78

RESULTS

1. COLLECTION OF PLANT SAMPLES

T e t o selected edicinal plants ere collected fro Irula tribe o en’s elfare

society (ITWSS) Thandarai, Chengalpet at four different seasons (June - Aug, Sep - Nov, Dec

- Feb, Mar - May) and their taxonomic characters were noted.

2. ISOLATION OF ENDOPHYTES - SEASONAL RECURRENCE STUDIES

2.1 Surface sterilization of plant material

The healthy plant materials were chosen for surface sterilization and process was carried

out as described in materials and methods. Ten different methods of treatment were adopted

for surface sterilization. The number of endophytic strains isolated by adopting 10 different

methods has been compared using the bar chart (Fig: 3). Surface sterilization method VII

yielded the largest number of endophytes. Methods II and IX yielded the second largest

number of endophytes as well as more effective in isolating sterile mycelium and yeasts.

Methods VIII and X does not produce any endophytes indicates that the endophytes within the

plant tissue have been destroyed. However the other methods yielded few numbers of

endophytes comparatively. Also methods V and VI might have produced certain epiphytes too

(Fig: 4 and 5).

2.2 Isolation of endophytes

A total of 338 leaf segments were inoculated per sampling per plant. The incubated

plates were observed for the growth of endophytic fungal colonies from the second day. The

endophytic fungal colonies growing on the leaf segments were noted and subcultured further

79

by hyphal tipping. Pure cultures of these fungal endophytes were later maintained on fresh

PDA plates and slants. These pure culture isolates were examined periodically for sporulation

and identified. Using Lactophenol cotton blue wet mounts these fungal isolates were

identified based on the morphology of the fungal culture, the mechanism of spore production

and characteristics of the spore following the standard mycological manuals. The

F

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3

I

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l

a

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i

on of endophytes using different surface sterilization methods

80

Fig: 4 Isolation of endophytes from Asparagus racemosus using different

surface sterilization methods on PDA plates

81

Fig: 5 Isolation of endophytes from Hemidesmus indicus using different

surface sterilization methods on PDA plates

identification of molds was based on the shape, method of production and arrangement of

spores (conidial ontogeny). The fungal endophytes growing on the leaf segments were

observed and the data was recorded for statistical analysis.

Generally mitosporic fungi and ascomycetes as well as some sterile fungi were recorded

as endophytes from the host plants. A total of 5400 leaf segments were inoculated from the

host plants sampled in different seasons that harbored 2457 isolates totally comprising 1489

isolates belonging to 38 fungal species of 17 different genera represents around 60.6%, 12

miscellaneous mycelia sterilia of 968 isolates that represents around 39.4%, and 13 had yeast

– like growth (Fig: 6, 7 and 8).

2.3 Identification of fungal isolates

The endophytic isolates obtained were subcultured by hyphal tipping and their pure

cultures were prepared and maintained using fresh PDA plates and slants. The pure cultures

were examined periodically for sporulation and identified.

82

Macroscopic appearance of the fungal colonies, their morphological appearance and the

mechanism of spore production and characteristics of the spores were noted using the standard

mycological manuals. The identification of molds was based on the shape, method of

production and arrangement of spores (conidial ontogeny). Microscopic appearance was

noted following wet mount preparation by lactophenol cotton blue staining and the

microscopic slides were mounted observing them under 4X, 10X and 40X objectives (Ellis,

1971; Sutton, 1980).

Following were the different fungal endophytes identified (Fig: 9) whose descriptive

morphological features are listed as follows:

Alternaria species

Colonies were fast growing, black to olivaceous-black or greyish, and were suede-like to

floccose. Microscopically branched acropetal chains (blastocatenate) of multicellular conidia

(dictyoconidia) were produced sympodially from simple, sometimes branched, short or

elongate conidiophores and were obclavate, obpyriform, sometimes ovoid and ellipsoidal,

often with a short conical or cylindrical beak, pale brown, smooth-walled or verrucose.

83

Fig: 6 Isolation of endophytes from A. racemosus and H. indicus on PDA plates

84

Fig: 7 Isolation of endophytes from A. racemosus on PDA plates

85

Fig: 8 Isolation of endophytes from H. indicus on PDA plates

86

Fig: 9 Fungal endophytes from A. racemosus and H. indicus

Aspergillus sp.

Curvularia sp.

Fusarium sp.

Acremonium sp.

87

Fig: 9 Fungal endophytes from A. racemosus and H. indicus

Colletotrichum sp.

Penicillium sp.

Trichoderma sp.

Nigrospora sp.

88

Fig: 9 Fungal endophytes from A. racemosus and H. indicus

Cladosporium sp.

Humicola sp.

Myrothecium sp.

Chaetomium sp.

89

Alternaria alternata

Produced black colour olivaceous colonies on PDA. Reverse side of the colonies were dark

brown. Colourless hyphae, Smooth conidiophores, 0 μ long, 3–6 μ t ick in size arise singly

or in groups often branched, straight and flexuous. The conidia were formed in long chains,

obclavate, obpyriform and ovoid in shapes. Beaks of conidia were short, cylindrical and smooth.

The overall length of conidia was 20–63 μ and t e t ickness as 9–1 μ .

Alternaria helianthi

The colony grows with greyish–white on media. The length and breadth of the conidia

produced on PDA edia aried fro 117 to 173 μ and 1 to 33.7 μ , respecti ely.

Acremonium strictum

Colonies were white to pale pink in colour usually slow growing often compact and moist

at first became powdery, suede-like with age. Hyphae were fine and hyaline and produced

mostly simple awl-shaped erect phialides of 20–40 μ . Conidia ere usually one-celled

(ameroconidia) hyaline and pigmented, globose to cylindrical, and mostly aggregated in slimy

heads at the apex of each phialide.

Aspergillus flavus

It produced light greenish–yellow color colony. Reverse side of the colonies were

yellowish at first turned brownish in mature age. Conodiophores were 400-1000 μ × -1 μ

in size, pitted, rough and uncolored. The conodial heads were hemispherical to subglobose. The

vesicles were dome-like and 10-30 μ in dia eter. T e pri ary sterig ata ere 7-10 μ × 3-4

μ and t e secondary sterig ata ere 10-1 μ × 3- μ in size.3-4 μ . Colorless conodia

were seen pyriform to globose in shape.

Aspergillus niger

Black colour colonies grew rapidly on PDA. Smooth thick walled unseptate conidiophores

200-100 μ long ere seen colourless near t e esicle. Conidial eads ere fuscuous, black and

globose with a diameter upto 300- 00 μ . Conidial c ains ere present o er t e entire surface

of colorless, globose and thick walled vesicles with rough, globose conidia.

90

Aspergillus fumigatus

The growth was more or less velvety, sporing tardily with heads bluish green. Columnar

conidial eads it arying lengt ere obser ed it 40 μ in breadt . Conidiop ores ere

smooth, short, greenish 2- μ in dia eter. P ialides ere borne directly on esicles ic ere

closely packed, lower ones deflected upwards 6-8 × 2-3 μ . Vesicles ere flask s aped and

fertile. Conidia were small globose smooth mostly 2.5-3 μ in dia eter.

Aspergillus flavipes

Silvery white or white colonies, reverse yellow to orange brown or reddish brown were

observed. Conidial heads columnar in size were observed on PDA media. Vesicles were globose

to ovate, metulae fertile over entire vesicle, conidial heads splitting over age. Conidia smooth,

globose, 2-3 μ in dia eter.

