Determination.of Carotene in Plant Materials ARUN.4CHALA SREENIVASAN AND K. > I . VIIUY.4 Department of Chemical Technology, I;niversity of Bombay, India
A rapid niethod for the routine determination of carotene in plant materials has been worked out by which the green tissues are triturated with a mixture of lime and anhydrous sodium sulfate. This results in their disintegration as well as adsorption of chlorophyll and other noncarotenoid pigments; the carotene itself is rendered readily extractable by petroleum ether or benzene and can be determined either as gross carotene or as its isomerides after chromatographic resolution. The method is particularly suitable for the analysis of green, leafy materials, feeds, and fodders and gives excellent agreement with standard procedures, but it can be applied with equal success to fresh fruits and vegetables.
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S THE determination of carotene in plant tissues, the two general methods of removal of noncarotenoids are phasic and chromatographic separations ( 4 , 30, 34). In view of the recognized variations in t,he extent of digestion and absorption of carotene and its interdependence on the presence of varying amounts of ant'ioxidants ( I , 7,14,15,16,39,do), complete chromatographic analysis, involving the separation of various carotene isomers, is neither practical nor necessary for rout,ine analytical purposes, particularly in the nutritional evaluat,ionof foods, feeds, and fodders (29j. The phase separation method, on the other hand, is not always quantit,ative (11, $3, 24, 26); certain biologically inert degradation products are also taken up in the epiphasic layer (36, 50). In addit'ion, a small percentage of the xanthophylls remains in the peti.oleum phase and appreciable amounts of carotene are taken up in t'he hypophasic methanol layer (11,46). The saponification step with hot alcoholic alkali which precedes phase separation (36) is inconvenient and causes inaccuracies due t.0 isomerization and, occasionally, loss of carotene (5,I d , 19,51). The possibility of a more direct method for obtaining carotene was indicated in the observations of Fremy ( I S ) that barium hydroxide could be used to remove chlorophyll from alcoholic extracts of green plant's. Tswett (46) had shown that, if a mixed solution of chlorophyll and carotene is shaken with an excess of finely divided calcium carbonate, the chlorophyll is completely adsorbed, leaving the carotene in solution. Kernohan (18) and Cooley et al. (6) suggested t'he use of soda ash for t,he selective adsorption of noncarotenoids, while Petering et d.(26, 32, 33) used baryta for removal of chlorophyll and saponifiable lipides from acetone extracts of alfalfa meal. lfore recently, technical hydrated lime has been employed for the adsorption of noncarotenoid pigments from petroleum ether extracts of vegetable leaf wastes (41)and directly from fresh plant tissues (28). Another adsorbent which is found efficient in preferentially adsorbing chlorophyll and other noncarotenoid plant pigments is dicalcium phosphate, originally Feeommended by Rloore (23, 24, 25) and since extensively used. A column of fat-free bone meal has been suggested as equally effective (20). In this communication is described the developiricnt of tl. niethod for the determination of carotene in which saponification and phasic separation are eliminated. h combination of freshly ignited lime and anhydrous sodium sulfate in one operation dehydrates green tissues and strongly adsorbs chlorophyll and non(Sarotene pigments, after which carotene can be quantitatively extracted with petroleum ether for estimat,ion as such or, where required, after chromatography. MATERIALS AND .METHODS
.ill preliminary trials were carried out with drumstick (JIuringu oleifera) or lucerne (Medicago sativa) leaves, two rich natural surcesof carotene (38, 43, 44). The method, as finally worked
out, has been tested on a large variety of fresh materials, listed in Table I. Carotene, referred to in this paper, includes both a- and p-isomerides although, in most cases, the a-isomer formed only a very small, if variable, fraction of the total carotene. Carotene determinations, for comparison, were made by an extraction procedure using a Waring Blendor and a solvent mixture of alcohol and petroleum ether similar to that of Moore and Ely (35),followed by adsorption and elution from a magnesia column according to the method of Wall and Kelley (46). This method, like the proposed method, does not involve saponification or phasic separation. Color measurement,s were made using a Kletb Summerson photoelectric colorimeter with a No. 42 filter in place. 9 calibration curve with a commercial source of carotene (goyo p- and 10% C Y - , kindly supplied by Barnett Laboratories, 6256 Cherry Ave., Long Beach 5 , Calif.), purified according to Frape and Kemmerer (11),and standardized a t 450 mp with a Billingham and Stanley visual spectrophotometer, was used to read off carotene contents. PRELIiMINARY OBSERVATIONS
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Xost plant materials, when fresh, contain about 80% of moiature; hence, any method of direct extraction of carotene should be preceded by drying, for which purpose preliminary blanching. and use of alcohol, acetone, and water-binding agents like anhydrous sodium sulfate, sodium carbonate, and calcium sulfate have been recommended (3, 8, 17, 26, 41, 42, 48). .Is freshly ignit'ed lime is a good desiccant as well as adsorbent for chlorophyll and sant,hophylls, a number of experiments were carried out in which known amounts of leaf were macerated in a mortar and further triturated with varying proportions of freshly prepared lime, chemically pure and ignited for a few minutes at a temperature of 250" t'o 300' C. before use. When drumstick or lucerne leaves were used, it was found that a proportion of leaf to lime of 1 to 3 gave a loose, fluffy mass, apparently dry and readily extractable wit,h petroleum ether or benzene. However, contrary to the observations of Ilurri (as),the percentage recovery of carotene from this meal was very low; partial heating for a few minutes inside a steam oven or, for longer periods, under vacuuni did not result in much improvement. But, practically all t'he carotene could be quantitatively recovered aft,er refluxing with 10% aqueous sodium carbonate, extraction with acetone, and phasic, separation between petroleum ether and rnet'hanol. I t would therefore follow that treatment n-ith lime in the cold did not. result, in destruction of carotene but t,hat, in the proportion used for efficient absorption of all the moisture in the leaf t,issue, the plant pigments, inclusive of carotene, xere adsorbed by the lime. Further trials with reduced quantities of lime together with varying amounts of anhydrous sodium sulfate showed that about equal weights of leaf, lime, and sodium sulfate gave, on grinding, a fluffy meal in which all the chlorophyll and practically all the xanthophylls were strongly adsorbed and where the carotene was so weakly adsorbed that it could be quantitatively eluted with petroleum et'her. I t was found best first to grind the leaf to a paste in a mort,ar and then add the lime-sodium sulfatr 720
V O L U M E 20, NO. 8, A U G U S T 1 9 4 8 T a h l e 1.
