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Fatty acids and sterols of coffee and mint suspension cultures Van de Voort, Frederik Robert 1974

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THE FATTY ACIDS AND STEROLS OP COFFEE AND MINT SUSPENSION CULTURES by F r e d e r l k Robert van de Voort B.Sc.(Agr.), U n i v e r s i t y of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Food Science We accept t h i s t h e s i s as conforming to the req u i r e d standard. THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 197^ In presenting t h i s thes is i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I fur ther agree that permission f o r extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representat ives . I t i s understood that copying or p u b l i c a t i o n of t h i s thes is f o r f i n a n c i a l gain s h a l l not be allowed without my wri t ten permission. Department of ^roo ^  '^ia.c.x <?tsi The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date yog 14 i i ABSTRACT The c e l l s of two p l a n t s , Coffea a r a b i c a and an unknown Mentha species , were grown as suspension c u l t u r e s i n l i q u i d media, i n order to analyse and compare the f a t t y a c i d s and s t e r o l s of the c e l l c u l t u r e s to those found i n the parent p l a n t s . The c e l l growth, the parameters of pH and c o n d u c t i v i t y of the media, and the composition of the n e u t r a l l i p i d f r a c t i o n were examined. In the case of the coffee c e l l c u l t u r e s , the c e l l growth and the media pH and c o n d u c t i v i t y were studied i n three d i f f e r e n t media, two defined and one undefined, wh i l e the mint c e l l c u l t u r e s were studied i n one other d e f i n e d medium. Both c o f f e e and mint c e l l c u l t u r e s were grown i n the absence of l i g h t (normal c u l t u r a l c o n d i t i o n s ) and i n the presence of l i g h t . The coffee c e l l s showed no d i f f e r e n c e s i n growth r a t e due to v a r i a t i o n s i n media composition. Exposure to l i g h t a f f e c t e d n e i t h e r the growth r a t e nor i n i t i a t e d c h l o r o p h y l l formation i n the coffee c e l l c u l t u r e s , although a d i s t i n c t green pigmentation formed i n the mint c e l l s . A p l o t of the i o n i c c o n d u c t i v i t y of coffee and mint c e l l suspension c u l t u r e s was e s s e n t i a l l y a m i r r o r image of the growth curve of the r e s p e c t i v e c u l t u r e . The f a t t y a c i d s present i n the n e u t r a l l i p i d f r a c t i o n of the c e l l s were stud i e d v i a gas chromatography, and were compared to the f a t t y acids found i n the seeds and tissues of the parent plants. Palmitate, stearate, oleate, l i n o l e a t e and linolenate were found i n a l l coffee and mint c e l l cultures, independent of the composition of the media and of the presence of absence of l i g h t . The appearance of short chain f a t t y acids (less than C - l6) occurred during the dying phases of culture. The f a t t y acid composition of the coffee c e l l cultures resembled the analyses of the l e a f and stem tissues of the coffee plant rather than the coffee bean. The c e l l cultures a l l contained linolenate, not found i n the coffee bean, and lacked arachidate, which was present i n the bean. In contrast to the coffee c e l l , the mint c e l l f a t t y acids resembled the f a t t y a c i d composition of the mint seed rather than the parent plant tissues which contained substantial quantities of the short chain f a t t y acids (less than C -16). The t o t a l f a t t y acid content of the coffee and mint c e l l cultures was lower than the seeds of the parent plant, but was comparable to parent plant tissues. A decrease i n the t o t a l f a t t y acid content of the neutral l i p i d f r a c t i o n of both cultures was noted during the death phase of culture. The t o t a l f a t t y acid content of the coffee c e l l cultures was not altered by changes i n media composition, nor by growth of the cultures i n the presence of l i g h t . However, the growth of mint c e l l cultures l n the presence of l i g h t had a marked e f f e c t on the f a t t y a c i d content, which increased approximately four f o l d i n comparison to cultures grown i n the dark. The s t e r o l composition of the unsaponifiable l i p i d found i n the extracts of coffee and mint c e l l cultures was i v i n v e s t i g a t e d v i a gas chromatography and t h i n l a y e r chromatography. The s t e r o l s present were compared to those found i n the seeds of the parent p l a n t s . The s t e r o l s found i n l a r g e c o n c e n t r a t i o n i n the coffee c e l l s werep - s i t o s t e r o l , s t i g m a s t e r o l and campesterol, while i n the mint c e l l s only JB - s i t o s t e r o l was conspicuous. The predominant s t e r o l s found i n the seeds of the parent p l a n t s and i n the pl a n t c e l l c u l t u r e s were i d e n t i c a l . However, the c e l l c u l t u r e s of both coffee and mint contained l a r g e r amounts of s t e r o l s i n t h e i r s a p o n i f i e d l i p i d e x t r a c t s than found i n the seeds. Furthermore, i n comparison to the seeds, the l i p i d e x t r a c t s of the c e l l c u l t u r e s contained greater q u a n t i t i e s of u n e s t e r i f i e d s t e r o l s . A wide v a r i e t y of s t e r o l s other than desmethyl s t e r o l s were located i n the seeds and c e l l c u l t u r e s , but were not i d e n t i f i e d as they c o n s t i t u t e d only a minor p o r t i o n of the t o t a l p l a n t s t e r o l s present. A l a r g e p o r t i o n of the u n s a p o n i f l a b l e s of the coffee bean was c h a r a c t e r i z e d as n o n - s t e r o i d a l . This n o n - s t e r o i d a l m a t e r i a l was i d e n t i f i e d as a mixture of two d i t e r p e n o l d a l c o h o l s , c a f e s t o i and kawheol, both known to be major c o n s t i t u e n t s of the u n s a p o n i f l a b l e s of coffee bean o i l . The co f f e e c e l l o i l u n s a p o n i f i a b l e s were a l s o found to c o n t a i n these two d i t e r p e n o i d a l c o h o l s . V ACKNOWLEDGEMENT I would l i k e to express my g r a t i t u d e to Dr. P.M. Townsley f o r h i s i n t e r e s t and guidance d u r i n g the course of t h i s study. Thanks are a l s o expressed to Dr. W.D. Powrie, Dr. J.P. Richards and Dr. CA. Hornby f o r t h e i r suggestions. I would a l s o l i k e to acknowledge the help given me by my w i f e . v i TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 I . S t e r o l s and t r i t e r p e n o i d s i n p l a n t t i s s u e c u l t u r e s . . . k I I . The f a t t y a c i d s of p l a n t t i s s u e c u l t u r e s . . 6 I I I . F a t t y a c i d s and s t e r o l s i n coffee bean o i l . 8 IV. F a t t y a c i d s and s t e r o l s of mint p l a n t s . . . 9 MATERIALS AND METHODS 12 I . P l a n t o r i g i n , media and the pr e p a r a t i o n of c a l l u s and s i n g l e c e l l suspensions . . . . 12 A. O r i g i n of the p l a n t s 12 B. Media used f o r the production of c a l l u s c u l t u r e s of coffee and mint 12 C. Media used f o r the suspension c u l t u r e of coffee and mint 12 D. C a l l u s p r e p a r a t i o n and formation . . . . Ik i . Coffee c a l l u s Ik i i . Mint c a l l u s Ik . E. P r e p a r a t i o n of suspension c u l t u r e s . . . 15 I I . Measurement of c e l l u l a r growth 15 I I I . Measurement of the pH and c o n d u c t i v i t y of the medium . 18 IV. M a t e r i a l s and methods f o r f a t t y a c i d analyses 18 v i i Page A. Neutral l i p i d extraction 1 8 3. Saponification of the glycerides i n the hexane extracts, and methylation of the component f a t t y acids 1 9 C. Gas chromatography of f a t t y acid methyl esters 1 9 i . Analysis of f a t t y acids 1 9 i i . Quantitation of glyceride f a t t y acids using an i n t e r n a l standard 20 V. Materials and methods f o r s t e r o l "analyses . . 22 A. L i p i d extraction 22 B. Saponification 22 C. I s o l a t i o n of unsaponifiables 22 D. Thin layer chromatography 23 E. S l l y l a t i o n of sterols f o r gas chromatography 23 P. Gas chromatography of s t e r o l s 24-G. Column chromatography - the i s o l a t i o n of the dlterpenoid alcohols c a f e s t o l and kawheol from coffee bean unsaponifiables 26 H. C r y s t a l l i z a t i o n of c a f e s t o l and kawheol . 26 I. U l t r a v i o l e t spectroscopy . 26 EXPERIMENTAL RESULTS 2? I. Growth studies 27 A. Growth study of the coffee c e l l suspension cultures 27 B. Growth study of the mint c e l l suspension cultures 28 C. Discussion 33 v i i l Page II . The f a t t y acid composition and content of coffee and mint 3^ A. The f a t t y acid composition and content of coffee tissues and coffee c e l l suspension cultures . . . 3k i . O i l extraction from Coffea arablca tissues 3k i i . The f a t t y acid composition of the green coffee bean, the leaf and the stem of the coffee plant . . . 35 i i i . The f a t t y acid content of the coffee bean, the leaf and the stem 36 i v . The f a t t y a c i d composition of the coffee c e l l suspension cultures . 37 v. The f a t t y acid content of the coffee c e l l cultures 38 v i . Summary of f a t t y acid composition and content of coffee . . . . . . 39 B. The f a t t y acid composition and content of mint tissues and mint c e l l suspension cultures 4-0 i . O i l extraction of Mentha tissues . . kO i i . The f a t t y acid composition of the mint seed and the mint plant tissues kl i i i . The f a t t y acid content of the mint seed and the mint f o l i a g e and stem tissues . . . . . . . . k2 i v . The f a t t y acid composition of the mint c e l l suspension cultures . . 4-2 v. The f a t t y a c i d content of the mint c e l l suspension cultures 4-3 v i . Summary of f a t t y acid composition and content of the mint plant and tissue cultures kk i x Page C. D i s c u s s i o n 44 I I I . The s t e r o l composition of coffee beans, mint seeds and coffee and mint c e l l c u l t u r e s . . 46 A. Thin l a y e r chromatography (TLG) 46 1. TLC of s e l e c t e d s t e r o l standards . . . 47 i i . TLC of coffee bean o i l and coffee c e l l c u l t u r e o i l 47 i i i . TLC of mint seed o i l and mint c e l l c u l t u r e o i l 49 i v . Summary of t h i n l a y e r a n a l y s i s . . . . 50 B. Gas chromatography (GLC) 50 i . GLC of s e l e c t e d s t e r o l standards . . . 51 i i . GLC of the t o t a l u n s a p o n i f i a b l e s of coffee bean o i l and c e l l c u l t u r e o i l . 51 C. I s o l a t i o n and i d e n t i f i c a t i o n of a c a f e s t o l and kawheol mixture 52 i . Column chromatography 52 i i . Thin l a y e r chromatography of the chloroform methanol eluent 53 i l l . U l t r a v i o l e t spectroscopy of the c r y s t a l l i z e d mixture suspected to be c a f e s t o l and kawheol 53 i v . GLC of the c r y s t a l l i z e d mixture suspected to be c a f e s t o l and kawheol 53 D. Gas chromatography of the i n d i v i d u a l i o d i n e p o s i t i v e spots i s o l a t e d from the prepa r a t i v e t h i n l a y e r p l a t e s of the coffee bean and the coffee c e l l o i l unsaponif l a b l e s 63 1. GLC of the suspected d l t e r p e n o i d a l c o h o l spot 64 i i . GLC of the s t e r o l spot having the same Rf value as the desmethyl standards . 64 X Page I i i . GLC of the s t e r o l spot which i s l o c a t e d between the d i and desmethyl standards . . . 6k E. GLC of the t o t a l u n s a p o n i f l a b l e s of mint seed o i l and mint c e l l c u l t u r e o i l . . 65 i . Mint seed unsaponif l a b l e s 65 i i . Mint c e l l unsaponif l a b l e s 65 F. GLC of i n d i v i d u a l i o d i n e p o s i t i v e spots i s o l a t e d from pr e p a r a t i v e t h i n l a y e r p l a t e s of s a p o n i f i e d mint seed and mint c e l l o i l 65 1 . GLC of the mint c e l l s t e r o l spot having the same Rf value as the desmethyl standards . 65 I i . GLC of the mint c e l l s t e r o l spot l o c a t e d between the d i and desmethyl s t e r o l standards 75 i i i . GLC of the mint seed s t e r o l spot which covers the regions occupied by the d i and desmethyl s t e r o l standards 75 G. Summary of gas chromatography of the t o t a l u n s a p o n i f l a b l e s of coffee and mint and of gas chromatography of TLC i s o l a t e d s t e r o l spots of co f f e e and mint 75 H. D i s c u s s i o n 77 CONCLUSION 80 LIST OF REFERENCES 82 LIST OP TABLES Table Page 1. D i s t r i b u t i o n of coffee s t e r o l s 10 2 . Composition of PRL-4 medium 13 3 . Composition of Modified Fox medium 16 4 . Composition of Murashige and Skoog medium . . . 17 5 . O i l content of v a r i o u s Coffea a r a b i c a t i s s u e s . 35 6 . The f a t t y a c i d composition of the stems, leaves and beans of Coffea a r a b l ca 36 7 . The f a t t y a c i d content of the l e a f , the stem and the bean of Coffea a r a b i c a 37 8. The f a t t y a c i d composition of the coffee c e l l suspension c u l t u r e s grown i n Medium #1 . . . . 37 9 . The f a t t y a c i d content of Coffea a r a b i c a c e l l c u l t u r e s 39 1 0 . O i l content of v a r i o u s Mentha t i s s u e s 4 l 1 1 . The f a t t y a c i d composition of the mint seeds and mint f o l i a g e and stem t i s s u e s 4 l 1 2 . The f a t t y a c i d content of the mint seeds and mint f o l i a g e and stem t i s s u e s 42 1 3 . The f a t t y a c i d composition of mint c e l l c u l t u r e s . 43 14. The f a t t y a c i d content of mint c e l l c u l t u r e s . . 43 1 5 . Retention times of s t e r o l standards 51 x i i LIST OF FIGURES F i g u r e Page 1. The s t r u c t u r e s of c a f e s t o l and kawheol 11 2. Graph of the standard curve 25 3. Growth curve of l i g h t grown c o f f e e c u l t u r e s . . 29 k. Growth curve of dark grown c o f f e e c u l t u r e s . . . JO 5. Growth curve of dark grown c o f f e e c u l t u r e s (low pH) . 31 6. Growth curve of dark grown mint c u l t u r e s . . . . 