Cladosporium cladosporioides

Effuse, velvety olivaceous to dark brown colour colonies grew on PDA. Reverse sides were

blackis bro n. ong s oot conidiop ores pale to oli aceous bro n ere upto 3 0 μ long,

yet shorter, 2-6 μ t ick. Conidia ere o oid to cylindrical and irregular in size. T e conidia

were formed in long branched chains, smooth and 5-30 μ × 3-6 μ in size.

Curvularia species

Woolly, blackish brown colour colonies grew rapidly on PDA. Reverse was blackish

brown. Brown colored conidia 8-14 μ × 1–3 μ , cur ed it t ree transverse septa with

central cell dark and enlarged compared to end cells were observed.

Curvularia lunata

Dematiaceous hyphomycete produced sympodial mid or dark brown colonies, middle septa

was not median, 3 septate and smooth walled, 18-32 × 8-16 μ , Conidia were pale brown,

cylindrical or slightly curved with one of the central cells being larger and darker, germination

was bipolar and some species were having a prominent hilum.

91

Colletotrichum species

Colonies were usually dark pigmented with white aerial mycelium consisting of numerous

black sclerotia and light brown coloured conidial masses, reverse was dark brown. Sclerotia were

usually abundant, spherical and were often confluent. Conidia were straight, fusiform, attenuated

at the ends 16-22 × 3-4 μ . Appressoria ere co on, cla ate, bro n, 11-16.5 × 6-9. μ ,

variable in shape.

Chaetomium species

Chaetomium produced darkly-pigmented, globose, ovoid, barrel to flask-shaped, ostiolate

ascocarps (perithecia) beset with dark-coloured terminal hairs (setae) which were straight,

branched or curved. Eight spored asci were clavate to cylindrical. Ascospores were one-celled,

darkly-pigmented, smooth-walled, of varying shape, mostly ovoid, ellipsoidal or lemon-shaped.

Fusarium moniliformae

White aerial mycelia at times tinged with purple reverse colorless turning purple, were

observed on PDA. Single celled, oval to club shaped microconidia 6 μ in size, ere for ed in

chains on monophialides. Sickle shaped thin walled macroconidia 23 – 4 μ long as

distinctly seen.

Fusarium oxysporum

Rapidly grown colonies of 4.5 diameter with white aerial mycelium, becoming purple, with

discrete orange sporodochia present in some strains, reverse hyaline to dark blue or dark purple

were just observed on the PDA media just after four days of inoculation. Conidiophores were

short, single, lateral monophialides in the aerial mycelium, later arranged in densely branched

clusters. Microconidia were abundant, never in chains, mostly non-septate, ellipsoidal to

cylindrical, straight and often curved, 5-12 × 2.3 - 3.5 μ . C la ydospores ere ter inal or

intercalary.

Fusarium solani

Fast grown colonies 4.5 cm in 4 days aerial mycelium white to cream, became bluish-

brown. Macroconidia were formed after 4-7 days from short multi-branched conidiophores.

Microconidia were abundant, cylindrical to oval one to two celled and formed from long lateral

92

phialides 8-16 × 2-4. μ . Microconidia ere 3 to septate (usually 3 septate), fusifor ,

cylindrical, often moderately curved, with an indistinctly pedicellate foot cell and a short blunt

apical cell.

Myrothecium species

White aerial mycelia grows after 10 days produced yellow color on PDA. The sporodochia

were olivaceous-black, flattened and cushion like, 60-7 0 μ in dia eter and conodiop ores

were subhyaline and repeatedly branched. Rod shaped to ellipsoidal conodia were produced at

the terminal of the conidiophores.

Penicillium species

The colonies were velvety, orangish green in color with thin white margin. The reverse

sides of the colonies were pale orange where mature colonies were deeply, radiantly and

wrinkled spores were abundant with grey-green shades. The conidiophores were mostly 70-80 ×

2 μ in size and s oot alled. Fi e to ten parallel sterig ata occasionally 1 μ in lengt

were present on verticals. The spores were arranged in a chain and conidial chains were up to

100 μ in lengt . T e ature conidia ere elliptical, s oot and 4-5 × 3 μ in size.

Penicillium funiculosum

White-coloured colonies, sometimes of a pale green-grey, slightly slimy and shiny with

erected funiculi at the centre, filamentous and lighter towards the edges were seen growing on

PDA. The penicillia were biverticillate and symmetrical. Smooth-walled conidiophores were 25-

60 × 2-3 µm in dimensions. The phialides were verticillate (5-8 per metulae), in dense groups

and cylindrical (10 × 2 µm) and conidia were smooth walled.

Phoma species

Irregular olivaceous grey with darker patches colonies were produced by Phoma sp on

PDA. Reverse side of the colonies were blackish brown and mycelia were immerse, branched,

septate and brown in color. The conodiomata were pycnidial, dark brown in color, semi-

93

immersed and thin walled. Pycnidia were developed separately or aggregately on media. The

conodia were straight, cylindrical, 5-6. μ in size.

Syncephalastrum species

Cottony to fluffy, white to light grey, became dark grey with the development of sporangia,

fast growing colonies were observed. Spore bearing sporangiophores were erect, stolon-like,

often producing adventitious rhizoids, and showed sympodial branching (racemose branching)

produced curved lateral branches. The main stalk bears finger-like merosporangia directly over

their entire surface. Merosporangia containing merospores which were thin-walled, evanescent

and contain 5-10 (18) globose to ovoid, smooth-walled spores.

Nigrospora species

Colonies were white, later brown to black during sporulation. Conidiophores

micronematous, branched, flexuous, colorless to brown and smooth. Conidia were solitary,

acregenous, simple, spherical and broadly ellipsoid, compressed dorsiventrally, black, shining,

smooth 10-16 μ in dia eter.

Humicola species

These colonies have grown hyaline, grey, greenish to dark brown and black in color.

Conidia were dark globose, smooth, producing singly at the end of the conidiogenous cells. The

latter cells were short hyaline, monoblastic, terminal. Spores were large, dark, spherical, single,

dry and smooth measuring 12-17 μ in dia eter.

Trichoderma species

Fast growing colonies with white and downy, latter developed yellowish-green to deep

green compact tufts, often only in small areas or in concentric ring-like zones on the agar

surface. Conidiophores were repeatedly branched, irregularly verticillate, bearing clusters of

divergent, often irregularly bent, flask-shaped phialides. Conidia were mostly green, sometimes

hyaline, with smooth or rough walls and were formed in slimy conidial heads (gloiospora)

clustered at the tips of the phialides.

94

Geotrichum species

Geotrichum sp. typically produced chains of hyaline, smooth, onecelled, subglobose to

cylindrical arthroconidia. Hyphal elements were progressively compartmentalised by

fragmentation of septa. The arthroconidia, which were quite variable in size germinated at one

end giving the appearance of a bud which latter developed into a septate mycelium.

Blastoconidia production was not found.

Gloeosporium species

The mycelium was greenish-grey in color, velvety in appearance, produced black sclerotia

in culture. Appressorium-like cells were formed in terminal and intercalary positions from

sclerotial hyphae. Conidia were hyaline, single celled, having parallel sides with rounded ends,

measuring 13.4-20.1 × 4.5-6.7 μ .

2.4 Seasonal recurrence of endophytes

In a geoclimatic context endophytic assemblages appear to be richer in tropical than in

temperate or cold zones of the world (Fisher et al., 1995; Arnold and Lutzoni, 2007). The

diversity of endophyte assemblages in these tropical plants was highest comprising various

hyphomycete and coelomycete members. Higher number of endophytes was isolated during wet

periods rather than dry periods. Different endophytic isolates were harbored in different seasons

whose relative colonization densities are tabulated in Tables. 5 and 6. The foliar endophyte

assemblage of A. racemosus and H. indicus were made up of Acremonium strictum,

Colletotricum sp., Geotrichum sp., Phomopsis sp., Trichoderma sp., Humicola sp., etc.

