WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Shukla et al. World Journal of Pharmacy and Pharmaceutical Sciences
SJIF Impact Factor 6.041
Volume 5, Issue 03, 1621-1631. Research Article ISSN 2278 – 4357
ANTIBACTERIAL ACTIVITY OF CAROM SEED/ THYME
(TRACHYSPERMUMAMMI), ANISE (PIMPINELLAANISUM) AND DRY
GINGER (ZINGIBER OFFICINAL) AGAINST V. CHOLERA, V.
PARAHAEMOLYTICUS AND V. VULNIFICUS
Naveen Kumar Shukla1* and Uma Shankar2
1
Division of Microbiology, Singhania University, Rajasthan.
2
Department of Biotechnology, CytoGene Research & Development, Lucknow (U.P.)-India.
ABSTRACT
Article Received on
11 Jan 2016, This study aims to highlight the antibacterial property of three common
Revised on 03 Feb 2016, spices which are- carom seed (Trachyspermumammi), anise
Accepted on 24 Feb 2016
(Pimpinellaanisum) and dry ginger (Zingiber officinal) against
epidemic strains of V. parahaemolyticus, V. cholera and V. vulnificus.
*Correspondence for Aqueous extracts of all three spices were obtained using solvents
Author
methanol, water and acetone. Antimicrobial effects of acetone extracts
Naveen Kumar Shukla
of Anise (Pimpinellaanisum) was observed to be highest against Vibrio
Division of Microbiology,
Singhania University, cholerae (20 ± 0.22 mm). The acetone extract of Dry ginger (Z.
Rajasthan. officinale) demonstrated moderate antibacterial activity (23 ± 0.35),
and water extract showed strong inhibitory effects (30 ± 0.34) against
Vibrio parahaemolyticus. A similar strong inhibitory effect was seen in acetone (30 ± 0.30)
and water extract (30 ± 0.34) of thyme (Trachyspermumammi) against Vibrio cholera and
Vibrio vulnificus respectively.
KEYWORDS: Antibacterial activity, thyme, anise and dry ginger, zone of inhibition,
therapeutic.
INTRODUCTION
Since Vibrio cholerae is autochthonous to the aquatic environment (Colwell and Spira.1992,
West and Lee, 1982; Xu et al., 1982), monitoring this bacterium in water sources is important
for control of cholera. A selective medium, such as thiosulfate citrate bile salts sucrose
(TCBS) agar, eliminates most nontarget bacteria in clinical samples but is not satisfactory for
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Shukla et al. World Journal of Pharmacy and Pharmaceutical Sciences
environmental samples because many bacteria present in natural water sources can produce
colonies on TCBS agar whose appearance is similar to the appearance of V. cholerae
colonies. Furthermore, the series of biochemical tests commonly used to identify V. cholerae
(Baumann and Schubert, 1984; Farmer et al., 1985; Kay et al., 1994; West and Colwell,
1983) was originally designed for clinical samples in order to specifically detect pathogenic
vibrios. Molecular methods, including PCR and DNA-DNA hybridization performed with
probes specific for V. cholerae, provide more reliable identification (Chun et al., 1995;
Nandi et al., 2000)
The beneficial health effects of many plants, used for centuries as seasoning agents in food
and beverages, have been claimed for not only preventing food deterioration but also acting
as antimicrobials against pathogenic microorganisms. A few studies have been carried out in
a systematic manner, although phytochemical and pharmacological investigations of several
plants have already led to the isolation of some of the natural antimicrobials (Iwu et al.,
1999). Scientists are searching for natural products that can be used in large scale to reduce
diarrhoea caused by vibrios. Some natural compounds have been examined to act against
bacterial growth whereas little is known about specific influence on their virulence regulation
(Shinji et al., 2011). Plant-derived drugs remain an important resource, especially in
developing countries, to combat serious diseases. Some plants contain bioactive compounds
such as glycosides, alkaloids and terpenes which may be used as drugs and antimicrobial
agents (Kurita et al., 1982). Many extracts and essential oils have been derived from plants
and found to have antibacterial, fungicidal and insecticidal properties (Hänsel et al., 1999).
