Natural Product Radiance, Vol. 8(5), 2009, pp.

483-487 Research Paper

In-vitro antioxidant activity of hot aqueous extract of

Helicteres isora Linn. fruits
P K Basniwal1*, M Suthar1, G S Rathore1, R Gupta1, V Kumar1, A Pareek2 and D Jain3
1
L B S College of Pharmacy, Udai Marg, Tilak Nagar, Jaipur-302 004, Rajasthan, India
2
L M College of Science and Technology (Pharmacy Wing), Shastri Nagar, Jodhpur-342 003, Rajasthan
3
School of Pharmaceutical Sciences, Rajiv Gandhi Technological University, Bhopal-462 036, M P, India
*Correspondent author, E-mail: pawanbasniwal@gmail.com; Tele Fax : 0141-2620517
Received 30 June 2008; Accepted 22 May 2009
Helicteres isora Linn.
Abstract
The antioxidant activity of the aqueous (hot) extract of Helicteres isora Linn. (Family-Sterculiaceae), commonly known
(AEHI) (FamilySterculiaceae) fruits was investigated in various in vitro models. The total as East Indian Screw Tree large shrub or
polyphenolic content of the extract was found 7.04% of AEHI, when compared to gallic acid and small tree occurs often gregariously,
total flavonoids content was 2.4 mg/g of AEHI, when compared to rutin. Hydrogen peroxide throughout India, South East Asia, the
radicals were inhibited at IC50 =165 g/ml, while ascorbic acid inhibited at 187.33 g/ml. AEHI
southern part of China and in dry
inhibited the nitric oxide radical at IC50 = 820 g/ml, when it was compared with rutin as standard
antioxidant with IC50 = 68.52 g/ml. Superoxide (SO) radicals inhibition was compared with deciduous forests, up to 1500 m on the
quercetin and IC50 value was found more than 1000 g/ml. Moreover, the results were observed in hill slopes6, 7. It is one of the Jamu raw
a concentration dependent manner. Present in vitro studies clearly indicated that the aqueous materials used in traditional folk
(hot) extract of H. isora has significant antioxidant activity. medicine in Indonesia; it is called Buah
Keywords: Helicteres isora, Indian Screw Tree, Total flavonoids, Superoxide, Antioxidant. Kayu Ules or Ulet-Ulet in Java island and
8
IPC code; Int. cl. A61K 36/00, A61P 39/06. is used for treating gastrospasm, as an
anthelmintic for tapeworm in Indonesia
and as an antispasmodic,
Introduction antipyretic, antidiarrhoeal and
Antioxidants are important in the antidysenteric in Saudi Arabia
prevention of human diseases. Naturally and as a tonic compound after
occurring antioxidants in leafy vegetables childbirth in the Malayan
and seeds, such as ascorbic acid, vitamin Islands8, 9. In traditional use,
E and phenolic compounds, possess the the root juice is claimed to be
ability to reduce the oxidative damage useful in cough, asthma,
associated with many diseases, including diabetes, empyema, intestinal
cancer, cardiovascular disease, cataracts, infections, a cure for scabies
atherosclerosis, diabetes, arthritis, when applied topically and a
Helicteres isora fruits
immune deficiency diseases and ageing1-3. favourite cure for snakebites.
Antioxidant compounds may function as in foods as they are suspected to be Fruits are demulcent, mildly astringent and
free radical scavengers, complexes of pro- carcinogenic. Therefore, the importance useful in griping and flatulence10-12.
oxidant metals, reducing agents and of search for natural antioxidants has A potent inhibitory activity of
quenchers of singlet oxygen formation4. greatly increased in the recent years. The aqueous (hot) extract of H. isora
They are often used in oils and fatty foods researchers have focused on natural fruits was reported against reverse
to retard their autoxidation. Synthetic antioxidants and numerous crude extracts transcriptase from avian myeloblastosis
antioxidants, such as butylated and pure natural compounds have been virus (AMVRT) 13 and anti-human
hydroxytoluene (BHT) and butylated reported to possess antioxidant immunodeficiency virus-type-1 (anti-
5
hydroxyanisole (BHA), have restricted use properties . HIV-1)14. Six neolignans, the Helicterins

