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The Effect of Freeze-Drying on the Glycosidal Content of Digitalis purpurea and Digitalis lutea L.*
JOURNAL OF THE AMERICAN PHARMACEUTICAL ASSOCIATION
Vol. XLIII, No. 2
REFJZRENCES (1) Gakenheimer, W. C., and Feller, B., THISJOURNAL, 38, 660(1049). (2) Trenner, N. R., Buhs, R. P., Bacher, F . A,, and Gakenheimer W. C . ibid. 39 36lfl950). (3) Lang,’C. A., hnd Cho;, B. F., Proc. SOC.ExpfZ. Bid. ~ , , i 7s i i~a--,. m” ..-I_., .-,m _ _ _ (4) Frost D. V. Armstrong, K. L., Lapidus M . , and Fricke, H. H., Am. khem. SOC.,Abst. 118th meethg. 1950. ( 5 ) Frost, D. V., Lapidns, M.. Plaut, K. A.. Scherfling, E., and Fricke, H. H., Science, 116, 119(1952).
(6) Campbell, J. A., McLaughlan, J. M., Chapman. D. G. THISJOURNAL 41 479(1952). (7j “The United btaies Pharmacopeia,” 14th rev., 3rd
Sup l., Mack Publishing Co., Easton, Pa., 1952. 83) Fantes K. H. Page J. E. Parker L. J. F . and Smith. E. I-.. hot. RO?).Soc.’ILond&~).B ld6. 592(19dO,. (9) ”TKeUnited States Pharmacop&a.” ldth rev., Mack Publishing Co., Easton, Pa. 1952 p. 660. M.,’Drucker, R., Tabenkin, B., J . (10) Cooperman. Bid. Chem. 191, 135&951).
The Effect of Freeze-Drying on the Glycosidal Content of DZgitaZis purpurea and Digitalis lutea L.” By F. P. COSGROVE? and E. P. GUTHS A study of the effects of freeze-drying on the glycosidal content of the leaves of Digitalis pulpurea L. and Di italis Zutea .I has been made. The results indicate that freeze-drying is applicabfe as a means of drying leaves of these two species of digitalis. It is su gested that this method of drying be ado ted for leaves of Digitalispnrpurea tfat are to be used as a source of material for t i e official preparations of this drug. The moisture content of the lyophilized material compares favorably with that found for the corresponding oven-dried samples. In regards to the toxicity tests, it was found that no significant differences were present with samples made from either the lyophilized or oven-dried material. The chemical assays indicate notable differences in the majority of samples compared. The types and quantities of glycosides present in the powdered samples are not under investigation. (I) has shown that fresh leaves of Digitalis purpurea have greater activity than dried leaves, but it is not always convenient to use the fresh leaves immediately after harvesting. Wijngaarden ( 2 ) has reported that in moist digitalis leaves, enzymes cause a slow but steady loss of activity. Stoll and Kreis (3) pointed out clearly the need of inactivating the digitalis enzymes. Studies by King and Gisvold (4) and Youngken, et al. ( 5 ) , have shown that freezing temperatures preserve the potency of the constituents of Digitalis purpurea leaves: In light of the literature which points to the destructive action of the enzymes in digitalis leaves as well as the disagreement concerning the optimum time and temperature required to dry these leaves, it follows that a method which could AMILTON
* Received August 21,1953, from the College of Pharmacy, The Ohio State University, Columbus, Ohio. Presented to the Scientific Seetion, A. PH. A., Salt Lake City meeting, August, 1963. Based upon a dissertation submitted to the Graduate School of The Ohio State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. t Present Address: University of Nebraska, College of Phnrmacy. 1Professor of Pharmacy. The Ohio State University, Collegc of Pharmacy.
remove moisture at low temperatures would be an ideal method for drying this drug. One process of desiccation used to dry material at freezing temperatures is called freeze-drying. Chambers and Nelson (6) have shown that freezedrying is applicable for drying belladonna leaves. With this process the moisture in the material is frozen and removed by means of reduced pressure directly from the frozen state to the vapor state. Although few data are available on the cheniical composition of the constituents of Digitalis lutea, it has been.used in place of the official digitalis and appears to be almost identical in activity with it. Leone (7), Boriani (8), and Welti (9) studied the activity of Digitalis lutea and found it t o be equal to Digitalis purpurea. White and Morris (lo), as well as Konnerth and Pickering (1I), found that Digitalis lutea grown in Minnesota was equal in activity to Digitalis purpurea. The objectives of this investigation are to determine the effects of freeze-drying on the glycosidal content of Digitalis purpztren L. and Digitalis lrrtea L., and to compare these resiilts with those
SCIENTIFIC EDITION
February, 1954
obtained with t h e leaves of the same source which were oven-dried at 50' for thirty-six hours.
