45

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he research work was carried out primarily on two aspects. Firstly,

screening of antifungal properties of oil and extracts (petroleum ether,

chloroform, methanol and butanol fraction of methanol) from pericarp of

fruits of S. mukorossi collected from different sources i.e. Forest Research

Institute, Dehradun and Gyarahdevi, Pithoragarh (Uttarakhand) and

Nainatikker, Sirmaur (Himachal Pradesh) against different forest fungi.

Secondly, isolation of pure hederagenin (sapogenin moiety) was

accomplished from the butanol soluble fraction of the methanol extract of

fruit pericarp through different chromatographic methods followed by its

screening for antifungal activity. The screening was based upon the IC50,

MIC, fungicidal/fungistatic and spore/conidial germination.

3.1. The Plant: S. mukorossi Linn.

S. mukorossi, a deciduous tree of North India, also known as ‘Areetha’ to

common man. It is one of the most important trees of tropical and sub-

tropical region of Asia. It is common tree in Shivaliks and the outer

Himalayas of Uttar Pradesh, Uttarakhand, Himachal Pradesh, Haryana and

Jammu and Kashmir. S. mukorossi fruits, commonly known as soapnuts,

are used medicinally as an expectorant, emetic, contraceptive, and for

treatment of excessive salivation, epilepsy, chlorosis and migranes. It is also

used in Ayurvedic medicine for treatment of eczema, psoriasis, and for

removing freckles. Soapnut is a popular ingredient in Ayurvedic shampoos

and cleansers (Suhagia et al., 2011).

3.2. Sampling Site

The details of sampling sites are given in Table3.2.1 and Fig.3.2.1.

Table3.2.1 Details of sampling sites

T Materials & methods

46

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Parameter Dehradun Gyarahdevi Nainatikker

Location Latitude 30° to 30°, 32 N’ 29°to 35°, N’ 30°to 33°, N’

Longitude 77°, 43’-78°, 24’ E 80°, 13’E 77°-21° E

Altitude 683 m (msl) 2,134 m (msl) >1,200 m (msl)

Climate Annual

Rainfall (mm)

1,261.5 to 2,060.0 367 1,500 to 1,800

Temperature

Minimum 0.2° 0°C 0°C

Maximum 36.9°C 30°C 27°C

3.3. Extraction of Plant Material

3.3.1. Collection

S. mukorossi seeds were collected from three different sources from December 2007

to January 2008.

47

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Himachal Pradesh

Uttarakhand

S

Fig.3.2.1. S. mukorossi sampling sites (S).

Distance not to be scaled.

S

S

48

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3.3.2. Preparation of fatty oil and extracts

The kernels were separated from the seed coat of S. mukorossi collected

from different sources. For oil extraction, kernels were cut into small pieces

and extracted with petroleum ether and concentrated on water bath to small

volumes. Further, for preparation of extracts, pericarp was separated from

seeds. The collected pericarp was spread on blotter paper sheet and air

dried in shade. The dried pericarp was then cut into pieces and extracted

successively with the solvents of increasing polarity viz. petroleum ether,

chloroform and methanol in a Soxhlet apparatus. These extracts were

concentrated on water bath to small volumes (Singh & Tripathi, 1993). Later

on, methanol extract was fractionated into ethyl acetate and butanol

fractions. The yield of extracts was determined on moisture free basis

(Table3.3.2.1.).

Table3.3.2.1. Yield of fatty oil and extracts (%) from different sources

of S. mukorossi

3.4. Acid Hydrolysis of Butanol Fraction of Methanol Extract of

S. mukorossi Pericarp

Oil/Extract Source/Yield (%)

Dehradun Gyarahdevi Nainatikker

Oil 41.0 42.0 33.0

Petroleum ether 0.2 0.3 0.3

Chloroform 2.5 3.1 5.6

Methanol 60.0 68.7 61.8

Butanol 38.0 40.0 42.0

49

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Based on higher IC50 at the lower concentrations, butanol fraction of methanol

extract was selected for acid hydrolysis, so that, it could be used in further

experiments. The butanol fraction is reported to contain glycosides with

hederagenin as sapogenin/aglycone moiety. To increase the antifungal efficiency of

the saponins, sugar moiety was removed through acid hydrolysis to get hydrolyzed

saponin containing hederagenin. In the indigenously standardized method, butanol

fraction was refluxed with aqueous 2N HCl under reflux for 2 h. After reflux, the

hydrolyzed mass was neutralized by adding NaOH. It was washed with chloroform

(3-4 times) and finally, with water (4-5 times) to remove extraneous impurities. The

hydrolyzed saponin containing hederagenin was obtained and later isolated in pure

form through column chromatography.

