Assessment of Biofilm formation among UTI isolates

and antimicrobial susceptibility pattern with modern

and traditional medicine

Final Report of a Minor Project

submitted to

The Member-Secretary

Tamilnadu State for Council for Higher Education Lady Willingdon College Campus, Kamarajar Salai, Chennai – 600 005.

Submitted by

Dr. S. RAJAN Assistant Professor in Microbiology

M. R. Government Arts College Reaccredited with ―B‖ Grade by NAAC

Affiliated to Bharathidasan University, Tiruchirappalli

Mannargudi – 614 001

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Introduction

Biofilms are the extracellular polymeric substances secreted by bacteria, when

it is growing on substratum. Biofilms are made up of polymeric substances especially

polysaccharides. The structure of the biofilm is not a mere homogeneous monolayer

of slime but is heterogeneous, both in space and over time, with ―water channels‖ that

allow transport of essential nutrients and oxygen to the cells growing within the

biofilm. Biofilms have a propensity to act almost as filters to entrap particles of

various kinds, including minerals and host components such as fibrin, RBCs, and

platelets. Biofilm-associated organisms grow more slowly than other organisms,

probably because the cells are limited by nutrient and/or oxygen depletion. Biofilm

producers cause a systemic infection. Biofilm formation becomes more complicated

when it is considered that any substratum placed into a fluid environment (whether

the open ocean, the bloodstream, or the urinary tract) acquires a conditioning film or

coating comprised of primarily proteinaceous material that is present in the fluid in

that environment. The presence of flagella, pili, fimbriae, or glycocalyx along with

biofilm may impact the rate of microbial attachment. This is because the microbial

cell, once drawn to the surface, must overcome the repulsive forces common to all

materials, and these appendages enable the cell to remain attached until more

permanent attachment mechanisms are in place. Korber et al. (1989) compared

mutant and wild-type organisms and showed that the presence of flagella facilitated

attachment of gram-negative bacteria to surfaces. Another study (Rosenberg et al.,

1982) demonstrated the importance of fimbriae (which are external proteinaceous

structures of bacteria) for attachment. Cell surface hydrophobicity has also been

shown to be very important for attachment (Christention, 1994).

It has been well documented that biofilms add to the virulence of the

pathogen. It has been estimated that the frequency of infections caused by biofilms,

especially in the developed world, lies between 65% and 80% as per reports from

Centres for Disease Control and Prevention (CDC) and National Institutes of Health

(NIH), respectively. The biofilm activity has been recorded in various infections, viz.,

dental caries, cystic fibrosis, osteoradionecrosis, urinary tract infections, native valve

endocarditis, otitis media and eye infections. The studies have shown association

of E. coli and Proteus mirabilis, important uropathogens, biofilms in patients with

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complicated catheter-associated urinary tract infections. These organisms can lead to

persistent infections as a result of periodic release from the said focus.

The microbial infection of the urinary system are called urinary tract infection

(UTI), which affects all parts of the system covering bladder (cystitis), urethra

(urethritis) and kidney (pyelonephritis). Urinary tract infections are a serious health

problem affecting millions of people each year. About 150 million cases of UTI

reported each year throughout the world (Ohieku and Magaj, 2013). In Asia,

Incidence of urinary tract infections estimated to be 8.3 million per year (Bamidele et

al., 2013). E. coli is the most important urinary pathogen which causes acute as well

as chronic infections (Jellheden et al., 1996). The strains of uropathogenic E. coli

(UPEC) are responsible for the majority of urinary tract infections that occur in 70-

90% of UTI cases. Ampicillin, chloramphenicol, kanamycin, nalidixic acid,

nitrofurantoin, streptomycin, norfloxacin, trimethoprim-sulfamethoxazole (SMP-

SMX) etc., are commonly used for the treatment of UTI and most of these are noted

to be not effective for the treatment. During the recent decades, the clinical

management of UTI is complicated by the increasing incidence of infections caused

by multidrug resistant E. coli strains. Pathogenic entry to the host cells are mediated

by virulent factors like bacterial enzymes, adherence factors like fimbriae/pili (Ofek

and Doyle, 1994), flagellin, urease, the haemolysin, metallo protease (Fraser et al.,

2002) and extended spectrum β-lactamases (ESBLs) (Coque et al., 2008). Antibiotic

susceptibility is correlated with the virulent factors in the pathogens (Yun et al.,

2013). Hence this study was taken up to determine the presentation and risk factors

associated with community-acquired urinary tract infection. The distribution of

bacterial strains isolated from these patients and their resistance pattern were also

studied. Molecular pattern of virulence determination, plasmid profile, RAPD pattern

were also assessed for better understanding of multidrug resistance and pathogenic

potentials of the UPEC.

A complete scientific evaluation of the pharmacological activity of the

proposed medicinal plants is significant before medicinal application. An attempt

must also be done to explain the importance of plant based cure to the humankind. By

doing so, traditional system of medicine will be supported and strengthened which in

turn will be used for the welfare of mankind. Against this background, much attention

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is accorded by the scientific community and design alternate therapy making use of

herbal medicines or traditional system of medicines (Essawi and Srour, 2000).

Accordingly, UTI also are being treated with many plants and plant products

(Yoganarasimhan, 2000). Mangifera indica Linn commonly called mango is

recognized to be an important herb in the Ayurvedic and indigenous medical systems

for over 4000 years, which belongs to the family Anacardiaceae. According to

ayurveda, a dried mango flower is used for the treatment of diarrhoea, dysentery,

cystitis and urethritis (Nadkarni, 1976; Yoganarasimhan, 2000).

Objectives

Isolation of bacterial pathogens from clinically evident cases of UTI.

Identification of bacteria -UPEC

Assessment of virulent factors – biofilm, cell surface hydrophobicity,

extracellular enzymes.

Antibiotic susceptibility of test isolates.

Comparison of antibiotic susceptibility pattern of biofilm producer and non

biofilm producing strains.

Plant material collection and processing of Mangifera indica seed kernel.

Partial purification of phenolic fraction.

Assessment of antibacterial activity using virulent strains.

Assessment of Antioxidant Power of extracts

Analysis of Phenolic compounds

Materials and Methods

Isolation and identification of Uropathogenic Escherichia coli (Koneman et al.,

1994)

Collection of Samples and transportation

A total of 50 mid stream urine samples were collected in each 30ml sterile

plastic bottle from the different age group of patients attending the Meenchi Hospital,

Thanjavur and Government and Private hospitals in and around Mannargudi. Method

of sample collection was instructed to the patients before sample collection for proper

sample collection. Patients are also indicated to clean external genitalia using dettol

solution, which reduce external contamination. The samples were properly labeled

indicating the source, date, time of collection, sex and age of patients. The urine

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samples were transported in cooler boxes to the Microbiology Laboratory, for

bacteriological investigations within 4 – 6 h of collection.

Primary Isolation of uropathogens (Koneman et al., 1994)

Direct inoculation of 0.1ml of urine sample was done on Hi-Chrome UTI agar.

Hi-Crome UTI Agar is a differential medium recommended for presumptive

identification of microorganisms mainly causing urinary tract infections. It permits

the growth of uropathogens like Escherichia coli. All the isolates were differentiated

by making use of the colour of the colony.

Isolation of Escherichia coli (Koneman et al., 1994)

Culturing of the organism in selective cum differential medium helps to isolate

and identify the bacterial etiology of urine samples. Particular group of

microorganisms were selected by adding selective ingredients to the medium, which

allows the growth of only specific microorganisms. Generally bile salts and

deoxycholate are used to inhibit gram positive microorganisms. Differentiation was

done by adding indicator to the medium. The indicator will differentiate the microbial

group based on its physiological characters. Table – 4.1 gives a clear picture about the

nature, purpose, principle and results of various enriched, selective and differential

media used to isolate uropathogenic Escherichia coli.

Differentiation of Escherichia coli (Koneman et al., 1994)

The following methods were adopted to differentiate E. coli from other

pathogens. Selective cum differential media like Eosin Methylene Blue Agar, Mac

Conkey Agar, SS agar, XLD agar, Haektoein enteric agar (Hi media, Mumbai, India)

were used for the isolation and preliminary identification of the urinary E. coli. The

selected cultures were streaked directly on the Mac Conkey agar plates and incubated

at 37°C for 24 hrs. After incubation, cultures were examined for significant growth.

The primary identification of the bacterial isolates was made based on colonial

appearance and pigmentation. Pink coloured LF colonies from Mac Conkey plates

were selected and maintained in nutrient agar slants for complete identification.

Pure cultures of urinary isolates were inoculated onto Eosin Methylene Blue

Agar, SS agar, XLD agar, Haektoein enteric agar (Hi-Media), incubated at 37°C for

24 hrs. Colour and morphology of the colonies were noted and partially confirm the

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isolates identity. Partially identified cultures were confirmed by making use of Hi-

Chrome UTI agar and biochemical tests.

Identification of clinical isolates

Selected colonies from selective and differential media were subjected to

macroscopy, microscopy and biochemical tests for identification. Characterization

and identification of the isolates was done using the methods of Cowan (1985),

Fawole and Oso’s (1988) and Cheesbrough (2004).

Confirmation of clinical isolates (Koneman et al., 1994)

Selected colonies from selective and differential media were subjected to

macroscopy, microscopy and biochemical tests for identification.

Macroscopic observation

Colony morphology on agar surface aids to identify the bacterial isolate. Each

and every individual species of microorganism form colonies of characteristic shape,

size and appearance (Presscott et al., 1999). Characteristic features of the organism

were observed by macroscopic observations.A loopful of culture from overnight

grown broth was streaked on the surface of nutrient agar and was incubated at 37°C

for 24 hours. Colony morphology, colour and consistancy were observed and

tabulated.

Microscopic observations

Microscopic observations like shape, grams nature and motility reveal the

availability of different morphological characters among microorganisms. Simple

staining, gram staining and hanging drop methods were done to look for their shape,

grams nature and motility of the isolate respectively (Henry, 1994).

Shape of the organism

Shape of an isolate was identified by making use of simple staining procedure

followed by its observation under light microscope. Bacterial smear was stained with

methylene blue dye and examined under bright field microscope (Nikon). Microbial

cells were observed for their shapes like rod, cocci or spiral.

