The presentation is a research work carried out at SRISTI and PERD for the development of the antidiarrhoeal product using Tridax procumbens.

1. Evaluation of antimicrobial activity and
phyto-chemistry of Tridax procumbens
Linn. for Anti-Diarrhoea
By: Rohit Satyam, B.Tech. 3rd Yr,
Noida Institute of Engineering & Technology, Greater Noida, UP
Innovator’s Name: Sukhdeb Rana
BIIS-2
SRISTI Honey Bee Network

2. The
BIGGER
Picture

5. 10-15%
5-9%
1-4%
<1%
No data

6. Common
infective agents
Bacteria Virus Parasites
Vibrio parahaemolyticus Noro Virus Cryptosporidium
parvum
Salmonella species Rota Virus Giardia intestinalis
Diarrheagenic E. Coli Sapo Virus E. Histolytica
Campylobacter jejuni Adenovirus Dientamoeba fragilis
Vibro cholera O1 Blstocystis hominis
Clostridium difficile
Yersenia enterocolitica
Y. pseudotuberculosis
C. coli
C. Upsaliensis
Shigella Sp.

7. Our Aim: To validate
Innovator’s practice and provide
it scientific backbone.
Use of Tridax paste with a spoon of sugar, twice a day

8. bjective

9. Assess
Antimicrobial
Potential of
Bisalyakarani
(Tridax
procumbens)
against Diarrhoea
using

10. Rank Scientific Name and Common Name
Kingdom Plantae – Plants
Subkingdom Tracheobionta – Vascular plants
Superdivision Spermatophyta – Seed plants
Division Magnoliophyta – Flowering plants
Class Magnoliopsida – Dicotyledons
Subclass Asteridae
Order Asterales
Family Asteraceae ⁄ Compositae – Aster family
Genus Tridax L. – tridax
Species Tridax procumbens L. – coatbuttons,
Tridax daisy, Bishalya karani (Orissa)
Plant Herb
Habitat A small, straggling, procumbent, perennial
Systematic
Classification
of Plant
Tridax procumbens

11. Phytochemical
Extraction
Identification of
Probable Lead
Compounds
Quantitative estimation
of
Total Phenolic
Content
Methodology
Antimicrobial
Activity of
Phytoconstituents
Pipeline
Followed

12. Phytochemical Analysis

13. 3
1
2
4
Qualitative Analysis
Sun drying of
Tridax procumbens
Powder Formation
Selection of Solvents
Extraction of
Phytoconstituents using Hot
Extraction/ Cold Extraction

14. Hexane Extract Ethyl Acetate Methanol Extract Refluxing Method

15. S. No. Plant
Material
Weight (gm)
Solvent Used Yield or Extract Value =
Difference in weights,
(W2 – W1)
(gm)
% Yield = (W2 – W1)/ W
1. 5 Hexane (50ml*3) 0.121 2.42
2. 5 Ethanol (50ml*3) 0.169 3.38
3. 5 Ethyl Acetate
(50ml*3)
0.605 12.10
4. 5 Methanol (50ml*3) 0.727 14.54
5. 5 Water (75ml*3) 0.402 8.04

16. Antimicrobial
Activity
 Well Diffusion Assay
 Minimum Inhibitory
Concentration
 Minimum Bactericidal
Concentration
 TLC-Bioassay

17. S.
No.
Bacterial
Strains
Source ZOI (aqueous extract)
(100 µl ) (200 µl
)
1. Staphylococ
cus aureus
(G+ve)
NCIM
2079
Nil Nil
2. Escherichia
coli (G-ve)
NCIM
2065
Nil Nil
A. Well Diffusion Assay: Zones of inhibition
were not seen in the Nutrient Agar plates
where different concentrations of Tridax
were tested against the standard strains of
nosocomial pathogens.
Hence in this study, aqueous extract of the
leaves showed no antibacterial activity. The
standard NCIM strains of the Gram positive
and Gram negative organisms did not show
any sensitivity to the different
concentrations of ethanolic extracts of
Tridax procumbens.
Similar results were obtained in the MIC
Gentamycin (500 µg/ml) was used as positive
control and Water as negative control
Stock Aq.
Soln 100
mg/ml
Stock Aq.
Soln 200
mg/ml
Pilot study for aqueous extract

