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The Influence of Bile Salts on Glycoside Biosynthesis in Digitalis purpurea I. Sodium cholate*
The Influence of Bile Salts on Glycoside Biosynthesis in Digitalis purpurea I.* Sodium Cholate By FRANK T. CHAN and DANIEL P. N . TSAOI Concentrations of 0.02, 0.10, 0.50, and 2.50% sodium cholate were injected into digitalis plants growing i n pots under controlled greenhouse conditions. Sodium cholate i n the concentrations of 0.50 and 2.50y0produced marked inhibition of total glycoside formation. A periodic assay of total glycoside biosynthesis following injections with 0 . 5 0 % sodium cholate injection indicated that the treated digitalis plants produced less glycosides than the controls.
many reports have been made on the growth of plants following t h e use of steroids as growth regulators. Controversial reports have been made on the influence of sex hormones on t h e growth of such plants (1, 2 ) . There have been several reports about t h e inhibiting effect of sodium cholate on plant metabolism (3-6). Deysson and Deysson (3, 4) reported that sodium cholate in concentrations between 0.01 and 0.25% exerted a marked toxic effect on meristematic cells of Atlium cepa. Deysson and Charbonnier ( 5 , 6) produced a retardation of both growth a n d mitotic activity of Allium cepa by treatment with concentration of 0.05y0 of sodium cholate. Much, el a2. ( 7 ) , found that a cholesterol mixture increased t h e growth of digitalis. A similar report b y Tsao and Youngken (8) indicated t h a t 0.5 a n d 2.0% cholesterol i n suspension produced a n increased glycoside production in digitalis plants grown in hydroponic media. However, sodium cholate, a bile salt possessiDg a basic structure similar to cholesterol and being more soluble in water than the latter, was employed in this study. This study deals with t h e influence of various concentrations of sodium cholate on glycoside biosynthesis and with the periodic investigation of the effect oi sodium cholate on glycoside formation in this plant. N THE LAST DECADE
MATERIALS AND METHODS Digitalis plants employed in this study were developed under carefully controlled greenhouse con-
* Received April 13, 1956, from the School of Pharmacy, Oregon State College. Corvallis. Presented to the Scientific Section, A . PH. A , , Detroit meeting, April, 1956. This study is based on a thesis presented to the Graduate School, Oregon State College, by Frank T. Chan. in partial fulfillment of the requirements for the Degree of Master of Science. This study has been supported in part by funds from General Research of the Graduate School of Oregon State College t Present address: Pharmacognosy Department, School of Pharmacy, Oregon State College, Corvallis. 228
ditions.' All seedlings and mature plants were grown from the strain of Digitalis purpurea LinnC which produced flowers of mixed colors, i. e., purple, white, lavender. Seeds were germinated in the greenhouse in culture flats containing soil composed of 2 parts of compost and 1part of peat moss. The seedlings were transplanted into pots containing the same soil mixture. These plants were subjected to artificial illumination for 12 hours daily for one month prior to the beginning of injections. The light was supplied by white 40-watt Sylvania fluorescent lamps, which were suspended 7 to 9 inches above the plants. Growth measurements of all plants were recorded daily during the experimental period. The two largest leaves were selected from each plant for measurement. The length and width of the blade and the length of petiole and blade were recorded. Sodium cholate solutions and distilled water were adjusted t o a pH of 6.8 by 10% sodium hydroxide or 10% hydrochloric acid. The solutions were administered into the plants by injection apparatus (Fig. 1). The outlet of a 10-ml. syringe was fitted with a No. 21-gauge needle by means of intravenous rubber tubing. The air bubbles were removed from the injection apparatus and a volume of specified solutions was injected into the petiole at a speed of about 1 ml. per hour exercising extreme care in order to avoid overinjury of the area of contact. The pressure applied to the plunger was supplied by 2 or 3 rubber bands x 33/4"). The plunger was adjusted occasionally during the injection period so that the required speed could be maintained. At the end of the treatment the plants were harvested and the weight of fresh leaves of each group was recorded. The fresh materials were then transferred immediately into a hot air circulating dryer and dried a t a temperature between 118 and 120 degrees F. Dried weight and moisture content were measured. The dried leaves were ground into a No. 60 powder and stored in an air-tight container for glycoside analyses. Digitalis tinctures were prepared according to U. S. P. X V method (9). The total glycoside was assayed by using a modified Knudson-Dresbach method (10). The absorbance was compared a t 1 The authors are indebted to Drs. H. W. Youngken, Jr. W. R . Welters, and Mr. M. Bakke of Drug Plant Lahora tories, College of Pharmacy of the University of Washington. Seattle, Washington, for their generosity in permitting the use of greenhouse facilities and help in preparing plants.
