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A study of the naringin content in California marsh seedless grapefruit

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A study of the naringin content in California marsh seedless grapefruit

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Content A STUDY OF THE MARINGIN CONTENT IN CALIFORNIA M ARSH SEEDLESS GRAPEFRUIT A T h e s is P re s e n te d t o th e F a c u lty o f th e D epartm ent o f C h em istry U n iv e r s ity o f S o u th e rn C a lif o r n i a I n P a r t i a l F u lf illm e n t o f th e R e q u irem en ts f o r th e D egree M a ste r o f A r ts by N. Y. Jam gochian S eptem ber 1959 This thesis, written by Nicholas Vahe Jaragochian under the direction of h Faculty Committee, and a p p r o v e d by all its m em bers, has been presented to and accepted by the Council on Graduate Study and Research in partial fulfill m ent of the requ irem en ts fo r the d eg ree of Master of Arts ................................ Dean Secretary D ate Sept e mb e r _ _ 19 39 Faculty Com m ittee TABLE OF CONTENTS CHAPTER _ • PAGE I . INTRODUCTION . . . . . . . . . . . . . . . . . 1 H i s t o r i c a l ...................................................................... 1 P u rp o se . ............................ 5 I I . NATURE A N D OCCURRENCE OF NARINGIN. . . . . . . 6 P r o p e r tie s . . . . .................................. . . . . . 6 F u n c tio n ................................................................... 8 I I I . M ETHODS OF EXTRACTION A ND QUANTITATIVE ANALYSIS............................ 12 E x tr a c tio n . . . . . . . . . . . . . . . . . 12 Q u a n tita tiv e m ethods . . . . . . . . . . . . 14 M o d if ic a tio n s . . . . . . . . . . . . . . . . 17 IV . EXPERIMENTAL W O R K A N D RESULTS..................................... 25 M ethods ..................................... 25 R e s u l t s .................................................................. 25 V. SU M M A R Y AND CONCLUSIONS.................................... 55 BIBLIOGRAPHY.......................................... 56 LIST OP TABLES TABLE PAGE I . P ro d u c tio n o f g r a p e f r u i t in 1937................................. 3 I I . S o l u b i l i t y o f n a r in g in in w a te r ................................. 6 I I I . P er c e n t o f n a r in g in in th e alb ed o o f M arsh S e e d le s s g r a p e f r u i t s to r e d f o r 6 weeks a t v a rio u s te m p e ra tu re s . ........................ . . . . . . 9 IV . N a rin g in c o n te n t in f r e s h and o ld g r a p e f r u i t . . 10 V. D ilu tio n f a c t o r s ............................... 16 V I. E x tr a c tio n o f n a r in g in u n d er v a rio u s c o n d itio n s o f tim e i and te m p e r a tu r e . ....................... 18 V II. E x tr a c tio n o f N a rin g in u n d e r v a r io u s c o n d i t i o n s ............................................................................ . . . . 18 V I I I . The in t e r f e r e n c e o f p o ta ssiu m d ic h ro m a te in th e s ta n d a r d ....................... 19 IX . The e x te n t o f d e e p e n i n g ................................................... 21 X. A verages o f n a r in g in c o n te n t in M arsh S e e d le s s g r a p e f r u i t ................................. 34 X I. A verages o f n a r in g in c o n te n t in f r u i t from th e same t r e e . 35 CHAPTER I INTRODUCTION H i s t o r i c a l . N a rin g in , th e b i t t e r p r i n c i p l e o f th e g r a p e f r u i t , w as d is c o v e re d by De V ry in th e b lo sso m s o f th e t r e e in 1 8 5 7 .1 De Vry re c o g n iz e d th e p re se n c e o f th e su b s ta n c e in a l l p a r t s o f th e t r e e b u t to th e g r e a t e s t e x te n t in th e f r e s h l y opened b lo sso m s. H offm ann2 and W ill con d u c te d e x te n s iv e r e s e a r c h on th e -s u b s ta n c e . . More r e c e n t l y Z o l l e r 4 and A sah in a and In u b u se 5 have s tu d ie d n a r in g in and i t s d e r i v a t i v e s . De Vry and Hoffm ann s e a rc h e d in v a in f o r n a r in g in in o th e r members o f th e c i t r u s f a m ily . N a rin g in i s noY/ known t o be one o f th e many n a t u r a l g lu c o s id e s p ro duced s o l e l y by one p la n t s p e c ie s . The trem en d o u s d e v e lo p m ent o f g r a p e f r u i t c u l t i v a t i o n in t h i s c o u n try h a s changed t h i s o b sc u re compound in to one o f p o t e n t i a l com m ercial i n t e r e s t . The to n s o f w astag e from th e ca n n in g p l a n t s a s w e ll a s th e c u l l s c o n ta in enough n a r in g in t o p roduce con s id e r a b le am o u n ts. As y e t , how ever, no im p o rta n t u se h as b een found f o r th e g lu c o s id e . ^•D e V ry, J a h r e s b e r io h te f u r Pharm acognos 132 (1866) . %Cd. H offm ann, " H e s p e rid in de V ry, A u ra n tin , M u rray in B e ric h te 9 690 (1 8 7 6 ). ®Wi{Ll, W ., "U eber d as N a r in g in ,” I an d I I , B e ric h te 18 , 20 (1 8 8 5 -1 8 8 7 ). 4Z o ll e r , H ., ” Some C o n s titu e n ts o f th e A m erican Grape f r u i t , ” J o u r , o f I n d u s t r i a l and E n g in e e rin g C h em istry 10 364 (1 9 1 8 ). : 5A sa h in a , Y. and In u b u se , M ., ”U eber d ie K o n s titu tio n d e s N a r in g e n in s ,” B e r ic h te 61B 1514 (1 9 2 8 ). 2 $he g r a p e f r u i t t r e e ( C itr u s decumana) i s p ro b a b ly a n a tiv e o f J a v a . H ere two v a r i e t i e s a re known, one w ith an i n t e n s e l y b i t t e r in e d ib le f r u i t known a s th e sh ad d o ck , and th e o th e r w ith a s w e e te r and j u i c e r f r u i t from w hich o u r d o m e stic s t r a i n s w ere d e v e lo p e d . The S p a n ish c o l o n is ts b ro u g h t th e g r a p e f r u i t to th e New W orld e a r l y in th e s ix t e e n t h c e n tu r y . The f r u i t was b ro u g h t to F lo r id a by way o f Mexico w here i t had b een grow ing f o r d e c a d e s . The e a r l i e s t re c o rd e d d a te o f i t s im p o rta tio n i n t o F lo r id a i s 1809,® when a S p a n ish g en tlem an b ro u g h t a few se e d s f o r h i s p r i v a t e g a rd e n s . I t i s g e n e r a lly b e lie v e d t h a t th e g r a p e f r u i t was in te rd u c e d in to F lo r id a much e a r l i e r . The e a r l y b o ta n ic a l w r i t e r s c o n s id e re d th e f r u i t i n e d i b l e , d u e, p e rh a p s , t o a c o n fu s io n o f th e two v a r i e t i e s . The t r e e rem ain ed a c u r i o s i t y u se d c h i e f l y f o r o rn a m e n ta l p u rp o se s u n t i l th e n a t iv e s o f F lo r id a s e p a r a te d th e d e s ir a b le s t r a i n s and began th e c u l t i v a t i o n o f th e f r u i t f o r l o c a l co n su m p tio n . The t o u r i s t s were s e rv e d th e f r u i t and soon a demand was c r e a te d o u ts id e o f th e s t a t e . By 1880 e x te n s iv e c u l t i v a t i o n was begun to su p p ly th e e v e r - in c r e a s in g m a rk e ts . S in ce t h a t tim e th e g r a p e f r u i t h a s become a common fo o d in a l l p a r t s o f th e c o u n try d u rin g m ost o f th e y e a r . F or y e a rs F lo r id a had a v i r t u a l m onopoly on th e p r o d u c tio n o f th e f r u i t . Today t h a t s t a t e p ro d u c e s ab o u t 50% o f th e n a t i o n ’ s 6Hume, H. H ., C u l tiv a tio n o f C itr u s F r u i t s . New Y ork: M cM illan Co. (1934) p . 