on the use of several plant by product which possess ...shodhganga.inflibnet.ac.in/bitstream/10603/19704/9/09_chapter 3.pdf · Bacterial diseases are responsible for heavy mortality

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Bacterial diseases are responsible for heavy mortality in wild and cultured fish. The

problems in the farms are usually tackled by preventing disease outbreaks or by

treating the actual disease with drugs or chemicals. The use of antimicrobial agents

has increased significantly in aquaculture practices (Alderman and Michel, 1992).

Antibiotics used in both human as well as veterinary medicines have been tried

experimentally to treat bacterial infections of fish. Problems including solubility,

palatability, toxicity, cost, delivery and governmental restrictions have limited the

available antibiotics to select few, especially in food fish culture. The indiscriminate

use of antimicrobials for disease control in animals increases the selective pressure

exerted on the microbial world and encourages the natural emergence of bacterial

resistance (Allsop, 1998; Verchuere et al., 2000). Decreased efficacy and resistance

of pathogens to antibiotics has necessitated development of new alternatives for

disease control in Aquaculture (Smith et al., 1994). Vaccination may be the most

effective method of controlling fish disease, even though disease caused by bacteria

like Aeromonas hydrophila has not been controlled by vaccination due to their

heterogeneity. It is therefore necessary to search for novel antibacterial compounds

with therapeutic potential for which the pathogens may not have resistance (Patil et

al., 2001). It is estimated that there are 250,000 to 500,000 species of plant on earth

(Borris, 1996). The effects of plant extracts on microbes have been studied by a very

large number of researchers in different parts of the world. For instance, water and

alcohol extracts of Ocimum sanctum and Ocimum gratissimum were highly toxic

against fungi after 15 days culture (Amadioha, 2000). Further extracts of garlic, onion

and green pepper have been reported to inhibit the growth of Escherichia coli,

Salmonella typhosa, Shigella dysenterae and Staphylococcus aureus (Johnson and

Vaughn, 1969; Sofowora, 1983; Arora and Kaur, 1999). Reports are also available

129

on the use of several plant by product which possess antimicrobial properties, on

several pathogenic bacteria and fungi (Rocio and Rion, 1989; Akobundu and

Agyakwa,1987; Almagboul et al., 1988; Deans and Svoboda, 1990; Diker et al.,

1991; Heisey and Gorham, 1992; Mishra et al., 1992; Oboh et al., 1992;

Hablemariam et al., 1993; De Pooter et al., 1995; Lis – Balchin and Deans, 1996;

Hilli et al., 1997; Oboh and Abula,1997; Reddy et al., 2001; Erdogrul, 2002; Atefl and

Erdogrul, 2003). In India much work has been done on ethno medicinal plants

(Maheswari et al., 1986, Rai, 1989; Negi et al., 1993). Interest in a large number of

traditional natural products has increased (Taylor et al., 1996). It has been

suggested that aqueous and ethanolic extracts from plants used in allopathic

medicine are potential sources of antiviral, antitumoral and antimicrobial agents

(Chung et al., 1995; Vlietinck et al., 1995). The selection of crude plant extracts for

screening programs has the potential of being more successful in initial steps than

the screening of pure compounds isolated from natural products (Kusumota et al.,

1995). The medicinal value of these plants lies in some chemical substances that

produce a definite physiological action on human body. The most important of these

bioactive constituents of plants are alkaloids, tannins, flavonoids and phenolic

compounds (Hill, 1952). Though literature speaks diverse studies of bioactivity of

plant extracts against human pathogens, our work on testing the antibacterial

efficacies of these on fish pathogens was comparatively a new concept and not

much attempt had been made earlier in this line. The three-selected medicinal flora

was screened for potential antibacterial activity against the pathogens isolated from

Labeo rohita affected by bacterial septicemia. This study also investigated the

fundamental scientific bases for the use of Azadirachta indica leaf extract, Piper

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betle leaf extract and Allium sativum clove extract by defining the phytochemical

constituents present in these plants.

Materials and methods

Description of Labeo rohita (Hamilton, 1822)

Rohu is the natural inhabitant of river systems of India, Nepal, Pakistan, Bangladesh

and Burma (Plate 1). In recent years it has been transplanted to many countries of

the world including Sri Lanka, Mauritius, USSR, Japan, Philippines, Laos, Malaysia

and Thailand. Normally it occupies the column region of the aquatic ecosystem and

feeds mostly on vegetable matter including higher plants, detritus, etc. It naturally

breeds in rivers and under special conditions in bundhs. Except by hypophysation to

which it responds quickly, it never breeds in ponds. It attains sexual maturity during

the second year. However, certain percentages of pond reared specimens mature

within one year. Fecundity varies from 2, 26,000 to about 28, 00,000 depending

upon the size (Khan and Jhingran, 1975). Rohu spawns during the monsoon (April—

September). Seeds collected from rivers or produced by bundh breeding or induced

breeding are reared with ease in seasonal or perennial undrainable ponds. Under

pond culture conditions it grows up to 900 g within one year.

Plate.1 LABEO ROHITA

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Taxonomy

Family : Cyprinidae

Order : Cypriniformes

Class : Actinopterygii

Genus : Labeo

Species : Rohita

DESCRIPTION OF TEST PLANTS

Azadirachta indica A. Juss.

Neem, Azadirachta indica is a fast growing sclerophyllous tree which is present in

the Indian subcontinent (National Research Council, 1992) and is now distributed

throughout Southeast- Asia, East and Sub-sahelian Africa, and some parts of

Central America (Schmutterer, 1990; Ascher, 1993). Azadirachta indica has been

used for centuries as the country store of developing nations. Earliest reference to it

is in Sanskrit writings that are over 4,000 years old (Larson, 1990). This broad-

leaved evergreen can reach heights of 30 meters with a trunk girth of 2.5 meters.

Neem matured at 3-5 years and fully reproductive at 10 years and live for over two

centuries. Its deep root system is well adapted to retrieving water and nutrients from

the soil profile, but this deep root system is very sensitive to water logging. The

neem tree thrives in hot, dry climates where shade temperatures often reach 50

degrees celsius and annual rainfall ranges from 400 to 1,200 millimeters. The tree

can withstand many environmental adversities including drought and infertile, stony,

shallow, or acidic soils (Verkerk and Wright, 1993). Neem has more than 100 unique

bio-active compounds, which have potential applications in agriculture, animal care,

public health, and for even regulating human fertility. The effective ingredients are

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present in all parts of the tree but are highly concentrated in the seeds (National

Research Council, 1992).

