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Phytoconstituents and Their Influence on Antimicrobial Properties of Morinda citrifolia L.



G. Sibi, Parul Chatly, Sayak Adhikari and K.R. Ravikumar
 
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ABSTRACT

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.

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  How 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.

DOI: 10.3923/rjmp.2012.441.448

URL: https://scialert.net/abstract/?doi=rjmp.2012.441.448
 
Received: December 28, 2011; Accepted: February 07, 2012; Published: March 15, 2012



INTRODUCTION

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 world’s 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).

Phytochemical assay
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 Dragendorff’s 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 Duncan’s Multiple Range test using the Statistical Analysis System (SAS, 1999) program. p-values less than 0.05 were considered to be statistically significant.

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).

Table 1: Phytochemical analysis of methanol extracts of M. citrifolia (mg g-1)
Image for - Phytoconstituents and Their Influence on Antimicrobial Properties of Morinda citrifolia L.
++: 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 stem extracts.

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, 2003).

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 infections.

Image for - Phytoconstituents and Their Influence on Antimicrobial Properties of Morinda citrifolia L.
Fig. 1: Antibacterial activity of M. citrifolia extract

Image for - Phytoconstituents and Their Influence on Antimicrobial Properties of Morinda citrifolia L.
Fig. 2: Fungal growth at different concentration of M. citrifolia extract

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.

CONCLUSION

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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