analytical characerization of some indiginous oils...
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Ph.D Thesis
ANALYTICAL CHARACERIZATION OF SOME INDIGINOUS OILS FOR THEIR COMMERCIAL
EXPLOITATION
A THESIS SUBMITTED TOWARDS THE PARTIAL FULFILMENT OF THE
REQUIRMENT FOR THE EWARD OF DOCTOR OF PHILOSPHY IN ANALYTICAL CHEMISTRY
SARFRAZ ISMAIL ARAIN
National Centre of Excellence in analytical chemistry,
University of Sindh, Jamshoro-Pakistan
2012
i
DEDICATTED
To My Affectionate Supervisors, Beloved Parents, Brother, Sisters and Friends Who’s Prayers,
Encouragement and Cooperation Enabled Me for this Achievements
ii
CERTIFICATE
This is to certify that Ms. SARFRAZ ISMAIL D/O Muhammad Ismail Arain has
carried out this research work on the topic “ANALYTICAL CHARACERIZATION
OF SOME INDIGENOUS OILS FOR THEIR COMMERCIAL EXPLOITATION”
under our supervision at the laboratories of National Centre of Excellence in analytical
chemistry, University of Sindh, Jamshoro-Pakistan. The work reported in this thesis is
original and distinct. Her dissertation is worthy of presentation to the University of Sindh
for the award of degree of Doctor of Philosophy in Analytical Chemistry.
Dr. Muhammad Tahir Rajput Professor Co-Supervisor Institute of Plant Sciences, University of Sindh, Jamshoro, Pakistan
Dr. Syed Tufail Hussain Sherazi Associate Professor Supervisor National Centre of Excellence in analytical chemistry, University of Sindh, Jamshoro, Pakistan
Dr. Muhammad Iqbal Bhanger Professor Co-Supervisor National Centre of Excellence in analytical chemistry, University of Sindh, Jamshoro, Pakistan
iii
ACKNOWLEDGEMENTS
I praise to The Almighty Allah (The Most Merciful, Gracious and The Most
Compassionate), Who is the entire and only source of every knowledge, Who guides me
in the obscurity and help me in difficulties, and His Prophet Hazrat Mohammad
Mustafa (Salallah-o-Alaihe Wasallim) gave us the spirit to learn the hidden and
unconcealed facts of nature.
I am highly grateful to my supervisors Dr. Syed Tufail Hussain Sherazi and Prof. Dr.
Muhammad Iqbal Banger (Director, NCEAC University of Sindh Jamshoro) and Dr.
Muhammad Tahir Rajput (Dean Faculty of Natural Sciences) for their deep interest,
support and sympathetic behavior during my studies that enabled me to complete my
research work successfully. I would like to express my uncountable thanks to
gratefulness and kindness.
I wish to express my sincere gratitude and appreciation to the people who have both
directly and indirectly contributed to this thesis.
I am particularly indebted to my teachers, especially Dr. Sarfaraz Ahmed Mahesar, Dr.
Farah Naz Talpur, Prof. Dr. Tasneem Gul Kazi, Dr. Sirajuddin, Dr. Shahabuddin
Memon, Dr. Najma Memon, Dr. Amber Rehana Solangi, Dr. Hassan Imran afridi,
Dr. Amna Baloch, Dr. Rana Shaid Iqbal who always offered their professional skills
whenever needed. I am grateful for their encouraging attitude in the solution of problems
faced during research work.
I would also like to thank the administrative and supporting staff of NCEAC specially Pir
Ziauddin, Nasrullah Kalhoro, Akhtar Vighio, Muddasar Arain, Mairaj Noorani and
Uncle Imran. I have no words to acknowledge the unconditioned support. They always
encouraged and cooperated with me and made every possible effort to provide the
invaluable input for the improvement of this study.
At last but not the least, I really acknowledge and offer my heartiest gratitude to my
parents; my entire family deserves my appreciation for their love tremendous moral
support, sacrifice, cooperation, encouragement, patience and valuable prayers for my
health and success during this work.
Ms. Sarfraz Ismail Arain
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ABSTRACT
Oil and fats whether for human consumption or for industrial purposes are largely derived
from plant sources. To meet the increasing demand for edible oils and oilcakes,
improvements are being made with conventional crops, as well as with other new sources of
plant species, that have the ability to produce unique desirable oils. Therefore, several plants
are now grown not only for food and fodder but also for a striking variety of products,
including oils with nutritional and pharmaceutical attributes. This necessitates the search of
new sources of indigenous oils. In the present study new native resource of oil i.e. Bauhinia
seeds and apple seeds have been explored.
The study is divided into five parts. In first and second part the physiochemical
characteristics, fatty acid composition, lipid bioactive, unsaponifiable content of extracted
oil of three locally grown Bauhinia species (B. purpurea, B. variegata and B. linnaei) were
evaluated. Analysis of fatty acid composition of oil samples revealed 13 fatty acids with
chain length C14 to C24. The major fatty acids were Myristoleic acid (C14:1) and lignoceric
acid (C24:0), linoleic, oleic and palmitic acid. Tocopherols (α-tocopherol, γ+β-tocopherol
and δ-tocopherols) were identified and α-tocopherol is reported first time in this study. The
unsaponifiable lipid fraction of Bauhinia species ranged 1.8-3.2%, β-sitosterol, campesterol
and stigmasterol were the major sterols which accounted for 84-92%. The proximate
compositions of meal residue of all samples were also analyzed to determine the suitability
of these seeds meal in animal feed formulations. The results revealed that Bauhinia species
could be helpful in understanding the influence of cultivar / variety on the quality of oil. The
study revealed that the seed oils of the Bauhinia species grown in Pakistan were found
nutritionally important with higher amount of PUFA, tocopherols and sterols.
In the third part of study the oxidative stability assessment was done by Differential
scanning calorimetry (DSC) and oxidative stability index (OSI) method among three
Bauhinia species (B. purpurea, B. variegata and B. linnaei), rice bran and cotton seed oil. B.
purpurea oil showed highest oxidative stability. Excellent calibration was achieved between
v
DSC T0 and OSI measurements. The coefficients of correlation were highly significant (P <
0.01) for each evaluation. The coefficient of the determination (R2) for analyzed oils was
above 0.9956, showing good linear regression, which revealed that oxidative stability of the
oils can be accurately determined by DSC in a short time as compared to OSI method.
In fourth part of study Infraspecific variation in composition of Bauhinia purpurea Linn. (B.
purpurea L.) seed oil was assessed for regional discrimination. Samples were collected from
five cities of Pakistan (Hyderabad, Tandojam, Multan, Pakpattan and Abbotabad). Linoleic
acid, α-tocopherol, and β-sitosterol contents were used to find variability and significant
difference among five regions and was found to be p<0.0001. On the basis of fatty acid
composition, five regions could not be discriminated using PCA, LDA on fatty acids
discriminated the regions and cross-validation was found to be 99%. Using tocopherols only
one PCA component was extracted and LDA on tocopherols discriminated within the
regions and cross-validation was found to be 100% perfect. PCA and LDA plots for sterol
composition showed five distinct groups for both statistical protocols and all cases were
100% correctly classified. The results of present study indicated that tocopherols and sterols
are better chemotaxonomic marker as compared to fatty acids for regional discrimination of
B. purpurea L.
In fifth part of study the extracted oil from four apple seed varieties (Royal Gala, Red
Delicious, Pyrus Malus and Golden Delicious) from Pakistan, total forty two samples were
investigated for their physiochemical characteristics, fatty acids profile and lipid bioactive
by GC-MS. The oil content in the seeds of apple varieties ranged from 26.8-28.7%. The
results revealed that linoleic acid (40.5-49.6%) was the main fatty acid. The unsaponifiable
lipid fraction of apple seed oils ranged from 1.8-2.1%, squalene, β-tocopherol, α-tocopherol,
campesterol, avenasterol, β-sitosterol, 9,19-Cyclolanost-24-en-3-ol and Stigmast-4-en-3-one
were identified, which accounted for 98- 100%. The variation among the results of both
fatty acids and lipid bioactive for four varieties was assessed by principal component
analysis, discriminant analysis and cluster analyses. The results conclude that both oil
fractions could be applied as a useful tool to discriminate the apple seed varieties.
vi
List of Contents
Dedication…………………………………………………………………….... i
Certificate………………………………………………………………………. ii
Acknowledgement……………………………………………………………… iii
Abstract………………………………………………………………………… iv
List of Contents………………………………………………………………… vi
List of Tables……………………………………………………………………. ix
List of Figures………………………………………………………………….. x
Abbreviations…………………………………………………………………… xii
Chapter - One INTRODUCTION 1-9
1.1. Background of Kachnar plant……………………………………........... 1
1.1.1. Seeds of Bauhinia species……………………………………… 3
1.1.2. Oil of Bauhinia species…………………………………………. 5
1.1.3.. Uses of Bauhinia species oil……………………………………. 5
1.2. Background of Malus plant……………………………………………... 6
1.2.1. Apple seeds……………………………………………………… 8
1.2.2. Uses of apple seed oil …………………………………………… 9
Chapter -Two LITERATURE REVIEW 10-17
2.1. Bauhinia………………………………………………………………. 10
2.1.1. Bauhinia seed ………………………………………………… 10
2.1.2. Bauhinia seed oil………………………………………………... 11
2.2. Oxidative stability of oils……………………………………………… 12
2.3. Apple seed…………………………………………………………….. 14
2.3.1. Apple seed oil………………………………………………….. 14
2.4. Chemometrics for Chemotaxonomic Classification of Bauhinia purpurea……………………………………………………………….
15
2.5. Chemometrics for apples varieties ……………………………………. 17
vii
Chapter -Three EXPERIMENTAL 18-30
3.1. MATERIALS AND METHODS………………………… 18
3.1.1. Collection of Samples…………………… 18
3.1.1.1. Seed Sampling for Bauhinia (B. purpurea, B. variegata and B. linnaei)…………………..................
18
3.1.1.2. Sampling for rice bran and cottonseed oil………….. 18
3.1.1.3. Seed sampling of B. purpurea for Chemotexonomic Study………………………………………………….
19
3.1.1.4. Seed sampling for Apples…………………………… 19
3.2. Reagents and standards ………………………………………… 19
3.3. Oil extraction………………………………………………………….. 20
3.4. Analysis of extracted oil…………………………………………… … 20
3.4.1. Physical and chemical parameters of extracted oil………….... 20
3.4.1.1. Refractive index…………………………………….... 20
3.4.1.2. Determination of peroxide value……………………... 21
3.4.1.3. Determination of Saponification value……………….. 21
3.4.1.4. Determination of iodine value…………………….. … 21
3.4.1.5. Determination of acid value………………………….. 22
3.4.1.6. Determination color of oil……………………………. 22
3.4.1.7. Determination of dienes and trienes (specific extinction)…………………………………...
22
3.4.2.Preparation of fatty acid methyl esters (FAMEs) official Method 22
3.4.2.1. Determination of fatty acid by GC-FID……………… 23
3.4.3. Preparation of samples for Tocopherol analysis………………. 24
3.4.3.1. Determination of tocopherols by HPLC……………… 24
3.4.4. Preparation of samples for Sterol analysis……………………………. 25
3.4.4.1. Determination of sterols by GC-MS…………………. 25
3.5. Oxidative stability…………………………………………………….. 26
3.5.1. Oxidative stability index……………………………………… 26
3.5.2. Differential scanning calorimetry analysis……………………. 26
3.6. Analysis of Oil seed Residue…………………………………………. 27
3.6.1. Determination of moisture content……………………………. 27
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3.6.2. Determination of protein content……………………………... 27
3.6.3. Determination of crude fiber …………………………………. 28
3.6.4. Determination of unsaponifiable matter………………………. 28
3.6.5. Determination of ash content…………………………………. 29
3.6.6. Determination of carbohydrate content……………………….. 29
3.7. Statistical analysis………………………………………… 29
3.8.1. Statistical analysis for Oxidative stability of B. purpure, B.variegata and B. linnaei ………
29
3.7.1. Statistical analysis for Chemotexonomic classifiction of B. purpurea……………………………………...…
30
3.8.3. Statistical analysis used for the classification of Apple seed oil 30
Chapter –Four RESULTS AND DISCUSSION 31-85
4.1. Physiochemical characterization of Bauhinia purpurea Seed Oil and Meal for Nutritional Exploration………………………………………..
31
4.1.1. Physiochemical characteristics of Bauhinia purpurea seed oil…… 31
4.1.2. Fatty acid profile of Bauhinia purpurea seed oil…………………. 33
4.1.3. Tocopherol profile of Bauhinia purpurea seed oil……………….. 35
4.1.4. Sterol composition Bauhinia purpurea seed oil …………………. 37
4.1.5. Characterization of Bauhinia purpurea seed meal………………. 38
4.2. Physicochemical Characteristics of Oil and Seed Residues of Bauhinia species (B.variegata and B. linnaei)……………………………………
40
4.2.1. Physicochemical Characteristics of B. variegate and B. linnaei seed oil ……………………………………………………………
40
4.2.2. Fatty acid profile of B.variegata and B. linnaei seed oil…………. 42
4.2.3. Tocopherol profile of B. variegata and B. linnaei seed
oil………..
45
4.2.4. Sterols profile of B. variegata and B. linnaei seed oil……………. 46
4.2.5. Proximate composition of B. variegata and B. linnaei seed oil…………………………………………………………….
47
4.3. Oxidative stability assessment of Bauhinia purpurea seed oil in comparison to two conventional vegetable oils by differential scanning calorimetry and Rancimate methods…………………………………..
49
ix
4.3.1. Oxidative stability………………………………………………… 49
4.4. Infraspecific variation in composition of Bauhinia purpurea Seed oil: Optimization of Chemotexonomic Indicators …..........................................
53
4.4.1. Fatty acids profile of B. purpurea seed oil of different origin…… 53
4.4.2. Tocopherols profile of B. purpurea seed oil of different origin….. 54
4.4.3. Sterols profile of B. purpurea seed oil of different origin………... 55
4.4.4. Chemometric…………………………………………………… 57
4.4.5. Fatty acids as markers of discrimination………………………… 58
4.3.6. Tocopherols as markers of discrimination…………………………. 58
4.3.7. Sterols as markers of discrimination……………………………….. 60
4.5. Prospects of Fatty Acid Profile and Bioactive composition from lipid seeds for the discrimination of Apple Varieties with the Application of Chemometrics……………………………………………………………….
63
4.5.1. Fatty acid composition of seed oil of apple seed oil……………….. 63
4.5.2. Lipid bioactive composition of apple seed oil …………………….. 65
4.5.3. Chemometrics……………………………………………………… 73
4.5.3.1. Principal component analysis for fatty acids…………….. 73
4.5.3.2. Principal component analysis for lipid bioactive………… 76
4.5.3.3. Linear discriminant analysis for fatty acids and unsaponifiable matter…………………………………….
78
4.5.3.4. Cluster analysis…………………………………………... 80
4.5.4. Physiochemical characteristics of apple seed varieties……………. 83
4.4.5. Proximate composition of apple seed varieties……………………. 85
Conclusion…………………………………………………………………… 87
Recommendation…………………………………………………………….. 89
References………………………………………………………………………… 90-102
x
LIST OF TABLES 32-85 Table. 4.1.1. Physicochemical characteristics of Bauhinia purpurea seed oil…… 32
Table. 4.1.2. Fatty acid profile of Bauhinia purpurea seed oil…………………… 34
Table. 4. 1.3. Tocopherol profile of Bauhinia purpurea seed oil………………… 36
Table. 4.1.4. Sterol profile of Bauhinia purpurea seed oil……………………… 38
Table. 4.1.5. Proximate composition of Bauhinia purpurea seed meal………… 39
Table. 4.2.1. Physiochemical characteristics of B. vareigata and B. linnaei seed oil 49
Table. 4.2.2. Fatty acid profile of B. vareigata and B. linnaei seed oil………… 44
Table. 4.2.3. Tocopherol profile of B. variegata and B. linnaei seed oil………… 45
Table. 4.2.4. Sterol profile of Bauhinia variegate and Bauhinia linnaei seed oil… 47
Table. 4.2.5. Analysis of B. variegata and B. linnaei seed meal……………… 48
Table. 4.3.1. Differential scanning calorimetry (DSC) oxidative induction time (T0) and oxidative stability index (OSI) values of B. purpurea, B. variegata and B. linnaei, rice bran and cotton seed oils……………
50
Table. 4.3.2. Pearson correlation coefficient matrix between differential scanning calorimetry (DSC) and oxidative stability index (OS I) method……
50
Table. 4.3.3. Relationships between oxidative stability index (OSI) values and differential scanning calorimetry (DSC) oxidative induction time (T0) at four different isothermal temperatures ………………………
51
Table. 4.3.4. Relationship between logarithm of DSC T0 values (log10 T0) and DSC isothermal temperature (T) of B. purpurea, rice bran and cotton seed oil………………………………………………………………
52
Table. 4.4.1. Fatty acid profile of B. purpurea seed oil of different origin……… 54
Table Table. 4.4.2. Tocopherol profile of B. purpurea of different origin………… 55
Table. 4.4.3. Sterol profile of B. purpurea………………………………………… 56
Table. 4.4.4. Statistical data of palmitic, stearic, oleic, linoleic acids, α-tocopherol, β-sitosterol and stigmasterol of B. purpurea oil……………………
57
Table. 4.4.5. Linear discriminant analysis of fatty acids, tocopherols and sterols… 58
Table. 4.5.1. Fatty acid compositional data (%) of apple seed oils………………… 64
Table. 4.5.2. Unsaponifiable compositional data (%) of apple seed oils with
xi
statistical analysis………………………………………………… 70
Table. 4.5.3. Linear discriminant analysis of fatty acids and unsaponifiables: statistics and classification of results…………………………………
80
Table. 4.5.4. Physiochemical chemical characterization of Apple seed oils………. 82
Table 4.5.5. Proximate composition of Apple seed residue……………………… 85
xii
LIST OF FIGURES Figure. .4.1.3. HPLC separation of tocopherol standards mixture (A) and tocopherol
isomers present in B. purpurea seed oil (B)……...
36
Figure. .4.2.2. Representative GC-FID Chromatogram of Fatty acids methyl esters for Bauhinia variegata oil. ……………………………..
43
Figure. 4.3.1. A representative differential scanning calorimetry oxidation curve of B. purpurea oil: (A) isothermal curve at 130 ◦C with nitrogen (99.99%) flowing at 50 ml/min; (B) isothermal curve at 130 ◦C with oxygen (99.99%) flowing at 50 ml/min………...
