Phytoconstituents and Their Influence on Antimicrobial Properties of Morinda citrifolia L.
Morinda citrifolia L., also known as noni or Indian mulberry is a small evergreen tree having antimicrobial, antitumor, antidepressant and immune enhancing effects. Various parts of M. citrifolia have been investigated for its phytochemical and antimicrobial properties. In the present study, methanolic extracts of leaf, stem and roots of M. citrifolia has been prepared and analyzed for their phytoconstituents. Qualitative analysis of the extracts revealed the presence of phenols, tannins, saponins, alkaloids, glycosides, flavonoids and steroids at various levels. Further, the extracts were tested against pathogenic bacterial and fungal strains at different concentrations to determine the influence of phytochemicals. The results revealed that root and leaf extracts has significant antimicrobial activities mainly due to phenolics and tannins. Staphylococcus epidermidis and Pseudomonas aeruginosa, major pathogens of nosocomial infections were effectively controlled by the extracts at various concentrations and root extract exhibited significant activity against Penicillium sp. and Fusarium sp. It has been revealed that, the bioactive substances have influenced the antimicrobial properties of M. citrifolia which could be exploited to formulate novel drugs from plant origin against bacterial and fungal infections.
to cite this article:
G. Sibi, Parul Chatly, Sayak Adhikari and K.R. Ravikumar, 2012. Phytoconstituents and Their Influence on Antimicrobial Properties of Morinda citrifolia L.. Research Journal of Medicinal Plants, 6: 441-448.
Received: December 28, 2011;
Accepted: February 07, 2012;
Published: March 15, 2012
In recent years, research has been focused to find novel compounds from plant,
animal and microbial origin. Increasing resistance development against frequently
used antimicrobial compounds by the micro organisms is urging to discover new
antimicrobial compounds, particularly from plant origin. Many plants synthesize
substances that are useful to control the growth of microorganisms and plants
are the possible source of antimicrobial agents (Adesina
et al., 2000). Plant-based, traditional medicine system continues
to play an essential role in health care, with about 80% of the worlds
inhabitants relying mainly on traditional medicines for their primary health
care (Owolabi et al., 2007). Though, synthetic
and semi synthetic drugs are available in todays market, there is need
for new ones from natural origin to cope up with the increased evolution of
multiple resistant strains.
Morinda citrifolia (Rubiaceae), also known as noni or Indian mulberry
is a small evergreen tree which is identifiable by its straight trunk, large,
bright green elliptical leaves with tubular flowers and its distinctive, ovoid
grenade-like yellow fruit. Apart from this appellation, there are many local
names that are also widely used in their respective countries namely, Noni apple,
Polynesia fruit, Indian mulberry (India), Bumbo (Africa), Mengkudu (Malaysia),
Cheeserut (Australia), Painkiller tree (Caribbean Islands), Nhau (Southeast
Asia), Morinda (Vietnam), Hai Ba Ji (China).
Various parts of the noni plant extracts have been reported to have significant
antimicrobial, antitumor, antidepressant and immune enhancing effects (Wang
et al., 2002; Gerson, 2002; McClatchey,
2002; Palu et al., 2008; Deng
and West, 2011). The plant has been studied for its insulinotropic (Hamid
et al., 2008), antioxidant (Krishnaiah et
al., 2007; Chanda et al., 2011; Krishnaiah
et al., 2011), wound healing (Nagori and Solanki,
2011), antiosteoporotic (Shirwaikar et al., 2011)
and antidiabetic (Dompeipen et al., 2011) activities.
Ridzwan et al. (2002) reported that, aqueous extracts
of M. citrifolia fruit has decreased coronary perfusion pressure and
developed tension in isolated rat heart. Due to the pharmaceutical values, M.
citrifolia has gained a great deal of interest (Hemwimon
et al., 2007).
The present study was conducted to evaluate the phytochemical properties of various parts of M. citrifolia and their influence on antimicrobial properties.
MATERIALS AND METHODS
Plant collection and extraction: Plant materials of M. citrifolia
such as whole leaves, roots and stem were collected, washed in running tap water
and finally with distilled water. The samples were shade dried for 2 days and
oven dried at 45°C for 24 h and ground to powder. The ground powder was
sieved (200 μm), extracted with methanol for 48 h at room temperature and
the solvent was removed by filtration using Whatman No.1 filter paper. Filtrates
were concentrated to dryness under reduced pressure for overnight and the final
extract was resuspended in methanol to make a stock solution (50 mg mL-1).
