You are on page 1of 6

Plant Cell Tiss Organ Cult (2010) 102:129134

DOI 10.1007/s11240-010-9712-x

RESEARCH NOTE

Efficient regeneration and antioxidant potential in regenerated


tissues of Piper nigrum L.
Nisar Ahmad Hina Fazal Bilal Haider Abbasi
Muhammad Rashid Tariq Mahmood
Nighat Fatima

Received: 12 November 2009 / Accepted: 11 February 2010 / Published online: 6 March 2010
Springer Science+Business Media B.V. 2010

Abstract The organogenic potential and antioxidant


potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper)
were investigated. Callus induction and shoot regeneration
were induced from leaf explants of potted plants cultured
on MS medium supplemented with different plant growth
regulators. The best callogenic response was observed on
explants cultured for 30 days on MS medium supplemented
with either 0.5 or 1.5 mg l-1 6-benzyladenine (BA) ?
1.0 mg l-1 a-naphthaleneacetic acid. Subsequent transfer
of the callogenic explants onto MS medium supplemented
with 1.5 mg l-1 BA ? 1.0 mg l-1 gibberellic acid (GA3)
achieved 85% shoot organogenesis after 30 days of culture.
The maximum number (7.2) of shoots/explant was recorded
for explants cultured in MS medium supplemented with
1.0 mg l-1 BA. Following the transfer of shoots to an
elongation medium, the longest shoots (5.4 cm) were
observed on MS medium supplemented with 1.0 mg l-1 BA
? 1.0 mg l-1 GA3. The elongated shoots were rooted on MS
medium supplemented with different concentrations of

N. Ahmad  B. H. Abbasi (&)  N. Fatima


Department of Biotechnology, Faculty of Biological Sciences,
Quaid-i-Azam University, Islamabad 45320, Pakistan
e-mail: bhabbasi@qau.edu.pk
H. Fazal  T. Mahmood
Department of Plant Sciences, Quaid-i-Azam University,
Islamabad 45320, Pakistan
M. Rashid
Department of Botany, Gomal University, Dera Ismail Khan,
Pakistan
H. Fazal
Pakistan Council of Scientific and Industrial Research (PCSIR)
Laboratories Complex, Peshawar 25100, Pakistan

indole butyric acid. An assay of the antioxidant potential of


the in vitro-grown tissues revealed that the antioxidant
activity of the regenerated shoots was significantly higher
than that of callus and the regenerated plantlets.
Keywords Antioxidant  6-Benzyladenine 
Gibberellic acid  Organogenesis  Piper nigrum
Abbreviations
BA
6-Benzyladenine
2,4 D 2,4 Dichlorophenoxyacetic acid
DPPH 1, 1-Diphenyl-2-picrylhydrazyl
GA3
Gibberellic acid
IBA
Indole butyric acid
MS0
MS medium without plant growth regulators
NAA
a-Naphthaleneacetic acid
PGRs Plant growth regulators

Piper nigrum L. (black pepper) is a flowering vine in the


family Piperaceae that is primarily cultivated for its fruit,
which is usually dried and used as a spice and seasoning
(Joseph et al. 1996). Black pepper is native to South India and
is extensively cultivated there and elsewhere throughout the
tropical regions. Dried ground pepper is one of the most
common spices in European cuisine and its descendants,
having been known and prized since antiquity for both its
flavour and its use as a medicine. The spiciness of black
pepper is due the presence of the chemical, piperine (1-piperoylpiperidine), which is a nitrogenous pungent substance
and a major alkaloid in black pepper. Piperine has been
shown to have anti-inflammatory, analgesic, anticonvulsant,
anti-ulcer and antioxidant activities and cytoprotective and

