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Chapter 5

Isolation and characterization of

phytoconstituents from seeds of B. juncea

Section I: Pharmacognosy and

physicochemical analysis

5.1 Introduction

Brassica vegetables are known to be very nutritive, providing nutrients and

health-promoting phytochemicals such as vitamins, carotenoids, fiber, soluble sugars,

minerals, glucosinolates and phenolic compounds (Soengas et al. 2011). The

beneficial effects of Brassica vegetables on health improvement have been partly

attributed to their complex mixture of phytochemicals possessing antioxidant activity.

Thus, the purpose of extraction was to isolate phytoconstituents from seeds

responsible for maximum antioxidant activity. The broad spectrum of beneficial

effects of the seeds observed in the earlier studies on B. juncea studies warrants

further exploration of B. juncea seeds as a potential source for obtaining

pharmacologically standardized phytotherapeutics, that could be potentially useful.

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The present study is one such attempt to rationalize the existing knowledge within the

framework of modern science principles, practices and the techniques. The flowchart

of the study is shown in the.

5.2 Materials and methods

5.2.1 Chemicals and reagents

The solvents used in the study include n-hexane, chloroform, ethyl acetate, toluene,

glacial acetic acid and methanol. They were of HPLC grade and purchased from

Thermo Fisher Scientific India Pvt. Ltd (Mumbai, India). L-ascorbic acid, gallic acid

pure, colchicine extra pure and N-acetyl glucosamine extra pure were purchased from

Sisco Research Laboratories (Mumbai, Inida); vitamin E ((±)-a-tocopherol, liquid)

from Himedia Laboratories (Mumbai, India); (+)-catechin from Natural remedies

(Banglore, India); Vanillin (99% purlss for synthesis) from Spectrochem Pvt Ltd,

(Mumbai, India) and bovine serum albumin (lyophilized powder, crystallized, ≥96%

GE), (-)-sinigrin hydrate (≥99.0% TLC) and quercetin (≥95% HPLC) were purchased

from Sigma Aldrich corporation (St. Louis, United States). DPPH was purchased

from Sigma Aldrich corporation (St. Louis, United States).

5.2.2 Sample collection and identification

The dried seeds of B. juncea were procured from the local departmental store

packaged in vacuum tight polythene bags and authenticated at Agharkar Research

Institute, Pune, India (voucher specimen number S-158, Annex I).

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5.2.3 Macroscopic and microscopic analysis

The macroscopic characters of the seeds were studied with reference to evaluating

organoleptic characteristics (Evans et al. 2002). For microscopic examination, the

seeds were taken and thin sections were cut with a sharp blade. The specimens were

stained with pholorglucinol (1% w/v in ethanol) and mounted with glycerol. The

photographs of the seeds and its morphology are presented in results.

5.2.4 Physicochemical Characterization

The physicochemical characterization of the seeds of B. juncea was carried out as per

the Ayurvedic Pharmacopoeia of India (The Ayurvedic Pharmacopoeia of India

1999).

Loss on drying

A clean crucible was dried to a constant weight in air-circulated oven at 105°C. 2 g of

the seed powder was placed in the crucible and dried in the oven at 105°C to constant

weight for 2 h. The crucible and its contents were cooled in a desiccator and weighed.

The moisture content was calculated and expressed in percentage.

Determination of ash content

Total ash content

A crucible was pre-heated in a muffle furnace at 450°C, cooled in desiccator and

weighed. 2 g of the seed powder was transferred into the crucible and re-weighed. The

crucible and its content was kept in the muffle furnace at 450°C till white ash was

obtained after eighteen hours. Ash contents were determined by weighing the crucible

and difference was determined.

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Water soluble ash value

The total ash was dissolved in 25 ml water and filtered through the ashless filter paper

(Whatman No. 41) to collect the water insoluble matter. It was ignited in an electric

furnace at 450°C in silica crucible till reached a constant value. The weight of

insoluble matter was subtracted from the weight of the total ash to indicate the weight

of water soluble ash.

Acid insoluble ash value

Total ash obtained was heated with addition of 25 ml of dilute HCl for 10 min. It was

filtered in an ashless filter paper and the residue was ignited in the furnace to get a

constant weight.

Determination of extractive values

Water soluble extractive value

4 gm of powder sample was macerated with 100 ml of distilled water in a glass

stopper closed flask for 6 hours. It was shaken frequently and allowed to stand for 18

hours. It was then filtered rapidly. 25 ml of filtrate was taken in a china dish,

evaporated to dryness on a water bath. The residue was weighed and the percentage of

water soluble extractive value was calculated with reference to the powder sample

taken initially.

Alcohol soluble extractive and ether soluble extractive values

To determine the alcohol soluble extractive and ether soluble extractive value, the

similar procedure was followed, using alcohol and ether respectively instead of water.

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5.3 Results

5.3.1 Mustard seeds showed characteristic morphology

The morphological characteristics of mustard seeds authenticated the plant illustrating

characteristic features. Seeds were reddish brown, 0.9-1 mm in diameter and bitter in

taste with characteristic pungent smell when crushed. The microscopic examination of

transverse section demonstrated certain specific characteristics of oil seeds. The testa

was dark reddish-brown to yellow and minutely pitted with the cells of the outer

epidermis containing mucilage. The embryo was oily and yellow in color, containing

two cotyledons folded against their midribs to enclose the radicals (Figure 5.1).

5.3.2 Physicochemical properties of mustard seeds

Further, the results obtained for the ash values and extractive values determined by

methods described in Ayurvedic Pharmacopeia, can be used for the quality control

purposes for mustard seeds, in various pharmacological interventions. The mean,

range and standard error values of ash contents and extractive values of Brassica

juncea seeds that resulted from analyses, are summarized in Table 5.1. The moisture

content of the seeds was also determined and found to be less than 2.1% which is

animportant quality control parameter indicating the stability and the susceptibility to

bacterial and fungal contamination.

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Figure 5.1 Microscopic examination of mustard seed sections stained with

phloroglucinol (100×)

(a) Mustard seeds, (b) seed coat (Sc) and cotyledon (Ct), (c) transverse section of seeds

passing through seed coat (Sc); testa (Te); palisade cells (Ps); and tegmen (Tg), (d) release of

mucilage (Mu), (e) parenchyma cells (Pc) and oil globules (B) with the inset (ei) showing

isolated oil globules (Ai), T. S. of seeds showing cotyledons (A); outer epidermis (Oe); inner

epidermis and (Ie) radical (Rd).

