Studies done on the hydro-ethanolic fruit extract of Parkia javanica plant to test and recognize its anti Vibrio cholerae activity.

1. PRESENTED BY-
RIKI NATH
M.SC. 4TH SEMESTER, 2018
ROLL NO.- 1606120021
REG. NO.- 07673 OF 2013-2014

2. Cholera is an acute diarrhoeal infection caused by ingestion of food or water
contaminated with the bacterium Vibrio cholerae.
Vibrio cholerae is a gram negative, facultative anaerobic, comma shaped bacterium.
It can kill a healthy individual within hours if they are not treated properly.
Cholera remains a global threat to public health and an indicator of lack of social
development.
Researchers have estimated that each year there are roughly 1.3 - 4 million cases and
21,000 – 143,000 deaths worldwide due to cholera.

3. • Vibrio cholerae are found in the
faeces of an infected individuals.
• Leakages in the sewage system may
contaminate the cleaned and potable
water making it unfit for drinking and
use.
• Dirty hands may transfer these
bacteria onto the food.
• Contaminated food or water serves as
medium for transmitting the disease
from one person to another.

4. Incubation period: 12 hours to 5 days
Symptoms:
 Acute watery diarrhoea
 Nausea and vomiting
 Mild to severe dehydration causing electrolyte imbalance
 Muscle cramps
 Hypovolemic shock
 Children are susceptible to hypoglycemia due to fluid loss leading to
seizures, coma

5. • British scientist John Snow hypothesized that the cholera
infections occurred from contaminated drinking water.
• In 1884 Robert Koch isolated the bacterium Vibrio cholerae
Robert KochJohn Snow

6. • The first pandemic cholera
occurred in 1817.
• To date, there have been 7
pandemics of cholera.
• The most recent epidemic
occurred following the Haiti
earthquake in 2010 which
caused over 9,000 deaths
from the disease.

7. Vibrio cholerae releases a toxin known as cholera toxin or choleragen.
Cholera toxin made up of 6 subunits:
• One A subunits (having A1 and A2 fragments)
• Five B subunits
A1 and A2 fragments are linked together by disulfide bonds.
When the toxin binds to the GM1 ganglioside receptor on the cell
membrane, the disulfide bridges are broken down .

9. B subunit of the cholera toxin
binds to the GM1 ganglioside
receptor on the cell membrane.

10. The binding causes breaking of the disulfide bonds between A1
and A2 fragments.
Endocytosis occurs.
The A1 fragment enters the cell.
A1 fragment binds to the G-protein in its activated GTP state
and catalyzes a covalent modification by adding an ADP- ribose
molecule to the G-protein.
This modification causes the G protein to be trapped in its
activated GTP state.

11.  Cholera can be treated with oral rehydration solution and antimicrobial
agents like tetracycline, ampicillin, nalidixic acid, erythromycin and
furazolidone.
 Antimicrobial resistance has developed against these antibiotics. There are
reports of multi drug resistant V. cholerae appearing with increasing
frequency.
 V. cholerae becomes drug resistant by exporting drugs through efflux
pumps, chromosomal mutations or developing genetic resistance via the
exchange of conjugative plasmids, conjugative transposons, integrons.
 WHO has suggested an urgent need to find new antimicrobial or new
approach to combat this serious issue.

12.  Parkia javanica is an ethnomedicinal plant in North- East India, widely used
by the people for the treatment of various ailments.
 The tribal people of Tripura (the Tripuri and Reang) use P. javanica extract to
cure stomach ache and cholera (Majumder et al., 2009).
 Although, Parkia javanica plant has antibacterial activity but there is no report,
on antibacterial activity against Vibrio cholerae strains and the molecular
mechanism behind this antibacterial activity.
 Therefore, this project has been undertaken to explore the anticholera
properties of Parkia javanica using three strains of Vibrio cholerae .

13. To determine the minimum inhibitory concentration
(MIC) of hydro-ethanolic extract of Parkia javanica fruit
(HEPJF) by serial dilution technique on Vibrio cholerae
strains.
To determine the minimum bactericidal concentration
(MBC) of the extract.
To determine the effect on reactive oxygen
species(ROS)generation.

