number of principal components of

different compounds found in essentials oils extracted from three citrus cultivars

collected from different localities


42.67%

9.61%8.10%7.59%

6.39%4.93%4.21%

3.22%2.84%2.75%2.06%1.87%1.46%1.40%.91%

-2 0 2 4 6 8 10 12 14 16 18

Eigenvalue number

-1

0

1

2

3

4

5

6

7

8E

igenvalu

e

42.67%

9.61%8.10%7.59%

6.39%4.93%4.21%

3.22%2.84%2.75%2.06%1.87%1.46%1.40%.91%

125

Table: 4.3.6 Eigenvalues of correlation matrix, and related statistics on the basis of percentage of

different compounds found in essentials oils extracted from three citrus cultivars

collected from different localities

Eigenvalue % Total - variance Cumulative -

Eigenvalue Cumulative - %

1 6.400887 42.67258 6.40089 42.6726

2 1.440788 9.60525 7.84167 52.2778

3 1.214833 8.09889 9.05651 60.3767

4 1.138648 7.59099 10.19516 67.9677

5 0.957818 6.38545 11.15297 74.3532

6 0.739641 4.93094 11.89262 79.2841

7 0.631497 4.20998 12.52411 83.4941

8 0.483160 3.22106 13.00727 86.7151

9 0.425372 2.83582 13.43264 89.5510

10 0.412194 2.74796 13.84484 92.2989

11 0.308669 2.05779 14.15351 94.3567

12 0.280280 1.86853 14.43379 96.2252

13 0.219285 1.46190 14.65307 97.6871

14 0.210137 1.40091 14.86321 99.0881

15 0.136792 0.91195 15.00000 100.0000

126

Table: 4.3.7 Principal components of different compounds on the basis of percentage

found in essentials oils extracted from three citrus cultivars collected from different

localities

Factor 1 Factor 2 Factor 3 Factor 4

GF_Abt -0.609980 0.005590 0.397107 0.021302

Kin_Abt -0.650603 0.132477 -0.379048 0.054526

Mus_Abt -0.294728 0.556339 0.213117 -0.406431

GF_Fsd -0.659820 -0.024756 0.176260 -0.319605

Kin_Fsd -0.617545 -0.252715 -0.423929 0.063077

Mus_Fsd -0.610897 0.433639 -0.456927 0.043530

GF_Lya -0.693308 -0.344039 0.088418 -0.089017

Kin_Lya -0.800160 -0.299148 -0.000835 -0.050234

Mus_Lya -0.776855 0.097165 0.369330 -0.032337

GF_RYK -0.795842 0.028546 0.303077 0.005494

Kin_RYK -0.015507 0.099516 0.331924 0.868948

Mus_RYK -0.350148 0.752625 -0.004783 0.106764

GF_Sgd -0.736856 -0.126301 0.054025 0.154324

Kin_Sgd -0.794966 -0.220734 -0.009331 -0.031355

Mus_Sgd -0.820470 0.045193 -0.314955 0.243359

127

Table: 4.3.8 Factor coordinates of cases on the basis of percentage of different compounds found in essentials oils extracted from three

citrus cultivars collected from different localities

Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Factor 7 Factor 8 Factor 9 Fact.10 Fact.11 Fact.12 Fact.13 Fact.14 Fact.15

(-) -a-Panasinsene 1.38113 2.05996 0.14148 -0.84070 0.69789 1.06491 0.62965 -0.35787 0.48871 -0.32548 0.42581 -0.33832 0.53148 -0.280989 0.001764

(-)a-Neoclovene 0.89650 -0.50747 0.81075 -0.85608 -1.59751 -0.40145 -0.87270 -1.06279 -0.13302 0.60449 -0.34338 -0.44204 -0.19781 -0.177524 -0.022183

(z) – Carveol 1.95654 -0.26838 0.45404 1.76186 0.48077 0.20747 -0.53587 0.20794 -0.25523 -0.07983 -0.19892 -0.27211 -0.18597 -0.094406 0.015729

a-caryophyllene 0.01275 -1.46954 0.43120 -0.36342 -1.06301 0.68528 0.55161 -0.38980 0.67543 -1.71810 -1.39193 1.05702 0.14243 0.306402 0.234045

ß-Citronellol 1.71066 0.56703 0.15182 -1.07893 1.05395 0.51113 -0.43897 0.37354 0.66322 0.22334 -0.05575 0.37419 0.26174 -0.198977 0.119489

Elemene 1.12671 0.52084 0.50989 -1.74958 0.30567 0.88219 -1.57746 0.04730 -0.11194 0.61156 -0.08920 -0.64042 -0.14107 -0.404621 0.044573

Elemol 1.48045 -0.46129 0.45267 -0.18543 -0.84923 -0.77252 0.26579 -0.73656 0.64214 0.21627 -0.30992 0.57257 0.20499 0.028121 0.052733

a-farnesol 1.38755 -0.51696 0.07694 -0.89674 -0.74072 0.34309 -0.95753 -0.24014 -1.02715 0.45304 0.18826 -0.87904 -0.23393 -0.212564 0.287620

a-gurjenene 1.28373 -1.19014 -0.07979 -0.43546 0.82067 -0.65447 1.20379 0.39485 0.13344 0.26281 0.02993 -1.16349 -0.17809 -0.982823 0.152190

?-Muurolene 1.20085 -0.26761 0.55987 -0.30215 0.66719 -0.69374 0.26670 -0.01775 -0.60735 -0.08671 0.90992 0.30860 -0.60424 1.072539 1.642634

ß-myrcene -3.60563 -1.85101 -3.56019 0.76184 0.59775 0.63615 0.64662 0.85730 1.13066 0.02573 -0.04360 -0.20991 -0.14659 -0.414007 0.230934

a -phellenderene -0.10650 -2.33439 -1.11207 -0.38667 1.81467 0.25950 1.13265 -0.84520 0.27833 -0.11254 -0.57037 -1.45335 0.55446 0.788542 -0.648365

a –Selinene 1.71066 0.56703 0.15182 -1.07893 1.05395 0.51113 -0.43897 0.37354 0.66322 0.22334 -0.05575 0.37419 0.26174 -0.198977 0.119489

1-Decanol 1.95654 -0.26838 0.45404 1.76186 0.48077 0.20747 -0.53587 0.20794 -0.25523 -0.07983 -0.19892 -0.27211 -0.18597 -0.094406 0.015729

2,6-Octadiene 1.27804 -0.72131 -0.16442 0.11826 -0.63328 0.74209 0.49005 1.04357 0.42180 1.00409 1.34017 0.27364 0.01507 0.412343 -0.051881

4-terpinenol 0.02060 2.08407 -1.27109 0.26157 -1.58965 -1.69171 0.70273 -0.38626 0.48005 -0.87896 -0.05688 -0.18776 -0.03514 0.184909 -0.653601

