Investigation of fresh vegetables and effects of vegetables extracts and to make it more effective than commercial antiobiotics by using Zingiber offinale (ginger) extract with vegetables against Bacterial strains.

1. M.Sc. Microbiology
(2019-2020)
Name –Roshni J.Darji
INVESTIGATION OF PHYTOCHEMICAL SCREENING,
ANTIMICROBIAL ACTIVITY AND ANTIOXIDANT
ACTIVITY OF FRESH VEGETABLES

2.  Contents:
 Introduction
Aim and Objectives
Materials and Methods
Results and Discussion
Summary and Conclusion
Future line of Investigation
References

3. Vegetables play an important role in human nutrition.
They supply dietary fiber and are important sources of essential vitamins, minerals, and trace
elements.
 When vegetables are included in the diet, there is found to be a reduction in the incidence
of cancer, stroke, cardiovascular disease, and other chronic diseases varies considerably; some vegetables
contains useful amounts of protein though generally they contain little fat, and varying proportions of
vitamins such as vitamin A, vitamin K, and vitamin B6 and carbohydrates.
Many nutritionists encourage people to consume plenty of fruit and vegetables, five or more portions a
day often being recommended.
Introduction

4. Vegetables possess immunomodulatory and antioxidant properties, leading to antibacterial activities.
The use of vegetable extracts and phytochemicals, both with known antimicrobial properties, can be of
great significance in therapeutic treatments.
They are known to have versatile immunomodulatory activity by stimulating both non-specific and specific
immunity.
Many vegetables have been used because of their antimicrobial traits, which are due to compounds
synthesized in the “secondary” metabolism of them.
Many plants belonging to the Tomato, Cucumber, cabbage , beetroot and carrot family, have a history
of health and fitness. Best known are beetroot,turmeric and tomato.

5. Aim and Objectives
The objectives of the present study are as follows:
 Extraction of Phytochemicals from Vegetables using Methanol and Chloroform.
To estimate phytochemicals of Brassica oleraceae, Solanum lycopersicum, Beta vulgaris, Daucus carota
and Cucumis sativus.
To determine Antibacterial effects of vegetables extracts and to make it more effective than commercial
antiobiotics by using Zingiber offinale (ginger) extract with vegetables against Bacterial strains.
To determine antioxidant activity of vegetable extract with and without Zingiber offinale.

6. Collection and Proceesing of Vegetables
Fresh Vegetables were collected and washed
properly with tap water
Rinsed with distilled water
Allowed the vegetables to dry for 2-3 days in shade
Crushed the vegetables with mechanical grinder.
Made dry powder of it
Stored the fine powder in airtight container in
refrigerator(4⁰C)
Prepararion of Vegetable Extracts
20 Grams dried powder + 150mL 90% Alcohol/
Chloroform
Kept on rotary shaker for 24 hours and than filterd
The liquid was stained off. The solid material was
pressed and than liquid was clarified using
muslin cloth
filtrates were air dried at room temperature.Moisture
was removed by vaccum oven( 50 ⁰C- 55 ⁰C)
Dried extracts were weighted to analyze the total
extract yield. Dissolved in Dimethyl sulfoxide
(DMSO). Stored in refrigerator for further use.

7. Microorganisms and Culture Conditions
• Grams positive bacteria:- Staphylococcus
aureus, Bacillis subtilis and Bacillus cereus.
• Gram negative bacteria:-Salmonella typhi,
Escherichia coli, Klebsiella pneumonia.
• All cultures were maintain by sub culturing on
nutrient agar slant and stored at 4°C in
refrigerator.
Culture media and Inoculums
preparation
• The different bacterial strain like gram positive
(staphylococcus aureus, Bacillissubtilis, Bacillus
cereus) and gram negative (Salmonella
typhi,Escherichia coli, Klebsiella pneumonia)
were inoculated in nutrient broth at 37°C for 24
hours.

8. Phytochemical analysis of different vegetable extracts
• Phytochemical analysis of methanol and chloroform extract were studied by following
methods, Steroids by Salkowski test, Flavonoids with the use of lead acetate, Saponins by foam test,
Tannins by ferric chloride test, Alkaloids by Mayer’s test, Carbohydrates by Molish test, Test for
Quinones and Reducing sugar by Benedict’s test
Test for Alkaloid:
•0.5mL extract+10mL
alcohol (boiled &filtered)
•5 ml filtrate+2mL dil
ammonia+5mL Chloroform
•Shake it gently& add mayers
reagent. Reddish brown preci
-pitates confirms positive
result
Test for Flavanoides:N
aOH Test:
•1-2 drops of aqueous NaO
H + few drops of HCL + 2
ml of vegetable extract
•yellow orange colour
Confirm positive result
Test for Tannins:Ferric
Chloride Test
•2 ml of vegetable extract+1
mL alcohol + 1 ml of neutral
ferric chloride
•blue or greenish colour
confirms positive result
Test for saponins:
Foam Test
•3 ml of water+ 2 ml of
vegetable extract(Shake it
vigorously)
•foam indicates the
presence of saponins
Test for Carbohydrates:
Molisch’s Test
•Few drops of Molisch’s
Reagent+ 1 ml extract followed
by 1mL of Conc. H2SO4
( Allowed
to stand for 2mins.)
•diluted with 5 ml of distilled
water . Red or dull violet
color at
Test for Steroids :
Salkowski Test
•2 ml of chloroform+ 0.5gm
extract+3mL Conc. H2SO4
to form layer
• reddish brown coloration
of the interface indicates
positive result
Test for Quinones:
Few drops of extracts+2 ml
of Con. HCL. yellow colour
precipitate indicates the
presence of quinones.
Test for Reducing sugar :
Benedicts test
•Few drops of extract+2ml
of Conc. HCL
•yellow color indicates the
presence of reducing sugar

