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Romanian Biotechnological Letters Vol.17, No.3, 2012 Copyright © 2012 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

6992 Romanian Biotechnological Letters, Vol. 17012 7340

Detection of elicitation effect on Hyoscyamus niger L. root cultures for the root growth and production of tropane alkaloids

Received for publication, March 7 2011

Accepted, July 9, 2012

MARVIN LOKE KAH HONG, ARVIND BHATT, NING SHU PING* AND CHAN LAI KENG School of Biological Sciences, University Sains Malaysia, 11800 Penang, Malaysia Corresponding author* Dr. Ning Shu Ping, Plant Tissue and Cell Culture Laboratory, School of Biological Sciences Universiti Sains Malaysia, 11800 Penang, Malaysia Tel: 604-6534629, Fax: 604-6565125, E-mail: [email protected]; [email protected]

Abstract

Hyoscyamus niger is an important medicinal plant and reported to be used for the treatment of various ailments. The present study tested the effect of different elicitor such as chitosan, casein hydrolysate, yeast extract and d-sorbitol on cell biomass, alkaloid production and root growth and proliferation in H. niger cell. Different concentration of chitosan, casein hydrolysate, yeast extract and d-sorbitol were added as elicitor to optimize cell biomass and alkaloid production. Addition of chitosan as elicitor in modified MS medium supplemented with 0.5 mg l–1 IBA suppressed the root growth and biomass production of H. niger, whereas addition of casein hydrolysate and d-sorbitol also did not show any significant increment in root production and accumulation of scopolamine and hyoscyamine. Addition of yeast extract in the root cultures of H. niger did not enhance root growth and hyoscyamine production but the addition of 0.5 g l–1 and 1.0 g l–1 yeast extracts enhanced the scopolamine production. As a whole, the results showed that MS medium supplemented with 0.5 mg l–1 IBA could be used for root culture of H. niger and elicitation was not necessary for the production of tropane alkaloids (scopolamine and hyoscyamine). The best root growth and proliferation was observed in 100 ml flask with maximum fresh biomass after three weeks of culture.

Keywords: Hyoseyamus niger, elicitor, tropane alkaloids, root culture, Solanaceae Introduction

Hyoscyamus niger L. (Family Solanaceae), commonly known as black henbane, is an important temperate medicinal plant. It is used for the treatment of various ailments such as ear and eye inflammation, rheumatism, treatment of ulcers, cough, motion sickness, asthma, rabies, fevers, bronchitis, renal colic, and spasms /1-2/. Two important tropane alkaloids, hyoscyamine and scopolamine have been detected from H. niger and showed strong potentials to use for either acute or chronic toxicity and acute urinary retention as well /3-4/.

Generally, secondary metabolites which are not essential to plant growth are produced in small quantity. However, these compounds are normally needed for natural defense. As the consumption of herbal medicines continues to increase, the dependency on wild plants to provide the active compounds is becoming more serious. Hence, plant in vitro culture techniques has been viewed as a major tool for the production of secondary metabolites. Production of tropane alkaloids via root culture has been reported in several other species of family Solanaceae such as Hyoscyamus albus and H. muticus /5/; Brugmansia candida /6-8/; Schizanthus species /9/. The research work of Lai /10/ also chronologically proved that tropane alkaloids could be obtained via micropropagated plantlets and the normal root cultures of H. niger.

Accumulation of secondary metabolites can be triggered by the application of elicitors to the culture medium. However, until now there is no detailed study available on the effect of


Romanian Biotechnological Letters, Vol. 17, No. 3, 2012 7341

elicitors on production of tropane alkaloids in untransformed root culture of H. niger. Therefore, the present study reports the elicitation effects on production of tropane alkaloids (hyoscyamine and scopolamine) from H. niger root cultures. Materials and methods

Plant materials and culture condition The petioles explants of 6 weeks old in vitro seedlings of H. niger were used for root

induction on MS supplemented with 2.0 mg l–1 IBA. The induced roots were subsequently multiplied in root proliferation medium, MS supplemented with 0.5 mg l–1 IBA in total darkness. The three weeks old root cultures were used as the plant materials for elicitation study.