EXPERIMENTAL The materials used in this investigation were the leaves from Digitalis purfiurea L., 1st and 2nd year 'plants, and Digitalis lutea L., 2nd and 3rd year plants. The leaves were collected a t random from the Drug Garden of the College of Pharmacy, The Ohio State University. Leaves from each of the plants were divided into two lots, A to be oven-dried a t 50" for thirty-six hours, and B t o be lyophilized at 250 p of pressure and a temperature of less than 35'. All samples to be lyophilized were packed in dry ice immediately after harvesting in order t o prevent, as far as possible, any change in the constituents by the enzymes or other reactants. Samples of Digitalis purpurea L., 1st and 2nd year plants, and Digitatis lutea L., 2nd and 3rd year plants, were oven-dried at 50' for thirty-six hours. Samples of Digitalis purgurea L., 1st and 2nd year plants, and Digitalis lutea L., 2nd and 3rd year plants, were lyophilized by means of the Stokes Freeze Drying Equipment Model 2004-L. The dried material was ground to a No. 40 powder and placed until used in tightly closed, lightresistant bottles, which contained a small bag of Drierite. Moisture Content.-In order t o determine whether the samples met the official requirements for moisture content, the powders were assayed according t o the U. S. P. XIV method for material containing no principles volatile at 100'. The freeze-drying process gave results comparable with those obtained with the oven-drying procedure. Results are given in Table I.
TABLE I.-PERCENTAGEOF MOISTURE~ Sample
Age of Plant, Years
Method of Drying
Percentage of Moisture
D . puriurea D. purpurea D. purpurea D. purpurea D. lutea D. lutea D. lutea D. lutea
1 1 2 2 2 2 3 3
Ovenb Freeze-drie& Ovenb Freeze-driedc Ovenh Freeze-driedc Ovenb Freeze-driedc
4.4 4.2 4.6 5.4 3.7 5.4 5.0 5.0
\
a As determined hv the Brabender Semi-Automatic Moisture Tester, Rochelic Park N. J. a Oven-dried a t 50° for t'hirty-six hours. Lyophilized at 250 p of pressure at a temperature,of less than 3 5 O by means of the Stokes Freeze Drying Equlpment Model 2004-L.
91
Alcohol, U. S. P. was added t o the percolate collected from each sample to bring the total volume to 1500 ml. The total glycoside content present in the percolate was determined by the modified Knudson-Dresbach method (12). A 25-ml. aliquot was concentrated spontaneously to approximately 5 ml. in order to eliminate possible interference from the ethanol. The determinations were made with the aid of a Fisher electrophotometer at 525 mp using a 23-ml. absorption tube, and readings were compared with those obtained with the U. S. P. Digitalis Reference Standard tincture. The total glycosides expressed as U. S. P. units of Reference Standard are given in Table 11.
TABLEII.-ToTAL GLYCOSIDES EXTRACTED FROM 10 GRAMSOF POWDERED DRUG
Sample
Age of Plant, Years
Method of Drying
D.purpurea D . purfiurea . .
1 1
D.@@urea D. burburea
- -
2 2
D. lutea D . lutea
2 2
D. lutea D . lutea
3 3
Oven0 Freezedried Ovene Freezedriedb Oven" Freezedriedb ovena Freezedried
Average Total Glycosides as Units of Reference Standard'
6 6
15.5 13.8
6 6
6.1 7.4
5 5
14.7
6 6
15.8 13.2
'
12.9
a Oven-dried at 50" for thirty-six hours. Lyophilized at 250 p of pressure at a temperature of less than 35' by means of the Stokes Freeze Drying Equipment Model 2004-1,. As determined by the Knudson-Dresbach method (12).
