Available online at
© Copyright protected. Unauthorised republication, reproduction, distribution, dissemination and copying of this document in whole or in part is strictly prohibited.
Saumya Singh, Himani Badoni, Vinay Kumar, Ayush Madan, Promila Sharma, Syed Mohsin Waheed*
Department of Biotechnology, Graphic Era University, 566/6 Bell Road, Clement Town, Dehradun, Uttarakhand, India.
*Corresponding author’s E-mail:
Accepted on: 15-04-2016; Finalized on: 31-05-2016.
Current work was undertaken with the aim of exploring the antioxidant properties of various plant extracts and their potential
therapeutic use. Methanolic extract of 4 plants namely Trapa bispinosa, Trigonella foenum-graecum, Syzygium cumini and Betula
utilis were investigated for their phytochemical composition and antioxidant properties. Trigonella foenum-graecum extract was
found to have the highest antioxidant activity demonstrated by DPPH inhibition assay and the extracts of all the four plants have
antioxidant properties. Besides, the extracts of these plants were tested for various classes of phytoconstituents present in these
plants to determine the phytochemical composition. All the four plant extracts have antioxidant property and at least one
compound from the class of phytocompounds like alkaloids, flavonoids reducing sugars and tannins as a constituent. The present
study is a part of larger project that would explore for the therapeutic potential of these extracts in various diseases resulting from
oxidative stress and especially of neuronal origin that would modulate a potential target in neurons and/or brain. The extracts
showing best antioxidant properties will potentially have good therapeutic properties for the diverse kinds of diseases resulting from
Keywords: Trapa bispinosa, Trigonella foenum-graecum, Syzygium cumini, Betula utilis, antioxidant, phytoconstituents, oxidative
raditional medicine has a long history since ancient
times. It is a bundle of knowledge, proficiencies and
practices based on theories, view points and
experiences native to different cultures, and inexplicable
and vague at times, nevertheless useful in protection of
health. Traditional medicine is the synthesis of beneficial
experience of generations of physicians practicing
medicine are used interchangeably as opposed to
conventional medicine in some countries. India is one of
the 12 mega biodiversity centers having 45, 000 species
of plant and its biodiversity is unmatched because it has
16 different agroclimatic regions including 10 vegetative
zones and 15 biotic regions
. The rich floral diversity
like Ayurveda, Unani, Siddha and Homeopathy. The
traditional medicine preparations include therapeutic
plants, minerals and organic matters etc. Even
therapeutic drugs used in conventional medicine are
derived from plants thus making them directly or
indirectly dependent upon the traditional medicine.
Trapa bispinosa, Trigonella foenum-graecum, Syzygium
cumini, and Betula utilis plants were chosen because of
their immense medicinal value in traditional medicine.
the family trapaceae. It grows throughout Africa and Asia
in lakes and ponds and is often cultivated for its edible
fruit. The therapeutic value of the whole herb and fruit
has long been documented in conventional medicine as a
cure for various ailments. The entire herb has been
antibacterial, antitumor, antioxidant and free radical
Trigonella foenum-graecum Linn. (Fenugreek) is an
India, Egypt, and Middle Eastern region. It is one of the
oldest medicinal plants, which is commonly used in
traditional medicine. The plant is reported to have anti-
hyperglycemic effect and used as antidiabetic agent. It
has diuretic, uterine & cardio tonic effects, hypotensive,
antidiabetic activities, and also antinociceptive and anti-
Jamun. S. cumini is an important medicinal plant in
various traditional systems of medicine. It is effective in
the treatment of diabetes mellitus, inflammation, ulcers
and diarrhea and preclinical studies show that it
antineoplastic properties (ref). The plant is rich in
acid, gallic acid, betulinic acid, quercetin, myricetin,
myricitin, myricetin. Myricetin works as a strong
antioxidant and quercetin shows protective effect against
birch is a moderate-sized tree that grows up to 20 m in
height. The bark is shining, reddish-white or white, with
A Comparitive Study of Antioxidant Properties and Phytochemical Composition of Trapa bispinosa,
Trigonella foenum-graecum, Syzygium cumini and Betula utilis
various pharmacological activities like antimicrobial, anti-
inflammatory, anticancer, antioxidant and anti-HIV
activities. The plant possesses various alkaloids having
diverse therapeutic effects
treatises of traditional medicines. The plants Syzygium
their antioxidant properties and there by their
therapeutic activity against various kinds of diseases
involving oxidative stress. Biological system during the
pathophysiologically, produces harmful intermediates
body can lead to damage of proteins, lipids, and DNA,
resulting in so-called oxidative stress. The excess
production of reactive oxygen species results in oxidative
stress, causing cellular damage, because ROS can react
with and damage cellular macromolecules, like DNA,
proteins and lipids
many age-related neurodegenerative diseases. Thus, the
phytoconstituents having antioxidant activity can mitigate
the effects of oxidative stress and therefore can be used
for therapeutic purpose in case of diseases occurring
through oxidative stress.