SCIENTIFIC EDITION
April 1957
229
7 groups; each group consisted of 10 plants. Two groups were designated as controls or untreated, i. e., groups A and B. The other five groups (groups C to G) were treated with specified solutions such as distilled water; 0.02, 0.10, 0.50, and 2.50 per cent of sodium cholate. Each plant of the treated groups received 5 ml. of the respective solutions at a rate of about 1 ml. per hour by injecting into the petiole. One such injection was given each day for three successive days. The f i s t injection was started on November 30,1955. The plants in Group G, injected with 2.50% of sodium cholate, showed severe toxicity, such as wilting and chlorosis, after the second injection. Plants in this group were harvested after the second injection with the control, Group B. The remaining 5 groups (A, C, D, E, F) were harvested on December 3, 1955. The plant material was dried and assayed for total glycosides. Results are shown in Tables I and I1 and Fig. 2.
525 mp in a 1-cm. cell by the use of a Beckman Model DU Photoelectric Quartz Spectrophotometer. A standard curve was constructed from a tincture prepared from U. S. P. Digitalis Reference Standard. Unknown tinctures were then compared with standard digitalis reference tincture and the glycoside yields were expressed in per cent potency of the latter reference standard, expressed as 100~o.
EXPERIMENTAL Part I. The Influence of Various Concentrations of Sodium Cholate on the Glycoside Biosynthesis.Seventy digitalis plants were transplanted into pots on September 8, 1955, and were grown under artificial illumination for one month before the experimental study. These plants were 151 days old from the date of germination and were divided into
TABLE L-ARRANGEMENTOF DIGITALISPLANTS DURINGEXPERIMENTAL FEEDING WITH SODIUM CHOLATE Plants Age, Days NO.
Part
Group
Experiment
I
A B C D E F G 1 2 3 4 5 6 7 8 9 10
Control Control Dist. water Sod. cholate 0.02% Sod. cholate 0.10% Sod. cholate 0.50% Sod. cholate 2.50% Dist. water Sod. cholate 0.50% Dist. water Sod. cholate 0.50% Dist. water Sod. cholate 0.50% Dist. water Sod. cholate o.5070 Dist. water Sod. cholate 0.50%
I1
151 151 151 151 151 151 151 189 189 190 190 191 191 192 192 193 193
10 10 10 10 10 10 10 6 6 6 6 6 6 6 6 6 6
c
PH of Soh.
Injection-Volume Rate (ml.) (hr.)
..
..
..
..
6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8
5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 6.0 5.0 5.0 5.0 5.0 5.0 5.0
.. ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5, ca. 5 ca. 5 ca. 5 ca. 5
No. of
Inject.
Leaf Yield Fresh Dry (Gm./Group)
220.0 210.0 238.0 218.0 202.0 225.0 125.0 170.0 146.0 150.0
.. 3 3 3 3
27.3 25.8 27.0 24.6 23.1
58.8
24.1 18.0 16.0 16.0 1m.o 13.0 754.0 i7.0 139.0 17.0 173.0 19.0 148.0 19.0 150.0 17.0 153.0 19.0
2 1 1 2 2
3 3 4 4
5 5
Moisture (per cent)
87.60 87.71 88.66 88.74 88.05 87.21 80.69" 89.41 89.04 89.33 87.73 88.98 87.77 89.02 87.18 88.67 87.57
~
a Plants showed severe wilting and chlorosis.
