118

6

SUMMARY AND CONCLUSION

• The study deals with a rapid and reproducible protocol for the

micropropagation of selected explants (nodal and shoot tip) of A. javanica and

A. lanata by direct and indirect organogenesis.

• Optimal regeneration was achieved in MS medium containing 0.5 mg/L SPM

hormone (A. javanica) and 0.5 + 0.5 mg/L BAP + KIN (A. lanata). In vitro

flowering was obtained in the same hormonal concentration.

• Somatic embryos were developed in the 2, 4 D + SPM at 2.0 + 0.5 mg/L. The

embryos were transformed into mature plantlets or sprouts in 1.0 + 0.5 mg/L

BAP + GA3 (A. javanica) and 1.0 + 0.5 mg/L BAP + TDZ (A. lanata)

• Higher concentrations of IBA + IAA (1.0 + 1.0 mg/L) produced maximum

root length and more roots in A. javanica and A. lanata was produced highest

number of roots at 2.0 mg/L IBA.

• The rate of successful acclimatization was 90% for both A. javanica and

A. lanata species.

• About 113 and 126 scorable bands (using four random primers) were observed

in the PCR amplified DNA of A. javanica, and A. lanata.

• About 98 monomorphic and 15 polymorphic bands were reported in A.

javanica with lowest percentage of polymorphic variations (13.27 % in all

samples of in vivo, in vitro leaf and callus).

• Percentage of polymorphic changes in the samples of A. lanata were 10.56 %

(110 bands were monomorphic and 13 were polymorphic bands).

119

• Jaccard’s similarity and distance coefficient values have shown similarity of

1.000 and distance of 0 scales among the samples.

• Based on the results of monomorphic bands occurred in the DNA samples, the

uniform distribution of secondary metabolites contents (for in vivo, in vitro

leaf and callus materials) is ensured.

• Little/few polymorphic variations occurred in gDNA within the samples

indicates the improved secondary metabolites contents in plants.

• The antimicrobial activity of A. javanica showed that most of the extracts

exhibited broad spectrum (higher to moderate range) of inhibition against the

tested bacterial and fungal pathogens.

• Highest inhibition was observed in the callus and in vitro leaf methanolic

extracts against S. aureus, E. faecalis, B. subtilis, and S. typhi followed by

chloroform, acetone and ethyl acetate extracts. A. niger and Fusarium was

found to be inhibited by the extracts tested.

• Callus and in vitro leaf extracts (ethyl acetate, methanol, and chloroform

solvents) of A. lanata was able to inhibit the tested pathogens i.e. S. aureus,

S. typhi, B. subtilis, E. coli, S. boydii, and P. vulgaris. Similarly, all the

extracts were found to have some inhibitory effects against the fungus namely

C. albicans and A. niger.

• The MIC values of methanolic extract of plant samples were found to be in the

range of 18 – 22 µg/mL (in vivo extracts), 15 – 22 µg/mL (in vitro extracts)

and 16 – 24 µg/mL (callus extracts) against the tested pathogens.

• MIC of methanolic extracts of A. lanata was reported in the range of 19 – 28

µg/mL (in vivo), 21 – 25 µg/mL (in vitro) and 12 – 17 µg/mL (callus). Colony

120

count of MBC plate from the respective MIC tubes also show clear or

negligible amount of colonies.

• Fractions separated from A. javanica and A. lanata by TLC was tested against

both Gram positive and negative organisms, (S. aureus and S. typhi). Fractions

of AJ-IL-F1, AJ-FL-F1, AJ-CA-F1 were found to inhibit the growth of

organisms.

• The qualitative phytochemical analysis of results shows the presence of

flavonoids, phenolic contents, tannins and carbohydrates in all the extracts of

tested plants. Alkaloids, saponins, terpenoids and fats and oils were found to

be present in most of the extracts of both plants.

• The methanol and aqueous extracts of A. javanica were reported to contain

higher amount of phenolics (196.68 ± 10.45 mg GAE/g), tannins (116.08 ±

3.71 mg GAE/g), flavonoids (177.54 ± 1.8 mg RE/g) and carbohydrates (139.0

± 5.1 mg GE/g).

• The extracts of A. lanata extracts was reported to contain higher amount of

phenols (193.98 ± 10.58 mg GAE/g), tannins (109.41 ± 9.42 mg GAE/g),

flavonoids (151.45 ± 6.97 mg RE/g) and carbohydrates (58.60 ± 3.6 mg GE/g)

from in vitro leaf extracts followed by in vivo and callus extracts.

• Antioxidant activity was tested by different assays. The DPPH assay results

showed lower IC50 values (87.02, 113.45, 71.54 µg/mL) which were recorded

in the methanol extracts (in vivo, in vitro leaf and callus) followed by acetone

and aqueous extracts in A. javanica.

121

• The antioxidant potential of A. lanata recorded least IC50 values (53.77 and

64.42 µg/mL) from in vivo and in vitro leaf methanol extracts followed by

aqueous extracts (DPPH assay).

• The total antioxidant potential of A. javanica was tested by

phosphomolybdenum, ferric reducing power and metal chelation assay. It was

observed that the methanolic extracts possessed higher antioxidant potential

followed by aqueous and ethyl acetate extracts.

• The in vitro leaf has higher antioxidant content based on FRAP and

phosphomolybdenum assay. In vivo leaf extracts has higher antioxidant

potential in metal chelation assay. Similarly, callus extracts also possessed

equal amount of total antioxidant content when compared with in vivo and in

vitro plants.

• Total antioxidant activity of A. lanata was tested by FRAP,

phosphomolybdenum and metal chelation assay which showed higher activity

in methanol extracts (in vitro and in vivo leaf extracts and callus extracts). Rest

of the solvent extracts (in vivo, in vitro leaf and callus) showed considerable

activity. Among them, the in vitro leaf and callus extracts were found to have

promising antioxidant property.

• The methanolic extract having higher antioxidant potential (by in vivo, in vitro

leaf and callus extracts) was further tested for anticancer activity on MCF – 7

cell line (cytotoxic potential) which showed better anticancer activity than the

commercial compound (5 - FU) with least IC50 values (AJ-FL = 114.43,

AJ-IL = 11.89, AJ-CA = 27.18 µg/mL) from A. javanica. The IC50 values of

A. lanata was recorded as AL-FL = 43.89, AL-IL = 167.77, and AL-CA =

122

22.45 µg/mL. Further DNA fragmentation assay results showed the digestion

of DNA damaged during the process of apoptosis.

• Preparative TLC of in vivo, in vitro leaf and callus methanol extracts produced

6 fractions (in vivo – 03, in vitro – 02, and callus 01) in A. javanica and

9 fractions (4 - in vivo leaf, 3 - in vitro leaf and 2 - callus) from A. lanata

extracts.

• Based on the Rf value, fractions separated from the extracts is found to belong

to the polyphenolic group of compounds.

• The fractions also confirmed the presence of phenolic and flavonoids

compounds as interpreted by the peaks obtained in the HPLC chromatogram

along with the standards (gallic acid, epicatechin, catechin, quercetin, vanillin,

chlorogenic acid, ferulic acid and resveratrol). Contents of the fractions were

quantified and expressed as the amount of standard equivalents.

• During UV spectral analysis, the absorbance peak was noticed at 248 nm (for

in vivo fraction) and 264 nm (for in vitro and callus fractions) for A. javanica.

• A. lanata also showed the highest absorbance peak at 283 nm (in vivo

fraction), 240 nm (in vitro fractions) and 268 nm (callus fraction) by UV

spectral analysis.

• GC – MS analysis of A. javanica showed 19 compounds from in vivo leaf and

9 from in vitro and callus extracts. About 16 compounds were identified (GC –

MS) in A. lanata, in vivo leaf and 6 compounds from in vitro and callus

extracts.

• The chemical library analysis results showed that the stigmasterol compound

(a polyphenolic group) was found in all the extracts and Methyl-methyl

123

deconate was the abundant compound present in the tested extracts, followed

by Hentriacontane, a major compound present in both plant extracts.

• Functional groups of the compounds were found and the presence of

C-H, C=O, O-N, -NH3, stretching, deformation and bending’s along with

aldehyde, ketone, amide and peptide linkages were found in the

identified compounds of A. javanica and A. lanata. Similar type of functional

group and chemical bonds were also reported in isolated fractions from both

plants.

• Based on the mass spectral analysis (by LC – MS) for the isolated

fractions, the molecular weight as follows (AJ FL F1 – 313.23, AJ IL F1 –

251.25, AJ CA F1 – 299.23, AL FL F1 – 343.22, AL IL F1 – 251.25, and

AL CA F1 – 439.18 kDa) from A. javanica and A. lanata respectively is

recorded.

• In a nutshell it is stated that micropropagation of A. javanica, anticancer

activity of in vivo, in vitro leaf and callus materials of both plants (A. javanica

and A. lanata) against human breast cancer cell line and LC – MS

spectral analysis of the fractions of both plants are reported for the first time.

• Based on the outcome of this study, it is confirmed that, the in vitro

leaf and callus materials from both the test plants can be efficiently used

for mass production of pharmaceutical drugs compared to the in vivo test

plant.

124

SUGGESTIONS AND FUTURE RECOMMENDATIONS

• Purification and in vivo trails (with animal model) for the isolated fractions.

• Analysis of Nuclear Magnetic Resonance of the compounds for

identification of its molecular structure i.e., elemental analysis.

• Molecular docking of the isolated compounds for prediction with

commercial compounds.

• Pharmacological aspects of isolated compounds.

with (Soliman, infections, swelling (Garg et al., 1975) 5-methylmellein

matted 2006) 1980; Qasim et al., 2013). (Claydon et al., 1985),

hairs Leaves Kaempferol-3-O-β-D-

Aerva javanica (Burm.f.) Juss. (Khare, Ulcer in animals, cattle fodder glucopyranosyl-(1→2)-α-

ex Schult. 2007) (Qureshi and Bhatti, 2009) L-rhamnopyranoside-7-O-

Seeds α-L-rhamnopyranoside

1.1. Description of plants

1.9.1. Description of Aerva javanica

Plant name/ Taxonomic

Botanical description Ethnobotanical/ pharmacological

position Habit Leaves Flowers aspects Phytochemical nature

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Caryophyllales

Family : Amaranthaceae

Genus : Aerva

Species : javanica (Burm. f.)

Juss. ex Schult

Perennial

herb,

Grows

up to

1.6m

(Khare,

2007)

Pale green,

20 – 40mm

long,

Alternate,

Lanceolate,

Shortly

petiolate,

Covered

White woody,

Raceme

inflorescence,

Flowering

period

January to

May

Roots and flowers

Rheumatism, Kidney problems,

tooth ache, wound bleeding, control

eye disease, (Mossa et al., 1987;

Abbas et al., 1992; Ghazanfar,

1994)

Whole plant

Dysentery, gonorrhoea, cutaneous Head ache, rheumatism (Srivivan

and Reddy, 2008)

Pharmacological properties

Antiplasmodial,

(Joanofarc and Vamsadhara, 2003;

Cytogentical and cytotoxic

(Soliman, 2006; Fatimi, 2007),

antihyperglycaemic (Reddy and

Reddy, 2009), antidiarrhoeal

properties (Ahmed et al., 2010)

Antimicrobial (Sharif et al., 2011).