4,11-Selinadiene 1.71066 0.56703 0.15182 -1.07893 1.05395 0.51113 -0.43897 0.37354 0.66322 0.22334 -0.05575 0.37419 0.26174 -0.198977 0.119489

a-citral 0.84118 0.58165 -1.99456 -0.01731 -0.37680 -1.50095 -0.45073 1.16779 -0.23956 -0.48159 -0.00680 0.08775 -0.34101 0.231880 -0.226072

a-pinene -5.78659 -0.33815 -0.63979 -1.12883 1.66190 -1.01552 -1.09860 -0.75253 -0.19408 -0.14663 -1.15970 0.27850 0.12185 -0.529042 0.551598

Anethol 1.35791 -0.01146 -0.34653 1.88082 0.28548 -0.82885 -1.43150 0.68285 0.20169 -1.12543 0.59295 -0.53358 0.12504 -0.224025 -0.282421

Aromadendrene 1.11028 1.81766 -1.20432 0.10196 -0.53758 0.08915 1.51353 -0.03852 -0.87099 0.01519 -0.31711 -0.36330 -0.38227 0.279487 -0.045648

Carveol 1.95654 -0.26838 0.45404 1.76186 0.48077 0.20747 -0.53587 0.20794 -0.25523 -0.07983 -0.19892 -0.27211 -0.18597 -0.094406 0.015729

Carvyol acetate (z) 1.48045 -0.46129 0.45267 -0.18543 -0.84923 -0.77252 0.26579 -0.73656 0.64214 0.21627 -0.30992 0.57257 0.20499 0.028121 0.052733

Caryophyllene -4.35050 -0.92968 2.25197 -1.69647 0.19408 -0.34451 -0.53305 1.52165 0.64648 -1.01098 0.86061 -0.17595 0.03119 -0.110671 -0.472818

Caryophyllene oxide 1.46549 -0.25890 1.18784 1.80252 -0.37602 -0.53707 -0.45103 -0.61471 0.63889 0.07162 -0.73055 0.16489 -0.14985 -0.059366 -0.294074

Cis-Carveol 1.95654 -0.26838 0.45404 1.76186 0.48077 0.20747 -0.53587 0.20794 -0.25523 -0.07983 -0.19892 -0.27211 -0.18597 -0.094406 0.015729

Cis, Trans-Farnesol 0.91051 0.59407 -0.31117 -0.05627 -0.52287 1.35292 1.39737 -0.65223 -0.53823 -1.63602 0.60565 0.37442 -0.52903 -0.963911 0.300751

Citronellal -3.66841 0.88868 -1.64947 0.42107 -3.20992 1.15965 -0.15206 0.26199 0.68983 -0.58841 -0.31944 -0.37803 0.27596 -0.308824 0.864761

Citronellol -2.36277 3.33691 1.03221 -1.32540 -0.30955 -1.82877 -0.81824 -0.21596 -1.06502 -0.26351 0.47711 0.00172 0.04338 0.454095 -0.437551

Copaene -2.92203 -1.97583 2.99670 1.69602 0.56446 0.01708 1.40417 1.20738 0.04627 -0.65677 -0.04097 0.45383 -0.32471 0.329162 -0.203512

d-cadinene -6.21921 -1.62649 -0.95604 0.06836 -0.02533 -0.78455 -0.16958 -0.08249 -0.43550 0.63412 0.23622 0.17795 -0.12481 0.082277 0.380228

D-Germacrene 0.79268 -1.18065 0.65401 -0.39480 -0.03612 -1.39901 1.28863 -0.42780 1.02756 0.41426 -0.50171 -0.72649 -0.14196 -0.947783 -0.157613

Decanal -6.21921 -1.62649 -0.95604 0.06836 -0.02533 -0.78455 -0.16958 -0.08249 -0.43550 0.63412 0.23622 0.17795 -0.12481 0.082277 0.380228

E-Nerolidol 1.71066 0.56703 0.15182 -1.07893 1.05395 0.51113 -0.43897 0.37354 0.66322 0.22334 -0.05575 0.37419 0.26174 -0.198977 0.119489

Estragole 0.58606 -1.46296 -1.33135 -0.13611 0.86663 1.08243 0.11957 -1.51456 -0.36387 -0.68021 0.50818 1.26399 -0.97130 -0.263794 -0.507450

Farnesol 1.71066 0.56703 0.15182 -1.07893 1.05395 0.51113 -0.43897 0.37354 0.66322 0.22334 -0.05575 0.37419 0.26174 -0.198977 0.119489

Farnesyl acetate 1.48045 -0.46129 0.45267 -0.18543 -0.84923 -0.77252 0.26579 -0.73656 0.64214 0.21627 -0.30992 0.57257 0.20499 0.028121 0.052733

Globulol 0.88182 -0.20437 -0.34790 -0.06648 -1.04451 -1.80884 -0.62984 -0.26164 1.09906 -0.82933 0.48194 0.31109 0.51600 -0.101498 -0.245416

Heneicosane -0.53321 -1.33817 0.92569 -0.62132 -2.27777 0.99930 -0.42557 0.24837 0.68579 0.76141 -0.20931 -0.77088 0.55347 1.394999 -0.311229

Heptacosane -0.52843 -1.18610 0.90774 -0.58013 -2.41270 1.19574 -0.41581 -0.11224 0.42902 0.39173 0.67746 0.64741 -1.31925 -0.633240 -0.380660

128

Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Factor 7 Factor 8 Factor 9 Fact.10 Fact.11 Fact.12 Fact.13 Fact.14 Fact.15

Hexahydrothymol 1.27804 -0.72131 -0.16442 0.11826 -0.63328 0.74209 0.49005 1.04357 0.42180 1.00409 1.34017 0.27364 0.01507 0.412343 -0.051881

Iso-Caryophyllene 1.43981 0.32472 -1.19398 -0.13626 -0.18152 -0.46463 0.44490 0.69288 -0.69648 0.56401 -0.79866 0.34922 -0.65201 0.361499 0.072078

Juniper Camphor 0.84944 2.85546 -0.77137 -0.75088 0.50881 0.62825 0.89360 0.24892 0.04422 0.17371 -0.59457 -0.12467 -0.28940 0.087430 -0.288695

Levoverbenone -1.80099 1.94664 0.22095 3.15128 -1.25700 0.22108 1.03112 0.20134 -0.35822 2.07895 -0.25977 0.79834 1.23706 -0.751899 -0.036375

Limonene -6.82454 1.10664 0.20175 1.44170 1.13821 0.54378 -0.43771 -0.40460 0.30195 0.09917 0.01968 -0.70361 -0.11704 -0.279278 -0.327352

Limonene oxide 0.84118 0.58165 -1.99456 -0.01731 -0.37680 -1.50095 -0.45073 1.16779 -0.23956 -0.48159 -0.00680 0.08775 -0.34101 0.231880 -0.226072