TABLEI ~.--CLYCOSIDE YIELDSOF DIGITALISPLANTS TREATED w i m VARIOUS CONCENTRATIONS OF SODIUM B Y THE ~NJECTION METHOD CHOLATE Part
I
I1
Experiment
A B C D
Control Control Dist. water Sod. cholate 0.02% (0.00005 M )
160.0 161.0 154.0 153.5
E
Sod. cholate 0.10% (0,00023 M )
140.0
F
Sod. cholate 0.50% (0.00116 M )
129.5
G 1 2 3
Sod. cholate 2.50% (0.00581 M ) Dist. water Sod. cholate 0.50% M) . - (0.00116 . Dist. water Sod. cholate 0.50% (0.00116 M ) Dist. water Sod. cholate 0.50% (0.00116 M ) Dist. water Sod. cholate 0.50% (0.00116 MI Dist. water Sod. cholate 0.50% (0.00116 M )
114.5 116.5 106.5 110.5 105.0
4 5 6
7 8 9 10 a
ActivityQ (Per Cent)
Group
In comparison with Digitalis U.
. I
~
n
Per Cent Difference
- 3.8 (us. Control A) - 4.1 (us. Control A) - 0.3 ( u s . Dist. water) - 12.5 (vs. Control A) - 9 . 1 ( v s . Dist. water) - 19.i (vs. Control A) - 16 .0 ( v s . Dist. water) '
-28.9 (us. Control B ) -8 6
-5.0
118 ---.l
109.8 113.0 104.5 120.0 110.5
-7.0
-7.5 -7.9
s. P. Reference Standard which was expressed as 100%.
230
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
This study indicated that sodium cholate in concentrations of 0.10, 0.50, and 2.50 per cent inhibited the glycoside biosynthesis in digitalis by 12.5, 19.1, and 28.9 per cent respectively as compared with the controls, and the concentrations of 0.10 and 0.50 per cent showed a decrease in glycosidc content of 9.1 and 16.0 per cent respectively as compared with the plants receiving the injection of distilled water (Table I1 arid Fig. 2). The lowest concentration of sodium cholate, O.OZ%, did not show significant inhibition on glycoside biosynthesis. There was no significant growth difference between treated and untreated groups except that the plants injected with 2.50% sodium cholate showed severe wilting and chlorosis.
Vol. XLVI, No. 4
160 -
3
150-
4
a
z
4
In 140IL
0
u)
W
130-
I-
-t 5
z
120-
W
I0
a
I- 110 -
z W 0
108-
90-
Fig. 2.-A4nalysis of total glycosides of digitalis feedings with sodium cholate. were assigned as experinientals (Groups 2, 4, 6, 8, 10). Each treated group had its own control group, according to their order of increasing numbers, such as Groups 1 and 2; 3 and 4; etc. The control groups were treated with distilled water while the experimental groups were treated with 0.50% sodium cholate. Each plant received 5 ml. of the respective solution a t a rate of about 1 ml. per hour by the injection method. The injection commenced on January Fig. 1.-Injection apparatus for the administration of sodium cholate in digitalis plants.
-
Part 11. Periodic Study of the Effect of Sodium Cholate on the Glycoside Biosynthesis.-Since 0.10, 0.50, and 2.50 per cent sodium cholate solutions inhibited the biosynthesis of digitalis glycosides, it was deemed important to further study the periodic effect of sodium cholate on the glycoside, formation. Sixty digitalis plants which had been grown in the cold frame since September 8, 1955, were transplanted into pots on December 7, 1955, and were allowed to grow in the greenhouse under artificial illumination for one month before the periodic study. These plants were 189 days old and were divided into 10 groups; each group consisted of 6 plants. Five groups were designated as controls (Groups 1, 3, 5, 7, 9j and the other five groups
CONTROL
_ _ TREATED
a
L
IOO~
o
1
I
2
3
4
5
NUMBER OF INJECTIONS
Fig. 3.-Periodic study of glycoside formation in digitalis following injections of 0.570 sodium cholate (one t o five day intervals).
SCIENTIFIC EDITION
April 1957
23 1
11, 1956. The plants in these 10 groups were injected with their respective solutions and on the second day after the first injection, the plants of Groups 1 and 2 were harvested while the remaining 8 groups were given a second injection. On the third day, Groups 3 and 4 were harvested while the remaining groups received the third injection. The injections and harvests were continued in the same manner until Groups 9 and 10 were collected on January 16, 1956. The plant material was weighed, dried, and assayed for the total glycosides. Results are shown in Tables I and I1 and Fig. 3. This periodic study of 0.50 per cent sodium cholate on digitalis plants showed a decrease in glycoside content as compared with the controls by 5.0 to 8.6 per cent, but the difference was not significant compared with the results obtained in the previous study in Part I (Table I1 and Figs. 2 and 3).
