Journal of Innovations in Pharmaceuticals and Biological Sciences JIPBS

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ISSN: 2349-2759

Research article
Antioxidant Activity: Root, Leaves and Fruits Aqueous Extracts of
MuntingiaCalabura

Mohamed Azmathulla Khan Y1, Subhas Chandrappa Mundasada2, Dinesha Ramadas*3

1Associate Professor, Department of Physiology, Shridevi Institute of Medical Sciences and Research
Hospital, Tumkur-572106, Karnataka, India.
2Professor, Department of Medical Biochemistry, Basaveshwara Medical College, Chitradurga-577

502, Karnataka, India.

3AIMS, Central Research Laboratory, Adichunchanagiri Biotechnology & Cancer Research Institute,

B.G. Nagara-571 448, Karnataka, India.

Abstract
Aim: To determine the antioxidant activity of the water extract of root, leaves and fruit of
MuntingiaCalabura were evaluated for their antioxidant activity. Materials& Methods:
The phytochemical analysis antioxidant activity of the extract was studies using plant’s
root, leaves and fruits. Results & Discussion: extracts showed that, each extract contains
rich with proteins, carbohydrates, polyphenols, flavonoids, ascorbic acid, chlorophyll and
negligible amount of α-tocopherol. The antioxidant activity of the above was evaluated by
DPPH (1,1-diphenyl-2-picryhydrazyl) radical scavenging activity. Standard antioxidants
like BHA, Curcumin and α-tocopherol were used as positive control. The leaf extract (52%)
showed more antioxidant activity in comparison to fruit extract (25%) and the root extract
(43%) showed less. Conclusion: The above antioxidant activity of the extracts may due to
the presence of phytochemicals like polyphenols, proteins, flavonoids, ascorbic acid and α-
tocopherol.

Key words: MuntingiaCalabura, Leaves, Root, Fruits, Antioxidant, DPPH, Phytochemical analysis

*Corresponding Author: Dinesha Ramadas, AIMS, Central Research Laboratory,

Adichunchanagiri Biotechnology & Cancer Research Institute, B.G. Nagara-571 448, Karnataka,
India.

1. Introduction

Oxidation is essential for the production of organisms at high concentration and

energy in biological system. During the damage all major cellular constituents in
normal course of producing energy, free our body [1]. In this scenario, the
radicals are generated. Though free medicinal properties of plants have been
radicals, radical derivatives and non investigated in several countries with a
radical reactive species are useful during focus on identifying key phytochemicals
oxidation but hazardous to living with potential therapeutic effects. The

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 Dinesha Ramadas et al., JIPBS, Vol 2 (4), 363-368, 2015

important active phytochemical founding roots, leaves and fruits were ground to
plant are alkaloids, tannins, flavonoids, powder, stored for further studies.
phenols, proteins and carbohydrates [2]
[3]. Phytochemicals are widely used to Aqueous extract preparation
treat many oxidative stress induced
diseases such as cancer, diabetes etc., Aqueous extract was prepared by
which are associated with ROS that dissolving 5g of powdered Muntingia
damage cellular biomolecules. Medicinal calabura root, leaf and fruits in 100ml of
plants, spices are often rich in phenols, double distilled water, vortexed for 4
flavonoids, proteins are known to have hours, centrifuged at 10000 rpm,
potent antioxidant properties that can supernatant collected, lyophilized and
prevent oxidative stress and provide stored at -10C.
health benefits [4] [5] [6]. Muntingia
calaburais an evergreen tree originally Phytochemical analysis
distributed in tropical America [7].
Literature shows that, the fruits, leaves The extract was tested for the presence of
and roots are used as medicine or part of bioactive compounds by adopting
food. The fruits can be processed into jams standard procedures.
and the leaves can be used for making tea.
Earlier studies have revealed flavones, Protein estimation
flavanones, flavans, and biflavans to be the
major constituents of this species, some of The protein estimation was carried
which have displayed anti-platelet according to Bradford’s method [9] using
aggregation and cytotoxic activities [8]. BSA as standard and hexane extracts of
different plant materials into a series of
2. Materials and Methods test tubes. Volume was made up to 100μl
with distilled water and 900μl of
Curcumin, BHA, Ascorbic acid, DPPH, α- Bradford’s reagent was added to each
tocopherol were purchased from the tube. Absorbance was read at 535nm.
Sigma Aldrich co. USA, Quercetin, A- Concentration of protein was calculated
tocopherol, Gallic acid, BSA were accordingly using standard graph.
purchased from the Himedia Co. All the
other chemicals and reagents were of α-tocopherol estimation
Analar grade were purchased from the
Merck Co., and S.d. fine chem., Mumbai, α-tocopherol estimation was carried out
India. according to Kivcak and Mer T [10]. 20μl-
100μlof standard α-tocopherol solution
Sample collection and 20 and 40 μl of the hexane extracts
was used for the estimation. Volume was
The plant MuntingiaCalabura’s root, leaf made up to 3ml using chloroform, 1 ml of
and fruits were collected from authentic 2, 2-dipyridyl, and 1 ml of FeCl3 solution,
source, Karnataka, India. The collected Incubated at 370C for 15 minutes, and the
roots, leaves and fruits were cleaned absorbance of the reaction mixture was
thoroughly with double distilled water read at 520nm, concentration was
and dried under the shade. Once the calculated accordingly by using the
drying process is complete, the dried standard graph.