The essential oil of thyme (Thymus vulgaris) is utilized as a flavour enhancer in a wide
variety of foods, beverages, confectionery products and in perfumery for the scenting of
soaps and lotions (Arora and Kaur, 1999). It possesses some antiseptic, bronchiolytic,
antispasmodic and antimicrobial properties that make it popular as a medicinal herb and as a
preservative for foods Briozzo et al; Cosentino et al., 1999). The therapeutic potential of
thyme rests on its contents of flavonoids, thymol, eugenol, aliphatic phenols as well as
saponins, luteolin and tetramethoxylated flavones (Deans and Ritchie, 1987; Dorman and
Deans, 2000).
Pimpinella anisum (aniseed) is an herbaceous and the composition of anise varies
considerably with foundation and farming method. The main constituents of aniseed are
protein(18%),fatty oil( 8-23%) ,essential oil( 2-7%), starch( 5%),N-free extract( 22-28%) and
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Shukla et al. World Journal of Pharmacy and Pharmaceutical Sciences
crude fibre (12-25%). (Pruthi, 1976; Gerard, 1997) Aniseed also contains anethole, a
phytoestrogen so aniseed has been used for treatment of menstrual cramps (Albert-Puleo,
1980). Pimpinella anisum oil exhibited in vitro strong inhibitory activities against the growth
of a wide spectrum of bacteria and fungi known to be pathogenic for man and other species
(Muller-Schwarze, 2006; Elgayyar et al., 2001).
Ginger (Zingiber officinale), a member of the Zingiberaceae family, is a well-known spice
used in the daily diet in many Asian countries (Demin and Yingying, 2010). It is a
rhizomatous plant grown throughout South‐eastern Asia, China and in parts of Japan, Austria,
Latin America, Jamaica and Africa. It has been used as a spice and medicine in India and
China since ancient times. It was known in Germany and France in the ninth century and in
England in 10th century for its medicinal properties (Sasidharan and Menon, 2010). Over
three quarters of the world population still rely on plants and plant extracts for health care
(Sasidharan and Menon, 2010). Ginger compounds are active against specific type of
diarrhea which is leading to cause death in infant in developing countries. Moreover, it has
been found that ginger is effective in treating nausea caused by sea sickness, morning
sickness and chemotherapy, though it was found superior over a place for post operative
nausea (Sebiomo et al., 2011). In addition, it has been reported that the main ingredients of
ginger like volatile oil, gingerol, shogaol and diarylheptanoids work as antioxidant, anti-
inflammatory, anti-lipid, anti-diabetic, analgesic, antipyretic and anti-tumor (Demin and
Yingying, 2010; Sasidharan and Menon, 2010; Sebiomo et al., 2011; Lee et al., 1986;
Penna et al., 2003; Kadnur and Goyal, 2005; Islam and Choi, 2008; Kim et al., 2008; Isa
et al., 2008; Wang et al., 2009; Shim et al., 2011).
MATERIALS AND METHOD
Preparation of spice extract
Three spices namely thyme (Trachyspermumammi), anise (Pimpinellaanisum) and dry ginger
(Zingiber officinal) were collected from vendors in Lucknow. Extraction was done at 10%
(w/v) of spices and solvents (RANKEM Chemicals) for 48 hours, the extract were filtered
using cheese cloth and filtrate was used for further work. The solvents used for extract
preparation were acetone, methanol and distilled water.
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Maintenance of bacterial culture
The stock culture of the identified three vibrio species (V. cholera, V. parahaemolyticus and
V. vulnificus) in previous part of this work (Shukla and Shankar, 2016) was maintained at
4°C in Tryptone soya agar slants.
Antimicrobial Assay of three spices against V. cholera, V. parahaemolyticus and V.
vulnificus
The antimicrobial analysis method was performed using Agar well diffusion method (Bauer
and Kirby, 1966) to evaluate the antimicrobial properties of three spices: thyme
(Trachyspermumammi), anise (Pimpinellaanisum) and dry ginger (Zingiber officinal).