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Table 5.1 Ash and extractive values of B. juncea seeds

Constituent Content (%)

Mean ± SE Range

Total ash 4.56 ± 0.14 4.42 – 4.7

Water-soluble ash 1.05 ± 0.01 1.04 – 1.06

Acid-insoluble ash 3.61 ± 0.11 3.5 – 3.72

Water-soluble extractives 5.21 ± 0.15 5.06 – 5.36

Alcohol-soluble extractives 9.43 ± 1.2 8.23 – 10.63

Ether-soluble extractives 25.7 ± 3.2 22.5 – 27.9

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Section II: Extraction and characterization of

phytoconstituents

5.4 Introduction

The premier steps to utilize the biologically active compound from plant resources are

extraction, pharmacological screening, isolation and characterization of bioactive

compound, toxicological evaluation and clinical evaluation. Extraction is the crucial

first step in the analysis of medicinal plants, because it is necessary to extract the

desired chemical components from the plant materials for further separation and

characterization. As the target compounds may be non-polar to polar and thermally

labile, the suitability of the methods of extraction must be considered. Various

methods, such as sonification, heating under reflux, soxhlet extraction and others are

commonly used (Sasidharan et al. 2011).

5.5 Methods

5.5.1 Preparation of plant extracts

Cold maceration

For the extraction by cold maceration, to 15 g mechanically grind plant powder

(coarse particles), 90 ml of solvent was added and kept on rotary shaker at 120 rpm/6

h at 28°C. After extraction, the extracts were filtered through filter paper (Whatman

No. 1) and the filtrate was concentrated by solvent evaporation under reduced

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pressure using Equitron rotovap (Medica Instruments Mgf. Ltd., Mumbai, India). The

condensed extract was then dried further in a pre-weighed silica crucible in hot air

oven at 45°C till constant weight; the yield was recorded and stored in glass vials in a

desiccator till use.

Soxhlet extraction

15 g plant powder was weighed to make a thimble using muslin cloth, which was then

inserted into the extractor attached to 150 ml round bottom flask in a heating mantle.

90 ml solvent was poured in to the extractor and the extraction was carried out at

64°C for 6 h. The extract was filtered, dried, weighed and stored.

Extraction by ultrasonication

For extraction, 15 g plant powder was weighed and 90 ml solvent was added in the

flask. The flask was then fixed in ultrasonic bath sonicator (Dakshin, Mumbai, India)

for 6 h. following extraction the extract as filtered, dried, weighed and stored.

Successive extraction of seeds by soxhlet method

Based on earlier results of the three techniques employed, the solvents for successive

extraction were selected in order to obtain the extract with more concentrated

antioxidants. For the extraction, 30 g plant powder was packed to form a thimble. The

powder was subjected to n-hexane, chloroform and methanol successively for soxhlet

extraction. The extract obtained at the end of the method following methanol

extraction, was then filtered, dried and weighed. The extract was then reconstituted in

methanol (1 mg/ml) and subjected to DPPH analysis (section 5.5.3).

Extraction of seeds by cold maceration using hydromethanol

Next, for the extraction of seeds, various proportions of hydromethanol were used

based on the results obtained from earlier experiments. The cold maceration of

powdered seeds of B. juncea was carried out using 80% methanol, 50% methanol and

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aqueous solution. The extracts obtained were dried and DPPH antioxidant activity

was determined (section 5.5.3). Based on the results, Brassica juncea 80%

hydromethanolic extract (BJHME) was found to be most effective and used for

bioassay guided column fractionation.

Fractionation of seed extract by column chromatography

10 g BJHME was loaded on to the glass column (450 × 18 mm) packed with 30 g

silica gel (60-120 mesh) dissolved in n-hexane. Stepwise gradient extraction was

carried out using the solvent scheme: 100% n-hexane (200 ml), 50% n-hexane + 50%

chloroform (200 ml), 100% chloroform (200ml), 50% chloroform + 50% methanol

(300 ml), 100% methanol (350 ml). Total of 125 fractions were collected of 10 ml

each, and pooled to total of 5 fractions, their TLC profile were developed using

mobile phase n-butanol: n-propanol: glacial acetic acid: water (3:1:1:1) and DPPH

autographic analysis by TLC was performed to screen the extract for potential

antioxidant activity.

5.5.2 TLC autographic assay

The HPTLC method was used to qualitatively determine the antioxidant activity of

extract by DPPH scavenging assay using 0.2% DPPH as a color developer. DPPH is a

paramagnetic purple colored compound with an odd electron. The color of the DPPH

reagent changes from purple to yellow due to the scavenging of free radicals by

antioxidants through donation of hydrogen to form the stable DPPH-H molecule,

visible on TLC plates. The method was used for the mobile phase system – toluene:

ethyl acetate: glacial acetic acid (4:4:1). Ascorbic acid – the water soluble vitamin and

α-tocopherol – the fat soluble vitamin were used as the positive control.

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5.5.3 DPPH Radical Scavenging Activity

The free radical scavenging activity of the BJHME was measured with stable DPPH

in terms of hydrogen donating or radical scavenging activity (Oyedemi et al. 2012).

100 µL of DPPH solution (0.36 mM DPPH in methanol) was added to 1 mL extract

(100-1000 µg/mL in methanol), vortexed thoroughly and kept in the dark at room

temperature for 30 min. Then, absorbance was measured at 517 nm using Lambda 25

uv/vis spectrometer (Perkin Elmer, Waltham, United States). Ascorbic acid was used

as the positive control. The percentage of inhibition was given by the formula: percent

inhibition (%) = [(A0-A1)/A0] x 100, where A0 was the absorbance of the control

solution and A1 was the absorbance in the presence of the sample and standards.

5.5.4 Characterization of phytoconstituents

Qualitative phytochemical screening

Various qualitative tests were performed to establish the biochemical profile of plant

extract with respect to its chemical composition using the standard procedures as

given in the Table 5.2 (Kokate et al. 2007).

Table 5.2 Standard methods for qualitative phytochemical analysis for detection

of broad class of phytoconstituents

Class of

compound

Test Reaction mixture Expected results Positive

control

Alkaloids Dragendorff’s

test

1 ml extract (1 mg/ml)

+ drop of

Dragendorff’s reagent

A prominent yellow

precipitate indicate

the test as positive

Colchicine

Carbo -

hydrates

Molish’s test 2 ml of extract (1

mg/ml) + two drops of

alcoholic solution of α-

naphthol + 1 ml of

concentrated sulphuric

acid

A red-violet layer at

the interface between

the acid (bottom) and

aqueous (upper)

layers is a positive

test

D-glucose

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Iodine test 0.01 M iodine in 0.12

M KI + 1 ml extract (1

mg/ml)

The immediate

formation of a vivid

blue color indicated

the presence of

amylose

Starch

Fats Solubility test To 1 g extract 1 ml

ether, chloroform,

ethanol or water

Solubility in ether

and chloroform and

insolubility in

ethanol and water

indicate positive test

Olive oil

Flavonoids Shinoda test 1ml 95% ethanol + 1 g

crushed seeds +

magnesium turnings +

concentrated

hydrochloric acid

Appearance of pink

color indicate

positive test

Quercetin

Lead acetate

test

1 ml extract (1 mg/ml)

+ 3 ml of 10% lead

acetate solution

A bulky white

precipitate indicate

the presence of

flavonoids

Quercetin

Saponins Foam test 0.5 ml extract (1

mg/ml) + 1.5 ml water.