95. su p p ly w ith e v e r - in c r e a s in g c o m p e titio n from T ex a s, C a l i f o r n ia , and A riz o n a . G r a p e f r u it c u l t i v a t i o n in C a li f o r n i a i s younger th a n in F l o r i d a . In 1890 some F lo r id a t r e e s w ere p la n te d h e re f o r t r i a l p u rp o s e s . The s t r a i n s w ere n o t s u i t e d to th e c l i m ate and s o i l , so t h i s f i r s t e x p e rim e n t d id n o t en co u rag e f u r t h e r d ev e lo p m e n t. L a te r th e M arsh S e e d le s s g r a p e f r u it was p la n te d in th e Im p e r ia l V a lle y w ith good r e s u l t s . From t h i s seco n d s t a r t g r a p e f r u i t c u l t i v a t i o n in C a l i f o r n i a h a s in c re a s e d r a p i d l y u n t i l in 1937 th e r e w ere 1 9 ,6 4 5 p la n te d a c r e s o f w h ich o n ly 4 ,0 0 0 w ere n o n - b e a r in g .7 The f o llo w in g t a b l e shows th e t o t a l g r a p e f r u i t p r o d u c tio n in 1 9 3 7 .8 TABLE I PRODUCTION OF GRAPEFRUIT IN 1937 S ta te - > 1 ,0 0 0 boxes F lo r id a 1 3 ,000 T exas 8 ,9 0 0 A rizo n a 2 ,3 0 0 C a lif o r n ia 1 ,8 9 0 7A M an u al■ f o r C itr u s G row ers. M utual Orange D i s t r i c t , R e d la n d s, C a l i f o r n i a , 1937. 8A g r i c u l t u r a l S t a t i s t i c s . Y fashington, D. C .: D ep t, o f A g r i c u ltu r e , 1938, p . 177. 4 Y ast am ounts o f th e f r u i t a re u sed in c a n n in g . D uring th e se a so n o f 1936 to 1937 F lo r id a p ack ed a lm o st f o u r m il lio n c a s e s o f can s and a s many o f th e j u i c e . 9 T h is in d u s tr y h a s s e r io u s p ro b lem s in d is p o s in g o f th e w a s te s . Some h a s b een d r ie d f o r c a t t l e f e e d . S e v e ra l b y -p ro d u c ts c o u ld be o b ta in e d from th e w a s te —p e c t i n , a ro m a tic o i l , c i t r i c a c i d , an d n a r i n g in . Of th e s e o n ly -th e o i l h a s b een p ro d u ced and t h i s in s m a ll q u a n t i t i e s due to a p r e ju d ic e a g a in s t th e g r a p e f r u i t p r o d u c t. S e v e ra l p a t e n ts have b een ta k e n o u t f o r th e com m e rc ia l e x t r a c t i o n o f p e c t i n , none o f w hich a re in u s e . The - c i t r i c a c id i s n o t p r e s e n t in s u f f i c i e n t am ounts to com pete w ith th e lem on p r o d u c t. N a rin g in h a s been c o n s id e re d a s a b y -p ro d u c t b ecau se o f i t s u n iq u e n e ss and i t s c o m p a ra tiv e ly e a s y e x t r a c t i o n . A l i t t l e h a s been s o ld f o r s ta n d a r d iz in g g r a p e f r u i t d r in k s . A few m in o r a p p l i c a t i o n s have b een su g g e s te d , m aking u se o f i t s b i t t e r t a s t e . S tu d ie s o f th e phy s i o l o g i c a l e f f e c t s o f th e g lu c o s id e have n o t been m ade, o r a t l e a s t have n o t b een p u b lis h e d . A r e c e n t e x p e rim e n te r h as found n a r in g e n in , th e n o n -s u g a r c o n s t i t u e n t o f th e g lu c o s id e , to lo w er th e b lo o d p r e s s u r e o f d o g s .10 A th e r a p e u ti c use f o r n a r in g in may y e t be fo u n d , b u t a t p r e s e n t th e much v a u n te d 9I b i d . . p . 179. 10C hem ical A b s tr a c ts 5073 1938 {Arm entano, L . , Z. Ges e x p t l Med. 102 1 9 3 8 ). 5 m e d ic in a l v a lu e p resu m ab ly due to i t s b i t t e r n e s s h as no s c i e n t i f i c f o u n d a tio n . N a rin g in h a s a l s o been s u g g e s te d a s a so u rc e o f rham nose s in c e t h i s s u b s ta n c e , w ith g lu c o se and n a r in g e n in , a r e p ro d u c ts o f h y d r o ly s is. ^ R e se a rc h on th e b y -p ro d u c ts o f th e g r a p e f r u i t in d u s tr y i s o f f a i r l y r e c e n t o r i g i n so t h a t much developm ent can be e x p e c te d in th e f u t u r e . P u rp o s e . The p u rp o se o f t h i s i n v e s t i g a t i o n i s to s tu d y th e c o lo r im e tr ic m ethod o f d e te rm in in g n a r in g in and to make a n a ly s e s o f th e d i f f e r e n t s e c tio n s o f th e M arsh S e e d le s s g r a p e f r u i t grown in C a lif o r n i a in v a r io u s s ta g e s o f d e v e lo p m ent to d e te rm in e a t w hich th e n a r in g in c o n te n t i s h ig h e s t. H p u l l e y , G eo ., and H. von L o e se c k e , " P r e p a r a tio n o f Rhamnose from N a r in g in ," J o u r . Am Chem S o c. , 61 175 (1 9 3 9 ). CHAPTER II THE NATURE AND OCCURRENCE OF NARINGIN P r o p e r t i e s . 12 N a r in g in , C27H32°14 * 2H2°» i s a n a t u r a l g lu c o s id e o f u n d ete rm in ed s t r u c t u r e o c c u rr in g in a l l p a r t s o f th e g r a p e f r u i t t r e e . N a rin g in c r y s t a l l i z e s from w a te r in f l u f f y cream c o lo re d n e e d le s w hich c o n ta in e i g h t m o le c u le s o f w a te r o f c r y s t a l l i z a t i o n and w hich m e lt a t 8 2 ° . On d ry in g a t 110° th e su b sta n c e lo s e s s i x o f th e w a te r m o le c u le s and m e lts a t 1 7 1 °. A lc o h o lic and w a te r s o l u t i o n s a re l e v o r a t a - t o r y t o th e e x te n t o f 8 2 .1 1 ° . N a rin g in g iv e s a y e llo w c o l o r a t i o n w ith a l k a l i e s and a b ro w n ish r e d one w ith f e r r i c c h lo r i d e . The g lu e o s id e i s v e ry s o lu b le in h o t a lc o h o l and a c e to n e , s o lu b le i n h o t w a te r , and s l i g h t l y s o lu b le in w a te r a t 2 0 ° . I t i s e a s i l y e x t r a c te d w ith w a te r due to i t s r a p id in c re a s e o f s o l u b i l i t y w ith th e r i s e in te m p e ra tu re : TABLE I I SOLUBILITY OF NARINGIN IN W ATER13 T em p eratu re G r a m s /lite r 200 C. .5 0 45 1 .9 6 55 7 .1 6 65 4 2 .2 1 75 1 0 8 .2 4 •^^H eilbron, I . M ., A D ic tio n a r y o f O rganic Compounds. New Y ork: O xford U n iv e r s ity P r e s s , 1938, p . 52, V o l. I I I . ^■^Pulley, 'S o l u b i l i t y o f N a rin g in in W a te r,” J o u r . I n d . and E ng. Chem ., A n a ly tic E d. 8 (1936) p . 360. 7 N a rin g in h y d ro ly z e s to form g lu c o s e , rh am n o se, and n a r in g e n in . The l a t t e r su b sta n c e h as b ee n i d e n t i f i e d by The d e r iv a tiv e n a r in g e n in c r y s t a l l i z e s in l i g h t y e llo w n e e d le s m e ltin g a t 2 5 1 °. I t p ro d u c e s a y e llo w c o lo r w ith a l k a l i e s and w ith s u lp h u r ic a c i d , th e l a t t e r tu r n in g r e d on s ta n d in g , N a rin g e n in h e a te d w ith p o ta s s iu m h y d ro x id e y i e l d s p h lo r o g lu e in o l and p -co u m a ric a c i d , N a rin g e n in i s s o lu b le i n a l c o h o l, e t h e r , and b e n z e n e , and in s o lu b le in w a te r . The c h a r a c t e r i s t i c t a s t e o f g r a p e f r u i t i s due to th e b i t t e r p r i n c i p l e . As Z o lle r p u ts i t , ’’Uponf g lu c o s id e s d e p en d s th e p rim a ry d i f f e r e n t i a t i o n o f th e s p e c i e s . p h © b i t t e r n e s s o f p u re n a r in g in may be n o tic e d in s o lu t io n s o f one p a r t in t e n th o u s a n d . T h is q u a l i t y h as le a d many to b e lie v e t h a t q u in in e o r o th e r b e n e f i c i a l a l k a l o i d s a re p r e s e n t in th e g r a p e f r u i t . No t e s t s have shown t h i s t o be t r u e . F u rth e rm o re a f t e r th e n a r in g in i s e x t r a c te d th e r e s id u e l o s e s i t s b i t t e r t a s t e . A sah in a an d Inubuse-^-4 a s 5 7 4* tr ih y d r o x y fla v o n o n e : oh II o 14A sah in a and Inubuse-, o p . c .i t . , p . 