Abstract: The crude extracts of Trianthema decandra L. (Aizoaceae) were studied for the antimicrobial and antioxidant potential. The antimicrobial property of the Trianthema decandra was studied against ten bacterial and two fungal strains using the disc diffusion method and minimum inhibitory concentration were determined for each strain, in which chloroform extract has shown bigger zone of inhibition (18.5 to 23.2 mm) at a concentration level of 1.5 mg disc-1 and MIC at 39 μg mL-1. Control studies with Chloramphenicol and Nystatin 30 μg disc-1 were used as standards for bacteria and fungus, respectively. The anti-oxidant activity was evaluated by DPPH free radical scavenging activity using spectrophotometric method and it was compared with the positive control as Butylated hydroxyl anisole. The total phenolic content was also determined. Total phenolic content was expressed as GAE (gallic acid equivalents) in which, chloroform extract has shown greater amount of GAE. The phytochemical analysis of the extract shows the presence of terpenoids, flavanoids, steroids and saponins.
INTRODUCTION
Emergence of new and resistant fungal pathogens, increasing incidence in immunocompromised patients and environmental and health problems of chemical fungicides dictate the search for novel antifungal agents for human diseases and plant protection (Sharma and Kumar, 2009). Consistency of biologic activity is essential requirements for the safe and effective use of therapeutic agents (Menghani et al., 2010).
Despite the existence of potent antibiotics, resistant or multiresistant strains are continuously appearing, imposing the need for a permanent search and development of new drugs. For Centuries plants have been used throughout the world as drugs and remedies for various diseases. These drugs serve as prototype to develop more effective and less toxic medicines (Sharma et al., 2009).
In developing countries and particularly in Colombia, low income people such as farmers, people of small isolate villages and native communities use folk medicine for the treatment of common infections. These plants are ingested as decoctions, teas and juice preparations to treat respiratory infections (Gonzalez, 1980). They are also made into a poultice and applied directly on the infected wounds or burns.
One way to prevent antibiotic resistance of pathogenic species is by using new compounds that are not based on existing synthetic antimicrobial agents (Shah, 2005). It is necessary to evaluate, in a scientific base, the potential use of folk medicine for the treatment of infectious diseases produced by common pathogens. Medicinal plants might represent an alternative treatment in non-severe cases of infectious diseases. They can also be a possible source for new potent antibiotics to which pathogen strains are not resistant (Fabricant and Farnsworth, 2001).
Trianthema decandra is a prostrate, glabrous, succulent and annual found almost throughout India as a weed in cultivated and waste land. The plant belongs to the family Aizoaceae. This family is well marked in their characteristics and cannot be confused with any other. The genus Trianthema consists of 20 species but only a few species have been phytochemically reported. Trianthema is a genus of annual or perennial plant characterized by usual fleshy, opposite, unequal, smooth-margined leaves; prostrate growth form; flowers with five perianth segments; flowers subtended by a pair of bracts; superior fruit a circumscissile capsule with a winged lid and stamens 5 or 10. It is commonly known as gadabani (Hindi) and vellai sharuni (Tamil) (Kirtikar and Basu, 1983).
Trianthema decandra has been used in various parts of Asia, Africa, Australia and South America for curing various diseases. In some African countries the plant has been popular use for skin diseases, wound healing, fever and tooth aches. In India it is used in the treatment of ophthalmic (Upadhay et al., 1998). The root applied to the eye cures cornesl ulcers, itching, dimness of sight and night blindness. The juice of leaves is used to treat the black quarter. The bitter roots are used for curing bacterial infections and its also given in combination with ginger as a cathartic. The leaves contains huge amount of vitamin C which is used to treat edema. The decoction of the herb is used as a vermifuge and is useful in rheumatitis. It is also an antidote to alcoholic poison.
Thus, the present investigation was carried out to evaluate the antimicrobial and antioxidant potential of Trianthema decandra.
