Article

Toxicology and Industrial Health

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Effect of different in vitro culture © The Author(s) 2013
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extracts of black pepper DOI: 10.1177/0748233713505126
tih.sagepub.com
(Piper nigrum L.) on toxic
metabolites-producing strains

Nisar Ahmad1, Bilal Haider Abbasi1 and Hina Fazal2,3

Abstract
In the present study, the effect of different in vitro cultures (callus, in vitro shoots) and commercially available
peppercorn extract was investigated for its activity against toxic metabolite-producing strains (Escherichia coli,
Pseudomonas aeroginosa, Salmonella typhi, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Candida albi-
cans). These in vitro cultures were extracted with ethanol, hexane, and chloroform, and the antipathogenic
activity was determined by well-diffusion method. Hexane extract of callus showed 22 mm zone of inhibition
against B. cereus, 23 mm against S. aureus, while regenerated shoots and seeds have shown 24.3 and 26 mm
zones of inhibition. The ethanolic extracts of regenerated Piper shoots have shown 25 mm activity against S.
aureus, 21 mm against B. cereus, and 16 mm in the case of C. albicans in comparison with standard antibiotics.
Peppercorn extracts in chloroform and ethanol had shown activities against B. cereus (23.6 mm) and B. subtilis
(23.5 mm). During in vitro organogenesis and morphogenesis, cells and tissues produced a comparable phyto-
chemicals profile like mother plant. Morphogenesis is critically controlled by the application of exogenous
plant-growth regulators. Such addition alters the hormonal transduction pathways, and cells under in vitro con-
ditions regenerate tissues, which are dependant on the physiological state of cells, and finally enhance the pro-
duction of secondary metabolites. To the best of our knowledge, this is the first report to compare the
antimicrobial potential of in vitro regenerated tissues and peppercorn with standard antibiotics. In conclusion,
most of the extracts showed pronounced activities against all the pathogenic microbes. This is a preliminary
work, and the minimum inhibitory concentration values needs to be further explored. Regenerated tissues
of P. nigrum are a good source of biologically active metabolites for antimicrobial activities, and callus culture
presented itself as a good candidate for such activities.

Keywords
P. nigrum, callus, in vitro shoots, peppercorn, pathogenic strains, antipathogenic activity

Introduction food additives (Srinivasan, 2007). Regarding medicinal

Piper nigrum L. (P. nigrum; black pepper) belonging to
the family Piperaceae is considered the king of spices
due to its spicy savor (Abbasi et al., 2010; Ahmad
et al., 2012a, 2013). P. nigrum is cultivated throughout
the world and is native to tropical and subtropical
regions of India (Ahmad et al., 2010, 2012a). High pun-
gency in black pepper fruits indicated the presence of
piperine. Piperine, the most active component in the
fruits of P. nigrum L., denotes the quality and value of
spiciness (Ahmad et al., 2011a; Bhat et al., 1995; Philip
et al., 1992). Peppercorn from P. nigrum can be used in
food processing, as crude drugs, and can also be used as
applications, P. nigrum has pronounced antibacterial,
1
Department of Biotechnology, Faculty of Biological Sciences,
Quaid-i-Azam University, Islamabad, Pakistan
2
Pakistan Council of Scientific and Industrial Research (PCSIR)
Laboratories Complex, Peshawar, Pakistan
3
Department of Plant Sciences, Faculty of Biological Sciences,
Quaid-i-Azam University, Islamabad, Pakistan
Corresponding author:
Nisar Ahmad, Department of Biotechnology, Faculty of Biological
Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan.
Email: nisarbiotech@gmail.com

