chapter 2: chapter 2 -...

Functional properties of banana rhizomeFunctional properties of banana rhizomeFunctional properties of banana rhizomeFunctional properties of banana rhizome

34

Chapter 2: Chapter 2: Chapter 2: Chapter 2:

Functional properties of pseudostem and Functional properties of pseudostem and Functional properties of pseudostem and Functional properties of pseudostem and

rhizome extracts of banana var. rhizome extracts of banana var. rhizome extracts of banana var. rhizome extracts of banana var.

Nanjanagudu RasbaleNanjanagudu RasbaleNanjanagudu RasbaleNanjanagudu Rasbale


35

Introduction

In Banana, underground corm is usually referred to a rhizome. It is closely

packed with leaf scars with short internodes. Rhizome has a central cylinder and

surrounding cortex separated by vascular bundles. Rhizome of banana is commonly used

as seed material for multiplication or propagation of plants in the commerce. Mass

production of disease free and high yielding variety of banana by tissue culture has

discouraged use of rhizome as seed material rendering it to underutilized and unexploited

biomass. Banana rhizome is often cooked and eaten as a vegetable mainly in tribal areas.

But indigenous ethno-medicine practices used its extracts to treat diabetes, piles,

intestinal worms, mental diseases, acidity, burns and wounds and pyorrhea

(Pushpangadan et al., 1989). Its extract is used as a coolant. In this study untapped

source of nutraceutical property of both rhizome and pseudostem was systematically

carried out to discover the underlying bioactive principles of var. Nanjanagudu Rasbale.

In addition, var. Nanjanagudu Rasbale is one of the exotic varieties fruits of

which have high demand due to their unique flavour and texture. Initial screening of

nutraceutical composition of eight commercial banana varieties revealed that highest total

phenolics and flavonoids were present in var. Nanjanagudu Rasbale. Concomitantly it

also exhibited highest antioxidant activities. Hence, detail investigations were carried

nutraceutical properties of pseudostem and rhizome of var. Nanjangudu Rasbale. A

systematic review of literature on research work on banana rhizome has revealed that,

there are no reports on bioactivity studies. Hence, different extracts from banana rhizome

were tested for an array of functional properties like antioxidant activity using different in

vitro assay models, antimicrobial activity using thirteen bacterial clinical isolates,

including seven Gram -ve bacteria and six Gram +ve bacteria and also six fungal strains.

The collagen induced platelet aggregation inhibitory activity by using chronolog dual

channel aggregometer and cytotoxicity by using HepG-2 cell lines. The details of the

work carried out are presented in this chapter.


36

Materials and methods

Plant material and preparation of extracts

Banana (Musa AAB var. Nanjanagudu Rasbale) plants (Wealth of India, 1962;

Venkatachalam, 2006) were identified and harvested from plantations in Mysore district

of Karnataka, India. After harvesting of fruit bunch, rhizome was removed from the soil,

washed, made into slices and dried in hot air oven at 45ºC for 24 hour and powdered to

60 mesh in an apex grinder. 1 Kg of powdered sample was then extracted serially using

serial extraction procedure as given in chapter 1 (materials and methods).

Identification and quantification of polyphenolic compounds

Quantification of polyphenolic compounds

Determination of total phenolic content (TPC)

As mentioned in chapter 1 (materials and methods).

Determination of total flavonoid content (TFC)

As mentioned in chapter 1 (materials and methods).

Separation of polyphenolic compounds

Reversed phase high performance liquid chromatography-diode array detector

(RPHPLC- DAD)

The polyphenolic compounds of acetone and methanol extract of banana rhizome

var. Nanjanagudu Rasbale were separated by RPHPLC, on C-18 column (model LC-10A,

Shimadzu Corporation, Japan), using a diode array detector (DAD) operating at 220, 280

and 320 nm. An isocratic solvent system, consisting of methanol: water: trifluoro acetic

acid (89.5:10:0.5), was used as a mobile phase at a flow rate of 1mL/min. Standards of

gallic acid, synergic acid, tannic acid, caffeic acid, gentisic acid, vanillic acid, trans


37

cinnamic acid, ferulic acid, protocatechuic acid, p-coumaric acid, catchol acid,

chlorogenic acid, pyrocatechol and epicatechin were used for identification of

polyphenolic compounds.

Direct infusion electrospray insertion mass spectrometry (ESI-MS)

The ESI-MS fingerprints of polyphenolic compounds of acetone and methanol

extract of banana rhizome var. Nanjanagudu Rasbale were obtained with an Alliance,

Waters 2695 mass spectrometer (Waters corporation, Micromass Ltd, UK) operating at

ESI (-ve mode). The capillary voltage was 3.0 kV; source and desolvation temperatures

were 120°C and 300°C, respectively; cone gas (argon) and desolvation gas (nitrogen)

flow rates were 50 1 h -1

and 500 l h-1

, respectively. The m/z values of the different

polyphenolic compounds available in the literature were used to match the m/z values in

the spectra obtained for the rhizome extracts.

Antioxidant activities (AOA) measurement

There is no simple universal method by which AOA can be measured accurately

and quantitatively and many methods have been developed based on different

mechanisms that generated different radicals and/or target molecules, oxidation

parameters, antioxidant reaction conditions and different endpoints. Considering the

multifaceted aspects of antioxidants and their reactivity, eight different antioxidant assays

viz., DPPH radical scavenging activity (DPPH RSA), superoxide radical scavenging

activity (SRSA), β-carotene bleaching inhibition (βCBI) assay, anti-lipid peroxidation

(ALPO) activity, metal chelating activity (MCA), hydrogen peroxide scavenging activity

(HPSA), nitric oxide scavenging activity (NOSA), total reducing power (TRP) assay

were applied. The antioxidant activity results of pseudostem and rhizome extracts were

expressed in EC50 value, which represents the sample concentration required to show

50% antioxidant activity. The methodology followed as described in the chapter 1.

(materials and methods), except for total reducing power (TRP) assay, which is given

below.