1514. 15Z o ll e r , o p . o i t . . p . 366. 8 F u n c tio n .-*-^ The r o le o f n a r in g in in th e l i v i n g t r e e and f r u i t i s n o t c l e a r l y u n d e rs to o d . Most g lu c o s id e s a r e accom panied by enzym es w hich have th e pow er to h y d ro ly z e them . The n a t u r a l g lu c o s id e s p ro b a b ly have many- f u n c t i o n s . They may s e rv e a s r e s e r v e fo o d s t o r e s . Some a re p o iso n o u s to i n s e c t s . O th e rs have b een fo u n d to h y d ro ly z e to s u b s ta n c e s o f a n t i s e p t i c q u a l i t i e s . When a b re a k i s made in th e baric o r e p id e rm is w hich c o n ta in s th e g lu c o s id e s o f t h i s ty p e th e a d jo in in g enzym es come in to c o n ta c t w ith th e g lu c o s id e hy d r o ly z in g i t in to th e s t e r i l i z i n g p ro d u c t w hich p r o t e c t s th e b re a k from b a c t e r i a . T lhether n a r in g in i s o f t h i s ty p e i s n o t known. The h y d r o ly s is p ro d u c t n a r in g e n in h as b een found by H arvey and Rygg to be to x ic to th e f r u i t . ^ I n j e c t i o n s o f t h i s s u b s ta n c e i n t o th e e p id e rm is o f f r e s h f r u i t p ro d u ced s o f t s p o ts and p in k c o l o r a t io n s . N a rin g in i s p r o b a b ly a b y -p ro d u c t o f r e s p i r a t i o n , l i k e p h l o r i d z i n , th e g lu c o s id e fo u n d in a p p le s h o o ts . I f t h i s i s th e c a se th e n a r in g in c o n te n t in th e f r u i t sh o u ld g r a d u a lly d e c re a s e w ith th e r e s p i r a t i o n . W hile m ost w o rk e rs have found t h i s to be t r u e , th e c a r e f u l work o f H arvey and Rygg-*-8 shows t h a t th e 16A rm stro n g , E . F . , The S im ple C a rb o h y d ra te s and th e G lu c o s id e s . New Y ork: Longmans G reen and C o ., 1910. ^ H a r v e y and Rygg, " F ie ld an d S to ra g e S tu d ie s on th e Changes in th e C o m p o sitio n o f th e R ind o f th e M arsh S e e d le s s G r a p e f r u it in C a l i f o r n i a ," J o u r , o f A g r i. R e se a rc h 52 723 (1 9 3 6 ). •^^tarvey and Rygg, "B eh a v io r o f P e c t ic S u b s ta n c e s and N a rin g in in G r a p e f r u it in F ie ld and S to r a g e ,” P la n t P h y s io lo g y 13 571 (1 9 3 8 ). n a r in g in c o n te n t does n o t alw ay s d e c re a s e w ith m a tu r i ty . As a r e s u l t o f t h e i r s tu d i e s th e y co n clu d e t h a t th e changes in th e g lu c o s id a l c o n te n t in th e f r u i t a f f i x e d to th e t r e e f o l low s no d e f i n i t e s e a s o n a l c o u r s e . In th e two s t u d i e s made, f r u i t from th e same t r e e s w ere p ic k e d ,e v e r y s i x .weeks and a n a ly z e d to d e te rm in e the*"amount o f n a r in g in p r e s e n t . The f i r s t ex p e rim e n t showed a d e c re a se w ith m a tu r ity and th e seco n d an in c r e a s e .' The n a r in g in seems to b a la n c e some e q u i lib r iu m w ith in th e f r u i t . The f a c t o r s in v o lv e d may be th e a c i d i t y , th e s u g a r c o n te n t, and th e te m p e r a tu r e . The e f f e c t s o f s to r a g e have a ls o b een s tu d ie d by H arvey and Rygg. They fo u n d t h a t th e n a r in g in c o n te n t had a te n d e n c y to in c re a s e when k e p t a t h ig h te m p e r a tu r e s . The f o llo w in g table-*-9 shows th e r e s u l t s o f s to r a g e f o r s i x w eeks a t f ix e d te m p e ra tu re s : TABLE I I I PERCENTAGE OF NARINGIN IN’ THE ALBEDO OF M ARSH SEEDLESS GRAPEFRUIT STORED FOR SIX W EEAS AT VARIOUS TEMPERATURES No S to ra g e 68° 56° 460 38° 2 .3 6 2 .7 1 2 .6 2 .5 2 2 .0 5 2 .5 1 2 .3 4 2 .2 4 2 .2 2 1 .6 4 1 .9 9 1 .7 6 1 .7 - * - 9H arvey and Rygg, i b i d . . p . 584. 10 A t o r d in a r y te m p e ra tu re s th e n a r in g in c o n te n t o f p lu c k e d f r u i t a p p a r e n tly alw ay s d e c re a s e s a lth o u g h enough a n a ly s e s have n o t been p u b lis h e d to a f f ir m t h a t t h i s i s a l ways th e c a s e . The a n a ly s e s o f Z o lle r show a d e c re a s e w ith a g e . The f r u i t s s e le c te d w ere n o t from th e same t r e e o r ev en fro m th e same g ro v e . The d if f e r e n c e s a re so m arked, how ever, t h a t i t i s e v id e n t t h a t a d e c re a s e in n a r in g in to o k p la c e in th e s e f r u i t : 20 TABLE IV NARINGIN content IN FRESH AND OLD GRAPEFRUIT V a r ie ty Age ~ T o tal* W t. N a rin g in P e r c e n t In d ia n R iv e r f r e s h 770 gms. .6 2 gms. .086 m ark et 715 .35 .049 W a lte rs f r e sh 682 .50 .0 7 3 4 m onths a t 15 692 .2 4 .0 3 5 M arsh S e e d le s s f r e s h 539 . 35 .067 m arket 570 .0 8 .0 1 4 T hese r e s u l t s w h ile i n t e r e s t i n g in a co m p arativ e way a r e r a t h e r lo w . Z o l l e r fs m ethod was an e s tim a tio n b a se d on th e d i r e c t w e ig h t o f th e c r y s t a l s . More r e c e n t a n a ly s e s show t h a t th e n a r in g in p e r c e n ta g e s ru n fro m .2 to .7 in o r d in a r y f r u i t . 20Z o l l e r , o £ . o i t . , p . 374. 11 W hile th e n a r in g in in th e f r u i t shows a te n d e n c y to d e c re a s e w ith m a tu r ity , t h i s i s n o t alw ay s th e c a se as shown by th e work o f H arvey and Rygg. The f a c t o r s in v o lv e d a re n o t p r o p e r ly u n d e rs to o d . CHAPTER III METHODS‘OF EXTRACTION AND QUANTITATIVE ANALYSIS E x t r a c t i o n . The e a r l y e x p e rim e n te rs u sed g r a p e f r u i t blossom s a s t h e i r so u rc e o f n a r in g in . The b lo sso m s v/ere h e ld by De Y ry to c o n ta in th e h ig h e s t p e rc e n ta g e o f th e g lu c o s id e . W ill o b ta in e d 600 gram s o f n a r in g in from 30 k i l o gram s o f d r ie d b lo sso m s, a y i e l d o f o n ly two p e r c e n t.* ^ The w h ite s e c tio n o f f r e s h f r u i t o f te n h a s a n a r in g in c o n te n t o f two p e r c e n t on th e f r e s h b a s i s , w h ich would be c o n s id e r a b ly h ig h e r in th e d r ie d p e e l . N a rin g in was f i r s t e x t r a c te d w ith a lc o h o l. The raw m a te r ia l was h e a te d w ith th e s o lv e n t and f i l t e r e d . On s ta n d in g th e n a r in g in c r y s t a l l i z e d o u t. Z o lle r d e v is e d a S o x h le t ty p e o f a p p a ra tu s t h a t was e v i d e n tl y q u ite e f f i c i e n t . 