123

130

anti-depressant effects (DHooge et al. 1996; Bai and Xu


2000; Gupta et al. 2000; Selven-diran et al. 2003; Lee et al.
2005). In a recent study, Wattanathorn et al. (2008) demonstrated that piperine also affects mood and cognitive disorder. Piperine can dramatically increase the absorption of
selenium, vitamin B and b-carotene as well as other nutrients
(Bhardwaj et al. 2007).
The germplasm of P. nigrum conserved in natural
repositories is under serious threat from various environmental stresses. In addition, germplasm conservation in a
seed bank is not pragmatic due to heterozygous nature
induced through cuttings (Nair and Gupta 2006). In vitro
propagation methods provide powerful tools for the mass
multiplication and germplasm conservation of this economically important species. To date, there have been no
reports on the successful in vitro regeneration of P. nigrum
from leaf explants of potted plants. However, several
protocols for regeneration and somatic embryogenesis in
P. nigrum from shoot tips and nodal explants have been
published (Philip et al. 1992; Bhat et al. 1995; Joseph et al.
1996; Nair and Gupta 2006).
Kapoor et al. (2009) recently demonstrated that the
protective effect of piperine is most likely due to its antioxidant activity. Although a few reports on the antioxidant
activity of cultivated P. nigrum are available in the literature (Singh et al. 2008; Topal et al. 2008; Aziz et al. 2009;
Misharina et al. 2009 ), there seems to be no mention of the
influence of organogenesis on antioxidant activity in
P. nigrum. In the study reported here, we established an in
vitro system for the production of P. nigrum from leaf
explants of potted plants and conducted 1, 1-diphenyl-2picrylhydrazyl (DPPH8)-based antioxidant assays to evaluate the antioxidant activity of the main secondary
metabolites in different in vitro-derived tissues.
Leaves were collected from potted plants of P. nigrum
maintained in the greenhouse. Leaves were sterilized
according to the method of Abbasi et al. (2010a). Briefly,
the leaves were immersed first in 70% (v/v) ethanol for 60 s
and then in a 0.2% (w/v) mercuric chloride (HgCl2) solution
for approximately 2 min, followed by three rinses with
sterile distilled water. These surface-sterilized explants
were placed on MS (Murashige and Skoog 1962) medium
containing 30 g l-1 sucrose, and solidified with 8 g l-1
agar (Agar Technical LP0013; Oxoid, Hampshire, England). Different plant growth regulators (PGRs) were added
to the medium, and the pH was adjusted to 5.8. All media
were autoclaved at 121C for 20 min. All cultures were
maintained in a growth room at 25 1C under a 16/8-h
light/dark photoperiod with light provided by cool-white
fluorescent tubes at an intensity ranging from approximately 40 to 50 lmol m-2 s-1 (Abbasi et al. 2010a).
For regeneration, leaf explants were cut into reasonably
sized pieces, and placed onto MS medium supplemented

123

Plant Cell Tiss Organ Cult (2010) 102:129134

with either 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (BA) or gibberellic acid (GA3) or with BA in
combination with 1 mg l-1a-naphthaleneacetic acid (NAA)
or 1 mg l-1 GA3. MS basal medium (MS0) without any PGR
was used as a control. About five to six explants were incubated in a 100-ml Erlenmeyer flask containing approximately 25 ml of medium. After about 4 weeks of culture, the
percentage callus induction was recorded. Yellowish-green
callus was excised and transferred to fresh MS medium
containing a similar composition of PGRs for organogenesis.
Data on percentage shooting, number of shoots per explant
and mean shoot length were recorded after 5 weeks following subculture of the callus cultures. Elongated shoots
were transferred to MS0 or MS containing indole butyric
acid (IBA) for rooting. After 5 weeks, the rooted plantlets
were removed, washed with water to remove any remaining
medium and transferred to a potting soil mixture where they
acclimatized and were grown under controlled conditions.
The antioxidant activity of the regenerated tissues, such
as callus, regenerated shoots and regenerated plantlets, was
determined using the method of Amarowicz et al. (2004)
with some modifications. Briefly, 10.0 mg of dried plant
tissue was dissolved in 4 ml of methanol and then added to
a methanol solution of DPPH (1 mM, 0.5 ml). The mixture was vortexed for 15 s and then left to stand at room
temperature for 30 min. The absorbance of the resulting
solution was read on a spectrophotometer at 517 nm.
A methanol solution of DPPH8 that had decayed and hence
no longer exhibited a purple colour [i.e. 2 mg of butylated
hydroxyanisole (BHA) dissolved in 4 ml of methanol with
0.5 ml of DPPH8 solution added] was chosen for background correction instead of pure methanol. The radical
scavenging activity was calculated as the percentage of
DPPH8 discolouration using the equation
% scavenging DPPH free radical 100  1  AE =AD
where AE is the absorbance of the solution when the extract
has been added at a particular level and AD is the absorbance of the DPPH8 solution with nothing added.
For the statistical analysis, each treatment consisted of
five culture flasks and data were collected from treatments
carried out in triplicate. Analysis of variance and Duncans
multiple range test were used to compare the means of the
different treatments.
The overall objective of current research was to develop
an efficient regeneration protocol for P. nigrum from leaf
explants (Fig. 1) and to evaluate the antioxidant activity of
the regenerated plant tissues. Several protocols for plant
regeneration have been reported for different species of
Piper, including P. nigrum (Philip et al. 1992; Bhat et al.
1995; Joseph et al. 1996; Kelkar et al. 1996; Kelkar and
Krishnamurthy 1998; Madhusudhanan and Rahiman

