ORIGINAL ARTICLE

Ecient in vitro elicitation of b-carboline alkaloidsin transformed r

Rawia Zayed *

Department of Pharmacognosy, FacDepartment of Pharmacognosy, Fac

R pted 2A 011

b-Carboline alkaloids;Hairy root culture and elici-

line alkaloids of Peganum harmala was established from P. harmala seedlings. Hairy root culture

was arised by inoculation of the excised seedling with Agrobacterium rhizogenes. Analysis of rolC

hydrogen peroxides (H O ), or biosynthetic precursor as tryptophane in order to stimulate the bio-

The results revealed that, treatment of the cultures with H2O2 (0.01%) induced the accumulation of

harmine, as the major b-carboline alkaloids in root, by approximately sixfold as compared to the

loid production approximately vefold (0.250.3 mg/g DW) within 24 h.

1. Introduction

and for treatment of skin disease.1 The smoke of the plant

has been used as a disinfectant.2 The alkaloids of P. harmala(harman alkaloids) have different and important pharmaco-logical actions. It is CNS stimulant and has hallucinogenic

activities at high doses.3 b-Carboline alkaloids affect a numberof molecular targets, ranging from DNA (intercalation) vianeuroreceptors to monoamine oxidase3 and therefore gureas potent defense compounds of P. harmala. DNA intercala-

tion, mutagenic, genotoxic and cytotoxic have been

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doi:10.1016/j.bfopcu.2011.07.002

Production and hosting by Elsevier

Bulletin of Faculty of Pharmacy, Cairo University (2011) 49, 711

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Open access under CC BY-NC-ND license.Peganum harmala plant has ancient reputations as antiseptic

E-mail address: rawiazayed@hotmail.com 2011 Faculty of Pharmacy, Cairo University. Production and hosting by Elsevier B.V.

* Corresponding author. Address: Department of Pharmacognosy,

Faculty of Pharmacy, Zagazig University, 44519 Zagazig, Egypt.

Open access under CC BY-NC-ND license.untreated control. The highest stimulation resulted in a harmine level of ca. 4.5 mg/g DW after

24 h of incubation. Concerning the biosynthetic precursor, tryptophane (2 mM) enhanced the alka-2 2

synthesis of b-carboline alkaloids, mainly harmine as the major one. Prole and amounts of b-carb-oline alkaloids were analyzed using capillary GLCMS.tation;

Peganum harmala

and virC genes by using polymerase chain reaction (PCR) has conrmed the transformation of

the root culture. Hairy root cultures were employed to study the production of harmine as the main

b-carboline alkaloids. The transformed cultures were incubated with potential elicitors, such aseceived 2 March 2011; accevailable online 26 August 2

KEYWORDS A

ooot cultures of Peganum harmala

ulty of Pharmacy, Sinai University, 44551 North Sinai, Egyptulty of Pharmacy, Zagazig University, 44519 Zagazig, Egypt

3 May 2011

bstract An efcient in vitro production of biomass hairy root cultures and elicitation of b-carb-niversity

rmacy, Cairo University

/locate/bfopcudirect.com

35

vated on antibiotic-free agar-medium. The best growthing

0 0

8 R. Zayedroots without Agrobacterium were transferred into hormone-

free WP liquid medium to which Claforan was added as antibi-otic (5 mg/10 ml medium). This procedure was repeated severaltimes until the Agrobacterium was completely eliminated. Then

the roots were subcultured every weak in fresh WP liquid med-ium without antibiotic for maintenance. HRC have a differentmorphology as compared to RC. They have many root hairs

and much more branches and the most overall importantadvantage of the transformed root cultures is their long-termstability. From the three tested strains of Agrobacterium there

was a success transformation only with 15834 strain.