49
Figure. 4.4.4.1. Linear discriminant function plot of fatty acids. Inset abbreviations:TJ (Tandojam), PP(Pakpattan), AA (Abbotabad), HYD (Hyderabad), M(Multan)…………………
59
Figure. 4.4.4.2. Linear discriminant function plot of tocopherols. Inset abbreviations: TJ (Tandojam), PP(Pakpattan), AA (Abbotabad), Hyd (Hyderabad) M(Multan)…………………..
60 Figure. 4.4.4.3. Principal component plot of sterols. Inset abbreviations: HYD
(Hyderabad), M (Multan), TJ (Tandojam), AA (Abotabad), PP(Pakpattan) and StS (Stigmasterol), CS (Compesetrol), SS (Sitosterol), AS (Avenasterol), StS3 (stigmasterol3), AV2(Avenasterol)…………………………………………
61
Figure. 4.4.4.4. Linear Discriminant plot of Bauhnia purpurea seed oil using fatty acids, tocopherols and sterols as chemical composition descriptors. Inset abbreviations: TJ (Tandojam), PP(Pakpattan), AA (Abotabad), Hyd (Hyderabad) M(Multan).
62
Figure. 4.5.2. Representative GC-MS chromatogram of the unsaponifiable lipid fraction of apple seed……………………………………
65
Figure. 4.5.3. Mass-spectrum of unsaponifiables compounds present in Apple seed oil………………………………………………….
66
Figure. 4.5.3.1a. PC1 verses PC2 of four varieties of apples based on fatty acid composition of RDA (Royal Gala apple), RDA (Red Delicious apple), PMA (Pyrus Malus apple), GDA (Golden Delecious apple)………………………………………………………….
74
Figure. 4.5.3.1b. PC3 verses PC4 of four varieties of apples based on fatty acid composition of RDA (Royal gala apple), RDA (Red delicious apple), PMA (Pyrus malus apple), GDA (Golden delecious apple)………………………………………………………….
75
xiii
Figure. 4.5.3.2a. PC1 verses PC2 of four varieties of apples based on unsaponifiable
composition of RDA (Royal gala apple), RDA (Red delicious apple), PMA (Pyrus malus apple), GDA (Golden delicious apple) and Eole (Ethyl oleate), Pht (Phytol), Squ (Squaline), sit ( β-Sitosterol), aToc (α-Tocopherol), bToc (β-Tocopherol), AV(Avenasterol), Stig (Stigmast-4-en-3-one), Cy (9,19-Cyclolanost-24-en-3-one)……………………………
76
Figure.. 4.5.3.2b. PC1 verses PC2 of four varieties of apples based on unsaponifiable composition of RDA (Royal gala apple), RDA (Red delicious apple), PMA (Pyrus malus apple), GDA (Golden delecious apple) and Eole (Ethyl oleate), Pht (Phytol), Squ (Squaline), sit ( β-Sitosterol), aToc (α-Tocopherol), bToc (β-Tocopherol), AV(Avenasterol), Stig (Stigmast-4-en-3-one), Cy (9,19-Cyclolanost-24-en-3-ol)……………………………..
77
Figure. 4.5.3.3. Discriminant function plots for four varieties of apples; (a) based on fatty acid composition, (b) based on unsaponifiable composition of GDA (Golden Delicious apple), RDA (Red Delicious apple), PMA (Pyrus Malus apple),), RGA (Royal Gala apple)……………………………………………………
79 Figure. 4.5.3.4. Dedrogram for four apple varieties using unsaponifiable and fatty
acid composition of RGA (Royal gala apple), RDA (Red delicious apple), GDA (Golden delicious apple), PMA (Pyrus malus apple)……………………………………………………
81
xiv
List of Abbreviations AOACS Association of Official Analytical Chemists a-Toc α-Tocopherol AA Abbotabad As Avenasterol b-Toc β-Tocopherol CS Compesterol CA Cluster analysis Cy 9,19-Cyclolanost-24-en-3-ol DF Degree of freedom DSC Differential scanning calorimetry Eole Ethyl oleate FFA Free Fatty acid FA Fatty acid FAMEs Fatty Acid Methyl Esters GDA Golden delicious apple GC-FID Gas chromatograph-flame ionization detector GC-MS Gas Chromatography-Mass Spectrometry HPLC High performance liquid chromatography hexa hexadecenal HYD Hyderabad IV Iodine value LDA Linear discriminant analysis M Multan MS Mean square meq of O2 /kg Milliequivalent of oxygen per kilogram of oil mg/kg Milligram per Kilogram μg/kg Microgram per kilogram μL Miro liter OSI Oxidative stability index Pht Phytol PMA Pyrus malus apple PC Principal component PCA Principal component analysis RGA Royal gala apple RDA Red delicious apple Squ Squaline Stig Stigmast-4-en-3-one SS sum of squares Sts Stigmasterol TJ Tandojam T0 Oxidative induction time Σ SFA Total saturated fatty acids Σ MUFA Total monounsaturated fatty acids Σ PUFA Total polyunsaturated fatty acids
1
Chapter - 01
INTRODUCTION
1.1. Background of Kachnar plant
Bauhinia consisting of more than 300 species belongs to the family Caesalpiniaceae
(Larson 1974; Wunderlin et al., 1987; Chopra, et al., 1996). In Pakistan Linn species of
Bauhinia are reported (Ali, 1973). Some members of Febaceae family are well
recognized for their valuable oilseeds such as soybeans, peanuts, ground nuts and some
trees nuts. Bauhinia is also known as orchid tree, purple camel’s foot, mountain ebony,
butterfly tree and Kachnar in Pakistan and India.
The Bauhinia genus is commonly found in south China (Hong Kong), southeastern Asia,
India, Pakistan, Hawaii, southern Texas, southwest Florida, coastal California, and
Australia. Linneaus in 1753, named this genus in honour of Casper and John Bauhin,
German botanist of sixteen century (Shiju Mathew, 2010). Bauhinia trees originated from
Hong Kong Island Botanic Gardens and then widely planted in several places (Lau, et al.,
2005).
Flowering periods of each species of Bauhinia may differ slightly. Bauhinia purpurea,
blooms from September to January while Bauhinia variegata and Bauhinia linnaei
blooms from autumn to spring (Little, et al., 1974). The seed ripens in late spring or early
summer (Little & Wadsworth 1964; 1974).
2
For the favorable growth of Bauhinia plant, reported parameters are altitude
approximately 1800 m; annual temperature 0-47ºC; annual rainfall 500-2500 mm; and
wide range of shallow, gravelly, loamy soil in the valleys, rocky hill slopes to sandy loam
(Bahuguna & Dhawan, 1990). Excellent germination of Bauhinia species without
scarification reported that the seeds contain less than 12% of moisture (Roberts, 1973),
99% of Bauhinia seeds germination recorded on moist blotting paper, and germination
start within four days (Francis & Rodríguez 1993).
Shoots, leaves, flower buds, and pods of Bauhinia species are consumed as vegetables in
native countries (Baily, 1941; Ramasatri and Shenolikar, 1974). Bauhinia species are rich
in polyphenolics (phytochemical constituents) have known for its medicinal uses (Patil,
2003), and all part of this plant are being used in traditional medicine for curing various
diseases, headache, fever, skin diseases, stomach tumor, blood diseases, dysentery,
bronchitis, leprosy, and diarrhea (Kirtika, & Basu, 1991, Parrota, 2001; Patil, 2003). B.
purpurea leaf extract possesses good anti-inflammatory, antipyretic and antinociceptive
properties (Zakaria et al., 2007). Antimalarial activity, regulation of thyroid hormone,
antilipidemic, antiobesity efficacy of B. purpurea bark extract and anthelmintic activity
of whole plant of B. purpurea have been reported in the literature (Vishal et al., 2009;
Panda 1999; Wahab et al., 1987; Kumar & Chandrasheker, 2011) while, B. variegata and
B. linnaei has chemoprevention and cytotoxic effect, sub chronic toxicity and antitumer
activity (Rajkapoor et al., 2006; 2004; 2003).
3
Several phytochemical constituents such as flavonoids, coumarins, steroids, stilbenes,
triterpene, flavnoids, phytol fatty esters, isoquercitin, lutein, and sterols have been
isolated from Bauhinia (Prakash & Kosha 1976; Gupta et al., 1979; Yadava et al., 2001;
Pettit et al., 2006; Verma et al., 2009; Reddy et al., 2003; Kumar & Chandrashekar,
2011).
1.1.1. Seeds of Bauhinia species
The seeds of B. purpurea, B. variegata and B. linnaei are enclosed in an elongated,
dehiscent, brown and flat seedpod varying in length 25-30cm, and width 1.5-2.5cm,
weighing approximately 7-8g, usually contain 8-10 seeds (Rajaram & Janardhanan
1991b; Vijayakumari et al., 1997a ). The seeds are shiny brown, flat, glabrous, rounded,
orbicular, 1-2mm thick and 13-16mm in diameter (Kirtika & Basu, 1991). The color of
the seeds turns to dark brown on ripening and storage.
The seeds of B. purpurea are considered to be rich source of essential dietary nutrients
such as oil content (12.4%), protein (27.2%), fiber (5.9%), carbohydrates (51.6%)
respectively and seed energy is 178449kJ kg-1 (Vijayakumari et al., 1997). The fiber of
seed contain hemicellulose (104g/kg), cellulose (78g/kg), lignin (8.6g/kg) and mineral
contents are potassium (8611.2 ppm), phosphorus (3647.0 ppm), calcium (1782.3 ppm),
magnesium (1094.8 ppm), sodium (186.4 ppm), iron (73.1ppm) in higher concentrations,
and manganese (7.7ppm), copper (7.4 ppm), zinc (21.0 ppm) are in lower concentration
as compared with other under-utilized legumes (Vijayakumari et al., 1997).
4
B. purpurea seeds contain essential amino acids in (g/16g) are aspartic acids 9.64g,
glutamic acid 14.47g, alanin 5.21g, valine 4.80g, glycine 4.58g, arginine 4.80g, serine
5.61g, cystine o.47g, methionin 1.43g, threonine 4.05g, phenylalanine 5.13g, tyrosine
2.57g, isoleucine 5.30g, leucine 6.81g, histadine 3.36g, lysine 5.58g and tryptophan 0.78g
of total proteins and the B. purpurea seeds protein fractions (albumins and globulins) are
the richest source of Aspartic acid (11.7-8.5), glutamic acid (10.8-17.6), serine (7.1-6.7)
and leucine (7.5-9.1). B. purpurea seeds also contain anti nutritional components such as
total free phenolics 12.5g/kg, tannin 18.6g/kg, hydrogen cyanide 65.2 mg/kg, tripsin
inhibitor activity 21.7 TIU/mg (TIU indicate trypsin inhibitor unit), erythrocytes for
human groups (A=21, B=42 O=5), and in vitro high protein digestibility 59.5% The
digestibility and protein utilization of processed seed was reported superior as compared
to raw seeds (Vijayakumari et al., 1997).
Chemical and biochemical characterization of B. variegata L. seeds, contains moisture
3.71%, ash 4.41%, lipids 16.41%, starch 19.08 %, reducing sugars 4.46%, protein
29.29%, carbohydrates 13.38% and fiber content 9.26% respectively. Mineral content in
B. variegata are phosphorus (2.5g/kg), potassium (11.5g/kg), calcium (2.9g/kg),
magnesium (5.1g/kg), copper (1.4g/kg), zinc (0.8 g/kg), cobolt and iron (1.3g/kg).
Protein fraction of B. variegata contains prolamin (13.5mg/g), glutelin acid (13.8mg/g),
albulin (13.1mg/g), glutalin basic (16.9mg/g) and globulin (146.1mg/g) have been
reported by Luciano et al., (2005).
5
1.1.2. Oil of Bauhinia species
The Bauhinia seed oil was considered a good source of lipid-soluble bioactives and
essential fatty acids. The high amount linoleic acids, sterols and tocopherols could be of
nutritional importance in the application of the seed oil. Bauhinia seeds may be
nutritionally considered as a new non-conventional supply for edible, cosmetics and
pharmaceutical purposes.
1.1.3. Uses of Bauhinia species oil
The fatty acid profile of Bauhinia (B. purpurea, B. variegata and B.linnae) seed oils
reveal that these could be used as edible oil. Furthermore, interest in Polly unsaturated
fatty acid (PUFA) as health-promoting nutrients has expanded dramatically in recent
years. The PUFAs are beneficial for human health in alleviating heart disease,
inflammatory conditions, diabetes, autoimmune disorders, and atherosclerosis
(Riemersma, 2001; Finley & Shahidi, 2001). Linoleic acid prevents high blood pressure,
also linoleic derivatives serve as precursors of some metabolic regulatory compounds and
structural components of the plasma membrane (Matos et al., 2009). PUFA with high
amount of linoleic acid makes the Bauhinia seed oil more valuable and suitable for
nutritional applications.
More than 100 plant sterols have been identified as these are valuable natural products,
members of the triterpen and represent major portion of un-saponifiable lipid fraction.
Biological activity of many phytosterols has been reported in literature especially as
preventives of many types of cancers (Canabate- Díaz et al., 2007; Awad et al., 2000).
6
The most common naturally occurring phytosterols are sitosterol, campesterol and
stigmasterol (Belitz and Grosch, 1999). They are of nutritional interest because of their
potential to lower both total low density lipoprotein and serum cholesterol in humans as
well as inhibiting the absorption of dietary cholesterol (Schwartz et al., 2008). Bauhinia
seed oil is also a significant source of phytosterols, i.e. β-sitosterols, campesterol and
stigmasterol and hence the oil could be used in functional foods and in dietary
supplements that protect from cancer diseases and help in lowering the blood cholesterol
levels.
Tocopherols are nutritionally important compounds due to their antioxidant and
biological activity (Ramadan & morsel 2006; Burton & Traber, 1990; Burton, 1994). It
has been reported that supplementation of antioxidants reduces the risk of degenerative
processes (Ramadan & morsel 2006; Kallio et al., 2002). The tocopherols protect cellular
components (proteins, DNA and lipids) from free radicals and reactive oxygen species
caused by UV radiation (Radak et al., 2011; Jari et al., 2006). Owing to the presence of
tocopherols, vegetable oils protect the PUFAs from peroxidation (Kamal-Eldin &
Andersson, 1996). Thus Bauhinia seed oil due to the appreciable level of tocopherols can
be used as a suitable ingredient in cosmetic formulations that help in preventing photo-
oxidation and also used to enhance the shelf life of other edible oils on blending with
reasonable ratio.
1.2. Background of Malus plant
Apple is usually considered to be the health complimentary table fruit of the world. It is
one of the most widely cultivated fruit. Botanically apple is called Malus pumila Mill and
7
it belongs to the family Rosaceae and sub family Maloideae. Apple is the most important
commercial fruit crop of North America and Europe. Apple trees; found throughout
temperate zones of the northern hemisphere (Rohrer et al., 1994).
Apples are grown in northwestern hilly tracts of Indo Pak sub-continent. In Pakistan,
Pishin Quetta, Mustang, Ziarat, Kalat, Chitral, Kashmir, Hunza, Swat, and other localities
over 1000m above the sea level are apple-growing regions (Tareen et al., 2003). In
Pakistan the total area under apple cultivation is 110.8 thousand hectares which includes
0.1 Sindh, 0.4 Punjab, 101.5 thousand hectare of Balochistan and 8.8 NWFP, while total
production of apple varieties in Pakistan is 333.8 thousand tons which includes 223.8
Balochistan, 106.3 NWFP, 3.6 Punjab and 0.1 Sindh (Agricultural Statistics of Pakistan,
2004; Iftikhar et al., 2009). Apple seeds are small and brown in color, found in the core
of every apple, about one inch long, 1/4-inch wide and 1/8-inch thick. The strong outside
seed coat, protects the embryo inside. Seeds constitute approximately 2-3% of the total
weight of apple pomace (Carson et al., 1994).
Apple is an extremely nutritive food containing sugar, protein, carbohydrates and
vitamins in a balanced form. Apple fruit is used in many products preparations like,
snacks, jellies, salads, marmalades, and jams. In many dishes, sweet meats, puddings,
pickles and other preserves including sauces, pie filling and slices. Sour varieties are used
for the preparation of fermented apple juice as cider (Hulme, 1970). The direct utilization
of apples associated with the prevention of various chronic diseases and apple juice
8
inhibits human low density lipoprotein oxidation (Hamauzu et al., 2005; Boyer and Liu,
2004; Pearson et al., 1999).
In fresh apple fruits, total phenolic content ranges from 110 to 357 mg/100 g (Eberhardt
et al., 2000; Podsedek et al., 2000; Liu et al., 2001; Sun et al., 2002). Data published
about the consumption of fruit phenolics show that in the United States, about 22% of the
phenolic compounds are obtained from apples (Boyer, & Liu, 2004; Sun et al., 2002;
Vinson et al., 2001). Apples contain high contents of flavonoids (Sun et al., 2002; Vinson
et al., 2001) and consumption of apple has been related with the reduction of lung cancer
incidence (Knekt et al., 1997; Le Marchand et al., 2000), cardiovascular disease (Knekt et
al., 1996), symptoms of chronic obstructive pulmonary disease (Tabak et al., 2001), and
the risk of thrombotic stroke (Knekt et al., 2000). It has been proved that phytochemical
extracts of apple exhibit potent antioxidant activity (Eberhardt et al., 2000; Sun et al.,
2002), antiproliferative activity against human cancer cells (Sun et al., 2002; Liu et al.,
2001) and prevent mammary tumors in rats (Liu et al., 2005). Apple peels had shown
higher antioxidant activity and antiproliferative activity than apple flesh (Eberhardt et al.,
2000; Wolfe et al., 2003; He & Liu, 2007).
1.2.1. Apple seeds
Apple seeds are non-endospermic embryo with fleshy cotyledons and a common
byproduct of apple processing industries. Apple seeds are good source of edible oil and
seedcake because of high protein content and significant amounts of potassium,
phosphorus, calcium, iron, and magnesium potentially serving as an animal feed
9
supplement (Yu, at al., 2007). Polar fraction of 70% aqueous acetone extract of apple
seeds contains two major compounds such as phloridzin and amygdalin. Minor
components are p-coumarylquinic acid, chlorogenic acid, phloretin-20-xyloglucoside, 3-
hydroxyphloridzin, and six quercetin glycosides such as galactoside arabinoside,
glucoside, rhamnoside, xyloside and rutinoside (Yinrong et al., 1999).