38

Total reducing power (TRP) assay

Reaction mixture, containing 10-300 µL (1 mg/mL stock) of pseudostem and

rhizome extracts/ascorbic acid in phosphate buffer (0.2 M, pH 6.6), was incubated with

K3Fe(CN)6 (1% w/v) at 50°C for 20 min. The reaction was terminated by the addition of

TCA solution (10% w/v) and the mixture was centrifuged at 3000 rpm/min speed for 10

min. The supernatant was mixed with distilled water and ferric chloride (0.1% w/v)

solution and left to incubate for another 10 min, and the formation of ferrous ion (Fe2+

)

was measured at 700 nm using UV-Vis Spectrophotometer (UV-160A, Shimadzu co.

Japan). Higher absorbance values indicative of greater reducing capacity of ferric (Fe3+

)

to ferrous (Fe3+

) ions. The sample concentration providing 0.5 OD of absorbance (EC50)

was calculated from the graph of absorbance at 700 nm against standard ascorbic acid

(Hsu et al., 2006).

Stability of the extracts

The pseudostem and rhizome extracts were stored at 27°C for 36 months. The

AOA of these stored extracts were compared with that of freshly prepared extracts. No

significant difference (p < 0.05) was observed using the different AOA assays mentioned

above.

In vitro screening for antimicrobial activity

Bacterial and fungal species and culture conditions

As described in the chapter 1, materials and methods section

Determination of antibacterial activity

Agar well-diffusion method



39

Determination of minimum inhibitory concentration (MIC)

The minimum inhibitory concentration was determined according to the method

described by Jones et al. (1985). Different concentrations (10-1000ppm) of crude extracts

of pseudostem and rhizome extracts, 100 µL of the bacterial suspension (105 CFU mL

-1)

was placed aseptically in 10 mL of nutrient broth separately and incubated for 24 h at

37ºC. The growth was observed both visually and by measuring OD at 600 nm at regular

intervals followed by pour plating as described earlier. The lowest concentration of the

test sample showing no visible growth was recorded as the minimum inhibitory

concentration. Triplicate sets of tubes were maintained for each concentration of the test

sample.

Antifungal activity assays


Minimum inhibitory concentration (MIC) agar dilution assay

MIC values of the fungal isolates were studied based on the agar dilution method

as described before (Gulluce et al., 2003). The pseudostem and rhizome extracts was

added aseptically to sterile melted PDA medium containing Tween 20 (0.5%, v/v) at the

appropriate volume to produce the concentration range of 10-1000 ppm. The resulting

PDA agar solutions were immediately poured into petri plates after vortexing. The plates

were spot inoculated with 10 µL of each fungi isolate. The inoculated plates were

incubated at 27°C for 7 days. At the end of incubation period, the plates were evaluated

for the presence or absence of growth. MIC values were determined as the lowest

concentration of the sample where absence of growth was recorded. Each test in this

study was repeated thrice.


40

Platelet aggregation inhibitory activity

Platelet preparation

Blood samples were taken from healthy volunteers who assured not to have taken

any drugs during the 2 weeks prior to the blood sampling. Blood was collected into

buffered sodium citrate (3.8% w/v) pH 6.5 as the anticoagulant at a ratio of 9:1 v/v and

used within 3 hr of collection. Platelet-rich plasma (PRP) was obtained by centrifugation

of the citrated blood at 1100 rpm for 20 min the residual blood was again centrifuged at

2500 rpm for 20 min to obtain the homologous platelet poor plasma (PPP). Platelet count

was adjusted to 1.6 x 107 platelets per µL of PRP.

Platelet-aggregation assay

Aggregation was measured turbidimetrically at 37°C with constant stirring at 1000

rpm in a Chronolog Dual Channel Aggregometer. About 0.45 mL of PRP was kept stirred

at 1200 rpm at 37°C, and aggregation was induced by collagen (10 µM). The change in

turbidity was recorded with reference to PPP using an omniscribe recorder for at least 5

min. The slope was calculated and it was used as control.

Similarly, different concentrations of pseudostem and rhizome extracts were added

to PRP, incubated for five min after which collagen (10 µM), was added. Platelet

aggregation was recorded using an omniscribe recorder for 5 min. The slope was

calculated. The difference in the slope between the control and the treated were expressed

as percent inhibition of platelet aggregation by banana pseudostem and rhizome extracts.

Cytotoxicity of banana pseudostem and rhizome extracts

Chemicals

Sulforhodamine B [SRB], 3-[4, 5-dimethyl thiazol-2-yl]-5-diphenyl tetrazolium

bromide [MTT], Fetal calf serum [FCS] were obtained from Sigma Aldrich Co, St Louis,


41

USA., Phosphate Buffered Saline [PBS], Dulbecco’s Modified Eagle’s medium [DMEM] and antibiotics from Hi-Media Laboratories Ltd., Mumbai., Trichloro acetic acid

[TCA] and tris buffer from SD fine chemicals Pvt. Ltd., Boisar, India., 25 cm2 and 75

cm2 tissue culture flasks, 96 well microtitre plates were procured from Tarson India Pvt.

Ltd., Kolkata, India, DMSO, glacial acetic acid and propanol from E-Merck Ltd.,

Mumbai, India.

Preparation of test solutions

For cytotoxicity studies, each extracts were dissolved in dimethyl sulphoxide

[DMSO] and volume was made up to 10 mL with DMEM, pH 7.4, supplemented with

2% inactivated FCS [maintenance medium] to obtain a stock solution of 1 mg/mL

concentration and sterilized by filtration and stored at -20°C till use. Serial two fold

dilution of the extracts was prepared from the stock solution to obtain lower

concentrations.