Elicitation effects on root cultures of H. niger Ten root pieces (1 cm) obtained from three weeks old root cultures were transferred into

100 ml Erlenmeyer flask containing 20 ml modified MS medium supplemented with 0.5 mg l–1 IBA and different concentration of chitosan (0, 50, 100, 200 and 300 mg l–1). The modified MS was made up of basic MS medium with modified macronutrients (15.0 mM ammonium nitrate, 33.1 mM potassium nitrate, 3.19 mM calcium chloride dihydrate, 1.9 mM magnesium sulphate heptahydrate and 1.4 mM potassium dihydrogen phosphate), 9.6 mM myo-inositol, 0.1 mM Fe-EDTA and 30 g l–1 sucrose.

The same procedure was repeated for casein hydrolysate (0, 0.5, 1.0, 2.0 and 3.0 g l–1), yeast extract (0, 0.5, 1.0, 2.0, 3.0 g l–1) and d-sorbitol (0, 5, 10, 15 and 20 g l–1). Six to eight replications were used for each elicitor treatment and each replication containing 10 root explants. Each elicitor effect was carried out separately using complete randomized block design. The fresh weight of roots was determined after three weeks of culture and the dried biomass was obtained after the culture was air-dried. The data were analyzed using one-way ANOVA followed by comparison of mean with Tukey’s HSD test at p≤0.05 to determine the best concentration of each studied elicitor for optimum root biomass production.

Effect of flask size on root proliferation of H. niger Four different flask sizes (100, 250, 500 and1000 ml) were used to study the effect of

flask size on root proliferation of H. niger. The study was carried out using complete randomized block design with six replications for each flask size. The root explants were cultured in modified MS medium supplemented with 0.5 mg l–1 IBA. The medium volume used was 1:5 of the flask volume. Ten root pieces (1.0 cm) were used for every 20 ml liquid medium. The cultures were continuously agitated at 120 rpm on rotary shaker under total darkness in culture room with temperature of 25 ± 2°C. The fresh weight of roots was determined after three weeks of culture and the dried weight was obtained after constant weight was achieved with air-drying.

The data were analyzed using one-way ANOVA followed by comparison of mean with Tukey Test at p≤0.05 to determine the best flask size for optimum production of root biomass.

Detection of Hyoscyamine and Scopolamine in H. niger root cultures Preparation of Hyoscyamine and Scopolamine standard curve Hyoscyamine and Scopolamine (Sigma Chemical Co.) were used as standard. The

standard was prepared by weighing 20.0 mg and dissolved in 10 ml methanol. Different concentrations ranging from 5 mg l–1 to 250 mg l–1 were prepared for each standard solution. One (1.0) μl of the standard injected into the GC-FID column. A gas chromatograph, Model

MARVIN LOKE KAH HONG, ARVIND BHATT, NING SHU PING, CHAN LAI KENG

7342Romanian Biotechnological Letter12 Romanian Biotechnological Letters, Vol. 17, No. 3, 2012

HP 5890 GC (Palo Alto, CA, USA) equipped with HP-5 Column (30 m X 0.25 mm ∅) and a flame ionization detector were used. Flow rate of carrier gas (nitrogen) of 0.8 ml/min and split ratio of 1:100 was maintained. The initial column temperature was 150˚C before it was increased at a rate of 6˚C /min until 270˚C. The FID detector and injector temperature was each maintained at 300˚C and 280˚C respectively /11/.

Chemical analysis using Gas Chromatography (GC-FID) Extraction of tropane alkaloids (hyoscyamine and scopolamine) and sample preparation

were done according to Berkov et al. /12/. The different sample materials used for determination of tropane alkaloids content were (i) roots cultured in basic MS medium and modified MS medium under total darkness and (ii) roots from modified MS medium elicited with chitosan, casein hydrolysate, yeast extract and d-sorbitol.