Aapple seeds are reported as a good source of phenolic antioxidants with amygdalin and
phloridzin, which are dominant polyphenols make up to 75% of the total polyphenols
(Yinrong et al., 1999). Due to the presence of amygdalin, apple seeds have tendency to
control the cancerous growths (http://www.livestrong.com/article/176867-medicinal-
uses-of-apple-seed/#ixzz3p3MNze5d)
1.2.2. Uses of apple seed oil
Apple seed oil is the byproduct of apple seeds having pale yellow color, odor similar to
almond oil with pleasant taste. Apple seed oil is a significant source of polyunsaturated
fatty acids therefore like soybean and sunflower it could be used in skin care applications
(Jari, et al., 2006). The fatty acid profile of apple seed oil makes it a strong candidate for
edible, pharmaceutical and cosmetic applications such as skin creams and lotions,
shampoo, massage oil, lipsticks and fancy soaps (Tian, et al., 2010; Yu, et al., 2007).
10
Chapter-02
LITERATURE REVIEW
2.1. Bauhinia
Bauhinia species like many other plants are natural source of vegetable protein, lipid,
carbohydrate and minerals. Almost every part of bauhinia has some applications. The
leaves, stems, wood and bark of Bauhinia species contain large amount of flavonoid
compounds.
2.1.1. Bauhinia seed
The seeds of B. purpurea contain significant amount of oil ranged from 15.0-17.5%
(Ramasastri & Shenolikar 1974; Sherwani, et al., 1982; Balogun and Fetuga 1985;
Ramadan et al., 2006; Sharanabasappa et al., 2007; Zaka et al., 1983), whereas protein,
fiber, carbohydrate, moisture, and energy are 25.6-27.2%, 4.6-5.8%, 51.6%, 7.3%, 2.9%
and 17844.9 KJ kg -1 DM, respectively (Rajaram & Janardhanan 1991; Vijayakumari et
al., 1997).
Data about nutritional and biological values including fatty acid composition and
phenolics profile of B. purpurea seeds is also reported in the literature (Bharatiya &
Gupta, 1981; Bharatiya, et al., 1979 and Badami & Daulatabad 1969). New galactoside
11
binding lectin isolated from B. variegata and B. purpurea and B. linnaei seeds have been
reported by Jose et al., (2007).
2.1.2. Bauhinia seed oil
The acid value (0.8%), iodine value (82.2 g of I2/100g of oil) and saponification value
(192.2 mg of KOH/g of oil) of B. purpurea seed oil have been reported by
Sharanabasappa et al., (2007) and the Physiochemical characteristics of B. variegata have
been reported by Zak et al., (1983).The major fatty acids in B. purpurea and B. variegata
seed oils are linoleic (C18:2), palmitic (C16:0), stearic (C18:0), and oleic acid (C18:1).
Palmitoleic (C16:1), α-linolenic (n-3and n-6), arachidic (20:0), eicosapentaenoic (C20:5),
behenic (C22:0) and lignoceric acid (C24:0) in B. purpurea and B. variegata are in lower
concentrations (Vijayakumari et al., 1997; Zaka et al., 1983; Badami et al., 1969;
Ramadan et al., 2006).
Neutral lipids, glycolipids and phospholipids were also determined by Ramadan et al.,
(2006). Linoleic (46.8%), palmitic (21.1%), stearic (15.4%) and oleic (16.4%) acids
were included in neutral lipid, linoleic (37.7%), palmitic (27.3%), stearic (15.4%) and
oleic (16.6%) acids were present in glycolipid, while linoleic (38.0%), palmitic (27.8%),
stearic (15.0%) and oleic (16.7%) acids were found in phospholipids of B. purpurea and
B. variegata seed oils. Minute amount of myristoleic, palmitoleic, margaric, α-linolenic
(n-3and n-6), arachidic, eicosapentaenoic, behenic and lignoceric acid (< 0.5%) were also
found in some samples of B. purpurea seed oil. The variations in the fatty acid profiles
12
were observed in B. purpurea and B. variegata seed oil produced in different regions of
India and Pakistan (Ramadan et al., 2006; Zaka, et al., 1983).
The determination of tocopherols by Normal-phase high performance liquid
chromatography (NP-HPLC) from B. purpurea seeds oil was reported by Ramadan et al.,
(2006). α -tocopherol (2.67g/kg) was the major tocopherol and δ-tocopherol (0.99g/kg)
was found to be in lower concentration similar to sunflower (Schwartz et al., 2008).The
level of sterols in B. purpurea seeds oil was quoted only in one study (Ramadan et al.,
2006). From the total unsaponifiable matter (11.9g/kg), 49% were phytosterols. β-
sitosterol (3.83g/kg) was the major sterol followed by stigmasterol (1.22g/kg),
campesterol (0.36g/kg), Δ7-stigmastenol (0.32g/kg), Δ7-avenasterol (0.01g/kg) and Δ5-
avenasterol (0.02g/kg). The sterols composition was found to be comparable with
soybean seed oil (Youk-meng et al 1999; Schwartz et al., 2008).
2.2. Oxidative stability of oil
The protection of oil quality, which remains suitable to consumers for longer time, is an
important objective of quality control in the oil and fat industry. Shelf life of vegetable
oils is the main characteristic that influences its suitability and market value (Smouse,
1995).The consequence of lipid oxidation leads to decrease in shelf life and has been
recognized as the big problem in the food industry (Jadhav et al., 1996).
Oxidative stability is one of the most important indications for maintaining the quality of
the vegetable oils (Tan et al., 2002). The resistance to oxidation is recognized as
13
oxidative stability under different conditions and is expressed as the period of time
necessary to accomplish an end point which can be selected according to diverse criteria,
but usually leads to rapid raise in the rate of lipid oxidation is a measure of oxidative
stability and is known as induction time (Cosgrove et al., 1987; Coppin & Pike., 2001). A
number of methods have been developed for the assessment of oxidative stability. There
are various accelerated stability tests to speedily confirm the stability of oils and fats as
oxidation is the major reason of oil degradation (White., 1991; Paul & Mittal., 1997).
Usually, the active oxygen (AOM) and Schaal oven test have been the most commonly
used tests to evaluate the stability of oil (Wan, 1997). It can also be determined by
oxidative stability index (OSI) method as recommended by AOAC (AOCS, 1997), which
is widely used in the fat and oil industry by using two commercially available
instruments, the Oxidative Stability Instrument from Omniom Inc. (Rockland, MA) and
Rancimat from Metrohm Ltd. (Herisau, Switzerland) (Akoh, 1994).
Recently, differential scanning calorimetry (DSC) has been used to determine the
oxidative stability (Cross, 1970) was the first investigator who used DSC, under
isothermal conditions with flow of oxygen. The induction period was taken as the time
where a fast exothermic reaction between oxygen and oil get started. Several researchers
have used the application of thermal analysis for accelerated oil stability test (Tan & Che
Man, 1999; Cebula & Smith, 1992).
14
Hassel’s results showed that oil samples, which required 14 days via AOM, were
appraised in less than 4 h by DSC (Hassel, 1976). Kowalski with his coworkers and other
researchers have also determined the oxidative stability of vegetable oils by DSC
(Kowalski et al 1997; Kowalski, 1989; Gloria & Aguilera, 1998; Raemy et al., 1987).
2.3. Apple seed
Apple seeds are a common by products of apple processing, Apple seeds contain oil from
17-29.5%, protein from 38.85-49.55 %, fiber from 3.92-4.32 %) and ash content from
4.31-5.20 % (Yu, et al., 2007; Tian et al., 2010; Marjan et al., 2007).
2.3.1. Apple seed oil
Fatty acid profile of apple seed oils includes linoleic (18:2,n-6), oleic (18:1,n-9), and
palmitic (16:0) as major fatty acids. While, stearic (C18:0), palmitic (C16:0), palmitoleic
(C16:1), linolenic (C18:3), arachidic (20:0), eicosenoic (20:1) eicosadienoic (20:2),
behenic (C22:0), lignoceric acid (C24:0) were present in small quantities (Yukui, et al.,
2009; Tian, et al., 2010; Marjan et al., 2007). Interest in apple seed oil is mainly due to
the presence of significant level of polyunsaturated fatty acid content (50-63%) and low
amount of saturated fatty acids (6-10%). The level of linoleic (49-62%), oleic (30-44%),
palmitic (6.5-8.1%), stearic (1.6-2.3%) and linolenic acid (0.4-0.7%) have been also
reported in the literature (Yu, et al., 2007; Yukui, et al., 2009; Tian, et al., 2010; Marjan
et al., 2007). The variations in the level of linoleic and oleic acid have been also
observed in apple seed oil produced in different geographic locations (Yu, et al., 2007;
15
Yukui, et al., 2009; Tian, et al., 2010; Marjan et al., 2007). Constitution of chemical
components of apple seed has been repoted by Lu et al (1998).
The apple seed oils Refractive index (1.465-1.466), density (0.902-0.903 mg/ml), iodine
value (94.14-101.15 g I2/100 g of oil), acid value (4.036-4.323 mg KOH/g of oil), and
the saponification value (179.01-197.25 mg KOH/g of oil) have been reported by Tian et
al., (2010).
2.4. Chemometrics for chemotaxonomic classification of B. purpurea
Literature reveals that there are multiple approaches in terms of chemical parameters and
statistical protocols to characterize the species/cultivars. Baraldi et al., (2007), have used
moisture, protein, lipid, glucide and ash components as chemical parameters to
characterize the species of Aesculus hippocastanum, while Arena et al (2007) have used
fatty acids and phytosterols as criteria to discriminate geographic origin of pistachio
seeds.
Vegetable oils are also subjected to multivariate study using tocopherols and fatty acids
as chemical descriptors (Kamal-Eldin et al., 1997; Giacomelli et al., 2006).Three varietal
olive oils are characterized chemometrically using fatty acids, tocopherols and
phytosterols (Matos et al., 2007).
Principal component analysis (PCA) is most commonly used chemometric procedure
applied to multiple data of samples to be investigated for variability. PCA allows the
16
number of variables to be reduced while maintaining most of the information by
simultaneously studying all of the variable relationships. It has been used in food science
and technology to classify foodstuffs according to their chemical composition, to group
samples with similar features, and to discriminate among different vegetable oils
(Giacomelli, et al., 2006). Other statistical procedures like linear discriminant function is
also reported to ascertain the quality of data obtained using PCA by assessing correct
classification of data (Arena et al., 2007).
Careful evaluation of the literature available for chemometric characterization of oils
using various chemical descriptors obtained by using a range of analytical techniques
suggests that chemical descriptors must be optimized for type of information/ variability
required. Matos et al., (2007), have used global PCA, which includes all the chemical
parameters studied and have used cluster analysis to further classify the varieties. Arena
et al., (2006) have plotted PCA and discriminant function of fatty acids and sterols
individually and their results showed that using fatty acids, 100% cases can be classified
correctly while using sterols the correctly classified cases are 95.83%.
Giacomelli et al., (2006) concluded that data obtained with tocopherols and CIELAB
provided better information. Data obtained by using LC-MS for acylglycerols,
tocopherols and sterols after subjecting to chemometric assessment provided 99%
prediction rate and 100% classification for olive oils obtained from Nocellara, Cerausola
and Biancolilla cultivars (Nagy et al., 2005).
17
2.5. Chemometrics for apple varieties
Two ancient, late-bearing apple varieties (cv. 'Diacciata' and 'Limoncella') were
characterized using micromorphological, genetic and biochemical approaches, and by
comparison with two commercial varieties, 'Gala' and 'Golden Delicious' (Chen et al.,
2011). Between apple juice discrimination produced from different varieties (Bramley,
Russet and Spartan) were evaluated by applying principal components analysis (PCA)
and linear discriminant analysis (LDA) to 1H NMR spectra of the juices (Belton et al.,
1998). Potential of visible/near-infrared (Vis/NIR) spectroscopy for its ability to
nondestructively differentiate apple varieties was explored by Yong et al., (2007).
The apple varieties used in their research included Fuji apples, Red Delicious apples, and
Copefrut Royal Gala apples. The chemometrics procedures applied to the Vis/NIR data
were principal component analysis (PCA), wavelet transform (WT), and artificial neural
network (ANN) and two ancient, late-bearing apple varieties (cv. 'Diacciata' and
'Limoncella') were characterized using micromorphological, genetic and biochemical
approaches, and by comparison with two commercial varieties, 'Gala' and 'Golden
Delicious'. There were significant differences between the two varieties (Minnocci et al.,
2010).
In the present study, four apple seed varieties were evaluated for lipid bioactives such as
fatty acids, sterols, tocopherols, hydrocarbons and other minor compounds by GC-MS
with the combination of principal component analysis (PCA), linear discriminant
analysis (LDA) and Hierarchical clustering analysis (HCA).
18
Chapter - 03
Experimental
3.1. MATERIALS AND METHODS 3.1.1. Collection of Samples Seed samples examined in the individual studies were harvested from different locations
and details of sampling have been discussed below
3.1.1.1. Seed Sampling for Bauhinia (B. purpurea, B. variegata and B. linnaei)
Approximately 2 kg seeds were collected from the each species (B. purpure, B. variegata
and B. linnaei) for this study. Bauhinia plants were grown in the campus of Sindh
University, Pakistan, and seeds harvested from five different locations. The plants/seeds
were identified by Professor Dr. Tahir Rajput, Head of Botany Department and the
voucher specimen deposited at the Herbarium Department of Botany, University of
Sindh, Jamshoro, Pakistan.
3.1.1.2. Sampling for rice bran and cottonseed oil
The rice bran and cottonseed oil samples were obtained from local oil industry,
Hyderabad, Sindh, Pakistan.
19
3.1.1.3. Seed sampling of B. purpurea for Chemotexonomic study
Bauhinia seed samples (~2kg) were collected from the B. purpurea plants grown in five
different region of Pakistan i.e. Hyderabad (25022'45''N68022'06''E), Tandojam
(25025'40.21''N68031'40.40''E), Multan (30012'00''N71027'00''E), Pakpattan
(30°21′0″N73°24′0″E) and Abbotabad (340 09'00''N13'00''E) during mid-February 2010.
3.1.1.4. Seed sampling for Apples
Four different varieties of apples (Royal Gala, Red Delicious, Pyrus Malus and Golden
Delicious) were selected for the study. Two samples of each variety were collected from
10 different locations of Pakistan; Quetta, Pishin, Ziarat, Mustang, Kalat, Kashmir,
Chitral, Swat, Hunza and Gilgit. Apples of selected varieties were picked from the trees
located in ten different locations from the beginning of August to the end of the
December, 2009. All collected samples were analyzed in duplicate. Apples were crushed
for the seed segregation and the seeds of each sample were stored in cellophane bag at 4
ºC prior to analysis. For the extraction of oil approximately 5g of seed from each variety
was used
3.2. Reagents and standards
Pure standards of fatty acids methyl esters were obtained from Sigma Chemical Co (St.
Llouis, MO, USA) Reagents and chemicals used were of the highest purity (HPLC grade)
purchased from Merck (Darmstadt, Germany). Sterol standards were purchased from
Fluka Chemie GmbH, Sigma-Aldrich (CH-9471, Buchs, Switzerland). Pure standard of
fatty acids methyl esters and tocopherols (dl-α- tocopherol, (+)-δ-tocopherol, γ-
20
tocopherol) were obtained from Sigma Chemical Co (St. louis, MO, USA). KOH, n-
hexane, ethyl alcohol, sodium chloride, methanol, anhydrous sodium sulphate, sodium
hydroxide, sodium thiosulphate, sulphuric acid, starch, iodine monochloride, glacial
acetic acid, potassium iodide, chloroform, carbon tetra chloride, acetonitrile and
methanol.
3.3. Oil extraction
Oil extraction of various seeds samples approximately (50 g) were ground and oil
extracted with n-hexane at 68–72 °C in a Soxhlet apparatus for quantitative determination
according to the method ISO 659 (1998). On water bath the extraction was carried out up
to 6 h with 0.5 L of n-hexane. After extraction the solvent was distilled under vacuum in
a rotary evaporator, the recovered oil was dried in oven for 1h at 75 °C. The oil was then
transferred to a desiccator and allowed to cool. The obtained oil was weighed to
determine the extraction yields. Solvent-free residual meal and extracted oils were stored
under nitrogen atmosphere at 5 °C for further analysis.
3.4. Analysis of extracted oil
3.4.1. Physical and chemical parameters of extracted oil
3.4.1.1. Refractive index
AOAC standard (1997) method no. 969.18 was used to measure the refractive index of
oil at 40 °C.
21
3.4.1.2. Determination of peroxide value
Peroxide value defined as the milliequivlents of active oxygen per kilogram of oil (meq
of O2 kg-1) expressed in the unit of milliequivalents, was determined, when potassium
iodide react with a mixture of oil and chloroform/acetic acid in dark according to AOCS
(1997) method Cd 8-5.
(g) sample of :Wt
1000ate thiosulphsodium of Nation)SampleTitr-ation(BlankTitrvaluePeroxide
)kg/O(meq 2
3.4.1.3. Determination of Saponification value
Number of KOH required to saponify 1 gram of oil is known as the Saponification value.
It is the hydrolysis of ester under alkaline condition and determined by the following
AOCS (1997) method Cd 3-25.
(g) sample of :Wt
56.01KOH of N titrationSample -ration(Blank tittion valueSaponifica
sample) of KOH/g of (mg
3.4.1.4. Determination of iodine value
According to AOAC (1997), the iodine value of oil was determined by Wijs method Cd
3d-63. In which dissolve oil sample (CCl4 used as solvent) was mixed with 25ml of Wij’s
(0.1mol/L) solution and reacted with freshly prepared (10%) potassium iodide solution.
The standard potassium thiosulphate (0.1 M) was used for titration with liberated iodine
from solution. Starch was used as an indicator in this procedure.
(g) sample of :Wt
69.12tethiosulphasodium of NTitration Sample-Titration(Blank value Iodinesample) of 100g/ I of (g 2
22
3.4.1.5. Determination of acid value
Acid value used to measure the free acids (total amount) found in a given quantity of fat.
Number of milligrams of KOH (potassium hydroxide) utilized to neutralizing the free
acids found in per gram of the oil sample determined by AOCS (1997) method Cd 3d-63.
(g) sample of :Wt
56.1 N Alkali of ml valueAcid
sample) of KOH/g of (mg
3.4.1.6. Determination color of oil
Lovibond Tintometer (Tintometer Ltd., Salisbury, U.K.), with a 1” in. cell was used to
measure the intensity of the color of oil.
3.4.1.7. Determination of diens and trienes (specific extinction)
Samples were diluted with n-hexane to measure the absorbance within limits (0.2–0.8)
and calculated by the following IUPAC method (1979).