3.5. Isolation of Pure Compound

The following analytical techniques have been used for qualitative and quantitative

isolation:

3.5.1. Column chromatography

The technique basically involves the application of the mixture of the compounds to a

homogenously packed glass column with adsorbent (silica gel, alumina, etc.) and

subsequent sequential elution of individual compounds with appropriate solvent or by

gradient elution. The column is simply a glass tube fitted with a tap at one end, with

dimensions such that the diameter to length ratio is in the range 1:10 to 1:30. The size

(volume) of the column required for any particular separation can be roughly

calculated once the weight of the mixture to be loaded to the column is known. It is

generally considered that the separation based on partitioning (most cellulose and

silica chromatography) the sample to packing ratio should be in the range of 1:50 to

1:500. The latter ratio being more appropriate to complex mixture than the former to

simple mixture (Ravindranath, 1989).

3.5.1.1. Column chromatography of hydrolyzed saponin

The hydrolyzed saponin was dissolved in minimum quantity of chloroform and

methanol mixture (4:1), adsorbed onto silica gel, dried and subjected to column

50

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chromatography over gel using chloroform and methanol in ratio of 3:1 as eluent. The

details of column are as follows:

Weight of extract = 15 g

Weight of silica gel for adsorbing extract = 54.95 g

Weight of silica gel used for packing column = 400 g

Solvent used for packing column = Chloroform

Retention volume of the column = 700 ml

Volume of each fraction collected = Fraction 1-110=500 ml

Fraction 111-125=2000 ml

Fraction 126- 215=100 ml

Fraction 216- 220=500 ml

Fraction 221- 280=500 ml

Different fractions of different volumes were collected and combined on the

basis of their TLC studies. The column on elution with chloroform and chloroform

methanol mixtures with increasing polarity afforded 280 fractions detailed in

Table3.5.1.1.

Table3.5.1.1. Fraction details collected from the hydrolyzed saponin

of S. mukorossi pericarp

Solvent system Fractions no. Volume (ml)

Pure Chloroform (100 %)

1-110 500

111-125 2000

Chloroform: Methanol (99:1) 126- 215 100

Chloroform: Methanol (97:3) 216- 220 500

Chloroform: Methanol (97:3) 221- 280 500

51

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The above fractions were examined on TLC using different solvent

systems indicating the presence of compounds present therein. The colour

of the spot was obtained by spraying with 5 per cent sulphuric acid and Rf

value was calculated as below:

Retention factor (Rf) value =

The compound i.e. hederagenin was eluted in fraction no. 216 to 220 and

was collected as colourless crystals in pure form. Later on, hederagenin

collected from different sources was used for evaluation of antifungal

properties.

3.6. Identification of Hederagenin

3.6.1. Ultraviolet Visible Spectroscopy Range

Ultraviolet and visible spectroscopy deals with the recording of the

absorption of radiations in the ultraviolet and visible region of the

electromagnetic spectrum. UV of colourless compounds are measured in the

range of 200-400 nm and for coloured compounds in the range of 400-700

nm. UV-VIS spectroscopy mainly used for detecting the presence and

elucidating the nature of the conjugated multiple bonds or aromatic rings. A

solvent widely used for UV spectroscopy is 95 per cent ethanol. Other

solvents employed are water, methanol, hexane, petroleum ether (Yadav,

1995 and Harborne, 1998).

Chemito, 2700 Spectrophotometer (India) was used to confirm the absence

of conjugated bonds in the pure compound through UV spectroscopy.

