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The Effect of Freeze-Drying on Chlorophyll in the Leaves of Some Selected Drug Plants
554
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
When these dosage ranges are compared with what is reported to be effective in man, it is noted that the rat requires relatively large doses. It may be that the rat is not the most suitable animal for determining the biological activity of licorice. SUMMARY 1. Spanish licorice extract, ammoniated glycyrrhizin, monoammonium glycyrrhizinate and glycyrrhetinic acid were tested for electrolytic activity similar to that of desoxycorticosterone acetate. 2. Spanish licorice extract, ammoniated glycyrrhizin, and monoammonium glycyrrhizinate, administered orally, exhibited an electrolytic activity when this activity is expressed as the K/Na ratio. It was observed that, first, there was an indication of quantitative decrease in sodium output and urine retention with increased drug dosage; second, there was little or no change in the potassium output.
VOl. XLIII, NO. 9
3. Spanish licorice extract, ammoniated glycyrrhizin and monoammonium glycyrrhizinate produce a K/Na ratio which follows that of desoxycorticosterone acetate with increased dosage. Glycyrrhetinic acid does not follow the desoxycorticosterone acetate pattern because of its greater potassium retention effect.
REEERENCES (1) Marcus F. Romanoff, L. P., and Pincus, G., Endocranol., 5 0 , 28’S(l652). (2) Revers, F. E., Ned. Tiidschr. Geneesk., 90, 135(1946). (3) I b i d . 92 2988(1948). G. G., Blomhert, G., Molhu sen, J. A,, (4) Bors;, Gerbraody, J., Turner. K. P., and DeVries, L. A c f o clrn. b d g . 5 405(1950). (5j Iholhuysen, J. A., Gerbrandy, J., DeVries, L. A,, deJong J. C. Lenestra J. B., Turner, K. P., and Borst, J. G. G., Lanlet, 2,3si(i950). (6) Groen, J.. Pelser, H., Willebrands. A. F., and Kamd o a C. E. New EWE[. J. Med. 244 471(1951). Kamminga, C. E., (77 Groeo,’J., Pelser, H., Freoiel, and Willebrands, A. F., J. C l m . Invesf., 31, 87(1952). (8) Cook, M . R.. and Elmadjian, F., THISJOURNAL, 42, 329(1953). (9) Simpsoo, S. A., Tait, J. F., Endocrinol., 5 0 , 150 (1952).
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The Effect of Freeze-Drying on Chlorophyll in the Leaves of Some Selected Drug Plants* By NOURI Y.MARY,t BERNARD V. CHRISTENSEN,$ and JACK L. BEAL.5 A comparative study of the effect of freeze-drying and oven-drying at 45-50’ on chlorophyll in the leaves of some selected drug plants was undertaken. The results indicated that more total chlorophyll was obtained from the freeze-dried leaves. The major difference was in the amount of chlorophyll a. Freeze-dryingfacilitated total chlorophyll extraction.
w
voluminous reports concerning the application of freeze-drying to biological substances are available, very little information concerning the application of freeze-drying as a possible artificial physical means of crude drug drying and preservation could be found in the HILE
* Received August 21 1953 from The Ohio State University College of Pharmaiy, Coiumbus, Ohio. Presented t o the Scientific Section, A. PH. A., Salt Lake City meeting, August, 1953. An abstract of a thesis presented t o the Graduate School of The Ohio State University by Nouri Y.Mary in partial fulfillment of the requirements for t h e degree of Master of Science. The authors gratefully acknowledge the assistance of the Statistics Laboratory of The Ohio State University in evaluating the d a t a listed in Tables I, 11, and 111. t Graduate student. Collece of Pharmacv. _ . The Ohio State University. . $ Dean, College of Pharmacy, The Ohio State University. 5 Assistant Professor, College of Pharmacy, T h e Ohio State University.
literature. Chambers and Nelson (1) were the first workers to investigate the use of high vacuum freeze-drying as a means of crude drug preservation. In order to adapt freeze-drying more extensively, further application of the process to other drug plants and their constituents was thought advisable. The chlorophyll pigments in some selected leaves of drug plants were chosen as the constituents of the investigation, because the leaves of plants readily lose their bright greenish color when dried a t elevated temperatures and kecome brownish after drying and storage. By freeze-drying at a lower temperature range it was thought that less chemical changes and alterations of the chlorophyll would occur, thus preserving the appearance of the leaves.