Cell lines and culture medium

HepG-2 a tumor cell line was procured from National Centre for Cell Sciences

[NCCS], Pune, India, and maintained at CFTRI cell culture facility. The cells were

cultured in DMEM medium supplemented with 10% FBS, anti-bacterial penicillin and

streptomycin (100 units/mL) and an anti-fungal agent amphotericin B [5 µg/mL] at 37°C in

a humidified atmosphere with 95% air and 5% CO2. Cells were grown up to confluency

in 25 cm2 flask. The cells were dissociated with TPVG solution [0.2% trypsin, 0.02%

EDTA, 0.05% glucose in PBS]. Further, the cells were plated on to 96 well microtitre

plates to study the cytotoxicity of extracts.

Determination of mitochondrial synthesis by MTT assay

The ability of the cells to survive a toxic insult has been the basis of most

cytotoxicity assays. This assay is based on the assumption that dead cells or their


42

products do not reduce tetrazolium. The assay depends both on the number of cells

present and on the mitochondrial activity per cell. The cleavage of MTT to a blue

formazan derivative by living cells is clearly a very effective principle on which the assay

is based. The principle involved is the cleavage of tetrazolium salt 3-[4, 5 dimethyl

thiazole-2-yl]-2, 5-diphenyl tetrazolium bromide [MTT] into a blue coloured product

[formazan] by mitochondrial enzyme succinate dehydrogenase. The extent of MTT

conversion requires the active mitochondrial succinate dehydrogenase enzyme and thus

the extent colour change indicates cell viability status of cells (Francis and Rita, 1986).

Procedure

Cells (~ 10,000 cells/well) were cultured with the DMEM medium containing

10% FCS in a 96 well microplate. After 24 h, when a partial monolayer was formed, the

supernatant was flicked off, washed the monolayer once with medium and 100 µl of

different extract concentrations were added to the cells in microtitre plates. The cells

without extracts are considered as control cells. The plates were then incubated at 37°C

for 3 days in 5% CO2 atmosphere, and microscopic examination was carried out and

observations were noted every 24 h. After 72 h, the extract solutions in the wells were

discarded and 50 µl of MTT in DMEM-PR [Dulbecco’s Modified Eagle’s medium

without phenol red, 2 mg/mL] was added to each well. The plates were gently shaken and

incubated for 3 h at 37°C in 5% CO2 atmosphere. The supernatant was removed and 50

µl of propanol was added and the plates were gently shaken to solubilize the formed

formazan. The absorbance was measured using a microplate reader at a wavelength of

540 nm. The percentage growth inhibition was calculated using the following formula

and CTC50 [concentration of drug or test extract needed to inhibit cell growth by 50%]

values were generated from the dose-response curves for each cell line. The pattern of the

cell lines as a group is used to rank compounds as toxic or non-toxic.

Growth Inhibition (%) = Mean OD of control- Mean OD of Sample

Mean OD of control x 100


43

Statistical analysis

Results were expressed as mean ± standard deviation of triplicate analysis. Data

are analyzed by one-way analysis of variance (ANOVA) and post-hoc mean separations

were performed by Duncan’s Multiple Range Test (DMRT) at p < 0.05. The data were

analyzed by using Microsoft Excel XPR (Microsoft Corporation, USA) software.

Results and Discussion

Percent contribution of different Bio-mass of var. Nanjanagudu Rasbale plant

Banana pseudostem occupies highest percentage (30.81%) of banana plant, while

the fruit bunch constitutes half the percent (15.95%) of banana plant. Rhizome occupies

the 5th

position with 12.67%, next to leaf and leaf base (23.14%), and leaf sheath

(15.05%). Pseudostem and rhizome together constitute 43.48% of the banana plant bio-

mass (Fig. 2.1). Which were underutilized and unexplored for their nutraceutical

importance. Currently < 2% of pseudostem production is used for human consumption

Figure 2.1: Biomass composition (per cent) of different parts of banana plant var.

Nanjanagudu Rasbale

Leaf & leaf base

23.14%

Flower & bract

1.41%

Fruit bunch

15.95%

Leaf sheath

15.05%

Pseudostem

30.81%

Rhizome

12.67%

Roots

0.97%


44

and for production of fiber (Uma et al., 2005), remaining are incinerated and wasted. The

systematic investigation carried out on bioactive composition of pseudostem and rhizome

of banana var. Nanjanagudu Rasbale is detailed below.

Yield of different solvent extracts

The results of using different solvents for the extraction of polyphenolic

compounds are given in table 2.1. From this table, it is clear that the different solvents

had different abilities to extract substances from rhizome and pseudostem. Methanol had

the highest extraction yield (2.26 and 2.23% w/w respectively) followed by hexane (2.02

and 1.53% w/w respectively), acetone (0.88 and 0.77% w/w respectively), chloroform

(0.110 and 0.117% w/w respectively) and ethyl acetate (0.095 and 0.088% w/w

respectively). Significant (p<0.05) differences in the yield of rhizome and pseudostem

extracts may be attributed to availability and solubility of extractable components in the

solvents used for extraction process (Hsu et al., 2006).

Table 2.1: Extract yield, total phenolic content and total flavonoid content of

different solvent extracts from pseudostem and rhizome of banana var. Nanjanagudu

Rasbale

Crude

extracts

Extract yield

(% w/w)

Total phenolic content

(mg GAE /g of extract)

Total flavonoid content

(mg CE /g of extract)