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Grams nature

Gram staining was performed to look for the grams nature of the isolate.

Purple coloured cells retains grams crystal violet and were called gram positive

bacterium. Pink coloured cells lost primary stain and picked up safranin colour and

were called as gram negative bacterium.

Motility

Bacteria were motile by their flagella. The number and location of which vary

among different species. Motility can be observed directly by hanging drop technique

i.e., by placing a drop of culture on a microscopic slide and looked under microscope

by keeping them inverted.

Biochemical tests

Physiological and metabolic characteristics of the microorganisms were

assessed through biochemical tests. These characteristics are very useful because they

are directly related to the nature and activity of microbial enzymes and transport

proteins. Proteins are gene products. Analysis of these characteristics provides an

indirect comparison of microbial genomes. The following tests were done to identify

the isolates. They are Indole test (I), Methyl red test (MR), Voges Proskauer test (VP),

Citrate utilization test (C), Urease production test (U), Nitrate reduction test (N),

Decarboxylation of lysine, ornithine and arginine, Phenylalanine deaminase test,

Oxidase test, Catalase test, TSI agar test and Carbohydrate fermentation test.

Assessment of virulent features of Uropathogenic Escherichia coli

Virulent factors are responsible for the pathogenicity of bacteria. Pathogen

must possess invasiveness, infectivity and pathogenic potential properties. The

availability of these properties directly proportional to the virulent properties.

Microbial virulence may be enhanced by surface factors, enzymatic factor, genomic

factors and plasmid factors. Assessment of virulent factors will provide the nature of

pathogens and helps to take specific precautions to handle the potent pathogens.

Biofilm detection

Tube method

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Two ml of trypticase soy broth in 12 x 75 mm borosilicate test tubes were

inoculated with a loopful of microorganisms from overnight culture plates and

incubated for 48 hours at 37°C, after which the contents were decanted and washed

with PBS (pH 7.3) and left to dry at room temperature. Afterward, the tubes were

stained with 4% solution of crystal violet (Merck, Darmstadt, Germany). Each tube

was then gently rotated to ensure uniform staining and then the contents were gently

decanted. The tubes were placed upside down to drain and then observed for biofilm

formation which was considered positive when a visible film lined the wall and

bottom of the tubes. Ring formation at the liquid interface was not regarded as

indicative of biofilm formation. The results were scored visually as 0-absent, 1-weak,

2-moderate, 3-strong (Mathur et al., 2006).

Haemolytic Activity

Blood Agar Plate Assay (Brenden and Janda, 1987)

The haemolytic activity of the E.coli was determined by blood agar plate

assay. Zone of haemolysis around the colonies on blood agar plates containing 5 %

(v/v) human blood, after 24 hour of incubation at 37oC was considered as positive.

Cell Surface Hydrobobicity

Microbial surface hydrophobicity was assessed with xylene according to

Rosenberg and Gutnick (1980). All isolates were grown into nutrient broth (50 ml) in

a 250 ml erlenmeyer flask with shaking in 200 rpm. Cells were harvested by

centrifugation (10000 × g, 15 min), washed twice in sterile phosphate-buffered saline

(pH 7.1) and suspended in the same buffer to an initial optical density (OD) of about

1.0 (A0) at 600 nm. Next, 300 μl of xylene was added to 3 ml of microbial suspension

and vortex for 2 min. After 10 min the OD of the aqueous-phase was measured (A1)

at 600 nm. The degree of hydrophobicity was calculated as 1-(a1/a2)100(%).

Assay for Beta Lactamase Production

Beta lactamase production was assayed using the method of Lateef (2004).

Broth culture of the test organism was spot inoculated on to Mueller-Hinton agar

containing penicillin and 1% starch then incubated overnight at 370C. The plates were

then flooded with freshly prepared phosphate buffered saline containing potassium

iodide. The presence of clear colourless zones around the bacterial growth is an

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indication of Beta lactamase production. Beta lactamase converts penicillin to

penicilloic acid, which reduces iodine to iodide monitored via decolourisation of the

starch iodine complex. All the bacterial isolates were tested for the production of beta

lactamases.

Antibiotic Sensitivity assay

All isolates were subjected into antibiotic stability test according to Roy et al.,

2006; Sharma et al., 2007 and Bauer et al., 1966. The susceptibility of isolates of

E.coli to antimicrobial agents was examined by an disc diffusion assay.

Antibiotics used for the assay

Gentamycin (Gen) - 30µg/disc

Ciprofloxacin (CF) - 30 µg/disc

Ampicillin (A) - 10 µg/disc

Erythromycin (E) - 15 µg/disc

Co-trimoxazole (Co) - 30 µg/disc

Cephalosporins (CE) - 10 µg/disc

Novobiocin (NV) - 05 µg/disc

Cefpodoxime (CPD) - 30 µg/disc

Tetracycline (T) - 30 µg/disc

Nalidixic acid (Na) - 30 µg/disc

Ceftizomine (CZX) - 15 µg/disc

Kanamycin (K) - 30 µg/disc

Vancomycin (Va) - 30 µg/disc

Bacitracin (B) - 15 µg/disc

Nitrofurantoin (NF) - 30 µg/disc

Imipenem (Im) - 15 µg/disc

All discs were purchased from Hi-Media, India.

Media used for the assay

Mueller Hinton Agar (Hi-Media). The Mueller Hinton Agar was prepared and

sterilized at 121°C and inoculated the isolates then incubated at 37°C for 24 hrs.

Determination of Antibacterial activity

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Disc diffusion method was followed (Bauer et al.,1966) to determine the anti

bacterial activity. Petriplates containing 20 ml of Mueller Hinton agar were seeded

with 4 hours old fresh culture of clinical isolates and referral strains. By making use

of template drawn discs were dispensed on the solidified Mueller Hinton agar with

test organisms. This was incubated at 37°C for 24 hours in an incubator (Rands SBC).

The test was performed in triplicates. The zone of inhibition was measured by making

use of Antibiotic zone scale (Hi - Media). The resistance patterns were interpretated

as per CDC recommendations.

Amplification of virulence factors from E.coli by multiplex PCR

Amplification of Eae gene (Feng, 1998)

The reaction mixture consists of 2 μl of template DNA, 1 μl of 300 nM of each

primer, 10 μl 2 X PCR master mixes (Promega, USA) and make up to 20 μl with

molecular grade water. Amplification was performed in a Bangalore Genei

thermocycler. After initial denaturation at 95°C for 1 min, the samples were

subjected to 25 cycles of denaturation at 94°C for 1min, annealing at 56°C for 1 min.

and extension at 72°C for 10 min. A final extension was performed at 72°C for 10

min. PCR products were examined by 1% (w/v) agarose gel electrophoresis in Tris

Borate EDTA buffer (pH 8·2).

The following primer were used for the amplification

5′-ATTACCATCCACACAGACGGT-3′

5′-ACAGCGTGGTTGGATCAACCT-3′

Identification of bacteria by molecular method

PCR Amplification of 16S rDNA

Pure culture of isolates was grown in Luria Bertani (LB) broth and genomic

DNA was extracted following the protocol given in Sambrook et al., (1989). PCR

amplification of the 16S rRNA gene was performed using the universal primers 27F

5´-AGAGTTTGATCCTGGCTCAG-3´ and 1492R 5´-

GGTTACCTTGTTACGACTT-3´ (Bioseve, Hyderabad). The reaction mixture

(50μL) contained 30mM Tris (pH 8.4), 50mM KCl, 1.5mM MgCl2, 50mM

concentrations of each deoxynucleoside triphosphate, 10pmol of primer, and 1 U of

Taq polymerase (Genei, Bangalore). PCR reaction conditions in MJ Research DNA

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Engine Tetrad were, 1 cycle of 95°C for 5min, 30 cycles of [94°C for 1min, 55°C for

1min, 72°C for 1min], 1 cycle of 72°C for 1min.

Purification of PCR Product and 16s rRNA gene sequencing

15μL of amplified DNA products were dissolved in 50μL of PCR cleanup

solution mixed well and incubated at 55°C for 15min. The mixture was centrifuged at

12000 rpm for 15min to remove the supernatant. DNA pellet was precipitated by the

addition of 600μL of 80% ethanol and centrifuged at 12,000rpm for 15min. Finally,

the DNA pellets were dried and dissolved in 15μL of Milli Q water (Millipore, USA).

Purified 16S rDNA product was extended using the primers 27F

5´AGAGTTTGATCCTGGCT- CAG3´ and 1492R 5´GGTTACCTTGTTA-

CGACTT3´. The extension products were purified by isopropanol precipitation. The

purified extension products were separated using Big Dye chemistry in the ABI

3730xl DNA Analyzer (Applied Biosystems Inc.) by capillary electrophoresis.

Sequence data analysis was done using Sequencing Analysis software.

Data Analysis

16S rRNA gene sequences of isolates were compared to the non-redundant

sequences database (GenBank, EMBL and DDBJ) using the BLASTn program in the

National Centre for Biotechnology Information (NCBI) website

(http://blast.ncbi.nlm.nih. gov /Blast.cgi.). Multiple sequence alignment was

performed for homologous sequences and a phylogenetic tree was constructed using

the neighbour joining method. The 16S rRNA gene sequences were also compared

with database sequences at the Ribosomal Database Project (RDP) using the RDP

classifier programme (http://rdp.cme.msu.edu/) for identification of the strains.

Sequences were submitted to GenBank and obtained accession number.

ANTIBACTERIAL STUDY OF PLANT EXTRACT

Plant materials selection, collection and extraction

Plant material – Selection and Collection

Seed kernel of Mangifera indica was selected to screen its bio potentials based

on its traditional usage. Seed kernel of Mangifera indica was collected as wild from

the thathachariar garden, Thiruvanaikoil, Tiruchirappalli, Tamil Nadu, India during

the month of June, 2015. Care was taken to select healthy fruit.

