Pharmacognosy and Phytochemical Analysis of Brassica juncea Seeds

Abstract

Introduction: Brassica juncea is an economically important plant that has been well-known in India for centuries for its medicinal and nutritive values. The broad spectrum of benefi cial effects of the seeds perceived with this plant warrants further exploration of B. juncea seeds as a potential source for obtaining pharmacologically standardized phytotherapeutics, which could be potentially useful. The objective of the present study was to perform the pharmacognosy of mustards seeds inclusive of qualitative and quantitative phytochemical analysis, fi ngerprinting by infrared spectroscopy and high performance thin layer chromatography analysis and toxicity assessment in vitro. Methods: Different sections of seeds were taken and stained with 0.1% phloroglucinol for microscopic examination. The seeds were extracted by 80% alcohol on a rotary shaker to perform phytochemical analysis and fi ngerprinting. The toxicity assessment of this extract was performed on human dermal fi broblast cells. Results: Microscopic examination of seeds showed characteristic features of mustard seeds. The extraction of these seeds by 80% alcohol resulted in IC50 value of 103 ± 3 μg/mL for 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl radical scavenging assay. The fingerprinting analysis of this extract indicated probable presence of sinigrin, quercetin, vanillin, catechin, vitamin E and sulfur-containing compounds. This extract exhibited 50% toxicity (IC50) at 1.79 mg/mL. Conclusion: The result achieved will be used to assess the therapeutic effi cacy of seed extracts for future pharmacological evaluations.

Full text

Phcog J | Sep-Oct 2014 | Vol 6 | Issue 5 i
Table of Contents
Vol 6, Issue 5, Sep-Oct, 2014
Original Article
Protective Effects of Beetroot Extract against Phenyl Hydrazine Induced Anemia in Rats
Anupam Jaiswal, Aditya Ganeshpurkar, Ankita Awasthi, Divya Bansal, Nazneen Dubey
DOI: 10.5530/pj.2014.5.1 1
Comparative Studies on Antioxidant Activity, Total Phenol Content and High Performance
Thin Layer Chromatography Analysis of Seabuckthorn (Hippophae rhamnoides L) Leaves
Amrit Kumar Singh, Prakash Deep, Suchita Dubey, Dharam Paul Attrey, Tanveer Naved
DOI: 10.5530/pj.2014.5.2 5
Hepatoprotective Effect of Calotropis procera in Isoniazid and Rifampicin Induced Hepatotoxicity
Noor Kamil, Hafi z Syed Imran-ul-Haque
DOI: 10.5530/pj.2014.5.3 9
Antihyperglycemic Activity and Standardization of the Bioactive Extract of Cleome droserifolia
Growing in Egypt
Amira Abdel Motaal, Shahira M. Ezzat, Hesham El-Askary
DOI: 10.5530/pj.2014.5.4 15
Comparative Morphological and Anatomical Study of Onosma caucasica Levin. ex M. Pop. and
Onosma sericea Willd. (Boraginaceae Juss.)
