ANTIBIOTIC RESISTANCE DUE TO SPICES FOODS (GARLIC AND TURMERIC)

1. THESIS REPORT
ON
ANTIBIOTIC RESISTANCE DUE TO SPICES FOODS (GARLIC AND
TURMERIC)
SUBMITTED TO:-
HOPE ONTERNATIONAL COLLEGE
SUBMITTED BY:-
UTTAM NEUPANE
SANTOSHI THAPA
23rd September 2014

2. Objectives
General
Observe antibiotic resistance due to spices food.
Specific
•To perform the physical properties of garlic and turmeric
•To perform the extraction by using suitable solvent system
and perform physiochemical assessment.
•To perform anti-microbial activity
•To determine the minimum concentration required for anti-microbial
activity.

3. Research question
1) Is there any relationship between antibiotic
resistance and spices food (Garlic and Turmeric)?
Hypothesis
•There is significant relationship between antibiotic
resistance and spices food (Garlic and Turmeric)

4. EXPERIMENTAL
1. Extraction
• Distilled water extraction
• Ethanol extraction of garlic and turmeric
and both in combination
• Chloroform extraction

5. Extraction procedure
Extraction of garlic and Turmeric by using suitable
solvent i.e. aqueous and non-aqueous solvent. There
are many processes of extraction but following only
(Aqueous and Organic solvents) extraction process
Aqueous extraction process (Both along also)
Garlic + Turmeric cut into small pieces
Peeled into fine powder sun drying
Add 95% ethanol Stored in 370 c for 72 hours
HPLC, GCMS, TLC filter (solution)
Antibiotic activity tests
Antibiotic resistance tests

6. • Organic extraction procedure, Powdered drug/
macerated plant 24hrs Light petroleum ether
Filtration Plant residue
CH3OH (72hrs), Filtration and Evaporation
Crude plant extract Filtrate Ether
Acidic solution Ether soluble (Evaporate)
Ether + NaOH Solution of Ether + NaOH
HPLC, GCMS, TLC Crude drugs
Antibiotic activity tests Antibiotic

7. 2. Phytochemical screening of ethanol extract
1) Alkaloid’s tests Result
Mayers tests ( no yellowish ppt) _
Wagnors test(no brown /reddish ppt) _
Hager’s test _
2) Carbohydrates tests
molisch’s test (voilet ring) +
Bendills test(red ppt) _
Fehling's test(red ppt) +
3) Glycosides tests
Bontrayers test(rose-pink colour) +
4) Saponin test
Froth Test(Formation of layer of foam) +
proteins and amino acids
Xanthoproteic Test +

8. + = compound detected
-= compound not detected
Phytochemical screening of non aqueous extract
Other are same expect alkaloid test. In non aqueous extract alkaloids also
detected. But carbohydrates is absent.
3. Antimicrobial Assay
a. Standard Antibiotic
•Amoxicillin (30 mcg: Hi media lab. Pvt. Ltd. Mumbi)
•Ciprofloxacin (30 mcg: hi media lab. Pvt. Ltd. Mumbi)

9. b. Micro-organisms Used
• Staphylococcus aureus
• Escherichia coli
c. Antimicrobial screening
Well diffusion method was employed for the
antimicrobial screening.
d. Preparation of the inoculums
The preparation of the bacterial suspension and
broth, solid broth media were prepared by
dissolving the 6.5 grams of broth in 500ml distilled
water and pouring 5ml of the medium in test

10. tubes. The Medias were let for cool down and
mixed for 15 minutes. Then with a loop, colonies of
each bacterium were put in the different broth and
then incubated for 24 hours at 37oC in an incubator.
In the broth 1ml of freshly prepared sterile saline
solution was added and the colonies formed on the
medium were scraped with an inoculating loop.
Turbid solution of each bacterium was obtained and
kept for future use. All these activities were carried
out in horizontal laminar flow.