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Authentication of plant material

The plant material was initially identified by the Gardner (variety Neelum) of

thathachariar garden, Thiruvanaikoil, Tiruchirappalli, Tamil Nadu, India. This

identity of the plant was confirmed by Dr. John Britto, Professor, Department of

Botany and Director, Rapinat herbarium, St. Joseph’s College, Thiruchirapalli,

Tamilnadu, India and also a member of doctoral committee Dr. P. Brindha, Associate

Dean, SASTRA University, Thanjavur. The voucher specimen of the plant material

has been submitted in the M. R. Government Arts College, Mannargudi for future

reference.

Processing of plant

Fresh Mangifera indica fruit was collected from thathachariar garden,

Thiruvanaikoil, Tiruchirappalli. Fruit was peeled off using sterile sharp knife. Flesh

part of the fruit was consumed and the seed was washed completely. Hard shell of the

seed was removed and the soft embryonic portion was collected and dried completely.

The dried seed kernel sample was powdered using electric grinder (Smith mixie,

India). Powder was stored in sterile container for further studies.

Preparation of Aqueous extract

The powdered plant material (150gm) was mixed with water and extracted

completely. The seed kernel powder was mixed with sterile water and kept for 72

hours and filtered with a muslin cloth and it was condensed in hot air oven at 50˚C.

The aqueous extracts were stored in a sterile container and refrigerated for future use

(Jonathan, 2009).

Preparation of Phenolic extract

Phenolic extract was collected by making use of soxhlet extraction. It was

performed by placing 50gm plant material with 1:1 ethanol and methanol. Extraction

was performed at 90˚C for 12 hours. The extracts were filtered under the vacuum

through Whatman filter paper (No. 1) under gravity. Extract was dried under vaccum

evaporator for removing the solvent. The remaining residues were stored in

refrigerator till further use (Shi et al., 2005).

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Preparation of disc with plant extracts

Known quantity of extracts of both aqueous and phemolic were dissolved in

DMSO: Methanol of 1:1 ratio. This in turn was diluted with equal volume of

phosphate buffered saline (PBS pH 7). It was then filter sterilized by making use of

sortorious syringe filter of pore size 0.22µm. Sterile discs of 6 mm diameter (Hi-

Media) were loaded with 50µg - 250 µg / disc concentration of the extract and were

dried. Dried discs were stored in sterile containers till use. Solvent loaded discs were

also prepared and used as negative control. Oxytetracycline loaded Hi-Media discs

were used as positive control.

Preparation of inoculums

Bacterial strains were inoculated in nutrient broth and incubated at 37ºC for 4

hours in a shaker (Orbitech, Scigenics, India) and was used for antibacterial activity

test.

Determination of Antibacterial activity

Disc diffusion method was followed (Bauer et al., 1966) to determine the

antibacterial activity of the seed kernel extract of Mangifera indica. Petriplates

containing 20 ml of Mueller-Hinton agar were seeded with 4 hours old fresh culture

of clinical isolates and referral strains. By making use of template drawn extracts and

fractions loaded discs were dispensed on the solidified Mueller Hinton agar with test

organisms. Oxytetracycline antibiotic disc (30µg/disc) obtained from M/s Hi-Media

laboratories Ltd, Mumbai was used as positive control for bacteria and solvent loaded

discs were used as negative control. The plates were incubated at 37°C for 24 hours in

an incubator (Rands SBC). The test was performed in triplicates. The zone of

inhibition was measured by making use of Antibiotic zone scale (Hi - Media).

Determination of Minimum Inhibitory Concentration

Agar dilution method was used to find out Minimal Inhibitory Concentration

(Anonymous, 1993a and b). Stock concentration of various plant extract was prepared

by making use of DMSO : Methanol, in the ratio of 1:1 which in turn was diluted

with equal volume of phosphate buffered saline, pH 7. Mueller Hinton agar was

prepared, sterilized and kept ready in molten condition. 20ml of the molten media

was taken and was mixed with known concentration of different extracts / fractions

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and were added in different tubes. This mixture was swirled carefully for complete

mixing of extract and media and poured on to the plate. After getting solidified it was

inoculated with the test organism and standard organism. The plates were incubated at

37°C for 24 hours. MIC was recorded based on the growth of the organisms.

In-vitro antioxidant assay

A great number of In Vitro methods have been developed to measure the

efficiency of natural antioxidants either as pure compounds or as plant extracts. α,α-

diphenyl-β-picrylhydrazyl radical scavenging assay (DPPH), Ferric reducing

antioxidant power (FRAP), Nitric oxide radical scavenging assay, Superoxide anion

radical scavenging assay, ABTS radical scavenging assay, Hydroxyl radical

scavenging assay, are the In Vitro antioxidant assay methods used to assess the

antioxidant activity of the seed kernel extract of M.indica.

DPPH assay: (α,α-diphenyl-β-picrylhydrazyl)

DPPH Radical Scavenging Activity (Spectrophotometric assay)

The free radical scavenging capacity of the extracts of Mangifera indica

aqueous and phenolic extracts was determined using DPPH. DPPH solution (0.004%

w/v) was prepared in 95% methanol. Extracts of Mangifera indica seed kernel was

mixed with 95% methanol to prepare the stock solution (10mg/100ml). The

concentration of extract solution was 10mg/100ml or 100g/ml. From stock solution

2ml, 4ml, 6ml, 8ml and 10ml of the solution were taken in five test tubes and serially

diluted, this was made up to final volume of each test tube to 10ml whose

concentration was then 20g/ml, 40g/ml, 60g/ml, 80g/ml and 100g/ml

respectively. Freshly prepared DPPH solution (0.004% w/v) was added in each of

these test tubes containing extracts and after 10 minutes, the absorbance was taken at

517nm using a spectrophotometer (Systronics UV-Visible Spectrophotometer 119,

INDIA). Ascorbic acid was used as a reference standard and dissolved in distilled

water to make the stock solution with the same concentration (10mg/100ml or

100g/ml) of extracts. Control sample was prepared containing the same volume

without any extract and reference ascorbic acid. 95% methanol was used as blank

(Soler Evans et al., 1997).

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Ferric reducing power assay

This experiment was carried out as described previously (Cuendet et al.,

1997). One ml of the plant extract solution (final concentration 100-500mg/L) was

mixed with 2.5ml phosphate buffer (0.2M, pH 6.6) and 2.5ml potassium ferricyanide

(K3Fe (CN6)) (10g/L), then the mixture was incubated at 50°C for 20 minutes. To this

2.5ml of trichloroacetic acid (100g/L) was added and centrifuged at 3000rpm for 10

minutes. Finally, 2.5ml of the supernatent solution was mixed with 2.5ml of distilled

water and 0.5ml Fecl3 (1g/L) and the absorbance was measured at 700nm in UV-

Visible Spectrophotometer (Systronics UV-Visible Spectrophotometer 119, India).

Ascorbic acid was used as standard and phosphate buffer as blank solution. The

absorbance of the final reaction mixture of two parallel experiments was expressed as

mean ± standard deviation. Increased absorbance of the reaction mixture indicates

stronger reducing power.

A test is the absorbance of test solution; A blank is absorbance of blank. The antioxidant

activity of the seed kernel extract was expressed as IC50 and compared with standard.

Nitric oxide radical scavenging activity

Nitric oxide (NO) was generated from sodium nitroprusside (SNP) and was

measured by the Griess reagent. SNP in aqueous solution at physiological pH

spontaneously generates NO, which interacts with oxygen to produce nitrite ions that

can be estimated by the use of Griess reagent. Scavengers of NO compete with

oxygen leading to reduce production of NO. Nitric oxide was generated from sodium

nitroprusside, which at physiological pH liberates nitric acid. This nitric acid gets

converted to nitrous acid and further forms nitrite ions (NO2 -) which diazotize with

sulphanilic acid and couple with naphthylethylenediamine (Griess reagent), producing

pink colour, which can be measured at 546 nm. Sodium nitroprusside (10 mM, 2 ml)

in phosphate buffer saline was incubated with the test compounds in different

concentrations at room temperature for 30 minutes. After 30 minutes, 0.5 ml of the

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incubated solution was added with 1ml of Griess reagent and the absorbance was

measured at 546 nm (Polshettiwar et al., 2007).

Superoxide radical scavenging activity (PMS-NADH System)

Superoxide anions were generated using PMS / NADH system. The

superoxide anions were subsequently made to reduce nitroblue tetrazolium, which

yielded a chromogenic product, which was measured at 560 nm. Phenazine

methosulfate-nicotinamide adenine dinucleotide (PMS-NADH) system was used for

the generation of superoxide anion. It was assayed by the reduction of nitroblue

tetrazolium (NBT). About 1ml of nitro blue tetrazolium (156μM), 1ml NADH

(468μM) in 100mM phosphate buffer of pH 7.8 and 0.1ml of sample solution of

different concentrations were mixed. The reaction was started by adding 100μl PMS

(60μM). The reaction mixture was incubated at 25°C for 5 minutes and absorbance of

the mixture was measured at 560nm against blank samples. The percentage inhibition

was determined by comparing the results of control and test samples (Kumarasamy et

al., 2007).

ABTS radical scavenging assay

ABTS (2, 2’-azinobis-3-ethylbenzothiozoline- 6-sulphonic acid) assay is based

on the scavenging of light by ABTS radicals. An antioxidant with an ability to donate

a hydrogen atom will quench the stable free radical, a process that is associated with a

change in absorption. The relatively stable ABTS radical was green and it was

quantified spectrophotometrically at 734 nm. ABTS radical cations were produced by

the reaction of ABTS and APS. The ABTS scavenging capacity of the extract was

compared with that of BHT and ascorbic acid and the percentage inhibition was

calculated. The stock solutions included were 7 mM ABTS solution and 2.4 mM

potassium per sulfate solution. The working solution was then prepared by mixing the

two stock solutions in equal quantities and allowed them to react for 14 hrs at room

temperature in dark. The solution was then diluted by mixing 1ml ABTS solution with

60ml methanol to obtain an absorbance of 0.706 ± 0.01 units at 734 nm using a

spectrophotometer. Fresh ABTS solution was prepared for each assay. Seed kernel

extract (1 ml) was allowed to react with 1ml of the ABTS solution and the absorbance

was taken at 734 nm after 7 minutes using a spectrophotometer. All determinations

were performed in triplicate (n = 3) (Blois et al., 1958).