22 s o l u b i l i t y b e h a v io r o f n a r in g in in w a te r makes r e c r y s t a l l i z a t i o n c o n v e n ie n t. How ever, in e x t r a c t i n g from th e raw m a te r ia l th e r e a re i n t e r f e r i n g f a c t o r s t h a t make w a te r a l e s s d e s ir a b le a g e n t th a n a l c o h o l. N a rin g in i s h y d ro ly z e d in b o il i n g w a te r and th e p r o p e c tin o f th e f r u i t p u lp i s h y d ro ly z e d to th e s o lu b le p e c t i n w hich makes p u r i f i c a t i o n more d i f f i c u l t . 23-Will, o p . c i t . , B e ric h te 20 p . 295. 22Z o l l e r , o p . c i t . . p . 370. 13 U sing th e m o d if ic a tio n s t h a t Poore w orked o u t w a te r may be u sed c o n v e n ie n tly . He fo u n d t h a t in u s in g f o u r p a r t s o f w a te r to one o f th e m a t e r i a l , 90° f o r f iv e m in u te s i s th e b e s t s e t o f c o n d itio n s to remove th e m ost n a r in g in and th e l e a s t p e c t i n . Two e x t r a c t i o n s w ere fo u n d t o remove 78 to 87$ o f th e s u b s t a n c e . 23 A lc o h o l p ro v ed more e f f i c i e n t . The p e c t i n was rem oved w ith n e u t r a l le a d a c e t a t e and th e e x c e s s le a d w ith h y d ro g en s u lp h id e . A summary o f th e com plete m ethod i s g iv e n below : To one p a r t o f f i n e l y ground m a te r i a l add f o u r o f w a te r . H eat to 9 0 °, h o ld in g t h i s te m p e ra tu re f o r f iv e m in u te s . F i l t e r p r e s s in g th e b u lk . Add two more p a r t s o f w a te r and h e a t to 8 0 ° . F i l t e r a t once and e v a p o ra te u n d e r re d u c e d p r e s s u r e to one n in th o f th e o r i g i n a l volum e. A f te r c o o lin g se e d w ith a few c r y s t a l s and s e t in an ic e box f o r two o r t h r e e < ? d a y s . F i l t e r th e c r y s t a l s and d is s o lv e them in a s m a ll am ount o f f o t w a te r . Add a s l i g h t e x c e s s o f n e u t r a l le a d a c e t a t e and f i l t e r h o t. Remove e x c e s s le a d by p a s s in g h y d ro g en s u lp h id e in th e h o t s o lu t io n and f i l t e r i n g . E v a p o ra te to a s m a ll volum e, c o o l and s e e d . R e c r y s t a ll i z e tw ic e from h o t w a te r , f i l t e r an d d ry a t 100° in an o v en . The a l c o h o lic m ethod w orked o u t by P o o re i s s im ila r to t h i s e x c e p t t h a t 300 m l. o f th e s o lv e n t i s added to 200 gram s o f th e m a te r ia l an d h e ld a t 70° f o r t h i r t y m in u te s . The n a r in g in u se d in t h i s s tu d y was e x t r a c te d b y a a im p le r, i f l e s s e x a c t , m ethod. The s u g g e s tio n o f P u lle y and von L o eseo k e2^ to c o v e r th e e x t r a c t w ith a la y e r o f 23h . d . P o o re , "R eco v ery o f N a rin g in and P e c t in from G r a p e f r u it R e s id u e ," J o u r , o f I n d . and E ng. Chem. 26 (1934) p . 639. ^ P u l l e y and von L o e se c k e , op,, c i t . , p . 175. 14 to lu e n e to p re v e n t m o u ld s-fro m fo rm in g was used* A lso tlie e x c e s s le a d was rem oved W ith p o ta s s iu m o x a la te a s s u g g e s te d b y H arvey and R y g g .25 The co m p lete m ethod u se d i s g iv e n below ; F iv e p a r t s o f w a te r w ere u sed t o c o v e r one o f th e raw m a t e r i a l . The ?<rhole was k e p t a t 80° f o r t e n m in u te s , f i l t e r e d p r e s s in g th e b u lk . The same was used f o r a f r e s h b a tc h , r e p e a tin g th e same p ro c e d u re . A f te r s e v e r a l e x t r a c t i o n s th e s o lu tio n was f i l t e r e d h o t th ro u g h a f i l t e r c e l l o f k ie s e lg u r and l e f t t o s ta n d a f t e r ad d in g a l a y e r o f to lu e n e . A f te r s e v e r a l day s th e c r y s t a l s w ere f i l t e r e d and d is s o lv e d in a s m a ll am ount o f h o t w a te r and f i l t e r e d . The c r y s t a l s w ere a g a in a llo w e d to fo rm and w ere f i l t e r e d . A f te r d is s o lv in g in h o t w a te r n e u t r a l le a d a c e ta te was added and th e s o l u t i o n f i l t e r e d . h o t. Lead o x a la te was ad d ed and th e w hole was f i l t e r e d h o t a g a in . The s o l u t i o n was l e f t to s ta n d f o r two d ay s a t th e end o f w hich th e c r y s t a l s w ere f i l t e r e d and d r i e d . Q u a n tita tiv e M ethods. N a rin g in h a s been d e te rm in e d q u a n t i t a t i v e l y b y f o u r m eth o d s, by g r a v im e tr ic e x t r a c t i o n , by g r a v im e tr ic e x t r a c t i o n o f th e d e r iv a tiv e n a r in g e n in , b y th e m easurem ent o f th e in c re a s e d re d u c in g pow er due to h y d ro l y s i s , and by a c o l o r im e tr ic m ethod u s in g th e c o lo r p ro d u ced w ith f e r r i c c h lo rid e * B ecause o f th e low n a r in g in p e r c e n ta g e , th e g r a v i m e tr ic m ethods n e c e s s i t a t e u s in g la r g e am ounts o f th e t e s t m a t e r i a l . Even u n d er th e b e s t c o n d itio n s c e r t a i n l o s s e s o c c u r in e x t r a c t i o n and, r e c r y s t a l l i z a t i o n w hich r e s u l t in low p e r c e n ta g e s . The h y d r o ly s is p ro d u c t n a r in g e n in h a s b een . 25,lR e p o rt o f th e Com m ittee on M ethods o f C hem ical A n a ly s is o f th e A m erican S o c ie ty o f R ia n t P h y s i o l o g i s t s , ” S e c tio n I I I , P la n t P h v s io lo g y 2 (1927) p . 197. 15 s u g g e s te d a s a b e t t e r s u b s ta n c e to e x t r a c t g r a v im e tr ic a lly s in c e i t i s in s o lu b le in Hot -w ater. The t h e o r e t i c a l y ie ld o f n a r in g e n in i s 44fo o f th e t o t a l g lu c o s id e , so th e lo s s e s o f h a n d lin g w o u ld rn a g h ify th e e r r o r in e x t r a c t i n g t h i s s m a lle r q u a n t ity . These m ethods may be s ta n d a r d iz e d w ith known q u a n t i t i e s an d an e r r o r f a c t o r in tro d u c e d t o g iv e f a i r l y a c c u r a te r e s u l t s . The t h i r d m ethod c o n s i s t s o f m e a su rin g th e in c re a s e d re d u c in g pow er o f a n a r in g in e x t r a c t a f t e r h y d r o l y s i s . Com p l e t e d i r e c t i o n s f o r t h i s m ethod have n o t b een p u b lis h e d , a lth o u g h H arvey and Rygg em ployed i t i n one o f t h e i r r e c e n t s t u d i e s . 2* * They c a lc u l a t e d th e n a r in g in by m u ltip ly in g th e su g a r c o n te n t (a s d e x tro s e ) tim e s th e f a c t o r 1 .6 . T h is m ethod e v i d e n tl y demands th e m ost s k i l l o f th e o p e r a to r to g iv e a c c u r a te r e s u l t s . The c o lo r im e tr ic ' m ethod.w as w orked o u t by H arvey and R y g g ,27 & nd was u se d by them in t h e i r l a t e s t s t u d y .^8 T h is m ethod i s th e q u ic k e s t a n d , a c c o rd in g to i t s o r i g i n a t o r s , p ro b a b ly th e m ost a c c u r a te e x c e p t in th e d e te r m in a tio n o f th e y e llo w s e c tio n o f th e p e e l in w hich c a se th e dye i n t e r - 2SH arvey and Rygg, J o u r , o f A g r i. R e se a rc h 52 (1936) p . 723. 27I b i d . , P la n t P h y s io lo g y 13 571 (1 9 3 8 ). 2 8 l b i d ., i i 463 (1 9 3 6 ). 