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

Cairo University

Bulletin of Faculty of Pharmacy, Cairo University

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ORIGINAL ARTICLE

Ecient in vitro elicitation of b-carboline alkaloids

in transformed root cultures of Peganum harmala
Rawia Zayed

Department of Pharmacognosy, Faculty of Pharmacy, Sinai University, 44551 North Sinai, Egypt
Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University, 44519 Zagazig, Egypt
Received 2 March 2011; accepted 23 May 2011
Available online 26 August 2011

KEYWORDS
b-Carboline alkaloids;
Hairy root culture and elicitation;
Peganum harmala

Abstract An efcient in vitro production of biomass hairy root cultures and elicitation of b-carboline 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
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 as
hydrogen peroxides (H2O2), or biosynthetic precursor as tryptophane in order to stimulate the biosynthesis of b-carboline alkaloids, mainly harmine as the major one. Prole and amounts of b-carboline alkaloids were analyzed using capillary GLCMS.
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
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 alkaloid production approximately vefold (0.250.3 mg/g DW) within 24 h.
2011 Faculty of Pharmacy, Cairo University. Production and hosting by Elsevier B.V.
Open access under CC BY-NC-ND license.

* Corresponding author. Address: Department of Pharmacognosy,

Faculty of Pharmacy, Zagazig University, 44519 Zagazig, Egypt.
Tel.: +20 0552343411, mobile: +20 0124989981.
E-mail address: rawiazayed@hotmail.com
1110-0931 2011 Faculty of Pharmacy, Cairo University. Production
and hosting by Elsevier B.V. Open access under CC BY-NC-ND license.
Peer review under responsibility of Faculty of Pharmacy, Cairo
University.
doi:10.1016/j.bfopcu.2011.07.002

Production and hosting by Elsevier

1. Introduction
Peganum harmala plant has ancient reputations as antiseptic
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 pharmacological actions. It is CNS stimulant and has hallucinogenic
activities at high doses.3 b-Carboline alkaloids affect a number
of molecular targets, ranging from DNA (intercalation) via
neuroreceptors to monoamine oxidase3 and therefore gure
as potent defense compounds of P. harmala. DNA intercalation, mutagenic, genotoxic and cytotoxic have been

8
investigated.35 They suggested that the antibiotic and toxic effects of b-carboline could be a function of DNA intercalation
and resulting mutations. These give strong indications for the
validity of harmine as an in vivo tracer for the assessment of
MAO-A enzyme binding in the brain.6 Trypanosomicidal
activity of several b-carboline alkaloids7 and larvicidal against
the larvae of the cotton leaf worm8 have stated that the biological activity of b-carboline alkaloids and a highly toxic effect
reasonably underlie the neurotoxicity.9
Since P. harmala plant grows as wild and not cultivated and
due to the biological importance of carboline alkaloids of the
plant.10 Khawar et al. (2005)11 and Neha et al. (2009)12 have
tried to micropropagate this species to facing the problem of
extinction. Most of P. harmala stable root cultures have been
established by transformation with A. rhizogenes13 to overcome on the difculties for production of long-stable root cultures as storage tissue. Hence, the present study was designed
to develop an efcient in vitro procedure to enhance the biosynthesis of b-carboline alkaloids in biomass of transformed
hairy root cultures of P. harmala by using potential elicitors.

2. Material and methods

2.1. Plant materials
2.1.1. Establishment of transformed root cultures
Mature seeds of P. harmala (maximum, 6 month old collected
from Southern Sinai, Egypt) were surface-sterilized in sodium
hypochlorite solution (1%, 15 min) then they were rinsed
twice with sterile distiled water. Afterwards the seeds were
treated with EtOH 70% for 5 min and rinsed twice with sterile
distiled water. The seeds were left overnight in sterile water and
then incubated on agar plates until germination. The germinated 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 old
seedlings with A. rhizogenes strains 15834, TR 105 and LBA
(kindly supplied by Prof. Dr. W. Alferman University of Duesseldorf, Germany). After 57 h the roots were surface-sterilized
with 1% sodium hypochlorite solution for 1 min and subcultivated on antibiotic-free agar-medium. The best growthing
roots without Agrobacterium were transferred into hormonefree WP liquid medium to which Claforan was added as antibiotic (5 mg/10 ml medium). This procedure was repeated several
times until the Agrobacterium was completely eliminated. Then
the roots were subcultured every weak in fresh WP liquid medium without antibiotic for maintenance. HRC have a different
morphology as compared to RC. They have many root hairs
and much more branches and the most overall important
advantage of the transformed root cultures is their long-term
stability. 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 conrm the transformation of the root cultures. PCR conditions:
96 C for 2 min, followed by 36 cycles of 94 C for 30 s, 60 C
for 30 s, 72 C for 2 min, and nally 15 min extension at
72 C. The primers for rolC DNA were 50 -ATGGCTGAA-