Flavonoids,

steroids, triterpenoids and

carbohydrates (Reddy

and Reddy 2009; Sharif et

al., 2011)

Isolated compounds

Apigenin 7-O-glucuronide

(Kenneth and Lawrence (Kucukislamoglu et al.,

2000), Isoquercetrin

(Guvenalp, and Demirezer,

2005), 7-(1’hydroxyethyl)-

2-(2″-hydroxyethyl)-3,4-

dihydrobenzopyran

(Donnell et al., 2006),

2-hydroxy-3-O-β-

primeveroside naphthalene-

1,4-dione (Ozgen et al.,

2009),

1.9.2. Description of Aerva lanata Botanical description Ethno botanical/ pharmacological

Plant name/ Taxonomic position

Habit Leaves Flowers aspects Phytochemical nature

Aerva lanata (L.) A. L. Juss. ex

Schultes

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Caryophyllales

Family : Amaranthaceae

Genus : Aerva

Species : lanata (L.) A. L. Juss. ex

Schultes

Prostrate,

decumbent,

sometimes

erect herb,

30-60 cm in

height,

woolly

tomentose

(Khare 2007;

Rajesh et al.,

2011)

Leaves are

simple,

alternate

and short,

petiolated,

densely

tomentose

Small and

hairy

white

colour,

sessile,

bisexual,

clustered

spikes

(Rajesh et

al., 2011)

Whole plant

Cough, sore throat,

wounds, cataract of

bladder, animal

constipation nasal

bleeding, fractures,

spermatohorrhoea,

cough, scorpion stings

(Mukerjee et al., 1984;

Sikarwar and Kaushik,

1993; Girach et al.,

1994), burning sensation

during urination

(Venkataramana, 2008).

Flowers and Roots

Headache, scabies,

jaundice, diarrhoea and

kidney disorders (Bedi

and Patel, 1978; Muthu

et al., 2006;

Soundararajan et al.,

2006; Khare, 2007).

Alkaloids, flavonoid, steroid,

terpenoids, phenolic compounds,

tannins and carbohydrates (Aiyar

et al., 1973; Chandra and Sastry

1990)

Isolated compounds

Alkaloids

Canthin-6-one beta-carboline,

aervine (10-hydroxycanthin- 6-

one), methylaervine (10-

methoxycanthin-6-one),

aervoside (10-β-

dglucopyranosyloxycanthin-6-

one) and aervolanine (3-(6-

methyoxy-β-carbolin-1-yl)

propionic acid) (Zapesochnaya,

1992),

Contd...

Leaves

Eye complaints, diarrhoea,

treatment against guinea worm

Roots

Snake bite, hepatitis, urinary

strangury (Yamunadevi et al.,

2011).

Pharmacological properties

Diuretic (Chopra et al., 1956;

lithiatic, urolithiasis (Rao,

1985), Udupihille and Jiffry,

1986), antitumor activities

(Zapesochnaya et al., 1992).

Diabetes, anthelmintic,

demulcent lithiasis (Kirtikar

and Basu, 1996), anti-

inflammatory (Vetrichelavan

et al., 2000), antimicrobial and

cytotoxic (Dulay, 2002)

hepatoprotective and nephro

protective (Shirwaikar et al.,

2004).

Glycosides

Kaempferol 3 –

rahamnogalactoside,

kaempferol 3 – 6” p –

coumaryl O – glucoside

(Mallabev et al., 1989)

Flavonoids

Tiliroside, coumaryl

tiliroside, isorhamnetin

(Mallabev et al., 1989).

Table 16: Shooting and in vitro flowering response of A. javanica in different concentrations of cytokinins and polyamines in direct

and indirect organogenesis

Node explants Regenerat Shoot tip explants Regenerat In vitro

Hormone

Concentration

(mg/L)

Shoot length

(cm)

No. of Multiple

shoot-1 explant

ion rate

(%) Shoot length

(cm)

No. of Multiple

shoot-1 explant

ion rate

(%)

Flowerin

g (%)

Basal 0 - - - - - - -

BAP 0.5 2.15 ± 0.36ab 3.0 ± 1.06 ab 57.14 2.27 ± 0.51 ab 2.14 ± 0.91 ab 92.8 -

1.0*

4.80 ± 0.92abc

5.71 ± 1.03abc

100 3.67 ± 0.61 ab

4.57 ± 2.09abc

92.85 -

1.5 2.83 ± 0.89a

2.78 ± 1.08a

85.71 2.83 ± 0.24a

2.50 ± 0.50a

92.85 -

2.0 1.30 ± 0.14cd

2.14 ± 0.14cd

85.71 1.51 ± 0.18cd

2.35 ± 0.61cd

82.71 -

BAP + KIN 0.5 + 0.5 1.95 ± 0.42cd 1.42 ± 0.49cd 35.17 1.51 ± 0.47cd 0.42 ± 0.49e 42.85 -

1.0 + 0.5 2.38 ± 0.21a 1.92 ± 0.96cd 35.17 2.47 ± 0.41ac 1.0 ± 0e 57.17 -

1.5 + 0.5 1.22 ± 0.15cd

2.28 ± 1.09ac

85.71 1.08 ± 0.46cd

0.14 ± 0.51e

85.75 -

2.0 + 0.5 1.24 ± 0.14cd

1.92 ± 1.16cd

85.71 1.53 ± 0.28cd

3.35 ± 1.44 85.71 -

2.5 + 1.0 2.50 ± 0.37a

2.35 ± 1.39ac

78.57 2.60 ± 0.25 ac

3.0 ± 0 ab

71.42 -

3.0 + 1.0 1.50 ± 0.70cd 1.78 ± 0.67cd 85.71 1.65 ± 0.23cd 2.42 ± 0.62ab 92.85 -

4.0 + 0.5 2.25 ± 0.13a 1.57 ± 0.49cd 85.71 2.49 ± 0.15ac 2.49 ± 0.15ab 92.85 -

BAP + SPM 1.0 + 0.5 3.81 ± 1.04ab

2.0 ± 0.65a

92.85 3.57 ± 0.15ab

3.0 ± 0ab

78.57 -

SPM 0.3#

2.76 ± 1.59a

2.42 ± 1.67ab

92.85 4.16 ± 0.54abc

3.0 ± 0ab

100 -

0.4 3.59 ± 1.22ab

3.42 ± 0.90abc

85.71 2.75 ± 0.16a

3.0 ± 0ac

85.71 -

0.5# 3.70 ± 1.77ab 3.0 ± 1.77ac 85.71 4.23 ± 0.27abc 3.57 ± 1.04ab 100 57.14

BAP + GA3^ 1.0 + 0.2 3.41 ± 1.04ab 3.81 ± 1.03ab 92.85 3.14 ± 0.55ac 2.82 ± 1.40ac 64.2 -

1.0 + 0.5 3.38 ± 0.37ab

3.2 ± 0.71ab

85.71 3.0 ± 0.19ac

2.70 ± 0.60c

64.2 -

BAP + TDZ^

1.0 + 0.2 3.40 ± 0.71ab

3.0 ± 0.10ac

78.57 3.0 ± 0.40ab

3.84 ± 1.28ab

85.71 -

1.0 + 0.5 3.01 ± 0.54ab 3.1 ± 1.06ac 78.57 3.0 ± 0.40ab 3.74 ± 1.81ab 85.71 -

Values in the table are the mean ± SE of triplicates, n=14 explants, Superscript in the each row carrying different letters are

significantly different at P<0.05 or 0.01, *-Significance at 0.05, # - Significance at 0.01 by Newman-Keuls Multiple Comparison Test.

^ - hormones used in shoot elongation of indirect organogenesis.

Figure 3: In vitro regeneration and embryogenesis of A. javanica in different concentration of cytokinins and spermidine, A. Regenerated

shoot tip explant, B. nodal explant, C. callus regeneration, D. multiple shooting, E. in vitro flowering, F. shoot elongation, G. in vitro roots,

H. matured plants (hardened).

Rate of callus Callus morphology

formation

Rate of

embryo

Frequency of

embryos

No. of shoots/embryo

cultures

++ Pale brown fragile - - -

++ Pale brown fragile - - -

++ Pale yellow fragile - - -

+++ Pale yellow fragile - - -

++ Yellow with pinkish

soft

+

10.33 ± 0.47ab

-

++ Pale yellow green soft + 11.33 ± 0.47ab

-

++++ Yellow green with -

pinkish soft +++ 88.40 ± 2.62cd

Table 17: Embryogenesis and indirect organogenesis from embryos of A. javanica callus

Hormone Concentrations

2, 4 - D 0.5

1.0

2.0

3.0

2, 4 - D +

NAA 2.0 + 0.5

2.0 + 1.0

2, 4 - D + SPM 2.0 + 0.5*

2, 4 – D +

ABA 0.5 + 0.2

* +++

Pale yellow green hard

++ 55.47 ± 1.04d

-

cd ab

0.5 + 0.5* +++ Pale yellow green with

pinkish hard

+++ 88.46 ± 2.62 1.2 ± 0.47

2, 4 - D + BAP # ++++ +++ 88.40 ± 2.62

cd 13.8 ± 1.20

a

+ SPM 2.0 + 2.0 + 0.5

Green

+ rate of callus and embryo development, - no embryo or shoot development. Values in the table are the mean ± SE of triplicates, n=14

explants, Superscript in the each row carrying different letters are significantly different at P<0.05 or 0.01, *-Significance at 0.05,

# - Significance at 0.01 by Newman-Keuls Multiple Comparison Test

Figure 4a: Embryogenesis of A. javanica at different concentrations of auxin and

spermidine, A. Greenish brown callus, B. embryo forming callus, C. matured

embryos, D. regeneration from embryo cells, E. sprouts/plantlets F. development of

multiple sprouts.

Figure 4b: Nature of different forms of callus in A. javanica, A. pale yellow green

callus, B. brown callus, C. yellow callus, D. greenish callus (Non embryo forming

cells)

Figure 6: In vitro regeneration and embryogenesis of A. lanata, A. regeneration of nodal explants, B. shoot tip explants, C. multiple

shooting, D. shoot elongation, E. in vitro flowering, F. in vitro rooting, G. acclimatized plants.

Table 19: Shooting and in vitro flowering response of A. lanata different concentrations of cytokinins and polyamines in direct and

indirect organogenesis

Hormones

Concentration

of hormones

L Shoot tip explants

Regeneration

Node explants Regener

ation

In vitro

Flowering

(mg/L) Shoot length

(cm)

No. of Multiple shoot

-1 explant

rate (%) Shoot length (cm)

No. of Multiple shoot

-1 explant

rate (%) (%)

Basal MS basal - - - - -

BAP 0.5 2.72 ± 0.41ab

3.10 ± 0.32bc

64.2 1.21 ± 0.31bc

1.33 ± 0.31bc

46.8 -

0.75 4.50 ± 0abc

3.94 ± 0.18b

64.2 1.25 ± 0.40bc

3.9 ± 0.10b

57.1 -

1.0* 5.33 ± 0.41abc

5.76 ± 0.91abc 85.7 4.83 ± 1.4abc

3.98 ± 1.19b 92.8 78.5

1.5 3.20 ± 0.55b 3.42 ± 0.6b

78.5 3.12 ± 1.4bc 1.24 ± 0.6bc

85.7 -

2.0 4.0 ± 0.63abc 4.0 ± 0.6ab

71.4 3.40 ± 1.2bc 2.81 ± 1.41bc

78.5 36.7

BAP + KIN 0.5 + 0.5# 5.76 ± 0.85

abc 4.91 ± 1.42

abc 92.8 4.93 ±0.84

abc 5.81 ± 0.1

abc 100 92.8

0.5 +0.75 2.72 ± 0.34ab

2.62 ± 0.11b

64.2 1.77 ± 0.33bc

3.26 ± 0.61bc

78.5 -

0.5 + 1.0 3.61 ± 0.24b 3.52 ± 0.6b

71.4 2.81 ± 0.22b 3.05 ± 0.14b

78.5 -

1.0 + 1.0 3.54 ± 0.61b 3.52 ± 0.6b

78.5 3.35 ± 2.51b 3.0 ± 0b

71.4 -

BAP + SPM 1.0 + 1.0 2.21 ± 1.04ab 2.0 ± 1.51ab

92.8 2.29 ± 1.5c 2.40 ± 0.47b

85.7 -

SPM 0.3 2.0 ± 0ab

2.42 ± 0.61ab

78.4 2.16 ± 3.58c 1.10 ± 0.21

bc 54.7 -

0.4 1.59 ± 1.53bc

1.28 ± 1.96bc

54.7 1.50 ± 2.16bc

1.8 ± 0bc

54.7 -

0.5 2.70 ± 1.27ab 2.90 ± 1.21ab

64.2 2.32 ± 2.71c 2.25 ± 0.44b

64.2 -

BAP + GA3^ 1.0 + 0.2 2.11 ± 2.0ab 2.81 ± 1.0ab

71.4 2.14 ± 1.55abc 2.32 ± 0.40bc

64.2 78.4

1.0 + 0.5* 2.33 ± 0.3

ab 2.42 ± 0.7

ab 64.2 1.96 ± 1.19

bc 1.75 ± 1.60

bc 64.2 -

BAP + TDZ^ 1.0 + 0.2 2.10 ± 1.71ab

2.20 ± 0.61ab

57.1 2.92 ± 0.65abc 2.84 ± 1.28

ac 64.2 -

1.0 + 0.5*

2.51 ± 1.54ab

2.81 ± 1.46ab

85.7 2.87 ± 1.40abc

2.74 ± 1.81ac

85.7 71.4

Values in the table are the mean ± SE of triplicates, n=14 explants, Superscript in the each row carrying different letters are

significantly different at P<0.05 or 0.01, *-Significance at 0.05, # - Significance at 0.01 by Newman-Keuls Multiple Comparison Test

