Bioactive Metabolites from Spilanthes acmella Murr.

Spilanthes acmella Murr. (Compositae) has been used as a traditional medicine for toothache, rheumatism and fever. Its extracts had been shown to exhibit vasorelaxant and antioxidant activities. Herein, its antimicrobial, antioxidant and cytotoxic activities were evaluated. Agar dilution method assays against 27 strains of microorganisms were performed. Results showed that fractions from the chloroform and methanol extracts inhibited the growth of many tested organisms, e.g. Corynebacterium diphtheriae NCTC 10356 with minimum inhibitory concentration (MIC) of 64-256 μg/mL and Bacillus subtilis ATCC 6633 with MIC of 128-256 μg/mL. The tested fractions all exhibited antioxidant properties in both DPPH and SOD assays. Potent radical scavenging activity was observed in the DPPH assay. No cytotoxic effects of the extracts against KB and HuCCA-1 cell lines were evident. Bioassay-guided isolation resulted in a diverse group of bioactive compounds such as phenolics [vanillic acid (2), trans-ferulic acid (5) and trans-isoferulic acid (6)], coumarin (scopoletin, 4) and triterpenoids like 3-acetylaleuritolic acid (1), β-sitostenone (3), stigmasterol and stigmasteryl-3-O-β-D-glucopyranosides, in addition to a mixture of stigmasteryl-and β-sitosteryl-3-O-β-D-glucopyranosides. The compounds 1–6 represent bioactive metabolites of S. acmella Murr. that were never previously reported. Our findings demonstrate for the first time the potential benefits of this medicinal plant as a rich source of high therapeutic value compounds for medicines, cosmetics, supplements and as a health food.


Introduction
Spilanthes acmella Murr. (Compositae) is the well known "toothache plant", also commonly used as a spice. It has a long history of use as a folklore remedy, e.g. for toothache, rheumatism and fever [1,2]. The plant has found applications in pharmaceuticals as an antitoothache formulation for pain relief [3], swelling and gum infections [3], periodontosis [4] and in mouthwashes [5]. In addition, its extract is an active component added to body and beauty care cosmetics as a fast acting muscle relaxant to accelerate repair of functional wrinkles [6]. The plant extract was also used for stimulating, reorganizing and strengthening the collagen network in anti-age applications, e.g. in antiwrinkle cream formulations [7,8]. As a nutritional supplement [9] small amounts of the plant extract have been used for taste improvement as a sweetener with high sweetness devoid of unpleasant aftertaste that does not affect the taste or odor of foods or drinks [10].
A number of constituents had been isolated from the S. acmella Murr., for example, spilanthol, isobutylamides [11,12] and triterpenoids [13]. Our recent studies have shown that the S. acmella Murr. exhibits vasorelaxant and antioxidant activities [14]. These results motivated us to further investigate potential new compounds exerting such activities. Moreover, we have found that compounds with antioxidant action also exhibit antimicrobial activity [15]. These facts led us to search for new types of bioactive metabolites present in the S. acmella Murr. and examine their antimicrobial and antioxidant activities. In addition, cytotoxic effects of the plant extracts was also tested.

Cytotoxic effects
The extracts of hexane, chloroform, ethyl acetate and methanol were tested against the KB and HuCCA-1 cell lines [18]. The results showed that all the extracts exhibited ED 50 values greater than 10 µg/mL and were consequently considered to be inactive.
Significantly, the ethyl acetate and methanol extracts displaying the most potent radical scavenging activity (DPPH) [14] afforded diverse antioxidants. There are phenolics (2, 5, and 6), coumarin (4), triterpenoids (1, 3 and MBSG). The most potent antioxidant fraction (SOD assay) of chloroform extract [14] afforded stigmasterol and stigmasteryl glucoside (SG). The former had been isolated previously from a light petrol extract of S. acmella Murr. along with β-sitosteryl-3-O-β-D-glucoside from the ethanol extract [19] and isolation of SG of the same plant had also been described [20]. Due to limited quantity of the isolates in those cases, they were not tested for antioxidants. However, all the isolates (except 3) were tested for antimicrobial activity, but no growth inhibition was observed at 64 µg/mL. The study indicates that compounds 1-6 are bioactive metabolites that have never been isolated from S. acmella Murr..
The isolated compounds had been reported to possess diverse bioactivities as follows: 3-acetylaleuritolic acid (1) had been shown to exhibit diverse bioactivities, e.g. antigrowth activity against S. aureus and S. typhimurium [21] and significant inhibition on vitality of adult male worms of O. gutturosa [22]. In addition, this compound showed strong inhibition of DNA topoisomerase II [23] and strong cytotoxic activity against human lung carcinoma A549 cells [23]. It had been reported that pentacyclic triterpenoids; oleanolic acid and erythrodiol exhibited vasorelaxant effect [24]. In our recent study, the observed vasorelaxant activity of S. acmella Murr. [14] could possibly be due to the pentacyclic, 3-acetylaleuritolic acid (1) isolated from fraction E5 of the ethyl acetate extract.
Isoferulic acid (6) has been known as a component of Chinese herbal medicine used for a pain killer and stomachic [52]. It is a main active compound of the rhizoma of Cimicifuga (Japanese traditional medicine used as an anti-inflammatory [53]).
Other isolates, vanillic acid (2), trans-isoferulic acid (6), stigmasterol and stigmasteryl glucoside had been reported to be strong antioxidants. 3-Acetylaleuritolic acid (1) displayed antimicrobial and strong cytotoxic activities. β-Sitostenone (3) showed significant hypoglycemic, antiarrhythmic and antitubercular actions. These compounds 1-6 represent the bioactive metabolites that were never previously isolated from the S. acmella Murr.. The chloroform extract with antioxidant and antimicrobial activities afforded fractions (C3, C4, C5) of strong antigrowth actions against C. diphtheriae NCTC 10356 with MIC 64-128 µg/mL. Interestingly, the inactive antimicrobial extracts (ethyl acetate and methanol) provided fractions mostly with strong growth inhibition against C. diphtheriae NCTC 10356 and B. subtilis ATCC 6633 with MIC 64-128 µg/mL. Moreover, strong or potent antioxidant fractions (F, M) of methanol extract exhibited antimicrobial activity. This relation was also observed for fractions of the chloroform extract. As a result, the data support the use of S. acmella Murr. as a rich source of compounds with high therapeutic values for medicines, cosmetics, food supplements and as a health food.