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H2O2 scavenging activity

H2O2 scavenging ability of aqueous and phenolic extracts of Mangifera indica

seed kernel was determined according to the method of Ali et al., (2009). A solution

of H2O2 (40mM) was prepared in phosphate buffer (pH 7.4). The aqueous and

phenolic extracts at 30µg/ml concentration in 3.4ml phosphate buffer were added to

H2O2 solution (0.6ml, 40mM). The absorbance value of the reaction mixture was

recorded at 230nm. Blank solution was containing the phosphate buffer without H2O2.

Assessment of % inhibition and IC50

Radical scavenging activity of the extract and standard were expressed in

terms of % inhibition. It was calculated using the formula ((AControl-ASample) /AControl) ×

100. Where AControl is the absorbance of the control and ASample is the absorbance in

the presence of the sample of aqueous and phenolic extracts. The IC50 value is defined

as the concentration (in μg/ml) of extracts that produced 50% antioxidant effect. IC50

= Concentration of extract / % inhibition X 50.

Phytochemical analysis

Qualitative phytochemical screening

Freshly prepared extracts were tested for the presence of phytochemical

constituents using standard methods (Anonymous, 2006; Lala, 1981).

Test for Alkaloids

i) Dragendorff’s test: To 1 ml of the extract, 1 ml of Dragendorff’s reagent

(potassium bismuth iodide solution) was added. An orange-red precipitate indicates

the presence of alkaloids.

ii) Mayer’s test: To 1 ml of extract, 1 ml of Mayer’s reagent (Potassium mercuric

iodide solution) was added. Whitish yellow or cream coloured precipitate indicates

the presence of alkaloids.

Test for steroids

2 ml of acetic anhydride was added to 0.5 g phenolic extract with 2 ml of

H2SO4. The colour change from violet to blue or green indicates the presence of

steroids.

17

Test for terpenoids

Salkowski test: To 0.5 g of the extract, 2 ml of chloroform was added and carefully

add 3 ml concentrated H2SO4 to form a layer. A reddish brown colouration of the

interface indicated the presence of terpenoids.

Test for flavonoids

i) Alkaline reagent test: Few drops of dilute ammonia and Con. HCl were added to

the portion of extract. A yellow colouration indicates the presence of flavonoids.

ii) Zinc Hydrochloride test: To few drops of extract, zinc dust and con. HCl was

added, the presence of red colouration indicates the presence of flavonoids.

iii) Aluminium test: To few drops of extract, 1 % Aluminium solution was added,

yellow colour indicates the presence of flavonoids.

Test for Saponins

About 2 g of the powdered sample was boiled in 20 ml of distilled water in a

water bath and filtered. 10 ml of the filtrate was mixed with 5 ml of distilled water

and shaken vigorously for a stable persistent froth. The frothing was mixed with 3

drops of olive oil and again shaken vigorously and observed for the formation of

emulsion.

Test for tannins

i) A little quantity of test solution was taken and mixed with basic lead acetate

solution. Formation of white precipitates indicates the presence of tannins.

ii) To 1 ml of the extract, add few drops of ferric chloride solution. Formation of a

blue- black or brownish green colour product shows the presence of tannins.

iii) A little quantity of the extract was treated with aqueous ammonia solution. A

deep green colour indicates the presence of tannins.

Quantification of metabolites

Secondary metabolites like flavonoids, tannins and phenols were quantified

employing standard methods (Ayoola et al., 2008). Coumarins was estimated by

making use of the method described by Kuster and Rocha (2004).

18

Determination of total tannins

The total tannin content in the plant extract was determined by modification of

a previous method (Polshettiwar et al., 2007). The water and phenolic extracts (0.1

ml) was mixed with 0.5 ml of Folin‐ Denis reagent followed by 1ml of Na2CO3 (0.5%

w/v) solution and made upto 10 ml with distilled water. The absorbance was

measured at 755 nm within 30 minutes of the reaction against the reagent blank.

Standard curve was prepared using 20, 40, 60, 80 and 100 μl of tannic acid. Total

tannins in extracts were expressed as equivalent to tannic acid (g TE/g extract).

Determination of total flavanoids

Aluminum chloride colorimetric method was used for flavonoids

determination (Chang et al., 2002). The seed kernel extract (0.5ml of 1:10g/ml-1) in

ethanol was mixed with 1.5ml of methanol, 0.1ml of 10% aluminum chloride, 0.1ml

of 1M potassium acetate and 2.8ml of distilled water. It remained at room temperature

for 30 minutes; the absorbance of the reaction mixture was measured at 415nm with a

single beam Systronics UV/Visible spectrophotometer (India). The calibration curve

was prepared by preparing quercetin solutions at concentrations 12.5 to 100g/ml-1 in

methanol.

Determination of total phenols

Total phenols were determined by making use of the method given by

McDonald et al., (2001). The diluted seed kernel extract (0.5 ml of 1:10 g/ml‐1) or

gallic acid (standard phenolic compound) was mixed with Folin Ciocalteu reagent (5

ml, 1:10 diluted with distilled water) and aqueous Na2CO3 (4 ml, 1M). The mixtures

were allowed to stand for 15 minutes and the total phenols were determined by

colourimetry at 765 nm. The standard curve was prepared using 0, 50, 100, 150, 200,

250 mg L-1

solutions of gallic acid in methanol: water (50:50, v/v). Total phenol

values are expressed in terms of gallic acid equivalent (mg g–1 of dry mass), which is

a common reference compound.

UV-FTIR analysis

Fourier Transform Infrared Spectroscopy analysis (FTIR) (Kannan et al., 2013)

The infrared (IR) spectra of phenolic extract M.indica seed kernel were

obtained using Fourier Transform Infrared Spectroscopy (Perkin Elmer Spectrum GX

19

FTIR, U.S.A). Samples discs were made by mixing 5 mg of dry biomass with 150 mg

of potassium bromide (KBr), and pressed them into tablet form. Infrared spectra were

recorded over 4000 - 500 cm-1

region with a resolution of 0.2 cm.

NMR analysis

1

H and 13

C NMR experiments were performed on a Bruker advance DPX300

spectrometer operating at 300 and 75 MHz respectively. Chemical shift values (δ)

were reported in parts per million (ppm) relative to appropriate internal solvent

standard and coupling constants given in hertz.

GC-MS

Make : Thermo GC- Trace Ultra ver: 5. Thermo MS DSQ IIII

Column : DB 35 - MS capillary standard non-polar column

Dimension : 30 Mts, ID: 0.25 mm, FILM: 0.25 μm

Carrier Gas : HE, FLOW: 1.0 ML/Min

Temp Prog : Oven temp 70° C raised to 250° C at 10 C/min

Statistical analysis

All the values were expressed as mean SD (standard Deviation). Statistical

analysis was carried out by using Origin software package (version 6.0). Statistical

significance of differences between the control and experimental groups was assessed

by One-way ANOVA. The value of probability less than 5% (P < 0.05) was

considered statistically significant (Panse & Sukhatme, 1978).

RESULT AND DISCUSSIONS

Urinary tract infections (UTIs) are among the most common bacterial

infection. UTIs are responsible for significant morbidity and health care costs

worldwide (Kudinha et al., 2012). UTIs were simply assessed by making use of

urine. In India, most of the UTI were assessed using mid stream urine. It is a simple

and easy method and represents actual infection status of the system. It was supported

by the report from Nepal (Acharya et al., 2011). They stated that clean catched mid

20

stream urine samples were simple and effective for proper recovery of urinary

pathogens. Mwaka et al., (2011) also stated that midstream urine was best suited for

the recovery of bacterial pathogens. A total of 50 urine samples were collected from

the clinically evident cases of UTI. All the urine samples were collected for a period

of two months. Samples were collected from both male and female cases and age wise

distribution of samples were from 1- above 60 years of age. About 60% (n=30) of

urine samples were from females and 40% (n=20) samples were from male (Fig. 1).

Higher incidence of female with UTI of this study was greatly supported by different

authors from all over the world. It was also in line with the report of Soto et al.,

(2011) from Spain. Ismaili et al., (2011) from Belgium indicated that 63% UTI cases

were from females and 37% were males. Mwaka et al., (2011) also expressed that

UTIs in women are a common problem in primary healthcare. Indian unpublished

data also revealed that females frequently visited health care units for UTI treatments

in all age groups. The urethra of female is very short and hence they are easily

encountered with pathogenic attack. Proper personnel hygiene and status of personnel

immunity may reduce the microbial burden.

Collected urine samples were transported to the microbiology laboratory for

processing. All the urine samples were processed properly as per the standard

methodology. Samples were visually examined for the change of colour and noted

properly. Colour variation in urine samples were described as dark yellow, brownish

yellow and pinkish red. Maximum number of urine samples collected from UTI cases

40%

60%

Figure 1

Incidence of UTI among Male and Female Population

Total Sample collection - 50

Male Female

21

were brownish yellow in nature (46%), followed by dark yellow (31%) and 23% urine

shown to be pinkish red in colour (Fig. 2). Colour of the urine samples were greatly

depends on the inflammatory and associated pathological condition of the urinary

track. Cystitis, pyelonephritis are the major inflammatory disease due to bacterial

interaction. Brownish yellow and pinkish red colour of the urine was due to

inflammatory response.

Microscopic examination of urine sample was done using standard methods.

On the basis of Gram staining & Giemsa staining of urine sample, it was found that

33 samples had pus cells and bacteria followed by 12 samples with pus cells,

inflammatory cells and bacteria and 5 urine showed the presence of bacteria only (Fig.

3). Cystitis and pyelonephritis like inflammatory diseases may influence on the

release of pus cells, neutrophils to the urine. Normal urine is free from these cells.

Presence of bacteria in urine is called bacteriuria. All the 50 urine samples were

considered as a UTI sample, which was evidenced in bacteriuria.

Dark yellow

31%

Brownish

yellow

46%

Pinkish red

23%

Figure 2

Colour description of Urine samples

22

All the urine samples were inoculated on Hi-Chrome UTI agar for the

complete recovery and differentiation of the urinary pathogens. Urinary pathogens

were differentiated based on the differential colour. E. coli produced pink-purple

colour colonies on Hi-Chrome UTI agar.