Glycosidal Content of Sample Tinctures.-Timtures were prepared from the lyophilized and ovendried material according t o the official directions and assayed for total glycosides by the modified Knudson-Dresbach method ( 12). Readings were made as described above. The results expressed as percentage potency of Reference Standard are shown in Table 111.
TABLEllI.--GLYCOSIDAL CONTENT TINCTURES
~
Extraction and Determination of Total Glycosides.-To extract the total glycosides without subjecting them to possible destruction by heat, the cold percolation method was employed. A 10-Gm. sample of each drug was packed in an 80-ml. percolator and macerated for twenty-four hours with 95% alcohol. Continuous percolation was carried out a t a rate of 1 ml. per minute until a portion of the percolate gave it negative Molisch test and a high transmittanry reading on an elcrtrophotonleter.
No. of Determinations
Method
Sample
Age of Plant, Years
D . purpurea D . gurpurea
1 1
D. purpurea D . purpurea
2 2
D . lutea D. lutea
3 3
Ovena Freezedriedb Ovena Freezedriedb Ovena Freezedriedb
of
Drying
6F
SAMPLE
No. of Determlnations
Average Potency of Reference Standard
9 9
119.8 111.2
9
9
74.7 69.7
6 6
102.0 94.5
Oven-dried at 50" for thirty-six hours. Lyophilized at 250 p of pressure at a temperature of less than 3 5 O by means of the Stokes\Freeze Drying E:quipment Model 2004-L. As determined by the modified Knudson-Dresbach method (12).
DB
JOURNAL OM THE
AMERICAN PHARMACEUTICAL ASSOC~ATION VOl. XL111,
NO.
2
TABLE IV.-EVALUATIONOF TOXICITY TESTS OF SAMPLE TINCTURES ON FROGS No. of Frogs Used
Range of Systolic Stoppa5e Times
Average Systolic Degrees Stoppage Standard of TimesC Error Freedom
Sample
Age of Plant, Years
D. purpurea D . burburea ~.)u;purea
1 1 2
Oveno Freeze-driedb Oven*
15 15 18
22-48 26-47 25-66
31 1 32.5 46.4
1.92 1.70 2.92
D.purpurea D. lutea
2 3
Freeze-driedb Ovena
18 17
24-82 24-59
50.6 43.8
4.21 2.82
D. lutea
3
Freeze-driedb
17
25-79
42.7
3.50
Method of Drying
Value
At 95% Level
28
0.546
2.048
34
1.113
2.032
32
0.244
2.048
f
a Oven-dried at 50° for thirty-six hours. Lyophilized a t 250 ~rof pressure at a temperature of less than 35” by means of the Stokes Freeze Drying Equipment Model 2004-L. Times are in minutes.
Toxicity Tests.-The toxicity of the samples was determined by diluting the tinctures to 1% with physiological salt solution and using the cup infusion method (13). While this method measures chiefly the toxicity reaction of digitalis, it gives satisfactory results for comparing whole leaf preparations such as tinctures and infusions. The time required to produce systolic stoppage of the frog’s heart was used as the basis for the toxicity of the samples. Results are given in Table
IV. Evaluation of the Toxicity Results.-The results of the toxicity tests were evaluated statistically. The t values were calculated as proposed by Snedecor (14), by pooling tRe variance of the results of the tinctures made with oven-dried material with the variance of the results of the corresponding tinctures made from lyophilized products using appropriate degrees of freedom, and testing the hypothesis that the means of the two samples being compared are the same. The t values that are greater than 95% probability t value, a t the appropriate degrees of freedom, are considered significant. Results are given in Table IV.
DISCUSSION The results of all the colorimetric assays with the exception of those for total glycosides of Digitalis purpurea, 2nd year plants, indicate that the ovendried samples contain supposedly higher content of glycosides than the corresponding lyophilized material. While this possibility might exist, the results of the toxicity tests when evaluated statistically indicate that no significant differences were present among the samples compared. No explanation is given for the behavior of Digitalis pur$urea, 2nd year plants, other than to statc that many of the leavcs contained small brown spots and the plants were flowering a t the time of harvcstitig.
