Carotene Vitamin C Treatment per pot Type of leaf produced fresh leaf of fresh leaf K deficiency 1 Small, dark, shrivelled 67 46 margin N deficiency 1.1 Small, light colour, erect 7 29 M
Trang 1THE CAROTENE AND VITAMIN C
FromtheLaboratoryofHygiene, University of Utrecht, Holland
(Received 14 October1936.) THE estimation of carotene and vitamin C in vegetables has generally been carried out without paying attention to the different conditions prevailing during growth of the plants analysed Therefore, the results obtained under these circumstances can only be looked upon as average values Although theseaveragevaluesaresufficient forpracticaluse, it isinteresting,withaview
to the quality of the crop, to examine ifthe caroteneand vitamin C contents canbe influenced by soiltreatmentand particularly by fertilizers
The purpose of the investigations was to decide if the concentration of the elements necessary in plant-growthhad any influenceonthe carotene and vitamin Ccontentsofthe plants
EXPERIMENTAL
The investigationswere carried out by means ofpot experiments, because only by this method of working can equal distribution of thesalts besecured, this being essential to produce a crop of sufficient uniformity In a number
of the experimentsthe pots contained pure washed quartz sand,in other cases
anexactlyanalysed sandy soil,which besidesKand Pdeficiency also showeda lowpH
The pots were supplied with a salt solution according to Kruger of the following composition:
Distilledwater 1000 g
pH=7-2 One or more components of this solution were omitted or administered in increased amounts, so that by the disturbance of the conditions of life an insight could begainedinto theconditions oftheformation ofvitamins inplants
K, Mg or N deficiency was produced by omitting the corresponding salt fromthesolution In orderto getCadeficiencyNa2HPO4was used instead of CaHPO4, 2H20 and P04 could be substitutedby SO4togive Pdeficiency. Pots ofindifferent material were used, such as glass, enamelled iron and glazed earthenware
Spinach wasused as a test plant; ithas the advantage ofgrowing rapidly andcontainingconsiderable quantities ofcaroteneand vitaminC
( 2307 )
Trang 2The plants were kept in a glasshouse The pots were weighed every day during theexperimentand theloss in weight was compensated bytheaddition
ofdistilled water
The full-grown plants were cut with scissors To avoid errors caused by differences in weight of the stalks, which contain only a very small quantityof ascorbic acid, thelatterwerediscardedand onlytheleaves were analysed
Table I
mg of ascorbic acid per 100 g of fresh material
Leaves 19
The plants had to be analysed rapidly, because at room temperature and even at ice-box temperature the leaves show a rather rapid decrease in vitamin Ccontent
Table II Storage of spinachleaves inthe ice-box (40)instopperedglass jars,
withapiece ofmoistcotton-woolto keep the leaves fresh
mg of ascorbic acid per 100 g.
No of days , A %
of storage Fresh small leaf Fresh large leaf
Table III Storage of spinach leavesatroomtemperature and in the ice-box
mg of ascorbic acid per 100 g.
No of days Fresh leaf at Fresh leaf in the
of storage room temp ice-box
Methods
Thevitamin C content ofleaveswasdetermined bythetitrationmethod of Birchetal.[1933],with2:6-dichlorophenolindophenolinacid medium
The acidextract of fresh spinachleaves shows the same vitamin C content before and aftertreatment with H2S or with mercuric acetate as used firstby Emmerie [1934]. Therefore this method (Emmerie) has only been used when estimating the loss of ascorbic acid during storage of the leaves, because in this case it ispossible that substancesother than vitaminCarepresent which reduce the indicatororthat, onthe otherhand, acertain amountof vitamin C
is reversibly oxidized and cannot be determined by titration according to
themethod of Birchetal
Carotene was extracted with ether, after saponification of the chlorophyll
etc by boiling the plant materialwith a saturated solution of KOH in 96% alcohol (20 ml per g of plant material) for half an hour [Guilbert, 1934] Besidescaroteneandxanthophyllthe ethercontainedsaponified chlorophylland flavones The latter were removed by washing the etherwith water Then the
J B H IJDO 2308
Trang 3ether was evaporated invacuo and theresidue dissolvedin lightpetroleum; the xanthophyll was then removed from the light petroleum by extraction with
85 and 90% alcohol