In the present study, twenty pure urinary isolates were isolated and identified.

Only 20% of the samples showed UPEC as a major pathogen. Mishra et al., (2013)

isolated 996 strains of bacteria belonging to 11 genus from 1245 samples. They

isolated Staphylococcus aureus, Enterococcus faecalis, Acinetobacter baumannii,

Citrobacter sp., Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae,

Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris and Pseudomonas aeruginosa.

Among these strains Acinetobacter baumannii, Citrobacter sp., Klebsiella oxytoca,

Proteus mirabilis were not recovered from the urine samples of Namakal patients,

Tamilnadu, India.

This study clearly stated the predominant nature of E. coli as a major urinary

pathogen. All (100%) urine sample had E. coli as the pathogen. Daoud and Afif

(2011) also reported that E. coli was the most frequent isolate throughout the 10 years

accounts for 60.64% of incidence at Lebanon. Mbanga et al., (2010) expressed from

Zimbabwe that 40.3% UTI pathogens were E. coli followed by 16.1% cogulase

negative Staphylococci, 11.2 % Klebsiella sp, 8% Staphylococcus aureus, 8% Group

A Streptococci and 8% were Klebsiella oxytoca. Present study revealed that E. coli as

Urine with

Pus, Inflammatory

cells and Bacteria

24%

Urine with

bacteria only

10%

Urine with Pus

cells and Bacteria

66%

Figure 3

Microscopic features of Urine sample

23

the predominant pathogen of UTI. This was backed by Siedelman et al., (2012),

Walters et al., (2012), Yamamichi et al., (2012), Acharya et al., (2011), Sharma et

al., (2011), Chlabicz et al., (2011) and different authors from different countries.

But there is no proper report from India especially from Tamilnadu. Virulent and

molecular profile study is needed for complete understanding of E. coli from UTI.

Though this study showed the presence of six different uropathogens, the

presence of E. coli indicates high significance as a UTI infective agent. A total of 50

E. coli were isolated from 100 urine samples. Among these 10 were isolated as single

isolate remaining 40 as mixed isolate. Hence in the present study, single or pure

isolate was selected for further analysis. Only 20% of samples had single urinary

isolate.

This study also indicated that 97% of UTI infections were due to bacteria (Fig.

4). No bacteria were recovered from 3% (n=3) of urine samples. This may be due to

infection other than bacteria.

Similar to this study report Chukwuenmeka Anyanwu et al., (2012) also

indicated the incidence of a single isolate of urinary pathogen. They recovered single

pure isolate from 65.4% of samples. In this study, only 20% of sample had single

pathogen. 10% pure bacterial isolates were indicated by Mwaka et al., (2011).

In the present study, 97% of samples showed the incidence of Escherichia

coli. This indicated that incidence of Escherichia coli in UTI was high during

22%

75%

3%

Figure 4

Recovery nature of E. coli and other bacteria in Urine

E. coli only Mixed bacteria No Bacteria

24

bacteriuria condition. Though different types of bacteria caused UTI, Escherichia coli

showed higher prevalence; thereby identification of pure isolates of E. coli was

carried out using standard methods. EMB agar, XLD agar, Rajhans medium,

Haektoein Enteric agar, SS agar and Hi-Chrome UTI agar were used for the selective

cum differential isolation of uropathogenic Escherichia coli. E. coli produced specific

colonies on these media (Table 1). Metallic sheen colony formation on Eosin

methylene blue (EMB) agar and salmon coloured colonies on Haektoein enteric agar

were the major identification features of E. coli. These two medium were considered

as a highly selective medium for the recovery of E. coli from the clinical samples.

This could be due to strong mixed acid fermentation which precipitates dyes available

in the medium and usage of lactose, glucose and sucrose as a carbon source for

oxidative and fermentative reactions (Willey et al., 2003). All these sugars were

utilized only by the E.coli and confirm its identity (Plate I-P. Figs 1to 7).

Table 1

Growth characteristics of Uropathogenic Escherichia coli on Different media

Table 2 expressed various staining, motility and biochemical nature of E. coli.

Acid and gas production from glucose and lactose fermentation by the test organisms

confirm the organisms identity as E. coli (Plate II-P .Figs 8 & 9). All the culturable

microorganisms were identified through growth on selective and differential media

along with biochemical tests (Koneman et al., 1994). Identification of E. coli based

on standard culture and biochemical features were also supported by Daoud and Afif

(2011).

S. No Media Growth Pattern

1 EMB Agar Metallic sheen colonies

2 Mac Conkey agar LF colonies

3 XLD aagar Yelow colour colonies

4 Haektoein enteric Agar Salmon Coloured colonies

5 Rajhans Medium Bluish Green Colonies

6 SS Agar Pink colour colonies

25

26

27

Table 2 - Microscopic and Biochemical Characterization of E. coli

Assessment of Virulent factors (Plate III)

Production of slime, lactamase and haemolytic activity are the major

virulent determinants of uropathogens. All the 10 E. coli isolates were subjected to

virulence factors determination. All the isolates under study showed the presence of

any one of virulent factor tested. Among the tested E. coli isolates 80% (n=8) showed

the production of slime factors, 90% (n=9) of the isolates showed betalactamase

enzyme producing ability, 90% (n=9) of the isolates showed cell surface

hydrophobicity and only 60% (n=6) of the strains indicated haemolytic activity (Fig.

5). Like the incidence rate the available virulent factors were also high among the E.

coli isolated from the female cases. Age wise incidence also proportionatly high as

S. No Test Result

1 Simple staining Rod

2 Gram Staining Gram negative

4 Catalase Positive

5 Motility Motile

6 Indole Positive

7 Oxidase test Negative

8 MR Positive

9 VP Negative

10 Citrate Negative

11 TSI A/A Gas positive H2S Negative

13 Urease Negative

14 Nitrate Positive

15 Phenylalanine deaminase Negative

16 Ornithine Decarboxylase Positive

17 Arginine Decarboxylase Negative

18 Lysine Decarboxylase Positive

19 Gelatin Hydrolysis Positive

20 Carbohydrate Fermentation

Glucose Acid and Gas

Sucrose Acid only

Lactose Acid and Gas

Mannose No Acid and Gas

Mannitol Acid only

Xylose Acid and Gas

28

like incidence of E. coli in different age groups. Slime production was directly related

to biofilm producing ability of the test pathogens.

Extra intestinal E. coli accounts for urinary tract infection. It contains several

putative virulence genes. Virulence genes were positively linked with the

pathogenicity of ExPEC (Pitout, 2012). Dhakal and Mulvey (2012) reported that UTI

producing E. coli able to produce haemolysis. It is a heat labile pore forming toxin. It

was produced by hlyA gene containing bacteria. HlyA induced proteolysis of host

proteins likely allows UPEC not only modulate epithelial cell function, but also

disable macrophages and suppress inflammatory responses.

UPEC exhibited multiple numbers of virulence factors. It facilitates

colonization of E. coli in the bladder (Hilbert et al., 2012). They reported that, 27

(35.1 %) and 50 (64.9 %) ESBL-producing UPEC strains in neonates and infants,

respectively. Of the 70 strains investigated for the presence of virulence factors,

adhesins were detected in 48.6 % strains (8.6 % in the neonate and 40 % in the infants

group) giving a statistically significant difference in adhesion expression between the

two groups. Bedenić et al., (2012) reported that 84.3% of uropathogenic E. coli strains

produce haemolysin producing strains. Slightly different report was expressed in this

study. Various conditions of the environment play a vital role in expression of the

89

6

9

0

1

2

3

4

5

6

7

8

9

10

Slime Production Beta lactamase

(ESBL)

Haemolysis Cell Surface

Hydrophobicity

No. of

UP

EC

Iso

late

s

Virulent Property

Figure 5

Incidence of Virulent factors in Uropathogenic

Escherichia coli

29

gene product. The pathogenic potential of E. coli strains is thought to be dependent on

the presence of virulence factors (VFs) (Johnson, 2003).

Antibiotic susceptibility assay (Plate III – P. Fig. 13)

Antibiotic sensitivity pattern of E. coli isolates were carried out by making use

of Kirby & Bauers disc diffusion method. All 20 pure E. coli isolates were subjected

to antimicrobial susceptibility screening. Among the isolates tested all the isolates

were resistant to minimum of 10 antibiotics (62.5%) out of 16 antibiotics tested. Only

5% (n=1) of the isolates were sensitive to all the antibiotics tested. This indicated that

all the isolates were resistant to multiple numbers of antibiotics. About 25% of

isolates were sensitive to gentamycin, which is one of the amyloglycoside groups of

30

antibiotics (Table 3). Similar kind of result was also given by Khan and Zaman

(2006). They reported that most of the isolates showed multiple antibiotic resistances.

All the organisms are completely resistant to antibiotics like CF, CPD, CE and B.

Among the antibiotics tested only imipenem showed good susceptibility. It yielded

90% sensitivity against all the UPEC tested. Imipenem acts as an antimicrobial

through inhibiting cell wall synthesis of various Gram-positive and Gram-negative

bacteria. It remains very stable in the presence of beta-lactamase produced by some

bacteria, and is a strong inhibitor of beta-lactamases from some Gram-negative

bacteria that are resistant to most beta-lactam antibiotics. As per report given by

Hoban et al., (2011) from USA imipenem were the most active agents inhibiting

>98% of all E. coli phenotypes. Similar to this, 90% of the isolates were also sensitive

to imipenem.

This result provides the present day’s microbial nature against multiple

numbers of antibiotics and stated the alarming situation of antimicrobial therapy.

People must develope new antimicrobial therapy with lesser side effects for better

treatment, otherwise few cases may not be treated properly that creates huge stress to

the urinary tract and also human mind, which leads to chronic infection in urinary

bladder, kidney etc., Combined antibiotic therapy may partly overcome this problem

for a shorter duration. 76.5% of community acquired infections were due to E. coli.

among them 60.6% of E. coli were ESBL producers (Siedelman et al., 2012; Walters

et al., 2012). The resistance rates of E.coli detected from urine culture was found to

be 100% for ciprofloxacin (CF), cefpodoxime (CPD) and Cephalosporin (CE), 98%

for erythromycin (E) and bacitracin (B), 94% for ceftizoxime(CZX) & kanamycin

(K), 93% for Novobiocin (NV) and tetracycline (T). 91% of strains were resistant to

norfloxacin (NA). Additionally, most sensitivity rates were reported for Imipenem

(90%) followed for gentamycin (33.3% ) (G). .