SCIENTIFIC EDITION
September, 1954
EXPERIMENTAL Collection and Drying of the Leaves.-The leaves of Podophyllum peltatum, Lime (podophyllum); Ricinus communis, Linn6 (castor), and Mentha spicata, LinnC (spearmint) were selected as the material of investigation in this work. Since chlorophyll is the substance of interest in this investigation, any kind of leaves could have been used, but the three mentioned leaves were chosen because they were available in the drug plant garden at the time of collection, and because, especially, podophyllum and castor leaves are comparatively large. The leaves were collected from the Medicinal Plant Garden of the College of Pharmacy of the Ohio State University, Columbus, Ohio. Each representative sample was divided into two groups. One group was oven-dried a t 45-50"
555
and the other group was freeze-dried. After drying, the samples were reduced to a No. 40 powder by means of a Wiley mill. It was noted that the oven-dried samples of the three kinds of leaves had a darker green color than the freeze-dried samples. Moisture Determination.-The moisture content of oven-dried and freeze-dried samples of the leaves was determined by the U. S. P. XIV method (2) for drugs containing no constituents volatile a t 100'. Freeze-drying removed more moisture from the leaves than oven-drying a t 45-50'. The purpose of performing the moisture determination was to obtain a percentage of moisture from which the succeeding calculations could be compared on a dry weight basis. Determination of Total Chlorophyll and the a and b Components.-Total chlorophyll and its a and b
TABLEDETERMINATION O F TOTAL CHLOROPHYLL AND THE A AND B COMPONENTS OF THE LEAVESOF MENTHA SPICATA, L I N N(SPEARMINT) ~ BY SPECTROPHOTOMETRIC METHOD
FreezeDried
Sample 1 Sample 2 Sample 3 Average
0.4077 0.4062 0.4004 0.4048
Sample 1 Sample 2 Sample 3 Average
0.2769 0.2759 0.2738 0.2755
Sample 1 Sample 2 Sample 3 Average
0.1317 0.1312 0.1268 0.1299
OvenDried
Difference
Total Chlorophyll0 0.3850 0.0227 0.3782 0.0280 0.3667 0.0337 0.3766 0.0282 Chlorophyll a" 0.2654 0.0115 0.2582 0.0177 0.2535 0.0203 0.2590 0.0165 Chlorophyll b* 0.1202 0.0115 0.1203 0.0109 0.1134 0.0134 0.1180 0.0119
Standard Deviation of the Difference
Ratio of Difference to its Standard Deviation
0.0311 0.0153 0.0208 0.0135
0.730
1.620 2.0896
0.0125 0.0073 0.0089 0.0057
0.920 2. 425b 2.281h 2. 895b
0.0211 0.0112 0.0153 0.0095
0.545 0.973 0.876 1.253
i ,830
-
Expressed as percentage. Indicates significant difference.
TABLE
II.-DETERMINATIONOF TOTAL CHLOROPHYLL AND THE A AND B COMPONENTS O F THE LEAVESO F RICINUSCOMMUNIS. LINN$ (CASTOR) BY SPECTROPHOTOMETRIC METHOD FreezeDried
Sample 1 Sample 2 Sample 3 Average
0.5874 0.5920 0.6048 0.5947
Sample 1 Sample 2 Samule 3 Average
0.4387 0.4421 0.4530 0.4446
Sample 1 Sample 2 Samule 3 Average
0.1487 0.1499 0.1518 0.1501
Expressed as percentage. Indicates significant difference.
OvenDried
Di5erence
Total Chlorophyllo 0.4905 0.0969 0.4984 0.0936 0.4986 0.1062 0.4958 0.0989 Chlorophyll an 0.0816 0.3571 0.3569 0.0852 0.3574 0.0956 0.3751 0.0695 Chlorophyll ba 0.0153 0.1334 0.0084 0.1415 0.1412 0.0106 0.1387 0.0114
Standard Deviation of the Di5erence
Ratio of Difference to its Standard Deviation
0.0210 0.0453 0.0229 0.0183
4. 614b 2. O6gb 4.638* 5.404D
0.0107 0.0225 0.0153 0.0097
7. 626b 3.7878 6. 24gb 7. 165b
0.0259 0.0323 0.0196 0.0153
0.591 0.260 0.541 0.754
5%
JOURNAL OF THE
AMERICANPHARM A C E U T I C A L AssocxmroN
VOl.
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NO.