The dried samples were macerated into powder using a mortar and pestle. The powdered samples of 0.3 g each was soaked overnight in 25 ml liquid mixture of chloroform-methanol-concentrated ammonium hydroxide (15:5:1), wrapped with aluminum foil to prevent exposure to light. They were then placed in sonicator bath for 10 minutes at 27 ± 2˚C before filtered through Whatman® filter paper No. 5. The filtrates were later added with 1.0g anhydrous Na2SO4 for drying. The extracts were then dried at 50˚C under pressure using a rotary evaporator (Eyela Rotary Vacuum Evaporator N-N Series and Eyela Digital Waterbath SB-651, Tokyo Rikikai Co. Ltd.) coupled with a water pump (Eyela Aspirator A-3S, Tokyo Rikikai Co. Ltd.). The dried residue was dissolved in 1 μl of methanol.

The sample solutions were analyzed by Gas Chromatography-Flame Ionization Detector (GC-FID). The condition used was the same with the establishment of hyoscyamine and scopolamine standard curves. For each sample, three replicates were used. Based on the width area of the respective chromatogram peaks obtained from each sample, the hyoscyamine and scopolamine content in the samples were determined from the calibration curves. Results and discussion

Both biotic and abiotic elicitors may enhance the secondary metabolite production in plant cell cultures. But no elicitor has been reported to have a general effect on many culture systems and no one system has been found to respond to all elicitors /13/. Therefore, screening of various elicitors and the concentration for a particular elicitor for enhancement and production of a desired compound is crucial and important.

Elicitation effects on root cultures of H. niger Results obtained from the present study indicated that the addition of chitosan as elicitor

in modified MS medium supplemented with 0.5 mg l–1 IBA suppressed the root growth and biomass production of H. niger. The control set of culture (without chitosan) induced the highest root biomass production (1.60 ± 0.07g fresh weight and 0.14 ± 0.00g dried weight in 20 ml medium) (Fig. 1). However, increasing concentration of chitosan (50 mg l–1 to 300 mg l–1) in the culture medium significantly reduced the root biomass production. Chitosan which is believed to act as an elicitor for other plant species only induced necrosis and suppressed root growth and biomass production in H. niger. Similarly the presence of chitosan inhibited the root growth in hairy root cultures of Ocimum basilicum /14/, Polygonum tinctorium /15/ and Panax ginseng /16/. However, addition of chitosan showed the positive effect in hairy roots of Hyoscyamus muticus /17/. In Azadirachta indica cell suspension culture, lower concentration (50 mg l–1) of chitosan did not inhibit biomass production but induce the azadirachtin accumulation, whereas higher concentration of chitosan (100-500 mg l–1) reduced the biomass production gradually /18/. This indicated that chitosan responds differently in different plant cultures and elicitation of chitosan on cell lines of a particular



plant species might result in an increase or decrease in cell growth or secondary metabolite content /17/.

Fig. 1. Effect of chitosan on production of root biomass of H. niger. (The mean values for each parameter followed by different alphabet were significantly different using Tukey test (p≤0.05).

Casein hydrolysate did not show any significant effect on root production and accumulation of scopolamine and hyoscyamine in H. niger root cultures. The control culture (without casein hydrolysate) induced maximum root biomass (2.50 ± 0.05 g fresh weight and 0.20 ± 0.00 g dried weight) after three weeks of culture. Addition of 0.5 g/L casein hydrolysate into the culture medium reduced the biomass production (2.37 ± 0.13 g fresh weight and 0.20 ± 0.01 g dried weight) (Fig. 2). Further increment in casein hydrolysate concentration (1.0 g l–1 to 3.0 g l–1) showed a gradual decreasing trend in biomass production. Ghosh et al. /19/ also reported that addition of casein hydrolysate in root culture of Gloriosa superba did not enhance the growth and colchicine content. Addition of casein hydrolysate in cell culture of Panax ginseng was found to be effective for saponin production but it did not increase the biomass /20/. However, casein hydrolysate improved the growth of Cardamine pratensis and Silene alba cell suspension cultures, only when the medium contained insufficient amount of phosphorus /21/. Casein hydrolysate could be a source of organic nitrogen, calcium, phosphate, several microelements, vitamins and mixture of different amino acids. Nonetheless, in plant tissue cultures, casein hydrolysate and amino acids are normally added when there is reduced nitrogen content. It is not needed when the medium contain adequate amounts of ammonium, nitrate and calcium ions like in MS medium /21/. The root proliferation medium in this study contained the optimized amount of ammonium, nitrate and calcium ions hence casein hydrolysate is not necessary for enhancing the biomass and tropane alkaloid production in H. niger.