There is evidence, but no scientific correlation, that trees grown in climates with

lower rainfall produce kernels with higher content of metabolites (Schmutterer,

1990).

Taxonomy

Phylum : Angiospermatae

Class : Dicotyledoneae

Order : Sapindales

Family : Meliaceae

Genus : Azadirachta

Species : Indica

Botanical description

Medium sized tree, up to 15 m tall, rarely 25 m, with short, straight bole and long

spreading branches, forming a dense, large, oval or rounded crown. Ever-green or,

under extreme heat and drought, deciduous. Old bark turning dark grey, thick and

furrowed. Leaves imparipinately compound with 7-17 pairs of leaflets, which are

ovate or lanceolate, falcate with uneven base and dentate margins, 6-8 cm long, 1-3

cm wide (Plate 2). Inflorescence, a 10-30 cm long panicle with many small white to

cream colored flowers (Plate 3). The neem produces ellipsoidal drupes (Plate 4) that

are about two centimeters in length, borne on axillary clusters. These fruits contain

kernels that have high concentrations of secondary metabolites (National Research

Council, 1992).

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Plate 2. Azadirachta indica sapling

Plate 3. Azadirachta indica Plate 4. Azadirachta indica

inflorescence branch showing ellipsoidal drupes

134

Special features

Azadirachta indica is known to possess antiinflammatory, antipyretic, antimicrobial,

anti-diabetic and diverse pharmacological properties (Satyanarayana et al., 1978;

Okpanyi and Ezeukwu, 1981; Rochanakij et al., 1985; Chopra and Chopra, 1955)

The aqueous extract of neem bark and leaf also possesses anticomplement and

immunostimulant activity. Neem oil has been shown to possess activity by selectively

activating the cell-mediated immune mechanisms to elicit an enhanced response to

subsequent mitogenic or antigenic challenge.

More than 135 compounds have been isolated from different parts of neem and

several reviews have also been published on the chemistry and structural diversity of

these compounds. The compounds have been divided into two major classes:

isoprenoids (like diterpenoids and triterpenoids containing protomeliacins, limonoids,

azadirone and its derivatives, gedunin and its derivatives, vilasinin type of

compounds and C- secomeliacins such as nimbin, salanin and azadirachtin ) and

non-isoprenoids, which are proteins (amino acids) and carbohydrates

(polysaccharides), sulphurous compounds, polyphenolics such as flavonoids and

their glycosides, dihydrochalcone, coumarin and tannins, aliphatic compounds, etc.

(Schmutterer et al., 1995).

Piper betle Linn.

The Betel (Piper betle) is a spice whose leaves have medicinal properties. The plant

is evergreen and perennial, with glossy heart-shaped leaves and white catkins, and

grows to a height of about 1 metre. The Betel plant originated in Malaysia and now

grows in India, Indonesia and Sri Lanka. The best Betel leaf is the "Magahi" variety

(literally from the Magadha region) grown near Patna in Bihar, India.

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The active ingredients of betel oil, which is obtained from the leaves, are betel-

phenol (or chavibetol or 3-hydroxy-4-methoxyalkylbenzene, which gives a smoky

aroma), chavicol and cadinene. The leaves are chewed together with mineral lime

(calcium oxide) and the areca nut.

Taxonomy


Class : Dicotyledoneae

Order : Piperales

Family : Piperaceae

Genus : Piper

Species : Betle


The betel leaf plant is a branching vine that may climb as high as 10-15ft (Plate 5

and 6), although it often grows as an understory ground cover. The plant prefers

warm, humid conditions, but can tolerate some drought. It is generally too tender to

grow outside of the tropics. Plants are climbers and dioecious. Stems rooted at

nodes, 2.5-5 mm thick, slightly woody. Petiole 2-5 cm, very finely powdery

pubescent; prophylls ca. 1/3 as long as petioles; leaf blade ovate to ovate-oblong,

those at apex of stem sometimes elliptic, 7-15 × 5-11 cm, papery to ± leathery,

abaxially densely glandular with very finely powdery pubescent veins, adaxially

glabrous, base cordate, sometimes rounded in leaf blades toward apex of stem,

symmetric or nearly so, apex acuminate; veins 7, apical pair arising 0.7-2 cm above

base, usually opposite, others basal; reticulate veins conspicuous. Spikes leaf-

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opposed. Male spikes nearly as long as leaf blades at anthesis; peduncle nearly as

long as petioles; rachis pubescent; bracts orbicular or suborbicular, rarely obovate,

1-1.3 mm wide, peltate, ± sessile. Stamens 2; filaments thick, ca. as long as anthers

or longer; anthers reniform. Female spikes 3-5 × ca. 1 cm, longer in fruit; rachis

fleshy, densely pubescent. Ovary partly immersed in and connate to rachis, apex

tomentose; stigmas usually 4 or 5, lanceolate, tomentose. Drupes fused to form

terete, fleshy, reddish, compound fruit, apices tomentose, prominent. Flower on May-

July (The Wealth of India, 1992)

Plate 5. Piper betle climbing on a twig Plate 6. Piper betle attached to a tree

Special features

P. betle is one such a plant of the several ingredients in a chew commonly known as

‘pan’. Usefulness of this plant against various dieases can be traced in the ancient

vedic literature Atharved as early as 3000 -2500 BC and its vedic name is Saptasira.

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Betel leaves with strong pungent and aromatic flavour are widely consumed as a

mouth freshener. The leaves are credited with wound healing digestive and

pancreatic lipase stimulant activities in the traditonal medicine (Santhanam and

Nagarajan, 1990; Chatterjee and Pakrashi, 1995) which has also been proved with

experimental animals (Prabhu et al., 1995).

Leaf extracts were reported to possess antioxidant property (Majumdar et al., 2002;

Majumdar et al., 2003), antimicrobial (Ramji et al., 2002) antifungal and anti

inflammatory activity (Ambarta, 1986).

P. betle is an aromatic, carminative, stimulant and astringent used as a preventive

for worms and in snake bite (The Wealth of India, 1992). Juice of the leaves is

dropped into eyes in painful infection and night blindness. Essential oil from leaves of

this plant has been used for the treatment of respiratory catarrhs and as antiseptic

and the fruit is employed with honey as a remedy for cough (Chandra et al., 1987).