31

Table 3

Antibioticsensitivity pattern of uropathogenic E. coli

S. No. Antibiotics Resistant Sensitive

1 GEN 070 30

2 CF 100 00

3 A 080 20

4 E 090 10

5 Co 080 20

6 NA 090 10

7 T 090 10

8 CZX 090 10

9 CPD 100 00

10 CE 100 00

11 K 090 10

12 B 100 00

13 Va 090 10

14 Nv 090 10

15 NF 090 10

16 Im 010 90

Relatively high level of resistance to antibiotics is a reflection of misuse or abuse of

these antibiotics during treatment of bacterial infections.

Bonkat et al.,(2013) expressed that the rate of ESBL-EC positive urine

samples increased significantly during the study period (3 in 2001 compared to 55 in

2010). The most active agents were imipenem, meropenem, and fosfomycin (100%),

followed by amikacin (99.1%) and nitrofurantoin (84%). The least active substances

were ampicillin-clavulanate (20%), sulfamethoxazole (28%), and ciprofloxacin

(29.6%).

Only one organism E8 was selected to study eae gene. This organism was

completely resistant to all antibiotics tested and possesss antibiotic resistant gene,

plasmid profile. Assessment of eae gene revealed the presence of eae gene in strain

No. E8. Figure 14 revealed 397 bp gene fragement, which could be an initimin

responsible for pathogenicity of the isolates (Plate IV – P. Fig. 14).

32

PLATE IV

P. Fig - 14

Amplification of eae gene in E8 isolate

This study stated that all the 10 E. coli isolates were considered as a MDR

pathogens. These strains are not able to treat effectively with the current antibiotics.

Imipenem is the only antibiotics effective for UTI treatment. To overcome this

problem, there is a urgent need of new therapy from the nature and are effective for

the MDR Pathogens.

Among the 10 organisms studied E8 strain was subjected for molecular

characterization by 16s rRNA analysis. The results of the Phylogenetic analysis are

presented in Plates IV and V. Escherichia coli isolate was characterized by

sequencing process. The results were confirmed the identity of the isolates. E. coli

was 95-100% similarity with respective species. E. coli sequences was submitted to

genbank and obtained accession number KT971133 (Plate V and VI).

33

34

35

M. indica seed kernel appears the whitish brown in colour and mild aromatic

odour with bitter taste. The shape of the seed kernel is looks like a 70 hours chick

embryo. The length of seed kernel was 5.4±0.88 µm and width was 2.11±0.6 µm.

Texture of the seed kernel was coarse and the fracture was very smooth (Table 4).

Table 4

Organoleptic characters of Mangifera indica seed kernel

S. No Character Observation

1 Colour Brownish White

2 Odour Mild aromatic

3 Taste Bitter

4 Texture Coarse

5 Fracture Smooth

36

Plate VII illustrated herbarium of Mangifera indica which was submitted for

authentification. Plate VIII provides anatomic features of various parts of Mangifera

indica. P. Figs. 24, 25 and 26 shows the pictures of seed kernel used for the study.

Table 5 exhibited the physiochemical analysis of the M. indica seed kernel

powder. Foreign matters are absent in MISK powder. The ash content of the powder

material is directly proportional to the quantity of foreign matter. Total ash, acid

37

insoluble ash, water soluble ash was within the limit of ayurvedic pharmacopeia of

India. M. indica seed kernel powder exhibited 7.2% total ash, 3.6% water soluble ash

and 2.9% acid soluble ash. Powder showed higher percentage of water soluble

extractive (25.9%) followed by alcohol soluble extractive (24%). Higher extractive

values indicated that the plant showed higher polar compounds like phenolic

compounds etc.

Table 5

Physiochemical Parameters of Mangifera indica Seed Kernel

The fluorescence analysis is also an important tool for the determination of

constituents in herbal drugs and it provides an idea about the chemical nature of the

plant material. The powder drug analysis was carried out by treating the samples with

various chemical reagents and observations were made in visible light and ultra violet

light of short and long wavelengths. M. indica seed kernel powder showed

characteristic brown, light brown, green, yellow, yellowish green, brown and black

colour (Table 6). This indicated that different chromophores are available in the

MISK.

S. No Parameter Results

1 Foreign matter Nil

2 Dry Powder Particle size 1.37µm

3 Wet Powder Particle size 2.2µm

4 Swelling index 0.5mL

5 Acid insoluble ash 2.9%

6 Water soluble ash 3.6%

7 Total ash 7.2%

8 Alcohol extractive 24%

9 Water extractive 25.9%

38

Table 6

Fluorescence Analysis of Mangifera indica Seed Kernel Powder

S.

No

Test 0 hours 24 hours 48 hours

Day light UV light Day light UV light Day light UV light

1 Plant Powder +

Chloroform

Brown Light

brown

Brown Greenish

Brown

Brown Green

2 Plant Powder +

Hexane

Pale

brown

Yellow Pale brown Pale brown Light

brown

Pale

brown

3 Plant Powder +

Benzene

Reddish

brown

Brown Reddish

brown

Green Reddish

brown

Brown

4 Plant Powder +

Aqueous NaOH

Reddish

brown

Yellowish

brown

Reddish

brown

Yellowish

brown

Reddish

brown

Reddish

brown

5 Plant Powder +

alcoholic NaOH

Greenish

yellow

Yellow Greenish

yellow

Green Yellowish

brown

Green

6 Plant Powder + 1N

HCl

Light

brown

Yellow Light

brown

Light

brown

Light

brown

Yellow

7 Plant Powder +

Ethanol

Light

brown

Greenish

yellow

Light

brown

Pale

yellow

Light

brown

Greenish

yellow

8 Plant Powder +

Ethyl acetate

Reddish

black

Black Black Black Black Black

9 Plant Powder +

Acetone

Light

brown

Yellow Light

brown

Pale green Light

brown

Greenish

yellow

10 Plant Powder + 50%

H2So

4

Meroon Black Black Black Black Black

Microbial limit assay is essential to check microbial load and to assess

pathogenic contaminations. Quality of microbiological analysis of seed kernel was

shown in Table 7. The extracts were subjected to microbiological quality analysis by

standard methods. Total bacteria and fungi and pathogens were analysed. There was

no enteric pathogen present in seed kernel. Total aerobic microbial load is within the

limits of Ayurvedic and international pharmacopoeial standards (Anonymous, 2006).

Table 7

Microbial limit assay of Mangifera indica seed kernel

S. No Test organism Microbial counts

CFU/g

1 Total aerobic Bacteria 39x102

2 Total Fungal count 4x101

3 Total Enteric Bacteria Nil

4 Total E. coli Nil

5 Salmonella Nil

Antibacterial Assay

The use of antibiotics becomes problematic due to the development of

antibiotic resistance among clinical isolates. Today human society needs new drug

with lesser side effect and should effective against MDR pathogens. To screen

39

antimicrobial potentials of MISK aqueous and phenolic extracts, five UPEC isolates

were selected and utilized for antimicrobial assay. MISKE produced 17±0.1 µg/Disc

concentration (Table 8).

Table 8

Antibacterial activity of Mangifera indica seed kernel aqueous extract

(MISKAE) (Test pathogen n=5) against UPEC

S.

No

Nature of

Isolate

Positive

control

(mm)

Negative

control

(mm)

Concentration in µg/Disc

200 400 600 800

1 Biofilm

Positive

21±0.1 - 13±0.12 15±0.11 17±0.1 19±0.15

2 Biofilm

Negative

24±0.6 - 15±0.1 17±0.12 18±0.11 22±0.6

Phenolic extract of Mangifera indica seed kernel showed good antimicrobial activity

at 400µg/ disc concentrations itself which was better than aqueous extract. It produced

18±0.11mm zone of inhibitions against E.coli, at 400 µg/Disc concentration (Table 9).

MISKPE produced antimicrobial at 50 µg/disc against E. coli. MISK extracts showed

good antimicrobial activity at higher concentrations. MISKPE showed better

antimicrobial activity than MISKAE.

Table 9

Antibacterial activity of Mangifera indica seed kernel phenolic extract

(MISKPE) against UPEC

S.No Nature of

Strain

Positive

control

(mm)

Negative

control

(mm)

Concentration in µg/Disc

50 100 200 400

1 Biofilm

Positive

21±0.1 - 14±0.1 16±0.12 17±0.12 18±0.1

2 Biofilm

Negative

24±0.6 - 15±0.12 16±0.13 18±0.11 23±0.3

40

Drug dilution method was followed to assess minimal inhibitory concentration

of different extracts. Growth inhibition study was assessed specifically by the way of

expressing MIC. MISK phenolic extract showed effective bacterial inhibition at lower

doses when compared to aqueous extract (Table 10). MISKPE inhibited biofilm

negative E. coli at 125 µg/ml concentration and biofilm positive strains produced 425

and 225 µg/ml whereas it was only at 175 µg/ml by aqueous extract. Plant extract

tested in this work revealed good antimicrobial activity. Maximum extracts produced

more zone of inhibition against different pathogen when compared to positive control.

This would suggest that plant drug source may consider or best replacement for the

current antibiotic therapy.

Table 10

Minimal inhibitory concentration of Mangifera indica aqueous and phenolic

extract against UPEC

S. No Nature of Isolate MIC value of µg/ml

Aqueous extracts Phenolic extract

1 Biofilm Positive 450 225

2 Biofilm Negative 175 125

41

There are many reports available that plants have been evaluated In Vitro for

their antibacterial potency against some important human pathogenic bacteria (Singh

and Singh, 2005). Gram positive bacteria were more susceptible than that of gram

negative bacteria in response to the MISK extract observed in the present study.

