IN_VITRO ANTI_OXIDANT ACTIVITY OF ERVATAMIA DIVERICATA LINN FLOWERS

1. 207
_________________________________
* Corresponding author:
K.Hemalatha
Malla Reddy College of Pharmacy,
Maisammaguda, Dhulapally, Secundrabad-14.
Hyderbad. Andhra Pradesh. India
E-mail address: kamurthy_18@yahoo.com
Available Online at: www.ijrpp.com Print ISSN: 2278 - 2648
Online ISSN: 2278 - 2656
(Research article)
PHYTOCHEMICAL EVALUATION AND IN-VITRO ANTI-OXIDANT
ACTIVITY OF ERVATAMIA DIVERICATA. LINN. FLOWERS
1
E.Srujana, *2
K.Hemalatha, 3
A.Ramya, 4
K. Priya darshini, 5
M.Sudhakar
Department of Pharmaceutical Chemistry, Malla Reddy College of Pharmacy, Andhra
Pradesh. India
________________________________________________________________________
ABSTRACT
One glycoside compound (Campestrol D glucose), one steroidal compound stigmosterol), two triterpinoidal
compounds(α-amyrin acetate, asiatic acid), one flavonoid compound(kaemferol),two alkaloidal compounds(11-
hydroxy voacristine, heyneanine) have been isolated from flowers of Ervatamia divericata, their structures were
established by spectral analysis and direct comparison with authentic samples. From the two in-vitro antioxidant
activity methods i.e. DDPH and nitric oxide inhibition methods, the isolated compound ED-5&7 i.e. kaemferol
and 11-hydroxy voacristine showed significant response of antioxidant activity (%inhibition-82.3% to 80.3%).
Due to the presence of these compounds kaemferol and 11-hydroxy voacristine in ethyl acetate and ethanol
extracts can show the nearly similar response compared with other extract. The present study revealed that E.
divertica flowers have significant anti-oxidant activity.
KEY WORDS: Ervatamia divericata, steroids, terpinoids, flavonoids, alkaloids, in-vitro anti-oxidant, DPPH,
nitric oxide.
INTRODUCTION
Ervatamia divericata (L) (Synonym:
Tabernaemontana divaricata)1
belongs to the
family Apocynaceae, is a glabrous, evergreen tree
indigenous to India and is cultivated in gardens for
its ornamental and fragrant flowers. This species
has been extensively investigated and a number of
chemical constituents such as alkaloids2-5
,
triterpenoids6-8
, steroids7,9
, flavonoids10
, phenyl
propanoids9
and phenolic acids10
were isolated
from leaves, roots and stems of the plant. In the
Indian traditional system of medicine the plant
material is widely used as a purgative, tonic to the
brain, the spleen and the liver; in the treatment of
cancer, wounds and inflammations11,12
. The plant
extract was also found to possess analgesic,
antipyretic, vasodilator and CNS depressant
effects113
, antispasmodic, hypotensive activity14
,
antiinflammatory, uterine stimulant15
effect and
cytotoxic activity16
. Furthermore, literature survey
of Ervatamia divericata flowers revealed that no
researcher has yet reported on phytoconstituents
and antioxidant activities. Therefore, it is worth
conducting an investigation on phyto constituents
of flowers and as well as in-vitro antioxidant
activities of different extracts of Ervatamia
divertica flowers.
MATERIALS AND METHODS
COLLECTION AND AUTHENTICATION
The whole plant of Ervatamia divericata Linn was
collected from Karimnagar, Dist, A.P. India It was
authenticated by Dr. B. Bhadraiah, HOD, Dept of
Botany, Osmania University, Hyderabad, A.P,
India.
International Journal of
Research in Pharmacology and
Pharmacotherapeutics

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PREPARATION OF EXTRACTS
The flower of Ervatamia divericata was extracted
(5kg) successively with n: hexane, ethyl acetate
and ethanol (70%) respectively. These extracts
were concentrated and remove the solvents. The
yields of n: hexane, ethyl acetate and ethanolic
extracts were found to be 31g, 25g, 33g
respectively.