Shake for 15 min

A layer of foam

indicate positive test

Proteins Millon’s test 2 ml of extract (1

mg/ml) + few drops of

Millon’s reagent

A white precipitate

indicate positive test

BSA

Ninhydrin test Two drops of ninhydrin

solution + 2 ml extract

(1 mg/ml)

A characteristic

purple indicate

positive test

Glycine

Phenolics

and tannins

Ferric

chloride test

5 ml extract (1 mg/ml)

+ 5% ferric chloride

solution

Dark green color

indicates a positive

test

Gallic

acid

Gums and

mucilage

1ml extract (1 mg/ml)

+ dil. HCl + Fehling’s

Test was performed

Development of red

color indicate the

positive test

Guggul

1 ml aq. KOH + 1 mg

extract

Swelling of the drug

indicate positive test

Adulsa

Determination of flavonoids content

The total flavonoid content was determined by the aluminum trichloride method

(Karamian & Ghasemlou 2013). Briefly, 1mL of extract (100–1000 µg/mL) or

quercetin standard solution (5–30 µg/mL) was mixed with 1.5 mL distilled water in

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the test tube, followed by 100 µL aluminum chloride (10%, w/v) and 100 µL

potassium acetate (1 M). The reaction mixture was then incubated at room

temperature for 45 min and the absorbance was measured at 415 nm by UV/Vis

spectrometer. The results of the plant sample were expressed as µg quercetin

equivalents/mg extract.

Determination of total phenolic content

The total phenolic content of the extract was determined by the Folin-Ciocalteu

reagent method (Karamian & Ghasemlou 2013). Briefly, 1 ml of extract or gallic acid

(2–10 µg/mL in methanol) was added to 5 mL Folin-Ciocalteu reagent (1:20) and

incubated for 5 min at room temperature. Next, 4 ml of sodium carbonate (10% w/v)

was added and further incubated for 15 min at room temperature for color

development. The absorbance was measured at 765 nm by UV/Vis spectrometer. The

amount of total phenolic content was expressed as µg gallic acid equivalent/mg

extract.

Determination of sugar and protein content

Sugar content was measured by 3, 5 dinitrosalicylic acid (DNS) method (Miller 1959)

and the estimation of proteins was carried out by Lowry Method (Lowry et al. 1951).

Fourier transform infrared (FTIR) fingerprinting

Approximately 1 mg dry powder of BJHME was pressed into a pellet with 200 mg of

potassium bromide and IR spectra were recorded with an accumulation of 45 scans on

IRPrestige-21 (Shimadzu Corporation, Kyoto, Japan).

Screening of phytoconstituents by TLC

BJHME was, checked by TLC on analytical plates over silica gel 60F254 (Merck &

Co., New Jersey, United States). The qualitative analysis for different class of

phytoconstituents was carried out by spotting the bands of BJHME using capillaries

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and using the mobile phase n-butanol: n-propanol: water: glacial acetic acid (3:1:1:1)

using different spray reagents.

HPTLC marker significant fingerprinting

The HPTLC study was carried out for detecting and quantifying the presence of

various marker compounds. HPTLC fingerprinting was performed at room

temperature on aluminum plates pre-coated with silica gel 60F254 (Merck, India).

Solutions of standards and sample were applied to the plates as bands 8.0 mm wide,

10.0 mm apart, and 10.0 mm from the bottom edge of the chromatographic plate

using a Camag (Muttenz, Switzerland) Linomat V sample applicator equipped with a

100µL Hamilton (India) syringe. Ascending development to a distance of 80 mm was

performed using a suitable mobile phase (Table 5.3) in a Camag glass twin-trough

chamber previously saturated with mobile phase vapor for 20 min. After

development, the plates were dried and then scanned with a CAMAG TLC scanner

with WINCAT software for quantification.

Table 5.3 Mobile phase systems for separation of marker compounds for HPTLC

marker-significant fingerprinting

Marker

compounds

Mobile phase

Catechin Toluene: ethyl acetate: glacial acetic acid (4:4:1)

Colchicine Toluene: ethyl acetate: glacial acetic acid (4:4:1) +

Toluene: ethyl acetate: diethyl amine (7:2:1)

Gallic acid Toluene: ethyl acetate: glacial acetic acid (4:4:1)

Quercetin Toluene: ethyl acetate: glacial acetic acid (4:4:1)

Quinine Toluene: ethyl acetate: glacial acetic acid (4:4:1) +

Toluene: ethyl acetate: diethyl amine (7:2:1)

Sinigrin n-butanol: n-propanol: water: glacial acetic acid (3:1:1:1)

Vanillin Toluene: ethyl acetate: glacial acetic acid (4:4:1)

Vitamin C Toluene: ethyl acetate: glacial acetic acid (4:4:1)

Vitamin E Toluene: ethyl acetate: glacial acetic acid (4:4:1)

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HPLC-DAD method development and quantification of

phytoconstituents

The study was performed to develop a sensitive and accurate HPLC method for the

profiling, identification and quantification of the chemical constituents in B. juncea

seed extracts. The standard solutions were prepared in methanol to obtain the required

concentration: sinigrin (25 ppm), catechin (100 ppm), vanillin (100 ppm), quercetin

(100 ppm) and vitamin E (1000 ppm). The respective standard stock solutions were

diluted to obtain the desired concentrations of sinigrin (0.01-25 ppm), catechin (0.1-

100 ppm), vanillin (0.1-100 ppm), quercetin (0.1-100 ppm) and vitamin E (0.1-1000

ppm). The calibration curve was plotted using the mean peak areas as the y-axis and

concentrations as the x-axis. Linear regression was used to evaluate y = mx + c and

the correlation co-efficient (r). 10 mg BJHME was reconstituted in the 10 mL HPLC

grade methanol and sonicated for 10 mins. The sample was filtered through 0.2 µ

filter and diluted 2 folds with methanol to obtain final concentration of 500 ppm to be

used for HPLC analysis.

Identification of polyphenols in the extract was carried out by UFLC Prominence

Gradient (Shimadzu Corporation, Kyoto, Japan) in Enable C18 G: 5µm 250 x 4.6 mm

column (Spinco Biotech Pvt. Ltd, Chennai, India) using diode array UV detector

(SPD-M20A). A binary gradient (pump: LC 20AD) of 10 mM KH2PO4 (pH: 7) in

deionized water (solvent A), and methanol (solvent B) was as follows: from 10% in 5

min to 50% solvent B over next 5 min, from 50% to 80% solvent B over 5 min, from

80% to 70% solvent B over 5 min, from 70% to 60% solvent B over 5 min, from 60%

to 50% solvent B over 5 min and then isocratic condition of 10% solvent B for a

further 5 min. The flow rate was 1 ml/min. The HPLC-DAD fingerprint analysis was

performed for the standards singrin, catechin, quercetin, vanillin and vitamin E.