32 7. TLC of s e l e c t e d s t e r o l standards 5k 8. TLC of two standards and of samples of c o f f e e bean o i l and c o f f e e c e l l c u l t u r e o i l 55 9 . TLC comparison of s a p o n i f i e d and n o n - s a p o n i f i e d c o f f e e bean o i l 56 10. TLC comparison of s a p o n i f i e d and n o n - s a p o n i f i e d c o f f e e c e l l c u l t u r e o i l 57 11. TLC comparison of mint seed o i l and mint c e l l c u l t u r e o i l 58 12. TLC o£ s a p o n i f i e d mint seed and mint c e l l c u l t u r e o i l 59 13. Gas chromatogram of the c o f f e e bean u n s a p o n i f l a b l e s 60 Ik. Gas chromatogram of c o f f e e c e l l c u l t u r e u n s a p o n i f i a b l e s 6 l 15. Gas chromatogram of the c r y s t a l l i z e d mixture of c a f e s t o l and kawheol i s o l a t e d from s a p o n i f e d c o f f e e bean o i l 62 16. Gas chromatogram of c a f e s t o l and kawheol i s o l a t e d by p r e p a r a t i v e TLC from the c o f f e e bean 66 x l i i Page 17. Gas chromatogram of c a f e s t o l and kawheol I s o l a t e d by p r e p a r a t i v e TLC from the coffee c e l l c u l t u r e . . . . . 67 18. Gas chromatogram of desmethyl s t e r o l s i s o l a t e d by pre p a r a t i v e TLC from the coffee c e l l c u l t u r e , 68 19. Gas chromatogram of the desmethyl s t e r o l s i s o l a t e d by pre p a r a t i v e TLC from the coffee bean 69 20. Gas chromatogram of s t e r o l s (Rf between d i and desmethyl s t e r o l standards) i s o l a t e d by prep a r a t i v e TLC from the co f f e e bean 70 21. Gas chromatogram of s t e r o l s (Rf between d i and desmethyl s t e r o l standards) i s o l a t e d by prep a r a t i v e TLC from the coffee c e l l c u l t u r e . 71 22. Gas chromatogram of the mint seed unsaponif i a b l e s . 72 23. Gas chromatogram of the mint c e l l c u l t u r e u n s a p o n i f i a b l e s 73 24. Gas chromatogram of s t e r o l s (Rf between d i and desmethyl s t e r o l standards) i s o l a t e d by pre p a r a t i v e TLC from the mint c e l l c u l t u r e . . 74 INTRODUCTION R e l a t i v e l y few I n v e s t i g a t i o n s regarding the t i s s u e c u l t u r e of e i t h e r coffee or mint have been reported i n the l i t e r a t u r e . In 1970, S t a r l t s k y ^ 3 ) described the f i r s t c a l l u s c u l t u r e of c o f f e e . K e l l e r et a l A z ^ K In 1972, reported the production and r e l e a s e of c a f f e i n e i n the c u l t u r e medium by the c a l l u s of Coffea a r a b i c a . In the same year, Buckland studied the c a f f e i n e , chlorogenic a c i d and amino a c i d content of the same coffee s p e c i e s . The c u l t u r e of peppermint and spearmint as c a l l u s e s and suspension c u l t u r e s was f i r s t described by L i n and Staba ( 3 D ± n 1961. Further i n v e s t i g a t i o n s were a l s o undertaken by Staba ( 1 2 > ^ » 5 2 ) concerning the growth c h a r a c t e r i s t i c s , c h l o r o p h y l l production and the e f f e c t of a n t i b i o t i c s on mint t i s s u e c u l t u r e s . However, no analyses of c e l l u l a r c o n s t i t u e n t s of mint c e l l c u l t u r e s have been made. I t has been s t a t e d by T a t t r i e and V e l i k y ^ ^ ) t h a t " i t i s p o s s i b l e t h a t the r o u t i n e c u l t i v a t i o n of p l a n t c e l l s i n r a p i d l y growing suspension c u l t u r e s w i l l provide a u s e f u l system f o r the study 6f l i p i d metabolism, membrane s t r u c t u r e , membrane tr a n s p o r t and p e r m e a b i l i t y " . This statement r e f l e c t s the b a s i c nature of the research r e q u i r e d to e x p l o i t the p o t e n t i a l s y n t h e t i c c a p a b i l i t y of the c e l l . However, i n order to determine the s u i t a b i l i t y of p l a n t c e l l s f o r the 2 study of l i p i d metabolism, comparative data on the l i p i d c o n s t i t u e n t s of the p l a n t c e l l s and the parent p l a n t t i s s u e s w i l l be u s e f u l . The o b j e c t i v e of t h i s study was, t h e r e f o r e , to determine the f a t t y a c i d and s t e r o l composition of coffee and mint c e l l c u l t u r e s , and compare t h i s data to the composition of the parent p l a n t t i s s u e s . 3 LITERATURE REVIEW Pl a n t c e l l c u l t u r e has advanced co n s i d e r a b l y s i n c e Haberlandt (17) f i r s t formulated the theory of p l a n t c e l l c u l t u r e i n an e a r l y attempt to. study the problems of c e l l d i f f e r e n t i a t i o n and c e l l i n t e r r e l a t i o n s h i p s . Although Haberlandt's attempts to c u l t u r e p l a n t t i s s u e s were un-s u c c e s s f u l , h i s work provided the impetus f o r other workers I n t e r e s t e d i n the f i e l d . Twenty years l a t e r , the a c t u a l c u l t u r e of p l a n t t i s s u e i n the form of excised r o o t t i p t i s s u e was accomplished by Kotte(26 , 2 7 ) and Robblns(37>38) f but f u r t h e r work was l i m i t e d by the i n a b i l i t y to continuously c u l t u r e the p l a n t m a t e r i a l . In 19 3^, White ( 5 *0 s u c c e s s f u l l y c u l t u r e d excised tomato roots by changing the carbon source from dextrose to sucrose. Two years l a t e r , he reported the continuous c u l t u r e of N l c o t l n a on agar s o l i d i f i e d medium i n the form of an u n d i f f e r e n t i a t e d mass of t i s s u e termed a c a l l u s . In the decade f o l l o w i n g 1950, a number of workers reported the growth or presence of e s s e n t i a l l y s i n g l e c e l l s i n submerged c u l t u r e of a number of p l a n t s p e c i e s . Since t h a t time a l a r g e number of p l a n t species have been success-f u l l y grown as c e l l suspensions. Prom i 9 6 0 onwards, the development of a v a r i e t y of d e f i n e d media made I t p o s s i b l e to study the metabolism and production of p l a n t c e l l m etabolites under c o n t r o l l e d c o n d i t i o n s , thus e l i m i n a t i n g the e f f e c t s of unknown c o n s t i t u e n t s . 4 Today, a major emphasis i n the plant c e l l f i e l d i s the study of the production or presence of secondary metabolites which are associated with the parent plant. N i c k e l l ^ S ) defined secondary metabolites (products) as those compounds which are not produced by a l l plants, whose functions are not known, and which, while not e s s e n t i a l metabolites, have considerable b i o l o g i c a l a c t i v i t y . Examples of secondary metabolites which have been found i n plant c e l l tissue cultures are glycosides, a l k a l o i d s , a n t i b i o t i c s , steroids, pigments and f l a v e n o i d s ^ 6 ) % I. Sterols and trlterpenoids In plant tissue cultures Numerous papers have appeared i n the l a s t few years concerning the presence of s t e r o l s and triterpenoids and t h e i r related derivatives i n plant c e l l cultures. Benveniste^^ separated the s t e r o l s and triterpenes of Nlcotlna c a l l u s cultures, and i d e n t i f i e d the major constituents a s y 9 - s i t o s t e r o l , campesterol, stlgmasterol and cholesterol, with these components accounting f o r 150 mg/100 g dry weight c e l l s . Two minor constituents, c i t r o s t a d l e n o l and 28-nor c i t r o s t a d i e n o l , were also found. Diosgenin, an important s t a r t i n g compound f o r the manufacture of pharmaceutically Important stero i d compounds, was i s o l a t e d by Kaul and Staba^ 2^) from Dloscorea  deltoldea, a culture also found to contain stlgmasterol and campesterol. S i m i l a r l y , Heble et a l . ( 2 1 ) reported the presence of diosgenin and ^ - s i t o s t e r o l i n the cultures of Solanum  xanthocarpum, noting that the s t e r o l content i n the tissue cultures was higher than i n the parent plant. In a l a t e r 5 p a p e r ^ 2 0 \ the same authors found l u p e o l to be the major t r i t e r p e n o i d c o n s t i t u e n t of these c u l t u r e s . Tomita et al. ( 5 0 ) looked f o r sesquiterpenes and s t e r o l s i n the t i s s u e c u l t u r e s of Llndera s t r y c h n l f o l l a , and found the p h y t o s t e r o l s campesterol, s t i g m a s t e r o l and ^ - s i t o s t e r o l i n a r a t i o of 6 «1 »53 ' They a l s o noted that the a b i l i t y of s t e r o l and sesquiterpene s y n t h e s i s was not i n h i b i t e d by one year of t i s s u e s u b c u l t u r e . In t h e i r study of nine p l a n t species l n suspension c u l t u r e , T a t t r l e and V e l i k y ^ ) reported that r o u t i n e t h i n l a y e r work showed the presence of f r e e s t e r o l s and s t e r o l e s t e r s l n a l l c u l t u r e s , with no obvious d i f f e r e n c e s i n the t h i n l a y e r p a t t e r n s . In a more thorough study of the s t e r o l s present i n one species (Ipornoea s p ) , these authors found the c e l l s to co n t a i n ^ - s i t o s t e r o l , campesterol and s t i g m a s t e r o l , i n a r a t i o of 71*12 : 1 7 . Laseter et a l . ( 2 9 ) s t u d i e d the s t e r o l s of Pinus e l l l o t t i c a l l u s t i s s u e s i n comparison to the s t e r o l s present i n the seeds and s e e d l i n g s . C h o l e s t e r o l , desmosterol, campesterol, s t i g m a s t e r o l , ^ - s i t o s t e r o l and c y c l o a r t e n o l were present i n a l l the t i s s u e s , but lophenol and 24--methylenophenol were found only i n the seeds and s e e d l i n g s , ^ - s i t o s t e r o l was the major s t e r o l i n a l l the t i s s u e s and comprised e i g h t y percent of the t o t a l seed s t e r o l s , t h i r t y -e i g h t percent of the c a l l u s s t e r o l s and for t y - s e v e n percent of the s e e d l i n g s t e r o l s . A new area of study concerning the s t e r o i d s of pl a n t c e l l s i s the b i o t r a n s f o r m a t i o n of extraneous s t e r o i d s added to the c u l t u r e medium. Furuya et a l . reported 6 the a b i l i t y of suspension cultures of Nlcotlna tabacum and Sophora a n g u s t l f o l l a to convert progesterone to 5 pregnanolone palmitate. These authors also noted that t h i s was the f i r s t report of the a b i l i t y of higher plants to convert an exogenous steroid to i t s ester form. The conversion of 4-androstene -3»17 dione to 5 ^-androstan 3>S-ol-17 one and 5<* -androstan-3 j&* 17 P dione, by Dloscorea  deltoidea suspension cultures was reported by Stohs and El-01emy(^) f a n d recently Furuya et.-.al. (2*5) have shown that progesterone can be converted to a number of pregnane type compounds and t h e i r glycosides by D i g i t a l i s purpurea. Research of t h i s nature opens up the p o s s i b i l i t y of u t i l i z i n g plant c e l l s f o r the conversion of r e a d i l y a v a i l a b l e steroids to more valuable compounds, i n the manner microorganisms are used today. I I . The f a t t y acids of plant tissue cultures In recent years, a number of studies have been carried out on the f a t t y acids present i n plant c e l l cultures i n r e l a t i o n to the f a t t y acids of the seeds and parent plant material. Staba et al.(41) studied the f a t t y acid composition of the t r i g l y c e r i d e s of rape and turnip rape cultures i n comparison to the roots, stems, seedlings and seeds of the parent plant. Erucic acid, a major f a t t y acid present i n the t r i g l y c e r i d e s of the seed o i l of both plants, was missing from the plant c e l l cultures. The t r i g l y c e r i d e content of the stems, roots, seedlings and plant c e l l cultures of both plants was found to be low, one to f i v e percent of the t o t a l 7 l i p i d s . Weete(53) s t u d i e d the f a t t y a c i d s of habituated (normal) and teratoma (tumor) c a l l u s c u l t u r e s of N l c o t l n a and i t s s e e d l i n g s . He found the f a t t y a c i d d i s t r i b u t i o n f o r a l l three t i s s u e s to be s i m i l a r , but noted t h a t the i n d i v i d u a l f a t t y a c i d content of the l i p i d was d i f f e r e n t ; w i t h the seedlings c o n t a i n i n g 4-2.03 mg» teratoma c u l t u r e 1.31 mg and habituated c u l t u r e 0.40 mg ( a l l per gram dry weight t i s s u e ) . Davydova et al.(9.10), w h i l e studying the f a t t y a c i d composition of f l a x endosperm and the t i s s u e c u l t u r e s d e r i v e d from f l a x , noted t h a t the t i s s u e c u l t u r e s had low l i p i d l e v e l s and a low l e v e l of f a t t y a c i d s i n the l i p i d . The l i p i d l e v e l of cu l t u r e s grown i n l i g h t and dark stayed the same, but the f a t t y a c i d content rose i n the c u l t u r e s grown i n l i g h t . T a t t r i e and Veliky(^9) s t u d i e d the f a t t y a c i d composition of nine species of pla n t s i n suspension c u l t u r e l n order to f i n d a pr e f e r a b l e c u l t u r e to study l i p i d metabolism and membrane s t r u c t u r e . Thin l a y e r s t u d i e s i n d i c a t e d no obvious d i f f e r e n c e s i n t o t a l l i p i d c o n s t i t u e n t s i n any c u l t u r e . In comparing the f a t t y a c i d s of the c e l l suspensions of Ipornoea sp and of Glycine max to t h e i r corresponding r o o t s , stems, leaves and seeds, the f a t t y a c i d p a t t e r n of the c e l l suspensions was found to resemble the f a t t y a c i d p a t t e r n of the leaves i n both c u l t u r e s . The amount of l i p i d i n Ipornoea and Glycine was 0.33$ and 0.50$ r e s p e c t i v e l y ( f r e s h weight b a s i s ) . Laseter et al.