18. Control Used Stock Concentration
Positive control (Gentamycin) 500 µg/ml
Hydro-alcoholic 1000 µg/ml
Ethyl Acetate 1000 µg/ml
Methanol 1000 µg/ml
Aqueous 2500 µg/ml
ZOI- Zone of Inhibition
Preliminary
(Pilot)
Antimicrobial
studies
PC-Positive
culture

19. Organism Source Extract Used
ZOI
(mm)
Control Used
ZOI
(mm)
ZOI of +ve
Control
Type of Diarrhoea
caused
Staphylococcus
aureus (G+ve)
NCIM 2079
Hydro-alcoholic Nil Hydro-alcoholic Nil 25
Staohylococcal
enteritis Diarrhoea
Ethyl Acetate Nil Ethyl Acetate Nil 25
Methanol 13 mm Methanol Nil 25
Aqueous Nil Aqueous Nil 25
Salmonella
abony
(G-ve)
NCIM 2257
Hydro-alcoholic Nil Hydro-alcoholic Nil 25
Salmonellosis
Diarrhoea
Ethyl Acetate Nil Ethyl Acetate Nil 25
Methanol Nil Methanol Nil 25
Aqueous Nil Aqueous Nil 25
Escherichia coli
(G-ve)
NCIM 2065
Hydro-alcoholic Nil Hydro-alcoholic Nil 20
Non-bloody
Diarrhoea
Ethyl Acetate 11 mm Ethyl Acetate Nil 20
Methanol Nil Methanol Nil 20
Aqueous Nil Aqueous Nil 20
Pseudomonas
argenosa
NCIM 2200
Hydro-alcoholic Nil Hydro-alcoholic Nil 25
Non-bloody
Diarrhoea
Ethyl Acetate Nil Ethyl Acetate Nil 25
Methanol Nil Methanol Nil 25
Aqueous Nil Aqueous Nil 25

20. Ethyl Acetate Methanol
Terpenoids Anthocyanins
Flavanoids Terpenoids
Saponins
Tannins
Xanthoxyllines
Totarol
Quassinoids
Lactones
Flavones
Phenones
Polyphenols
The above tabulated compounds could be
expected to show up during the detailed
phytochemical analysis. Phenols, polyphenols,
Quinones, Flavonoids, Saponins, Alkaloids and
Tannins are reported to have as antimicrobial
and anti-diarrhoeal activity according to the
literature available.

21. B. Minimum Inhibitory Concentration (MIC)
MIC
Staphylococcus
aureus (G+ve)
in (mg/ ml)
Salmonella abony
(G-ve)
in (mg/ ml)
Escherichia coli
(G-ve)
in (mg/ ml)
Pseudomonas
argenosa
in (mg/ ml)
Hydro-
alcoholic
500 500 1000 1000
Ethyl Acetate 500 125 250 250
Methanol 62.5 31.25 62.5 250
Aqueous ND ND ND ND
The above mentioned MIC propose antimicrobial activity of extracts except aqueous
extract. The cidal or static activity was checked by carrying out MBC.
MBC was performed for
these three organisms

22. Minimum
Bactericidal
Concentration
RESULTS
Though the extract projected antimicrobial activity, bacterial growth
was observed during MBC. This shows Bacteriostatic property of
Tridax against representative bacteria.