5.5.5 Statistical analysis

All the experiments were performed in triplicates and data are expressed as mean ±

standard error. The IC50 values of the extracts for antioxidant assays were calculated

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by performing regression analysis of the data points. One way ANOVA followed by

Tukey’s test was used to compare the methanolic extracts obtained by three different

methods Figure 5.2.

5.6 Results

5.6.1 Results of extraction of B. juncea seeds

Successful determination of biologically active compounds from plant material is

largely dependent on the type of extraction procedure used. Three extraction methods

were used - cold maceration, soxhlet and sonication to obtain maximum antioxidants.

For optimal extraction, different solvent schemes were employed ranging from non-

polar to polar. The specific polarity of solvent results in extraction of molecules of the

similar polarity. In the present work, extraction procedure was standardized based on

evaluations of DPPH activity as a preliminary tool. The results were expressed as IC50

values and compared.

Extraction of powdered seeds of B. juncea was carried out using non-polar solvent n-

hexane, mid-polar solvent chloroform and polar solvent methanol employing three

different extraction techniques. Cold maceration results in the extraction of readily

extractable solutes in the solvent inclusive of heat-labile compounds. Extraction by

soxhlet method involves boiling the solvents and passing this heated solvent through

the plant powder and extracting the solutes, which generally results in loss of heat-

labile phytoconstituents. Lastly, extraction using ultrasonication involves the forced

degradation by breaking of cells as a result of penetration of ultrasonic waves and

release of compounds. The extraction carried out using these methods, were evaluated

based on yield and DPPH antioxidant activity and the results are shown in Figure 5.2.

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Figure 5.2 Extraction of B. juncea seeds employing soxhlet, cold maceration and

ultrasonication

(a) extraction by cold maceration; (b) soxhlet; (c) ultrasonication; (d) graphical represents of

the extraction by three methods

Table 5.4 Comparison of B. juncea seed extracts

Method Solvent Activity Yield

Cold n-hexane 1153 ± 78.37 24.2 ± 0.81

chloroform 702 ± 92.53 9.53 ± 0.77

methanol 173.33 ± 11.39 8.67 ± 0.67

Soxhlet n-hexane 883.33 ± 72.65 27.5 ± 1.65

chloroform 445.33 ± 11.85 11.1 ± 0.26

methanol 153.33 ± 15.03 12.4 ± 1.13

Sonication n-hexane 1350 ± 76.38 26 ± 0.58

chloroform 877 ± 105 9.67 ± 0.67

methanol 186 ± 15 4.67 ± 0.88

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It was observed that, extraction by methanol resulted in DPPH activity at lowest

concentrations versus chloroform and n-hexane. Also, the activity of seeds when

extracted by methanol employing three methods was not significantly different

(Figure 5.2 d). However, the yield in all three methods was significantly different with

very low yield using ultrasonication was obtained. Hence, further extraction was

carried out by soxhlet method using n-hexane, chloroform and methanol in successive

manner. Since, n-hexane and chloroform result in good yield with poor activity, it was

postulated that the successive extraction using these solvents would result in removal

of largely inactive proportions. The extraction of residue thereafter with methanol

should result in better activity than methanol alone. However, it was observed that,

there was a significant difference between the activities of the methanolic extract

obtained from successive extraction method versus single solvent extraction. On

successive extraction the activity of the extract reduced, with increase in IC50 to 268 ±

9.87 µg/ml. Since, hot extraction by successive soxhlet method did not increase the

activity, the cold extraction method was further explored. To obtain, the maximum

antioxidant compounds from seeds, various proportions of hydroalcohol were

employed. It was observed that, in case of extraction by 50% methanol in water IC50

was 255.33 ± 15.5 µg/ml, 80 % methanol in water was 102.67 ± 3.18 µg/ml and

aqueous was 488.33 ± 20.22 µg/ml. Thus, extraction of seeds using 80% hydroalcohol

was significantly better than other extraction methods employed. Hence, gradient

column extraction of BJHME was carried out. Column extraction of BJHME was

carried out using gradient system of solvents n-hexane, chloroform and methanol and

total of 5 different fractions were collected. The fractions were then checked for the

appearance of yellow bands on TLC plates following DPPH spraying which is an

indicator of antioxidant activity. However, it was observed that, none of the fractions

showed the distinct yellow color development when TLC was developed using the

mobile phase system -butanol: n-propanol: glacial acetic acid: water (3:1:1:1) sprayed

with DPPH reagent. These results indicated that separation of phytoconstituents

employing column separation technique, resulted in loss of activity of seeds. Hence,

BJHME with maximum antioxidant activity was used in the study.

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5.6.2 Characterization of BJHME

5.6.3 B. juncea seeds showed presence of broad classes of

phytoconstituents

The preliminary phytochemical analysis of mustard seeds indicated the presence of

various phytoconstituents in seeds as depicted in Table 5.5.

Quantitative analysis of total flavonoids, phenolics, sugars

and proteins in BJHME

The phenolic content of BJHME was estimated to be 107 ± 0.03 µg GAE/mg extract

(R2 = 0.9914) and that of flavonoids 4 ± 0.02 µg QE/mg extract (R2 = 0.9744).

Flavonoids are class of secondary plant metabolites with significant antioxidant and

chelating properties. Antioxidant activity of flavonoids depends on the structure and

substitution pattern of hydroxyl groups. The glucose concentration in the extract was

calculated using the equation y = 0.0002x – 0.0033 (R2 = 0.9695) and was found to be

78.95 ± 6.71 µg N-acetyl glucosamine equivalent/mg extract whereas the protein

content was estimated from the equation y = 0.0028x + 0.013 (R2 = 0.9961), to be

377.77 ± 00.68 µg bovine serum albumin equivalent/mg extract.

FTIR detected the presence of several functional groups

In FTIR spectroscopy, infrared (IR) radiation is passed through the extract, from

which part of the IR radiation is absorbed by the extract and part of it is transmitted.

The resulting spectrum represents the molecular absorption and transmission, creating

a molecular fingerprint of the extract representing absorption peaks that correspond to

the frequencies of vibrations between the bonds of the atoms present in it. The IR

fingerprint of BJHME also showed presence of multiple peaks (Figure 5.3), with

relatively few however very diagnostic peaks in the region above 2000 cm-1 in

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Table 5.5 Preliminary phytochemical analysis of seeds of B. juncea

Phytoconstituent Inference

Alkaloids +

Carbohydrates +

Fats +

Flavonoids +

Saponins -

Proteins +

Phenolics & tannins +

Gums and mucilage +

+ = phytoconstituent present in seed, - = phytoconstituent absent in seed

Figure 5.3 IR fingerprint of BJHME

90

contrast the other half contains many peaks with varying shapes and intensities. With

the absorption peaks of stretching at 2930, 1665 and 2123.72 cm-1 and bending

vibrations at 795.67 cm-1, BJHME shows the presence of alkanes, alkenes C=C and

alkynes C≡C. BJHME also showed a broad peak at 3333.14 cm-1 for O-H stretching

and 795.67 cm-1 for O-H bending and ring puckering, indicating the presence of

alcohols and phenols. Peaks for amines and carboxylic acid O-H bond stretching, C-

O-H bending were observed at 1053.18, 2930 and 1426 cm-1 respectively. The sharp

peak of 2123.72 cm-1 is a probable indication of presence of isocyanates,

isothiocyanates, diimides, azides and ketenes. The spectra also indicated the presence

of nitroso and nitro compounds with the peaks at 1514.19, 1514.19 and 1334.8 cm-1.