9. Determination of Antimicrobial activity
Antibacterial activity of individual Vegetable extract,
mixture of three vegetable extract and mixture of all five
vegetable extract mixed with and without Zingiber offiinale
(ginger) against pathogen
• Well difusion method was performed for
antimicrobial activity of vegetable extract
• The lawn culture test organisms on nutrient agar
media were used and wells were made on
imoculated plates.
• Individual extract, mixture of three extracts and
mixture of five extracts with and without
Zingiber offiinale (ginger) were added in three
different wells (50 mg/ml,500 µl).
• Allowed to diffuse extract for 24hours and
incubated at 37˚C
• Determination was done by measuring
diameterof zone size
Antibacterial activity of Antibiotic against
pathogen
• Twelve different Antibiotics were used for
screening
• The lawn culture of test organisms on nutrient
agar media were used and wells were made on
imoculated plates.
• Antibiotics (50mg/ml,500 µl) was added into the
well
• Allowed to diffuse extract for 24hours and
incubated at 37˚C
• Determination was done by measuring
diameterof zone size
Nutrient broth was used as the media for culturing of bacterial strains . A loop full of bacterial culture were
inoculated in nutrient broth and incubated at 37˚C for 24 hours.

10. MIC (Minimum inhibitory concentration) determination: Agar macro-dilution method for
various vegetable extracts (Indiviual vegetable extract, mixture of three vegetable extract
and mixture of all five vegetable extract)without Zingiber offiinale (ginger) and with
Zingiber offiinale (ginger)
Microbes inoculated in flask containing 50mL nutient broth
flask incubated at 37˚C for 24 hour .Referred as seeded broth.
Muller hinton agar plates were made and inoculated.
Swabbing of test organisms from the seeded broth was done on
Muller hinton agar plates
After solidification of agar, 6mm wells were made by punching.
from 2,00,000 µg/ml to 195.31µg/ml concentration, extracts
were added in wells
Incubated for 24hours at 37˚C
MIC was defined as lowest concentration of extract in which no
growth was observed after incubation .

11. Antioxidant activity of various vegetable extracts (Indiviual vegetable extract,
mixture of three vegetable extract, mixture of all five vegetable extract)without
Zingiber offiinale (ginger) and with Zingiber offiinale (ginger)
A) Reducing power activity
• Extract (20, 40, 60, 80, 100 μg)+1.0 mL deionised
water +1.0 mL of 0.2 M phosphate buffer (pH
6.6)+1.0 mL of 1% potassium ferricyanide
Incubated at 50 °C for 20 min
1.0 ml of 10% trichloroacetic acid (TCA)
was centrifuged at 3000 rpm for 10 min.
2.5 ml supernatent+2.5 mL deionised water+0.5mL 0.1%
ferric chloride
absorbance was measured at 700 nm
higher absorbance indicates a higher reducing power.
B) Ames test
• 500µl (50 mg/ml) of extract was applied at centre of
glucose minimal agar plate seeded with test
organisms.
• Extract diffusion into the agar concentration gradient
is formedIf the chemical is mutagenic it will give
rise to a ring of revertant colonies surrounding the
area where the extract was applied. If the extract is
toxic it will give rise to zone of inhibition of growth.
Ethydium bromide was used as positive control to
determine the mutagenic effect of the extract

12. RESULT AND DISCUSSION
Phytochemical analysis:
⁕Table1 Phytochemical analysis of methanol extract of Vegetables:
Compounds
Methanol Extract
Brassica
oleracae
Solanum
lycopersicum
Beta
vulgaris
Daucus
carota
Cucumis
sativus
Alkaloids + + _ _ +
Flavonoids + + + + _
Steroids + _ + + +
Saponins _ + + + +
Tannins + + + + +
Carbohydrates + + _ + +
Quinones + + + _ _
Reducing
Sugar
+ + + + _

13. ⁕ Table2 Phytochemical analysis of Chloroform extract of Vegetables:
Compounds
Chloroform Extract
Brassica
oleracae
Solanum
Lycopersicum
Beta
vulgaris
Daucus carota Cucumis sativus
Alkaloids + _ + _ +
Flavonoids _ + + + +
Steroids _ + + _ _
Saponins + + _ _ +
Tannins _ _ + _ +
Carbohydrates _ _ _ _ +
Quinones + _ _ + _
Reducing
Sugar
+ + _ + +

14. Antibacterial activity
⁕Table 3 Antibacterial activity of Methanol extract of individual vegetables
Test organisms
Brassica
Oleracae
Solanum
lycopersicm
Beta
vulgaris
Daucus
carota
Cucumis
sativus
Escherichia coli - 25 13 - 15
Klebsiella
pneumonia
20 - 10 - 23
Salmonella typhi - 17 17 18 21
Staphylococccus
aureus
13 16 16 10 20
Bacillus cereus 10 13 18 12 14
Bacillus subtilis - 13 20 14 10
Zone of inhibition in mm