Janna Vladimirovna Daironas, Fatima Kazbekovna Serebryanaya, Ifrat Nazimovich Zilfi karov
DOI: 10.5530/pj.2014.5.5 22
Pharmacognostic and Phytochemical Studies on Flowers of Aerva lanata [L.] Juss. ex. Schult
Netala Silvia, C. H. Rajeswari, D. Mounica, R. Manasa, D. S. N. B. K. Prasanth
DOI: 10.5530/pj.2014.5.6 29
Pharmacognostic Investigation of Valeriana hardwickii Wall. A Threatened Herb
Anant V. Bhandarkar, S. Shashidhara, M. Deepak
DOI: 10.5530/pj.2014.5.7 33
Evaluation of Wound Healing Potential of Some Indian Herbal Extracts and it’s Formulation
in Acne Vulgaris
S. A. Thube, M. J. Patil
DOI: 10.5530/pj.2014.5.8 37
Contents | Contd...... |

ii Phcog J | Sep-Oct 2014 | Vol 6 | Issue 5
Pharmacognosy and Phytochemical Analysis of Brassica juncea Seeds
Harita Parikh, Aparna Khanna
DOI: 10.5530/pj.2014.5.9 47
Immunomodulatory Effect of Water Soluble Polysaccharides Isolated from Metroxylon sagu in
Animal Models of Immunosuppression
Sireesha Pulla, Nagarjuna Sannithi, Siva Reddy Challa
DOI: 10.5530/pj.2014.5.10 55
Letter to Editor
Development and Antifungal Evaluation of Cinnamaldehyde Containing Silver Nanoparticles
against Candida albicans
Sharada L. Deore, Sharad Chaudhari, Bhushan A. Baviskar, Somshekhar S. Khadabadi
DOI: 10.5530/pj.2014.5.11 63
Contents | Contd...... |

Phcog J | Sep-Oct 2014 | Vol 6 | Issue 5 47
PHCOG J ORIGINAL ARTICLE
Pharmacognosy and Phytochemical Analysis of
Brassica juncea Seeds
Harita Parikh, Aparna Khanna*
Department of Biological Sciences, School of Science, NMIMS University, Vile Parle (West), Mumbai, Maharashtra, India
ABSTRACT
Introduction: Brassica juncea is an economically important plant that has been well-known in India for centuries for
its medicinal and nutritive values. The broad spectrum of benefi cial effects of the seeds perceived with this plant
warrants further exploration of B. juncea seeds as a potential source for obtaining pharmacologically standardized
phytotherapeutics, which could be potentially useful. The objective of the present study was to perform the
pharmacognosy of mustards seeds inclusive of qualitative and quantitative phytochemical analysis, fi ngerprinting
by infrared spectroscopy and high performance thin layer chromatography analysis and toxicity assessment in vitro.
Methods: Different sections of seeds were taken and stained with 0.1% phloroglucinol for microscopic examination.
The seeds were extracted by 80% alcohol on a rotary shaker to perform phytochemical analysis and fi ngerprinting. The
toxicity assessment of this extract was performed on human dermal fi broblast cells. Results: Microscopic examination
of seeds showed characteristic features of mustard seeds. The extraction of these seeds by 80% alcohol resulted
in IC50 value of 103 ± 3 μg/mL for 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl radical scavenging assay. The
fi ngerprinting analysis of this extract indicated probable presence of sinigrin, quercetin, vanillin, catechin, vitamin E
and sulfur-containing compounds. This extract exhibited 50% toxicity (IC50) at 1.79 mg/mL. Conclusion: The result
achieved will be used to assess the therapeutic effi cacy of seed extracts for future pharmacological evaluations.
Keywords: Antioxidant, cytotoxicity, Fourier transform infrared spectroscopy, high performance thin layer
chromatography, microscopy, phenolics
INTRODUCTION
Brassica juncea Czern. and Coss., also known as Indian
mustard, Chinese mustard, oriental mustard, leaf mustard,
or mustard green, is a species of mustard family of
Brassicaceae (cruciferous) plants.1 B. juncea is an economically
important plant that has been well known in India for
centuries for its medicinal and nutritive values. Various
parts of the plants are edible and used in a range of folk
medicines and spices. The mustard seeds have been used
traditionally for the treatment of muscular rheumatism,
infl ammatory neuralgic affections, vomiting and dengue.2
The seeds have also used as folkloric medicine against
jaundice in the Jalgoan district of Maharashtra and by
the Sugali tribes of the Yerramalais forest of the Eastern
Ghats of Andhra Pradesh, India.3,4 There are various
polyherbal formulations of mustard discussed in ayurveda
medicine. A decoction of Moringa oleifera root (1 in 20) with
the addition of bruised mustard seed is useful in doses
of 1-2 ounces in ascites due to liver and spleen diseases.5
A fresh root of M. oleifera mixed with mustard seeds and
green ginger is used as a counter-irritant and blistering
agent.5 A paste of equal parts mustard, horseradish seeds,
hemp seeds and barley mixed with sore buttermilk is a
useful application to the scrofulous glands of the neck.
According to ayurveda medicine, mustard preparations
are mild laxatives, diuretics, and liver-bile stimulators6
and have been also documented to purge the toxins out
of the body.7 The leaves and seeds of these plants are
edible and diverse medicinal uses of seeds are also well-
known in other countries. In China, mustard seed is a folk
remedy for arthritis, foot ache, lumbago and rheumatism.