Limoneneglycol 1.95654 -0.26838 0.45404 1.76186 0.48077 0.20747 -0.53587 0.20794 -0.25523 -0.07983 -0.19892 -0.27211 -0.18597 -0.094406 0.015729

Linalool -6.13677 1.82601 0.00041 1.65107 0.32510 1.17027 -1.46055 -0.71336 -0.08347 -0.09882 0.21146 0.59544 0.22991 0.696625 -0.117006

Menthadien-1-ol 1.95654 -0.26838 0.45404 1.76186 0.48077 0.20747 -0.53587 0.20794 -0.25523 -0.07983 -0.19892 -0.27211 -0.18597 -0.094406 0.015729

Menthol 1.71066 0.56703 0.15182 -1.07893 1.05395 0.51113 -0.43897 0.37354 0.66322 0.22334 -0.05575 0.37419 0.26174 -0.198977 0.119489

Nerolidol 0.89650 -0.50747 0.81075 -0.85608 -1.59751 -0.40145 -0.87270 -1.06279 -0.13302 0.60449 -0.34338 -0.44204 -0.19781 -0.177524 -0.022183

Nootkatone -4.61308 1.10183 2.12936 -1.48737 -0.15578 0.80893 1.69515 0.92212 -0.42944 -0.01501 -0.47008 -0.41334 -0.83095 0.305354 -0.582853

Octacosane -0.84807 -1.46715 0.72675 -1.06216 -0.63215 1.41942 -0.51999 0.41956 -2.32361 -1.26107 -0.73704 0.39783 0.86507 -0.346854 -0.277152

Octanal 1.31752 -1.03487 -1.31164 -0.06771 1.04099 0.25531 -0.02822 -1.50763 -0.25212 0.47181 0.60580 0.31262 -0.00366 0.611185 -0.563390

Perillaldehyde 1.37287 -0.21385 -1.08170 -0.10714 -0.18773 -1.06430 -0.71468 0.56101 0.20493 -0.98078 1.01358 -0.12591 0.47988 -0.136538 0.064387

Perillyl acetate 1.38755 -0.51696 0.07694 -0.89674 -0.74072 0.34309 -0.95753 -0.24014 -1.02715 0.45304 0.18826 -0.87904 -0.23393 -0.212564 0.287620

Sabinene 1.17897 1.36253 -0.76104 -0.98910 0.86488 0.07448 -0.17503 0.98032 0.21873 0.72253 -1.07612 0.58785 -0.55915 0.169442 -0.170969

ß-Citral 1.43981 0.32472 -1.19398 -0.13626 -0.18152 -0.46463 0.44490 0.69288 -0.69648 0.56401 -0.79866 0.34922 -0.65201 0.361499 0.072078

ß-cubebene -0.74589 -0.91943 2.16629 -0.45829 0.94587 -2.44624 1.52608 -0.21871 -0.65756 0.48298 0.34321 0.23897 0.24649 -0.280895 0.376401

ß-pinene 0.10446 2.47500 2.45146 1.11185 0.97394 -0.10995 0.08340 -1.16252 1.02422 -0.47022 0.16175 -0.13596 -0.56035 0.746024 0.625250

Terpinene-4-ol 1.31752 -1.03487 -1.31164 -0.06771 1.04099 0.25531 -0.02822 -1.50763 -0.25212 0.47181 0.60580 0.31262 -0.00366 0.611185 -0.563390

Trans-A-Terpineol 1.64198 1.02216 -0.29146 0.01214 -0.34851 0.52580 1.24958 -0.64531 -0.42649 -0.48400 0.70326 -0.57695 0.43861 -0.088932 0.244811

Trans-Anethol 1.23526 -1.05094 -0.28289 -0.33568 -0.03187 0.60270 0.31899 0.43978 -0.10695 -0.71752 -0.76267 -0.33135 1.07394 1.146340 0.487907

Trans-carane 1.20359 -0.41272 0.39015 -0.21352 0.32992 -0.40945 0.33267 0.39904 -1.58174 0.28508 0.37842 0.92799 1.33044 -0.419491 -0.383547

Trans-Carveol 1.95654 -0.26838 0.45404 1.76186 0.48077 0.20747 -0.53587 0.20794 -0.25523 -0.07983 -0.19892 -0.27211 -0.18597 -0.094406 0.015729

Trans-nerolidol 1.64198 1.02216 -0.29146 0.01214 -0.34851 0.52580 1.24958 -0.64531 -0.42649 -0.48400 0.70326 -0.57695 0.43861 -0.088932 0.244811

Valencene -6.80958 0.90425 -0.53342 -0.54625 0.66500 0.30833 0.27911 -0.52645 0.30519 0.24382 0.44031 -0.29594 0.23780 -0.191792 0.019455

129

Fig: 4.3.21 Two dimensional ordination of three citrus cultivars collected from different

localities on PC1 and PC2


GF_Abt

Kin_Abt

Mus_Abt

GF_Fsd

Kin_Fsd

Mus_Fsd

GF_LyaKin_Lya

Mus_Lya

GF_RYK

Kin_RYK

Mus_RYK

GF_Sgd

Kin_Sgd

Mus_Sgd

-1.0 -0.5 0.0 0.5 1.0

Factor 1 : 42.67%

-1.0

-0.5

0.0

0.5

1.0

Fa

cto

r 2 :

9.6

1%

GF_Abt

Kin_Abt

Mus_Abt

GF_Fsd

Kin_Fsd

Mus_Fsd

GF_LyaKin_Lya

Mus_Lya

GF_RYK

Kin_RYK

Mus_RYK

GF_Sgd

Kin_Sgd

Mus_Sgd

130

Fig: 4.3.22 Two dimensional ordination of different compounds found in essentials oils

extracted from three citrus cultivars collected from different localities on the basis of

percentage PC1 and PC2



(-) -a-Panasinsene

(-)a-Neoclovene

(z) – Carveol

a-caryophyllene

ß-CitronellolElemene

Elemola-farnesol

a-gurjenene

?-Muurolene

ß-myrcene

a -phellenderene

a -Selinene

1-Decanol

2,6-Octadiene

4-terpinenol

4,11-Selinadienea-citral

a-pinene

Anethol

Aromadendrene

CarveolCarvyol acetate (z)