Determination of total phenolics: Total phenol content in the extracts was
determined using a modified Folin-Ciocalteu method (McDonald
et al., 2001). One milliliter of the extract was added to 10 mL deionized
water and mixed with 5 mL of Folin-Ciocalteu reagent and 4 mL of 1 M sodium
carbonate. The tubes were vortexed and allowed to stand for 30 min at room temperature.
The resulting blue complex was then measured at 765 nm using UV-VS spectrophotometer.
Determination of total flavonoids: Total flavonoids were estimated by
mixing 0.5 mL of extract with 0.5 mL of 2% AlCl3 ethanol solution
(Ordonez et al., 2006). The tubes were allowed
to stand for 1 h at room temperature and the resulting yellow colour was measured
at 420 nm.
Determination of tannins, alkaloids and saponins: The presence of tannins
in the extract was identified by mixing 0.5 mL of extract with 1 mL of water
and 1-2 drops of ferric chloride solution (Iyengar, 1995).
For the presence of alkaloids, about 0.5 mL of the extract was stirred with
5 mL of 1% aqueous hydrochloric acid on a steam bath for 5 min and filtered.
A few drops of Dragendorffs reagent were used to treat 1 mL of the filtrate
and turbidity or precipitation with this reagent was taken as evidence for the
presence of alkaloids. To detect saponins in the extract, 0.5 mL of the extract
was dissolved in distilled water in a test tube and frothing which persisted
on warming was taken as preliminary evidence for saponins.
Determination of terpenoid and steroid: 0.5 mL each of acetic anhydride
and chloroform was added to 4 mL of extract. The mixture was then added with
concentrated sulphuric acid for colour formation (Siddiqui
and Ali, 1997).
Determination of glycosides: To the solution of the extract in glacial
acetic acid, few drops of ferric chloride and concentrated sulphuric acid were
added and observed for the coloration (Siddiqui and Ali, 1997).
Antibacterial assay: The disc diffusion method was used to study the
antibacterial activity (Bauer et al., 1966) of
M. citrifolia extracts. Bacterial isolates viz., Staphylococcus aureus,
Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia
coli, Serratia marcescens, Pseudomonas aeruginosa and Klebsiella
pneumoniae were tested against the extracts. Each bacterial culture was
diluted as 1:100 with fresh sterile nutrient broth and inoculated (2.5x103
CFU mL-1) on sterile Mueller Hinton Agar (MHA) plates by swabbing.
Different concentrations (100, 200, 300, 400 and 500 μL) of extract were
loaded in sterile filter paper disc and placed on the MHA plate. The plates
were incubated at 37°C for 18-24 h and after incubation; the diameter of
the zone of inhibition was measured by using HiMedia antibiotic scale.
Antifungal assay: The disc diffusion method was used to study the antifungal activity by mixing different concentrations (100, 200, 300, 400 and 500 μL) of various parts of plant extracts into potato dextrose agar in triplicates with dimethyl sulfoxide (DMSO) as control. The plates were inoculated with 5 days old fungal cultures viz., Penicillium, Cladosporium, Fusarium, Alternaria and Aspergillus at the center as point inoculation and incubated at 25°C for 5 days. After incubation, the diameter of the colonies were measured for the inhibitory activity of the extracts and compared with control. Complete suppression of growth by a specific concentration was considered as significant.
Statistical analysis: The data were subjected to one way Analysis of
Variance (ANOVA) and differences between samples were determined by Duncans
Multiple Range test using the Statistical Analysis System (SAS,
1999) program. p-values less than 0.05 were considered to be statistically
RESULTS AND DISCUSSION
Information on chemical constituents of plants helps for the discovery of novel
drugs. Qualitative phytochemical investigation revealed that the extracts of
M. citrifolia contained phytoconstituents viz., phenols, alkaloids, flavonoids,
glycosides, tannins, saponins and steroids at various levels (Table
1). These bioactive components are naturally occurring in most plant materials,
known to be bactericidal and fungicidal, thus conferring the antimicrobial property
to plants (Van der Watt and Pretorius, 2001; El-Astal
et al., 2005).