It is also used in the treatment of tumors; leaves are
used in soups for bladder infections, infl ammation or
haemorrhage. In Korea, the seeds are used for abscesses,
colds, lumbago, rheumatism and stomach disorders. The
seeds are also reported to be hypoglycemic,8 antioxidant,9
anti-diabetic,10 hyperglycemic,11 anxiolytic,12 goiterogenic,13
and hepatoprotective.2 The seed paste is used to treat
*Corresponding author:
Dr. Aparna Khanna,
Department of Biological Sciences, School of Science,
NMIMS University, V. L. Mehta Road, Vile Parle (West),
Mumbai - 400 056, Maharashtra, India.
Phone No.: +91 22 42199981, Fax +91 22 26114512,
E-mail: aparna.khanna@nmims.edu
DOI: 10.5530/pj.2014.5.9

Parikh and Khanna: Pharmacognosy and phytoanalysis of B. juncea seeds
48 Phcog J | Sep-Oct 2014 | Vol 6 | Issue 5
backache, arthritis, paralysis, styes, edema of the lungs
and liver, aperient, stimulant and emmenagogue.7,14 The
leaves, seeds, and stems have been shown to reduce the
severity of asthma and high blood pressure, restore normal
sleep attacks and prevent heart attack in patients suffering
from atherosclerosis or patterns in women experiencing
symptoms of menopause, and reduce the frequency of
migraine diabetic heart disease.15 The hepatoprotective
activity of aqueous extract of mustard leaves have been
evaluated against carbon tetrachloride-induced hepatic
damage in albino rats.16
Taken together, these reports strongly suggest the
therapeutic potential of seeds against various disorders.
However, no definitive evaluation of nature of
phytoconstituents involved in their observed effects
has yet been made. This inability arises not only from
the diverse types of extracts and experimental design
used in different studies but also due to variations in the
agronomic conditions used in cultivation and harvest.
The broad spectrum of benefi cial effects of the seeds
observed in these studies warrants further exploration
of B. juncea seeds as a potential source for obtaining
pharmacologically standardized phytotherapeutics,
that could be potentially useful. To address the lack of
literature on standardization, our work focuses on the
pharmacognosy of these seeds inclusive of fi ngerprinting
by infrared (IR) spectroscopy and high performance thin
layer chromatography (HPTLC) analysis and toxicity
assessment in vitro.
MATERIALS AND METHODS
Identifi cation and authentication
B. juncea seeds were identifi ed and authenticated at Agharkar
Research Institute, Pune, India (Voucher specimen
number: S-158).
Macroscopic examination of seeds
The macroscopic characters of the seeds were studied with
reference to evaluating organoleptic characteristics.
Microscopic examination of seeds
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.
Determination of ash content and extractive value
Total, water-soluble and acid-insoluble ash contents
and water, alcohol and ether soluble extractive values of
the powdered seeds was determined as per the standard
procedure.17
Extraction of phytoconstituents
The extraction of B. juncea seeds was carried out by 80%
methanol using a rotary shaker for 6 h and the antioxidant
activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging assay was measured.
DPPH radical scavenging activity
The free radical scavenging activity of the extract was
measured with stable DPPH in terms of hydrogen donating
or radical scavenging activity. 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. Next, the
absorbance was measured at 517 nm using ultraviolet-visible
(UV-VIS) spectrometer (Perkin Elmer Lambda 25, Perkin
Elmer India, Thane, India). Ascorbic acid was used as the
positive control. The percentage of inhibition was given by
the formula: Percent inhibition (%) = ([A0-A1]/A0) × 100,
where A0 is the absorbance of the control solution and A1 is
the absorbance in the presence of the sample and standards.
Qualitative evaluation of phytoextract
Phytochemical analysis of crude extracts were carried out
to determine the presence of the various biomolecules
using standard procedures.18 Qualitative tests for tannins,
fl avonoids, steroids, alkaloids, sugars, proteins, and fats
were performed.