Caryophyllene

Caryophyllene oxideCis-Carveol

Cis, Trans-Farnesol

Citronellal

Citronellol

Copaene

d-cadinene

D-Germacrene

Decanal

E-Nerolidol

Estragole

Farnesol

Farnesyl acetate

Globulol

HeneicosaneHeptacosane

Hexahydrothymol

Iso-Caryophyllene

Juniper Camphor

Levoverbenone

Limonene

Limonene oxide

Limoneneglycol

Linalool

Menthadien-1-ol

Menthol

Nerolidol

Nootkatone

Octacosane

Octanal

Perillaldehyde

Perillyl acetate

Sabinene

ß-Citral

ß-cubebene

ß-pinene

Terpinene-4-ol

Trans-A-Terpineol

Trans-Anethol

Trans-caraneTrans-Carveol

Trans-nerolidolValencene

-8 -6 -4 -2 0 2 4

Factor 1: 42.67%

-3

-2

-1

0

1

2

3

4F

acto

r 2: 9

.61%

(-) -a-Panasinsene

(-)a-Neoclovene

(z) – Carveol

a-caryophyllene

ß-CitronellolElemene

Elemola-farnesol

a-gurjenene

?-Muurolene

ß-myrcene

a -phellenderene

a -Selinene

1-Decanol

2,6-Octadiene

4-terpinenol

4,11-Selinadienea-citral

a-pinene

Anethol

Aromadendrene

CarveolCarvyol acetate (z)

Caryophyllene

Caryophyllene oxideCis-Carveol

Cis, Trans-Farnesol

Citronellal

Citronellol

Copaene

d-cadinene

D-Germacrene

Decanal

E-Nerolidol

Estragole

Farnesol

Farnesyl acetate

Globulol

HeneicosaneHeptacosane

Hexahydrothymol

Iso-Caryophyllene

Juniper Camphor

Levoverbenone

Limonene

Limonene oxide

Limoneneglycol

Linalool

Menthadien-1-ol

Menthol

Nerolidol

Nootkatone

Octacosane

Octanal

Perillaldehyde

Perillyl acetate

Sabinene

ß-Citral

ß-cubebene

ß-pinene

Terpinene-4-ol

Trans-A-Terpineol

Trans-Anethol

Trans-caraneTrans-Carveol

Trans-nerolidolValencene

131

4.3.7 Correlation of environmental factors with compounds in citrus peel essential oils

Environmental factors play a significant role in occurrence of compounds in the essential oils.

The correlation coefficients estimated between environmental conditions and compounds

given in Table 4.3.9 in Grapefruit, Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and

Musambi is given the Tables 4.3.10 ,Table 4.3.11 and Table 4.3.12 respectively. Fifteen

compounds (ß-Citronellol, Elemol, α –Selinene, 4-terpinenol, 4,11-Selinadiene, Carvyol

acetate (z), Citronellol, Decanal, E-Nerolidol, Farnesol, Farnesyl acetate, Globulol,

Limonene, Menthol and Perillaldehyde) have statistically significant correlation with

elevation for the three citrus cultivars in Table 4.3.9 . All these compounds except limonene

have a positive correlation with the elevation. Limonene has a negative correlation with

elevation. Four compounds have statistically negatively correlation with maximum

temperature for three citrus cultivars in Table 4.3.9 (4-terpinenol, Citronellol, Decanal and

Globulol). Fifteen compounds (ß-Citronellol, Elemol, α –Selinene, 4-terpinenol, 4,11-

Selinadiene, Carvyol acetate (z), Citronellol, Decanal, E-Nerolidol, Farnesol, Farnesyl acetate,

Globulol, Linalool, Menthol and Perillaldehyde) have statistically negative correlation with

minimum temperature for three citrus cultivars. Limonene has the statistically positive

correlation with minimum temperature. Fifteen compounds(ß-Citronellol, Elemol, α –

Selinene, 4-terpinenol, 4,11-Selinadiene, Carvyol acetate (z), Citronellol, Decanal, E-

Nerolidol, Farnesol, Farnesyl acetate, Globulol, Linalool, Menthol and Perillaldehyde) have

significant negative correlation with average temperature and Citronellol and Globulol have

highly significantly negative correlation with average temperature(Table 4.3.9).Three

compounds (Citronellol, Decanaland Globulol) have statistically significant positive

correlation with rainfall in essential oils of all three citrus cultivars (Table 4.3.9). Globulol has

highly significant correlation with rainfall. Globulol have negatively significant correlation

with morning wind speed (Table 4.3.9) No compound was significantly correlated with

evening wind speed (Table 4.3.9).

132

Table: 4.3.9 Correlation for environmental condition with compounds of essential oils

of three citrus cultivars

Correlations WS8am WS5pm Avg.WS Rainfall Min.tm

p

Max.tm

p Avg.tmp

Elevatio

n

ß-Citronellol -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

Elemol -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

α –Selinene -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

4-terpinenol -0.244 -0.159 -0.200 0.513 -0.567* -0.520* -0.545* 0.576*

4,11-Selinadiene -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

Carvyol acetate (z) -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

Citronellol -0.256 -0.132 -0.191 0.617* -0.696** -0.634* -0.667** 0.689**

Decanal -0.242 -0.122 -0.180 0.627* -0.570* -0.636* -0.609* 0.570*

E-Nerolidol -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

Farnesol -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

Farnesyl acetate -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

Globulol -0.517* -0.409 -0.462 0.740** -0.767** -0.738** -0.757** 0.773**

Limonene 0.255 0.191 0.223 -0.418 0.515* 0.424 0.469 -0.520*

Linalool -0.170 -0.060 -0.112 0.487 -0.524* -0.503 -0.516* 0.511

Menthol -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

Perillaldehyde -0.356 -0.282 -0.319 0.510 -0.529* -0.509 -0.522* 0.533*

* = Significant (P<0.05); ** = Highly significant (P<0.01)

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4.3.8 Correlation for environmental condition with compounds of essential oils extracted from

Grapefruit

In grapefruit peel essential oil thirteen compounds ((-)α-Neoclovene, α-caryophyllene,

Elemol, 4-terpinenol, Carvyol acetate (z), Caryophyllene oxide, Decanal, Farnesyl acetate,

Globulol, Heneicosane, Heptacosane, ß-pinene) were statistically significant correlation with

rainfall. Heneicosane has highly significant correlation with rainfall (Table: 4.3.10). Fourteen

compounds in the grapefruit peel essential oil ((-)α-Neoclovene, α-caryophyllene, Elemol, 4-

terpinenol, Carvyol acetate (z), Caryophyllene oxide, Decanal, Farnesyl acetate, Globulol,

Heneicosane, Heptacosane, Linalool and ß-pinene) were in significant negative correlation

with minimum temperature (Table: 4.3.10). Thirteen compounds in the grapefruit peel

essential oils ((-)α-Neoclovene, α-caryophyllene, Elemol, 4-terpinenol, Carvyol acetate (z),

Caryophyllene oxide, Decanal, Farnesyl acetate, Globulol, Heneicosane, Heptacosane, ß-

pinene) were negatively correlated with maximum temperature (Table: 4.3.10). Fourteen

compounds from grapefruit peel essential oils ((-)α-Neoclovene, α-caryophyllene, Elemol, 4-

terpinenol, Carvyol acetate (z), Caryophyllene oxide, Decanal, Farnesyl acetate, Globulol,

Heneicosane, Heptacosane, Linalool and ß-pinene) have statistically negative correlation with

the average temperature (Table: 4.3.10). Fourteen compounds in grapefruit peel essential oils

((-)α-Neoclovene, α-caryophyllene, Elemol, 4-terpinenol, Carvyol acetate (z), Caryophyllene

oxide, Decanal, Farnesyl acetate, Globulol, Heneicosane, Heptacosane, Linalool and ß-pinene)

have statistically positive correlation with elevation(Table: 4.3.10.)

