International Journal of Pharmacy and Biological Sciences-IJPBSTM (2019) 9 (2): 1335-1340

Online ISSN: 2230-7605, Print ISSN: 2321-3272

Research Article | Biological Sciences | Open Access | MCI Approved
UGC Approved Journal

Phytochemical and Antioxidant Studies on

Leaf Extracts of Muntingia calabura L.
Ninan Jisha a, A. Vysakh a, V. Vijeesha and MS Lathaa, *
aSchool of Biosciences, Mahatma Gandhi University, Priyadarshini Hills, Kottayam,
Kerala, India.

Received: 10 Jan 2019 / Accepted: 9 Mar 2019 / Published online: 1 Apr 2019
*Corresponding Author Email: mslathasbs@gmail.com

Abstract
Medicinal plants act as a storehouse of ingredients which can be exploited for its therapeutic
values. Hence, they serve as an asset for pharmaceutical industries. The present investigation
aims to describe the qualitative and quantitative phytochemical analysis and in vitro antioxidant
activity of Muntingia calabura L. leaf extracts. The phytochemical analysis revealed the presence
of phenolics, flavonoids, steroids, terpenoids and alkaloids in EEMC, MEMC and AEMC. The total
phenolic and flavonoid content was found to be higher in MEMC. The antioxidant activity of
various leaf extracts of Muntingia calabura L. was determined mainly by DPPH, ABTS + and FRAP
assays. In all the three antioxidant assays MEMC exhibited a greater potential to scavenge free
radicals at its lower concentration. Muntingia calabura L. acts as an excellent source of
biomolecules with highest free radical scavenging potential and hence very effective against
oxidative stress related disorders.

Keywords
Muntingia calabura; Antioxidant; Phytochemicals; Free radical.

*****

1. INTRODUCTION known and unknown phytochemicals are found in

Plant based traditional system of medicine still plants which are meant to protect the plant from
provides health benefits for the mankind (1). Active disease and damage. Phytochemicals can offer a
pharmaceutical compounds isolated from plants protective role against various human diseases (5).
possessing significant medicinal properties can be Phytochemicals are not an inevitable component for
used in drug development and synthesis (2). Plant the sustenance of human life but have an importance
offers a good source of nutrients in spite of its in disease prevention. The most common cause of
therapeutic values (3). Plants played a significant role many diseases is the oxidative cell damage by free
in human culture development worldwide. Since radicals (6). Oxidative stress related diseases are very
historical times, people use medicinal plants very common now a day. So, it is very essential to
frequently for various ailments. Phytomedicine are neutralize the free radicals generated by reactive
safe because they are either in combined or pooled oxygen species. Antioxidants can reduce the
form of more than one molecule. oxidative stress in cells by scavenging the free
Phytochemicals are naturally occurring; biologically radicals (7). Plants acts as reservoir of natural
active chemical compounds present in plants which antioxidants (8). It is very much essential to
provide health benefits for humankind (4). Many understand the biomolecules from plants that are