Plant parts, methanol, DPPH, Fehling’s solution A and
Fehling’s solution B, ethanol, distilled water, HCl,
chloroform, conc. H
hexane, alpha naphthol, NaOH , CuSO
The plant Syzygium cumini (leaves), Trigonella foenum-
India, and Trapa bispinosa (fruit) collected from Roorkee,
district of Uttarakhand India.
The plants Trapa bispinosa, Trigonella foenum-graecum,
(Trigonella foenum graecum Acc No. 114996, Trapa
114993, Betula utilis Acc. No. 114994).
TB, TF, SC and BU plant parts were dried under shade and
powdered in laboratory. The prepared powder forms
were soaked overnight in methanol prior to extract
preparation in Soxhlet apparatus.
The powders (200 grams) of TF, SC, BU and TB put in 7:3
methanol water mixtures for boiling using Soxhlet
apparatus for 8 cycles at 68°C for ~8 hours with
intermittent shaking. The prepared extract was filtered
and evaporated by rotary evaporator at 60°C. The dried
extract powder was kept in a glass container in a
refrigerator and used subsequently for the preliminary
screening of phytochemicals and experimental studies.
Preparation of aqueous extracts
TF, SC, BU and TB powders (200 grams) were boiled in
distilled water (1000 ml) for 15-20 minutes, then left
overnight at room temperature and filtered next
morning. The filtrate was evaporated to concentrate in
hot air oven at 80°C and concentrate was stored in
refrigerator. The concentrated extracts were used for
The DPPH radical scavenging capacity of each extract was
determined using method of Miliauskas
maximum absorption at 515 nm wavelength, the color
disappears by reduction of antioxidant compound. The
DPPH solution was prepared in methanol (6 × 10
and 2 ml of this solution was mixed with 100 µl of
Methanolic extracts using different concentrations (20,
40, 80µg/ml). The samples were incubated for 30 min at
37 °C, and then absorbance was measured at 515 nm
(AE). A blank sample was prepared with 100 µl of
methanol and 2 ml of DPPH solution and absorbance was
measured (AB). Standard was prepared using 1% ascorbic
acid and serially diluted to 3.125% of original. Ascorbic
acid matched concentration used as control. The
experiment was carried out in triplicate and each
experiment was repeated three times. % inhibition was
calculated using the following formula:
(% inhibition) = [(AB-AE)/AB] *100
Where AB absorbance of the blank sample and AE is
absorbance of the plant extract.
Molisch's test (general test): to 2-3 ml extract, add few drops of
alpha naphthol solution in alcohol. Shake & add concentrated
from the side of a test tube.
of two liquids.
Carbohydrates are present.
Biuret test (general test): To 3 ml extract add 4% NaOH & few
drops of 1% CuSO
Proteins/peptides are absent.
Test for amino acids
Ninhydrin test (general test): heat 3 ml extract and 3 drops of
5% ninhydrin solution in boiling water bath for 10 min.
Purple or bluish colour appears.
Amino acids are present.
Place a small amount of extract on glass slide. Make a smear.
Add a drop of Sudan Red III reagent. After 2 min. wash with 50%
alcohol mount in glycerin observe under microscope.
No oil globules are observed.
Fats and oils are absent.
Test for steroid:
Liebaermann-Burchard reaction: mix 2 ml extract with
chloroform. Add 1-2 ml acetic anhydride and 2 drops of conc.
from the side of the test tube.
acid layer shows greenish yellow
Steroids are present.
Test for cardiac glycosides: (Keller- Killiani test): to 2 ml extract,
add glacial acetic acid, 1 drop 5% FeCl
and conc. H
Reddish brown colour appears at
the junction of two liquid layers and
upper layer appears bluish green.
Cardiac glycosides are present.
Foam test: shake the drug extract or dry powder vigorously with
Persistent foam observed.
Test for alkaloids
Wagner’s test: 2-3 ml filtrate with few drops of Wagner’s
Reddish brown ppt.
Alkaloids are present.
Add few drops of 5% FeCl
solution in extracts.
