The ethnobotanical and pharmacological evaluation of plant based chemicals have shown rapid strides in the last few decades. Plants have been a rich source of important therapeutic agents and form the basis of herbal systems of medicine, like ayurveda, resulting in the revival of ancient traditions of medicine. The present study was carried out to investigate the anti oxidant potential of the inflorescence and fruits of Alstonia scholaris using an in vitro model system like DPPH assay and Beta carotene Assay. The methanol extract of the flower showed powerful antioxidant activity by DPPH and Beta-carotene assays in comparison with the standard butylated hydroxy toluene (BHT), l- ascorbic acid. For DPPH assay the IC-50 value was also calculated and was found to have a significant correlation between benzene extract of flower and methanol extract of fruits. Overall, the methanol extracts of flower showed higher anti oxidant activity than the fruit. Keywords: Alstonia scholaris ,anti oxidant potential, DPPH, Beta Carotene.

James, Joel

Misra, Chandra Shekhar

Pratyush, Kumar

Sahadevan, Lipin Dev Mundur

Thankamani, V

Veettil, Arun Kumar Thaliyil

English

Advanced Research Journals

International Journal of Phytomedicine 3 (2011) 475-479 
http://www.arjournals.org/index.php/ijpm/index 
 
Original Research Article 
           
In Vitro Antioxidant Activity of Flowers and Fruits of Alstonia scholaris 
Joel James1, Arun Kumar Thaliyil Veettil1, Kumar Pratyush1, Chandra Shekhar Misra1, Lipin Dev Mundur  Sahadevan1, 
Thankamani V1* 
 
*Corresponding author: 
 
Dr. V. Thankamani  
1School of Biosciences and 
Technology 
VIT University,  
Vellore-632014 
Tamil Nadu, India. 
M: +919677464881,       
+919447733366 
 
 
 
 
 
 
 
 
A B S T R A C T  
The ethnobotanical and pharmacological evaluation of plant based chemicals 
have shown rapid strides in the last few decades. Plants have been a rich 
source of important therapeutic agents and form the basis of herbal systems of 
medicine, like ayurveda, resulting in the revival of ancient traditions of 
medicine. The present study was carried out to investigate the anti oxidant 
potential of the inflorescence and fruits of Alstonia scholaris using an in vitro 
model system like DPPH assay and Beta carotene Assay. The methanol 
extract of the flower showed powerful antioxidant activity by DPPH and Beta-
carotene assays in comparison with the standard butylated hydroxy toluene 
(BHT), l- ascorbic acid. For DPPH assay the IC-50 value was also calculated 
and was found to have a significant correlation between benzene extract of 
flower and methanol extract of fruits. Overall, the methanol extracts of flower 
showed higher anti oxidant activity than the fruit.  
Keywords: Alstonia scholaris, anti oxidant potential, DPPH, Beta Carotene.   
 
 
Introduction 
The free radicals have been found to be the cause of 
some of the major diseases such as heart diseases, 
diabetes, gout and most recently, cancer. Various 
pathological conditions have been found in connection 
with cellular necrosis because of the auto oxidation of 
cellular membrane lipids caused by free radicals [1]. 
Therefore reduction of these radicals by suitable anti 
oxidant molecules is crucial [2]. Nature provides 
answers to many ailments and is an excellent store 
house of remedies. Presence of flavonoids and other 
polyphenolic compounds in the plant parts have been 
reported to show their remarkable biological effects like 
anti oxidant property and anti cancer activity [3]. 
The plant Alstonia scholaris which belongs to 
Apocynaceae is an evergreen tree found in India and 
other parts of south Asia. Being one of the most 
important medicinal plant in the family, it is used 
against fever, malarial, abdominal disorders, 
dyspepsia, leprosy, skin diseases, pruritus, tumours, 
chronic and foul ulcers, asthma, bronchitis, 
cardiopathy, helminthiasis, agalactia and debility [4, 5]. 
Folkloric use of the plant parts have been known since 
time immemorial and is used as a bitter tonic, 
aphrodisiac, febrifuge, stimulant, expectorant, 
alterative, carminative, anti- periodic, astringent and 
stomachic[6]. The bark is used as a remedy for treating 
asthma, lung cancer, hypertension, and pneumonia 
while the extracts of the leaf are used to treat fever [7]. 
The preliminary studies on in vitro nitric oxide 
scavenging activity have been reported in parts of 
Alstonia scholaris plant [8]. Several natural 
antioxidants have been sourced from plants for treating 
diseases like cancer. However owing to the paucity of 
the clinical data available for the anti oxidant property 
of Alstonia scholaris, the present study was 
undertaken to explore the in vitro anti oxidant property 
of the flowers and fruits of Alstonia scholaris using 
DPPH assay and Beta Carotene Assay. 
 
