Isolation, purification & characterization of BGS-Haridrinshodhganga.inflibnet.ac.in/bitstream/10603/76614/6/chapter 3.pdfdamages caused by oxidative stress and to fortify the defense

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CHAPTER -3

Isolation, purification & characterization of BGS-Haridrin

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Introduction

The use of antioxidants has been known to prevent harmful oxidation

reactions in biological systems. Antioxidants are of more interest because they may

help to protect the human body against reactive oxygen species (ROS) which are said

to be inductive factor for many diseases (Halliwell et al., 1995, Suleka Mandal et al.,

2009). The addition of antioxidants to food is the most effective way for delaying

lipid peroxidation, which is the reason for the unpleasant flavors.

In the food industry, synthetic antioxidants, such as butylated hydroxyanisole

(BHA), propyl gallate and tert-butyl hydroquinone are widely used. However, at

more than 0.1% doses, toxicity of the above may also be encountered (Van and Van,

1997). Their safety issues, however, are highly discussed, thus generating the need to

search for substitute materials from natural and safe sources as food antioxidants. The

natural antioxidants can be used in the food industry, and there is evidence that these

substances may exert their antioxidant effects within the human body (Bjelakovic et

al., 2012). Scientists have suggested that dietary antioxidants such as ascorbate,

tocopherol, and carotenoids from fruits and vegetables could help to protect cells from

damages caused by oxidative stress and to fortify the defense systems against

degenerative diseases. Biological function of natural antioxidants has been carried

out with numerous plant materials worldwide, including those used as foods (Seung et

al., 2013, Ames, 1983). Antioxidants from dietary sources have been identified as

safer and effective antioxidants in the context of their efficiency, availability,

abundance, inexpensive nature and non-toxicity.

Earlier studies in our laboratory on the purification of novel water soluble,

antioxidant peptides from turmeric (Curcuma longa) of 6 kDa and 14 kDa have been

reported that they exhibit excellent potency in modulation of cellular oxidative stress

and antiinflammatory activity (Srinivas and Shalini, 1991; Srinivas et al., 1992,

Mukunda Chethan kumar and Leela Srinivas, 2008). the studies on biological activity

of turmerin, provide evidence for effective anti HIV therapy at less toxic doses of

AZT could be possible in the presence of turmerin when compared to curcumin

(Cohly et al., 2003). Another glycoprotein of 34 kDa β-turmerin which was isolated,

purified and characterized in our laboratory from Curcumin Industry generated

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turmeric waste grits. The glycoprotein protected DNA from UV, Fenton reactants

(Smitha et al., 2009). Proteins from Curry leaves (Murraya koeneigii) and Sundakai

(Solanum torvum) were also reported from our laboratory showing excellent

antioxidant and antimicrobial activity. (Ningappa and Leela Srinivas, 2008,

Mylarappa et al., 2010, Sivapriya and Leela Srinivas, 2007, Sivapriya et al., 2011). It

was also reported by us that, dietary components such as turmeric and vegetable extracts

effectively modulate ROS induced lipid peroxidation and DNA damage (Shalini and

Srinivas, 1987; Srinivas and Shalini, 1991; Sujatha and Srinivas, 1995).

The study on inhibition of linoleic acid peroxidation by Soya bean protein

hydrolysate (Yamaguchi et al., 1997) suggests that a protein from dietary sources

especially of plants origin exhibits modulatory effects against oxidative stress. It was

also reported that, wild edible fruits are as a potential source of phytochemicals with

capacity to inhibit lipid peroxidation (Morales et al., 2013). Our pioneering effectors

led to the search of a new protein antioxidant in Turmeric which is nontoxic, easily

available, cost effective, better than available synthetic antioxidants. It has been

shown in Chapter-2 that boiling water extract of turmeric, exhibited potential

antioxidant properties at much lower doses than other standard antioxidants. This

suggests the possible presence of active components that explain the protective role

against oxidative stress. Therefore, present investigation was carried out to identify a

potent, new protein antioxidant from Turmeric. A thorough search was conducted to

isolate, purify and characterize a new antioxidant glycoprotein (28 kilo daltons) from

boiling water extract of Turmeric, which is referred as BGS-Haridrin. The

antioxidant properties of BGS-Haridrin are studied in comparison with known

antioxidants such as α-tocopherol, Curcumin, ascorbate and synthetic antioxidant like

BHA.

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3.1 Materials

The Turmeric powder obtained from local sources cleaned with dilute

Potassium Permanganate solution and dried in shade and powdered (mesh). Butylated

hydroxyl anisole, α-tocopherol, acrylamide, bisacrylamide, tetra methyleneamine

tetra acetic acid (TEMED), thiobarbituric acid, calf thymus DNA, ferrous sulphate,

ascorbic acid, polyvinyl pyrrolidone (PVP), 2-deoxy ribose, ferric chloride, sodium

carbonate, sodium tartarate, linoleic acid, coomassie blue R-250, SDS, trypsinogen

were purchased from Sigma, US. Sephadex G 10 and G 25, DEAE were from

Pharmacia, Sweden. All other chemical unless otherwise mentioned were purchased

from E. Merck, Germany. The 0.45μm and 0.22μm filter were purchased from

Sartorius, India. The spectrophotometric analysis was done using Shimadzu UV-1601

Spectrophotometer. All organic solvents were of analytical grade and were distilled

prior to use.

3.2 Methods

Isolation and purification of BGS-Haridrin from Curcuma longa L.

rhizome powder Turmeric tubers are purchased from local market. Tubers are washed with

water, rinsed in 0.5% KMnO4 and again thoroughly washed with distilled water, dried

in shade and powdered. Five grams of Curcuma longa L. powder, sieved through

B.P.100 mesh was added to 300 ml of boiling double distilled water. The resultant

solution was vortexed thoroughly in a magnetic stirrer for 2 hours at 40C and allowed

to stand overnight at 40C. The resultant suspension was centrifuged at 10,000 rpm, 20

min, at 40C. The supernatant was filtered through Whatmann No.1 filter paper and

Sartorius filter (0.22μm) and the volume was measured. To the pellet 300ml of boiling

double distilled water was again added, mixed well and processed as mentioned

above. The two supernatants thus obtained were pooled, mixed. To the above

mixture, ammonium sulphate to 65% concentration was added, vortexed for 24 hours

at 4C. Then the precipitated protein was separated by centrifuging at 10000 rpm, 20

minutes at 4C. The separated precipitated protein was dialyzed through 2000MWCO

against double distilled water for 72 hours and concentrated by lyophilization. It was

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then passed through 0.22µ Spinex filter. The dialysate was tested for total protein

yield. The residue (pellet) obtained was weighed separately after drying to constant

weight to calculate the solubility (Sujatha and Srinivas, 1995) of the root powder. The

solubility was found to be 22%. The protein (220mg in 4ml of water) from the

concentrated dialysate was desalted on Sephadex G-10 with water as eluent

(Vt=57ml, V0=19ml) at 40C. The fractions were collected at a flow rate of 18ml/hr.

The eluted fractions were monitored at 280nm.

3.2.1 Sephadex G-25 column chromatography of Turmeric protein extract

Sephadex G25 was equilibrated with water for 48 hours and packed on to

column (Vo= 46ml, Vt= 138ml).The protein fractions obtained from Sephadex G-10

column were pooled and lyophilized. The concentrated protein (80mg in 2ml of

water) was loaded on to Sephadex G-25 column (Vo= 46ml, Vt= 138ml) with water as

eluent at 40C. The fractions were collected at 18ml/hour. The eluted fractions were

monitored at 280nm. Three peaks were obtained and they were designated as peak I,

peak II and peak III were pooled, concentrated and suspended in 10mM phosphate

buffer, pH7.4.