9
TABLE III.-DETERMIXATIONOF TOTAL CHLOROPHYLL AND THE A AND B COMPONENTS O F THE LEAVESO F PODOPHYLLUM PELTATUM, LINNS(PODOPHYLLUM) BY SPECTROPHOTOMETRIC METHOD OvenDried
FreezeDried
Sample 1 Sample 2 Sample 3 Average
0.4500 0.4548 0.4615 0.4554
Sample 1 Sample 2 Sample 3 Average
0.3089 0,3096 0.3161 0.3115
Sample 1 Sample 2 Sample 3 Average
0.1411 0.1452 0.1454 0.1439
Difference
Total Chlorophylla 0.4073 0.0427 0.4102 0.0446 0.3748 0.0867 0.0580 0.3974 Chlorophyll a" 0.2743 0,0346 0.2680 0.0416 0.2451 0.0710 0.2624 0.0491 Chlorophyll bR 0.1330 0.0081 0.1422 0.0030 0.1297 0.0157 0.1350 0,0089
Standard Deviation of the Difference
Ratio of Difierence t o Its Standard Deviation
0.0994 0.0728 0.0610 0.0458
0.430 0.613 1.421 1.266
0.0265 0.0240 0.0141 0.0128
1.306 1.733 5.035* 3. 83Bh
0.0772 0.0527 0.0536 0.0359
0.105 0.057 0.293 0.248 __ ~~
Expressed as percentage. b Indicates significant difference.
components were determined according to the A. 0. A. C. method (3). The results are listed in Tables I, 11, and 111. The percentages are mean values of a minimum of 12 determinations. The results were statistically evaluated by the Statistics Laboratory of The Ohio State University. The hypothesis tested was that the means of the two compared samples, oven-dried and freeze-dried, are the same. A ratio value greater than 1.950, obtained by dividing the mean difference by the standard deviation, was considered significant, since according to a table of normal distribution (4) the chances of obtaining a greater value is less than 5%. The Rate of Chlorophyll Extraction.-When cxtracting chlorophyll from the leaves, a greater difficulty was encountered and more time consumed in extracting the oven-dried samples as compared with the freeze-dried samples. The object of run-
ning the extraction studies was therefore t o substantiate this finding by numerical data. A Fisher electrophotometer, AC Model, was first calibrated according to the A. 0. A. C. method (3), with 1.0 cm. absorption cells and a No. 650(A) light filter. A calibration curve that rigidly followed Beer's law was constructed. This was employed for calculating the data shown in Table IV. The extraction procedure followed was a s follows: about 5 Gm. accurately weighed, of each of the ovendried and freeze-dried powdered material of all the three leaf samples were packed lightly in 80-cc. percolators of the same size and diameter. The powders were moistened with 35 cc. of 85% acetone C. P., and when the liquid was about to drip from the percolators, the lower orifice was closed and the drugs were allowed t o macerate for twenty-four hours. The percolation was then allowed t o proceed at a moderate rate, gradually adding a new
TABLE IV.-THE RATEOF EXTRACTION OF TOTAL CHLOROPHYLL FROM PODOPHYLLUM, CASTOR, A N D SPEARMINT LEAVES" Podophyllum Leaves OveriFreezeDried Dried
Successive 50-cc. I'ractions
1 2 3 4 5 6 7
b
9 10 11 12 13 Total
330.0 3.8 1.6 0.8 0.5 0.4 0.3 0.3 0.2 0.2
260.0 6.2 3.5 2.0 1.8 1.6 1.6 1.4 1.2 1.o
...
... ... ...
__
280.2
...
_
360. o 18.0 9.0 6.6 6.4 5.4 4.1 3.4 3.0 2.8 2.5 2.3 2.0 425.0
285.0 9.0 4.8 3.4 2.8 2.5 1.7 1.5 I .3 1.3 1.1 1.1
...
338.1
Total Chlorophyll in mg./L.Castor I.eaves OvenFreezeDried Dried
_
The results are the average of three determinalions.
1.0 316.5
~
~
___~ __
\
Spearmint Leaves OvenFreezeDried Dried
230.0 10.0 7.0 (5.5 5.3 5.2 4.4 4.0 3.9 3.6 2.6 1.8 1.8 286.1
280.0 15.0 7.4 5.2 3.8 3.2 2.0 1.4 1.4 1.2 0.8 0.4 0 .4
322.2
__
-
SCIENTIFIC EDITION
September, 1954
menstruum in 25-cc. portions t o the percolators. Successive 50-cc. fractions of percolate were collected, their per cent transmission immediately determined on the Fisher electrophotometer, AC Model, and the chlorophyll content of each fraction read directly on the calibration curve. Percolation was stopped in each case when the transmission of the last fractions of percolates approached minimum. The results of the extraction studies of chlorophyll in the three kinds of leaves appear in Table IV.
