[Advances in Food Research] Advances in Food Research Volume 4 Volume 4 || The Chemistry of Chlorophyll (With Special Reference to Foods)

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<ul><li><p>The Chemistry of Chlorophyll (with Special Reference to Foods)l </p><p>BY S . ARONOFF Iowa State College. Ames. Iowa </p><p>CONTENTS </p><p>Page I . Introduction . . . . . . . . . . . . . . . . . . . . . 134 </p><p>I1 . Nomenclature . . . . . . . . . . . . . . . . . . . . . . 135 I11 . The Chemistry of Chlorophyll . . . . . . . . . . . . . . . . 138 IV . Extraction and Isolation . . . . . . . . . . . . . . . . . . 150 </p><p>155 </p><p>1 . Determination . . . . . . . . . . . . . . . . . . . . 155 2 . Spectrophotometry . . . . . . . . . . . . . . . . . . 155 3 . Criteria of Purity . . . . . . . . . . . . . . . . . . . 162 </p><p>a . Extracted Material . . . . . . . . . . . . . . . . 163 (1) Chromatographic Homogeniety . . . . . . . . . . 163 (2) Oxygen Uptake: Allomerization and the Phase Test . . 163 (3) Methanolysis . . . . . . . . . . . . . . . . 164 (4) Absence of Chlorophyllides . . . . . . . . . . . 164 (5) The Cleavage Test . . . . . . . . . . . . . . 164 </p><p>b . Isolated Material . . . . . . . . . . . . . . . . . 164 (1) Ratio of Heights of Absorption Bands . . . . . . . 164 </p><p>4 . Absorption Coefficients . . . . . . . . . . . . . . . . . 165 5 . Colorimetric Analysis . . . . . . . . . . . . . . . . . 165 6 . Fluorimetric Analysis . . . . . . . . . . . . . . . . . 172 </p><p>VI . By-products of Chlorophyll . . . . . . . . . . . . . . . . . 174 1 . Industrial Uses . . . . . . . . . . . . . . . . . . . 174 </p><p>a . Pigments and Paints . . . . . . . . . . . . . . . . 174 b . Chlorophyll and Oil Oxidation . . . . . . . . . . . . 175 c . Chlorophyll as a Deodorizer . . . . . . . . . . . . . 175 </p><p>2 . Medical Applications . . . . . . . . . . . . . . . . . 175 a . Therapeutic Action . . . . . . . . . . . . . . . . 175 b . Antibiotic Action . . . . . . . . . . . . . . . . . 176 c . Gonadotropic Effeet . . . . . . . . . . . . . . . . 177 d . Photodynamic Aspects . . . . . . . . . . . . . . . 177 </p><p>178 </p><p>References . . . . . . . . . . . . . . . . . . . . . . . 179 The others are: Absorption </p><p>spectra of chlorophyll and related compounds. Chem . Revs . 47. 175 (1950), and Chlorophyll, Botan . Rev . 16. 525 (1950) . </p><p>133 </p><p>V . Analytical Methods and Criteria of Purity . . . . . . . . . . . . </p><p>e . The Fa te of Chlorophyll on Mammalian Ingestion . . . . . . </p><p>This is the last of three reviews on chlorophyll . </p></li><li><p>134 S. ARONOPP </p><p>I. INTRODUCTION </p><p>It is not the purpose of this review to summarize the changes in the chlorophyll moeity in various products as the result of technological operations. These are the practical problems associated with the par- ticular worker in food research. Rather it is an attempt to survey those aspects of chlorophyll which should make an investigation into these changes more understandable or easier. Thus, it will be apparent that those processes which release free organic acids from the cell (e.g., blanching) will result in pheophytin formation ; that alkaline oxidations will cause pclrpurins to arise ; that cooking in copper kettles may cause the substitution in the chlorophyll molecule of magnesium by copper, etc. I n other words, here we assume a general approach, from which particular, practical applications may be deduced. </p><p>The importance of chlorophyll to the research worker in food chem- istry arises from three sources : (1) the degradation of chlorophyll dur- ing food processing; (2) the fate of chlorophyll in biological systems; and (3) the possible utilization of chlorophyll as a raw material for industrial and pharmacological purposes. </p><p>Consideration of the first point invites a brief survey of the pertinent chemistry of chlorophyll, analytical methods used in its determination, and industrial and medical aspects of possible