11. e. Preparation of the culture media
The MHA culture media was prepared at the Range
of 38 gram media in 1000ml distilled water (DW).
The solution obtained after mixing the media in
distilled water was heated to boiling in order to
ensure the proper mixing. The media in the conical
flask was then covered with cotton. The media was
sterilized in autoclave at 1210C for 15 minutes at
15lbs pressure. 5ml of agar media were poured in
sterilized perti plates having the diameter of 9 cm

12. After the solidification of the media, each bacterium
was spread in each petri plates with the help of
cotton swab, after 30 minutes five bores were
made in each plate with the help of borer having
the diameter of six mm for the plant extract while a
single borer was made for the standard antibiotics.
For an extract 30 plates were prepared in order to
investigate the sensitivity in nine different
concentrations against different organism. Then
100μl of each extract were poured in the respective
bores and plates. The plates were then incubated
for 48 hours at 37oC for bacteria. After the
respective time the zone of inhibition of the extract
and the antibiotic were measured

13. f. Procedure for TLC behavior
• 250ml beaker was used as TLC developing chamber;
stationary media was prepared TLC plate of 0.25
mm thickness (silica gel 254F grade). After the
saturation of developing chamber with the solvent
system, the TLC plate was developed in the solvent
system for 20 minutes maximum 9cm length, by
ascending technique. The plates were removed
from the beaker after the development and dried.
• The detection of spots in the TLC plates was carried
out by visualization under UV chamber in UV light
before spraying reagents. After Spray the plate was
left for 5-10 minutes, and observed

14. g. UV- Visible Spectrophotometry behavior of
extract
Preparation of sample
• Extract were dissolved in 100 ml of respective
solvent system (Ethanol). Then the solution was
made different concentration (10ppm, 20ppm,
30ppm, 40ppm, 50ppm, 60ppm, 70ppm, 80ppm
and 90ppm). Then the extract were visualized at
330nm to 570nm. The spectrum of each extract was
observed

15. g. HPLC test
Preparation of sample
• In a 50-ml flask, garlic powder one gram was taken and
added to 25 ml of 95% ethanol solution containing 0.01
N HCl, and the mixture was well shaken for 45minutes.
95% ethanol solution containing 0.01 N HCl was added
to the mixture to make accurately 50 ml. The mixture
was centrifuged at 5000for 5 min and the obtained
sample was analyzed by HPLC.
• HPLC conditions were as follows: column, Symmetry
C18 (5 μm, 150 mm _ 3.9 mm,; column temperature,
25 °C; flow rate, 0.8 mL/min; mobile phase, 50
mMphosphate buffer (pH2.6)/methanol (85:15, v/v);
wavelength, UV 205 nm; injection volume, 10 μL.

16. i. GC-MS
The extract was store at - 80C. Add 10% Acetone
solution to the ethanolic extract to wash was carried
out for the tested ethanolic extract before
introduction to Gas Chromatography with Mass
Spectrometer.
(GC-MS): with inlet temperature: 250°C, 1 μl
injection, stationary phase 5% phenyl methyl
siloxane. Dimension 30 m × 0.25 μm ID × 0.25 mm
film thickness, Helium gas with 1.3 ml/min flow. Oven
programme: 90ºC (2 min), ramp 20ºC / min, 150 (0
min), ramp 6ºC /min, 270°C (10 min); total run time is
25 min. The used MSD temperature was 290ºC, quad
temperature was 150ºC, and ion source temperature
was 230ºC.

17. RESULTS

18. Sample Yield % of
Ethanol
Yield % of
DS
Yield % of
Cholorofor
m
Garlic 42.6% 45% 1.5%
Turmeric 22.5% 33.5% 1.5%
Mixed 58.57% 70.85 4.07%

19. Anti-bacterial Assay
The zone of inhibition (ZOI) was used for the
screening of antibacterial properties. E. coli and
Staphylococcus aureus was found to be sensitive to
the plant extract

20. S.N Standard antibiotic used
AMOX % CIP %
Bacterial strain used
E. coli S. aureus
1 10 10 0
2 20 20 1, 1.1 0.8, 1
3 30 30 1.2, 1.3 1.4, 1.4
4 40 40 1.5, 1.7 1.7, 1.7
5 50 50 2, 2.2 2.2, 2.2
6 60 60 2.5, 3 3, 3.5
7 70 70 3.3, 4.3 3.5, 4.6
8 80 80 Above 5 Above 5
9 90 90 Above 5 Above 5

21. The MIC of the Working standard antibiotics was
determined by the well diffusion method the MIC
for Amoxicillin is 0.002μg/ml to 0.009μg/ml was
found. And the MIC of Ciprofloxacillin for both
strains was 0.002μg/ml to 0.009μg/ml was found
from the table
The MIC and Zone of Inhibition (ZOI) of extract
on E. coli and S.aureus