DISCUSSION
557
in the temperature will retard the activity of the enzyme. Since less chlorophyll was retained in the oven-dried samples than in the freeze-dried samples of all the three leaves, it is apparent that enzymatic activity was retarded to a greater extent in the freeze-dried samples than in the oven-dried ones, hence the less destruction and breakdown of chlorophyll in the freeze-dried samples. The ratio of chlorophyll a and b in the oven-dried samples was lower than the ratio of these two components in the freeze-dried samples of the leaves. This, of course, i s due t o the fact that chlorophyll a was more sensitive t o oven-drying than freezedrying temperatures and chlorophyll b was equally sensitive t o the temperatures of both drying procedures. The ratio of chlorophyll a t o b in the leaves investigated, both oven-dried and freeze-dried, compare favorably with the ratio of these two components as reported by other investigators. That chlorophyll is extracted more rapidly from the freeze-dried samples than the oven-dried samples of the three kinds of leaves is apparent from the data obtained in Table IV. This may possibly be attributed t o the fact that high temperatures caused more shrinkage of the tissue cells than low temperatures, thus exposing less area to the effect of the menstruiim.
In reference to Tables I and I1 (castor and spearmint leaves) it can be observed that there is more total chlorophyll in the freeze-dried samples than in the oven-dried samples. This difference is a significant amount. The major difference lies in the amount of chlorophyll a in the two samples since this difference is of a significant magnitude, while the difference in chlorophyll b is not a significant amount. In the case of podophyllum leaves, as can be observed in Table 111, there is an unusual result. The experiment showed no significant difference between the total chlorophyll of the freeze-dried and oven-dried samples. However, the amount of chlorophyll a in the freeze-dried sample was signifiCONCLUSIONS cantly greater than in the oven-dried sample. This result can be attributed to several apparent The leaves of Podophyllum peltutum, Linn6 factors. The podophyllum leaves were the first Ricinus communis, LinnC (cas(podophyllum), leaves of the experiment t o be assayed, and the laboratory technique involved in the experiment tor), and M w t h spicutu, Linne (spearmint) was improved in the latter experiments. This fact were used in this investigation. The results is evident from the larger standard deviation for showed that: the podophyllum’ experiment in comparison t o the 1. More total chlorophyll was obtained from standard deviation of the other experiments. The the freeze-dried leaves of podophyllum, castor, fact that the ratio of chlorophyll a t o chlorophyll b in podophyllum leaves is approximately 2 to 1 and spearmint than from the oven-dried leaves. while in the castor plant is approximately 2.8 to 1 2. The total difference was largely due to the is another factor. Although the difference in difference in chlorophyll a since the difference was chlorophyll a is significant, the large amount of of a significant magnitude, whereas the difference chlorophyll b, which does not show a significant difference, offsets somewhat the difference in chloro- in chlorophyll b was not a significant amount. 3. The ratio of chlorophyll u to b in the phyll a. It is true that the ratio of chlorophyll a t o chlorophyll b in spearmint leaves is also approxioven-dried samples was lower than the ratio of mately 2 t o 1, and that the large amount of chloro- these two pigments in the freeze-dried samples phyll b did not offset the difference in chlorophyll of the leaves. The ratio of chlorophyll a to b in a enough t o keep the difference in total chlorophyll the leaves investigated, both oven-dried and from being significant. But there is a difference in the two cases of podophyllum leaves and spear- freeze-dried, compares favorably with the ratio mint leaves. I n podophyllum leaves the amount of of these two components as reported by other chlorophyll b in the two compared samples, freezeinvestigators. dried and oven-dried leaves, is almost the same 4. Freeze-drying Eacilitated chlorophyll exwhile in the case of spearmint leaves the freezcdried sample has more chlorophyll b than the ovcn- tractions. dricd, even though it is not a significant amount. In summary, the total chlorophyll results were REPERENCES possibly influenced by the large standard deviation, (1) Chambers, M . A., and Nelson, J. W., THISJOURNAL; the high proportion of chlorophyll b t o chlorophyll 36, 326(1950). a and the fact that there was a very minute dif(2) “The Pharmacopeia of the United States ” 14th rev., ference in the amount of chlorophyll b in the two Maok Publishin Company Easton Pa. 1950 b. 714 (3) “Official hethods oi An,$& of ’the Association of compared samples. Official Agricultural Chemists George Banta Publishing Company, Menasha, Wisconsi& 1950, p. 112. The optimum temperature of chlorophyllase, a n (4) Snedecor G. W. “Statistical Method,” The Iowa enzyme that occurs alongside its substratum State College Pkess. Ambs. Iowa, 1946, p. 180. (5) Willstatter, R., and Stoll, A,;, “Investigations on “chlorophyll” in the green parts of all the plants, Chlorophyll: Methods and Results The Science Press is about 20” (5). Either an increase or a decrease Printing Company, Lancaster, Pa., I9iS.