ISSN: 0975-0185 
 
  
This work is licensed under a Creative Commons Attribution 3.0 License.  
 Joel  et al. International Journal of Phytomedicine 3 (2011) 475-479 
 
476 
 
Materials and Methods 
Collection and Processing of Plant Sample 
The Alstonia scholaris plant was collected from VIT 
University campus. The plant parts viz. leaves, stem 
bark, roots, flowers and fruits were then shade dried 
and pulverized to obtain a fine powder. Flowers and 
fruits were kept separate for further analysis. 
 
Extraction 
The powdered flower and fruit parts were subjected to 
successive extraction using different solvents in the 
increasing order of their polarity- hexane, benzene, 
methanol and water. The extracts were dried and 
concentrated to be used for further analysis. 
 
Anti-Oxidant Activity 
All the extracts were subjected to antioxidant activity 
assays using 1,1-diphenyl-2-picryl-hydrazil (DPPH) 
and β-carotene methods. Ascorbic acid and Butylated 
Hydroxy Toluene (BHT) were used as the reference 
standards. IC50 (μg) values of the extracts for DPPH 
and Hydroxy radical scavenging methods were 
determined using Mat-lab software. 
 
DPPH Radical Scavenging Assay 
Free radical scavenging activity of the extracts was 
measured using the procedure described by Blois [9]. 
The stock solutions of crude extracts at 1mg/mL were 
prepared in DMSO. To each of the reaction mixtures of 
different concentrations, 3.0 mL of freshly prepared 
solution of 2, 20-diphenyl-1-picrylhydrazyl (DPPH) at 
concentration 19g/500ml was added. Absorbance was 
measured at 515 nm after 30min. The remaining 
amounts of DPPH-radical were calculated from 
calibration curve. The scavenging activity of hydroxyl 
radical (%) was calculated according to the equation: 
[(A515blank - A515sample)/A515blank]*100. IC50 
values were further calculated using Mat-lab software. 
 
Beta Carotene Bleaching Assay 
10 ml of Linoleic acid solution dissolved in ethanol (2 
mg/ml) and 10 ml of β-carotene solution (2 mg/ml) in 
acetone were added to 10 ml of the molten agar (1.2% 
27 solutions in boiling water). The mixture was then 
shaken and then poured into Petri dishes (25 ml per 
dish). The plates were kept away from light. Wells (8 
mm diameter) were then created using sterile agar 
borer and 100μl of each extract (1 mg) in DMSO were 
transferred into the wells and the Petri dishes were 
incubated at 45 ºC for 4 h. A zone of orange color 
around the well indicated positive for antioxidant 
activity. The diameter of the zone was measured in 
mm[10]. 
 
 
Table 1: DPPH Assay [IC50 value = Ascorbic Acid - 10.65, BHT – 32.06] of flower extracts of Alstonia scholaris  
Extract 
Concentration 
of Extract(µg) 
Extracts 
Hexane Benzene Methanol Water 
 10 0 10 26 35 
 50 0 35 56 75 
FLOWER 100 0 51 76 86 
 250 0 61 89 87 
 IC 50(µg) NA 95 29 16 
 
Table 2: DPPH Assay [IC50 value = Ascorbic Acid - 10.65, BHT – 32.06] of fruit extracts of Alstonia scholaris  
Extract 
Concentration 
of Extract(µg) 
Extracts 
Hexane Benzene Methanol Water 
 10 22 46 22 14 
 50 43 69 63 50 
FRUIT 100 62 87 90 79 
 250 93 92 93 80 
 IC 50(µg) 60 11.5 34.75 50 
 
 Joel  et al. International Journal of Phytomedicine 3 (2011) 475-479 
 
477 
 
 
Figure 1: Inhibition of DPPH by the flower extracts of Alstonia scholaris 
[IC50 value = Ascorbic Acid - 10.65, BHT – 32.06] 
 
 
Figure 2: Inhibition of DPPH by the fruit extracts of Alstonia scholaris 
[IC50 value = Ascorbic Acid - 10.65, BHT – 32.06] 
 