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 Dinesha Ramadas et al., JIPBS, Vol 2 (4), 363-368, 2015

Total phenolics distilled water. 1ml of 5% phenol was

added to each tube followed by 5ml of
Total phenolics were determined 96% sulphuric acid, intensity of the colour
according to the method of FolinCiocalteu was read at 520 nm. The amount of total
reaction [11] with minor modifications sugar present in the given unknown
using gallic acid as a standard (0-100µg). sample solution was calculated using the
Various concentrations of hexane extracts standard calibration curve.
ranging from 0-100μg were taken in
series of test tubes & the volume was Flavonoid estimation
made up to 500μl with distilled water.
500μl of the Folin-ciocalteu reagent was Flavonoid estimation was done according
added to each tube, the mixture was to Cheon et al [14] by using Quercitin as a
allowed to stand for 10 minutes followed standard. Various concentrations (0-
by addition of l.0ml of 20% Sodium 100μg) of hexane extracts were taken in
carbonate, incubated at 10 minutes at test tubes. Made up the volume to 1.5 ml
37C. Absorbance was read at 750nm and with 95% ethanol. Then 100 μl of 10% of
the concentration was calculated using the aluminium chloride, 100μl of 0.1M of
standard graph accordingly. potassium acetate was added to each tube.
The total volume was made up to 2.8ml of
Ascorbic estimation by using distilled water. Optical Density
(O.D) was measured at 415 nm and the
Ascorbic estimation was carried out concentration was calculated accordingly.
according to Sadasivam S., Manickam [12].
Different concentrations (0-100μg) of Estimation of Total chlorophyll
hexane extracts were taken along with
standard ascorbic acid. A drop of thiourea Chlorophyll estimation was determined
solution and 1ml of 2,4dinitrophenyl according to the method of Sadasivam and
hydrazine reagent was added to each tube Manickam[12]with minor modifications.
and the volume as made up to 100μl with In brief, 0.5ml of the hexane extract was
4% oxalic acid and incubated at 37C for 3 mixed with the 20ml of 80% acetone.
hours. Then tubes cooled on ice water and Centrifuged at 5000 rpm for 5 minutes
5ml of 85% sulphuric acid was added to and supernatant was collected. The
each tube. Mix the reaction mixtures process was repeated for several times till
thoroughly. The orange color developed the clear supernatant was obtained. All
was read against a reagent blank at the supernatants were combined and
540nm. The concentration was calculated volume was made up to 1 mL with 80%
on the basis of the standard curve. acetone. The absorbance of the solution
was read at 645 and 663 nm. The amount
Total sugar estimation of the total chlorophyll present in the
extract, mg chlorophyll/gram extract was
Sugar estimation was done according to calculated accordingly using the
Dubois method [13]. 10 - 100 μg of the calibration curve.
working standard solution was pipetted
into a series of test tubes 200μl of the Antioxidant activity
extracted sample was pipetted into two 1,1-Diphenyl-2-picrylhydrazyl (DPPH)
separate test tubes. The volume in each radical scavenging activity
tube was made up to 1000μl with double