Quantitative estimation
Determination of the total fl avonoid content
The total flavonoid content was determined by the
aluminum trichloride method.19 Briefl y, 1 mL of extract
(100-1000 μg/mL) or quercetin standard solution
(5-30 μg/mL) was mixed with 1.5 mL distilled water in
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.

Parikh and Khanna: Pharmacognosy and phytoanalysis of B. juncea seeds
Phcog J | Sep-Oct 2014 | Vol 6 | Issue 5 49
Total phenolic content
The total phenolic content of the extract was determined
by the Folin–Ciocalteu reagent method.19 Briefl y, 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
The sugar content was measured by the 3,5-dinitrosalicylic
acid method and the estimation of proteins was carried out
by the Lowry method.15,16
Fourier transform infrared (FTIR) spectroscopic
analysis of plant extract
Approximately, 1 mg of dried extract was pressed into
a pellet with 200 mg of potassium bromide and IR
spectra were recorded with an accumulation of 45 scans
and a resolution of 4/cm on IRPrestige-21 (Shimadzu
Corporation, Kyoto, Japan).
Qualitative profi ling of extract by TLC
The seed extract was checked by TLC on analytical
plates over silica gel 60F254 (Merck and Co., New Jersey,
USA). The qualitative analysis for different class of
phytoconstituents was carried out by spotting the bands
of extract using capillaries and using the mobile phase
n-butanol:n-propanol: water:glacial acetic acid (3:1:1:1)
using different spray reagents.
HPTLC fi ngerprinting
DPPH HPTLC 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.18
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.
HPTLC marker signifi cant fi ngerprinting
The HPTLC study was carried out for detecting the
presence of vanillin, quercetin and catechin sinigrin.
HPTLC fi ngerprinting was performed at room temperature
on aluminum plates pre-coated with silica gel 60F254
(Merck and Co., New Jersey, USA). 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
100 μL Hamilton (India) syringe. Ascending development
to a distance of 80 mm was performed using a suitable
mobile phase (Table 1) 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 quantifi cation.
Cytotoxicity assessment on human dermal fi broblast
(HDF) cells
HDF cells were obtained from Scientifi c Research Center,
V. G. Vaze College, Mumbai, India, were grown in 10%
fetal bovine serum in Dulbecco’s minimal essential medium
(DMEM) containing 100 U/mL penicillin and 100 mg/mL
streptomycin at 37°C in a humidifi ed CO2 incubator.
(3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide assays of the seed extract on HDF cells were
performed. Toxicity toward HDF was assessed with cells
Table 1: HPTLC marker signifi cant fi ngerprinting of
mustard seed extracts
Chemical
constituents
Mobile phase Rf
aQuantifi cation
(mg/g%)b
Vanillin Toluene:ethyl
acetate:glacial
acetic acid (4:4:1)
0.62 2.57±0.1
Sinigrin n-butanol:
n-propanol:glacial
acetic acid (3:1:1:1)
0.47 0.7±0.001
Catechin Toluene:ethyl
acetate:glacial
acetic acid (4:4:1)
0.15 0.06±0.01
Quercetin Toluene:ethyl
acetate:glacial
acetic acid (4:4:1)
0.58 0.13±0.02
aResults based on densitometry-HPTLC, bvalues expressed as the mean±standard error.
HPTLC: High performance thin layer chromatography

Parikh and Khanna: Pharmacognosy and phytoanalysis of B. juncea seeds
50 Phcog J | Sep-Oct 2014 | Vol 6 | Issue 5
plated in 96-unit well plates at a density of 5 × 104 cells/well.
After adherence, the medium was removed and replaced by
serum-free media containing seed extract (0.1-6.4 mg/mL)
and incubated for 24 h at 37°C in a humidifi ed CO2
incubator. Doxorubicin (0.001-10 μM) was used as a
positive control. Control cells were incubated with DMEM.
Cell viability was determined by measuring the absorbance
at 570 and 655 nm. Results were expressed as percentage
cellular viability.
RESULTS
Macroscopic and microscopic examination of seeds
The seeds were reddish brown with a smooth texture and
approximately 0.9-1 mm in diameter; they had a bitter taste
and characteristic pungent smell when crushed. The testa
was dark reddish-brown to yellow and minutely pitted. The
cells of the outer epidermis of the testa contained mucilage.