^ - hormones used in shoot elongation of indirect organogenesis

Figure 7: Embryogenesis of A. lanata in different concentration of auxins and spermidine A. Embryo forming callus, B. greenish embryo

cells, C. yellow brown embryo cells, D. yellow embryo cells, E. initiation of sprouts from yellow brown embryo cells, F. sprout maturation.

abc

Table 20: Embryogenesis and indirect organogenesis from A. lanata No. of

Hormone Concentrations Rate of callus

formation Callus morphology

Rate of

embryo

Frequency of embryos

shoots/embryo

cultures

2, 4 – D 0.5 +++ Green soft - - -

1.0 +++ Greenish yellow soft - - -

1.5 +++ Yellow hard - - -

2.0 +++ Pale yellow soft - - -

2, 4 – D + NAA 0.5 + 0.5 ++ Pale yellow soft - - -

0.5 + 1.0 ++ Pale yellow soft - - -

0.5 + 1.5 +++ Pale yellow green hard - - -

0.5 + 2.0 ++++ Pale yellow green hard - - -

2, 4 - D + SPM 2.0 + 0.5*

+++

Yellow green with pinkish -

soft +++ 88.40 ± 2.62abc

ab

2,4 – D + ABA 0.5 + 0.2 +++ Pale yellow green hard ++ 45.74 ± 1.94 -

0.5 + 0.5#

++++ Pale yellow green with

pinkish hard

+++ 87.56 ± 1.28 -

2,4 – D + BAP + # +++ 92.40 ± 1.62

abc 58.82 ± 2.20

ab

SPM 2.0 + 2.0 + 0.5

++++ Green

+ rate of callus and embryo development, - no embryo or shoot development. Values in the table are the mean ± SE of triplicates, n=14

explants, Superscript in the each row carrying different letters are significantly different at P<0.05 or 0.01, * - Significance at 0.05,

# - Significance at 0.01 by Newman-Keuls Multiple Comparison Test

Table 21: In vitro rooting of A. lanata in different concentrations of auxins

Hormones

Concentration

of hormones

(mg/L)

Mean of root

length (cm)

Number of

multiple root/

stem

Rate of roots

formation (%)

IBA 0.5 2.7 ± 0.6c

2.5 ± 0.7c

50

1.0*

3.4 ± 0.9abc

3.3 ± 0.9ab

64.2

1.5 4.8 ± 1.1bc

5.0 ± 1.2abc

64.2

2.0#

6.8 ± 1.4abc

7.6 ± 0.4abc

100

IBA + IAA 0.5 + 0.5 4.1 ± 0.7bc

3.8 ± 0.6bc

64.2

0.5 + 1.0*

3.3 ± 0.5abc

2.9 ± 0.7bc

42.8

0.5 + 1.5*

3.2 ± 0.3abc

2.7 ± 0.5c

42.8

0.5 + 2.0*

3.2 ± 0.2abc

3.2 ± 0.5abc

42.3

IBA + NAA 0.5 + 0.5 2.6 ± 0.9c

2.5 ± 0.6c

57.14

0.5 + 1.0 3.0 ± 0.7bc

2.8 ± 0.7c

57.14

0.5 + 1.5 3.0 ± 0.8bc

3.0 ± 0.7bc

42.8

0.5 + 2.0 3.3 ± 0.4ab

3.2 ± 0.4ab

42.8

Values in the table are the mean ± SE of triplicates, n=14 explants, Superscript in

the each row carrying different letters are significantly different at P<0.05 or 0.01,

*-Significance at 0.05, # - Significance at 0.01 by Newman-Keuls Multiple

Comparison Test

Figure 8: In vitro regenerated plants in polycups (after 4 weeks of acclimatization)

Plants

Primer

Total no. of

scorable

bands

No. of

monomorphic

bands

No. of

polymorphic

bands

Oligo 01 27 23 4

Oligo 02 28 25 3

A. Oligo 03 30 26 4

javanica Oligo 04 28 24 4

Table 24: RAPD-PCR amplification products of DNA extracted from different

samples (in vivo, in vitro leaf and callus) of A. javanica

% of

polymorphism

Overall 98

total 113

14.81

10.71

13.33

14.28 15 13.27

% of polymorphism = no. of polymorphic bands/ total no. of scorable bands X 100.

Table 25: Distribution and size of polymorphic bands of different samples (in vivo,

in vitro leaf and callus) materials of A. javanica

Primer Size of polymorphic

band (kb)

Distribution of polymorphic bands

In vivo leaf In vitro leaf callus

Oligo 01

Oligo 02

Oligo 03

Oligo 04

5.0 - + +

2.5 + - +

2.0 - - -

1.5 - - -

1.0 - - -

500 bp - - -

5.0 + - -

2.5 - - +

2.0 - - +

1.5 - - -

1.0 - - -

500 bp - - -

5.0 - - +

2.5 - - -

2.0 - - -

1.5 - - -

1.0 + - +

500 bp - + -

5.0 - - -

2.5 - - +

2.0 - - -

1.5 - - -

1.0 - + +

500 bp + - -

+ Present, – Absent of polymorphic band

Figure 10: RAPD Profile of A. javanica DNA using different primers (Oligo 01, Oligo 02, Oligo 03, and Oligo 04), Sample I – in vivo

leaf, sample II – in vitro leaf, sample III – callus, L – Marker

Table 26: Similarity Matrix computed with Jaccard’s coefficient of A. javanica

Sample 1 Sample 2 Sample 3 Sample 1R Sample 2R Sample 3R

Sample 1 1 0.204 0.416 1.000 0.204 0.416 Sample2 1 0.400 0.204 1.000 0.400

Sample 3 1 0.416 0.400 1.000

Sample 1R 1 0.204 0.416

Sample 2R 1 0.400

Sample 3R 1

Table 27: Distance Matrix based on Jaccard’s coefficient of A. javanica

Sample 1 Sample 2 Sample 3 Sample 1R Sample 2R Sample3R

Sample 1 0 0.216 0.190 0.000 0.216 0.190

Sample 2 0 0.200 0.216 0.000 0.200

Sample 3 0 0.190 0.200 0.000

Sample 1R 0 0.216 0.190

Sample 2R 0 0.200

Sample 3R 0

Table 28: Distance matrix based on RMSD coefficient of A. javanica

Sample 1 Sample 2 Sample 3 Sample 1R Sample 2R Sample3R

Sample 1 0 0.313 0.207 0.000 0.313 0.207

Sample 2 0 0.192 0.313 0.000 0.192

Sample 3 0 0.207 0.192 0.000

Sample 1R 0 0.313 0.207

Sample 2R 0 0.192

Sample 3R 0

Sample 1R - 3R – Repeat of Sample 1 – 3, Sample 1 = in vivo leaf;

Sample 2 = in vitro leaf; Sample 3 = in vitro callus

Figure 11: Phylogenetic Tree Analysis (Phenogram) of A. Javanica, Sample 1 = in

vivo plant leaf, Sample 2 = in vitro plant leaf, Sample 3 = in vitro callus.

band (bp) In vivo plants In vitro plants callus

600 - + -

RBA 10 300 - - +

200 - + -

600 + - -

RBA 13 300 - + +

200 - - +

OPS 01 600 - + -

300 + - +

200 - - -

OPS 12 600 - - -

300 + - -

200 - + +

Table 29: RAPD-PCR amplification products of DNA extracted from different

samples (in vivo, in vitro leaf and callus) of A. lanata using four random primers

Plants Primer

Total no. of

scorable

bands

No. of

monomorphic

bands

No. of

polymorphic

bands

% of

polymorphism

A. lanata

RBA 10 28 25 3 10.71

RBA 13 34 30 4 11.76

OPS – 01 33 30 3 9.09

OPS – 12 28 25 3 10.71

Overall total 123 110 13 10.56

% of polymorphism = no. of polymorphic bands/ total no. of scorable bands X 100.

Table 30: Distribution and size of polymorphic bands of different samples from A. lanata

Primer Size of polymorphic Distribution of polymorphic bands

+ Present – Absent of polymorphic band

Figure 12: RAPD Profile of A. lanata DNA using different primers, S1 – in vivo leaf, S2 – in vitro leaf, S3 – callus, M – Marker

Table 31: Similarity matrix computed with Jaccard’s coefficient of A. lanata

Sample 1 Sample 2 Sample 3 Sample 1R Sample 2R Sample3R

Sample 1 1 0.414 0.237 1.000 0.414 0.237

Sample 2 1 0.200 0.414 1.000 0.200

Sample 3 1 0.237 0.200 1.000

Sample 1R 1 0.414 0.237

Sample 2R 1 0.200

Sample 3R 1

Table 32: Distance matrix based on Jaccard’s coefficient of A. lanata

Sample 1 Sample 2 Sample 3 Sample 1R Sample 2R Sample3R

Sample 1 0 0.586 0.763 0.000 0.586 0.763

Sample 2 0 0.800 0.586 0.000 0.800

Sample 3 0 0.763 0.800 0.000

Sample 1R 0 0.586 0.763

Sample 2R 0 0.800

Sample 3R 0

Table 33: Distance matrix based on RMSD coefficient of A. lanata

Sample 1 Sample 2 Sample 3 Sample 1R Sample 2R Sample 3R

Sample 1 0 0.636 0.831 0.000 0.636 0.831

Sample 2 0 0.816 0.636 0.000 0.816

Sample 3 0 0.831 0.816 0.000

Sample 1R 0 0.636 0.831

Sample 2R 0 0.816

Sample 3R 0

Sample 1R - 3R – Repeat of Sample 1 – 3, Sample 1 = in vivo plant leaf; Sample 2 = in vitro

plant leaf; Sample 3 = in vitro callus

Figure 13: Phylogenetic Tree Analysis (Phenogram) of A. lanata, Sample 1 = in vivo

leaf, Sample 2 = in vitro leaf, Sample 3 = in vitro callus.