16 f e r e s w ith, th e c o lo r m a tc h in g . The m ethod c o n s i s t s o f m aking a 500 c c . e x t r a c t of a 10 gram sam ple and d i l u t i n g t h i s w ith th e p ro p e r am ounts o f w a te r to ap p ro x im ate th e n a r in g in c o n t e n t o f th e s ta n d a r d s o l u t i o n . One d ro p o f 50$ f e r r i c c h lo r id e i s added to £5 c c . o f e a c h and a r e a d in g ta k e n w ith th e c o lo r im e te r . The c o lo r p ro d u ced h as th e d is a d v a n ta g e o f d e e p e n in g , so t h a t th e s ta n d a r d s o lu t io n m ust he made up f r e s h f o r e a ch r e a d in g . The s ta n d a r d i s made up o f 1 c c . o f an a lc o h o lic n a r in g in s o l u t i o n ( .£ gram in 100 c c . o f 95$ a lc o h o l) d i l u t e d w ith w a te r to 35 c c . T h is m akes a f i n a l s t r e n g t h o f 8 m illig ra m s in 100 c c . The n a r in g in c o n te n t i s e a s i l y c a lc u l a te d by th e in v e rs e r e l a t i o n s h i p o f th e co n cen t r a t i o n s w ith th e d e p th s o f th e c o lo r im e te r tu b e s . The am ounts o f d i l u t i o n and o f th e o r i g i n a l volume m ust be ta k e n in to a c c o u n t to o b ta in th e t o t a l n a r in g in c o n te n t. A con v e n ie n t im y to f i n d th e d i l u t i o n s n eed ed i s to em ploy th r e e s ta n d a rd d i l u t i o n s , 10 c c . in 2 5 , 5 c c . i n 25, and 5 c c . in 50. Then th e r e s u l t o b ta in e d m ust be m u lt ip lie d b y th e f o llo w in g f a c t o r s : TABLE V DILUTION FACTORS T e s t s o l u t io n ta k e n s t r a i g h t X 5 ft tl d i l . 10 cc in 25 cc X 1 2 .5 ft f? ,f 5 cc in 25 cc X 25 r r n ” 5 cc in 50 ce X 50 The m ost a c c u r a te r e s u l t s a re o b ta in e d when the- c o lo r im e te r < > r e a d in g s a re c lo s e t o g e t h e r . 17 M o d if ic a tio n s . The s o u rc e s o f e r r o r o f th e c o lo rim e t r i c m ethod o f d e te rm in in g n a r in g in a re due to th e i n s t a b i l i t y o f th e c o lo r p ro d u ced w ith f e r r i c c h l o r id e , to th e i n t e r f e re n c e o f th e y e llo w dye o f th e o u te r p e e l , and to any weak n e s s e s o f th e o p e r a to r * s ju d g m en t. I f th e s e e r r o r s can be made c o n s i s t e n t l y , co m p ariso n s tu d i e s w i l l be a c c u r a te . The f o llo w in g s tu d i e s w ere made to overcom e, a s much a s p o s s i b l e , th e s e s o u re e s o f e r r o r , a s w e ll a s to f i n d th e b e s t g e n e ra l p ro c e d u re t o u s e . H arvey and Rygg s u g g e s t p r e p a r in g th e t e s t e x t r a c t by b o il i n g te n gram s o f th e sam ple in 500 c c . o f w a te r f o r th r e e m in u te s and s t r a i n i n g th ro u g h c h e e s e c lo t h .29 To d e te rm in e i f any l o s s due to h y d r o ly s is o c c u rre d in b o i l i n g th e s o l u t i o n s e v e r a l e x t r a c t i o n s w ere c a r r i e d o u t u n d er d i f f e r e n t c o n d itio n s and th e n a r in g in c o n te n t o f e a c h d e te rm in e d . The same sam ple and s o lu t i o n was u se d in each o f th e s e t e s t s . In e a c h case a f t e r th e f i r s t e x t r a c t i o n th e volume was m arked on th e n e c k o f th e f l a s k and th e s o l u t io n made up t o i t s o r i g i n a l volume i f a l t e r e d . The s o l u t i o n was o f s u c h . s tr e n g t h t h a t a 5 c c . p o r tio n was d i l u t e d to 25 c c . and t e s t e d w ith th e f o llo w in g r e s u l t s : 29H arvey and Rygg, P la n t P h y s io lo g y 11 (1936) p . 464. 18 TABLE VI EXTRACTION OF NARINGIN UNDER VARIOUS CONDITIONS OF TIME AND TEMPERATURE C o n d itio n s Mg; N a rin g in in 100 e c . 80° f o r 5 m in. 6 .6 5 80° f o r 10 m in. 7 .7 B o ile d f o r 5 m in. 8 .0 B o ile d f o r 10 m in. 8 .0 To d u p lic a te th e c o n d itio n s u n d e r w hich a c t u a l e x t r a c t i o n s a r e made th e same t e s t s w eve p e rfo rm e d u s in g f r e s h sam p les f o r e a c h . The m a te r ia l u se d was th e w h ite p o r tio n o f th e p e e l w e ll g round and m ixed so t h a t hom ogeneous sam ples c o u ld he ta k e n ; TABLE V II EXTRACTION OF NARINGIN UNDER VARIOUS CONDITIONS C o n d itio n s Mg. N a rin g in in 100 c c . 80° f o r 10 m in. 7 .7 5 B o ile d f o r 3 m in. 8 .9 B o ile d f o r 30 m in. 8 .2 5 (same s o lu tio n ) From th e s e t e s t s i t ¥/as d e c id e d t h a t b o i l i n g f o r th r e e m in u te s rem oved th e m ost n a r in g in . W hile b o i l i n g f o r te n m in u te s showed no l o s s , th e s o l u t i o n becam e somewhat t u r b i d , 19 due to th e p e c t i n e x t r a c t e d . Some n a r in g in was l o s t when th e s o l u t i o n was b o ile d f o r 30 m in u te s . The in te r f e r e n c e o f th e .y e llo w dye i s a t a maximum when t e s t s a re made o f th e y e llo w s e c tio n o f th e p e e l a lo n e . The n a r in g in c o n te n t i s su ch t h a t no d i l u t i o n i s n e c e s s a r y . W hile a m a tch in g o f c o lo r s i s o u t o f th e q u e s tio n , a m a tch in g o f sh a d e s o r o f i n t e n s i t i e s o f l i g h t i s p o s s i b l e . By t h i s means th e e x t e n t o f in t e r f e r e n c e o f th e y e llo w c o lo r o f a .2 5 fo p o ta s s iu m d ic h ro m a te s o l u t i o n in th e s ta n d a r d s o lu t io n was a s c e r t a i n e d . The d ic h ro m a te vms p la c e d d ro p b y d ro p in a s ta n d a r d s o lu t io n and com pared w ith a n o th e r s ta n d a r d . The s e t t i n g s o f th e c o lo rim e te r w ere a s fo llo w s : TABLE V III THE INTERFERENCE OF POTASSIUM DICHROMATE IN THE STANDARD D rops . o f p o ta s s iu m . d ic h ro m a te S e t ti n g o f th e C o lo rim e te r 0 25 t25 1 2 5 -2 4 .2 2 2 5 -2 2 .5 3 25-20 4 2 5 -1 8 .1 5 25-17 I f i t can be s a f e l y 'assumed t h a t th e d ic h ro m a te s o lu t i o n i n t e r f e r e s in a s i m i l a r way to th e n a t u r a l d y e, a ro u g h c o r r e c t i o n can be made by m a tc h in g th e y e llo w a s w e ll a s th e b ro w n ish re d c o l o r . S in c e .th e n a r in g in i t s e l f g iv e s a y e llo w c o lo r in s o l u t i o n , a m a tch in g o f th e dye w ith d ic h ro m a te was f i r s t t r i e d a f t e r th e f e r r i c c h lo r id e was a d d e d . A f t e r an i n i t i a l r e a d in g was ta k e n th e two s o l u t io n s w ere h e ld up to th e l i g h t and th e d ic h ro m a te s o lu t io n added d ro p b y d ro p u n t i l th e s ta n d a r d a p p e a re d th e same a s th e t e s t s o l u t i o n . Then .by com paring th e i n i t i a l - r e a d i n g w ith th e r e a d in g o f th e s ta n d a rd w ith th e same number o f d ro p s o f th e d ic h ro m a te s o l u t io n a s was u se d in m a tc h in g , a rough c o r r e c t i o n was m ade: E xam ple: I f th e i n i t i a l s e t t i n g i s 25-19 and two d ro p s w ere n eed ed to m atch th e y e llo w d y e, th e c o r r e c t io n i s a p p lie d by com paring w ith th e r e a d in g o f th e s ta n d a r d w ith two d ro p s a s shown ahove to be 2 5 -2 2 .5 . 