Fig. 2. Effect of casein hydrolysate on production of root biomass of H. niger. (The mean values followed by different alphabet for each parameter were significantly different using Tukey test (p≤0.05)).

The addition of yeast extract in the root cultures of H. niger did not increase the root

growth and hyoscyamine production but the addition of 0.5 g l–1 and 1.0 g l–1 yeast extracts enhanced the scopolamine production. All the roots produced were normal and healthy in culture medium with or without yeast extract. There was no significant different in root production in culture medium supplemented with different amount of yeast extract. However, further increment of yeast extracts into the culture medium gradually reduced the biomass production in H. niger roots (Fig. 3). Other studies also found that yeast extract did not affect the biomass production but increased the secondary metabolite production of some plant species such as Brugmansia candida /7/, Coleus forskohlii /22/, Gloriosa superba /19/ and Artemisia annua /23/. In present study, the yeast extract did not favor root growth and hyoscyamine production but did enhance scopolamine production. The elicitation effect on scopolamine production could be attributed to the possible contribution of some cations like Zn, Ca and Co which act as abiotic elicitors /24/. In addition, yeast extract is consisted with various compounds other than amino acids, vitamins, and minerals. Hence, similarly like casein hydrolysate, it can enhance tissue growth in a culture medium containing relatively low amount of vitamins or nitrogen but its exact effect on plants has yet to be determined /25/.



Fig. 3. Effect of yeast extract on production of root biomass of H. niger. (The mean values followed by different alphabet for each parameter were significantly different using Tukey test (p≤0.05)).

Results revealed that increasing concentration of d-sorbitol into the culture medium caused gradual reduction in biomass production (Fig. 4).

Fig. 4. Effect of d-sorbitol on production of root biomass of H. niger. (The mean values followed by different alphabet for each parameter were significantly different using Tukey test (p≤0.05).

The addition of different concentration of d-sorbitol into H. niger root culture did not

showed any effect on root production and accumulation of scopolamine and hyoscyamine. D-



sorbitol was used widely as osmoticum in plant cell cultures to induce osmotic pressure and to have positive effects on the production of root biomass and tropane alkaloids when the sucrose content or other carbon sources are insufficient in the culture /26-27/. In the present study, the proliferation medium contained the optimized amount of sucrose, which was a more favorable carbon source than sorbitol had also taken up the role to initiate osmotic pressure and carbon role /26/. Therefore, the osmotic pressure in medium induced by the d-sorbitol was not needed to elicitate H. niger normal root culture for biomass and tropane alkaloids production.

Detection of Hyoscyamine and Scopolamine in H. niger root cultures In the study, both the tropane alkaloids (hyoscyamine and scopolamine) were successfully

detected from the studied samples using GC-FID. Results obtained revealed that hyoscyamine was detected at retention time 7.221 minutes (Fig. 5a) and scopolamines at 7.951 minutes (Fig. 5b). While blank sample (methanol), was detected at 1.322 minute (Fig. 5c).

Fig. 5a. Gas chromatogram of Hyoscyamine standard at retention time of 7.221 minute.

H. niger roots cultured in basic MS medium supplemented with 0.5 mg l–1 IBA

contained more hyoscyamine as compared to those cultured in modified MS medium supplemented with 0.5 mg l–1 IBA. Basic MS medium supplemented with 0.5 mg/L IBA enhanced the hyoscyamine production (31.29 ± 1.43 mg/g) which was significantly higher than modified MS medium supplemented with 0.5 mg l–1 IBA (23.65 ± 1.86 mg/g). However there was no significant difference in scopolamine content and root biomass production in both basic MS medium supplemented with 0.5 mg l–1 IBA and modified MS medium supplemented with 0.5 mg l–1 IBA (Table 1).