721
C a r o t e n e \-slues lloisture, Proi>u,ed m Method
Sample
-/g f
l y a t h i leaf (S'rsbania grandiflora) Amaranth iA marant hus gan get - Zscsheile and Whitmore (621, \vert' treated with ali(potu of a standard carotene solution in petroleum et,her and t,he solvclnt \vas evaporated off by a hart period of drying in LI s1e;tni o v t ' r i hc9foi-e trituration or ding. .Is observed by \Tall and Kellqy (,i?),cwotene is not nffwted tiy evaporation on a stc:xni hnth under these conditions. Thil i~t~sults for cwottme obtained hy the proposed method coriiably with those given hy the standard procedure (Table I ) , Quantitat,ive recovery of carotene is also obtained with rai'otenp adrlt:d to plant sample* (Table 11); in the latter case, the xliyht~ly lxtter recovery resultiiig from blanched tissues, par-. ticularly n-ith drunistick leaf anti lucerne, is evidently due to in-. activation of cai,otcne-destioyiIig systems in rhese wurces irf. 23). Thrl difft.rencc.h. tioivwer, are .sinal1 ( . 5 L ) . 1
Table IT.
Hecover!
of (:aroteue Added to P l a n t Samples
~ a r o - Caro.tene in tene
Proposed
Wall and Kelley
- " I P L h L _ . _____ Method
Carotene,
Recov-
Carotene
Recoy-
~arotenl~.
Occasionally. \Tit11 w n i t b ot' (,hi, plant iiiatciials exaniiiicvl, small ,Juantities of xanthophylls \\-ere extracted along with the carotene' 1)y thc petrolttuni ether solvent. In oiily one inst,aiic.ta-viz., hitter gourd (Table I)-& small amount of chlorophyll rernaintd iinadsorbed by thr. lime and could not be roniovrd e w n hy in(creasing the propoi,tion of hi(:. Evidently, fhi. oh\orophyll an0 ?he xarithophylls from tlir other s o u r ~ e sare iiot c:ompletcly adsorbed by lime. They could, ho\\-vver, he readily frcw-l bl- passing the estracts through a column of dicdciuni phosphate as recorninend(d hy l l o o r i ~( 2 3 ) . I!ttnioval of such tracvs of c.hloroyhyll 01' \anthopliyll, whert, present, could he c+€ecteti equally n-oll ivlicn rhr adsorhcrit \vas actded along with t h c s lime to tht, giuund nieal ,)r ~ h c n as , \vas preferred. t h r pttrolr!nrn i ~ t h v i(>ytrart , \\'a-J u - t 41aktlii with a *ni:i11 qiiantity of i t .
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PROCEDURE
A Hciglied aniouiiI oi the material (2 to 5 gi'anis, coiwspoiiding 100 to 500 micrograms of carotene) is trans(erred to a glass or porcelain mortar and ground intimately with 1 to 2 grams of quartz sand and 1 or 2 drops of a 2% solution of sodium cyanide. A quantity of anhydrous sodium sulfate, equal to the weight of the material taken, is now added and the mixture triturated again. This is followed by the addition of a n equal amount of freshly ignited lime and further maceration when a loose, fluffy meal with a light greenish yellow tint results. This is transferred to a hcaker and dried in the steam oven for 15 to 20 minutes. The mixture is next extracted in the cold or, preferably, over a water bath maintained at 50" to -60" C. with successive sniall quantities of low-boiling petroleum ether till no more carotene is extracted. Three or four extractions are usually sufficient. Tliv extract, after decantation each time, is passed through a filter paper. For routine purposes, some type of continuous extractor may be used, if preferred. The combined petroleum et,her extract is shaken with a small quantity (0.5 to 1 gram) of dicalcium phosphate to remove a n y chlorophyll and xanthophylls that, mav be present in solution. T h e extract, if made turbid by suspended materials, is filtered and ma& up to volume, a-hen ir is ready for -0 approximately
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