In A. racemosus, Colletotrichum sp.1 and Phomopsis sp.1, were found to be dominant with

overall relative colonization densities (R.C.D) of 7.11% and 5.44% followed by A. strictum and

Trichoderma sp.1 of 4.83% and 4% respectively. However considering their seasonal recurrence,

A. strictum was recurring the most except in June-August, following Colletotrichum sp.1 but not

the Phomopsis sp. Among the mycelia sterilia, Morphotype sp.7

Table. 5 Relative Colonization Densities (%R.C.D) of fungal endophytes isolated

in different seasons from Asparagus racemosus

95

S.No. Endophyte

Year I Year II Overall

%R.C.D June-

Aug

Sep -

Nov

Dec -

Feb

Mar -

May

June -

Aug

Sep -

Nov

Dec -

Feb

Mar -

May

1 Alternaria helianthi - 1.78 - - - 0.89 1.78 - 0.56

2 F. moniliformae 4.44 4.44 1.78 - 8.44 3.56 - - 2.83

3 F. oxysporum - - - - 7.11 3.11 4.00 - 1.78

4 P. funiculosum 2.67 - - 4.89 - - - - 0.94

5 Phomopsis sp.1 11.11 5.78 9.33 - 12.44 4.89 - - 5.44

6 Phomopsis sp.2 13.78 7.56 8.00 - - 5.78 - - 4.39

7 A. strictum - 4.89 4.00 9.33 - 4.89 7.11 8.44 4.83

8 A. niger 1.33 2.22 - 3.11 3.56 1.78 - 4.00 2.00

9 A. flavus - 0.89 - - - 0.44 - 2.22 0.44

10 Cladosporium sp.1 2.22 1.78 - 8.00 - - - - 1.50

11 Humicola sp. 0.44 4.00 1.33 - - - - - 0.72

12 Trichoderma sp.1 - 2.67 3.11 5.78 - 2.67 8.44 9.33 4.00

13 Myrothecium sp. - - - - 6.22 0.44 0.89 - 0.94

14 Geotrichum sp.1 - - - - - 4.44 5.33 6.22 2.00

15 Geotrichum sp.2 - - - - 4.00 2.22 - 0.78

16 Colletotrichum sp.1 3.56 6.67 6.67 12.00 - 5.33 10.67 12.00 7.11

17 Colletotrichum sp.3 7.56 8.89 4.89 - 11.11 6.67 - - 4.89

18 Morphotype sp.1 4.00 - - 3.56 4.00 - -- 2.22 1.72

19 Morphotype sp.2 4.89 5.33 4.44 - - 4.00 4.89 - 2.94

20 Morphotype sp.3 3.11 0.89 - 0.89 3.11 - - - 1.00

21 Morphotype sp.5 5.78 4.89 6.22 - 8.44 7.56 8.00 - 5.11

22 Morphotype sp.6 - 1.78 0.89 - - - - 0.33

23 Morphotype sp.7 12.89 13.33 - 7.56 12.00 12.44 - 7.56 8.22

24 Morphotype sp.8 - 6.67 7.56 - - - - 1.78

25 Morphotype sp.9 - 3.56 2.22 2.22 - 2.67 3.11 1.33 1.89

26 Morphotype sp.10 8.44 9.78 8.44 - 9.33 10.67 10.22 - 7.11

27 Morphotype sp.11 1.33 - - - - 6.22 - - 0.94

Table. 6 Relative Colonization Densities (%R.C.D) of fungal endophytes isolated

in different seasons from Hemidesmus indicus

96

and Morphotype sp.10 were recurring in most seasons dominantly among other morphotypes

with a overall R.C.D of 8.22% and 7.11%, respectively (Fig. 10).

S.No. Endophyte

Year I Year II

Over all

% R.C.D June -

Aug

Sep -

Nov

Dec -

Feb

Mar -

May

June -

Aug

Sep -

Nov

Dec -

Feb

Mar -

May

1 A. alternata - 4.44 4.00 5.33 - 4.44 4.44 - 2.83

2 Alternaria sp.1 2.67 - 4.89 - - 4.00 - 1.44

3 F. moniliformae 5.78 4.89 4.44 - 5.78 4.89 - 4.89 3.83

4 F. solani 5.33 4.00 - - 5.33 4.44 - - 2.39

5 P. funiculosum - - - - - 0.44 0.89 - 0.17

6 Penicillium sp.1 - 0.89 0.44 - - - - - 0.17

7 Penicillium sp.2 - - - - 2.22 0.44 - 2.22 0.61

8 A. strictum 7.11 5.78 4.44 - 6.67 5.33 5.78 5.33 5.06

9 Acremonium sp.1 - 3.56 - 6.22 - - - - 1.22

10 A. flavipes - 0.44 3.56 - 0.44 0.44 1.78 0.83

11 A. fumigatus 2.22 0.44 0.44 - - - - - 0.39

12 Syncephalastrum sp. 5.33 2.67 2.67 - 4.00 2.67 - - 2.17

13 Cladosporium

cladosporioides - 2.67 0.44 4.89 - 2.22 3.11 - 1.67

14 Cladosporium sp.2 - - - - 3.11 1.78 - - 0.61

15 Nigrospora sp. - 2.67 3.11 - - 3.11 3.11 4.00 2.00

16 Trichoderma sp.2 3.56 0.89 0.89 - 3.56 0.89 - - 1.22

17 Gleoesporium sp.1 - 3.11 4.00 - 4.89 4.00 4.00 - 2.50

18 Gleoesporium sp.2 - - - - - 3.56 3.56 - 0.89

19 Curvularia lunata 4.89 1.78 1.78 - 4.00 1.78 2.22 - 2.06

20 Curvularia sp.1 - 0.89 1.78 - - - - - 0.33

21 Curvularia sp.2 4.00 - - 4.00 - - - 1.00

22 Curvularia sp.3 - - - - - 0.89 1.78 3.11 0.72

23 Chaetomium sp. - 1.33 1.33 - - 0.89 - - 0.44

24 Colletotrichum sp.2 - 6.67 5.33 - - 6.22 - 5.78 3.00

25 Morphotype sp.1 - 3.56 4.89 - 3.56 3.11 - - 1.89

26 Morphotype sp.2 6.22 6.67 - 5.78 - 5.33 - 2.22 3.28

27 Morphotype sp.4 5.78 5.33 - - 5.33 4.89 - - 2.67

28 Morphotype sp.5 - - 5.78 6.67 - - 4.00 4.89 2.67

29 Morphotype sp.6 - 4.89 7.11 - - 4.00 6.67 - 2.83

30 Morphotype sp.7 4.89 5.33 - - 4.00 - - 1.33 1.94

31 Morphotype sp.9 2.67 3.11 - - - 2.22 2.67 - 1.33

32 Morphotype sp.10 4.00 - - 4.89 6.22 6.22 - 4.00 3.17

33 Morphotype sp.12 4.44 4.00 5.33 - 4.89 - 4.89 - 2.94

97

In H. indicus the dominant species was A. strictum having higher overall R.C.D of 5.06%

followed by F. moniliformae and Colletotrichum sp.2 of 3.83% and 3%, respectively (Fig: 10).

Further A. strictum was the most recurring isolate in almost all seasons followed by F.

moniliformae and Curvularia lunata having R.C.D of 2.06%. Among the sterile mycelia,

Morphotype sp. 2 and Morphotype sp. 10 were dominant recurring mostly among other

morphotypes with overall R.C.D of 3.28 and 3.17, respectively.