The leaves, known as Sirih in Malayan, provide an oil that contains a number of

phenolic substances, many of which are allylphenols. Chavicol (4-hydroxyallyl-

benzene) is a major component and is strongly antiseptic (Ueda and Sasaki., 1951).

Allium sativum Linn.

Garlic (Allium sativum), a member of the lily family, is a perennial plant that is

cultivated worldwide. The garlic bulb is composed of individual cloves enclosed in a

white skin. It is the bulb, either fresh or dehydrated, that is used as a spice or

medicinal herb.

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Garlic contains 0.1-0.36% of a volatile oil composed of sulfur-containing compounds:

allicin, diallyl disulfide, diallyl trisulfide, ajoene and minor amounts of other di- and

polysulphides. These volatile compounds are generally considered to be responsible

for most of the pharmacological properties of garlic. Other constituents of garlic

include: alliin (S-allyl-L-cysteine sulfoxide), S-methyl- L-cysteine sulfoxide, protein

(16.8%, dry weight basis), high concentrations of trace minerals (particularly

selenium), vitamins, glucosinolates, and enzymes (alliinase, peroxidase, and

myrosinase) (Leung, 1980; Raj and Parmar, 1977).

Garlic is believed to stem from Central Asia, although no wild form is known. Of the

about 700 species of genus Allium, many are native to Central Asia, the center of

diversity ranging from the Himalayas to Turkestan.

Garlic has a very long history of use as a food and a medicine (Phillips and Foy,

1995). It was given to the Egyptian laborers when building the pyramids because it

was believed to confer strength and protect from disease, it was also widely used by

the Romans (Phillips and Foy, 1995).


The leaves are long, narrow and flat like grass (Plate 7). The bulb (the only part

eaten) is of a compound nature, consisting of numerous bulblets, known technically

as 'cloves,' grouped together between the membraneous scales and enclosed within

a whitish skin, which holds them as in a sac (Plate 8). The flowers are placed at the

end of a stalk rising direct from the bulb and are whitish, grouped together in a

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globular head, or umbel, with an enclosing kind of leaf or spathae, and among them

are small bulbils.

1.

Plate 7. Young garlic plant Plate 8. Garlic bulb

Taxonomy


Class : Monocotyledoneae

Order : Liliales

Family : Liliaceae

Genus : Allium

Species : Sativum

140

Special features

Garlic has a very long folk history of use in a wide range of ailments, particularly

ailments such as ringworm, Candida and vaginitis where its fungicidal, antiseptic,

tonic and parasiticidal properties have proved of benefit (Sharma, 1977; Amer et al.,

1980; Prasad and Sharma, 1980; Adetumbi and Lau, 1983; Hughes and Lawson,

1991). The fresh bulb is much more effective medicinally than stored bulbs;

extended storage greatly reduces the anti-bacterial action.

Garlic has strong antioxidant properties and its role in preventing age-related

diseases like cardiovascular diseases, cancer, arthritis, cataract formation etc. had

been investigated for the past 10-15 years (Rahman, 2003).

Pharmacological research on garlic has shown for the thiosulfinates free radical

scavenging, inhibition of lipid peroxidation (Phelps and Harris, 1993; Harris et al.,

1995), inhibition of platelet aggregation (Barrie et al., 1987; Kiesewetter et al., 1993),

stimulation of fibrinolysis (Kiesewetter et al., 1990), and reduction of serum

cholesterol and lipid levels (Bordia, 1981; Brosche et al., 1990; Mader, 1990;

Rotzsch et al., 1992). In vitro and in vivo animal studies have also demonstrated

garlic's ability to inhibit tumor formation (Weisberger and Pensky, 1958; Belman,

1983) and reduce blood pressure (Elbl, 1991; Koch, 1992). Other in vitro studies

have concluded that garlic possesses direct anti-atherosclerosis effects and

inhibition of cholesterol biosynthesis by allicin and ajoene. In vivo studies in animals

have concluded that garlic powder, fresh garlic, and garlic oil reduced experimentally

induced hyperlipidemia and atherosclerosis (ESCOP, 1997).

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Collection of plant material

Azadirachta indica leaf were collected from Sri Paramakalyani Centre for

Environmental Sciences Campus, Piper betle leaf were collected from a farm at Attur

and Allium sativum cloves were collected from local market. The plants were

identified and classified by Dr. M.Vishvanathan (Taxonomist), of Sri Paramakalyani

centre for Environmental Sciences, Manonmaniam Sundaranar University,

Alwarkurichi and the voucher specimens were deposited in the laboratory of Sri

Paramakalyani Centre for Environmental Sciences, Alwarkurichi.

Extraction

The extraction method used in this study was a modification of Ann (1985), Azoro

(2000), Nair et al. (2005) and Akinyemi et al. (2005). The plant materials were

washed several times in running water and allowed to air dry. This was done to

reduce the microbial load of the plant material due to handling and stress. The outer

covering of garlic was manually peeled and sliced into cutlets. These sliced cutlets

and leaves of Azadirachta indica and Piper betle having initial weight of 200g were

separately oven dried at temperature of 60ºC for 6 days. Using a milling the plant

material was pulverized into powder. The powder was weighed using electronic

weighing machine.

Weights of the powder

Azadirachta indica - 54.02g

Piper betle - 37.24g

Allium sativum - 63.48g

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Aqueous extraction

5g of the Azadirachta indica leaf powder was dissolved in 250 ml conical flask using

200 ml distilled water and maintained at 60ºC for 3 hours. The mixture was filtered

through Whatman No.1 filter paper. The precipitate was discarded and the filtrate

was poured into pre weighed Petri plates; evaporated to dryness in rotary evaporator

and the dried extract was used for the experiment. The same procedure was

followed for the preparation of water extract of Piper betle leaf and Allium sativum

clove. After evaporation the extracts were recovered and weighed.

Ethanol extraction

5g of the Azadirachta indica leaf powder was dissolved in 250 ml conical flask using

200 ml 80% ethanol, plugged with cotton and kept on a rotary shaker at 190 -220

rpm for 24 hours. The mixture was filtered through Whatman No.1 filter paper. The

precipitate was discarded and the filtrate was poured into pre weighed Petri plates;

evaporated to dryness in rotary evaporator and the dried extract was used for the

experiment. The same procedure was followed for Piper betle leaf and Allium

sativum clove ethanol extract preparation. After evaporation the extracts were

recovered and weighed.