Table 1. Effect of culture medium on hyoscyamine and scopolamine production in H. niger root culture



Medium Fresh root

biomass

(g)±s.e.

Hyoscyamine

content (mg/g) ±

s.e.

Scopolamine

content (mg/g) ±

s.e.

hyoscyamine (%)

scopolamine (%)

Basic MS medium

+

0.5 mg l–1 IBA

1.06 ± 0.06 a 31.29 ± 1.43 a 39.71 ± 3.05 a 0.26 0.33

Optimized MS

medium + 0.5 mg

l–1 IBA

1.06 ± 0.06 a 23.65 ± 1.86 b 35.93 ± 0.55 a 0.20 0.30

Means within the same column followed by the same alphabet were not significantly different using student t-test (p≤0.05).

Fig. 5b. Chromatogram of Scopolamine standard at a retention time of 7.951 minute.

Fig. 5c. Gas chromatogram of the blank sample (methanol).

Addition of casein hydrolysate in the culture medium as an elicitor did not showed any

effect on hyoscyamine and scopolamine accumulation. The culture medium (without casein hydrolysate) induced the highest amount of hyoscyamine (16.36 ± 0.38 mg/g) and



scopolamine (35.06±3.07 mg/g) from the H. niger root cultures. However, increasing amount of casein hydrolysate into the culture medium reduced the accumulation of hyoscyamine and scopolamine (Table 2).

Table 2. Effect of casein hydrolysate elicitation on hyoscyamine and scopolamine production in H. niger root culture

Casein hydrolysate

Mean of fresh biomass

(g) ± s.e.

Hyoscyamine content (mg/g)

± s.e.

Scopolamine content (mg/g) ±

s.e.

Hyoscyamine (%)

Scopolamine (%)

Control

2.50 ± 0.05 a

16.36 ± 0.38 a

35.06 ± 3.07 a

0.14

0.29

5.0 g l–1

2.37 ± 0.13 a

11.07 ± 1.01 b

25.08 ± 2.52 ab

0.09

0.21

1.0 g l–1

1.92 ± 0.13 b

9.21 ± 0.73 bc

19.63 ± 0.86 b

0.08

0.16

2.0 g l–1

1.65 ± 0.04 b

5.50 ± 0.18 c

17.11 ± 0.69 b

0.05

0.14

3.0 g l–1

1.54 ± 0.08 b

5.46 ± 0.78 c

14.22 ± 0.35 b

0.05

0.12

Means within the same column followed by the same alphabet were not significantly different using Tukey test (p≤0.05).

Root cultures treated with yeast extract did not show higher accumulation of both

hyoscyamine and root biomass of H. niger as compared to the control. The range of hyoscyamine content was between 9.31 ± 1.19 mg/g to 13.26 ± 0.89 mg/g in the medium with or without yeast extract elicitation. However, addition of 0.5 g l–1 and 1.0 g l–1 yeast extract into the culture medium increased the scopolamine production of 30.40 ± 1.97 mg/g and 24.37 ± 0.43 mg/g respectively (Table 3).

Table 3. Effect of yeast extract elicitation on hyoscyamine and scopolamine production in H. niger root culture

Yeast extract Mean of

fresh biomass (g) ± s.e.

Hyoscyamine content (mg/g) ±

s.e.

Scopolamine content (mg/g) ±

s.e. Hyoscyamine

(%) Scopolamine

(%)

Control 2.62 ± 0.24 a 10.36 ± 0.75 a 18.98 ± 2.06 b 0.09 0.16

0.5 g l–1 2.63 ± 0.32 a 13.26 ± 0.89 a 30.40 ± 1.97 a 0.11 0.25

1.0 g l–1 2.42 ± 0.17 a 10.87 ± 0.16 a 24.37 ± 0.43 a 0.09 0.20

2.0 g l–1 2.30 ± 0.27 a 9.31 ± 1.19 a 16.05 ± 0.68 b 0.08 0.13

3.0 g l–1 2.03 ± 0.24 a 10.57 ± 1.94 a 16.71 ± 1.23 b 0.09 0.14




Similarly, addition of d-sorbitol also did not enhance the production of hyoscyamine and

scopolamine in H. niger root cultures. The culture medium without d-sorbitol produced 16.42 ± 0.40 mg/g hyoscyamine and 16.38 ± 1.25 mg/g scopolamine. Addition of 5.0 g l–1 d-sorbitol in the culture medium accumulated 20.04 ± 1.35 mg/g hyoscyamine and 22.75 ± 1.61 mg/g scopolamine. However, further increased in d-sorbitol concentrations caused reducing effect on hyoscyamine and scopolamine production (Table 4).