A variation of the amount of primary and secondary glycosides may be a possible reason for differences in the majority of samples compared colorimetrically. Bell and Krantz (15) and Eastland, et at. (16), have shown that secondary glycosides and aglycones give greater optical density to the color reaction of this test than do primary glycosides when equal concentrations of these constituents are coinpared. Therefore, it seems that the degree of complexity of the glycoside mixture, namely the relative proportions of primary glycosides t o secondary glycosides and aglycones, has ati important effect on the colorimetric assay. If primary glycosides are broken down to secondary glycosides and aglycones by heat and enzymatic action, then this may partly explain why the oven-dried samples show supposedly higher glycosidal content when assayed by the modified Knudson-Dresbach method.
REFERENCES (1) Hamilton, H., J . Am. Chem. So;., 41, 25(1919). (2) Wijogaarden, D. deL. Van. Arch. E r p f l . Path. Phormakol. 113 59(1926) (3)’Stoli, A,, and’Kreis, W., Helu. Chim. Acla, 18, 120 (1935). (4) King, R., and Gisvold, 0..THISJOWRNAL, 39. 109 (1950). (5) Youngken, H. W., Jr., Djao, E. H.. and Tsao, D. P,’, ibid., 40, 569(1951). (6) Chambers, M., and Nelson, J. W., ibid., 39, 823 (1Y5U).
(7) Leone,,G., Boll. soc. ifal. biol. speu., 12. 751(1937). (8) Bortani, A,, ibid., 14, 380(1939). (9) Welt], C.. Arch. intern. pharmacodynamie, 37. 250
,- --,.
(ism 1
(10) White, S., and Morris, R . , Arch. Infernal M e d . . 21, 740(1918). (11) Konnerth, R. A., and Pickering, E., This Journal, 15, 1069(1926). (12) Bell, F . K., and Krantz, J. C.,Jr., J . Phurrnacol. E x p f l . T h u a p . , 83, 213(1946). (13) Hiner, L. D . . A m . J , Pharm., 3, 79(1938). 114) Snedecor, G . W., “Statistical Methods,” 4th ed., The Iowa State College Press, Ames, Iowa, 1946, p. 77. ( 1 5 ) Bell, F. K., and Krantz, J. C., Jr., TIIISJ O U R N A L . 38, 107(1949). (16) Eastland, C. J., Lowday. D. P., and Sellwood, E. I%., J . Pharm. and Pharmacol., 4, Sll(1962).
Vol. XLIII, No. 2
REFJZRENCES (1) Gakenheimer, W. C., and Feller, B., THISJOURNAL, 38, 660(1049). (2) Trenner, N. R., Buhs, R. P., Bacher, F . A,, and Gakenheimer W. C . ibid. 39 36lfl950). (3) Lang,’C. A., hnd Cho;, B. F., Proc. SOC.ExpfZ. Bid. ~ , , i 7s i i~a--,. m” ..-I_., .-,m _ _ _ (4) Frost D. V. Armstrong, K. L., Lapidus M . , and Fricke, H. H., Am. khem. SOC.,Abst. 118th meethg. 1950. ( 5 ) Frost, D. V., Lapidns, M.. Plaut, K. A.. Scherfling, E., and Fricke, H. H., Science, 116, 119(1952).
(6) Campbell, J. A., McLaughlan, J. M., Chapman. D. G. THISJOURNAL 41 479(1952). (7j “The United btaies Pharmacopeia,” 14th rev., 3rd
Sup l., Mack Publishing Co., Easton, Pa., 1952. 83) Fantes K. H. Page J. E. Parker L. J. F . and Smith. E. I-.. hot. RO?).Soc.’ILond&~).B ld6. 592(19dO,. (9) ”TKeUnited States Pharmacop&a.” ldth rev., Mack Publishing Co., Easton, Pa. 1952 p. 660. M.,’Drucker, R., Tabenkin, B., J . (10) Cooperman. Bid. Chem. 191, 135&951).