Tryptone soya agar plates were prepared to evaluate the antimicrobial activity of spices
extract, against selected Vibrio species.
Bacterial culture was uniformly spread on nutrient agar plates, after five minutes 6mm
diameter well was bored in the plates, spices extract; standard antibiotic solution (as positive
control) and solvents (as negative control) were poured into the well. The plates were
incubated at 370C for 24hr. and after incubation plates were observed for the zone of
inhibition (mm).
RESULT AND DISCUSSION
The first part of this work has described the identification, using multiplex PCR, of three of
the pathogenic species i.e., V. cholera, V. parahaemolyticus or V. vulnificus in different pond
waters collected from different regions of Uttar Pradesh.Also three common spices, Black
cardamom (Amomumsubulatum), Mustard seed (Brassica nigra) and Liquorice
(Glycyrrhizaglabra) extracts were analyzed for their antimicrobial activity against these three
vibrio species.
The antimicrobial assay result of spices showed a maximum activity by aqueous extract
Liquorice (Glycyrrhizaglabra) of 23 ± 0.52 (mm) against V.vulnificus followed by Mustard
seed (Brassica nigra) of 22.1 ± 0.60 (mm) against V.vulnificus. Methanolic and water
extracts of mustard seed also showed significant activity against V.cholera of 20.1 ±0.62
(mm) and 19 ± 0.59 (mm) respectively (Shukla and Shankar, 2016).
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Shukla et al. World Journal of Pharmacy and Pharmaceutical Sciences
Table 1: Identified vibrio species- V.cholerae, V.parahaemolyticus and V.vulnificus in
different pond water samples as reported in earlier part of this work (Shukla and
Shankar, 2016)
S.No. Location GC* YC* GC and YC V.V. V.C. V.P.
1 Indira Nagar - - -
2 Vaishnopuram P A - ✓
3 Sidhari P A -
4 Charbagh A P - ✓
5 Kakadeo A P - ✓
6 Khatima chauraha P A - ✓
7 Rawatpur P A -
8 Geetanagar P A - ✓
9 Kalyanpur - - -
10 Pratapgarh A P - ✓
A- Colony absent; P-Colony present
V.V.-Vibrio vulnificus; V.C.-Vibrio cholera; V.P.-Vibrio parahaemolyticus
The second part of this study aims to highlight the antibacterial property possessed by three
more spices which includes thyme (Trachyspermumammi), anise (Pimpinellaanisum) and dry
ginger (Zingiber officinal). Extracts were prepared in three solvent- Acetone, methanolic and
water and agar well diffusion method (Bauer and Kirby, 1966) was used for the assay. The
results observed are shown below in Table 1.
Figure 1: Antibacterial activity of spices by agar well diffusion method; Media used-
Tryptone
Soya Agar media; well size-4 mm. (a) carom seed extract activity against V.parahaemolyticus
(b)Anise extract against V.cholerae (c) Anise extract against V.haemolyticus
Antimicrobial effects of acetone extracts of Anise (Pimpinellaanisum) was observed to be
highest against Vibrio cholerae (20 ± 0.22 mm) contrary to activity reported by Gulcin et al.
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Shukla et al. World Journal of Pharmacy and Pharmaceutical Sciences
where study was done against 10 bacterial species and also Candida albicans with disc
diffusion method. In the study, ethanolic extract showed significant inhibitory activity against
all tested bacteria but not effective on Candida albicans. However, the antimicrobial effect of
water extract was not detected against Gram-negative bacteria, Pseudomonas aeruginosa, and
Escherichia coli, but it was effective against Candida albicans (Gulcin et al., 2003). The
alcoholic extracts of Pimpinella anisum seeds also showed antibacterial activity against
Micrococcus luteus and Mycobacterium smegmatis (Ates and Erdogrul, 2003).It was also
observed that methanolic extract showed no significant effect against any of the test bacterial
strains of vibrio, with no inhibitory zone against Vibrio parahaemolyticus and Vibrio
vulnificus (0.0 ± 0.0) and low inhibitory zone against Vibrio cholera.