Rhizome Pseudostem Rhizome Pseudostem Rhizome Pseudostem

Hexane 2.02±0.05b 1.53±0.03b 23.3±1.90e 12.7±1.10e 11.2±1.70e 4.8±0.90e

Chloroform 0.110±0.03d 0.117±0.02d 108.9±10.70c 36.6±2.24e 63.1±4.70cd 9.6±1.1e

Ethyl acetate 0.095±0.01e 0.088±0.01d 86.7±3.40d 49.0±3.9c 47.2±3.10cd 16.0±0.6c

Acetone 0.88±0.02c 0.77±0.02c 685.3±20.17a 291.0±22.47a 401.7±22.17a 80.0±4.0a

Methanol 2.26±0.06a 2.33±0.04a 285.8±13.46b 92.0±4.1b 200.4±12.58b 32.0±2.8b

Data expressed as mean ± standard deviation of triplicate measurements. Mean values with different superscripts differ

significantly at p<0.05; GAE-gallic acid equivalents, CE-catechin equivalents


45

Total phenolic and total flavonoid content of banana rhizome extract

The acetone extract of rhizome showed higher TPC and TFC (685.3 mg GAE and

401.7 mg CE/g of extract respectively) followed by methanol (285.8 mg GAE and 200.4

mg CE/g of extract respectively), chloroform (108.9 mg GAE and 63.1 mg CE/g of

extract respectively), ethyl acetate (86.7 mg GAE and 47.2 mg CE/g of extract

respectively) and hexane (23.3 mg GAE and 11.2 mg CE/g of extract respectively) and

also these values were statistically (p<0.05) significant (table 1). Whereas, in pseudostem

extracts also acetone showed higher TPC and TFC (291 mg GAE and 80 mg CE/g of

extract respectively), followed by methanol (92 mg GAE 32 mg CE/g of extract

respectively). The recovery of polyphenols from plant materials is reported to be

influenced by the solubility of the phenolic compounds in the solvent used for the

extraction process, chemical nature of the phenolic compounds, extraction method and

the assay method employed (Naczk and Shahidi, 2006). The quantity and quality of the

phenolic compounds present in plant can vary significantly due to different factors, such

as plant genetics and cultivar, soil composition and growing conditions, maturity state,

and post harvest conditions, and others (Jaffery et al., 2003).

Characterization of polyphenolic compounds

Identification and quantification of polyphenolic compounds by RPHPLC–DAD

and ESI-MS analysis

RPHPLC–DAD chromatogram of acetone and methanol extracts monitored at

280 nm (Figure 2.2) revealed the presence of polyphenolic compounds such as tannic,

catechol, gentisic, (+) catechin, protocatechuic, gallic, caffeic, chlorogenic and cinnamic

acids along with some unidentified peaks. These compounds have been identified

according to their retention time and the spectral characteristics of their peaks compared

to those of standards. Protocatechuic acid, (+) catechin and gentisic acid were detected to

be the major polyphenolic compounds detected in the acetone extract of banana rhizome,

with about 56.42, 49.91 and 41.23 µg/mg, respectively. Other important polyphenolic


46

compounds recorded were caffeic, chlorogenic, catechol and cinnamic acids at the

concentration of 39.06, 32.55, 30.38 and 28.21 µg/mgm respectively (Table 2.2). Results

demonstrated a quantitative and qualitative difference in polyphenolic compounds in

acetone extract of banana rhizome was observed.

Table 2.2: Identification and quantification of polyphenolic compounds of acetone

extract from banana var. Nanjanagudu Rasbale by RPHPLC-DAD and ESI-MS analysis

Peak

No

Polyphenolic

compounds

Retention

Time (min)

RPHPLC-DAD

(µg/mg of extract)

ESI-MS

[M–H]¯ (m/z)

1 Synergic acid 2.016 - -

2 Tannic acid 2.955 13.02 -

3 Pyrocatechol 3.542 - -

4 Catechol acid 5.618 30.38 -

5 Gentisic acid 6.848 41.23 153.12

6 (+)-Catechin 11.701 49.91 289.73

7 Vanillic acid 12.601 - -

8 Protocatechuic

acid 14.517 56.42 153.12

9 Gallic acid 16.971 15.19 -

10 P-Coumaric acid 20.501 - -

11 Caffeic acid 21.077 39.06 179.24

12 Chlorogenic acid 22.910 32.55 354.03

13 Ferulic acid 26.605 - 193.31

14 Cinnamic acid 30.872 28.21 147.16

Direct infusion electrospray ionisation mass spectrometry (ESI-MS) were used to

identify the polyphenols that were detected in the RPHLPC-DAD chromatogram. The

ESI-MS fingerprint with different molecular weight ranging from 100–500 Da of acetone

extract of banana rhizome var. Nanjanagudu Rasbale is shown in fig. 2.3 The presence of

polyphenolics compounds like, cinnamic acid, gentisic acid, protocatechuic acid, caffeic

acid, (+) catechin and chlorogenic acid in RPHPLC-DAD chromatogram were also

confirmed by their m/z values from ESI-MS fingerprints (Table 2.2).


47

Figure 2.2: RPHPLC-DAD chromatograms of polyphenolic compounds from

acetone extract of banana rhizome var. Nanjanagudu Rasbale. Peak no. 2-

Synergic acid, 4-Catechol acid, 5-Gentisic acid, 6-Catechin, 8-Protocatechuic acid,

9-Gallic acid, 11-Caffeic acid, 12-Chlorogenic acid, 13- Ferulic acid and 14-

Cinnamic acid

Figure 2.3: ESI-MS fingerprints (zoomed spectra) of acetone extract of banana

rhizome var. Nanjanagudu Rasbale


48

Antioxidant activities of different extracts of banana rhizome

DPPH radical scavenging activity (DPPH RSA)

A DPPH radical-generating system offers a convenient and accurate method for

titrating the oxidizable groups of natural and synthetic antioxidants. Unlike laboratory-

generated free radicals such as hydroxyl radical and superoxide anion, DPPH radical has

the advantage of being unaffected by side reactions, such as metal ion chelation and

enzyme inhibition (Amarowicz et al., 2004). The scavenging activity of pseudostem and

rhizome extracts were tested using a methanol solution of the ‘stable’ free DPPH radical

(Table 2.3). Rhizome extracts exhibited a strong ability to quench DPPH radicals when

compared to pseudostem extracts. Acetone extract of rhizome and pseudostem presented

highest antioxidant activity (EC50 value of 16 and 76 µg mL−1

respectively), followed by

methanol extract (72 and 141 µg mL−1

respectively). This radical scavenging ability of

rhizome and pseudostem extracts could be related to the nature and concentration of

polyphenolic compounds, thus contributing to their electron transfer/hydrogen donating

ability.