Other fungal endophytes isolated from both plants includes Syncephalastrum sp.

Nigrospora sp., Humicola sp., Cladosporium sp., Geotrichum sp., Gloesporium sp., etc. among

which A. flavus and Penicillium sp., were possessing least overall R.C.D of 0.44 and 0.17,

respectively.

2.5 Statistical analysis

Fungal occurrence was measured by calculating colonization density, colonization rates

and isolation rates. The data obtained was compared for all the four seasons between the host

plants represented graphically (Fig: 11). The mean over all colonization rates and isolation

rates of endophytes from A. racemosus were 73.89% and 76.22% and from H. indicus were

64.5% and 60.28%, respectively (Tables. 7 and 8). Further the overall colonization and

isolation rates were higher during the wet periods of Sep-Nov in both A. racemosus (92.22%

and 95.11%) and H. indicus (82% and 77.11%) compared to other seasons.

One way ANOVA was further performed to compare the isolation rates and colonization

rates of fungal endophytes of each plant occurring from four different seasons (Kumar and

Hyde, 2004). The test reveals significant differences that overall colonization rate and isolation

rate was significantly higher in A. racemosus in the month of Sep-Nov than that of H. indicus

(p < 0.05) as well as other seasons (Table. 9). Therefore it can be concluded that significant

differences in the total number of isolates and their means obtained in each season with their

corresponding colonization and isolation rates.

98

Fig: 10 Overall % R.C.D of recurring fungal endophytes in A. racemosus

and H. indicus

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1

O

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l

colonization and isolation rates of endophytic fungi from

different seasons in A. racemosus and H. indicus

1 - Alternaria alternata 2 - F. moniliformae 3 - Phomopsis sp.1 4 - Acremonium strictum

5 - Aspergillus niger 6 - Trichoderma sp.1 7 - Trichoderma sp.2 8 – Cladosporium cladosporioides

9 - Syncephalastrum 10 - Gleoesporium sp.1 11 - Colletotrichum sp.1 12 - Colletotrichum sp.3

13 - Curvularia lunata

99

Table 7 Over all colonization and isolation rates of endophytic fungi from different

seasons in A. racemosus

Colonization rate and

Isolation rate

June - Aug Sep - Nov Dec - Feb Mar - May

Year I Year

II

Year I Year

II

Year I Year

II

Year I Year II

Overall colonization rate (%)

85.33

84.00

95.11

89.33

66.22

63.56

56.00

51.56

Overall isolation rate (%) 87.56

85.78

97.78

92.44

68.89

66.6

57.33

53.33

Mean Overall colonization

rate (%) 84.6 92.2 64.8 53.7

Mean Overall isolation rate

(%) 86.6 95.1 67.7 55.3

Table. 8 Overall colonization and isolation rates of endophytic fungi from different

seasons in H. indicus

Colonization rate and

Isolation rate

June - Aug Sep - Nov Dec - Feb Mar - May

Year I Year

II

Year I Year

II

Year I Year

II

Year I Year II

Overall colonization rate (%) 72.00 67.56 86.22 77.78 62.22 56.89 48.89 44.44

Overall isolation rate (%) 68.89 63.56 80.00 74.22 58.22 51.56 46.22 39.56

Mean Overall colonization

rate (%) 69.78 82.00 59.56 46.67

Mean Overall isolation rate

(%) 66.22 77.11 54.89 42.89

100

. Table. 9 Statistical analysis – One way ANOVA analysis

ANOVA: Single Factor Analysis of colonization rates for Asparagus racemosus

SUMMARY

Groups Sum Average Variance

June - Aug 169.3333 84.66667 0.888889

Sep - Nov 184.4444 92.22222 16.69136 Dec - Feb 129.7778 64.88889 3.555556 Mar - May 107.5556 53.77778 9.876543

ANOVA

Source of

Variation

SS df MS F P-value F crit

Between Groups 1875.457 3 625.1523 80.6327 0.000493 6.591382

Within Groups 31.01235 4 7.753086

Total 1906.469 7

June - Aug Sep - Nov Dec - Feb Mar -

May

87.56 97.78 68.89 57.33

85.78 92.44 66.67 53.33

ANOVA: Single Factor analysis of isolation rates for Asparagus racemosus

SUMMARY

Groups Sum Average Variance

June - Aug 173.3333 86.66667 1.580247

Sep - Nov 190.2222 95.11111 14.22222

Dec - Feb 135.5556 67.77778 2.469136

Mar - May 110.6667 55.33333 8

ANOVA

Source of

Variation

SS df MS F P-value F crit

Between Groups 1947.062 3 649.0206 98.81704 0.000331 6.591382

Within Groups 26.2716 4 6.567901

Total 1973.333 7

June - Aug Sep - Nov Dec - Feb Mar -

May

72.00 86.22 62.22 48.89

67.56 77.78 56.89 44.44

101

ANOVA: Single Factor Analysis of colonization rates for Hemidesmus indicus

SUMMARY

Groups Sum Average Variance

June - Aug 139.5556 69.77778 9.876543

Sep - Nov 164 82 35.65432

Dec - Feb 119.1111 59.55556 14.22222

Mar - May 93.33333 46.66667 9.876543

ANOVA

Source of

Variation

SS df MS F P-value F crit

Between Groups 1353.16 3 451.0535 25.91158 0.004417 6.591382

Within Groups 69.62963 4 17.40741

Total 1422.79 7

June - Aug Sep - Nov Dec - Feb Mar -

May

68.89 80.00 58.22 46.22

63.56 74.22 51.56 39.56

ANOVA: Single Factor Analysis of isolation rates for Hemidesmus indicus

SUMMARY

Groups Sum Average Variance

June - Aug 132.4444 66.22222 14.22222

Sep - Nov 154.2222 77.11111 16.69136

Dec - Feb 109.7778 54.88889 22.22222

Mar - May 85.77778 42.88889 22.22222

ANOVA

Source of

Variation

SS df MS F P-value F crit

Between Groups 1300.222 3 433.4074 23.00524 0.005523 6.591382

Within Groups 75.35802 4 18.83951

Total 1375.58 7

102

3. SECONDARY METABOLITE PRODUCTION OF ACREMONIUM STRICTUM

A. strictum was the chosen endophytic fungi for further studies due to their common

prevalence in both plants recurring mostly. Prior to the extraction of secondary metabolites low

molecular weight metabolites preliminary growth studies were performed.

3.1 Preliminary growth studies of Acremonium strictum

The culture flasks inoculated with A. strictum was evaluated for culture turbidity by

recording the optical density (OD) from 0th

hour at 405 nm using UV-VIS spectrophotometer.

The portions (1 ml) of broth cultured were aseptically transferred into cuvette and their optical

density was recorded against the blank. Sequential OD measurements for every 24 h were

recorded until the decline phase was reached to generate growth curves.

The following phases in the growth curve (Fig: 12) could be characterized based on

changes in OD the lag phase in which no changes in OD were measured. A rapid increase in

OD was later observed that maximal slope indicating maximum growth at an OD of 3.26 was

reached on 18th

day during log phase. Following which the stationary phase is reached where

there is no change in the OD leading to decline phase.