Plant Cell Tiss Organ Cult (2010) 102:129134

2000Nair and Gupta 2006). However, to the best of our


knowledge, there has been no published report on successful regeneration from leaf explants of P. nigrum due to
the recalcitrance of leaf tissue to in vitro conditions.
There has been mention of endogenous microbial contamination causing severe setbacks to the in vitro establishment of aseptic cultures of P. nigrum (Philip et al.
1992; Bhat et al. 1995). To avoid such issues, we used the
valuable protocol of Abbasi et al. (2010a) to decontaminate
leaf explants and observed no contamination in subsequent
experiments. The application of this decontamination protocol significantly decreased the levels of contamination by
approximately 75% (B5%; data not shown). In many of the
protocols used for decontaminating leaf explants, the
explants have shown a sensitivity to the decontaminating
agents (Eeswara et al. 1999; Debnath 2009; Makunga et al.
2003; Atak and Celik 2009). However, we did not observe
any inhibitory effect of the decontamination protocol on
regeneration (data not shown).
The effects of various PGRs, such as BA, GA3, 2,4-D
alone or BA in combination with 1 mg l-1 GA3 or 1 mg l-1
NAA on indirect organogenesis were evaluated (Figs. 2, 3, 4,
5). The leaf explants of P. nigrum used in our study
responded to all of the PGRs used (Fig. 1). The best callus
induction was recorded on MS medium supplemented with
0.5 mg l-1 BA alone (93%) and with 1.5 mg l-1 BA ?
1.0 mg l-1 NAA (90%; Fig. 1). Callus induction on medium
containing only 2, 4-D (42%) or GA3 (32%) was significantly
lower than that in medium containing the other PGRs, and no
callus induction was observed on MS0 medium. However,
the addition BA to GA3-containing medium enhanced callus
induction (70%) to levels comparable to those obtained with
1.0 mg l-1 BA alone and 2.0 mg l-1 BA ? 1 mg l-1 NAA.
In a recent study on Silybum, we reported that the addition of
NAA to medium containing BA/GA3 enhanced callus
induction (Abbasi et al. 2010a). Bhat et al. (1995) did not
observe any response of leaf explant of P. nigrum to PGRs;
however, other species of Piper have been successfully
regenerated from leaf explants on a similar medium. The
findings of Philip et al. (1992) on shoot-tip explants of
P. nigrum are in agreement with our data. Madhusudhanan

131

and Rahiman (2000) reported that the addition of activated


charcoal to the culture medium induced callus formation
but failed to produce regenerants from leaf explants of
P. nigrum. Differences in the data among these studies are
likely due to (pre-)existing genetic diversity between
explants (Abbasi et al. 2010b).
Callogenesis is considered to be a significant feature of
indirect organogenesis and essential for research on biologically active molecules in medicinal plant species
(Abbasi et al. 2007, 2010a). Data on organogenesis was
determined after 5 weeks of subculture. The best percentage shooting was recorded for explants cultured on medium containing the combination of 1.5 mg l-1 BA ?
1 mg l-1 GA3 (85%). However, the combination
2.0 mg l-1 BA ? 1 mg l-1 GA3 produced a shooting
percentage of 78%, which was similar that obtained on
medium containing 0.5 mg l-1 BA (75%). In contrast, the
addition of NAA to medium already containing BA significantly inhibited shooting. Similar values have been
reported for Capsicum species (Rubluo and Barroso 1992).
However, in another study on Silybum, Abbasi et al.
(2010a) made different observations regarding the incorporation of auxin in cytokinin-containing medium. Bhat
et al. (1995) obtained a maximum number of differentiated
shoot buds for P. longum at low concentrations of BA;
nonetheless, BA was more effective than kinetin. Philip
et al. (1992) found that BA at concentrations [5 mg l-1
suppressed the proliferation and growth of shoots in shoottip cultures of P. nigrum. We recorded 7.2 shoots/explant
on medium containing 1.0 mg l-1 BA. Based on our
results and those reported earlier, the overall response of
medicinal plant species to BA is quite positive (Kelkar and
Krishnamurthy 1998; Liu et al. 2003). Kelkar and Krishnamurthy (1998) found that moderate concentrations of BA
induced the optimum number of shoots per explant (6.1) in
P. colubrinum. In our study, the incorporation of BA in
medium already containing GA3 produced a reasonable
number of shoots/explant (Fig. 3), while the presence of 2,
4-D was not beneficial for either callogenesis or shoot
organogenesis (Figs. 2, 3, 4, 5). The addition of NAA to
the medium significantly inhibited shoot organogenesis.

