Bioactive triterpenoids , antimicrobial , antioxidant and cytotoxic activities of Eclipta prostrata Linn

Rungrot Cherdtrakulkiat, Somchai Boonpangrak, Ratchanok Pingaew, Supaluk Prachayasittikul, Somsak Ruchirawat, Virapong Prachayasittikul* Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand. Center for Innovation Development and Technology Transfer, Faculty of Medical Technology,Mahidol University, Bangkok 10700, Thailand. Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand. Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand. Laboratory of Medicinal Chemistry, Chulabhorn Research Institute and Program in Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210, Thailand. Center of Excellence on Environmental Health and Toxicology, Commission on Higher Education (CHE), Ministry of Education, Thailand.


INTRODUCTION
Eclipta prostrata Linn.(Compositae), a medicinal plant, has been used for treatment of hyperlipidemia, atherosclerosis, hepatic disorders (Kim et al., 2008, Chokotia et al., 2013), inflammatory conditions, ophthalmic and digestive disorders (Arunachalam et al., 2009) as well as skin diseases.The plant species is well recognized as the best remedy for hair dying and hair growth (Tewtrakul et al., 2007).The extract of E. prostrata was reported to inhibit toxic or lethal action of snake venom (Mors et al., 1989;Melo et al., 1994;Pithayanukul et al., . .recorded on a Bruker AVANCE 300 NMR spectrometer (operating at 300 MHz).Infrared spectra (IR) were obtained on a Perkin Elmer System 2000 FTIR.Column chromatography was carried out using silica gel 60 (0.063-0.200 mm).Analytical thin layer chromatography (TLC) was performed on silica gel 60 PF 254 aluminum sheets (cat. No. 7747 E.,Merck).

Plant material
E. prostrata (aerial parts) were collected from Nakornratsrima province, Thailand.The plant has been identified (BKF 075505) by The Forest Herbarium, Royal Forestry Department, Bangkok.

Extraction
The dried powder of E. prostrata (1.2 kg) was extracted with hexane (4L × 5days × 3) followed by filtration.The filtrates were combined and evaporated in vacuo to give a hexane extract (25 g).Similar extraction was performed using chloroform and ethyl acetate to give the corresponding chloroform (33 g) and ethyl acetate (29 g) extracts, respectively.In this study, chloroform and ethyl acetate extracts were investigated.

Isolation
Isolation of the extracts was performed by a silica gel column chromatography using gradient elution with increasing polarity of the solvents.Fractions were collected and combined based on TLC chromatograms and evaporated to dryness.

Antimicrobial Assay
Antimicrobial activity of the tested compounds was performed using the agar dilution method as previously described (Srisung et al., 2013).The tested compound (10.24 mg) was dissolved in DMSO (0.2 mL) and then added to the Müller Hinton (MH) broth (1.8 mL).A two-fold dilution was prepared, and 1 mL of each diluted compound was transferred to the MH agar (19 mL) to give the initial concentration of 256g/mL.The DMSO (0.5%) as a control, was added to the MH agar.Microorganisms cultured in the MH broth at 37C for 24 h, were diluted with 0.9% normal saline solution to adjust the cell density of 1.5×10 8 CFU/mL.The microorganisms were inoculated onto each plate and further incubated at 37 o C for 18-48 h.Compounds which exerted high efficacy to inhibit cell growth of the microorganisms were determined.Twenty-eight strains of tested microorganisms were gram negative bacteria:

Antioxidant assay
The compounds were tested for antioxidant property using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.The DPPH (a stable purple color radical) reacts with an antioxidant to form a light-yellow colored of diphenylpicrylhydrazine, the reduced product that can be spectrophotometrically detected.The assay (Prachayasittikul et al., 2012) was initiated by adding 1 mL solution of DPPH in methanol (0.1 mM) to a sample solution (0.45 mL, 1 mg/mL dissolved in DMSO).The reaction mixture was incubated for 30 min in a dark 100 . .