DOI: https://doi.org/10.21276/ijpbs.2019.9.2.161 MS Latha* et al 1335

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responsible for disease prevention and its (0.1 ml), 80% methanol (1.5 ml) and distilled water
antioxidant efficacy. Muntingia calabura L. is a (2.8 ml) to the extract (0.5 ml) and standard (0.5 ml)
medium sized evergreen tree native to American kept in different test tubes and shaken well. A blank
continent and is locally known as Jamaican cherry was also prepared in the same manner but the
tree (9). In Peruvian folklore medicine, it is difference is that 0.5 ml of distilled water was used
documented that M.calabura L. possesses so many instead of the sample or standard and the aluminum
medicinal values (10). The current study aimed to chloride used in the assay was replaced by distilled
report the phytochemical composition of Muntingia water. All tubes were kept for an incubation period
calabura L. leaf samples and also to evaluate its of 30 min at room temperature. Using UV-Visible
antioxidant efficacy. spectrophotometer, the absorbance was read at 415
nm. The flavonoid concentration was expressed in
2. MATERIALS AND METHODS mg quercetin equivalent (QE) per gram of extract.
2.1. Plant material 2.5. In vitro antioxidant assays
Muntingia calabura L. leaf samples were collected 2.5.1 DPPH radical scavenging assay
during March – April 2018 from kottayam district, The DPPH radical scavenging assay was done by the
Kerala, India. The plant material was authenticated method adopted by Sanchez-Moreno et al (14). 10 ml
by a taxonomist and the specimen is maintained in of test sample at different concentrations (0-200
the institute. µg/mL) were added to 190 ml DPPH (150 mM) which
2.2. Preparation of various extracts. was prepared in methanol solution. The solutions
Plant leaf powder (40g) was extracted with 400 mL of were shaken well and kept at 37 0Cfor 20 min. The
different solvents with increasing polarities in solvent used for the assay was taken as blank.Due to
soxhlet apparatus. The solvents used are petroleum the quenching of DPPH free radicals there will be a
ether, chloroform, ethyl acetate, methanol and decrease in absorbance of test samples. Absorbance
water. The solvent was evaporated using a rotary at 517 nm using UV-Visible spectrophotometer was
evaporator and obtained the dried petroleum ether documented. Inhibition percentage was calculated
extract (PEMC), chloroform extract (CEMC), ethyl as an indication of radical scavenging activity.
acetate extract (EEMC), Methanolic extract 2.5.2. ABTS cation free radical-scavenging activity
(MEMC)and aqueous extracts (AEMC). Free radical scavenging potential of Muntingia
2.3. Qualitative phytochemical analysis calabura leaf extracts by means of ABTS + radical
The qualitative phytochemical analysis of various scavenging activity was evaluated by the method
extracts of Muntingia calabura L. leaf samples were followed by Re et al (15), with minor modifications.
carried out by the method described by Evans (11). ABTS is a colourless compound which is transformed
2.4. Quantitative phytochemical analysis into its blue–green counterpart ABTS + by losing an
2.4.1. Determination of total phenolics electron by nitrogen molecule on the parent
The EEMC, MEMC and AEMC were taken to quantify compound. A stock solution of ABTS mixed with 2.45
the total phenolic content by the method described mM potassium persulfate and allows the mixture to
by Singleton and Rossi with slight modifications stand in the dark at room temperature for 12–16 h
(12).To the 100 ml crude extract (1 mg/ml) add 0.2 which generates ABTS +. Different concentrations of
mlof Folin-Ciocalteu reagent, 2 ml distilled water and Muntingia calabura leaf extracts (1.562–100 μg/ml)
1 ml 15% Na2CO3. This reagent mixture was taken for were taken to react with 180 µl of ABTS + solution and
measuring its absorbance at 765 nm using UV-Visible kept for 12 min in dark at room temperature. The
spectrophotometer (T60U, PG Instruments Limited, absorbance of the sample extracts was monitored at
UK) after an incubation period of 2 h at room 734 nm.
temperature. The values obtained were compared 2.5.3. FRAP Assay
with Gallic acid standard and were expressed in mg FRAP assay was followed to estimate the reducing
equivalents of Gallic acid (mg GAE) per g dry weight potential of Muntingia calabura by method
of extract. described by Benzie and Strain (16). The FRAP assay
2.4.2. Determination of total flavonoids depends on the principle that the ferricyanide (Fe 3+)
Total flavonoid content of EEMC, MEMC and AEMC got reduced in accordance with the antioxidant
was determined using quercetin as standard by the potential of the extract. 2.5ml of 10 mM TPTZ
procedure followed by Chang et al with minute solution in 40 mM HCl, 2.5ml of 20 mM Fecl 3. 6H2O
modifications (13). A calibration curve of quercetin and 25ml of 300 mM acetate buffer (pH 3.6)
was prepared in the range of 10-100 µg/ml. Add 10% constitutes the FRAP reagent. To 30µl of the extract
aluminum chloride (0.1 ml), 1 M potassium acetate 900 µl FRAP reagent and 70 µl water were added. The

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reaction mixture was kept at 37 0C for a 30 min time 3. RESULTS

period. The absorbance of the reaction mixture was 3.1. Qualitative phytochemical analysis
taken at 593nm and IC50 values were calculated. The phytochemical analysis showed the presence of
various phytoconstituents like alkaloids, phenolics,
flavonoids, steroids, terpenoids etc., in ethyl acetate,
methanol and aqueous extracts (Table 1).
Phytochemical screening revealed the presence of
phenolics and flavonoids as its principal components.