Table 2: Antimicrobial activity of thyme (Trachyspermumammi), anise
(Pimpinellaanisum) and dry ginger (Zingiber officinal) extracts in 3 different solvents
against pathogenic Vibrio species
Zone of Inhibition (mm)
Spices used V.C V.P V.V
Act. 30 ± 0.30 0.0 ± 0.0 7 ± 0.21
Thyme
Met. 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
(Trachyspermumammi)
Wat. 20 ± 0.24 19 ± 0.20 30 ± 0.34
Act. 20 ± 0.22 16 ± 0.14 8 ± 0.11
Anise
Met. 11 ±0.32 0.0 ± 0.0 0.0 ± 0.0
(Pimpinellaanisum)
Wat. 12 ± 0.34 18 ± 0.32 0.0 ± 0.0
Act. 23 ± 0.35 9 ± 0.12 19 ± 0.21
Dry ginger (Zingiber
Met. 14 ± 0.16 0.0 ± 0.0 19 ± 0.24
officinal)
Wat. 15 ± 0.57 30 ± 0.34 20 ± 0.52
The acetone extract of Dry ginger (Z. officinale) demonstrated moderate antibacterial activity
(23 ± 0.35), and water extract showed strong inhibitory effects (30 ± 0.34) against Vibrio
parahaemolyticus. The effect of ethanolic and methanolic extracts of ginger were studied by
Bhargava et al. (2012), and have demonstrated similar MIC values of ethanolic extract
against tested bacteria. Taura et al. (2014) have showed that the ethanolic extract of ginger
was more effective on S. aureus (MIC at 100 µg/ml), but it did not act against E. coli and P.
aeruginosa. In another study done by Naji and Jassemi (2010) ethanolic extract of ginger
showed the best effect against P. aeruginosa and E. coli. It was also observed that methanolic
extract showed no significant effect against these gram negative bacteria, with no inhibitory
zone against Vibrio parahaemolyticus (0.0 ± 0.0) and low inhibitory zone against Vibrio
cholera (14 ± 0.16).
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Shukla et al. World Journal of Pharmacy and Pharmaceutical Sciences
Ivanovic et al. (2012) reported significant activity of the extract and essential oil of thyme
against E. coli and Salmonella strains, with MIC of 640 µg mL-1. Such activity was
attributed to the high concentration of thymol in the extract (39.7%) and in the essential oil
(48.49%). It was also reported antimicrobial activity of the essential oil of thyme against E.
coli 5% (V/V) and other food borne bacteria (Silva et al., 2013). A similar strong inhibitory
effect was seen in acetone (30 ± 0.30) and water extract (30 ± 0.34) of thyme
(Trachyspermumammi) against Vibrio cholera and Vibrio vulnificus respectively. Methanolic
extract showed no such activity against vibrio spp.
CONCLUSION
It was showed that the different solvent extracts of Thyme (Trachyspermumammi), Anise
(Pimpinellaanisum) and Dry ginger (Zingiber officinal) had variation in their antibacterial
effect against three pathogenic vibrio species i.e.., Vibrio cholera, Vibrio parahaemolyticus
and Vibrio vulnificus. Water extract in all the three spices had shown moderate to strong
inhibitory effect against these gram negative bacteria. Methanolic extract showed poor
activity with minimum or no zone of inhibition. These finding also highlights the potential
antibacterial activity against pathogenic species which are majorly responsible for water
borne diseases. The bacterial activity against pathogenic strains of bacteria was performed
using the agar well diffusion technique and showed that significant antibacterial activity.
Therefore focus must be shifted to identification of active compounds in these potentially rich
spices which are commonly used for their flavours and taste.
ACKNOWLEDGMENT
I am highly indebted to my advisor Dr. Uma Shankar for his guidance and constant
supervision as well as for providing necessary information regarding the study.
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