|| Phytochemical analysis of methanol extracts
of M. citrifolia (mg g-1)
|++: Quantitatively present, +: Present, -: Absent
Phenolic compounds have been found to be the major group of functional micronutrients
in noni plant (Morton, 1992; Dixon
et al., 1999; Wang and Su, 2001). The present
study revealed that significant levels of phenolic compounds were present in
leaves and roots followed by stem extracts of M. citrifolia. Phytochemical
analysis of root extract of M. citrifolia as determined by the total
phenol, flavonoids, tannins and steroids were higher than that of the leaf and
On the other hand, the leaf extract of the plant has higher level of total alkaloids and glycosides. Saponins were present in leaf and root extracts and quantitative amount of terpenoids were present in leaf extract. Glycosides were present in all the extracts.
Quercetin, isoflavones, epigallocatechin represents a group of flavonoids and
their antimicrobial properties has been well documented (Cushnie
and Lamb, 2005). Antibacterial and antifungal activity of saponins isolated
from plant materials have been reported by previous studies (Campbell,
1993; Soetan et al., 2006; Barile
et al., 2007). However, saponins have ineffectiveness on Gram negative
bacteria due to poor penetration on cell membranes (Soetan,
The last two decades has witnessed increased investigations on plants as a
source of human disease management (Aiyelaagbe, 2001;
Prashanth et al., 2001) due to the genetic variability
of microorganisms against the antibiotics. The antibacterial activity of M.
citrifolia has been evidenced by many reports and is due to the presence
of phenolic compounds (Atkinson, 1956).
The antibacterial activities of M. citrifolia extracts were tested by
the presence or absence of inhibition zones and zone diameter (Fig.
1). The effect of plant extract was different with different bacterial strains.
Among the different extracts tested, roots extract exhibited significant antibacterial
activity with larger zones of inhibition followed by the leaf extract and the
most susceptible microbes to all extracts were S. epidermidis and P.
aeruginosa. Leaf extract has significant levels of alkaloids which attributed
in control the growth of bacteria. Antibacterial activity of alkaloids particularly
against gram positive bacteria was reported by Karou et
al. (2006). S. epidermidis and P. aeruginosa are known
pathogens of biomaterial infections and their inhibition by the M. citrifolia
root extracts might suggest their possible use in the treatment of nosocomial
|| Antibacterial activity of M. citrifolia extract
|| Fungal growth at different concentration of M. citrifolia
The overall result suggests that S. epidermidis is the most susceptible
strain and the resistant was S. marcescens in the presence of root extract
and S. pyogenes with leaf extract. Sharma and Smita
(2010) reported that, ethyl acetate extract of M. citrifolia exhibited
broad spectrum of inhibition against gram negative bacteria, whereas alcoholic
extract of M. citrifolia has exhibited potent antimicrobial activity
(Kumar et al., 2010). Although, the antibacterial
properties of stem extracts of M. citrifolia are not as effective as
the leaf and root extracts, it still possesses some activity against bacterial
strains used in this study. Apart from Gram positive and Gram negative bacteria,
antibacterial activity of M. citrifolia against mycoplasma and plant
pathogen was revealed by previous studies (Rivera et
al., 2011; Sunder et al., 2011).
All the concentrations of the extract inhibited the fungal species with varying
degree of sensitivity (Fig. 2). The stronger and broader spectrum
of antifungal activity was observed in root extract of M. citrifolia against
Penicillium and Fusarium followed by leaf extract. In higher concentrations,
prominent antifungal activity against was observed which supports the earlier
investigations (Banso and Adeyemo, 2007; Chung
et al., 1993; Bele et al., 2010) that
the tannins isolated from the medicinal plants possess remarkable toxic activity
against bacteria and fungi. However, Aspergillus was able to tolerate
higher concentrations (500 μL) of leaf extract and Curvularia in
the presence of root extract. Weak antifungal activity of stem extract was seen
throughout the study.
In general, the order of antimicrobial activity follows the sequence: root extract>leaf extract>stem extract. Thus, the methanolic root extract of M. citrifolia can be used as the active constituent of antimicrobial agents. Bioactive substances from M. citrifolia plant could be employed in the formulation of antimicrobial agents for the treatment of various bacterial and mycotic infections. The presence of antimicrobial activity in M. citrifolia plant extracts give support to their traditional use for treating conditions associated with microorganisms in humans and consequently seems to fight against multi-resistant microbes.
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