Table 34: Antibacterial activity of different solvents extracts A. javanica (in vitro, field leaf and callus)

Org name Method Hexane Chloroform Ethyl acetate

IL FL CA C IL FL CA C IL FL CA C S. aureus WELL 11.0±0 12.0±0 10.0±0 21.0±1.0 9.0±0 8.0±0 9.0±0 21.0±1.0 12.0±0 12.0±0 12.0±0 26.0±1.0

DISC 9.0±0 8.0±0 8.0±0 10.0±1.0 9.0±1.0 9.0±1.0 9.0±1.0 10.0±1.0 7.0±0 6.0±0 12.0±0 14.0±1.0

S. typhi WELL - 11.0±1.0 12.0±1.0 21.0±1.0 - - - 11.0±1.0 8.0±0 9.0±0 9.0±0 26.0±1.0

DISC - - - 21.0±1.0 9.0±1.0 9.0±1.0 9.0±0 13.0±1.0 9.0±1.0 9.0±0 9.0±0 14.0±1.0

C. diphtheriae WELL 10.0±1.0 8.0±1.0 8.0±1.0 26.0±1.0 12.0±1.0 12.0±0 14.0±1.0 26.0±1.0 11.0±0 11.0±1.0 11.0±1.0 26.0±1.0

DISC 9.0±0 6.0±0 8.0±1.0 21.0±1.0 6.0±0 8.0±0 8.0±0 17.0±1.0 8.0±0 9.0±0 12.0±1.0 14.0±1.0

P. vulgaris WELL 11.0±0 9.0±1.0 8.0±0 26.0±1.0 8.0±1.0 12.0±0 12.0±0 16.0±1.0 12.0±0 12.0±0 11.0±1.0 26.0±1.0

DISC 7.0±0 7.0±0 9.0±1.0 12.0±1.0 9.0±1.0 10.0±0 9.0±1.0 15.0±1.0 8.0±0 8.0±0 8.0±0 14.0±1.0

V. vulnificus WELL 9.0±1.0 9.0±1.0 - 26.0±1.0 8.0±1.0 8.0±0 9.0±1.0 26.0±1.0 - - - 26.0±1.0

DISC 7.0±1.0 7.0±0 8.0±0 12.0±1.0 8.0±0 8.0±0 8.0±0 15.0±1.0 - - - 10.0±1.0

S. dysenteriae WELL 8.0±0 12.±0 7.0±0 15.0±1.0 9.0±1.0 13.0±1.0 8.0±0 15.0±1.0 12.0±0 9.0±1.0 9.0±0 26.0±1.0

DISC - - - 17.0±1.0 8.0±0 8.0±0 7.0±0 15.0±1.0 10.0±0 9.0±1.0 10.0±0 12.0±1.0

E. coli WELL 8.0±0 13.0±1.0 8.0±0 21.0±1.0 9.0±1.0 13.0±0 9.0±1.0 21.0±1.0 - - - 21.0±1.0

DISC 6.0±0 8.0±0 12.0±1.0 16.0±1.0 7.0±0 7.0±0 7.0±0 15.0±1.0 - - - 16.0±1.0

E. faecalis WELL 11.0±1.0 12.0±0 - 26.0±1.0 10.0±1.0 12.0±0 12.0±0 26.0±1.0 14.0±0 13.0±1.0 12.0±0 26.0±1.0

DISC 6.0±0 6.0±0 6.0±0 12.0±1.0 - - - 15.0±1.0 9.0±1.0 8.0±1.0 12.0±0 12.0±1.0

L. acidophilus WELL 8.0±0 11.0±0 12.0±0 16.0±1.0 11±1.0 12.0±0 12.0±0 26.0±1.0 8.0±0 10.0±1.0 12.0±0 16.0±1.0

DISC 7.0±0 8.0±0 8.0±0 12.0±1.0 6.0±0 8.0±0 12.0±0 12.0±1.0 9.0±0 7.0±0 7.0±0 25.0±1.0

P. aeruginosa WELL 12.0±1.0 12.0±0 8.0±0 26.0±1.0 14.0±1.0 13.0±1.0 12.0±0 16.0±1.0 9.0±0 10.0±0 10.0±1.0 26.0±1.0

DISC 8.0±0 7.0±0 6.0±0 14.0±1.0 6.0±0 8.0±0 12.0±0 12.0±1.0 8.0±0 8.0±0 8.0±0 21.0±1.0

S. flexneri WELL 11.0±1.0 11.0±1.0 12.0±0 26.0±1.0 11±1.0 10.0±0 15.0±1.0 16.0±1.0 11.0±1.0 10.0±1.0 12.0±1.0 26.0±1.0

DISC 8.0±0 8.0±0 8.0±0 14.0±1.0 8.0±0 8.0±0 13.0±1.0 12.0±1.0 9.0±0 8.0±0 9.0±0 22.0±1.0

B. subtilis WELL 7.0±0 11.0±0 12.0±0 21.0±1.0 7.0±0 8.0±0 6.0±0 21.0±1.0 7.0±0 11.0±1.0 12.0±0 21.0±1.0

DISC 8.0±0 8.0±0 8.0±0 14.0±1.0 9.0±1.0 9.0±0 10.0±1.0 12.0±1.0 9.0±0 9.0±0 8.0±0 12.0±1.0

S. boydii WELL 12.0±1.0 12.0±0 12.0±0 21.0±1.0 14.0±1.0 15.0±1.0 14.0±1.0 26.0±1.0 14.0±0 12.0±0 12.0±0 21.0±1.0

DISC 9.0±1.0 8.0±0 7.0±0 16.0±1.0 8.0±0 8.0±0 8.0±0 12.0±1.0 9.0±0 9.0±0 9.0±0 12.0±1.0

– Nil activity, IL – in vitro leaf, FL – field leaf, CA – callus and C – Control. Contd...

Org name Method Acetone Methanol Aqueous IL FL CA C IL FL CA C IL FL CA C

S. aureus WELL 8.0±0 8.0±0 8.0±0 26.0±1.0 11.0±1.0 12.0±1.0 12.0±1.0 16.0±1.0 12.0±0 10.0±0 10.0±0 23.0±1.0

DISC 10.0±1.0 11.0±1.0 11.0±0 14.0±1.0 12.0±1.0 16.0±1.0 14.0±1.0 16.0±1.0 6.0±0 6.0±0 6.0±0 12.0±1.0

S. typhi WELL 8.0±0 12.0±1.0 12±0 26.0±1.0 12.0±1.0 11.0±1.0 11.0±1.0 21.0±1.0 - - - 23.0±1.0

DISC - - - 25.0±1.0 13.0±1.0 12.0±1.0 11.0±1.0 14.0±1.0 8.0±0 7.0±0 8.0±0 12.0±1.0

C. diphtheriae WELL 11.0±0 12.0±1.0 10.0±0 26.0±1.0 10.0±0 8.0±0 8.0±0 21.0±1.0 11.0±0 12.0±0 11.0±0 23.0±1.0

DISC 8.0±0 9.0±0 10.0±0 12.0±1.0 14.0±1.0 14.0±0 10.0±1.0 15.0±1.0 8.0±0 10.0±0 12.0±0 11.0±1.0

P. vulgaris WELL - 9.0±0 11.0±0 21.0±1.0 11.0±1.0 9.0±0 9.0±0 21.0±1.0 10.0±0 10.0±0 13.0±0 23.0±1.0

DISC - 7.0±0 7.0±0 10.0±1.0 16.0±1.0 16.0±0 12.0±0 16.0±1.0 9.0±0 10.0±0 11.0±0 11.0±1.0

V. vulnificus WELL 7.0±0 7.0±0 9.0±0 21.0±1.0 9.0±0 9.0±0 9.0±0 21.0±1.0 8.0±0 9.0±0 10.0±0 16.0±1.0

DISC 6.0±0 6.0±0 - 15.0±1.0 21.0±0 20.0±0 20.0±0 20.0±1.0 8.0±0 9.0±0 9.0±0 11.0±1.0

S. dysenteriae WELL 10±1.0 9.0±0 8.0±0 21.0±1.0 9.0±0 11.0±0 12.0±0 18.0±1.0 9.0±0 12.0±0 11.0±0 16.0±1.0

DISC 6.0±0 6.0±0 - 12.0±1.0 8.0±0 7.0±0 8.0±0 12.0±1.0 - - - 25.0±1.0

E. coli WELL - - - 21.0±1.0 10.0±0 8.0±0 13.0±0 26.0±1.0 8.0±0 9.0±0 12.0±0 16.0±1.0

DISC 9.0±0 10.0±1.0 10.0±0 12.0±1.0 8.0±0 8.0±0 8.0±0 11.0±1.0 9.0±0 8.0±0 8.0±0 10.0±1.0

E. faecalis WELL 10.0±0 10.0±1.0 10.0±0 26.0±1.0 16.0±0 13.0±0 15.0±0 26.0±1.0 - - - 16.0±1.0

DISC - - - 14.0±1.0 14.0±1.0 17.0±1.0 11.0±0 19.0±1.0 - 9.0±0 10.0±0 10.0±1.0

L. acidophilus WELL 8.0±0 8.0±0 7.0±0 26.0±1.0 10.0±1.0 12.0±0 15.0±0 21.0±1.0 9.0±0 9.0±0 9.0±0 23.0±1.0

DISC 9.0±0 8.0±0 8.0±0 10.0±1.0 13.0±1.0 12.0±0 12.0±0 12.0±1.0 12±0 8.0±0 12.0±0 10.0±1.0

P. aeruginosa WELL 9.0±0 10.0±1.0 11.0±1.0 26.0±1.0 17.0±1.0 16.0±1.0 13.0±1.0 26.0±1.0 8.0±0 12.0±0 11.0±0 23.0±1.0

DISC 6.0±0 6.0±0 6.0±0 14.0±1.0 15.0±1.0 15.0±1.0 13.0±1.0 10.0±1.0 10.0±0 8.0±0 12.0±0 10.0±1.0

S. flexneri WELL 8.0±0 12.0±1.0 11.0±1.0 16.0±1.0 10.0±1.0 9.0±1.0 11.0±1.0 21.0±1.0 - - - 26.0±1.0

DISC 7.0±0 7.0±0 6.0±0 14.0±1.0 9.0±1.0 9.0±1.0 13.0±1.0 20.0±1.0 12.0±1.0 8.0±0 12.0±0 10.0±1.0

B. subtilis WELL 9.0±0 12.0±0 11.0±0 16.0±1.0 14.0±1.0 13.0±1.0 11.0±1.0 21.0±1.0 - - - 21.0±1.0

DISC 9.0±0 7.0±0 7.0±0 14.0±1.0 10.0±0 9.0±0 9.0±0 15.0±1.0 10.0±0 8.0±0 8.0±0 12.0±1.0

S. boydii WELL 8.0±0 8.0±0 8.0±0 16.0±1.0 9.0±0 8.0±0 7.0±0 21.0±1.0 - - - 23.0±1.0

DISC 9.0±0 8.0±0 8.0±0 14.0±1.0 9.0±0 8.0±0 9.0±0 15.0±1.0 8.0±0 8.0±0 10.0±0 12.0±1.0

– Nil activity, IL – in vitro leaf, FL – field leaf, CA – callus and C – Control.

Figure 15a: Antibacterial activity of different solvent extracts of A. javanica (well

diffusion method C – control, IL – in vitro leaf, FL – field leaf, CA – callus),

A. C. diphtheriae, B. P. vulgaris – hexane extract, C. S. dysenteriae – chloroform

extract, D. S. boydii, E. P. aeruginosa – ethyl acetate extract, F. C. diphtheriae –

acetone extract, G. P. aeruginosa, H. E. faecalis – methanol extract, and

I. S. flexneri – aqueous extract.

Figure 15b: Antibacterial activity of different solvent extracts of A. javanica (disc