PRELIMINARY PHYTOCHEMICAL
SCREENING
Conventional standard protocols17,18
for detecting
the presence of different chemical constituents in
the plant extracts were employed. The tests for the
secondary metabolites viz. alkaloids, tannins,
sterols, saponins, amino acids, glycosides, proteins,
sterols/terpenes, reducing sugars, non-reducing
sugars, resins flavonoids and phenols were carried
out with the all the different extracts of Ervatamia
divericata flowers using preliminary
phytochemical screening.
ISOLATION OF CONSTITUENT FROM
N:HEXANE EXTRACT
The concentrated n:hexane extract (25gm) was
dissolved in n:hexane (20ml) and chromatographed
through a column of silica gel 60-120 mesh LR
(diam.4cm X 45cm). The column being
successively eluted with n:hexane:ethyl acetate in
graded mixture, i.e. 95:05, 90:10, 85:15, 80:20 up
to 100 % ethyl acetate. From above elution’s, four
different fractions were collected (i.e. fraction A,
B, C & D).
Fraction A was eluted from n: hexane: ethyl
acetate (95:05) and it gave a single spot by using n:
hexane: ethyl acetate (8:2) as mobile phase. On
evaporation gave a waxy compound. The product
was designated as compound ED1.
Compound ED1
IR (KBr cm-1
, λ max): 3553.11 cm-1
,2919.21 cm-
1
,2850.80 cm-1
,1637.38 cm-1
,1465.83 cm-1
,1379.08
cm-1
,1134.66 cm.-1
1
HNMR δ values ppm: 5.4(m,1H vinyl proton
C-6), 5.1(d,1H proton of C-2′ glucose),3.5 to
3.6(m,4H protons of glucose),3.7(m, 7H protons of
glucose), 1.2 to 2.3(m,10 X CH2 and 6CH
protons),0.8 to 1.1(m,18H, 6 X CH3).
13
CNMR (CDCl3): 110(C-2′), 74.1(C-3′),
76.8(C-4′),71.5(C-5′), 81.5(C-6′), 74.3(C-7′),
37.2(C-1), 31.7(C-2), 71.6(C-3), 41.8(C-4),
140.8(C-5), 121.8(C-6),32.0(C-7), 31.8(C-8),
56.8(C-9),37.7(C-10), 21.1(C-11), 39.5(C-
12),42.7(C-13), 56.4 (C-14), 26.3(C-15),25.9(C-
16), 56.2(C-17), 12.0(C-18),19.3(C- 19), 36.1(C-
20), 19.4(C-21),33.6(C-22), 32.4(C-23), 39.1(C-
24),15.4(C-25), 32.2(C-26), 20.7(C-27),20.7(C-28).
Mass Spectrum (MS) showed M+
at m/z 562,
corresponding to the molecular formula [M +
, C34
H58O6]. The maximum peak was that of the ion M-
14 due to solitting off of CH2 groups. It was found
to be identical with Campesterol -D-glucose on
comparison with authentic sample (mixed M.p.
CO-TLC and super impossible IR).
C H 3
C H 3CH 3
CH 3
C H 3
C H 3
O
O
OH
OH
O H
O H
1
2
3
4
5
6
7
8
9
10
11
12
13
14 15
16
17
18
19
20
21
22
23
24
25
26
27
28
1'
2'
3' 4'
5'
7'
6'
Fraction B was eluted from n hexanen:ethyl
acetate (85:15), gave single compound, confirmed
by using TLC plate in acetone: : methanol (8:2) as
mobile phase, On evaporation gave a sticky mass
compound. The product was designated as
compound ED2.
Compound ED2
IR (KBr cm-1
, λ max): 3446.24 cm-1
, 2926.59
cm-1
, 2855.5 cm-1
, 1740.37 cm-1
, 1460.77 cm-1
,
1376.21 cm-1
, 1169.43 cm-1
.
1
HNMR δ values ppm: 5.4(d,1H, vinylic proton
C-6), 5.2 and 5.1(m,2H olifinic protons C-22,C-
23),3.1(m,1H,CH-OH hydroxyl group), 2.4 to

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10
5
1
4
2
3
8
7
9
6
16
15
OCCH3
O
CH3
CH3
CH3
CH3
CH3
CH3 CH3
H
27
CH3
11
12
13
14
17
18
19
20
21
22
23 24
25
28
29
30
26
1.12(m,18H, 9xCH2 and 1H of methane group,1.0
to 0.8(m,18H, 6xCH3 group).