3.6.2. Infrared Spectroscopy

IR spectra may be measured on plant substances in an automatic recording

IR Spectrophotometer either in solution in chloroform or carbon

tetrachloride (1-5%), as a mull with nujol oil or in the solid state, mixed with

potassium bromide. The range of measurement is from 4,000 to 667 cm-1

Distance travelled by solvent

Distance travelled by spot

52

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shows spectral bands or peaks due to the vibrations of individual bonds or

functional groups in the molecule under examination. The region below

1,200 cm-1 shows bands due to the vibrations of the whole molecule and

because of its complexity is known as the ‘fingerprint region’. IR

spectroscopy can also usefully contribute to structural elucidation, when

new compounds are encountered in plants (Harborne, 1998).

Shimadzu, FTIR 8400 Spectrophotometer (Japan) was used to detect

the characteristic absorptions of pure compound in IR spectrum.

3.6.3. Nuclear Magnetic Resonance Spectrosopy (NMR)

NMR spectroscopy is the conventional method for the structure

determination of natural products. The 1H and 13C NMR spectra reflect the

distribution of electrons surrounding the hydrogen and carbon nuclei and

are sensitive techniques to provide evidences for configurational and

conformational characteristics. The introduction of Fourier transformational

(FT) technique in the pulsed NMR in 1966 by Ernst and Anderson (Ernst,

1966) started a new era in this branch of spectroscopy.

3.6.3.1. 1H-NMR Spectroscopy

Proton NMR spectroscopy essentially provides a means of determining the

structure of an organic compound by measuring the moments of its

hydrogen atoms. In most compounds, hydrogen atoms are attached to

different groups (as –CH2, -CH3, -CHO, -NH2, >CHOH, etc.) and the proton

NMR spectrum provides a record of the number spectrum appears in the

range 0-10 ppm downfield from the reference signal of tetramethylsilane

(TMS). The structural information that proton NMR provides is based on two

physical phenomena, chemical shift (ppm) and scalar coupling constants

(J). These data are frequently applied to assign relative stereochemistry and

to analyze the conformation of natural products.

3.6.3.2. 13C-NMR Spectroscopy

The most abundant isotope 12C has no overall nuclear spin, having an equal

number of protons and neutrons. The 13C isotope, however, does have spin

53

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1/2, but is only 1 per cent abundant. 13C chemical shift has a range of about

220 ppm relative to the resonance of TMS. 13C-NMR spectra are more highly

resolved and, in most cases, each carbon within the molecule can be

assigned to a separate signal. Different substituted carbon atoms give shifts

within specific ranges, for example, aliphatic carbon atoms give shifts

between 0 to 40 ppm and aromatic carbon atoms give shifts between 110 to

150 ppm. This procedure is widely used in structural analysis, although the

need to have a sample weighing about 10 mg is still a limitation (Harborne,

1998).

The δ values of the 1H-NMR and 13C NMR spectrum of pure compound

were detected through NMR spectroscopy.

3.7.High-Performance Thin-layer Chromatography Densitometric

Quantification of Hederagenin

HPTLC is a versatile, powerful but basically a simple separation technique.

HPTLC provides the rapid and positive analysis of plant constituents leading

to semi quantitative/quantitative information on chief constituents of the

formulation. It can provide fingerprinting for monitoring the identity and

purity of plant constituents.

Today HPTLC has a firm place among various analytical techniques as

a reliable method for quantification at micro, nano and even at pictogram

level, even when present in complex formulations. HPTLC has its own

specific value, the economic considerations do weigh in favor of HPLTC

(Sethi, 1996).

3.7.1. Apparatus

(a) Spotting device-Linomat 5 automatic sample applicator (CAMAG,

Switzerland).

(b) Syringe-100 µL.

(c) HPTLC chamber-20 x 10 cm plates; HPTLC aluminium sheet pre-coated with

silica gel 60F254, Merck KGaA, Germany).

54

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(d) Densitometer- TLC scanner3 linked to win CATS software (CAMAG). Scanner

mode- Scanning wavelength: 254 nm; lamp: Tungsten; measurement type:

remission; measurement mode: absorption; optical filter: second-order; detection

mode: automatic.

Scanner settings- Slit dimension: 5.00 x 0.45 mm, micro; optimize optical system

for max: light; scanning speed: 20 mm/s; data resolution: 100 µm/step.

Experimental conditions : Temperature, 25°C± 2°C.