This method can be simplified by extracting the carotene with light petroleum immediately after saponification Thus,no chlorophyll or flavones and only a little of the xanthophyll dissolve in the light petroleum; the xanthophyll can be removed by washing once with 85% methyl alcohol The saponification must be carried out with only half of the amount of alcoholic KOH used by Guilbert In this case, the concentration of the alcohol is reduced to about 90%by the water in theplant material; at this concentration carotenedissolvessparingly and isextracted easily, whilexanthophyll dissolves better in the alcohol than in thelightpetroleum By 4 to 5 extractions all the carotene can be removed from thealcohol; furtherlightpetroleum extractions contain xanthophyll only, which can be washed out again with 85% methyl alcohol Therefore the extraction of carotene is complete when in the light petroleum extract all the yellow colouring matter can be removed with 85% methylalcohol
The amount of carotene was determined with the ZeissStufenphotometer
ResuU8
A The influence of Ca, K, N and Mg deficiencies on the carotene and vitamin C contents of spinach The experiment included 23 pots, filled with quartz sand
Table IV Carotene Vitamin C
Treatment per pot Type of leaf produced fresh leaf of fresh leaf
K deficiency 1 Small, dark, shrivelled 67 46
margin
N deficiency 1.1 Small, light colour, erect 7 29
Mg deficiency 2 Light-coloured spots 34 31
B Theinfluenceof addednitrogenoun fertilizersonthe caroteneandvitamin C contents of spinach grown in pots,filled with a sandy soil and supplied with increasing quantities ofP205
Eachresult is themean of two analyses
Table V Vitamin C inmg.per100g.offresh leaf
mg.P20,per pot Organic N _
mg per pot 0 15 30 45 60
125 119 116 103 125 130
Table VI Carotene in y perg. offresh leaf
mg.P205per pot Organic N
mg per pot 0 15 30 45 60
Trang 4J B H IJDO Table VII Vitamin C in mg.per100g. of fresh leaf
15 72 74 94 81
mg.P206per pot 30 74 72 75 116
45 58 76 90 105
60 61 86 113 92
Table VIII Carotene in y per g offresh leaf
mg.P205per pot 15
36 40 107 96
30 34 46 73 103
45 35 51 88 63
60 27 49 73 71
C The influenceof addednitrogenous fertilizersonthecaroteneandvitamin C contents of spinach grown in pots filled with a sandy soil and sUpplied with increasing quantities of potassium
Each resultis the mean of two analyses
Table IX Vitamin C in mg.per100 g of fresh leaf
mg K20 per pot 40
124 124 148
80 113 126 150
120 114 122 140
Table X Carotene in y per g offreshleaf
mg K20 per pot 40
86 101 92
80 45 94 82
120 49 104 94
Table XI Vitamin Cinmg.per100 g of freshleaf.
mg K20 per pot 40
105 121 70
80 119 136 142
120 122 134 140
TableXII Carotene in y per g offreshleaf
mg K20 per pot 40
86 106 104
80 62 96 108
120 65 81 113
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0 56 73 90 90
Inorganic N
mg per pot
0
25
75
125
Inorganic N
mg per pot
0
25
75
125
0 37 65 80 67
0 64 100 92 81
Organic N
mg per pot
0
25
75
125
Organic N
mg per pot
0
25
75
125
r
0 100 124 93 100
10
121
Inorganic N
mg per pot
0
25
75
125
Inorganic N
mg per pot
0
25
75
125
r
0 99 113 80 113
Trang 5D Addition ofphosphate had noeffect.
E From the results of theexperiments recorded under C, the influence of added potassium fertilizer at different concentrations of nitrogen in the soil also becomes evident (Tables IX-XII)
DISCUSSION
The results of the analyses show that the carotene andascorbicacidcontents
ofthe test plants largelydepend on the amounts ofnitrogenand potassium in the soil
A larger amount of nitrogen results in greater carotene and vitamin C contents
An increasing potassium content of the soil causes a decrease in carotene and an increase invitaminC Diminutionofthecarotenecontent is onlyobvious
in the plants grown in pots with little nitrogen Generally the increase in vitamin C content is most distinct in plants grown with a high concentration
ofnitrogen Under theconditions prevailingin theexperiments,theinfluenceof
P, Caand Mgsalts is small
Apart from the practical results, it is interesting to make an attempt to drawsomephysiologicalconclusionsand to view the data in the light of what is knownabout theinfluenceoffertilizersonphotosyntbesisandchlorophyll content Briggs [1922] was the first to investigate the influence of K, Mg and Fe Gregory & Richards [1929] stated that the assimilation of nitrogen-deficient plants ofHordeum is "subnormal" Muller [1932] found the same for Sinapis alba Gaszner& Goeze [1934] stated thatlarger additions of nitrogenresulted
in anincreased assimilationand transpirationand inincreased chlorophylland albumin contents both with rye and wheat; thedifference betweendifferently treatedplantsbecame greaterwhen the plants grew older
Fromthe data oftheexperiments with Nfertilizerdescribed in this paper,