Tannins are present
Test for flavonoids
Ferric chloride test – test solution treated with few drops of
ferric chloride solution
of Trigonella foenum-graecum extract extract, (2) DPPH
radical scavenging activities of Trapa Bispinosa extract,
(3) DPPH radical scavenging activities of Syzygium cumini,
(4) DPPH radical scavenging activities of Betula utilis
The results of our laboratory prepared extracts from the
four medicinal plants are presented in table 2. The
percentage yield of the extracts were 26 gm (13 %) and
27 gm (13.5 %) for Trapa bispinosa, 30 gm (15 %) and
29.8 gm (14.9 %) for Trigonella foenum-graecum, 23.92
gm (11.6 %) and 27.2 gm (13.6 %) for Syzygium cumini
and 24 gm (12 %) and 25.8 gm (12.9) for Betula utilis
aqueous and methanolic extracts respectively.
Botanical Servay of India with accession number
(Trigonella foenum graecum Acc No. 114996, Trapa
bispinosa Acc No. 114995, Syzygium cumini Acc. No.
Important medicinal phytochemicals such as reducing
sugars, flavonoids, alkaloids, tannins, proteins, amino
acids, steroids and cardiac glycosides were tested. The
result of the phytochemical analysis shows that the all
four plants are rich in at least one of proteins, amino acid,
steroid, cardiaglycoside, tannin, alkaloids, flavonoids
reducing sugars and tannins.
DPPH radical scavenging activities of plant extracts varied
from 56% to 92%. Trigonella foenum-graecum extract
showed the highest antioxidant activity of 92%, 90% and
86% inhibition in methanolic extract and in aqueous
extract inhibition of 90%, 88% and 88% of DPPH was
recorded. In Trapa bispinosa plant methanolic extract
DPPH inhibition of 88%, 87% and 81%, and in aqueous
extract 87%, 75% and 53% was observed. Syzygium
cumini methanolic extract showed DPPH inhibition of 87
%, 71% and 65% and in aqueous extract inhibition
observed was 89%, 75% and 60%. Inhibition in Betula
utilis methanolic extract was 88%, 87% and 81%; and in
aqueous extract DPPH inhibition was 56%, 66% and 75%
inhibition at 80, 40 and 20µg/ml concentrations
The extracts of four plants that were part of this study
gave yields in the range of approximately 12-15% (Table
1) and are rich in various classes of phytocompounds like
alkaloids, tannins, flavonoids, cardiac glycosides, steroids,
amino acids and carbohydrates (Table 2). All these classes
of phytoconstituents exert diverse effects on physiology
depending upon their composition.
toxicity, heart diseases, cancer, obesity, and are known to
be hypoglycemic, neuroprotective and beneficial for liver
function, blood lipids, proapoptosis, cardiovascular
health. In addition their phytoconstituents act as
cytotoxic agents against malignant brain-tumors but their
role in curing diseases of neuronal origin is explored to
lesser extent. Most studies provide only the superficial
information, lacking depth and targets to cure the
diseases. Traditional awareness of plants for medicine is
based on personal experiences and the knowledge
handed down from one generation to next generations
mostly by word of mouth or also in the form written
treatise or manuscripts in various Indian vernacular
languages. By 19th century active principles of medicinal
plants were isolated based on such knowledge base and
discovery of quinine from Cinchona bark was the first
active principle isolated and characterized
. Extract of TF
gitogenin, neotigogens 4-hydroxyisoleucine. whereas
methanolic extract TF is composed of caparine, trignollin,
fenugrikine, gentianine, tigogenin, neotigogens, 4-
hydroxyisoleucine and the aqueous extract of TB contains
pyridoxine, thiamine, nicotinic acid, D amylase,
pantothenic acid, phosphorylase, 2β,3α,23-trihydroxyurs-
amylase, pantothenic acid, phosphorylase, 2β,3α,23-
trihydroxyurs-12-en-28-oic acid (Pubchem).
Both BU and SC plants are known for their medicinal
value for curing various pathophysiological conditions.
The phytoconstituents of BU are betulin, lupeol, oleanolic
acid, acetyloheanolic acid, betulitc acid, lupenone,
sitosterol, methyle betulonate, methyl betulate and
karachic acid. BU is known for its antiseptic, proapatotic
properties. Oleanic acid have anti-inflammatory and anti-
tumor properties and it prevents from cerebral
. Betulinic acid is well-known as a
neuroectodermal and malignant tumor cells and it was
also reported to stimulate apoptosis in these cells.
Betulinic acid acts as an anticancer agent operating
through different mechanisms and has been reported to
activate apoptotic pathways in cancer cells
. The plant SC
hypolipidemic, antianaemic, antioxidant properties. SC
works as strong antioxidant and thus it has oxygen radical
scavenging capacity. The SC contains phytochemicals
namely malic acid, oxalic acid, gallic acid, betulic acid,
quercetin, myricetin, myricitin. Myricetin works as strong
antioxidant and quercetin shows protective effect in
. Owing to their antioxidant rich
therapeutic potential to fight against various types of
diseases of diverse origin and nature viz., infectious
diseases of bacterial, fungal and viral nature, life style
diseases like diabetes and hypertension, diseases of
neurodegenerative diseases like AD and PD, besides
various forms and types of cancers.