(30) compared the f a t t y a c i d s of c a l l u s c u l t u r e s o f > Pinus e l l l o t t l to the f a t t y a c i d s of the seeds, needles and s e e d l i n g s . The f a t t y a c i d p a t t e r n of the c a l l u s e s 8 resembled t h a t of the needles and s e e d l i n g s , although the general f a t t y a c i d composition was s i m i l a r i n a l l m a t e r i a l s . The amount of l i p i d e x t r a c t e d from the i n d i v i d u a l t i s s u e s was 62 .02$ f o r the seeds, 5 .03$ f o r the stems, 7.4-3$ f o r the leaves and 4 , 0 1 $ f o r the c a l l u s e s . The authors make note of the f a c t t h a t the amount of l i p i d e x t r a c t e d from the Pinus c a l l u s was approximately two times higher than t h a t e x t r a c t e d from tobacco and soybean c u l t u r e s . I I I . F a t t y a c i d s and s t e r o l s i n co f f e e bean o i l The f a t t y a c i d composition of co f f e e bean (seed) o i l has been known f o r many years ( 5 » 7 » 1 3 » 1 9 ) . The major f a t t y a c i d c o n s t i t u e n t s of coffee bean o i l are p a l m i t a t e , s t e a r a t e , o l e a t e , l i n o l e a t e , l i n o l e n a t e and ar a c h i d a t e . Behenate i s present i n minor amounts and tr a c e s of m y r i s t a t e , p a l m i t o l e a t e , margarate and gadoleate have been detected. The f a t t y a c i d compositions of var i o u s c o f f e e species are a l l v ery s i m i l a r , w i t h no major d i f f e r e n c e s being observed due to c l i m a t i c or s o i l c o n d i t i o n s ( 7 ) . The o i l content of cof f e e beans ranges from seven to s i x t e e n percent, w i t h s e v e n t y - f i v e percent of the o i l being t r i g l y c e r i d e s ( 1 9 ) . Coffee o i l contains an unusually high content of u n s a p o n i f i a b l e matter (up to twelve p e r c e n t ) , the main c o n s t i t u e n t s of which are two d i terpenoid a l c o h o l s , c a f e s t o l (C20H28O3) a n d -kawheol (C20H24O3) ^ - ^ ' J . The s t r u c t u r e s of c a f e s t o l and kawheol have been e l u c i d a t e d by D j e r a s s i et al. ( 1 1 ), and are as shown i n Figure 1 . C a f e s t o l ( c a f e s t e r o l ) , which has been used as an a n t i - i n f l a m a t o r y agent i n the treatment of 9 rheumatoid a r t h r i t i s , and kawheol, also known as tetrahydro-cafestol, are present i n coffee o i l mainly as mono esters of fatty acids, with a small amount present as free alcohols. Two minor constituents of the unsaponiflables are phosphotides and phytosterol esters. Although the sterol composition of coffee o i l has been studied by several workers, the most comprehensive study i s a co-operative work done by the two groups of Nagasampagl and Rowe and Simpson and Goad, entitled "Sterols of Coffee"(35). The distribution of sterols i n coffee beans, as found by these authors, i s illustrated i n Table 1^35). The results of the work done by Nagasampagl et a l . show that the sterols, the majority of which are i n the esterlfied form, make up 5*b% of the li p i d s . The major sterols are campesterol, ^ - s i t o s t e r o l and stigmasterol, with the rest being present in comparatively minor amounts. IV. Fatty acids and sterols of mint plants There is apparently no literature available on the fatty acid composition of mint. In a textbook by Bonner <5). the l i p i d content of mint leaves i s stated as being S%* and ln a work by Vidal ( 5 1 ) f the fat content of Japanese mint seeds i s stated to be 25,28$ (w/w). Two papers mention the sterol content of mint plants. Shadakova et al.(**Q) analysed for sterols in Mentha piperita leaves, rejected plant material and macerated tissue l e f t over after d i s t i l l a t i o n of essential o i l s . Vitamin D2 and JB -sitosterol were identified using 10 t h i n l a y e r chromatography, and the I s o l a t e d c r y s t a l form of / 3 - s l t o s t e r o l and i t s benzoate were comparatively i d e n t i f i e d by spectrophotometry and m e l t i n g p o i n t . The authors mentioned tha t one s t e r o l was not i d e n t i f i e d . B attu et al.(3) detected the presence of y S - s l t o s t e r o l i n Mentha p i p e r i t a v i a t h i n l a y e r chromatography and v i s u a l i z a t i o n by spray reagents, but doubted i t to be a pure compound due to a l a r g e m e l t i n g p o i n t spread. Table 1. D i s t r i b u t i o n of c o f f e e s t e r o l s % from beans % from o i l S t e r o l Occurring Occurring T o t a l T o t a l as e s t e r s f r e e C y c l o a r t e n o l 1.2 0.2 1.4 8 24—Methylenecycloartanol 3.0 0.7 3.7 4 Cycloeucalenol 0.4 0.1 0.5 O b t u s i f o l l o l 0.4 0.1 0.5 24-Methylenelophenol 0.3 0.1> 0.4 1 C i t r o s t a d i e n o l 0.5 0.1 0.6 1 Campesterol 11.3 6.5 17.8 11 S i t o s t e r o l 33.5 P29.3 52.8 53 S t i g m a s t e r o l 9.3 12.8 22.1 21 59.9 . 39.9 99.8 99 11 C a f e s t o l K a w h e o l F i g u r e 1. The s t r u c t u r e s o f c a f e s t o l a n d k a w h e o l 12 MATERIALS AMD METHODS I. Plant o r i g i n , media and the p r e p a r a t i o n of c a l l u s and  s i n g l e c e l l suspensions A. O r i g i n of the p l a n t s The two p l a n t s used throughout t h i s study were Coffea a r a b i c a and an u n i d e n t i f i e d Mentha species. Coffee t i s s u e m a t e r i a l was r e a d i l y a v a i l a b l e from the U n i v e r s i t y of B r i t i s h Columbia B o t a n i c a l Garden greenhouse where a number of coffee t r e e s are growing. Mint p l a n t s were not a v a i l a b l e but seeds and d r i e d p l a n t m a t e r i a l ( r e c e n t l y gathered f o l i a g e and stems) of w i l d mint from the Blue Mountains i n Colorado were obtained from the U n i v e r s i t y seed c o l l e c t i o n . B. Media used f o r the production of c a l l u s c u l t u r e s of coffee and mint The b a s a l agar s o l i d i f i e d medium PRL-4, as devised by Gamborg^ 1^, was used to I n i t i a t e t i s s u e c a l l u s c u l t u r e s of both p l a n t s . The composition of t h i s medium i s l i s t e d i n Table 2. C. Media used f o r the suspension c u l t u r e of coffee and mint Coffee suspension c u l t u r e s were i n i t i a t e d i n l i q u i d PRL-4 media. A f t e r suspension formation, the coffee c u l t u r e s were t r a n s f e r r e d i n t o two v a r i a t i o n s of Modified Fox medium.03). These two a l t e r e d media are termed Medium #1 and Medium #2, and d i f f e r from Modified Fox only i n t h e i r hormone composition. 13 Table 2. Composition of PRL-4 Medium^ 1 6^ Ingredient mg/l NaH2PO^ H 20 90 NagHPO^ 30 KCL 300 ( N H 4 ) 2 S 0 4 200 MgS0 4 • ?H 20 250 KNO^ 1000 C a C l 2 • 2H 20 150 KI 0.75 I r o n * 28 Micronutrlents+ 1.0 ml Vitamins++ 10.0 ml Sucrose 20.0 g N-Z Amine type A 2.0 mg 2,4-D 2.0 mg F i n a l pH 6.2 *Sequestrene 330 Fe (Gelgy A g r i c . Chem., Saw M i l l R i v e r Bd., Ardsley, N.Y.) •Stock s o l u t i o n . D i s s o l v e d i n 100 ml water* 1 g MnSO^ "H^O, 300 mg H^BO^, 300 mg ZnSO^ • 7H"20, 25-mg Na2MoO^ • 2H20, 25 mg CuSO^, 25 mg C o C l 2 • 6H20. ++Stock s o l u t i o n . D i s s o l v e d l n 100 ml H20« 10 mg n i c o t i n i c a c i d , 100 mg thiamine, 10 mg p y r i d o x i n e , 1 g m y o i n o s i t o l . 14 The composition of Modified Pox medium i s presented i n Table 3 i w i t h the hormone changes l i s t e d beneath the t a b l e . Mint suspension c u l t u r e s were grown o n l y i n Murashige and Skoog tobacco medium ( 3 4 ) , the composition of which i s shown i n Table 4 . D. C a l l u s p r e p a r a t i o n and formation i . Coffee c a l l u s Unripe coffee c h e r r i e s and r a p i d l y growing branch t i p s were taken from the co f f e e t r e e s , a t the U n i v e r s i t y of B r i t i s h Columbia B o t a n i c a l Garden. The t i s s u e s were cut i n t o small s e c t i o n s and placed i n t o a 5% h y p o c h l o r i t e s o l u t i o n to s t e r i l i z e any n a t u r a l m i c r o b i a l f l o r a . The t i s s u e pieces were a s e p t i c a l l y t r a n s f e r r e d i n t o a sequence of p e t r i p l a t e s c o n t a i n i n g s t e r i l e d i s t i l l e d water to remove r e s i d u a l hypo-c h l o r i t e from the t i s s u e s . The t i s s u e pieces were t r a n s f e r r e d i n t o s t e r i l e 100 ml d i l u t i o n b o t t l e s c o n t a i n i n g 20 ml of agar s o l i d i f i e d PHL -4 medium and the c u l t u r e s were then placed i n a temperature c o n t r o l l e d room, kept a t 28 °C, w i t h no l i g h t present. Within two weeks, c a l l u s formation was r e a d i l y apparent, and w i t h i n one month the c a l l u s e s were l a r g e enough to t r a n s f e r i n t o l i q u i d medium. C a l l u s propagation was f a c i l i t a t e d by monthly t r a n s f e r s . i i . Mint c a l l u s E x a c t l y the same s t e r i l i z a t i o n procedure described f o r coffee was used f o r the mint seeds, which were a l s o c u l t u r e d on s o l i d PRL - 4 medium. Most mint seeds d i d not germinate but went d i r e c t l y i n t o c a l l u s . Good c a l l u s 15 formation took approximately the same length of time as i n the case of the co f f e e t i s s u e s . E. P r e p a r a t i o n of suspension c u l t u r e s £11 suspension c u l t u r e s were grown i n 250 ml erlenmeyer f l a s k s c o n t a i n i n g 100 ml l i q u i d medium. C a l l u s c u l t u r e s of coffee were i n o c u l a t e d i n t o PRL -4 l i q u i d medium. The c u l t u r e s were grown and maintained i n the dark a t a temperature of 28°C and a r e l a t i v e humidity of 95%- A g i t a t i o n and a e r a t i o n of the c u l t u r e s were provided v i a a gyratory shaker 1 o p e r a t i n g a t 110 r.p.m. i n a one i n c h c i r c u l a r o r b i t . Suspension c u l t u r e formation evolved s l o w l y as the c e l l s were t r a n s f e r r e d weekly, and a f t e r two months the co f f e e c a l l u s e s formed a suspension of an "apple sauce" consistency. The mint suspension c u l t u r e never a t t a i n e d t h i s s t a t e but remained i n a r a t h e r granular form. I I . Measurement of c e l l u l a r growth C e l l u l a r growth of both c o f f e e and mint suspension c u l t u r e s was measured by the f o l l o w i n g procedure. A sequence of ei g h t d u p l i c a t e or t r i p l i c a t e f l a s k s was Inoculated w i t h a known inoculum, and harvested a t convenient I n t e r v a l s (every two to three days) by f i l t e r i n g out the c e l l s through M y r a c l o t h 2 , u s i n g a Buchner f u n n e l . The harvested c e l l s were free z e d r i e d i n a V i r t is3 f r e e z e d r i e r to a constant weight. xNew Brunswich S c i e n t i f i c Co., Inc. 2Chicopee M i l l s Inc., 1450 Broadway, New York 3vir t is Research Equipment Co., Gardiner, New York 16 Table 3... Composition of Modified Fox Medium(33) Ingredient NH^N03 KNO^ Ca ( N 0 3 ) 2 • 4 H 20 MgS0 4 • 7H 2 0 KH 2P0 i + KCL Na2EDTA • 2R"20 F e 2 ( S 0 4 ) 3 ZnSO^ • 7H20 MnSO^ • H 20 H 3 B 0 3 KI Glycine N i c o t i n i c a c i d P y r idoxine Thiamine I n o s i t o l Indole-3-acetate K i n e t i n Sucrose mg/1 1000 1000 500 300 250 50 35 25 7 . 5 5 . 0 5 . 0 0 .8 3 .0 0 . 5 0 . 1 0 . 1 100 2 0.05 30,000 Medium #1 - 2,4-D was added i n a con c e n t r a t i o n of 2 mg/1 Medium #2 - 2,4-D was added as the only hormone a t 2 mg/1 ( k i n e t i n and indole-3-acetate were e l i m i n a t e d ) Table .4.. .Composition .of Murashige and Skoog Medium(3^) Ingredient mg/l NH 4N0 3 1650 KN0 3 1900 H3B 0 3 6.2 KH 2P0 4 170 KI O.83 Na MoO, • 2R_0 2 4 2 0.25 C o C l 2 . 6H 2O 0.025 C a C l 2 • 2H 20 440 MgSO^ • ?H 20 370 MnSO^ * 4H 20 22.3 ZnSO^ • 7H 20 8.6 CuSO^ • 5H 2o 0.025 Na2EDTA 37.5 FeSOj,, • 7H20 27.85 Thiamine h y d r o c h l o r i d e 0.1 N i c o t i n i c a c i d 0.5 P y r i d o x l n e h y d r o c h l o r i d e 0.5 Glycine 2.0 Addendum: Sucrose 30 g / l i t e r M y o i n o s i t o l 100 mg Indole-3-acetic a c i d 10 mg K i n e t i n 0.04mg 1 8 The c e l l dry weights were p l o t t e d a g a i n s t Incubation time to o b t a i n the growth curve. I I I , Measurement of the pH and c o n d u c t i v i t y of the medium A f t e r the removal of the c e l l s f o r growth measurements, the e l e c t r o l y t i c c o n d u c t i v i t y of the medium was measured u s i n g a YSI model 31 C o n d u c t i v i t y Bridge. The hydrogen i o n a c t i v i t y was recorded u s i n g a pH meter. This procedure was c a r r i e d out a t each harvest i n t e r v a l . IV. M a t e r i a l s and methods f o r f a t t y a c i d analyses A. N e u t r a l l i p i d e x t r a c t i o n The n e u t r a l l i p i d f r a c t i o n of the p l a n t m a t e r i a l s was i s o l a t e d by e x t r a c t i o n w i t h hexane (b.p. 66-69"C), i n order to study the f a t t y a c i d composition of the g l y c e r l d e s present I n the l i p i d m a t e r i a l . Mint seeds and mint pla n t m a t e r i a l (a mixture of f o l i a g e and stem) were e x t r a c t e d w i t h petroleum e t h e r / d l e t h y l ether (50:50 v / v ) . P r i o r to l i p i d e x t r a c t i o n , a l l the p l a n t t i s s u e s , w i t h the exception of mint seeds and c o f f e e beans, were f r e e z e d r i e d . To f a c i l i t a t e the e x t r a c t i o n , the t i s s u e s were ground to pass through a 4-0 mesh s i e v e . A l l e x t r a c t i o n s were performed u s i n g a Goldberg e x t r a c t i o n apparatus. A f t e r the e x t r a c t i o n was completed (one hour), the e x t r a c t i n g solvent c o n t a i n i n g the p l a n t l i p i d s was f i l t e r e d or c e n t r i f u g e d a t room temperature to remove any p a r t i c u l a t e matter, i n c l u d i n g any i n s o l u b l e waxes. The solvent was removed usi n g a stream of dry n i t r o g e n and the r e s i d u a l l i p i d ( o i l ) weighed i n a t a r r e d v i a l . The 19 o i l weight was expressed as a percentage of the dry weight of the t i s s u e . B. S a p o n i f i c a t i o n of the g l y c e r i d e s i n the hexane e x t r a c t s , and m e t h y l a t i o n of the component f a t t y a c i d s S a p o n i f i c a t i o n and methylation of g l y c e r i d e f a t t y a c i d s were performed acco r d i n g to the method of Luddy et a l ( 3 2 ) . Their procedure, which i s recommended f o r the p r e p a r a t i o n of samples f o r gas chromatographic a n a l y s i s , i s a r a p i d and q u a n t i t a t i v e method of concurrent s a p o n i f i c a t i o n and methylation using sodium methoxide. The procedure r e q u i r e s only 1 to 3° mg of o i l to be placed i n a small v i a l , to which i s added 0 .25 ml of k% anhydrous sodium methoxide s o l u t i o n . The v i a l i s f l u s h e d with n i t r o g e n , sealed and heated to 65°C f o r two minutes, w i t h i n t e r m i t t e n t shaking. The v i a l i s cooled to room temperature and a 0 .6 g mixture of s i l i c a g e l and calcium c h l o r i d e (1»1 w/w) i s added and s t i r r e d i n t o the reactant s o l u t i o n . The s i l i c a g e l absorbs