15. ⁕Table 4 Antibacterial activity of Methanol extract of individual vegetables mixed with
Zingiber offiinale (ginger).
Test organisms
Brassica
Oleracae+G
Solanum
lycopersicm+
G
Beta
vulgaris+G
Daucus
carota+G
Cucumis
sativus+G
Escherichia
coli
16 33 17 11 34
Klebsiella
pneumonia
22 13 12 14 28
Salmonella
typhi
11 20 23 24 26
Staphylococccus
aureus
16 22 19 15 23
Bacillus cereus 13 19 25 17 21
Bacillus subtilis 17 13 26 20 15
Zone of inhibition in mm

16. ⁕ Table 5 Antibacterial activity of Methanol mixture of three vegetable extract
Test organisms SL+DC+BV DC+BV+BO BV+BO+CS BO+CS+SL DC+CS+SL CS+BV+DC
Escherichia
coli
19 23 27 23 22 22
Klebsiella
pneumonia
22 20 30 26 23 23
Salmonella
typhi
24 20 19 25 20 20
Staphylococcus
aureus
24 25 28 23 20 20
Bacillus cereus 26 28 30 35 20 20
Bacillus subtilis 25 24 24 30 21 21
Zone of inhibition in mm

17. ⁕Table 6 Antibacterial activity of Methanol mixture of three vegetable extract mixed with
Zingiber offiinale (ginger)
Test organisms SL+DC+BV
+G
DC+BV+BO
+G
BV+BO+CS
+G
BO+CS+SL
+G
DC+CS+SL
+G
CS+BV+DC
+G
Escherichia
coli
25 31 35 29 28 32
Klebsiella
pneumonia
30 33 37 34 30 31
Salmonella
typhi
29 27 26 31 30 35
Staphylococcus
aureus
35 34 34 31 31 35
Bacillus cereus 33 36 37 40 30 33
Bacillus subtilis 32 31 31 36 29 28
Zone of inhibition in mm

18. Test organism
SL +BO +BV +DC +
CS
Escherichia coli 29
Klebsiella pneumonia 34
Salmonella typhi
36
Staphylococcus aureus
28
Bacillus cereus
30
Bacillus subtilis
33
⁕Table 7 Antibacterial activity of methanol extract
of mixture of all five vegetables
⁕Table 8 Antibacterial activity of methanol extract
of mixture of all five vegetable extract mixed with
Zingiber offiinale (Ginger)
Zone of inhibition in
mm
Test organism
SL +BO +BV +DC + CS +G
Escherichia coli 29
Klebsiella pneumonia 34
Salmonella typhi 36
Staphylococcus aureus 28
Bacillus cereus 30
Bacillus subtilis 33
Zone of inhibition in mm

19. Fig.1 Methenol extract of Cucumis
sativus mixed with Zingiber offiinale
(ginger) and Cucumis sativus
individual against Escherichia coli
Fig.2 Methanol extract of mixture
of BV+BO+CS and BV+BO+CS+
G against Bacillus cereus
Fig.3 Methanol extract Mixture of all
five vegetables with and without
Zingiber offiinale)

20. Table 9 Antibacterial activity of Chloroform extract individual vegetables
Test organisms Zone of inhibition in mm
Brassica
Oleracae
Solanum
lycopersicm
Beta
vulgaris
Daucus
carota
Cucumis
sativus
Escherichia coli - 20 13 10 15
Klebsiella pneum
onia
14 14 10 18 23
Salmonella typhi 13 13 12 17 21
Staphylococccus
aureus
10 11 11 13 20
Bacillus cereus 10 10 14 14 14
Bacillus subtilis 11 11 16 13 10

21. ⁕Table 10Antibacterial activity of chloroform extract of individual vegetables with
Zingiber offiinale (ginger)
Test organisms Zone of inhibition in mm
Brassica
Oleracae+
G
Solanum
lycopersicm
+G
Beta
vulgaris+
G
Daucus
carota+
G
Cucumis
sativus+
G
Escherichia
coli
16 33 17 11 34
Klebsiella pneu
monia
22 13 12 14 28
Salmonella
typhi
11 20 23 24 26
Staphylococccus
aureus
16 22 19 15 23
Bacillus cereus 13 19 25 17 21
Bacillus subtilis 17 13 26 20 15

22. ⁕Table 11 Antibacterial activity of chloroform mixture of three vegetable extract
Test organisms
Zone of inhibiton in mm
SL+D
C+BV
DC+B
V+BO
BV+B
O+CS
BO+C
S+SL
DC+C
S+SL
CS+B
V+DC
Escherichia coli 13 17 23 24 23 21
Klebsiella pneumonia 20 20 28 24 22 23
Salmonella typhi 23 22 21 24 19 27
Staphylococcus aureus 23 24 24 25 24 25
Bacillus cereus 25 27 32 30 22 27
Bacillus subtilis 27 26 25 28 21 22