Extraction and analysis of secondary metabolites

The inoculated flask cultures of A. strictum were harvested after 18 days when it has the

attained maximum growth. The culture filtrate and the mycelial mat separated were extracted

using different organic solvents that gave extracts amounted to varying yields as listed in

Table.10

The extracts were then subjected to preliminary studies like UV and IR

spectrophotometry. UV spectra 200–400 nm using UV-VIS spectrophotometer was recorded

for all the extracts obtained using different solvents (Table. 11). The Infra Red spectra (IR)

were also obtained on the Fourier Transform Infra Red Spectrometer (Nicolet, model 670,

USA) using pellets of KBr (Fig: 13). These results infer the physical properties of the active

compounds present in the biologically active extracts. The inference of 2954-2869 is prominent

in all the IR spectras indicating C-H stretching which denotes the presence of

103

Fig: 12 Growth curve of A. strictum

104

Table. 10 Yield of extracts from A. strictum

NAME OF THE

SOLVENT USED

YIELD (mg/L )

CULTURE

FILTRATE

MYCELIAL

MAT

Hexane

Dichloromethane

Diethyl ether

Acetone

350

479

282

314

1700

1540

1320

1490

105

Table. 11 UV Spectroscopic Absorbance of Different Solvent Extracts

S.NO NAME OF THE

SOLVENT

CULTURE FILTRATE MYCELIAL MAT

WAVELENGTH

(nm) ABSORBANCE

WAVELENGTH

(nm) ABSORBANCE

1.

Hexane

204

210

221

273

354

1.6978

2.7601

2.9316

2.4547

0.1512

204

210

221

273

354

2.3009

2.258

2.5120

0.9264

0.5752

2.

Dichloromethane

310

317

0.4071

0.3235

310

317

0.9412

0.3816

3.

Diethyl ether

285

233

227

222

217

212

210

203

0.5125

0.4396

0.2712

0.2806

0.3237

0.3799

0.4028

0.2148

304

232

288

236

225

215

210

203

2.1974

1.9976

2.0448

1.3761

0.4774

0.4969

0.5715

1.4349

4.

Acetone

300

354

1.4634

0.4005

300

354

0.5908

0.1563

Fig: 13 IR spectral analysis

106

a) IR spectrum of Diethyl ether extract of A. strictum – Culture filtrate

4000

.036

0032

0028

0024

0020

0018

0016

0014

0012

0010

0080

060

045

0.0

0.05101520253035404550556065707580859095

100.

0

cm-1

%T

A-E

THE

R

3395

3072

292528

55

1927

1688

1605

1521

1464

1349

1288

1210 11

91

1080

972

906

849 81

3772

706

645

107

b) IR spectrum of Dichloromethane extract of A. strictum – Culture filtrate

40

00

.03

60

03

20

02

80

02

40

02

00

01

80

01

60

01

40

01

20

01

00

08

00

60

04

50

.0

0.05

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

10

0.0

cm-1

%T

A-D

CM 3

26

5

29

2328

53

25

75 24

89

21

50

20

9820

27

19

40

18

66

17

79

16

84

15

201

46

4

13

78

13

47

13

13

12

80

10

631

01

197

3

90

0

81

5

74

370

5

65

1

108

c) IR spectrum of Hexane extract of A. strictum – Culture filtrate

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 0.0

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

100.0

cm-1

%T

A-HEXANE

3704

2956 2928

2873

2731 2673

2608 2383

2026

1462

1378

1294 1247

1136 1065

1038 984

957 903 889

815 757

725

659 522

109

d) IR spectrum of Acetone extract of A. strictum – Culture filtrate

110

e) IR spectrum of Diethyl ether extract of A. strictum – Fungal mat

111

f) IR spectrum of Dichloromethane extract of A. strictum – Fungal mat

112

g) IR spectrum of Hexane extract of A. strictum – Fungal mat

113

h) IR spectrum of Acetone extract of A. strictum – Fungal mat

114

alkanes. The following spectral data of 1712, 1606, 1462, 1377 & 1222, indicates the

presence of C=O stretching denoting ketones, asymmetric nitro group, O-CH3 stretching

denoting methyl/methylene groups, C-H stretching because of R-CH3 methyl groups

respectively to be present in the extracts. The existence of absorption peaks 1683, 1684, 1688

indicates C=C stretc ing ibration indicating t e presence of β Unsaturated a ines bonds,

can be related to the presence of unsaturated fatty acids in the organic extract being focused

in further part of the study.

Qualitative analysis revealed the presence of various secondary metabolites like

alkaloids, terpenoids, flavonoids etc. The quality of chemical compounds (secondary

metabolites) extracted however varied depending on the polarity of the organic solvent used

to obtain metabolites of hydrophilic and hydrophobic nature. Accordingly the results indicate

the presence of different metabolites extracted using different organic solvents among which

acetone and dichloromethane were better solvents in extraction of various metabolites

followed by hexane and diethyl ether (Table. 12) (Belofsky et al., 1998; Holler, 1999 and Lin

et al., 2000).

4. FATTY ACIDS PRODUCTION FROM A. STRICTUM

Growing the fungi in different media amended with varying carbon and nitrogen

sources optimization of process parameters like pH, temperature, light and dark conditions

etc. can affect their growth and the lipid accumulation. Hence media standardization for

strain improvement necessitates obtaining higher yield of fatty acids.

4.1 Standardization of media for strain improvement

The inoculated flask cultures of A. strictum grown in different media amended with

varying substrates as well as the cultures subjected to different conditions of varying pH and

temperature with 12:12 hours cycle of light and dark conditions were taken to study their

growth phases by taking optical density (OD) at 405 nm using UV-VIS spectrophotometer.

The sequential OD measurements were taken for every 24 h that was used to construct their

growth curves. The different phases in the growth curves could be characterized as initial lag

phase, the log phase showing maximal slope indicating maximum growth and the stationary

phase where there is no change in the OD leading to

115

Table. 12 Qualitative analysis of secondary metabolites

S.No.

CHEMICAL GROUP

& TEST

FILTRATE FUNGAL MAT

HEXANE DCM DE ACETONE HEXANE DCM DE ACETONE

1.

2.

3.

4.

5.

6.

Alkaloids (Mayer’s

test)

Carbohydrates

(Fe ling’s test)

Steroids (Lieberman

Burchand test)

Tannins(Ferric

chloride test)

Flavonoids (Shinoda

test)

Saponins (Frothing

test)

-

-

+

-

+

+

+

-

+

+

-

+

-

+

-

-

+

+

+

+

-

-

+

+

-

-

+

+

+

+

+

-

+

+

+

+

-

+

+

-

+

+

+

+

+

+

+

+

116

the decline phase.

As shown in Fig: 14, growth curves of the A. strictum culture obtained from different

media substrates were compared. The graph shows maximal slope reaching highest peak with

higher OD value of 3.35 for the Potato dextrose broth culture amended with yeast extract (PDB-

YE) compared to other media cultures.

Comparative studies among the growth curves shown in (Fig: 15 and 16) also reveals

highest peak for A. strictum culture grown in pH 6, room temperature with 12 hrs cycle of light

and dark with OD of 3.26 and 3.21 indicating the optimal conditions required to achieve

maximal growth of the A. strictum. These studies were evaluated along with their biomass yield

concurrently to standardize the optimal conditions for the growth of A. strictum for higher yield

of fatty acids.

4.2 Biomass production

The inoculated flask cultures of A. strictum grown in different media amended with varying

substrates as well as the cultures subjected to different conditions of varying pH and temperature

were harvested after 18 days of culture and their biomass was obtained (Yongmanitchai and

Ward, 1991). The biomass of the dried cultures was weighed and the results are represented in

(Fig: 17 and 18). Higher yield of nearly 0.87, 0.85 and 0.65 g of biomass per 100 ml of culture

media were obtained with the cultures grown in PDB-YE under conditions of pH 6 and room

temperature with 12 h cycle of light and dark conditions.