Table 2.3: Antioxidant activity (EC50 value µg mL−1

) of pseudostem and rhizome

extracts of banana var. Nanjanagudu Rasbale and standards

Sl.

No

Antioxidant

assays

Rhizome extracts Pseudostem extracts Standards

CHCl3 Et.Ace AcO MeOH Et.Ace AcO MeOH

1 DPPH RSA 152±2.3b 176±4.2

a 16±0.6

d 72±1.3

c 260±4.8

c 76±3.2

a 141±1.3

b BHT <10

2 SRSA 209±3.7b 248±6.2

a 27±1.8

d 120±2.0

c - 85±2.5

a 165±2.5

b BHT <10

3 βCBI assay 118±2.5b - 33±1.3

c 187±5.1

a - 119±1.9

a 290±6.2

b BHT <10

4 ALPO activity 134±4.0b - 61±1.8

c 181±3.9

a - 141±2.6

a 235±4.9

b BHT <10

5 MCA - - 92±2.6a 24±1.1

b - 156 ±3.7

b 85±2.1

a EDTA <10

6 HPSA - - 54±2.0a 202±4.5

b - 155±3.9

a 260±5.2

b AA <10

7 NOSA - - 30±1.6a - - 120±3.6

a 240±5.6

b CN <10

8 TRP♣ 90±2.0

c - 6.5±0.2

a 21±1.5

b - 26±1.8

a 44±2.2

b AA 24±2.0

Each value represents means ± SD (n = 3) and values with different superscripts differ significantly at p<0.05. EC50: Effective concentration of

the sample to show 50% of antioxidant activity. Hex-Hexane, CHCl3-Chloroform, Et.Ace-Ethyl acetate, AcO-Acetone and MeOH-methanol;

EDTA- ethylenediamine tetra-acetic acid, AA-ascorbic acid, CN-curcumin,; DPPH RSA-1,1-diphenyl-2-picrylhydrazyl radical scavenging

activity, SRSA-superoxide radical scavenging activity, βCBI-β-carotene bleaching inhibition assay, ALPO-Anti-lipid peroxidation activity,

MCA-metal chelating activity, HPSA-hydrogen peroxide scavenging activity, NOSA- Nitric oxide scavenging activity and TRP-total reducing

power assay; ♣sample concentration to get 0.5 of absorbance at 700 nm


49

Superoxide radical scavenging activity (SRSA)

Superoxide radicals are generated during the normal physiological process mainly

in mitochondria. Although superoxide anion is by itself a weak oxidant, it gives rise to

the powerful and dangerous hydroxyl radicals as well as singlet oxygen both of which

contribute to the oxidative stress (Dahl and Richardson, 1978). Superoxide anion plays an

important role in the formation of reactive oxygen species [ROS] such as hydrogen

peroxide, hydroxyl radical, and singlet oxygen, which induce oxidative damage in lipids,

proteins and DNA (Dahl and Richardson, 1978; Halliwell and Gutteridge, 1991).

Therefore superoxide radical scavenging by antioxidants has physiological implications.

The data presented in table 2.3, indicates that rhizome and pseudostem extracts especially

acetone is a strong superoxide anion quencher (EC50 value of 27 and 85 µg mL−1

respectively). The second most active extract is methanol, which showed EC50 value of

120 and 165 µg mL−1

respectively. These results suggest that the polyphenolic

constituents of rhizome and pseudostem extracts, notably phenolics and flavonoids

display scavenging effect on superoxide anion radical generation that could help to

prevent oxidative damage of the major bio-molecules like proteins and lipids.

Β-carotene bleaching inhibition (βCBI) assay

The antioxidant activity of the pseudostem and rhizome extracts determined by

the β-carotene-linoleic acid model system was presented in table 2.3. The oxidation of

linoleic acid generates peroxyl free radicals due to the abstraction of hydrogen atom from

diallylic methylene groups of linoleic acid (Kumaran and Karunakaran, 2006). This

conjugated diene hydroperoxides attack the β-carotene molecule and as a consequence it

undergoes rapid decolorization. The corresponding decrease in absorbance can be

monitored spectrophotometrically at 470 nm. The presence of an antioxidant can reduce

the extent of β-carotene destruction by reacting with the linoleate free radical or any other

free radical formed within the system. Activity of the samples at the concentrations of 10-

300 µg/mL was reflected in their ability to inhibit the bleaching of β-carotene. Acetone


50

extract again displayed highest βCBI inhibition activity (EC50 value of 33 and 119 µg

mL−1

for rhizome and pseudostem respectively). Interestingly, chloroform extract from

banana rhizome also possessed better βCBI inhibition activity (EC50 value of 118 µg

mL−1

) than methanol extract (EC50 value of 187 µg mL−1

). Although chloroform extract

contained a lower TPC and TFC than methanol extract, proportionally higher antioxidant

activity demonstrated that the type of polyphenols rather than the amounts is responsible

for antioxidant activity. Differences in antioxidant activity of plant extracts could be due

to structural differences of phenolic and flavonoid compounds and their derivatives.

Antioxidant activity in this complex heterogeneous medium suggests that rhizome and

pseudostem extracts have a potential use as an antioxidant preservative in emulsion type

systems.

Anti-lipid peroxidation (ALPO) activity

Lipid peroxidation involves in the genesis of several pathophysiological

conditions, such as atherosclerosis, ischemia, liver disorder, neural disorder, and pesticide

toxicity (Ames et al., 1993). In this method, lipid peroxidation is initiated in the rat liver

microsomes, which leads to the production of MDA. The lipid peroxidation can be

indirectly quantified by derivatising MDA with TBA at high temperature and acidic

conditions resulting in the formation of diadduct, which is a pink chromogen and can be

detected spectrophotometically at 532 nm. The effect of pseudostem and rhizome extracts

on lipid peroxidation is presented in (Table 2.3). The rhizome and pseudostem extracts

showed ALPO activity with EC50 value between 61 and 235 µg mL−1

. The acetone

extract of banana rhizome showed better ALPO activity as compared to other extracts

(EC50 value between 61 µg mL−1

). Even though the TPC and TFC of chloroform extract

is quite low, it showed comparatively higher ALPO activity (EC50 value of 134 µg mL−1

)

than methanol extract of rhizome and pseudostem (EC50 value of 181 and 235 µg mL−1

respectively). This may be attributed to the type of compounds extracted by chloroform

extract, which may possess higher activity as compared to the compounds extracted by

other solvents.


