Presentation

Areeba Shaikh | M.Sc.-II Biotechnology 1

BY AREEBA SHAIKHR.D. National College,

M.Sc. Part-II (Biotechnology)Roll no.: 263

PHYTOCHEMICAL ANALYSIS OF PLANT EXTRACTS AND THEIR ACTION AGAINST

PATHOGENS ISOLATED FROMMASTITIS SUSPECTED MILK SAMPLES

A DISSERTATION SUBMITTED TO THE UNIVERSITY OF MUMBAIFOR THE PARTIAL FULFILMENT OF THE DEGREE OF

MASTER OF SCIENCE IN BIOTECHNOLOGYUNDER THE GUIDANCE OF DR. VIKAS KARANDE, ASSISTANT PROFESSOR,

DEPARTMENT OF VETERINARY PHARMACOLOGY AND TOXICOLOGY,BOMBAY VETERINARY COLLEGE, PAREL,

MUMBAI – 400012.

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INTRODUCTION

• S. aureues, E.coli, K. pneumoniae, Salmonella typhi.

Normal flora of raw milk

• Causes | Symptoms | Economic losses every year

Mastitis – Inflammation of mammary gland

• Pros and Cons | Antibiotic resistance in pathogens

Treatment of Mastitis by Antibiotics

• Resistant gene transfer | Alternative traditional therapy

Effects of Bovine Mastitis on Human health

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INTRODUCTION

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• Ethnic medicine | Plant derivatives

Plants and Plant products as Antimicrobial agents

• Aromatic substances | Secondary metabolites

Major groups of Antimicrobial compounds on Plants • Bacteriostatic and bacteriocidal effects

Mechanism of action of Phytochemicals

PHENOLS QUINONES FLAVONOIDS

TANNINS COUMARINES TERPENOIDS

ALKALOIDS

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INTRODUCTION

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Hydroxylated phenols, shown to be toxic to microorganisms. The sites and number of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to microorganisms, with evidence that increased hydroxylation results in increased toxicity.

Quinones provide a source of stable free radicals, and are known to complex irreversibly with nucleophilic amino acids in proteins, often leading to inactivation of the protein and loss of function. For that reason, the potential range of quinone antimicrobial effects is great. Probable targets in the microbial cell are surface-exposed adhesins, cell wall polypeptides, and membrane-bound enzymes.

The mode of action of Tannins’ antimicrobial action is related to their ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins, etc.

Alkaloids are heterocyclic nitrogen compounds and have been found to have microbiocidal effects, the major antidiarrheal effect is probably due to their effects on transit time in the small intestine.

INTRODUCTION

OBJECTIVES

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To isolate organisms from mastitis suspected milk samples from different regions of Mumbai.

To identify the organisms using biochemical and morphological test

To explore the in vitro antibacterial activity of Clove (Syzygium aromaticum), Cinnamon (Cinnamon zeylanicum),

Cumin (Cuminum cyminum) and Chirayita (Swertia chirayaita) against isolates of S. aureus and E. coli.

To study the phytochemical properties of the given plant products

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MATERIALS AND METHODS

MATERIALS AND METHODS

Dehydrated media, chemicals and reagents.

Petri plates, Pipettes, Test tubes, beakers, flasks and

measuring cylinders.

Clove, Cinnamon,

Cumin, Chirayita

Laminar Air flow hood, Incubator, Refrigerator,

Bunsen burners, gas cylinder, weighing

balance, Autoclave, Microscope.

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MATERIALS AND METHODS

Standard strains

Standard strains of E. coli and S. aureus were procured.

All the isolates were confirmed through biochemical tests by comparing with the results of standard strains.

Collection of samplesFor this study, 14 milk samples were collected during the period of 1 month from dairy farms in 3 different regions of Mumbai, namely Andheri, Malad and Marol, from cattle exhibiting signs of mastitis.

Location of dairy farm

Sample number Physical appearance pH of milk sample

ANDHERI

1 Normal 7.0

2 Normal 7.0

3 Normal 7.0

MALAD

4 Yellow, watery 5.5

5 Off white in colour, watery 4.0

6 Normal 7.0

7 Yellowish in colour 5.5

MAROL

8 Normal 4.0

9 Off-white, watery 4.0

10 Yellow, watery 5.0

11 Normal 7.0

12 Normal 6.5

13 Normal 6.5

14 Normal 6.0

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MATERIALS AND METHODS

Organism to be

Isolated

Medium used Method of

streaking

Incubation

conditions

Escherichia coli EMB AgarHexagon

Method

37°C for 24

hoursStaphylococcus aureus MSA

Klebsiella pneumonia MacConkey’s Agar

Salmonella Bismuth Sulphite Agar

1) Enrichment of microorganisms1 ml of each sample was extracted aseptically and homogenized with 9 ml sterile enrichment broth (lactose broth for E. coli, K. pneumonia and Salmonella and peptone water for S. aureus) and incubated at 37 °C for 24 hours, for further analysis.2) Media and growth conditions

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Physiological and biochemical examinationColony Characteristics were observed from all the streaked media plates.