3.7.2. Preparation of sample solution

Two grams of the powdered pericarp of S. mukorossi from different sources

were hydrolyzed with 50 ml of 3.5 M aqueous sulfuric acid under reflux for

6 h at 100°C on a water bath. The solution was then allowed to cool to room

temperature and filtered. The marc was washed with water, neutralized with

10 per cent sodium carbonate solution, washed with water again, and dried

in hot air oven at a temperature not exceeding 50°C. After drying, the marc

was extracted with chloroform under reflux (25 ml X 4) on a water bath for a

period of 1 h. The combined chloroform extract was concentrated to dryness

under vacuum (Kalola et al., 2008).

3.7.3. Preparations of sample solutions of hederagenin

Different solutions of hederagenin as obtained above from different sources

were prepared by dissolving 10 mg of hederagenin in 5 ml (Dehradun) and

10 ml (Gyarahdevi and Nainatikker) of mixture of 4 per cent of methanol in

chloroform. Ten grams of standard hederagenin was also dissolved in 10 ml

of mixture of 4 per cent of methanolic chloroform.

3.7.4. Preparation of the calibration graph of hederagenin

Ten microlitres of the standard solution of hederagenin (200 to 300 ng/spot) from

Dehradun source and 16 micro litres from Gyarahdevi and Nainatikker sources

were applied in duplicate alternatively with 2 to 8 micro litres of standard solution

of hederagenin (200 to 800 ng/spot) onto a pre-coated HPTLC plate using the

automatic samples plotter (band length: 6 mm, distance between the tracks: 13

mm). The plate was developed in an HPTLC chamber containing the mobile phase

55

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i.e. methanolic chloroform (8%), at 25° ±2°C. The plate was developed to a distance

of 80 mm. It was dried at room temperature. The bands were scanned at 254 nm

and a calibration graph was constructed by plotting area under the peaks vs. the

corresponding standard concentrations of hederagenin.

3.7.5. Quantification of hederagenin in the samples

The peak areas were recorded. The amount of hederagenin in samples from

different sources was calculated using the linear regression equation derived from

the calibration graph of hederagenin.

3.8. Test Fungi

Eight forest fungi were selected for bioassay viz. Alternaria alternata, Colletotrichum

gloeosporioides, Phoma sp., Phomopsis dalbergiae, Ganoderma lucidum, Fusarium

oxysporum, Rhizoctonia solani and Trichoderma piluliferum. The rationale for short-

listing these fungi is ecological, host specificity, plant part infectivity, wider

presence, economic loss, etc. The cultural details are as follows:

3.8.1. Alternaria alternata

Family: Pleosporeaceae

Host: Populus deltoides

Disease symptoms: Leaf spot

Characteristics: Alternaria is a genus of ascomycete fungi. Alternaria species are

known as major plant pathogen. It is commonly recognized as a ubiquitous,

cosmopolitan species. Colonies are dark olive green to brown, floccose to velvety

(heavily sporulating, Fig.3.7.1.). Colonies become pleomorphic over time, and lose

the ability to sporulate with subsequent transfer. Plant pathogen most commonly

found on weakened plants, soil, dead organic debris, on food stuffs and textiles

(Rotem, 1994).

3.8.2. Colletotrichum gloeosporioides

Family: Glomerallaceae

56

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Host: Dalbergia sissoo Roxb.

Disease symptoms: Leaf spot and blight

Characteristics: Colletotrichum and its telemorph Glomerella have been implicated

in plant diseases. Colonies variable, greyish white to dark grey (Fig.3.7.1.); aerial

mycelium even and felted or in tufts associated with conidiomata, reverse unevenly

white to grey or darker especially with age; appressoria clavate, ovate, obovate,

sometimes lobed, sepia brown, 6-20 x 4-12 m; conidia formed in pale salmon

masses, straight, cylindrical, apex obtuse, base truncate, 12-17 x 3.5-6.0 m;

conidia typically elongated with rounded ends and characteristically, slightly

narrow in the middle than at the end; produced dark brown long setae in the

acervulus (Sutton, 1992).

3.8.3. Phoma sp.

Family: Sphaeropsidaceae

Host: P. deltoides

Disease symptom: Leaf spot

Characteristics: It is prominent during late monsoon (September– October).