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Table: 4.3.10 Correlation for environmental condition with compounds of essential oils extracted

from Grapefruit.

Correlations WS8am WS5pm Avg.WS Rainfall Min.tmp Max.tmp Avg.tmp Elevatio

n

(-)α-Neoclovene -0.314 -0.117 -0.211 0.899* -0.919* -0.925* -0.928* 0.900*

α-caryophyllene -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Elemol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

4-terpinenol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Carvyol acetate (z) -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Caryophyllene oxide -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Decanal -0.417 -0.249 -0.330 0.951* -0.893* -0.961** -0.935* 0.902*

Farnesyl acetate -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Globulol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Heneicosane -0.660 -0.525 -0.592 0.985** -0.962** -0.978** -0.977** 0.983**

Heptacosane -0.411 -0.234 -0.319 0.955* -0.924* -0.969** -0.954* 0.925*

Linalool -0.302 -0.152 -0.224 0.861 -0.884* -0.876 -0.885* 0.902*

ß-pinene -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**


135



In Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel essential oils Citronellal has

statistically highly significant negative correlation with wind speed at morning and Limonene

has statistically significant positive correlation with wind speed at morning (Table: 4.3.11).In

Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel essential oil only Citronellal has

statistically significant negative correlation with wind speed at evening (Table: 4.3.11). In

Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel essential oil Citronellal has statistically

significant negative correlation with average wind speed (Table: 4.3.11). Nine compounds

present in essential oils of Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel (4-

terpinenol, α-citral, Citronellol, Decanal, Globulol, Limonene oxide, Linalool, Perillaldehyde,

Valencene) showed the statistically significant positive correlation with rainfall (Table:

4.3.11). Eight compounds in Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel essential

oil (4-terpinenol, α-citral, Citronellol, Globulol, Limonene oxide, Linalool, Perillaldehyde,

Valencene) showed the highly significant negative correlation with minimum temperature

(Table: 4.3.11). Nine compounds in the Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel

essential oil (4-terpinenol, α-citral, Citronellol, Decanal Globulol, Limonene oxide, Linalool,

Perillaldehyde, Valencene) showed the significant negative correlation with maximum

temperature (Table: 4.3.11). Seven compounds in Kinnow (C.nobilis Loureiro×C. deliciosa

Tenore) peel essential oil (4-terpinenol, α-citral, Citronellol, Globulol, Limonene oxide,

Perillaldehyde, Valencene) showed the highly significant negative correlation with average

temperature and two compounds ( Decanal and Linalool) were significantly negative

correlated with average temperature (Table: 4.3.11). Eight compounds in Kinnow (C.nobilis

Loureiro×C. deliciosa Tenore) peel essential oils (.4-terpinenol α-citral Citronellol Globulol

Limonene oxide Linalool Perillaldehyde Valencene) were statistically highly significantly

correlated with elevation (Table: 4.3.11).

136

Table: 4.3.11 Correlation for environmental condition with compounds of essential oils

extracted from Kinnow(C.nobilis Loureiro×C. deliciosa Tenore).

Correlations WS8am WS5pm Avg.WS Rainfall Min.tmp Max.tmp Avg.tmp Elevation

4-terpinenol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

α-citral -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Citronellal -0.964** -0.885* -0.927* 0.813 -0.794 -0.785 -0.794 0.794

Citronellol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Decanal -0.368 -0.202 -0.281 0.912* -0.820 -0.922* -0.879* 0.826

Globulol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Limonene 0.898* 0.841 0.873 -0.807 0.841 0.777 0.812 -0.868

Limonene oxide -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Linalool -0.777 -0.633 -0.705 0.932* -0.974** -0.929* -0.956* 0.961**

Perillaldehyde -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Valencene -0.692 -0.550 -0.621 0.970** -0.993** -0.967** -0.985** 0.999**


137


Musambi

In musambi peel essential oils Citronellal has statistically highly significant positive

correlation with wind speed at morning and Aromadendrene has statistically significant

positive correlation with wind speed at morning (Table: 4.3.12).In musambi peel essential oil

Citronellal has statistically significant positive correlation with wind speed at evening (Table:

4.3.12). In musambi peel essential oil two compounds (Aromadendrene and Citronellal) have

statistically significant positive correlation with average wind speed (Table: 4.3.12). Seven

compounds present in essential oils of musambi peel (ß-Citronellol, Elemene, α –Selinene,

4,11-Selinadiene, E-Nerolidol, Farnesoland Menthol) showed the statistically significant

positive correlation with rainfall (Table: 4.3.12). Seven compounds present in essential oils of

musambi peel (ß-Citronellol, Elemene, α –Selinene, 4,11-Selinadiene, E-Nerolidol,

Farnesoland Menthol) showed the highly significant negative correlation with minimum

temperature (Table: 4.3.12). Seven compounds present in essential oils of musambi peel (ß-

Citronellol, Elemene, α –Selinene, 4,11-Selinadiene, E-Nerolidol, Farnesoland Menthol)

showed the significant negative correlation with maximum temperature (Table: 4.3.12). Seven

compounds available in essential oils of musambi peel (ß-Citronellol, Elemene, α –Selinene,

4,11-Selinadiene, E-Nerolidol, Farnesoland Menthol) showed the highly significant negative

correlation with average temperature (Table: 4.3.12). Seven compounds found in essential oils

of musambi peel (ß-Citronellol, Elemene, α –Selinene, 4,11-Selinadiene, E-Nerolidol,

Farnesoland Menthol) showed the highly significant positive correlation with elevation (Table:

4.3.12).

138

Table: 4.3.12Correlation for environmental condition with compounds of essential oils extracted from Musambi.

Correlations WS8am WS5pm Avg.WS Rainfall Min.tmp Max.tmp Avg.tmp Elevat.