Table 4. Effect of d-sorbitol elicitation on hyoscyamine and scopolamine production in H. niger root culture.

d-sorbitol

Mean of fresh

biomass (g) ±

s.e.

Hyoscyamine

content (mg/g) ±

s.e.

Scopolamine

content (mg/g) ±

s.e.

Hyoscyamine

(%)

Scopolamine

(%)

Control 3.36 ± 0.26 a 16.42 ± 0.40 a 16.38 ± 1.25 a 0.14 0.14

5.0 g l–1 3.50 ± 0.20 a 20.04 ± 1.35 a 22.75 ± 1.61 a 0.17 0.19

10.0 g l–1 3.21 ± 0.25 a 18.92 ± 0.72 a 22.01 ± 0.26 a 0.16 0.18

15.0 g l–1 2.89 ± 0.15 a 18.96 ± 0.63 a 23.16 ± 2.14 a 0.16 0.19

20.0 g l–1 2.65 ± 0.17 a 16.98 ± 0.24 a 16.46 ± 1.84 a 0.14 0.14


Effect of flask size on root proliferation of H. niger Results indicated that the flask size of the shake flask system affected the root growth of

H. niger significantly. The best root growth and proliferation was observed in 100 ml flask with an increase of maximum fresh biomass (190.0 ± 1.2 mg / root explant) after three weeks of culture. While increasing flask size suppressed the root growth. However, flasks size did not affect the dried root biomass (Table 5). Min et al. /28/ also reported similar results in adventitious root culture of Scopolia parviflora. In liquid-phase culture like the shake flask system, oxygen deficiency was possible due to mass transport limitation /29-30/. As shake flasks size increased using different medium volumes in the system, the oxygen limitation problem also increased and would affect the in vitro growth and morphogenesis /31/. Lee et al. /3/ reported that aeration at 1 vvm in shake flask system increased tropane alkaloid and biomass production in Atropa belladonna root cultures. For successful cultivation of plant cell suspensions, the shake flask has to provide good mixing at low shear stress levels with a moderate oxygen supply. Large vessel sizes are more accommodating but it is impossible to recommend the best vessel sizes for any specific in vitro culture procedure without trial experimentation. Shake flask system did not prove to be a suitable growth system for up-scaling of H. niger root culture possibly resulted from the inadequate supply of nutrients and oxygen in big culture vessel. The best size and shape of vessel vary from one plant species to another and other factors like volume of medium and the density of inoculation should also be taken into account.



Table 5. Effect of flask size on H. niger root production

Flask size Fresh biomass increase per root explant

(mg) ± s.e. Dried biomass per root

explant (mg) ± s.e.

100 ml 190.0 ± 1.2 a 10.0 ± 0 a

250 ml 120.0 ± 0.4 b 10.0 ± 0 a

500 ml 80.0 ± 0.7 c 10.0 ± 0 a

1000 ml 80.0 ± 0.7 c 10.0 ± 0 a

Means within the same column followed by the same alphabet were not significantly different using Tukey test (p≤0.05). Conclusion

MS medium supplemented with 0.5 mg l–1 IBA could be used for root culture of H. niger

and elicitation was not necessary for the production of tropane alkaloids (scopolamine and hyoscyamine). Only the addition of low concentration of yeast extract (0.5 g l–1 and 1.0 g l–1) enhanced scopolamine production in the root cultures. Acknowledgement

We thank University Sains Malaysia fellowship scheme for financial support and School of Biological Sciences for research facilities.

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