4.3 Extraction of fatty acid

Total lipids were extracted from the biomass obtained according to the Bligh and Dyer

method (Bligh and Dyer, 1959) with little modifications by using hexane and acetone instead of

chloroform and methanol (Fig: 19). The extracts obtained were subjected for TLC profiling.

4.4 TLC profiling

Using palm oil as standard, the lipid extracts obtained were subjected for TLC profiling on

precoated silica gel plates with the solvent system - diethyl ether: hexane: acetic acid (4:13:0.4).

After the run was complete the spots obtained were identified using potassium permanganate.

Fatty acid composition of palm oil constitutes: Palmitic acid,

117

Fig: 14 Effect of media on growth of A. strictum

Fig: 15 Effect of pH on growth of A. strictum

118

Fig: 16 Effect of temperature on growth of A. strictum

119

Fig: 17 Biomass production from different media under different conditions.

Fig: 18 Effect of media, pH and temperature on biomass of A. strictum

PSB PDB Richard’s Oat meal Corn meal PDB-YE

3 4 5 6 7 8

21oC 37oC 26oC

120

Fig: 19 Extraction of fatty acids

Tube A – 37ºC

Tube B – 26ºC

Tube O – 21ºC

Tube H – pH 3

Tube G – pH 4

Tube F – pH 5

Tube E – pH 6

Tube D – pH 7

Tube C – pH 8

Tube I – Ric ard’s

Tube J – PSB

Tube K – Oat meal

Tube L – PDB

Tube M – Corn meal

Tube N – PDB-YE

Lane O – pH 7

A B C D E F G H I J K L M N O

121

Stearic acid, Oleic acid and triglycerides (Rf: 0.54, 0.41, 0.32 and 0.7). The TLC profile

varied among the extracts obtained from biomasses of varying media, pH and revealed

nearly 3–4 spots with Rf values of 0.54, 0.41, 0.32 and 0.7 indicating the presence of

Palmitic acid, Stearic acid, Oleic acid and triglycerides compared with the palm oil

standard (Fig: 20).

4.4 Estimation of free fatty acids

Free Fatty acid content is known as the acid number/acid value. Based on the

methanolic KOH titration method (Cox and Pearson, 1962) using the titre value the amount

of free fatty acids present in the different extracts was calculated. The results indicate that

the extract obtained from the biomass harvested under the following culture conditions of

PDB-YE cultured under pH 6, room temperature produced higher amount of 36.3, 27.6 and

15.2 percentage of free fatty acids with an acid number of 72.23, 54.9 and 30.24 compared

to other cultures (Fig: 21, 22 and 23).

4.5 Preparation of FAMEs

Owing to increased acid value of 72.23, the fatty acid extract obtained from the PDB-

YE culture was subjected for the preparation of FAMEs. Once the FAMEs were prepared

they were analyzed using GC-FID.

4.6 Gas Chromatographic conditions

The FAMEs prepared were subjected for gas chromatography using GC–FID that the

FAME profiling revealed the presence of 14 fatty acids from A. strictum, with high

percentage of Tricosanoic acid of 28.6 g/100 g followed by Caprylic acid of 12 g/100 g and

Linoleic acid of 8.98 g/100 g of fat (Table. 13, Fig: 24 and 25).

Hence fatty acids extracted from various seed cultures being optimized for various

parameters were subjected for acid value estimation of fatty acids and quantified. The

culture harvested from PDB-YE optimized at pH 6 in room temperature showed increased

biomass of 0.87 g/100 ml with 36.3% of free fatty acids production was considered for

further studies.

122

* OA – Oleic Acid

,

Fig: 20 TLC profiling of fatty acids from different media under different

conditions of pH and temperature.

OA OA

A B C D E F G H I J K L M N O P

Lane A – Palm oil Std.

Lane B – PSB

Lane C – Corn meal

Lane D – PDB-YE

Lane E – Oat meal

Lane G – PDB

Lane H – Ric ard’s

Lane F – 21 ºC

Lane I – 26 ºC

Lane J – 37 ºC

Lane K – pH 3

Lane L – pH 4

Lane M – pH 5

Lane N – pH 6

Lane O – pH 7

Lane P – pH 8

123

Fig: 21 Effect of media on percentage of free fatty acids and acid value

Fig: 22 Effect of temperature on percentage of free fatty acids and acid value

124

Fig: 23 Effect of pH on percentage of free fatty acids and acid value

125

Fig: 24 GC chromatogram showing fatty acid profile of A. strictum

126

127

Fig:25 Fatty acid Profile of A.strictum

Fig: 25 Fatty acid profile of A. strictum

Table. 13 Fatty Acid Profile of A. strictum

S.No. Fatty acid Yield (g)/100 g

1. Caprylic acid 12

2. Lauric acid 1.23

3. Tridecanoic acid 3.34

4. Myristoleic acid 2.47

5. Pentadecanoic acid 3.99

6. Palmitic acid 2.37

7. Palmetoleic acid 5.47

8. Heptadecenoic acid 4.61

9. Elaidic acid 6.52

10. Oleic acid 2.63

11. Linoelaidic acid 8.06

12. Linoleic acid 8.98

13. Tricosanoic acid 28.6

14. Lignoceric acid 7.6

15. Saturated fat 0.202

16. Mono unsaturated fat 0.072

17. Poly unsaturated fat 0.06

18. Transfat 0.05

1 - Caprylic acid 2 - Lauric acid 3 - Tridecanoic acid 4 - Myristoleic acid

5 - Pentadecanoic acid 6 - Palmitic acid 7 - Palmetoleic acid 8 - Heptadecenoic acid

9 - Elaidic acid 10 - Oleic acid 11 - Linoelaidic acid 12 - Linoleic acid

13 - Tricosanoic acid 14 - Lignoceric acid

1 2 3 4 5 6 7 8 9 10 11 12 13 14

128

5. ENHANCEMENT OF FATTY ACID PRODUCTION USING ELICITORS

Metallic salts are used as elicitors in inducing the production of metabolites in

microorganisms where these metal ions play as co-factors for key enzymes involved in

biosynthesis of lipids. Hence fatty acid production in the endophytic fungi, A. strictum was

enhanced using different metallic salts as elicitor compounds under the cultural conditions

optimized for the strain improvement.

5.1 Preparation of culture media and conditions

PDB-YE culture media amended with different metal ion salt concentrations as elicitors

ranging from 1 µg – 500 mg of Magnesium sulphate (MgSO4.7H2O), Manganese sulphate

(MnSO4.5H2O), Copper sulphate (CuSO4.5H2O), Zinc sulphate (ZnSO4.7H2O) and Ferric

chloride (FeCl3.6H2O) was prepared. Cultures grown in PDB-YE served as control. The

cultures were harvested after 18 days of inoculation being maintained at room temperature

with 12 h cycle of light and dark conditions.

5.2 Biomass production

The inoculated flask cultures of A. strictum grown in different media amended with

varying metal ion salt concentrations and the control were harvested after 18 days of culture,

and their biomass was obtained (Yongmanitchai and Ward, 1991). The biomass of the dried

cultures was weighed and the results were represented in Fig: 26. The biomass yield from the

control was 0.87 g/100 ml. Minimal increase in salt concentration fro 1 μg to 00 μg yielded

elevated levels of biomass that maximum yield of nearly 1.5 g of biomass was obtained with

the cultures grown in PDB-YE a ended it 00 μg of etallic salts (MgSO4.7H2O,

MnSO4.5H2O, CuSO4.5H2O, ZnSO4.7H2O and FeCl3.6H2O) under conditions of pH 6, room

te perature it 1 cycle of lig t and dark conditions. Ho e er ig er t an 00 μg of salt

concentration yielded lower levels of biomass interfering with their growth. The following

media composition for A. Strictum was standardized for higher biomass and fatty acid

production.

