INT J CURR SCI 2012, 262-267

SHORT COMMUNICATION ISSN 2250-1770

Phytochemical analysis, antioxidant and antimicrobial studies of fruit rind of
Couroupita guianensis (AUBL)
Regina V and K.M.Uma Rajan
PG & Research Department of Botany, Pachaiyappa’s college, Chennai-600 030, India
Corresponding author: E-mail: regi.prem@yahoo.in; Phone: +91-8754482049
Abstract
Couroupita guianensis possesses antibiotic, antifungal, antiseptic and analgesic qualities). The present work
was undertaken to know more about its phytochemical analysis, compound isolation antimicrobial activity and
antioxidant status to ensure its usage in Pharmaceutical industries. The different solvent extracts were loaded on cut
wells with the volume of 100ul of pre-sterilized medium of each petriplate containing swabbed pathogens such as
Staphylococcus aureus, E.coli, Micrococcus, Cornybacterium diptheria, Candida albicans, were incubated and
observed zone of inhibition rate and determined MIC. Phytochemical analysis were carried out by TLC and HPLC
methods and antioxidant activity by 1,1-diphenyl-2picryhydrazyl. chloroform, hexane and ethanol extract of fruit rind
of Couroupita guianensis Aubl. showed its significant antibacterial and antifungal activity at the various conc. against
major microbes. Qualitative phytochemical observation showed main components like Rutin,Quercetin, Kaempherol,
Farmaricetin, Lutolin, Ursolic acid Saponines derivatives like Hopanes, which have anticancer activity. Number of
molecules of DPPH scavenged per molecule of the tested compounds was found to be the strongest antiradical activity.
Keywords: antimicrobial, antioxidant, Couroupita guianensis, fruit rind, phytochemical
Received: 24th December; Revised: 19th January; Accepted: 28thJanuary; © IJCS New Liberty Group 2012
Introduction
Medicinal plants represents a rich source of antioxidant and antimicrobial analysis of ethanolic
antibiotic, antifungal, antiseptic and analgesic qualities extract from the rind of the fruit is the rich source of
(Nelson and Wheeler, 1937). They are used medicinary Saponin (Hamed et al., 2005).
in different countries (Ghillean et al., 1986; Mori and Materials and methods
Prance, 1978). Couroupita guianensis (Aubl) belongs to Plant materials
the family Lecythidaceae, commonly known as cannon
The fresh fruits of Couroupita guianensis
ball tree. A truly amazing tree does not grow branches
(Aubl) Lecythidaceae were collected in the month of
that reach out from the straight trunk, it bears large,
August 2010 from Pachaiyappa’s college, Chennai-30
showy flowers, almost through the year, on the trunk and
Near Hostel block and identified with the help of the
not on braches like most other trees.The tree also
flora of Tamilnadu, India, and authenticated.
produces globular brown woody, indehiscent,
(No;PARC/2010/949) (Nair and Henry, 1983).
amphisarcun (double fleshy) fruits of an astonishing size,
Extract preparation
almost the size of a human head (Mitre, 1998). Size of a
mature fruit-24 cm in diameter, weight of a mature fruit- The rind of the fruit was separated by breaking
1450 gms, weight of the shell (fruit rind) from a fruit-545 the fruit and dried at room temperature in the laboratory
gms. It is widely planted in trophical and subtrophical for a period of two weeks sunshade, coarsely powered,
botanical gardens as an ornamental throughout the tropics weighed then in hot continuous extraction (soxhlet)
and sub tropics, it does well under cultivations and it is (Agbafor, 2004) with water, ethanol, chloro-form and
used to feed animals. To investigate the phytochemical, hexane. Each time before extracting with next solvent
 Regina and Uma Rajan, 2012
(successive extraction) (Harborne, 1973) the powdered
material was dried at room temperature. After the
effective extraction solvent were concentrated using
rotary flash evaporator and water was removed by freeze
drying and extract obtained were subjected to
phytochemical, antioxidant and antimicrobial analysis
(Raghvendra et al., 2006).
Phytochemical analysis
Thin Layer Chromatography (TLC) studies
were carried out for various extracts to confirm the
presence of different phytoconstituents (Paulami Mandal
and Tapan Ganguly, 2009). TLC is a mode of liquid
chromatography, in which, the extract is applied as a
small spot or band at the origin of thin sorbent layer
supported on a glass plastic metal plate. The mobile
phase migrates through the stationary phase by capillary
action. The separation of solutes takes place due to their
differential adsorption (Lazarowych and Pekos, 1998)
partition coefficient with respect to both mobile and
stationary phases. Each separated component has same
migration time but different migration distance.The
mobile phase consists of a single solvent or a mixture of
solvents. Although, number of sorbents like silica gel,
cellulose, polyamide, alumina, chemically modified silica
gel etc. is used, Silica gel (type 60) is most commonly
used adsorbent. Handmadeplates are prepared by using
techniques like, pouring, dipping or spraying. Now-a-
days, readymade precoated plates are also available. The
plates need to be activated at 110C for 1 hr. This removes
water / moisture loosely bound to silica gel surface.
Adsorbent : Silica gel GF 254 (activated)Thickness : 0.4
mm Plate size : 10 x 20 cm activation temp: 1100C for
1hrVolume of spot: 20 ml Solvent system: Hexane: Ethyl
acetate (2:3). The spots were observed in iodine chamber
yellow to brown spot. The aqueous extract, which
showed the presence of saponins was subjected to
column chromatography. Adsorbent: Silica gel 60-120
Ethonolic extract column length : 20 cm × 8 cm Sample
loaded : 1 gm Mobile phase : Ethyl acetate: ethanol (5:5)
flow rate : 20 drops/ min. Detection: Iodine chamber or
spray with phenol sulphuric acid. Preparative TLC plates
of (approx.) 0.5 mm layer thickness were prepared using
slurry of Silica Gel GF254 by pouring technique. The
plates were activated at 110C for 1 h. Sample of AqE of
bark was prepared in distilled water and applied as a band
on the plate. Sample overloading was avoided to reduce
tailing effect. Then the plates were dried in air and
developed in the pre-saturated developing chamber using
the same solvent system as used for qualitative TLC
(Careri et al., 2001). The substances separated as distinct
bands. HPTLC of isolated compound was performed
using CAMAG TLC Scanner 3 and Linomat-V. The
FTIR spectrum of isolated compound has shown
characteristic peaks(Gordon et al., 1994), IR spectra were
recorded in KBr on a Perkin-Elmer infrared-283
spectrophotometer as nujol and are expressed in cmG1.
1H NMR spectra were obtained on the AMX-400 liquid
state spectrometer at 440 MHz in DMSO were showed in
fig. 1.
Antioxidant analysis
The effect of the fruit rind of Couroupita
guianensis in scavenging free radicals and oxidants were
analysed against a battery of known standard radicals and
oxidants, in vitro (Naznin Ara and Hasan Nur, 2009).
Due to the presence of an odd electron it gives a strong
absorption maximum at 517 nm. As this electron
becomes paired of in the presence of an hydrogen
donor,ie a free radical scavenging antioxidant (Irina
Koleva et al., 2002), the absorption strength is decreased
and the resulting decolorization is stoichiometric with
respect to the number of electron captured. DPPH (Ju
Dong Yeo et al., 2010) radical quercetin, kaemferol,
resveratrol, myricetin, catechin, fistein, ellagic acid and
narringenin at different concentrations were compared
with respect to trolox and α-tocopherol.
Qualitative analysis of micro-bicidal inhibitory activity
(Well diffusion method)
The antibacterial activities of Couroupita
guyanensis (Sanjay et al., 2007). aqueous, ethanol,
chloroform and hexane extracts were tested by standard
well diffusion method using SCDA (Soya bean Casein
Digest Agar) media.
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 Regina and Uma Rajan, 2012