13
CNMR (CDCl3): 37.6(C-1), 31.2(C-2), 76.5(C-
3),41.5(C-4), 145.1(C-5), 121.5(C-6),32.8(C-7),
31.9(C-8), 50.3(C-9),38.4(C-10), 23.1(C-11),
39.9(C-12),46.2(C-13), 59.0(C-14), 26.5(C-
15),24.8(C-16), 55.2(C-17), 14.0(C-18),21.2(C-19),
42.0(C-20), 15.6(C-21),129.6(C-22), 124.9(C-23),
47.1(C-24) 33.6(C0-25), 21.3(C-26), 22.6(C-
27)28.0(C-28), 15.9(C-29). MS mass spectrum
showed M+
at m/z 412, corresponding to the
molecular formula (C29H48O). It was found to be
identical with Stigmosterol on comparison with
authentic sample (mixed m.p. CO-TLC and super
impossible IR).
OH
CH3
CH3
CH3
CH3
CH3
CH3
1
2
3
4
5
6
7
8
9
10
11
12
13
14 15
16
17
18
19
20
21 22
23
24
25
26
28
29
27
Fraction C was eluted from nhexane : ethyl
acetate (70:30), gave single compound, conformed
by using TLC plate in acetone : methanol (8:2) as
mobile phase. It was designated as compound ED3
Compound ED3
IR (KBr cm-1
, λ max): 2944.77 cm-1
,2871.34
cm-1
, 1715.42 cm-1
, 1463.42 cm-1
, 1380.62 cm-1
1
HNMR δ values ppm: 5.3(d,1H, vinylic proton
C-12), 3.3 (m,1H proton C-3), 2.3(m,3H acetyl
group), 2.0 to 1.5(m, CH2 CH group protons), 96 to
0.93(m,6H, 2x CH3 groups, C-29,C-230), 1.3 to
0.99(m,18H, 6 x CH3 C-23,C-24,C-25,C-26,C-
27,C-28).
13
CNMR (CDCl3): 40.3(C-1), 24.6(C-2), 78.8(C-
3),35.4(C-4), 52.1(C-5), 18.1(C-6)32.7(C-7),
48.6(C-8), 47.8(C-9)33.9(C-10), 24.7(C-11),
129.5(C-12),139.8(C-13), 45.1(C-14), 31.(8C-
15)25.9(C-16), 46.9(C-17), ,30.0(C-18),40.2(C-19),
29.7(C-20), 29.6(C-21),29.4(C-22), 22.5(C-23),
22.5(C-24),17.9(C-25), 14.0(C-26), 26.6(C-
27),19.1(C-28), 14.2(C-29), 13.9(C-30),178.9(C-
31), 21.0(C-32). MS mass spectrum showed M+
at
m/z 468, corresponding to the molecular formula
(C32H52O2). It was found to be identical with α-
amyrin acetate on comparison with authentic
sample (mixed M.p. CO-TLC and super impossible
IR).

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Fraction D was eluted from n hexane: ethyl
acetate (40:60), gave single compound, conformed
by using TLC plate in acetone : methanol (8:2) as
mobile phase. It was designated as compound
ED4.
Compound ED4
IR (KBr cm-1
, λ max): 3478 cm-1
,2954 cm-
1
,1723cm-1
, 1380 cm-1
1
HNMR δ values ppm: 10.1(s, 1H,COOH group
C-28 ), 5.3(s,1H vinylic protons C-12), 3.5(s, 3H,
OH protons of C-2,C-3,C-23), 2.8(m,2H, CH
group protons at C-2,C-3), 2.3 to 1.2(m, CH and
CH2 protons), 1.1 to 0.8(m,6 x CH3 groups of 18
protons at,C-24,C-25,C-26,C-27,C-29,C-30).
13
CNMR (CDCl3): 44.9 (C-1), 68.5 (C-2), 79.6 (C-
3), 39.5 (C-4), 54.7 (C-5), 17.9 (C-6), 32.1 (C-7),
33.9 (C-8), 48.7 (C-9), 36.9 (C-10), 22.8 (C-11),
128.2 (C-12), 140.1 (C-13), 42.8 (C-14), 28.2 (C-
15), 23.5 (C-16), 47.8 (C-17), 51.7 (C-18), 42.2 (C-
19), 29.1(C-20), 30.7(C-21), 35.4 (C-22), 71.5(C-
23), 29.2(C-24), 15.7(C-25), 17.3(C-26), 26.7(27),
178.9(C-28), 20.9(C-29), 24.6(C-30). MS mass
spectrum showed M+
at m/z 488, corresponding to
the molecular formula (C30H48O5). It was found to
be identical with Asiatic acid on comparison with
authentic sample (mixed M.p. CO-TLC and super
impossible IR.