23. *Table 12 Antibacterial activity of Chloroform mixture of three vegetable extract mixed with
Zingiber offiinale (ginger)
Test organisms
Zone of inhibition in mm
SL+DC
+BV+G
DC+ BV+
BO+ G
BV+BO
+CS+ G
BO+CS
+SL+G
DC+CS+
SL+G
CS+BV
+DC+G
Escherichia coli 25 24 24 25 29 31
Klebsiella pneumonia 29 31 35 37 31 30
Salmonella typhi 28 28 27 33 28 34
Staphylococcus aureus 33 30 31 30 30 32
Bacillus cereus 31 33 36 38 27 31
Bacillus subtilis 30 30 32 34 26 27

24. Test organism
SL +BO +BV +DC + CS
Escherichia coli 29
Klebsiella pneumonia 34
Salmonella typhi 36
Staphylococcus aureus 28
Bacillus cereus 30
Bacillus subtilis 33
Test organism Zone of inhibition in mm
SL +BO +BV +DC + CS +G
Escherichia coli 29
Klebsiella pneumonia 34
Salmonella typhi 36
Staphylococcus aureus 28
Bacillus cereus 30
Bacillus subtilis 33
Zone of Inhibition in mm
*Table 13 Antibacterial activity of Chloroform
extract of mixture of all five vegetables
*Table 14 Antibacterial activity of Chloroform
extract of mixture of all five vegetables mixed with
Zingiber offiinale (Ginger) .

25. Brassica Oleracae, Solanum
lycopersim, Beta vulgaris and
Cucumis sativus against
Escherichia coli
FigChloroform extract of SL+DC+
BV, DC+BV+BO, BV+BO+CS and
BO+CS+SL mixed with Zingiber
offiinale (ginger) against
Escherichia coli
Chloroform extract of Mixture
of all five vegetables with and
without Zingiber offiinale
against Bacillus cereus

26. ⁕ Table 9 Antibacterial activity of antibiotics
Test
organism
Zone of inhibition in mm
LE OX AM PIP GE CEF ME CL VA TE NE CP
Escherichia coli 27 - - - 21 - - - - 40 28 -
Klebsiella
pneumonia
27 - - - 14 - - - 26 32 19 36
Salmonella typhi 41 - 40 - 24 - - - - 36 - 40
Staphylococcus
aureus
40 40 38 - - - - - - - - -
Bacillus cereus 39 38 29 - 19 24 - - - - - 35
Bacillus subtilis 35 32 27 - 22 29 - - - - - 38
LE= Levofloxacin, OX=Oxacillin, AM= Ampicillin, PIP= Piperacillin, GE= Gentamycin, CEF= Cefluzidin,
ME= Methicillin, CL= Clindamycin, VA=Vancomycin, TE= Tetracyclin, NE= Neomycin, CP= Chloramphenicol.

27. • The antimicrobial activity of antibiotics against test organisms were performed and sensitivity of organisms
was known. Test organisms were sensitive to Levofloxacin(100%), Oxacillin (50%), Ampicillin (66.66%),
Gentamycin ( 83.3%), Cefluzidin (33.3%), Vancomycin (16%), Tetracyclin (50%), Neomycin (33.3%) and
Chloramphenicol (66.66%). Organisms were found resistant against Piperacillin, Methicillin and
Clindamycin.
Bacillus cereus is
sensitive to
Levofloxacin, Oxacillin,
and Gentamycin and is
ressistent to Piperacillin
Bacillus cereus is sensitive
to Cefluzidin and resistant
to Methicillin,
Clindamycin and
Vancomycin

28.  MIC of Methanol extract of. Brassica Oleracae for various microorganisms
 The MIC value of methanol extracts of Brassica Oleracae was found to be 100,000 µg/ml against
Bacillus subtilis, Escherichia coli and Klebsiella pneumonia.
 MIC of chloroform extract of. Brassica Oleracae for various microorganisms
 The MIC value of methanol extracts of Brassica Oleracae was found to be 100,000 µg/ml against Bacillus
subtilis and Klebsiella pneumonia.
 MIC of Methanol extract of. Brassica Oleracae with Zingiber offiinale for various microorganisms
 The MIC value of methanol extracts of Brassica Oleracae with Zingiber offiinale was found to be 50,000
µg/ml against Bacillus cereus, Staphylococcus aureus, Escherichia coli and Salmonella typhi.
 MIC of chloroform extract of Brassica Oleracae with Zingiber offiinale for various microorganisms
 The MIC value of methanol extracts of Brassica Oleracae was found to be 50,000 µg/ml against Bacillus
subtilis and Klebsiella pneumonia.
 MIC of Methanol extract of Solanum lycopersicum for various microorganisms
 The MIC value of methanol extracts of Solanum lycopersicum was found to be 100,000 µg/ml against
Bacillus subtilis and Klebsiella pneumonia.
 MIC of chloroform extract of Solanum lycopersicum for various microorganisms
 The MIC value of chloroform extracts of Solanum lycopersicum was found to be 100,000 µg/ml against
Bacillus subtilis..
 MIC of Methanol extract of Solanum lycopersicum with Zingiber offiinale for various microorganisms
 The MIC value of methanol extracts of Solanum lycopersicum with Zingiber offiinale was found to be
50,000 µg/ml against, Escherichia coli and Klebsiella Pneumonia