Fig. 1. 1H NMR spectra

The test bacteria such as Staphylococcus aureus,
Escherichia coli, Corynebacterium diphtheriae,
Micrococcus sp. and Candida albicans are skin
pathogens were included in this study (Minakshi et al.,
2008). 10 mg/ml concentration of the different solvent
extracts were loaded on cut wells with the volume of 100
µl of presterilized medium of each petriplate containing
swabbed pathogens and plates were incubated at 37ºC for
24-48 hrs for observing zone of inhibition rate. Zone of
inhibition (Mahesh and Satish, 2008) was measured using
standard scale in millimeter.
Minimum Inhibitory Concentration (MIC) for the
different extracts of the Couroupita guyanensis
The microbial activity of Couroupita
guyanensis aqueous, ethanol, chloroform and hexane
extracts was checked for determining its MIC against
tested pathogens. Known bacterial population of bacterial
suspension was prepared by serial dilution of culture such
as Staphylococcus aureus, Escherichia coli,
Corynebacterium diphtheriae, Micrococcus sp. and
Candida albicans and certain specific known population
of culture was used for specific pathogens to determine
MIC (Black, 1996).
Different concentration of 5, 10, 15, 20, 25, 30,
35, 40, 45, 50 and 55 mg/ml of Couroupita guianensis
specific extract was incorporated onto SCDA agar plates
with 20 ml of medium. Specific known populations of
pathogens were spread plated on the agar and all plates
were incubated at 37°C for 24 hr in order to determine
inhibitory growth and reduction colonies.