The peaks at 795.67 and 879.58 cm-1 indicated the presence of sulfane esters, whereas

1053.18 cm-1 an indicative of thiocarbonyl were also present. The extract also showed

the peaks for sulfoxide and sulphate at 1053.18 and 1334.8 cm-1, phosphorous

containing compounds phosphine, esters and phosphoramide with the peaks at

1052.18, 926.84, 1053.18 and 1272.11 cm-1. Oxidized nitrogen is present in the form

of oxime and aromatic amine oxides with absorption peaks at 1665.6, 926.84 and

1272.11 cm-1. Thus FTIR spectroscopy indicated the presence of numerous

compounds like alcohols, phenols, sulphur containing compounds, nitrogen

containing compounds which are present in plant in abundance and are known to

exert various pharmacological effects.

TLC profile demonstrated the presence of several classes of

phytoconstituents

The fingerprinting of BJHME by TLC was carried out to detect the presence of

various class of phytoconstutients that could be present in BJHME that are reported to

be antioxidants and hepatoprotective. Using different spray reagents, large classes of

compounds were detected and observed. Total of 7 distinct bands were observed

under different spraying conditions. Based on these observations of TLC profile,

various classes of phytoconstituents were identified (Table 5.6).

91

Table 5.6 TLC profile of seed extract indicating the presence of various classes of

phytoconstituents

Reagent Observation Inference

Visible UV Short UV Long

Anisaldehyde-

H2SO4

red brown

coloration,

Blue-violet,

Blue, Red

Quenching

Blue, Violet,

Green

fluorescence

Essential oils,

Pigments, Triterpenes,

Saponins

DPPH Yellow band

against purple

background

- - Antioxidant activity

Dragendorff Orange-brown - Blue Alkaloids

Ethanolic-

H2SO4

Brown-black - - Total number of

bands

NP-PEG - - Orange, Green,

Blue, Blue-

green

Bitter drugs

Flavonoids

Anthracene

Vanillin-

H2SO4

Lemon yellow,

Blue, Blue-violet,

Red, Yellow

brown

-

- Pungent principle,

Saponins

Without

spraying

- Quenching Dark yellow,

Green , Blue

Red

Dark-blue

Bitter drugs,

Flavonoids,

Pigments,

Pungent principle

92

HPTLC fingerprinting demonstrated presence of specific

markers in BJHME

In the study of identification of bioactives, silica plates were used to establish the

TLC fingerprint. The samples were identified by comparing the Rf values of bands in

their fingerprints with those of marker compounds. For positive identification, the

sample should show bands with chromatographic characteristics, including color and

position, similar to those of reference compounds. The behaviour of the extract was

observed under UV (254 and 366 nm) and visible light and was compared with the

spectra of each component (Figure 5.4-Figure 5.8). Sinigrin, vanillin, catechin and

quercetin were detected in BJHME and quantified Table 5.7.

Further, the evidence of antioxidant efficacy was acquired by DPPH HPTLC

Autographic analysis. The extract showed multiple yellow bands against purple

background when sprayed with DPPH reagent, an indication of antioxidant activity

similar to the positive controls: Vitamin C and Vitamin E (Figure 5.9).

HPLC-DAD analysis of BJHME detected the presence of

phytoconstituents

Due to the relatively high-molecular mass and intrinsic features of hydrophobic and

hydrophilic mixtures of plant bioactives, HPLC technique was used. All the

components were separated simultaneously and analyzed along with determination of

low concentrations of analytes. A sensitive and accurate HPLC method was

developed for the profiling and identification of markers in BJHME.

In the course of optimizing the conditions of separation, the influence of the stationary

and mobile phases were firstly investigated. In order to achieve a satisfactory

separation of the chemical compounds in the extract, different HPLC parameters,

including column temperature, detection wavelength and mobile phases were

examined. It was found that the column oven temperature of 30°C gave optimal

separation. Furthermore, the mobile phase was optimized through comparison of

different solvents, solvent ratios and gradient profiles.

93

Figure 5.4 HPTLC chromatogram (254 nm) and densitogram of BJHME with

sinigrin

Track (T) 1 = BJHME (200 µg), 2 = BJHME (300 µg), 3 = sinigrin (6 µg), 4 = sinigrin (14

µg).

94

Figure 5.5 HPTLC chromatogram (366 and 254 nm) and densitogram of

BJHME with catechin

Tract (T) 1 = BJHME (200 µg), 2 = BJHME (300 µg), 3 = catechin (250 ng), 4 = sinigrin

(500 ng)

95

Figure 5.6 HPTLC chromatogram (visible, 254 and 366 nm) and densitogram of

BJHME with quercetin

Track (T) 1 = BJHME (200 µg), 2 = BJHME (300 µg), 3 = quercetin (50 ng), 4 = quercetin

(100 ng), 5 and 6 = spiked (200 µg + 50 ng quercetin)

96

Figure 5.7 HPTLC chromatogram (254 nm) and densitogram of BJHME with

gallic acid and vanillin

Track (T)1 = BJHME (150 µg), 2 = BJHME (200 µg), 3 = BJHME (250 µg), 4 = gallic acid

(5 µg), 5 = vanillin (50 µg), 6 = BJHME (200 µg)

97

Figure 5.8 HPTLC chromatogram (254 and 366 nm) of BJHME with quinine &

colchicine

Track (T) 1: BJHME (150 µg), 2: BJHME (200 µg), 3: quinine (500 ng), 4: quinine (1 µg), 5:

colchicine (10 µg), 6: colchicine (20 µg).

Table 5.7 Quantification of markers in BJHME by HPTLC analysis

Marker compounds Rfa Quantification in BJHME (mg/g %)b

Vanillin 0.62 2.57 ± 0.1

Sinigrin 0.47 0.7 ± 0.001

Catechin 0.15 0.06 ± 0.01

Quercetin 0.58 0.13 ± 0.02

a results based on densitometry-HPTLC, b values expressed as the mean ± standard error

98

Figure 5.9 DPPH autographic analysis of BJHME

Track (T) 1 = BJHME (200 µg), 2 = BJHME (250 µg/mL), 3 = Vitamin C (2 µg/mL), 4 =

Vitamin C (5 µg/mL), 5 = Vitamin E (4 µg/mL), 6 = Vitamin E (10 µg/mL)

99

Since the isocratic elution mode had some of the components in the sample had too

long retention times and not all the compounds in the sample could be eluted, thus

linear gradient elution was used.