MATERIALS AND METHODS
Plant Materials
The plant Trianthema decandra was collected from the Salem District,
Tamil Nadu, India during June 2008. The plant was taxonomically identified and
authenticated by the Botanical survey of India, Coimbatore (Tamil Nadu) and
voucher specimen was deposited in our laboratory for future reference.
Source of Microorganisms
To study the antimicrobial activity of various extracts of Trianthema
decandra, the strains of bacteria, yeast and fungi were collected from Microbiology
Laboratory at PSG Hospitals, Coimbatore-641 004. The selected microorganisms
included bacteria such as Staphylococcus aureus, Streptococcus faecalis,
Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia,
Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae,
Proteus vulgaris, Bacillus subtilis, Yersinia enterocolitica
and fungi such as Candida albicans and Cryptococcus neoformans.
They were clinical isolates from different pathologic medium from patients diagnosed
as having various infections at the Microbiology Laboratory of PSG Hospitals,
Coimbatore-641 004.
Extraction Procedure and Phytochemical Analysis
Around 200 g of the whole plant was dried in shade, pulverized by a mechanical
grinder and passed through a 40-mesh sieve to get the fine powder and stored
in an airtight container. The dried powder (25 g) was extracted with petroleum
ether (60-80°C), remove wax and then extracted three times with chloroform
(CHCl3), ethyl acetate (EtoAc), 100% ethanol (EtoH), 70% ethanol(EtoH)
and water sequentially with soxhlet apparatus. The solvents from various extracts
were then concentrated in rotavapour at reduced pressure below 40°C.
Phytochemical Analysis
The presence of phytochemicals, alkaloids (Draggendorff’s), flavanoids
(Shibata’s reaction), saponins (Frothing test), tannins (5% ferric chloride),
terpenoides (2,4 dinitrophenyl hydrazine), steroids (Libermann’s Burchard
test) and phenols were evaluated according to the methods described by Edeoga
et al. (2005).
Pharmacological Evaluation
Antioxidant Activity
Total Phenolic Content Estimation (TPC)
The total phenolics in extracts were determined according to Folin-Ciocalteu
procedure by Singleton and Rossi (1965). Four hundred
microlitres of sample (two replicates) were taken in test tubes; 1.0 mL of Folin-Ciocalteu
reagent (diluted 10 fold with distilled water) and 0.8 mL of 7.5% sodium carbonate
were added. The tubes were mixed and allowed to stand for 30 min and the absorption
at 765 nm was measured against a blank, which contained 400 μL of ethanol
in place of sample. The total phenolic content was expressed as gallic acid
equivalents in mg g-1 of chloroform extract.
DPPH Free Radical Scavenging Activity
The antioxidant activity of the plant extract was estimated using a slight
modification of the DPPH radical scavenging protocol reported by Chen
et al. (1999). For a typical reaction, 2 mL of 100 μM DPPH solution
in ethanol was mixed with 2 mL of 100 μg mL-1 of plant extract.
The effective test concentrations of DPPH and the extract were therefore, 50
μM and 50 μg mL-1, respectively. The reaction mixture was
incubated in the dark for 15 min and thereafter the optical density was recorded
at 517 nm against the blank. For the control, 2 mL of DPPH solution in ethanol
was mixed with 2 mL of ethanol and the optical density of the solution was recorded
after 15 min. The assay was carried out in triplicate. The decrease in optical
density of DPPH on addition of test samples in relation to the control was used
to calculate the antioxidant activity, as percentage inhibition (%IP) of DPPH
radical.
