The International journal of analytical and experimental modal analysis ISSN NO:0886-9367

Studies of Antibacterial Activity of Tribulus Terrestris L. (Zygophyllaceae)

Mahesh Pathak and Ganesh Kulkarni
JES College, Jalana, Dist. Jalana (M.S.), India

Abstract
Antibacterial activity in the crude extracts of roots, stem, leaves and fruits of Tribuluss terrestris
L. was examined against the different human pathogenic bacteria viz. E. coli, Salmonella typhi,
Staphylococcus aureus, Pseudomonas aeruginosa and Shigella flexneri by 96 well-plate
methods. The results revealed that, the methanol fruit extract noticed minimum inhibitory
concentration (MIC) at 2µl for bacterium S. typhi, and S. flexneri while it was 4 µl for S. arueus,
P. aurignosa and S. flexneri whereas 8µl for E. coli. Stem extracts has 2µl for S. flexneri, P.
aurignosa and E. coli, 6µl for S. typhi and S. arueus whereas 10µl for E. coli. The MIC for leaf
extracts was found to be effective at 2µl for S. flexneri, and S. typhi, 6µl for P. aurignosa and S.
arueus whereas 4µl for E. coli. Root extracts showed the MIC at 8 and 10µl for S. typhi and S.
arueus, 2, 4, 6 and 8µl for P. aurignosa, 4 and 8µl for E. coli and 2µl for S. flexneri. Among
these root extract showed the maximum inhibition at 2µl for S. flexneri and 2, 4, 6 and 8µl for P.
aurignosa respectively compared to other bacterium.

Key words: Tribulus terrestris, antibacterial, 96 well-plates, MIC

Introduction:
Medicinal plants are an indispensable part of the traditional medicine system practiced all
over the world due to its low cost, easy access and ancestral experience (Jindal et al., 2012).
Indian ayurvedic system is one of the noteworthy systems of traditional medicine practice that
uses mainly plants in the treatments of ailments in man as well as other animals. In the past few
decades interest in the subject of antimicrobial plant extracts has been immensely growing. The
use of herbal medicines in Asia has a long history of human interactions with the environment.
Plants are frequently used for traditional medicine which contains a wide range of substances
that can be used to treat chronic as well as infectious diseases (Abtahi et al., 2014). T. terrestris
has some valuable medicinal properties which are used in sexual disorders, cardiac diseases,
antimicrobial activity, anthelmintic activity and anti-cancerous activity (Saima Hashim et al.,

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2014). It has been traditionally used as analgesic, anti-hypertensive, and diuretic agents. It is
also useful in the treatment of urinary tract infections (Sara Batoei et al., 2006). In the Chinese
traditional medicine system, T. terrestris has been used for treatment of respiratory tract
infections, mastitis and different kinds of eye infections (Wu et al., 1999). Dastagir et al., (2012)
studied the antibacterial activity of some selected plants of family Zygophyllaceae and
Euphorbiaceae, methanolic and n-hexane extracts of T. terrestris were tested against the
bacterium namely Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus
aureus and Klebsiella pneumoni extract showed the highest inhibition against E. coli (zone of
inhibition 10 mm). Screening of medicinal plants for antimicrobial activities and phytochemicals
is important for finding potential new compounds for therapeutic use.

Tribulus terrestris L. is belongs to family Zygophyllaceae, distributed throught the world

which is popularly known as Sarata (Marathi), Caltrop root (English), Gokhru (Hindi), Gokshura
(Bengali), Nerinjil (Tamil), Palleruveru (Telgu). Procumbent annual herb, prostrate; stem 30-
75cm long, terete, pubescent or glabrate. Leaves opposite, abruptly pinnate, in unequal pairs, 2.5-
5 cm long; petioles 5-10 mm long; stipules subulate, hairy; leaflets 3-6 in pairs, 5-10 mm long,
cuneate or rounded at base, mucronate, pubescent on both surface; petioles very short. Flowers
yellow, axillary solitary or leaf opposed, 6-10 mm across; pedicels 0.5-1.5 cm long, hairy. Sepals
lanceolate 4-5 mm long, acute, pubescent outside. Petals yellow or golden, obovate, twice as
long as sepals; Ovary is pubescent; style very short. Fruits globose, capsule, schizocarpic, 6-10
mm in diameter, with 5 woody mericarps; lateral spines much longer than basal ones, seed single
per mericarp.