ß-Citronellol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Elemene -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

α -Selinene -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

4,11-Selinadiene -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Aromadendrene 0.887* 0.869 0.883* -0.529 0.436 0.488 0.467 -0.439

Citronellal 0.959** 0.945* 0.957* -0.506 0.542 0.469 0.507 -0.535

E-Nerolidol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Farnesol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**

Menthol -0.667 -0.527 -0.596 0.955* -0.989** -0.952* -0.976** 0.997**


139

Chapter 5

DISCUSSION

Citrus is the major fruit crop of Pakistan. Its production is approximately over than

2150 Metric tons annually. Its peel is treated as a wasted material. The outer surface of citrus

fruit known as flavedo which contains the numerous glands and carry different types of

essential oils. These essential oils can be extracted from citrus peel with different methods and

a good income can be earned. These essential oils can convert a waste into a wealth (Ezejiofor

et al., 2011). Citrus peel essential oils have very useful aroma profiling and therapeutic

compounds. These compounds are being used in the industries such as cosmetics,

pharmaceuticals, foods, beverages etc. Citrus peel essential oils are environment friendly and

can be a source of income. Therefore this research was planned and executed to find the best

method a best cultivar regarding the essential oils, some new compounds and the climatic

effects in these compounds.

In the first experiment steam distillation method of extraction was applied.

Temperature plays an important role in steam distillation. Thus three levels of temperature

were used in this experiment and it was found that 105oC gave the best results in terms of

essential oil yield and chemical components. A negative correlation was found between

temperature and compounds as temperature increased the yield of compound decreased. The

effect of heat in steam distillation process in this research work also confirmed by the results

of Kasuan et al., (2010) who described that a controlled steam temperature is advantageous in

order to avoid the deterioration of the quality of essential oils. The results are also supported

by Tajjudin et al., (2012) who concluded that higher temperature in the steam distillation

affected the quality of the essential oils and reduced the yield percentage of the essential oils.

The present study was also supported by the findings of Zakiah et al., (2013) in which they

found that the yield of essential oil was more at a controlled lower temperature as compared to

those where higher temperature was maintained. Majeed et al., (2013) also reported the similar

observations and concluded that temperature just above the boiling point of water was the best

for essential oil extraction in steam distillation method of extraction. These results were also

confirmed by the work of Eikani et al., (2007). They used a temperature range of 100oC, 125oC,

150oC and 175oC and found that 100oC is the best for coriander essential oil extraction in steam

distillation. Density of the essential oils was also affected by the higher temperature during the

140

distillation by steam. Decreased density of essential oils at higher temperature in present study

was also due to the same reason as described above. These findings are supported by the work

of Thavanapong et al., (2011) who reported that higher temperature of extraction reduced the

physical properties of products such as density and refractive index of the essential oils. .

The citrus peel essential oils are the complex mixture of different chemical constituents

and each compound has its own importance. It is because these different compounds can be

used for different purposes and amount which is mostly spent on it to purchase can be saved

or by selling income can be earned. Therefore the separation and recognition was done by the

help of GC-MS analysis. GC-MS is a specialized and relatively modern technique fulfilling

the essential oil analysis requirements efficiently. Therefore, the pure citrus peel essential oils

were subjected to Gas chromatography – Mass Spectrometry analysis. Retention time for citrus

peel essential oils was observed to be 32 minutes. This time is meant for the run time of GC-

MS apparatus for the isolation of the compounds present in the essential oil samples. As the

purity of the essential increases the number of the peaks or availability of the more compounds

increases. The maximum number of peaks in the citrus peel essential oils were formed in

between 6-20 minutes. This time retention results were in accordance with Ou et al., (2015)

who observed that retention time for isolation of compounds in peel essential oils of Citrus

grandis and Citrus paradisi were at the range of 3.6 to 23.3 minutes and maximum peaks were

formed during 5-20 minutes. Terada et al., (2010) also found the maximum number of peaks

in the citrus peel oil during 10-20 minutes. These results are also in accordance with the results

of Kamal et al., (2011) in which they found that maximum peaks in citrus peel essential oils

in GC-MS were formed during 5-20 minutes after running the GC-MS. Peaks obtained in the

result of GC-MS analysis of citrus peel essential oils were matched separately according to

NIST and other international chemistry libraries.

Citrus peel essential oils are characterized by large number of components those are

named as terpenes, sesquiterpenes, esters and alcohols. In the present work the citrus peel

essential oils showed that Limonene was the major component of all the citrus peel essential

oils. This result is confirmed by the findings of Lota et al., (2001) in which they reported the

chemical composition of 15 mandarine species in their peel and leaf oil constituents and also

found that among these Limonene was the major constituent of all the samples which is

according to the present work. The results are also confirmed by the findings of Zakiah et al.,

141

(2013) those concluded that limonene was the most prominent constituent of the citrus peel

essential oil. The findings of Bourgou et al., (2012) also confirmed the results of the present

study and found that limonene was the major constituent of the citrus peel essential oil. In the

present study it was found that there were some other compounds common in most of the

essential oils samples extracted at different temperature levels in steam distillation which were

negatively correlated with the temperature such as α- pinene, sabinene and β-pinene. Similarly

some of compounds showed positively correlation with temperature; as with rise in

temperature Limonene, citronellal, terpinene-4-ol, α-terpineol and caryophyllene also

increased .The results are also supported by the findings of Mahalwal and Ali (2001) who

analyzed the essential oil of orange by GC-MS and found that limonene was the most

dominated followed by β-myrcene, α-terpinolene and β-pinene. The results are also similar to

the findings of Droby et al., (2008) who observed the composition of peel essential oil of Citrus

sinenis and found that limonene was the predominant compound with percentage varying from

72.41 to 94.77%. Besides the limonene some other compounds in the citrus peel essential oils

were also also found in good yield percentage i.e., linlool, α-pinene, caryophyllene, nootkatone

and valencene. Ou et al., (2015) reported the similar ratio of limonene and other major

constituents of the citrus essential oils. Similar type of compounds were also found by the

Kirbaslar et al., (2006) who reported the yield % of such compounds such as limonene

-caryophyllene (0.4%), d-cadinene (0.2%), nootkatone (0.2%),

-terpineol

(0.1%), neryl acetate (0.1%) and geranyl acetate (0.1%). The results of the present study are

also confirmed by Kamal et al., (2011) who found Limonene and ß-myrcene were the main

constituents in the oils from fresh, ambient-dried and oven-dried peels of C. sinensis. In the

present study it was observed that a considerable amount of α-pinene was also determined.

This comparison of common compounds makes it easy to find out the optimum temperature

during extraction, method of essential oil extraction and cultivar to be adopted for the

extraction and separation of the particular compounds. The results were confirmed by the

results of Lota et al., (2001) who found the major constituents comprising of limonene upto

90-94 % but a different percentage of linalool, sabinene, limonene ϒ-terpenine in the different

citrus cultivars essential oils in France.

142

Some of the compounds in citrus peel essential oils were found in very little percentage

such as α-neocleovene, 1-decene, α-caryophyllene, aromadendrene, citronellol, citronellal, d-

cadinene, levoverbenone, and β-pinene. Ou et al., (2015) and some other researchers reported

their work and comparison of these reports showed some variation. This variation could be due

to the method of extraction, variety selected, analysis technique, identification tools,

application of fertilizers, pesticides usage and other soil inputs or even cultural practices.