diffusion method C – control, IL – in vitro leaf, FL – field leaf, CA – callus),

A. S. dysenteriae – chloroform extract, B. P. vulgaris – hexane extract,

C. E. coli – ethyl acetate extract, D. L. acidophilus – acetone extract, E. E. faecalis

- methanol extract, F. P. aeruginosa – aqueous extract. G. S. flexneri,

H. L. acidophilus, and I. S. typhi - methanol extracts

Table 35: Antifungal activity of the extracts of A. javanica

Org name Method Hexane Chloroform Ethyl acetate

IL FL CA C IL FL CA C IL FL CA C

C. albicans WELL 8.1±0 11.0±0 13.3±1.0 16.0±1.0 9.0±0 8.0±0 9.0±0 21.0±1.0 12.0±0 8.0±0 8.0±0 16.0±1.0

DISC - - - 16.0±1.0 - - - 14.0±1.0 - - - 14.0±1.0

A. niger WELL - 11.0±0 12.2±1.0 15.0±1.0 - - - 14.0±1.0 12.0±0 8.0±0 8.0±0 16.0±1.0

DISC - - - 14.0±1.0 - - - 13.0±1.0 9.0±0 9.0±0 8.0±0 14.0±1.0

M. racemosus WELL 9.0±1.0 11.0±1.0 11.4±1.0 16.0±1.0 - - - 16.0±1.0 9.0±0 10.0±0 8.0±0 16.0±1.0

DISC - 9.0±0 - 21.0±1.0 6.0±0 8.0±0 8.0±0 17.0±1.0 8.0±0 9.0±0 - 14.0±1.0

Fusarium sp WELL - - - 14.0±1.0 8.0±0 7.0±0 12.0±1.0 16.0±1.0 - - - 16.0±1.0

DISC 7.0±0 7.0±0 9.2±0 14.0±1.0 9.0±0 7.0±0 9.0±1.0 15.0±1.0 8.0±0 8.0±0 8.0±0 14.0±1.0

Org name Method Acetone Methanol Aqueous

IL FL CA C IL FL CA C IL FL CA C

C. albicans

A.niger

WELL

DISC

WELL

-

-

8.0±0

-

-

8.0±0

-

-

8.0±0

16.0±1.0

16.0±1.0

16.0±1.0

8.0±0

12.0±0

-

8.0±0

13.0±1.0

-

8.0±0

8.0±0

-

14.0±1.0

16.0±1.0

14.0±1.0

8.0±0

-

8.0±0

8.0±0

-

8.0±0

8.0±0

-

8.0±0

26.0±1.0

24.0±1.0

26.0±1.0

DISC - - - 16.0±1.0 - - - 14.0±1.0 9.0±0 9.0±0 8.0±0 24.0±1.0

M. racemosus WELL 8.0±0 9.0±0 - 16.0±1.0 6.0±0 8.0±0 8.0±0 16.0±1.0 9.0±0 10.0±0 8.0±0 26.0±1.0

DISC - 9.0±0 - 16.0±1.0 - - - 16.0±1.0 - - - 24.0±1.0

Fusarium sp WELL - - - 14.0±1.0 8.0±0 7.0±0 12±0 16.0±1.0 - - - 26.0±1.0

DISC - - - 14.0±1.0 9.0±0 7.0±0 9.0±1.0 15.0±1.0 8.0±0 8.0±0 8.0±0 24.0±1.0

– Nil activity, IL – in vitro leaf, FL – field leaf, CA – callus and C – Control.

Figure 16: Antifungal activity of A. javanica well and disc diffusion method,

C – control, IL – in vitro leaf, FL – field leaf, CA – callus, A. C. albicans,

B. Fusarium sp, C. A. niger well diffusion method, D. C. albicans, E. Fusarium sp,

F. A. niger disc diffusion method

Figure 17: Minimum Bactericidal Concentration of A. javanica extract, A – Positive

control, B – in vivo leaf, C – in vitro leaf extract and D – callus extract against

S. aureus.

Table 37: Antibacterial activity of different solvent extracts of A. lanata extracts (in vitro, field leaf and callus)

Org name Method Hexane Chloroform Ethyl acetate

IL FL CA C IL FL CA C IL FL CA C

S. aureus WELL 8.0±0 8.0±0 9.0±0.1 13.0±1.0 9.0±0 8.0±0 8.0.0±0 14.0±1.0 12.0±0 11.0±0 12.0±0 13.0±1.0

DISC 8.0±0 8.0±0 8.0±0 13.0±1.0 8.0±0 8.0±0 8.0±0 13.0±1.0 11.0±0 11.0±0 8.0±0 14.0±1.0

S. typhi WELL 7.0±0 8.0±0 6.0±0 21.0±1.0 7.0±0 8.0±0 6.0±0 11.0±1.0 8.0±0 8.0±0 6.0±0 26.0±1.0

DISC 9.0±0 9.0±0 - 21.0±1.0 9.0±0 9.0±0 9.0±0 13.0±1.0 7.0±0 7.0±0 8.0±0 14.0±1.0

C. diphtheriae WELL 8.0±0 7.0±0 8.0±0 26.0±1.0 11.0±0 11.0±0 11.0±0 26.0±1.0 12.0±0 12.0±0 11.0±0 26.0±1.0

DISC 8.0±0 8.0±0 10.0±0 21.0±1.0 8.0±0 8.0±0 13.0±0 17.0±1.0 7.0±0 9.0±0 8.0±0 14.0±1.0

P. vulgaris WELL 10.0±0 - 11.0±0 26.0±1.0 7.0±0 - - 17.0±1.0 11.0±0 11.0±0 11.0±0 26.0±1.0

DISC 9.0±0 9.0±0 9.0±0 12.0±1.0 8.0±0 8.0±0 9.0±0 15.0±1.0 10.0±0 10.0±0 8.0±0 14.0±1.0

V. vulnificus WELL 10.0±0 10.0±0 - 26.0±1.0 10.0±0 11.0±0 9.0±0 26.0±1.0 10.0±0 7.0±0 - 26.0±1.0

DISC 8.0±0 8.0±0 8.0±0 12.0±1.0 11.0±0 10.0±0 8.0±0 16.0±1.0 12.0±0 14.0±0. - 20.0±1.0

S. dysenteriae WELL 9.0±0 9.0±0 7.0±0 15.0±1.0 8.0±0 6.0±0 8.0±0 15.0±1.0 10.0±0 10.0±0 9.0±0 26.0±1.0

DISC 6.0±0 6.0±0 6.0±0 17.0±1.0 8.0±0 8.0±0 7.0±0 15.0±1.0 7.0±0 7.0±0 9.0±0 12.0±1.0

E. coli WELL 7.0±0 8.0±0 10.0±0 21.0±1.0 8.0±0 8.0±0 10.0±0 21.0±1.0 12.0±0 12.0±0 11.0±0 21.0±1.0

DISC 9.0±0 9.0±0 10.0±0 16.0±1.0 8.0±0 8.0±0 - 15.0±1.0 - - - 16.0±1.0

E. faecalis WELL 6.0±0 6.0±0 - 26.0±1.0 8.0±0 8.0±0 - 26.0±1.0 11.0±0 12.0±0 - 26.0±1.0

DISC 7.0± 12.0±0 10.0±0 12.0±1.0 8.0±0 8.0±0 9.0±0 15.0±1.0 11.0±0 11.0±0 11.0±0 12.0±1.0

L. acidophilus WELL - 11.0±0 7.0±0 18.0±1.0 9.0±0 - 13.0±0 26.0±1.0 10.0±0 8.0±0 8.0±0 16.0±1.0

DISC 9.0±0 8.0±0 8.0±0 11.0±1.0 10.0±0 9.0±0 12.0±0 12.0±1.0 11.0±0 8.0±0 12.0±0 25.0±1.0

P. aeruginosa WELL 8.0±0 8.0±0 10.0±0 26.0±1.0 12.0±0 11.0±0 10.0±0 16.0±1.0 8.0±0 10.0±0 12.0±0 26.0±1.0

DISC 8.0±0 8.0±0 9.0±0 14.0±1.0 9.0±0 9.0±0 10.0±0 12.0±1.0 7.0±0 8.0±0 6.0±0 21.0±1.0

S. flexneri WELL 9.0±0 8.0±0 8.0±0 26.0±1.0 - - 8.0±0 16.0±1.0 8.0±0 - - 26.0±1.0

DISC 12.0±0 12.0±0 11.0±0 14.0±1.0 12.0±0 11.0±0 8.0±0 12.0±1.0 7.0±0 9.0±0 9.0±0 22.0±1.0

B. subtilis WELL 7.0±0 8.0±0 9.0±0 21.0±1.0 10.0±0 9.0±0 11.0±0 21.0±1.0 8.0±0 12.0±0 12.0±0 21.0±1.0

DISC 9.0±0 9.0±0 10.0±0 14.0±1.0 12.0±0 12.0±0 11.0±0 12.0±1.0 8.0±0 8.0±0 7.0±0 12.0±1.0

S. boydii WELL 9.0±0 10.0±0 11.0±0 21.0±1.0 10.0±0 8.0±0 10.0±0 26.0±1.0 15.0±0 10.0±0 12.0±0 21.0±1.0

DISC 10.0±0 9.0±0 11.0±0 16.0±1.0 8.0±0 9.0±0 10.0±0 12.0±1.0 10.0±0 10.0±0 13.0±0 12.0±1.0

– Nil activity, IL – in vitro leaf, FL – field leaf, CA – callus and C – Control. Contd…

Org name Method Acetone

IL FL

CA

C

Methanol

IL FL

CA

C

Aqueous

IL FL

CA

C

S. aureus WELL 10.0±0 10.0±0 9.0±0 26.0±1.0 12.0±0 11.0±0 14.0±0 16.0±1.0 - - - 23.0±1.0

DISC 10.0±0 8.0±0 9.0±0 14.0±1.0 7.0±0 7.0±0 6.0±0 16.0±1.0 8.0±0 8.0±0 7.0±0 12.0±1.0

S. typhi WELL 7.0±0 7.0±0 6.0±0 26.0±1.0 12.0±0 12.0±0 11.0±0 21.0±1.0 7.0±0 7.0±0 7.0±0 23.0±1.0

DISC 9.0±0 9.0±0 9.0±0 25.0±1.0 11.0±0 11.0±0 11.0±0 14.0±1.0 6.0±0 7.0±0 8.0±0 12.0±1.0

C. diphtheriae WELL 7.0±0 7.0±0 8.0±0 26.0±1.0 7.0±0 7.0±0 8.0±0 21.0±1.0 8.0±0 8.0±0 11.0±0 23.0±1.0

DISC 8.0±0 8.0±0 7.0±0 12.0±1.0 9.0±0 9.0±0 8.0±0 10.0±1.0 12.0±0 12.0±0 10.0±0 11.0±1.0

P. vulgaris WELL 11.0±0 - 8.0±0 11.0±1.0 16.0±0 14.0±0 12.0±0 21.0±1.0 8.0±0 - 8.0±0 23.0±1.0

DISC 11.0±0 10.0±0 - 10.0±1.0 10.0±0 10.0±0 10.0±0 16.0±1.0 12.0±0 10.0±0 11.0±0 11.0±1.0

V. vulnificus WELL 8.0±0 11.0±0 9.0±0 21.0±1.0 - 8.0±0 9.0±0 21.0±1.0 16.0±0 13.0±0 10.0±0 16.0±1.0

DISC - - - 15.0±1.0 - - - 20.0±1.0 - - - 11.0±1.0

S. dysenteriae WELL 9.0±0 - 8.0±0 21.0±1.0 12.0±0 8.0±0 12.0±0 18.0±1.0 - - 11.0±0 16.0±1.0

DISC 8.0±0 8.0±0 7.0±0 21.0±1.0 9.0±0 9.0±0 8.0±0 12.0±1.0 12.0±0 12.0±0 11.0±0 25.0±1.0

E. coli WELL 8.0±0 8.0±0 11.0±0 21.0±1.0 7.0±0 7.0±0 8.0±0 26.0±1.0 6.0±0 6.0±0 7.0±0 16.0±1.0

DISC 9.0±0 9.0±0 11.0±0 12.0±1.0 11.0±0 11.0±0 9.0±0 11.0±1.0 12.0±0 11.0±0 9.0±0 10.0±1.0

E. faecalis WELL 6.0±0 6.0±0 - 26.0±1.0 14.0±0 16.0±0 - 26.0±1.0 6.0±0 6.0±0 6.0±0 16.0±1.0

DISC - - - 14.0±1.0 - - - 19.0±1.0 - - - 10.0±1.0

L. acidophilus WELL 13.0±0 11.0±0 12.0±0 26.0±1.0 16.0±0 15.0±0 18.0±0 21.0±1.0 10.0±0 10.0±0 10.0±0 23.0±1.0

DISC 10.0±0 12.0±0 11.0±0 10.0±1.0 18.0±0 11.0±0 8.0±0 12.0±1.0 8.0±0 8.0±0 8.0±0 10.0±1.0

P. aeruginosa WELL 8.0±0 8.0±0 13.0±0 26.0±1.0 8.0±0 8.0±0 8.0±0 26.0±1.0 10.0±0 11.0±0 11.0±0 23.0±1.0

DISC 9.0±0 9.0±0 9.0±0 14.0±1.0 8.0±0 9.0±0 8.0±0 10.0±1.0 7.0±0 8.0±0 8.0±0 10.0±1.0

S. flexneri WELL - - 11.0±0 16.0±1.0 - - - 21.0±10 - - - 26.0±1.0

DISC 13.0±0 13.0±0 12.0±0 16.0±1.0 14.0±0 13.0±0 12.0±0 20.0±1.0 13.0±0 14.0±0 10.0±0 10.0±1.0

B. subtilis WELL - 6.0±0 8.0±0 16.0±1.0 - - 8.0±0 21.0±1.0 8.0±0 8.0±0 11.0±0 21.0±1.0

DISC 12.0±0 8.0±0 8.0±0 14.0±1.0 8.0±0 9.0±0 9.0±0 15.0±1.0 12.0±0 9.0±0 9.0±0 12.0±1.0

S. boydii WELL 6.0±0 6.0±0 8.0±0 16.0±1.0 8.0±0 8.0±0 7.0±0 21.0±1.0 9.0±0 7.0±0 7.0±0 23.0±1.0

DISC 11.0±0 11.0±0 12.0±0 14.0±1.0 12.0±0 11.0±0 11.0±0 15.0±1.0 10.0±0 9.0±0 9.0±0 12.0±1.0

– Nil activity, IL – in vitro leaf, FL – field leaf, CA – callus and C – Control.