3.4.2. Preparation of fatty acid methyl esters (FAMEs) official Method
IUPAC standard method (1979) was used for the preparation of FAMEs, in which oil or
fat (50 mg) was weighed into 100ml ground-necked round bottom flask, 20ml of
methanol was then added and content of the flask were refluxed for 30 minute until the
droplets of the oil disappeared. On cooling methanolic fraction was gently transferred to a
separating funnel, and was extracted with 10 ml of n-hexane. Separating funnel was
shaken gently by rotating several times and upper layer (n-hexane) was removed, wash
thrice with distilled water (10ml). This n-hexane solution was dried over anhydrous
23
sodium sulphate, filtered and used for GC analysis. The dry and solvent free methyl
esters were preserved under nitrogen atmosphere in a sealed sample tube in deep freezer
and used for further analysis.
3.4.2.1. Determination of fatty acid by GC-FID
Gas liquid chromatography was used for the determination of Fatty acid composition
after derivatization to methyl esters according to IUPAC (1979) standard method
Analysis of FAMEs were carried out using Perkin Elmer gas chromatograph model 8700,
equipped with flame ionization detector and a methyl lignocerate coated polar capillary
column SP-2340 (60m х 0.25 mm) 0.2 µm film thickness from Supelco (Bellefonte, PA,
USA). Oxygen-free nitrogen was used as a carrier gas at a constant pressure 33.5 psi.
Other conditions were as follows: initial oven temperature, 130 °C; ramp rate, 4°C/min;
final temperature, 220 °C; injector temperature, 260 °C; detector temperature, 270 °C. A
sample volume of 1.0 μl was injected with split ratio of 1:40.
FAMEs were identified by comparing their relative and absolute retention times to those
of authentic standards of FAMEs obtained from Sigma Chemical Co. All of the
quantification was done by a built-in data-handling program provided by the
manufacturer of the gas chromatograph (Perkin Elmer). The FA composition was
reported as a relative percentage of the total peak area.
24
3.4.3. Preparation of samples for Tocopherol analysis
Determination of tocopherols of different vegetable oils was carried using (Gliszczynska-
Swiglo & Sikorska, 2004). Tocopherols (α, g and δ) analysis was carried out using
reverse phase HPLC method. Stock and working standard solutions of tocopherol were
prepared in 2-propanol and injected 20 µl into the column. Peak areas versus
concentration were plotted to generate standard calibration curve. Similarly 0.12 g of B.
purpurea, B.variegata and B.linnaei oil were weighed and dissolved in 1 ml of 2-
propanol.
3.4.3.1. Determination of tocopherols by HPLC
A 20 µl portion was injected onto Hitachi model 6200 HPLC unit equipped with
Licrosorb Octadecylsilane (ODS) column, a mobile phase consisting of 50% acetonitrile
and 50% methanol was used with a flow rate of 1ml/min. The eluent were detected using
a Hitachi F-1050 scanning florescence detector set at emission wavelength of 325 nm
with an excitation at 295 nm. Tocopherols were identified by comparing their relative
retention times with those of corresponding standards and were quantified on the basis of
peak areas of the unknowns with those of pure standards (Sigma Chemica Co., St Louis,
Mo, USA). All quantitation was carried out by using CSW32 chromatographic integrator.
All the experiments were repeated at least thrice when the variation on any one was
routinely less than 5%.
25
3.4.4. Preparation of samples for Sterol analysis
Extraction and separation of total sterols was performed after saponification of the oil
sample without derivatization according to the method (Ramadan & Morsel, 2003).Oils
or fats (250 mg) was weighed into 100ml of round bottom flask and refluxed for 60 min
with 5ml of ethanolic (6% w/v) potassium hydroxide solution with few anti-bumping
granules.The unsaponifiable fraction was extracted three times with 10 ml of petroleum
ether, the extracts were combined and washed three times with 10 ml of neutral
ethanol/water (1:1 v/v), and then dried overnight with anhydrous sodium sulphate. The
extract was evaporated in a rotary evaporator at 25 °C under reduce pressure, and then
ether was completely evaporated under nitrogen atmosphere and reconstituted with
hexane for injection into GC/MS.
3.4.3.1. Determination of sterols by GC-MS
The GC-MS analysis of sterol was performed on Agilent 6890 N gas chromatography
instrument coupled with an Agilent MS-5975 inert XL mass selective detector and an
Agilent autosampler 7683-B injector (Agilent Technologies, Little Fall, NY, USA). A
capillary column HP-5MS (5% phenyl methylsiloxane) with dimension of 30m x 0.25mm
i.d x 0.25 micron film thickness (Agilent Technologies, Palo Alto, CA, USA) was used
for the separation of sterols. Sample was injected at injector temperature 280 °C. The
initial temperature was 150 °C and ramped to 250 °C at 15 °C/ min and maintained for 2
min, raised to 310 °C at the rate of 15 °C /min, and kept at 310 °C for 10 min. The split
ratio was 1:50, helium was used as a carrier gas with a flow rate of 1.2 ml/min. The mass
spectrometer was operated in the electron impact (EI) mode at 70 eV; ion source
26
temperature 230 °C; quadrupole temperature 150 °C; translating line temperature 270 °C;
the mass scan ranged from 50-550 m/z; Em voltage 1035 V.
The identification of sterols was based on the comparison of their relative retention times
with those of authentic standards. The sterols were also identified and authenticated using
their MS spectra compared to those from the NIST mass spectral library. The
quantification was done by Chemstation data handling software Agilent-Technologies
3.5. Oxidative stability
3.5.1. Oxidative stability index
Oxidative stability index was evaluated by OSI instrument (automated Metrohm
Rancimat model 679) following AOCS Official Method (Cd 12b-92 AOCS 1997). The
instrument was run at 110 °C and an air flow rate of 20 l/h was bubbled through the oil
(2.5 g). The volatile degradation products were trapped in distilled water, increasing the
water conductivity. The oxidative stability index was the time necessary to reach the
conductivity curve inflection point.
3.5.2 Differential scanning calorimetry analysis
The oxidative stability of conventional oils was determined by a Mettler Toledo
differential scanning calorimeter DSC-820 (Schwerzenbach, Switzerland). The
instrument was calibrated with pure indium and the baseline was obtained with an empty
open aluminum crucible. The weighed amount of samples (5.0±0.25 mg) were taken into
open aluminum DSC crucible and placed in the sample compartment of the instrument.
27
The four different isothermal temperatures were used (110, 120, 130, and 140°C) and
purified oxygen (99.99%) was passed through the sample at 50 ml/min.
3.6. Analysis of Oil seed Residue
3.6.1. Determination of moisture content
Moisture content of seed meal was determined by the method AOCS (1993). Five grams
of test portion was taken in dish container and dry it in an oven at 130°C for 2h. Heated
portion cool in desiccator at room temperature and loss of weight was determined by the
following equation.
sample wetof :Wt
100 sampledry of wt -sample wet of Wt (wt/wt) Moisture %
3.6.2. Determination of protein content
Kjeldahl digestion method (distillation and acid digestion) was used to determine total
protein from seed residues as the nitrogen content of the sample multiplied by nitrogen
factor. For the protein calculation nitrogen conversion factor was 6.25 used according to
the official standard method (AOCS 1993). Following formula was used for the
determination of Percentage of protein in seed meal individually.
% Protein= (VAcid-VBlank) X 1.4007 X N X 6.25/g sample
%N=[(N Acid)(ml Acid)-(ml blank)(N NaOH)-(ml NaOH)(N NaOH)][1400.67] /mg
sample
%Protein = [6.25 X %N]
28
3.6.3. Determination of crude fiber
According to the AOAC official standard method (1993), fiber content was determined
using 2.5g seed meal defatted and extracted with n-hexane (15 ml). The meal residue for
digestion boiled with sulfuric acid solution (0.3mol/L), followed by washing and
separation of insoluble residue, after digestion the residue + sodium hydroxide
(0.3mol/L), was boiled followed by washing and separation, with distilled water, and
drying. The residue was dried, ashed at 600 °C in a muffle furnace and loss in mass was
calculated by the following formula.
sample of :Wt
100 ignition on in wt Loss fiber crude of %
3.6.4. Determination of unsaponifiable matter
According to the AOAC official standard method (1993).Weigh accurately 5g of the
sample into a 200ml Erlenmeyer flask. Add 20ml of alcohol (95%) and 50ml of 50%
KOH solution. Boil gently under a reflux condenser for 1 hour. Add 100ml of distilled
water, after cooling transfer the solution in separating funnel and extract the saponifiable
solution with n-hexane/diethyl ether three times. Wash the extracted solution with water
three times and wish with 40ml of aqueous KOH solution then washed with 40ml of
distilled water. Evaporate the solvent by distillation on water bath. Add 5ml of acetone
and remove the volatile solvent completely and dry the residue in the oven at 103 ◦C for
15 minute and cool residue in desiccator and weigh accurately. Loss of mass in
unsaponifiables was calculated by the following formula.
(g)sample of :Wt
100acids)fatty of Weight -residue of(Weight matter ableUnsaponifi%
29
3.6.5. Determination of ash content
Powdered seeds samples about 0.5 g was ignited and incinerates at 550 oC for about 12 h
in muffle furnace, determined according to AOCS (1993) standard method. Ash content
was determined by the following formula.
(g)sample of :Wt
100 ash of Wt Ash %
3.6.6. Determination of carbohydrate content
By the difference of mean values, Carbohydrate was estimated, i.e.
Carbohydrate content = 100 - [%Lipids + %Proteins + %Ash + %Moisture].
3.7. Statistical analysis
Statistical analysis of individual studies have been discussed below
3.8.1. Statistical analysis for Oxidative stability of B. purpure, B. variegata and B. linnaei
Statistical data were analyzed by using SAS 8.2 software (SAS institute, Cary,NC,USA).
Duncan multiple range test to compare differences among means and SAS REG
procedure was used between DSC T0 and OSI values. The relationship between DSC T0
and DSC isothermal temperaturewas also determined by the SAS REG procedure which
is used for a simple linear equation. All the experiments were carried in triplicate and
reported as mean ± standard deviation.
30
3.7.2. Statistical analysis for Chemotexonomic classifiction of B. purpure
For One Way ANOVA, Minitab Scan, Release 1, (1995) was used (Minitab Scan,
Release 1, 1995). Multivariate analysis of experimental data including principal
component analysis (PCA) and linear discriminant analysis (LDA) were performed for
the classification of B. purpurea, by using Statgraphic Plus software (Manugistic Inc.
Rockville, MD, USA).
3.8.3. Statistical analysis used for the classification of Apple seed oil
In the present work a simple chemometric criteria was used on the bases of fatty acid
composition and lipid bioactives present in the apple seed oil to distinguish the varieties
of apple. An analytical study was carried out for each variable individually used to test
the differences between varieties, with One Way ANOVA (Minitab Scan, Release 1,
1995). Multivariate test of significance was also applied for the determination of Wilk’s
lambda (measure of group differences among twenty nine variables).
The principal components analysis (PCA) was performed to identify design for the
interaction between variables, categorization and division of each variety. Variables used
in PCA were selected on the basis of ANOVA results. Eigenvalues were also examined
for each analysis; three factors were enough to explain all the variability. Cluster analysis
was also applied in order to explore the grouping of samples according to the similarities
occur in discriminant parameters using the specific software (Statgraphics Plus software,
1998). Principal component analysis (PCA), hierarchical cluster analysis (HCA), and
linear discriminant analysis were examined with using the Statgraphic Plus software
(Manugistic Inc Rockville MD USA).
31
Chapter-04
RESULTS AND DISCUSSION
Part 1
4.1. Physiochemical characterization of Bauhinia purpurea Seed Oil and Meal for Nutritional Exploration
Work of this part has been published and cited as: Arain et al., Polish. J. Food Nutr. Sci. (2010), Vol. 60, pp. 343-348.
4.1.1. Physiochemical characteristics of Bauhinia purpurea seed oil
The seeds contain a higher percentage of total lipids (18.16%) compared to the reported
value (12.45%). This disagreement in oil yield may be due to the differences in natural
soil texture and environmental effects (Leilah & Al-Khateeb, 2003). However, the
average oil content of B. purpurea seeds is more or less equivalent to the two
conventional oil seed crops: cottonseed, and soybean (Pritchard & Rossell, 1991).
The main physicochemical characteristic of B. purpurea seeds oil was presented in Table
4.1.1. Refractive index is a characteristic parameter which may indicate the purity of
particular oil. The determined value of refractive index of B. purpurea seed oil was found
with a mean value of 1.4645 at 40°C. The colour of the extracted crude oils was golden
yellow with 2.52 red and 50.5 yellow by the lovibond tintometer in 5.25 inch cell, which
is in the normal range for the good quality of crude oil. The intensity of the color of
vegetable oils depends mainly upon the presence of various pigments like carotenoids
32
and chlorophyll, which are effectively removed during the degumming, chemical refining
and bleaching process.
Table 4.1.1. Physicochemical characteristics of Bauhinia purpurea seed oil.
Content Characteristics
Oil (%) 18.13 ± 0.12
Iodine value(g of I2 /100 g of oil) 99.19 ± 0.79
Saponification value(mg of KOH/g oil) 189.02 ± 1.39
Acidity (as oleic acid g /100 g) 0.16 ± 0.02
Unsaponifiable matter (g /100 g) 1.81 ± 0.34
Peroxide value (meq /kg of oil) 0.5 ± 0.05
Refractive index (40°C) 1.4645 ± 0.00
Color (red unit) 2.52 ± 0.07
Color (yellow unit) 50.5 ± 0.16
Conjugated dienes (λ232) 0.8 ± 0.2
Conjugated triens (λ270) 0.03 ± 0.1
* values are means ± standard deviation of triplicate determinations
The iodine value (99.19 g of I2/100 g of oil) and saponification value (189.02 mg of
KOH/g of oil) suggested that the B. purpurea oils could be fine for soap making and in
the manufacturing of lather shaving creams. As a result it could be explored for cooking
and may find other uses as well as raw material in industries for the preparation of
vegetable oil-based ice-creams. The mean acid value (AV) was found to reach 0.16 g/100
g of oil. The acidity of the oil was significantly lower, and to some extent the nutritional
value depends on oil’s acidity. The AV of the non processed crude B. purpurea oil was
within the range reported for edible oil (Rossell, 1991). A very low acidity of B. purpurea
oil indicates its good quality and stability (Norman, 1979). Unsaponifiable matter was
determined with a mean value of 1.81 g/100 g oil, which is comparable with that of olive
33
oil (Ojeh, 1981), and not so significantly different from those of corn, soybean, and
sunflower and safflower oil (Van Niekerk & Burger, 1985). The peroxide value, which
measures hydroperoxides present in the oil, was found to reach 0.5 (meq/kg of oil). The
oils with high peroxide values are not so stable and easily become rancid with an
undesirable odour. The specific extinction at 232 nm 0.8 and 270 nm was 0.07 which
shows deterioration and purity of the oil (Anwar et al., 2006).
4.1.2. Fatty acid profile of B. purpurea seed oil
According to the results are shown in Table 4.1.2. Fourteen fatty acids were identified;
the analysis of FAMEs showed that B. purpurea seed oil contained a significant amount
of saturated fatty acids (30.27%).
Among individual saturated fatty acids, palmitic acid was found to predominate with a
mean value of 17.47%, followed by stearic acid 11.40%. The other saturated fatty acid
i.e. arachidic, behenic, and lignoceric were detected at levels lesser than 1%.i.e. arachidic,
behenic, and lignoceric were detected at levels The oil was found to contain a high level
of unsaturated fatty acids up to 69.73%. Along with the content of monounsaturated fatty
acid (MUFA), oleic acid was the major contributor 11.84%, the other MUFAs were
determined at the level lesser than 1%. In the case of polyunsaturated fatty acids (PUFA),
linolic acid (n – 6)
34
Table 4. 1.2. Fatty acid profile of Bauhinia purpurea seed oil.
Fatty acid Content* (%)
Myristoleic acid (C14:1) 0.18 ± 0.02
Palmitic acid (C16:0) 17.47 ± 0.98
Palmitoleic acid (C16:1) 0.16 ± 0.01
Stearic acid (C18:0) 11.4 ± 0.64
Oleic acid (C18:1) 11.84 ± 0.97
Linoleic acid (C18:2) 55.34 ± 0.72
Alpha linolenic (C18:3) 0.47 ± 0.02
Gama linolenic (C18:3) 0.36 ± 0.02
Arachidic acid (C20:0) 0.92 ± 0.01
Eicosadienoic acid (C20:2) 0.36 ± 0.01 Eicosapentaenoic (C20:5) 0.38 ± 0.02
Lignoceric acid (C24:0) 0.14 ± 0.02
Nervonic acid (C24:1) 0.51 ± 0.04
Σ SFA 30.27
Σ MUFA 12.79
Σ PUFA 56.94
*values are means ± satandard deviation of triplicate determination
was the predominant fatty acid, i.e. 55.34% of the total fatty acids. The concentration of
linoleic acid was relatively high, while that of other fatty acids like C16:0, C18:0, C18:1
of the oil investigated in the present study was lower than the reported values (Ramadan
et al., 2006). The stearic, oleic and linoleic acid contents of B. purpurea constituting
about (78.58%) of the total fatty acids were corresponding to those of cottonseed, corn,
and soybean oil (Pritchard & Rossell, 1991). The arachidic acid (C20:0), behanic acid
(C22:0), lignoceric acid (C24:0), nervonic acid (C24:1), eicosadienoic acid (C20:2), γ-
linolenic acid (C18:3 n-6), α-linolenic acid (C18:3 n-3) and eicosapentaenoic acid
(C20:5), were also determined in minor quantities (<1%).
35
A good combination of SFA 30.27%, MUFA 12.79%, and PUFA 56.94% with a
significant level of essential fatty acids was found in B. purpurea seed oil and thus it
could be explored as special oil for nutritional applications and fuctional foods. For over
two last decades, several physiological and clinical investigations have focused on the
metabolism of polyunsaturated fatty acids (PUFAs). Their outcomes confirm the
beneficial effects of these acids on both normal health and chronic diseases,
4.1.3. Tocopherol profile of B. purpurea seed oil
The nutritionally important components, such as tocopherols (vitamin E) are the major
fat-soluble membrane-localized antioxidant in humans and also contribute the stability of
the oil (Kallio et al., 2002). α-tocopherol has the highest vitamin E activity; it prevents
cardiovascular disease, cancer, infection, inflammation, and decreases the risk of
degenerative diseases (Brigelius-Flohe & Traber, 1999). Results of HPLC separation of
tocopherol standards mixture (A) and tocopherols present in B. purpurea seed oil (B) are
shown in Figure 4.1.3.