Vol 8(5) September-October 2009 483

Research Paper

AF were isolated and reported in aqueous of solvent. The yield of extract (AEHI) was rutin in the range of 1-10g/ml. The total
(hot) extract of the fruits15 and the plant found 1.8%. The dried extract thus flavonoid content was expressed as
also reported with flavonoid obtained was used for the assessment of equivalent to rutin in mg/g or % w/w of
glucuronides16. antioxidant activity by various in vitro the extracts.
However, no data are available in assays.
the literature on the antioxidant activity Nitric oxide radical scavenging
of H. isora fruits. Therefore, the present Total phenolic content activity
work was aimed to investigate its Total soluble phenolic of AEHI Nitric oxide was generated from
antioxidant activity by various in vitro was estimated by Folin-Ciocalteau reagent sodium nitroprusside and measured by the
models. method17 using gallic acid as a standard Griess reaction. Sodium nitroprusside in
phenolic compound. One ml of stock aqueous solution at physiological pH
Materials and Methods solution (1g/ml) of AEHI was diluted up spontaneously generates nitric oxide20-22
Instruments and chemicals to 46 ml with distilled water and 1.0 ml which interacts with oxygen to produce
Shimadzu UV-160A spectro- of Folin Ciocalteau reagent was added and nitrite ions which can be estimated by use
photometer (Shimadzu Corporation, mixed thoroughly. 3.0 ml of 2% sodium of Griess reagent. Scavengers of nitric
Japan) was used for the measurement of carbonate was added to solution mixture oxide compete with oxygen leading to
absorbance of solution mixtures. after 3 min and allowed to stand for 2h reduced production of nitric oxide21,22.
L-Ascorbic acid, gallic acid, quercetin, with intermittent shaking. The absorbance Sodium nitroprusside (5M) in
rutin, potassium ferricyanide, of the blue colour was observed at phosphate-buffered saline (PBS) was
ethylenediaminetetra acetic acid (EDTA), 760 nm. The concentration of total mixed with 3.0 ml of different
nitroblue tetrazolium (NBT), phenazine phenolic compounds in the extract was concentrations (10320g/ml) of the
methosulphate (PMS), nicotinamide estimated as gallic acid which was drug and incubated at 25C for 150 min.
adenine dinucleotide (NADH), obtained by extrapolating the absorbance This solution was treated with Griess
napthylethylenediamine dihydrochloride of gallic acid on standard gallic acid graph reagent (1% sulphanilamide, 2% H3PO4
(NEDD), sulphanilamide, sodium (absorbance = 0.0008 X gallic acid). The and 0.1% napthylethylenediamine
nitroprusside, aluminum chloride and concentration of total phenols was dihydrochloride). The absorbance of
ferric chloride were purchased from expressed as mg/g of dry extract18. resulting chromophore from diazotization
Sigma Chemical Co. (St Louis, MO, USA). and subsequent coupling with
All other chemicals and reagents used Total flavonoid content napthylethylenediamine was observed at
were of analytical grade. Total soluble flavonoid of AEHI 546nm. Control reaction was also carried
was estimated by aluminium chloride out in between potassium nitrite and
Plant material and extract colorimetric method19. 0.5ml of stock Griess reagent and absorbance was
preparation solution (1g/ml) of AEHI, 1.5 ml measured. The percentage of nitric oxide
The fruits were purchased, from methanol, 0.1ml aluminium chloride radical scavenging was calculated as, NO
the local market of Ootacamund and (10%), 0.1 ml potassium acetate (1M) scavenged (%) =[(Acont - Atest)/Acont ] X 100,
authenticated by Director, Survey of were added to reaction test tube and (Acont = absorbance of control reaction &
Medicinal Plants and Collection Unit, volume was made up to 5ml with distilled Atest = absorbance in presence of sample
Ootacamund, Tamil Nadu, India and were water. After incubation at room of extract). Rutin was used as standard
shade dried, coarsely powdered and temperature for 30 min, the absorbance for nitric oxide radical scavenging activity.
extracted with hot water by maceration of the reaction mixture was measured at
process. The extract was filtered and 415 nm. Total phenolic content was Superoxide anion radical
concentrated in vacuum and kept in a calculated by extrapolating the absorbance scavenging activity
vacuum desiccator for complete removal of reaction mixture on standard curve of Measurement of superoxide