Peak I was selected based on its higher antioxidant activity and the

homogeneity was analyzed by polyacrylamide gel electrophoresis (Native gel). The

native gel, two closely bound proteins bands were found and hence it was further

subjected to Anion exchange chromatography.

3.2.2 Ion exchange chromatography

20 mg of lyophilized Peak I protein was subjected to Ion exchange using

DEAE A-25 (Vo=5ml, Vt=15ml , flow rate 0.5ml/5 min) elution was carried out with

Tris HCl buffer ( ranging from 20 mM to 100mM of pH 8). Fractions were monitored

at 280nm using Shimadzu spectrophotometer (1601A). Five peaks are obtained, Peak

IV showed the antioxidant activity and other peaks were inactive. Peak IV fractions

were pooled, dialyzed against water and concentrated. The protein obtained named

as “BGS-Haridrin”

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3.2.3 Determination of nature of BGS-Haridrin by basic native polyacrylamide gel electrophoresis (PAGE)

Basic native PAGE was carried out according to the method of Laemmeli et al

(Lammeli et al., 1970). This separates the acidic proteins on the basis of net charge of

proteins under basic condition. Two volume of protein sample was mixed with one

part of sample buffer [0.3 M Tris, glycerol (20%) and bromophenol blue (2 mg)]. The

samples were loaded on to gel with 4% stacking (pH 6.8) and 10% separating gel (pH

8.9). Electrophoresis was carried out with running buffer (25mM Tris, 190mM

glycine, pH 8.3) at 40 volts for stacking gel and 80 volts for separating gel. Proteins

were fixed and stained in a solution containing 50% methanol, 20% acetic acid and

0.025% Coomassie brilliant blue. The gels were destained with mixture of methanol:

acetic acid (10%).

3.2.4 SDS polyacrylamide gel electrophoresis

Homogeneity and the molecular weight of BGS-Haridrin were determined by

10% SDS- PAGE using the method of Laemmeli et al. (Lammeli et al 1970). Along

with one part of sample buffer [(0.3 M Tris, pH 6.8, SDS (5%), glycerol (30%), β-

Mercaptoethanol (5%) and Bromophenol blue (2 mg)] the samples were incubated at

950C for 5 minutes and aliquots were loaded on to the gel containing 3% poly

acrylamide in stacking gel and 10% in separating gel. Electrophoresis was carried out

with running buffer (25mM Tris, 190mM glycine, 0.1% SDS) at 40 volts for stacking

gel and 80volts for separating gel. Proteins were stained with Coomassie brilliant

blue.

3.2.5 PAS staining

PAS staining was carried out according to the method of Leach (Leach et al,

1990) as follows: SDS – PAGE was performed in slab gels. The gel was placed in a

glass baking dish and submerged in 5% phosphotungstic acid in 2N HCl. Rocked

gently for 90 minutes at room temperature and drained. Soaked the gel in 500ml of

7% methanol +14% acetic acid for 1 hour, repeated this step and drained. Oxidized

the oligosaccharides with 1% periodic acid in 7% TCA for 1 hour and drained.

Removed excess periodic acid with 250ml of 0.5% sodium metabisulfite 0.1N HCl at

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room temperature, finally, stained with Schiff’s reagent by submerging the gel in the

reagent and allowing the pink colour to develop in the dark on ice.

3.2.6 Reverse phase high-pressure liquid chromatography (rp HPLC)

Reversed Phase HPLC (RP-HPLC) of BGS-Haridrin was done using

Shimadzu double pump HPLC system. 5ml of HPLC quality water was injected and a

blank gradient was run to identify any background noise and to balance baseline.

BGS-Haridrin (5mg in 50ml HPLC quality water) was injected onto C18 µ Bondpak

column. The fractions were collected at the flow rate of 1ml/min and eluted by

gradient elution using (0–90%) acetonitrile : water as the mobile phase (with

0.1%TFA). The peaks were monitored by fluorescence emission at 345nm (Excitation

at 285nm).

3.2.7 Molecular mass of BGS-Haridrin by Voyager Spec #1 Mass Spectrometer

Electro spray ionization spectra of BGS-Haridrin recorded on Voyager Spec

#1 (BP 4635.6, 4264) electro spray mass spectrometry. Electro spray ionization

carried out using capillary with ID of 0.1mm. The tip held at 5000V in a positive ion

detection mode. Nebulization assisted by N2 gas (99.8%) at a flow rate of 10l/min.

The spray chamber held at 3000C. Data was acquired over suitable mass range with

cycle time of 3s.

3.2.8 Isolation of human erythrocyte ghosts

Human blood was collected from healthy donors. Erythrocyte ghost which is

free of hemoglobin and superoxide dismutase was prepared by method of Dodge et al

(Dodge et al., 1963). The blood was centrifuged at 2500 rpm 15 minutes, the

supernatant was discarded and the pellet was washed 3 to 5 times with isotonic

phosphate buffer (310 isomilli osmolar, pH 7.4), centrifuged at 2500 rpm at 4C for

20 minutes. RBC cell pellet was suspended in hypotonic phosphate buffer (201 milli

osmolar) and incubated for overnight at ambient temperature for hypotonic hemolysis

to take place. Contents were centrifuged at 12,000 rpm at 4C for 20 minute, the pellet

was dislodged gently with rubber policeman and washed twice with hypotonic

phosphate buffer, centrifuged at 1500 rpm for 20 minutes to remove unlysed RBC

cells. Supernatant was collected and centrifuged at 12,000 rpm for 20 minutes and

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washed twice in 0.9% NaCl. Erythrocyte ghost was suspended in 0.9% NaCl in

aliquots and stored at –20C for further use. The protein content of ghost was

estimated by Bradford’s method (Bradford, 1976).

3.3 Biochemical characterization of Protein Antioxidant BGS- Haridrin 3.3.1 Estimation of Protein The protein content of the extracts was determined by Bradford’s method

(Bradford, 1976) using bovine serum albumin as standard. Different aliquots of the

extract ranging from 0-50μg were made up to 0.1ml with distilled water. To this 0.9ml

of Bradford’s reagent (dissolve 100mg of Coomassie blue G250 in 50ml of 95%

ethanol, and then mixed with 100ml of 85% Orthophosphoric acid and made up to

1000ml with distilled water) was added. The blue color developed was read at 595nm.

The total protein content was calculated according to the standard calibration curve.

3.3.2 Estimation of total sugar The total sugar concentration of the extracts was estimated by Dubois method

(Dubois et al., 1956). Different aliquots of extract (0-50μg) were made up to 1ml with

distilled water. To this 1ml of 5% phenol and 5ml of concentrated 36N sulphuric acid

were added. Orange colour developed was read at 520nm immediately. The total

sugar concentration was calculated according to the standard glucose calibration

curve.

3.3.2 Determination of total phenolic compounds

Total phenolics content was determined according to the method of Kujala

(Kujala et al., 2000) with minor modifications, using Gallic acid as standard. The

extracts ranging from 0-100 μg and Gallic acid were dissolved in 0.5ml of water and

were mixed with 500 μl of 50% Folin-ciocalteau reagent. The mixture was then

allowed to stand for 10min followed by the addition of 1ml of 20% Na2CO3. After 10

min incubation at ambient temperature, the absorbance of the supernatant was

measured at 730 nm. The total phenolics content was expressed as gallic acid

equivalents (GAE) in milligrams per gram of powder.

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3.3.4 Estimation of Total Flavonoid Total flavonoid content was determined by modifying the aluminum chloride

colorimetric method from the procedure reported by Woisky and Salatino (Woisky

and Salatino, 1998). Standard calibration curve was prepared using quercetin. 10mg

of Quercetin was dissolved in 80% ethanol and then diluted solutions were separately

mixed with 1.5ml of 95% ethanol, 0.1ml of 10% aluminum chloride, 0.1ml of 1M

potassium acetate and 2.8ml of distilled water. After incubation at room temperature

for 30min, the absorbance of the reaction mixture was measured at 415 nm. Proper

controls were also done. The total flavonoid content was calculated according to the

standard quercetin calibration curve.