The embryo was oily and yellow in color, containing two
cotyledons folded against their midribs to enclose the
radicals (Figure 1).
Physicochemical characterization of seeds
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 B. juncea seeds that
resulted from analyses, are summarized in Table 2. The
moisture content of the seeds was also determined and
found to be <2.1%, which is an important quality control
parameter indicating the stability and the susceptibility to
bacterial and fungal contamination.
Preliminary characterization of seeds
The preliminary phytochemical study of B. juncea seeds
was carried out to characterize the chemical constituents
present in the extracts following standard procedures.
The results indicated the presence of polyphenols such
as phenolic acids, fl avonoids, alkaloids, and tannins in
methanolic extract. The qualitative analysis also indicated
the presence of fi xed oil content in n-hexane extract of
seeds. The calculated values of oils were 30%, similar
to reported quantities of 24-35%. Preliminary data were
extended to isolate phytoconstituents enriched with
antioxidant properties from the seeds.
Extraction of seeds
Mustard seeds were extracted by 80% methanol in water to
obtain an antioxidant-rich extract. The yield was calculated
Figure 1: Microscopic examination of mustard seeds stained with phloroglucinol (×100) (full page width). (a) Mustard seeds, (b) seed
coat (Sc) and cotyledon (Ct), (c) transverse section of seeds passing through 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),
(f) T. S. of seeds showing Ct; outer epidermis (Oe); inner epidermis (Ie) and radical (Rd).
Table 2: Ash and extractive values of seeds of
B. juncea
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
The data is represented as mean±SE, where n=3. B. juncea: Brassica juncea,
SE: Standard error

Parikh and Khanna: Pharmacognosy and phytoanalysis of B. juncea seeds
Phcog J | Sep-Oct 2014 | Vol 6 | Issue 5 51
to be of 7.24 ± 0.45% and the IC50 value for DPPH radical
scavenging capacity was determined to be 103 ± 3 μg/mL.
Quantitative estimations of biomolecules
The fl avonoid content of the extract was found to be
4 ± 0.02 μg quercetin equivalent/mg extract (R2 = 0.9744)
and the phenolic content was 107 ± 0.03 μg gallic
acid equivalent/mg extract (R2 = 0.9914). 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, 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 fi ngerprint of the extract
representing absorption peaks that correspond to the
frequencies of vibrations between the bonds of the atoms
present in it. The IR fi ngerprint of extract also showed
presence of multiple peaks, with relatively few however
very diagnostic peaks in the region above 2000/cm in
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 and bending
vibrations at 795.67/cm, BJHAE shows the presence
of alkanes, alkenes C=C and alkynes C≡C. BJHAE also
showed a broad peak at 3333.14/cm for O-H stretching
and 795.67/cm 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
respectively. The sharp peak of 2123.72/cm 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. The peaks at
795.67 and 879.58/cm indicated the presence of sulfane
esters, whereas 1053.18/cm an indicative of thiocarbonyl
were also present. The extract also showed the peaks
for sulfoxide and sulfate at 1053.18 and 1334.8/cm,
phosphorous containing compounds phosphine, esters
and phosphoramide with the peaks at 1052.18, 926.84,
1053.18 and 1272.11/cm. 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. Thus
FTIR spectroscopy indicated the presence of numerous
compounds such as alcohols, phenols, sulfur containing
compounds, nitrogen-containing compounds, which are
present in plant in abundance and are known to exert
various pharmacological effects.
TLC qualitative profi le showed the presence of several
classes of phytoconstituents
The fi ngerprinting of extract by TLC was carried out to
detect the presence of various class of phytoconstutients
that could be present in the extract that are reported to be
antioxidants and hepatoprotective. Using different spray
reagents, large classes of compounds were detected and
observed. A total of seven distinct bands were observed
under different spraying conditions with Rf 0.19, 0.26,
0.28, 0.4, 0.43, 0.5 and 0.57. Based on these observations
of TLC profi le, various classes of phytoconstituents were
identifi ed (Table 3).