Abs
Abs room.The absorbance at 517 nm was measured using UV-visible spectrophotometer (UV-1610, Shimadzu),and the percentage of radical scavenging activity (RSA) was calculated from the following equation: where Abs.control is the absorbance of the control reaction and Abs.sample is the absorbance of the tested compound.

Cytotoxic assay
Cytotoxic assay was performed using the modified method as previously described (Prachayasittikul et al., 2008).Briefly, the confluent cell monolayers were trypsinized and diluted with appropiate culture medium to a final concentration of 3×10 5 cells/mL.Portions (100 µL) containing approximately 3×10 4 cells were added into 96-well flat-bottomed tissue plates and incubated overnight at 37C in a humidified 5% CO 2 incubator.Solution (100 µL) containing various concentrations of tested compounds, positive control (etoposide) or negative control (DMSO) were added to each well and the plates were incubaed for an additional 48 h.After the incubation, each well was washed three times with phosphate-buffered saline (pH7.2) and then stained with crystal violet.The excess dye was removed, the stained cells were lysed with 100 mM HCl (100 µL) in absolute methanol and the optical density was determined at 540 nm by a microtitre plate reader (Titertek, Multiskan MCC/340).All assays were carried out in quadruplicate and the mean values were calculated.The cytotoxic activity was expressed as an ED 50 (the effective dose that inhibits 50% of cell growth).

Isolation
The extracts (chloroform and ethyl acetate) of E. prostrata were isolated by the column chromatography.Isolated fractions (C1-C6) of the chloroform extract were mainly found to be a mixture of triterpenoids and long chain hydrocarbons together with trace amount of aromatic components as seen by 1 H NMR and IR spectra (data not shown).Repeated chromatographic separation or purification was performed on some selected fractions in which the fraction C6 was further isolated to provide eight fractions (C6.1-C6.8) of yellow solids.Fraction C6.4 was purified by recrystallization to give 3acetylaleuritolic acid (1).Purification of fraction C6.6 gave stigmasteriol (2), chemical structures are shown in Figure 1.Additionally, compound 1 was also found in fraction E2.2 of the ethyl acetate extract.Their structures were confirmed by comparison of 1 H NMR and IR spectra with the authentic samples.

Antimicrobial activity
The plant extracts (chloroform and ethyl acetate) and isolated fractions (C2-C7) were evaluated for their antimicrobial potency against twenty-eight strains of microorganisms using the agar dilution method.Results (Table 1) showed that the bioactive fractions (C3, C4, C6, and C7) exhibited inhibiroty activity against both gram positive and gram negative bacteria with their minimum inhibitory concentrations (MIC) of 64 g/mL.The chloroform and ethyl acetate extracts displayed activity against S. cerevisiae ATCC 2601 with the MIC value of 256 g/mL.Frations C2 and C5 were found to be inactive antimicrobials.However, the DMSO (0.5%) was tested in parallel with the compounds and showed no effect on the tested microorganisms.

Antioxidant activity
Radical scavenging activity of the plant extracts and fractions (C2-C7) was carried out using the DPPH assay.It was found that (Table 2) ethyl acetate extract displayed the highest RSA (IC 50 151.7 g/mL) as compared to fractions C4, C5 and C6 with IC 50 range of 223.9 -467.7 mg/mL.However, chloroform extract and fractions C2,C3 and C7 were shown to be inactive antioxidants.