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
When these dosage ranges are compared with what is reported to be effective in man, it is noted that the rat requires relatively large doses. It may be that the rat is not the most suitable animal for determining the biological activity of licorice. SUMMARY 1. Spanish licorice extract, ammoniated glycyrrhizin, monoammonium glycyrrhizinate and glycyrrhetinic acid were tested for electrolytic activity similar to that of desoxycorticosterone acetate. 2. Spanish licorice extract, ammoniated glycyrrhizin, and monoammonium glycyrrhizinate, administered orally, exhibited an electrolytic activity when this activity is expressed as the K/Na ratio. It was observed that, first, there was an indication of quantitative decrease in sodium output and urine retention with increased drug dosage; second, there was little or no change in the potassium output.
VOl. XLIII, NO. 9
3. Spanish licorice extract, ammoniated glycyrrhizin and monoammonium glycyrrhizinate produce a K/Na ratio which follows that of desoxycorticosterone acetate with increased dosage. Glycyrrhetinic acid does not follow the desoxycorticosterone acetate pattern because of its greater potassium retention effect.
REEERENCES (1) Marcus F. Romanoff, L. P., and Pincus, G., Endocranol., 5 0 , 28’S(l652). (2) Revers, F. E., Ned. Tiidschr. Geneesk., 90, 135(1946). (3) I b i d . 92 2988(1948). G. G., Blomhert, G., Molhu sen, J. A,, (4) Bors;, Gerbraody, J., Turner. K. P., and DeVries, L. A c f o clrn. b d g . 5 405(1950). (5j Iholhuysen, J. A., Gerbrandy, J., DeVries, L. A,, deJong J. C. Lenestra J. B., Turner, K. P., and Borst, J. G. G., Lanlet, 2,3si(i950). (6) Groen, J.. Pelser, H., Willebrands. A. F., and Kamd o a C. E. New EWE[. J. Med. 244 471(1951). Kamminga, C. E., (77 Groeo,’J., Pelser, H., Freoiel, and Willebrands, A. F., J. C l m . Invesf., 31, 87(1952). (8) Cook, M . R.. and Elmadjian, F., THISJOURNAL, 42, 329(1953). (9) Simpsoo, S. A., Tait, J. F., Endocrinol., 5 0 , 150 (1952).
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The Effect of Freeze-Drying on Chlorophyll in the Leaves of Some Selected Drug Plants* By NOURI Y.MARY,t BERNARD V. CHRISTENSEN,$ and JACK L. BEAL.5 A comparative study of the effect of freeze-drying and oven-drying at 45-50’ on chlorophyll in the leaves of some selected drug plants was undertaken. The results indicated that more total chlorophyll was obtained from the freeze-dried leaves. The major difference was in the amount of chlorophyll a. Freeze-dryingfacilitated total chlorophyll extraction.
w
voluminous reports concerning the application of freeze-drying to biological substances are available, very little information concerning the application of freeze-drying as a possible artificial physical means of crude drug drying and preservation could be found in the HILE
* Received August 21 1953 from The Ohio State University College of Pharmaiy, Coiumbus, Ohio. Presented t o the Scientific Section, A. PH. A., Salt Lake City meeting, August, 1953. An abstract of a thesis presented t o the Graduate School of The Ohio State University by Nouri Y.Mary in partial fulfillment of the requirements for t h e degree of Master of Science. The authors gratefully acknowledge the assistance of the Statistics Laboratory of The Ohio State University in evaluating the d a t a listed in Tables I, 11, and 111. t Graduate student. Collece of Pharmacv. _ . The Ohio State University. . $ Dean, College of Pharmacy, The Ohio State University. 5 Assistant Professor, College of Pharmacy, T h e Ohio State University.
literature. Chambers and Nelson (1) were the first workers to investigate the use of high vacuum freeze-drying as a means of crude drug preservation. In order to adapt freeze-drying more extensively, further application of the process to other drug plants and their constituents was thought advisable. The chlorophyll pigments in some selected leaves of drug plants were chosen as the constituents of the investigation, because the leaves of plants readily lose their bright greenish color when dried a t elevated temperatures and kecome brownish after drying and storage. By freeze-drying at a lower temperature range it was thought that less chemical changes and alterations of the chlorophyll would occur, thus preserving the appearance of the leaves.