This process of extraction was repeated to recover larger quantity of the extracts and

they were stored in the refrigerator at 0ºC for further use of antimicrobial sensitivity

testing. The yields were recovered as percentage of the quantity of the initial plant

material (5g) used.

Yield in g × 100

--------------------- = Yield (%)

5

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Bacterial species

All the highly virulent and moderately virulent strains of Aeromonas hydrophila and

Pseudomonas fluorescens isolated from Labeo rohita and stored in the laboratory

after pathogenicity study were retrieved for antibacterial study. The stock was

cultured onto TSA (Tryptic soy agar medium) incubated for 24 hours at 28ºC and

used for the study.

Antibacterial activity Assay

The antibacterial activity of the samples was assayed by the standard Nathan’s Agar

Well Diffusion (NAWD) technique (Nathan et al., 1978) against the test strains on

Tryptic soy agar (TSA) in Petri plates with drilled wells of 6 mm diameter. A constant

amount of 0.7g of extracts in 50 µl Dimethyl Sulfoxide (DMSO) was placed on to

each well. The well at the centre without the extract serves as control. After 22 -24

hour of incubation at room temperature, the susceptibility of the test organisms was

determined by measuring the diameter of the zone of inhibition around each well.

Oxytetracycline disc was used as positive control. The test was repeated three times

and the average of the zones was recorded in millimeters and results were reported

as mean ± standard deviation of treatment. The antibacterial spectra of the selected

plants against the test microorganisms were also reported as percentage, calculated

by taking positive control oxytetracycline as 100% inhibition. The data was analysed

by analysis of variance (ANOVA).

Determination of Minimum Inhibitory Concentration (MIC)

Minimum inhibitory concentration (MIC) was determined by serially diluting the

extracted fractions in DMSO so that concentrations of 100 µg, 125 µg, 150µg,

175µg, 200µg, 225µg, 250µg, 275µg and 300µg/50 µl DMSO were loaded into each

well for testing the susceptibility of the organisms. MIC is regarded as the

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concentration giving the least inhibitory activity and below which there is no further

inhibition.

PHYTOCHEMICAL SCREENING

Chemical tests were carried out on the aqueous extract and on powdered specimens

using standard procedures to identify the constituents of the selected plant materials

as described by Harborne (1973); Trease and Evans (1989); Sofowara (1993).

Aqueous extract was prepared by soaking 100g of dried powdered samples in 200

ml of distilled water for 12 hours. The extracts were filtered using Whatman filter

paper No 42 (125mm) and the filtrate was used as aqueous extract.

Test for tannins

About 0.5g of the dried powdered samples of different plants were boiled in 20 ml of

water in a test tube and then filtered. To the filtrate a few drops of 0.1% ferric

chloride was added and observed for brownish green or a blue black colouration.

Test for saponin

About 2g 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 shaken vigorously, then observed for the formation of emulsion.

Test for flavonoids

Three methods were used to determine the presence of flavonoids in the plant

samples. 5 ml of dilute ammonia solution was added to a portion of the aqueous

filtrate of each plant extract followed by addition of concentrated H2SO4 . A yellow

colouration observed in each extract indicated the presence of flavonoids. The

yellow coloration disappears on standing.

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Few drops of 1% aluminium solution were added to a portion of each filtrate. A

yellow coloration was observed indicating the presence of flavonoids.

A portion of the powdered plant sample was heated with 10 ml of ethyl acetate over

a steam bath for 3 min. The mixture was filtered and 4 ml of the filtrate was shaken

with 1 ml of dilute ammonia solution. A yellow colouration was observed indicating a

positive test for flavonoids.

Test for steroids

About 0.5g of the powdered sample is mixed with ethyl alcohol and added with two

ml of acetic anhydride and 2 ml H2SO4. The colour changed from violet to blue or

green in samples indicating the presence of steroids.

Test for terpenoids

Five ml of each extract was mixed in 2 ml of chloroform, and concentrated H2SO4 (3

ml) was carefully added to form a layer. A reddish brown coloration of the interface

was formed to show positive results for the presence of terpenoids.

Test for phenolic compounds

3 ml of the extract was treated with a few drops of neutral ferric chloride solution and

observed for blackish red colouration.

Test for sugar/glycoside

0.5g of the dried powdered sample in a watch glass was mixed thoroughly with 0.5

ml of anthrone. Two drops of concentrated H2SO4 was added and mixed well using a

glass rod. It was then heated over a steamed water bath. Formation of dark green

colour indicated the presence of sugar/glycoside.

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Test for alkaloids

Two ml of acetic anhydride was added to 0.5 g of the dried powdered sample with 2

drops of Dragendroff’s reagent. Formation of reddish orange precipitate indicated the

presence of alkaloids.

Test for Quinone

About 3 ml of the extract was treated with a few drops of 0.5N alcoholic potassium

hydroxide solution. Dark coloration ranging from red to blue indicated the presence

of quinones.

Test for coumarin

About 0.5 g of the powdered dried sample in ethyl alcohol was treated with 0.5N

alcoholic NaOH and colour changes were recorded. Dark yellow colour formation

indicated positive for coumarin.

Test for carboxylic acid

About 3 ml of the sample was mixed with 3 ml of sodium bicarbonate solution.

Effervescence indicated the presence of carboxylic acid.

Detection of proteins and free amino acids

One ml of the extract was gently warmed with equal volume of 10% NaOH. After

cooling a drop of diluted CUSO4 solution was added to the mixture. Formation of

reddish violet colour indicated the presence of proteins.

One ml of the extract was added with few drops of ninhydrin solution. Formation of

purple colour indictated the presence of aminoacids

Detection of resins

One ml of the extract was treated with few drops of acetic anhydride solution

followed by one ml of concentrated H2SO4 solution. Coloration ranging from orange

to yellow indicated the presence of resins.

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Detection of fixed oils and fats

Few drops of 0.5N alcoholic KOH was added to one ml of the extract with a few

drops of phenolphthalein and the mixture was heated for 1 -2 hours. Soap formation

showed the presence of fixed oils and fats.

Detection of xanthoproteins

About one ml of the extract was added with few drops of concentrated HNO3 and

ammonia solution. Formation of reddish orange precipitate indicated the presence of

xanthoprotein.