Fingerprinting analysis of the seed extract
In the study of identifi cation of bioactives, silica plates were
used to establish the TLC fi ngerprint. The characteristic
of the extract was observed under UV (254 and 366 nm)
and VIS light was compared with the spectra of each
component (Table 1). Sinigrin, vanillin, catechin and
quercetin were found to be present and were quantifi ed.
Of these, vanillin, catechin and quercetin were phenolic
acids and reported to be antioxidants. The evidence of
antioxidant efficacy was acquired by DPPH HPTLC
autographic analysis. The extract showed multiple yellow
bands against a purple background when sprayed with
DPPH reagent, an indication of antioxidant activity similar
to the positive controls: Vitamins C and E (Figure 2).
Non-toxic nature of seed extract on HDF cells
The cytotoxic effect of the seed extract of B. juncea was
evaluated on HDF cells and it was observed that the extract
showed a dose-dependent cytotoxicity (Figure 3) with IC50
at 1.79 mg/mL in vitro.
DISCUSSION
Brassicaceae vegetables represent an important part of the
human diet worldwide and are considered important food
crops in China, Japan, India and European countries.
The macroscopic and microscopic examination identifi ed
the seeds of B. juncea by showing typical morphological

Parikh and Khanna: Pharmacognosy and phytoanalysis of B. juncea seeds
52 Phcog J | Sep-Oct 2014 | Vol 6 | Issue 5
characteristics. Furthermore, the total, water soluble
and acid-insoluble ash contents of the seeds of B. juncea
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 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 which
may change the ash content depending upon presence
or absence of various contaminants thus becoming an
important parameter of quality assessment.
B. juncea are known to produce several classes of bioactive
phytochemicals including glycosides, fl avonoids, phenolic
compounds, sterols, triterpene alcohols, glucosinolates
(GLSs), 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 benefi cial
effects of Brassica vegetables on health improvement
have been partly attributed to their complex mixture of
phytochemicals possessing antioxidant activity.20 Various
classes of phytoconstituents from seeds of B. juncea were
detected via qualitative analysis. 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.20 For optimal extraction of antioxidants, 80%
methanol was used employing rotary shaker for 6 h, since
hydroalcoholic mixtures are the most versatile and widely
employed solvent system. Alcohol is at in penetrating cell
walls and seed degradation and causes polyphenols to
be released from cells; water is a non-toxic solvent with
higher polarity. Thus it can solubilize polar compounds to
the highest degree, acting as an agent most suited to the
extraction of the active principles from plant drugs.21 This
technique translated into a good DPPH scavenging activity
(IC50 = 103 ± 3 μg/mL) of the extract.
Considering the health benefi ts, establishing the therapeutic
potential of the seeds, phenolics and fl avonoids is critical; it
was observed that the hydroalcoholic extract that showed
promising antioxidant activity characteristic of these
phytoconstituents. It has been reported that the fl avonoid
content of B. juncea is not very high, but the spectrum of
fl avonoids observed for this plant is wider than that of any
other plant in the Brassicaceae family.22 Mustard meal has
been reported to be a good source of phenolic compounds.
Table 3: TLC profi le of seed extract indicating the presence of various classes of phytoconstituents
Reagent Observation Inference
Visible UV short UV long
Anisaldehyde-H2SO4Red brown coloration, blue-violet,
blue, red
Quenching Blue, violet, green
fl uorescence
Essential oils, pigments,
triterpenes, saponins
DPPH Yellow band against purple background - - Antioxidant activity
Dragendorff Orange-brown - Blue Alkaloids
Ethanolic-H2SO4Brown-black - - Total number of bands
NP-PEG - - Orange, green,
blue, blue-green
Bitter drugs, fl avonoids,
anthracene
Vanillin-H2SO4Lemon yellow, blue, blue-violet, red,
yellow brown
- - Pungent principle, saponins
Without spraying - Quenching Dark yellow, green,
blue, red, dark-blue
Bitter drugs, fl avonoids,
pigments, pungent principle
DPPH: 2, 2-diphenyl-1-picrylhydrazyl, UV: Ultra violet, TLC: Thin layer chromatography
Figure 2: 2,2-diphenyl-1-picrylhydrazyl (DPPH) autographic
analysis of mustard seed extract (column width). Key:
lane 1 = seed extract (200 μg), lane 2 = seed extract (250 μg/mL),
lane 3 = vitamin C (2 μg/mL), lane 4 = vitamin C (5 μg/mL),
lane 5 = vitamin E (4 μg/mL), lane 6 = vitamin E (10 μg/mL)
when the mobile phase of toluene: ethyl acetate: glacial acetic
acid (4:4:1) was used. Antioxidant compounds scavenge the free
purple-colored DPPH radicals to the yellow-colored compound.