Figure 18a: Antibacterial activity of A. lanata (well diffusion method C – control,

IL – in vitro leaf, FL – field leaf, CA – callus), A. C. diphtheriae, B. S. flexneri,

C. L. acidophilus – methanol extract, D. S. boydii, E. S. typhi, F. S. dysenteriae –

ethyl acetate extract, G. B. subtilis, H. S. aureus, I. S. dysenteriae – acetone extract

Figure 18b: Antibacterial activity of A. lanata (disc diffusion method C – control,

IL – in vitro leaf, FL – field leaf and CA – callus), A. S. flexneri, B. L. acidophilus,

C. S. typhi – methanol extract, D. S. aureus, E. S. dysenteriae, F. B. subtilis –

acetone extract, G. S. typhi – ethyl acetate extract, H. C. diphtheriae – aqueous

extract, I. P. aeruginosa – methanol extract.

Table 38: Antifungal activity of the extracts of A. lanata

Org name Method Hexane Chloroform Ethyl acetate

IL FL CA C IL FL CA C IL FL CA C

C. albicans WELL 8.0±0 8.0±0 8.0±0 21.0±1.0 8±1.0 8.0±0 8±1.0 21.0±1.0 9.0±0 9.0±0 9.0±0 26.0±1.0

DISC 9.0±0 9.0±0 9.0±0 10.0±1.0 9.0±0 9.0±0 9.0±0 10.0±1.0 8.0±0 9.0±0 9.0±0 14.0±1.0

A. niger WELL 10.0±0 11±0 11.0±0 21.5±1.0 12.0±0 11.0±0 11.0±0 15.0±1.0 8.0±0 8.0±0 8.0±0 26.0±1.0

DISC 9.0±0 9.0±0 9.0±0 21.0±1.0 9.0±1.

0

9±1.0 9±1.0 13.0±1.0 9.0±0 9.0±0 9.0±0 14.0±1.0

M.

racemosus

WELL - - - 26.0±1.0 - - - 26.0±1.0 - - - 26.0±1.0

DISC - - - 21.0±1.0 - - - 17.0±1.0 - - - 14.0±1.0

Fusarium

sp.

WELL - - - 26.0±1.0 - - - 10.0±1.0 - - - 26.0±1.0

DISC - - - 12.0±1.0 - - - 15.0±1.0 - - - 14.0±1.0

Org name Method Acetone Methanol Aqueous

IL FL CA C IL FL CA C IL FL CA C

C. albicans WELL 8.0±0 8.0±0 - 21.0±1.0 8.0±0 8.0±0 12.0±0 21.0±1.0 - - - 26.0±1.0

DISC 11.0±0 10.0±0 10.0±0 10.0±1.0 10±1.0 10.0±0 10±1.0 10.0±1.0 9.0±0 9.0±0 10.0±0 14.0±1.0

A. niger WELL - - - 21.0±1.0 9±0.1 9.0±0 9.0±0 15.0±1.0 12.0±0 8.0±0 8.0±0 26.0±1.0

DISC 8.0±0 8.0±0 8.0±0 21.0±1.0 7±0.1 7.0±0 7.0±0 13.0±1.0 8.0±0 8.0±0 8.0±0 14.0±1.0

M.

racemosus

WELL 8.0±0 7.0±0 7.0±0 26.0±1.0 8±1.0 9.0±0 7.0±0 26.0±1.0 8.0±0 7.0±0 7.0±0 26.0±1.0

DISC - - - 21.0±1.0 - - - 17.0±1.0 - - - 14.0±1.0

Fusarium

sp.

WELL 8.0±0 8.0±0 7.0±0 26.0±1.0 9±1.0 9.0±0 9.0±0 16.0±1.0 9.0±0 8.0±0 8.0±0 26.0±1.0

DISC - - - 12.0±1.0 - - - 15.0±1.0 - - - 14.0±1.0

– Nil activity, IL – in vitro leaf, FL – field leaf, CA – callus and C – Control.

Figure 19: Antifungal activity of A. lanata (C – control, IL – in vitro leaf, FL – field

leaf, CA – callus) A. A. niger, B. Fusarium sp, C. M. racemosus well diffusion

method D. A. niger, E. Fusarium sp, F. C. albicans disc diffusion method

Figure 20: Minimum Bactericidal Concentration of A. lanata extracts, A – Positive

control, B – in vivo leaf, C – in vitro leaf extract and D – callus extract against E.coli

Figure 21: Bioautography of TLC fractions against S. aureus & S. typhi, A – AJ IL

F1, B – AJ FL F1, C – AJ CA F1 against S. aureus, D – AJ FL F1, E – AJ IL F1,

and F – AJ CA F1 against S. typhi

Figure 22: Bioautography of TLC fractions of A. lanata, A – AL FL F1, B – AL IL

F1, C – AL CA F1 (S. aureus), and D – AL CA F1 (S. typhi)

Figure 23: Preliminary phytochemical of A. javanica extracts

Table 40: Preliminary phytochemical analysis of extracts of A. javanica

HX CL EA AC MT AQ

Tests FL IL CA FL IL CA FL IL CA FL IL CA FL IL CA FL IL CA

Alkaloids + + - + + - + + + + + + - + - + + +

Flavonoids + + + + + + + + + + + + + + + + + +

Phenolic + + + + + + + + + + + + + + + + + +

Terpenoids + + - - - - - - - - - - - - - - - -

Tannins + + + + + + + + + + + + + + + + + +

Saponins + + + + + - - - - + + + + + + + + +

Carbohydrates + + + + + + + + + + + + + + + + + +

Amino acids - - - - - - + + - + - - + + + + - -

Glycosides + + - + + + + + - + + - + + + + + +

Fixed oils - - - - - - - - - + + + + + + + - -

` HX – hexane; CL – chloroform; EA – ethyl acetate; AC – acetone; MT – methanol; AQ – aqueous; FL – in vivo leaf; IL – in vitro

leaf; CA – callus; + = indicates presence; – = indicates absence.

Table 41: Quantification of phytochemicals (total phenols, tannins) from different extracts of A. javanica

Total phenolic(mg GAE/g) Tannins (mg GAE/g)

Solvents In vivo leaf In vitro leaf Callus In vivo leaf In vitro leaf Callus

Hexane 105.64 ± 1.79c 115.32 ± 14.21

c 83.44 ± 2.40

b 15.7 ± 4.11

f 16.32 ± 6.30

f 8.23 ± 1.37

f

Chloroform 54.19 ± 1.98d 41.21 ± 0.50

e 61.44 ± 4.05

c 29.03 ± 6.69

e 31.98 ± 4.31

e 4.9 ± 1.57

f

Ethyl acetate 30.21 ± 0.82e 65.39 ± 4.64

d 55.24 ± 1.32

c 36.17 ± 4.74

e 30.63 ± 2.86

e 10.51 ± 0.76

f

Acetone 33.24 ± 2.91e 73.95 ± 7.90

d 111.54 ± 6.93

c 34.37 ± 5.98

e 31.38 ± 4.09

e 13.65 ± 4.05

f

Methanol*

150.25 ± 5.29a 155.51 ± 4.72

a 145.23 ± 1.68

a 82.01 ± 2.76

b 87.60 ± 4.24

b 30.84 ± 4.9

a

Aqueous*

156.68 ± 1.45a 139.20 ± 5.44

a 124.65 ± 4.16

a 76.08 ± 3.71

b 78.23 ± 7.02

b 34.71 ± 5.7

a

GAE – Gallic acid equivalents, values in the table are the mean ± SE of triplicates, Superscript in the each row carrying different letters

are significantly different at P<0.05 or 0.01, *-Significance at 0.05, by Newman-Keuls Multiple Comparison Test

Table 42: Quantification of phytochemicals (flavonoids and carbohydrates) from different extracts of A. javanica

Flavonoids (mg RE/g) CHO (mg GE/g)

Solvents In vivo leaf In vitro leaf Callus In vivo leaf In vitro leaf Callus

Hexane 61.59 ± 0.7d 30.58 ± 1.8

e 41.01 ± 2.2

e 126.95 ± 4.4

a 110.54 ± 2.3

a 88.66 ± 3.7

b

Chloroform 57.25 ± 6.8c 44.49 ± 4.9

e 40.14 ± 5.3

e 120.00 ± 3.4

a 113.59 ± 4.3

a 75.16 ± 4.8

b

Ethyl acetate 88.84 ± 2.2b 93.77 ± 2.4

b 83.62 ± 6.4

b 116.87 ± 0.6

a 116.29 ± 5.0

a 80.15 ± 4.6

b

Acetone*

105.94 ± 2.3a 67.97 ± 7.2

d 145.65 ± 0.9

ab 117.23 ± 3.0

a 121.43 ± 0.3

a 88.99 ± 3.6

b

Methanol#

141.01 ± 2.1ab

153.19 ± 2.0abc

177.54 ± 1.8abc

139.00 ± 2.1ab

127.28 ± 4.3a 107.72 ± 1.6

a

Aqueous*

109.42 ± 3.6a 104.49 ± 3.8

a 104.20 ± 2.0

a 127.77 ± 1.1

a 114.12 ± 1.3

a 102.85 ± 1.2

a

RE – Rutin equivalents, GE – glucose equivalents, values in the table are the mean ± SE of triplicates, Superscript in the each row

carrying different letters are significantly different at P<0.05 or 0.01, *-Significance at 0.05, # - Significance at 0.01 by Newman-Keuls

Multiple Comparison Test

Figure 24: Preliminary phytochemicals of A. lanata extracts

HX CL EA AC MT AQ FL IL CA FL IL CA FL IL CA FL IL CA FL IL CA FL IL CA

+ + - + + - + + - + + - + + + + + -

+ + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + +

- + - - - - + - - + + + - - - - - -

+ + + + + + + + + + + + + + + + + +

- - - - - - - - - + + - + + + + - -

+ + + + + + + + + + + + + + + + + +

+ - - + - - + + - + + - + + - + + -

+ - - + - - - - - + + - + + + + + +

- - - - - - - - - - - - - - - - - -

Table 43: Preliminary Phytochemical analysis of A. lanata extracts

Tests

Alkaloids

Flavonoids

Phenolic

Terpenoids

Tannins

Saponins

Carbohydrates

Amino acids

Glycosides

Fixed oils

HX – hexane; CL – chloroform; EA – ethyl acetate; AC – acetone; MT – methanol; AQ – aqueous; FL – in vivo leaf; IL – in vitro leaf;

CA – callus; + = indicates presence; – = indicates absence.