36
(A) (B)
Fig. 4.1.3. HPLC separation of tocopherol standards mixture (A) and tocopherol isomers present in B. purpurea seed oil (B) with peak identity:1 – α-tocopherol; 2 – (β+γ)-tocopherol; and 3 – δ tocopherol.
Levels of different tocopherols present in the B. purpurea seed oil are summarized in
Table 4.1.3. Three isomers of tocopherols were identified in B. purpurea seed oil, i.e. α-
tocopherol,
Table 4. 1. 3. Tocopherol profile of Bauhinia purpurea seed oil
Tocopherols Content* (mg/100 g)
α-tocopherol
89.60 ± 6.48
(β+γ)-tocopherol
64.49 ± 3.98
δ-tocopherol
1.73 ± 0.13
*values are means ± standard deviation of triplicate determination
1
2
3
2
3
1
37
Β+γ-tocopherol, and δ-tocopherol constituting 58%, 41%, and 1% of the total
tocopherols, respectively. The level of predominant tocopherols (α-tocopherol and γ+β-
tocopherol) for investigated B. purpurea oil indigenous to Pakistan was greatly different
as compared to the values reported for β-tocopherol 72.2% and δ-tocopherol 27.8% of the
total tocopherol contents (Ramadan & Morsel, 2006). In the present study, levels of α-
and γ-tocopherol were significantly higher than those reported for soybean, groundnut,
cottonseed, and sunflower (Rossell, 1991).
4.1.4. Sterol profile of B. purpurea seed oil The levels of phytosterols in vegetable oils have been used for the identification of oils,
oil derivatives and also for the determination of oil quality (De-Blas & Del-Valle, 1996;
Nyam et al., 2009). The composition of sterols in B. purpurea oil was determined by the
GC-MS (Table 4.1.4). The total sterol fraction of the oil mainly consisted of six sterols,
with β-sitosterol (662.4 mg/100 g of oil) and stigmasterol (178.3 mg/100 g of oil)
predominating. These two major components constituted 84% of the total sterols.
The total sterol fraction of the oil mainly consisted of six sterols, with β-sitosterol (662.4
mg/100 g of oil) and stigmasterol (178.3 mg/100 g of oil) predominating. These two
major components constituted 84% of the total sterols. Among other determined sterols
were compesterol and Δ5-avenasterol (12% of the total sterols).
38
Table 4.1.4. Sterol profile of Bauhinia purpurea seed oil.
Sterols Content* (mg/100 g)
Compesterol 77.0±5.5
Stigmasterol 178.3±7.4
β-sitosterol 662.4±11.3
Δ5-avenasterol 40.5±4.7
Δ7-avenasterol 16.2±2.4
Δ7-stigmasterol 25.3±5.8
*values are means ±standard deviation of triplicate determination
The Δ7-stigmasterol and Δ7-avnasterol were at a lower levels 4%, while brassicasterol,
lanosterol, sitostenol and 24-stigmastadinol were not detected in B. purpurea sterol
fraction. The contents of major sterols, β-sitosterol and stigmasterol, of the investigated
oil were comparable whereas the level of compesterol and Δ5-avenasterol Δ7-stigmasterol
and Δ7-avenasterol varied to some extent with the reported values (Ramadan & Morsel,
2006).The sterol composition of the major fraction of B. purpurea oil was greatly
different from most of the conventional edible oils (Rossell, 1991).
Many beneficial effects have been shown for the sitosterol as described by Yang et al.
(2001). Plant sterols due to their antioxidant activity and impact on health have been
added to edible oils as a successful functional food (Nyam et al., 2009; Ramadan &
Morsel, 2006).
4.1. 5. Characterization of B. purpurea seed meal.
The proximate analysis of B. purpurea seed residue (Table 4.1.5) after oil extraction
(meal) revealed high protein content (43.72 g/100 g). In terms of percentage,
39
carbohydrates (33.93 g/100 g) were found the major contributor after protein. While, the
mean value of moisture, fiber, and ash contents were 5.69, 8.04 and 5.78 g/100 g,
respectively.
Table 4.2. 5. Proximate composition of Bauhinia purpurea seed meal.
Constituent Content* (%)
Moisture 5.69 ± 7.9
Protein 43.72 ± 10.1
Fiber 8.04 ± 4.3
Ash 5.78 ± 7.2
Carbohydrates 33.93 ± 15.2
*values are means ± standard deviation of triplicate determination.
The results determined for protein, fiber and ash contents have clearly shown that B.
purpurea seed meal could serve as a good source of protein, for the manufacturing of
poultry and animal feeds.
40
Part 11
4.2. Physicochemical Characteristics of Oil and Seed Residues of Bauhinia species (B.variegata and B. linnaei).
4.2.1. Physicochemical characteristics of Bauhinia seed oil
Table 4.2.1 shows the results of physicochemical characteristics of extracted oils (B.
variegata and B. linnaei). The refractive indices (40 ˚C) of the oils B. variegata and B.
linnaei found in the present study 1.4589-1.4588 were comparable with those of olive oil
(Rudan-Tasic et al., 1999). No earlier reported literature values for refrective indices of
apple seed oil are available to compare the results of present study.
Table 4.2.1. Physiochemical characteristics of B. vareigata and B. linnaei oil
Constituents B. variegata B. linnae
Refractive index (40 ºC) 1.4589 ± 0.0 1.4588 ± 0.0
Iodine value (g of I2/100g of oil) 84.5 ± 1.6 92.2 ± 1.2
Free fatty acids (%) 0.6 ± 0.1 0.9 ± 0.6
Saponification values (mg of KOH /g of oil ) 191.3 ± 1.9 195.5 ± 2.1
Peroxide value (meq O2 / kg of oil) 1.9 ± 0.6 2.4 ± 0.9
Unsaponifiable matter (%) 0.9 ± 0.4 1.2 ± 0.1
Color (1”cell) (red unit) 2.2 ± 0.5 2.9 ± 0.4
Yellow unit 30.0 ± 1.1 25.0 ± 1.8
Conjugated dienes (λ232) 1.2 ± 0.1 2.2 ± 0.3
Conjugated triens (λ270)
0.2 ± 0.0 0.5 ± 0.1
All values are means ± SD, analyzed individually in triplicate.
Peroxide value and free fatty acids are the measure of oil quality. The levels of FFA 0.6-
0.9%, and peroxide value 1.9-2.4 (meq O2/kg of oil) were found to be comparable than
41
those commonly suggested level for commercial vegetable oils (Norman, 1997). The
specific extinction at 232 nm 1.2 and 270 nm, which revealed the oxidative deterioration
and purity of the oil (Anwar et al., 2006) of Bauhinia seed oils, were 1.2-2.2 and 0.2-0.5,
respectively. No reported data for specific extinctions of apple seed oil are available to
compare the results of present study.
The results regarding to the lower concentration of peroxide value and free fatty acids
content indicate that B. variegata and B. linnaei seed oils could be used for edible
purposes. The iodine values of these two species were in the range of 84.5-92.2 (g of
I2/100g of oil), lower iodine value confers, to B. variegata oil, more stability and
comparable with the iodine value of olive oil (Eskin et al., 1996). Iodine value correlated
with the degree of unsaturation present in the oil of both varieties.
The saponification value were found in the range of 191.3-195.5 (mg of KOH/g of oil),
were close in agreement with those of olive oil and canola oil (Eskin et al., 1996),
indicating the presence of very high proportion of low molecular weight triacylglycerols
in B. variegata and B. linnaei oils, were comparable to the saponification value of canola
and olive oil (Eskin et al., 1996).
The unsaponifiable matters of both varieties ranged 0.9-1.2% were in close agreement
with those of corn, olive, sunflower and soybean (Norman, 1979).The color of extracted
crude oil of both varieties representing red unit ranged 2.2-2.9 and in yellow unit 30.0-
42
25.0 respectively. The red and yellow units of investigated oil were found be comparable
with those of suggested for good quality commercial vegetable oils (Norman, 1979).
4.2.2. Fatty acid composition of B. variegata and B. linnaei seed oil
Fatty acid composition of Bauhinia varieties (B. varigata and B. linnaei) are shown in
Table 4.2.2.The representative GC-FID chromatogram Bauhinia variegata seed oil was
presented in (Fig 4.2.2.). Thirteen fatty acids were identified in Bauhinia varieties; in
which the linoleic acid was the predominant fatty acids 42.1% for B. varigata and 45.8%
for B. linnaei seed
The dietary fat (lipid), rich in linoleic acids are beneficial in alleviating the cardiovascular
disorders, arteriosclerosis, high blood pressure and coronary heart diseases (Vles et al.,
1989).The linoleic acids derivatives are the precursors of some metabolic regulatory
compounds and also serve as constituent of the plasma membrane (Vles et al., 1989).
The content of total saturated fatty acids present in both varieties including palmitic
(C16:0), stearic (C18:0), arachidic (C20:0), behenic (C22:0) and nervonic (C24:1) acids
in the oil were 41.7-37.9% for B. variegata and B. linnaei respectively
43
Figure 4.2.2. Representative GC-FID Chromatogram of Fatty acids methyl esters for Bauhinia variegate oil. Note: Elution order of fatty acids with respect to retention time of fatty acids: C16:0, C16:1, C17:0, C18:0, C18:1 cis 9, C18:1 cis 7, C18:2, C20:0, C18:3 n-3, C18:3 n-6, C22:0, C20:5, C24:1. Retention time. 11.05, 11.84, 12.23, 14.07, 14.82, 14.90, 16.25, 16.95, 17.20, 17.62, 19.86, 21.92, 22.72
44
Table 4.2.2. Fatty acid profile of B. vareigata and B. linnaei seed oil
All values are means ± SD, analyzed individually in triplicate. ∑SAFA, total saturated fatty acids; ∑MUFA, total monounsaturated fatty acids; ∑PUFA, total polyunsaturated fatty acids
in which the palmitic acid 22.1-16.8% was the dominant fatty acid. The total
monounsaturated fatty acids (C18:1n-9) were found ranging from 15.1-14.7%, whereas
palmitoleic C16:1, eicosapentaenoic C20:5 and nervonic C24:1 acids were identified in
both varieties with lower concentration (<1) and arichidic acid (1.3-1.2%) The linolenic
acid (C18:3 n-3, n-6) were also found in lower concentration (<1) in both varieties.
The results of fatty acid composition of B. variegata were found to be quite comparable
with the results of previous study (Zaka et al., 1983).The major fatty acids were Linoleic,
oleic, stearic and palmitic acids in seeds oil of Bauhinia in which B. linnaei contributing
to 45.8%, 12.6%, 18.8% and 17.3% of the total fatty acids and showed relatively high
percentage 47.4% of polyunsaturated fatty acids as compared to the B. variegata about
Fatty acids B.variegata B. linnaeiPalmitic C16:0 22.1 ± 1.5 16.8 ± 0.9 Palmitoleic C16:1 0.4 ± 0.1 0.5 ± 0.03 Margaric C17:0 0.3 ± 0.04 0.5 ± 0.02 Stearic C18:0 17.5 ± 1.7 18.8 ± 1.2 Oleic C18:1 cis 9 13.4 ± 0.8 12.6 ± 1.3 Oleic C18:1 cis 7 0.5 ± 0.1 0.7 ± 0.2 Linoleic C18:2 42.1± 1.8 45.8 ± 1.4 Linolenic C18:3 n-3 0.6 ± 0.4 0.9 ± 0.3 Linolenic C18:3 n-6 0.5 ± 0.1 0.7 ± 0.2 Archidic C20:0 1.3 ± 0.6 1.2 ± 0.4 Behenic C22:0 0.5 ± 0.2 0.6 ± 0.3 Eicosapentaenoic C20:5 EPA 0.2 ± 0.4 0.4 ± 0.5 Nervonic C24:1 0.6 ± 0.6 0.5 ± 0.7 ∑SAFA 41.7 37.9 ∑MUFA 15.1 14.7 ∑PUFA 43.2 47.4
45
43.2% respectively. The fatty acid composition of Bauhinia seed oils (B.variegate and B.
linnaei) shows that the oil is a good source of the nutritionally essential fatty acids. Both
oils varieties were found to be contained high level of polyunsaturated fatty acids.
Interest in health-promoting nutrients such as polyunsaturated fatty acids has expanded
dramatically in recent years, and a rapidly growing literature illustrates their benefits
(Riemersma, 2001). Results revealed that this special fatty acid composition makes the
Bauhinia (B.variegata and B. linnaei) seeds oil a unique constituent for nutritional
application.
4.2.3. Tocopherol profile of B. variegata and B. linnaei seed oil
Tocopherols are the important quality criterion to elucidate the identity of vegetable oils.
Tocopherols possess an antioxidant activity, which protects polyunsaturated fatty acids of
oil against oxidation (Kamal-Eldin & Andersson 1997; Szymanska and Kruk, 2008).
Furthermore, biological activity protects cells against oxidative stress (Bertrand and
Mehmet, 2006). Epidemiologic studies recommend that deficiency of vitamin E in
humans may causes to increased risk for certain types of cancer and for atherosclerosis
(Rimm et al. 1993).
Table 4.2.3. Tocopherol profile of B. variegata and B. linnaei seed oil
Tocopherols (mg/kg) B. variegata B.linnaei
α-tocopherol 663 ± 6.48 369 ± 3.6
(β+γ)-tocopherol 486 ± 3.98 415 ± 1.8
δ-tocopherol 5.2 ± 0.5 2.4 ± 0.7
*values are means ± standard deviation of triplicate determination
46
It is also suggested that the supplementation of tocopherols (antioxidants) may prevent
the risk of degenerative processes (Kallio et al. 2002). α-tocopherol has reported to
improve the lability of the blood to carry oxygen, to prevent and dissolve blood clots and
is effective in preventing scar formation (Giller, and Matthews, 1986).
The data for the quantification of tocopherols (α, β+γ and δ) of seed oil of different
Bauhinia species; B. variegata and B. linnaei are shown in Table 4.2.3. The content
(mg/kg) of α- tocopherol in the seed oils of Bauhinia species investigate varied widely.
There were significant differences (p<0.05) in the levels of α-tocopherol which are highly
variety-dependent (Owen et al., 2000b). The level of α- tocopherol, (β+γ)-tocopherol and
δ-tocopherol in B. variegata oil were 663, 486 and 5.2mg/kg of oil quite higher as
compared to the B. linnaei which contained the level of α- tocopherol 369, (β+γ)-
tocopherol 415 and δ-tocopherol 2.4 mg/kg respectively. The level of α- tocopherol in B.
variegata and B. linnaei seed oils, was higher than those reported for groundnut (178),
maize (282), cottonseed (338), soybean (99), palm (89) in mg/kg of oil, and low erucic
acid rapeseed oils (Rossell, 1991). The level of (β+γ)-tocopherol (415-486) in the Bauhinia
species seed oils examined was lower than those reported for corn (592), comparable
with soybean (494), and higher than olive (12.3), sunflower (25.2), grape seed (15.00)
and rapeseed (280mg/kg) of oils (Gliszczynska-Swiglo, et al., 2007).
4.2.4. Sterols profile of B. variegata and B. linnaei seed oil.
Sterols are the most important class of the minor components and comprise a major
portion of the unsaponifiable matter of most vegetable oils (Kiritsakis and Christie.
47
2000). The composition of the sterols of B. variegata and B. linnaei as determined by
GC-MS are shown in Table 4.2.4.
The sterol fraction of the B. varigata oil mainly consisted of β-sitosterol 62.6%,
stigmaterol 17.1% and compesterol 12.3% while the sterol fraction of B. linnaei
constituted of β-sitosterol 58.3%, stigmaterol 22.1% and compesterol 11.8% respectively.
The level of Δ5-avenasterol, Δ7-avenasterol, and Δ7-stigmasterol ranged from 5.5-3.5%,
1.3-1.9% and 1.2-2.4% were found in both species. The level of β-sitosterol in both
investigated species found (62.6-58.3%) was
Table 4.2.4. Sterole profile of B. variegata and B. linnaei seed oil.
Sterols (%) B. variegata B. linnaei
Compesterol 12.3 ± 0.8 11.8 ± 0.7
Stigmasterol 17.1 ± 1.8 22.1 ± 1.2
β-sitosterol 62.6 ± 2.1 58.3 ± 1.3
Δ5-avenasterol 5.5 ± 0.6 3.5 ± 0.9
Δ7-avenasterol 1.3 ± 0.3 1.9 ± 0.2
Δ7-stigmasterol 1.2 ± 0.1 2.4 ± 0.1
*values are means ±standard deviation of triplicate determination
significantly (p<0.05) higher than those reported for groundnut, cottonseed, soybean,
maize, and low erucic acid rapeseed oils (Rossell, 1991).
4.2.5. Proximate composition of Bauhinia seed meal
Table 4.2.5 shows the proximate compositions of B. variegata and B. linnaei seed meal.
The results revealed that high amount of protein content of the seeds ranging from 41.9-
48
38.6%, where as fiber, moisture, ash and carbohydrates content were found to be 6.9-
7.3%, 6.7-6.3%, 4.8-4.2% and 28.4-33.8% respectively. The protein content of B.
variegata 41.9% was higher as compared to B. linnaei 38.6% and closely comparable to
the previous reported data (Zaka et al., 1983). This analysis showed, that the meal of
Bauhinia seeds varieties with other essential nutrients (fiber, ash and carbohydrets) could
be an excellent source of protein, which can be added to the chicken diets as a source of
energy (calories) and it is a good substitute of (sunflower and soybean) meal for the local
poultry feed industry.
Table 4.2.5. Analysis of B. variegata and B. linnaei seed meal
Constituents (%) B. variegata B.linnaei Oil content 18.0 ± 0.9 17.4 ± 0.6 Moisture 6.7 ± 0.46 6.3 ± 0.4 Protein 41.9 ± 1.67 38.6 ± 1.7 Ash 4.8 ± 0.1 4.2 ± 0.3 Fiber 6.9 ± 0.8 7.3 ± 0.6 Carbohydrates 28.4 ± 1.6 33.8 ± 1.0
All values are means ± SD, analyzed individually in triplicate.