SCIENTIFIC EDITION
September, 1954
EXPERIMENTAL Collection and Drying of the Leaves.-The leaves of Podophyllum peltatum, Lime (podophyllum); Ricinus communis, Linn6 (castor), and Mentha spicata, LinnC (spearmint) were selected as the material of investigation in this work. Since chlorophyll is the substance of interest in this investigation, any kind of leaves could have been used, but the three mentioned leaves were chosen because they were available in the drug plant garden at the time of collection, and because, especially, podophyllum and castor leaves are comparatively large. The leaves were collected from the Medicinal Plant Garden of the College of Pharmacy of the Ohio State University, Columbus, Ohio. Each representative sample was divided into two groups. One group was oven-dried a t 45-50"
555
and the other group was freeze-dried. After drying, the samples were reduced to a No. 40 powder by means of a Wiley mill. It was noted that the oven-dried samples of the three kinds of leaves had a darker green color than the freeze-dried samples. Moisture Determination.-The moisture content of oven-dried and freeze-dried samples of the leaves was determined by the U. S. P. XIV method (2) for drugs containing no constituents volatile a t 100'. Freeze-drying removed more moisture from the leaves than oven-drying a t 45-50'. The purpose of performing the moisture determination was to obtain a percentage of moisture from which the succeeding calculations could be compared on a dry weight basis. Determination of Total Chlorophyll and the a and b Components.-Total chlorophyll and its a and b
TABLEDETERMINATION O F TOTAL CHLOROPHYLL AND THE A AND B COMPONENTS OF THE LEAVESOF MENTHA SPICATA, L I N N(SPEARMINT) ~ BY SPECTROPHOTOMETRIC METHOD
FreezeDried
Sample 1 Sample 2 Sample 3 Average
0.4077 0.4062 0.4004 0.4048
Sample 1 Sample 2 Sample 3 Average
0.2769 0.2759 0.2738 0.2755
Sample 1 Sample 2 Sample 3 Average
0.1317 0.1312 0.1268 0.1299
OvenDried
Difference
Total Chlorophyll0 0.3850 0.0227 0.3782 0.0280 0.3667 0.0337 0.3766 0.0282 Chlorophyll a" 0.2654 0.0115 0.2582 0.0177 0.2535 0.0203 0.2590 0.0165 Chlorophyll b* 0.1202 0.0115 0.1203 0.0109 0.1134 0.0134 0.1180 0.0119
Standard Deviation of the Difference
Ratio of Difference to its Standard Deviation
0.0311 0.0153 0.0208 0.0135
0.730
1.620 2.0896
0.0125 0.0073 0.0089 0.0057
0.920 2. 425b 2.281h 2. 895b
0.0211 0.0112 0.0153 0.0095
0.545 0.973 0.876 1.253
i ,830
-
Expressed as percentage. Indicates significant difference.
TABLE
II.-DETERMINATIONOF TOTAL CHLOROPHYLL AND THE A AND B COMPONENTS O F THE LEAVESO F RICINUSCOMMUNIS. LINN$ (CASTOR) BY SPECTROPHOTOMETRIC METHOD FreezeDried
Sample 1 Sample 2 Sample 3 Average
0.5874 0.5920 0.6048 0.5947
Sample 1 Sample 2 Samule 3 Average
0.4387 0.4421 0.4530 0.4446
Sample 1 Sample 2 Samule 3 Average
0.1487 0.1499 0.1518 0.1501
Expressed as percentage. Indicates significant difference.
OvenDried
Di5erence
Total Chlorophyllo 0.4905 0.0969 0.4984 0.0936 0.4986 0.1062 0.4958 0.0989 Chlorophyll an 0.0816 0.3571 0.3569 0.0852 0.3574 0.0956 0.3751 0.0695 Chlorophyll ba 0.0153 0.1334 0.0084 0.1415 0.1412 0.0106 0.1387 0.0114
Standard Deviation of the Di5erence
Ratio of Difference to its Standard Deviation
0.0210 0.0453 0.0229 0.0183
4. 614b 2. O6gb 4.638* 5.404D
0.0107 0.0225 0.0153 0.0097
7. 626b 3.7878 6. 24gb 7. 165b
0.0259 0.0323 0.0196 0.0153
0.591 0.260 0.541 0.754
5%
JOURNAL OF THE
AMERICANPHARM A C E U T I C A L AssocxmroN
VOl.
XLIII,
NO.