10
5
1
4
2
3
8
7
9
6
13
14
12
11 17
16
18
15
21
22
20
19
OH
23 24
26
27
25 COOH
30
OH
OH
29
28
ISOLATION OF CONSTITUENT FROM
ETHYL ACETATE EXTRACT
The concentrated ethyl acetate extract (23gm) was
dissolved in ethyl acetate (20ml) and chromate
graphed through a column of silica gel 60-120
mesh LR (diam.4cm X 45cm). The column being
successively eluted with ethyl acetate : methanol in
graded mixture i.e. 95:05, 90:10, 85:15, 80:20., up
to 50: 50ethyl acetate :methanol. From above
elutions, single fraction was collected (i.e. fraction
E).
Fraction E was eluted from ethyl acetate:
methanol (80:20) and it gave single spot by using
TLC plate in chloroform: methanol (3.5:1.5) as
mobile phase. The product was designated as
compound ED5.
Compound ED5
IIR (KBr cm-1
, λ max): 3421.4 cm-1
,2817 cm-
1
,1660 cm-1
,1570.0cm-1
,1225.1 cm-1
1
HNMR δ values ppm: 10.80(s,1H 0H of C-
3),7.95(d,2H protons of C-2′
, C-6′
),6.9(d,2H
protons of , C-3′
, C-5′
), 6.5 to 6.2(d, 2H protons of
C-6,C-8),5.3(s, 3H protons of OH groups).
13
CNMR (CDCl3): 145.9(C-2), 137.52(C-3),
176.79(C-4),160.61(C-5), 97.3(C-6), 163.78(C-7),
93.36(C-8), 157.69(C-9), 107.2(C-10),127.3(C-1),
127.5(C-2′
,C-6′
), 128.9(C-3′
,C-5′
), 137.8(C-4′
). MS
mass spectrum showed M+
at m/z 286,
corresponding to the molecular formula
(C15H10O6). It was found to be identical with
kaemferol on comparison with authentic sample
(mixed M. p. CO-TLC and super impossible IR).

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OOH
OH O
OH
OH
1
2
34
5
6
7
8
9
10
1'
2'
3'
4'
5'
6'
ISOLATION OF CONSTITUENT FROM
ETHANOLIC EXTRACT
The concentrated ethanolic extract (25gm) was
dissolved in ethanol (20ml) and chromate graphed
through a column of silica gel 60-120 mesh
LR(diam.4cm X 45cm). The column being
successively eluted with chloroform: methanol in
graded mixture i.e 90:10, 80:20, 70:30., up to
50:50. From the above elution’s two fractions were
collected (i.e, fraction F&G).
Fraction F was eluted from chloroform: methanol
(90:10) and gives single spot by using chloroform:
methanol (7:3) as mobile phase. The compound
was designated as compound ED6.
Compound ED6
IIR (KBr cm-1
, λ max): 3442.01 cm-1
, 3364.30
cm-1
, 2944.77cm-1
, 1740.44 cm-1
, 1463.42 cm-1
1
HNMR δ values ppm: 10.1(s, br 1H,N-H group
N-14 ),7.2 to 6.8(m,2H Aromatic protons C-9,C-
12),5.3(s, br, 1H, OH proton C-11),3.8(s,1H, -
OCH3 group at C-23),3.7(s,1H, -OCH3 group at C-
22),3.5(s, 1H,OH group at C-12),3.4 to 1.5(m,-
CH3- methenine protons),1.18(s,3H,CH3 at C-20).
13
CNMR (CDCl3): 137.3(C-1), 31.8(C-2),
55.6(C-3), 57.4(C-5), 21.0(C-),109.9(C-7),
121.2(C-8), 100.7(C-9), 153.9(C-10),137.3(C-
11),107.9(C-12),129.9(C-13),52.5(C-15),34.1(C-
16),26.5(C-17),36.4(C-18),76.5(C-19), 76.5(C-20),
22.9(C-21),54.9(C-22), 53.1(C-23), 54.9(C-24).