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2 Toxicology and Industrial Health
antifungal, antiviral, antimutagenic, and antioxidant
activities (Abbasi et al., 2010; Ahmad et al., 2010,
2012a; Dorman and Deans, 2000; Saxena et al., 2007).
Nerolidol and b-caryophyllene isolated from P. nigrum
have anesthetic activity (Santra-Mantra et al., 2005).
The fruits of this species are also used to treat respiratory
tract diseases and infections such as asthma, cold extre-
mities and sore throat, digestive problems including
chronic indigestion, colon toxins, colic, and diarrhea,
and fevers including congestion fever and intermittent
fever and also control obesity (Ahmad et al., 2011a;
Ravindran, 2000). P. nigrum also claimed to possess
antiapoptotic activity, antidepressant, analgesic, anti-
inflammatory, antimetastatic, antispasmodic, antisper-
matogenesis, antithyroid, hepatoprotective, insecticidal,
larvicidal, and pesticidal activities (Balkrishna,
1995; Kumar et al., 2007; Li et al., 2007; Mishra and
Singh, 2009; Pathak and Khandlewal, 2006; Scott
et al., 2008).
Plants produced a variety of active metabolites that
act as a defense system against various pathogenic
agents (Ahmad et al., 2011b, 2012b, 2012c). Accord-
ing to the literature cited, to our knowledge, the anti-
microbial activity in regenerated tissues in this
species has not been reported. The overall thrust of the
present study was to investigate and compare the anti-
microbial potential of regenerated tissues with piper
fruits and with standard antibiotics.
Materials and methods
Plant materials
Reasonable amount of callus and regenerated shoots
were taken from the experiment of Ahmad et al. (2013)
and peppercorn was purchased from local markets.
Extract preparation from regenerated tissues
The extracts were prepared according to the method
described by Fazal et al. (2010, 2012). Briefly, the
callus, in vitro regenerated shoots, and peppercorn
of P. nigrum were dried in an oven (Thermo Scien-
tific, Germany) at 50 + 2 C, ground, and sieved to
get fine powder from which the extracts were pre-
pared (Figure 1). An ethanol extract of the plant was
obtained by taking 5 g of dried powder in a separate
container. To this, 50 ml ethanol was added and kept
for 1 week with periodic shaking (The soaked mate-
rial was stirred every 18 h using a sterilized glass rod),
filtered, and the filtrate was collected. This procedure
was repeated three times with fresh volume of
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ethanol. The filtrates were pooled. The same proce-
dure was used for hexane and chloroform extracts.
The final extracts were passed through Whatman fil-
ter paper No 1 (Whatman Ltd, Maidstone, UK). The
pooled ethanol, hexane, and chloroform extracts were
concentrated separately using rotary vacuum evapora-
tor at 40 C and stored at 4 C in an air-tight bottle. The
extract obtained from each plant was dissolved in
each solvent independently to get stock solutions.
Microbial strains used
Bacterial strains, namely, Escherichia coli (ATCC #
25922), Pseudomonas aeroginosa (ATCC # 9721),
Staphylococcus aureus (ATCC # 6538) were obtained
from PCSIR Laboratories (Lahore, Pakistan). Clinical
isolates of Salmonella typhi, Bacillus subtilis, and
Bacillus cereus were obtained from Microbiology
Laboratory, Quaid-i-Azam University, Islamabad,
Pakistan, and the fungal strain Candida albicans was
obtained from the Hayatabad Medical Complex
(Peshawar, Pakistan). These microorganisms were pro-
cured and maintained on nutrient agar medium at 4 C.
Assay procedures
The antibacterial potential of ethanol, hexane, and
chloroform extracts of various tissues of P. nigrum was
determined by well-diffusion method (Fazal et al.,
2010; 2012; Parekh and Chanda, 2007). Nutrient agar
media plates were seeded with a sterile swab moistened
with the bacterial suspension of 18- to 24-h cultures of
microbial inoculums (a standardized inoculum of 1–2 
107 CFU ml1 0.5 McFarland standard). Wells (8 mm
in diameter) were boarded into the agar media with
the help of sterilized cork borer. Then, 50 l each of
all solvent extracts were poured in the wells of the
inoculated plates. Antibiotics (24 ml per well) and
dimethyl sulfoxide (DMSO; 24 ml per well) were also
poured into the wells as positive and negative con-
trols, respectively. Inoculated plates were then incu-
bated at 37 C for 24 h and zones of inhibition were
measured in millimeter on the following day.
Results and discussion
The antimicrobial activities of the extracts of regener-
ated tissues and commercially available peppercorn
were investigated and given in terms of diameters for
the zones of inhibition (Figure 2). Samples were
applied against Gram negative bacterial strains (E. coli,
P. aeroginosa, and S. typhi), Gram positive bacterial
Ahmad et al. 3

Callus In vitro shoots In vitro plantlets Peppercorn

15 g (FW) 13 g (FW) 14 g (FW) 21 g (FW)

Dried

Thermo Scientific oven (50±2 ºC)

Grind

Callus (5g) Shoots (5g) Plantlets (5g) Fruits (5g)

50 ml Periodic shaking 50 ml

Ethanol Ethanol Ethanol Ethanol

+ + + +

Hexane Hexane Hexane Hexane

+ + + +
Chloroform Chloroform Chloroform Chloroform

Filter and rotary vacuum evaporator

Crude extract

Antipathogenic activities

Figure 1. Schematic representation of extract preparation.

strains (B. subtilis, B. cereus, and S. aureus), and a
fungal strain (C. albicans). Hexane extract of callus
showed 22 mm zone of inhibition against B. cereus
(Figure 3), while regenerated shoots and seeds have
shown 24.3 and 26 mm zones of inhibition. The pep-
percorn showed better activity against B. cereus
when the solvent was hexane. Chloroform extracts
of regenerated Piper shoots showed 16.9 mm activ-
ity against S. aureus (Figure 4). The ethanol extracts
of regenerated Piper shoots have shown 25 mm inhi-
bitory zone against S. aureus, 21 mm against B. cereus,
11.6 mm against S. typhi, and 16 mm in the case of C.
albicans. Peppercorn extracts in hexane, chloroform,
and ethanol had shown activities against B. cereus

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4 Toxicology and Industrial Health

Figure 2. Best zones of inhibition exhibited by different extracts against selected pathogenic microorganisms.