15. • Fresh green fruits of Parkia javanica were grinded into 500 ml of 50% ethanol and
50% water mixture (hydro-ethanolic).
• Then the solution was filtered through Whatman filter paper no. 1 for 3 times and
dried in rotary evaporator at 70°C to prepare the hydro-ethanolic extract of Parkia
javanica fruit (HEPJF).
•
• Finally the extract was freeze- dried and stored at - 20°C.
• 100 mg of dried extract was dissolved in 1ml of 25% DMSO in water and filtered by a
0.22 µm syringe filter and stored at -20°C (Nikolic et al., 2014).
•

16. Vibrio cholerae strains Vibrio cholerae Classical Y 1254 (VC 01),
Vibrio cholerae 06 VTE 2523 (VC 02) and Vibrio cholerae 097
VTE 2357 (VC 03) were grown, cultured and maintained on Muller
Hinton Broth and stored at 4 ͦ C.

17. % of Inhibition = [1- {(Exp. - Blank) / (Positive Control – Negative Control)}* 100]
• MIC was determined by serial dilution technique, with
an inoculum of 106 CFU/ml of Vibrio cholerae strains
in separate 96 well plate, in presence of increasing
concentrations of HEPJF.
• The bacterial cultures were incubated at 37 ͦ C and
shaken at 200 rpm for 24 hours. Then the bacterial cell
viability was determined by measuring the OD value at
600 nm.
• Here, HEPJF with media, used as blank; Media,
HEPJF and bacterial culture, used as experiment;
media with bacterial culture and 25% DMSO, used as
positive control; and media with only 25% DMSO,
used as negative control. Then, % of Inhibition was
calculated by following formula:

18. • After determining the MIC values, MBCs for each
bacterial strains were determined by treating the
bacteria with 3 different doses, IC100, < IC100 and
>IC100 dose.
• After incubation with these 3 doses, one loop full
bacterial culture from each tube was streaked on
Muller Hinton agar plate in respective zone and
again these plates were incubated at 37 ͦ C for
overnight
• IC100 value indicates the concentration which
inhibits 100% of bacterial growth, whereas, MBC
value indicates the concentration at which a drug
can kill the bacterial species.

19. • 0.1 ml of each bacterial suspension (where
OD600 = 1.0) in Hank’s balanced salt solution
(HBSS) was incubated with respective IC50
dose of HEPJF for 3 hours with 15 min
interval at 37 ͦ C. Then 500 μl of 1 mg/ml
NBT was added and again incubated for 30
min at 37 ͦ C.
• After incubation, 0.1 (M) HCl was added and
tubes were centrifuged at 3000 rpm for 10
min. The pellets were treated with 0.6 ml of
DMSO to extract the reduced NBT.
• Then, 0.5 ml of HBSS was added and OD
was measured at 575 nm (intracellular ROS)
(Pramanik et al., 2012).

20. • The phytochemical screening of crude HEPJF was assessed by standard
method (Brinda et al., 1981; Siddiqui and Ali, 1997; Savithramma et
al., 2011).
• Phytochemical screening was performed to identify the important natural
chemical groups such as alkaloids, flavonoids, phenolic compounds,
steroids, terpenoids etc.
• Biochemical reactions in this screening revealed the presence and
absence of these compounds in the study plant (Anbukkarasi et al.,
2017).

22. Antibacterial activity of HEPJF on Vibrio cholerae strains were
obtained by determining the minimum inhibitory concentrations.
As shown in Table 1, the growth of all three Vibrio cholerae
strains were inhibited completely at 10 mg/ml concentration of
HEPJF, however the order of observed IC50 dose on three
bacterial strains were:
Vibrio cholerae 097 VTE 2357 (VC 03) < Vibrio cholerae 06 VTE
2523 (VC 02) = Vibrio cholerae classical Y 1254 (VC 01).