2.1.2. Polymerase chain reaction (PCR)Total DNA was extracted from the hairy root cultures of P.harmala, non transformed root and from A. rhizogenes. PCR(thermal cycler; Biometra, TGradient) was employed to con-rm the transformation of the root cultures. PCR conditions:

96 C for 2 min, followed by 36 cycles of 94 C for 30 s, 60 Cfor 30 s, 72 C for 2 min, and nally 15 min extension at72 C. The primers for rolC DNA were 50-ATGGCTGAA-investigated. They suggested that the antibiotic and toxic ef-

fects of b-carboline could be a function of DNA intercalationand resulting mutations. These give strong indications for thevalidity of harmine as an in vivo tracer for the assessment ofMAO-A enzyme binding in the brain.6 Trypanosomicidal

activity of several b-carboline alkaloids7 and larvicidal againstthe larvae of the cotton leaf worm8 have stated that the biolog-ical activity of b-carboline alkaloids and a highly toxic effectreasonably underlie the neurotoxicity.9

Since P. harmala plant grows as wild and not cultivated anddue to the biological importance of carboline alkaloids of the

plant.10 Khawar et al. (2005)11 and Neha et al. (2009)12 havetried to micropropagate this species to facing the problem ofextinction. Most of P. harmala stable root cultures have been

established by transformation with A. rhizogenes13 to over-come on the difculties for production of long-stable root cul-tures as storage tissue. Hence, the present study was designedto develop an efcient in vitro procedure to enhance the bio-

synthesis of b-carboline alkaloids in biomass of transformedhairy root cultures of P. harmala by using potential elicitors.

2. Material and methods

2.1. Plant materials

2.1.1. Establishment of transformed root culturesMature seeds of P. harmala (maximum, 6 month old collectedfrom Southern Sinai, Egypt) were surface-sterilized in sodium

hypochlorite solution (1%, 15 min) then they were rinsedtwice with sterile distiled water. Afterwards the seeds weretreated with EtOH 70% for 5 min and rinsed twice with steriledistiled water. The seeds were left overnight in sterile water and

then incubated on agar plates until germination. The germi-nated seeds were transferred to hormone-free WP solid-medium14 to produce the seedlings. Hairy root cultures

(HRC) of P. harmala were initiated by infecting 4-weeks oldseedlings with A. rhizogenes strains 15834, TR 105 and LBA(kindly supplied by Prof. Dr. W. Alferman University of Dues-

seldorf, Germany). After 57 h the roots were surface-sterilizedwith 1% sodium hypochlorite solution for 1 min and subculti-GACGACCTGTGTT-3 ; and 5 -TTAGCCGATTGCAAA

CTTGCAC-3.15 The primers for virC DNA were 50-AT-CATTTG TAGCGACT-30; and 50-AGCTCAAACCTGvCTTC-30.16 Gelelectrophoresis of PCR products from trans-formed and non-transformed root cultures as well as from

A. rhizogenes was carried out to identify the presence orabsence of rolC and virC genes.

2.2. Chemicals

Solvents (Riedel-deHaen, SeelzeGermany):Hydrogenperoxide

(H2O2), dichloromethane, 70% ethanol, 1% sodium hypochlo-rite (NaClO), hydrochloric acid (HCl) and sodium hydroxide(NaOH) were obtained from Sigma-Aldrich-Chemie (Ger-

many). Agar (Bacto-Agar, Difco Laboratories USA) and gelrite(Carl Roth GmbH Karlsruhe, Germany). Isolute-Sorbent (Iso-lute HM-N, International Sorbent Technology Ltd. UK).

2.3. Cultures media

Hormone free WP liquid and solid media were used for opti-

mal growth and production for HRC of P. harmala. The pHof the medium was adjusted to 5.7 before autoclaving. Forsubculture we have used 50 ml medium in 200 ml asks. In

induction experiments 10 ml in 100 ml asks were employed.The YeastMannitol-Broth-medium (YMB) was used forgrowth and maintenance of A. rhizogenes at 39 C. It containsmannitol (10 g/l), yeast extract (0.4 g/l), NaCl (0.1 g/l),

MgSO47H2O (0.2 g/l), K2HPO4 (0.5 g/l) and agar (1%) atpH 7.2.

