ANTIBACTERIAL ACTIVITY OF ISOLATED PHYTOCHEMICALS(MICRO-591)

Maninderjeet kaurL-2008-BS-09-IM

M.Sc. Int. (Hons.) Microbiology

Punjab Agricultural University,Ludhiana

PHYTOCHEMICALS

“Phyto” - plant. Naturally occurring, biologically active chemical

compounds in plants. Phytochemicals are beneficial to human health as

responsible for the disease protection through various modes of action.

More than 4000 of these compounds have been discovered to date and it is expected that scientists will discover many more.

TYPES OF PHYTOCHEMICALS Alkaloids Glycosides Flavonoids Phenolics Tannins Terpenes Essential Oils Phyto-estrogens Phytosterols

ALKALOIDS

Largest group of secondary chemical constituents

Made from ammonia compounds Basically of nitrogen bases synthesized from

amino acid

Plant-derived alkaloids in clinical use are Morphine

Codeine

SOME SCREENING METHODS FOR THE DETECTION OF ALKALOIDSReagents/Test Composition Result

Meyer’s Reagent Potassium mercuric iodide solution

Cream precipitate

Wagner’s Reagent Iodide in potassium iodide

Reddish-brown precipitate

Tannic Acid Tannic acid Precipitate

Hager’s Reagent A saturated solution of picric acid

Yellow precipitate

GLYCOSIDES Condensation products of sugars Colorless, crystalline carbon, hydrogen and

oxygen- containing water-soluble phytochemicals

Found in cell sap

Plant-derived glycosides are:- α- Terpineol

Cinnamyl acetate

β- glucosid

FLAVONOIDS Important group of polyphenols Widely distributed among the plant flora Found in almost all plant based food and

beverages Level depending in degree of ripeness,

variety and processing >4000 distinct flavonoids identified

Nearly present in 70% of plants. Quercetin, Kaempferol and QuercitrinOther group include Flavans Flavonods Anthocyanidins Catechin

Anthocyanidins

PHENOLICS Chemical compounds occur as natural color

pigments Responsible for the color of fruits of plants Have multiple functionsClassified into- Phenolic acids Flavonoid polyphenolics (flavones,

flavonones) Non-flavonoid polyphenols

CAFFEIC ACID is regarded as most common phenolic compound distributed in plant flora.

TANNINS Widely distributed in plant flora Phenolic compounds of high molecular

weight Found in root, bark, stem and outer layers of

plant tissues Gallic acid- plant derived

TERPENES

Most widespread Chemically diverse group of natural productsMajor plant derived- β- Caryophyllene Terpenolen α- cubebene

ESSENTIAL OILS Odorous and volatile productsMajor plant derived are- Eugenol Gein Senigrin Amygdalin

PHYTOESTROGENS

Naturally occurring Resemble mammalian oestrogen Iso- flavonone -associated with cancer

prevention, improved cardio-vascular health and improved bone health.

PHYTOSTEROLS Most abundant in the seeds of green and

yellow vegetables. Important in human diet-reduce cholesterol

and facilitate excretion from the body

TOP TWELVE PHYTO FOODS

Soy Tomato Broccoli Garlic Flax seeds Citrus fruits Melons: cantaloupe,

watermelon Pink grapefruit Blueberries Sweet potatoes Chili peppers Legumes: beans, and

lentils

MECHANISM OF ACTION OF PHYTOCHEMICALS

Inhibit microorganisms Interfere with metabolic processes Modulate gene expression Modulate signal transduction pathways Chemotherapeutic with chemo-prevention

referring to the use of agents to inhibit, reverse or retard tumourgensis.

MECHANISM OF ACTION

SOME SPECIFIC MODES OF ACTIONS

Anti-oxidants Anti-carcinogenic Anti-ulcer Anti-diabetic Anti-inflammatory Antimicrobial activity

ANTIMICROBIAL ACTIVITY

Protect against pathogenic insects, bacteria, fungi or protozoa.

Phenolics helping in the reduction of particular adherence of organisms to the cells lining the bladder which reduces UTIs.

Exert bacteriostatic or bactericidal activity on microbes.

Volatile gas combinations of cinnamon and clove oil inhibit growth of spoilage fungi, yeast and bacteria.

Antimicrobial activity varies for the same plant part tested due to geographical location, nutrient content and extraction methods.

ANTIBACTERIAL ACTIVITY MECHANISMS OF VARIOUS

PHYTOCHEMICALS

1.Inhibition of nucleic acid- DNA synthesis strongly inhibited by

flavonoids in Proteus vulgaris RNA synthesis most affected in

Staphylococcus aureus. B-ring intercalates with the hydrogen

bonding with the stacking of nucleic acid bases and lead to inhibitory action.

DNA gyrase was inhibited in Escherichia coli. by different phytochemicals.

2. Inhibition of cytoplasmic membrane functionSophoraflavanone G have antibacterial activity against Methicilin-resistant Staphylococcus aureus and streptococci.Studied through liposomal model membranes lead to the alteration of membrane fluidity in hydrophilic and hydrophobic regions so reduce the fluidity of outer and inner layers.

A strong antibacterial catechin found in green tea. Catechin have greater activity against Gram-

positive than Gram-negative bacteria. Catechin perturb lipid bilayers by directly

penetrating them and disrupting the barrier function.

3. Inhibition of energy metabolism Licochalcone (flavonoids) have inhibitory activity

against Staphylococcus aureus and Micrococcus luteus but not against E.coli.

Interferes with the energy metabolism as energy is required for active uptake of various metabolites.