Antimicrobial Activity Assay
Disc Diffusion Test
The in vitro antimicrobial activity of the sample solution was studied
by Disc diffusion method. Plates were prepared by pouring 20 mL of sterile nutrient
agar (Himedia) into sterile petridishes and were inoculated with a loopful broth
culture of each organism. Sterile Whatmann filter paper discs (6 mm diameter)
impregnated with 20 μL (1.5 mg disc-1) quantity of dimethyl
sulfoxide solution of various extracts were air dried and placed on the agar
plates. The plates were incubated at 37°C for 24 h. Control studies with
Chloramphenicol and Nystatin 30 μg were used as standards for bacteria
and fungus. The solvent DMSO control were done concurrently (Umadevi
et al., 2003). Growth inhibition of each microbial strain was calculated
as the percentage of inhibition of radial growth relative to the control. The
plates were used in triplicate for each treatment. The relative growth inhibition
of treatment compared to control was calculated by percentage, using the following
formula:
Minimal Inhibitory Concentration Determination (MIC)
Minimum inhibition concentration assay was performed in nutrient broth containing
0.05% phenol red and supplemented with 10% glucose (NBGP), method described
by Zgoda and Porter (2001). All the test extracts including
standard drugs were initially dissolved with in DMSO and the solution obtained
was added to NBGP to a final concentration of 5000 μg mL-1 for
each crude extract. This was serially diluted by twofold, to obtain concentration
ranging from 5000 μg to 1.22 μg mL-1. One hundred microlitres
of each concentration was added to a well (96-well micro plate) containing 95
μL of NBGP and 5μL of standard inoculums, the appropriate inoculum
size for standard MIC is 2x104 to 105 cfu mL-1.
The final concentration of DMSO in the well was less than 1%.The negative control
well consisted of 195 μL of NBGP and 5 μL of the standard inoculum.
The plates were covered with a sterile plate sealer, then agitated to mix the
content of the wells using a plate shaker and incubated at 37°C for 24 h.
The assay was repeated twice and microbial growth was determined by observing
the change of colour in the wells (red when there is no growth and yellow when
there is growth). The lowest concentration showing no colour change in the well
was considered as the MIC.
RESULTS
Extraction Yield and Phytochemical Estimation
Around 25 g of whole plant yielded in petroleum ether (2.56%), chloroform
(1.44%), ethyl acetate (1.36%), 100% ethanol (0.16%), 70% ethanol (21.08%) and
water (3.24%).
In the TLC of chloroform extract of Trianthema decandra gave 8 spots at the following Rf values: 0.12,0.14,0.26,0.43,0.59,0.75,0.89 and 0.96, respectively, in the solvent system of petroleum ether: ethyl acetate (75:25).
The phytochemical analysis of chloroform extract has shown the presence of flavanoids, terpenoids, steroids and absence of tannins, alkaloids, glycosides. The phytochemical analysis of water and ethanol extract has shown the presence of saponins and terpenoids, respectively.
Antioxidant Activity
Total Phenolic Content
Total phenolic content was expressed as GAE (gallic acid equivalents) in
which, chloroform extract has shown greater amount of GAE. Total phenolic contents
of chloroform extract were 74.6±1.26 mg g-1 weight of dry
extract. Values are Mean±SD of six individual experiments.
Antioxidant Activity (DPPH Free Radical Scavenging)
The control and test samples were compared for the determination of percentage
of inhibition. The petroleum ether extract, chloroform extract, ethyl acetate
extract, ethanol extract, 70% ethanol extract, water extract and butylated hydroxyl
anisole have shown 2.99±0.08, 15.34±0.05, 2.73±0.1, 46.94±0.05,
13.52±0.08, 33.55±0.13 and 85.32±0.24, respectively.
Antimicrobial Activity
Disc Diffusion Test
The chloroform and ethyl acetate extracts from the Trianthema decandra
has shown inhibition effects on the growth of all the organisms tested, but
their efficiency in inhibition was varied from one organism to another. In almost
all, the tested organisms growth was inhibited by the chloroform and ethyl acetate
extracts. Chloroform extract has shown higher range of inhibition diameter (IDZ)
from 18.5 to 23.2 mm, whereas ethylacetate extract has shown inhibition range
of 16.5 to 23.4 mm. Chloramphenicol and Nystatin have shown IZD ranged from
18±0.08 to 23.6±0.12 mm at a concentration of 30 μg disc-1.
All IZD corresponding to test organisms are shown in Table 1.