3.3.5 Estimation of Curcumin The curcumin content was estimated by Rubro Curcumin method

(Vijayalakshmi and Chandrashekara, 1982). Different aliquots of Curcumin were

made up to 1ml with acetone. To this 1ml of oxalic acid (0.02%), 1ml of boric acid

(0.013%) was added. Tubes were kept in boiling water bath till the solvent

evaporated completely. Rubro curcumin or red colour complex formed after heating

was dissolved in 2ml of distilled ethanol. Absorbance was read at 480nm within

10min. The curcumin concentration in the extract was calculated according to the

standard curcumin calibration curve.

3.3.6 Estimation of ascorbic acid Ascorbate was determined according to the method of Sadasivam and

Manickam (Sadasivam and Manickam, 1997) with minor modifications. 1g of each

extract was taken in polypropylene tube containing 25ml of 4% oxalic acid, mixed

well, centrifuged at 10,000 rpm for 10min. the supernatant was transferred to conical

flask and added bromine water (1 to 2 drops of liquid bromine dissolved in 100ml

cold water) drop wise till the solution turned yellow. The excess bromine was

removed by aeration (the beaker was kept open until disappearance of yellow color)

and the solution was made up to 50ml with 4% oxalic acid. Similarly, L-ascorbate

(10mg/100ml) stock was converted into dehydro form by bromination.

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The brominated extracts (0-100μg) or brominated L-ascorbate (10-100μg)

was made up to 5.9ml with 4% oxalic acid. 100μl of 10% thiourea solution was added

to each tube and mixed. Then, 1ml of 2,4-dinitrophenyl hydrazine reagent (2%

aqueous solution of 2, 4-dinitrophenyl hydrazine in 9N sulphuric acid) was added to

each tube and mixed incubated for 3hrs at 370C. The tubes were cooled on ice water

and added with 4ml of 80% sulphuric acid and cooled again on ice water. Color

developed was read against reagent blank at 540nm. From the calibration curve of

dehydro ascorbate, the total ascorbic acid content of the extracts was calculated.

3.3.7 Estimation of α–tocopherol Alpha tocopherol was determined according to the method of Kivcak and Mert,

2001). It is based on the reduction of ferric iron to ferrous iron by -tocopherol,

which forms a red colour with 2,2’ dipyridyl. One gram of each extract was taken in

10 ml of hexane and transferred to conical flask further extracted with 200ml of

hexane with stirring for 24hrs. The next day, contents of the flasks were shaken

vigorously and filtered through a Whatmann No.1 filter paper. The hexane extract was

distilled in vacuum to get dry extract and stored at –200C. 10mgof dry hexane extract

was dissolved in 10ml of chloroform. Aliquots of hexane extract solution (20–100μl)

and α–tocopherol solution, 20–100μl (10mg of α–tocopherol (Sigma) was dissolved in

10ml of absolute alcohol) was transferred to separate volumetric flask and the volume

was made up to 3ml with chloroform. Then 1ml of 2,2’ dipyridyl reagent (125mg of

2,2’ dipyridyl dissolved in 25ml of absolute ethanol and stored in dark bottle at 40C)

was added to each tube and mixed. Then, 1ml of FeCl3 solution (200mg of

FeCl3.6H2O in 100ml of ethanol and stored in brown bottle and kept in refrigerator

until used) was added and mixed well. After 15min, read against the blank at 520nm.

A blank was run, using 3ml of chloroform, 1ml of 2,2’-dipyridyl reagent and 1ml of

FeCl3 solution. The amount of α–tocopherol was calculated using the calibration

curve for α-tocopherol.

3.3.8 Estimation of amino sugar 100μg of BGS-Haridrin was treated with 100μl of 10mM potassium

ferricyanide solution and the absorbance was measured at 420nm (Imoto and

Yagashita, 1971). The sugar content was expressed on total protein content basis. A

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standard calibration curve was prepared using N-acetyl glucosamine and proper

controls were included.

3.3.9 Estimation of SH groups 5-5’ Dithiobis (2- nitro benzoic acid) reacts with free –SH side chain of

cysteine to form an s-s bond between protein and a thio-nitro benzoic acid residue,

which can be measured by spectral analysis (Masuda et al., 2005). 100μl of the BGS-

Haridrin protein solution (0.5mg) was taken in 2ml of phosphate buffer of 0.1M, pH

8.0, 20μl of 0.4% DTNB of aqueous solution was added and mixed well. Absorbance

was measured at 412 nm after 2min, 10 min, 20th, 60th and 120th minute and the

highest value was used to calculate SH content, using the molar extinction coefficient

of 13,600 M-1cm-1

The concentration of sulfhydrals was determined by using the following

formula.

Where A = Absorbance at 420 nm

= Molar extinction coefficient = 13600/M/cm

D = Dilution factor

3.3.10 Stability to proteases The BGS-Haridrin was tested for its ability to withstand with proteases like

trypsin, and pepsin. 500μg of BGS-Haridrin was incubated at 370C for 1 hour with

20μg of trypsin in 20mM phosphate buffer pH 8.0 or 20μg of pepsin in 20mM sodium

acetate buffer, pH 2.0. The reaction was arrested by keeping the tubes in ice. The

incubation mixture contained BGS-Haridrin in the presence or absence of proteolytic

enzymes in a ratio of 25:1 w/w was used. Aliquots of samples were then subjected to

determine its hydroxyl radical scavenging activity by deoxy ribose assay.

3.3.11 Shelf life of BGS-Haridrin

To check the shelf life of BGS-Haridrin was stored at ambient temperature,

4C and -20C for about 30 days. For one day, one week, 15 days and 30 days, BGS-

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Haridrin was taken from each group and tested for its antioxidant activity by free

radical scavenging assay (Halliwell and Gutteridge, 1985).

3.3.12 Thermal stability of BGS-Haridrin The stability of the BGS-Haridrin protein was tested against heat treatment at

1000C up to 60 minutes. The solubility was tested in water, tris buffer (10mM, pH

7.4), phosphate buffer (10mM, pH 7.4) and saline.

3.4 Biological characterization:

3.4.1 Hydroxyl radical scavenging activity of BGS-Haridrin

The hydroxyl radical scavenging activity of BGS-Haridrin was done according

to the method of Halliwell and Gutteridge 1985. The reaction mixture containing

FeCl3

(100 μM), EDTA (104 μM), H2O

2 (1 mM) and 2-deoxy- D-ribose (2.8 mM)

were mixed with or without BGS-Haridrin (0.1 µM) in 1 ml final reaction volume

made with potassium phosphate buffer (20 mM pH 7.4) and incubated for one hour at

37°C. α-tocopherol (400µM), BHA (400µM) and Curcumin (400μM) were used as

positive control. The mixture was heated at 95°C in water bath for 15 min followed by

the addition of 1 ml each of TCA (2.8%) and TBA (0.5% TBA in 0.025 M NaOH

containing 0.02% BHA). Finally the reaction mixture was cooled on ice and

centrifuged at 5000 rpm for 15 min. Absorbance of supernatant was measured at 532

nm using the negative control without any antioxidant was considered 100%

oxidation. The percentage hydroxyl radical scavenging activity of protein was

determined.