23. Quantitative
Analysis
Preparation of
Gallic Acid Samples
Add Folin-Clocalteu
Reagent
Incubate for 5 min
Add 20% Na2CO3
Incubation for 30
min
Determination of
Mean Optical
Density
Methodology for
plotting calibration
curve for Gallic Acid

24. S. No Aliquot of
Gallic Acid
(ml)
Concentration
of Gallic Acid
(µg/ml)
MQ
(ml)
Folin
Ciocalteu
Reagent
(ml)
Incubationfor5minutes
20%
Na2CO3
(ml)
Incubationfor30minutes
Mean OD
(at 765
nm)
1. 0.1 2mg 10 1.5 4 0.035
2. 0.5 10mg 10 1.5
4 0.126
3. 1.0 20mg 10 1.5
4 0.241
For Methanolic Extract (20 mg/ml)

25. y = 0.0037x + 0.0162
R² = 0.9982
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 50 100 150 200 250 300
OD
CONCENTRATION
OD Linear (OD)
S. No.
Concentration
of Gallic Acid
(µg/ml)
Mean
Optical
Density
(OD)
1. 0 0
2. 50 0.205
3. 100 0.405
4. 150 0.593
5. 200 0.747
6. 250 0.941
(µg/ml)

26. Total Phenolic Content = 57.06 µg/ml
or
2.853 mg/ g
of Tridax powder (in terms of Gallic Acid
equivalents)
INFERENCE: Evidently, phenolic constituents have ability to inhibit enterpooling and delay gastro-
intestinal transit. Therefore, they are very useful in controlling Diarrhoea. Standardization of method of
preparation and estimation if they are present in sufficient is therefore required to decide if the plant
could be commercially exploited.

27. DPPH Assay for Radical
Scavenging Ability
determination
(2, 2 Diphenyl-1-picrylhydrazyl)

28. 54.48
49.39
45.52
0
10
20
30
40
50
60
1000 µg/ml 500 µg/ml 100 µg/ml
%INHIBITORYCONC.
CONCENTRATION
1000 µg/ml 500 µg/ml 100 µg/ml
The plant was
found to have
considerable
antioxidant activity
Antioxidants have free radical scavenging activity that are released during
Non-infectious diarrhoea such as in case of oxidative stress (irritable
bowel Syndrome), Lactose intolerance, and overdose of other drugs.

29. Conclusion & Value Addition

30. • While the Agar well diffusion study and MIC
revealed the antimicrobial activity of Ethyl
Acetate extract against E. Coli and
Methanolic extract activity against
Methanolic Extract, the aqueous extract did
not.
• The difference in activity between the
aqueous and alcoholic extracts can be
explained by the fact that different
solvents have varying capacities to extract
phytoconstituents based on their solubility
and polarity.
 Enzyme Inhibition
 Substrate Depriviation
 Complex with cell wall
 Membrane Disintegration
 Metal-ion complexation
 Intercalation into cell wall or
DNA of parasites
The possible mechanism of
cytic or cidal activity could
be due to:

31. The plant is preferable to treat Non-Infectious Diarrhoea that is caused by
Oxidative stress occurring inside subject’s body due to over production of Reactive
Oxygen Species (ROS)
Tridax showed considerable antioxidant activity. % Inhibition did not change
drastically with concentration.
From the Microbial studies carried out, it is conclusive that Tridax procumbens of
Orissa have minimal bacteriostatic properties and no bactericidal activity in the
present studies we carried out. But it possess considerable antioxidant properties
and could be used in symptomatic treatment rather than curative treatment.
Also, administration of Sugar twice a day with paste might reduce the Diarrheal
symptoms, sing sugar is found to have a role in Ion Balance and Osmoregulation.