it canbe observed that nitrogen has aninfluenceon the carotene and vitamin C contents similar to its influence on assimilation and chlorophyll content It seems plausible to suppose that there exists a direct relationship between photosynthesis andvitamin C content, in viewof the fact that the vitaminC content ofleaves increases when theyare irradiated with neon-lightand that etiolated plants contain no vitamin C Another argument in support of this theorycan befound in theexperiment ofRandoinet al [1935], whofoundless vitaminCinwhite than in greenportions ofplants
This connexion with photosynthesis does not hold good in the case of carotene Etiolatedplantsshow noassimilationand possesscarotene Moreover Willstatter & Stoll [1913], Scherz [1929], Pfuitzer& Pfaff [1935]state that the carotene content of plants fluctuates with the chlorophyll content Further-more, Karrer & Helfenstein [1931] pointed out that carotene is derived from phytol or phytolaldehyde, establishing a chemical relationship between the nitrogenous chlorophylland theN-freecarotene
From thedataconcerningtheinfluenceofnitrogenousfertilizer itseemsthat there exists a relationship between photosynthesis and vitamin C content on the onehand and between chlorophyll contentand carotene onthe other The data obtained from the experiments with potassium fertilizer confirm thissupposition
Gaszner& Goeze[1934]findanincreasedphotosynthesis underthe influence
of more potassium fertilizer in wheat and rye, 25 days old and grown in a medium of comparatively high N content Plants grown with little N show practicallynodifference in assimilation underinfluence ofKfertilizer
Trang 6J B H IJDO
Ata low Nlevel thechlorophyllcontent ofK-deficient plants is much higher thanof plantsrichlysupplied with potassium; at a high N level this difference does not exist
Concerning the influence of potassium on photosynthesis, similar results were obtained by Briggs [1922], Gregory & Richards [1929] and Lundeg'ardh [1932]; the decrease of green colouring matter as aresult of increasing quantities
ofKfertilizeris awell-known facttoevery farmer and isfrequently mentioned in theliterature [seeMaiwald, 1923; Remy & Dhein, 1932; Remy& Liesegang, 1926] From the data concerning the influence of K fertilizer on the carotene and vitamin C contents of spinach, grown on a sandy soil with increasing quantities
of nitrogen, it can be readily seen that carotene shows the same fluctuations as chlorophyll, whereas obviously vitamin C is a product of assimilation or at least closelyconnectedwith the process Carotene content decreases at low N levels
asaresult of increasing quantities of K, whereas this decrease ispracticallyzero
at high N levels; vitamin C content increases but little at low N levels as a result of the addition of K fertilizers, whereas at high N levels a rapid increase can beobserved
From theresults of the experiments it can also be concluded that nitrogen and potassium stand in close interrelation physiologically; K deficiency has theeffectofN excess and K excess acts like N deficiency
Therefore, fertilizerexperiments concerning one of these two elements only give accurate results if made at different concentrations of the other element
SUMMARY
1 Details aregiven ofthe methods of growing spinach for analysis
2 Tablesare presented showing lossinvitamin C content during storage
3 A modified method forestimating the carotene content ofvegetables is given
4 Tablesarepresented showingtheinfluenceofK, N,CaandMgfertilizers
onthe carotene and vitamin C contents ofspinach Ahigherlevelofnitrogen results in a greater carotene and vitamin C content An increasing potassium content of the soil causes a decrease in carotene content and an increase in
vitamin C
5 An attempt is made to show a relationship between chlorophyll and carotene; on the other hand the suggestion that ascorbic acid only can be a
product ofphotosynthesisisdiscussed
REFERENCES.
Birch, Harris & Ray (1933) Biochem J 27, 590.
Briggs (1922) Proc roy Soc B 94, 20.
Emmerie (1934) Biochem J 28, 268.
Gaszner & Goeze (1934) Z Bot 27, 255.
Gregory & Richards (1929) Ann Bot., Lond., 43, 119.
Guilbert (1934) J indu8tr Engng Chem (Anal Ed.), 6, 452.
Karrer & Helfenstein (1931) Helv chim Acta, 14, 78.
LundegArdh(1932) Die Nahrstoffaufnahme der Pflanzen (Jena.)
Maiwald (1923) Z angew Bot Charkiv, 5, 33.
Miiller (1932) Planta, 16, 1.
Pfutzer & Pfaff (1935) Z angew Chem 36, 581.
Randoin, Giroud & Leblond (1935) C.R Soc Biol., Pari8, 31, 297.
Remy & Dhein (1932) Landw Jb 76, 953.
& Liesegang (1926) Landw Jb 64, 213.
Scherz(1929) PlantPhysiol.4, 269.
Willstaitter & Stoll (1913). Untersuchungen iuberChlorophyll; Methoden und Ergebnisse (Berlin.)
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