51

Metal chelation activity (MCA)

Ferrous ions (Fe2+

) are the most powerful pro-oxidants among the various species

of metal ions (Halliwell and Gutteridge, 1999) and these free transition metals react with

either hydrogen or lipid peroxides to produce hydroxyl and alkoxyl radical compounds

(fenton reaction). These radicals are extremely reactive and contribute to oxidative stress.

Minimizing ferrous ion may afford protection against oxidative damage by inhibiting

production of ROS. The results demonstrate that the effectiveness of pseudostem and

rhizome extracts in inhibiting the formation of ferrous and ferrozine complex and

thereby, indicates the iron binding capacity. Unlike in other antioxidant activities,

methanol extracts from rhizome and pseudostem showed high chelating activity (EC50

value of 24 and 85 µg mL−1

respectively) followed by acetone (EC50 value of 92 and 156

µg mL−1

respectively) and others (Table 2.3). This might be due to presence of high TPC,

TFC and polyphenolic compounds.

Hydrogen peroxide scavenging activity (HPSA)

The H2O2 scavenging ability of rhizome and pseudostem extracts is shown in

table 2.3 and compared with standard ascorbic acid. The result explains that rhizome and

pseudostem extracts were capable of scavenging H2O2. High H2O2 scavenging activity

with low EC50 value of 54 and 155 µg mL−1

were observed in acetone extract of rhizome

and pseudostem respectively, followed by methanol extract (EC50 value of 202 and 260

µg mL−1

respectively). These results show that rhizome extracts had strong H2O2

scavenging activity than pseudostem extracts. In the present study the polyphenolic

compounds in the rhizome and pseudostem extracts may probably be involved in H2O2

scavenging activity. Polyphenols have been shown to protect mammalian and bacterial

cells from cytotoxicity induced by hydrogen peroxide, especially compounds with the

orthodihydroxy phenolic structure quercetin, catechin, gallic acid, caffeic acid and its

esters (Halliwell, 1991).


52

Nitric oxide scavenging activity (NOSA)

NO or RNS, such as NO2, N2O4, N3O4, NO3¯ , and NO2¯ , are very reactive and

considered to be potentially cytotoxic and capable of injuring the surrounding cells, and

also responsible for altering the structural and functional behavior of many cellular

components. NO is a defence molecule with cytotoxic, microbiocidal, and microbiostatic

activities, however, it is also implicated in inflammation, cancer, and other pathological

conditions (Halliwell and Gutteridge, 1984). As shown in table 2.3 the rhizome and

pseudostem extracts showed nitric oxide scavenging activity with the EC50 value in the

range of 30-240 µg mL−1

. In particular, NOSA of acetone extract from banana rhizome

was found to be highest (EC50 value of 30 µg mL−1

), indicating that the high polyphenols

present acetone extract showed high scavenging activity by inhibition on nitrite

production. Whereas, among pseudostem extracts, acetone and methanol extracts showed

NOSA with EC50 value of 120 and 240 µg mL−1

respectively. Surprisingly, methanol

extract of banana rhizome failed to show EC50 value even upto the concentration of 300

µg/mL. The rhizome and pseudostem extracts rich in polyphenols may have the property

to counteract or preventing the ill effects of excessive NO generation in the human body.

Further, the NO scavenging activity may also help to arrest the chain of reactions

initiated by excess generation of NO that are detrimental to human health.

Total reducing power (TRP) assay

The reducing power of acetone and methanol extracts from banana rhizome (EC50

value of 6.5 and 21 µg mL−1

, respectively) and acetone extract from banana pseudostem

(EC50 value of 26 µg mL−1

) were higher than that of natural antioxidant ascorbic acid

(EC50 value of 24 µg mL−1

) (Table 2.3). The reducing power capacity of a rhizome and

pseudostem extracts may serve as a significant indicator of antioxidant property by

donates electrons to reactive free radicals species, thus promoting the termination of free

radical chain reactions. The ability of the rhizome and pseudostem extracts to reduce Fe3+

to its more active Fe2+

form might also be indicative of its ability to act as a prooxidant.


53

Rhizome polyphenols and their multiple antioxidant activities

These data established that the antioxidant activities of rhizome and pseudostem

extracts could be attributed to their polyphenolic compounds as reported in various plant

extracts (Kamatou et al., 2010; Kubola and Siriamornpun, 2008). In this study, rhizome

extracts showed good antioxidant activity in all the eight models tested. However, the

magnitude of antioxidant activity varies with the type of extracts. This could be due to the

difference in concentrations and type of antioxidative compounds present in these

extracts. Polyphenols (phenolics, flavonoids and phenolic acids) are considered to be the

most active natural antioxidants and act as antioxidants by hydrogen/electron donators,

reducing agents, scavenging free radicals and terminate radical chain reactions, and

chelating transition metal ions (Karaman et al., 2010). The superiority of acetone extract

of banana rhizome antioxidant activities as compared to other extracts may be explained

by the amount and nature of individual polyphenolic compounds. Chromatogram

obtained from RPHPLC-DAD analysis depicts higher concentration of protocatechuic

acid, (+) catechin, gentisic acid, caffeic acid, chlorogenic acid, catechol acid and

cinnamic acids that were detected in acetone extract and conferred strong antioxidant

activities. ESI-MS fingerprint depict the presence of cinnamic acid, gentisic acid,

protocatechuic acid, caffeic acid, ferulic acid, (+) catechin and chlorogenic acid which

were detected by RPHPLC-DAD chromatogram.