Scherrer and Gerhardt (1971) reported that gram positive bacteria have outer

peptidoglycan layer which is not an efficient barrier. The gram negative bacteria have

an outer phospholipidic membrane that makes the cell wall impermeable to lipophilic

solutes, while the prune constitute a selective barrier to hydrophilic solutes with an

exclusion limits of about 600 Da. Many results confirmed these observations that

most plant extracts were found to be more active against gram positive bacteria than

gram negative ones (Kelmanson et al., 2000).

Antimicrobial property of a plant depends on its biologically active

phytoconstituents. A wide range of antiinfective actions have been assigned to tannins

(Haslam, 1996). Some authors have found that more highly oxidized phenols are

inhibitorier (Scalbert, 1991). Flavonoids are synthesized by plants in response to

microbial infection (Dixon et al., 1983). Terpenoids are active against bacteria

(Ahmed et al., 1993), fungi (Ayafor et al., 1994), viruses (Fujioka and Kashiwada,

1994) and protozoa (Ghoshal et al., 1996).

Antioxidant Assay

The antioxidant effect of aqueous and phenolic extracts of Mangifera indica

seed kernel was described in Table 11. DPPH radical is a widely used method to

detect antioxidants activities in a relatively short time compares to others methods

(Soares et al., 1997). The percentage of DPPH radical scavenging activity of phenolic

extract of M.indica seed kernel were 93.87±0.03 and aqueous extract showed the

53.8±0.01 at 100µg/ml concentration. The standard ascorbic acid produced

52.15±0.76% DPPH scavenging power at 50 µg/ml concentration. DPPH antioxidant

assay is based on the ability of 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free

radical, to decolourize in the presence of antioxidants (Table 11).

42

Table 11

DPPH Radical scavenging activity of Mangifera indica seed kernel Concentration 20µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100 µg/ml IC

50

MISKAE 26.55±0.02 27.9±0.02 29.99±0.08 30.58±0.03 33.87±0.02 97.01±0.02

MISKPE 15.57±0.01 17.58±0.01 32.54±0.02 39.00±0.02 50.10±0.03 90.55±0.03

Concentration 10 µg/ml 20 µg/ml 30 µg/ml 40 µg/ml 50 µg/ml IC50

Ascorbic acid 0.73±0.08 14.57±0.81 31.07±1.35 41.75±0.08 52.15±0.76 35.5±1.15

The reducing capacity of a compound may serve as a significant indicator of

its potential antioxidant activity. Antioxidants present in the sample reduce Fe3+

to

Fe2+

by donating amount of electrons and can be assessed by measuring OD at 700

nm. Percentage of inhibitance increased with increased dose dependent manner.

About 93.33% free radical scavenging power was exhibited by Phenolic extract at

100µg/mL concentration with 15.1±0.03 µg/ml IC50 (Table 12). The reducing power

of extract of M.indica was found remarkable and the reducing power of the extract

was observed to rise as the concentration of the extract gradually increased.

Table 12

Reducing power radical scavenging activity of Mangifera indica seed kernel

Concentratio

n

20µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100

µg/ml

IC50

MISKAE 13.3±0.01 26.4±0.02 36.9±0.03 44.3±0.01 53.8±0.01 86.4±0.02

MISKPE 23.8±0.03 54.8±0.02 89.7±0.04 90.1±0.05 93.3±003 15.1±0.03

Concentration 10 µg/ml 20 µg/ml 30 µg/ml 40 µg/ml 50 µg/ml IC50

Ascorbic

acid

5.4±0.01 13.3±0.05 26.9±0.08 37.5±0.09 53.6±0.08 64.9±0.02

Table 13 showed superoxide radical scavenging power of the extracts. Super

oxide radical scavenging assay was carried out to describe the antioxidant or super

oxide radical scavenging power of plant extracts which was found to be more than

51.75% for aqueous extracts and 52.81% for phenolic extracts at 100µg/ml

concentration with 63.4±0.05 µg/mL and 60.01±0.03 µg/mL of IC50 respectively.

Table 13

Superoxide radical scavenging activity of Mangifera indica seed kernel

Concentration 20µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100 µg/ml IC50

MISKAE 38.7±0.03 41.98±0.05 43.12±0.03 44.53±0.02 51.75±0.01 63.4±0.05

MISKPE 42.2±0.01 43.34±0.1 47.47±0.01 48.33±0.01 52.81±0.01 60.01±0.03

Concentration 10 µg/ml 20 µg/ml 30 µg/ml 40 µg/ml 50 µg/ml IC50

Ascorbic acid 66.3±0.80 66.5±0.01 68.12±0.80 70±0.61 71±0.6 40.2±0.93

43

Nitric oxide scavenging activity was observed with aqueous and phenolic of

M.indica seed kernel and it is significantly good when compared to standard (Table

14). MISKPE produced 80.27% nitric oxide scavenging activity and MISKAE

showed 46.34% at 100 µg/ml concentrations and only 53.67% inhibition were noted

in standard. Reactive oxygen species and Nitric oxide are available in inflammatory

cells and cancer cells and other pathological conditions (Moncada et al., 1991). It

was found that the reducing power of the extract increased based on their

concentration.

Table 14

Nitric Oxide scavenging activity of Mangifera indica seed kernel

Concentration 20µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100 µg/ml IC50

MISKAE 8.34±0.02 15.53±0.03 35.74±0.45 42.9±0.52 46.34±0.54 69.97±0.61

MISKPE 48.75±0.01 58.56±0.02 67.45±0.01 77.56±0.04 80.27±0.12 66.47±0.15

Concentration 10 µg/ml 20 µg/ml 30 µg/ml 40 µg/ml 50 µg/ml IC50

Ascorbic acid 5.4±0.21 13.39±0.31 26.97±0.36 37.5±0.39 53.67±0.66 64.9±0.68

ABTS method also depicted antioxidant power of MISK. 74.75% of

scavenging power was noted in MISKPE whereas 47.64 % for MISKAE at 100

µg/ml concentration (Table 15). The extracts efficiently scavenged ABTS radicals

generated by the reaction between 2, 2’‐azinobis (3‐ ethylbenzothiazolin‐6‐sulphonic

acid) (ABTS) and ammonium persulfate.

Table 15

ABTS radical scavenging activity of Mangifera indica seed kernel

Concentration 20µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100 µg/ml IC50

MISKAE 9.2±0.12 16.7±0.15 35.4±0.18 43.1±0.19 47.6±0.12 67.7±0.19

MISKPE 54.5±0.12 62.7±0.15 65.4±0.21 71.6±0.23 74.7±0.27 66.4±0.28

Concentration 10 µg/ml 20 µg/ml 30 µg/ml 40 µg/ml 50 µg/ml IC50

Ascorbic acid 5.4±0.01 13.3±0.03 26.9±0.02 37.5±0.06 53.6±0.05 64.9±0.06

H2O2 act as a recipient and receive the electrons from extracts/standard of

antioxidant compound. High antioxidant activity was noted for phenolic extracts

(72.57%) as well as aqueous extracts (46.34%) at 100 µg/ml concentration with more

than 66µg/ml of IC50 value (Table 16). H2O2 act as a recipient and receive the

electrons from extracts/standard of antioxidant compound.

44

Table 16

H2O2 scavenging activity of Mangifera indica seed kernel

Concentration 20µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100 µg/ml IC50

MISKAE 8.34±0.01 16.23±0.05 36.24±0.02 42.2±0.05 46.34±0.06 60.97±0.09

MISKPE 54.75±0.02 59.96±0.03 62.58±0.05 71.3±0.05 72.57±0.06 66.47±0.09

Concentration 10 µg/ml 20 µg/ml 30 µg/ml 40 µg/ml 50 µg/ml IC50

Ascorbic acid 5.4±0.02 13.39±0.05 26.97±0.06 37.5±0.02 53.67±0.01 64.9±0.05

Overall, all the methods expressed good antioxidant power which was

expressed with reference to percentage of inhibitance. Amomg the extract MISKPE

showed better result (Fig. 6).

DPPH is one of the free radicals widely used for testing preliminary radical

scavenging activity of the plant extract. Scavenging of DPPH radical is related to the

inhibition of lipid peroxidation (Rekka and Kourounakis 1991). DPPH is usually used

as a substance to evaluate the antioxidant activity (Tara et al., 2012). Antioxidants

either transfer an electron or a hydrogen atom to DPPH, thus neutralizing its free

radical character. The reducing capacity of compounds could serve as indicator of

potential antioxidant property (Meir et al., 1995). It is claimed that phenolic

compounds are powerful chain breaking antioxidants.

33.87

53.8 51.746.3 47.6 46.3

50.1

93.3

52.8

80.374.7 76.5

0

20

40

60

80

100

120

DPPH

Radical

Scavenging

Reducing

Power

Assay

Superoxide

Radical

Scavenging

Nitric

Oxide

Scavenging

ABTS

Radical

Scavenging

H2O2

Scavenging

Per

cen

tage

Antioxidant Method

Figure 6

In - Vitro Antioxidant power of MISK extracts

MISKAE

MISKPE

45

Tanaka et al., (1988) have observed a direct correlation between antioxidant

activity and reducing power of certain plant extracts. Reducing power activity is often

used to evaluate the ability of natural antioxidant to donate electron (Dorman et al.,

2003).

Superoxide anion is oxygen centered iron complexes such as cytochrome.

Superoxide radicals are known to be very harmful to cellular components as a

precursor of oxygen species (Halliwell & Gutreridge, 1999). Nitric oxide (NO), being

a potent pleiotropic mediator in physiological processes and a diffusible free radical in

the pathological conditions, reacts with superoxide anion and form a potentially

cytotoxic molecule, the 'peroxynitrite (ONOO‐)'. Its protonated form, peroxynitrous

acid (ONOOH), is a very strong oxidant (Malinski, 2007).ABTS assay is an excellent

tool for determining the antioxidant activity of hydrogen‐donating antioxidants and of

chain‐breaking antioxidants (Sreejayan, 1997). In the total antioxidant activity, ABTS

is blue chromophores produced by the reaction between ABTS and potassium per

sulfate and in the presence of the M.indica seed kernel extracts. Hydrogen peroxide is

a weak oxidizing agent that inactivates a few enzymes directly, usually by oxidation

of essential thiol (-SH) groups. It can cross cell membranes rapidly; once inside the

cell, it can probably react with Fe²+ and possibly Cu²+ ions to form hydroxyl radicals

and this may be the origin of many of its toxic effects (Kumaran and Karunakaran,

2007). The antioxidative activities observed can be attributed to either the different

mechanisms exhibited by different phenol and polyphenolic compounds that is,

tocopherols, Gallic acid, tannins, flavonoids and other organic acids and to the

synergistic effects of different compounds. Many studies have shown that many

phenolic compound contribute significantly to the antioxidant activity (Demla et al.,

2012) and act as highly effective free radical scavengers which is mainly due to their

redox properties, which can play an important role in adsorbing and neutralizing free

radicals, quenching singlet and triplet oxygen or decomposing peroxides.