484 Natural Product Radiance


anion scavenging activity of the AEHI was
based on the method described by Liu
el al23 with slight modification. Super-
oxide radicals are generated in PMS-NADH
systems by oxidation of NADH and assayed
by the reduction of nitro blue tetrazolium
(NBT). The superoxide radicals were
generated in 3.0 ml of Tris-HCl buffer
(16 M, pH 8.0) containing 1.0 ml of
NBT (50 M) solution, 1.0ml NADH
(78 M) solution and sample solution of
the extract (50-300 g/ml) in water. The
reaction initiated by addition of 1.0ml of
phenazine methosulphate (PMS) solute on
(10M) to the mixture. The reaction
mixture was incubated at 250C for 5 min
and the absorbance was measured at 560
nm against blank samples. L-Ascorbic acid
was used as a control. Decreased
absorbance of the reaction mixture
indicates increased superoxide anion
scavenging activity. The % inhibition of
superoxide anion generation was
calculated as, SOD scavenged (%) =
[(A cont - A test )/A cont ] X 100, (A cont =
absorbance of control reaction & Atest =
absorbance in presence of sample of
extract). Quercetin was used as standard
for superoxide anion radical scavenging
activity.
H2O2 radical scavenging activity
The ability of AEHI to scavenge
H2O2 was determined according to the
method of Ruch R J et al24. A solution of
H 2O 2 was prepared in phosphate
buffer (pH 7.4). H2O2 concentration was
determined spectrophotometrically
by measuring absorption with
extinct coefficient for H 2O 2. AEHI
(10-320 g/ml) in distilled water was
added to H2O2 solution (0.6 ml, 40 M).
After 10 min, absorbance of H2O2 was
Vol 8(5) September-October 2009
measured at 230 nm against blank
solution containing the phosphate buffer
without H2O2. The % of H2O2 scavenging
effect of both extract and standard
compound was calculated as, H 2 O 2
scavenged (%) = [(Acont - Atest)/Acont ] X
100, (A cont = absorbance of control
reaction & Atest = absorbance in presence
of sample of extract). L-Ascorbic acid was
used as standard for H 2 O 2 radical
scavenging activity.
Results and Discussion
Total phenolic compounds and
flavonoids
Phenolic compounds are known
as powerful chain breaking antioxidants25
and phenols are very important
plant constituents because of their
scavenging ability due to their hydroxyl
groups 26. These compounds have an
important role in stabilizing lipid
oxidation and are associated with
antioxidant activity 27 . The phenolic
compounds may contribute directly to
antioxidative action. It was suggested
that polyphenolic compounds have
inhibitory effects on mutagenesis and
carcinogenesis in humans, when ingested
up to 1g daily from a diet rich in fruits
and vegetables 28. The total phenolic
content of AEHI was found 7.04%, which
was equivalent of gallic acid, one of the
components responsible for the
antioxidant activity. Flavonoids present in
food of plant origin are also potential
antioxidants29, 30. Most of the beneficial
effects of flavonoids are attributed to their
antioxidant and chelating abilities31 .
Studies have shown that certain flavonoids
exhibit hypoglycaemic effect32. The total
flavonoids content of AEHI was found to
be 2.4 mg/g and equivalent to rutin.
Research Paper
Inhibition of nitric oxide and
H2O2 radical
Nitric oxide radical generated
from sodium nitroprusside at
physiological pH was found to be
inhibited by content of AEHI. IC50 value of
AEHI was found to be 825 g/ml with 3.68
standard deviation while rutin showed
IC 50 =68.5g/ml with 2.27 standard
deviation. Comparison of nitric oxide
radical inhibitory activity with rutin, AEHI
showed mild antioxidant activity.
Hydrogen peroxide itself is not
very reactive, but it may be toxic to cell
due to increase in hydroxyl radical
concentration in the cells33. Thus, removal
of H2O2 as well as O2- leads to survival of
the cell life and its components. The
scavenging ability of AEHI on hydrogen
peroxide is comparable with that of
standard ascorbic acid. IC50 value of AEHI
was 165g/ml with 2.95 standard
deviation for inhibitory activity of hydrogen
peroxide radical, while its standard
ascorbic acid showed IC50=187g/ml with
3.45 standard deviation. These finding
shows that AEHI is more potent than its
standard ascorbic acid for hydrogen
peroxide radical inhibitory activity.
Superoxide anion radical
In the PMS/NADH-NBT system,
superoxide anion derived from dissolved
oxygen by PMS/NADH coupling reaction
reduces NBT. The decrease of absorbance
at 560 nm with antioxidants indicates the
consumption of superoxide anion in the
reaction mixture. AEHI has exhibited mild
superoxide radical scavenging activity
having IC50 more than 1000 g/ml, while
quercetin was found more potent having
IC 50 =155g/ml with 3.16 standard
deviation.
485
Research Paper
Conclusion
The aqueous (hot) extract of
H. isora Linn. (AEHI) exhibited strong
antioxidant activity by inhibiting nitric
oxide and scavenging superoxide anion
and hydrogen peroxide radicals when
compared with different standards such
as L-ascorbic acid, quercetin and rutin.
In addition to this, the AEHI contains a
significant amount of phenols and
flavonoids, which play a major role in
controlling oxidation. Thus, this easily
available plant can be used as a potential
antioxidant. However, further work is
required on the isolation and
identification of the antioxidant
components present in it.
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