the f r e e f a t t y a c i d s and g l y c e r o l , while the calcium c h l o r i d e complexes any excess methanol. To d i s s o l v e the methyl e s t e r s , 3 ml of CS 2 are added and the s o l u t i o n i s c e n t r i f u g e d . The CS 2 supernatant i s then t r a n s f e r r e d to a new v i a l , the CS 2 volume i s reduced under a stream of n i t r o g e n gas to an approximate volume of 0 .9 ml, and an i n t e r n a l standard, octadecane, Is added. C. Gas chromatography of f a t t y a c i d methyl e s t e r s i . A n a l y s i s of f a t t y a c i d s The a n a l y s i s of the f a t t y a c i d methyl e s t e r s , d e r i v e d by the s a p o n i f i c a t i o n and e s t e r i f i c a t i o n of the o i l 20 e x t r a c t e d from the p l a n t c e l l s , seeds and t i s s u e s , was performed on a Becker Research Gas Chromatograph 3810 provided w i t h a flame i o n i z a t i o n d e t e c t o r . F a t t y a c i d s e p a r a t i o n was obtained by u s i n g a 15% d i e t h y l e n e g l y c o l succinate l i q u i d phase (HI-EFF IBP 1) on 80/100 mesh Gas Chrom P*- support. The column was made of s t a i n l e s s s t e e l , 6' long by 1/4" l . d . Commercially p u r i f i e d n i t r o g e n was used as the c a r r i e r gas a t a r a t e of 54 ml/mln, w i t h an isothermal column temperature of 180°C. The component f a t t y a c i d s were i d e n t i f i e d by co-chromatography w i t h the appropriate standards and by t h e i r corresponding r e t e n t i o n times. The areas under the peaks were c a l c u l a t e d by u s i n g the formula i height x width a t 1/2 peak height i i . Q u a n t i t a t i o n of g l y c e r i d e f a t t y a c i d s u s i n g an i n t e r n a l standard Method A — Q u a n t i t a t i o n of the g l y c e r i d e f a t t y a c i d s was performed by u s i n g octadecane as an I n t e r n a l standard, since octadecane does not i n t e r f e r e w i t h major f a t t y a c i d methyl e s t e r peaks. A standard curve was determine* by the f o l l o w i n g procedure. Two standard s o l u t i o n s , composed of 100 mg of octadecane i n 10 ml CS 2 and 10 mg methyl palmitate i n 1 ml CS 2 t were prepared. Standards were mixed i n known concentrations and 5 M! of mixed standard were subjected to gas chromatographic s e p a r a t i o n . A r a t i o , which was determined by d i v i d i n g the area under the p a l m i t a t e peak by the height 1» 2Applled Science L a b o r a t o r i e s , Inc. 21 of the octadecane peak, was p l o t t e d a g a i n s t the c o n c e n t r a t i o n of methyl palmitate (y a x i s ) , producing a l i n e a r r e l a t i o n s h i p passing through zero at zero c o n c e n t r a t i o n (Figure 2 ) . The equation of the l i n e was found to be: y=136x. Using an equimolar mixture of f a t t y a c i d methyl e s t e r s ^ , the areas under the v a r i o u s f a t t y a c i d peaks were found to be p r o p o r t i o n a l to t h e i r c oncentrations. The p l o t obtained from the recorder response was found to be l i n e a r , and thus the use of t h i s gas chromatographic q u a n t i t a t i o n technique allowed r e p r o d u c i b l e r e s u l t s to be obtained w i t h i n an acceptable e r r o r (* 10%). Method B — Q u a n t i t a t i o n was performed by c a r r y i n g out the s a p o n i f i c a t i o n and methylation r e a c t i o n , described i n Method A, on 1 mg of t r i p a l m i t a t e to which had been added 10 j j i l of octadecane, the i n t e r n a l standard. The methyl e s t e r s and the i n t e r n a l standard were taken up i n 3 ml of GS^. and i n j e c t e d i n t o the gas chromatograph. From the r e s u l t i n g gas chromatogram, the r a t i o of the height of the octadecane peak to the area under the palmitate peak was c a l c u l a t e d . The constant r a t i o obtained was considered to be equal to 1 mg of g l y c e r l d e f a t t y a c i d s . E x a c t l y the same procedure described above f o r t r i p a l m i t a t e was c a r r i e d out on mint t i s s u e l i p i d s , and the t o t a l area under the f a t t y a c i d peaks was r e l a t e d to the height of the octadecane standard, i n order to c a l c u l a t e the amount of g l y c e r l d e f a t t y a c i d s present i n the e x t r a c t e d 1 A p p l i e d Science L a b o r a t o r i e s , Inc. 22 l i p i d . When c a l c u l a t e d , the amount of f a t t y a c i d was converted to a percentage of the t o t a l weight of the l i p i d ipidergoing the r e a c t i o n . V. M a t e r i a l s and methods f o r s t e r o l analyses A. L i p i d e x t r a c t i o n A l l p l a n t m a t e r i a l s used f o r the analyses were ground to a s i z e s u f f i c i e n t to pass through a 4-0 mesh s i e v e . The e x t r a c t i o n of the s t e r o l s was performed u s i n g a 50/50 v/v mixture of petroleum ether and d i e t h y l ether i n a Goldberg e x t r a c t i o n apparatus. The sol v e n t c o n t a i n i n g the e x t r a c t e d o i l was c e n t r i f u g e d to remove any I n s o l u b l e matter, then the solv e n t was removed us i n g a dry stream of n i t r o g e n . The o i l residue y i e l d was recorded. B. S a p o n i f i c a t i o n The crude l i p i d e x t r a c t was added to 95$ ethanol c o n t a i n i n g 10$ of a 60$ KOH s o l u t i o n . The KOH-ethanol s o l u t i o n was r e f l u x e d f o r a minimum of one hour to saponify the l i p i d , and the p r e p a r a t i o n was cooled to room temperature. C. I s o l a t i o n of u n s a p o n i f i a b l e s The s a p o n i f i e d mixture was placed i n a separatory f u n n e l and d i l u t e d with f o u r volumes of water. This s o l u t i o n was e x t r a c t e d w i t h three successive a l l q u o t s of ether, and the ether p o r t i o n s were pooled and washed wi t h water three times to remove any r e s i d u a l base. Most of the ether was d i s t i l l e d o f f , and the remaining s o l u t i o n was put under vacuum i n a rotoevaporator to remove the remaining s o l v e n t and water. The r e s i d u a l m a t e r i a l was taken up i n d i e t h y l ether, f i l t e r e d 23 and. placed in a tarred v i a l . A stream of dry nitrogen was used to remove the solvent, and the weight of the un-saponif iable residue was recorded. D . Thin layer chromatography A l l thin layer chromatography was done using Polygram1 s i l G precoated plates, with a layer thickness of 0.25 mm. Unsaturated compounds were visualized by msing iodine vapour. The major solvent system used was chloroform: ethyl-acetate (9:1 v/v). Qualitative and semi-quantitative thin layers were run on both unsaponifiable material and the petroleum ether/diethyl ether l i p i d extracts of the various plant materials. Preparative thin layer plates were used to separate the unsaponifiable constituents. The areas corresponding to the iodine positive spots were eluted and derivatlzed for gas chromatography. E. Silylation of sterols for gas chromatography The sterols and diterpene alcohols in the unsaponifiable matter were converted to trimethylsllyl ether derivatives via the method of Sweeley et a l . ). Their method uses anhydrous pyridine as the reaction solvent, hexamethyldisilazane as the reactant and chlorotrimethylsllane as a catalyst. This reaction system converts easily accessible hydroxyl groups to trimethylsllyl ethers in the following manner: 3R0H + Me3SlNHSlMe3 + Me^SiCl »- 3R0SlMe + NH^Cl Macherey-Nagel and Co., Duren 24 A f t e r f i v e minutes of r e a c t i o n , a heavy p r e c i p i t a t e (HH^Cl) i s apparent. The r e a c t i o n v i a l i s then c e n t r i f u g e d to c l e a r the s o l v e n t f o r d i r e c t i n j e c t i o n i n t o the gas chromatograph. S a t i s f a c t o r y conversion was a t t a i n e d f o r the s t e r o l s by u s i n g a t o t a l of 0.5 ml reagents (8 mg sample or l e s s ) and 1.0 ml t o t a l reagents (greater than 8 mg sample) i n a constant r a t i o of 10i2:1 (v/v) of p y r i d i n e , hexamethyldisllazane and c h l o r o t r i m e t h y l s l l a n e r e s p e c t i v e l y . In some cases the p y r i d i n e solvent was removed wi t h a stream of dry n i t r o g e n a f t e r the r e a c t i o n was completed, and replaced w i t h another s o l v e n t such as carbon d i s u l p h i d e or iso-octane to reduce the solvent peak found i n gas chromatography. P. Gas chromatography of s t e r o l s Gas chromatography of s t e r o l s was performed u s i n g the Becker Research Gas Chromatograph type 3810. The column used f o r s t e r o l analyses was a ten f o o t , s t a i n l e s s s t e e l column c o n t a i n i n g a 5% OV-1 l i q u i d phase adsorbed on Gas Chrom s o l i d support. Nitrogen was used as the c a r r i e r gas a t a f l o w r a t e of 31*2 ml/min. The i n j e c t i o n p o r t temperature was 300°C, and i n order to f a c i l i t a t e the best p o s s i b l e s e p a r a t i o n of the p l a n t s t e r o l s , a temperature program of 240°C f o r 1.5 hours w i t h an increase i n temperature to 280°C f o r the remainder of the run, was f o l l o w e d . A constant chart speed of 0.1 cm/min was used due to the long d u r a t i o n of the run. I d e n t i f i c a t i o n of f o u r s t e r o l s was p o s s i b l e through the use of a v a i l a b l e standards and t h e i r corresponding r e t e n t i o n times. ^ A p p l i e d Science L a b o r a t o r i e s , Inc. R a t i o o f t h e a r e a under p a l m i t a t e p e a k : h e i g h t o f octadecane peak F i g u r e 2. Graph o f the s t a n d a r d c u r v e 26 G. Column chromatography - the i s o l a t i o n of the d i t e r p e n o i d a l c o h o l s , c a f e s t o l and kawheol, from coffee bean u n s a p o n i f l a b l e s F o l l o w i n g a s i m p l i f i c a t i o n of the procedure used by Nagasampagl et a l . (^ 5) f A ZIQ C M x i c m column c o n t a i n i n g 6 gm of Brockman grade I I I alumina i n petroleum ether was made, and a 100 mg sample of coffee o i l u n s a p o n i f l a b l e s was a p p l i e d to the top of the column. The column was e l u t e d w i t h 100 ml of petroleum ether to remove any hydrocarbons and then e l u t e d w i t h 100 ml of petroleum e t h e r i d l e t h y l ether (60:40 V / V ) to remove the s t e r o l s . Samples of the f i n a l drops of t h i s eluent were subjected to t h i n l a y e r chromatography to ensure complete removal of the s t e r o l s . To r e l e a s e the d i t e r p e n o i d a l c o h o l s ( 3 5 ) . the column was eluted w i t h chloroformimethanol (99:1 v / v ) . This f r a c t i o n was kept f o r f u r t h e r examination. H. C r y s t a l l i z a t i o n of c a f e s t o l and kawheol The chloroform:methanol f r a c t i o n obtained from alumina column chromatography was placed i n a rotoevaporator to remove the s o l v e n t s . The r e s i d u a l m a t e r i a l was r e f l u x e d i n petroleum ether f o r f i v e minutes, the s o l v e n t was t r a n s f e r r e d to another f l a s k and the petroleum ether was removed under vacuum. The residue was used f o r u l t r a v i o l e t spectroscopy. I . U l t r a v i o l e t spectroscopy The c r y s t a l l i z e d m a t e r i a l obtained from petroleum ether was run i n d i e t h y l ether, w i t h a d i e t h y l ether reference, on a Unlearn Sp. 800 B u l t r a v i o l e t spectrophotometer. 27 EXPERIMENTAL RESULTS I . Growth Studies A. Growth study of the c o f f e e c e l l suspension c u l t u r e s Growth s t u d i e s of c o f f e e c e l l suspension c u l t u r e s were I n i t i a t e d i n order to c h a r a c t e r i z e the growth p a t t e r n of the c u l t u r e s . The coffee c e l l s were grown i n three media, PRL-4 (an undefined medium) and two defined media, Medium #1 and Medium #2, which d i f f e r from each other only i n hormone composition. A sequence of e i g h t d u p l i c a t e or t r i p l i c a t e f l a s k s of each media was i n o c u l a t e d with 10 ml (0.2 g dry weight c e l l s ) of stock c u l t u r e s grown up i n t h e i r corresponding media. Two sets of c u l t u r e s were Inoculated as described above, one grown i n the dark (normal c u l t u r a l c o n d i t i o n s ) and the other grown i n the presence of l i g h t (9»840 lumens). C e l l s were harvested every two to three days, f r e e z e d r i e d and weighed. The pH and c o n d u c t i v i t y of the c u l t u r e medium were measured a t each harvest. The changes over a period of f o u r t e e n days i n c e l l d ry weight, pH and c o n d u c t i v i t y of the f r e e c e l l suspension c u l t u r e s of coffee grown In Medium #1, under c o n d i t i o n s of l i g h t or dark, are i l l u s t r a t e d i n Figures 3 and 4. The c e l l growth ciairves shown f o r Medium #1 i l l u s t r a t e the trends observed i n a l l three media, si n c e s i m i l a r r e s u l t s were obtained f o r c e l l s grown i n Medium #2 and i n PRL-4. Maximum c e l l y i e l d i n a l l c u l t u r e s occurred around day 8, w i t h the 28 " e x p o n e n t i a l " phase of growth o c c u r r i n g between days 4- and 8. The pH of a l l three media fo l l o w e d s i m i l a r p a t t e r n s , with a decrease o c c u r r i n g d u r i n g the f i r s t two days. During the "expon e n t i a l " phase of growth the pH of the supporting medium increased. This r i s e i n pH was noted i n a l l growth s t u d i e s . Adjustment of the pH of the medium t o pH 5 p r i o r to s t e r i l i z a -t i o n , i n order to give an i n i t i a l c u l t u r e pH of 4, produced a pronounced l a g i n the growth of the c u l t u r e s (Figure 5 ) . This l a g was e l i m i n a t e d by adjustment of the pH of the u n s t e r i l i z e d medium to provide a f i n a l pH of 5 to 6 f o l l o w i n g steam s t e r i l i z a t i o n . The c o n d u c t i v i t y data obtained f o r a l l coffee c u l t u r e s showed that c o n d u c t i v i t y was e s s e n t i a l l y a m i r r o r image of the growth curve. B. Growth study of the mint c e l l suspension c u l t u r e s The growth of the mint c e l l c u l t u r e s i n stock media was fo l l o w e d under the c o n d i t i o n s of l i g h t or dark, and the c o n d u c t i v i t y and pH were measured a t harvest I n t e r v a l s . The growth of the mint c e l l suspension c u l t u r e s i n the dark i s presented i n Figure 6. The r e s u l t i n g growth curve i l l u s t r a t e d i n Figure 6, along w i t h the parameters of pH and c o n d u c t i v i t y , were e x a c t l y the same as f o r the c u l t u r e grown i n the presence of l i g h t (Figure not shown). The only d i f f e r e n c e observed between l i g h t and dark grown mint c u l t u r e s was the development of a green pigmentation i n the c e l l c u l t u r e s grown l n the presence of l i g h t . As i n the co f f e e c u l t u r e s , the c o n d u c t i v i t y of the media i n v e r s e l y r e f l e c t e d the growth r a t e of the c u l t u r e . 