Compared with other solvent systems, methanol: 10 mM KH2PO4, pH – 7, mobile-

phase system presented more powerful separation ability for the investigated

compounds with sharper peak and better peak symmetry. As a result, the solvent

system consisting of 10 mM KH2PO4, pH: 7 (a) and methanol (b) was ultimately

selected as the mobile phase system. In BJHME, not all the peaks but most of the

main peaks could be well separated by the optimum gradient elution. Since it is not

practically feasible to strive for baseline separation of all components in one run to

fulfil the identification objectives of fingerprinting of herbal medicines; the resolution

under the optimized conditions was acceptable. All marker polyphenols were

successfully separated in a single HPLC run. The peak purity of each polyphenol in

the BJHME extract was assessed by diode array detection. HPLC fingerprinting for

simultaneous detection of all compounds in BJHME was obtained at 273 nm (Figure

5.10). Selection of detection wavelength was one of the key factors contributing to a

reliable and reproducible HPLC fingerprint of BJHME. Diode array detector (DAD)

detector was applied to select the optimum wavelength.

100

Figure 5.10 HPLC-DAD fingerprint of BJHME and standards sinigrin, catechin,

vanillin, quercetin and vitamin E at 273 nm

(a) Mixture of standards; (b) BJHME showing peaks of standards sinigrin, catechin, vanillin,

quercetin and vitamin E at 5.669, 20.92, 23.538, 26.011 and 26.984 min respectively

101

Section III: Assessment of antioxidant activity of BJHME in vitro

5.7 Introduction

Due to the complexity of the composition of foods, separating each antioxidant

compound and studying it individually is costly and inefficient, notwithstanding the

possible synergistic interactions among the antioxidant compounds in a food mixture.

Since performing only one assay is not a true reflection of the antioxidant capacity of

food, there is an emerging view that a combination of rapid, sensitive and

reproducible methods should be used wherever an antioxidant screening is desired.

5.8 Methods

5.8.1 FRAP assay

The reducing power of BJHME was quantified by FRAP assay (Song et al. 2010). 1

mL extract of various concentrations (100-500 µg) prepared in distilled water. It was

then mixed with 2.5 mL of phosphate buffer (0.2M, pH 6.6), 2.5 mL of 1 % potassium

ferricyanide and incubated at 500C for 30 minutes. After incubation, 2.5 mL of

trichloroacetic acid (10% w/v) was added and centrifuged for 10 min at 6500 rpm. 2.5

mL of the supernatant was diluted with 2.5 mL water and 0.5 mL freshly prepared

ferric chloride (0.1% w/v) was added to it. The absorbance was measured at 700 nm.

The sample was replaced with water for blank. Ascorbic acid was used as positive

control. Increase in absorbance of the reaction mixture as compared to reference

solution, correspond to the reducing power. The reducing power of BJHME was

calculated using formula: percent inhibition (%) = [(A0-A1)/A0] x 100, where A0 was

102

the absorbance of the control solution and A1 was the absorbance in the presence of

the sample and standards.

5.8.2 Superoxide radical scavenging assay

The superoxide radical scavenging activity of BJHME was measured towards

superoxide anion radicals generated in a non-enzymatic phenazinemethosulfate -

nicotinamide adenine dinucleotide (PMS-NADH) system through the reaction of

PMS, NADH, and oxygen (Lau et al. 2002). Its scavenging activity was assayed by

the reduction of nitrobluetetrazolium (NBT). The superoxide anion was generated in 3

mL of Tris-HCl buffer (100 mM, pH 7.4) containing 0.75 mL of NBT (300 μM)

solution, 0.75 mL of NADH (936 μM) solution and was mixed with 0.3 mL of

different concentrations of the extract (100-500 µg). The reaction was initiated by

adding 0.75 mL of PMS (120 μM) to the mixture. After 5 min of incubation at room

temperature, the absorbance at 560 nm was measured. Ascorbic acid was used as the

positive control. The super oxide anion radical scavenging activity was calculated

using the formula: percent inhibition (%) = [(A0-A1)/A0] x 100, where A0 was the

absorbance of the control solution and A1 was the absorbance in the presence of the

sample and standards.

5.8.3 ABTS Radical Scavenging Assay

ABTS assay is based on the scavenging of light by ABTS radicals and was performed

at Natural remedies, Bangalore, India. The relatively stable ABTS radical has a green

color and is quantified spectrophotometrically. An antioxidant with an ability to

donate a hydrogen atom will quench the stable free radical, a process which is

associated with a decrease in absorbance of the radical. The assay is performed as per

method described by (Auddy et al. 2003). ABTS radical cations were produced by

reacting ABTS and APS and incubating the mixture at room temperature in dark for

16 hours. Briefly, the total reaction volume contained 10mM PBS pH 7.4, positive

control, and test solutions of various concentrations. ABTS radical solution was added

103

to a final concentration of 0.219 mM. The reaction mixture was mixed and

immediately read at 734 nm using VersaMax ELISA microplate reader (Molecular

devices, Sunnyvale, United States). A control reaction was carried out without the test

sample. The % inhibition was calculated by formula: percent inhibition (%) = [(A0-

A1)/A0] x 100, where A0 was the absorbance of the control solution and A1 was the

absorbance in the presence of the sample and standards.

5.8.4 ORAC-Hydrophillic Assay

This assay was performed at natural remedies, Bangalore, India; as per Dávalos et al.

method (Dávalos et al. 2004). A pre-incubation mixture of 140 µl contained – 20 µl of

test solution, Trolox of various concentrations, 75 mM Sodium phosphate buffer (pH

7.4); 120 µl of Sodium fluorescein (117 nM). The mixture was incubated at 370C for

10 mins. Following pre-incubation, 60 µl of AAPH (40 mM) is added and mixed for

15 seconds. The reaction was carried out for 90 minutes at 370C. The fluorescence

measurements were taken at 485 nm excitation and 520 nm emission filters. Data

reduction and ORAC value calculation was done as per Davalos et al.

5.9 Results

Antioxidant compounds from plant extract can act either by free radical scavenging,

chelating of transitional metal, as reducing agents and as activators of antioxidant

defence enzyme systems to suppress radical damage in biological system (Halliwell &

Gutteridge 2007). Thus, on the basis of the type of chemical reactions involved, major

in vitro antioxidant capacity assays can be divided into HAT reaction based assays

and ET reaction based assays (Huang et al. 2005).