1 9 /2 2 .5 = 8/X X = 9 .5 I n com paring r e a d in g s l i k e t h i s th e same c o n d itio n s m ust be o b se rv e d a s i t was fo u n d t h a t ch an g es in th e ty p e and am ount o f l i g h t u se d c a u se d v a r i a t i o n s in th e r e a d in g s .. B ecause o f th e d i f f i c u l t y in m a tch in g th e dye c o lo r w ith th e re d p r e s e n t i t was f i n a l l y d e c id e d to m atch th e c o lo r o f th e s o lu t i o n s b e fo re ad d in g th e f e r r i c c h l o r id e . A f t e r t h i s was done th e r e a d in g s w ere ta k e n in th e norm al way m a tc h in g c o lo r s i n th e c o lo r im e te r and c a l c u l a t i n g th e r e s u l t s d i r e c t l y from t h i s r e a d in g . T h is m ethod p ro d u ced somewhat h ig h e r r e s u l t s th a n th e o t h e r . B oth can be c o n s id e r e d o n ly a s ro u g h c o r r e c t iv e m ethods to overcom e t h i s i n t e r f e r e n c e . 21 To d e te rm in e th e e x te n t o f d e e p e n in g o f th e f e r r i c c h lo r id e r e a c t i o n c o lo r a f r e s h s ta n d a r d s o lu t io n was com p a re d e v e ry f o u r m in u te s w ith one t h a t was a llo w e d t o s ta n d . The change i s shown "by th e f o llo w in g s e t t i n g s o f th e c o l o r i m e te r: TABLE IX THE EXTENT OF DEEPENING S e tti n g s Time in m in u te s 2 5 -2 4 .9 0 2 5 -2 4 .2 4 2 5 -2 3 .6 8 25-23 12 2 5 -2 2 .6 16 2 5 -2 2 .2 20 25-22 24 O th er t e s t s showed t h a t s o l u t i o n s o f th e same s tr e n g t h deep en ed a t th e same r a t e . I t i s im p o rta n t t h a t r e a d in g s he ta k e n a t u n ifo rm i n t e r v a l s o f tim e a f t e r th e f e r r i c c h lo r id e i s ad d e d . No t e s t s w ere made to f i n d o u t to w hat e x t e n t t h i s c o lo r r e a c t i o n fo llo w e d B e e r T s Law, s in c e th e o b je c t o f th e p ro c e d u re was to ap p ro x im a te as, n e a r l y a s p o s s ib le th e n a r i n - g in c o n te n t o f th e s ta n d a r d by means o f d ilu tio n * A ll r e a d in g s o f w ide d if f e r e n c e w ere r e j e c t e d and th o s e c l o s e s t t o g e th e r h e ld a s th e m ost a c c u r a te . I n th e a n a ly s e s m ade, th e e x t r a c t i o n was done by b o i l i n g f o r th r e e m in u te s and s t r a i n i n g . I n c e r t a i n c a s e s w here th e n a r in g in c o n te n t was h ig h , th e p r e s s e d sam ple was w ashed a g a in w ith a s m a ll p o r tio n o f b o il i n g w a te r and p r e s s e d . A lso i n th o s e c a s e s w here th e m a te r ia l was ta k e n w hole b e cau se o f d i f f i c u l t y in g e t t i n g r e p r e s e n t a t i v e sa m p le s , a l i t e r was u se d to e x t r a c t in s te a d o f th e cu sto m ary h a l f l i t e r . CHAPTER IV EXPERIMENTAL W O R K AN D RESULTS M ethod. The c o l o r im e tr ic m ethod a s o u t l i n e d i i n th e l a s t s e c tio n was u se d t o make a n a ly s e s o f th e n a r in g in con t e n t o f C a lif o r n i a grown g r a p e f r u i t in s e v e r a l s ta g e s of- d e v elo p m e n t. The f r u i t was a l l o f th e M arsh S e e d le s s v a r i e t y e x c e p t f o r some g re e n p e a r-s h a p e d f r u i t w ith v e ry t h i c k r i n d s . G reen and f r e s h l y p ic k e d r i p e f r u i t from th e same l o c a l i t y , and m a rk et f r u i t o f unknown age were s e le c te d t o r e p r e s e n t th r e e s ta g e s o f d ev e lo p m e n t, g re e n , f r e s h , and o ld . F o r a " b e tte r co m p ariso n in th e n a r in g in c o n te n t f r u i t o f s t i l l g r e a t e r d if f e r e n c e s w ere o b ta in e d from one t r e e , y e a r - o ld r ip e n e d f r u i t l e f t a tta c h e d t o th e t r e e , f r e s h r ip e f r u i t o f th e c u r r e n t c ro p , and s m a ll g re e n f r u i t o f a few weeks o f a g e . In e a ch c a s e , e x c e p t f o r th e v e r y sm a ll g re e n f r u i t w here th e s e c tio n s w ere n o t s u f f i c i e n t l y d e v e lo p e d f o r se p a r a t i o n , th e f r u i t was w eighed w hole and th e n p e e le d . The w h ite s e c t i o n o f th e h in d was c a r e f u l l y s e p a r a te d from th e . o u te r y e llo w and b o th w eig h ed . Each s e c t i o n was th o ro u g h ly gro u n d up and m ixed f o r sa m p lin g . Ten gram s o f e a c h were w eighed o u t f o r e x t r a c t i o n . The m em branes o f th e ju ic y p a r t o f th e f r u i t were p re s s e d d ry w ith a h y d r a u lic p r e s s and a ls o j w eig h ed . T h is p u lp was ta k e n w hole f o r e x t r a c t i o n , s in c e t h i s £4 was fou n d to be more c o n v e n ie n t th a n p r e p a r in g i t f o r a c c u r a te sa m p lin g . E x tr a c tio n was done by b o i l i n g f o r th r e e m in u te s in h a l f a l i t e r o f w a te r and s t r a i n i n g th e s o lu t i o n th ro u g h a c h e e s e c lo th f i l t e r . A f te r c o o lin g , th e e x t r a c t was made up to e x a c t l y 500 c c . in a v o lu m e tric fla s k * The s m a ll g re e n f r u i t w ere ta k e n w hole f o r e x t r a c t i o n and w ere o f su c h h ig h n a r in g in c o n te n t t h a t a l i t e r was u sed in e x t r a c t i o n . I n m ost c a s e s th e sam ple was w ashed w ith a s m a ll am ount o f b o i l i n g w a te r a f t e r b e in g p r e s s e d d ry o f th e f i r s t s o l u t i o n . In com paring th e t e s t e x t r a c t w ith th e s ta n d a r d s o lu t i o n , a p ro p e r d i l u t i o n was so u g h t so t h a t th e i n t e n s i t y o f r e a c t i o n c o lo r was a b o u t th e same i n e a c h . D ilu tio n s were made b y d i l u t i n g 5 and 10 c c . p o r tio n s to £5 c c . In e x t r a c t s o f h ig h n a r in g in c o n te n t 5 cc. p o r tio n s w ere made up t o 50 c c . A f te r s e v e r a l d e te r m in a tio n s w ere m ade, i t was fo u n d t h a t th e y e llo w p e e l e x t r a c t c o u ld be u sed s t r a i g h t an d th e w h ite p e e l and p u lp e x t r a c t s c o u ld be d i l u t e d one p a r t to f i v e . The y e llo w dye o f th e o u te r r in d was b a la n c e d w ith a d ic h ro m a te s o l u t i o n , a s e x p la in e d e ls e w h e re . The t o t a l n a r in g in c o n te n t was C a lc u la te d from th e r e a d in g s , ta k in g in to a c c o u n t th e e x t e n t o f d i l u t i o n and th e t o t a l w e ig h t o f th e w h o le . From t h i s were c a lc u l a te d th e p e rc e n ta g e o f n a r in g in i n th e w hole f r u i t , th e p e rc e n ta g e in e a c h s e c t i o n , and th e d i s t r i b u t i o n p e rc e n ta g e o f e a c h . £5 Thanks a r e due to th e S i e r r a M adre-Lam anda C itr u s A s s o c ia tio n o f P a sa d e n a , C a l i f o r n i a , w hich s u p p lie d p a r t Of th e f r u i t u se d in t h i s s tu d y . O th er f r u i t s w ere p u rc h a se d a t m a rk e ts , o r w ere ta k e n d i r e c t l y fro m th e t r e e s . R e s u lts The M a rin g in C o n ten t o f C a li f o r n i a M arsh S e e d le s s G r a p e f r u it F re s h F r u i t W e ig h t in gram s Grams o f n a r in g in P e r e e n t n a r in g in D i s t r i b u t i o n % n a r in g in 1 Whole 460 1 .9 8 .4 4 1 0 0 .0 Ye H ow 78 .40 .5 1 2 0 .2 W hite 84 : 1 .3 6 1 .6 2 6 8 .2 P u lp 65 .2 2 .3 7 1 1 .6 £ Whole 424 1 .8 5 .4 4 1 0 0 .0 Y ellow 66 .3 5 .5 3 1 8 .9 W hite - 60 1 .1 1 1 .8 7 6 0 .0 P u lp 65 .39 .60 2 1 .1 3 Whole 380 1 .8 6 .4 9 1 0 0 .0 Y ellow 64 .3 2 .50 1 7 .2 W hite 62 , 1 .3 8 2 .2 2 7 4 .1 P u lp 50 .1 6 .3 2 8 .7 4 Whole 410 2 .3 0 .5 6 100.0 Y ellow 60 .4 1 .6 8 1 7 .8 W hite 72 1 .7 2 2 .3 9 7 4 .6 P u lp 61 .17 .2 8 7 .6 5 Whole 5 .0 2 .3 7 .4 8 1 0 0 .0 Y ellow 77 .39 .50 1 6 .4 W hite 84 1 .6 2 1 .9 3 6 8 .0 P u lp 57 .3 6 .6 3 1 5 .6 6 -Whole 393 2 .0 5 ' .5 2 1 0 0 .0 Y ellow 58 .3 5 .60 1 7 .0 W hite 82 1 .4 1 1 .7 2 6 9 .0 P u lp 40 • .29 .7 2 1 4 .0 Av Whole 4 2 9 .5 2 .0 7 .49 1 0 0 .0 e r Y ellow 67 .37 .5 5 1 7 .9 age W hite 74 1 .4 3 1 .9 6 6 9 .0 P u lp 5 6 .3 3 .27 .49 1 3 .1 26 M arket F r u i t W e ig h t i n gram s Grams o f n a r in g in P e r c e n t n a r in g in D i s t r i b u t i o n fo n a r in g in 1 -Whole 360 1 .5 7 .435 1 0 0 .0 Y ellow 58 .39 .6 7 2 4 .8 ‘ -W hite 37 .9 8 2 .6 5 6 2 .4 P u lp 33 .20 .6 1 1 2 .8 2 Whole 420 1 .4 6 .35 1 0 0 .0 Y ellow 54 .37 .7 1 2 5 .5 W hite 30 .86 2 .8 5 5 9 .3 P u lp 53 .2 2 .4 2 1 5 .2 3 Whole 325 1 .3 1 .40 1 0 0 .0 Y ellow 65 .45 .69 3 4 .3 W hite 45 .67 1248 5 1 .2 P u lp 42 .19 .45 1 4 .5 4 Whole 424 1 .6 4 .3 9 1 0 0 .0 Y ellow 62 .36 .5 8 2 1 .9 W hite 55 1 .0 5 1 .9 1 6 4 .1 P u lp 45 . .2 3 .5 1 1 4 .0 Av- Whole 3 8 2.25 1 .6 9 .3 9 1 0 0 .0 e r - Y ellow 5 9 .7 5 .39 .66 2 6 .6 3 age W hite 4 1 .7 5 .89 2 .2 2 5 9 .2 5 P u lp 4 3 .2 5 .2 1 .50 14*12 27 G reen M arsh S e e d le s s V a r ie ty W e ig h t i n gram s Grams o f n a r in g in P e r c e n t n a r in g in D i s t r i b u t i o n fo n a r in g in 1 Whole 304 1 .9 2 .6 3 1 0 0 .0 Y ellow . 4 2 .5 .3 9 .9 2 2 0 .3 W hite 43 1 .1 0 2 .5 6 5 7 .3 P u lp 66 .4 3 .6 7 . 2 2 .4 2 Whole 391 2 .1 6 .5 5 1 0 0 .0 Y ellow 51 .50 .9 8 2 3 .6 W hite 5 6 .5 1 .1 7 2 .0 7 5 4 .2 P u lp 79 .4 8 .6 1 2 2 .2 Av Whole 3 4 7 .5 2 .0 4 .5 9 1 0 0 .0 e r Y ellow 4 6 .7 5 .45 .9 5 2 1 .9 5 age W hite 4 9 .7 5 1 .1 4 2 .5 1 5 5 .7 5 P u lp 7 2 .5 .4 6 .6 4 2 2 .3 0 28 G reen P e a r-s h a p e d V a r ie ty 1 W eight in gram s Grams o f n a r in g in P e r c e n t n a r i n g i ^ D is t r i b u t i o n $ n a r in g in Whole Y ellow W hite P u lp 3 2 1 .5 72 117 3 5 .7 3 .7 0 .9 5 2 .4 5 .30 1 .1 6 1 .3 2 2 .1 8 .8 4 1 0 0 .0 2 5 .7 ’6 6 .2 8 .1 2 Whole 382 4 .6 4 1 .2 1 1 0 0 .0 Y ellow 83 1 .0 3 1 .4 8 2 2 .2 W hite 154 3 .1 8 2 .3 2 6 8 .5 P u lp 4 4 .5 .4 3 .97 9 .3 3 Whole 320 3 .1 3 .9 8 1 0 0 .0 Y ellow 69 .57 .8 2 1 8 .0 W hite 109 2 .2 2 2 .0 2 7 1 .0 P u lp 4 3 .6 .35 1 .2 6 1 1 .0 4 Whole 291 3 .8 3 1 .3 1 100.0 Y ellow 63 .5 6 .8 9 1 4 .6 IThite 116 2 .9 5 2 .5 4 7 7 .0 P u lp 46 .3 2 1 .4 2 8 .4 Av Whole 3 2 8.62 3 .8 3 1 .1 9 1 0 0 .0 e r Y ellow 7 1 .7 5 .7 8 1 .1 3 2 0 .1 2 age W hite 1 2 4 .0 2 .7 0 2 .2 9 7 0 .7 8 P u lp 4 2 .4 5 .3 5 1 .1 2 9 .2 0 G r a p e f r u it fro m th e Same T ree Y ear Q ia n F ru it 29 W eight in gram s Grams o f n a r in g in P e r c e n t n a r in g in D i s t r i b u t i o n fo n a r in g in 1 . Whole 297 1 .3 3 .4 5 1 0 0 .0 Y ellow 31 .1 0 .3 1 7 .2 W hite 36 .9 4 2 .6 0 ' 7 0 .0 P u lp 58 .2 9 .51 2 2 .8 2 Whole 266 1 .3 2 .50 1 0 0 .0 Y ellow 26 .0 9 .3 3 6 .5 W hite 33 .9 4 2 .8 7 7 1 .5 P u lp 50 .2 9 . .5 8 2 2 .0 Av Whole 2 8 1 .5 1 .3 3 .4 8 1 0 0 .0 e r Y ellow 2 8 .5 .10 .3 2 6 .8 5 age W hite 3 4 .5 . 94 2 .7 4 7 0 .7 5 P u lp 5 4 .0 .2 9 .55 2 2 .4 0 F re s h F r u i t 1 ?/hole 329 1 .7 5 .77 1 0 0 .0 Y ellow 3 6 .5 .0 7 .1 9 4 .0 W hite 51 1 .4 3 2 .8 0 8 1 .7 P u lp 28 .25 .8 8 1 4 .3 2 Whole • ... 248 - . 1 .6 7 .67 1 0 0 .0 Y ellow 38 .06 .1 6 3 .6 W hite 53 1 .2 9 2 .4 4 7 7 .3 P u lp 40 .3 2 .80 1 9 .1 Av Whole 288.00 1 .7 1 .72 1 0 0 .0 e r Y ellow 3 7 .2 5 .07 .1 8 3 .8 age W hite 5 2 .0 0 1 .3 6 2 .6 2 7 9 .5 P u lp 3 4.00 .2 9 .8 4 1 6 .7 30 F r u i t from Same T re e , c o n t. S m all G reen F r u i t 'r > W eight in gram s Grams o f n a r in g in P e r c e n t n a r in g in 1 ' Whole 4 7 .5 2 .5 8 5 .4 3 • S Whole 4 0 .8 2 .6 6 6 .5 3 3 Whole S 35.2 2 .3 9 6 .6 8 4 Whole 30 2 . 24 7 .4 5 At - Whole e ra g e 3 8 .3 8 2 .4 7 6 .5 2 31 A verage P e rc e n ta g e s o f M arsh S e e d le s s G ra p e f ru it S e c tio n Age o f f r u i t o f f r u i t W eight in gram s N a r in g in , in gram s P e r c e n t n a r in g in Y/hole f r u i t G reen 3 4 7 .5 2 .0 4 .59 F resh. 4 2 9 .5 2 .0 7 .4 9 O ld' * : 3 8 2.25 1 .6 9 .3 9 Y ellow G reen 4 6 .7 5 .45 s e c t i o n F re s h 67 .37 Old 5 9 .7 5 .39 W hite s e c ti o n G reen F re s h Old ' 4 9 .7 5 7 4 .0 0 4 1 .7 5 1 .1 4 1 .4 3 .89 2 .3 1 1 .9 6 2 .2 2 P u lp G reen 7 2 .5 .46 .6 4 F re sh 5 6 .3 3 .2 6 .49 Old 4 3 .2 5 .21 .50 32 A verage P e rc e n ta g e s o f F r u i t from th e Same T ree • S e c tio n Age W e ig h t N a rin g in P e r c e n t o f f r u i t o f f r u i t in grains m gram s n a r in g in Whole G reen 3 8 .3 8 2 .4 7 6 .5 2 f r u i t F re s h 288.00 1 .7 1 .7 2 Old 2 8 1 .5 1 .3 1 .4 8 Y ellow : :e t : F r e s h 3 7 .2 5 .0 7 .1 8 s e c t i o n Old 2 8 .5 .10 .3 2 W hite F re s h 52 1 .3 6 2 .6 2 s e c tio n Old , 3 4 .5 .9 4 2 .7 4 P u lp F re s h 34 .29 .84 Old 54 .29 .5 5 CHAPTER V SU M M A RY AN D CONCLUSIONS I n s tu d y in g th e c o l o r im e tr ic m ethod o f d e te rm in in g n a r i n g i n , th e fo llo w in g a s p e c ts w ere ta k e n u n d e r c o n s id e r a t i o n , e x t r a c t i o n , th e in t e r f e r e n c e o f th e y e llo w dye o f th e p e e l , and th e d e e p e n in g o f th e r e a c t i o n c o lo r* To f i n d th e b e s t m ethod o f e x t r a c t i o n v a r io u s c o n d i t i o n s w ere t r i e d o u t, k e e p in g th e te m p e ra tu re a t 80° f o r f iv e and f o r te n m in u te s , and b o i l i n g f o r t h r e e and f o r te n m in u te s . The two b o ili n g te m p e ra tu re s showed th e h ig h e s t p e rc e n ta g e o f e x t r a c t i o n fro m w hich was co n c lu d e d t h a t b o i l - in g f o r th r e e m in u te s was th e b e s t s e t o f c o n d itio n s to u s e . No l o s s due to h y d r o ly s is was n o te d u n t i l th e s o l u t i o n was b o ile d f o r t h i r t y m in u te s . To f i n d th e e x te n t o f d e e p e n in g o f th e r e a c t i o n , c o l o r a f r e s h l y p r e p a re d s ta n d a r d s o l u t i o n was com pared e v e ry f o u r m in u te s w ith one t h a t was a llo w e d to s ta n d . The d e e p e n in g was found t o be c o n s id e r a b le o v e r a p e r io d o f tw e n ty -f o u r m in u te s . I t was co n c lu d e d t h a t an u n ifo rm p e r io d o f tim e s h o u ld be ta k e n in p r e p a r in g th e sam p les f o r r e a d in g . The in t e r f e r e n c e o f th e dye c o lo r was c o r r e c te d a p p r o x i m a te ly by a d d in g d ro p s o f a .2 5 fo p o ta s s iu m d ic h ro m a te s o l u t i o n to th e s ta n d a r d u n t i l th e dye c o lo r was m a tch e d . Two m ethods w ere t r i e d th e l a s t o f w hich was c o n s id e re d th e more a c c u r a te . 