Antimicrobial activities of different extracts from banana pseudostem and rhizome

Hexane extract of banana rhizome appears to inhibit the six Gram +ve bacteria

tested. Whereas, choloroform, acetone and methanol extracts also inhibit the five Gram

+ve bacteria tested, except S. aureus. High activity against Gram +ve bacteria with low

MIC values were observed in chloroform extract of banana rhizome (90, 200 and 220

ppm against L. monocytogenes, M. luteus and E. fecalis respectively.) followed by

acetone, hexane and methanol extracts. In banana pseudostem extracts, chloroform

exhibited wide range of inhibition against six Gram +ve bacteria tested. Acetone extract

showed antibacterial against selected Gram +ve bacterias like M. luteus, B. Subtilis and


54

L. Monocytogenes. Surprisingly, methanol extract did not show inhibitory effect against

any of the six Gram +ve bacteria tested. Interestingly, among Gram +ve bacteria, ethyl

acetate extract from banana rhizome and pseudostem was found to inhibit B. cereus only.

In general, banana rhizome extracts showed higher inhibitory activities against Gram +ve

baceteria when compared to banana pseudostem extracts (Table 2.4).

Table 2.4 Antimicrobial activity (MIC in ppm) of different extracts from rhizome and

pseudostem of banana var. Nanjanagudu Rasbale

Microbial species Rhizome extracts Pseudostem extracts

Hex Chl Et.AC AcO MeoH Hex Chl Et.AC AcO MeoH

Bacterial strains

Gram +ve

M. luteus 380 200 - 260 500 360 450 - 650 -

S. aureus 700 - - - - - 580 - - -

E. fecalis 280 220 - 770 750 360 400 - - -

B. cereus 400 290 650 300 940 500 370 750 - -

B. subtilis 530 280 - 450 580 440 390 - 550 -

L. monocytogenes 310 90 - 350 420 - 430 - 420 -

Gram -ve

P. aeruginosa - - - - 740 850 - - - -

E. coli - - - - - 380 220 - 460 -

S. typhi - 350 - - 800 350 - 540 - -

K. pneumoniae - - - - - - 250 - - -

E. aerogenes 330 - - - - 600 - - - -

P. mirabilis - - - - - - - - - -

Y. enterocolitica - - - - - - - - - -

Fungal strains

A. niger 840 350 - 620 - 200 650 - 730 -

A. flavus 460 710 - 700 - 220 580 - - 850

A. fumigatus - 530 - 460 - - - - - -

A. parasiticus - 750 - 450 - - - - - -

P. rubrum 900 - - - - - 500 - 820 -

F. moniliforme - - - - - 360 370 - - -

Each value represents means ± SD (n = 3) and values. Hex-Hexane, CHCl3-Chloroform, Et.Ace-Ethyl acetate, AcO-Acetone and

MeOH-methanol


55

Among the seven Gram -ve bacteria tested only P. aeruginosa and S. typhi were

found to be inhibited by methanol extract of banana rhizome. While hexane and

chloroform extracts showed higher inhibitory activity against E. aerogenes and P.

aeruginosa respectively. Among pseudostem extracts, hexane showed inhibitory activity

against maximum of four Gram -ve bacteria tested viz., P. aeruginosa, E. coli, S. typhi

and E. Aerogenes. Whereas, chloroform showed against two (E. coli and K. pneumoniae),

ethyl acetate and acetone extracts one each (S. typhi and E. coli respectively). .

Surprisingly, methanol extract did not show inhibitory effect against any of the six Gram

-ve bacteria tested. Results clearly indicate that Gram +ve bacteria are the most sensitive

microorganisms tested when compare to Gram -ve bacteria. The reason for different

sensitivity between Gram-positive and Gram negative bacteria could be ascribed to the

morphological differences between these microorganisms. The Gram +ve bacteria

contain an outer peptidoglycan layer, which is an ineffective permeability barrier

(Scherrer and Gerhardt, 1971), whereas Gram-negative bacteria have an outer

phospholipidic membrane carrying the structural lipopolysaccharide components. This

makes the cell wall impermeable to lipophilic solutes, while porins constitute a selective

barrier to the hydrophilic solutes with an exclusion limit of about 600 Da (Nikaido and

Vaara, 1985).

Antifungal activity of different solvent extracts of pseudostem and rhizome were

tested against six fungal strains reveals that Aspergillus species are the most susceptible

organisms than P. rubrum and F. moniliforme. Chloroform and acetone extract of banana

rhizome found to be effective against all the four Aspergillus species tested viz., A. niger,

A. flavus, A. fumigatus and A. parasiticus. Ethyl acetate and methanol extracts of banana

rhizome failed to inhibit any of the six fungal strains tested. Interestingly, P. rubrum was

inhibited only by hexane extract and all the five extracts form banana rhizome failed to

inhibit F. moniliforme. Among pseudostem extracts, chloroform and hexane extracts

showed better activity against fungal stains tested followed by ethyl acetate (against A.

niger and P. rubrum) and methanol (only against A. flavus). High antifungal activity with

low MIC values of 200 and 220 ppm were observed in hexane and chloroform extract of

pseudostem against A. niger and A. flavus respectively (Table 2.4).


56

The result obtained in this study indicates that the different extracts from rhizome

and pseudostem of banana var. Nanjanagudu Rasbale exhibits varying levels of

antibacterial activity. These differences could be due to the nature and level of the

antimicrobial agents present in the extracts, their mode of action on the different test

microorganisms, antimicrobials or active compounds of different polarity were present in

the extracts. This could indicate the influence of the soil nature, elevation, and other

environmental factors on the nature and level of antimicrobials synthesized by this

banana var. Nanjanagudu Rasbale. This antimicrobial activity results from crude extracts

of banana rhizome and pseudostem may be more beneficial than isolated constituents,

since a bioactive individual component can change its properties in the presence of other

compounds present in the extracts (Borchers et al., 2004).