9
TABLE III.-DETERMIXATIONOF TOTAL CHLOROPHYLL AND THE A AND B COMPONENTS O F THE LEAVESO F PODOPHYLLUM PELTATUM, LINNS(PODOPHYLLUM) BY SPECTROPHOTOMETRIC METHOD OvenDried
FreezeDried
Sample 1 Sample 2 Sample 3 Average
0.4500 0.4548 0.4615 0.4554
Sample 1 Sample 2 Sample 3 Average
0.3089 0,3096 0.3161 0.3115
Sample 1 Sample 2 Sample 3 Average
0.1411 0.1452 0.1454 0.1439
Difference
Total Chlorophylla 0.4073 0.0427 0.4102 0.0446 0.3748 0.0867 0.0580 0.3974 Chlorophyll a" 0.2743 0,0346 0.2680 0.0416 0.2451 0.0710 0.2624 0.0491 Chlorophyll bR 0.1330 0.0081 0.1422 0.0030 0.1297 0.0157 0.1350 0,0089
Standard Deviation of the Difference
Ratio of Difierence t o Its Standard Deviation
0.0994 0.0728 0.0610 0.0458
0.430 0.613 1.421 1.266
0.0265 0.0240 0.0141 0.0128
1.306 1.733 5.035* 3. 83Bh
0.0772 0.0527 0.0536 0.0359
0.105 0.057 0.293 0.248 __ ~~
Expressed as percentage. b Indicates significant difference.
components were determined according to the A. 0. A. C. method (3). The results are listed in Tables I, 11, and 111. The percentages are mean values of a minimum of 12 determinations. The results were statistically evaluated by the Statistics Laboratory of The Ohio State University. The hypothesis tested was that the means of the two compared samples, oven-dried and freeze-dried, are the same. A ratio value greater than 1.950, obtained by dividing the mean difference by the standard deviation, was considered significant, since according to a table of normal distribution (4) the chances of obtaining a greater value is less than 5%. The Rate of Chlorophyll Extraction.-When cxtracting chlorophyll from the leaves, a greater difficulty was encountered and more time consumed in extracting the oven-dried samples as compared with the freeze-dried samples. The object of run-
ning the extraction studies was therefore t o substantiate this finding by numerical data. A Fisher electrophotometer, AC Model, was first calibrated according to the A. 0. A. C. method (3), with 1.0 cm. absorption cells and a No. 650(A) light filter. A calibration curve that rigidly followed Beer's law was constructed. This was employed for calculating the data shown in Table IV. The extraction procedure followed was a s follows: about 5 Gm. accurately weighed, of each of the ovendried and freeze-dried powdered material of all the three leaf samples were packed lightly in 80-cc. percolators of the same size and diameter. The powders were moistened with 35 cc. of 85% acetone C. P., and when the liquid was about to drip from the percolators, the lower orifice was closed and the drugs were allowed t o macerate for twenty-four hours. The percolation was then allowed t o proceed at a moderate rate, gradually adding a new
TABLE IV.-THE RATEOF EXTRACTION OF TOTAL CHLOROPHYLL FROM PODOPHYLLUM, CASTOR, A N D SPEARMINT LEAVES" Podophyllum Leaves OveriFreezeDried Dried
Successive 50-cc. I'ractions
1 2 3 4 5 6 7
b
9 10 11 12 13 Total
330.0 3.8 1.6 0.8 0.5 0.4 0.3 0.3 0.2 0.2
260.0 6.2 3.5 2.0 1.8 1.6 1.6 1.4 1.2 1.o
...
... ... ...
__
280.2
...
_
360. o 18.0 9.0 6.6 6.4 5.4 4.1 3.4 3.0 2.8 2.5 2.3 2.0 425.0
285.0 9.0 4.8 3.4 2.8 2.5 1.7 1.5 I .3 1.3 1.1 1.1
...
338.1
Total Chlorophyll in mg./L.Castor I.eaves OvenFreezeDried Dried
_
The results are the average of three determinalions.
1.0 316.5
~
~
___~ __
\
Spearmint Leaves OvenFreezeDried Dried
230.0 10.0 7.0 (5.5 5.3 5.2 4.4 4.0 3.9 3.6 2.6 1.8 1.8 286.1
280.0 15.0 7.4 5.2 3.8 3.2 2.0 1.4 1.4 1.2 0.8 0.4 0 .4
322.2
__
-
SCIENTIFIC EDITION
September, 1954
menstruum in 25-cc. portions t o the percolators. Successive 50-cc. fractions of percolate were collected, their per cent transmission immediately determined on the Fisher electrophotometer, AC Model, and the chlorophyll content of each fraction read directly on the calibration curve. Percolation was stopped in each case when the transmission of the last fractions of percolates approached minimum. The results of the extraction studies of chlorophyll in the three kinds of leaves appear in Table IV.