interest to the food in- dustry. The second problem inquires essentially whether the ingestion of chlorophyll is harmful, to which we may answer that, to the best of our knowledge, under normal circumstances i t is not. Never- theless, we are aware that, a t times, degradation products of chloro- phyll may result in pathological conditions in stock, such as that caused by photosensitization in light-colored animals. One inquires, further, whether on the other hand chlorophyll or a solubilized derivative may be helpful nutritionally. Because of conflicting evidence, there is no clear- cut evidence to support such a claim. Considering the third point above, there are consistent reports of the possible value of chlorophyll in treatment of wounds, etc., and, on the industrial side, it has long been used, although probably not to its greatest extent, as a coloring matter. </p><p>Of the various chlorophylls now known to exist in nature (Aronoff 1950b), by far the most important are the chlorophylls a and B , the common green matter of all higher and most lower photosynthetic plants. It is possible that, as our technology makes more use of oceanic flora, we will be concerned more than academically with the chlorophylls c and d, the bilins, and the bacterial chlorophylls. IIowever, in this discussion we will restrict ourselves to the a and b forms. </p></li><li><p>CHLOROPHYLL 135 </p><p>There is uncertainty as to the actual mode of existence of chlorophyll (a and b ) in plants; the product characterized to date is that ex- tracted from plant tissues. Any difference must be a subtle one, as very mild methods may be used. Even so, there is no single absolute method of determining the purity of the extracted material, and under certain conditions the crude chlorophyll may be only a minor part of the yield. By suitable methods of refinentent we may obtain products which appear to undergo no further change in properties with addi- tional manipulations. It is these products we call chlorophylls a and b, and which we use as our standards. The chemistry of these compounds is essentially complete, and there is no reaeon why the chlorophylls can- not be used with confidence as raw materials of known purity f o r a variety of purposes. </p><p>11. NOMENCLATURE </p><p>Every subject with a large history of research has developed its own language. A variety of terms and structures native to chlorophyll chem- istry is appended below : </p><p>Porphyrin. The general class of con,iugated, cyclic, tetrapyrrole </p><p>Porphine. A porphyrin in which all four nitrogens are equivalent compounds, including porphines, chlor ins, azoporphines, etc. </p><p>except for differences caused by @-substitutions. </p><p>3c 2c(2d I </p><p>A numbering system based on the c1as:sical system used by Fischer is given in formula I. Since the carbons adjacent to the nitrogens have heretofore not been numbered, it has recently been suggested (Wittenberg and Xhemin, 1950) that a revision of the nomenclature of the porphyrin ring is desirable to permit identification of the indi- </p></li><li><p>136 S. ARONOFF </p><p>vidual carbons. With the above method there appears to be no neces- sity to dispense with the commonly used Fischer system. Pyrrole. The four cyclic components of the porphyrin nucleus; they </p><p>result from reductive degradation of porphyrins. </p><p>I! </p><p>Maleic imides. as in 111. </p><p>The products of oxidative degradation of porphyrins, </p><p>H I </p><p>III </p><p>Etioporphine III. 1,3,5,8-Tetramethyl-2,4,6,7-tetraethyl porphine. Rhodoporphine. Same as etioporphine 111, except 6-carboxy-7-pro- </p><p>pionic acid. Pyrroporphine. Same as etioporphine 111, except 6-desethyl-7-pro- </p><p>pionic acid. Phylloporphine. Same as etioporphine 111, except 6-desethyl-7-pro- </p><p>pionic acid, y-methyl. Chlorin. A dihydro- (@,r) -porphine. I n chlorophyll terminology, </p><p>this usually implies a 2-vinyl substitution in addition. Phorbin. A chlorin containing an isocylic ring connecting