Effect of Adding Cholesterol and Different Sugars to the Nutrient Medium on Production the Steroidal Saponin " Digitonin " From Digitalis Purpurea L Iraqi J Pharm Sci, Vol.19(1) 2010 In Vitro Digitonin production in Digitalis plant 31 In Vitro Effect of Cholesterol and Different Sugars on Digitonin Production in Multiplied Shoots of Digitalis purpurea L. Plant Zainab J.Awad *,1 * Department of Pharmacognosy ,College of Pharmacy, University of Baghdad ,Baghdad, Iraq. Abstract Production of the steroidal saponin digitonin in multiplied shoots of Digitalis purpurea , (var. Excelsior Mixed) has been achieved in vitro by two experiments. In the experiment 1, shoot tips ( 1cm length ) explants from the sterilized seedlings were excised and cultured on MS medium ( Murashige and Skoog medium) supplemented with 0.5 mg/L TDZ (thidiazuron) and cholesterol at the concentrations 0.0, 0.1, 0.3, 0.5, 1.0, 1.5, 2.0 or 4.0 mg/L. After 45 day, results showed that the treatment with 0.5 mg/L TDZ and 2.0mg/L cholesterol had a positive effected on increasing the dry weight of multiplied shoots and their production of digitonin when compared with other treatments, where this treatment gave 2.98 g dry weight of multiplied shoots and digitonin at amount of 64.42 g/g dry weight, while the other studied characteristics of multiplied shoots (content the total chlorophyll, soluble sugars and starch.) also this treatment had appositive effected and was gave the following values:- (3.51 mg/g fresh weight, 5.06% ,6.09% ) respectively. After that experiment 2 was carried out . The objective of this experiment was to increase the degree of digitonin production in multiplied shoots compared with the experiment 1.Therfore in the experiment 2, we were selected supplements the best treatment of experiment 1(0.5mg /L TDZ and 2.0mg/ L cholesterol ) and supplemented to the MS medium with the sugars glucose, fructose, sucrose or maltose at the concentrations 30,50,70 or 90 g/L for each sugar. After 45 days results showed that the treatment with 0.5 mg/L TDZ, 2.0 mg/L cholesterol and 50g/L maltose was the best a compared with other treatments, where this treatment gave 4.52 g dry weight of multiplied shoots and digitonin at amount of 191.87 g/g dry weight . The content of multiplied shoots from the total chlorophyll, soluble sugars and starch also this treatment gave highest values and they are 4.97 g/g fresh weight , 5.91% and 8.30% respectively . Key words: Digitalis purpurea, Cholesterol, Sugars, Digitonin . الخالصة Digitalis purpuseaفٓ االفسع اىمخضاعفت ىىباث اىدٔضٕخاىس (Digitonin)اوخاس اىسٕخسَئٕد اىصابُوٓ اىدٔضٕخُوٕه سم 1( حم اوضاشي خازس اىضسم اىحٓ بُاسطت حضسبخٕه فٓ اىخضسبت األَىّ، فُصيج اطساف االفسع بطُه Excelsior Mixed)ضسب ميغم/ىخس َاىنُىسخسَه باىخسامٕص 5.0بخسمٕص TDZ)مساشٕل َسنُك( مضافاً اىًٕ MSَشزعج عيّ َسظ مه اىبادزاث اىُمعقمت TDZميغم/ىخس 5.0ُٔماً أظٍسث اىىخائش بان اىمعاميت اىمخاىفت مه 00ميغم/ىخس، بعد 0.5اَ 0.5، 1.0، 1.5، 5.0، 5.0، 5.1، 5.5 ٓ عيّ شٔادة اىُشن اىضاف ىألفسع اىمخضاعفت َمحخُاٌا مه اىدٔضٕخُوٕه مقازوتً مع ميغم/ىخس مُىسخسَه ماوج ذاث حأرٕس أضاب 0.5مع مأنسَغم/غم َشن 20.00غم محخُٔت عيّ اىدٔضٕخُوٕه بمقداز 2..0اىمعامالث االخسِ، حٕذ بيغ اىُشن اىضاف ىألفسع اىمخضاعفت اىنيُزَفٕو اىنيٓ، اىسنسٔاث اىرائبت َاىىشاء، فقد ماوج صاف. اما اىصفاث االخسِ اىخٓ ُدزسج ىالفسع اىمخضاعفت ٌَٓ محخُاٌا مه %، 0.52ميغم/غم َشن طسْ , 0.01) -أفضو اىقٕم ٌَٓ : حٕذ أعطج ذاث حأرٕس إٔضابٓ عيّ ٌري اىصفاث أٔضاٌري اىمعاميت إوخاس اىدٔضٕخُوٕه فٓ األفسع %( َعيّ اىخُاىٓ. بعد ذىل أصسٔج اىخضسبت اىزاوٕت َاىٍدف مىٍا ٌُ إمناوٕت إحداد شٔادة أمزس فٓ .2.5 ميغم /ىخس 5.0اضافاث أفضو معاميت فٓ اىخضسبت األَىّ ٌَٓ MSاىمخضاعفت مقازوت مع اىخضسبت االَىّ، ىرىل اضٕف اىّ َسظ TDZ َ0.5 غم/ىخس ىنو 5.