Fig. 1 Plant regeneration in Piper nigrum L. a Callus b regenerated shoots c shoot multiplication d shoot elongation e rooting f regenerated
plantlets

123

Plant Cell Tiss Organ Cult (2010) 102:129134

c
cd
e

-1

2.0BA+GA3
1.5 BA+GA3
1.0 BA+GA3
0.5 BA+GA3
2.0 GA3
1.5 GA3
1.0 GA3
0.5 GA3
2.0BA+NAA
1.5 BA+NAA
1.0 BA+NAA
0.5 BA+NAA
2.0 BA
1.5 BA
1.0 BA
0.5 BA
2.0 2,4-D
1.5 2,4-D
1.0 2,4-D
0.5 2,4-D
MS0
0

Plant Growth Regulators (mg l )

-1

Plant Growth Regulators (mg l )

132

ef

h
g
bc
a
b
de

d
d

bc
a

h
gh

g
f

10

20

30

40

50

60

70

80

90

100

2.0BA+GA3
1.5 BA+GA3
1.0 BA+GA3
0.5 BA+GA3
2.0 GA3
1.5 GA3
1.0 GA3
0.5 GA3
2.0BA+NAA
1.5 BA+NAA
1.0 BA+NAA
0.5 BA+NAA
2.0 BA
1.5 BA
1.0 BA
0.5 BA
2.0 2,4-D
1.5 2,4-D
1.0 2,4-D
0.5 2,4-D
MS0

bc
bc
cd
gh
h
g

cd

a
c
ij
i

h
gh

-1

a
bc

d
h

i
h
h

gh

e
d
c
b

i
i
10

h
h

20

30

40

50

60

2.0BA+GA3
1.5 BA+GA3
1.0 BA+GA3
0.5 BA+GA3
2.0 GA3
1.5 GA3
1.0 GA3
0.5 GA3
2.0BA+NAA
1.5 BA+NAA
1.0 BA+NAA
0.5 BA+NAA
2.0 BA
1.5 BA
1.0 BA
0.5 BA
2.0 2,4-D
1.5 2,4-D
1.0 2,4-D
0.5 2,4-D
MS0

ab

70

80

90

100

d
g

de

hi

c
c
d
f

h
f

e
d

g
h
hi
i
0

Fig. 4 Effects of various concentrations of 2,4-D, BA, GA3, BA ?


1 mg l-1 NAA and BA ? 1 mg l-1 GA3 on the number of shoots per
explant in P. nigrum. Data were collected after 5 weeks of subculture
to MS media with a similar composition of plant growth regulators.
Values are the means of five replicates. Columns with the same lowercase letters are not significantly different at P \ 0.05

Plant Growth Regulators (mg l )

-1

Plant Growth Regulators (mg l )

ij

ab

Number of shoots/explant

Fig. 2 Effects of various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (BA) or gibberellic acid (GA3)
alone, BA ? 1 mg l-1a-naphthaleneacetic acid (NAA) and BA ? 1
mg l-1 GA3 on percentage callus induction of P. nigrum. Data were
collected after 4 weeks of culture. Values are the means of five
replicates. Columns with the same lower-case letters are not
significantly different at P \ 0.05

fg
f

% Callus Induction

2.0BA+GA3
1.5 BA+GA3
1.0 BA+GA3
0.5 BA+GA3
2.0 GA3
1.5 GA3
1.0 GA3
0.5 GA3
2.0BA+NAA
1.5 BA+NAA
1.0 BA+NAA
0.5 BA+NAA
2.0 BA
1.5 BA
1.0 BA
0.5 BA
2.0 2,4-D
1.5 2,4-D
1.0 2,4-D
0.5 2,4-D
MS0

