I= STUDIES ON COMPOSITION AND METABOLISM OF LIPIDS IN SEEDS OF CASSIA SPECIES
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Title of Thesis
STUDIES ON COMPOSITION AND METABOLISM OF LIPIDS IN SEEDS OF CASSIA SPECIES
Author(s)
Shahina Zaka
Institute/University/Department Details
University of the Punjab, Lahore
Session
1988
Subject
Chemistry
Number of Pages
260
Keywords (Extracted from title, table of contents and abstract of thesis)
lipids, cassia species, cassia absus, cassia fistula, cassia occidentalis, carotenoids, tocopherols, triacylglycerol, methyl esters

Abstract
Variation in the total lipid contents, 1ipid class and their fatty acid composition were studied in the seeds of Cassia absus, Cassia fistula and Cassia occidentalis during the various stages of maturation and germination. The structure of triacylglycerols and the position of the ethylenic double bonds in the fatty acids were also determined. Variation in the contents of carotenoids, tocophero1s sand activities of lipase and phosphalipase enzymes were also studied during maturation.

The seeds of C.absus and C.occidentalis matured in 12 weeks after flowering (WAF) and of C.fistula in 24 WAF. The total lipid contents increased along with maturation from 0.71% and 0.60% (both at 2 WAF) and 0.48% (at 4 WAF) to 4.4% and 3.4') % (both at 12 WAF) in C.absus, C.occidentalis and 3.78% (at 24 WAF) in C.fistula on a dry weight basis.

The total lipids obtained at various maturity stages were fractionated into total neutral and total polar lipids in a silicic acid column with hexane:diethyl ether (70:30) followed by diethyl ether alone. The contents of total neutral lipids increased in the given maturity stages of the three Cassia species while contents of total polar lipids decreased during this period accordingly.

The total neutral lipids were fractionated by TLC using hexane:diethyl ether:acetic acid (80:20:2) as developing sol vent. The 1ipid classes separated were hydrocarbon, wax esters, triacylglycerol, free fatty acids and partial glycosides. These fractions were quantified by combined TLC-GC analysis. Triacylglycerol, the major class of neutral lipids, increased from 4.65% to 27.29% in C.absus 3.21% to 19.9% in C.fistula and 2.35% to 21.11% in C.occidentalis. The contents of all the other classes of neutral lipids decreased during seed maturation.

The total po1ar Lipids were separated into various fractions by TLC by double development with the solvent system chloroform: methanol: water (65:30:5) to 1/4he of plate, followed by development with hexane: diethyl ether: acetic acid (80:20:1.5). The polar lipid classes separated were glyco lipids, phospholipids and their lysoderivatives. During seed maturation the contents of phosphatidyI choline increased whereas the contents of the other polar lipid: classes were decreased in the three species.

During the initial stages of seed germination, the total 1ipid contents decreased rapid1y in the cotyledons of C.absus, C.fistula and C.occidentalis. However, the total lipid contents increased gradually in the cotyledons of the three Cassia species as the root length increased to 30 mm.

The total lipid contents were reduced in the primary root of different lengths 5, 10,15,20,25 and 30 mm during germination. Lipid contents in various parts of the primary root grown to 30 mm were lower in the regions close to the cotyledons than in the parts towards the growing tips of the roots. During germination the contents of the total neutral lipids decreased from 49.89% at the resting stage to 45.49% in the cotyledons of C.absus as.30 mm root length. The corresponding variations observed in C.fistula and C.occidentalis was similar. The contents of the total polar lipids increased accordingly in the three species. As compared to the cotyledons the total neutral lipids decreased while those of total polar lipids increased very significantly in the primary roots. The contents of the total neutral lipids were high in the growing tips of the primary root and their levels decreased towards the base of the root whereas the levels of total polar lipids) increased accordingly in the primary roots of the three species.