Although very little percentage of trace compounds was present in the essential oils but also

contributed an important share in the total. Thus the total share of the minor or trace compounds

also seemed noticeable. These trace compounds provides the clear, specific odor or fragrance

to the citrus essential oils

In the second experiment the essential oil was extracted from the peel with two

methods of extraction i.e., steam distillation and supercritical fluid extraction system only to

compare the method regarding to yield of essential oil and its constituents. It was observed that

oil recovery percentage was low in the supercritical fluid extraction system but the number of

these compounds was more in quantity. This can safely decided that supercritical fluid

extraction system is more sophisticated which can extract some unusable waxes and other

compounds but it need more expertise and expenditure. Boutekedjiret et al., (2003) reported

that more essential oil yield percentage was obtained by steam distillation. Steam distillation

and supercritical fluid extraction system have their own advantages and disadvantages.

Although the number of chemical constituents found in the essential oil extracted by

supercritical fluid extraction system was more than the steam distillation but steam distillation

method of essential oil extraction was better as it was much easier to operate and essential oil

yield is more. This finding is also confirmed by the statement of Masango (2005) who stated

that operation of steam distillation improved recovery of valuable essential oils, energy saving

and environmental friendly and extraction of undesired compounds was also noted during

supercritical fluid extraction of coriander essential oil (Kerrola and Kallio, 1993).

Climatic and environmental factors affect the chemical composition of the essential

oils. No exact reference for citrus fruit in this regards could not found but in other crops it is

previously reported that essential oil yield and their chemical components are highly affected

by climatic and genetic factors (Rahimmalek et al., 2009). Moreover, they found that leaf

essential oil composition of fennel collected from different geographical regions of Iran

143

resulted in variation in terms of oil yield and chemical compounds. They also found a negative

correlation of oil yield with climate temperature. These findings are not directly related to

citrus cultivars but these can be correlate with the findings of present results because the

composition of compounds could be similar in crops and fruits plants regarding the effect of

climatic zones.

The results of present study indicated that the presence of Limonene in citrus peel of

citrus fruits grown in different regions showed negative correlation with the elevation as the

elevation increased the quantity of limonene decreased. The reason could be such as rainfall,

minimum and maximum temperatures etc. In the present work it was found that compounds

for example ß-Citronellol, Elemol, α –Selinene, 4-terpinenol, 4,11-Selinadiene, Carvyol

acetate (z), Citronellol, Decanal, E-Nerolidol, Farnesol, Farnesyl acetate, Globulol, Menthol

and Perillaldehyde have positive correlation with elevation. In this research it was found that

the compounds such as ß-Citronellol, Elemol, α –Selinene, 4-terpinenol, 4,11-Selinadiene,

Carvyol acetate (z), Citronellol, Decanal, E-Nerolidol, Farnesol, Farnesyl acetate, Globulol,

Linalool, Menthol and Perillaldehyde were significantly in negative correlation with average

temperature. Similar results were observed by Melito et al., (2016) who found that altitude

level and climatic condition affected the chemical composition of essential oil of Helichrysum

italicum subsp. microphyllum collected from different locations of Sardinia, Italy. This result

was also supported by the findings of Fanciullino et al., (2006) who found two distinguished

chemotypes, limonene and limonene/γ-terpinene, for mandarin peel essential oils of the fruit

collected from different locations. The results of the present study was also confirmed by the

findings of Dhouioui et al., (2016) who observed the variation in the chemical components of

the essential oils of Aristolochia longa ssp. Paucinerv is due to the climate and seasonal change;

they found the different number and percentages of the chemical compounds in essential oils

collected from different climatic conditions. The results of present study is also supported by

the findings of Edinardo et al., (2016) who observed the different chemical percentages of the

compounds in the essential oil of Cordia verbenacea collected at different seasons and

climates. In this work it was found that some compounds such as Citronellol, Decanal and

Globulol had statistically significant positive correlation with rainfall in citrus peel essential

oils. The result of this study are also supported by the results of Apotosoaie et al., (2010) who

concluded that climate temperature and rainfall directly affect the composition of the

compounds in the essential oils of Foeniculun vulgare. The results were also confirmed by the

144

findings of Salamon et al., (2010) who found biodiversity in chamomile essential oils was due

to influence of eco-physiological conditions i.e., biotic and abiotic factors.

The citrus peel oil and the major compounds found in the citrus peel essential oils have

a precious market value. These compounds are being used in different industries such as

cosmetics, perfumes, backing and medicines. Limonene is commonly used in many ways such

as used in a perfume to mask the bitter taste of alkaloids, aftershave lotions, bath products and

other similar products (Matsura et al., 2002). The other uses of limonene are as an alternative

to botanical insecticides, natural and alternative medicines for asthma, paint remover, solvent

for 3-D printing, a number of model airplane glue and as biofuel. (Hirota et al., 2010).

Nootkatone is nontoxic to humans, an approved food additive, and commonly used in foods,

cosmetics, and pharmaceuticals. It is effective repellent or insecticide against mosquitos, red

bugs, head lice and some other insects as these are environment friendly insecticide (Dolan et

al., 2009). α-Pinene is an anti-inflammatory and a broad spectrum antibiotic so its medicinal

value is also important (Nissen et al., 2010). Caryophyllene is an approved food ingredient

and is antinociceptive, neuroprotective, anxiolytic and antidepressant (Katsuyama et al., 2013).

Linalool is used as a scent in 60–80% of perfumed hygiene products and cleaning agents

including soaps, detergents, shampoos, and lotions. In addition, linalool is used as an

insecticide against flea, fruit fly and cockroach .

The uses of these compounds as mentioned above showed the importance of that huge

material that is considered a wastage. Therefore, this research has great importance and

recommend to the citrus processing industries that they can increase their income if small units

of steam distillation systems are installed in their industries or combined system of different

industries can be managed and the selling of extracted essential oil can increase their income.

Skaria et al., (2007) stated that Germany earned an annual turnover of more than six billion

dollars in the field of their essential oil industry and India earned foreign exchange from this

sector more than Rs. 1300 million. Gunkel et al., (2010) viewed the high diversity of aromatic

oils for economic development share. More over this peel oil can be packed in different sizes

of bottles mentioning the benefits of these compounds to export in the world market. These

processing industries can use their own fruit peel as well as they can buy the peel from different

industries as well as individuals. This will also create self employment opportunity in country

side. It will certainly improve the economic status of people and enable them to put forth their

more share in the uplift of the nation.

https://en.wikipedia.org/wiki/Perfume

https://en.wikipedia.org/wiki/Soap

https://en.wikipedia.org/wiki/Detergent

https://en.wikipedia.org/wiki/Shampoo

https://en.wikipedia.org/wiki/Insecticide

https://en.wikipedia.org/wiki/Flea

https://en.wikipedia.org/wiki/Cockroach

145

Chapter-5 SUMMARY

Citrus belongs to family Rutaceae and has a very important place in plant kingdom.