Gram Staining was performed for all the isolates.

Biochemical tests were performed to confirm E. coli, K. pneumonia and Salmonella using Gram staining, Indole, Methyl red, Voges- Proskauer test, Simon citrate agar, and various sugar fermentation tests. Confirmation of the genus, Staphylococcus was done by Gram staining and various biochemical tests including Catalase test, and different sugar fermentation tests.

Extract preparation of the herbal samplesThe herbal samples were ground into a fine powder in a mortar and pestle and an extraction was made by soaking the 5g of each herb in 50ml of 50% ethanol for 24 hours, and making a final concentration of 100mg/ml. The extraction was filtered aseptically and sterilized using syringe filter.

Antibiotic sensitivity testing using Agar cup methodS. aureus and E. coli were spread on the MH Agar plates, and 4 wells on each plate were made with the help of a sterile borer. The filtrate of the extraction was then inoculated in the 4 wells made on MH Agar plate, and kept at 37 for 24 hours.

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Species Family Local Name Part used Ethanolic extractCumin

(Cuminum cyminum)

Umbelliferae Jeera Fruit CMN

Clove(Syzygium

aromaticum)

Myrtaceae Lavanga Flower stalk and bud

CLV

Cinnamon(Cinnamomu

m zeylanicum)

Lauraceae Darchini Stem bark CIN

Chiretta(Swertia chirata)

Gentianaceae Chirayita Stem CHI

Four different Plant Extracts; Chirayita,

Cinnamon, Clove and Cumin.

Antibacterial activity of different spices and herbs

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Plant Secondary metabolites

Method To observe

Alkaloids5 mg plant extract in 10 ml methanol;a portion of 2 ml extract + 1% HCl + steam, 1 ml filtrate + 6 drops of Mayor’s reagent.

Creamish precipitate indicates presence of Alkaloids.

Anthocyanins 5 mg plant extract in 10 ml methanol; a portion 2 ml + 1% HCl +heating.

Orange color indicates the presence of Anthocyanins.

Anthraquinones5 mg plant extract in 10 ml methanol; a portion of 2 ml + 2 ml ether-chloroform 1:1 (v/v) + 4 ml NaOH 10% (w/v).

Red color indicates the presence of Anthraquinones.

Flavonoids5 mg plant extract in 10 ml methanol; a portion of 2 ml + conc.HCl + magnesium.

Ribbon pink-tomato red color indicates the presence of flavonoids.

Phenols5 mg plant material in 10 ml methanol; a portion of 2 ml + 2 ml FeCl3.

Violet-blue or greenish color indicates the presence of phenols.

Saponins Frothing test: 0.5 ml filtrate + 5 ml distilled water.

Frothing persistence indicates presence of Saponins.

Tannins5 mg plant extract in 10 ml distilled water; a portion of 2 ml + 2 ml FeCl3.

Blue-black precipitate indicates the presence of tannin.

Preliminary phytochemical screening procedure.

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RESULTS

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RESULTS

Enrichment broths (A: Lactose Broth; B: MSB) showing growth.

A B

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RESULTS

Negative and positive controls of MSA

Showing no growth (left) and growth of

Yellow colonies (right)

Negative and positive controls of EMB

Showing no growth (left) and growth of

Green metallic sheen colonies (right)

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RESULTS

Pink colonies observed on MacConkey’s Agar

No colonies observed onBismuth Sulfite Agar

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RESULTS

Media used for streaking of culture

Samples showing growth Total number of

samples

Type of colonies

Mannitol Salt Agar 1, 2, 3, 5, 7, 9, 11, 12, 13, 14 10 Yellow colonies turning the medium yellow

EMB Agar 1, 2, 3, 9, 10, 12 6 Colonies with metallic green sheen

MacConkey’s Agar 1, 2, 3, 9, 11 5 Pink Colonies

Bismuth Sulphite Agar None 0 -

Number of samples from which the isolates were observed after streaking on respective selective media.