29.  MIC of chloroform extract of Solanum lycopersicum with Zingiber offiinale for various
microorganisms
 The MIC value of methanol extracts of Solanum lycopersicum was found to be 50,000 µg/ml against
Bacillus cereus and Salmonella typhi.
 MIC of Methanol extract of Beta vulgaris for various microorganisms
 The MIC value of methanol extracts of Beta vulgaris was found to be 100,000 µg/ml against Bacillus
subtilis, Staphylococcus aureus, Salmonella typhi and Klebsiella pneumonia.
 MIC of chloroform extract of Beta vulgaris for various microorganisms
 The MIC value of chloroform extracts of Beta vulgaris was found to be 100,000 µg/ml against Bacillus
subtilis, Escherichia coli and Salmonella typhi.
 MIC of Methanol extract of Beta vulgaris with Zingiber offiinale for various microorganisms
 The MIC value of methanol extracts of Beta vulgaris with Zingiber offiinale was found to be 50,000 µg/ml
against Bacillus subtilis, Staphylococcu aureus Escherichia coli, Salmonella typhi and Klebsiella
Pneumonia.
 MIC of chloroform extract of Beta vulgaris with Zingiber offiinale for various microorganisms
 The MIC value of chloroform extracts of Beta vulgaris was found to be 50,000 µg/ml against
Staphylococcus aureus and Salmonella typhi.
 MIC of Methanol extract of Daucus carota for various microorganisms
 The MIC value of methanol extracts of Daucus carota was found to be 100,000 µg/ml against Bacillus
cereus, bacillu subtilis, Escherichia coli and Salmonella typhi
 MIC of chloroform extract of Daucus carota for various microorganisms
 The MIC value of chloroform extracts of Daucus carota was found to be 100,000 µg/ml against Bacillus
cereus, bacillus subtilis and Salmonella typhi

30.  MIC of Methanol extract of Daucus carota with Zingiber offiinale for various microorganisms
 The MIC value of methanol extracts of Daucus carota with Zingiber offiinale was found to be 50,000 µg/ml
against Bacillus cereus, Staphylococcu aureus, Salmonella typhi, and Klebsiella Pneumonia.
 MIC of chloroform extract of Daucus carota with Zingiber offiinale for various microorganisms
 The MIC value of chloroform extracts of Daucus carota with Zingiber offiinale was found to be 50,000
µg/ml against Bacillus cereus, Staphylococcu aureus, Salmonella typhi, and Klebsiella Pneumonia.
 MIC of Methanol extract of Cucumis sativus for various microorganisms
 The MIC value of methanol extracts of Daucus carota was found to be 100,000 µg/ml against Bacillus
cereus, bacillu subtilis, Staphylococcus aureus Escherichia coli, Klebsiella pneumonia and Salmonella typhi
.
 MIC of chloroform extract of Cucumis sativus for various microorganisms
 The MIC value of chloroform extracts of Cucumis sativus was found to be 100,000 µg/ml against Bacillus
cereus and Salmonella typhi
 MIC of Methanol extract of Cucumis sativus with Zingiber offiinale for various microorganisms
 The MIC value of methanol extracts of Cucumis sativus with Zingiber offiinale was found to be 50,000
µg/ml against Bacillus cereus, Staphylococcu aureus and Klebsiella Pneumonia.
 MIC of chloroform extract of Cucumis sativus with Zingiber offiinale for various microorganisms
 The MIC value of chloroform extracts of Cucumis sativus with Zingiber offiinale was found to be 50,000
µg/ml against Staphylococcu aureus and Klebsiella Pneumonia.
 MIC of Methanol extract of mixture of (Solanum lycopersicum, Daucus carota and Beta vulgaris )for
various microorganisms
 The MIC value of methanol extracts of mixture of (Solanum lycopersicum, Daucus carota and Beta
vulgaris ) was found to be 25,000 µg/ml against bacillus subtilis and Klebsiella pneumonia

31.  MIC of chloroform extract of mixture of (Solanum lycopersicum, Daucus carota and Beta vulgaris)
for various microorganisms
 The MIC value of chloroform extracts of mixture of (Solanum lycopersicum, Daucus carota and Beta
vulgaris ) was found to be 25,000 µg/ml against Klebsiella pneumonia.
 MIC of Methanol extract of (Solanum lycopersicum, Daucus carota and Beta vulgaris )with Zingiber
offiinale for various microorganisms
 The MIC value of Methanol extracts of Solanum lycopersicum, Daucus carota and Beta vulgaris )with
Zingiber offiinale was found to be 12,500 µg/ml against Bacillus cereus, Bacillus subtilis, Escherichia coli
and Salmonella typhi.
 MIC of Methanol extract of (Solanum lycopersicum, Daucus carota and Beta vulgaris )with Zingiber
offiinale for various microorganisms
 The MIC value of chloroform extracts of Solanum lycopersicum, Daucus carota and Beta vulgaris )with
Zingiber offiinale was found to be 12,500 µg/ml against Bacillus cereus, Bacillus subtilis and Escherichia
coli.
 25 MIC of Methanol extract of mixture of (Daucus carota, Beta vulgaris and Brassica Oleracae )for
various microorganisms
 The MIC value of methanol extracts of mixture of (Daucus carota, Beta vulgaris and Brassica Oleracae )
was found to be 25,000 µg/ml against bacillus subtilis Escherichia coli and Klebsiella pneumonia.
 MIC of Chloroform extract of mixture of (Daucus carota, Beta vulgaris and Brassica Oleracae )for
various microorganisms
 The MIC value of methanol extracts of mixture of (Daucus carota, Beta vulgaris and Brassica Oleracae )
was found to be 25,000 µg/ml against bacillus subtilis Escherichia coli and Klebsiella pneumonia.