The percentage of triacylglycerol, the major class of natural lipids decreased in the cotyledons of C.absus from 28.20% at the resting stage to 25.09% when the primary root length was 30 mm. Similar variations in C. fistula and C. occidentalis were observed. The contents of free fatty acids increased from 3.82%, 1.43% and 2.76% at resting stage to 5.14%, 2.06% and 4.1% in the germinated cotyledons of C.absus, C.fistula and C.occidentalis at 30 mm root length whereas the contents of all the other neutral lipid classes showed reduction during the process of germination. the quantities of phosphatidyl choline i.e. 19.40% at the resting stage of C.absus reached upto 21.31% when the primary root increased to 30 mm. The corresponding variations in C.fistula and C.occidentalis were 23.41% to 25.50% and 23.47% to 27.77%, respectively. The contents of other polar lipids showed a decrease in the germinating cotyledons of the three Cassia species.

The variations observed in the neutral and polar lipids of the whole primary roots were quite similar to the cotyledons of the three species. The amounts of triacylglycerols remained constant in various parts of the primary root of the three C1ssia species but the quantities of free fatty acids decreased in the growing tip of the root as compared to the base of the root of the three Cassia species. Polar lipids showed low quantities in the growing tip of the root but their levels increased gradually towards the base of the root in the three cassia species.

The major fatty acids in the total lipid of the developing seeds of C.absus, C.fistula and C.occidentalis as determined by GC were palmitic (C 16: 0), oleic (C 18: 1) and lino leic (C 18: 2). The contents of C 16: 0 were reduced at 12 WAF in C.absus and C. occidentalis at 24 WAF in C. fistula. The percentages of C 18: 2 i.e. 26.10% and 10.50% in C. fistula and C. occidentalis (both at 2 WAF) and 14.60% in C. fistula (at 4 WAF) were increased to 51.50% and 31.58% (both at 12 WAF) and 28.2% (at 24 WAF) in C. sbsus, C. occidentalis and C. fistula respectively.

During germination C16:0 showed an increase in the cotyledons of the three species at 5 mm root length. The contents of C18: 2 were reduced from 51.50% at the resting stage to 51.09% in the cotyledons of C.absus when the primary root increased to 5 mm. The corresponding reduction was 28.20% to 20.0% and 31.58% to 29.17% in C.fistula and C.occidentalis respectively. The quantities 15f C16:0and C18:2 did not change significantly as the root length increased to 30 mm. Changes in C16:0 and C18:2 were manifold in the whole primary roots. An increase in C16:0 and reduction in C18:2 contents were observed from the tip to the base of the primary root of the three species. The triacylglycerols of the matured seeds of the three species were separated into six fractions (I-VI) according to the degree of instauration by argentation-TLC and their quantities were determined in C.absus, C.fistula and C.occi dentalis. The major fatty acids detected were C16:0, C18:1, C18:2, and C18:3. The more saturated fractions contained higher amounts of palmitic and oleic acids while the quantities of C18:2 and C18:3 increased with increase in the degree of unsaturation.

The seed oils of C.absus, C.fistula and C.occidentalis have been studied for the oxidative degradation of their unsaturated fatty acids by Von Rudloff's oxidation technique. The composition of the unsaturated fatty acids found by GC techniques showed quantities of monoenoic acid as 13.21% in C.absus, 22.41% in C.fistula and 15.80% in C.occidentalis. The percentages of dienoic acid found were 51.50%, 28.20% and 31.58%) respectively and 'trienoic acid contents were 2.32%, 1.50% and 3.14% in the seed oils of the three species. Total fatty acids were converted to their methyl esters and were separated into various fractions by argentation TLC. Each fraction was oxidised by modified Von Rudloff's method of oxidation to mono and dicarboxylic fatty acids which were analysed by TLC and GC analysis to determine their percentages and chain lengths. Thus, the position of double bonds in the constituent fatty acids were concluded as ∆ 9: 10 in monoenoic, ∆ 9: 10 and ∆ 12: 13 in dienoic and ∆ 9: 10 ∆ 12: 13 and ∆ 15: 16 in trienoic-fatty –acids. The position of ethylenic double bonds, therefore, showed the presence of usual fatty acids and there was not any positional isomer present in any of the three seed oils.