RESULTS

Effect of extraction methods on percentage yield

The yields of the extracts of Azadirachta indica, Piper betle and Allium sativum with

respects to solvents are shown in Table.1

Table.1 Yield of extracts of plants with respect to solvents

Plants Solvents Yield (g) Yield (%)

Azadirachta indica Aqueous 3.30 66.00

Ethanol 3.70 74.00

Piper betle Aqueous 3.50 70.00

Ethanol 3.90 78.00

Allium sativum Aquoeus 2.80 56.00

Ethanol 3.00 60.00

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Plate 9. Aqueous extract of A. Piper betle, B. Allium sativum and C. Azadirachta indica

In all the cases it was found that the yield recorded for the selected plants in ethanol

extract was higher than the aqueous extract (Table 1). Among the three plants

ethanol extract of Piper betle recorded the highest yield (78%) followed by

Azadirachta indica (74%) and Allium sativum (60.0%). For water extracts also Piper

betle recorded the maximum yield (70%) followed by Azadirachta indica (66%) and

Allium sativum (56%).

Two –way ANOVA for the data on percentage yield as a function of difference between selected plants and deployed solvents

ANOVA

Source of Variation SS df MS F P-value F crit

Rows 66.66666667 1 66.66667 25 0.03775 18.51276

Columns 277.3333333 2 138.6667 52 0.018868 19.00003

Error 5.333333333 2 2.666667

Total 349.3333333 5

It could be noted from statistical analysis that there was a significant difference in

yield between the three plants and within the various solvents (p<0.05).

ANTIBACTERIAL ACTIVITY OF DIFFERENT EXTRACTS

The results of the antibacterial activity of the aqueous and ethanol extracts of

Azadirachta inidca against the Aeromonas hydrophila are given in Table 2.

A B C

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Table 2. Zone of inhibition for various extracts of Azadirachta indica compared to

reference drug: activity against Aeromonas hydrophila. Each value is a mean of three

individual observations with standard deviation

Pathogen Zone of inhibition as mm Zone of inhibition as percentage

Oxytetra

cycline

Aqueous

Mean (mm)

Ethanol

Mean(mm)

Oxytetra

cycline

Aqueous

Mean(mm)

Ethanol

Mean(mm)

Aeromonas hydrophila (ref strain) 18.88±0.08 6.00±0.00 11.98±0.05 100 00 46.43

Aeromonas hydrophila (A-12) 18.9±0.07 6.00±0.00 11.98±0.05 100 00 46.36















Aeromonas hydrophila ( A-21) 18.88±0.08 6.00±0.00 11.88±0.08 100 00 45.65





F –Value 0.994 - 1.294 - - 3.809

Significance 0.488NS

- 0.235 NS

- - 0.0001 S

Mean: Mean value of diameter of inhibition zone with standard deviation As the diameter of well was 6 mm, 6mm diameter included in the table is indicative of no activity Percentage was calculated after subtracting disc diameter from all observations

Among the different extracts of Azadirachta indica tested, the ethanol crude extract

of Azadirachta indica showed good inhibitory activity against all the pathogenic

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strains of Aeromonas hydrophila with zone diameter ranged between 11.80 ± 0.07

and 11.98 ± 0.05 (Plate 11). The reference strains recorded a zone diameter of

11.98 ± 0.05. Statistical analysis of the data revealed that the difference in inhibition

zone diameter between different Aeromonas hydrophila isolates for crude ethanol

extract of Azadiracta indica was not significant (p>0.05). None of the pathogens were

inhibited by crude aqueous extract of Azadirachta indica in the present study. On

the other hand oxytetracycline was able to produce a zone diameter ranged between

18.84± 0.06 and 18.96±0.06 mm (Plate 10). Statistically the difference in zone

diameter among the twenty selected Aeromonas hydrophila isolates against

oxytetracycline was not significant (P>0.05).

Among the 20 isolates, maximum percentage inhibition was recorded for Aeromonas

hydrophila strain A-12 (46.36%) and the minimum was recorded for A-10 (45.10%).

Single factor ANOVA of the results obtained for antibacterial activity in terms of

percentage inhibition of different Aeromonas hydrophila isolates showed that there

was a significant difference in inhibition percentage recorded by Azadirachta indica

crude ethanol extract for different Aeromonas hydrophila isolates suggesting that the

antibacterial potential of crude ethanol extract of Azadirachta indica was different for

all the Aeromonas hydrophila isolates selected for the study.

151

Table.3 Zone of inhibition for various extracts of Azadirachta indica compared to

reference drug: activity against Pseudomonas fluorescens. Each value is a mean of

three individual observations with standard deviation


Oxytetra

cycline

Aqueous

Mean (mm)

Ethanol

Mean(mm)

Oxytetra

cycline

Aqueous

Mean(mm)

Ethanol

Mean(mm)

Pseudomonas fluorescens ref strain 17.88±0.08 6.00±0.00 10.98±0.05 100 00 41.92

Pseudomonas fluorescens (P-1) 17.9±0.07 6.00±0.00 10.98±0.05 100 00 41.85
















F –Value 1.077 - 1.45 - - 1.89


- 0.179 NS

- - 0.059 NS

Mean : Mean value of diameter of inhibition zone with standard deviation As the diameter of well was 6 mm, 6mm diameter included in the table is indicative of no activity Percentage was calculated after subtracting disc diameter from all observations

The crude ethanol extract of Azadirachta indica exhibited higher inhibition activity for

Pseudomonas fluorescens strains (Table 3) and the zone of inhibition ranged

between 10.80±0.07 and 10.98±0.05 mm (Plate 13). Statistical analysis of the data

showed that the difference in zone diameter for the selected Pseudomonas

152

fluorescens strains against crude ethanol extract of Azadirachta inidca was not

significant (P>0.05). The antibiotic Oxytetracycline was able to inhibit Pseudomonas

fluorescens strains with a maximum zone diameter of 17.96±0.06 mm and a

minimum of 17.84±0.06 mm respectively (Plate 12). The results of inhibition zone

diameter obtained for different isolates of Pseudomonas fluorescens for

oxytetracycline were also not significantly different even at 5% level.

Among the 16 isolates, maximum percentage inhibition was recorded for

Pseudomonas fluorescens strain P-1 (41.85%) and the minimum was recorded for

P-15 (40.47%). Single factor ANOVA of the results obtained for antibacterial activity

in terms of percentage inhibition of different Pseudomonas fluorescens isolates

showed that there was no significant difference in inhibition percentage recorded by

crude ethanol extract of Azadirachta indica for different Pseudomonas fluorescens

isolates suggesting that the antibacterial potential of crude ethanol extract of

Azadirachta indica was same for all the Pseuodomonas fluorescens isolates

selected for the study.