Cy and C6 </p><p>with two additional carbons (9 and 10). Purp&amp;n. A phorbin with an ether linkage of Cg or Cl0, e.g., </p><p>I V </p><p>Chlorins, phorbins, and purpurins are often provided with suf- fixes denoting the number of oxygen atoms in the molecule, e.g., chlorin e6, with six atoms of oxygen. An exception is purpurin 18, whose name is derived from its acid number, i.e., the percentage </p></li><li><p>CHLOROPHYLL 137 </p><p>of aqueous HC1 required to extract two-thirds of the pigment from ether if equal volumes of ether and aqueous HC1 are used. Meso compounds. Chlorophyll derivatives in which the 2-vinyl group </p><p>has been reduced to 2-ethyl. One thus speaks of mesopheophorbide, or mesochlorin e5, etc. </p><p>Chlorophyll a. Mg chelate of 1,3,5,8-tetramethyl-4-ethyl-2-vinyl-9- keto-10-carbomethoxyphorbin phytyl-7-propionate. </p><p>Chlorophyll b. Corresponds to chlorophyll a, except that the 3-posi- tion is substituted by a formyl group rather than a methyl group. It is therefore 1,5,8-tetramethyl-3-formyl-4-ethyl-2-vinyl-9-l~eto-l0- carbomethoxyphorbin phytyl-7-propionate. </p><p>Pheophytin. Chlorophyll minus Mg. Pheophorbide. Pheophytin minus phytol. Pheoporphyrin us. </p><p>Phytol. </p><p>Isomeric with pheophorbide, but the two labile Hs (7 and 8 ) have migrated to the vinyl, converting it to an ethyl. </p><p>An alcohol of the following structure : </p></li><li><p>138 S. ARONOFF </p><p>111. THE CHEMISTRY OF CHLOROPHYLL </p><p>The chemistry of chlorophylls a and b is complete in the sense that the molecules may be synthesized in stepwise fashion from simpler mole- cules of well-known structure. It is not complete in two other senses: (1) we are not yet certain that the extracted substances are not in some subtle manner different from those existing in the natural state; and (2) there is some doubt as to the fine structure of the chlorophyll- i.e., hydrogen tautomerism in the free bases, and the contribution of the particular substituents to the absorption spectra. </p><p>The fundamental chemical similarity of chlorophyll and heme have been recognized for almost a century. Early studies were concerned with drastic oxidative and reductive degradation products of chlorophyll and hemin. Thus, hydriodic acid reacting on chlorophyll forms a variety of pyrroles : opso- (VIII) , hemo- ( IX), crypto- (X) , and phyllo-pyrroles (XI) : </p><p>H </p><p>VIIl </p><p>Chromate oxidation resulted in the formation of acidic and basic imides. The acid substances were hematinic acid (XII) and carbon dioxide. The basic imides were methylethylmaleic imide (XIII) , citra- conimide (XIV), and hemotricarboxylic imide (XV) . </p><p>The origin of these imides and their relation to the chlorophyll struc- ture is indicated in the diagram for pheophorbide (XVI). </p><p>The correlation of products formed by chromate oxidation with their origin is given in Table I. Table I1 summarizes the oxidation products of a variety of compounds related to chlorophyll or resulting from its partial degradation. </p></li><li><p>CHLOROPHYLL 139 </p><p>i '? H3C i H COOH </p><p>i </p><p>R XIV </p><p>O X 0 H3C H H C,H, </p><p>xv </p><p>Carbon dioxide </p><p>acid imide XVI </p></li><li><p>140 S. ARONOFB </p><p>TABLE I </p><p>Correlation of the Products Fornied by Chromate Oxidation and Their Orlgin </p><p>Product formed Origin </p><p>Acid substances 1. Carbon dioxide Primarily from methylene carbons. </p><p>2. Hematinic acid Obtained only from porphines, not from chlorins and rhodins; therefore arises from ring IV, not 111. </p><p>Basic substances 3. Hemotricarboxylic imide Obtained only from chlorins and phorbins, no t </p><p>from porphines, therefore arises only from ring IV. </p><p>4. Methylethylmaleic imide Not from chlorins and phorbins of the chlorophyll b series; therefore only from ring 11, not I. </p><p>5. Citraconimide Only from those compounds possessing a b-methyl- p-H, such as deuteroporphine or pliyllochlorin, therefore not from ring I. </p><p>TABLE I1 </p><p>Oxidation