34 The f i r s t c o n s is te d o f m aking a r e a d in g by m a tch in g th e shade r a t h e r th a n c o lo r and a d d in g th e d ic h ro m a te s o lu t io n to ap p ro x im ate th e dye c o l o r . By r e f e r r i n g to e f f e c t of th e same num ber o f d ro p s on th e s ta n d a r d s o l u t i o n , a c a l c u l a t i o n was m ade. The second m ethod c o n s is te d o f m a tch in g th e y e llo w dye b e fo re a d d in g th e f e r r i c c h lo r id e . The r e a d in g w as ta k e n in th e u s u a l way. T h is was c o n s id e re d th e b e t t e r m ethod s in c e some d i f f i c u l t y was e x p e rie n c e d i n m a tc h in g th e dye i n th e p re s e n c e o f th e f e r r i c c h lo r id e r e a c t i o n c o l o r . B o th m ethods w ere c o n s id e re d o n ly a s ro u g h c o r r e c t i o n s . A n a ly s e s of C a l i f o r n i a M arsh S e e d le s s g r a p e f r u i t in v a r io u s c o n d itio n s w ere made— g re e n , f r e s h , an d o ld o r m ark et A n a ly se s o f th e w h ite and y e llo w s e c tio n s o f th e r in d and o f th e in n e r p u lp w ere made t o f i n d th e p e r c e n ta g e s o f n a r i n g in in e a c h . The r e s u l t s showed a g ra d u a l d e c re a s e in n a r in g in c o n te n t w ith a g e . The fo llo w in g a re a v e ra g e s o f th e p e r c e n ta g e s o f n a r in g in i n th e w hole f r u i t ; TABLE X AVERAGES OF NARINGIN CONTENT IN M ARSH SEEDLESS-GRAPEFRUIT G reen F re s h Old .5 9 $ ' -.49 .3 9 35 F r u i t p ic k e d from th e same t r e e showed ev en g r e a t e r v a r i a t i o n s , s in c e t h e i r d if f e r e n c e s in age w ere a l s o g r e a t e r . TABLE XI AVERAGES OF NARINGIN CONTENT IN FRUIT FROM THE SA M E TREE G reen 6 .5 2 $ F re sh .7 2 Old .4 8 The w h ite s e c t i o n o f th e r in d was fo u n d to be th e r i c h e s t i n n a r in g in o f any o f th e s e c t i o n s . The a v e ra g e p e rc e n ta g e s r a n from .8 9 to o v e r 2$ in th e g re e n f r u i t . The d if f e r e n c e in th e n a r in g in c o n te n t o f th e g re e n M arsh S e e d le s s f r u i t and th e g re e n p e a r sh a p ed f r u i t was due to th e d if f e r e n c e in th e r e l a t i v e am ounts o f th e w h ite p e e l . The w h ite s e c t i o n o f th e r i n d o r p e e l o f th e M arsh S e e d le s s v a r i e t y c o n s t i t u t e d a b o u t 14$ o f th e w hole by w e ig h t, w h ile t h a t o f th e o th e r was 36-40$ o f th e w h o le . The p e rc e n ta g e s o f n a r in g in in ea ch w ere 2 .3 1 and 2 .2 6 . T hese r e s u l t s a p p a r e n tly d is p ro v e th e c o n te n tio n o f th e e a r l y w o rk ers t h a t th e f r e s h b lo sso m s c o n ta in th e h ig h e s t p e rc e n ta g e o f n a r in g in , g iv e n b y W ill* ^ a s 2$ i n th e d r ie d b lo s s o m s . 30W ill, B e ric h te 2 0 , p . 295 36 The h ig h p e rc e n ta g e o f n a r in g in in th e s m a ll g re e n f r u i t th e s iz e o f b i l l i a r d b a l l s was s u r p r i s i n g . The s m a ll e r th e f r u i t th e h ig h e r th e p e rc e n ta g e o f n a r in g in i t was fo u n d to h a v e . T hese f r u i t h ad more n a r in g in by w e ig h t th a n any o th e r e x c e p t th e g re e n p e a r sh ap ed o n e s . T h is s u g g e s ts t h a t sh o u ld g r a p e f r u i t e v e r be grown e x c lu s iv e ly f o r th e p r o d u c tio n o f n a r in g i n , an u n l i k e l y th in g a s lo n g as ca n n in g w a ste s a r e a v a i l a b l e , t h a t f r u i t o f t h i s ty p e w ould b e th e m ost p r o f i t a b l e f o r p r o c e s s in g . B I B L I O G R A P H Y BIBLIO GRAPHY A g r i c u l t u r a l S t a t i s t i c s . U. S . D ep t, o f A g r ., f^ a sh in g to n , D. C* 1938. A rm stro n g , E . F . , S im ple G a rb o h y d ra te s and th e G lu e o s id e s . New York: Longmans G reen and C o ., 1 9 1 0 . A s a h in a ,,Y . and M. U. In u b u se , "U eber d ie K o n s tit u tio n d e s N a r in g e n in s , ” B e rio h te 61B p l5 1 4 1 928. De V ry , J a h r e s b e r ic h t e f u r Pharm acognos 152 1 8 6 6 . H arv ey , E . M. and G. L . Rygg, ’’B e h av io r o f P e c tio S u b s ta n c e s and N a rin g in in G r a p e f r u it i n F ie ld and S t o r a g e ,” ■ Bia-ftt P h y s io lo g y 13 571 1938. ________t " F ie ld and S to ra g e S tu d ie s on th e C hanges i n th e . C o m p o sitio n o f th e R ind o f th e M arsh S e e d le s s G rape f r u i t i n C a l i f o r n i a , ” J o u r , o f A g r. R e se a rc h 52 p p . 725- 746 1936. ________ , ’’C o lo rim e tr ic D e te rm in a tio n o f N a r in g in ,” P la n t P h y s io lo g y 11 p . 463 1936. H e ilb ro n , I . M ., A D ic tio n a r y o f O rganic Compounds. New Y ork: O xford U n iv e r s it y P r e s s , 1938, p . 5 2 , v o l . I I I . H offm ann, E d ., ’’H e s p e rid in de V ry , A u ra n tin , M u rra y in ,” B e rio h te 9 690 1876. Hume, H. H ., C u l tiv a tio n o f C itr u s F r u i t s . New Y ork: M cM illan, 1934 (R e p rin t o f 1926TI A M anual f o r C itr u s G row ers. M utual Orange D i s t . , R e d la n d s , C a l i f o r n i a , 1937. P o o re , H. D.., ’’R eco v ery o f N a rin g in an d P e c t in from G r a p e f r u i t , : R e s id u e ,” J o u r . I n d . E ng. Chem. 26 637 1 9 3 4 . P u lle y , G e o ., " S o l u b i l i t y o f N a rin g in in W a te r .” •J o u r . In d . E ng. Chem. 28 360 1936. P u lle y , G eo ., a#d von L o e se c k e , H ., " P r e p a r a tio n o f Rhamnose fro m N a r in g in ,” J o u r . Am. Chem. S o c. . J a n . 1939 p . 175. "R e p o rt o f th e Com m ittee on M ethods o f C hem ical A n a ly s is o f th e A m erican S o c ie ty o f P la n t P h y s i o l o g i s t s , ” S e c tio n I I I , P la n t P h y s io lo g y 2 (1927) p . 1 9 7 . 3© W ill, W .t "U eber d as N a r in g in ," B e rio h te 18 1311-1335 1885. _, "U eber d as N a rin g in II," B e rio h te 20 294-304 1 8 8 7 . _, "U eber den Z ucker au s H e s p e rid in und N a r in g in ," B e rio h te 20 1186 1887. Z o l l e r , H ., "Some C o n s titu e n ts o f th e A m erican G r a p e f r u i t ," J o u r . In d . E ng. Chem. 10 364 1 9 1 8 . p UMI Number: EP41515 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI Dlsssrtstion Publishing UMI EP41515 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 -1346

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Creator Jamgochian, Nicholas Vahe (author)

Core Title A study of the naringin content in California marsh seedless grapefruit

School Department of Chemistry

Degree Master of Arts

Degree Program Chemistry

Degree Conferral Date 1939-09

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag agriculture, food science and technology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor [illegible] (committee chair), [illegible] (committee member), Johnstone, George R. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-788207

Unique identifier UC11348077

Identifier EP41515.pdf (filename),usctheses-c17-788207 (legacy record id)

Legacy Identifier EP41515.pdf

Dmrecord 788207

Document Type Thesis

Rights Jamgochian, N. V.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

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