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 Regina and Uma Rajan, 2012

Results and discussion Table 1. DPPH Radicals scavenging activity

The Retardation Factor (Rf) is calculated using Standar Dry Dry
following formula, d (1 (10
Reagents Blank
(0.16 mg/ml mg/ml)
Rf = Distance travelled by solute from the origin mg/ml) )
Distance travelled by solvent from the origin Methano
3.8 ml 3.7 ml 3.7 ml 3.7 ml
l
The preliminary phytochemical investigation
BHT Nil 100 µl Nil Nil
revealed the presence of alkaloids, carbohydrates,
glycosides, saponins and triterpenoids. The ethonolic Sample Nil Nil 100 µl 100 µl
extract of fruit rind showed the presence of saponins. The DPPH 200 µl 200 µl 200 µl 200 µl
isolated compound is hopane which is the derivatives of
saponin which showed in figs. 2 and 3. Table 2. Qualitative analysis of microbicidal inhibitory
Fig. 6. Hopane molecule activity

Zone of inhibition (mm)

Microbes (at
Ethano Hexa
10 mg/ml) H2O CHCl3
l ne
S. aureus Nil 9 6 3
E. coli Nil 9 3 8
Micrococcus Nil 9 nil 2
sp.
C. diptheriae Nil 9 5 7
C. albicans 18 14 22 12

Qualitative analysis of microbicidal inhibitory

activity of the different extracts of Couroupita guianensis
at the concentration of 10 mg/ml showed good inhibitory
activity in which the ethanol extract showed good
inhibitory activity against S. aureus E. coli, C. diptheriae
and Micrococcus sp. among the other tested extracts
whereas chloform extracts showed good inhibitory

Number of molecules of DPPH scavenged per activity against C. albicans. Apart from the ethanol

molecule of the tested compounds like quercetin, extracts which showed good activity for most of the

myricetin was found to have the strongest antiradical pathogens, chloroform showed good activity against most

activity. Each molecule of quercetin and myricetin of the pathogens next to ethanol extracts. Based on the

scavenged 10 molecules of DPPH; catechin and fistein extracts which showed good inhibitory activity against

scavenged 5 molecules; resveratrol scavenged 3.6 particular pathogens and minimum inhibitory

molecules and ellagic acid could scavenge 3.3 molecules concentration of particular extracts which showed good

of DPPH. Quercetin, myricetin, catechin, fistein, inhibitory activity was determined. In which the ethanol

resveratrol and ellagic acid were stronger than trolox and extracts at different concentration were tested against

-tocopherol which scavenged 2 molecules of S. aureus at 105 CFU/ml, among the tested different

DPPH. Kaemferol and naringenin were found to be the concentrations the 30 mg/ml concentration showed

weakest antiradicals, which scavenged 1.7 and 0.5 complete reduction of bacterial colonies in tested media

molecules of DPPH per molecule respectively, showed in and fixed as MIC for S. aureus. As far as the C.diptheriae

table 1. and E. coli is concerned the population used is 102

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 Regina and Uma Rajan, 2012

CFU/ml and 104 CFU/ ml respectively and among tested
concentration of ethanolic extracts 45 mg/ml showed
complete inhibition i.e., complete reduction of colonies in
tested media for both the pathogens and fixed as MIC for
C. diptheriae and E. coli The same ethanol extracts were
showed minimum inhibitory concentration against
Micrococcus sp. at 106 CFU/ml with the concentration of
20 mg/ml finally the Candida albicans is completely
inhibited at the concentration of 15 mg/ml at 106
CFU/ml, showed in tables 2 and 3.
Conclusions
The Phytochemical constituents were analyses
by TLC, HPTLC, HPLC, UV, NMR and IR techniques
were carried out to gather an idea about the nature and
structure of the active compound present in the fruit rind
is hopane which has anticancerous activity and the crude
extract showed good antioxidant agent and also the
extract showed good antimicrobial activity against
specified microbes. The present work has shown that the
fruit rind of Couroupita guianensis Aubl. studied is
potentially good source of many diseases and that further
investigations are carried to support the view that
traditional use in medicine and also assisting primarily
health care.
Acknowledgements
The authors would like to thank Dr Prasanth,
Junior Research Scientist, Cholayil Research and
Development Centre, Chennai for providing experimental
facilities and information of traditional use and for his
kind assistance in the course of this research.