153

Table 4. Zone of inhibition for various extracts from Piper betle compared to reference

drug: activity against Aeromonas hydrophila. Each value is a mean of three individual

observations with standard deviation


Oxytetra

cycline

Aqueous

Mean (mm)

Ethanol

Mean(mm)

Oxytetra

cycline

Aqueous

Mean(mm)

Ethanol

Mean(mm)

Aeromonas hydrophila (ref

strain)

18.84±0.06 6.00±0.00 12.9±0.07 100 00 53.74
















Aeromonas hydrophila ( A-21) 18.88±0.08 6.00±0.00 12.92±0.08 100 00 53.73





F –Value 0.994 - 0.452 - - 0.893


- 0.971 NS

- - 1.83 NS

Mean : Mean value of diameter of inhibition zone with standard deviation As the diameter of disc /well was 6 mm, 6mm diameter included in the table is indicative of no activity Percentage was calculated after subtracting disc diameter from all observations

154

Antibacterial activity of crude water and ethanol extracts of Piper betle were

tabulated in table 4. From the results obtained, it is found that crude water extract of

Piper betle was totally inactive against Aeromonas hydrophila and did not produce

zone in the lab trials for all the tested isolates (Plate 14). On the other hand ethanol

extract of Piper betle was having appreciable antibacterial activity against various

Aeromonas hydrophila isolates selected for the study (Plate 15). The maximum zone

diameter recorded was 12.94 ± 0.06 mm with the isolate A-3 and the minimum was

reported for A-28 which recorded a zone diameter of 12.82±0.08 mm. Single factor

ANOVA of the results on zone of inhibition by crude ethanol exract of Piper betle

against different Aeromonas hydrophila showed that the difference in zone diameter

within individual strains of Aeromonas hydrophila against crude ethanol extract of

Piper betle was not significant and hence it could be concluded that all the tested

isolates were inhibited uniformly by Piper betle crude ethanol extract. Percentage

inhibition potential of Piper betle crude ethanol extract for various Aeromonas

hydrophila isolates was also calculated. The results showed that the percentage

inhibition values of crude ethanol extract of Piper betle varied between 53.01 –

53.74%. Statistically these values were not differed significantly, suggesting that all

the Aeromonas hydrophila isolates were uniformly inhibited by crude ethanol extract

of Piper betle.

155

Table 5. Zone of inhibition for various extracts from Piper betle compared to reference

drug: activity against Pseudomonas fluorescens. Each value

is a mean of three individual observations with standard deviation


Oxytetra

cycline

Aqueous

Mean (mm)

Ethanol

Mean(mm)

Oxytetra

cycline

Aqueous

Mean(mm)

Ethanol

Mean(mm)

Pseudomonas ref 17.88±0.08 6.00±0.00 6.00±0.00 100 00 00

Pseudomonas fluorescens (P-1) 17.9±0.07 6.00±0.00 6.00±0.00 100 00 00
















F –Value 1.077 - - - - -


- - - - -


From the results reported in table 5, it is found that either of the two extracts of Piper

betle tested in the study was not able to inhibit Pseudomonas fluorescens isolates.

156

All the Pseudomonas fluorescens isolates were resistance to both crude water and

ethanol extracts of Piper betle.

Table6. Zone of inhibition for various extracts from Allium sativum compared to

reference drug: activity against Aeromonas hydrophila. Each vale is a mean of three

individual observations with standard deviation


Oxytetra

cycline

Aqueous

Mean (mm)

Ethanol

Mean(mm)

Oxytetrac

ycline

Aqueous

Mean(mm)

Ethanol

Mean(mm)

Aeromonas hydrophila (ref strain) 18.84±0.06 13.9±0.07 31.98±0.05 100 61.53 202.34

Aeromonas hydrophila (A-12) 18.88±0.08 13.88±0.08 31.98±0.05 100 61.18 201.71















Aeromonas hydrophila ( A-21) 18.88±0.08 13.88±0.08 31.94±0.06 100 61.18 201.41





F –Value 0.994 0.994 0.921 - 0.3295 2.557


0.488 NS

0.565 NS

- 0.995 NS

0.005 S


157

The results on antibacterial activity of crude aqueous and ethanol extract of Allium

sativum against different Aeromonas hydrophila isolates were presented in Table 6.

Among the two extracts, ethanol extract of Allium sativum exhibited better

antibacterial activity with a maximum zone diameter of 31.98 ±0.05 mm which

recorded a percentage inhibition of 201.71%. Crude aqueous extract of Allium

sativum inhibited Aeromonas hydrophila isolates with zone diameter ranged between

13.84 ± 0.06 and 13.96 ± 0.06 mm. Statistically the variation in zone diameter

between various Aeromonas hydrophila strains was not significant even at 5% level.

Crude ethanol extract of Allium sativum was highly active against all the strains of

Aeromonas hydrophila selected for the study. Antibacterial activity in terms of zone

diameter varied between 31.84 ± 0.06 and 31.98±0.05 mm respectively (Plate 16).

The difference in zone diameter within strains of Aeromonas hydrophila was not

significantly different (p>0.05). Percentage inhibition of Aeromonas hydrophila

isolates by crude aqueous extract ranged between 60.78% and 61.42% and the

difference in percentage inhibition potential of various Aeromonas hydrophila isolates

were not significantly different (P>0.05). On the other hand, the variations in

percentage inhibition potential of ethanol extract of Allium sativum against different

Aeromonas hydrophila strains was highly significant (P<0.005).