3.4.2 Lipid peroxidation inhibition assay

The evaluation of antioxidant activity of BGS-Haridrin based on the inhibition

of peroxidation in RBC ghost was done according to Shimazaki et al., 1984. The

evaluation of oxidation was done by measuring the TBA reactive substances (Dahle et

al, 1962). The human erythrocyte ghost was isolated according to the method of

Dodge et al. 1963. 100 μl of ghost suspension (300 μg membrane protein equivalent)

was subjected to peroxidation by ferrous sulphate and ascorbic acid (10:100 μmol) in

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final volume of 1 ml of Tris buffered saline (20 mM, pH 7.4,150 mM NaCl). The

reaction mixture was treated with or without BGS-Haridrin (0.1 µM), BHA, α-

tocopherol and Curcumin (400μM) were used as positive control. The contents were

incubated for 1 h at 37°C. The reaction was terminated by the addition of 10 μl of 5%

phenol and 1 ml of 1% TCA. To each system 1 ml of 1% TBA was added, the

contents were kept in a boiling water bath for 15 min, cooled and centrifuged at 6000

rpm for 10 min. The absorbance of supernatants was measured colorimetrically at 535

nm. Appropriate blanks were included for each measurement. The negative control

without any test sample was considered as 100% peroxidation. The % inhibition of

lipid peroxidation was determined accordingly by comparing the absorbance of the

test samples with negative control.

3.4.3 DPPH radical scavenging activity of BGS-Haridrin

DPPH radical scavenging activity was assessed according to the method of

Shimada et al. (1992). The BGS-Haridrin at concentrations ranging from 2 µg to 14

µg was mixed in 1 ml of freshly prepared 0.5 mM DPPH ethanolic solution and 2 ml

of 0.1 M acetate buffer pH 5.5. The resulting solutions were then incubated at 37°C

for 30 min and measured colorimetrically at 517 nm. Ascorbic acid, BHA and

Curcumin (2 to 14 µg) was used as positive control under the same assay conditions.

Negative control was without any inhibitor or BGS-Haridrin. Lower absorbance at

517 nm represents higher DPPH scavenging activity. The % DPPH radical scavenging

activity of BGS-Haridrin was calculated from the decrease in absorbance at 517 nm in

comparison with negative control.

3.4.4 Nitric oxide radical scavenging activity

Nitric oxide radical scavenging activity was determined according to the

method reported by Marocci et al (Marocci et al., 1994). BHA (400µM), α-

tocopherol (400µM) and Curcumin (400µM) standards in the range of 2 –10 µl were

taken in respective tubes containing phosphate buffer saline, so that the volume in

each tube was made up to 1ml. Spice extracts were taken in the volume of 30µl

(25g) to the tubes containing phosphate buffer. For controls, volume was made up to

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3ml with phosphate buffer saline and for the tests 1ml. Then 2ml of 10mM sodium

nitroprusside was added to all the tubes except the controls. Nitric oxide radicals were

generated from the samples spontaneously during the incubation period of 150 min.

0.5ml of the solution was taken from each tube to their respective tubes. To this 1ml

of 0.33% sulphanilamide was added and were incubated at room temperature for 5

min., followed by the addition of 0.1% of NED to each tube and incubated all the

tubes under diffused light for 30 min. The nitrite ions released were measured

spectrophotometrically at 516nm.

3.4.5 Superoxide scavenging activity

The Superoxide radical (O2•-) scavenging activity of BGS-Haridrin was

measured according to the method of Lee et al. (Lee et al., 2002). The reaction

mixture containing 100μl of 30mM EDTA (pH 7.4), 10μl of 30mM hypoxanthine in

50mM NaOH, and 200μl of 1.42mM nitro blue tetrazolium with or without BGS-

Haridrin and SOD serving as positive control at various concentrations ranging from

50-300μg. After the solution was pre-incubated at ambient temperature for 3min,

100μl of xanthine oxidase solution (0.5U/ml) was added to the mixture and incubated

for one hour at 37C, and the volume was made up to 3ml with 20mM phosphate

buffer (pH 7.4). The solution was incubated at room temperature for 20 min,

absorbance was measured at 560 nm. Appropriate controls were included to rule out

the artifacts induced reaction. The control was without any inhibitor. Inhibitory effect

of BGS-Haridrin on superoxide radicals was calculated as

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3.4.6 Reducing power and iron chelating activity of BGS-Haridrin

Test for ferric ion reducing power (Fe3+ to Fe2+) The ferric ion reducing power activity of BGS-Haridrin was determined

according to the method of Wang et al. (Wang et al., 2003). 100μl of potassium

ferricyanide solution (4mM) was mixed with 200μl of 20mM phosphate buffer pH 6.5

in the presence or absence of BGS-Haridrin or BHA at various concentrations ranging

from 0–100μg. The contents were incubated at 50°C for 20 mins. 200μL of 10%

trichlro acetic acid was added to the reaction mixture and centrifuged at 5000rpm. The

resulting supernatant was taken and mixed with 100μl of ferric chloride (FeCl3)

solution (2mM) and final volume was made up to 1ml with water and then incubated

at 37°C for 10minutes. The absorbance was recorded at 700nm in Shimadzu

spectrophotometer. Absorbance increases with increase in reducing power.

Test for ferrous ion chelating ability (binds Fe2+) Ferrous ion chelating activity of BGS-Haridrin was measured according to the

method of Suter and Richter (Suter and Richter, 2000). The reaction solution

containing ferrous chloride (FeCl2) (200μM) and potassium ferricyanide (400μM)

with or without BGS-Haridrin or EDTA at various concentrations of ranging from 0–

100 μg were added in final volume of 1ml water and mixed. The reaction mixture was

incubated at 20C for 10 minutes. Formation of the potassium hexacyanoferrate

complex was measured at 700 nm in Shimadzu spectrophotometer. The assay was

carried out at 20C to prevent Fe2+ oxidation. Lower absorbance indicated higher iron

chelating capacity. The control was without any chelating compound or test sample.

The percent ferrous ion chelating effect was calculated from the following equation:

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3.4.7 Determination of reactive carbonyls in proteins (Protein carbonyl assay)

The most common products of protein oxidation in biological samples are the

protein carbonyl derivatives of Pro, Arg, Lys, and Thr (Cadenas et al, 1977). These

derivatives are chemically stable and serve as markers of oxidative stress for most

types of ROS. Many of the current assays involve derivitization of the carbonyl group

with dinitrophenylhydrazine (DNPH), followed by an anti-DNP antibody detection.

When DNPH reacts with protein carbonyls, the amount of protein-hydrozone

produced can also be measured spectrophotometrically at 375 nm (Wakeyama et al,

1982). Protein carbonyls in BSA standards (1-10 mg/mL) are derivatized with

DNPH first. Then the BGS-Haridrin protein also precipitated in ice-cold

trichloroacetic acid (TCA) (final concentration 10% v/v). The protein pellet was re-

suspended in 0.5 ml of 10 mM 2,4-dinitrophenylhydrazine (DNPH) / 2 M HCL. They

were vortexed continuously at room temperature for 1 hour. The proteins were

precipitated with 0.5 mL of 20% TCA, centrifuged at 8000 rpm for 3 min. The pellet

was washed with 1 mL of ethanol-ethyl acetate (1:1;v/v) to remove free DNPH

reagent, and allowed to stand for 10 min. The protein was centrifuged for 5 min at

8000 rpm and the supernatant was discarded. The washing procedure was repeated

two times for a total of three washes. The resulting protein pellet was re-suspended in

1.0 mL of 6M guanidine with 2 mM potassium phosphate buffer (pH 2.3, adjusted

with trifluoroacetic acid). The proteins were incubated at 37oC for 15 to 30 min to aid

dissolution of the protein. All proteins were then centrifuged to remove any insoluble

material remaining in suspension. The concentration of DNPH was determined at its

maximum wavelength (375 nm) and the molar absorption coefficient of 22, 000 M-

1cm-1 was used to quantify the levels of protein carbonyls. Samples were

spectrophotometrically analyzed against a blank of 1 mL of guanidine solution (6M

guanidine with 2mM potassium phosphate). Protein concentration was determined in

the samples by determining the absorbance at 280 nm. Protein carbonyl content was

expressed as nmol/mg.