The oil content (Table 4.2.5) of B. linnaei and B. variegata seeds was in the range of
17.4-18.0 %. B. variegata contained 18.0% of oil which was higher than those reported in
previous study data (Zaka et al., 1983). Such type of variations in the concentrations of
nutrients within the country between varieties and species may be associated to the
probable changes in cultivated regions (climatic and geographical differences) where the
seeds had been grown (Atta, 2003). The average oil contents of B. variegata and B.
linnaei seed in the present study were found to be comparable with those of two
conventional oilseed crops: of soybean (17.0-21.0%) and cotton (15.0-24%), grown in the
Asian and European countries (Pritchard, 1981).
49
Part 111
4.3. Oxidative stability assessment of Bauhinia purpurea seed oil in comparison to two conventional vegetable oils by differential scanning calorimetry and Rancimate methods
The work of this part has been published and cited as: Arain et al., Thermochimica Acta
2009, 484, 1–3.
4.3.1 Oxidative stability
Straight line was observed with the stream of nitrogen (99.99%) flowing at 50 ml/min by
the differential scanning calorimetry for B. purpurea oil at 130 ◦C as shown in Fig. 4.3.1,
curve A clearly indicates that peak is not exothermic. Whereas exothermic oxidation
curve obtained when oil samples were run under oxygen atmosphere (99.99%) flowing at
50 ml/min (Fig. 4.3.1, curve B).
Fig. 4.3.1. A representative differential scanning calorimetry oxidation
curve of B. purpurea oil: (A) isothermal curve at 130 ◦C with nitrogen (99.99%) flowing at 50 ml/min; (B) isothermal curve at 130 ◦C with oxygen (99.99%) flowing at 50 ml/min.
50
Oxidation process is a principally exothermic reaction which occurs in between the oil
and oxygen. The comparative stability of the B. purpurea, B. variegata, B. linnaei, rice
bran and cotton seed oil towards oxidation was analyzed by the extrapolated T0 values.
Oxidative induction time (T0), oxidative stability index (OSI) and Differential scanning
calorimetry (DSC) values of B. purpurea, B. variegata, B. linnaei rice bran and cotton
seed oils are depicted in Table 4.3.1.
Table 4.3.1.. Differential scanning calorimetry (DSC) oxidative induction time (T0)
and oxidative stability index (OSI) values of B. purpurea, rice bran and cotton seed oils.
Oil DSC T0 (min) OSI (min) 110 ˚C 120 ˚C 130 ˚C 140 ˚C 110 ˚C B. purpurea 483.33 269.77 99.11 48.44 1339.31 Rice baran 132.89 72.74 36.06 18.66 217.34 Cotton seed 172.41 92.00 42.74 20.30 427.28 B. variegata 294.82 138.84 69.26 31.85 538.56 B. linnaei 171.33 78.82 41.91 23.56 461.59
OSI instrument at the isothermal temperature (110 ◦C) gave significantly (P < 0.05)
higher oxidative induction time than DSC technique. This variation could be due to a
smaller sample size which was used in the DSC analysis as compared to OSI instrument
(5mg vs. 5 g).
Table 4.3.2. Pearson correlation coefficient matrix between differential scanning calorimetry (DSC) and oxidative stability index (OS I) methods
DSC110 DSC120 DSC130 DSC140
OSI110 -------- --------- --------- ---------- OSI130 0.998 --------- --------- --------- OSI140 0.985 0.994 --------- --------- OSI 110 0.977 0.999 0.987 ---------
1.000 0.987 0.981 0.999 a Significance at 0.01 level (P < 0.01). DSC at isothermal temperature 110 ◦C, DSC120; DSC at isothermal temperature 120 ◦C, DSC 130; DSC at isothermal temperature 130 ◦C, DSC140; DSC at isothermal temperature140 ◦C, OSI 110 ◦C.
51
The results of oxidative stability measured as induction time (IT) value by Rancimat
assay for B. purpurea (Table 4.3.1) shows oxidative stability up to 1339.31min, higher
than B. variegata (638.5 min), B. linnaei (458.8 min), rice bran (217.34min) and cotton
seed oil (427.28 min). The extraordinary stability of B. purpurea oil may be due to the
presence of higher amount of tocopherols (Yoshida, 1994; Kamal-Eldin & Appelqvist,
1996). Each DSC isothermal temperature was found to have a significant effect (P <
0.01) on the DSC T0 (Table 4.3.1) measurements. For the analyzed oils, with increasing
isothermal temperature a significant (P < 0.05) decrease was observed for T0. Generally,
with an increase in 10 ◦C from 110 to 140 ◦C, the T0 value was reduced approximately to
half of its earlier appraisal (Table 4.3.1). This detail is in agreement with Q10 law for the
association among the rate of chemical reaction and temperature (Tan et al., 2002; Mark
Sewald & Jon Devries, 2008). An excellent coefficient correlation was found between the
DSC T0 and OSI measurements as shown in Table 4.3.2.
The coefficients of correlation were also highly significant (P < 0.0001) for each
evaluation. In observation of the high association between DSC T0 and OSI time linear
regression equations were calculated (Table 4.3.3).
Table 4.3.3. Relationships between oxidative stability index (OSI) values and differential
scanning calorimetry (DSC) oxidative induction time (T0) at four different isothermal temperatures.
Indicator
(Y) Indicator
(X) Regression equation P-values
OSI 110 DSC110 T0(OSI110) = 0.4899T0(DSC110)−129.89 0.0001 OSI 120 DSC120 T0(OSI120) = 0.4834T0 (DSC120)−333.95 0.0001 OSI 130 DSC130 T0(OSI130) = 0.3276T0 (DSC130)−313.46 0.0001 OSI 140 DSC140 T0(OSI140) = 0.4668 T0 (DSC140)−538.8 0.0001
52
Table 4.3.4. Relationship between logarithm of DSC T0 values (log10 T0) and DSC isothermal temperature (T0) of B. purprea, B. variegata, B. linnaei, rice bran and cotton seed oil.
Oil Regression equation Coefficient of determination B. purpurea T = 54.323−0.0351 log10 T0 0.9997 Rice bran T = 12.273−0.0487 log10 T0 0.9996 Cotton seed T = 14.789−0.0305 log10 T0 0.9956 B. variegata T= 21.195−0.0301 log 10 T0 0.9932 B. linnaei T= 28.33−0.025 log 10 T0 0.9994
The regression equations of logarithm DSC T0 values against DSC isothermal temperature
were established and given in Table 4.3.4. The coefficient of the determination (R2) for
analyzed oils was above 0.9956, showing good linear regression which revealed that oxidative
stability of the oils can be accurately determined by DSC in a short time as compared to OSI
method.
53
Part 1V 4.4. Infraspecific Variation in Composition of Bauhinia purpurea Seed oil:
Optimization of Chemotexonomic Indicators
4.4.1. Fatty acids profile of B. purpurea of different origin Fatty acid composition in seed oils varied widely and fatty acid often predominates as
characteristic of its particular plant origin. The fatty acid profiles are considered as
chemotaxonomic markers to define groups of various taxonomic ranks in flowering
plants (Mongrand et al., 2005; Spitzer, 1999).
The extracted oil of B. purpurea seeds samples contained significant amounts of palmitic,
stearic, oleic and linoleic acids, which were the major usual fatty acids. Palmitic (C16:0)
ranged from 15.35% to 19.45% (Table 4.4.1). It was highest in the B. purpurea seed oil
samples from Multan (19.45%), Pakpattan (18.49%), Abotabad (18.01%) and lowest in
Tandojam oil samples (15.35%) with little variations. linoleic acid ranged from 46.85%
to 56.78%%, the highest values of linoleic acid was found in B. purpurea oil samples
from Tandojam (56.78%) and Hyderabad (53.91%) as compared to Multan (49.41%),
Pakpattan (46.85%) and Abotabad (50.25%) respectively.
In addition to linoleic acid, the seed oils have highest level of oleic acid ranged from
12.61 to 13.86%, the oleic acid was found in nearly equal amount in oil samples from
Hyderabad (12.67%) and Tandojam (12.61%), Pakpattan (13.86%), Multan (13.44%) and
Abbotabad (13.45%) respectively. Alpha linolenic, gamma linolenic acids and other
minor fatty acids were also identified in B. purpurea oil samples of different origin in
lower concentrations.
54
Table 4.4.1. fatty acid profile of B.purpurea seed oil of different origin.
Fatty acids (%) Hyderabad Tandojam Multan Pakpattan Abbotabad Myristolic (C14:1) 0.2 ± 0.1 0.3 ± 0.2 ND ND ND
Palmitic (C16:0) 17.2 ± 0.8 15.3 ± 0.62 19.8 ± 1.0 18.9 ± 0.8 16.1 ± 0.9
Palmetoleic(C16:1) 0.2 ± 0.1 0.2 ± 0.1 ND ND ND
Stearic (C18:0) 11.8 ± 0.7 12.3 ± 0.5 12.9 ± 0.9 13.4 ± 0.5 13.3 ± 0.6
Oleic (C18:1) 12.7± 0.9 12.4 ± 0.6 13.5 ± 0.6 13.8 ± 0.6 13.4 ± 0.8
Linoleic (C18:2) 53.8 ± 2.2 55.8 ± 2.1 49.4 ± 1.7 48.9 ± 1.6 51.7 ± 1.8
Alpha linolenic (n-3) 0.5 ± 0.1 0.4 ±0.1 0.5 ± 0.2 0.6 ± 0.1 0.8 ± 0.1
Gama linolenic (n-6) 0.4 ± 0.2 0.4 ± 0.2 0.4 ± 0.1 0.5 ± 0.2 0.5 ± 0.1
Arachidic (C20:0) 1.2 ± 0.3 1.1 ± 0.1 1.04 ± 0.2 1.1 ± 0.4 1.4 ± 0.2
Eicosadienoic (C20:2) 0.4 ± 0.1 0.5± 0.2 0.6 ± 0.1 0.8 ± 0.2 0.7 ± 0.3
Eicosapentaenoic (C20:5) 0.5 ± 0.2 0.4 ± 0.3 0.4 ± 0.1 0.5 ± 0.2 0.4 ± 0.2
Behenic acid (C22:0) 0.4 ± 0.1 0.3 ± 0.1 0.4 ± 0.2 0.6 ± 0.2 0.6 ± 0.1
Lignoceric (C24:0) 0.2 ± 0.1 0.1 ± 0.2 0.3 ± 0.1 0.3 ± 0.1 0.5 ± 0.1
Nervonic |(C24:1) 0.5 ± 0.1 0.5 ± 0.1 0.6 ± 0.2 0.6 ± 0.1 0.6 ± 0.2
Each value is an average of eight samples, with its standard deviations. ND: Not Detected Since the FA patteron of B. purpurea seed oils showed a remarkable uniformity in terms
of their content of oleic, palmitic and minor fatty acids. It is proposed that this could be
of chemotaxonomical interest and their quantity may indicate closer or more distant
relationships among the different origin in B. purpurea seed oils.
4.4.2. Tocopherols profile of B. purpurea of different origin
Tocopherol profile is an important quality criterion for the assessment of the seed oils,
the composition of tocopherol of B. purpure seed oils is shown in Table 4.4.2.
55
Table 4.4.2. Tocopherol profile of B. purpurea seed oil from different origin
Tocopherols (mg/kg)
Hyderabad Tandojam Multan Pakpattan Abbotabad
-tocopherol 889 23.1 727 27.9 675 27.5 535 18.9 795 13.4
β-tocopherl 448 19.3 493 13.2 545 25.3 548 14.2 525 23.4
-tocopherol 175 15.5 194 9.8 157 16.2 149 12.0 146 12.1
Each value is an average of eight samples, with its standard deviations.
There are certain differences in the tocopherol composition of the different seed oils, the
α-tocopherol (535-889mg/kg) and β-tocopherol (448-578mg/kg) were the major vitamin
E active isomers in B. purpurea seed oils of different origin. The variation in the content
of δ-tocopherol (0.87-194mg/kg) was relatively small in all samples. Remarkably high
amount of α-tocopherol in B. purpurea seed oils could be motivating for the production
of naturally occurring tocopherols for the stabilization of oils and fats against oxidative
deterioration and for applications in pharmaceutical and dietary products (Dunford &
King, 2000). High amount of α-tocopherol and β-tocopherol and low amount of δ-
tocopherol with little variations may be useful as chemotaxonomic maker to differentiate
the origin of B. purpurea seed oils.
4.4.3. Sterols profile of B. purpurea seed oil of different origin
Phytosterols are minor constituents of all vegetable oils comprising major portion of the
unsaponifiable matter of most vegetable oils. All phytosterols in humans blood and
tissues are derived from the diet because humans cannot synthesize phytosterols
(Mushtaq et al., 2007). They are of interest due to their impact on health. Recently,
56
sterols have been added to vegetable oils as an example of a successful functional food
(Ntanios, 2001).
Table 4.4.3. Sterols profile of B. purpurea seed oil from different origin
Sterol (%) Hyderabad Tandojam Multan Pakpattan Abbotabad
Compesterol 8.7 0.6 8.4 0.7 7.9 0.7 8.9 0.8 8.3 0.8
Stigmasterol 16.7 1.2 18.1 0.8 25.8 0.8 22.7 1.2 29.3 0.9
-Sitosterol 66.3 2.4 64.3 1.9 57.2 2.6 60.5 1.8 54.5 2.1
5 Avenasterol 3.6 0.6 2.6 0.5 2.9 0.5 2.3 0.7 3.1 0.5
7Avenasterol 2.1 0.7 3.7 0.8 3.6 0.6 3.2 0.6 2.5 0.5
7 Stigmasterol 2.6 0.3 2.9 0.5 2.3 0.6 2.4 0.4 2.3 0.2
Each value is an average of eight samples, with its standard deviations.
The content of phytosterols determined in the B. purpurea seed oils is shown in Table
4.4.3. β-sitosterol (53.5-66.3 %), compesterol (7.9-8.9%) and stigmasterol (17.7-29.3 %)
were the major component of the total sterols of B. purpurea seed oils. 5 Avenasterol
(1.7-3.1 %), 7Avenasterol (2.1-3.7 %) and 7 Stigmasterol (1.6-2.9 %) of total sterols
were present in lower concentration with little variatons. Hyderabad (66.3%), Tandojam
(64.3%) and Pakpattan (60.5%) seed oils were characterized by the high amount of β-
sitosterol. Main components of B. purpurea seed oils were β-sitosterol and stigmasterol
with higher variations. Interesting is the remarkably high amount of β-sitosterol and
stigmasterol in all samples of B. purpurea seed oils of different origin. The high amount
57
of β-sitosterol and stigmasterol (Table 4.4.3) could make these components as
chemotaxonomic markers to differentiate the origin of B. purpurea seed oils.
4.4.4. Chemometric
First an analytical study was carried out for each variable individually used to test the
differences between B. purpurea seed from different origin, with One Way ANOVA.
Table 4.4.4 shows, DF, SS, MS, F and, Pvalues of all variables (C18:2, α-tocopherol, β-
sitosterol and stigmasterol), explain highly significant differences (P< 0.0001) were
observed among different regions.
Table 4.4.4 . Statistical data of palmitic, stearic, oleic, linoleic acids, α-tocopherol, β-
sitosterol and stigmasterol of B. purpurea oil
Components DF SS MS F P<0.0001
Palmitic acid (C16:0) 4 113.0 14.0 94.7 0.000
Linoleic acid (C18:2) 4 481.4 120.3 196.9 0.000
α-Tocopherol 4 122.1 23.1 156.0 0.000
β-sitosterol 4 382.4 208.1 219.6 0.000
Stigmasterol 4 45.8 21.6 143.6 0.000
DF, Degree of freedom, SS, sum of squares, MS, Mean squares
To assess the variability within the regions, multivariate analysis of experimental data
including principal component analysis (PCA) and linear discriminant analysis were
performed for the classification of B. purpurea. To identify the variability in seed oils,
principal component analysis and linear discriminant analysis were used as statistical
tools. Data for three groups of molecules; fatty acids, sterols and tocopherols were
subjected to analysis individually and also all together to find best combination of
markers
58
4.4.4.1. Fatty acids as markers of discrimination
Principal component analysis of eight fatty acids has shown five linear combinations in
which only two components were extracted, accounts to 77.4 % of the variability with the
eigenvalue greater than one (Fig 4.4.4.1). Five different origins were separated in two
groups; the samples from Hyderabad and Tandojam (group 1) provinces were on negative
plane of principal component 1, while Multan, Abbotabad and Pakpattan (group 2) were
on positive plane.
Table 4.4.5. Linear discriminant analysis of fatty acids, tocopherols and sterols
Eigenvalue Varience (%) Canonical
correlation
p-value
Fatty acids 1 106.2 96.18 0.99533 0.0000 2 3.0 2.77 0.86810 0.0166 Tocopherols 1 40.4 93.14 0.98785 0.0000 2 2.9 6.81 0.86425 0.0000 Sterols 1 64.5 88.81 0.99234 0.0000 2 6.6 9.13 0.93215 0.0000 3 1.2 1.65 0.73881 0.0000 4 0.3 0.41 0.47750 0.0340 Alltogether 1 156.2 74.65 0.996 0.0000 2 41.9 20.07 0.988 0.0000 3 7.5 3.61 0.939 0.0000 4 3.5 1.67 0.882 0.0000
Linear discriminate analysis which characterize or separate two or more classes of objects
to model the difference between the classes of data. Fatty acid composition of B.
purpurea L. seed oil was subjected to LDA. Two discriminating functions were used with
59
p values <0.05 accounts for 99% variability. The plot of discriminate analysis (Fig.
4.4.4.1) confirms the diversity in the samples of different botanical origin. However,
when all the samples were treated as unknown on the basis of leaving one out and
assessing the model for identification of regions, only 60% of cases were identified
correctly on the basis of fatty acids as discriminating parameter. Hence, merely fatty
acids can not be used as markers for regional identification of B. Pupurea L.
PP
M
TJ
HYD
AA
Fig. 4.4.4.1. Linear discriminant function plot of fatty acids. Inset abbreviations: TJ
(Tandojam), PP (Pakpattan), AA (Abbotabad), HYD (Hyderabad), M (Multan).
4.4.4.2. Tocopherols as markers of discrimination
The average values of three tocopherols assayed for five regions are shown in Table 4.4.2
PCA of tocopherols for five different regions on 40 samples provided 78.8% variance
(first component) for eigenvalue of 2.3 and 15.6% variance (second component) for
eigenvalue 0.47. Here, in this case only one component could be extracted with
60
eigenvalue greater than one and PCA plot for tocopherols is not possible. However, linear
discriminant analysis provides 93.1% (linear discrimination function 1) and 6.8% (linear
discrimination function 2) variance for eigenvalues of 40.4 and 2.9, respectively.