The Effect of Freeze-Drying on the Glycosidal Content of DZgitaZis purpurea and Digitalis lutea L.” By F. P. COSGROVE? and E. P. GUTHS A study of the effects of freeze-drying on the glycosidal content of the leaves of Digitalis pulpurea L. and Di italis Zutea .I has been made. The results indicate that freeze-drying is applicabfe as a means of drying leaves of these two species of digitalis. It is su gested that this method of drying be ado ted for leaves of Digitalispnrpurea tfat are to be used as a source of material for t i e official preparations of this drug. The moisture content of the lyophilized material compares favorably with that found for the corresponding oven-dried samples. In regards to the toxicity tests, it was found that no significant differences were present with samples made from either the lyophilized or oven-dried material. The chemical assays indicate notable differences in the majority of samples compared. The types and quantities of glycosides present in the powdered samples are not under investigation. (I) has shown that fresh leaves of Digitalis purpurea have greater activity than dried leaves, but it is not always convenient to use the fresh leaves immediately after harvesting. Wijngaarden ( 2 ) has reported that in moist digitalis leaves, enzymes cause a slow but steady loss of activity. Stoll and Kreis (3) pointed out clearly the need of inactivating the digitalis enzymes. Studies by King and Gisvold (4) and Youngken, et al. ( 5 ) , have shown that freezing temperatures preserve the potency of the constituents of Digitalis purpurea leaves: In light of the literature which points to the destructive action of the enzymes in digitalis leaves as well as the disagreement concerning the optimum time and temperature required to dry these leaves, it follows that a method which could AMILTON
* Received August 21,1953, from the College of Pharmacy, The Ohio State University, Columbus, Ohio. Presented to the Scientific Seetion, A. PH. A., Salt Lake City meeting, August, 1963. Based upon a dissertation submitted to the Graduate School of The Ohio State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. t Present Address: University of Nebraska, College of Phnrmacy. 1Professor of Pharmacy. The Ohio State University, Collegc of Pharmacy.
remove moisture at low temperatures would be an ideal method for drying this drug. One process of desiccation used to dry material at freezing temperatures is called freeze-drying. Chambers and Nelson (6) have shown that freezedrying is applicable for drying belladonna leaves. With this process the moisture in the material is frozen and removed by means of reduced pressure directly from the frozen state to the vapor state. Although few data are available on the cheniical composition of the constituents of Digitalis lutea, it has been.used in place of the official digitalis and appears to be almost identical in activity with it. Leone (7), Boriani (8), and Welti (9) studied the activity of Digitalis lutea and found it t o be equal to Digitalis purpurea. White and Morris (lo), as well as Konnerth and Pickering (1I), found that Digitalis lutea grown in Minnesota was equal in activity to Digitalis purpurea. The objectives of this investigation are to determine the effects of freeze-drying on the glycosidal content of Digitalis purpztren L. and Digitalis lrrtea L., and to compare these resiilts with those
SCIENTIFIC EDITION
February, 1954
obtained with t h e leaves of the same source which were oven-dried at 50' for thirty-six hours.
EXPERIMENTAL The materials used in this investigation were the leaves from Digitalis purfiurea L., 1st and 2nd year 'plants, and Digitalis lutea L., 2nd and 3rd year plants. The leaves were collected a t random from the Drug Garden of the College of Pharmacy, The Ohio State University. Leaves from each of the plants were divided into two lots, A to be oven-dried a t 50" for thirty-six hours, and B t o be lyophilized at 250 p of pressure and a temperature of less than 35'. All samples to be lyophilized were packed in dry ice immediately after harvesting in order t o prevent, as far as possible, any change in the constituents by the enzymes or other reactants. Samples of Digitalis purpurea L., 1st and 2nd year plants, and Digitatis lutea L., 2nd and 3rd year plants, were oven-dried at 50' for thirty-six hours. Samples of Digitalis purgurea L., 1st and 2nd year plants, and Digitalis lutea L., 2nd and 3rd year plants, were lyophilized by means of the Stokes Freeze Drying Equipment Model 2004-L. The dried material was ground to a No. 40 powder and placed until used in tightly closed, lightresistant bottles, which contained a small bag of Drierite. Moisture Content.-In order t o determine whether the samples met the official requirements for moisture content, the powders were assayed according t o the U. S. P. XIV method for material containing no principles volatile at 100'. The freeze-drying process gave results comparable with those obtained with the oven-drying procedure. Results are given in Table I.