Protein Carbonyl (M) = A375 nm / 22,000 M-1 Protein Carbonyl (nmol/mL) = A375 nm x 45.45 (nmol/mL)

75

3.5 Synergistic study between BGS-Haridrin, BGS-Turmerin and Curcumin 3.5.1 Synergistic activities of BGS-Haridrin and BGS-Turmerin

Hydroxyl radical scavenging activity of BGS-Haridrin (28kDa) and BGS-

Turmerin (14kDa) was done to study the interaction or synergistic antioxidant

activities between BGS-Haridrin and BGS-Turmerin both are isolated from boiling

water of Turmeric extract. Antioxidant activity was studied by keeping BGS-Haridrin

as constant (50µM) and varying concentrations of BGS-Turmerin (0 to 0.175µM).

The reverse was studied by keeping BGS-Turmerin as constant (0.09µM) and varying

concentrations of BGS-Haridrin (0 to 0.1µM) to study the possibility of synergy

between the two related molecules present in Turmeric.

3.5.2 Synergistic activities of BGS-Haridrin and Curcumin Hydroxyl radical scavenging activity of BGS-Haridrin and Curcumin was

done to study the interaction or synergistic antioxidant was done to study the

interaction or synergistic activities between BGS-Haridrin- antioxidant protein

isolated from boiling water extract of Turmeric and Curcumin – the

diferuloylmethane – a lipid soluble compound from Turmeric. Antioxidant activity

was studied by keeping BGS-Haridrin as constant (0.05µM) and varying

concentrations of Curcumin (0 to 140µM). In the same way, the antioxidant activity

was studied by keeping Curcumin as constant (200µM) and varying concentrations of

BGS-Haridrin (0 to 0.1µM).

3.6 Statistical Analysis Results are expressed as Means ± S.D of five experiments. Student’s t-test was

used for statistical significance using SPSS (statistical presentation system software)

for windows version 10.0.1 software, Inc. New York. A probability level of P < 0.05

was considered as statistical significance in comparing with relevant controls.

76

RESULTS:

FLOW CHART -3.1

EXTRACTION OFBGS-HARIDRIN

Turmeric tubers are washed with water, rinsed with 0.5% KMnO4 and again thoroughly washed with double distilled water

Cleaned turmeric tubers dried and powdered to fine powder and 5 g of turmeric powder was weighed

300ml of double distilled boiling water was added and boiled for five minutes

Vortexed for two hours, Centrifuged at 10,000 rpm for 20 minutes at 4C

Concentrate the dialyzed extract and passed through 0.22 micron filter paper, filtrate collected, stored in -20C

To the supernatant, ammonium sulphate TO 65% was added to precipitate proteins, vortexed for 24 hours at 4C, centrifuged at 10000 rpm at 4C

Precipitated proteins collected, dialyzed (2000 MWCO) for 72 hours against water and finally lyophilized

77

Fig. 3.01: G-25 purification of Crude Turmeric protein

Gel filtration profile of boiling water crude dialyzed (2 MWCO) extract of

Turmeric (Curcuma longa L) on Sephadex G-25 column. (Vo= 46ml, Vt= 138ml,

flow rate 1ml/5 min, 100mM Tris HCl buffer, pH 7.4, 100mM NaCl) Three peaks (PI,

PII and PIII) eluted from the column and Peak I (Fraction from 35 to 50) was selected

for further studies.

78

Fig. 3.02: DEAE A25 chromatography of Peak I protein

The active peak I fractions subjected to ion exchange chromatography using

DEAE A-25. (Vo= 5ml, Vt=15ml, flow rate 0.5ml/5 min Tris HCl buffer ranging

from 20mM to 100mM). The chromatogram shows five peaks. The peak IV was

more active and selected for further studies.

79

Fig. 3.03 SDS-PAGE of BGS-Haridrin

Electrophoretic profile of BGS-Haridrin on SDS-PAGE, BGS-Haridrin (15µg)

and low molecular weight markers (15µg) loaded on gel, electrophoresis carried at

80V for 5% stacking gel, 40V for 10% resolving gel. After electrophoresis the gel

stained with Coomassie brilliant blue R250 stain for 8h. Lane 1 is medium range

molecular weight marker, and Lane 2 is BGS-Haridrin (~28kDa) respectively.

80

Fig. 3.04 Reversed Phase HPLC (rp-HPLC) of BGS-Haridrin

Reversed Phase HPLC (RP-HPLC) of BGS-Haridrin (5g in 50l HPLC

quality water), injected to C18 Bondpak column. Fractions collected at the flow rate

of 1ml/min, eluted by gradient elution using (0–90%) acetonitrile : water as mobile

phase (with 0.1%TFA).

81

Fig. 3.05 Determination of molecular mass of BGS-Haridrin by Voyager Spec #1 Mass Spectrometer

Electro spray ionization spectra of BGS- Haridrin recorded on a Voyager Spec

#1 electrospray mass spectrometry. Electrospray ionization carried out using capillary

with ID of 0.1mm. The tip held at 5000V in a positive ion detection mode.

Nebulization assisted by N2 gas (99.8%) at a flow rate of 10l/min. The spray chamber

held at 3000C. Data was acquired over suitable mass range using conventional

quadrupole detector with cycle time of 3 seconds.

82

Table-3.1 Proximate analysis of boiling water extract of Curcuma longa L. rhizome powder.

Protein (mg/g %)

5.51 ± 0.56

Sugars (mg/g%)

26.03 ± 0.09

Curcuminoids (mg/g%)

0.52 0.02

Polyphenols (mg/g%)

0.48 ± 0.03

Flavonoids (mg/g%)

0.33 ± 0.02

Ascorbic acid (mg/g%)

-

Vitamin E (mg/g%)

0.10 ± 0.01

Amino sugars (mg/g%)

0.49 ± 0.01

SH groups (µmole /g%)

1.20 ± 0.10

Protein, sugars, Curcuminoids, polyphenols, flavonoids, vitamin E, vitamin C,

amino and sugars present in the boiling water extract of Turmeric (Curcuma longa)

were estimated as described in materials and methods and values are expressed in

mg/g% of turmeric and sulfydral groups in µmole/g.

Results are shown as mean ± SD (n = 5).

83

Table-3.2 Proximate analysis of BGS-Haridrin

Protein (mg/g%)

0.51 ± 0.01

Sugars (mg/g%)

0.02 ± 0.001

Curcuminoids (mg/g%)

Nil

Polyphenols (mg/g%)

Nil

Flavonoids (mg/g%)

Nil

Ascorbic acid (mg/g%)

Nil

Vitamin E (mg/g%)

Nil

Amino sugars (mg/g%)

0.004± 0.001

SH groups (µmole /g%)

0.01 ± 0.002

Protein, sugars, Curcuminoids, polyphenols, flavonoids, vitamin E, vitamin C,

amino and sugars present in purified antioxidant glycol protein BGS-Haridrin isolated

from boiling water extract of Turmeric (Curcuma longa) were estimated as described

in materials and methods and values are expressed in mg/g% of turmeric and

sulfydral groups in µmole/g.


84

Table 3.3 Summary of purification of BGS-Haridrin from boiling water extract of Curcuma longa L rhizome

Steps in isolation of

BGS-Haridrin

Total protein

Concentration (mg)

Total protein

recovered (mg)

Crude aqueous extract

220

220

Sephadex G-10

210

180

Sephadex G-25 Peak I Peak II Peak III

010

003

003

004

DEAE A25 of Peak I

003

001

BGS-Haridrin, isolated from boiling water extract of Curcuma longa L.

rhizome powder fractionated through Sephadex G-25, DEAE A25 and the net protein

yield


85

Fig. 3.06 BGS-Haridrin resists Pepsin and Trypsin the proteolytic enzymes

0102030405060708090

100

Pepsintreatment

Trypsintreatment

% in

hibi

tion

of h

ydro

xyl

radi

cal a

ctiv

ity

DR + FR +BGS-Haridrinassay

DR + FR +BGS-Haridrin +Enzyme assay

BGS-Haridrin (500μg) was treated with 20μg of pepsin/trypsin. Hydroxyl

radical scavenging activity was done by deoxyribose assay as described in methods.