DISCUSSION
557
in the temperature will retard the activity of the enzyme. Since less chlorophyll was retained in the oven-dried samples than in the freeze-dried samples of all the three leaves, it is apparent that enzymatic activity was retarded to a greater extent in the freeze-dried samples than in the oven-dried ones, hence the less destruction and breakdown of chlorophyll in the freeze-dried samples. The ratio of chlorophyll a and b in the oven-dried samples was lower than the ratio of these two components in the freeze-dried samples of the leaves. This, of course, i s due t o the fact that chlorophyll a was more sensitive t o oven-drying than freezedrying temperatures and chlorophyll b was equally sensitive t o the temperatures of both drying procedures. The ratio of chlorophyll a t o b in the leaves investigated, both oven-dried and freeze-dried, compare favorably with the ratio of these two components as reported by other investigators. That chlorophyll is extracted more rapidly from the freeze-dried samples than the oven-dried samples of the three kinds of leaves is apparent from the data obtained in Table IV. This may possibly be attributed t o the fact that high temperatures caused more shrinkage of the tissue cells than low temperatures, thus exposing less area to the effect of the menstruiim.
In reference to Tables I and I1 (castor and spearmint leaves) it can be observed that there is more total chlorophyll in the freeze-dried samples than in the oven-dried samples. This difference is a significant amount. The major difference lies in the amount of chlorophyll a in the two samples since this difference is of a significant magnitude, while the difference in chlorophyll b is not a significant amount. In the case of podophyllum leaves, as can be observed in Table 111, there is an unusual result. The experiment showed no significant difference between the total chlorophyll of the freeze-dried and oven-dried samples. However, the amount of chlorophyll a in the freeze-dried sample was signifiCONCLUSIONS cantly greater than in the oven-dried sample. This result can be attributed to several apparent The leaves of Podophyllum peltutum, Linn6 factors. The podophyllum leaves were the first Ricinus communis, LinnC (cas(podophyllum), leaves of the experiment t o be assayed, and the laboratory technique involved in the experiment tor), and M w t h spicutu, Linne (spearmint) was improved in the latter experiments. This fact were used in this investigation. The results is evident from the larger standard deviation for showed that: the podophyllum’ experiment in comparison t o the 1. More total chlorophyll was obtained from standard deviation of the other experiments. The the freeze-dried leaves of podophyllum, castor, fact that the ratio of chlorophyll a t o chlorophyll b in podophyllum leaves is approximately 2 to 1 and spearmint than from the oven-dried leaves. while in the castor plant is approximately 2.8 to 1 2. The total difference was largely due to the is another factor. Although the difference in difference in chlorophyll a since the difference was chlorophyll a is significant, the large amount of of a significant magnitude, whereas the difference chlorophyll b, which does not show a significant difference, offsets somewhat the difference in chloro- in chlorophyll b was not a significant amount. 3. The ratio of chlorophyll u to b in the phyll a. It is true that the ratio of chlorophyll a t o chlorophyll b in spearmint leaves is also approxioven-dried samples was lower than the ratio of mately 2 t o 1, and that the large amount of chloro- these two pigments in the freeze-dried samples phyll b did not offset the difference in chlorophyll of the leaves. The ratio of chlorophyll a to b in a enough t o keep the difference in total chlorophyll the leaves investigated, both oven-dried and from being significant. But there is a difference in the two cases of podophyllum leaves and spear- freeze-dried, compares favorably with the ratio mint leaves. I n podophyllum leaves the amount of of these two components as reported by other chlorophyll b in the two compared samples, freezeinvestigators. dried and oven-dried leaves, is almost the same 4. Freeze-drying Eacilitated chlorophyll exwhile in the case of spearmint leaves the freezcdried sample has more chlorophyll b than the ovcn- tractions. dricd, even though it is not a significant amount. In summary, the total chlorophyll results were REPERENCES possibly influenced by the large standard deviation, (1) Chambers, M . A., and Nelson, J. W., THISJOURNAL; the high proportion of chlorophyll b t o chlorophyll 36, 326(1950). a and the fact that there was a very minute dif(2) “The Pharmacopeia of the United States ” 14th rev., ference in the amount of chlorophyll b in the two Maok Publishin Company Easton Pa. 1950 b. 714 (3) “Official hethods oi An,$& of ’the Association of compared samples. Official Agricultural Chemists George Banta Publishing Company, Menasha, Wisconsi& 1950, p. 112. The optimum temperature of chlorophyllase, a n (4) Snedecor G. W. “Statistical Method,” The Iowa enzyme that occurs alongside its substratum State College Pkess. Ambs. Iowa, 1946, p. 180. (5) Willstatter, R., and Stoll, A,;, “Investigations on “chlorophyll” in the green parts of all the plants, Chlorophyll: Methods and Results The Science Press is about 20” (5). Either an increase or a decrease Printing Company, Lancaster, Pa., I9iS.