Citrus is grown worldwide in more than 140 countries. Citrus fruits, such as oranges, grapefruit

and limes can be eaten fresh, but citrus fruits are in the global processing and utilization.

Orange juice comprises for one third of 85% of the total processed citrus consumption and its

peel is mostly considered as wasted material in our country and in some other parts of the

world as well. Citrus peel has the oil sacs in the flavedo and these essential oils could be

considered suitable alternatives to chemical additives for use in the food industry. The citrus

peel essential oil contains pleasant sensory characteristics components that are popular in food,

pharmaceuticals and cosmetics industries. Therefore this research was planned and executed

to investigate the effect of temperature on the yield and quality of essential oil extracted from

citrus peel, method of extraction, effects of growing regions on the essentials oils of citrus peel

and cultivars having the maximum essential oil in their peel.

First experiment was to determine the effect of different temperature on the citrus peel

essential oils extracted by the steam distillation method. Three citrus cultivars namely Citrus

paradisi, Citrus reticulata and Citrus sinensis were used in this experiment and three different

temperatures such as 105, 110 and 120oC were used in Steam distillation method. It was

observed that 1050C was the most suitable temperature where maximum essential oil from the

peel of different cultivars can be extracted by Steam distillation method. A negative correlation

was found between the temperature and essential oil the yield percentage, oil density and

refractive index. As temperature increased all these parameters decreased. Maximum number

of chemical constituents were also obtained at this temperature. . Chemical characterization of

the essential oils was done by GC-MS and maximum total 57 compounds were founds in all

cultivars in all temperatures but maximum 30 compounds were found in Grapefruit at 105oC.

The results of second experiments showed that maximum yield (0.311%) of essential

oil in Steal distillation method while the yield was 0.234% in SCFE method in Grapefruit peel.

. SCFE extracted 34 Chemical compounds while 25 compounds were extracted from the peel

of Grapefruit by steam distillation. Oil density in SCFE system recovered oil was 0.835

gm/cm3 and in steam distillation was 0.831 gm/cm3. . Refractive index of essential oil extracted

in SCFE was 1.474 and in steam distillation it was 1.471. Steam distillation method was found

better for the essential oil extraction to get the maximum yield but more number of compounds

can be extracted by SCFE method.

146

Effect of growing regions (climatic conditions) showed positive response on the

essential oils in three selected citrus cultivars. Similar trend was found in all cultivars

regarding the oil yield. Maximum yield was found in peel of fruits those were grown in hot

climate. Maximum oil percentages of 0.321 %, 0.309% and 0.294% were found from the peels

of Grapefruit, Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and Musambi respectively

those were harvested from Rahim Yar Khan district (Hot climate). Minimum oil yield was

found from the fruits of cooler climate (Abbotabad) which were 0.295, 0.294%, 271% in

Grapefruit, Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and Musambi respectively.

Refractive index was highest (1.471) in Grapefruit of Abbotabad and minimum in Layyah

(1.461). Similar trend was found in other both cultivars with little changes; in Kinnow

(C.nobilis Loureiro×C. deliciosa Tenore) and Musambi. Maximum refractive index in

Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) peel essential oil was recorded in

Abbotabad (1.476) and minimum was recorded in the Kinnow (C.nobilis Loureiro×C.

deliciosa Tenore) peel oil from Rahim Yar Khan (1.463). Maximum refractive index in

Musambi peel essential oil was recorded in Abbotabad (1.472) and minimum was recorded in

the Musambi peel oil from Rahim Yar Khan (1.462). Maximum oil densities of 0.834 gm/cm3,

0834 gm/c m3 and 0.833 gm/c m3 from in Rahim Yar Khan district and minimum of 0.829

gm/cm3, 0.827 gm/cm3 and . 0.822 gm/cm3 were found from Abbotabad from the peels of

Grapefruit, Kinnow (C.nobilis Loureiro×C. deliciosa Tenore) and Musambi respectively.

Chemical composition of these essential oils was observed in the different pattern. The

cultivars from the climates with high rain fall and low average temperature resulted in more

number of chemical components in their essential oils. Limonene is the major part of the citrus

peel essential oils in all cultivars which showed negative correlation with elevation. Many of

the compounds such as ß-Citronellol, Elemol, α –Selinene, 4-terpinenol, 4,11-Selinadiene,

Carvyol acetate (z), Citronellol, Decanal, E-Nerolidol, Farnesol, Farnesyl acetate, Globulol,

Linalool, Menthol and Perillaldehyde were negatively correlated with average temperature..

Data collected from Pakistan Meterological Department showed that rainfall, relative humidity

and other climatic factors also correlate with the availability of chemical components of the

essential oils.

147

Conclusion

It is concluded that steam distillation method is the best method of essential oil extraction as it

is easier, economical and environment friendly. The temperature of 105oC was found most

suitable for extraction of more yield of essential oil from Steam distillation method. . The citrus

fruits of different climatic zones showed significant variation in their volatile compounds. The

peel of fruits from cooler climate (Abbotabad) showed more number of compounds while

maximum yield was in fruits of hot climate (Rahim Yar Khan). Moreover comparison of three

cultivars showed that the peel of Grapefruit had maximum essential oil components as

compared to other both cultivars.

Recommendations and Future Prospects

1. Juice industries in the country should install the essential oil unit at their premises. This

will not only help the industrialists to increase their income but also the farmers will fetch

more earnings.

2. Small essential oil units using steam distillation method can be installed at a low

investment cost. Even the juice venders in the cities may use this technique for essential

oil production. The Grapefruit peel has more essential oils therefore more attention should

be given to collect the peels of Grapefruit.

3. Essential oil extraction units should be installed at the places with cooler climate.

4. GC-MS should be used for the analysis of the essential oils for research purposes.

Future Prospects

The citrus peel essential oils can be used for the researches to compete the different disease

such as cancer, Alzheimer, asthma etc. Postharvest losses of the fruits can also be minimized

by the use of these natural essential oils with no harmful effect on human health. Biofuel is the

talk of the day and these essential oils may be a good initiative for the purpose. Backing

industries can enhance their products’ shelf life by using the citrus peel essential oils.

Researches for animal feed in addition with citrus peel essential oil can improve the animal

health.

Disclaimer:

These recommendations are purely based on the lab experiments conducted under controlled

conditions. The discrepancies may arise in the outcomes under commercial situations. The

project investigator Mr. Rizwan Mahmood and University of Agriculture, Faisalabad will

accept no liability whatsoever reason of negligence or otherwise arising from the reliance or

use of these recommendations.

148

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