Minimal Inhibitory Concentration Determination (MIC)
The chloroform and ethyl acetate extracts of Trianthema decandra were
tested at different concentrations for antimicrobial activity, the extent of
their inhibitory activities against the test organisms could be well understood
only by comparing the MICs values obtained. The results indicated that Candida
albicans (MIC: 39 μg mL-1) is most sensitive to chloroform
extract and Streptococcus faecalis (MIC: 312.5 μg mL-1)
and Escherichia coli (MIC: 156.2 μg mL-1). The MIC values
for Chloramphenicol and Nystatin were ranged from 3.13 to 62.5 μg mL-1.
All MIC corresponding to tested organisms are shown in Table 2.
| Table 1: | Antimicrobial activity of various extracts of Trianthema decandra.on different microbes and their corresponding IZD |
| Values are Mean±SD, n = 6. -: Indicates no inhibition
PE: Petroleum ether extract, EtoH: Ethanol extract, EA:Ethyl acetate extract, -ve control: DMSO control, +ve control: Chloramphenicol/ystatin, CHCL3: Chloroform extract |
|
| Table 2: | Antimicrobial activity of various extracts of Trianthema decandra.on different microbes and their corresponding MIC |
| Standard: Chloramphenicol/Nystatin | |
DISCUSSION
Medicinal plants are an important target of patent claims since they have proved to be of great interest to the International drug and cosmetic industry. Several plants have been identified for their antimicrobial properties. The investigation of the herbal extracts and herbal mixtures for their antimicrobial properties is still in its infancy. Diet and physical exercise are the two important factors linked to free radical generation and the antioxidants (Souri et al., 2004). In general, there are two major strategies which convey partial resistance against oxidative stress to most cell types: small antioxidant molecules like ascorbate, polyunsaturated fatty acids or sugars (mainly mannitol) and ROS-scavenging enzymes, such as superoxide dismutase (SOD), catalase and various peroxidases (Steiling et al., 1999). The role of free radicals reactions in biology has become an area of intense interest. It is generally accepted that free radicals play an important role in the development of tissue damage and pathological events in the living organisms (Velazquez et al., 2003). Hence, if a compound having antioxidant potentials and antimicrobial activity additionally; it can be a good therapeutic agent for accelerating then wound healing process. Hence the present study was carried out to analyse the antimicrobial and antioxidant activity of Trianthema decandra.
Results obtained in this study conform the antimicrobial activity of Trianthema decandra, which also possesses antioxidant activity. Antioxidant activity was assessed by DPPH scavenging method where ethanolic extract was found to be most potent antioxidant, for which values are 46.94±0.05 as inhibition percentage. The higher phenolic content of chloroform extract (TPC: 74.6±1.26 mg g-1) might have contributed to higher antioxidant activity of ethanolic extract of Trianthema decandra. Candida sp. cause 6.2% of human infections, ranking as the 4th most prevalent infectious agent (Sanasam and Ningthoujam, 2010). In the preliminary screening of antimicrobial activity, using disc diffusion method, chloroform extract has shown higher zone of inhibition which ranged from 18.5 to 23.2 mm. The same is reproduced in the MIC where as the lowest MIC obtained for chloroform extract is 39 μg mL-1.
CONCLUSION
It would also be interesting to study the effects of leaf extracts of Trianthema decandra on medically important microbes for development of new antimicrobial agents for preventive treatment of serious microbial disease infections in animals and human beings along with plant fungal diseases. In this regard, we have started a program aimed at the evaluation of antimicrobial activity of various leaf extracts of Trianthema decandra, in hope to find out new natural products to be used in the bioactive compounds of certain important drug for human. In conclusion, Trianthema decandra was taken for further study to isolate the novel antimicrobial compounds. However, the components responsible for the antimicrobial activities of its extracts are currently unclear, further works should be performed on the isolation and identification of the components in the extracts, especially in the chloroform, ethyl acetate and ethanol extract.
ACKNOWLEDGMENTS
The authors are very grateful to SRM University, Tamil Nadu, India for granting permission, financial support and executing the work at Department of Biotechnology, School of Bioengineering. Our sincere thanks to Dr. P. Manivasakam, USA for providing the guidance, encouragement and valuable suggestions during the various stages in research work.