MS mass spectrum showed M+
at m/z 400,
corresponding to the molecular formula (C22
H28O5N). It was found to be identical with 11-
hydroxyl voacristine on comparison with
authentic sample (mixed M. p. CO-TLC and super
impossible IR).
N
H
N
CH3
OH
O
O
O
OH
CH3
CH3
1 2 3
56
78
9
10
11
12
13
15
16
17
18
19
20
21
22
23
24
Fraction G was eluted from chloroform:
methanol (70:30) and it gave single spot by using
TLC plate, chloroform : methanol (7:3) as mobile
phase . The product was designated as compound
ED-7.
Compond ED7
IIR (KBr cm-1
, λ max): 3359.79 cm-1
, 2927.10
cm-1
, 2872.99 cm-1
, 1732.37, 1602.77cm-1
, 1462.
91 cm-1
, 1235.53 cm-1

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1
HNMR δ values ppm: 10.2(s, 1H,N-H group
N-14 ), 7.3 to 5.8(m,4H Aromatic protons C-9,C-
12), 5.3(s, 1H, NH proton of indole nucleus N-11),
4.1(s,3H, -COOCH3 protons C-23), 3.7(s, 1H,OH
proton at C-19), 2.8 to 1(m ,CH and CH2 protons),
0.09(m,3H,CH2 protons at C-20)
13
CNMR (CDCl3): 62.1(C-1), 47.4(C-2), 36.6(C-
3), 55.0(C-5), 31.7(C-6),37.5(C-7), 121.5(C-8),
124.1(C-9), 114.4(C-10),126.9(C-11), 109.2(C-
12),139.4(C-13),27.9(C-15),31.1(C-16), 33.8(C-
17), 41.9(C-18),76.5(C-19), 23.8(C-20), 55.1(C-
21),175.5(C-22), 58.9(C-23). MS mass spectrum
showed M+
at m/z 354, corresponding to the
molecular formula (C21 H26O3N2) It was found to
be identical with heyneanine on comparison with
authentic sample (mixed M. p. CO-TLC and super
impossible IR).
N
H
N
CH3
OH
O
OCH3
1 2 3
56
78
9
10
11
12
13
15
16
17
18
19
20
21
22
23
In-vitro anti oxidant activity:
Free radical scavenging activity by DPPH
method:
The free radical scavenging activity of MEEC was
measured by 1,1-diphenyl-2-picryl-hydrazil
(DPPH) using the method of Blois19
. Three
different extracts and three isolated compounds i.e.
ED-2, 5 & 7 (100-500 µg/ ml) was prepared and in
a test tube 2.5ml of methanol was taken and to this
solution add 0.5ml of 0.2mM DPPH reagent and
add 0.1ml of sample solution. The sample was
incubated at room temperature for 20 minutes. At
the end of 20 minutes the optical density (OD) of
the incubated mixture was read at 515 nm against
alcohol using UV spectrophotometer. The optical
density of sample blank was subtracted from the
sample reading. A positive control was maintained
without adding any sample. Ascorbic acid was
maintained as standard drugs. The percentage
protection was calculated using the following
formula.
% inhibition = [(Acont- Atest)/Acont] X 100
Nitric oxide radical scavenging method
Nitric oxide generated from sodium nitroprusside
in aqueous solution at physiological pH interacts
with oxygen to produce nitrite ions, which were
measured by the Griess reaction 20,21
. Sodium
nitroprusside (10mM, 4ml)in phosphate buffer pH-
7.4, 1ml was mixed with different concentrations
(100-500µg/ml) of each extract was dissolved in
ethanol incubated at 250
C for 150 minutes. The
same reaction mixture without the sample but the
equivalent amount of ethanol serves as control.
After the incubation period adds 0.25 ml of
sulfanilic acid reagent was added. Mix it well and
allow standing for 5min for completion of
diazotization. Then 0.25ml of NEDD was added
and mixed well allowed to stand for 30min. Pink
colour chromophore was formed. The absorbance
of chromophore was read at 540nm using UV-
spectrophotometry. Ascorbic acid is used as
standard drug.