2.4. Induction experiment

HRC were treated by different inducers as alternative method

to induce the accumulation of the secondary metabolite inplants. Hydrogen peroxides (H2O2) and tryptophane (T) wereused as inducers. Under aseptic conditions (Laminar Flow,

Ceag Shirp- Reinraum technik, Germany), the inducers wereadded to 10 ml fresh medium in 100 ml Erlenmeyer asks.The cultures were incubated in a gyrator shaker 110 rpm at25 C in an illuminated culture room.

2.5. Analytical methods

After the incubation times with inducers, HRC was harvestedby Buchner vaccum ltration, dried, weighed and homoge-nized in mortar with 1 M HCl (20 ml). The homogenate was

left at room temperature overnight, then made alkaline withNH4OH (pH 10). Solid phase extraction on Isolute column(20 g) was carried out with dichloromethane (three times,20 ml each). The eluates were concentrated by rotary evapora-

tor, the residue was kept at 4 C until analysis. For analysis theresidue was taken up in MeOH and was injected into the gaschromatography. Medium samples were treated accordingly

but the initial 1 M HCl step was omitted . Using GLCMS,the alkaloids were identied. Harmine and harmaline wereused as external standards for quantitative interpretation.

2.6. GLCmass spectrum (GLCMS)

The GLCMS analysis were carried out on a Carlo ErbaHRGC 4160 gas chromatography equipped with a Fused silica

DB1 (30 m 0.25 mm i.d.) column. The capillary column was

alkaloid in HRC of P. harmala. The results showed that theaccumulation of harmine was an inducer-dose dependantand harmine biosynthesis was enhanced app. vefold

(Fig. 2). The maximum level was 0.250.3 mg/g DW harminewith 2 mM tryptophane.

These results with P. harmala are representing example forthe power of H2O2 to induce the formation of alkaloids as

defense system in the plant. Since the production of b-carbo-line alkaloids in in vitro cultures is of biotechnological impor-tance, the elicitation via elicitors offers a chance to improve

this yield.

4. Discussion

The rst important factor which switched our investigationwith P. harmala, was the production of long-stable hairy root

culture. Seeds from different sources were investigated toobtain good germinating seedlings and high producing cul-tures. This work results showed a variation in the germination

Efcient in vitro elicitation of b-carboline alkaloids in transformed root cultures of Peganum harmala 9directly coupled with a quadruple mass spectrometer, a Finn-igan MAT 4500 operating at 45 eV. Conditions: injector250 C, temperature program: 150300 C 15 C/min, 300 C;20. Split ratio 1:5; carrier gas helium, 0.5 bar (ow rate2 ml/min).

3. Results

3.1. Establishment of hairy root cultures

To establish a productive strain of root cultures of plants, itmust select the best producing strain and optimize the culture

conditions for growth and production of secondary com-pounds. Obtaining stable root cultures from P. harmala wasvery difcult. Different media have been tried (MS, MS med-

ium supplied with 2,4-D, WP and WP medium with 2,4-Dand kinetin) and different conditions (e.g. light and dark),but each time the root cultures easily broken. After a certaintime, RC acquired brownish-red color and more over the med-

ium. At the end of four growth cycles, growth of the culturesslowly stopped and they nally died. While looking for a suit-able medium for the root cultures, WP liquid and solid media

proved to be optimal for growth and maintenance of P. harmal-a cultures as well as for secondary metabolite accumulation.

HRC of P. harmala was established by infecting sterilized

growing seedlings with A. rhizogenes strain 15834. These weremaintained as stable root cultures in hormone-free WP liquidmedium. The most important advantage of the transformed

root cultures is their stability, which give suitable chance forfurther investigations. Among 15 infected seedlings two trans-formed cultures were obtained.