Table 3. Minimum Inhibitory Concentration (MIC) for the different extracts of the Couroupita guyanensis
Total no of colonies in different concentrations (mg/ml)
Control
Microbes Control 5 10 15 20 25 30 40 45
Staphylococcus
aureus Ethanol 72 # # # 14 4 nil # #
(105CFU/ml)
Escherichia coli
Ethanol * # # # 45 # # 9 nil
(104CFU/ml)
Micrococcus sp.
Ethanol 70 # 43 7 nil # # # #
(106CFU/ml)
Corynebacterium
diptheriae Ethanol 45 # # # 7 # # 4 nil
(102CFU/ml)
Candida lbicans
Chloroform * 101 2 nil # # # # #
(106CFU/ml)

CFU- Colony forming unit, # -not applicable , * -too numerous to count

References
Nelson EK, DH Wheeler (1937). Some constituents of
the cannonball fruits J.Am.Chem.Soc.
59(12), pp 2499-2500.
Ghillean T Prance, Scott A Mori (1986). Annals of the
Missouri Botanical Garden,73:99-101.
Mori SA, GT Prance (1978). Additional notes on the
floral biology of Neotropical Lecythidaceae.
Brittonia 30:113-341.
Mitre (1998). Couroupita guianensis 2006.
Hamed Al, Piacente S, Autore G, Marzocco S, Pizza C
Oleszek W (2005). Antiproliferative hopane
and oleanane glycosides from roots of Glinus
lotoides. Planta Med.71 (6) 554-560.
Nair NC, Henry AN (1983). Flora of Tamil Nadu, India
158.1983.
Agbafor KN (2004). The effect of aqueous and organic
extracts of fresh leaves of Baphia nitida on
tissue acetylcholinestenase in guinea pigs.
Journal of Science and Technology, 10, 1-8.
Harborne IB (1973). Phytochemical methods: A Guide to
Modern Techniques of plant Analysis,
Chapman and Hall, New York, USA, 2nd
edition.

5
 Regina and Uma Rajan, 2012

Raghvendra MP, Sathyish S, Raveesha KA (2006). Wound Healing and Antioxidant potential of
Phytochemical analysis and antibacterial Couroupita guianensis in rats.
activity of Oxalis corniculata: a known Pharmacologyonline, 3(3):269-281.
medicinal plant. My Science, 1(1)72-78. Minakshi G Joshi, DV Kamat, SD Kamat (2008).
Paulami Mandal, Tapan Ganguly (2009). Flourescence Evaluation of herbal handwash formulation
spectroscopic characterization of the Nat. Pro.Rad..7(5), 413-415.
interaction of Human adult Hemoglobin and Mahesh B, Satish S (2008). Antimicrobial Activity of
Two isatin. J.Phys Chem.B.2009, 113 (45) some Important Medicinal Plant Against
14904-14913. plant and Human Pathogens. World Journal
Lazarowych NJ, Pekos P (1998). Use of finger printing of Agricultural Sciences 4(s):839-843.
and marker compounds for identification and Black JG (1996). Microbiology: Principles and
rd
standardization of botanical drugs:Strategies Applications, 3 Edn, Prentice Hall, New
for applying pharmaceutical,HPLC analysis Jersey, USA, pp.436-443.
to herbal products. Drug information Journal,
32, 497-512.
Careri M, Elviri L, Mangia A (2001). Liguid
Chromatography-atmospheric pressure
chemical ionization mass spectrometric
characterization of sitosterol and
stigmasterol in soyabean oil. Jr. of
Chromatographic Analysis, 935(1-2), 249-
257.
Gordon W, Kirby Garry T, Walker (1994).
Configurational purity of Capsaicin olefin
Phytochemistry 36 (1) 185-187
Naznin Ara, Hasan Nur, (2009). In vitro antioxidant
activity of methanolic leaves and flowers
extracts of ‘Lippia alba’. Research Journal of
Medicine and Medical Sciences, 4: 107-110
Irina L Koleva, Teris A Van Beek, Jozef P. H Linssen,
Aede de Groot, Lyuba N. Evstatieva (2002).
Screening of plant extracts for Antioxidant
Activity:a comparative study on three testing
metods. Phytochemical Analysis 13 (1) 8-17.
Ju Dong Yeo, Min Kyu Jeong, Jae Hwan Lee (2010).
Application of DPPH absorbance method to
monitor the degree of oxidation in thermally-
oxidized oil model system with antioxidants.
Food science and Biotechnology 19 (1) 253-
256
Sanjay, Prahlad Umachigi, Jayaveera KN, Ashok Kumar
CK, Kumar GS (2007). Antimicrobial,