158

Table 7. Zone of inhibition for various extracts from Allium sativum compared

to reference drug: activity against Pseudomonas fluorescens Each value is a

mean of three individual observations with standard deviation


Oxytetra

cycline

Aqueous

Mean (mm)

Ethanol

Mean(mm)

Oxytetra

cycline

Aqueous

Mean(mm)

Ethanol

Mean(mm)

Pseudomonas ref 17.84±0.06 9.9±0.07 22.92±0.08 100 32.94 142.91

Pseudomonas fluorescens (P-1) 17.88±0.08 9.9±0.07 22.96±0.06 100 32.83 142.76
















F –Value 1.077 0.270 0.799 0.437 3.788


0.996 NS

0.677 NS

0.961 NS

0.0005 S


The results on antibacterial activity of crude aqueous and ethanol extract of Allium

sativum against different Pseudomonas fluorescens strains were presented in Table

7. Among the two extracts ethanol extract of Allium sativum exhibited maximum

antibacterial activity with a higher zone diameter of 22.96 ± 0.06 mm (Plate 17)

which recorded a percentage inhibition of 142.76%. Crude aqueous extract of Allium

159

sativum inhibited Pseudomonas fluorescens isolates with zone diameter ranged

between 9.86 ± 0.06 and 9.94 ± 0.06 mm. Statistically the variation in zone diameter

between various Pseudomonas fluorescens strains was not significant even at 5%

level. Crude ethanol extract of Allium sativum was highly active against all the strains

of Pseudomonas fluorescens selected for the study. Antibacterial activity in terms of

zone diameter varied between 22.82 ± 0.05 and 22.96±0.06 mm respectively. The

difference in zone diameter within strains of Pseudomonas fluorescens was not

significantly different at 5% level Percentage inhibition of Pseudomonas fluorescens

isolates by crude aqueous extract ranged between 32.44% and 33.05% and the

difference in percentage inhibition potential of various Pseudomonas fluorescens

isolates were not statistically significant (P>0.05). On the other hand, the variation in

percentage inhibition potential of ethanol extract of Allium sativum against different

Pseudomonas fluorescens strains was highly significant (P<0.005), the values being

ranged between 141.11 and 142.76%.

Minimum Inhibitory Concentration of crude water and ethanol extract of test

plants

As there was no statistical significance in zone diameter produced by different

strains of Aeromonas hydrophila and Pseudomonas fluorescens selected for the

study, highly virulent strains designated as Aeromonas hydrophila (A-10) and

Pseudomonas fluorescens (P-3) during phenotypic characterization of the isolates

were chosen for determination of minimum inhibitory concentration (MIC) of ethanol

extract of Azadirachta indica, Piper betle and water and ethanol extract of Allium

sativum.

160

Table 8. Values of Minimum inhibitory concentration of ethanol extract of Azadirachta

indica and Piper betle and water and ethanol extract of Allium sativum against

Aeromonas hydrophila (A-10) and Pseudomonas fluorescens (P-3). (Zone diameter

were expressed as values with excluded well diameter)

Concentrations

(µg/50µl)

Zone diameter (mm)

Aeromonas hydrophila (A-10) Pseudomonas fluorescens (P-3)

Az.Et Pi.Et Al.Wa Al.Et Az.Et Pi.Et Al.Wa Al.Et

300 5.5 6 7 24 4 - 3 15

275 5 5 6.5 23.5 3.5 - 2.5 14

250 4.5 4 6 23 3 - 2 13

225 4 3 5.5 22.5 2.5 - 1.5 12.5

200 3.5 2 5 22 2 - 1 11

175 3 1 4.5 21.5 1.5 - - 10.5

150 2 - 4 21 1 - - 9.5

125 1 - 3.5 20.5 - - - 9

100 - 3 20 - - - 8.5

75 - - 2 18.5 - - - 7.5

50 - - 1 17 - - - 6.5

25 - - - 15 - - - 6

2.5 - - - 8 - - - 2

2.0 - - - 7 - - - 1

1 - - - 5 - - - -

0.5 - - - 3 - - - -

0.3 - - - 2 - - - -

0.25 - - - 1 - - - -

Az.Et – Azadirachta indica ethanol extract, Pi.Et – Piper betle ethanol extract

Al.W – Allium sativum water extract, Al.Et – Allium sativum ethanol extract

Table 8 shows that the minimal inhibitory concentration (MIC) of the crude ethanol

extracts of Allium sativum was found to be lower for the pathogen Aeromonas

hydrophila (0.25µg) than that of Pseudomonas flurorescens (2µg). The MIC of

161

ethanol extract of Azadirachta indica against Aeromonas hydrophila was 125 µg and

that of Pseudomonas fluorescens was 150 µg. On the other hand, ethanol extract of

Piper betle recorded a MIC value of 175 µg for Aeromonas hydrophila and

completely inactive against Pseudomonas fluorescens. Water extract of Allium

sativum also recorded lower MIC value for Aeromonas hydrophila which was 50 µg,

whereas Pseudomonas fluorescens was found to be resistant and showed a MIC

value of 200 µg respectively.

Table 9. Pytochemical analysis of the medicinal plants, selected for the study

Constituents Azadirachta indica Piper betle Allium sativam

Triterpenoids + + -

Steroids + + +

Phenolic compounds + + +

Sugar + + +

Alkaloid + + -

Tannin + - -

Quinones - - -

Coumarin + - -

Flavanoid + + +

Carboxylic acid - - -

Fixed oils and fats + + +

Protein + + +

Free amino acids + + +

Resin + - +

Saponin + + +

Xanthoprotein + + -

The phytochemical characters of the selected plants were summarized in Table 9. All

the three plants were pytochemically differ from one another. The results on

phytochemical screening showed that the three plants exhibited positive reaction for

steroids, phenolic compounds, sugars, flavonoids, protein, free amino acid, fixed oils

162

and saponin. However tannin and coumarin were present only in Azadirachta indica

leaf. Xanthoprotein, alkaloids and triterpenoids were found in Azadirachta indica and

Piper betle leaf. None contained quinines and carboxylic acids. Resin was reported

for Azadirachta indica leaf and Allium sativum clove.

Discussion

The yield of all the three selected plant materials in aqueous solution was found to

be lesser than ethanol extraction suggesting the higher extractable capacity of

ethanol as reported by Ekwenye and Elegalam (2005) for Zingiber officinale and

Allium sativum. The present study was designed to obtain preliminary information on

the antimicrobial effect of three medicinal plants on fish pathogenic microorganisms.

The hole/well plate diffusion method was preferred to be used in this study since it

was found to be better for testing plant extracts than the disc diffusion method

(Essawi and Srour, 2000).

Ethanol extract of Azadirachta indcia was active against both Aeromonas hydrophila

and Pseudomonas fluorescens and the activity was higher for Aeromonas hydrophila

than Pseudomonas fluorescens (Table 2 and 3). On the other hand aqueous and

ethanol extract of Piper betle was inactive against Pseudomonas fluorescens. Both

water and ethanol extracts of Allium sativum were active against Aeromonas

hydrophila and Pseudomonas fluorescens. Among the two extracts, ethanol extracts

exerting stronger antibacterial activity than water extract. Among the two pathogens,

Pseudomonas fluorescens was found to be resistant to both water and ethanol

extract of Piper betle.