late had a toxic effect on the meristematic cells, and the retardation of both growth and mitotic activity in Allium cepa. The plants in Group G, injected with 2.50y0 sodium cholate solution, showed severe toxic symptoms as evidenced by wilting and chlorosis. Wang (12) and Pihar (13) showed that bile salts inhibited succinic oxidase. Kiese and Reinwein (14) reported a depression of oxygen uptake in heart muscle after treating with sodium cholate solution. Manni (15) showed that bile salts inhibited the adenosine triphosphate ( ATPase) activity of myosin. I t is concluded that the inhibition of glycoside formation by sodium cholate in digitalis may be attributed, partially or wholly, to the above factors.
DISCUSSION
A study has been made on the influence of sodium cholate on the glycoside biosynthesis in Digitalis pur@irea. T h e following salient d a t a were observed: 1. A modification has been described for introducing chemical compounds into digitalis leaf tissues. 2. Concentrations of 0.02, 0.10, 0.50, and 2.50 per cent sodium cholate were injected into digitalis plants growing in pots under controlled greenhouse conditions. Sodium cholate in the concentrations OP 0.10, 0.50, and 2.50 per cent produced marked inhibition of total glycoside formation. 3. A periodic assay of total glycoside biosynthesis following injections with 0.50 per cent sodium cholate indicated t h a t the treated digitalis plants produced less glycosides than the controls.
The data on total glycoside yield in Part I1 revealed that 0.50% sodium cholate inhibited glycoside formation by 5.0 t o 8.6 per cent in comparison with those plants treated with distilled water, while the same concentration in Part I produced an inhibition of glycoside production by 16.0% in comparison with plants treated with distilled water (Table 11). The variance of results on glycoside inhibition may be attributed to such factors as age of digitalis plants, temperature, and season. The plants used in Part I were young (151 days old) and were grown in the greenhouse all of the time while those employed in Part I1 were firstly, older (189 days old) and secondly, were grown in the cold frame for 62 days (from September 8, 1955 to December 9, 1955). Tsao and Youngken (11) indicated that the age of plants and season of growth have a great influence on the glycoside formation. It is, therefore, concluded that the variance in results on glycoside inhibition in Parts I and IT can be attributed t o the age of pIant and the environmental conditions which interfere with the anabolism of the digitalis plants in the production of glycosides. The possibility of physical factors, like osmotic pressure, which could exert a marked inhibition or toxic symptoms has been eliminated in this study. The osmotic pressure of 2.50 per cent (0.00116 M ) sodium cholate, if it were completely dissociated, would be less than the osmotic pressure required to rupture plant cells. Tsao and Youngken (8) indicated that 0.04 M potassium chloride in Hoagland solution did not show any inhibition effect on glycoside formation in digitalis plants. Since digitalis plants, injected with distilled water, showed no significant difference in glycoside formation from the untreated plants, hypotonicity was eliminated. Various workers (3-6, 12-15) showed the inhibiting effect of bile salts on plants and animals. The reports by Deysson and Deysson (3,4)and Deysson and Charbonnier (5, 6) indicated that sodium cho-
SUMMARY
REFERENCES (1) Chouard, P., Gynaeco[ogia, 34, 253(1935). ( 2 ) Thimann, K . V.. Ann. Rev. Biochem., 4, 545 (1935). (3) Deysson, G., and Deysson, M., Compt. rend., 229,
1 Z K 7 /,."=",. 1QAQ\ *"".
(4) Deysson G. and Deysson M. ibid. 230, 121(1950). (5) Deysson: G.', and Charbohnie;, A.,'ibid., 236, 1912
(19.53). \ - - - - , -
(6) Deysson, G., and Charbonnier, A., B d l . s o ~ .chim. France, 35, 1007(1953). (7) Much, V. H., Nyren, T . , and Schubert, J., Zentr. Bakteriol. Porisitenk., Abt. I, 116, l(1930). (8) Tsao, D. P. N., and Youngken, H. W., Jr., THIS JOURNAL, 41, 407(1952). (9) :'United States Pharmacopeia," 15th rev., Mack Publishing Co Easton Pa. 1955 p. 212. (10) Bell, $. K., an'd KLantz,' J. C., Jr., J . Phormacol. Erptl. Thcrap. 83 213(1945). (11) Tsao, b. 5. N., and Youngken, H. W., Jr., THIS JOURNAI, 41, 19(1952). 12) Wang Y L. Chinese J . Physiol. 17 231(1950). [13) Pihar;O.: C/&:cm. Listy, 47, 1401(i953;. (14) Kiese, M., and Reinwein, D., Biochem. Z.,324, 51
-- .