22. S.N Concentration % ZOI of Garlic ZOI of Turmeric ZOI of Mixed
1 10 0.0 0.0 0.0
2 20 0.0 0.0 0.0
3 30 0 0 1.0
4 40 1.1 0.9 1.9
5 50 1.8 1.2 2.4
6 60 2.6 1.9 3.8
7 70 3.5 2.2 5.0
8 80 5.0 3.0 5.0
9 90 5.0 5.0 5.0

23. S.N Concentration % ZOI of Garlic ZOI of Turmeric ZOI of Mixed
1 10 0.0 0.0 0.0
2 20 0.0 0.0 0.0
3 30 0 0 1.1
4 40 1.1 0.9 1.9
5 50 1.8 1.2 2.8
6 60 2.2 1.9 3.9
7 70 2.7 2.2 4.8
8 80 3.9 3.0 5.0
9 90 5.0 5.0 5.0

26. Fig: Mean Rf value of extract
UV- Spectroscopy showed that the mixed of this two
samples formed new compound may be.
DISCUSSION

28. It was seen that the extraction yield in distilled
water was higher than ethanol and chloroform.
Ethanol extraction was higher than that of
chloroform. The highest extraction yield for
ethanol, distilled water and chloroform as follows

29. In the present investigation, the antibacterial
activity of the plant extract were tested against two
micro-organisms S. aureus and E. coliat different
concentration, all solvent extract ( ethanol, distilled
water and chloroform). All extract found to be
effective against tested strains. Ethanol and
Distilled Water extract showed more pronounced
antibacterial activity against both strains and
chloroform showed less antibacterial activity than
ethanol and distilled water. Ethanol and distilled
water showed nearly similar antibacterial activity.
Distilled water extract showed more effective
against both strains than other. The extracts were
found to be effective against Gram positive as well
Gram negative strain

30. • The antibacterial activity of the plant extract were
tested against resistant bacteria and found more
effective than market antibiotics.
• The present investigation showed that garlic caused
the antibiotic resistance to the bacteria. When
strains are treated with extract less than the MIC
value, and left for 24 hour incubation time the
culture the strain again and treated with the MIC of
market antibiotic (Amoxicillin and Ciprofloxacin).
But the standard MIC values of both antibiotics
were found 0.010 to 0.3 for E. coli and o.1 to 1 were
found for the Methicillin Resistant S. aureus and
S.aureus

31. The phytochemical screening of the crude extracts
of the Garlic, turmeric and mixed revealed the
presence of the some bioactive compounds as
tannins, phenolic, saponins, glycosides, alkaloids
and sugar
The appearance of different spectrum of garlic,
turmeric and mixed extract on UV-Visible
Spectrometer indicates that the mixed chemical is
different from the principle compounds of garlic
and turmeric. In UV visible spectrometer mixed
extract appearance different of observances than
garlic and turmeric it indicates it formed new
compounds.

33. The mixed extract showed more antibacterial
activity than that of garlic and turmeric, it indicates
that the mixed extract was either new compound or
enhance the activity of garlic or turmeric when
mixed together.
GC-MS

35. CONCLUSOON
• The phytochemical screening study revealed
that plants possess many chemical
constituents which are responsible for the
different therapeutic and pharmacological
effect on animals.

36. Our study allow us to conclude that the crude
extracts of ethanol, distilled water and chloroform
of the plants containing the many phytochemical
constituents alkaloids, carbohydrate, saponins,
glycosides, tannins and reducing compounds. The
results of the present study are encouraging as the
tested extracts revealed potential antibiotic activity,
although the inhibitory activity was strains specific
as well as concentration and extraction solvents
used. The study confirms that Gram-positive and
Gram-negative bacteria are more susceptible
towards the extracts

37. The study allow to us to conclude that the spices
foods Garlic and Turmeric causes the antibiotic
resistance when intake daily in small quantity which
causes the bacteria to developed mutation or to
produced neutralizing enzyme or changing the
binding site for the antibiotic.

38. Recommendations
In order to promote , proper utilization of
antibiotic as well as spices, followings
recommendation and future research areas
are forwarded
• Also regarding the antibacterial activity of the
extracts against other bacteria, which are
susceptible towards extracts?

39. • for future study regarding the chemical structure
determination of mixed extract of these plants
Garlic and turmeric.
• the crude extract of these plants need to be further
purified to obtain the pure compounds which was
responsible for the antibiotic activity.
• Regarding the antibiotic resistance mechanism of
bacteria.
• To regarding the in-vitro and in-vivo relationship of
extract in many animals.

40. THANK YOU