33 C. D i s c u s s i o n Although B u c k l a n d g r e w coffee c e l l s as a suspension c u l t u r e i n B-5 medium, the author*s attempts to use t h a t medium were un s u c c e s s f u l . When PRL-4 and Medium #1 and #2 were employed, r a t e of growth and y i e l d of c e l l s were s u p e r i o r to Buckland*s(°") r e s u l t s . A l l three media used produced s i m i l a r r e s u l t s , i n d i c a t i n g that the v a r i a t i o n s i n composition d i d not i n f l u e n c e growth of the c u l t u r e . The f a c t that c o f f e e c e l l s grew continuously through successive t r a n s f e r s i n Medium #2 i n d i c a t e d t h a t 2,4-D was the major growth s t i m u l a t o r of the c u l t u r e s , and th a t the other two hormones were superfluous. This p o s t u l a t e was v e r i f i e d when the c u l t u r e was grown i n the presence of i n d o l e - 3 - a c e t i c a c i d and k i n e t i n alone, or without any of the hormones. Under those c o n d i t i o n s , growth ceased a f t e r the second t r a n s f e r . The growth r a t e of both the coffee and mint c e l l s was not a f f e c t e d by the presence of l i g h t . In both c u l t u r e s the c o n d u c t i v i t y data i n d i c a t e d an inverse r e l a t i o n s h i p between growth and c o n d u c t i v i t y , an observation noted by Hahlbrock et a l . (18) l n t h e i r study of p a r s l e y and soybean suspension c u l t u r e s . A comparable r a t e of growth was observed i n both c u l t u r e s , even though the mint c e l l s grew as granules r a t h e r than as " s i n g l e c e l l s " , as i n the case of c o f f e e . The only major d i f f e r e n c e observed d u r i n g the growth of co f f e e and mint c u l t u r e s was the development of a stro n g green pigmentation l n the mint c e l l s when the c u l t u r e was grown i n the presence of l i g h t . Dobberstein and S t a b a ^ 1 2 ^ reported a s i m i l a r pigmentation i n Japanese mint suspension c u l t u r e s grown i n the presence of l i g h t . 34 I I . The f a t t y a c i d composition and content of coffee and mint A. The f a t t y a c i d composition and content of c o f f e e t i s s u e s and coffee c e l l suspension c u l t u r e s A f t e r s a p o n i f i c a t i o n and methylation of the coffee t i s s u e l i p i d , the f a t t y a c i d composition of three t i s s u e s of the coffee p l a n t (the bean, the stem and the l e a f ) was examined v i a gas chromatography i n order to provide a comparative reference to the f a t t y a c i d composition of the c o f f e e c e l l c u l t u r e s . The f a t t y a c i d compositions of c o f f e e c e l l c u l t u r e s grown In three d i f f e r e n t media (PRL-4, Medium #1 and #2) were i n v e s t i g a t e d . Each of these c u l t u r e s grown i n d i f f e r e n t media was grown i n both continuous l i g h t and i n continuous dark, and analysed f o r f a t t y a c i d composition to see whether these two environmental c o n d i t i o n s caused any changes i n f a t t y a c i d c o n s t i t u e n t s . Furthermore, the e f f e c t of c u l t u r e Incubation time on the f a t t y a c i d composition was s t u d i e d by h a r v e s t i n g the c u l t u r e s on day 7 (maximum c e l l y i e l d ) and on day 14 ( d e c l i n i n g c e l l y i e l d ) . The f a t t y a c i d contents of the t i s s u e s s t u d i e d were c a l c u l a t e d u s i n g Method A. The t o t a l weight of the f a t t y a c i d s accounted f o r was derived from the standard curve, i . O i l e x t r a c t i o n from Goffea a r a b l o a t i s s u e s Hexane e x t r a c t i o n s were performed on the coffee bean, the leaves and the stems, and on the coffee c e l l c u l t u r e s grown i n the three d i f f e r e n t media under the c o n d i t i o n s of l i g h t or dark and harvested on day 7 or day 14. The o i l 35 content of these v a r i o u s t i s s u e s i s recorded i n Table 5» The o n l y d i f f e r e n c e which could be noted from the data was the decrease i n o i l content of the day 14- harvested c e l l s . The data obtained for' the o i l contents of the c e l l s grown i n the three media and under c o n d i t i o n s of l i g h t or dark was a l l very s i m i l a r . In g e n e r a l , the o i l content of the c e l l s compared w i t h the o i l content of the l e a f and stem of the coffee p l a n t r a t h e r than the bean. Table 5 . O i l content of v a r i o u s Coffea a r a b l o a t i s s u e s Coffee t i s s u e Percent o i l e x t r a c t e d Green bean 12 . 0 Leaf 3 . 0 Stem 1.9 P l a n t c e l l c u l t u r e - day 7 2.4* P l a n t c e l l c u l t u r e - day 14 1 . 3 * •Average of separate analyses f o r c e l l s grown i n a l l three media under the c o n d i t i o n s of l i g h t o r dark i i . The f a t t y a c i d composition of the green coffee bean, the l e a f and the stem of the c o f f e e p l a n t Gas chromatography of the methylated hexane e x t r a c t s of the c o f f e e bean, the leaves and the stems was performed to i d e n t i f y the component f a t t y a c i d s , i n order to compare t h e i r r e s p e c t i v e compositions to that of the p l a n t c e l l c u l t u r e s . The major f a t t y a c i d s found to be present i n the three c o f f e e p l a n t t i s s u e s are i l l u s t r a t e d i n Table 6. The f a t t y a c i d composition of the coffee bean was found to be palmitate (39%), s t e a r a t e (6%), o l e a t e (7%)» l i n o l e a t e (44$) and a r a c h i d a t e (3%), with t r a c e s of m y r i s t a t e and p a l m l t o l e a t e . 36 These r e s u l t s e s s e n t i a l l y f o l l o w e d the r e s u l t s obtained by Carisano et a l . (7 ) j . n t h e i r study of the Coffea a r a b i c a coffee bean. The f a t t y a c i d composition of the l e a f and stem t i s s u e s i l l u s t r a t e d a g e n e r a l l y s i m i l a r f a t t y a c i d composition to the bean, w i t h the notable exception of the presence of l i n o l e n a t e , which was absent from the bean, and the l a c k of a r a c h l d a t e , which was present i n the bean. Table 6 . The f a t t y a c i d composition of the stems, leaves and beans of Coffea a r a b i c a Component f a t t y a c i d . Stems Leaves Beans £C-l4 ~ $ ~ $ Tr$ C-l6 36 40 39 C-18 8 13 6 C-I81I 6 Tr 7 C-18J2 31 20 44 C-18 s 3 20 26 mm mm C -20 — . —— 3 i i i . The f a t t y a c i d content of the coffee bean, the l e a f and the stem Through the use of the standard curve (Method A), the weight of the f a t t y a c i d s d e r i v e d from the g l y c e r i d e s of the hexane e x t r a c t , could be c a l c u l a t e d . By c o n s i d e r i n g the amount of o i l methylated, an approximate percentage of the f a t t y a c i d content of the o i l could be d e r i v e d . On the b a s i s of these c a l c u l a t i o n s , the f a t t y a c i d contents of the coffee bean, the l e a f and the stem of the coffee p l a n t are recorded i n Table 7 . The f a t t y a c i d content of 7 0 . 0 $ (derived from g l y c e r i d e s ) corresponds q u i t e w e l l with the value of 7 5 . 0 $ t r i g l y c e r i d e reported l n the l i t e r a t u r e ^ 1 ? ) f o r the coffee 37 bean. The f a t t y a c i d content values obtained f o r the l e a f and stem were much lower than those obtained f o r the co f f e e bean. Table 7 . The f a t t y a c i d content of the l e a f , the stem and the bean of Coffea a r a b i c a Tissue Percent f a t t y a c i d Green bean 7 0 . 0 Leaf 5 . 0 Stem 2 0 . 0 i v . The f a t t y a c i d composition of the co f f e e c e l l suspension c u l t u r e s Gas chromatography of the methylated hexane e x t r a c t s of coffee c e l l s grown i n each of the three media, under the co n d i t i o n s of t o t a l l i g h t or t o t a l dark, revealed no d i f f e r e n c e s i n f a t t y a c i d composition. The r e l a t i v e f a t t y a c i d compositions of the coffee c e l l c u l t u r e s grown i n Medium #1, i n l i g h t or dark, and harvested on day 7 or day 14 are presented i n Table 8. Table 8. The f a t t y a c i d composition of the coffee c e l l suspension c u l t u r e s grown i n Medium #1 Component Day 7 Day 14- Day 7 Day 14 f a t t y a c i d dark dark l i g h t l i g h t *C-14 — % 16% —% 20% C -16 42 36 39 30 C-18 8 5 6 4 C-18J1 Tr 2 Tr 3 C-18«2 33 22 33 20 C-18.3 - 15 18 22 22 38 The f a t t y a c i d composition of the c u l t u r e grown i n the dark and harvested on day 7, as i l l u s t r a t e d i n Table 8, was t y p i c a l of a l l the coffee c e l l c u l t u r e s , r e g a r d l e s s of media composition. V a r i a t i o n s i n the r e l a t i v e percentages of i n d i v i d u a l f a t t y a c i d s were present i n each c u l t u r e , but no c o n s i s t e n t d i f f e r e n c e s were observed. The only n o t i c e a b l e e f f e c t on the composition of the f a t t y a c i d s of the c u l t u r e s was an increase i n short chain f a t t y a c i d s (C-14 and l e s s ) i n the c u l t u r e s when they were harvested on day 14, as I l l u s t r a t e d i n Table 8. The f a t t y a c i d composition of the coffee c e l l s e s s e n t i a l l y p a r a l l e l e d the f a t t y a c i d composition of the leaves and stems of the coffee p l a n t , but d i f f e r e d from the coffee bean. The coffee c e l l c u l t u r e s contained l i n o l e n a t e , which was not found i n the coffee bean and lacked a r a c h i d a t e , which was present l n the bean. v. The f a t t y a c i d content of the coffee c e l l c u l t u r e s The f a t t y a c i d contents were determined f o r the c u l t u r e s grown l n the three d i f f e r e n t media, i n the presence of l i g h t or i n t o t a l darkness, and harvested on day 7 or day 14. The data obtained f o r the f a t t y a c i d contents of the c e l l s grown i n the d i f f e r e n t media and i n the presence or absence of l i g h t was a l l very s i m i l a r . However, a d i f f e r e n c e i n the f a t t y a c i d content was observed between c u l t u r e s harvested on day 7 and on day 14, as shown i n Table 9. Although there was some variance i n the amount of Hatty a c i d present i n each i n d i v i d u a l c u l t u r e , a c o n s i s t e n t l y higher amount of f a t t y a c i d was present i n c u l t u r e s harvested on 39 day 7. The r e s u l t s of the data i n d i c a t e d t h a t a decrease i n f a t t y a c i d l e v e l s occurred d u r i n g the death phase of c u l t u r e . G e n e r a l l y , f a t t y a c i d l e v e l s i n the co f f e e c e l l c u l t u r e s resembled the a n a l y t i c a l values obtained f o r the stems of the co f f e e p l a n t . Table 9. The f a t t y a c i d content of Coffea a r a b l c a c e l l c u l t u r e s Day of Harvest Percent F a t t y A c i d * Day 7 30.0 Day 14 11.0 •Average of separate analyses f o r c e l l s grown i n a l l three media under c o n d i t i o n s of l i g h t or dark v i . Summary of f a t t y a c i d composition and content of coffee The t o t a l q u a n t i t y of o i l found i n the c e l l c u l t u r e s was comparable to the o i l content found i n the leaves and stems of the coffee p l a n t . S i m i l a r l y , the component f a t t y a c i d s of the co f f e e c e l l c u l t u r e o i l resembled the f a t t y a c i d s found i n the leaves and stems of the coffee p l a n t . The coffee c e l l c u l t u r e s and the leaves and stems of the coffee p l a n t a l l contained l i n o l e n a t e , a f a t t y a c i d not found i n the g l y c e r i d e s of the coffee bean. The f a t t y a c i d arachidate was found to be unique to the bean t i s s u e . A l l other f a t t y a c i d s found l n the va r i o u s t i s s u e s were common to a l l the t i s s u e s . No d i f f e r e n c e i n the t o t a l f a t t y a c i d content or composition was observed between co f f e e c e l l c u l t u r e s grown i n the three media. S i m i l a r l y , no changes l n f a t t y a c i d content 40 or composition due to growth i n the presence or absence of l i g h t were noted. The only apparent f a c t o r which a f f e c t e d both the f a t t y a c i d content and composition of the c u l t u r e s was the time of harvest (or c e l l m a t u r i t y ) . The appearance of short chain f a t t y a c i d s and a decrease i n the f a t t y a c i d l e v e l i n the hexane e x t r a c t a b l e o i l were noted i n c e l l s harvested i n the dying phase of c u l t u r e . B. The f a t t y a c i d composition and content of mint t i s s u e s and mint c e l l suspension c u l t u r e s The f a t t y a c i d composition and content of the n e u t r a l l i p i d s e x t r a c t e d from the mint seeds, p l a n t m a t e r i a l ( f o l i a g e and stems) and mint c e l l c u l t u r e s were I n v e s t i g a t e d . Only two parameters, the e f f e c t of l i g h t and the time of har v e s t , were considered. The mint c e l l suspension c u l t u r e s grown i n Murashige and Skoog medium were compared to the parent seeds and pl a n t m a t e r i a l . i . O i l e x t r a c t i o n of Mentha t i s s u e s The mint c e l l suspension c u l t u r e s were ex t r a c t e d with hexane s o l v e n t , whereas the mint p l a n t m a t e r i a l and the mint seeds were ext r a c t e d w i t h petroleum e t h e r i d i e t h y l ether (50»50 v / v ) . The t o t a l e x t r a c t a b l e o i l of the t i s s u e s i s presented i n Table 10. No d i f f e r e n c e