Platelet aggregation inhibitory activity of banana rhizome and pseudostem extracts

Platelets readily aggregate in response to a variety of endogenous substances and

they can initiate thrombus formation, leading to ischemic diseases. In addition, the

interactions between platelets and blood vessel walls are important in the development of

thrombosis and cardiovascular diseases (Dinerman and Mehta, 1990). Therefore, the

inhibition of platelet function represents a promising approach for the prevention of

thrombosis. Regulation of platelet activity by using plants, which contains various

phytochemical constituents, has proven to be a successful strategy for the prevention of

thrombosis. Antiplatelet agents, such as aspirin, dipyridamole, thienopyridines, and

platelet glycoprotein IIb/IIIa antagonists have amply demonstrated their utility in

preventing and treating coronary artery thrombosis (Van De and Steinhubl, 2000;

Calverley, 2001). Plant extracts may be an alternative to currently used antiplatelet

agents, because they constitute a rich source of bioactive chemicals (Cho et al., 2004;

Lim et al., 2004) and free from adverse effects and have excellent pharmacological

actions, they could lead to the development of new classes of possibly safer and

antiplatelet agents (Cho et al., 2004; Lim et al., 2004; Tsai et al., 2000). The results on the

inhibition of platelet aggregation induced by the agonist collagen with extracts of

rhizome and pseudostem were presented in table 2.5. Among the different extracts, ethyl


57

acetate extract from rhizome and pseudostem showed higher activity in inhibiting

collagen induced human platelet aggregation (EC50 value 80 and 121 µg mL−1

respectively), followed by acetone (EC50 value 112 and 130 µg mL−1

respectively),

methanol (EC50 value 162 and 221 µg mL−1

respectively) and chloroform (EC50 value

274 and 298 µg mL−1

respectively). Whereas, hexane extract failed to show EC50 value

even upto the concentration of 300 µg/mL. The high platelet-aggregation inhibitory

activity of ethyl acetate, acetone and methanol extracts may be due to the presence of

polyphenols and other phytochemicals. Polyphenols like phenolics and flavonoids (Lin

and Lu, 1996; Tsai et al., 2000), and terpenoid compounds (Shen et al., 2000) were

reported to have effective inhibitory activities of platelet aggregation collagen induced by

various agonists like ADP, collagen and arachidonic acid). Thus, the active components

present in the rhizome and pseudostem extracts might exert their effects in platelet

aggregation inhibition.

Table 2.5 Platelet aggregation inhibitory activity (EC50

value* µg mL−1

) of different extract from rhizome and

pseudostem of banana var. Nanjanagudu Rasbale

Extracts Rhizome Pseudostem

Hexane - -

Chloroform 274±5.4d 298±6.0

d

Ethyl acetate 80±1.8a 121±3.2

a

Acetone 112±1.8b 130±2.4

b

Methanol 162±2.4c 221±4.6

c

Each value represents means ± SD (n = 3) and values with different superscripts differ

significantly at p<0.05. *EC50: Effective concentration of the sample to show 50% of platelet

aggregation inhibitory activity


58

Cytotoxicity of banana rhizome and pseudostem extracts

Cytotoxicity assays are widely used in vitro toxicology studies. In this present

study, the MTT (3-[4, 5- imethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay

was employed for the detection of cytotoxicity of HepG2 (human liver hepatocellular

carcinoma) cell line following exposure to different extracts of banana rhizome and

pseudostem (Table 2.6). Among the five extracts from banana rhizome tested, the

chloroform extract showed more toxicity (EC50 value 76 µg mL−1

), followed by acetone

(EC50 value 96 µg mL−1

), ethyl acetate (EC50 value 120 µg mL−1

), methanol (EC50 value

172 µg mL−1

) and hexane (EC50 value 204 µg mL−1

). Whereas, among pseudostem

extracts, chloroform extract showed more toxicity with low EC50 value of 82 µg mL−1

,

followed by ethyl acetate (106 µg mL−1

), acetone (126 µg mL−1

), methanol (135 µg

mL−1

) and hexane (284 µg mL−1

). The cytotoxicity results of different extracts from

banana rhizome and pseudostem indicate that, different type and quantity of active

principles present in these extracts might have responsible for difference in cytotoxicity.

The phytochemical rich extracts from rhizome and pseudostem of banana var.

Nanjanagudu Rasbale merit further investigations to identify the active principles

responsible for the cytotoxicity properties and to further prove the activity in animal

models.

Table 2.6 Cytotoxicity (EC50 value* µg mL−1

) of

different extract from rhizome and pseudostem of

banana var. Nanjanagudu Rasbale

Extracts Rhizome Pseudostem

Hexane 204±4.4e 284±5.7

e

Chloroform 76±1.0a 82±1.7

a

Ethyl acetate 120±1.9c 106±2.2

b

Acetone 96±1.2b 126±2.0

c

Methanol 172±2.6d 135±3.6

d

Each value represents means ± SD (n = 3) and values with different superscripts differ

significantly at p<0.05. *EC50: Effective concentration of the sample to show 50% of

cytotoxicity


59

Conclusion

The banana rhizome var. Nanjanagudu Rasbale extracts displayed various

bioactive properties like antioxidant activity, antimicrobial activity, platelet-aggregation

inhibitory activity and cytotoxicity. The current approach of serial extraction process of

rhizome powders with different solvents of increasing polarity has yielded fascinating

results. These results explain and validate the use of banana rhizome extracts in their

native form for therapeutic purpose emphasized in traditional medicine practices in India

and elsewhere. The acetone extract of banana rhizome showed high total phenolic and

total flavonoid content and multiple antioxidant activity in all the eight in vitro assays

tested, and chloroform extract displayed high antimicrobial activity against wide

spectrum of bacterial and fungal strains tested. Whereas, both extract exhibited platelet

aggregation inhibitory activity and cytotoxicity properties. Hence, acetone and

chloroform extract were selected for the isolation, purification and characterization of

bioactive molecules.

chapter 2: chapter 2 -...

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