اَ 05، 05، 05ميغم/ىخس مُىسخسَه مع اىسنسٔاث سنسَش، ميُمُش، فسمخُش اَ ماىخُش َباىخسامٕص غم/ىخس 05ميغم/ىخس مُىسخسَه مع TDZ ،0.5ميغم/ىخس 5.0ُٔماً، اظٍسث اىىخائش بان اىمعاميت اىمخاىفت مه 00عيّ حدي. بعد ُسنس 1.1.20غم محخُٔت عيّ مادة اىدٔضٕخُوٕه بمقداز 0.00ماىخُش ماوج ٌٓ األفضو، حٕذ بيغ اىُشن اىضاف ىألفسع اىمخضاعفت ألفسع اىمخضاعفت مه مادة اىنيُزفٕو، اىسنسٔاث اىرائبت َاىىشاء فقد أعطج ٌري اىمعاميت أضاً مأنسَغم/غم َشن صاف، أما محخُِ ا %( عيّ اىخُاىٓ.2.05%، 1..0ميغم/غم َشن طسْ، 0..0أفضو اىقٕم ٌَٓ: ) Introduction Digitalis purpurea ( Fam: Scrophularaceae ) is a biennial herb plant (1) . In the medicinal and pharmacy fields this plant has a great benefit because it contains the cardiac glycosides like" Digitoxin" and steroidal saponin glycosides like" Digitonin" both of these groups representing compounds of considerable importance to the pharmaceutical industry (2) . As yet, these compounds can not be synthesized economically by chemical or microbiological methods (3) . 1Corresponding author E- mail : merciful2003@ yahoo.com Received : 24/3/2009 Accepted : 9/1/2010 Iraqi J Pharm Sci, Vol.19(1) 2010 In Vitro Digitonin production in Digitalis plant 32 In the field of pharmacy and medicine, the steroidal saponin digitonin had a great importance because it was used to determine the ratio of cholesterol in the blood, plasma and bile acids but the great importance and interest of this compound and some steroidal saponins like the Diosgenin owing to their relation ship with some important substances as progesteron hormone, cortisone and vitamin D where the structures of these steroidal saponins have the same nucleus of these substances , then these steroidal saponins can be used as a precursors for the production of these substances . Originally these substances were isolated from expensive and limit sources like adrenal cortex and bile acids of cattle (4 ,5) For this important many studies using modern technology like tissue culture technique ( in vitro) to increasing the production of steroidal saponins in some plants . Where via the usage of this technology different explants could be cultured and inducted to different stage of growths as a source for these important medical compounds and in a high concentrations without being restricted to the environmental conditions as well as cleans of the pharmaco which could be obtained (6 ) .Therefore the objective of our study using this technique to inducted the multiplication shoots of Digitalis purpurea (var exlecior mixed) and increasing their production from the digitonin compound . Material and method First :- Tissue culture of the plant Digitalis purpurea; this work went through the following steps:- 1. Preparation of sterilized seedlings : That was, initiation a specific culture of seedlings free from any contamination which it will be a source for shoot tips and that was performed by:- A. Medium preparation : Basic medium composition was that of MS (7) with 30g/L sucrose. The pH of this medium was adjusted to 5.5 then gelled with 8g/L Difco Bacto agar . Ten milliliters of this medium was dispensed into 25x125 ml tubes. After capped with clear autoclavable lids, tubes were autoclaved at 121 0 C for 20 min . B. Seeds germination : Seeds of the plant Digitalis purpurea ( var exlecior mixed) were surface sterilized in 70% ethyl alcohol for 30 second. This was followed with three sterilize distilled rinses ( 8) , after that the seeds were cultured on MS medium , and allowed to germinate by incubated in growth room at 25  2 C with artificial illumination given by fluorescent light of about 1600 lux at 16 hour / day ( 9) . Incubation was for30 days and then the seedlings are ready to take the explants shoot tips which excised and cultured in step 2. 