3.2. Conrmation of the transformed nature of the hairy rootcultures by PCR analysis

The results from agarose gel electrophoresis of PCR-ampliedproducts from genomic DNA (Fig. 1) showed that the rolCgene was detected in HRC but not detected in non-trans-

formed root cultures. The band corresponding to a fragmentof the virC gene was found only in the mixture prepared withDNA from A. rhizogenes (Fig. 1). This indicates that the hairyroot cultures of P. harmala were actually transformed and not

contaminated with A. rhizogenes.

3.3. Induction of alkaloid accumulation

Due to the physiological and pharmacological importance ofP. harmala alkaloids, and because the collection of intact plant

is difcult and unreliable in the eld, we have explored whetherthe tissue culture technique could provide an alternative tosupply these high value natural compounds. The biomass of

the root culture was induced by transformation withA. rhizogenes, then the transformed root cultures representedthe alternative biomass reserve for further investigation.

Accumulation of secondary compounds may in some cases

require different manipulation as a change in cultural condi-tions or the use of different elicitors to induce the biosynthesisof the secondary products. Hydrogen peroxide (H2O2) func-

tions as a signaling molecule in plants. A wide range of abioticand biotic stresses result in H2O2 generation from a variety ofsources.17 Dai and Ane (1995)18 reported that kinetic analysis

for chloramphenicol acetyltransferase activity and mRNAlevel in transgenic tobacco (Nicotiana tabacum L.) plantsshowed that induction of nopaline by H2O2 was similar to thatof methyl-jasmonate. Zayed and Wink (2005)19 reported that

b-carboline alkaloids in hairy root cultures of P. harmala wereinduced approximately by sevenfold when incubated withmethyl-jasmonate. Therefore, we have tried to investigate the

accumulation of b-carboline alkaloids in hairy root cultureof P. harmala by treatment with potential elicitors as H2O2and tryptophane.

After 24 h of incubation, the alkaloids were extracted anddetermined quantitatively by GLC. The results showed thatH2O2 (0.01%) induced the accumulation of harmine, as themajor b-carboline alkaloids, by approximately sixfold as com-pared to the untreated control. The highest stimulationresulted in harmine level of ca. 4.5 mg/g DW after 24 h of incu-bation (Fig. 2).

Since tryptophane is consider as a precursor for the biosyn-thesis of b-carboline alkaloids.20 So in the present work it wastested as an external inducer for biosynthesis of b-carboline

Figure 1 Agarose gel electrophoresis for PCR products from

genomic DNA of transformed root culture of P. harmala (T), non-

transformed root culture (N) and A. rhizogenes (A). PCR was

performed with rolC primers (Tr &Ar) and virC primers (Tv &Av).

21

10 R. Zayed0

1000

2000

3000

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2500

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Band the maintenance of the produced cultures. Hence, the

good selection of the seeds was an important factor in thisstudy. After selection of the best productive seeds, it has beentried to come up with a suitable media (WP solid- and liquid-

medium) and the conditions for maintenance of the culturesand also for production of secondary compounds.16

Thus an important question therefore seems to be how tostabilize RC. One of the trials was the transformation with

A. rhizogenes that produced stable productive HRC. Of thethree different A. rhizogenes strains only with 15834 straingood hairy roots were produced. Among 15 infected seedlings,

two transformed root cultures were obtained.Analytical results by GLC showed that harmine is the main

alkaloid constituent in HRC of P. harmala accounting ca. 90%

of the total alkaloids in WP liquid-medium. The nding byKuzovkina et al. (1990)13 proved that MS-medium with nitrogen concentration (MS-medium with KNO3 and

NH4NO3 concentration reduced by 50%) was optimal mediumfor growth and higher alkaloid yield in HRC of P. harmala.Their TLC analysis showed that harmine was the mainalkaloid followed by harmalol in HRC of P. harmala. Berlin

and other molecular targets. J Chem Ecol 1998;(11):1881937.

carboline alkaloids on intact Trypanosoma cruzi epimastigotes.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol

0

500

1000

1500

con T1 T2 T3

Ha

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Figure 2 Dose dependent induction of b-carboline alkaloid inhairy root culture of Peganum harmala by: (A) H2O2 at concen-

trations: 1 = 0.01%, 2 = 0.1% and 3 = 1%, con = control. (B)

Tryptophane at concentrations: T1 = 0.5 mM, T2 = 1 mM, and

T3 = 2 mM, con = control. Root cultures were harvested at 24 h

after induction. The content of alkaloid was determined by GLC

MS, and represented by harmine as the major one. The experiment

was performed in triplicate and the values are represented by the

mean SD.1999;122(1):2731.