Phytochemicals PEMC CEMC EEMC MEMC AEMC

Alkaloids ++- ++- ++-- +-- ---
Flavanoids --- --- +++ +++ +++
Phenolics --- ++- +++ +++ +++
Tannins --- --- --- +++ +--
Cardiac glycosides --- --- --- --- ---
Triterpenoids --- --- +-- ++- ++-
Steroids +-- +-- ++- ++- +++
Saponins --- --- --- --- +++

Table 1. Qualitative phytochemical analysis of various extracts of Muntingia calabura leaf. Key words: +
indicates presence; - indicates absence.
Sample Total phenolic content Total flavonoid content
(mg of gallic acid/g (mg of quercetin/g
Dry weight of the sample) Dry weight of the sample)
EEMC 51.27± 0.42 22.38± 0.12
MEMC 82.56± 0.25 31.45± 0.27
AEMC 11.23± 0.68 16.59± 0.15
Table 2. Quantitative phytochemical analysis Muntingia calabura leaf extracts

3.2. Quantitative phytochemical analysis exhibited its highest potential to scavenge free
The ethyl acetate, methanol and aqueous extract radicals at its lowest concentration in all the three
were found to contain rich amount of bioactive antioxidant assays.
molecules like phenolics and flavonoids. These 3.3.1. DPPH assay
phytochemical agents present in the ethyl acetate, The DPPH radical scavenging activities of various
methanol and aqueous extracts of Muntingia extracts of Muntingia calabura were studied and
calabura leaf samples were quantified and was were shown in Figure 1. IC50 values of all the extracts
shown in Table 2. The total phenolic content of EEMC were estimated and it was noted that all the extracts
is 51.27± 0.42, MEMC is 82.56 ± 0.25 and AEMC is of Muntingia calabura exhibited a significant DPPH
11.23 ± 0.68 mg equivalents of gallic acid/ g dry radical scavenging activity. Among all the extracts
weight of the sample. The total flavonoid content of studied, MEMC exhibited highest DPPH radical
EEMC is 22.38 ± 0.12, MEMC is 31.45 ± 0.27 and quenching activity with an IC 50 value of 76.43±0.01
AEMC is 16.59 ± 0.15 expressed as mg equivalents of μg/ml. The IC50 values of other extracts are EEMC
Quercetin/g dry weight of the sample. having 97.99 ± 0.02 μg/ml, AEMC having 180.6 ± 0.08
3.3. In vitro antioxidant assays μg/ml, CEMC having 361.1 ± 0.04 μg/ml, PEMC
In vitro antioxidant activities of various extracts were having 418.2 ± 0.06 μg/ml. The IC50 value of standard
analyzed using three different assays. The MEMC ascorbic acid was observed as 54.38 μg/ml.

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PEMC

CEMC
Percentage of inhibition
EEMC

Concentration (μg/ml )
Figure 1. DPPH assay of various extracts of Muntingia calabura leaf

PEMC
CEMC
EEMC
MEMC
Percentage of inhibition

AEMC
Ascorbic acid

Concentration (μg/ml )
+
Figure 2. ABTS assay of various extracts of Muntingia calabura leaf

3.3.2. ABTS+ assay 3.3.3. FRAP assay

ABTS assay relies on the principle that the measure FRAP assay is one of the powerful in vitro antioxidant
of extracts needed to quench 50% of ABTS. + radicals assays which focuses on the reduction of ferric iron
by electron donation. The IC 50 value of MEMC is (III) into ferrous iron (II). Among all the leaf extracts
found to be 61.67±0.03 μg/ml in ABTS assay which of Muntingia calabura MEMC showed a maximum
showed highest activity than the other extracts. The activity in FRAP assay and its IC 50 value was found to
IC50 value of EEMC was 113.07 ± 0.08 μg/ml, AEMC be 82.42±0.03 μg/ml (Figure 3). The IC 50 values of
was 152.09 ± 0.07 μg/ml, CEMC was 314.30 ± 0.07 other extracts were 127.11 ± 0.02 μg/ml (EEMC),
μg/ml, and PEMC was 396.01 ± 0.03 μg/ml, which 132.44 ± 0.02 μg/ml (AEMC), 205.33 ± 0.07 μg/ml
also showed a potential to scavenge ABTS .+ radicals (CEMC), and 247.71 ± 0.03 μg/ml (PEMC) which was
against the standard ascorbic acid with the IC50 value shown in Figure 3. In this assay the ferrous sulphate
37.45 μg/ml. was taken as the standard (IC50 value 41.26 μg/ml).