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RESULTSColony Characteristics and Grams Nature

10 Isolates

found on MSA

Characteristics Observation

Size Small

Shape Circular

Colour Yellow

Margin Round and completeElevation Slightly elevatedOpacity OpaqueConsistency SmoothGrams Nature Gram positive cocci in present

in chains

Colony characteristics of the isolates observed on MSA andthe Grams staining image from microscope at 100x of the same

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RESULTSColony Characteristics and Grams Nature

6 Isolates found on

EMB

Characteristics Observation

Size SmallShape CircularColour Green and purpleMargin Round and completeElevation FlatOpacity OpaqueConsistency ButteryGrams Nature Gram negative rods in

chains

Colony characteristics of the isolates observed on EMB andthe Grams staining image from microscope at 100x of the same

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RESULTSColony Characteristics and Grams Nature

5 Isolates found on

MAC

Characteristics ObservationSize MediumShape CircularColour PinkMargin Round and completeElevation Slightly elevatedOpacity OpaqueConsistency ButteryGrams Nature Gram negative bacilli in

clusters

Colony characteristics of the isolates observed on MAC andthe Grams staining image from microscope at 100x of the same

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RESULTSBiochemical tests for identification and confirmation of organisms

TEST TO OBSERVE INFERENCESUGAR FERMENTATION TEST

Colour of the phenol red broth containing all the three sugars changes from red to yellow indicating acid production(Figure A and B)

1. Mannitol Acid production1. Lactose Acid production1. Sucrose Acid productionCATALASE TEST(Colony + H2O2)

Strong effervescence Positive for Catalase(Figure C)

MANNITOL TEST Media turns yellow Positive for Mannitol fermentation(Figure D)

Identification of Staphylococcus aureus

(A) Before culture inoculation; (B)-After fermentation showing yellow colour in Tryptone broth; (C)- Effervescence observed; (D) - Yellow

colouration of Mannitol indication Mannitol fermentation.

A B

D

C

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RESULTSBiochemical tests for identification and confirmation of organisms

TEST TO OBSERVE INFERENCESUGAR FERMENTATION TEST

Colour of the phenol red broth containing all the three sugars changes from red to yellow indicating acid production(Figure A and B)

1. Glucose Acid production1. Lactose Acid production1. Sucrose Acid productionIMViC TESTS1. Indole test

(Sample + xylene+10 drops of Kovac’s reagent)

Red layer at the top of the solution

Positive(Fig. C)

1. Methyl Red Test(Sample + 10 drops of Methyl Red)

Solution turns red Positive(FigC)

1. VP Test(Sample + KOH +α-Naphthol)

Mahogany Red colour(Negative)

Negative(Fig. C)

1. Citrate Utilization Test Colour change from green to blue (negative)

Negative(Fig. C)

Identification of E. coli

BA

C

A- Before culture inoculation; BAfter fermentation showing yellow colouration of Tryptone broth; C- IMViC Tests- Tube-1: Indole ring test, Tube-2: MR test showing red coloration of solution, Tube-3: No change observed in VP Test, Tube-4: Citrate Utilization test showing no colour change in Media.

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RESULTS

TEST TO OBSERVE INFERENCESUGAR FERMENTATION TEST

Colour of the phenol red broth containing all the three sugars changes from red to yellow indicating acid production(Figure 9A and 9B)

1. Glucose Acid production1. Lactose Acid production1. Sucrose Acid production

IMViC TESTS1. Indole test

(Sample + xylene+10 drops of Kovac’s reagent)

Red layer at the top of the solution

Positive(Fig. 9C)

1. Methyl Red Test(Sample + 10 drops of Methyl Red)

Solution turns red Positive(Fig9C)

1. VP Test(Sample + KOH +α-Naphthol)

Mahogany Red colour(Negative)

Negative(Fig. 9C)

1. Citrate Utilization Test Colour change from green to blue (negative)

Negative(Fig. 9C)

A- Before culture inoculation; BAfter fermentation showing yellow colouration of Tryptone broth; C- IMViC Tests- Tube-1: Indole ring test, Tube-2: MR test showing red coloration of solution, Tube-3: No change observed in VP Test, Tube-4: Citrate Utilization test showing no colour change in Media.

Biochemical tests for identification and confirmation of organisms

BA

C

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RESULTSAntibacterial activity of different plant extracts

1: Influence of Plant extracts against S. aureus.A)No inhibition by CMN; (B) ZDI by CIN=18mm;(C) ZDI by CLV=14mm; (D) No Inhibition by CHI.