129

Fig: 26 Effect of metallic salt elicitors on biomass production

130

Potato dextrose broth with yeast extract (PDB-YE) and with added salt elicitors

Potato - 20 g

Dextrose - 2 g

Ammonium tartarate - 0.1 g

Pottassium dihydrogen phosphate - 0.7 g

Magnesium sulphate - 0.15 g

Disodium hydrogen phosphate - 0.2 g

Yeast extract - 0.15 g

Calcium chloride - 0.01 g

Cobalt nitrate - 0.00001 g

Magnesium sulphate - 500 µg

Manganese sulphate - 500 µg

Copper sulphate - 500 µg

Zinc sulphate - 500 µg

Ferric chloride - 500 µg

Distilled water - 100 ml

pH - 6

5.3 Extraction of fatty acid

Total lipids were extracted from the biomass obtained according to the Bligh and Dyer

method (Bligh and Dyer, 1959) with little modifications by using hexane and acetone instead

of chloroform and methanol. The extracts obtained were subjected for TLC profiling.

5.4 TLC profiling

Using palm oil as standard the lipid extracts obtained were subjected for TLC profiling

on precoated silica gel plates with the solvent system diethyl ether: hexane: acetic acid

(4:13:0.4). After the run was complete the spots obtained were identified using potassium

permanganate. Fatty acid composition of palm oil constitutes: Palmitic acid, Stearic acid,

Oleic acid and triglycerides (Rf: 0.54, 0.41, 0.32 and 0.7). The TLC profile varied among the

extracts obtained from biomasses of mother culture and the media amended with different

metal ion salt concentrations that revealed nearly 3–4 spots with Rf values of 0.54, 0.41, 0.32

131

and 0.7 indicating the presence of Palmitic acid, Stearic acid, Oleic acid and triglycerides

compared with the palm oil standard (Fig: 27).

5.5 Estimation of free fatty acids

Free Fatty acid content is known as the acid number/acid value. Based on the methanolic

KOH titration method (Cox and Pearson, 1962) using the titre value the amount of free fatty

acids present in the different extracts was calculated. The results indicate that the extract

obtained from the control produced 36.3% of free fatty acids with an acid number of 72.2

(Fig: 28). Further the extract harvested from the biomass of culture amended with metal ion

salt concentration of 00 μg produced ig est a ount of .34% of free fatty acids it an

acid number of 116.10 compared to other cultures (Fig: 28).

Owing to increased biomass production, higher percentage of free fatty acids and the

acid value, the strain grown in PDB-YE amended with 500 µg salt concentration is considered

to be the improved strain of A. strictum and thereafter referred to as ASIS01 strain.

5.6 Preparation of FAMEs

The fatty acid extracted from the biomass harvest from the ASIS01 strain grown in

PDB-YE amended it etal ion salt concentration of 00 μg as subjected for t e

preparation of FAMEs. Once the FAMEs were prepared they were analyzed using GC-FID.

5.7 Gas Chromatographic conditions

The FAMEs prepared were subjected for gas chromatography using GC–FID that the

FAME profiling revealed the presence of 7 fatty acids from the improved strain of A. strictum

ASIS01 with high percentage of Oleic acid of 22.99 g/100 g. (Table. 14, Fig: 29 and 30).

Comparing the results of fatty acid profiling of the control strain with ASIS01 it revealed the

production of 6 fatty acids in common, yielding higher percentage of Oleic acid of 22.99

g/100 g followed by pentadecenoic acid of 19.27 g/100 g of fat (Fig: 30). This reveals a 10

fold increase in the production of Oleic acid in the strain improved. Further the overall

percentage of total monounsaturated fat reveals 80% increase of 87.86 g/100g and

Polyunsaturated fat reveals 12% increase of 12.14 g/100 g in improved strain though the fatty

acid profile shows comparative qualitative difference in producing the following six

132

* OA – Oleic Acid

Fig: 27 TLC profiling of fatty acids from media containing different metallic

salt elicitor concentrations

S A B C S D E F S G H I S J K L

S M N O P Q S R T U V W X Y

Lane S – Palm oil Std.

Lane A – CNTRL

Lane B – 500 µg

Lane C – 5 mg

Lane D – 100 µg

Lane E – 1 mg

Lane F – 10 mg

Lane G – 25 µg

Lane H – 50 µg

Lane I – 75 µg

Lane J – 1 µg

Lane K – 5 µg

Lane L – 10 µg

Lane S – Palm oil Std.

Lane M – 15 mg

Lane N – 20 mg

Lane O – 25 mg

Lane P – 50 mg

Lane Q – 75 mg

Lane R – 100 mg

Lane T – 125 mg

Lane U – 150 mg

Lane V – 200 mg

Lane W – 300 mg

Lane X – 400 mg

Lane Y – 500 mg

OA

133

Fig: 28 Effect of salt concentration on percentage of free fatty acids and acid value

134

Fig: 29 GC chromatogram showing fatty acid profile of A. strictum ASIS01

135

136

Fig: 30 Fatty acid Profile of A. strictum (Control and ASIS01)

Table. 14 Fatty Acid Profile of A. strictum-ASIS01

S.No. Fatty acid Yield (g)/100 g

1 Caprylic acid 0

2 Lauric acid 0

3 Tridecanoic acid 0

4 Myristoleic acid 14.15

5 Pentadecanoic acid 19.27

6 Palmitic acid 0

7 Palmetoleic acid 17.94

8 Heptadecenoic acid 13.51

9 Elaidic acid 0

10 Oleic acid 22.99

11 Linoelaidic acid 0

12 Linoleic acid 12.14

13 Tricosanoic acid 0

14 Lignoceric acid 0

15 Saturated fat 0

16 Mono unsaturated fat 87.86

17 Poly unsaturated fat 12.14

18 Transfat 0

137

fatty acids: Myristoleic acid, Pentadecenoic acid, Heptadecenoic acid, Palmetoleic acid,

Oleic acid and Linoleic acid.

6. PURIFICATION OF OLEIC ACID

Fractions were collected via HPLC that the methyl ester of Oleic acid was eluted at

13.7 min and thus purified by HPLC under the conditions as described in materials and

methods (Fig: 31). The fraction that contained Oleic acid was thus collected and

concentrated. Following comparisons with the standard via GC-FID, the purified oleic acid

was checked further for its biological activity.

7. ANTICANCER STUDY OF OLEIC ACID AGAINST MCF-7 AND HEP-

G2 CELL LINES

The effect of Oleic acid on the viability of human breast cancer (MCF-7) and liver

cancer (Hep-G2) cell lines was evaluated to investigate the cytotoxicity and antiproliferative

activity. MTT assay, a sensitive simple and reliable practice which measure cell viability was

used to evaluate the cytotoxicity and antiproliferative activity of the Oleic acid. It induced a

dose dependent inhibitory effect against the cell lines tested. IC50, which represent the

concentration of oleic acid inhibiting 50% of the cells are presented in Table 15 and 16. The

IC50 values of Oleic acid were found to be 1.25 mg/ml against MCF-7 and 6 . μg/ l

against Hep–G2 cell lines respectively (Tab. 15 and 16, Fig: 32, 33, 34, 35 and 36).