Septate hyphae, pycnidia, conidia, and sometimes, chlamydospores are present.

Hyphae are septate to brown while pycnidia are the fruiting bodies that are large,

dark in colour, round to pyriform in shape with size ranging from 70-100 µm in

diameter, and with one to several openings called the ostioles on their surface from

which the conidia are released outside (Fig.3.7.1.). Conidia are hyaline, oval-

shaped, unicellular and each conidum usually has two oil droplets inside;

chlamydospores are brown, may appear in long chains or solitary, and may either

be unicellular or multicellular and alternarioid, which resembles Alternaria in

appearance (Boerema, et al., 2004).

3.8.4. Phomopsis dalbergiae

Family: Valsaceae

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Host: D. sissoo

Disease symptom: Leaf spot

Characteristics: Pycnidia dark olivaceous to dark brown, globose, innate, finally

erumpent, glabrous, 96.9-220.4 m in diameter, average 136 m, ostiole round to

somewhat oval, centric, usually about 20 m in diameter, occasionally up to 40 m

diameter; stylospores hyaline, single-celled, smooth, elongated filiform usually bent

or hooked, and acute to obtuse 6.0-13.2 x 1.2-2.5 m, average 11.1 x 1.5 m;

conidiophores simple; conidia single celled, hyaline ovoid to cylindrical, smooth,

ends usually obtuse, 4.0-6.7 x 1.3-3.33 m, average 5.7 x 2.3 m (Fig.3.7.1.;

Rensberg et al., 2006).

3.8.5. Fusarium oxysporum

Family: Nectriaceae

Host: Acacia nilotica (L.) Willd. ex Delile

Disease symptom: Wilt

Characteristics: Colonies fast growing on PDA; mycelium delicate white or peach

but usually with a purple tinge, sparse to abundant than floccose, becoming felted

and sometimes wrinkled in older cultures (Fig.3.7.1.); conidia of two types,

microconidia generally abundant, variable, oval – ellipsoid cylindrical, straight to

curved, 5-12 x 2.2-3.5 m, borne on simple philades arising laterally on the

hyphae or from short sparsely branched conidiophores; macroconidia thin walled,

generally 3-5 septate, fusoid- subulate and pointed at both ends; occasionally

fusoid-falcate macroconidia are found with a somewhat hooked apex and a

pedicellate base: 3-septate 27-46 x 3-5 m, 5-septate 35-0 x 3-5 m; pointed at

both ends; chlamydospores, both smooth and rough walled, generally abundant,

terminal and intercalary, generally solitary but occasionally formed in pairs or in

chains (Shanmugam et al., 2006).

3.8.6. Ganoderma lucidum

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Family: Ganodermataceae

Host: D. sissoo

Disease symptom: Root rot

Characteristics: Growth slow to rapid; advancing zone: white, even appressed;

hyphae hyaline, thin-walled branched; aerial mycelium; hyphae

as in advancing zone, thin to slightly thick walled; fibre hyphae hyaline, branched

aseptate, with narrow lumen, 1.4–2.9 m in dia; with frequent clamp connections

(Fig.3.7.1.); chlamydospores hyaline, slightly thick-walled, terminal to intercalary,

ellipsoid, sometime in chains, 8.8–11.8 m x 3.7–5.9 m; cuticular cells from

crustose layer hyaline to light brown, round to irregular in shape, closely packed;

staghorn hyphae with projections, hyphal system is usually trimitic, occasionally

dimitic, generative hyphae, hyaline, thin walled, branched, septate or not, and

clamped; basidiospores, ovoid or ellipsoid-ovoid, occasionally cylindric-ovoid and

always truncate at the apex; colony, white to pale yellow and even, felty to floccose

at optimum temperature on PDA (Flood et al., 2000).

3.8.7. Rhizoctonia solani

Family: Ceratobasidiaceae

Host: Eucalyptus hybrid

Disease symptom: Post - emergence damping - off

Characteristics: Colony white, hyphae broad and coarse (8-12 m in dia),

colourless when young, but turning dark brown with age (Fig.3.7.1.); young

branches inclined at angles of 45°– 90° from the direction of growth of the parent

hyphae and usually constricted at the point of origin; a septum or cross-wall

always present near the base of the branch; sclerotia germinate by hyphal growth;

persists in soil as hyphae or sclerotia; infection often starts from sclerotia; hyphae

penetrate living cells through susceptible dead tissues or invade directly through

cracks and wounds; invading hyphae grow rapidly through the host tissue causing

it to turn brown and collapse (Garcia et al., 2006).