DPPH scavenging activity was estimated to be 103.37 ± 4.2 µg/mg at 50% BJHME

concentration, as discussed. Ferric reducing capacity of BJHME was checked to

evaluate the reducing power. The assay involves the change of yellow color of

BJHME test solution to green depending upon its reducing power. IC50 for ferric

104

reducing power of BJHME was calculated to be 83.26 ± 1.11 µg/mg. The reducing

properties have been shown to exert antioxidant action by donating hydrogen atom to

break the free radical chain. It has been reported that, compounds with structures

containing -OH, -SH, -COOH, -PO3H2, C=O, -NR, -S- and/or -O- in a favourable

structure-function configuration show chelation activity (Yuan et al. 2005). Thus,

molecules including phenolic acids like vanillin, catechin, gallic acid, tannic acid;

glucosinolates like thiocyantaes; alkaloids like sinapic acid, sinigrin and the

flavonoids quercetin, rutin, kaempherol, isorhamnetin, and its glycoside derivatives,

many of which reported to be present in B. juncea (Kumar et al. 2011) that could be

present in the native or modified form in BJHME are noted to chelate metal ions.

Superoxide scavenging activity of BJHME in the assay was found to be 345.22 ± 5.15

µg/mg. Superoxide is generated from O2 by multiple pathways like NADPH oxidation

by NADPH oxidase, oxidation of xanthine or hypoxanthine by xanthine oxidase, or

oxidation of reducing equivalents (e.g., NADH, NADPH, and FADH2) via the

mitochondrial electron transport system. Although superoxide anion is a weak

oxidant, it gives rise to generation of powerful and dangerous hydroxyl radicals as

well as singlet oxygen, both of which contribute to oxidative stress. Scavenging of

these radicals by BJHME may contribute to stop the chain reactions initiated by ROS

species. BJHME also has an IC50 of 83.05 µg/mL in ABTS radical scavenging assay

and ORAC value of 1115 µmoles TE/g of extract. ORAC measures antioxidant

inhibition of peroxyl and hydroxyl radical induced oxidation and reflects classical

radical chain breaking antioxidant activity by H-atom transfer (Karadag et al. 2009).

5.10 Discussion

The macroscopic and microscopic examination authenticated the seeds of B.

juncea by showing typical morphological characteristics. Also, the total ash, water

soluble ash and acid-insoluble ash contents of the seeds of B. juncea has been

reported which are important indices to illustrate the quality as well as purity of herbal

drug. Total ash includes physiological ash, which is derived from the plant tissue

itself and non-physiological ash, which is often from environmental contaminations

105

such as sand and soil. B. juncea, like other herbal materials, show a variation in the

variety and contents of compounds according to differences in growing conditions,

such as soil type, climate, etc which may change the ash content depending upon

presence or absence of various contaminants thus becoming an important parameter

of quality assessment.

Further, it is well documented that phytoextracts contain free radical

scavenging molecules, such as vitamins, terpenoids, phenolic acids, lignins, stilbenes,

tannins, flavonoids, quinones, coumarins, alkaloids, amines, betalains, and other

metabolites, which are rich in antioxidant activity. The preliminary phytochemical

study of B. juncea was carried out to characterize the chemical constituents present in

the extracts following standard procedures.

The results obtained from qualitative phytochemical analysis revealed the

presence of plant biomolecules like fats, proteins, amino acids along with other

important secondary metabolites like alkaloids, flavonoids, phenolics and tannins

which are known for their varied pharmaceutical roles. The use of preliminary data

obtained was extended to extract the relevant biomolecules from the seeds.

The APAP hepatotoxicity, as discussed in the chapter 4, was due to excessive

generation of ROS resulting in damage to hepatocyte membrane, DNA fragmentation

and extensive necrosis leading to alteration of cell cycle. In APAP toxicity, the

binding of active metabolite NAPQI with GSH, resulting in glutathione depletion,

which is an intracellular antioxidant compound. This sulphur containing biomolecule

in reduced form reduces reactive oxygen species undergoing oxidation. The depletion

of GSH results in oxidative stress within the cells, which then binds to various

proteins and lipid bilayer, propagating the toxicity. The strategy behind the

phytotherapy against APAP toxicity was hence to extract phytoconstituents with

maximum antioxidant activity that can act as reducing agents against ROS

diminishing the deleterious effects. Further, phytoconstituents have been reported to

be nutritious owing to the presence of various vitamins, carotenoids, phenolics, etc

which has an added advantage along with the therapeutic effect. For the extraction

and isolation of active phytoconstituents from seeds of B. juncea, the bioassay guided

approach was used. For the preliminary antioxidant screening of the extract, a simple,

106

easy, reproducible and widely used method of DPPH scavenging assay was

performed; followed by extensive characterization of the selected extract for various

radicals scavenging potential by FRAP, ABTS, ORAC and superoxide scavenging

assays.

Mustard seeds are the biggest source of edible oil in northern part of India.

Thus, extraction using n-hexane was performed to check, if the oils released by the

method show antioxidant activity. But the fixed oils, alkanes, some terpenoids,

alkaloids, and coumarins that get extracted in non-polar solvents showed no activity

till the highest concentration of 1000 µg/ml in any of the methods. Chloroform is a

mid-polar solvent which usually extracts waxes, steroids, carotenoids, pigments and

resins. Extraction with chloroform showed very little activity with IC50 at high

concentration of 445.33 ± 11.85 µg/ml with soxhlet method; whereas, method of cold

maceration and ultrasonication resulted in even lesser activity. It is also known, that

flavonoid glycosides, tannins, some alkaloids and other pharmacologically active

compounds are usually extracted in polar solvent systems. Extraction with methanol

gave moderate activity of 153.33 ± 15.03 µg/ml with soxhlet extraction, 173.33 ±

11.39 µg/ml with cold maceration and 186 ± 15 µg/ml with method of ultrasonication.

Extraction of seeds by alcohol resulted in significantly higher activity as compared to

n-hexane and chloroform (p≤0.05). Alcohols are more efficient in penetrating cell

walls and seed degradation and cause polyphenols to be released from cells (Tiwari et

al. 2011). It was observed, that with methanol (polarity index 5.1) all the three method

resulted in non-significant difference in activity. Hence to isolate the antioxidant

fraction, the method of soxhlet was further extended to be used for successive

extraction. It was observed that with soxhlet method of extraction, the yield of n-

hexane extract was 27.5 ± 1.65% and chloroform extract was 11.1 ± 0.26% with

minimal activity (Table 5.4). Thus, it was presumed that the successive extraction of

seeds by n-hexane and chloroform, would result in removal of approximately 35-40%

inactive fraction, and final extraction by methanol to obtain the active fraction with

higher activity. However, it was observed that, with the removal of inactive fraction,

the activity of methanol fraction reduced. The activity of methanolic fraction obtained

as a result of successive extraction (IC50 = 268 ± 9.87 µg/ml) was lower than the

activity of methanolic extract obtained after the single solvent extraction (IC50 =

153.33 ± 15.03 µg/ml). This could be due to possible synergism among the

107

phytoconstitents. It has been well reported in the study of plant extracts, that the

effects observed with plants is usually due to the combined effect of all the

phytoconstituents present in it. Thus, with successive extraction, the removal of other

fractions could have resulted in the loss of combined effect contributed by the other

fractions resulting in reduction of activity. Further, the low activity derived with

soxhlet method could be because of thermal degradation of parent compounds present

in seeds, generated because of successive exposure to hot solvent vapors. Since, the

hot successive extraction method did not improve the antioxidant capacity

significantly; the next approach was to modify solvents used in cold maceration

technique. To improve the activity of extract obtained using methanol by cold

maceration, methanol was combined with different proportions of distilled water.