was noted i n the amount of o i l ex t r a c t e d from the mint c e l l s grown i n the l i g h t or grown i n the dark. Furthermore, no d i f f e r e n c e was observed i n o i l content between c e l l s harvested on day 7 or day 15. The o i l content found i n the pl a n t c e l l c u l t u r e s was i n the same range as t h a t found l n the mint p l a n t f o l i a g e and stem m a t e r i a l , c o n t r a s t i n g s h a r p l y with the high o i l content found 41 In the seed. Table 10. O i l content of v a r i o u s Mentha t i s s u e s Mint t i s s u e Percent o i l e x t r a c t e d Mint seed 19.0 F o l i a g e and stem 1.2 Mint c e l l c u l t u r e s 1.8* •Average of separate analyses f o r c e l l s grown i n l i g h t or dark, and harvested on day 7 or day 14. i i . The f a t t y a c i d composition of the mint seed and the mint p l a n t t i s s u e s Gas chromatography of the methylated n e u t r a l l i p i d e x t r a c t s of the mint seed and mint p l a n t m a t e r i a l ( f o l i a g e and stem) was performed i n order to I d e n t i f y the component f a t t y a c i d s of these mint t i s s u e s . T h e i r r e s p e c t i v e compositions were compared to that of the p l a n t c e l l c u l t u r e . The major f a t t y a c i d s found i n the mint seed and mint plant t i s s u e s are shown i n Table 11. Table 11. The f a t t y a c i d composition of the mint seeds and . mint f o l i a g e and stem t i s s u e s Component f a t t y a c i d Seed P l a n t t i s s u e s ( F o l i a g e and Stem) C-12 ~ $ 10$ C-14 1 10 C-15 — 5 C-16 7 10 C-18 Tr 4 C-18si 8 11 C-18I2 20 14 C-18s3 . . _ .64 34 42 The f a t t y a c i d composition of the mint seed was found to be m y r i s t a t e ( 1 $ ) , palmitate (7%), s t e a r a t e (Trace), o l e a t e (8$ ) , l l n o l e a t e (20$) and l i n o l e n a t e ( 6 4 $ ) . Although both the seed and the f o l i a g e and stem t i s s u e s contained l i n o l e n a t e as the major f a t t y a c i d , a more complex f a t t y a c i d composition was apparent i n the l a t t e r t i s s u e s . A major p r o p o r t i o n of the t o t a l f a t t y a c i d s found i n the f o l i a g e and stem occurred as short c hain f a t t y a c i d s (C-16 and l e s s ) . i i i . The f a t t y a c i d content of the mint seed and mint f o l i a g e and stem t i s s u e s The f a t t y a c i d content of the mint seed and t i s s u e s was c a l c u l a t e d u s i n g Method B, and the r e s u l t s are as shown i n Table 1 2 . The r e s u l t s i n d i c a t e d that a much lower content of f a t t y a c i d was present i n the p l a n t t i s s u e s than i n the seed. Table 1 2 . The f a t t y a c i d content of the mint seeds and mint . f o l i a g e and stem t i s s u e s Tissue Percent f a t t y a c i d Seed 7 0 . 0 F o l i a g e and stem 2 3 . 0 i v . The f a t t y a c i d composition of the mint c e l l suspension c u l t u r e s Gas chromatography of the methylated hexane e x t r a c t s of the mint c e l l s grown i n l i g h t or l n dark, and harvested on day 7 or day 1 5 . revealed no changes i n the c o n s t i t u e n t f a t t y a c i d s . The r e l a t i v e f a t t y a c i d composition of the mint c e l l c u l t u r e s i s presented i n Table 1 3 . 43 Table 13. The f a t t y a c i d composition of mint c e l l c u l t u r e s Component f a t t y a c i d Day 7 dark Day 7 l i g h t Day 15 dark Day 15 l i g h t C -16 C-18 C-18 j l C-18i2 C-18t3 34$ 4 7 19 34 22$ 2 5 17 51 23$ 2 6 18 49 25$ 1 3 15 54 The f a t t y a c i d composition of the mint p l a n t c e l l c u l t u r e s c l o s e l y resembled the composition of the mint seed, r a t h e r than the f o l i a g e and stem. The f o l i a g e and stem t i s s u e s appeared to have a more complex f a t t y a c i d composition. v. The f a t t y a c i d content of mint c e l l suspension c u l t u r e s The f a t t y a c i d content of the mint c e l l c u l t u r e s , c a l c u l a t e d v i a Method B, i s presented i n Table 14. Table 14. The f a t t y a c i d content of mint c e l l c u l t u r e s Day of harvest and exposure to l i g h t Percent f a t t y a c i d (w/w) $ Day 7 l i g h t Day 7 dark Day 15 l i g h t Day 15 dark 40.0 10.0 25.0 5 . 0 The values presented i n Table 14 i l l u s t r a t e a n o t i c e a b l e d i f f e r e n c e l n the f a t t y a c i d content of the mint c e l l c u l t u r e s grown l n the presence of l i g h t , as compared to s i m i l a r t i s s u e grown i n the dark. In both cases, a decrease i n the f a t t y a c i d content i s apparent when the c e l l s were harvested on day 44 v i . Summary of the f a t t y acid composition and content of the mint plant and tissue cultures The o i l content of the mint c e l l cultures was comparable i n quantity to the content found i n the mint f o l i a g e and stem. The mint c e l l cultures had a s i m i l a r f a t t y a c i d composition to the mint seed. The mint f o l i a g e and stem d i f f e r e d from the mint c e l l cultures and mint seed by the presence of a substantial ammount of f a t t y acids which were of chain length equivalent to C-16 or l e s s . The parameters of absence or presence of l i g h t and harvest time did not a f f e c t the o i l content of the mint c e l l cultures. However, growth of the cultures i n the l i g h t resulted i n higher quantities of f a t t y acid i n the c e l l culture o i l . Exposure to l i g h t did not a f f e c t the quantity of f a t t y acid produced i n the coffee c e l l cultures. C. Discussion The experimental studies have shown that the amount of neutral l i p i d found i n both the mint and coffee c e l l suspension cultures was generally comparable i n amount to that found l n the parent plant l e a f and stem tissues, rather than to that found l n the seeds. .The average amount of l i p i d recovered from the coffee c e l l s (2.4$) and from the mint c e l l s (1.8$) f a l l s within the range reported i n the literature ( 5 3i49,41,3 0 , 1 0 ) f o r various plant c e l l cultures. The f a t t y a c i d composition of the coffee c e l l s resembled the composition of the coffee leaves and stems rather than that of the bean. The presence of l i n o l e n i c acid and the absence of arachidic acid i n the c e l l s , leaves and stems distinguished 45 these tissues from the bean. The f a t t y acid composition of the mint c e l l cultures was found to resemble the mint seed rather than the f o l i a g e and stems. However, most Investigators have stated that although minor differences i n f a t t y a c i d composition may e x i s t , the composition of plant c e l l cultures tends to resemble the composition c h a r a c t e r i s t i c to the parent plant. In t h i s study a si m i l a r conclusion concerning f a t t y a c i d composition can be drawn f o r coffee and mint c e l l cultures. Growth of the coffee c e l l s l n three d i f f e r e n t media did not a f f e c t the f a t t y acid composition of the c e l l . Therefore, i t would appear that the f a t t y acid composition of the cultures was not a function of the ingredients present i n the growth media. Both coffee and mint c e l l cultures were grown i n the presence of l i g h t , and i n both cases l i g h t d i d not a f f e c t the f a t t y acid composition. In general, the f a t t y a c i d content of the plant c e l l cultures was found to be very low, an observation which has been reported by a number of dther investigators( 1 0»3 0»4l,49,53) # The f a t t y acid content of both coffee and mint c e l l cultures was shown to decrease i n the l a t e r stages of c e l l culture (day 14 and 15). This decrease was probably due to the u t i l i z a t i o n of the glycerides as an energy source a f t e r the sugars of the medium had been exhausted. Light d i d not a f f e c t the f a t t y acid content found i n the coffee c e l l s . However, the culture of the mint c e l l s i n the presence of l i g h t resulted In an increase i n the amount of f a t t y a c i d . A 46 s i m i l a r phenomenon was reported by Davydova^ 1 0^ who noted that f l a x t i s s u e c u l t u r e s grown l n the presence of l i g h t contained l a r g e r amounts of f a t t y a c i d than the same c u l t u r e s grown i n the dark. This increase i n f a t t y a c i d content was a s s o c i a t e d with the development of green pigmentation i n the c u l t u r e s . S i m i l a r l y , i n the case of the mint c e l l c u l t u r e s , a strong green pigmentation developed which was l a c k i n g i n the coffee c e l l c u l t u r e s . Therefore, i t would appear that the Increase i n f a t t y a c i d content of the c e l l c u l t u r e s was r e l a t e d i n some manner to the production of c h l o r o p h y l l or the a c t i v a t i o n of c h l o r o p h y l l s y n t h e s i s . Analyses of the green pigment i n the mint c e l l c u l t u r e s exposed to l i g h t has been done by P l e a r (personal communication) u s i n g t h i n l a y e r chromatography. His analyses revealed the presence of c h l o r o p h y l l a and b i n these c u l t u r e s but not i n s i m i l a r c u l t u r e s grown i n the dark. I I I . The s t e r o l composition of coffee beans, mint seeds  and coffee and mint c e l l c u l t u r e s The s t e r o l s present i n the u n s a p o n i f i a b l e f r a c t i o n of the e x t r a c t e d l i p i d of the seeds and c e l l c u l t u r e s of coffee and mint were analysed v i a the methods of t h i n l a y e r chromatography and gas chromatography. A. Thin l a y e r chromatography (TLC) A l l t h i n l a y e r p l a t e s were developed u s i n g a chloroform:ethyl acetate mixture as the s o l v e n t . This solvent allowed the s e p a r a t i o n of the s l i g h t l y p o l a r s t e r o l s , i n the order of t h e i r methyl s u b s t i t u t i o n ^ »**2), from the 47 non-polar t r i g l y c e r i d e s and s t e r o l e s t e r s . The separated compounds were i n d i c a t e d by exposure to i o d i n e vapour. The p e r t i n e n t data i s reproduced to s c a l e i n Figures 7 - 1 2 . i . TLC of s e l e c t e d s t e r o l standards Three desmethyl s t e r o l s ( c h o l e s t e r o l , s t i g m a s t e r o l and ^ - s i t o s t e r o l ) and one dimethyl s t e r o l ( l a n o s t e r o l ) were chosen as standards. The sepa r a t i o n obtained f o r these s t e r o l s i s presented l n Figure 7 . Although the desmethyl s t e r o l s could not be separated from each other, l a n o s t e r o l , the dimethyl s t e r o l , separated r e a d i l y . A mixture of a l l f o u r s t e r o l s d i d not i n t e r f e r e w i t h the se p a r a t i o n of the dimethyl s t e r o l from the desmethyl s t e r o l s . The Rf values f o r the dimethyl s t e r o l and the desmethyl s t e r o l s were 0.47 and O.36 r e s p e c t i v e l y . I t i s known t h a t the l i p i d f r a c t i o n from p l a n t t i s s u e s contains t r i g l y c e r i d e s and e s t e r i f i e d s t e r o l s ( 2 2 , 4 5 ) . In order to c h a r a c t e r i z e these l i p i d c o n s t i t u e n t s , t r i o l e i n and c h o l e s t e r o l acetate were run as r e p r e s e n t a t i v e compounds. The r e s u l t i n g t h i n l a y e r a n a l y s i s of these compounds i s shown i n Figure 8 , samples 3 and 4 . Both these compounds ran to the upper part of the t h i n l a y e r p l a t e and had e s s e n t i a l l y the same Rf v a l u e s , I.e. O .85. i i . TLC of co f f e e bean o i l and co f f e e c e l l c u l t u r e o i l (a) Non-saponified o i l a n a l y s i s Ten m i l l i g r a m s of coffee bean o i l and coffee c e l l o i l were d i s s o l v e d s e p a r a t e l y i n 1 ml of petroleum ether. A l i q u o t s of 2 0 j u l of the s o l u t i o n were spotted on a t h i n l a y e r p l a t e f o r development. The r e s u l t i n g chromatograms are presented i n Figure 8 , samples 1 and 2 . Both o i l s i n d i c a t e d s u b s t a n t i a l amounts of i o d i n e p o s i t i v e m a t e r i a l l n the t r i g l y c e r i d e ! s t e r o l e s t e r r e g i o n , Rf O .85. In a d d i t i o n , the p l a n t c e l l o i l produced two spots i n the s t e r o l r e g i o n , (Hf 0 .44 and 0 . 3 5 ) . which were absent l n the bean o i l sample a t the con c e n t r a t i o n t e s t e d . Furthermore, the bean o i l showed the presence of a spot not a s s o c i a t e d w i t h the s t e r o l r e g i o n or the t r i g l y c e r i d e t s t e r o l e s t e r r e g i o n (Rf 0 . 