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3.8.8. Trichoderma piluliferum

Family: Hypocreaceae

Host: Isolated from rhizosphere of D. sissoo

Characteristics: Colonies grow rather slowly at room temperature and form a

smooth surfaced and almost translucent or watery white sparse mycelial mat over

the surface of the medium (Fig.3.7.1.); mycelium composed of smooth-walled,

septate and colourless hyphae up to 10 m dia; chlamydospores infrequent; main

branches of the conidiophores, 5- 7 m dia, produce many side branches; the apex

of each branch terminated by a phialide; highly ramified conidiophores usually

arise in compact tufts; phialides short and plump, flask shaped to almost pyriform,

narrower at the base than the middle and attenuate abruptly into slender conical

necks, 4.5-6.5 x 2.8–3.5 m but the terminal phialide may attain 10 m; arise in

somewhat irregular whorls of two to five immediately beneath the terminal

phialides; the phialospores globose; 2.5–3.5 m dia; base of the spore usually

appear as a truncate apiculus (El-Meleigy et al., 2010).

3.9. Testing of Fatty Oil, Extracts and Hederagenin

The essential oil extracts and hederagenin of S. mukorossi were tested for their

toxicity against fungal pathogens by the Poisoned Food Technique (Grover &

Moore, 1962) on the basis of Inhibitory Concentration (IC50). Minimum Inhibitory

Concentration (MIC) of hederagenin was worked out by same technique. The

nature of the toxicity (fungicidal & fungistatic) of the hederagenin was determined

following methodology of Iqbal et al. (2004). Hederagenin also exhibited marked

effect on germination of fungal spores/conidia by the slide germination method

(Ogbebor & Adekunle, 2005).

60

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Fig. 3.7.1. Pictorial

details of Test Fungi.

(A) Front view of the

culture, (B) back view

of the culture and (C)

spores and mycelium.

Test Fungi A B C

A. alternata

C. gloeosporioides

Phoma sp.

P. dalbergiae

F. oxysporum

G. lucidum

R. solani

T. piluliferum

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3.9.1. Inhibitory Concentration (IC50)

The toxicity of extracts (petroleum ether, chloroform, methanol & butanol) of

pericarp of S. mukorossi from different sources was determined against eight test

fungi. Further, the toxicity of hederagenin from different sources was determined

against three test fungi i.e. Phoma sp., R. solani and T. piluliferum.

A culture of the test fungi was grown on Potato Dextrose Agar (PDA) medium

for certain period (generally 7 days) at the optimum temperature (25°±1°C) for

growth. Petroleum ether and chloroform extracts from all three sources were

dissolved in acetone solvent to prepare the concentration (per cent). Methanol and

butanol extracts from all three sources were dissolved in sterilized distilled water to

make concentration (per cent). The solvents used for dissolving were taken on the

basis of polarity. PDA supplemented with different plant extracts at four

concentrations (0.5, 1.0, 1.5 & 2.0 %) and hederagenin at five concentrations (200,

400, 600, 800 & 1000 ppm) were poured in the petri plates under aseptic

conditions.

During experimentation, it was observed that hederagenin has severe

solubility problem. It was practically insoluble in water and soluble only in

chloroform-alcohol mixture. Further, it precipitated soon after pouring into

medium, PDA. The dissolution was also attempted in acetone in which, it was

insoluble. The compound was soluble in DMSO, however, it precipitated out in

PDA. After repeated trails in different solvents it was observed that the compound

is soluble in ethyl alcohol after constant shaking at a temperature of 78oC for 1 hr.

Therefore, hederagenin was dissolved into ethyl alcohol in following ratio i.e. 200

ppm in 1 ml, 400 ppm in 2 ml and so on. As hederagenin is slowly soluble in ethyl

alcohol, therefore, vial containing the mixture was placed in water bath for few

minutes at 90°C. Then, it was mixed with PDA. These plates were left in laminar air

flow for two days, so that, the solvent evaporated over time. This precaution was

observed to avoid any adverse effect of the solvent on the fungal growth. After that,

inoculation of test fungi was done.