Products of Chlorophyll and Related Compounds </p><p>Acid fraction Basic fraction </p><p>Compound </p><p>Chlorins a Phyllochlorin (6-free) Phorbins a Chlorins b Phorbins b Porphines, from chlorophyll Pyrroporphine (6-free) Porphines, from hemin Deuteroporphine (2,4-free) Protoporphine </p><p>Hema- tinic acid </p><p>0 0 0 0 0 </p><p>+ + + + + </p><p>Hemotri- carboxylic </p><p>acid C </p><p>+ + + + + 0 0 0 0 0 </p><p>Citra- 'onimide </p><p>0 + 0 0 0 0 </p><p>+ 0 </p><p>+ + </p><p>Methylethyl- mn!cic imide </p><p>+ + + 0 0 </p><p>+ + + 0 0 </p><p>Although chlorophyll degradations of this type have not been studied by isotopic techniques, they have for hemin (Wittenberg and Shemin, 1950 ; Muir and Neuberger, 1949). Hemin is reduced to mesoporphyrin, so that hematinic acid is obtained from rings I11 and IV, and methylethyl- maleic imide from rings I and 11. Wittenberg and Shemin (1950) car- ried out the further degradation of the imides as follows. </p></li><li><p>CHLOROPHYLL 141 </p><p>NH,OH, E tOH (1) Hematinic acid p methylethylmaleic imide +CO, </p><p>175" C. </p><p>NaCIO, </p><p>080, </p><p>( 2 ) Methylethylmaleic imide - + methylethyltartaric imide </p><p>(3) Metliylethyltartaric h i d e HIO, pyruvic acid + a-ketobutyric acid acid </p><p>H </p><p>H H XVII </p><p>Get' (4a) a-Ketobutyric acid </p><p>(4b) Pyruvic acid + CO, + acetic acid propionic acid + CO, </p><p>If desired, both the propionic and the acetic acids may be degraded further for the individual atoms. </p><p>The knowledge of the degradation products plus the synthesis of various porphyrins from these degradation products identical with </p></li><li><p>142 S. ARONOFF </p><p>known degradation products of chlorophyll have been the primary basis for the elucidation of chlorophyll chemistry. </p><p>The reactive positions in the chlorophyll molecule are depicted above (XVIII). I n this diagram an attempt has been made to segregate the main types of reactions, and each of these reactions has been given a number (Roman numeral) which serves as a basis of classification for the succeeding discussion. Obviously not all these reactions may be of biological significance, but, should chlorophyll become a prominent raw material industrially, the degradation products may assume significance. (For a more extensive summary of similar scope, see Fischer and Stern, 1940). </p><p>I. Metal Complexing Next to the alkali metal complexes (e.g., disodium pheophytin) those </p><p>of the alkaline earths [e.g., Mg-pheophytin (chlorophyll) ] are dissoci- ated most readily. The reaction occurs easily with carboxylic acids (e.g., oxalic or acetic) and is the most common cause of discoloration of green food products due to the natural presence of plant organic acids. Neutral or alkaline solutions are required to retain this chelation during processing. </p><p>The rate of removal of the Mg from chlorophyll a (in aqueous acetone) exceeds that of chlorophyll b by ninefold (see Mackinney and Joslyn, 1940). A possible explanation of this difference in rate has been ascribed to the inductive effect of the formyl group, increasing the bond strength of the Mg (Aronoff, 1950a). </p><p>A variety of metals may be complexed with the porphyrin ring; an example is given below. These result in compounds of varying stability. It should be noted that it is possible to prepare doubly complexed por- phyrins, e.g., Mg-Cu porphyrins. Presumably in this case the additional complex involves the carboxyl groups as well as the central portion of the ring. The monovalent elements form complexes in which there are two alkali metal ions within each porphine ring. </p></li><li><p>CHLOROPHPLL 143 </p><p>Example. Substitution of Mg by Cu in chlorophyll and related compounds. Dissolve 100 mg. chlorophyll in 13 ml. chloroform. Add 2 ml. of a boiling metha- </p><p>nolic solution of cupric acetate (ca. 30 mg.). Boil the mixture 2 min., and then wash out the alcohol and exces...</p></li></ul>

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