32.  MIC of Methanol extract of (Daucus carota, Beta vulgaris and Brassica Oleracae )with Zingiber
offiinale for various microorganisms
 The MIC value of Methanol extracts of (Daucus carota, Beta vulgaris and Brassica Oleracae )s )with
Zingiber offiinale was found to be 12,500 µg/ml against Bacillus cereus, Escherichia coli, Salmonella
typhi. and lkebsiella pneumonia
 MIC of Chloroform extract of (Daucus carota, Beta vulgaris and Brassica Oleracae )with Zingiber
offiinale for various microorganisms
 The MIC value of chloroform extracts of (Daucus carota, Beta vulgaris and Brassica Oleracae ) with
Zingiber offiinale was found to be 12,500 µg/ml against Bacillus cereus, Salmonella typhi. and klebsiella
pneumonia.
 MIC of Methanol extract of mixture of (,Beta vulgaris, Brassica Oleracae and cucumis sativu s)for
various microorganisms
 The MIC value of methanol extracts of mixture of (Beta vulgaris, Brassica Oleracae and cucumis sativas)
was found to be 25,000 µg/ml against bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Escherichia
coli, and Klebsiella pneumonia.
 MIC of Chloroform extract of mixture of (Beta vulgaris, Brassica Oleracae and cucumis sativus)for
various microorganisms
 The MIC value of chloroform extracts of mixture of (Beta vulgaris, Brassica Oleracae and cucumis
sativas) was found to be 25,000 µg/ml against bacillus subtilis, Bacillus cereus, Staphylococcus aureus,
Escherichia coli, and Klebsiella pneumonia.

33.  MIC of Methanol extract of (Beta vulgaris, Brassica Oleracae and cucumis sativus) with Zingiber
offiinale for various microorganisms
 The MIC value of Methanol extracts of (Beta vulgaris, Brassica Oleracae and cucumis sativus)with
Zingiber offiinale was found to be 12,500 µg/ml against Bacillus cereus, Salmonella typhi. and klebsiella
pneumonia
 MIC of Chloroform extract of (Beta vulgaris, Brassica Oleracae and cucumis sativus) with Zingiber
offiinale for various microorganisms
 The MIC value of Methanol extracts of (Beta vulgaris, Brassica Oleracae and cucumis sativus)with
Zingiber offiinale was found to be 12,500 µg/ml against Bacillus cereus, Escherichia coli, Salmonella
typhi. and klebsiella pneumonia
 MIC of Methanol extract of mixture of (Brassica Oleracae, cucumis sativus and (Solanum
lycopersicum )for various microorganisms
 The MIC value of methanol extracts of mixture of (Brassica Oleracae, cucumis sativus and (Solanum
lycopersicum ) was found to be 25,000 µg/ml against bacillus subtilis, Staphylococcus aureus, Escherichia
coli, and Klebsiella pneumonia.
 MIC of Chloroform extract of mixture of (Brassica Oleracae, cucumis sativus and (Solanum
lycopersicum )for various microorganisms
 The MIC value of chloroform extracts of mixture of (Brassica Oleracae, cucumis sativus and (Solanum
lycopersicum ) was found to be 25,000 µg/ml against Staphylococcus aureus, Escherichia coli, and
Klebsiella pneumonia.
 MIC of Methanol extract of (Brassica Oleracae, cucumis sativus and (Solanum lycopersicum ) with
Zingiber offiinale for various microorganisms
 The MIC value of Methanol extracts of (Brassica Oleracae, cucumis sativus and (Solanum
lycopersicum)with Zingiber offiinale was found to be 12,500 µg/ml against Bacillus cereus, Staphylococcus
aureus, Salmonella typhi. and klebsiella pneumonia

34.  MIC of Chloroform extract of (Brassica Oleracae, cucumis sativus and (Solanum lycopersicum ) with
Zingiber offiinale for various microorganisms
 The MIC value of Chloroform extracts of (Brassica Oleracae, cucumis sativus and (Solanum
lycopersicum)with Zingiber offiinale (table 4.51) was found to be 12,500 µg/ml against Bacillus cereus,
Staphylococcus aureus and Salmonella typhi.
 MIC of Methanol extract of ( Daucus carota, cucumis sativus and solanum lycopersicum) for various
microorganism.
 The MIC value of methanol extracts of ( Daucus carota, cucumis sativus and solanum lycopersicum) ( table
4.52) was found to be 25,000 µg/ml against bacillus subtilis, bacillus cereus, Staphylococcus aureus,
Escherichia coli, Salmonella typhi and Klebsiella pneumonia.
 MIC of Chloroform extract of ( Daucus carota, cucumis sativus and solanum lycopersicum) for various
microorganism.
 The MIC value of chloroform extracts of ( Daucus carota, cucumis sativus and solanum lycopersicum) (
table 4.52) was found to be 25,000 µg/ml against Staphylococcus aureus, Escherichia coli, Salmonella typhi
and Klebsiella pneumonia.
 MIC of Methanol extract of ( Daucus carota, cucumis sativus and solanum lycopersicum) with
Zingiber offiinale for various microorganisms
 The MIC value of Methanol extracts of ( Daucus carota, cucumis sativus and solanum lycopersicum) with
Zingiber offiinale (table 4.50) was found to be 12,500 µg/ml against Bacillus cereus, Staphylococcus
aureus, Salmonella typhi. and klebsiella pneumonia
 MIC of Chloroform extract of ( Daucus carota, cucumis sativus and solanum lycopersicum) with
Zingiber offiinale for various microorganisms
 The MIC value of Chloroform extracts of (( Daucus carota, cucumis sativus and solanum lycopersicum)
with Zingiber offiinale (table 4.51) was found to be 12,500 µg/ml against Bacillus cereus, Staphylococcus
aureus and Salmonella typhi.