(10FAi , --,
(15) Manui, E., Arch. sci. b i d . (Italy), 38, 295(1954).
many reports have been made on the growth of plants following t h e use of steroids as growth regulators. Controversial reports have been made on the influence of sex hormones on t h e growth of such plants (1, 2 ) . There have been several reports about t h e inhibiting effect of sodium cholate on plant metabolism (3-6). Deysson and Deysson (3, 4) reported that sodium cholate in concentrations between 0.01 and 0.25% exerted a marked toxic effect on meristematic cells of Atlium cepa. Deysson and Charbonnier ( 5 , 6) produced a retardation of both growth a n d mitotic activity of Allium cepa by treatment with concentration of 0.05y0 of sodium cholate. Much, el a2. ( 7 ) , found that a cholesterol mixture increased t h e growth of digitalis. A similar report b y Tsao and Youngken (8) indicated t h a t 0.5 a n d 2.0% cholesterol i n suspension produced a n increased glycoside production in digitalis plants grown in hydroponic media. However, sodium cholate, a bile salt possessiDg a basic structure similar to cholesterol and being more soluble in water than the latter, was employed in this study. This study deals with t h e influence of various concentrations of sodium cholate on glycoside biosynthesis and with the periodic investigation of the effect oi sodium cholate on glycoside formation in this plant. N THE LAST DECADE
MATERIALS AND METHODS Digitalis plants employed in this study were developed under carefully controlled greenhouse con-
* Received April 13, 1956, from the School of Pharmacy, Oregon State College. Corvallis. Presented to the Scientific Section, A . PH. A , , Detroit meeting, April, 1956. This study is based on a thesis presented to the Graduate School, Oregon State College, by Frank T. Chan. in partial fulfillment of the requirements for the Degree of Master of Science. This study has been supported in part by funds from General Research of the Graduate School of Oregon State College t Present address: Pharmacognosy Department, School of Pharmacy, Oregon State College, Corvallis. 228
ditions.' All seedlings and mature plants were grown from the strain of Digitalis purpurea LinnC which produced flowers of mixed colors, i. e., purple, white, lavender. Seeds were germinated in the greenhouse in culture flats containing soil composed of 2 parts of compost and 1part of peat moss. The seedlings were transplanted into pots containing the same soil mixture. These plants were subjected to artificial illumination for 12 hours daily for one month prior to the beginning of injections. The light was supplied by white 40-watt Sylvania fluorescent lamps, which were suspended 7 to 9 inches above the plants. Growth measurements of all plants were recorded daily during the experimental period. The two largest leaves were selected from each plant for measurement. The length and width of the blade and the length of petiole and blade were recorded. Sodium cholate solutions and distilled water were adjusted t o a pH of 6.8 by 10% sodium hydroxide or 10% hydrochloric acid. The solutions were administered into the plants by injection apparatus (Fig. 1). The outlet of a 10-ml. syringe was fitted with a No. 21-gauge needle by means of intravenous rubber tubing. The air bubbles were removed from the injection apparatus and a volume of specified solutions was injected into the petiole at a speed of about 1 ml. per hour exercising extreme care in order to avoid overinjury of the area of contact. The pressure applied to the plunger was supplied by 2 or 3 rubber bands x 33/4"). The plunger was adjusted occasionally during the injection period so that the required speed could be maintained. At the end of the treatment the plants were harvested and the weight of fresh leaves of each group was recorded. The fresh materials were then transferred immediately into a hot air circulating dryer and dried a t a temperature between 118 and 120 degrees F. Dried weight and moisture content were measured. The dried leaves were ground into a No. 60 powder and stored in an air-tight container for glycoside analyses. Digitalis tinctures were prepared according to U. S. P. X V method (9). The total glycoside was assayed by using a modified Knudson-Dresbach method (10). The absorbance was compared a t 1 The authors are indebted to Drs. H. W. Youngken, Jr. W. R . Welters, and Mr. M. Bakke of Drug Plant Lahora tories, College of Pharmacy of the University of Washington. Seattle, Washington, for their generosity in permitting the use of greenhouse facilities and help in preparing plants.