23. IC50 (mg/ml) IC100 (mg/ml)
Vibrio cholerae classical Y 1254 (VC 01) 5 ± 0.43 10 ± 0.22
Vibrio cholerae 06 VTE 2523 (VC 02) 5 ± 0.18 10 ± 0.24
Vibrio cholerae 097 VTE 2357 (VC 03) < 5 ± 0.34 10 ± 0.27
0
10
20
30
40
50
60
70
80
90
100
110
%ofInhibition
Concentration of HEPJF
VC 01
VC 02
VC 03
Table 1 : MIC values for Vibrio cholerae strains.
% of Inhibition of Vibrio cholerae strain in presence of P. javanica fruit extract (HEPJF)

24. MBC(mg/ml) MBC/MIC
Vibrio cholerae Classical Y 1254 (VC 01) 10 1
Vibrio cholerae 06 VTE 2523 (VC 02) 10 1
Vibrio cholerae 097 VTE 2357 (VC 03) 10 1
Table 2: MBC values for Vibrio cholerae strains.
Fig. 3: Muller Hinton Agar Plate showing MBC of HEPJF on Vibrio cholerae strains. A- Vibrio
cholerae Classical Y 1254, B- Vibrio cholerae 06 VTE 2523, C- Vibrio cholerae 097 VTE 2357

25. • Finally, to understand the mechanism of antibacterial activity of HEPJF, intracellular
reactive oxygen species (ROS) was estimated after treatment with HEPJF at IC50
dose.
• As shown in Fig 4, after treatment of HEPJF, the production of ROS was increased
drastically with time.
• It was highest in VC 06 VTE 2523 (VC 02), in which ROS production increased
about 60% in 3 hours compared to control, whereas in VC 097 VTE 2357 (VC 03),
ROS production increased about 35%.
• The order of observed ROS production on 3 Vibrio cholerae bacterial strains were:
VC 06 VTE 2523 (VC 02) > VC Classical Y 1254 (VC 01) > VC 097 VTE 2357 (VC 03).

26. 0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
%ofNormalizedROS
Time in min
VC 01
VC 03
VC 02
Fig.4. Effect of HEPJF on normalized % of ROS produced by Vibrio cholerae strains;
VC 01: Vibrio cholerae Classical Y 1254; VC 02: Vibrio cholerae 06 VTE 2523; VC 03:
Vibrio cholerae 097 VTE 2357.

27. • The phytochemical screening
was performed by testing the
extract in presence of certain
chemicals according to the
protocols (Brinda et al., 1981;
Siddiqui and Ali, 1997;
Savithramma et al., 2011).
• The extract showed the presence
of major classes of secondary
metabolites such as alkaloids,
flavonoids, phenolic
compounds, steroids and
terpenoids etc (Table 3).
Chemical Constituent Crude HEPJF
Alkaloids +
Flavonoids
+++
Phenolic Compounds +++
Steroids ++
Terpenoids
++
+++: abundant; ++: average; +: trace
Table 3: Chemical composition of crude HEPJF

28. The hydro-ethanolic extract of Parkia javanica fruit had antibacterial
activity against three Vibrio cholerae strains at 10 mg/ml concentration.
After treatment of HEPJF, the production of ROS in VC 06 VTE 2523
(VC 02) increased about 60% in 3 hours compared to control. The
phytochemical screening of the extract showed the presence of major
classes of secondary metabolites such as alkaloids, flavonoids, phenolic
compounds, steroids and terpenoids.
From this study we can conclude that, the hydro-ethanolic extract of
Parkia javanica may be used as a potent source of anti- Vibrio cholerae
agent.
Therefore, this study is justified to explore the proper use of this plant in
traditional medicine and this may be a promising plant and the result
confirms the use of this plant in traditional medicine for the treatment of
cholera infections.

29. It is an honour, a privilege and great pleasure for me to extend my sincere gratitude to
Prof. Samir Kumar Sil, Department of Human Physiology, Tripura University, who
encourage me and gave me the golden opportunity to do this wonderful project.
I am highly indebted to all my teachers Dr. Debasish Maiti , Dr. Sudipta Pal and Dr.
Dipayan Choudhuri, Department of Human physiology, Tripura University.
I am grateful to Smt. Supriya Sengupta and all other laboratory staff of the departments
for their co-operation and help in completion of my work.
At last I cordially express my special thanks to all research scholars of Molecular
Genetics and Cell Physiology Lab. For their kind support and help.
I would like to express my heartfelt gratitude to my parents for their unflagging love
and support throughout my life.

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