Water is a good solvent to extract plant polyphenols as it offers high solubility, it is

less toxic and inexpensive compared to other organic solvents. It was observed that,

extraction with 80% methanol in water improved the antioxidant activity of the

extract (section 5.9). The higher activity of the hydroalcoholic extract could be due to

the presence of higher amounts of polyphenols that could be extracted due to

combined effect of high solubility offered by water and high cell-penetration power of

alcohol. Hydroalcoholic mixtures are also completely miscible further suited for the

extraction of the active principles from the seeds.

This extract obtained with 80% methanol by cold maceration was termed

Brassica juncea hydroalcoholic extract referred to as BJHME. Next, BJHME was

subjected to column extraction following a gradient of n-hexane, 50% n-hexane +

50% chloroform, chloroform, 50% chloroform + 50% methanol and methanol. The

similar results were observed with the separation of phytoconstituents by column

chromatography, the activity of the extract reduced, and became undetectable by

DPPH autographic analysis. Thus, for the analysis of hepatoprotective potential,

BJHME was used, since it resulted in maximum activity at minimum concentration

(IC50 = 102.67 ± 3.18 µg/ml).

Dietary antioxidants, including polyphenolic compounds, vitamins e and c and

carotenoids, are believed to be the effective nutrients in the prevention of these

oxidative stress related diseases (Huang et al. 2005). With the objective of

supplementing the cells with natural antioxidants under the situations of GSH

108

depletion, an important effort was dedicated to identify and characterize BJHME for

its antioxidant activity, which may aid in understanding the probable effect it has on

HepG2 cells under APAP induced oxidative stress. It was observed that seeds possess

variety of radical scavenging potential in ET and HAT based assays. Also, earlier

studies on mustard seed meal report similar findings on mustard seeds exhibiting

antioxidant potential (Singh & Malik 2011; Dubie et al. 2013). The leaves of mustard

are also reported to possess antioxidant activity (Nouairi et al. 2008; Kim et al. 2003)

and has been reported to be related other therapeutic effects it exerts on

streptozotocin-induced diabetic rats (Yokozawa et al. 2002). Peroxynitrite radical

scavenging potential of mustard has been used in isolation of active phytoconstituents

like isorhamnetin (Choi et al. 2002) and sinapic acid (Zou et al. 2002). BJHME also

exhibited radical scavenging and reducing potential in in vitro assays. Cabbage,

broccoli and cauliflower belongs to the family Brassicaceae as that of mustard Table

5.8.

Brassica vegetables show antioxidant activity of 3-8.9 µmol TE/g as versus

BJHME (1115µmol TE/g). Also these plants possess almost one fifth or lesser content

of total phenolics compared to Indian mustard seeds as reported by Podsedek, 2005.

When compared with other spices and condiments used in dressings in variety of

Indian cuisines such as cumin and ginger, BJHME also showed strong antioxidant

capacity, justifying the use of seeds for various ailments by traditional practitioners.

Further, phenolics are not sole the source of antioxidant activity supported by the fact

that the plants known for exhibiting hepatoprotective potential like green tea,

pomegranate, mango and amla show high antioxidant activity, in spite of having

lesser phenolic content Table 5.8. Mustard meal has been reported to be a good source

of phenolic compounds. More than a dozen phenolic acid conjugates have been

reported and the spectrum of phenolics is also unique and broad (Cartea et al. 2011).

The antioxidant capacity of Brassica species has been related to their phenolic profile

and content, particularly flavonoids, since phenolic compounds have demonstrated a

higher antioxidant activity than vitamins and carotenoids (Cartea et al. 2011).

109

Table 5.8 Comparison of phenolic content and antioxidant activity of B. juncea

with other plants of Brassicaceae family, condiments and reported

heptoprotective plants

Scientific

Name

Folk Name Antioxidant Activity References

Total

phenolics

(µg GA/ g)

ORAC

(µmol TE/ g)

Other members of Brassicaceae family

B. juncea Indian

mustard

107 ± 0.03 1115

B. oleracea var.

oleracea

Cabbage 15.3 ± 2.1 3 (Podsedek 2005)

B. oleracea Broccoli 34.5 ± 1.0 3.8 (Podsedek 2005)

B.oleracea

var.botrytis

Cauliflower 27.8 ± 1.5 8.9 (Podsedek 2005)

Other condiments

Cuminum

cyminum

Cumin 750 ± 69 76800.2 ±

7500

(Ninfali et al. 2005)

Zingiber officinale Ginger 200.5 ± 19 14840.2 ±

1060

(Ninfali et al. 2005)

Other hepatoprotective plants

Camellia sinensis

(leaves)

Green tea 21.02 ± 1.54 8777 ±

35.36

(Hiraganahalli et al.

2012; Anesini et al.

2008)

Punica granatum

(fruit peel)

Pomegranate 11-21

(range)

3481 ±

206.5

(Hiraganahalli et al.

2012; Shams

Ardekani et al. 2011)

Mangifera indica

(Bark)

Mango 43.33 8450 (Cai et al. 2004)

Phyllantus

emblica (fruit)

Amla 62-439

(range)

3944 ±

128.7

(Hiraganahalli et al.

2012; Liu et al. 2008)

110

Brassicaceae vegetables represent an important part of the human diet

worldwide and are considered important food crops in China, Japan, India and

European countries. Various classes of phytoconstituents from seeds of B. juncea

were also detected via qualitative analysis. B. juncea are known to produce several

classes of bioactive phytochemicals including glycosides, flavonoids, phenolic

compounds, sterols, triterpene alcohols, glucosinolates, proteins and carbohydrates.

The available pre-clinical information on this easily cultivable and edible plant

strongly suggests that it could be a sustainable source of affordable nutraceuticals or

drugs. The beneficial effects of Brassica vegetables on health improvement have been

partly attributed to their complex mixture of phytochemicals possessing antioxidant

activity(Cartea et al. 2011). In the present work, the extraction procedure was

standardized based on evaluations of DPPH activity as a preliminary tool. Recent

reports suggest that cruciferous vegetables act as a good source of natural antioxidants

due to their high levels of carotenoids, tocopherols and ascorbic acid (Cartea et al.

2011).