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Research outline

Molecular insight into anti-infective effects of selected Nova Scotian fruit phytochemicals against Streptococcus pyogenes

Supervisor: Prof. Dr. Vasantha Rupasinghe

In general fruits and especially berries are good source of phytochemicals, particularly

anthocyanins, proanthocyanidins, and terpenoids. These phytochemicals known as secondary

metabolites, are considered to be anti-inflammatory, anti-carcinogenic, and anti-microbial

agents, as well as antioxidants. My proposed research project will focus on ten selected

berries/fruits of Nova Scotia and their anti-infective effects against a common infectious

pathogen called streptococcus pyogenes, still the leading cause of bacterial pharyngitis (strep.

throat) especially among children up to 36%. Rheumatic fever, rheumatic heart disease, and

glomerulonephritis (kidney inflammation) are the post-streptococcal sequelae followed by

unattended/untreated acute pharyngeal infections that could cause death.

The selected fruit crops contain substantial amount of these secondary metabolites and therefore

suggesting that these fruits probably possess anti-infective properties.

The fruits that will be tested in my proposed research are:

Cultivated berries: Blackcurrant (Ribes nigrum), Blueberry (Vaccinium corymbosum), Cranberry

(Vaccinium macrocarpon), Honeysuckle (Lonicera caerulea), Partridgeberry (Vaccinium vitis-

idaea)

Wild berries and fruits: Bayberry (Myrica pensylvanica), Bearberry (Arctous alpines),

Chokeberry (Aronia arbutifolia), Rosehip (Rosa rugosa), Staghorn (Rhus typhina)

There is fair scientific evidence that phytochemicals for example proanthocyanidins of cranberry

exert significant anti-streptococcal effects at as low concentration as 500 microgram per ml by

85 percent, in vitro. Some terpenoids such as ursolic acid, a penta-cyclic triterpene acid, found in

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rosehip, bayberry and juniper berry have been reported to completely inhibit the adherence of

streptococcus mutans, main cause of dental caries, at 1024 microgram per ml, in vitro. Various

medicinal plants have recently been tested for antimicrobial activity and all have proven that

phytochemicals particularly polyphenols exhibit significant antibacterial activity against different

strains of S. pyogenes and clinical isolates such as Wild maracuja (Passiflora foetida), white

weed (Ageratum conyzoides), Calabash tree (Crescentia cujete), bush-banana (Uvaria chamae),

ginger (Zingiber Officinale). Proanthocyanidins of the South African Geranium (Pelargonium

sidoides) have been shown to successfully suppress S. pyogenes growth.

1. Research hypothesis and objectives

Phytochemicals of the Nova Scotia’s cultivated and wild fruits can suppress or inhibit the growth

of S. pyogenes in a specific approach of adherence and biofilm formation inhibition under

experimental conditions.

1.1. General objective

Identification and understanding of mode of action of phytochemicals of the selected cultivated

edible berries and wild fruits of Nova Scotia with inhibition activity against S. pyogenes.

1.2. Specific objectives 1.2.1. Preparation of fruit extract by 4 different extraction techniques with 3 different

extracting solvents 1.2.2. Assessment of inhibition activity of polyphenol-rich and isoprenoid-rich extracts

of ten selected fruit crops against S. pyogenes by Broth dilution method, Disc diffusion assay, Bacterial growth assay

1.2.3. Chemical characterization of extracts with inhibition activity against S. pyogenes and fractionating the effective crude extracts

1.2.4. Investigation of mechanism of inhibition and disruption of adherence and biofilm formation of selected extracts/phytochemicals by Adherence inhibition assay, Biofilm formation inhibition assay, ATPase inhibition assay

1.3. Expected outcome

• Discovery and characterization of antimicrobial compounds of Nova Scotia fruits

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• Incorporation of most effective identified Nova Scotian fruit phytochemical(s) into

dehydrated bee honey lozenges for the prevention & treatment of strep. throat

• Contribution to scientific community by elucidating phytochemicals’ mode of action

1.4. Time line

• Winter 2014 course work, proposal writing

• Summer 2014 ATC completion, crude extraction & extracts fractionation

• Fall 2014 course work, crude extraction & fractionation will continue, initial micro-

biological screening phase

• Winter 2015 teaching assistantship, fractionation will continue, mode of action

(MOA) study

• Summer 2015 MOA study will continue, data analysis, article writing

• Fall 2015 data analysis, thesis writing & defense