M
PP
AA
TJ
Hyd
Fig. 4.4.4.2. Linear discriminant function plot of tocopherols. Inset abbreviations:
TJ (Tandojam), PP(Pakpattan), AA (Abbotabad), Hyd (Hyderabad), M(Multan).
4.4.4.3. Sterols as markers of discrimination
Principal component analysis performed for sterols is shown in Fig. 4.4.4.3. Sterol
composition is the most useful parameter for discrimination (Ruiz-Mendez et al., 2008).
Very clear combinations for five regions are observed with variance of 38.4% for first
principal component, 30.6% in second and 14.8% in third. Classification of data for
suitability of data for unknowns shows that all the cases can be classified correctly.
Similarly, LDA also showed well-separated groups for all five regions and correct
classifications of unknowns. The sterols used for chemometric discrimination among the
61
types of oils from different origin (Ruiz-Mendez et al., 2008). The classification of data
to fit in this model shows that all the cases 100% correctly classified.
HYD
TJ
M
AA
PP
Fig. 4.4.4.3. Principal component plot of sterols. Inset abbreviations: HYD (Hyderabad), M
(Multan), TJ (Tandojam), AA (Abotabad), PP (Pakpattan) and StS (Stigmasterol), CS (Compesetrol), SS (Sitosterol), AS (Avenasterol), StS3 (stigmasterol3), AV2(Avenasterol).
To further evaluate the data, PCA and LDA were performed on the all data for fatty
acids, tocopherols and sterols (all together). Processing all the data provides four
components to be extracted with eigenvalues greater than one with variance of 37.1%,
13.4%, 11.7% and 8.3% on PCA. The eigenvalues and related parameters for linear
discriminant analysis are summarized in Table 4.4.5. LDA plot (Fig. 4.4.5) for first two
factors shows very distinct linear combinations with Hyderabad and Tandojam laying on
the negative plane for first factor and near to origin on the second factor, while Multan is
on positive plane of first function and on origin of second and Abbotabad on positive
plane of first and negative of the second while both planes are positive for Pakpattan.
Classification of data shows that all the cases 100% classified correctly using this model.
62
AA
M
PP
TJ
Hyd
Fig. 4.4.4.4. Linear Discriminant plot of Bauhnia purpurea seed oil using fatty acids,
tocopherols and sterols as chemical composition descriptors. Inset abbreviations: TJ (Tandojam), PP(Pakpattan), AA (Abotabad), Hyd (Hyderabad), M(Multan)
It is evident from the statistical analysis that B. pupurea L. is susceptible to changes in
environment in terms of its chemical composition of oil. LDA shows (Figure 4.4.6) that
sample from Hyderabad and Tandojam (both from Sindh province Pakistan) lies close to
each other for their chemical composition, in fact two cities experience much similar
environment geographically. The Abbotabad, pakpattan and Multan belong to Punjab
province of Pakistan; they are located at much greater distance from each other as
compared to Hyderabad and Tandojam.
63
PART V
4.5. Prospects of Fatty Acid Profile and Bioactive composition from lipid seeds for the discrimination of Apple Varieties with the Application of Chemometrics
The data produced from the two classes of the compounds were interpreted by using
summarized descriptive statistics with F and p values for four apple seed varieties of oils
are arranged in Tables 4.5.1and 4.5.2. In the examination of the data in Tables 4.5.1and
4.5.2, the large differences and clear similarities among the four varieties of oils and the
level of the variations were observed depending on the class of compounds considered.
4.5.1. Fatty acid composition
The oil content in seeds of four varieties of apples ranged from 26.8-28.9% (Table 4.5.1).
From the data presented it could be seen that highest oil content 28.9 ± 0.9% was found
in seeds of apple variety Pyrus Malus, while the lowest 26.8 ± 0.7% was found in the
seeds of Golden Delicious. The oil content of apple seeds varieties obtained in this work
agreed with that reported by Yu et al, (2007), Marjan et al, (2007) and Yukui et al.,
(2009).
The principal fatty acid components in the apple seed oils were linoleic and oleic acids
(Table 4.5.1). The linoleic acid was found to be the dominant fatty acid in Royal Gala
45.1 ± 3.6%, Red Delicious 47.8 ± 3.5%, and Pyrus Malus 49.6 ± 3.3% respectively,
while the Golden Delicious contained linoleic acid 40.5 ± 2.1% comparatively in lower
concentration. It is notable that Golden Delicious oil could be easily distinguished from
other varieties by the high level of oleic acid 45.5 ± 1.4 %. Relatively lower percentage
of oleic acid 39.3 ± 2.7% was found in Red Delicious with respect to other varieties.
64
Table 4.5.1. Fatty acid compositional data (%) of apple seed oils
amean values in percentage within each class of fatty acids
bstandard deviation statistical analysis canalysis of varieance, probability < 0.001
The principal fatty acids i.e. linoleic and oleic acid in present study were found to be
quite comparable with the results of previous reported studies (Yu et al, 2007, Marjan et
al, 2007; Yukui et al., 2009).The palmitic 6.1 ± 0.4%, stearic 3.1 ± 0.3% and ecosenoic
acid 1.0 ± 0.1 were found in Pyrus Malus with lower concentrations. Linolenic,
palmitoleic, heptadecanoic, 11-ecosenoic and docosanoic acids were also identified in
traces level (< 1%). The results revealed that the oil obtained through apple seeds are the
richest source of linoleic and oleic acids. The dietary lipids, rich in linoleic and oleic
acids are beneficial for human health (Finley and Shahidi. 2001). Due to the appreciable
content of oil and favorable fatty acid composition, the apple seeds oils have potential use
as edible oil (Yukui et al., 2009). The variation among the fatty acid composition of the
oil might be to genetic features (Minnocci et al., 2010).
Classes of Compounds (Variables)
Royal Gala
Red Delecious
Pyrus Malus Golden
Delicious ANOV
Ac
Mean(%)
SDb
(±)
Mean(%)
SD (±)
Mean (%)
SD (±)
Meana
(%) SD
(±) F-
observed
Oil content 27.2 1.1 27.6 0.8 28.9 0.9 26.8 1.7 3.4 Palmitic ( C16:0) 7.4 0.5 6.7 0.3 6.1 0.4 7.1 0.4 15.6 Palmitoleic (C16:1) 0.1 0.0 0.1 0.0 0.2 0.0 0.1 0.0 18.7 Heptadecanoic (C17:0) 0.1 0.0 0.1 0.0 0.0 0.0 0.1 0.0 64.4 Stearic (C18:0) 2.5 0.6 2.3 0.3 2.0 0.4 3.1 0.3 14.7 Oleic (C18:1) 41.7 1.1 39.3 2.7 38.7 1.7 45.5 2.1 36.9 Linoleic (C18:2) 45.1 3.6 47.8 3.5 49.6 2.2 40.5 1.6 25.4 Linolenic (C18:3) 0.3 0.1 0.3 0.1 0.4 0.0 0.3 0.0 8.8 Ecosanoic (C20:0) 1.7 0.2 2.0 0.2 0.9 0.1 2.0 0.2 51.2 11-Ecosenoic( C20:1) 0.7 0.2 1.0 0.1 0.6 0.0 0.7 0.0 14.6 Docosanoic (C22:0) 0.4 0.1 0.5 0.0 0.7 0.0 0.6 0.1 12.3
65
4.5.2 Lipid bioactive composition
Fig. 4.5.2. Represents GC-MS chromatogram of the unsaponifiable lipid fraction of apple seed oil.
Fig. 4.5.2. Representative GC-MS chromatogram of the unsaponifiable lipid fraction of apple seed. (1)Hexadecanoic acid, ethyl ester (2) phytol, (3) ethyloleate, (4) hexadecenal, (5) 3-ecosene, (6) octadecanoic acid, ethyl ester, (7) 1-docasene, (8) docasene, (9) 1-hexacosane, (10) octacosane, (11)squalene, (12) nonacosene, (13) β-tocpherol, (14) α-tocopherol, (15) compesterol, (16) avenasterol, (17) β-sitosterol, (18) 9,19-Cyclolanost-24-en-3-ol, (19) Stigmast-4-en-3-one
66
Figure 4.5.3. Mass-spectrum of unsaponifiables compounds present in Apple seed oil
-tocopherol Mw 430
Phytol Mw 296
Squaline Mw 410
67
β -tocopherol Mw 416
β-sitosterol Mw 414
Stigmasterol Mw 412
68
Compesterol Mw 400
Avenasterol Mw 412
Ethyloleate Mw 310
69
Stigmasta-4-en-3-one Mw 412
9,19-Cyclolanost-24-en-3-ol Mw 426
70
Table 4.5.2. Unsaponifiable compositional data (%) of apple seed oils with statistical analysis.
amean values in percentage within each class of fatty acids bstandard deviation canalysis of varieance, probability < 0.001
The composition of unsaponifiable lipid fraction of four apple seed varieties is shown in
Table 4.5.2. The sterols are important constituents that help for the stability of the oil at
high temperature and act as inhibitors in polymerization reactions (Velesco and
Dobarganes, 2002). β-sitosterol is responsible for the preventive effects on diseases due
to reactive oxygen species (Vivacons and Moreno, 2005).
Classes of compounds (Variables)
Royal Gala Red Delicious
Pyrus Malus
Golden Delicious
ANOVAC
Meana SD (±)
Meana SD (±)
Meana SD (±)
Meana SD (±)
F- observed
Hexadecanoic acid, ethyl ester
7.2 0.5 7.4 0.4 6.6 0.3 5.7 0.2 23.2
Phytol 3.5 0.7 1.6 0.2 0.6 1.1 1.1 0.5 28.6
Ethyl Oleate 39.2 1.6 38.6 2.1 34.6 3.1 35.5 1.9 15.6
9-hexadecenal 3.2 0.7 1.5 0.2 0.7 0.4 0.8 0.2 32.8
3-Eicosene 0.9 0.1 0.8 0.1 0.8 0.2 0.7 0.3 8.5
Octadecanoic acid, ethyl ester
1.3 0.1 0.9 0.1 2.9 1.1 1.8 0.9 34.8
1-Docasene 2.7 0.2 2.8 0.2 2.4 0.3 2.6 0.1 10.3
Docosane 0.9 0.1 0.8 0.1 0.9 0.5 0.9 0.4 8.6
1-Hexacosene 1.1 0.1 1.0 0.1 1.2 1.2 0.8 0.6 37.2
Octacosane 0.8 0.1 0.9 0.2 0.8 0.4 0.9 0.3 8.7
Squalene 6.7 1.2 5.7 0.5 5.8 0.8 6.4 1.2 9.4
Nonacosane 1.4 0.2 1.2 0.2 0.9 0.3 1.5 0.5 8.5
β-Tocopherol 1.4 0.2 1.7 0.4 1.7 0.5 1.8 0.7 17.2
α-Tocopherol 6.4 1.1 5.4 0.8 6.1 0.6 5.6 0.6 8.6
Campesterol 0.8 0.1 0.5 0.0 0.7 0.2 0.9 0.3 9.6
Avenasterol 0.6 0.0 0.6 0.0 0.6 0.1 0.8 0.2 13.4
β-sitosterol 16.2 1.1 14.8 1.1 13.6 1.4 15.9 1.2 69.3
9,19-Cyclolanost-24-en-3-ol
3.2 0.3 3.7 0.5 3.6 1.1 4.6 0.6 26.5
Stigmast-4-en-3-one 2.8 0.4 1.9 0.6 3.8 0.5 3.2 0.9 31.9
71
In all varieties, β-sitosterol was found to be a predominant as compare to other detected
sterols. In Royal Gala highest level of β-sitosterol 16.17 ± 0.7% was evaluated and
followed by Red Delicious 14.77 ± 1.1%, Pyrus Malus 13.6 ± 1.1% and Goleden
Delicious 15.9 ± 1.2% respectively.In the mass spectrum of β-sitosterol (Fig 4.5.6)
characteristic fragment ion established at m/z 414, 396, 381, 329, 303 and 273.
Cycloartinol (9, 19-Cyclolanost-24-en-3-ol) is well-known intermediate in the
biosynthetic pathways of plant sterol (Wasuke et al., 1987). Highest percentage of 9, 19-
cyclolanost-24-en-3-ol was observed in Golden Delicious 4.8 ± 0.6% as compared to
other varieties. The mass spectrum of 9, 19-cyclolanost-24-en-3-ol (Fig 4.5.2b) showed
characteristic fragment ion occurred at m/z 426, 406,393, 365, 355, 341 and 281.
The hypoglycaemic effects of stigmast-4-en-3-one have been reported (Alexander-Lindo
et al., 2004). The mass spectrum of stigmast-4-en-3-one revealed characteristic fragment
ion occurred at m/z 412, 397, 355, 341, 327, 295 and 281 are shown (Fig. 4.5.2b) Higher
Level of stigmast-4-en-3-one was observed in Pyrus Malus 4.6 ± 0.6 % as compared to
other varieties which were shown relatively lower amount of Stigmast-4-en-3-one (< 4.00
%). The diagnostic fragments in the mass spectrum of Avenasterol at m/z 412, 397, 370,
355, 341, 327, 295 and 281are shown in (Fig. 4.5.2b) and the fragments in the mass
spectrum of stigmasterol (Fig. 4.5.2b) exhibited characteristic fragment ion at m/z 412,
397, 379, 367, 355, 346, 328, 314 and 299 and mass spectrum of compesterol showed
characteristic fragment ion at m/z 400, 382, 367, 355, 341, 327, 315and 281(Fig. 4.5.2b).
Avenasterol, stigmasterol and compesterol were found in minor quantities (<1%) in apple
seed varieties.
72
Tocopherols are nutritionally most important components, which are responsible to
enhance the stability of the oil in addition to other health benefits due their antioxidant
activity (Herrera and Barbas, 2001; Traber and Atkinson, 2007). The fragment ion in the
mass spectrum α-tocopherol established at m/z 430, 415, 396, 381, 367, 355, 302, and
281 whereas the fragment ion in the mass spectrum of β-tocopherol were identified (Fig.
4.5.2b) at m/z 416, 405, 389, 377, 355, 341, and 281. In all samples (Table 4.5.2a), the
highet level of α-tocopherol was observed in Royal Gala 6.4 ± 1.1% and β-tocopherol
was found in highest level in Golden Delicious 1.8 ± 0.7% respectively.
Ethyl oleate was found to be a major constituent of unsaponifiable lipid fraction of apple
seed varieties. Ethyl oleate is rapidly hydrolyzed to oleic acid, then absorbed and
distributed within the body in the similar manner of oleic acid (Robert et al., 2003). Mass
spectra displayed a major fragment ion of ethyl oleate (Fig. 4.5.2b) at m/z 310, 264, 222,
180, 155 and 137. As shown in Table 4.5.2 ethyl oleate was found in Royal Gala 39.2 ±
1.6% comparatively in higher concentration.
Hexadecanoic acid and octadecanoic acid and 9-hexadecenal were also identified.
Squalene, is the major hydrocarbon and present more than 90% of the total hydrocarbon
in unsaponifiable lipid fraction of vegetable oils (Lanzon et al., 1994). Squaline protect
human skin from lipid peroxidation, and reduce low-density lipoprotein (LDL) as well as
triglyceride levels in hypercholesterolemia (Kohno et al., 1995; Kelly et al., 1999). Mass
spectra of squaline exihibited fragment ion at m/z 410, 396, 386, 367, 341, 281, 207 and
73
149.Table 4.5.2 clearly shows that Royal Gala contained 6.7 ±1.2 % higher concentration
of squaline, and hydrocarbons such as 3-eicosene, octacosane, 1-docasene, docosane, 1-
hexacosene were also detected in oil.
Phytol is commonly found in all plants and it is the minor constituent of human diet,
precursor for vitamins E and K1. Number of studies explored the various cellular and
biological effects of phytols (Christiane et al., 1986; Hibasami et al., 2002).In mass
spectra of phytol a identified fragment ion occure at m/z 288, 278, 267, 206, 191, 179 and
123 (Fig. 4.5.2b). The lowest concentration of phytol was observed in Pyrus Malus 0.6 ±
1.1% as compared to other varieties. Results indicated that variations in minor
components were found among the different apple varieties.
4.5.3. Chemometrics
4.5.3.1. Principal component analysis for fatty acids
In this study, Principal component analysis (PCA) was used on fatty acid data matrix in
order to identify a small number of factors that explain most of the variance observed in
the variables and that could differentiate the apple seed varieties. According to the
eigenvalues (>1), the first three principal components were selected which correspond to
46.51, 20.93 and 18.80% variance of the original data, collectively sum up to 86.24% of
the variance in the original data.. Four groups of selected apple seed varieties were
clearly discriminated in the scatter plot (Fig 4.5.3.1a and b) by principal components (PC)
1 and 2. The majority of samples from Royal Gala, Red Delicious and Golden Delicious
74
varieties were clustered together quite closely, only one cluster of Pyrus Malus was
located away from these three groups.
From the loading and biplot (Fig. 4.5.3.1a and b), Golden Delicious apple confirmed the
correlation of C18:0, C18:1 and C16:0, whilst Royal Gala and Red Delicious were
correlated with C17:0, C20:0 and C20:, while Pyrus Malus samples correlated with the
C22:0, C16:1 and C18:2 which were supported by the previous study (Bianchi et al.,
2001).
Fig. 4.5.3.1a. PC1 verses PC2 of four varieties of apples based on fatty acid composition of
RDA (Royal Gala apple), RDA (Red Delicious apple), PMA (Pyrus Malus apple), GDA (Golden Delicious apple)
75
Fig. 4.5.3.1b. PC3 verses PC4 of four varieties of apples based on fatty acid composition of RDA
(Royal gala apple), RDA (Red delicious apple), PMA (Pyrus malus apple), GDA (Golden delicious apple)
The results of the PC1 and PC2 (Fig. 4.5.3.1a) as well as PC1 and PC3 (Fig. 4.5.3.1b) plots
were proved excellent differentiations among the four groups of oils. Samples of Pyrus
Malus varieties were differentiated from the others, but there is a partial overlapping
between Royal Gala, Red Delicious and Golden Delicious apple seed varieties. Royal
Gala, Red Delicious and Golden Delicious apple seed oil samples were characterized by
positive score on PC1, whereas Pyrus Malus samples were prominently differentiated
from the others groups due to their negative loading on PC2. The results revealed that all
the variables at the same time, getting more satisfactory results in the differentiations of
the four varieties. Different scientists applied PCA by selecting different approaches and
chemical variables. For example acidity (PC1) and phenolic compounds (PC2) were
found to be the most relevant parameters for discrimination of apple varieties (Del
Campo et al., 2006).