The appropriate controls were included in all the experiments. The control was

without any BGS-Haridrin or enzyme and the % hydroxyl radical scavenging activity

was calculated accordingly.


Footnote: DR- Deoxyribose; FR- Fenton reactants.

86

Table 3.4: Shelf life of BGS-Haridrin as measured by Hydroxyl radical scavenging activity

No. of

days

Hydroxyl radical scavenging activity

27C

4C

-20C

01st day

80.1 2.6

80.1 2.6

80.1 2.6

07th day 76.2 1.2 79.3 1.6 79.2 1.6

15th day 70.4 1.8 76.4 1.3 77.7 1.4

30th day 55.2 2.1 71.4 1.5 73.1 1.9

The antioxidant protein BGS-Haridrin which was stored at ambient

temperature, 4C and -20C for about 30 days, checked for its shelf life by hydroxyl

radical scavenging assaying antioxidant activity for one day, one week, 15 days and

30 days by free radical scavenging assay.


87

Fig 3.07: Stability of antioxidant activity of BGS-Haridrin against temperature.

0

10

20

30

40

50

60

70

80

30 min 60 min

Minutes

% in

hibi

tion

BGS-Haridrin (0.1µM) was heated in boiling water bath and at the end of 30

minutes and 60 minutes, the antioxidant activity was estimated by TBARS method.

The antioxidant activity is expressed as % inhibition.


88

Table 3.5 Hydroxyl radical scavenging activity of Turmeric and BGS-Haridrin - A comparison

Sl.No

Antioxidants Concentration % Inhibition of Hydroxyl radical

1

α-tocopherol

400 M

76.5 2.2

2 Curcumin 400 M 68.5 2.8

3 BHA 400M 71.3 2.3

4 Crude extract of

Turmeric

25 g 63.5 2.2

5 BGS Haridrin 0.1µM (0.003g)

80.1 2.6

Deoxy D-ribose (2.8mM) + FeCl3 (100µM) + EDTA (104 μM) + H2O

2 (1

mM) at indicated concentrations in 0.1ml potassium phosphate buffer (20mM, pH

7.4), incubated at 37C, 1hr. The hydroxyl radicals were measured as described in

methods. The control was without any antioxidant and the % inhibition of hydroxyl

radicals was calculated accordingly.


89

Table 3.6 Antioxidant activity in two model systems

% Inhibition of lipid peroxidation

Sl. No

Antioxidants Concentration

Linoleic acid micelles

Erythrocyte ghost

1

tocopherol

400 M

87.5 2.5

72.3 ± 1.5

2 Curcumin 400 M 77.3 3.1 57.2 ± 4.9

3 BHA 400M 80.2 1.8 64.3 ± 4.1

4 Crude boiling water

extract of Turmeric

25 g 75.3 2.1 61.6 ± 2.1

5 BGS Haridrin 0.1µM (0.003g)

76.4 2.6 65.5 ± 2.1

Linoleic acid micelles (1.8μmole) or ghost (300μg) + ferrous sulphate:

ascorbate (10:100μmole) ± extracts or antioxidants at indicated concentrations in

0.5ml TBS pH 7.4, incubated at 37C, 1hr.The TBARs were measured as described

in methods. The control was without any antioxidant and the % inhibition of lipid

peroxidation was calculated as described in methods.


90

Table 3.7 Specific antioxidant activity in various stages of isolation and purification of BGS-Haridrin

Sl.

No.

Purification steps

Protein concentration

(µg)

% Antioxidant

activity

Specific antioxidant activity of

protein

01

Boiling water extract of Turmeric

4

63.5±1.2

15.8±0.6 / µg

02 Peak I from Sephadex G-25 column fractionation

2 70.1±0.8 17.5±0.5 / µg

03 Peak IV from DEAE –A 25 column fractionation

0.0028 80.3±1.6 40.1±0.5 / ng

Deoxy D-ribose (2.8mM) + FeCl3 (100µM) + EDTA (104 μM) + H2O

2 (1

mM) at indicated concentrations in 0.1ml potassium phosphate buffer (20mM, pH

7.4), incubated at 37C, 1hr. The hydroxyl radicals were measured as described in

methods. The control was without any antioxidant and the % inhibition of hydroxyl

radicals was calculated accordingly.


91

Fig. 3.08 DPPH radical scavenging activity of BGS-Haridrin – dose response

0102030405060708090

0 2 4 6 8 10 12 14

Dose (mg)

% in

hibi

tion

Ascorbic acid

Curcumin

BGS-Haridrin

BHA

Dose-dependent DPPH radical scavenging activity of BGS-Haridrin glyco

protein isolated from Turmeric (Curcuma longa L). The control was without BGS-

Haridrin or Ascorbic acid or Curcumin or BHA. The DDPH radical scavenging

activity was calculated as described in methods.


92

Fig. 3.09

Nitric oxide radical scavenging activity of BGS-Haridrin – dose response

0

10

20

30

40

50

60

70

80

90

0 2 4 6 8 10

Dose (mg)

% in

hibi

tion

Curcumin

BHA

BGS-Haridrin

Ascorbic acid

Dose-dependent Nitric oxide radical scavenging activity of BGS-Haridrin

glyco protein isolated from Turmeric (Curcuma longa L). The control was without

BGS-Haridrin or BHA or Curcumin or Ascorbic acid. The Nitric oxide radical

scavenging activity was calculated as described in methods.


93

Fig. 3.10 Super oxide radical scavenging activity of BGS-Haridrin – dose response

0102030405060708090

100

0 1 2 3 4 5 6 7 8

Dose (mg)

% in

hibi

tion

Curcumin

BHA

BGS-Haridrin

Ascorbic acid

Dose-dependent Super oxide anions scavenging activity of BGS-Haridrin

glyco protein isolated from Turmeric (Curcuma longa L). The control was without

BGS-Haridrin or BHA or Curcumin or Ascorbic acid. The Super oxide radical

scavenging activity was calculated as described in methods.


94

Fig. 3.11 Ferric ion reducing activity of BGS-Haridrin – dose response

Ferric ion reducing activity of BHA (72µg), EDTA (60µg) and BGS-Haridrin

(20 to 80µg) was determined as described in methods. Increase in ferric ion reducing

activity shows higher reducing power.

The results are mean ± SD (n = 5)

95

Fig. 3.12 Ferrous ion chelating activity of BGS-Haridrin – dose response

Does response iron chelating effect of EDTA (60µg), BHA (72µg) and BGS-

Haridrin (20, 50 and 100µg) at different concentrations was determined as described

in methods. The lower absorbance at 700nm indicated higher chelating effect.


96

Fig. 3.13 Estimation of protein carbonyl formation

Formation of protein carbonyl by BGS-Haridrin was estimated. Bovine

Serum Albumin (BSA) taken as standard (0 to 10 µg). Quantity BGS-Haridrin was

estimated against its O.D 0.38 nm is 5µg.


97

Fig. 3.14 Antioxidant synergy between BGS-Haridrin and BGS-Turmerin

BGS-Haridrin kept constant 0.05µM (IC-50 % of inhibition is 40) and

increasing amounts of BGS-Turmerin added 0 to 0.175 µM. BGS-Turmerin increases

the antioxidant activity of BGS-Turmerin by 49% at 0.175µM.