% inhibition = [(Acont- Atest)/Acont] X 100
RESULT AND DISCUSSION
The results of preliminary qualitative
phytochemical investigation of different extracts ns
of Ervatamia divericata indicated the presence
sterols, triterpenoids, flavonoids and alkaloids. The
phytoconstiuents isolated and charactersed by
spectral data, of different extracts of Ervatamia
divericata flowers namely, campesterol-D-
glucoside, stigmosterol, α-amyrin acetate, Asiatic
acid (n:hexane extract), kaemferol(ethyl acetate

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extract), 11- hydroxyl voabacristine and
heyneanine (ethanolic extract). The results were
represented for in-vitro anti-oxidant activities for
both the models (DPPH and nitric oxide) in the
form of table no 1 and 2, graphs and data was
subjected for anti-oxidant activity. All the extracts
and isolated compounds (ED2, 5 &7) of Ervatamia
divaricate flowers in graded concentrations was
tested for antioxidant activity in two different in-
vitro models. It was observed that the test
compounds scavenged free radicals in a
concentration dependent manner in the models
studied. Maximum percentage inhibition of DPPH
radicals by the ethyl acetate and ethanolic extracts
were shown 70% ND 68 % respectively. Whereas
isolated compounds ED5 &7 exhibited 71% and
69% respectively. (Table no 1) (Graph: 1).
Standard drug ascorbic acid showed about 74%
inhibition of the DPPH radicals.
Table no 1: Percentage free radical scavenging activity (DPPH method)
Sl.
NO
Different extracts and
isolated compounds
Percentage
inhibition (µg/ml)
Activity
1 n:hexane extract 35 poor
2 Ethyl acetate extract 70.04 good
3 Ethanol extract 68.25 good
4 Compound ED-2 39 poor
5 Compound ED-5 71.9 good
6 Compound ED-7 69.32 good
7 Ascorbic acid (standard) 74.9
Graph: 1
In the nitric oxide radical scavenging model, the
maximum percentage inhibition of nitric oxide
radicals by was ethyl acetate and ethanolic extracts
were shown 81% and 68 % respectively. Whereas
isolated compounds ED5 &7 exhibited 82% and
80% respectively. (Table no 2) (Graph:2). Ascorbic
acid exhibited 85% inhibition.

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Table no 2: Percentage nitric oxide radical scavenging method
Sl. NO
Different extracts
and isolated compounds
Percentage
inhibition ( µg/ml)
Activity
1 n:hexane extract 43 Poor
2 Ethyl acetate extract 81.5 Good
3 Ethanol extract 78 Good
4 Compound ED-2 49.7 Poor
5 Compound ED-5 82.3 Good
6 Compound ED-7 80.3 Good
7 Ascorbic acid (standard) 85.6
Graph: 2
5
15
25
35
45
55
65
75
85
95
% inhibition
% inhibition
The different extracts and selected isolated
compounds from Ervatamia divericata flowers
were found to have anti-oxidant activity in both
models in the present study. On comparison, it was
found that, ethyl acetate and ethanolic extracts and
their isolates of compounds i.e. ED5 and ED7
shown significant anti-oxidant activity in both
models. The results obtained in present study
indicate that extracts of Ervatamia divericata
inhibit free radical scavenging activity. The overall
antioxidant activity of these extracts might be
attributed to its sterols, triterpenoids, flavonoids,
alkaloids, phenolic and other phytochemical
constituents. These could be a source of natural
antioxidant that could have greater importance as
therapeutic agent in preventing or slowing
oxidative stress related degenerative diseases.
Polyphenol, tannins and flavonoids are very
valuable plant constituents in the scavenging action
due to their several phenolic hydroxyl groups22
.
The exact constituents of Ervatamia divericata
flowers, which show free radical scavenging
action, are unclear. However, the phyto
constituents like sterols, triterpenoids, alkaloids,
polyphenol and flavonoids present in the flower
extracts may be responsible for antioxidant activity.

9. 215
K. Hemalatha. et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-1(2) 2012 [207-215]
www.ijrpp.com
Thus, the radical scavenging activity and nitric
oxide radical scavenging activities are strongly
suggested that Ervatamia divericata flowers has
antioxidant activity. Further studies are needed to
evaluate the in-vivo antioxidant potential of this
extract in various animal models and to isolate the
active component.
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