TABLE I.-PERCENTAGEOF MOISTURE~ Sample
Age of Plant, Years
Method of Drying
Percentage of Moisture
D . puriurea D. purpurea D. purpurea D. purpurea D. lutea D. lutea D. lutea D. lutea
1 1 2 2 2 2 3 3
Ovenb Freeze-drie& Ovenb Freeze-driedc Ovenh Freeze-driedc Ovenb Freeze-driedc
4.4 4.2 4.6 5.4 3.7 5.4 5.0 5.0
\
a As determined hv the Brabender Semi-Automatic Moisture Tester, Rochelic Park N. J. a Oven-dried a t 50° for t'hirty-six hours. Lyophilized at 250 p of pressure at a temperature,of less than 3 5 O by means of the Stokes Freeze Drying Equlpment Model 2004-L.
91
Alcohol, U. S. P. was added t o the percolate collected from each sample to bring the total volume to 1500 ml. The total glycoside content present in the percolate was determined by the modified Knudson-Dresbach method (12). A 25-ml. aliquot was concentrated spontaneously to approximately 5 ml. in order to eliminate possible interference from the ethanol. The determinations were made with the aid of a Fisher electrophotometer at 525 mp using a 23-ml. absorption tube, and readings were compared with those obtained with the U. S. P. Digitalis Reference Standard tincture. The total glycosides expressed as U. S. P. units of Reference Standard are given in Table 11.
TABLEII.-ToTAL GLYCOSIDES EXTRACTED FROM 10 GRAMSOF POWDERED DRUG
Sample
Age of Plant, Years
Method of Drying
D.purpurea D . purfiurea . .
1 1
D.@@urea D. burburea
- -
2 2
D. lutea D . lutea
2 2
D. lutea D . lutea
3 3
Oven0 Freezedried Ovene Freezedriedb Oven" Freezedriedb ovena Freezedried
Average Total Glycosides as Units of Reference Standard'
6 6
15.5 13.8
6 6
6.1 7.4
5 5
14.7
6 6
15.8 13.2
'
12.9
a Oven-dried at 50" for thirty-six hours. Lyophilized at 250 p of pressure at a temperature of less than 35' by means of the Stokes Freeze Drying Equipment Model 2004-1,. As determined by the Knudson-Dresbach method (12).
Glycosidal Content of Sample Tinctures.-Timtures were prepared from the lyophilized and ovendried material according t o the official directions and assayed for total glycosides by the modified Knudson-Dresbach method ( 12). Readings were made as described above. The results expressed as percentage potency of Reference Standard are shown in Table 111.
TABLEllI.--GLYCOSIDAL CONTENT TINCTURES
~
Extraction and Determination of Total Glycosides.-To extract the total glycosides without subjecting them to possible destruction by heat, the cold percolation method was employed. A 10-Gm. sample of each drug was packed in an 80-ml. percolator and macerated for twenty-four hours with 95% alcohol. Continuous percolation was carried out a t a rate of 1 ml. per minute until a portion of the percolate gave it negative Molisch test and a high transmittanry reading on an elcrtrophotonleter.
No. of Determinations
Method
Sample
Age of Plant, Years
D . purpurea D . gurpurea
1 1
D. purpurea D . purpurea
2 2
D . lutea D. lutea
3 3
Ovena Freezedriedb Ovena Freezedriedb Ovena Freezedriedb
of
Drying
6F
SAMPLE
No. of Determlnations
Average Potency of Reference Standard
9 9
119.8 111.2
9
9
74.7 69.7
6 6
102.0 94.5
Oven-dried at 50" for thirty-six hours. Lyophilized at 250 p of pressure at a temperature of less than 3 5 O by means of the Stokes\Freeze Drying E:quipment Model 2004-L. As determined by the modified Knudson-Dresbach method (12).
DB
JOURNAL OM THE
AMERICAN PHARMACEUTICAL ASSOC~ATION VOl. XL111,
NO.
2
TABLE IV.-EVALUATIONOF TOXICITY TESTS OF SAMPLE TINCTURES ON FROGS No. of Frogs Used
Range of Systolic Stoppa5e Times
Average Systolic Degrees Stoppage Standard of TimesC Error Freedom
Sample
Age of Plant, Years
D. purpurea D . burburea ~.)u;purea
1 1 2
Oveno Freeze-driedb Oven*
15 15 18
22-48 26-47 25-66
31 1 32.5 46.4
1.92 1.70 2.92
D.purpurea D. lutea
2 3
Freeze-driedb Ovena
18 17
24-82 24-59
50.6 43.8
4.21 2.82
D. lutea
3
Freeze-driedb
17
25-79
42.7
3.50
Method of Drying
Value
At 95% Level
28
0.546
2.048
34
1.113
2.032
32
0.244
2.048
f
a Oven-dried at 50° for thirty-six hours. Lyophilized a t 250 ~rof pressure at a temperature of less than 35” by means of the Stokes Freeze Drying Equipment Model 2004-L. Times are in minutes.
