Antimicrobial , antioxidant and cytotoxicity potential of Manihot multifida ( L . ) Crantz ( Euphorbiaceae )

Manihot multifida (L.) Crantz (Euphorbiaceae) is widely used in popular medicine for the treatment of infected wounds. This study evaluated the in vitro antioxidant and antimicrobial potential of this species against strains of Gram-positive and Gram-negative bacteria and fungi, known to cause infections in humans. The extracts showed minimal inhibitory concentration (MIC) varying from 39 to 2500 μg/mL for antimicrobial activity. The methanolic extract of fruits, aqueous and hexane extracts of leaves showed a very strong activity against Candida albicans (ATCC 18804) with MIC of 39 μg/mL. Furthermore, the methanolic extract of M. multifida leaves exhibited DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging potential with inhibitory concentration (IC50) values of 46.9 μg/mL, followed by hexane extract of leaves with IC50 values of 59.2 μg/mL. The cytotoxic activity against brine shrimp was stronger for the methanolic extract of leaves (lethal concentration LC50 of 15.6 μg/mL). These results suggest that M. multifida has interesting antimicrobial and antioxidant activities. Moreover, these results corroborate the popular use of this specie in treating fungal infections since it demonstrates significant activity against C. albicans.


INTRODUCTION
Manihot multifida (L.) Crantz (Euphorbiaceae) (= Jatropha multifida L.) is commonly used in Brazilian folk medicine to treat wounds and gastrointestinal ulcers (Buch et al. 2008).In Asia and Africa it is used for the treatment of infected wounds, skin infections and scabies (Kosasi et al. 1989).Martins et al. (2005) developed a study with eight plant species, including M. multifida and found antiseptic and antifungal activity for this species.Another study developed by Aiyelaagbe et al. (2008) in Nigeria, with methanol and hexane extracts of leaves, roots and stems of M. multifida also showed the antimicrobial activity of this species, and for some micro-organisms this effect was even greater than the control drug.The methanol and ethyl acetate extracts of stem wood and bark showed higher activity against Proteus mirabilis than gentamicin, and among the extracts, stem and root methanolic extracts showed antimicrobial activity against Pseudomonas aeruginosa resistant to gentamicin.Hamza et al. (2006) verified the accuracy of the biological activities of these species according to ethnopharmacology and concluded that the methanolic extracts of leaves, stems and roots from M. multifida have strong activity against Candida sp. and Cryptococcus neoformans.
In order to validate the popular use of Manihot multifida (L.), this study examined the in vitro antioxidant and antimicrobial potential of this species against strains of Gram-positive and Gram-negative bacteria and fungi, known to be causing infections in humans.A preliminary phytochemical screening fir the identification of the major components of the special metabolism was also conducted and cytotoxicity was determined by brine shrimp lethality bioassay.

PLANT MATERIAL
The leaves and fruits were collected in Juiz de Fora, Minas Gerais, Brazil, in June of 2009 (23°35ʼ45ˮS 46°43ʼ45ˮW).Voucher specimens were deposited in the Herbarium Leopoldo Krieger (CESJ) of the Federal University of Juiz de Fora (number 54865).

PREPARATION OF THE EXTRACT
The leaves (136.6g) were powdered and separated into two fractions.One of them (66.1g) was macerated with hexane and methanol, successively (3 x 200mL) for five days at room temperature.After evaporation of the solvent under reduced pressure at 45°C, the respective hexane (6.5g) and methanolic (15.5g) extracts were obtained.The other fraction (70.5g) was submitted to infusion and subsequent lyophilization to obtain the aqueous extract (23.8g).
The fruits (47.8g) were powdered and macerated with hexane and methanol, successively (3 x 200mL) for five days at room temperature.
After evaporation of the solvent under reduced pressure at 45°C, the respective hexane (6.2g) and methanolic (3.4g) extracts were obtained.
All the extracts were kept in tightly stoppered bottles under refrigeration until used for the biological testing and phytochemical screening.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) ANALYSIS
These analyses were performed on a Shimadzu LC-10 Instrument, two LC-10 AT pumps, a Rheodyne 7725 injector with 20µl loop and a photodiode array detector SPD-M10A.The column employed was a Zorbax SB-18; 250 x 4.6mm, 5μm particle size.A linear gradient of a binary solvent system, A:B, which varied from 0 to 100% B was run at a flow rate of 1 mL/min over sixty minutes where A consisted of acetonitrile: H 2 O, 5:95, with 0.05% TFA and B consisted of acetonitrile: H 2 O, 65:35, with 0.05% TFA.The mobile phase was returned to its original composition over the course of 60 min, and an additional 10 min were allowed for the column to re-equilibrate before injection of the next sample.The sample volume was 20μl at a concentration of 1mg/mL and the temperature was maintained at 25°C during the analysis.Detection was performed simultaneously at 220, 270, 335 and 360nm.