Cytotoxic activity
Cytotoxic assay was performed toward HuCCA-1 and KB cells.The chloroform and ethyl acetate extracts of E. prostrata showed inhibitory effect on the two tested cells with ED 50 > 100 g/mL.
In addition, C3 and C4 also displayed antimicrobial activity against C. diphtheriae NCTC 10356; and fraction C6 exhibited activity against S. pyogenes with the same MIC (64 g/mL) value.Previously, ethyl acetate extract of E. alba (E.prostrata) was reported to exhibit antimicrobial activity against many strains of microorganisms with MICs of 1.56-25.00mg/mL (Borkataky et al., 2013).These investigated microorganisms i.e., M. catarrhalis, S. pyogenes and C. diphtheriae are pathogenic bacteria causing various kinds of diseases.M. catarrhalis has been found to be a true pathogen in respiratory tract of human, and is the most common bacteria that causes ototis media in children after Haemophilus influenzae and Streptococcus pneumoniae.However, it can cause chronic obstructive pulmonary disease in adults (De Vries et al., 2009;Buskirk et al., 2014).C. diphtheriae is a causative agent of Diphtheria which can be spread from human to human.Although this disease can cause high fatality rate, however, DPT vaccine has been generated for a successful protection.Nevertheless, this disease is not disappear because of the unvaccinated people in some developing countries (Adler et al., 2013;Meera et al., 2014).S. pyogenes is the most common causative agent of bacterial pharyngitis.However, this bacteria can cause a wide range of disease such as scarlet fever, impetigo, necrotizing fasciitis, streptococcal toxic shock syndrome and also cause the autoimmune diseases, acute poststreptococcal glomerulonephritis as well as acute rheumatic heart disease (Morefield et al., 2014;Walker et al., 2014).The results could suggest the potential use of E. prostrata as traditional medicine for these infectious diseases.
Antioxidant activity (DPPH assay) of E. prostrata was noted for the ethyl acetate extract while the other tested samples were found to be slightly active to inactive antioxidants.In our previous study, the hexane extract showed relatively weak antioxidant effect (Prachayasittikul et al., 2010).It was reported that the whole plant extract (ethyl acetate) of E. prostrata diplayed antioxidant activity (ferric reducing ability) (Chauhan et al., 2012) Both of the aerial plant extracts (chloroform and ethyl acetate) exerted cytotoxic activity against the HuCCA-1 and KB cells with ED 50 values > 100 g/mL.Previously, the hexane extract (aerial part) of the plant species was reported to show cytotoxic activity (ED 50 = 100 g/mL) against the two human cancer cell lines (Prachayasittikul et al., 2010).The whole plant extract (ethyl acetate) of E. alba was documented to exert moderate cytotoxic activity against human lung epithelial adenocarcinoma cell line (HCC-827) (Chauhan et al., 2012).Regarding biological activities, compound 1 isolated from other plant species was reported to exhibit antibacterial activity against S. aureus and S. typhimurium (Peres et al., 1997) , and to show significant inhibition on vitality of adult male worm of Onchocerca gutturosa (Nyasse et al., 2006).Furthermore, 3-acetylaleuritolic acid (1) displayed strong inhibition of DNA topoisomerase II, and strong cytotoxic activity against human lung carcinoma A549 cells (Wada et al., 2006).Compound 2 was previously reported to exert antioxidant activity as determined by the thiocyanate method (Hung et al., 2001) and by the lipid antioxidant property (Ramadan et al., 2007).In addition, stigmasterol (2) significantly suppressed HMG-CoA reductase activity (11% reduction) in plasma cholesterol levels in Wistar and WKY rats feeding 0.5% of compound 2 (Batta et al., 2006).A mixture of -sitosterol and stigmasterol exhibited antimicrobial activity against S. cerevisiae ATCC 2601 with MIC of 64 g/mL (Prachayasittikul et al., 2009).
In conclusion, the E. prostrata exerts antimicrobial and antioxidant activities, and constitutes bioactive triterpenoids (1 and 2) with diverse biological effects.The results demonstrate benificial effects of the plant species as antimicrobials and bioactive compounds for medicinal uses.

CONFLICT OF INTEREST STATEMENT
We declare that we have no conflict of interest.

Table 1 :
Antimicrobial activity (MIC) of E. prostrata a Ampicillin at 10 g/mL was used as a control of the antimicrobial testing system; it showed 100% inhibition againstS.aureus ATCC 25923, B. subtilis ATCC 6633, S. epidermidis ATCC 12228, S. pyogenes, E. tarda, N. mucosa and M. catarrhalis.b MIC: Minimum inhibitory concentration was the lowest concentration that inhibited the growth of microorganisms.c At 64 g/mL, C3 showed 75% inhibition against N. mucosa.d At 128 g/mL, C4 showed 75% inhibition against S. pyogenes.