This investigation supports that four plants are promising
source of natural antioxidants. Antioxidant properties
differ significantly among the four selected plant extracts
depending upon the content and composition of the
phytoconstituents. Among these plant extracts, Trigonella
antioxidant properties. The plant BU shows less
antioxidant activity in inverse dose dependant manner in
contrast with other three plants taken in this study, but
that does not undermine its therapeutic value since it has
strong proapoptotic nature due to the presence of
betulinic acid. Our results are in conformation with a
previous study on B. utilis
Chopra R.N., Nayar S.L. and Chopra I.C. In Glossary of
Willcox ML, Bodeker G. Traditional herbal medicines for
Phytochemistry, 56, 2001, 237-243.
Swerdlow Joel L. Nature’s Medicine: Plants That Heal,
carcinogenesis, Cancer Cells, 3(1), 1991, 1-7.
Ames BN, Shigenaga MK. Oxidants are a major contributor
Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants
National Academy of Sciences, 90, 1993, 7915–22.
Miliauskas, G, Venskutonis, PR, Van Beek, TA. Screening of
plant extracts, Food Chemistry, 85, 2004, 231–237.
Brand-Williams, W Cuvelier ME, Berset C. Use of a free
Science and Technology, 28, 1995, 25–30.
Harborne JB. Phytochemical Methods. New Delhi: Springer
Kim JH, Park SH, Kim YW, Ha JM, Bae SS, Lee GS. The
Traditional Herbal Medicine, Dangkwisoo-San, Prevents
Cerebral Ischemic Injury through Nitric Oxide-Dependent
Mechanisms, Evidence Based Complementary Alternative
Medicine 2011. Journal of Ethnopharmacol, 140(1), 2012,
Khosla P, Gupta DD, Nagpal RK. Effect of Trigonella foenum
graecum (Fenugreek) on blood Glucose in normal and
Das PK, Bhattacharya S, Pandey JN, and Biswas M.
against streptozotocin induced diabetic rats. Global Journal
of Pharmacol, 5(3), 2010, 186-190.
Dakshinamurtia K, Sharma SK, Jonathan A, Geiger D.
Neuroprotective actions of pyridoxine. Biochim Biophys
Acta, 1647, 2003, 225-229.
Vyawahare NS, and Ambikar DB. Evaluation of
fruits of Trapa bispinosa in laboratory animals.
International Journal Pharma Science, 22, 2010, 32-35.
Fulda S, Friesen C, Los M, Scaffidi C, Mier W, Benedict M,
Nunez G, Krammer PH, Peter ME, Debatin KM. Betulinic
acid triggers CD95 (APO 1/Fas)-and p53-independent
apoptosis via activation of caspases in neuroectodermal
tumors. Cancer Research, 57, 1997, 4956–4964.
Fulda S, Jeremias I, Steiner HH, Pietsch T, Debetulinic
malignant brain-tumor cells. International journal of
cancer, 82, 1999, 435–41.
Kumar A, Ilavarasan R, Jayachandran T. Antiinflammatory
activity of Syzygium cumini seed. African Journal of
Biotechnology, 7, 2008, 1932-40.
Laura Caltana, María Luisa Nieto, and Alicia Brusco
cerebral ischemia Neural Regenration Research, 10(4),
Caltana L, Rutolo D, Nieto ML, Brusco A. Further evidence
for the neuroprotective role of oleanolic acid in a model of
focal brain hypoxia in rats. Neurochemestry International,
79, 2014, 79-87.
Fisher DE. Apoptosis in cancer therapy: crossing the
threshold, Cell, 78(4), 1994, 539–542.
Choi DH, Li C, Choi JS. Effects of myricetin, an antioxidant,
on the pharmacokinetics of losartan and its active
metabolite, EXP-3174, in rats: possible role of cytochrome
P450 3A4, cytochrome P450 2C9 and P-glycoprotein
inhibition by myricetin. Journal of Pharmacy and
Pharmacology, 62(7), 2010, 908-14.
Kumaraswamy M. V. and Satish S. Free radical scavenging
activity and lipoxygenase inhibition of Woodfordia
fructicosa Kurz and Betula utilis Wall, African Journal of
Biotechnology, 7(12), 2008, 2013-2016.
Aqil F, Gupta A, Munagala R, Jeyabalan J, Kausar H, Singh
IP, Gupta RC. Antioxidant and antiproliferative activities of
Syzygiumcumini L. (‘jamun’, the Indian Blackberry) Nutr
Source of Support: Nil, Conflict of Interest: None.