Table 44: Quantification of phytochemicals (total phenols, tannins) from different extracts of A. lanata

Total phenolic (mg GAE/g extract) Tannins (mg GAE/g extract)

Solvents Field leaf In vitro leaf Callus Field Leaf In vitro leaf Callus

Hexane 23.23 ± 1.14d 27.91 ± 2.14

d 24.81 ± 1.66

d 8.71 ± 1.39

d 20.32 ± 4.31

d 8.23 ± 1.37

d

Chloroform 20.00 ± 1.89 d 34.10 ± 2.25

d 17.76 ± 0.69

d 5.69 ± 2.33

d 15.31 ± 2.77

d 4.90 ± 1.57

d

Ethyl acetate 28.23 ± 0.82 d 55.18 ± 1.37

b 15.72 ± 1.32

d 9.50 ± 3.21

d 13.30 ± 4.57

d 10.51 ± 0.76

d

Acetone 28.63 ± 2.50 d 54.19 ± 2.90

b 34.62 ± 2.24

e 11.04 ± 1.61

d 26.71 ± 3.42

d 13.65 ± 4.05

d

Methanol#

193.98 ± 0.44abc

137.85 ± 2.44abc

69.37 ± 3.59b 109.41 ± 3.59

abc 82.60 ± 2.24

b 36.84 ± 2.12

b

Aqueous*

120.72 ± 0.58abc

137.65 ± 2.60abc

60.67 ± 2.97b 87.01 ± 1.42

b 78.23 ± 2.02

b 36.05 ± 2.13

b

GAE – gallic acid equivalents, values in the table are the mean ± SE of triplicates, Superscript in the each row carrying different letters

are significantly different at P<0.05 or 0.01, *-Significance at 0.05, # - Significance at 0.01 by Newman-Keuls Multiple Comparison

Test

Table 45: Quantification of phytochemicals (flavonoids and carbohydrates) from different extracts of A. lanata

Flavonoids (mg RE/g) Carbohydrates (mg GE/g)

Solvents In vivo Leaf In vitro leaf Callus in vivo leaf In vitro leaf Callus

Hexane 29.71 ± 3.05c 21.88 ± 1.81

c 23.62 ± 2.19

c 31.07 ± 4.8

b 14.73 ± 6.3

b 5.70 ± 0.6

d

Chloroform 28.26 ± 2.30c 23.91 ± 0.87

c 25.65 ± 1.74

c 36.14 ± 4.1

c 9.66 ± 2.9

c 6.03 ± 0.1

c

Ethyl acetate 54.06 ± 3.53b 35.22 ± 2.30

c 41.30 ± 3.14

b 26.92 ± 0.4

c 15.65 ± 4.4

c 5.18 ± 0.2

c

Acetone*

92.03 ± 2.53a 88.26 ± 3.48

b 150.58 ± 4.29

abc 27.31 ± 0.5

c 14.66 ± 4.9

c 6.10 ± 0.2

c

Methanol#

135.80 ± 4.46ab

118.41 ± 3.92ab

151.45 ± 4.97abc

46.28 ± 3.6b 58.60 ± 3.2

ab 9.57 ± 0.5

c

Aqueous*

110.00 ± 1.74ab

96.38 ± 4.10ab

90.00 ± 2.70ab

20.92 ± 0.3c 17.96 ± 3.7

c 7.09 ± 1.0

c

RE – rutin equivalents, GE – Glucose equivalents, values in the table are the mean ± SE of triplicates, Superscript in the each row

carrying different letters are significantly different at P<0.05 or 0.01, *-Significance at 0.05, # - Significance at 0.01 by Newman-Keuls

Multiple Comparison Test

Table 47: FRAP, phosphomolybdenum and metal chelation assay of A. javanica extracts

Solvents

FRAP (mM (Fe)II/g extract) Phosphomolybdenum (mg AAE/g) Metal chelation (mg EDTAE/g)

In vivo leaf In vitro leaf Callus In vivo leaf In vitro leaf Callus In vivo leaf In vitro leaf Callus

Hexane 43.90 ± 1.9b

15.22 ± 1.0c

10.66 ± 6.4c

86.95 ± 2.4c

100.54 ± 2.3bc

78.66 ± 3.7c

89.32 ± 3.0c

93.16 ± 3.7b

98.38 ± 1.5bc

Chloroform 23.61 ± 4.6d

09.20 ± 0.7c

17.97 ± 0.7c

90.00 ± 3.4b

103.59 ± 2.3bc

85.16 ± 2.8c

110.36 ± 4.4bc

118.81 ± 2.0bc

95.35 ± 1.6b

Ethyl acetate 27.58 ± 4.1c

16.72 ± 1.7c

14.70 ± 0.5c

76.87 ± 0.6c

106.29 ± 2.0bc

83.15 ± 4.6c

133.70 ± 0.8a

112.69 ± 4.9bc

99.39 ± 0.5bc

Acetone 20.93 ± 2.7c

33.22 ± 3.3b

15.92 ± 0.4c

217.23 ± 2.0ab

121.43 ± 0.3b

87.99 ± 3.6c

167.27 ± 3.6a

43.27 ± 1.2c

97.71 ± 0.5b

Methanol* 124.26 ± 1.7a 157.01 ± 2.7a 130.18 ± 0.1a 379.00 ± 1.1a 377.28 ± 2.3a 167.72 ± 1.6b 178.96 ± 1.8a 126.75 ± 5.3b 98.63 ± 2.2bc

Aqueous 92.00 ± 6.7b

71.39 ± 0.3b

78.53 ± 2.2a

227.77 ± 1.1ab

344.12 ± 3.3a

142.85 ± 1.2b

127.22 ± 1.1ab

114.18 ± 1.0bc

17.32 ± 2.0c

AAE – ascorbic acid equivalents, EDTAE – ethylene diamine tetra acetic acid equivalents, values in the table are the mean ± SE of

triplicates, Superscript in the each row carrying different letters are significantly different at P<0.05 *-Significance at 0.05, Newman-

Keuls Multiple Comparison Test

Table 49: Total antioxidant (FRAP, phosphomolybdenum, metal chelation) potential of the extracts of the A. lanata extracts

Samples

FRAP (mM (Fe)II/g) Metal chelation (mg EDTAE/g) Phosphomolybdenum (mg AAE/g)

In vivo Leaf In vitro leaf Callus In vivo Leaf In vitro leaf Callus In vivo Leaf In vitro leaf Callus

Hexane 5.58 ± 4.04c

11.74 ± 4.15c

3.70 ± 0.47c

87.10 ± 1.01c

8.73 ± 3.88c

14.21 ± 1.52c

40.89 ± 3.11cd

33.18 ± 5.25cd

26.85 ± 3.69c

Chloroform 9.69 ± 2.06c 11.29 ± 3.41c 10.32 ± 4.10c 112.69 ± 6.36b 34.63 ± 7.04c 11.18 ± 1.59c 107.27± 3.40bc 74.80 ± 4.02c 77.10 ± 4.01c

Ethyl acetate 5.10 ± 0.22c

7.74 ± 1.76c

4.88 ± 0.58c

193.27 ±1.05ab

28.52 ± 4.93c

15.22 ± 0.54c

80.90 ± 2.22c

94.78 ± 5.00c

42.85 ± 2.40cd

Acetone 3.53 ± 0.30c

28.00 ± 1.89c

13.38 ± 1.91c

186.85 ± 4.41ab

127.45 ± 1.18b

13.53 ± 0.46c

356.95 ± 0.78a

111.07 ± 5.68bc

70.71 ± 3.64c

Methanol# 112.88 ±2.80a 73.48 ± 2.75b 33.29 ± 0.12b 282.19 ± 6.35a 242.57 ± 5.28a 114.46 ± 2.15a 400.48 ± 4.10a 345.90 ± 4.32a 102.55 ± 0.80b

Aqueous*

64.02 ± 3.82b

67.22 ± 3.30b

25.77 ± 0.78b

186.68 ± 3.37ab

185.56 ± 1.01ab

94.02 ± 3.95ab

206.29 ± 1.12b

204.57 ± 6.74b

91.50 ± 3.20c

AAE – ascorbic acid equivalents, EDTAE – ethylene diamine tetra acetic acid equivalents, values in the table are the mean ± SE of

triplicates, Superscript in the each row carrying different letters are significantly different at P<0.05 or 0.01, *-Significance at 0.05, # -

Significance at 0.01 by Newman-Keuls Multiple Comparison Test

Figure 25a: Different concentrations of A. javanica in vivo leaf extract in MTT assay

Figure 25b: Different concentrations of A. javanica in vitro leaf extract

Figure 25c: Different concentrations of A. javanica callus leaf extract

Figure 26: DNA fragmentation of MCF – 7 cell line, M – Marker, L1 Control, L2 -

in vivo sample, L3 - in vitro sample and L4 - callus sample

Figure 27a: Different concentrations of A. lanata in vivo leaf extract

Figure 27b: Different concentrations of A. lanata in vitro leaf extract

Figure 27c: Different concentrations of A. lanata callus leaf extract

Figure 28: DNA fragmentation of MCF – 7 cell lines by A. Lanata, M – Marker,

C – control, L1 – in vivo, L2 – in vitro, L3 – callus (IC50 concentration).

Table 52: Rf values of the compounds separated from A. javanica extracts

Extract No. of Rf value Interaction Fractions

AJ – IL – MT 02 0.888, 0.944 polyphenols and polysaccharides (Martin, et.al, 1986)

AJ – FL – MT 03 0.866, 0.877, 0.9 polyphenols and proteins, (Bate-Smith, 1981)

AJ – CA – MT 01 0.877 Polyphenol (Bate-Smith, 1981)

AJ – IL – MT = A. javanica in vitro leaf Methanol extract, AJ – FL – MT = A. javanica

in vivo leaf Methanol extract, AJ – CA – MT = A. javanica callus Methanol extract

Table 53: Rf values of the compounds separated from A. lanata extracts

Extract No. of Rf value Interaction Fractions

AL – IL – MT 03 0.800, 0.811, 0.854 polyphenols (Martin, et.al, 1986)

AL – FL – MT 04 0.245, 0.249, 0.414,

0.870,

Fatty acids, amino acids, and polyphenols (Bate-

Smith, 1981)

AL – CA – MT 02 0.214, 0.865 polysaccharides and Polyphenol (Bate-Smith,

1981)

AL – IL – MT = A. lanata in vitro leaf Methanol extract, AL – FL – MT = A. lanata in

vivo leaf Methanol extract, AL – CA – MT = A. lanata callus Methanol extract

Figure 29a: TLC fraction of A. javanica extracts, A – In vivo leaf, B – In vitro leaf,

C – Callus extract

Figure 29b: TLC fraction of A. lanata extracts, A – In vivo leaf, B – In vitro leaf, C

– Callus extract

Figure 30a: HPLC chromatogram of AJ-FL-F1 (A. javanica field leaf fraction 1)

Figure 30b: HPLC chromatogram of AJ-IL-F1 (A. javanica in vitro leaf fraction 1)

Figure 30c: HPLC chromatogram of AJ-CA-F1 (A. javanica callus fraction 1)

Figure 30d: HPLC chromatogram of standards 1 – Gallic acid, 2 – Catechin, 3 – Epicatechin, 4 – Quercetin

Figure 31a: HPLC chromatogram of AL– FL– F1 (A. lanata field leaf fraction 1)

Figure 31b: HPLC chromatogram of AL–IL–F1 (A. lanata in vitro leaf fraction 1)

Figure 31c: HPLC chromatogram of AL–CA–F1 (A. lanata callus fraction 1)

Figure 31d: HPLC chromatogram of standards 1 – Chlorogenic acid, 2 – Vanillin, 3 – Ferulic acid, 4 – Resveratrol