Mean Shoot Length (cm)

% Shooting

Fig. 3 Effects of various concentrations of 2,4-D, BA or GA3 alone,


BA ? 1 mg l-1NAA and BA ? 1 mg l-1 GA3 on percentage shooting
in P. nigrum. Data were collected after 5 weeks of subculture to MS
media with a similar composition of plant growth regulators. Values
are means of five replicates. Columns with the same lower-case letters
are not significantly different at P \ 0.05

Kelkar et al. (1996) found that the combination of BA ?


2,4-D induced an optimum shooting response in other
species of Piper. Shooting was recorded for all of the PGRs
tested in our experiments (Figs. 3, 4, 5). Data on mean
shoot length (Fig. 5), however, showed that the incorporation of BA into medium containing GA3 produced the
longest shoots (5.4 cm), and in contrast to the data on
number of shoots/explant, the addition of NAA to medium
containing BA increased mean shoot length. Similar
observations were reported by Makunga et al. (2003) for
Thapsia spp. However, Lucchesini et al. (2009) found
optimum shoot length for Echinacea angustifolia on

123

Fig. 5 Effects of various concentrations of 2,4-D, BA, GA3 alone,


BA ? 1 mg l-1 NAA and BA ? 1 mg l-1 GA3 on mean shoot length
in P. nigrum. Data were collected after 5 weeks of subculture to MS
media with a similar composition of plant growth regulators. Values
are the means of five replicates. Columns with the same lower-case
letters are not significantly different at P \ 0.05

medium containing only BA. Magyar-Tabori et al. (2010)


concluded from their work that the type and concentration
of cytokinins that result in optimum shoot production is
largely dependent on genotype, and interactions have
proven to be significant.
Shoots grown on shoot organogenesis medium were
transferred to MS0 and MS medium supplemented with
different concentrations of IBA for rooting (Table 1).
Rooting frequency was found to be directly positively
correlated with increasing concentrations of IBA in the
medium. The optimum percentage rooting (89%), number
of roots/shoot (7.8) and root length (11.5 mm) were
obtained shoots cultured on medium containing 2 mg l-1

Plant Cell Tiss Organ Cult (2010) 102:129134

133

Table 1 Effects of different concentrations of indole butyric acid on


percentage rooting, number of roots per shoot and approximate root
length after 20 days of culture on rooting media
Indole butyric
acid (mg l-1)

Rooting
(%)

Number of
roots/shoot

Root length
(mm)

0.5

40 d

2.1 d

5.1 d

1.0

62 c

4.6 c

6.9 b,c

1.5

78 b

6.2 b

8.3 b

2.0

89 a

7.8 a

11.5 a

Values followed by different letters are significantly different at


P \ 0.05

IBA. We obtained healthy roots (Fig. 1e). White roots


appeared after 15 days of culture, turning brown thereafter.
In contrast, Philip et al. (1992) obtained rooted plantlets
through shoot-tip cultures of P. nigrum on medium containing 1 mg l-1NAA. In a study on another Piper species,
rooting was achieved on medium containing indole acetic
acid (IAA) (Kelkar et al. 1996). It would therefore appear
that Piper species are not recalcitrant to commonly used
auxins for rooting. However, some medicinal species have
shown rooting in MS0 (Abbasi et al. 2010a). Rooted
plantlets were successfully transferred to pots for further
growth under controlled conditions (Fig. 1f).
Regenerated plantlets are able to accumulate secondary
metabolites similar to those found in the mother plant
(Shilpa et al. 2010). The antioxidant potential of the
regenerated tissues of P. nigrum was determined using the
DPPH8 free radical (Fig. 6). The shoot culture was found to
have a significantly higher antioxidant potential than the
other regenerated tissues. Singh et al. (2004) reported that

Antioxidant activity (%)