R. Zayed
GACGACCTGTGTT-30 ; and 50 -TTAGCCGATTGCAAA
CTTGCAC-3.15 The primers for virC DNA were 50 -ATCATTTG TAGCGACT-30 ; and 50 -AGCTCAAACCTGv
CTTC-30 .16 Gelelectrophoresis of PCR products from transformed and non-transformed root cultures as well as from
A. rhizogenes was carried out to identify the presence or
absence of rolC and virC genes.
2.2. Chemicals
Solvents (Riedel-de Haen, Seelze Germany): Hydrogen peroxide
(H2O2), dichloromethane, 70% ethanol, 1% sodium hypochlorite (NaClO), hydrochloric acid (HCl) and sodium hydroxide
(NaOH) were obtained from Sigma-Aldrich-Chemie (Germany). Agar (Bacto-Agar, Difco Laboratories USA) and gelrite
(Carl Roth GmbH Karlsruhe, Germany). Isolute-Sorbent (Isolute HM-N, International Sorbent Technology Ltd. UK).
2.3. Cultures media
Hormone free WP liquid and solid media were used for optimal growth and production for HRC of P. harmala. The pH
of the medium was adjusted to 5.7 before autoclaving. For
subculture 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 for
growth and maintenance of A. rhizogenes at 39 C. It contains
mannitol (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%) at
pH 7.2.
2.4. Induction experiment
HRC were treated by different inducers as alternative method
to induce the accumulation of the secondary metabolite in
plants. Hydrogen peroxides (H2O2) and tryptophane (T) were
used as inducers. Under aseptic conditions (Laminar Flow,
Ceag Shirp- Reinraum technik, Germany), the inducers were
added to 10 ml fresh medium in 100 ml Erlenmeyer asks.
The cultures were incubated in a gyrator shaker 110 rpm at
25 C in an illuminated culture room.
2.5. Analytical methods
After the incubation times with inducers, HRC was harvested
by Buchner vaccum ltration, dried, weighed and homogenized in mortar with 1 M HCl (20 ml). The homogenate was
left at room temperature overnight, then made alkaline with
NH4OH (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 evaporator, the residue was kept at 4 C until analysis. For analysis the
residue was taken up in MeOH and was injected into the gas
chromatography. Medium samples were treated accordingly
but the initial 1 M HCl step was omitted . Using GLCMS,
the alkaloids were identied. Harmine and harmaline were
used as external standards for quantitative interpretation.
2.6. GLCmass spectrum (GLCMS)
The GLCMS analysis were carried out on a Carlo Erba
HRGC 4160 gas chromatography equipped with a Fused silica

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

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

directly coupled with a quadruple mass spectrometer, a Finnigan MAT 4500 operating at 45 eV. Conditions: injector
250 C, temperature program: 150300 C 15 C/min, 300 C;
20. Split ratio 1:5; carrier gas helium, 0.5 bar (ow rate
2 ml/min).
3. Results
3.1. Establishment of hairy root cultures
To establish a productive strain of root cultures of plants, it
must select the best producing strain and optimize the culture
conditions for growth and production of secondary compounds. Obtaining stable root cultures from P. harmala was
very difcult. Different media have been tried (MS, MS medium supplied with 2,4-D, WP and WP medium with 2,4-D
and kinetin) and different conditions (e.g. light and dark),
but each time the root cultures easily broken. After a certain
time, RC acquired brownish-red color and more over the medium. At the end of four growth cycles, growth of the cultures
slowly stopped and they nally died. While looking for a suitable medium for the root cultures, WP liquid and solid media
proved to be optimal for growth and maintenance of P. harmala 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 were
maintained as stable root cultures in hormone-free WP liquid
medium. The most important advantage of the transformed
root cultures is their stability, which give suitable chance for
further investigations. Among 15 infected seedlings two transformed cultures were obtained.
3.2. Conrmation of the transformed nature of the hairy root
cultures by PCR analysis
The results from agarose gel electrophoresis of PCR-amplied
products from genomic DNA (Fig. 1) showed that the rolC
gene was detected in HRC but not detected in non-transformed root cultures. The band corresponding to a fragment
of the virC gene was found only in the mixture prepared with
DNA from A. rhizogenes (Fig. 1). This indicates that the hairy
root 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 of
P. harmala alkaloids, and because the collection of intact plant
is difcult and unreliable in the eld, we have explored whether
the tissue culture technique could provide an alternative to
supply these high value natural compounds. The biomass of
the root culture was induced by transformation with
A. rhizogenes, then the transformed root cultures represented
the alternative biomass reserve for further investigation.
Accumulation of secondary compounds may in some cases
require different manipulation as a change in cultural conditions or the use of different elicitors to induce the biosynthesis
of the secondary products. Hydrogen peroxide (H2O2) functions as a signaling molecule in plants. A wide range of abiotic
and biotic stresses result in H2O2 generation from a variety of