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 Dinesha Ramadas et al., JIPBS, Vol 2 (4), 363-368, 2015

DPPH radical scavenging activity was plant was done and its phytochemical
assessed according to the method of analysis was done. The leaves extract was
Shimada et al. with minor modifications rich with Carbohydrates (194mg/g),
[15] [16]. The Root, leaves and fruit proteins (6.21mg/g), Flavonoids
extracts of MuntingiaCalabura at a (42.61mg/g), chlorophyll (79.12mg/g)
concentration of 25µg each was mixed in 1 and Ascorbic acid (11.21mg/g) when
ml of freshly prepared 0.5 mM DPPH compared to root and fruits. Where as
ethanolic solution and 2 ml of 0.1 M fruits extract rich with polyphenols
acetate buffer pH 5.5. The resulting (29.03mg/g) when compared to root and
solutions were then incubated at 37C for leaves extract. The root extract rich with
30 min and measured spectrophoto α-tocopherol when compared to leaves
metrically at 517 nm. BHA and Ascorbic and fruits extract. When these extracts
acid (400 μM) was used as positive are subjected to antioxidant analysis using
control under the same assay conditions. DPPH method (Figure 1), where
Negative control was without any Curcumin, α-tocopherol and Ascorbic acid
inhibitor or Root, leaves and fruit extracts are used as positive control at a dosage of
of MuntingiaCalabura. Lower absorbance 400µM concentration. The extracts of
at 517 nm represents higher DPPH Muntigacalabura used at a dosage of 25µg.
scavenging activity. The % DPPH radical The positive controls Curcumin (61%), α-
scavenging activity of extracts of tocopherol (63%), Ascorbic acid (58%)
MuntingiaCalabura was calculated from inhibited the DPPH radicals. The leaf
the decrease in absorbance at 517 nm in extract (52%), fruit extract (25%) and
comparison with negative control. root extract (43%) inhibited the DPPH
radicals. The above results indicated that,
3. Results and Discussion all the three extract of Muntigiacalabura
plant showed good antioxidant activity
As shown in Table 1, the water extract of when compared to positive controls.
root, leaf and fruits of Muntigiacalabura

Table 1. Phytochemical analysis of Muntingiacalabura aqueous extract

Root Leaves Fruits
(mg/g) (mg/g) (mg/g)

Carbohydrates 138.0± 1.36 194.0±1.44 65.33±1.62

Protein 2.63±0.05 6.21±0.11 2.11±0.09
Polyphenols 14.06±0.35 23.06±1.55 29.03±1.25
Flavonoids 14.34±0.01 42.61±1.02 21.71±0.31
Ascorbic acid 09.13±0.04 11.21±0.12 13.03±0.03
α-tocopherol 0.63±0.02 0.41±0.01 0.13±0.01
Chlorophyll 0.82±0.01 79.12±0.02 0.11±0.01

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 Dinesha Ramadas et al., JIPBS, Vol 2 (4), 363-368, 2015

Figure 1. DPPH radical scavenging activity by Muntingiacalabura aqueous extract of leaf,

root and fruit

70

60

50
% inhibition

40

30

20

10

0
No Ascorbic Alpha Curcumin Leaf Root Fruit
treatment acid tocopherol (400µM) extract extract extract
(400µM) (400µM)

DPPH (0.5mM) + with or without extract of Muntingiacalabura (25μg) /α-tocopherol / Ascorbic

acid / Curcumin (400 μM). Mixture incubated at 37C for 30 min and the absorbance read at 517
nm using spectrophotometer.
Values are means ± SD of triplicates.

Conclusion antibacterial potential of Elaeagnuskologa

These results indicated that, the (Schlecht.) leaf, Asian Pac J Trop Med
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Mathé G: Polyphenols: do they play a role
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acid, polyphenols and α-tocopherol
Biomed Pharmacother2002; 56: 200-207.
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Antioxidant activity, total phenolic and
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