100

80

60

b
c

40

20

R
eg
an ene
tle ra
ts ted
Pl

eg

al

lu

en
e
sh rat
oo ed
ts

Fig. 6 Antioxidant activity in regenerated tissues of P. nigrum. The


activity was determined using 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
as free radical. Values are the means standard deviation of triplicate
experiments. Means with the same lower-case letters are not significantly different at P \ 0.05

the antioxidant activity of Piper extracts is comparable to


that of butylated hydroxyanisole and butylated hydroxytoluene. Irradiation and steam have been reported to be
damaging to the antioxidant activity of piperine (Waje
et al. 2008). Andarwulan and Shetty (1999) found that
differentiated tissues of Pimpinella anisum had a higher
antioxidant activity and higher total phenolic contents. We
also found a lower antioxidant activity in callus cultures.
However, Abbasi et al. (2010a) recently reported that in
their system, the callus culture of Silybum had a higher
antioxidant activity. This variation in data demonstrates
that different components are accumulated during different
growth phases. Hence, the regeneration protocol reported
here obtained a higher content of physiologically active
antioxidants. This protocol can be scaled up to bioreactor
level to produce chemically consistent P. nigrum plantlets.
Acknowledgements The authors acknowledge the support of
Higher Education Commission (HEC) of Pakistan and University
Research Fund (URF) of Quaid-i-Azam University (QAU), Islamabad, Pakistan.

References
Abbasi BH, Saxena PK, Murch SJ, Liu CZ (2007) Echinacea
biotechnology: challenges and opportunities. In Vitro Cell Dev
Biol-Plant 43:481492
Abbasi BH, Khan MA, Mahmood T, Ahmad M, Chaudhary MF,
Khan MA (2010a) Shoot regeneration and free-radical scavenging activity in Silybum marianum L. Plant Cell Tissue Org Cult.
doi 10.1007/s11240-010-9692-x
Abbasi BH, Ahmad N, Fazal H, Mahmood T (2010b) Conventional
and modern propagation techniques in Piper nigrum. J Med
Plant Res 4:007012
Amarowicz R, Pegg RB, Rahimi-Moghaddan P, Barl B, Weil JA
(2004) Free radical scavenging activity and antioxidant activity
of selected plant species from the Canadian Prairies. Food Chem
84:551562
Andarwulan N, Shetty K (1999) Phenolic content in differentiated
tissue cultures of untransformed and Agrobacterium-transformed
roots of Anise (Pimpinella anisum L.). J Agric Food Chem 47:
17761780
Atak C, Celik O (2009) Micropropagation of Anthurium andraeanum
from leaf explants. Pak J Bot 41:11551161
Aziz N, Mehmood MH, Mandukhal SR, Bashir S, Raoof S, Gilani AH
(2009) Antihypertensive, antioxidant, antidyslipidemic and
endothelial modulating activities of a polyherbal formulation
(POL-10). Vascul Pharm 50:5764
Bai YF, Xu H (2000) Protective action of piperine against experimental gastric ulcer. Acta Pharmacol Sin 21:357359
Bhardwaj RK, Glaeser H, Becquemont L, Klotz U, Gupta SK, Fromm
MF (2007) Piperine, a major constituent of black pepper, inhibits
human P-glycoprotein and CYP3A4. J Pharmacol Exp Therapeut
302:645650
Bhat SP, Chandel KPS, Malik SK (1995) Plant regeneration from
various explants of cultivated Piper species. Plant Cell Rep
14:398402
DHooge R, Pei YQ, Raes A, Lebrun P, Bogaert PP, De Deyn PP
(1996) Anticonvulsant activity of piperine on seizures by
excitatory amino acid receptior agonists. Arzneimittelforschung
46:557560