Material and Methods

The plant material of T. terrestris L., was collected from Gogababa foot hill of university campus
of Dr. Babasaheb Ambedkar Marathwada University, Aurangabad district of Maharashtra state,
during rainy season and identified by using authentic literature (Naik, 1998, Singh et al., 2000).
The collected material was shade dried and used for the further study.

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Preparation of crude extracts

Dried powder of root, stem, leaf and fruit was extracted in Soxhlet extractor apparatus at 650 C
for 18-24 hours; methanol was used as solvent the prepared sample was stored in amber colour
bottle and used for the antibacterial studies.

Test microorganisms
The authentic human pathogenic bacteria culture namely E. coli, Pseudomonas aeruginosa,
Shigella flexneri, Salmonella typhi and Staphylococcus aureus were obtained from Indubai Patel
College of Pharmacy and Research Centre, Dharamraj, Dist. Anand, Gujarat.

Antibacterial Activity
96-well plates method
The antibacterial activity was carried out by micro dilution method on tested bacteria in order to
determine the antibacterial activity of root, stem, leaf and fruit extract. 100μl sterile Mueller-
Hinton broths was added onto each well, 2μl serial diluted bacterial suspension was loaded in
each well, different concentrations as 2, 4, 6, 8 and 10μl of root, stem, leaf and fruit extract was
loaded into each well. The prepared 96-well plate was sealed with parafilm and placed in
incubator at 37 °C for 24 hours, finally optical density was measured at 540 nm on the
spectrophotometer (Getachew, et al., 2015).

Results and Discussion

Methanolic fruit extracts of Tribulus terrestris L. in various concentrations were tested
against different human pathogenic bacteria, it was observed from the results that maximum
inhibition of S. typhi and S. flexneri were found in 2µl concentration, and in case of P.
aurignosa, S. arueus and S. flexneri it was 4µl whereas for E. coli it was 8µl. Regarding
minimum inhibitory concentration (MIC) it was 2 µl for S. typhi, and S. flexneri while it was 4µl
for S. arueus, P. aurignosa and S. flexneri whereas 8µl for E. coli (table 1, fig. 1). For the stem
extracts maximum inhibition of S. flexneri, P. aurignosa and E. coli were found in 2µl
concentration, and in case of S. typhi and S. arueus it was 6µl whereas for E. coli it was 10µl.

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Regarding minimum inhibitory concentration (MIC) it was 2µl for bacterium of S. flexneri, P.
aurignosa and E. coli while it was 6µl for S. typhi and S. arueus whereas 10µl for E. coli (table
2, fig. 2).

Leaf extracts showed the maximum inhibition for S. flexneri, and S. typhi were found in
2µl concentration, and in case of P. aurignosa and S. arueus it was 6 µl whereas for E. coli it
was 4µl. The minimum inhibitory concentration (MIC) was 2µl for S. flexneri, and S. typhi while
it was 6µl for P. aurignosa and S. arueus whereas 4µl for E. coli (table 3, fig. 3). For root
extracts maximum inhibition of S. typhi and S. arueus were found in 8 and 10µl concentration, in
case of P. aurignosa it was 2, 4, 6 and 8µl at this concentrations root extract were showed the
maximum inhibition against the bacterium whereas for E. coli it was 4 and 8µl and 2µl for S.
flexneri. Regarding minimum inhibitory concentration (MIC) it was 8 and 10µl for S. typhi and
S. arueus while it was 2, 4, 6 and 8µl for P. aurignosa respectively, whereas 4 and 8µl for E.
coli and 2µl for S. flexneri (table 4, fig 4).

Table 1: Antibacterial MIC activity of T. terrestris fruit on various human pathogens

T. terrestris Bacterial suspension (2µl conc.)

Sr.
fruit
No S. typhi S. flexneri P. aurignosa S. arueus E. coli
(conc. µl)
1 2 µl 0.06 0.06 0.08 0.08 0.06
2 4 µl 0.07 0.06 0.06 0.06 0.08
3 6 µl 0.08 0.09 0.08 0.25 0.09
4 8 µl 0.08 0.10 0.18 0.12 0.05
5 10 µl 0.08 0.09 0.22 0.10 0.19
MIC 2 µl 2, 4 µl 4 µl 4 µl 8 µl
S. E. ± 0.004 ±0.0083 ±0.0318 ±0.0335 ±0.0250