The broad antimicrobial action of the ethanol extract of all the tested plants could be

ascribed to aromatic or saturated compounds, such as tannin, phenolic compounds,

163

flavonoids, terpenoids, steroids and alkaloids present in the plant materials are better

liberated by ethanol extraction (Cowman, 1999). Aqueous extract of Azadirachta

indica and Piper betle was totally inactive against all the bacterial strains of

Aeromonas hydrophila and Pseudomonas fluorescens. The results are in agreement

with that of Satish et al. (1999), who reported aqueous leaf extract of Azadirachta

indica was totally inactive against phyto pathogenic bacterium Xanthomonas

campestris. Piper betle was reported to possess good antibacterial activity and

maintained a broad spectrum antibacterial activity against pathogens, such as

Ralstonia, Xanthomonas, and Erwinia. It was also revealed that the ethanol extract

of P. betle had more superior action than streptomycin (Ueda and Sasaki, 1951). But

in the present study the bacterial strains did not respond to Piper betle extract.

Sastry and Rao (1994) reported that some of the fish pathogens did not respond to

marine algae and mangroves extracts, whereas the purified fractions showed broad-

spectrum activity against multiple strains and suggested the masking of antibacterial

activity by the presence of some inhibitory compounds or factors in the extract as the

reason. Similar observations were made by Vlachos et al. (1997), who found that

fractionation of crude extracts tested enhanced their activity against both gram

negative as well as the resistant gram positive pathogens. Antibacterial activity of

Piper betle was due to allylpyrocatechol (APC) classically known as betel phenol.

Ramji et al. (2002) while fractioning methanol extract of Piper betle reported that all

the antibacterial activity was resided on the ether fraction. Moreover the variation of

antibacterial activity of our extracts might be due to distribution of antimicrobial

substances, which varied from species to species as suggested by Lustigman and

Brown (1991).

164

In the present study, the most promising antibacterial activity was recorded for

Allium sativum. Findings of this study are similar to those reported by Elnima et al.

(1983); Singh and Shukla (1984); Chowdhury et al. (1991); Perez (1993), Cellini et

al. (1996); Arora and Kaur (1999), Krishna and Banerjee (1999). The maximum zone

diameter of 32 mm and 23 mm against Aeromonas hydrophila and Pseudomonas

fluorescens obtained in the present study (Table 6 and 7) is comparable with the

zone diameter reported for 100% fresh juice of garlic against human pathogens

(Kivan and Kunduhoglu, 1997), which suggested the efficacy of ethanol on extraction

of potential antimicrobial compounds from the plant. The highest zone diameter of

30.67 mm has been recorded for Aeromonas hydrophila against Hasandede seed

extract of grapes at 10% concentration (Bayard et al., 2005). Singh and Shukla

(1984) reported that garlic was more effective than any of the test antibiotics

(penicillin, ampicillin, doxycycline, streptomycin and cephalexin) against clinical

strains of Staphylococcus, Escherichia, Proteus, Pseudomonas and Klebsiella

bacteria. In the present study ethanol extract of Allium sativum exert potential

antibacterial activity than the test antibiotic Oxytetracycline.

The antibacterial activity of garlic is reported to be due to the action of allicin or diallyl

thiosulphinic acid or diallyl disulphide (Avato et al., 2000). It is postulated that the

antibacterial and antifungal properties of garlic juice are due to the inhibition of

succinic dehydrogenase via the inactivation of thiol group. Our results revealed

differences in the sensitivity of different Aeromonas hydrophila strains to garlic

extract, suggesting the heterogeneity of the organism (Indhu et al., 2006).

The findings that Aeromonas hydrophila is susceptible to extracts obtained from the

three plants studied are also similar to the susceptibility of that microbe to different

165

plant extracts reported by several researchers (Turk et al., 2003; Mamtha et al.,

2004; Hori et al., 2006; Indhu et al., 2006). In all the cases Pseudomonas fluoresens

was found to be resistant to each of ethanolic and aqueous extracts of three

medicinal plants. The resistance attributed to Pseudomonas could be due to the

presence of capsule in those bacteria as noted by Morse et al. (1986) and

Padmakumar and Ayyakkannu (1997) in Pseudomonas aeruginosa against

Azadirachta indica stem bark extract and micro algae extracts.

The minimum inhibitory concentration (MIC) of garlic ethanol extract for Aeromonas

hydrophila was 5µg/ml, which was in parallel with the report of Chowdhury et al.

(1991) against Shigella dysenteriae, Sh.flexneri, Sh.sonnei and Escherichia coli.

The MIC value obtained for Aeromonas hydrophila by Allium sativum was found to

be very low when compared with the MIC of other plant extracts as reported by Turk

et al. (2003). The MIC value of ethanol extract of Cetraria aculeate for the bacteria

Aeromonas hydrophila was 607µg/ml. The MIC of ethanol extract of Azadirachta

indica against Aeromonas hydrophila and Pseudomonas fluorescens was 2500µg/ml

and 3000µg/ml, which was very high when compared to the MIC of ethanol extract of

neem leaves against Trichophyton rubrum and Microsporum nanum (250µg/ml) as

reported by Natarajan et al. (2003). Some extracts had discordant results with the

disc diffusion and microdilution methods. A good explanation for this variation would

be the difference in the technique used (tube dilution and cup diffusion). For

instance, extracts of X. caffra gave no inhibition zone for some organisms with the

disc diffusion whereas the microdilution method gave MICs of 6 mg/ml for the same

organisms. Similar results were obtained previously (Rios et al., 1988; Silva et al.,

1996). This might be due to the difference in solubility of possible active compounds.

In the disc diffusion method, the limited diffusion of the less polar active compounds

166

in solid media might explain the lack of inhibition zone around the disc, whereas in

the microdilution method the compounds in solution get easily in contact with the

organisms.

The results of pytochemical screening of three medicinal plants indicated the

presence of medically active substances such as flavanoids, phenolic compounds,

saponin, steroids etc. Azadirachta indica leaf extract showed the presence of large

number of medically active constituents followed by Piper betle and Allium sativum.

But antibacterial activity was pronounced for Allium sativum extract than the two

other plants tried hence it was suggested that Allium sativum crude extract can be

used as potential antibacterial against Aeromonas hydrophila and Pseudomonas

fluorescens strains whereas Azadirachta indica and Piper betle extracts required

fractioning for better results.

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