123

134
Debnath SC (2009) A two-step procedure for adventitious shoot
regeneration on excised leaves of lowbush blueberry. In Vitro
Cell Dev Biol Plant 45:122128
Eeswara JP, Allan EJ, Mordue J, Stuchbury T (1999) A procedure for
sterilizing leaf explants collected from wild neem (Azadirachta
indica) trees. J Natl Sci Found Sri Lanka 27:131136
Gupta SK, Bansal P, Bhardwaj RK, Velpandian T (2000) Comparative anti-nociceptive, anti-inflammatory and toxicity profile of
nimesulides vs nimesulide and piperine combination. Pharmacol
Res 41:657662
Joseph B, Joseph D, Philip VJ (1996) Plant regeneration from somatic
embryos in black pepper. Plant Cell Tiss Organ Cult 47:8790
Kapoor IPS, Singh B, Singh G, De Heluani CS, De Lampasona MP,
Catalan CAN (2009) Chemistry and in vitro antioxidant activity
of volatile oil and oleoresins of Black pepper (Piper nigrum).
J Agric Food Chem 57:53585364
Kelkar SM, Krishnamurthy KV (1998) Adventitious shoot regeneration from root, internode, petiole and leaf explants of Piper
colubrinum Link. Plant Cell Rep 17:721725
Kelkar SM, Deboo GB, Krishnamurthy KV (1996) In vitro plant
regeneration from leaf callus in Piper colubrinum link. Plant Cell
Rep 14:398402
Lee SA, Hong SS, Han XB, Hwang JS, Oh GJ, Lee KS, Lee MK, Hwang
BY, Ro JS (2005) Piperine from the fruits of Piper nigrum with
inhibitory effect on monoamine oxidase and antidepressant like
activity. Chem Pharm Bull 53:832835
Liu CZ, Murch SJ, El-Demerdash M, Saxena PK (2003) Regeneration
of the Egyptian medicinal plant Artemisia judaica L. Plant Cell
Rep 21:525530
Lucchesinia M, Bertolib A, Mensuali-Sodic A, Pistelli L (2009)
Establishment of in vitro tissue cultures from Echinacea
angustifolia D.C. adult plants for the production of phytochemical compounds. Sci Hortic 122:484490
Madhusudhanan K, Rahiman BA (2000) The effect of activated
charcoal supplemented media browning of in vitro cultures of
Piper species. Biol Plant 43:297299
Magyar-Tabori K, Dobranszki J, da Silva JAT, Belley SM, Hudak I
(2010) The role of cytokinins in shoot organogenesis in apple.
Plant Cell Tiss Organ Cult. doi 10.1007/s11240-010-9696-6
Makunga NP, Jager AK, van Staden J (2003) Micropropagation
of Thapsia garganicaa medicinal plant. Plant Cell Rep 21:
967973

123

Plant Cell Tiss Organ Cult (2010) 102:129134


Misharina TA, Terenina MB, Krikunova NI (2009) Antioxidant
properties of essential oils. Appl Biochem Microbiol 45:642647
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol Plant 15:473497
Nair RR, Gupta SD (2006) High-frequency plant regeneration through
cyclic secondary somatic embryogenesis in black pepper (Piper
nigrum L.). Plant Cell Rep 24:699707
Philip VJ, Joseph D, Triggs GS, Dickinson NM (1992) Micropropagation of black pepper (Piper nigrum Linn) through shoot tip
cultures. Plant Cell Rep 12:4144
Rubluo M, Barroso M (1992). In vitro morphogenetic responses and
cytokinin-auxin interaction for callus production in pepper. An
Inst Biol Univ Nacional Autonoma Mexico Ser Bot 63(2):
195201
Selven-diran K, Singh JP, Krishnan KB, Saktisekaran D (2003)
Cytoprotective effect of piperine against benzole [a]pyrene
induced lung cancer with reference to lipid peroxidation and
antioxidant system in Swiss albino mice. Fitoterapia 74:109115
Shilpa K, Selvakkumar C, Senthil AK, Lakshmi BS (2010) In vitro
root culture of Ocimum sanctum L. and evaluation of its free radical
scavenging activity. Plant Cell Tiss Org Cult. doi 10.1007/
s11240-009-9661-4
Singh G, Marimuthu P, Catalan C, de Lampasona MP (2004)
Chemical antioxidant and antifungal activities of volatile oil of
black pepper and its acetone extract. J Sci Food Agric 84:1878
1884
Singh R, Singh N, Saini BS, Rao HS (2008) In vitro antioxidant
activity of pet ether extract of black pepper. Ind J Pharmacol 40:
147151
Topal U, Sasaki M, Goto M, Otles S (2008) Chemical compositions
and antioxidant properties of essential oils from nine species of
Turkish plants obtained by supercriticial carbon dioxide extraction and steam distillation. Int J Food Sci Nutr 59:619634
Waje CK, Kim H-K, Kim K-S, Todoriki S, Kwon J-H (2008)
Physicochemical and microbiological qualities of steamed and
irradiated ground black pepper (Piper nigrum L.). J Agric Food
Chem 56:45924596
Wattanathorn J, Chonpathompikunlert P, Muchimapura S, Priprem A,
Tankamnerdthai O (2008) Piperine, the potential functional
food for mood and cognitive disorders. Food Chem Technol 46:
31063110