SCIENTIFIC EDITION
April 1957
229
7 groups; each group consisted of 10 plants. Two groups were designated as controls or untreated, i. e., groups A and B. The other five groups (groups C to G) were treated with specified solutions such as distilled water; 0.02, 0.10, 0.50, and 2.50 per cent of sodium cholate. Each plant of the treated groups received 5 ml. of the respective solutions at a rate of about 1 ml. per hour by injecting into the petiole. One such injection was given each day for three successive days. The f i s t injection was started on November 30,1955. The plants in Group G, injected with 2.50% of sodium cholate, showed severe toxicity, such as wilting and chlorosis, after the second injection. Plants in this group were harvested after the second injection with the control, Group B. The remaining 5 groups (A, C, D, E, F) were harvested on December 3, 1955. The plant material was dried and assayed for total glycosides. Results are shown in Tables I and I1 and Fig. 2.
525 mp in a 1-cm. cell by the use of a Beckman Model DU Photoelectric Quartz Spectrophotometer. A standard curve was constructed from a tincture prepared from U. S. P. Digitalis Reference Standard. Unknown tinctures were then compared with standard digitalis reference tincture and the glycoside yields were expressed in per cent potency of the latter reference standard, expressed as 100~o.
EXPERIMENTAL Part I. The Influence of Various Concentrations of Sodium Cholate on the Glycoside Biosynthesis.Seventy digitalis plants were transplanted into pots on September 8, 1955, and were grown under artificial illumination for one month before the experimental study. These plants were 151 days old from the date of germination and were divided into
TABLE L-ARRANGEMENTOF DIGITALISPLANTS DURINGEXPERIMENTAL FEEDING WITH SODIUM CHOLATE Plants Age, Days NO.
Part
Group
Experiment
I
A B C D E F G 1 2 3 4 5 6 7 8 9 10
Control Control Dist. water Sod. cholate 0.02% Sod. cholate 0.10% Sod. cholate 0.50% Sod. cholate 2.50% Dist. water Sod. cholate 0.50% Dist. water Sod. cholate 0.50% Dist. water Sod. cholate 0.50% Dist. water Sod. cholate o.5070 Dist. water Sod. cholate 0.50%
I1
151 151 151 151 151 151 151 189 189 190 190 191 191 192 192 193 193
10 10 10 10 10 10 10 6 6 6 6 6 6 6 6 6 6
c
PH of Soh.
Injection-Volume Rate (ml.) (hr.)
..
..
..
..
6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8 6.8
5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 6.0 5.0 5.0 5.0 5.0 5.0 5.0
.. ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5 ca. 5, ca. 5 ca. 5 ca. 5 ca. 5
No. of
Inject.
Leaf Yield Fresh Dry (Gm./Group)
220.0 210.0 238.0 218.0 202.0 225.0 125.0 170.0 146.0 150.0
.. 3 3 3 3
27.3 25.8 27.0 24.6 23.1
58.8
24.1 18.0 16.0 16.0 1m.o 13.0 754.0 i7.0 139.0 17.0 173.0 19.0 148.0 19.0 150.0 17.0 153.0 19.0
2 1 1 2 2
3 3 4 4
5 5
Moisture (per cent)
87.60 87.71 88.66 88.74 88.05 87.21 80.69" 89.41 89.04 89.33 87.73 88.98 87.77 89.02 87.18 88.67 87.57
~
a Plants showed severe wilting and chlorosis.
TABLEI ~.--CLYCOSIDE YIELDSOF DIGITALISPLANTS TREATED w i m VARIOUS CONCENTRATIONS OF SODIUM B Y THE ~NJECTION METHOD CHOLATE Part
I
I1
Experiment
A B C D
Control Control Dist. water Sod. cholate 0.02% (0.00005 M )
160.0 161.0 154.0 153.5
E
Sod. cholate 0.10% (0,00023 M )
140.0
F
Sod. cholate 0.50% (0.00116 M )
129.5
G 1 2 3
Sod. cholate 2.50% (0.00581 M ) Dist. water Sod. cholate 0.50% M) . - (0.00116 . Dist. water Sod. cholate 0.50% (0.00116 M ) Dist. water Sod. cholate 0.50% (0.00116 M ) Dist. water Sod. cholate 0.50% (0.00116 MI Dist. water Sod. cholate 0.50% (0.00116 M )
114.5 116.5 106.5 110.5 105.0
4 5 6
7 8 9 10 a
ActivityQ (Per Cent)
Group
In comparison with Digitalis U.