2. Shoots multiplication and the production of steroidal saponin "Digitonin": These processes were inducted by two experiments:- Experiment 1 This experiment was carried out to inducted multiplication the cultured shoot tips and their digitonin production and by the following steps:- 1. Preparation of the nutrient medium:- Basic medium MS was used supplemented with 30g/L sucrose plus 0.5 mg/L TDZ (10) (for multiply the cultured shoot tip) and Cholesterol at a concentrations of 0.0, 0.1, 0.3 , 0.5, 1.0, 1.5, 2.0 or 4.0mg/L ( for induct the production of digitonin ). The pH of the media were adjusted to 5.5 and gelled 8g/L Difco Bacto agar. Twenty five milliliter medium was dispensed into 25x125 ml tubes. After capped with clear autoclavable, tubes were autoclaved at 121 0 C for 25 min . 2. Cultured the shoot tip:- Excised the shoot tips ( 1cm length ) from the sterilized seedlings and one shoot tip in every tube was cultured, then allowed to multiplication by incubated in a growth room at the same condition of seeds germination ( 11) . Experiment 2 This experiment was carried out after experiment 1 .The objective of this experiment was to increasing the degree of digitonin production in multiplied shoots compared with the experiment 1. Therefore we were selected supplements the best treatment of experiment 1 (0.5 mg/L TDZ and 2.0 mg/L cholesterol) and supplemented to MS medium with the sugars glucose, fructose, sucrose or maltose at a concentrations of 30,50,70 or 90 g/L for each sugar , the pH of these media and their gelled , distribution, autoclaving , cultured the shoot tip and condition the incubated in a growth room as in the experiment 1. Second :- Analysis of the multiplied shoots . After 45 days in cultures, multiplied shoots are ready for studying the following characteristics:- Iraqi J Pharm Sci, Vol.19(1) 2010 In Vitro Digitonin production in Digitalis plant 33 1. Total chlorophyll content:- This characteristic was estimated according to goodwing method (12 ) . 2. Dry weight estimation:- The multiplied shoots were taken out of the tubs and washed in water to remove agar from them, after wiped with clean cloth ,the shoots were separated and differentiated on filter paper to be dried in an oven at 40C until they reached constant weight (13 ) , later, shoots were grinded to hard powder and kept in paper sacks in dry condition and dark place . 3. Soluble sugars and starch content:- These characteristics were estimated according to joslyn method (14 ) . 4. Extraction of the steroidal saponin "digitonin" according to the procedure of Brain et al (15 ) 5. HPLC analysis of digitonin:-This analysis was carried out by using ( 16) C 18 – column and the mobile phase used was acetonitril: water (25:75). The flow rate was 1.5 ml/min .The measurements were carried out , using uv detector at 205 nm. Statistical analyses in our study were made by using complete randomized design. The mean differences were tested utilizing LSD test. ( 17) Results Multiplication of cultured shoot tip and yielded the highest number of shoots in the experiment 1 and 2 could be inducted by supplemented 0.5 mg/L TDZ to MS medium . In experiment 1, supplemented different concentrations of cholesterol to MS medium with o.5 mg/L TDZ , showed positive effect on digitonin production in multiplied shoots and other studied characteristics for these shoots ( total chlorophyll content, soluble sugar, starch and dry