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

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.

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)

EXPERIMENTAL1. Extraction

• Distilled water extraction

• Ethanol extraction of garlic and turmeric

and both in combination

• Chloroform extraction

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

• 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

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 +

+ = compound detected

-= compound not detected

Phytochemical screening of non aqueous extractOther 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)

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

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.

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

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

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

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

g. HPLC testPreparation 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.

i. GC-MSThe 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.

RESULTS

Sample Yield % of Ethanol

Yield % of DS

Yield % of Choloroform

Garlic 42.6% 45% 1.5%

Turmeric 22.5% 33.5% 1.5%

Mixed 58.57% 70.85 4.07%

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

S.N Standard antibiotic used AMOX % CIP %

Bacterial strain usedE. 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

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

S.N Concentration % ZOI of Garlic ZOI of Turmeric ZOI of Mixed

1 10 0.0 0.0 0.02 20 0.0 0.0 0.03 30 0 0 1.0

4 40 1.1 0.9 1.95 50 1.8 1.2 2.4

6 60 2.6 1.9 3.87 70 3.5 2.2 5.08 80 5.0 3.0 5.09 90 5.0 5.0 5.0

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

Fig: Mean Rf value of extract

UV- Spectroscopy showed that the mixed of this two samples formed new compound may be.

DISCUSSION

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

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

• 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

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.

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

CONCLUSOON

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

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

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.

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?

• 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.

THANK YOU