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Table 2: Antibacterial MIC activity of T. terrestris stem on various human pathogens

T. terrestris Bacterial suspension (2µl conc.)

Sr.
Stem
No S. typhi S. flexneri P. aurignosa S. arueus E. coli
(conc. µl)
1 2 µl 0.05 0.04 0.05 0.13 0.07
2 4 µl 0.05 0.05 0.06 0.07 0.08
3 6 µl 0.04 0.05 0.06 0.06 0.09
4 8 µl 0.06 0.05 0.07 0.07 0.08
5 10 µl 0.07 0.06 0.07 0.08 0.07
MIC 6 µl 2 µl 2 µl 6 µl 2,10 µl
S. E. ± 0.0050 ± 0.0031 ± 0.0037 ±0.0124 ± 0.0037

Table 3: Antibacterial MIC activity of T. terrestris leaf on various human pathogens

T. terrestris Bacterial suspension (2µl)

Sr.
Leaf
No S. typhi S. flexneri P. aurignosa S. arueus E. coli
(conc. µl)
1 2 µl 0.22 0.22 0.21 0.26 0.22
2 4 µl 0.26 0.26 0.23 026 0.21
3 6 µl 0.27 0.29 0.18 0.23 0.24
4 8 µl 0.25 0.37 0.24 0.27 0.29
5 10 µl 0.22 0.32 0.21 0.31 0.26
MIC 2, 10µl 2 µl 6 µl 6 µl 4 µl
S. E. ± 0.0102 ± 0.0255 ± 0.0102 ± 0.0102 ± 0.0143

Table 4: Antibacterial MIC activity of T. terrestris root on various human pathogens

T. terrestris Bacterial suspension (2µl)

Sr.
Root
No S. typhi S. flexneri P. aurignosa S. arueus E. coli
(conc. µl)
1 2 µl 0.02 0.00 0.00 0.02 0.01
2 4 µl 0.02 0.03 0.00 0.04 0.00
3 6 µl 0.03 0.04 0.00 0.02 0.01
4 8 µl 0.01 0.03 0.00 0.01 0.00
5 10 µl 0.01 0.01 0.03 0.01 0.01
MIC 8, 10 µl 2 µl 2,4,6,8 µl 8, 10 µl 4, 8 µl
S. E. ± 0.0037 ± 0.0073 ± 0.006 ± 0.0054 ± 0.0024

MIC = Minimum Inhibitory Concentration

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2 µl
0.25 4 µl
6 µl
8 µl
0.2 10 µl

0.15

0.1

0.05

0
S. typhi S. flexneri P. aurignosa S. arueus E. coli

Fig. 1: Antibacterial activity of T. terrestris fruit on human pathogen

0.14 2 µl
4 µl
0.12 6 µl
8 µl
0.1 10 µl

0.08

0.06

0.04

0.02

0
S. typhi S. flexneri P. aurignosa S. arueus E. coli

Fig. 2: Antibacterial activity of T. terrestris stem on human pathogen

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2 µl
0.4
4 µl
0.35 6 µl
8 µl
0.3 10 µl
0.25

0.2

0.15

0.1

0.05

0
S. typhi S. flexneri P. aurignosa S. arueus E. coli

Fig. 3: Antibacterial activity of T. terrestris leaf on human pathogen

0.04 2 µl
4 µl
0.035 6 µl
8 µl
0.03
10 µl
0.025

0.02

0.015

0.01

0.005

0
S. typhi S. flexneri P. aurignosa S. arueus E. coli

Fig. 4: Antibacterial activity of T. terrestris root on human pathogen

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Conclusion:
The root, stem, leaf and fruit extract of Tribulus terrestris L. has potential antibacterial agent as
it showed the significant activity against tested bacterial human pathogens. The results of present
study will form the basis for selection of Tribulus terrestris L. for further investigation in the
potential discovery of new herbal bioactive compounds. However, further studies are needed to
better evaluate the potential effectiveness of the crude extracts to find out the novel antibacterial
drugs.

Acknowledgement
Authors are grateful to the principal of Indubai Patel College of Pharmacy and Research Centre,
Dharamraj, Dist. Anand, Gujarat for providing all the necessary facilities for the present work.

References

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Dastagir Ghulam, Hussain Farrukh and Khan Abid Ali (2012). Antibacterial activity of some
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