Component 1 (48.27%)
Com
pon
ent
3 (
14.1
0%)
C16:0
C16:1
C17:0
C18:0C18:1
C18:2
C18:3
C20:0
C20:1
C22:0
-4 -2 0 2 4-2.9
-1.9
-0.9
0.1
1.1
2.1
RGA
RDA
GDA
PMA
76
In another reported study examination of the principal component loadings showed that
the levels of malic acid and sucrose were two important variables, but variations in the
composition of the minor constituents were also found to make a significant contribution
to the discrimination (Belton et al., 1998). In the present study PCA was used to
summarize the information of the data matrix in a more reduced way. Oleic acid (PC1)
and linolenic acid (PC2) were the key contributors to the discrimination of apple
varieties.
4.5.3.2. Principal component analysis for unsaponifiable
PCA was also applied to the unsaponifiable data matrix of the all samples. In first four
PCs correspond to 46.31, 16.75, 13.18 and 9.37 % variance with eigenvalues (>1),
together they account for 85.60% of the variance in the original data.
Fig. 4.5.3.2a. PC1 verses PC2 of four varieties of apples based on unsaponifiable composition of
RDA (Royal gala apple), RDA (Red delicious apple), PMA (Pyrus malus apple), GDA (Golden delicious apple) and Eole (Ethyl oleate), Pht (Phytol), Squ (Squaline), sit ( β-Sitosterol), aToc (α-Tocopherol), bToc (β-Tocopherol), AV(Avenasterol), Stig (Stigmast-4-en-3-one), Cy (9,19-Cyclolanost-24-en-3-ol).
Component 1 (46.31%)
Com
pon
ent
2 (
16.7
5%)
Hex
Pht
Eole
hexa
Eic
Oct
T
Docm Hex
mOct
SquNonaToc
bToc
Com
AV
sit
Cy
Stig
-7 -5 -3 -1 1 3 5 -3.9
-1.9
0.1
2.1
4.1 RDA
RGA
GDA
PMA
77
Com ponent function 1 (46.31% )
Com
pon
ent
3 (
13.1
8%)
Hex
Pht
Eolehexa
Eic
Oct
T
Doc
m Hexm Oct
Squ
NonaToc
bTocCom
AV
sit
Cy
Stig
-7 -5 -3 -1 1 3 5 -2.9
-1.9
-0.9
0.1
1.1
2.1
3.1
PM AR DA
GDA R GA
PCs plots for unsaponifiable data matrix revealed excellent differentiations of the four
groups of oils. In PC 1, 2 and 3, Red Delicious apple samples, correlared with ethyl
oleate and hexadecenal, Royal Gala correlated with squalene, Phytol and β-sitosterol, and
Golden Delicious correlated with Avenasterol, Compesterol and β-tocopherol whereas
Pyrus Malus was correlated with stigmasterol and α-tocopherol. Using PCA, Golden
Delicious and Pyrus Malus apple seed varieties were well differentiated from the others
by the all variables while there was a partial overlapping between samples of Royal Gala
and Red Delicious apple seed varieties as shown in score plot (Fig. 3a and b). PCA was
applied by some other authors on the composition of the minor constituents to
discriminate the apple varieties (Chen et al., 2011; Belton et al., 1998). Their results also
indicated that minor components could play considerable role to differentiate the apples
varieties.
Fig. 4.5.3.2b. PC1 verses PC2 of four varieties of apples based on unsaponifiable composition
of RDA (Royal gala apple), RDA (Red delicious apple), PMA (Pyrus malus apple), GDA (Golden delecious apple) and Eole (Ethyl oleate), Pht (Phytol), Squ (Squaline), sit ( β-Sitosterol), aToc ( α-Tocopherol), bToc (β-Tocopherol), AV(Avenasterol), Stig (Stigmast-4-en-3-one), Cy (9,19-Cyclolanost-24-en-3-ol).
78
4.5.3.3. Linear discriminant analysis for fatty acids and unsaponifiable matter
Linear discriminant analysis was applied to the fatty acid composition data of the apple
seed varieties, grouping them on the basis of the four linear associations (Fig. 4.5.3.3a).
The eigenvalues, cumulative and the canonical correlation values were presented in Table
4.5.3. The first two discriminant functions explained 96.25% of the total variance.
Four linear combinations were examined, because LDA selects directions which give
maximum separation between the studied groups. LDA plot (Fig. 4.5.3.3a) for first two
functions discriminated the Golden Delicious and Red Delicious apple samples on
negative half with partial overlapping, and Pyrus Malus apple samples were located on
positive half. Red Delicious apple variety was discriminated along linear function one
and two (Fig. 4.5.3.3b). Eigenvalues, percent of variance, cumulative percentage and
canonical correlation for discriminant functions revealed that all the groups were
classified correctly by the model summarized in Table 4.5.3. The first two discriminant
functions explained 96.43% of the total variance. LDA plot.
(Fig. 4.5.3.3b) for first two functions were discriminated the Red Delicious apple seed
variety on negative half, the Golden Delicious apple on positive half, Royal Gala apple
sample lie in between positive and negative half (Ranalli et al., 2002), whereas Pyrus
Malus samples was differentiated along linear function one and two as shown in Fig.
4.5.3b.
79
Fig. 4.5.3.3a. Discriminant function plots for four varieties of apples; (a) based on
Fig. 4.5.3.3b. fatty acid composition, (b) based on unsaponifiable composition of GDA (Golden Delicious apple), RDA (Red Delicious apple), PMA (Pyrus Malus apple),), RGA (Royal Gala apple).
The model for the classification (fatty acid and unsaponifiables) shows (Table 4.5.3) that
all the cases were 100% correctly classified The same apple varieties grown in different
Function 1 (75.47%)
Fun
ctio
n 2
(20.
96 %
)
GDA
PMA
RDA
RGA
- 13 -8 -3 2 7 12 17
-6
-3
0
3
6
9 GDA
Function 1 (76.43%)
Fu
nct
ion
2 (
18.8
2 %
)
GDA PMA RDA RGA
- 29 - 9 11 31 51
- 19
- 9
1
11
21
A
B
80
locations exhibited almost similar fatty acids and unsaponifiable contents indicated that
genotypic changes may cause discrimination in apple varieties.
Table. 4.5.3. Linear discriminant analysis of fatty acids and unsaponifiables:
statistics and classification of results
Discriminant function Eigenvalues Percent of variance
Cumulative percentage
Canonical correlation
Fatty acid composition 1 2 3
Unsaponifiables
1 2 3
74.03
18.76 1.48
640.44 157.74 39.78
76.43 18.82 4.75
75.47 20.96 3.57
76.43 95.25 100.00
75.47 96.43 100.00
0.99 0.97 0.77
0.99 0.99 0.98
4.5.3.4. Cluster analysis
In hierarchical cluster analysis (HCA), distances between pairs of samples were
calculated and compared, elatively short distances between samples indicated similarity;
dissimilar samples are separated by the large distances. The apple samples were classified
into four groups. Cluster analysis was used to determine the pattern of clustering between
the apple varieties (Del Campo et al., 2006).
Results of the present study indicated that differentiation and combination of the groups
were totally based on the similarities among the samples (Fig. 4.5.3.4). The major group
contained the Royal Gala, Red Delicious, and Golden Delicious apple samples (Mildner-
Szkudlarz et al., 2003), while Pyrus Malus apple was separated by the large distances as
an individual group which is very clear in Fig. 4.5.3.4.
81
Nearest Neighbor Method,Squared Euclidean
Dis
tanc
e
0
5
10
15
20
25
RG
AR
GA
RG
AR
GA
RG
AR
GA
RG
AR
GA
RG
AR
GA
RD
AR
DA
RD
AR
DA
RD
AR
DA
RD
AR
DA
RD
AR
DA
PM
AP
MA
PM
AP
MA
PM
AP
MA
PM
AP
MA
PM
AP
MA
GD
AG
DA
GD
AG
DA
GD
AG
DA
GD
AG
DA
GD
AG
DA
Fig. 4.5.3.4. Dedrogram for four apple varieties using unsaponifiable and fatty acid composition
of RGA (Royal Gala apple), RDA (Red Delicious apple), GDA (Golden Delecious apple), PMA (Pyrus Malus apple).
82
Table 4.5.4. Physico-chemical characteristics of seed oils of different Apple varieties
Values are mean ± SD of three seed oils of each apple varieties, analyzed individually in triplicate Different letters in superscript indicate significant differences within apple varieties
Components Royal. Gala Red Delicious Pyrus Malus Golden. Delicious
Refractive index (40°C) 1.466 ± 0.0a 1.465 ± 0.1b 1.473 ± 0.0b 1.471 ± 0.0ab
Density 0.918 ± 0.0 a 0.912 ± 0.0a 0.909 ± 0.0a 0.925 ± 0.0a
Color (red unit) 1.4 ± 0.2 a 1.7 ± 0.1a 1.6 ± 0.0a 1.8 ± 0.0a
Color (yellow unit) 15.00 ± 0.3a 16.00 ± 0.4a 17.00 ± 0.3b 18.00 ± 0.5b
Saponification values (mg of KOH /g of oil) 181.2 ± 4.2b 186.3 ± 3.2b 198.6 ± 4.6ab 193.7 ± 5.2a
Iodine value (g of I2/100g of oil) 98.9 ± 3.5b 109.7 ± 1.2c 115.4 ± 2.3a 96.3 ± 1.4b
Acidity (as oleic acid %) 1.2 ± 0.2c 0.5 ± 0.1c 1.5 ± 0.4b 0.9 ± 0.1b
Unsaponifiable matter (%) 1.1 ± 0.2c 0.9 ± 0.5c 1.2 ± 0.5c 1.4 ± 0.4bc
Peroxide value (meq O2 / kg of oil) 1.9 ± 0.4b 2.8 ± 0.6a 2.9 ± 0.4c 1.8 ± 0.3a
Conjugated Dienes (λ232) 0.6 ± 0.1a 0.8 ± 0.3b 1.1 ± 0.2a 0.8 ± 0.3a
Conjugated Trienes (λ270) 0.2 ± 0.1a 0.2 ± 0.1a 0.5 ± 0.1a 0.1 ± 0.0a
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4.5.4. Physiochemical characteristics of apple seed varieties
The results of various physiochemical characteristics of the extracted apple seed oils of
different varieties are depicted in Table 1. The apple seed varieties investigated, exhibited
no significant (p<0.05) variations with regard to the value of Refractive indices (40 °C )
and density (24°C), which ranged from 1.466-1.473 and 0.909-0.925 mg/ml,
respectively. The present results were quite comparable with those of reported by Tian, et
al., (2010) from China apple seed oils, refractive index (1.465-1.466) and density (0.902-
0.903 mg/ml) respectively. The refrective indices (1.466-1.473) determined in the present
analysis of apple
seed oils, agreed well with those reported for mustard seed (1.461–1.469), cottonseed
(1.458–1.466), almond kernel (1.462–1.465), groundnut (1.460–1.465), kapok seed
(1.460–1.466) oils, sunflower (1.467–1.469), rapeseed (1.465–1.469), safflower (1.468–
1.469), and grape seed (1.473–1.477) oils (Rossell, 1991). Thus the degree of variation of
typical oil from true values of refractive index and density could be indicating its relative
purity.
The color of apple seed oils (1.4-1.8R + 15.0-18.0Y), which varied significantly (p<0.05)
within the varieties analyzed. The results indicate that these oils could be used for edible
applications. Color intensity of vegetable oils is mainly attributed to the presence of
various pigments such as carotenoids and chlorophyll which can remove along with the
oil during extraction. Such pigments are successfully removed during bleaching and
refining processing of oils. The vegetable oils with minimum color are more acceptable
84
for domestic and edible applications. No earlier reported literature values for color index
of apple seed oil are available to compare the results of present study. Free fatty acids and
Peroxide values have been commonly used as most important parameters to monitor the
quality of edible oil.
The acidity and peroxide values of apple seed oils were in the range of 0.5-1.4% and 1.1-
2.9 meq O2 / kg of oil. These values were found to be significantly (p<0.05) lower than
that reported in literature (Tian et al., 2010). The lower acidy and peroxide values of
apple seed oil showed that these oils could have long shelf life and used as edible oils.
Iodine value is the measure of degree of unsaturation of the oil, the iodine values of apple
seed oil was in the range of (96.3-115.4 of I2/100g oil) and comparable with the values
reported by Tian et al., (2010). The iodine value of Pyrus malus apple (115g of I2/100g
oil) was higher indicated that this oil contained a considerable amount of unsaturated
fatty acids as shown by its high level of linoleic and oleic acids (Table 4.5.1.).
The seed oil of different apple seed varieties from Pakistan had relatively low oxidative
measures (Table 1). The specific extinctions at 232 and 270 nm, which showing the
oxidative deterioration and purity of the oil (Anwar et al., 2006), of apple seeds, were
1.9-3.4 and 0.4-0.8, respectively. No reported data for specific extinctions of apple seed
oil are available to compare the results of present study.
In present study the saponification values (181.2-198.6 mg of KOH/g of oil) of apple
seed oils, differed significantly (p<0.05) among the varieties analyzed and comparable
85
with those reported by Tian et al., (2010). When compared with some non-conventional
and conventional oilseed crops, the saponification values in apple seed oils were found to
be quite similar to those of rice bran (179–195) oils, corn (187–195), olive (184–196),
cottonseed (189–198), soybean (188–195) and pumpkin (185–198) oils.
The unsaponifiable matter (0.9–1.4%) determined in present study of apple seed oils were
comparable with those of olive (0.7–2.5%), corn (0.5–2.8%) and cotton seed (0.5–1.5%)
oils, but within the range of groundnut (0.2–0.8%), safflower (0.3–1.5%), palm (kernel)
(0.2–0.8%), palm fruit (0.3–1.2%), low erucic acid rapeseed(0.2–1.8%) and high-erucic
acid rapeseed (0.2–2.0%) oils (Rossell, 1991). No earlier reports are available on the
quantification of unsaponifiable matter of apple seed oils to compare the results of
present analysis
4.5.5. Proximate composition of apple seed varieties
Proximate composition of the apple oilseed residues (Table 4.4.5) revealed a high protein
content of the seeds, ranging from 34.8 to 39.8%, vary significantly (p<0.05) among
analyzed varieties were comparable with literature values (Yu et al., 2007; Tian at al.,
2010). The Pyrus malus exhibited the highest protein contents (38.9%), whereas, Royal
gala had lowest protein contents (34.8%). Yu et al., (2007) and Tian at al., (2010)
reported the protein contents of apple seeds from China to be 33.8-34.5% and 38.8-49.5%
respectively.
86
Table 4.4.5. Proximate composition of Apple seed residue
Constituents (%)
Royal Gala Red Delicious Pyrus Malus Golden Delicious
Oil content 27.2±0.8 27.6±0.5 28.9±0.6 26.8±0.4 Moisture 5.2±0.1 4.9±0.2 5.3±0.1 4.7±0.2 Protein 34.8±1.8 36.5±1.2 39.8±1.7 37.7±1.4 Ash 3.5±0.4 3.8±0.5 4.2±0.9 3.4±0.7 Fiber 3.3±0.5 3.5±0.3 4.1±0.5 3.7±0.5 Carbohydrates 29.3±1.4 27.2±2.1 21.8±1.3 27.4±1.7
Values are mean ± SD of three seed oils of each apple varieties, analyzed individually in triplicate, Different letters in superscript indicate significant differences within apple varieties
The fiber and ash content of the apple seeds of different apple varieties ranged from 3.3-
4.1% and 3.5-4.2%, respectively. Tian et al. (2010) reported the fiber (3.9-4.3%) and ash
content (4.3-5.2%) respectively. Such variation in the concentrations of nutrients among
varieties and species may be associated to the variations of maturity stage, cultivated
regions and storage conditions. It could also be due to the climatic and geographical
differences where apple seeds had been grown (Atta et al., 2003)
87
Chapter -05
CONCLUSION
The present study provides quantitative and qualitative nutritional data of B. purpurea oil
and meal, which assess their potential for the useful applications. The results of present
study indicated that Bauhinia seed varieties contained significant amount of oil which is
comparable to soybean and cotton seeds. The extracted oil showed a reasonable ratio of
saturated and unsaturated fatty acids. The presence of appreciable level of essential fatty
acids, tocopherols, sterols and other favorable physiochemical characteristic make the
Bauhinia oil nutritionally viable for human health. The Bauhinia seed residue (meal)
could be used as a source of protein in the manufacturing of poultry and animal feeds.
The results of present study for the determination of the oxidative stability of Bauhinia
along with two conventional vegetable oils have shown a high correlation between DSC
T0 values and OSI values. Both methods confirmed that Bauhinia oil is very stable oil
when compared to rice bran and cottonseed oil. Due to considerable oxidative stability,
Bauhinia oil may find some appropriate applications in future. Furthermore, the DSC
method offers simplicity without using any chemical and time saving nature. Therefore,
DSC method can be easily used as an alternative technique for the measurement of
oxidative stability in edible oil processing industries where mostly OSI technique is used.
The data subjected to chemometric analysis on minor constituents shows better
discriminate within regions than fatty acids. Increasing the number of variables by using
88
fatty acids, sterols and tocopherols altogether further improve the interpretability. LDA
was found better model than PCA for discrimination of B. pupurea seed oil. Results of
the current study indicated that discrimination patterns are composition dependant and
must be optimized to explore better chemotaxonomic marker using chemometric
techniques.
The composition of the apple seed oil was performed on GS-MS and gave valuable
information. The results of the present study confirmed that fatty acids and
unsaponifiable components are genuine parameters and indicators of the quality of oil
and also suitable in chemometric techniques for the classification of apple seed varieties.
Fatty acid composition of the apple seeds oil revealed that a considerable amount of
essential fatty acids and lipid bioactives are present in the apple varieties. The high level
of linoleic acid content makes the oil nutritionally and industrially viable. Due to the
significant level of unsaponifiable components such as tocopherols and sterols the apple
seed oil could be used in the functional foods.
89
Chapter -06
RECOMMENDATION
1. Generally, due consideration should be given to plantation throughout the country for a better green environment.
2. Attention should be focused on the production of new quality oilseeds especially on legume trees to obtain maximum yield per acre and to reduce heavy import bill of edible oils and oilseeds in Pakistan. .
3. Apple juice factory wastes containing seeds should not be discarded. Its proper
utilization could be beneficial in the value addition of their product.
4. Growers should grow best available varieties of oilseeds to obtain maximum yield of oil per acre.
5. Over all edible oil consumption should be decreased by general public for better health and to decrease huge amount of foreign exchange utilized on its import.
90
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