Table 56: Bioactive compounds detected in A. javanica extracts by using GC – MS

Plant Sample RT Compound MW Formula Reported property Author

A. In vivo 15.73 MYO-INOSITOL, 4-C-METHYL- 194 C7H14O6 Antitumor Hudlicky et al., 2002

javanica leaf 28.704 STIGMASTEROL 83-48-7 412 C29H48O Antioxidant, anti-inflammatory,

immunomodulatory

Hassanein et al, 2012,

Norman et al., 1972

29.359 3BETA-HYDROXY-5-CHOLEN-24-

OIC ACID 5255-17-4

374 C24H38O3 Antibacterial Batte et al., 1990

30.37

HENTRIACONTANE

436

C31H64

Antimicrobial

Milen georgiev et al.,

2010

In vitro

15.839

MYO-INOSITOL, 4-C-METHYL-

194

C7H14O6

Antitumor

Hudlicky et al., 20002

leaf 16.424 .BETA.-D-GLUCOPYRANOSIDE,

METHYL

194 C7H14O6 Antiallergic, Cytotoxic,

antidiabetic

Kim et al., 2000, Lee &

Sohn 2008

28.679 STIGMASTEROL 412 C29H48O Antioxidant, anti-inflammatory,

immunomodulatory

Hassanein et al, 2012,

Norman et al., 1972

Callus 15.34 .BETA.-D-GLUCOPYRANOSIDE,

METHYL

194 C7H14O6 Antiallergic, Cytotoxic,

antidiabetic

Kim et al., 2000, Lee &

Sohn 2008

19.810 Olyl alcohol 268 C18H36O Antifungal Dawn et al., 1962

20.025 Olic acid 282 C18H34O2 Antioxidant Visioli & Galli 2002

28.67 STIGMASTEROL 83-48-7 412 C29H48O Antioxidant, anti-inflammatory,

immunomodulatory

Hassanein et al, 2012,

Norman et al., 1972,

Figure 32a: GC – MS chromatogram of A. javanica in vivo leaf methanol extract

Figure 32b: GC – MS chromatogram of A. javanica in vitro leaf methanol extract

Figure 32c: GC – MS chromatogram of A. javanica callus methanol extract

Table 57: Bioactive compounds detected in A. lanata extracts by using GC – MS

Plant Sample RT Compound MW Formula Reported property Author

A. In vivo 17.85 METHYL 11-METHYL-DODECANOATE 228 C14H28O2 Antibacterial Ntougias et al., 2000

lanata leaf 20.32

3-METHYL-2-(2-OXOPROPYL)FURAN

138

C8H10O2

Anti-inflammatory

Berger et al., 2002

30.37

HENTRIACONTANE

436 C31H64

Antimicrobial

Milen georgiev et al., 2010

In vitro

29.37

10,12,14-NONACOSATRIYNOIC ACID

426 C29H46O2

Antioxidant

Yang et al., 2011

leaf 16.85

Z,Z-6,28-HEPTATRIACTONTADIEN-2-ONE

530

C37H70O

Antimicrobial

Milengeorgiev et al., 2010

23.11

1,2-BENZENEDICARBOXYLIC ACID,

278

C16H22O4

Antianderogenic property,

Koch et al., 2003,

MONO(2-ETHYLHEXYL) ESTER Antidiabetic Maruthupandian and Mohan,

2011

Callus

16.94

3,7,11,15-TETRAMETHYL-2-HEXADECEN-1-

296

C20H40O

Antibacterial, antifungal

Ogunlein et al., 2009

OL

23.54

PHENOL, 3,5-BIS(1,1-DIMETHYLETHYL)-

206

C14H22O

Antiinflammatory

Unangst et al., 1992

25.05

CYCLOTRISILOXANE, HEXAMETHYL-

222

C6H18O3Si3

Biohealth function

Xie and Li 2010

Figure 33a: GC – MS chromatogram of A. lanata methanol in vivo leaf extract

Figure 33b: GC – MS chromatogram of A. lanata methanol in vitro leaf extract

Figure 33c: GC – MS chromatogram of A. lanata methanol callus leaf extract

Table 58: FT – IR analysis of A. javanica samples Vibrations

O-H stretch.

N-H stretch (amines)

cm-1

AJ-FL-MT AJ –FL-F1 AJ-IL-MT AJ-IL-F1 AJ-CA-MT AJ-CAF1

3396 3411 3371 3395 3396 3415

C-H stretch. 2946 2948 2946 2947 2943 2969

-O-CH3 asym. stretch.

C-H stretch (aldehydes)

2834 2838 2834 2839 2828 2842

S-H stretch. 2531 2524 2528 2523 2522 2523

C=C stretch.

C=N stretch

N-H defor.

O=C-O asym. stretch.

NO2 stretch.

C=C stretch.

C=N stretch.

-N=N stretch.

C=O stretch. (phenols, ketones, aldehydes, carboxylic

acids, carboxylic ester, lactones,amides)

C-H defor.

O-H bending in plane

C=O stretch.

2048 2048 2046 2074 2053 2075 1567 1649 1570 1648 1571 1647 1404 1412 1404 1414 1406 1412

Contd...

R-O-R stretch.

C-OH stretch.

C-H stretch. (amine)

1118 1112 1117 1112 1118 1112

C-OH stretch.

P-O strerch.

C-OC asym. Stretch (ether)

1027 1052 1026 1053 1027 1031

O-H deform.

C-H stretch.

- - - - 953 1015

C-N vib.

N-O stretch.

C-H bending (out of plane). (aromatic hydrocarbones)

- - - - 829 -

O-H bending (out of plane)

C-O stretch.

C-N stretch.

C-H bending (out of plane), (aromatic hydrocarbones)

751 - 740 - 752 -

C-H bending. (alkynes)

N-H bending out of plane (amide, lactone)

696 - 696 - - -

C-H bending (out of plane). (aromatic hydrocarbones) - - - - 702 691

O-N=O defor. 652 660 653 659 653 -

AJ – IL – MT = A. javanica in vitro leaf Methanol extract, AJ – FL – MT = A. javanica in vivo leaf Methanol extract, AJ – CA – MT =

A. javanica callus Methanol extract; AJFLF1 – A. javanica field leaf fraction 1, AJILF1 – A. javanica in vitro leaf fraction 1, AJCAF1 –

A. javanica callus fraction 1.

Figure 34a: FT – IR spectrum for crude sample of A. javanica (AJ MT FL – A. javanica methanol field leaf)

Figure 34b: FT – IR spectrum for fraction of A. javanica (AJ FL F1 – A. javanica field leaf fraction 1)

Figure 35a: FT – IR spectrum for crude sample of A. javanica (AJ MT IL A. javanica methanol in vitro leaf)

Figure 35b: FT – IR spectrum for fraction of A. javanica (AJ IL F1 – A. javanica in vitro leaf fraction 1)

Figure 36a: FT – IR spectrum for crude sample of A. javanica (AJ MT CA A. javanica methanol callus)

Figure 36b: FT – IR spectrum for fraction of A. javanica (AJ CA F1 – A. javanica callus fraction 1)

Table 59: FT – IR analysis of A. lanata samples

Vibrations cm-1

AL FL MT AL FL F1 AL IL MT AL IL F1 AL CA MT AL CA F1

O-H3 Trans stretch (amines) 3433 3372 3370 3368 3408 3412

C-H stretch. 2923 2946 2947 2946 2922 2948

-O-CH3 asym 2858 2866 - 2867 - 2836

C-H stretch (aldehydes) 2360 2221 2213 2221 2360 -

C=C stretch, C=O stretch 2098 2046 - 2047 - 2074

NO2 stretch, O=C-O asym 1717 - - - 1717 -

O-H bending in plane, C=O stretch. (Ketones, aldehydes, carboxylic acids, carboxylic ester, lactones,amides)

1629 1659 1571 1662 1616 1650

C-H defor. O-H bending in plane C=O stretch.

1455 1453 1477 1453 1457 1454

C-H stretch. (amine) 1368 1415 1403 1414 1373 1411

R-O-R stretch. 1274 1113 - 1113 1219 1113

C-OH stretch. 1219 - - - - -

P-O stretch 1088 - 1120 - 1092 -

S=O Str - 1027 1026 1028 - 1026

C-H def (benzene ring with 1 free H) - - 927 - - -

C-H def (Benzene ring) - - 830 - - -

C-H bending (out of plane), (aromatic hydrocarbones) 770 - 753 - 771 -

C-H def (R1CH=CHR2) - - 696 - - 690

N-H bending out of plane (amide, lactone) 663 660 651 658 666 -

C-H - - 620 - - -

O-N=O defor. 598 - - - 595 -

O-N stretch 459 - - - 457 -

AL – IL – MT = A. lanata in vitro leaf Methanol extract, AL – FL – MT = A. lanata in vivo leaf Methanol extract, AL – CA – MT = A. lanata

callus Methanol extract, ALFLF1 – A. lanata field leaf fraction 1, ALILF1 – A. lanata in vitro leaf fraction 1, ALCAF1 – A. lanata callus fraction 1.

Figure 37a: FT – IR spectrum for crude sample of A. lanata (AL MT FL – A. lanata methanol field leaf)

Figure 37b: FT – IR spectrum for fraction of A. lanata (AL FL F1 – A. lanata field leaf fraction 1)

Figure 38a: FT – IR spectrum for crude sample of A. lanata (AL MT IL – A. lanata methanol in vitro leaf)

Figure 38b: FT – IR spectrum for fraction of A. lanata (AL IL F1 – A. lanata in vitro leaf fraction 1)

Figure 39a: FT – IR spectrum for crude sample of A. lanata (AL MT CA – A. lanata methanol callus)

Figure 39b: FT – IR spectrum for fraction of A. lanata (AL CA F1 – A. lanata callus fraction 1)

Re

lative A

bu

nd

an

ce

100 313.23

90

80

70

60

50

40 212.08

30

20

251.28

10 198.08 359.19

180.17

0

298.26

387.36

456.72 512.02 56 0.17 637.60 670.74 751.67

824.77

885.67 960.52

100 200 300 400 500 600 700 800 900 1000

m/z

Figure 42a: LC - MS spectrum of fractions of A. javanica (AJ-FL-F1 – A. javanica field leaf fraction 1)

Re

lative A

bu

nd

an

ce

100 251.26

90

80

70

60

50

40

30

208.23 20

180.26 10

313.22

149.09 276.48 334.99 0

404.58 462.69

560.96

652.34 707.92

752.18 827.88

917.04

981.35

100 200 300 400 500 600 700 800 900 1000

m/z

Figure 42b: LC - MS spectrum of fractions of A. javanica (AJ-IL-F1 – A. javanica in vitro leaf fraction 1)

Re

lative A

bu

nd

an

ce

100 299.23

90

80

70

60

50

40

30

269.23

20

10

139.09 0

241.22

221.04

371.60

447.48

489.28

615.37

664.76

756.99

837.93

910.26 961.53

100 200 300 400 500 600 700 800 900 1000

m/z

Figure 42c: LC - MS spectrum of fractions of A. javanica (AJ-CA-F1 – A. javanica callus fraction 1)

Re

lative A

bu

nd

an

ce

100 251.25

90

80

70

60

50

40

30

20

10

149.11

0

208.24

236.19 180.28

283.21 343.20

391.09

447.34 469.15

523.17

566.56 5 88.00 6 26.48

100 150 200 250 300 350 400 450 500 550 600

m/z

Figure 43a: LC - MS spectrum of fractions of A. lanata (AL-FL-F1 – A. lanata field leaf fraction 1)

Re

lative A

bu

nd

an

ce

100 343.22

90

80

70

60

50

40

30

20

10

192.13 0

313.22

285.21

256.31

234.84

391.12 430.95

513.69 579.33

649.31

697.69

759.82

831.30 916.06

985.99

100 200 300 400 500 600 700 800 900 1000

m/z

Figure 43b: LC - MS spectrum of fractions of A. lanata (AL-IL-F1 – A. lanata in vitro leaf fraction 1)

Re

lative A

bu

nd

an

ce

100 439.18

90

80

70

60

50

40

30

20

212.09 10

198.09 274.36 313.26 424.23

489.14 712.96

755.05

560.94 0

622.40 788.89 907.73 999.17

100 200 300 400 500 600 700 800 900 1000

m/z

Figure 43c: LC - MS spectrum of fractions of A. lanata (AL-CA-F1 – A. lanata callus fraction 1)

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