weight ) as show in table (1). But a significant effect was observed with the concentration 2.0 mg/L cholesterol and 0.5 mg/L TDZ. Therefore we were selected this treatment and was used in the experiment 2 with different sugars . In this experiment , all concentrations of maltose sugar had signification effect on digitonin production, but the best concentration was 50 g/L which had more effect on the production of this compound and other studied characteristics of multiplied shoots as compared with other treatments of this experiment (Table 2 ,3 ,4 and 5 ). Table 1 : Effect of different concentrations of Cholesterol. with the presence of 0.5 mg/L TDZ on the content of multiplied shoots of Digitalis purpurea from the total chlorophyll , soluble sugars , starch , digitonin and on their dry weight. Concentration mg/L Total Chlorophyll mg/g fresh weight Soluble Sugars (%) Starch(%) Digitonin g/g dry weight Dry weight (g) 0.0 3.31 4.36 5.11 33.13 2.11 0.1 3.39 4.44 5.14 33.60 2.09 0.3 3.33 4.42 5.31 35.71 2.13 0.5 3.47 4.54 5.24 37.56 2.36 1.0 3.49 4.61 5.66 43.49 2.41 1.5 3.47 4.88 5.84 49.46 2.71 2.0 3.51 5.06 6.09 64.42 2.98 4.0 3.48 4.41 5.86 44.40 2.31 L.S.D.5% 0.05 0.21 0.17 6.19 0.23 Table 2 : Effect of different concentrations of sugars with the presence of 0.5mg/L TDZ and 2mg/L Cholesterol on total chlorophyll content of multiplied shoots of Digitalis purpurea . Total Chlorophyll mg/g fresh weight. Concentration g/L Sugars Maltose Fructose Glucose Sucrose 4.69 3.18 3.41 3.51 30 4.97 3.85 3.51 3.90 50 4.31 3.84 3.56 3.96 70 3.60 3.61 3.15 3.42 90 0.23 L.S.D 5% Iraqi J Pharm Sci, Vol.19(1) 2010 In Vitro Digitonin production in Digitalis plant 34 Table 3 :- Effect of different concentration of sugars with the presence of 0.5 mg/L TDZ and 2mg/L Cholesterol on soluble sugars and starch content of multiplied shoots of Digitalis purpurea . Table 4 :- Effect of different concentrations of sugars with the presence of 0.5 mg/L TDZ and 2mg/L Cholesterol on digitonin content of multiplied shoots of Digitalis purpurea . Table 5 :- Effect of different concentrations of the sugars with the presence of 0.5 mg/L TDZ and 2mg/L Cholesterol on the dry weight of multiplied shoots of Digitalis purpurea . Dry weight (g) Concentration g/L Sugars Maltose Fructose Glucose Sucrose 3.14 2.28 2.42 2.96 30 4.52 2.91 3.61 3.18 50 4.03 2.71 3.72 3.51 70 3.90 2.51 2.31 2.40 90 0.25 L.S.D 5% Discussion In the field of tissue culture, shoots cultures (multiplied shoots) of Digitalis plant was considered one of the main sources to obtain some medicinal compounds like the cardiac and saponin glycosides (18) because the site of biosynthesis of these medicinal compounds is the shoots of this plant (19) for this reason , in the present study we were inducted multiplication the cultured shoot tip and formation the shoots cultures by supplemented the growth regulator TDZ ( cytokinin – like ) to MS medium at a concentration 0.5 mg/L ( 10) , and because the strong effects of this growth regulator (20) , shoot cultures in our study were appeared as leaf masses consisted from a high number of small and dwarf shoots as shown in figure (1). Soluble Sugars (%) Concentration g/L Sugars Maltose Fructose Glucose Sucrose 5.69 4.59 4.78 5.06 30 5.91 4.55 4.81 5.31 50 5.49 4.38 4.20 5.54 70 4.44 3.33 3.08 4.01 90 0.30 L.S.D 5% Starch(%) Concentration g/L Sugars Maltose Fructose Glucose Sucrose 7.91 5.74 5.79 6.09 30 8.30 5.82 5.87 6.81 50 7.30 5.50 5.55 6.76 70 6.12 4.49 5.01 5.65 90 0.41 L.S.D 5% Digitonin g/g dry weight. Concentration g/L Sugars Maltose Fructose Glucose Sucrose 113.11 42.13 51.01 64.42 