Inhibit strongly oxygen-consumption in Micrococcus luteus and Staphylococcus aureus but not in E.coli.

ANTIBACTERIAL ACTIVITY OF AQUEOUS EXTRACT OF ALLICIN AGAINST METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS Allicin main biologically active

antimicrobial phytochemical produced in garlic extracts The allicin liquid tested for

antimicrobial activity against MRSA using an agar well diffusion method.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).

A TYPICAL ZONE OF INHIBITION PRODUCED BY 500MG/ML ALLICINAGAINST MRSA.

Allicin liquid was active against S. aureus strains at or above 62.5 μg/mL.

No activity was detected below 62.5 μg/mL

Concentrations of 250 μg/mL and above are most active.

88% of strains have MICs for allicin at 16 μg/mL, and all strains were inhibited by allicin at 32 μg/mL.

The present study demonstrated that the majority (88%) of strains have MBCs for allicin at 128 μg/mL and all the strains were killed by allicin at 256 μg/mL.

PHYTOCHEMICALS OF CITRUS FRUITS

Different solvent extracts (ethyl acetate, ethanol, petroleum ether and water) showed the presence of flavonoids, terpenoids, tannins and alkaloids from citrus fruit peels and Citrus limon.

ANTIBACTERIAL ACTIVITY Against four pathogenic bacteria organisms

(S. aureus, E.coli, S. typhi and K. pneumonia )

Acetone extract of Citrus sinensis shows maximum zone of inhibition against E.coli (16mm) followed by S. typhi (15mm), K. pneumonia (14mm) and S. aureus (13mm) whereas the ethanol and aqueous extract of Citrus sinensis not show such high antibacterial activity.

Not all phytochemicals have equal antibacterial activity. This is due to the difference in extraction process and difference in plant parts.

PHYTOCHEMICALS OF MENTHA PIPERITA L.(PEPPERMINT)

Perennial and strongly scented herb.

Crude extracts from different plant part is rich in at least one of the alkaloids, flavonoids, phenols, tannins.

Higher concentrations of alkaloids were present in the ethanol, methanol and ethyl acetate leaf extracts when compared to the stem and root extracts.

The antibacterial potential of six extracts from leaf, stem and root are effective against bacteria such as B. subtilis, S. pneumonia, S. aureus, E. coli, P. vulgaris and K. pneumonia.

Among all the extracts, ethyl acetate, chloroform and ethanol leaf extracts showed high activity (7.2 - 15.3 mm of zone of inhibition) on all organisms.

The root extracts (chloroform, hexane and petroleum ether) did not show any activity on S. pneumonia, S. aureus, E. coli, P. vulgaris and K. pneumonia.

PHYTOCHEMICALS OF MEDICINAL PLANTS(TULSI, CLOVE, NEEM AND

DATIWAN)

The percentage yield of the phytochemicals in these plants are-

Tulsi-29.08% Clove-19.58% Datiwan- 21.07% Neem- 17.15%Basically, alkaloids, glycosides, terpenoids, steroids, flavonoids, tannins and reducing sugars were there.

Among four plants Clove is most effective against S. typhi.

All the plants were ineffective against E. coli and K. pneumonia.

The largest zone of inhibition (22 mm) obtained with clove against S. typhi and Minimum Bactericidal Concentration (MBC) value of 5 mg/l effective with Neem against S. typhi

K. pneumoniae and E. coli were found to be resistant with all the plant extracts.

PHYTOCHEMICALS OF AMLA (EMBLICAOFFICINALIS)

The phytochemicals from the solvent free extract of Emblica officinalis fruit as well leaf are:-

Isolated phytochemicals show antibacterial activity against three respiratory pathogens i.e. Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae.

Synergistic interactions are also important.

+++ : Highest synergistic activity ++ : Moderate synergistic activity, - : Indifference

PHYTOCHEMICALS OF SWEITNIA MAHAGONI

Large deciduous and economically timber tree. The crude extract of this consists of

The seed extract is active against 5 Gram positive and 9 Gram negative bacteria and 5 fungal strains.

The extract is more active as compared to amoxicillin against Bacillus thuringienesis and Pseudomonas aeruginosa at 1mg/L.

The MIC and MBC ranging form 12.5 to 50 mg/L and 25 to 50 mg/L for the SMCM seed extract respectively.

PHYTOCHEMICALS OF MORINGA OLEIFERA

Commonly known as “Drumstick”.

Highly nutritive vegetable in many countries.

Leaves contains phytochemicals having potent anticancer activity.

Antimicrobial activity of Chloroform, Ethanol, Water extract of Moringa oleifera against human pathogens

The antibacterial activity of chloroform extract-

Maximum zone of inhibition (6 mm) against Escherichia Coli, Salmonella typhii, No zone of inhibition against Pseudomonas aeruginosa, Staphylococcus aureus.

The antibacterial activity of ethanol extract

Maximum zone of inhibition (14 mm) against Salmonella typhii and minimum inhibitory zone (8 mm) against Escherichia Coli .

The antibacterial activity of aqueous extract-

Maximum inhibitory zone (8 mm) against Staphylococcus aureus. No inhibitory zone against Salmonella typhii, Pseudomonas aeruginosa, Escherichia Coli.

CONCLUSION Future optimization of these compounds

through structure alteration allow the development of a pharmacologically acceptable antimicrobial agent and group of agents.

The increasing acceptance of the chemical diversity of natural products is well suited to provide the core scaffolds for future drugs.

There is need to be further developments in the use of novel natural products and chemical libraries based on natural products in drug discovery campaigns