Result and Discussion 
DPPH Free Radical Scavenging Activity 
DPPH, a relatively stable organic radical has been 
widely used in the determination of antioxidant activity 
of single compounds, as well as of different plant 
extracts. The IC50 results of the DPPH scavenging 
activity of Alstonia scholaris flower and fruit extracts 
are presented in Table 1 and 2. The scavenging ability 
of all the extracts was compared with the standards 
ascorbic acid and BHT. Except hexane extract of 
flower all the other crude extracts exhibited significant 
DPPH scavenging efficacy as shown in Figure 1 and 2. 
Benzene extract of flower followed by methanol and 
water extract of fruit exhibited significant DPPH radical 
scavenging activity with IC50 values 95 μg/ml, 
34.75μg/ml and 50μg/ml respectively compared to 
vitamin C (IC50 10.65 μg/ml). When the extracts were 
compared with the standards Ascorbic Acid and BHT, 
Benzene extract of flower and methanol extract of fruit 
showed significant correlation. 
0
10
20
30
40
50
60
70
80
90
100
10 50 100 250 IC 50
Conc. of Flower Extract(µg)
%
 In
hi
bi
tio
n 
of
 D
PP
H
Hexane
Benzene
Methanol
Water
0
10
20
30
40
50
60
70
80
90
100
10 50 100 250 IC 50
Conc. of Fruit Extract(µg)
%
 In
hi
bi
tio
n 
of
 D
PP
H
Hexane
Benzene
Methanol
Water
 Joel  et al. International Journal of Phytomedicine 3 (2011) 475-479 
 
478 
 
Table 3: β-carotene Assay [BHT – 40mm (1mg) & 25mm (0.5mg)] of flower and fruits extracts of Alstonia scholaris 
      Extract   Concentration 
of Extract(mg) 
Extracts 
Hexane Benzene Methanol Water 
Flowers 0.5 8 12 15 11 
 1 9 14 17 13 
Fruits 0.5 9 8 14 14 
 1 12 9 17 16 
 
 
 
Figure 3: Antioxidant activity in terms of means of zone of Beta carotene color retention (mm) of flowers and fruits of 
Alstonia scholaris. 
 
Beta Carotene Bleaching Assay 
In a β-carotene/linoleic acid model system, β-carotene 
undergoes rapid decolorization in the absence of an 
antioxidant. All extracts except hexane extract of 
flower, were able to retain the color of beta carotene. 
Methanol extract of flower showed highest color 
retention with a mean zone of 17 mm. The values of all 
the extracts are shown in Table 3. It is interesting to 
note that the methanol and water extracts of both the 
flower and fruit showed equal potential as shown in 
Figure 3. Where as in all the other antioxidant assays 
methanol extract showed higher activity irrespective of 
the flower or fruit. In the present study, the anti oxidant 
potential of the flowers ad fruits are evaluated. 
Previous studies on A scholaris have suggested the 
presence of nitric oxide scavenging activity [11]. Further 
findings on the anti oxidant results of ethanolic extract 
of A scholaris [12] prompted us to evaluate the anti 
oxidant potential of the inflorescences by 2 methods: 
DPPH and Beta Carotene assays as it is impossible to 
evaluate the anti oxidant activity by one method due to 
oxidative processes involved and the variable nature of 
the anti oxidants[13,14]. The effect of probable anti 
oxidants present in plant on DPPH radical scavenging 
was supposed to be due to their hydrogen donating 
ability. DPPH being a stable free radical accepts an 
electron on the free hydrogen radical to become 
stabilised diamagnetic molecule [15]. The scavenging 
of the radical by hydrogen donation is caused by the 
anti oxidants, because of the reaction between anti 
oxidant molecule and free radical. The present in vitro 
anti oxidant study is in agreement with the findings of 
Arulmozhi et al. on the ethanolic extract of A scholaris. 
The presence of the flavanoids in the preliminary 
investigation [16] can be correlated with its potential anti 
oxidant activity, however further investigation is 
needed to validate such correlation.  However the 
report by Juhi et al.[17] showed no correlation between 
0
2
4
6
8
10
12
14
16
18
0.5 1 0.5 1
Flower                                 Fruits 
Concentration of Extract(µg)    
Re
te
nt
io
n 
of
 ß
 C
ar
ot
en
e 
Co
lo
r(
m
m
) Hexane
Benzene
Methanol
Water
 Joel  et al. International Journal of Phytomedicine 3 (2011) 475-479 
 
479 
 
anti oxidant activity and the quantitative phytochemical 
screening i.e. total polyphenolic content and tannin 
fractions which is opposite to many published literature 
where anti oxidant activity is closely related to 
polyphenolic content[18,19]. 
 
Conclusion 
The findings of the present study clearly indicate the 
potential antioxidant properties of the flowers and fruit. 
The promising results of this plant extract with 
antioxidant assay models proved the plant to be a 
potential antioxidant agent. This efficacy of the plant 
could be attributed to the synergistic effect of various 
phytochemicals like flavanoids, steroids and 
polyphenolic compounds. Further detailed investigation 
needs to be undertaken in order to evaluate the mode 
of action of these plant extracts at the molecular level. 
 
Acknowledgement 
The authors are thankful to VIT University for providing 
necessary facilities and support to carry out this 
research work. 
 
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In Vitro Antioxidant Activity of Flowers and Fruits of Alstonia scholaris

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In Vitro Antioxidant Activity of Flowers and Fruits of Alstonia scholaris

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