GAS CHROMATOGRAPHY / MASS SPECTROMETRY (GC/MS)
This analysis was carried out using a Hewlett Packard 6890 gas chromatograph equipped with a fused silica capillary column (HP-5, 30m × 0.25mm, 0.25μm film thickness), helium as carrier gas with a flow rate 1.0mL/min; temperature programming from 70°C to 290°C (2°C/min), coupled to a Hewlett-Packard 5972 mass spectrometer.The MS operating parameters were: 70eV, ion source 250°C equipped with EI.The compound identification was carried out by comparison of their retention indices (RI) with literature values; and the MS data with those from Wiley 275.1 mass spectral data base.

DPPH RADICAL SCAVENGING
The free radical scavenging activity of sample solutions in methanol was determined based on their ability to react with stable 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical (Govidarajan et al. 2003).The plant samples at various concentrations (7.8 to 250 μg/mL) were added to a 152 μM solution of DPPH in methanol.After incubation at 37°C for 30 min, the absorbance of each solution was determined at 517 nm.The antioxidant activity of the samples was expressed as IC 50 (inhibitory concentration), which was defined as the concentration (in µg/mL) of sample required to inhibit the formation of DPPH radicals by 50%.As positive control, rutin was used.

CYTOTOXICITY ASSAY
Brine shrimp lethality bioassay (Meyer et al. 1982) was carried out to investigate the cytotoxicity of the extracts.Brine shrimp (Artemia salina Leach) eggs were hatched in a beaker filled with seawater under constant aeration.After 48h the nauplii were collected by pipette and were counted macroscopically in the stem of the pipette against a lighted background.Solutions of the extracts were made in seawater containing 1% DMSO, at varying concentrations (10 to 1000μg/mL) and incubated in triplicate vials with 10 brine shrimp larvae.After 24h of incubation, the nauplii were examined against a lighted background, with a magnifying glass and the number of survivors in each vial was counted and noted.Both positive (thymol) and negative (sea water containing 1% DMSO) control assays were carried out in order to verify the susceptibility of A. salina under assay conditions employed.LC 50 was determined from 24h counts.The general toxicity activity was considered weak when LC 50 was above 250 µg/mL.

MICROBIAL STRAINS
The samples were evaluated against a panel of micro-organisms, including the bacterial strains The minimal inhibitory concentration (MIC) of each extract was determined by using the broth microdilution techniques as described by the National Committee for Clinical Laboratory Standards (NCCLS/CLSI 2002).MIC values were determined in RPMI 1640 buffered to pH 7.0 with MOPS for yeast and in Mueller Hinton broth (MHB) for bacteria.C. albicans was cultured at 30°C for 48h in SDA, and bacteria were cultured at 37°C for 24h in MHA.Sample stock solutions were diluted from 5000 to 2.5µg/ mL (final volume = 80µl) with a final DMSO concentration 1%.Then, RPMI or MHB (100 µl) was added to microplates.Lastly, 20µl of 10 6 CFU/mL of standardized yeast and bacterial suspensions were placed in microplates and the test was performed in a volume of 200µl.Plates were incubated at 30°C for 48h for yeasts and at 37°C for 24h for bacteria.The same tests were performed simultaneously for growth control (RPMI + yeast and MHB + bacteria) and sterility control (RPMI or MHB + extract).As positive control, amphotericin B and chloramphenicol, were used.The MIC values were calculated as the highest dilution showing complete inhibition of the tested strain.