32. The extractive value for Bioactive components could be enhanced by using Tridax of different region
besides that of Orissa. With commercialization point of view, the plant should be procured locally since it
is easily available weed.
The extractive value are expected to be high at the time of flowering period. Therefore time of
procurement should be considered for better yield.
The propagation of plant inside a separate facility will aid in maintaining standards and quality.
In-silico studies on Adenovirus, Norovirus can be performed to search therapeutic and prophylactic
candidates for Vaccine development against Viral Diarrhoea. This is advantageous since the preliminary
studies for virus in wet lab would be sumptuous and would require higher BSL level and skills.
The antimicrobial effects can be further studied in causative microorganisms with the high BSL facility, if
desired. Clinical trials of controlled subjects are suggested.
A comparative study of Tridax of different region can reveal metabolic pathways that are responsible for
phytochemicals that were found to be altogether absent in our plant of Orissa.
Future Scope of the present study

33. Thank You

34. Mobile Phase Ratio Solvent
No of Bands
(At 254 nm)
No of Bands
(At 366 nm)
Standards
Used
RESULTS
Toluene : Ethyl acetate : Formic acid : Methanol 3 : 3 : 0.8 : 0.2
Hexane 1 1
Ethyl acetate 3 2
Methanol 1 2
Toluene : Ethyl acetate : Formic acid : Methanol 4 : 8 : 0.5 : 0.2
Hexane 0 2
Ethyl acetate 3 3
Methanol 1 1
Chloroform : Acetic acid : Methanol 9 : 0.5 : 0.5
Hexane 4 3
Ethyl acetate 6 7
Methanol 4 4
Chloroform : Acetic acid : Methanol 7 : 0.5 : 0.5
Hexane 0 0
Ethyl acetate 0 0
Methanol 0 0
Chloroform : Acetic acid : Methanol 7 : 0.5 : 0.3
Hexane 0 0
Ethyl acetate 0 0
Methanol 0 1
Toluene : Ethyl acetate 9:01
Hexane 3 3
ß-sitosterol
& Luepiol
developeed
Ethyl acetate 5 6
Methanol 6 3
Toluene : Ethyl acetate 7:03
Hexane 1 2
Ethyl acetate 3 6
Methanol 3 4
Toluene : Ethyl acetate : Methanol 8:01:01
Hexane 2 2
Ethyl acetate 3 7
Methanol 2 4
Toluene : Ethyl acetate : Methanol 8:01:01
Hexane 0 0
Catechin &
Aigenin
Not
developed
Ethyl acetate 4 5
Methanol 4 4

36. 11 mm
13 mm
Pilot Study
Only Ethyl Acetate extract showed
activity against E. coli
Only Methanol extract showed activity
in Staphylococcus aureus

37. Extract
Wells
A
B
C
D
E
F
G
H
1000 mg/ml
500 mg/ml
250 mg/ml
125 mg/ml
62.5 mg/ml
31.25mg/ml
15.625 mg/ml
7.8125 mg/ml
Minimum Inhibitory Concentration of various Extracts of Tridax procumbens against Staphylococcus
aureus
Methanol Ethyl Acetate Hydroalcholic Aqueous
MIC

38. Methanolic
Samples
concentration
Absorbance pf
samples, As
Absorbance of
Control, Ac
% Inhibition =
100*(Ac – As )/ Ac
1000 mg/ml 0.5995 1.3170 54.48
500 mg/ml 0.6665 1.3170 49.39
100 mg/ml 0.7175 1.3170 45.52

39. S. No Test Method Observation Inference
Ethanol Ext.
1. Alkaloids (Dragendroff’s Test) Filtrate (400 µl) + Dragendroff’s Reagent (2-3
drops)
Reddish Brown ppt.
+
2. Triterpenoids (Salkowski’s
Test)
Filtrate (400 µl) + CHCl3 + of conc. H2SO4 +
Shake + Allow to stand
Greenish Yellow
color +
3. Flavanoids (Shinoda Test) and
(Lead Acetate Test)
Filtrate (400 µl) + Magnisium Ribbon + conc.
HCl
Filtrate (400 µl) + Lead acetate (10%) + NaOH
(10%) + Dil. HCl
No Change
White ppt.
-
+
4. Phenols and Tannins (Ferric
chloride Test)
Filtrate (400 µl)+ FeCl3 solution (3-4 drops) No Change
-
5. Saponins (Foam Test) Filtrate (400 µl) + Equivalent amount of H2O No change
-