35.  MIC of Methanol extract of ( Daucus carota, cucumis sativus and Beta vulgaris for various
microorganism.
 The MIC value of methanol extracts of (Daucus carota, cucumis sativus and Beta vulgaris was found to be
25,000 µg/ml against bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella typhi and
Klebsiella pneumonia.
 MIC of Chloroform extract of (Daucus carota, cucumis sativus and Beta vulgaris) for various
microorganism.
 The MIC value of chloroform extracts of (Daucus carota, cucumis sativus and Beta vulgaris was found to
be 25,000 µg/ml against bacillus cereus, Salmonella typhi and Klebsiella pneumonia.
 MIC of Methanol extract of ( Daucus carota, cucumis sativus and Beta vulgaris) with Zingiber
offiinale for various microorganisms
 The MIC value of Methanol extracts of (Daucus carota, cucumis sativus and solanum lycopersicum) with
Zingiber offiinale was found to be 12,500 µg/ml against Bacillus cereus,Bacillus subtilis, Staphylococcus
aureus,Escherichia coli Salmonella typhi. and klebsiella pneumonia
 MIC of Chloroform extract of ( Daucus carota, cucumis sativus and Beta vulgaris) with Zingiber
offiinale for various microorganisms
 The MIC value of chloroform extracts of ( Daucus carota, cucumis sativus and solanum lycopersicum)
with Zingiber offiinale was found to be 12,500 µg/ml against Bacillus cereus,Bacillus subtilis, Escherichia
coli and klebsiella pneumonia
 MIC of Methanol extract of all five vegetables ( Daucus carota, cucumis sativus, Beta vulgaris, Solanum
lycopersicum and Brassica oleracae for various microorganism
 The MIC value of methanol extracts of all five vegetables (Daucus carota, cucumis sativus, Beta vulgaris,
Solanum lycopersicum and Brassica oleracae) was found to be 6,250 µg/ml against bacillus cereus and
bacillus subtilis.

36.  MIC of Chloroform extract of all five vegetables ( Daucus carota, cucumis sativus, Beta vulgaris,
Solanum lycopersicum and Brassica oleracae for various microorganism
 The MIC value of chloroform extracts of all five vegetables (Daucus carota, cucumis sativus, Beta vulgaris,
Solanum lycopersicum and Brassica oleracae) was found to be 6,250 µg/ml against bacillus subtilis.
 MIC of Methanol extract of all five vegetables ( Daucus carota, cucumis sativus, Beta vulgaris, Solanum
lycopersicum and Brassica oleracae) with Zingiber offiinale for various microorganism
 The MIC value of methanol extracts of all five vegetables (Daucus carota, cucumis sativus, Beta vulgaris,
Solanum lycopersicum and Brassica oleracae) was found to be 3,125 µg/ml against bacillus cereus,bacillus
subtilis, Staphylococcus aureus,Escherichia coli, Salmonella tyhi and Klebsiella pneumonia..
 MIC of Chloroform extract of all five vegetables ( Daucus carota, cucumis sativus, Beta vulgaris,
Solanum lycopersicum and Brassica oleracae) with Zingiber offiinale for various microorganism
 The MIC value of chloroform extracts of all five vegetables (Daucus carota, cucumis sativus, Beta vulgaris,
Solanum lycopersicum and Brassica oleracae) was found to be 3,125 µg/ml against bacillus cereus,bacillus
subtils, Escherichia coli and Klebsiella pneumonia..

37. Antioxidant activity of various Vegetable extracts:(Reducing power activity (Individual
extracts with and without Zingiber offiinale)
a) b) c)
d) e) e) f) (f)
(d) Daucus carota+ Zingiber offiinale (e) Beta vulgaris
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
20 40 60 80 100
Reducingpoweractivity(ODat
700nm)
Concentration
Methanol
Chloroform
Reducing power activity of
(a) Brassica oleracae
b) Brassica oleracae + Zingiber (c) Daucus carota
offiinale
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
20 40 60 80 100
Reducingpoweractivty
(ODat700nm)
Concentration
Methanol
Chloroform
0
0.02
0.04
0.06
0.08
20 40 60 80 100
Reducingpower
activity(ODat700nm)
Concetration
Methanol
Chloroform
0
0.02
0.04
0.06
0.08
0.1
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
0
0.01
0.02
0.03
0.04
0.05
20 40 60 80 100
Reducingpower
activity(ODat700nm)
Concentration
Methanol
Chloroform
0
0.02
0.04
0.06
0.08
0.1
0.12
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
Chloroform
f) Beta vulgaris+ Zingiber offiinale