. I
~
n
Per Cent Difference
- 3.8 (us. Control A) - 4.1 (us. Control A) - 0.3 ( u s . Dist. water) - 12.5 (vs. Control A) - 9 . 1 ( v s . Dist. water) - 19.i (vs. Control A) - 16 .0 ( v s . Dist. water) '
-28.9 (us. Control B ) -8 6
-5.0
118 ---.l
109.8 113.0 104.5 120.0 110.5
-7.0
-7.5 -7.9
s. P. Reference Standard which was expressed as 100%.
230
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
This study indicated that sodium cholate in concentrations of 0.10, 0.50, and 2.50 per cent inhibited the glycoside biosynthesis in digitalis by 12.5, 19.1, and 28.9 per cent respectively as compared with the controls, and the concentrations of 0.10 and 0.50 per cent showed a decrease in glycosidc content of 9.1 and 16.0 per cent respectively as compared with the plants receiving the injection of distilled water (Table I1 arid Fig. 2). The lowest concentration of sodium cholate, O.OZ%, did not show significant inhibition on glycoside biosynthesis. There was no significant growth difference between treated and untreated groups except that the plants injected with 2.50% sodium cholate showed severe wilting and chlorosis.
Vol. XLVI, No. 4
160 -
3
150-
4
a
z
4
In 140IL
0
u)
W
130-
I-
-t 5
z
120-
W
I0
a
I- 110 -
z W 0
108-
90-
Fig. 2.-A4nalysis of total glycosides of digitalis feedings with sodium cholate. were assigned as experinientals (Groups 2, 4, 6, 8, 10). Each treated group had its own control group, according to their order of increasing numbers, such as Groups 1 and 2; 3 and 4; etc. The control groups were treated with distilled water while the experimental groups were treated with 0.50% sodium cholate. Each plant received 5 ml. of the respective solution a t a rate of about 1 ml. per hour by the injection method. The injection commenced on January Fig. 1.-Injection apparatus for the administration of sodium cholate in digitalis plants.
-
Part 11. Periodic Study of the Effect of Sodium Cholate on the Glycoside Biosynthesis.-Since 0.10, 0.50, and 2.50 per cent sodium cholate solutions inhibited the biosynthesis of digitalis glycosides, it was deemed important to further study the periodic effect of sodium cholate on the glycoside, formation. Sixty digitalis plants which had been grown in the cold frame since September 8, 1955, were transplanted into pots on December 7, 1955, and were allowed to grow in the greenhouse under artificial illumination for one month before the periodic study. These plants were 189 days old and were divided into 10 groups; each group consisted of 6 plants. Five groups were designated as controls (Groups 1, 3, 5, 7, 9j and the other five groups
CONTROL
_ _ TREATED
a
L
IOO~
o
1
I
2
3
4
5
NUMBER OF INJECTIONS
Fig. 3.-Periodic study of glycoside formation in digitalis following injections of 0.570 sodium cholate (one t o five day intervals).
SCIENTIFIC EDITION
April 1957
23 1
11, 1956. The plants in these 10 groups were injected with their respective solutions and on the second day after the first injection, the plants of Groups 1 and 2 were harvested while the remaining 8 groups were given a second injection. On the third day, Groups 3 and 4 were harvested while the remaining groups received the third injection. The injections and harvests were continued in the same manner until Groups 9 and 10 were collected on January 16, 1956. The plant material was weighed, dried, and assayed for the total glycosides. Results are shown in Tables I and I1 and Fig. 3. This periodic study of 0.50 per cent sodium cholate on digitalis plants showed a decrease in glycoside content as compared with the controls by 5.0 to 8.6 per cent, but the difference was not significant compared with the results obtained in the previous study in Part I (Table I1 and Figs. 2 and 3).
late had a toxic effect on the meristematic cells, and the retardation of both growth and mitotic activity in Allium cepa. The plants in Group G, injected with 2.50y0 sodium cholate solution, showed severe toxic symptoms as evidenced by wilting and chlorosis. Wang (12) and Pihar (13) showed that bile salts inhibited succinic oxidase. Kiese and Reinwein (14) reported a depression of oxygen uptake in heart muscle after treating with sodium cholate solution. Manni (15) showed that bile salts inhibited the adenosine triphosphate ( ATPase) activity of myosin. I t is concluded that the inhibition of glycoside formation by sodium cholate in digitalis may be attributed, partially or wholly, to the above factors.