General
Melting points were determined on an Electrothermal 9100 melting point apparatus and are uncorrected. 1 H-and 13 C-NMR spectra were recorded on a Bruker AM 400 instrument with a 400/100 MHz operating frequency using CDCl 3 or CD 3 OD solution with tetramethylsilane as internal standard. Mass spectra were determined using a Finnigan MAT INCOS 50 mass spectrometer. Infrared spectra (IR) were obtained on Perkin Elmer System 2000 FTIR. Ultraviolet (UV) spectra were measured with Milton Roy Spectronic 3000 Array. Column chromatography was carried out using silica gel 60 (0.063 -0.200 mm) and silica gel 60 (<0.063 mm). Thin Layer Chromatography (TLC) and preparative TLC were carried out on silica gel 60 PF 254 (cat. No. 7747 E., Merck).

Cell cultures
HuCCA-1 cells were established from chlolangiocarcinomas experimentally induced in hamsters. The cell lines were characterized and have been maintained in CRI laboratory ever since 1994 in Ham's F12 culture medium (GIBCO Laboratories, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, USA), 100 U/mL penicillin and 100 μg/mL streptomycin. The KB cell lines, originally derived from epidermoid carcinoma of the floor of the oral cavity and commonly used as a reference laboratory standard for cytotoxicity assay, have been maintained in CRI laboratory in DMEM (Dulbecco's modified Eagle medium).

Isolation
Isolation was performed using conventional (gravity) column chromatography otherwise stated. A ratio of 1:30 for separated materials and silica gel was used for the chromatography. The separation was carried out using gradient elution with increasing polarity. Fractions were combined based on TLC chromatograms.

Biological evaluations
Antimicrobial assay: Antimicrobial activity of the plant extracts, fractions and isolates (1, 2, 4, 5, 6, stigmasterol, SG and MBSG) was investigated using the agar dilution method [16]. Briefly, the test compounds dissolved in either CH 2 Cl 2 or MeOH were individually mixed with Müller Hinton (MH) broth to obtain a final volume of 2 mL. A two-fold dilution was prepared and the solution was then transferred to the MH agar solution to yield the final concentrations ranging from 4-256 μg/mL.
Twenty seven strains of microorganisms (Table 4), cultured in MH broth at 37 °C for 24 h, were diluted with 0.9 % normal saline solution to adjust the cell density of 10 8 CFU/mL. The organisms were inoculated onto each plate using a multipoint inoculator and further incubated at 37 °C for  h. Compounds which possessed high efficacy to inhibit bacterial cell growth were analyzed. Antioxidative assay: The antioxidative activity of the extracts was elucidated by the DPPH radical scavenging assay [16]. Experiments were initiated by preparing a 0.1 mM solution of DPPH in methanol. One mL of this solution was added to a sample solution (0.5 mL, 1 mg/mL dissolved in methanol). After 30 min, absorbance at 517 nm was measured and the percentage of radical scavenging activity was calculated from the following equation: % Radical scavenging = (1-Abs.sample/Abs.cont)×100 where Abs.cont is the absorbance of the control reaction and Abs.sample is the absorbance of the tested sample. The SOD activity was assayed by measuring inhibition of the photoreduction of nitro blue tetrazolium (NBT) [17]. The indirect assay is comprised of several reactions: the photochemically excited riboflavin was first reduced by methionine into a semiquinone, which donated an electron to oxygen to form a superoxide source. The superoxide readily converted NBT into a purple formazan product. As a result, the SOD activity was inversely related to the amount of formazan formed.
Cytotoxic assay: Cytotoxic activity of the plant extracts was determined by a slightly modified method described previously [18]. Briefly, the confluent cell monolayers were trypsinized and diluted with appropriate culture medium to a final concentration of 3×10 5 cells/mL. Portions (100 μL) containing approximately 3×10 4 cells were distributed into 96-well flat-bottomed tissue culture plates and incubated overnight at 37°C in a humidified 5% CO 2 incubator. Solutions (100 μL) containing different concentrations of tested extracts (0.001-10 μg/mL) or taxol (0.012-1.2 μg/mL) were added to each well and the plates were incubated as above for an additional 48 h. After the incubation, each well was washed (x 3) with phosphate-buffered saline (pH 7.2) and then stained with Crystal Violet. After 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 by a microtitre plate reader (Titertek, Multiskan MCC/340) set to read at a wavelength of 540 nm. All tests were carried out in quadruplicate and the mean value was calculated. The activity was expressed as ED 50 (the effective dose that inhibits 50% of cell growth).