38. g) h)
(g) Cucumis sativus (h) Cucumis sativus+ Zingiber offiinale
i) j)
(i) Solanum lycopersicum
0
0.005
0.01
0.015
0.02
0.025
20 40 60 80 100
Reducingpoweractivity(oD
at700nm)
Concentration
Methanol
Chloroform
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
20 40 60 80 100
Reducingpoweractivity(OD
at700nm)
Concentration
Methanol
Chloroform
0
0.01
0.02
0.03
0.04
0.05
0.06
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
concentration
Methanol
Chloroform
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
(j) Solanum lycopersicum+
Zingiber offiinale
In order to evaluate reducing pow
er activity, all the extract were incu-
bated with ferricyanide complex an
d observed for their efficiency to
reduce this complex in ferrous form.
 The results obtained from the sets
of experiments showed that,
maximum reduction of ferricyanide
complex was observed with Beta vu
lgaris+ Zingiber offiinale (0.08-0.1)

39. Reducing power activity (Mixture of three Vegetable extracts with and without Zingiber
offiinale)
0
0.01
0.02
0.03
0.04
0.05
20 40 60 80 100
Reducingpower
activity(ODat700nm)
Concentration
Methanol
Chloroform
a)
0
0.1
0.2
0.3
0.4
0.5
0.6
20 40 60 80 100
Reducingpoweractivity
(ODAT700NM
Concentration
Methanol
Chloroforrm
b)
0
0.05
0.1
0.15
0.2
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
Chloroformc)
0
0.05
0.1
0.15
0.2
0.25
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
Chloroformd)
0
0.05
0.1
0.15
0.2
0.25
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methan…
Chlorof…
e)
0
0.05
0.1
0.15
0.2
0.25
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
Chlorof…f)
(a)Solanum lycopersicum+ Daucus carota
+ Beta vulgaris
(b) Solanum lycopersicum+ Daucus carota
+Beta vulgaris + Zingiber offinale
(c) Daucus carota + Beta vulgaris
+ Brassica oleracae
(d) Daucus carota + Beta vulgaris +
Brassica oleracae+ Zingiber offinale
(e)Beta vulgaris + Brassica oleracae
+ Cucumis sativus
(f) Beta vulgaris + Brassica oleracae +
Cucumis sativus+ Zingiber offinale

40. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
Chloroform
g)
0
0.05
0.1
0.15
0.2
0.25
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
Chloroform
h)
0
0.1
0.2
0.3
0.4
0.5
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
0
0.05
0.1
0.15
0.2
0.25
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
Chloroform
i)
j)
0
0.02
0.04
0.06
0.08
0.1
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
0
0.05
0.1
0.15
0.2
20 40 60 80 100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
Chlorofor…
k) l)
(g) Brassica oleracae + Cucumis sativus+
Solanum lycopersicum
(h) Brassica oleracae + Cucumis sativus+
Solanum lycopersicum+ Zingiber offinale
(i) Daucus carota+Cucumis sativus+
Solanum lycopersicum
(j) Daucus carota+Cucumis sativus+
Solanum lycopersicum+ Zingiber offinale
(k) Cucumis sativus+ Beta vulgaris+
Brassica oleracae
(l) Cucumis sativus+ Beta vulgaris+
Brassica oleracae+ Zingiber offinale

41. 0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
20
40
60
80
100
Reducingpoweractivity
(ODat700nm)
Concentration
Methanol
Chlorofo…
0.98
1
1.02
1.04
1.06
20 40 60 80 100
Reducingpower
activity(ODat700nm)
Concentration
(a)Brassica oleracae + Beta vulgaris+ Daucus carota+
Solanum lycopersicum+ Cucumis sativus
(b) Brassica oleracae + Beta vulgaris +Daucus carota
+Solanum lycopersicum + Cucumis sativus +
Zingiber offinale
•In order to evaluate reducing power activity, all the extract were incubated with ferricyanide complex and observed for their
efficiency to reduce this complex in ferrous form.
•The results obtained from the sets of experiments showed that, maximum reduction of ferricyanide complex was observed with
Brassica oleracae + Beta vulgaris +Daucus carota +Solanum lycopersicum + Cucumis sativus + Zingiber offinale (1.056).
•In order to evaluate reducing power activity, all the extract were incubated with ferricyanide complex and observed for their
efficiency to reduce this complex in ferrous form.
•The results obtained from the sets of experiments showed that, maximum reduction of ferricyanide complex was observed with
(j) Daucus carota+Cucumis sativus+ Solanum lycopersicum+ Zingiber offinale (0.493).
Reducing power activity of five mixture of vegetables with and without Zingiber offinale

42. Ames test
• The spot test was done as described by Ames and Whitfield., (1966) by applying 500µl
(50 µg/ml) of the Vegestable extracts with and without zingiber offinale to the centre
of a glucose minimal agar plate seeded with test organisms.
• If the chemical is mutagenic it will give rise to a ring of revertant colonies surrounding
the area where the extract was applied.
• The results observed shows that no ring of revertant colonies were observed
surrounding the area of extracts in glucose minimal agar plate.
• Thus, the extract of both all the vegetables can be considered as non carcinogenic
compound.