6 l ) , which was absent from the c e l l o i l a t the co n c e n t r a t i o n t e s t e d . (b) S a p o n i f i e d o i l a n a l y s i s A comparative a n a l y s i s of coffee bean o i l , before and a f t e r s a p o n i f i c a t i o n , was performed u s i n g TLC. A desmethyl and a dimethyl standard were run along side the s a p o n i f i e d and non-saponified c o f f e e o i l s , i n order to note p o s s i b l e changes l n Rf values due to changes i n sol v e n t composition. The r e s u l t i n g t h i n l a y e r chromatogram i s presented i n Figure 9 . S a p o n i f i c a t i o n of the coffee bean o i l r e s u l t e d i n the disappearance of spots *a* and 'b' (sample 2) and the appearance of three spots as shown i n sample 3» a l l of which have lower Rf values than t h e i i o d i n e p o s i t i v e m a t e r i a l present i n the non-saponified coffee bean o i l . Two of the spots i n the s a p o n i f i e d sample have m o b i l i t y s i m i l a r to the s t e r o l s . One spot compares f a v o r a b l y w i t h the desmethyl s t e r o l s . The other i o d i n e p o s i t i v e spot i s l o c a t e d between the desmethyl and dimethyl s t e r o l standards. A t h i r d 49 spot, w i t h an Rf of 0 .03 (spot 'a') remains c l o s e to the o r i g i n . A t h i n l a y e r a n a l y s i s was made of coffee c e l l o i l and s a p o n i f i e d c o f f e e c e l l o i l (Figure 1 0 ) . The s a p o n i f i e d c e l l o i l produced s i m i l a r i o d i n e p o s i t i v e compounds as the s a p o n i f i e d coffee bean o i l . The two spots t e n t a t i v e l y l a b e l l e d as s t e r o l s i n the unsaponlfied c e l l o i l were i n t e n s i f i e d l n the s a p o n i f i e d c e l l o i l chromatogram. A t h i r d spot (sample 3» spot ' a ' ) , s i m i l a r i n Rf to that of the slow running component (Figure 9 . sample 3 . spot '§.'), which was present i n s a p o n i f i e d c o f f e e bean o i l , a l s o occurred i n s a p o n i f i e d c e l l o i l . i i i . TLC of mint seed o i l and mint c e l l c u l t u r e o i l (a() Non-saponified o i l a n a l y s i s Ten m i l l i g r a m s of mint seed o i l and 10 mg of mint c e l l o i l were each d i s s o l v e d i n 1 ml of petroleum ether. Twentyjxl of each sample were spotted on a t h i n l a y e r p l a t e and developed s i d e by side with the s t e r o l standards and with the c o f f e e bean sample (Figure 1 1 ) . The mint seed o i l showed the presence of only one i o d i n e p o s i t i v e area which was In the t r i g l y c e r i d e s s t e r o l e s t e r r e g i o n . The mint c e l l o i l a l s o contained t h i s m a t e r i a l , along w i t h two spots running i n the s t e r o l r e g i o n . Neither of the mint o i l s contained a component s i m i l a r to the compound(s) which produced spot *a' found i n the c o f f e e bean o i l . (b) S a p o n i f i e d o i l a n a l y s i s The chromatograms of the s a p o n i f i e d mint o i l s are shown i n Figure 12 . The s a p o n i f i e d mint c e l l o i l chromatogram 50 shows the presence of two s t e r o l spots s i m i l a r to those found i n the unsaponifled mint c e l l o i l , one spot concurrent to the desmethyl s t e r o l standard, and the other between the d i and desmethyl standards. The saponified seed o i l produced one large spot which almost encompasses the whole s t e r o l region. No spots are present l n the lower regions of the thin layer plate, such as are found i n the saponified coffee bean o i l . i v . Summary of t h i n layer analysis The non-saponified o i l chromatograms of both the coffee and mint c e l l cultures indicated the presence of free s t e r o l components, l n detectable amounts, which were not located i n the corresponding seed o i l chromatograms. After saponification of the seed and c e l l culture o i l s , the r e s u l t i n g chromatograms Indicated the presence of two s t e r o l spots i n a l l samples, with the exception of the o i l of the mint seed. One of the ste r o l s had an Rf value s i m i l a r to the desmethyl s t e r o l s , and the other had an Rf value between the di and desmethyl s t e r o l regions. The mint seed produced only one spot which encompassed both s t e r o l regions. The coffee bean o i l chromatogram showed a component between the s t e r o l and t r i g l y c e r i d e tsterol ester regions which was absent from the coffee c e l l o i l at the concentration tested. This component was detectable i n the saponified coffee c e l l o i l when higher concentrations were analysed on a thi n layer plate. B. Gas chromatography (GLC) GLC of the st e r o l s was performed on a 5% 0V-1 column. The ste r o l s were derivatized to form t h e i r respective 51 t r l m e t h y l s l l y l ethers and I n j e c t e d i n t o the gas chromatograph. A c a r r i e r n i t r o g e n f l o w r a t e of 3 2 . 1 ml/min and a chart fspeed of 0.1 cm/min were used. The column temperature was kept a t 240°C f o r 1.5 hours, to separate the more v o l a t i l e f r a c t i o n , and then increased to 280*0. 1. GLC of s e l e c t e d s t e r o l standards The f o u r s t e r o l standards, c h o l e s t e r o l , s t i g m a s t e r o l , campesterol and / 3 - s l t o s t e r o l , were d e r i v a t l z e d s e p a r a t e l y i n 1 ml of r e a c t a n t s , and 10 j*! of each s o l u t i o n were i n j e c t e d i n t o the gas chromatograph, which was set a t an a t t e n u a t i o n of xlOOO. The corresponding r e t e n t i o n times of the s t e r o l standards are recorded i n Table 15. A mixture of the f o u r s t e r o l standards i n j e c t e d i n t o the gas chromatograph d i d not r e s u l t i n any a l t e r a t i o n of r e t e n t i o n times. Table 1 5 . Retention times of s t e r o l standards S t e r o l Retention time (minutes) C h o l e s t e r o l 109 Campesterol 123 S t i g m a s t e r o l 126 > 3 - S i t o s t e r o l 135 I i . GLC of the t o t a l u n s a p o n i f l a b l e s of coffee bean o i l and c e l l c u l t u r e o i l (a) Coffee bean o i l u n s a p o n i f i a b l e s Ten mg of coffee bean u n s a p o n i f i a b l e s were s i l y l a t e d i n 1 ml reagent f o r f i v e minutes, c e n t r i f u g e d , and the solvent volume reduced to 0 . 5 ml u s i n g a stream of dry n i t r o g e n gas. Twenty JJJ. of the reactant mixture were 52 i n j e c t e d i n t o the gas chromatograph which was programmed i n the manner described p r e v i o u s l y . The r e s u l t i n g chromatogram i s presented i n Figure 13. Two major peaks appeared i n the n o n - s t e r o i d a l r e g i o n ( p r i o r to B) and are i d e n t i f i e d as peaks 'a' and *b'. Eight peaks appeared i n the s t e r o i d a l r e g i o n ( a f t e r P) of the chromatogram. Three of these peaks, • f ' , 'g' and 'h' were t e n t a t i v e l y i d e n t i f i e d as campesterol, s t l g m a s t e r o l and y g - s i t o s t e r o l r e s p e c t i v e l y , s i n c e they had r e t e n t i o n times i d e n t i c a l to the corresponding standards. The remaining peaks were not i d e n t i f i e d . (b) Coffee c e l l c u l t u r e o i l u n s a p o n i f i a b l e s In the same manner described f o r c o f f e e bean o i l u n s a p o n i f i a b l e s , 10 mg of coffee c e l l o i l were s i l y l a t e d , and 10 jm.1 were i n j e c t e d i n t o the gas chromatggraph. The r e s u l t i n g chromatogram, Figure 14-, was q u i t e s i m i l a r to t h a t of the coffee bean o i l u n s a p o n i f i a b l e s . Two peaks, 'a' and 'b', which appeared p r i o r to the s t e r o i d a l r e g i o n , had i d e n t i c a l r e t e n t i o n times to the peaks 'a' and 'b' found i n the c o f f e e 011 u n s a p o n i f l a b l e s . Peaks 'c', 'd', 'e', ' f and 'g' corresponded i n r e t e n t i o n times and r e l a t i v e r a t i o s to the peaks ' f , 'g', 'h', * i ' and ' j * found l n the coffee bean o i l u n s a p o n i f i a b l e s . C. I s o l a t i o n and i d e n t i f i c a t i o n of a c a f e s t o l and kawheol mixture 1. Column chromatography Hydrocarbons and s t e r o l s were e l u t e d from a 100 mg sample of ooffee bean u n s a p o n i f l a b l e s adsorbed on a 6 g alumina column, by passing petroleum ether f o l l o w e d by a mixture of petroleum e t h e r s d i e t h y l ether (60:40 v/v) through the column. The d i t e r p e n o i d a l c o h o l s , c a f e s t o l and kawheol, were e l u t e d with chloroform methanol (99»1 v / v ) , and the eluent was c o l l e c t e d . 11. TLC of the chloroformmethanol eluent A sample of the eluent was spotted on a t h i n l a y e r p l a t e and developed using the standard solvent system. The r e s u l t i n g p l a t e , when exposed to i o d i n e vapour, revealed a s i n g l e spot w i t h an Rf value of 0 . 0 3 , i d e n t i c a l to spot 'a' (Figure 9» sample 3) produced by the development of the coffee bean u n s a p o n i f i a b l e s . i l l . U l t r a v i o l e t spectroscopy of the c r y s t a l l i z e d mixture suspected to be c a f e s t o l and kawheol Upon removal of the chloroformmethanol solvent by rotoevaporation, the r e s i d u a l m a t e r i a l was r e f l u x e d i n petroleum ether and c r y s t a l l i z e d from the s o l v e n t . The c r y s t a l s were d i s s o l v e d i n d i e t h y l ether and the ab s o r p t i o n maxima of the s o l u t i o n was determined on an u l t r a v i o l e t © spectrophotometer. Maximum ab s o r p t i o n occurred a t 2220 A, I d e n t i c a l to the value f o r caf e s t o l ^ ^ ) . i v . GLC of the c r y s t a l l i z e d mixture suspected to be c a f e s t o l and kawheol The c r y s t a l l i z e d m a t e r i a l was s i l y l a t e d l n the usual manner and i n j e c t e d i n t o the gas chromatograph, producing two peaks, ('a' and 'b* Figure 15) with the same r e t e n t i o n times as peaks 'a* and 'b' present i n the coffee bean u n s a p o n i f i a b l e s (Figure 13). 54 Rf .47 Rf .36 Origin I Sample 1 2 3 Figure 7. TLC of selected s t e r o l standards Sample: 1- Cholesterol 2- Cholesterol, stigmasterol and / J - s i t o s t e r o l 3- Cholesterol, stigmasterol, p-sitosterol and lanosterol 4- Lanosterol 55 Rf .85 e 0 Rf .61 Rf .44 Rf .35 0 O r i g i n n u • • Sample 1 2 3 4 Figure 8. TLC of two standards and of samples of coffee bean o i l and coffee c e l l cul ture o i l Sample: 1- Coffee c e l l cul ture o i l 2- Coffee bean o i l 3- T r i o l e i n 4- Choles terol acetate 56 R f . 8 4 0 R f . 6 5 R f . 5 0 R f . 4 5 R f . 3 6 0 0 © 0 R f . 0 3 O r i g i n • • © O • S a m p l e 1 2 3 4 F i g u r e 9 . TLC c o m p a r i s o n o f s a p o n i f i e d a n d n o n - s a p o n i f i e d c o f f e e b e a n o i l S a m p l e : 1 - C h o l e s t e r o l 2 - C o f f e e b e a n o i l 3 - S a p o n i f i e d c o f f e e b e a n o i l ( e t h e r s o l u b l e s ) 4 - L a n o s t e r o l 57 R f . 9 4 0 R f . 6 0 R f . 5 2 0 R f . 4 0 0 © R f . 03 O r i g i n • 0 • S a m p l e 1 2 3 4 F i g u r e 10... TLC c o m p a r i s o n o f s a p o n i f i e d a n d n o n s a p o n i f i e d c o f f e e c e l l c u l t u r e o i l S a m p l e : 1 - C h o l e s t e r o l 2 - C o f f e e c e l l c u l t u r e o i l 3 - S a p o n i f i e d c o f f e e c u l t u r e o i l ( e t h e r s o l u b l e s ) 4 - L a n o s t e r o l 58 R f . 9 3 0 0 0 R f . 7 1 © R f . 4 8 R f . 4 2 0 b 0 0 O r i g i n « • 0 • S a m p l e 1 2 3 4 F i g u r e 1 1 . TLC c o m p a r i s o n o f m i n t s e e d o i l a n d m i n t c e l l c u l t u r e o i l S a m p l e : 1 - C h o l e s t e r o l 2 - M i n t s e e d o i l 3 - M i n t c e l l c u l t u r e o i l 4 - C o f f e e b e a n o i l 59 Rf .60 Rf .51 Rf .44 Origin Sample Figure 12. TLC of saponified mint seed and mint c e l l culture o i l Sample: 1- Cholesterol 2- Saponified mint seed o i l (ether solubles) 3- Saponified mint c e l l culture o i l (ether solubles) 4- Lanosterol 60 I5o 5f 63 • o R e t e n t i o n t i m e ( m i n u t e s ) F i g u r e 1 3 . Gas c h r o m a t o g r a m o f t h e c o f f e e b e a n u n s a p o n i f i a b l e s I n d i v i d u a l p e a k r e t e n t i o n t i m e s ( m i n u t e s ) ( a ) - 56 ( c ) - 93 ( e ) - 106 ( g ) - 126 ( i ) - 143 ( b ) - 6 1 ( d ) - 98 ( f ) - 123 ( h ) - 135 ( j ) - 154 A t t e n u a t i o n x2 000 u n t i l p r o g r a m t e m p e r a t u r e c h a n g e ( P ) , t h e n i n c r e a s e s e n s i t i v i t y t o x l O O 61 R e t e n t i o n time (minutes) F i g u r e 14. Gas chromatogram o f c o f f e e c e l l c u l t u r e u n s a p o n i f i a b l e s I n d i v i d u a l peak r e t e n t i o n t i m e s ( m i n u t e s ) (a) - 56 (c) - 123 (e) - 135 (g) - 154 (b) - 61 (d) - 126 ( f ) - 143 A t t e n u a t i o n x500 120 P*J 6*0 R e t e n t i o n t i m e ( m i n u t e s ) F i g u r e 1 5 . Gas c h r o m a t o g r a m o f t h e c r y s t a l l i z e d m i x t u r e o f c a f e s t o l a n d k a w h e o l i s o l a t e d f r o m s a p o n i f i e d c o f f e e b e a n o i l I n d i v i d u a l p e a k r e t e n t i o n t i m e s ( m i n u t e s ) ( a ) - 56 ( b ) - 61 A t t e n u a t i o n x lOOO 63 Cafestol and kawheol are known from the l i t e r a t u r e to be the major constituents of the unsaponifiables of the coffee bean o i l . In t h i s study, gas chromatography of the unsaponiflables of coffee bean o i l indicated the presence of two major components which appeared p r i o r to the s t e r o l region on the gas chromatogram. Thin layer chromatography also indicated the presence of a component not associated with the s t e r o l constituents. The procedure followed f o r the i s o l a t i o n of c a f e s t o l and kawheol was e s s e n t i a l l y the same as that used by Nagasampagi et a l ( 3 5 ) . The i s o l a t i o n procedure produced a c r y s t a l l i n e material which had an o absorption maxima at 2220 A, the c h a r a c t e r i s t i c absorption of c a f e s t o l . GLC of the c r y s t a l l i n e material produced two peaks with retention times matching the two component peaks •a' and *b' of the coffee bean unsaponifiables. From th i s data i t was concluded that the material producing peaks •a* and 'b* was most probably c a f e s t o l and kawheol. D. Gas chromatography of the i n d i v i d u a l iodine positive spots i s o l a t e d from the preparative t h i n layer plates of the coffee bean and the coffee c e l l o i l unsaponifiables Preparative t h i n layer plates were developed f o r a l l the saponified o i l samples. The separated constituents along with the s i l i c a gel were scraped off the plates and were eluted from the gel with an appropriate solvent. The solvent was removed v i a a stream of nitrogen gas, and the r e s i d u a l material s i l y l a t e d i n the standard manner. In some cases pyridine was removed a f t e r s i l y l a t i o n and replaced with 64 lso-octane or carbon d i s u l f i d e , i n order to reduce the solvent peak produced on the gas chromatogram. i . GLC of the suspected diterpenoid alcohol spot GLC of the suspected diterpenold alcohol spot (Rf 0.03), present i n thi n layer chromatograms of coffee bean and coffee c e l l o i l unsaponiflables, resulted i n the chromatograms shown i n Figures 16 and 1?. As was expected, only two peaks were present i n the chromatograms, and they were the peaks previously i d e n t i f i e d as c a f e s t o l and kawheol. The only difference between the two chromatograms i s the r a t i o of the two components. i i . GLC of the s t e r o l spot having the same Rf value as the desmethyl standards Figures 18 and 19 i l l u s t r a t e the chromatograms produced by GLC of the s t e r o l spot present on bean and c e l l t h i n layers. The c e l l s t e r o l spot showed three components, •a', 'b' and 'c', with retention times i d e n t i c a l to those of campesterol, stigmasterol and ^ - s i t o s t e r o l . The bean s t e r o l spot contained the same sterols as the c e l l s t e r o l spot, plus an a d d i t i o n a l two unknowns. In both cases, the r e l a t i v e r a t i o s of the concentrations of the i d e n t i f i e d s t e r o l s were the same. i l l . GLC of the s t e r o l spot which i s located between the d i and desmethyl standards The r e s u l t i n g chromatograms f o r the bean and c e l l are given i n Figures 20 and 21. In the case of the bean s t e r o l s , two major components were dominant. In contrast, a more even d i s t r i b u t i o n of components was apparent i n the 65 c e l l s t e r o l s . None of the peaks were i d e n t i f i e d , but are presented f o r comparative purposes, to show the differences i n composition between the bean and c e l l s t e r o l s which appeared on the th i n layer plate at the same Rf value. E. GLC of the t o t a l unsaponifiables of mint seed o i l and mint c e l l culture o i l i . Mint seed unsaponifiables In the usual manner 12.3 mg of mint seed un-saponif iables were derivatlzed, the solvent was reduced to 0.5 ml and 20 jxl were injected into the gas chromatograph. The r e s u l t i n g chromatogram i s presented i n Figure 22. Five minor peaks were produced ('a','b*,'c*,'d• and ' e'). plus one ma$or peak ('f') which had a retention time corresponding to .e-sitosterol. 