Figure 1 Agarose gel electrophoresis for PCR products from

genomic DNA of transformed root culture of P. harmala (T), nontransformed root culture (N) and A. rhizogenes (A). PCR was
performed with rolC primers (Tr & Ar) and virC primers (Tv & Av).

sources.17 Dai and Ane (1995)18 reported that kinetic analysis

for chloramphenicol acetyltransferase activity and mRNA
level in transgenic tobacco (Nicotiana tabacum L.) plants
showed that induction of nopaline by H2O2 was similar to that
of methyl-jasmonate. Zayed and Wink (2005)19 reported that
b-carboline alkaloids in hairy root cultures of P. harmala were
induced approximately by sevenfold when incubated with
methyl-jasmonate. Therefore, we have tried to investigate the
accumulation of b-carboline alkaloids in hairy root culture
of P. harmala by treatment with potential elicitors as H2O2
and tryptophane.
After 24 h of incubation, the alkaloids were extracted and
determined quantitatively by GLC. The results showed that
H2O2 (0.01%) induced the accumulation of harmine, as the
major b-carboline alkaloids, by approximately sixfold as compared to the untreated control. The highest stimulation
resulted in harmine level of ca. 4.5 mg/g DW after 24 h of incubation (Fig. 2).
Since tryptophane is consider as a precursor for the biosynthesis of b-carboline alkaloids.20 So in the present work it was
tested as an external inducer for biosynthesis of b-carboline
alkaloid in HRC of P. harmala. The results showed that the
accumulation of harmine was an inducer-dose dependant
and harmine biosynthesis was enhanced app. vefold
(Fig. 2). The maximum level was 0.250.3 mg/g DW harmine
with 2 mM tryptophane.
These results with P. harmala are representing example for
the power of H2O2 to induce the formation of alkaloids as
defense system in the plant. Since the production of b-carboline alkaloids in in vitro cultures is of biotechnological importance, the elicitation via elicitors offers a chance to improve
this yield.
4. Discussion
The rst important factor which switched our investigation
with P. harmala, was the production of long-stable hairy root
culture. Seeds from different sources were investigated to
obtain good germinating seedlings and high producing cultures. This work results showed a variation in the germination

R. Zayed

6000

Harmine conc. (g/g DW)

10

5000

et al. (1992)21 had found that B50 is the productive medium

for growth and accumulation of b-carboline alkaloid and
HPLC analysis showed that harmine was the main alkaloid
in root cultures under the tested conditions. These variable results 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 accumulation of secondary metabolites. One of these trials was the
induction of the accumulation of these compounds by application of elicitors. The results of the present work revealed that
the efcient inducer to alkaloid accumulation in P. harmala
hairy root was H2O2 followed by tryptophan, where a maximum accumulation of harmine was app. 4.5 and 2.5 mg/g after
24 h, respectively.

4000
3000
2000
1000
0

3000

Harmine conc. ( g/g DW)

con

2500

Acknowledgements
The author deeply thanks to Prof M. Wink director of the
IPMB, Heidelberg, Germany for his allowing carrying out this
work in the laboratory of the institute. Also, the author gratefully acknowledge the help of Mrs. Backhaus (IPMB,
Heidelberg, Germany) for GLCMS measurements and
Mrs. H. Staudter (IPMB, Heidelberg, Germany) for maintaining of the tissue cultures.

2000
1500
1000
500

References
0
con

T1

T2

T3

Figure 2 Dose dependent induction of b-carboline alkaloid in

hairy root culture of Peganum harmala by: (A) H2O2 at concentrations: 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.

and the maintenance of the produced cultures. Hence, the

good selection of the seeds was an important factor in this
study. After selection of the best productive seeds, it has been
tried to come up with a suitable media (WP solid- and liquidmedium) and the conditions for maintenance of the cultures
and also for production of secondary compounds.16
Thus an important question therefore seems to be how to
stabilize RC. One of the trials was the transformation with
A. rhizogenes that produced stable productive HRC. Of the
three different A. rhizogenes strains only with 15834 strain
good 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 by
Kuzovkina et al. (1990)13 proved that MS-medium with
nitrogen concentration (MS-medium with KNO3 and
NH4NO3 concentration reduced by 50%) was optimal medium
for growth and higher alkaloid yield in HRC of P. harmala.
Their TLC analysis showed that harmine was the main
alkaloid followed by harmalol in HRC of P. harmala. Berlin

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