8. EI-Gengaihi E, Dimetry Z, Mohamed M. Chemical and biological

investigation of harmala plant. 2. Alkaloidal investigation. J Appl

Ent 1997;121:1657.

9. Albores R, Neafsey J, Drucker G, Fields Z, Collins A. Mitochon-

drial respiratory inhibition by N-methylated b-carboline deriva-tives structurally resembling N-methyl-4-phenylpyridine. Proc

Natl Acad Sci 1990;87(23):936872.

10. Sobhani M, Ebrahimi A, Mahmoudian M. An in vitro evaluation

of human DNA topoisomerase I inhibition by Peganum harmala

L. seeds extract and its beta carboline alkaloid. J Pharm Pharm Sci

2002;5:1923.

11. Khawar M, Ozel A, Balci S, Ozcan S, Arslan O. Efcient shoot

regeneration in Syrian Rue (Peganum harmala L.) under in vitro

condition. Int J Agric Biol 2005;7:7903.4. Wakabayashi K, Totsuka Y, Fukutome K, Oguri A, Ushiyama H,

Sugimura T. Human exposure to mutagenic/carcinogenic hetero-

cyclic amines and comutagenic beta-carbolines. Mutat Res

1997;376(12):2539.

5. Picada N, da Silva V, Erdtmann B, Henriques T, Henriques A.

Genotoxic effects of structurally related beta-carboline alkaloids.

Mutat Res 1997;379(2):13549.

6. Bergstrom M, Westerberg G, Langstrom B. 11C-harmine as a

tracer for monoamine oxidase A (MAO-A): in vitro and in vivo

studies. Nucl Med Biol 1997;24(4):28793.

7. Rivas P, Cassels K, Morello A, Repetto Y. Effects of some beta-et al. (1992) had found that B50 is the productive medium

for growth and accumulation of b-carboline alkaloid andHPLC analysis showed that harmine was the main alkaloidin root cultures under the tested conditions. These variable re-sults may be due to the differences in the nature of the culture

strain or the media.In vitro, there were different trials to induce the accumula-

tion of secondary metabolites. One of these trials was the

induction of the accumulation of these compounds by applica-tion of elicitors. The results of the present work revealed thatthe efcient inducer to alkaloid accumulation in P. harmala

hairy root was H2O2 followed by tryptophan, where a maxi-mum accumulation of harmine was app. 4.5 and 2.5 mg/g after24 h, respectively.

Acknowledgements

The author deeply thanks to Prof M. Wink director of theIPMB, Heidelberg, Germany for his allowing carrying out thiswork in the laboratory of the institute. Also, the author grate-

fully acknowledge the help of Mrs. Backhaus (IPMB,Heidelberg, Germany) for GLCMS measurements andMrs. H. Staudter (IPMB, Heidelberg, Germany) for maintain-

ing of the tissue cultures.

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Efcient in vitro elicitation of b-carboline alkaloids in transformed root cultures of Peganum harmala 11

Efficient in?vitro elicitation of -carboline al1 Introduction2 Material and methods2.1 Plant materials2.1.1 Establishment of transformed root cultures2.1.2 Polymerase chain reaction (PCR)

2.2 Chemicals2.3 Cultures media2.4 Induction experiment2.5 Analytical methods2.6 GLCmass spectrum (GLCMS)

3 Results3.1 Establishment of hairy root cultures3.2 Confirmation of the transformed nature of the hairy root cultures by PCR analysis3.3 Induction of alkaloid accumulation

4 DiscussionAcknowledgementsReferences