DISCUSSION
A study has been made on the influence of sodium cholate on the glycoside biosynthesis in Digitalis pur@irea. T h e following salient d a t a were observed: 1. A modification has been described for introducing chemical compounds into digitalis leaf tissues. 2. Concentrations of 0.02, 0.10, 0.50, and 2.50 per cent sodium cholate were injected into digitalis plants growing in pots under controlled greenhouse conditions. Sodium cholate in the concentrations OP 0.10, 0.50, and 2.50 per cent produced marked inhibition of total glycoside formation. 3. A periodic assay of total glycoside biosynthesis following injections with 0.50 per cent sodium cholate indicated t h a t the treated digitalis plants produced less glycosides than the controls.
The data on total glycoside yield in Part I1 revealed that 0.50% sodium cholate inhibited glycoside formation by 5.0 t o 8.6 per cent in comparison with those plants treated with distilled water, while the same concentration in Part I produced an inhibition of glycoside production by 16.0% in comparison with plants treated with distilled water (Table 11). The variance of results on glycoside inhibition may be attributed to such factors as age of digitalis plants, temperature, and season. The plants used in Part I were young (151 days old) and were grown in the greenhouse all of the time while those employed in Part I1 were firstly, older (189 days old) and secondly, were grown in the cold frame for 62 days (from September 8, 1955 to December 9, 1955). Tsao and Youngken (11) indicated that the age of plants and season of growth have a great influence on the glycoside formation. It is, therefore, concluded that the variance in results on glycoside inhibition in Parts I and IT can be attributed t o the age of pIant and the environmental conditions which interfere with the anabolism of the digitalis plants in the production of glycosides. The possibility of physical factors, like osmotic pressure, which could exert a marked inhibition or toxic symptoms has been eliminated in this study. The osmotic pressure of 2.50 per cent (0.00116 M ) sodium cholate, if it were completely dissociated, would be less than the osmotic pressure required to rupture plant cells. Tsao and Youngken (8) indicated that 0.04 M potassium chloride in Hoagland solution did not show any inhibition effect on glycoside formation in digitalis plants. Since digitalis plants, injected with distilled water, showed no significant difference in glycoside formation from the untreated plants, hypotonicity was eliminated. Various workers (3-6, 12-15) showed the inhibiting effect of bile salts on plants and animals. The reports by Deysson and Deysson (3,4)and Deysson and Charbonnier (5, 6) indicated that sodium cho-
SUMMARY
REFERENCES (1) Chouard, P., Gynaeco[ogia, 34, 253(1935). ( 2 ) Thimann, K . V.. Ann. Rev. Biochem., 4, 545 (1935). (3) Deysson, G., and Deysson, M., Compt. rend., 229,
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(4) Deysson G. and Deysson M. ibid. 230, 121(1950). (5) Deysson: G.', and Charbohnie;, A.,'ibid., 236, 1912
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(6) Deysson, G., and Charbonnier, A., B d l . s o ~ .chim. France, 35, 1007(1953). (7) Much, V. H., Nyren, T . , and Schubert, J., Zentr. Bakteriol. Porisitenk., Abt. I, 116, l(1930). (8) Tsao, D. P. N., and Youngken, H. W., Jr., THIS JOURNAL, 41, 407(1952). (9) :'United States Pharmacopeia," 15th rev., Mack Publishing Co Easton Pa. 1955 p. 212. (10) Bell, $. K., an'd KLantz,' J. C., Jr., J . Phormacol. Erptl. Thcrap. 83 213(1945). (11) Tsao, b. 5. N., and Youngken, H. W., Jr., THIS JOURNAI, 41, 19(1952). 12) Wang Y L. Chinese J . Physiol. 17 231(1950). [13) Pihar;O.: C/&:cm. Listy, 47, 1401(i953;. (14) Kiese, M., and Reinwein, D., Biochem. Z.,324, 51
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(15) Manui, E., Arch. sci. b i d . (Italy), 38, 295(1954).