0

10

20

30

40

50

60

70

80

90

100

0 0.05 0.075 0.1 0.125 0.15 0.175

Dose of BGS-Turmerin (mM)

% in

hibi

tion

BGS-Haridrin (0.05mM)

20* 28* 32* 41* 44* 49*

98

Fig-3.15 Antioxidant synergy between BGS-Turmerin and BGS-Haridrin

BGS–Turmerin (0.09µM) kept as constant (IC-50 % of inhibition is 38) and

increasing amounts of BGS-Haridrin added up to 0.1µM. BGS- Haridrin increases

antioxidant activity by only 58%.


0

20

40

60

80

100

120

0 0.01 0.02 0.04 0.06 0.08 0.1

Dose of BGS-Haridrin (mM)

% in

hibi

tion

BGS-Turmerin (0.m)

20* 31* 38* 45* 53* 58*

99

Fig. 3.16: Antioxidant synergy between BGS-Haridrin and Curcumin

BGS–Haridrin - 50nM kept as constant (IC-50 % of inhibition is 40%) and

increasing amounts of Curcumin added 0 to 400µM. Curcumin increases antioxidant

activity by 38%.


0

10

20

30

40

50

60

70

80

90

0 50 100 200 250 300 400

Dose of Curcumin (mM)

% in

hibi

tion

BGS-Haridrin (0.m)

09* 15* 21* 26* 33* 38*

100

Fig-3.17 Antioxidant synergy between Curcumin and BGS- Haridrin

Curcumin - 200µM kept as constant (IC-50 % of inhibition is 34%) and

increasing amounts of BGS-Haridrin (0 to 0.1µM). BGS-Haridrin increases

antioxidant activity by 55%.


0

10

20

30

40

50

60

70

80

90

100

0 0.01 0.02 0.04 0.06 0.08 0.1

Dose of BGS-Haridrin (mM)

% in

hibi

tion

09* 14*

Curcumin m

25* 33* 42* 55*

101

Discussion

Herein, the isolation, purification and characterization of 28 kDa antioxidant

glycoprotein antioxidant BGS-Haridrin from boiling water extract of Turmeric

(Curcuma longa L) is discussed. The present study demonstrates in vitro modulation

of oxidative stress by this glycoprotein in comparison with known antioxidants in

different model systems.

The Flow chart-1 gives the stepwise procedure of ammonium sulphate

precipitation of Turmeric proteins. The precipitated protein pellet dissolved in PBS

was dialyzed against same buffer and further passed through sephadex G 10 column

to remove ammonium salts if any and other low molecular components such as free

sugars or polyphenols and the like which could contribute to the antioxidant activity.

The proteins were concentrated by lyophilization at –370C to required volume or

protein concentration and it was referred as crude protein of Turmeric. As represented

in the Table 3.1, the composition of proteins, total sugar, curcuminoids, phenolics,

flavonoids, ascorbic acid, vitamin E and sulphydral groups in turmeric extract were

investigated. Fractionation of crude protein of Turmeric (10mg) on sephadex G 25

column by eluting with water resulted in three optical density peaks as monitored at

280nm (Fig 3.1). The peaks were designated as Peak I, Peak II and Peak III in the

order. The peak I (Fig 3.1) was selected as active peak. The fractions of each peak

were pooled separately, concentrated by lyophilization to a final volume of 2ml.

Protein estimation was done in each stage of extraction as in the Table 3.2. The

Native page of peak I showed presence of more than one protein and also its acidic

nature. Further it was subjected to anion exchange chromatography using DEAE

A25 (Fig 3.2). Five active peaks were found and peak IV was found to be more

active. The peak IV appeared as homogenous single band on SDS-PAGE of 10%

separating gel with the approximate molecular weight of 28 kilo Daltons (Fig 3.3).

The purity was further confirmed by rp-HPLC, which showed single peak (Fig 3.4)

followed with molecular mass detection (Fig. 3.5). It was confirmed that, its

molecular weight 28.3 kDa. The characteristic glycoprotein nature of BGS-Haridrin

was confirmed by staining the gel with PAS reagent. 50μg of BGS-Haridrin was

separated by SDS-PAGE and then stained with PAS to detect glyco-proteins, which

showed appearance of pink colored band.

102

The effect of digestive proteases like Trypsin and Pepsin on BGS-Haridrin

determined accordingly. The trypsin and Pepsin are the two major proteases present

in the human digestive track. In order to find the stability of BGS-Haridrin against

these proteases, the study was done as shown in the Figure 3.6. The pepsin and the

pepsin treatment did not much affect the antioxidant activity of BGS-Haridrin,

however, pepsin treatment resulted in 5% loss of initial antioxidant activity of BGS-

Haridrin and trypsin treatment resulted in 4% loss of initial antioxidant activity of

BGS-Haridrin. The Pepsin and Trypsin are specific proteases which require restricted

sequences in order to act. In general, treatment of BGS-Haridrin with proteases

resulted in negligible loss of antioxidant activity of BGS-Haridrin. This result shows

the anti-protease nature of BGS-Haridrin and probably exhibits its propensity to resist

digestive proteases and thus available to the system as an antioxidant.

Table 3.4 shows that, the shelf life of BGS-Haridrin protein. As shown in

table, the % of antioxidant activity of BGS-Haridrin at 27C decreases at 30 day and

it could offer only 55% inhibition against hydroxyl radicals (as against 80% inhibition

seen at day 1). At 4C, the loss of activity was less and by 30th day, BGS-Haridrin

still shows the % inhibition of hydroxyl radicals up to 71% (as against 80% inhibition

seen at day 1). At -20C, the loss of activity was negligible when compared to the

protein stored at room temperature. At 30th day, BGS-Haridrin still shows antioxidant

activity up to 73% (as against 80% inhibition seen at day 1). It is not much difference

in the antioxidant activity of BGS-Haridrin protein stored at 4C. There is no

appreciable antioxidant activity of BGS-Haridrin stored in ambient temperature shows

the usual loss of stability of protein. The antioxidant activity of BGS-Haridrin seems

to be reasonable stored at 4C and -20C.

The antioxidant protein BGS-Haridrin heat stability capacity was estimated as

shown in Fig. 3.7. BGS-Haridrin (0.1µM) was kept in boiling water bath for 30

minutes and 60 minutes. As shown in the figure, BGS-Haridrin shows 70% inhibition

of lipid peroxidation after 30 minutes of heat treatment and shows 55% inhibition of

lipid peroxidation after 60 minutes of heat treatment. This shows that, even at 100C,

the protein shows reasonable stability as compared to other protein like albumin.

The effective hydroxyl radical scavenging activity of BGS-Haridrin

antioxidant protein was studied in comparison with other well known antioxidants

like BHA, Curcumin and alpha tocopherol. As shown in Table 3.5, BGS-Haridrin

103

scavenges hydroxyl radicals to the tune of 80.1 2.6 % at 0.1µM dosage. The

hydroxyl radical scavenging activity of BGS-Haridrin was higher than when

compared to α-tocopherol (76.5 2.2%) at (400µM), BHA (71.3 2.3 %), Curcumin

(68.5%) at 400µM dosage and the crude boiling water extract of turmeric which also

contains phenolics compounds, sugars and flavonoids scavenges the hydroxyl radicals

as shown in Table 3.1 at a dosage of 25µg shows hydroxyl radical scavenging activity

of 63.5%. Hydroxyl radicals may cause damage to membrane lipids and especially

bind to double of DNA (Srinivas and Shalini, 1991, Susumu et al., 2012). Thus BGS-

Haridrin could be a good antioxidant to prevent the toxicity of OH° radical to

deoxyribose than that of synthetic antioxidant BHA and other natural antioxidants like

Ascorbic acid and Curcumin.

The lipid peroxidation inhibition nature of BGS-Haridrin was studied. As

shown in the Table 3.6, BGS-Haridrin was found to effectively inhibit ferrous

sulphate: ascorbate (inducer of lipid peroxidation) induced linoleic acid and

membrane lipid peroxidation dose dependently at 0.1µM by 76.4 2.6% and

65.5 ± 2.1 % respectively. The antioxidant activity of BGS-Haridrin was comparable

to α-tocopherol (400μM), Curcumin (400μM) and BHA (400μM) which offered

87.5 2.5 %, 77.3 3.1%, 80.2 1.8 % inhibition against linoleic acid oxidation and

72.3 ± 1.5 %, 57.2 ± 4.9 %, 64.3 ± 4.1 % respectively against ghost lipid peroxidation.