Changes in carotenoids, estimated by the solvent extraction and taking 0.D at 450 mu, declined from 0.68% to 0.05% in C.absus, 0.32% to 0.11% in C.occidentalis between 2-12 WAF. The decrease in C.fistula seeds during 4-24 WAF was 0.74% to 0.15%. The tocopherol contents in the unsaponifiable increased in the three species from 0.09% to 0.47% in C.absus, 0.12% to 0.60% in C.fistula and 0.90% to 0.61% in C.occidentalis between the stages mentioned above.

Both the mature and developing seeds showed the presence of two acids lipases with pH optima 3.0 and 4.0 and a phospholipase with pH optimum 5.6. During maturation the lipase activities decreased from 7.6 uU and 6.13 uU per 20 seeds in C.absus and C.occidentalis (both at 2 WAF) to 3.3 uU and 3.82 uU per 20 seeds between 4-24 WAF) In C.fistula the reduction was from 5.64 uU to 3.84 uU per 20 seeds between 4-24 WAF. The activities of phospholipase were increased in the maturing seed of the three species. The relative activities of the two enzymes studied in the various solvent systems were found to be similar.
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S. No. Chapter Title of the Chapters Page Size (KB)
1 1 ABSTRACT 1
77.41 KB
2 2 Introduction 8
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  2.1 General 8
  2.2 Lipids And Fatty Acids During Seed Maturation 16
  2.3 Lipids And Fatty Acids During Seed Germination 23
  2.4 Fatty Acids Distribution In Triacylglycerols 28
  2.5 Characterisation Of Ethylenic Double Bonds 34
  2.6 Carotenoids And Tocopherols During Seed Maturation 37
  2.7 Lipase And Phospholipase Activities 40
3 3 Materials And Methods 47
151.62 KB
  3.1 Plant Materials 47
  3.2 Lipid Extraction 48
  3.3 Characterisation Of Lipids By Chromatography 49
  3.4 Fractionation Of Triacylglycerol 51
  3.5 Enzymatic Hydrolysis Of Triacylglycerol 53
  3.6 Saponification Of Cassia Lipids 54
  3.7 Fractionation Of Methyl Esters 54
  3.8 Oxidation Of Methyl Esters 55
  3.9 Isolation And Estimation Of Carotenoids And Tocopherols 56
  3.10 Isolation Of Lipase And Phospholipase 57
  3.11 Lipase Activity 58
  3.12 Phospholipase Activity 59
  3.13 Phospholipase Specificity 60
4 4 Results 62
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  4.1 Lipids Of Matured And Developing Seeds 62
  4.2 Lipid Classes Of Matured And Developing Seeds 72
  4.3 Lipids Of Cotyledons And Primary Roots 81
  4.4 Lipid Classes Of Cotyledons And Primary Roots 87
  4.5 Fatty Acids Of Resting Seeds 108
  4.6 Fatty Acids Of Whole Lipids During Seed Maturation 109
  4.7 Fatty Acids Of Lipid Classes 111
  4.8 Acids Of Cotyledons And Primary Roots 144
  4.9 Fractionation Of Triacylglycerol 162
  4.10 Quantification Of 2-Monoacylglycerol 166
  4.11 Fractionation Of Methyl Esters 171
  4.12 Oxidation Of Methyl Esters 173
  4.13 Carotenoids And Tocopherols During Seed Maturation 175
  4.14 Effect Of Ph On Lipase Activity 178
  4.15 Lipase Activity During Seed Maturation 182
  4.16 Effect Of Solvents On Lipase Activity 182
  4.17 Effect Of Ph On Phospholipase Activity 186
  4.18 Phospholipase Activity During Seed Maturation 189
  4.19 Effect Of Solvents On Phospholipase Activity 189
  4.20 Phospholipase Specificity 192
5 5 Discussion 194
334.77 KB
6 6 References
219.31 KB