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26
S.aureus C S.aureus C
B.cereus C B.cereus C
Regenerated shoot extracts (mg/ml)
B.subtilis C B.subtilis C
S.typhi C S.typhi C
P.aeroginosa C P.aeroginosa C
Callus extracts (mg/ml)

E. coli C E. coli C
C.albicans E C.albicans E
S.aureus E S.aureus E
B.cereus E B.cereus E
B.subtilis E B.subtilis E
S.typhi E S.typhi E
P.aeroginosa E P.aeroginosa E
E. coli E E. coli E
S.aureus H S.aureus H
B.cereus H B.cereus H
B.subtilis H B.subtilis H
S.typhi H S.typhi H
P.aeroginosa H P.aeroginosa H
E. coli H E. coli H
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26
In vitro inhibitory activity (mm) In vitro inhibitory activity (mm)

Figure 3. Antimicrobial activities of callus extracts (H, E,
and C) against Escherichia coli, Pseudomonas aeroginosa, Sal-
monella typhi, Candida albicans, Bacillus subtilis, Bacillus cereus,
and Staphylococcus aureus. Mean values were taken from
three independent experiments. Mean values (+SD) are
significantly different when p < 0.05. H: hexane; E: ethanol;
C: chloroform.
Figure 4. Antimicrobial activities of regenerated shoots
extracts (H, E, and C) against Escherichia coli, Pseudomonas
aeroginosa, Salmonella typhi, Candida albicans, Bacillus subtilis,
Bacillus cereus, and Staphylococcus aureus. Values are mean of
three replicates along with SD. Column with each value are
significantly different at p < 0.05. H: hexane; E: ethanol; C:
chloroform.

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Ahmad et al. 5

Table 1. Standard antibiotic activities against selected microorganisms, values (mean + SD þ LSD) were taken in dupli-
cate from three independent experiments.a
Test organisms Ciprofloxacin Azithromycin Clotrimazole
b,*
Escherichia coli 43.16 + 1.04 0 0
Pseudomonas 34.36 + 0.62c 0 0
aeroginosa
Salmonella typhi 42.53 + 0.45b 0 0
Bacillus subtilis 0 25.50 + 0.50b 0
Bacillus cereus 0 20.16 + 0.62c 0
Staphylococcus 0 22.40 + 1.24c 0
aureus
Candida albicans 0 0 31.93 + 1.00b
a
Mean values with common alphabets are significantly different at p < 0.05; *Significance level is 0.05.

pathogenic microbes (Chaudhry and Tariq, 2006;

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
S.aureus C Ghori and Ahmad, 2009; Karsha and Lakshmi, 2010;
B.cereus C
B.subtilis C Pundir and Jain, 2010; Sasidharan and Menan, 2010;
Peppercorn extracts (mg/ml)

S.typhi C
P.aeroginosa C Yasmeen et al., 2012). These results showed that in
E. coli C
S.aureus E vitro culture extracts of this species possess efficient
B.cereus E
B.subtilis E and good activities against pathogenic strains. Patho-
S.typhi E genic agents from different sources enter into the
P.aeroginosa E
E. coli E human bodies and produce toxic metabolites causing
C.albicans E
S.aureus H different diseases and infections. Synthetic antibiotics
B.cereus H
B.subtilis H are used frequently against these pathogenic strains,
S.typhi H
P.aeroginosa H but nowadays, these microorganisms started resistance
E. coli H
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 due to mutations and other factors. To overcome such
In vitro inhibitory activity (mm) problem, plant-based products are more effective than
synthetic products and are also very ecofriendly.
Figure 5. Antimicrobial activities of peppercorn extract Under in vitro conditions, in vitro cultures produced
(H, E, and C) against toxic metabolites producing species. different active compounds that are free from patho-
Values are means of three replicates along with SD. Col- gens, safe, and that can be easily collected from con-
umn with each value are significantly different at p < 0.05.
tainers. In this study, we have used different extracts
H: hexane; E: ethanol; C: chloroform.
of in vitro cultures to check their efficacy against
(inhibition zone diameter of 26 mm, 23.6 mm, and 19.3 pathogenic strains. The results showed that these
mm, respectively) and B. subtilis (inhibition zone dia- extracts of in vitro cultures are nearly same in effi-
meter of 11.5 mm, 17.2 mm, and 23.5 mm, respec- cacy as standard antibiotics. These results suggested
tively; Figure 5). However, hexane, ethanol, and that such approaches should be upscaled into bior-
chloroform extracts of callus had shown activities eactor for higher biomass production and commer-
against B. cereus (22 mm, 19.7 mm, and 9.7 mm, cial purposes.
respectively) and S. aureus (15.3 mm, 23 mm, and
14 mm, respectively). Moreover, hexane extracts of Acknowledgement
peppercorn have also shown 8.7 mm zone against fun- The present work was financially supported by Higher Edu-
gal strain, C. albicans. It is interesting to note that cation Commission (HEC) of Pakistan.
extracts were more effective against bacteria than
fungi. The activities of standard antibiotics are given
in Table 1. However, there are no reports available References
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