11. Mint c e l l unsaponifiables Afte r d e r i v a t i z a t l o n of 11.4 mg of the mint c e l l unsaponifiables, the solvent was reduced to 0.5 ml, and 25 >xl were injected into the gas chromatograph. The r e s u l t i n g chromatogram, presented i n Figure 23, shows only two peaks, a minor unidentified peak and a major peak with a retention time corresponding to £-sitosterol. F. GLC of i n d i v i d u a l iodine positive spots Isolated from preparative thin layer plates of saponified mint seed and mint c e l l o i l 1. GLC of the mint c e l l s t e r o l spot having the same Rf value as the desmethyl standards The r e s u l t i n g chromatogram (not presented) showed only one peak with a retention time Id e n t i c a l to the yB-sitosterol standard. 66 120 Pt Retention time (minutes) I T Figure 16. Gas chromatogram of c a f e s t o l and kawheol is o l a t e d by preparative TLC from the coffee bean • Individual peak retention times (minutes) (a) - 56 (b) - 61 Attenuation xlOOO Retention time (minutes) Figure 17. Gas chromatogram of c a f e s t o l and kawheol i s o l a t e d by preparative TLC from the coffee c e l l culture Individual peak retention times (minutes) (a) - 56 (b) - 61 Attenuation xlOOO 68 c 1 1 1 120 P f 60 0 Retention time (minutes) Figure 18. Gas chromatogram of desmethyl s t e r o l s i s o l a t e d by prep a r a t i v e TLC from the coffee c e l l c u l t u r e I n d i v i d u a l peak r e t e n t i o n times (minutes) (a) - 123 (c) - 135 (b) - 126 Att e n u a t i o n xlOOO 69 e n 1 « r 120 Pf 60 0 Retention time (minutes) Figure 19. Gas chromatogram of the desmethyl s t e r o l s i s o l a t e d by preparative TLC from the coffee bean I n d i v i d u a l peak re tent ion times (minutes) (a) - 100 (c) - 123 (e) - 135 (b) - 109 (d) - 126 Attenuation x200 70 R e t e n t i o n t i m e ( m i n u t e s ) F i g u r e 2 0 . Gas c h r o m a t o g r a m o f s t e r o l s ( R f b e t w e e n d i a n d d e s m e t h y l s t e r o l s t a n d a r d s ) i s o l a t e d b y p r e p a r a t i v e TLC f r o m t h e c o f f e e b e a n I n d i v i d u a l p e a k r e t e n t i o n t i m e s ( m i n u t e s ) ( a ) - 100 ( c ) - 1 3 2 ( e ) - 152 ( b ) - 109 ( d ) - 1 4 1 A t t e n u a t i o n x 10 0 R e t e n t i o n t i m e ( m i n u t e s ) 7b" F i g u r e 2 1 . Gas c h r o m a t o g r a m o f s t e r o l s ( R f b e t w e e n d i a n d d e s m e t h y l s t e r o l s t a n d a r d s ) i s o l a t e d b y p r e p a r a t i v e TLC f r o m t h e c o f f e e c e l l c u l t u r e I n d i v i d u a l p e a k r e t e n t i o n t i m e s ( m i n u t e s ) ( a ) -( b ) -93 102 ( c ) -( d ) -1 1 1 1 2 4 ( e ) -( f ) -130 137 ( g ) -( h ) -144 153 A t t e n u a t i o n x l O O 1 • r 120 P t 60 0 Retention time (minutes) Figure 22. Gas chromatogram of the mint c e l l cul ture unsaponifiables I n d i v i d u a l peak re tent ion times(minutes) (a) - 98 (c) - 110 (e) - 122 (b) - 106 (d) - 115 (f) - 135 Attenuation x200 73 b Retention time (minutes) Figure 23. Gas chromatogram of the mint c e l l culture unsaponifiables Individual peak retention times\(minutes) (a) - 124 (b) - 135 Attenuation xlOOO 74 F i g u r e 2 4 . Gas c h r o m a t o g r a m o f s t e r o l s ( R f b e t w e e n d i a n d d e s m e t h y l s t e r o l s t a n d a r d s ) i s o l a t e d b y p r e p a r a t i v e TLC f r o m t h e m i n t c e l l c u l t u r e I n d i v i d u a l p e a k r e t e n t i o n t i m e s ( m i n u t e s ) ( a ) - 91 ( c ) - 110 ( e ) - 136 ( g ) - 153 ( b ) - 100 ( d ) - 1 2 1 ( f ) - 142 A t t e n u a t i o n x l O O 75 I i . GLC of the mint c e l l s t e r o l spot l o c a t e d between the d i and desmethyl s t e r o l standards The r e s u l t i n g chromatogram i s presented i n F i g u r e 2k. Seven u n i d e n t i f i e d peaks appeared on the chromatogram, i n d i c a t i n g t h a t a complex mixture of s t e r o l components e x i s t s . i i i . GLC of the mint seed s t e r o l spot which covers the r e g i o n s occupied by the d i and desmethyl s t e r o l standards As expected, the r e s u l t i n g chromatogram (not presented) was e x a c t l y the same as t h a t of the t o t a l mint seed u n s a p o n i f i a b l e s ( F i g u r e 22). G. Summary of gas chromatography of the t o t a l u n s a p o n i f i a b l e s of c o f f e e and mint and of gas chromatography of TLC I s o l a t e d s t e r o l spots of c o f f e e and mint Although no q u a n t i t a t i o n of the gas chromatographic d a t a was performed, some g e n e r a l statements can be made with regard to the r e l a t i v e amounts of s t e r o i d a l and n o n - s t e r o i d a l m a t e r i a l present i n the t o t a l c o f f e e bean and c e l l un-sa p o n i f l a b l e s . A r e l a t i v e e s t i m a t i o n was p o s s i b l e through c o n s i d e r a t i o n of the amount of sample i n j e c t e d and of the r e c o r d e r a t t e n u a t i o n r e q u i r e d to produce peaks on the chromatogram. In the case of the c o f f e e bean o i l un-sap o n i f i a b l e s , the n o n - s t e r o i d a l m a t e r i a l (peaks 'a' and 'b') accounted f o r the m a j o r i t y of the sample. The c o f f e e c e l l u n s a p o n i f l a b l e s contained r e l a t i v e l y more s t e r o i d a l m a t e r i a l . Both the c o f f e e bean and the c o f f e e c e l l u n s a p o n i f i a b l e s contained three s t e r o l s , l n the same r e l a t i v e r a t i o s , t e n t a t i v e l y i d e n t i f i e d as £-sitosterol, s t l g m a s t e r o l and 76 campesterol i n order of decreasing concentration. A general comparison of the r e l a t i v e quantities of s t e r o l s present i n the t o t a l mint seed and mint c e l l o i l unsaponifiables was possible through the use of the same semi-quantitative method described above f o r coffee. Both sources of mint o i l s contained /3-sitosterol as the major s t e r o l component, but the plant c e l l s contained substantially-more of t h i s s t e r o l . Gas chromatography of s t e r o l components of the coffee bean and coffee c e l l o i l Isolated by preparative TLC revealed that the desmethyl s t e r o l spot was composed of the desmethyl s t e r o l s , ^ - s i t o s t e r o l , campesterol and stigmasterol. This r e s u l t v e r i f i e d the i n i t i a l assumption, based on GLC of the t o t a l unsaponifiables, that these st e r o l s were present. The coffee bean sterols located between the d i and desmethyl s t e r o l standards were d i f f e r e n t i n composition and r e l a t i v e concentration than the s i m i l a r l y located coffee c e l l s t e r o l s . Sterols found i n th i s region of the TLC plate are probably monomethyl ste r o l s because of the order of separation i n the chloroform iethyl acetate solvent system (des, mono, dimethyl ste r o l s ) ^ 8 ) . Gas chromatographic analysis of the TLC isolated' s t e r o l spots of mint confirmed the premise that p-sitosterol was the major s t e r o l of the mint seed and mint c e l l o i l unsaponiflables. Although a number of minor sterols were present i n the mint seed, ^ - s i t o s t e r o l was overwhelmingly dominant i n the mint c e l l . Analysis of the c e l l s t e r o l 77 material which had an Rf value between the d i and desmethyl s t e r o l standards indicated a complex mixture of components. H. Discussion The sterols and diterpenold alcohols of the coffee bean and the coffee c e l l cultures were studied i n a q u a l i t a t i v e and semi-quantitative manner. The major st e r o l s of the coffee bean were shown to be ^ - s i t o s t e r o l , stlgmasterol and campesterol i n order of decreasing concentration. These results agree with those obtained by Nagasampagi et al.(35) i n t h e i r study of the coffee bean. The coffee c e l l cultures were found to contain the above mentioned st e r o l s i n the same r e l a t i v e concentrations present i n the coffee bean. The s t e r o l content of the coffee c e l l cultures was found to be considerably higher than that found i n the coffee bean, although no absolute quantitative values were obtained. Furthermore, comparative t h i n layer chromatographic studies of the non-saponified o i l s of the bean and of the c e l l i l l u s t r a t e d the presence of more free s t e r o i d a l material i n the c e l l culture o i l than i n the bean o i l . Both the coffee bean and coffee c e l l unsaponifiables contained minor amounts of s t e r o l s other than the desmethyl s t e r o l s . Gas chromato-graphic examination of these minor s t e r o l s i l l u s t r a t e d differences i n composition and concentration between the bean and the c e l l . Two diterpenold alcohols, c a f e s t o l and kawheol, were i s o l a t e d from coffee bean o i l unsaponifiables, and i d e n t i f i e d by U.V. spectroscopy, TLC and GLC. Gas 78 chromatography of the t o t a l unsaponifiable coffee bean o i l revealed that the two diterpenoid alcohols were the major constituents, a r e s u l t i n agreement with the l i t e r a t u r e ^ ^ . 35) > Cafestol and kawheol were also found to be present i n the coffee c e l l unsaponifiable matter, but i n lower concentrations. As stated previously, studies of the s t e r o l s i s o l a t e d from the seeds and plant c e l l cultures of the unknown Mentha species showed ^ - s i t o s t e r o l to be the major and commonly occurring s t e r o l i n both tissue sources. This data i s i n agreement with the l i t e r a t u r e ( 3 » ^ ° ) f a s A - s i t o s t e r o l has been noted to be a major s t e r o l i n the Mentha p i p e r i t a plant. The mint c e l l cultures contained more free s t e r o i d a l l i p i d components than found i n a corresponding analysis of the seeds. Gas chromatography of the sterols which were located between the d i and desmethyl s t e r o l regions on the t h i n layer plate of the mint c e l l unsaponiflables, revealed the presence of a complex un-i d e n t i f i e d mixture of s t e r o l s . Thin layer chromatography of the mint seed unsaponifiables revealed only one large iodine positive spot which covered almost the complete s t e r o l region of the plate. This encompassing spot indicated the possible presence of s t e r o l s of a l l three degrees of methyl sub s t i t u t i o n within the mint seed. The t o t a l s t e r o l content of the mint c e l l cultures was found to be considerably higher than that found i n the mint seed. Various i n v e s t i g a t o r s ^ ' 2 0 ' 2 1 ' 2 9 ' ' * ' 9 ' 5 0 , 5 5 ) have found sterols i n a wide v a r i e t y of plant c e l l cultures. 79 When the s t e r o l composition of each of the c e l l c u l t u r e s was compared to that of the r e s p e c t i v e parent p l a n t t i s s u e s , i t t has been stated t h a t , i n general, no major q u a l i t a t i v e d i f f e r e n c e s were discerned. In t h i s study, a s i m i l a r c o n c l u s i o n was drawn f o r coffee and mint c e l l c u l t u r e s , i . e . the major s t e r o l s found l n coffee and mint c e l l c u l t u r e s were s i m i l a r to those found i n the parent p l a n t s . 80 CONCLUSION C e l l suspensions of Coffea arabica and an unknown Mentha species were cultured i n l i q u i d media, and the s t e r o l s and f a t t y acids were analysed. These l i p i d constituents were also investigated i n the parent plant tissues, i n order to provide comparative data which could be of use to other workers investigating l i p i d metabolism i n plant c e l l cultures. The o i l content of the plant c e l l cultures f e l l within the range found l n the leaves and stems of the parent plants. In contrast, the seed had a higher o i l content. The f a t t y acid composition of the coffee c e l l cultures resembled the composition of the leaves and stems. In the study of mint, the pattern of f a t t y acids found i n the c e l l cultures was s i m i l a r to that found i n the seed. However, although differences i n f a t t y acid composition between plant c e l l cultures and various parent plant tissues were noted, i t can be stated that the major f a t t y acids c h a r a c t e r i s t i c of the parent plant were found i n the plant c e l l s . Growth of the coffee c e l l cultures i n three d i f f e r e n t media did not r e s u l t i n changes i n the f a t t y acid composition or content. Exposure to l i g h t did not a f f e c t the f a t t y acid composition or content of coffee c e l l cultures, but did cause the development of a green pigmentation and a concurrent increase i n the f a t t y acid content i n the mint c e l l cultures. 81 The major s t e r o l s of the plant c e l l cultures were found to be the same as those present i n the parent seeds. The free s t e r o l content of the extracted l i p i d from the plant c e l l cultures and the s t e r o l content of the unsaponifiables of the l i p i d were found to be higher than the corresponding contents found i n the seed. 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