The boiling water extract of turmeric at a dosage of 25µg shows lipid peroxidation

inhibition activity of 61.6 ± 2.1 %. Thus from the above results, BGS-Haridrin is a

effective inhibitor of lipid peroxidation like BHA, Curcumin and α-tocopherol but at a

much lower dose (Mancuso et al., 2012). As shown in Table 3.7, Peak IV fraction of

DEAE A-25 column exhibits maximum scavenging of hydroxyl radicals upto

80.3 ± 1.6 which is one fold higher than sephadex G25 fraction and one and half fold

higher than crude extract. It is worthy mentioning that, the dose of BGS-Haridrin is

200 to 400 folds lesser than sephadex G-25 column fraction and crude extract of

Turmeric.

It is confirmed using a direct approach with DPPH radicals, a stable radical

being used to evaluate free radical scavenging capacities of natural antioxidants. As

shown in Fig. 3.8, at 2 to 14µg dose dependent DPPH radical scavenging activity of

BGS-Haridrin with other antioxidants like Ascorbic acid, BHA and Curcumin, the

BGS-Haridrin significantly inhibited the DPPH radicals. The positive control

104

Ascorbic acid shows maximum scavenging activity of 75% at the dosage of 56.77

nM (10 µg) and BGS-Haridrin shows maximum of DPPH radical scavenging activity

of 73% at 0.43 nM (12µg) dosage, where as other two positive controls BHA at

77.7nM (14µg) and Curcumin at 38nM (14µg) showed the scavenging activity of

64% and 62% respectively (Tuba, A.K., 2008). The above results indicate that, the

BGS-Haridrin is a powerful protein antioxidant towards scavenging DPPH radicals

and comparable with Ascorbic acid. The advantage is that, it is a chain breaking

antioxidant which does not turn into a radical itself at the end of the chain reaction.

The superoxide radicals are the free radicals and are generated in a variety of

biological systems by auto-oxidation processes or by enzymatic activities. The

concentration of superoxide anions increases under conditions of oxidative stress and

related situations (Lee et al., 2002). Moreover, superoxide anions produce other

kinds of cell damaging free radicals and oxidizing agents. Herein, we used the NBT

assay system to check BGS-Haridrin scavenge superoxide radicals. In the system,

xanthine oxidase, which is one of the main enzymatic sources of ROS in vivo,

generated superoxide radicals, which consequently reduced NBT to yield insoluble

blue formazan. As shown in Fig. 3.10, a dose dependent study was done towards

scavenging superoxide radicals by BGS-Haridrin along with other antioxidants like

Curcumin, BHA and Ascorbic acid. Here BGS-Haridrin inhibited NBT reduction by

92% by a maximum dose of 0.285 nM (8µg). Curcumin shows maximum inhibition

of 90% at a dosage of 19nM (7µg). BHA shows a maximum inhibition of 65% at a

dosage of 38.8 nM (7µg) and Ascorbic shows maximum inhibition of reduction of

NBT of 60% at a dosage of 45.4 nM (8µg). This observation indicates that BGS-

Haridrin is efficient superoxide scavenger like Curcumin. Similar studies by other

researchers have reported that, Curry leaves, Methi leaves and Sundakai could

significantly scavenge superoxide anions (Ningappa and Leela Srinivas, 2007,

Sivapriya and Leela Srinivas, 2007, Thammannagowda and Leela Srinivas, 2010).

The results of ferric ion reducing power of BGS-Haridrin are as shown in

Figure 3.11. The % of ferric ion reducing power of BGS-Haridrin at 2.86nM (80μg)

was found to be 90% which was comparable to BHA at 399nM (72µg) was 85% and

EDTA at 161.2 nM (60µg) was 74%. Further, the Ferrous ion chelating effect of

BGS- Haridrin was tested, as summarized in Fig. 3.12, the % of ferrous ion chelating

effect of BGS-Haridrin at a dose of 3.57nM (100µg) was found to be 92% indicating

that it effectively chelates iron when compared to EDTA at a dose of 161.2nM (60µg)

105

was found to be 72% and BHA at a dose of 399nM (72µg) was found to be 90%.

Fenton reaction could cause damage to all the classes of biological molecules (Nathan

and Julia, 2009).

As shown in the Fig. 3.13, proteins carbonyls formed are estimated by using

proteins carbonyl assay. Spectrophotometrically at 375 nm, the concentration of

DNPH determined. As shown in the materials and methods, amount of carbonyl

formed was estimated using formula and BSA was used as standard protein. The

spectrophotometric reading for 5 µg/10µl of BGS-Haridrin concentration was 0.38

nm. Finally the amount protein carbonyl formed was 90.9 nmol/mg. The above

results shows that, BGS-Haridrin chemically stable as estimated by Crabonyl

formation to establish whether a synergy of complimentary antioxidant activity exists

between the three antioxidant components of Turmeric namely BGS-Turmerin, BGS-

Haridrin and Curcumin.

The Figure 3.14 shows that, the antioxidant synergy between BGS-Haridrin

and BGS-Turmerin. Hence, BGS-Haridrin was kept as constant at its IC-50 dose

50nM. It provided inhibition of only 40% by scavenging hydroxyl radicals. The

BGS-Turmerin was added in increasing dose of 0 to 0.175 µM and addition of BGS-

Turmerin 0.1µM to BGS-Haridrin increases the antioxidant activity by 49%. To

understand the phenomenon better, the reverse was studied by keeping BGS-Turmerin

dose as constant at its IC-50 value is 0.09µM. It gave an inhibition of 38% as shown

in the figure 3.15. BGS-Haridrin was added in increasing dosage of 0 to 0.1µM.

Addition of BGS-Haridrin 0.1µM to BGS-Turmerin 0.09µM increases the antioxidant

activity by 58%. These combination forms the multi potent antioxidant activity and

ability of scavenging free radicals in a lower dosage and it also shows protein-protein

interaction.

The Figure 3.16 shows that, the antioxidant synergy between BGS-Haridrin

and Curcumin. The BGS-Haridrin was kept as constant and its IC-50 value is

0.05µM. It gave an inhibition of 40% by scavenging hydroxyl radicals. The

Curcumin was added in increasing amount of 0 to 400 µM. The cocktail of BGS-

Haridrin 0.05µM and Curcumin 400µM increases the antioxidant activity by 38%.

Similarly, the reverse was studied by keeping Curcumin as constant and its IC-50

value is 200µM. It gave an inhibition of 34% as shown in the figure 3.17. BGS-

Haridrin was added in increasing dosage of 0 to 0.1µM. The cocktail of Curcumin

200µM and BGS-Haridrin 0.1µM increases the antioxidant activity by 55%. These

106

combination forms the multi potent antioxidant activity and ability of scavenging free

radicals in a lower dosage.

These combinations give strong antioxidant activity and protection to cell or

cytoplasm from free radicals. This combination helps to stabilizes and protects the

cell membrane and other bioorganic components like other antioxidants (Vitamin E

and Vitamin C) (Samir A.E.B., 2006). These components are not only effective in

prevention, but also active after the free radicals react to form other toxic compounds

like secondary products.

The above studies shows that BGS-Haridrin exhibits potent antioxidant

activities such as inhibition of lipid peroxidation, hydroxyl, DPPH, nitric oxide and

superoxide radical scavenging activities. From the above facts, we could conclude

that the BGS-Haridrin is an effective protein antioxidant and a synergy exists between

the other proteins to enhance the antioxidant activity.

107

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