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After solidification, small disc (0.7 cm dia) of the 10-day-old fungus culture

was cut with a sterile cork borer and transferred aseptically upside down at the

centre of a Petri dish. Suitable checks were maintained where the culture discs

were grown under same conditions on PDA without extract/hederagenin. Solvent

checks (a solvent which is used for dissolving extract i.e. acetone for extracts and

alcohol for hederagenin) were maintained to check out the inhibitory effect of

solvent on fungi. Petri plates were incubated at 25°±1°C. The radial growth of

fungus colony was measured after every twenty-four hours till the fungus in the

control plate completely occupied it. Three replications were maintained. The

antifungal activity was evaluated by measuring the relative growth of fungus in

treatment vis-a-vis control.

The percent growth inhibition over control was worked out using the

formula of Vincent (1927).

I = C-T/C x 100

Where,

I is inhibition per cent,

C is colony diameter in control (mm) and

T is colony diameter in treatment (mm).

3.9.2. Minimum Inhibitory Concentration (MIC)

The MIC was determined as that concentration above which the fungal

growth was totally suppressed and below which the fungus resumed growth.

Hederagenin isolated from Gyarahdevi source was used to find out the

minimum inhibitory concentration. Experiments were carried out by above

mentioned poisoned food technique using different concentrations of

compound i.e. hederagenin against all eight fungi. Also, hederagenin from

rest of the two sources i.e. Dehradun and Nainatikker were tested at the

MIC achieved by Gyarahdevi source.

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3.9.3. Fungicidal and fungistatic activity

The nature of the toxicity (fungicidal & fungistatic) of hederagenin isolated

from different sources against the eight test fungi was determined The

fungal discs from the MIC Petri plates of all three sources were transferred

to PDA and incubated at 25°±1°C for seven days. Plate showing growth after

the incubation period represented fungistatic activity and no growth on

plate indicated fungicidal nature of toxicity of the hederagenin.

3.9.4. Spore/conidial germination

Spores/conidia from 10- day- old culture of A. alternata, C. gloeosporioides,

Phoma sp., P. dalbergiae, G. lucidum, F. oxysporum, R. solani and T.

piluliferum on PDA plates were taken. Conidial suspensions were made in 10

ml sterilized distilled water. The spores/conidia 30µmlwere examined and

counted with the help of haemocytometer (Aneja, 2003). The number of

spores/conidia of fungi in one ml of sterilized distilled water were as follows:

Table3.8.4. Count of spores/conidia (no./ml of sterilized distilled

water) of different test fungi

Fungus Spores/Conidia (no./ml)

A. alternata 2.50 x 106

C. gloeosporioides 1.25 x 106

Phoma sp. 2.25 x 106

P. dalbergiae 1.05 x 106

F. oxysporum 1.85 x 106

G. lucidum 1.40 x 106

T. piluliferum 2.05 x 106

Hederagenin isolated from of S. mukorossi pericarp of Gyarahdevi

source was dissolved in alcohol. Different concentrations of hederagenin

were placed in the cavity of the depression slide and allowed to air dry.

Spore suspension of fungi was prepared in sterilized distilled water. The

suspension was added to the dried hederagenin and thoroughly mixed. The

cavity slides were incubated in Petri dish moist chamber. Three replications

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were maintained for each treatment. The spore suspension in solvent

(alcohol) and sterilized distilled water (without compound) served as control.

The spores were observed for germination in four different microscopic fields

(under 40X) and recorded after twenty-four and forty-eight hours to

calculate percent inhibition of spores/conidia germination using Vincent

formula (1927).

I = C-T/C x 100

I = Inhibition of spore germination,

C= germination in control and

T = germination in treatment.

3.10. Statistical Analysis

Data were analyzed using Genstat software (GEN532-2). Complete

Randomized Design (CRD) was followed for statistical analysis. Two-way

analyses were used for the data of extracts, hederagenin and spore

germination. All the data was analyzed after angular (arc-sin)

transformation of values. Treatment means were compared using CD at 5

per cent level of significance.