The preliminary phytochemical screening of the aqueous extract revealed the

presence of terpenoids, flavonoids, saponins, phenolic compound tannins, Lignin,

protein and total carbohydrates where as phenolic extract showed alkaloids,

terpenoids, flavonids, phenolic compound and Tannins. This test is required to elicit

46

the primary nature of phytochemicals available in the extract. This study indicated

that Phenoilc compounds flavonoids, terpenoids and tannins may responsible for the

biological activity (Table 17).

Table 17

Phytochemical analysis of Mangifera indica seed kernel

S. No Test Aqueous

extract

Phenolic

extract

1 Alkaloids Negative Positive

2 Steroids Negative Negative

3 Terpenoids Positive Positive

4 Flavonoids Negative Positive

5 Saponins Positive Positive

6 Tannins Positive Positive

7 Lignin Positive Negative

8 Phlobatannins Negative Negative

9 Fat and Oil Negative Negative

10 Test for Glycosides Positive Positive

11 Inulin Negative Negative

12 Anthroquinones Negative Negative

13 Cardiac glycosides Positive Positive

14 Proteins Positive Positive

15 Carbohydrates Positive Positive

16 Amino acids Positive Positive

17 Phenolic compounds Positive Positive

In order to quantify important phenolic compounds available in the extracts,

standard spectrophotometric methods were used and the results indicated that aqueous

extract yielded 1.7% flavonoids,2.4% Tannins and 43% phenolic compound.

Phenolic extracts showed higher a concentration of these phytochemical’s such

flavonoids 6.1%, Tannins 12% and 48.6% of phenols. Presence of such diverse

phytochemical’s was indicated by different authors (Table18).

Table 18

Quantitative phytochemical analysis of Mangifera indica seed kernel

S. No Phytoconstituents % Yield (water) % Yield

(Phenolic)

1 Flavonoids 1.7±0.35 6.1±0.45

2 Tannins 2.4±0.23 12.±0.62

3 Polyphenols 43±0.81 48.6±0.76

UV-FTIR Analysis

The vibration of functional group of phenolic extract of M. indica could be

evaluvated from FTIR spectra. A characteristic peak obtained at 1665.9 cm-1

and

1535.08 cm-1

pertains to aromatic shift and major strectch at 3433 cm-1

contributes to

47

OH group. 2997cm-1 denotes the CH2 shift and also the significant shift at 1039 cm-1

and 952.98 cm-1

contributes the C-O vibration. The existence of one or more aromatic

rings is normally determined from C-H stretching and C=C ring vibration which was

observed from 1316 cm-1

,1429cm-1

and 2913cm-1

. The presence of C-H and OH

group above 3750cm-1

implies presence of phenolic compounds in M.indica seed

kernel phenolic extract (Table 19).

Table 19

FTIR analysis of MISKPE

S. No Peaks Groups

1 3433.96 OH

2 2997.86,2913.68 CH2

3 2449.43,2098.66,1992.17,1904.74 Enolic (C=C-OH)

4 1655.9,1535.08 Aromatic ring

5 1429,1316 C=C

6 1039,952.98 C-O

GC-MS Analysis

GC–MS analysis of the aqueous and phenolic extract of the M.indica seed

kernel showed the phenolic profile compounds including Benzene -1,3,5-triol, and

1,6,7-trihydroxy-2-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-

xanthone -9-one, Cholesta-4-6-dien-3-ol, were tentatively identified on the basis of

spectral data (Plate XXV and XXVI). These chemicals are belongs to phenolic

compounds. They may responsible for biopotentials of MISK.

48

P. Fig 29- structure of 1,6,7- Trihydroxy-2-……

OHO

HO

O OH

O

OH

OHHO

OH

1,6,7-Trihydroxy-2-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl)-xanthen-9-one

P. Fig.30 – Benzene -1, 3, 5-triol

HO OH

OH

Benzene-1,3,5-triol

The FTIR spectrum was used to identify the functional group of the active

components based on the peak value in the region of infrared radiation. The FTIR

spectrum confirmed the presence of alcohols, phenols, alkanes, alkynes, alkyl halides,

aldehydes, carboxylic acids, aromatics, nitro compounds and amines in different

extracts. Hence, the phenolic extracts subjected to UV-VIS and FTIR analysis for the

Plate X

P. Fig. 28– GCMS analysis of MISKPE

49

identification of chemical constituents present in M. indica. In addition, UV-VIS and

FTIR spectroscopy is proved to be a reliable and sensitive method.

The current pioneering study suggests that phenolic extract is a potent

therapeutic agent. It paves the way for the development of several treatment regimens

based on this extract. In addition, further research is necessary to identify and purify

the active compounds responsible for therapeutic activity. This was substantiated

from the FTIR studies. The Characterstc peak showed notable functional groups for

phenolic compounds.

Analysis of the extract under FTIR technique showed that the presence of

phenolic compound which can be isolated and further screened for different kind of

biological activities depending their therapeutic uses. Further research will be needed

to find out the structural analysis of flavonoid compound by use of different analytical

methods such as NMR and GC-Mass spectrophotometer.

Phenolics can enhance the body's immune system to recognize and destroy

cancer cells as well as inhibiting the development of new blood vessels (angiogenesis)

that is necessary for tumour growth. They also attenuate adhesiveness and

invasiveness of cancer cells thereby reducing their metastatic potential. Augmentation

of the efficacy of standard chemo- and radiotherapeutic treatment regimes and the

prevention of resistance to these agents is another important effect of plant phenolics

that warrants further research. Plant phenolics appear to have both preventative and

treatment potential in combating cancer and warrant further, in-depth research (Dai

and Mumper 2010).

Phenolics are compounds possessing one or more aromatic rings with one or

more hydroxyl groups. The structure diversity is a result of the variation in

hydroxylation pattern, stereochemistry at the three chiral centers. This result was

substantiated with the present NMR results where, the peaks showed aromatic rings

with hydroxyl groups.

Quantification of phenolic compounds in plant extract is influenced by the

chemical nature of the analyse, as well as assay method, selection of standards and

50

presence of interfering substances. Because of the heterogeneity of natural phenolics

and the possible interference from other readily oxidized substances in the plant

materials, it is not surprising that several methods have been used for determination of

total phenolics. The chemical structures of the purified phenolic acids were

confirmed by GC-MS and NMR (Khoddami et al., 2013).Phenolic extract of M.

indica shows the phenol and poly phenolic compound such as mangiferin (1, 6, 7-

trihydroxy-2-(3, 4, 5 – trihydroxy – 6 –hydroxymethyl – tetrahydro – pyran – 2 - yl) -

xanthone – 9 - one) and poly phenol (Benzene -1, 3, 5 - triol). In present study both

the compounds was carried out antioxidant, antiulcer, antidiarrhoeal and anticancer

activities.

Flavonoids, such as quercetin, are antioxidants. They scavenge damaging

particles in the body known as free radicals, which damage cell membranes, tamper

with DNA and even cause cell death. Antioxidants can neutralize free radicals and

may reduce or even help prevent some of the damage they cause. They also help in

keeping LDL ("bad") cholesterol under control, which scientists think may contribute

to heart disease. Flavonoids act like an antihistamine and an antiinflammatory drug

and may help protect against heart disease and cancer.

Polyphenolic compounds might inhibit cancer cells by xenobiotic

metabolizing enzymes that alter metabolic activation of potential carcinogens, while

some flavonoids could also alter hormone production and inhibit aromatase to

prevent the development of cancer cells. The mechanism of action of anticancer

activity of phenols could be by disturbing the cellular division during mitotis at the

telophase stage. It was also reported that phenols reduce the amount of cellular protein

and mitotic index and colony formation during cell proliferation of cancer cells (Zhao

et al., 2007). The Elrich tumour cells are one of the rapidly growing carcinoma with

very aggressive behavior and are able to grow in almost all strains of mice (Segura et

al., 2000). Ascitic tumour implantation promotes local inflammatory reactions leading

to increase in vascular permeability and results in intense edema formation, cellular

migration and progressive ascetic fluid formation. Ascetic fluid is essential for tumour

growth. Since it was constitutes the direct nutritional source for tumour cells. Thus

MISK extracts decrease the tumour volume, tumour weight and packed cell volume of

solid tumour. Reduction in the viable cell count and increased dead cell count towards

51

normal in tumour host suggest antitumour effect against EAC cells in mice (Bala et

al., 2010).

P. Fig. 31 - Cholesta-4-6-dien-3-ol.

Flavonoids are most dommonly known for their antioxidant activity and

reduce LPO not only by preventing or slowing the onset of cell necrosis, but also by

improving vascularity. Phenolic compounds are commonly known for their

antioxidant, anti-inflammatory and antimicrobial activities, while, tannins have been

reported to possess antioxidant, wound healing and antimicrobial activities.

Phytochemical study of AME indicated the presence of phenolic compounds,

flavonoids, tannins, saponins and phytosterols, etc., which have got important

pharmacological effects. Recent research has also shown that, through overlapping or

complementary effects, the complex mixture of phytochemical in fruits and

vegetables provides a better protective effect on health than single phytochemical.

The protective effects of MISKPE may therefore, be attributed to the complex

mixture of phytochemical present in MISKPE, which have been reported to have

antidiarrhoeal, antiulcer, anti-inflammatory, immunomodulatory and antimicrobial

properties (Ghatule et al., 2014).

Summary and Conclusion

52

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