8. ANTIOXIDANT ACTIVITY OF OLEIC ACID USING DPPH ASSAY

The Radical scavenging activity of Oleic acid fraction was determined using the α,α-

diphenyl-β-picrylhydrazyl (DPPH) assay. Free radical scavenging capacity of the Oleic acid

was noted to be increased in a concentration dependent manner. In the DPPH assay, the

ability of the examined fatty acid to act as donor of hydrogen atoms or electrons in

transformation of DPPH into its reduced form DPPH-H was investigated. The sample was

able to reduce the stable, purple-colored radical DPPH into yellow-colored DPPH-H. At a

concentration of 200 µg/ml the scavenging activity of Oleic acid reached 51.2%, which was

comparable to that of reference standard BHT (Table. 17, Fig: 37 and 38).

138

Fig: 31 HPLC chromatogram for the purification of Oleic Acid (OA)

OA

139

Table. 15 Anticancer activity of Oleic acid against MCF-7 cell lines

S.No Concentration

(mg/ml) Dilutions

Absorbance

(O.D)

Cell viability

(%)

1 10 Neat 0.04 10.52

2 5 1:1 0.1 26.31

3 2.5 1:2 0.16 42.1

4 1.25 1:4 0.19 50

5 0.625 1:8 0.24 63.15

6 0.312 1:16 0.28 73.68

7 0.156 1:32 0.33 86.84

8 Cell control - 0.38 100

Table. 16 Anticancer activity of Oleic acid against Hep-G2 cell lines

S.No Concentration (µg/ml) Dilutions Absorbance

(O.D)

Cell viability

(%)

1 1000 Neat 0.06 13

2 500 1:1 0.1 21.7

3 250 1:2 0.16 34.7

4 125 1:4 0.2 43.4

5 62.5 1:8 0.23 50

6 31.2 1:16 0.28 60.8

7 15.6 1:32 0.31 67.3

8 Cell Control - 0.46 100

140

Normal Vero cell line Cytotoxicity of Oleic acid

Fig: 32 Cytotoxicity effect of Oleic acid on Vero cell lines

141

Fig: 33 Anticancer activity of Oleic acid against MCF-7 cell lines

Normal MCF-7cell line Toxicity- 10 mg/ml Toxicity- 1.25 mg/ml

Toxicity- 0.625 mg/ml Toxicity- 0.312 mg/ml

Fig: 34 Anticancer activity of Oleic acid against MCF-7 cell lines

142

Fig: 35 Anticancer activity of Oleic acid against Hep-G2 cell lines

Normal Hep-G2 cell line Toxicity- 1000 µg/ml Toxicity- 125 µg/ml

Toxicity- 62.5 µg/ml Toxicity- 31.2 µg/ml

Fig: 36 Anticancer activity of Oleic acid against Hep-G2 cell lines

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Table. 17 Antioxidant activity of Oleic acid using DPPH assay

S.No Concentration

(µg)

Sample

(µl)

DMSO

(µl)

DPPH

(ml)

Absorbance

(O.D)

(%) of

test

(%) of

BHT Std

1 0 0 0 0 0.614 0 0

2 20 2 38 2.96 0.596 6.5 12

3 40 4 36 2.96 0.581 9.2 18

4 60 6 34 2.96 0.577 11.3 22

5 80 8 32 2.96 0.576 12.1 27

6 100 10 30 2.96 0.555 13.3 34

7 120 12 28 2.96 0.528 15.4 41

8 140 14 26 2.96 0.494 19.6 45

9 160 16 24 2.96 0.415 24.8 50

10 180 18 22 2.96 0.336 36.8 53

11 200 20 20 2.96 0.656 51.2 57

144

Fig: 37 Antioxidant activity of Oleic acid using DPPH assay

Fig: 38 Antioxidant activity of Oleic acid using DPPH assay

145

9. APOPTOTIC DNA FRAGMENTATION ASSAY

The cell death by DNA fragmentation was examined by conducting agarose gel

electrophoresis of DNA samples isolated from Hep – G2 and MCF – 7 cell lines treated

with Oleic acid sample. Healthy control cells maintained in culture medium alone did not

show DNA fragmentation. However the DNA isolated from Oleic acid treated cells

revealed DNA ladders consisting of fragments when stained with ethidium bromide.

Consistent with the MTT assay the cell lines treated with higher concentrations of Oleic

acid showed DNA smearing (slight DNA degradation) that the IC50 concentration of Oleic

acid - 1. g/ l and 6 . μg/ l as opti u enoug to induce frag entation in MCF –

7 and Hep – G2 cell lines respectively (Fig: 39 and 40).

10. MOLECULAR STUDY USING PCR

Initial olecular studies ere done to isolate t e δ-9 fatty acid desaturase gene

mediating Oleic acid production from A. strictum using PCR amplification technique.

10.1 Isolation of Genomic DNA

Genomic DNA was isolated from pure cultures of A. strictum – mother culture and

the improved strain ASIS01 using lysis buffer and the samples were electrophoresed in

0.8% agarose gel. The DNA bands were visualized using a UV transilluminator and the

images were captured using Gel documentation system. Intact fungal DNA bands were

isolated from the mother culture and the improved strain ASIS01 of A. strictum is shown in

Fig: 41, later e ployed for PCR a plification of δ -9 fatty acid desaturase gene.

10.2 Amplification using PCR

T o sets of degenerati e oligonucleotide pri ers ere generated for δ -9 fatty acid

desaturase gene responsible for Oleic acid production. Following PCR the products were

electrophoresed in 1% agarose gel and the amplified bands were observed after Ethidium

bromide staining. The bands were visualized using a UV transilluminator and the images

were captured using Gel documentation system. The amplification resulted in generation of

multiple bands indicative of binding of degenerative primers with the DNA apart from the

specified locus. A plification using first set of pri ers (δ -9, FAS Primer 1) revealed 3

146

Lane 1: 1 kb ladder Lane 2: 1 kb ladder

Lane 2: 10 mg/ml Lane 3: 1000 µg/ml

Lane 3: 1.25 mg/ml Lane 4: 125 µg/ml

Lane 4: 0.625 mg/ml Lane 5: 62.5 µg/ml

Lane 5: 0.312 mg/ml Lane 6: 31.2 µg/ml

Fig: 39 and 40 Apoptotic DNA fragmentation assay in MCF-7 cell lines and

Hep-G2 – cell lines

1 2 3 4 5 1 2 3 4 5 6 7

147

bands of nearly 350 bp, 470 bp and 600 bp in mother culture (Lane S1) and 4 bands of

nearly 350 bp, 470 bp, 560 bp and 650 bp (Lane S2) in improved strain when compared

with the molecular ladder respectively (Fig: 42, S1 and S2). Amplification using second set

of pri ers (δ -9, FAS Primer 2) revealed 2 bands of nearly 680 bp and 850 bp (Lane 1A) in

mother culture and 4 bands of nearly 590 bp, 810 bp and 900 bp (Lane 2A) in improved

strain when compared with the molecular ladder, respectively (Fig: 43, 1A and 2A).

A ong t e t o sets of pri ers t e first set of pri er (δ -9, FAS Primer 1), produced the

amplified products of expected size of approx. 469 bp and therefore can further be

employed in future for PCR optimization and gene sequencing studies.

148

Fig: 41 Isolation of Genomic DNA from A. strictum

Lane MW: 1000 bp ladder Lane MW: 1000 bp ladder

Lane 1A: PCR bands – mother culture Lane 1A: PCR bands – mother culture

Lane 2A: PCR bands – ASIS01 Lane 2A: PCR bands – ASIS01

Fig: 42 Amplification of δ -9 fatty acid Fig: 43 Amplification of δ -9 fatty acid

desaturase using FAS Primer 1 desaturase using FAS Primer 2

Genomic DNA of

A. strictum

Molecular

Marker