2: Synergistic action of plant extracts against S. aureus.(A)Ethanol control; (B) Zone diameter inhibition by CIN=26mm;

(C) ZDI by CIN=14mm; (D) ZDI by synergistic effect of CIN+CLV=31mm

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26

RESULTS

S. aureus E. coli S. aureus

Cinnamon 18 0 26

Clove 14 0 14

Cumin 0 0 0

Chirayita 0 0 0

CIN + CLV NaN NaN 31

2.5

7.5

12.5

17.5

22.5

27.5

32.5

ZDI of plant extracts against S. aureus and E. coli

Zone

Inhi

bitio

n Di

amet

er

(mm

)

Zone diameter of inhibition (ZDI) of the four plant extracts for S. aureusCIN= Cinnamomum zeylanicum; CLV= Syzygium aromaticum;

CMN= Cuminum cyminum. CHI=Swertia chirayaita

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RESULTS

CINNAMON CLOVECUMIN

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RESULTS

Plant

Secondary

metabolites

To observe CIN CLV CMN

Alkaloids Creamish precipitate - + +

Anthocyanins Orange color - - +

Anthraquinones Red color - + +

Flavonoids Ribbon pink / tomato red color - - -

Phenols Violet-blue or greenish color - + +

Saponins Frothing + - -

Tannins Blue-black precipitate + + +

Phytochemical analysis of the plant extracts

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DISCUSSIONSThe isolates were checked for their susceptibility against plant extracts such as Clove (Syzygium aromaticum), Cinnamon (Cinnamonum zeylanicum), Cumin (Cuminum cyminum) and Chirayita (Swertia chirayaita), out of which Cinnamon showed the highest inhibitory activity against S. aureus followed by Clove.

The inhibitory action of Cinnamon was higher than that of Clove. Cumin and Chirayita did not show any inhibition.

The synergistic action of Cinnamon and Clove together showed a higher inhibitory activity. No inhibitory activity was seen of these plant extracts against E. coli isolates.

Since phytochemicals are said to be responsible for the antibacterial activity of plants and plant products, these samples were then tested for the presence of phytochemicals by basic preliminary phytochemical screening tests, by which, presence of Alkaloids, Phenols, Tannins, Quinones and Saponins was observed, which may be the reason for the antibacterial activity of the plants against S. aureus.

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Cinnamon Clove Cumin0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Plants with phytochemicals

Num

ber

of p

hyto

chem

ical

s

DISCUSSIONS

Number of Phytochemicals present in CIN, CLV and CMN respectively

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Cinnamon was found to have Saponins and Tannins, while Clove showed positive results for presence of Alkaloids, Anthraquinones, Phenols and Tannins. Cumin, though did not have any antibacterial effect against S. aureus, was studied for presence of phytochemicals as well. It showed positive results for Alkaloids, Anthrocyanins, Anthraquinones, Phenols and Tannins.

It can be concluded that the ethanol-extractable phytochemicals from the plants possess in-vitro antimicrobial activity against the test microorganisms. The ability of these phytochemicals depends on not only qualitative but also quantitative content.

Since Cumin had the maximum number of five phytochemicals present, still it did not show any antibacterial activity, unlike in the case of Clove and Cinnamon which showed the presence of four and two phytochemicals respectively.

This means that the amount or the measure of phytochemicals present in the plant is responsible to elicit antimicrobial response.

DISCUSSIONS

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CONCLUSION

The inhibitory action of Cinnamon was higher than that of Clove. Cumin and Chirayita did not show any inhibition. The synergistic action of Cinnamon and Clove together showed a higher inhibitory activity in comparison to the extracts when used alone.

Presence of Alkaloids, Phenols, Tannins, Quinones and Saponins was observed, which may be the reason for the antibacterial activity of the plants against S. aureus.

Not only the number of phytochemicals, but also the amount of a single phytochemical present in the plant is responsible to elicit an antimicrobial response.

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SIGNIFICANCEand

FUTURE SCOPE

Cinnamon and Clove along with a combination of different herbs with antibacterial property, like Senna alexandrina and Cassia angustifolia can be used as traditional herbs, to obtain a synergistic effect MDR phenotypes of S. aureus or E. coli and

may hold a promise for potential application in the pharmaceutical industry as a source of useful drugs.

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REFERENCES

Prescott LM. Microbial nutrition, growth and control and microbial diseases and their control. In: Prescott LM, Harley JP, Klein DA. (eds.) Microbiology. Boston: WCB/McGraw Hill; 1999, p. 107-110.

Oliver SO, Jayarao BM, Almeida RA. Foodborne pathogens in milk and dairy farm environment: food safety and public health environment. Foodborne Pathog Dis 2005; 2: 115-129.

Khan R, Islam B, Akram M, Shakil S, Ahmad A, Ali SM, et al. Antimicrobial activity of five herbal extracts against multi drug resistant (MDR) strains of bacteria and fungus of clinical origin. Molecules 2009;14(2):586-597.

Journal of Dairy Science, 1994 J Dairy Sci 77:2103-2112

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