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PEMC

CEMC

EEMC
Percentage of inhibition

MEMC

AEMC

Ferrous
Sulphate

Concentration (μg/ml )
Figure 3. FRAP assay of various extracts of Muntingia calabura leaf

4. DISCUSSION have well documented medicinal and physiological

Medicinal plants played a central role in human and actions.
community health. Frequent usage of herbal The process of oxidation is an important step in
medicines by a large population is possibly due to its various biological processes of living organisms for
efficacy, safety and cost effectiveness. Plant derived releasing energy. This causes the generation of free
compounds have a significant role in novel drug radicals. Higher level of free radicals, radical
development. The medicinal value of a plant was derivatives and non-radical reactive species can
offered by chemical substances present in it, which damage all the cellular components in our body
elicits a definite physiological action on the human leading to oxidative stress related disorders and
body (17). The most important bioactive constituents hence they are hazardous (19). The reactive oxygen
of plants are alkaloids, tannins, flavonoids and species is the key factor behind various diseases like
phenolic compounds. cancer, diabetes, heart diseases and they damage
Muntingia calabura L. is a shrub widely grown as the body organs like eyes, lungs, brain, kidney, heart,
roadside tree and its leaves have well documented pancreas, etc (20). Antioxidants on the other hand
medicinal uses. In Peruvian folk remedies, the protect our body from free radical damage by giving
antiseptic property of Muntingia calabura L. leaves up their electrons and it can also stimulate wound
and its effectiveness in treating swellings of lower healing (21). Thus, antioxidants act as a panacea for
extremities are well reported. In South America the various diseases. Therefore, it is worthwhile to
leaf decoctions of Muntingia calabura L. were used evaluate the antioxidant efficacy of Muntingia
to reduce gastric ulcers (18). calabura L. leaf extracts. The in vitro antioxidant
The different extracts derived from the leaves of evaluation of various extracts revealed its capability
Muntingia calabura L. revealed the presence of to scavenge free radical at a lower concentration.
various phyto constituents such as alkaloids, The Methanolic extract of Muntingia calabura L.
flavonoids, steroids, terpenoids etc in ethyl acetate, leaves exhibited higher antioxidant activity. The
methanol and aqueous extracts. The total flavonoid higher antioxidant efficacy of MEMC may be due to
and phenolic content in ethyl acetate, methanol and the rich phytochemicals present in it. The antioxidant
aqueous extracts were quantified and it was found properties of flavonoids are due to the chelating of
that a conspicuous amount of secondary metabolites metal ions, such as iron and copper. It has been
were present in it. The phenolics and flavones recognized that, phenols and flavonoids showed
primarily act as antioxidants and therefore it is antioxidant activity and their effects on human
advantageous to determine the amount present in nutrition and health are considerable.
those extracts. These plant secondary metabolites

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CONCLUSION disease prevention. Asia Pacific Journal of Clinical

The phytochemical screening of Muntingia calabura Nutrition 12 (1): 9-22(2003).
L. leaf extracts provides evidence that it contains 7. Nikolova M, Evstatieva L, Nguyen TD, Screening of
plant extracts for antioxidant properties. Botanica
some important bioactive molecules which exhibits a
Serbica 2011 35(1): 43-48 (2011).
strong antioxidant potential. Different extracts
8. Erkan, N., Ayranci, G., Ayranc,i E, Antioxidant activity
exhibited variations in their antioxidant potential of rosemary (Rosmarinus officinalis) extract, Black
and that may be due to the differences in their seed (Nigella sativa) essential oil , carnosic acid,
chemical composition. From the present study it was rosmarinic acid and sesamol. Food Chem 110: 76-82
noted that the antioxidant potential increases with (2008).
an increase in the sample concentration. Thus, we 9. Huda-Faujan N, Noriham A, Norrakiah AS and Babji
can conclude that Muntingia calabura L. leaves are a AS, Antioxidant activity of plants ethanolic extracts
new alternative source for free radical scavengers containing phenolic compounds. African J Biotech 8:
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development of new drugs against oxidative stress
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Compliance with ethical standards
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