The appearance of yellow color in the and high performance thin
layer chromatography plate after developing with 0.2% DPPH
reagent indicates antioxidant activity.

Parikh and Khanna: Pharmacognosy and phytoanalysis of B. juncea seeds
Phcog J | Sep-Oct 2014 | Vol 6 | Issue 5 53
More than a dozen phenolic acid conjugates have been
reported, and the spectrum of phenolics is also unique
and broad.20 The antioxidant capacity of Brassica species
has been related to their phenolic profi le and content,
particularly fl avonoids, since phenolic compounds have
demonstrated a higher antioxidant activity than vitamins
and carotenoids.20
Glucosinolates (GLSs) are found in Brassica vegetables.
Among many such vegetables, the GLS content of B. juncea
has been reported to be the highest.23 In fact, the majority of
cultivated plants that contain GLSs belong to the family of
Brassicaceae. In mustard seeds, sinigrin (a GLS) gets converted
to allyl isothiocyanate (an organo sulfur compound),
releasing glucose under the infl uence of water by the action
of the enzyme myrosinase.24 The IR spectrum indicated the
probable presence of sulfur-containing compounds. Further,
the extract also showed the presence of many such functional
groups that can result in conjugation reactions within the
compounds that can aid in reduction of reactive oxygen
species and antioxidant activity of the extract.
Fingerprinting and quantification of phenolics and
flavonoids such as quercetin, vanillin, vitamin E and
catechin in the hydralcoholic extract was carried out since
these phenolics and fl avonoids have been reported to be
present in mustard seeds. The antioxidant activity of these
phytoconstituents has also been well documented.25-30
Vitamin E competes for scavenging peroxyl radicals
much faster than polyunsaturated fatty acids and almost
200 times faster than commercial antioxidant butylated
hydroxytoluene31 since only a small amount of vitamin E
can protect a large amount of polyunsaturated fat in the
membranes. Quercetin detected in mustard seeds stops
propagation of lipid peroxidation, increases glutathione
(GSH) levels, antioxidant enzyme function and prevents
Ca2+ - dependent cell death.32,33 Catechin is known to reduce
lipid peroxidation and increase GSH production.34 Mustard
Figure 3: Cytotoxic effect of mustard seed extract on human dermal fi broblast cells (full page width). The cytotoxicity of mustard
seeds was assessed by (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay using doxorubicin as a positive control.
The dose-dependent reduction in the viability of cells was observed with seed extract and doxorubicin.
seeds reported to contain a high content of cysteine
residues,35 may even bind with oxygen electrophiles, further
assisting the antioxidant potency. Altogether, we postulate
that these phyconstituents contribute to the protective
effi cacy of extract in the suppression of the elevation of
reactive oxygen species generation.
The cytotoxicity assessment of hydroalcoholic extract
of B. juncea seeds was carried out in vitro, since toxicity
forms a crucial part in pre-clinical studies resulting in drug
failure. In this work, the toxicity assessment was carried
out in vitro on the cells derived from HDF cells that are
most abundant cells in humans; the results revealed that the
extract exhibit minimal toxicity in vitro that can be further
explored for in vivo study. Also, it has been reported in a
recent work that mustard seeds fed to rats at doses equal
to normal human intake do not cause any adverse effects
on histopathological parameters.36 These result can be used
to assess the therapeutic effi cacy of seed extracts for future
pharmacological evaluations.
ACKNOWLEDGMENT
The authors express their sincere thanks to Anchrom
Laboratories, Mumbai, for providing HPTLC facility
required for this study.
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Source of Support: None, Confl ict of Interest: None declared.