Toxicity Tests.-The toxicity of the samples was determined by diluting the tinctures to 1% with physiological salt solution and using the cup infusion method (13). While this method measures chiefly the toxicity reaction of digitalis, it gives satisfactory results for comparing whole leaf preparations such as tinctures and infusions. The time required to produce systolic stoppage of the frog’s heart was used as the basis for the toxicity of the samples. Results are given in Table
IV. Evaluation of the Toxicity Results.-The results of the toxicity tests were evaluated statistically. The t values were calculated as proposed by Snedecor (14), by pooling tRe variance of the results of the tinctures made with oven-dried material with the variance of the results of the corresponding tinctures made from lyophilized products using appropriate degrees of freedom, and testing the hypothesis that the means of the two samples being compared are the same. The t values that are greater than 95% probability t value, a t the appropriate degrees of freedom, are considered significant. Results are given in Table IV.
DISCUSSION The results of all the colorimetric assays with the exception of those for total glycosides of Digitalis purpurea, 2nd year plants, indicate that the ovendried samples contain supposedly higher content of glycosides than the corresponding lyophilized material. While this possibility might exist, the results of the toxicity tests when evaluated statistically indicate that no significant differences were present among the samples compared. No explanation is given for the behavior of Digitalis pur$urea, 2nd year plants, other than to statc that many of the leavcs contained small brown spots and the plants were flowering a t the time of harvcstitig.
A variation of the amount of primary and secondary glycosides may be a possible reason for differences in the majority of samples compared colorimetrically. Bell and Krantz (15) and Eastland, et at. (16), have shown that secondary glycosides and aglycones give greater optical density to the color reaction of this test than do primary glycosides when equal concentrations of these constituents are coinpared. Therefore, it seems that the degree of complexity of the glycoside mixture, namely the relative proportions of primary glycosides t o secondary glycosides and aglycones, has ati important effect on the colorimetric assay. If primary glycosides are broken down to secondary glycosides and aglycones by heat and enzymatic action, then this may partly explain why the oven-dried samples show supposedly higher glycosidal content when assayed by the modified Knudson-Dresbach method.
REFERENCES (1) Hamilton, H., J . Am. Chem. So;., 41, 25(1919). (2) Wijogaarden, D. deL. Van. Arch. E r p f l . Path. Phormakol. 113 59(1926) (3)’Stoli, A,, and’Kreis, W., Helu. Chim. Acla, 18, 120 (1935). (4) King, R., and Gisvold, 0..THISJOWRNAL, 39. 109 (1950). (5) Youngken, H. W., Jr., Djao, E. H.. and Tsao, D. P,’, ibid., 40, 569(1951). (6) Chambers, M., and Nelson, J. W., ibid., 39, 823 (1Y5U).
(7) Leone,,G., Boll. soc. ifal. biol. speu., 12. 751(1937). (8) Bortani, A,, ibid., 14, 380(1939). (9) Welt], C.. Arch. intern. pharmacodynamie, 37. 250
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(10) White, S., and Morris, R . , Arch. Infernal M e d . . 21, 740(1918). (11) Konnerth, R. A., and Pickering, E., This Journal, 15, 1069(1926). (12) Bell, F . K., and Krantz, J. C.,Jr., J . Phurrnacol. E x p f l . T h u a p . , 83, 213(1946). (13) Hiner, L. D . . A m . J , Pharm., 3, 79(1938). 114) Snedecor, G . W., “Statistical Methods,” 4th ed., The Iowa State College Press, Ames, Iowa, 1946, p. 77. ( 1 5 ) Bell, F. K., and Krantz, J. C., Jr., TIIISJ O U R N A L . 38, 107(1949). (16) Eastland, C. J., Lowday. D. P., and Sellwood, E. I%., J . Pharm. and Pharmacol., 4, Sll(1962).