30 191.87 83.78 68.18 98.93 50 171.22 81.91 73.28 108.55 70 165.61 51.67 67.33 61.72 90 17.35 L.S.D 5% Iraqi J Pharm Sci, Vol.19(1) 2010 In Vitro Digitonin production in Digitalis plant 35 Figure 1 : Multiplication shoot tip of Digitalis purpurea (Var. excelsior Mixed) in the treatment 0.5 mg/L TDZ and 2.0 mg/L cholesterol after 45 days. So it was difficult to distinguish the stem tips of these shoots then it was not possible to count their number and arrange the statistical analysis for them.Total chlorophyll content, soluble sugars and starch of the shoot cultures was studied as an indicator to degree of the changes in quantity of digitonin in these shoots, where if the content of chlorophyll was high this mean the green plastids have high degree of differentiation and that reflected positively on the ability of green plastids to provide some necessary enzymes to produce these compounds (21, 22) . As for the soluble sugars and starch , these compounds are the main source for energy and carbon element which are necessary to formation the great carbon structure of steroidal compounds (23) . Thus the rich places of chlorophyll , sugars and starch will be also rich in these medicinal compounds.In experiment 1, Presence of the cholesterol with 0.5 mg/ TDZ in MS medium had effected on increasing the internal level of digitonin inside the tissue of multiplied shoots especially in the concentration 2.0 mg/L , because this material was considered as a precursor to form the digitogenin ( aglycon part of digitonin ) (24) as shown in figure (2). Also presence the cholesterol in MS medium was led to increasing the content of multiplied shoots from the total chlorophyll, soluble sugars and starch. This increasing may be due to analysis this precursor and supply of some elements for these shoots like the carbon element which is necessary to formation the chlorophyll , soluble sugars and starch (25) and reflected that positively on shoots production from the digitonin. All of these increases reflected positively on increasing the dry weight of these shoots ecpically in the concentration 2.0 mg/L . Therefore we were selected this concentration with 0.5 mg/ L TDZ and used in the experiment 2 with different sugars. In the experiment 2, the treatment 50 g /L maltose with 2.0 mg/ L cholesterol and 0.5 mg/L TDZ significantly increased the digitonin , total chlorophyll, soluble sugars and starch in multiplied shoots and their dry weight as compared with other treatments of this experiment . The reason may be due, the maltose sugar is disaccharide and consist of two molecule of glucose . Therefore presence of this sugar in MS medium was led to increasing the number of glucose molecules inside the tissue of these shoots and that reflected positively on different bioprocesses of these shoots like: 1- increasing the supply of sugar part (glucose) which is necessary to building the digitonin where this compound consist from 2 molecules of glucose, 2 molecules of galctose and 1 molecule of xylose (25 ) . 2 – increasing the supply of energy which is necessary to growth and development of the shoots ( 23) , 3- increasing the supply of carbon element which is necessary to building the great carbon structure of chlorophyll, different sugars, starch and digitonin (5,25 ) . Iraqi J Pharm Sci, Vol.19(1) 2010 In Vitro Digitonin production in Digitalis plant 36 digitogenin Figure 2 : The biosynthesis of steroidal sapogenin digitogenin (24) Iraqi J Pharm Sci, Vol.19(1) 2010 In Vitro Digitonin production in Digitalis plant 37 References 1. 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