QUANTITATIVE EVALUATION OF ANTIMICROBIAL ACTIVITY
The antimicrobial activity of plant extracts may be expressed in different ways based on technique used.The micro-dilution method yields MIC values, the minimum concentration at which inhibition is observed (µg/mL).In this work, methods other than numerical values were used to express antimicrobial efficiency.Beside results being recorded in terms of MIC (mg/mL), total activity values were employed, as well as percent activity values.These demonstrate the total antimicrobial potency of particular extracts and the microbial susceptibility index (MSI), which is used to compare the relative susceptibility among the microbial strains (Eloff 2004):

Total number of tested microial strains
The percent activity demonstrates the total antimicrobial potency of particular extracts.It shows the number of microbes found susceptible to one particular extract.MSI = 100 x number of extracts effectve against each microbial strain of total samples MSI is used to compare the relative susceptibility among the microbial strains.MSI values ranges from '0' (resistant to all samples) to '100' (susceptible to all samples).

STATISTICAL ANALYSIS
The IC 50 for antioxidant activity and the LC 50 for cytotoxicity activity were calculated using Grafit 5 software and Probit analysis, respectively.Statistical differences between the treatments and the control were evaluated by ANOVA test.
GC/MS analyses for the hexanic leaves extract presented a major signal (54.53%) at 47.67 min, which mass spectrum (m/z 424) suggested that the compound is a pentacyclic triterpenoid with a lupane basic type.This compound was identified by comparison of the MS data with those from Wiley 275.1 mass spectral data base.

ACTIVITY
The methanolic extract of M. multifida leaves exhibited the lowest DPPH scavenging potential with IC 50 values of 46.9μg/mL, followed by the hexane extract of leaves with IC 50 values of 59.2μg/ mL (Table II), indicating that these fractions have a good potential as free radical scavengers.The control rutin showed IC 50 values of 1.4μg/mL.Polyphenols, particularly flavonoids, which were found in all extracts of M. multifida, and are also widely distributed in the plant kingdom and present in considerable amounts in fruits, vegetables, spices, medicinal herbs and beverages, have been used to treat many human diseases, such as diabetes, cancers and coronary heart diseases (Broadhurst et al. 2000).
Moreover, flavonoids have been shown to exhibit antioxidative, antiviral, antimicrobial and antiplalet activities (Middleton and Kandaswami 1992).The biological activities of these polyphenols in different systems are believed to be due their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Osawa 1994).

CYTOTOXICITY
The methanolic extract of leaves exhibited significant cytotoxicity activity against Artemia salina (LC 50 15.6µg/mL),followed by hexane and methanol extracts of fruits (LC 50 34.1 and 89.8µg/mL, respectively) (Table II) as LC 50 values < 250µg/mL are considered significant for crude extracts (Rieser et al. 1996).The brine shrimp lethality assay is based on the ability to kill laboratory-cultured Artemia salina nauplii and is considered to be one of the most useful tools for the preliminary assessment of general toxicity (McLauglin et al. 1991).
McLaughlin et al. ( 1998) correlated the toxicity against A. salina with the toxicity against human solid tumor cell lines.Those results suggest that this method can be employed as a preliminary analysis of cytotoxicity of novel substances.The diterpenoid multifidone isolated from M. multifida showed significant decrease in cell viability in four different cancer cell lines (Das et al. 2009).

ANTIMICROBIAL ACTIVITY
The MIC values obtained from this study for all samples tested ranged from 2500 to 39µg/mL.Aligiannis et al. (2001) proposed a classification for plant materials, based on MIC results as follows: strong inhibitors -MIC up to 500µg/mL; moderate inhibitors -MIC between 600 and 1500µg/mL; weak inhibitors -MIC above 1600µg/mL.The methanolic extract of fruits, aqueous and hexane extracts of the leaves showed very strong activities (MICof 39 µg/mL).The antimicrobial activity for the samples is presented in Table III.
Total activity values (Table III) revealed that the aqueous extract of M. multifida was the most active against C. albicans (ATCC 18804) as its antifungal component can be diluted in 8666.7mL of solvent

Figure 1 -
Figure 1 -The HPLC profiles of the extracts of M. multifida.(A) methanolic leaves extract; (B) aqueous leaves extract and (C) methanolic fruits extract.Column Zorbax SB-C18; linear gradient of a binary solvent system, A:B, which varied from 0 to 100% B was run at a flow rate of 1 ml/min over sixty minutes where A consists of acetonitrile: H 2 O, 5:95, with 0.05% TFA and B consists of acetonitrile: H 2 O, 65:35, with 0.05% TFA.The mobile phase was returned to its original composition over the course of 60 minutes.The sample volume was 20μl at a concentration of 1mg/ml and the temperature was maintained at 25°C during the analysis.Detection was performed at 220nm.
. All samples presented phenols and flavonoids.