A new furanocoumarin from the fruits of Scaevola taccada and antifungal activity against Pythium insidiosum

Abstract A new coumarin, scataccanol (1) and 10 known compounds were isolated from the fruits of Scaevola taccada (Gaertn.) Roxb. All compounds were evaluated for antifungal activity against Pythium insidiosum. Compounds 5 and 7 showed strong antifungal activity with minimum inhibitory concentration values of 5 and 10 μg/mL, respectively. Structural determination of all compounds was accomplished by 1D and 2D-NMR, IR and MS.


Introduction
Scaevola taccada (Gaertn.) Roxb. (synonym, Scaevola taccada var. sericea (Vahl) Merr.; Family Goodeniaceae), a flowering plant, is commonly found in beach scrubland around the Arabian Sea, the tropical Indian Ocean and tropical Islands of the Pacific. It is known as Naupaka Kahakai (Hawaiian), Magoo (Divehi), Merambong (Malay), Ngahu (Tongan) and Ruk Ta-lay (Thai). The flowers of this plant have a fan-like shape, and hence, a fan flower or half flower was given. In Thailand, the roots of S. taccada are used to cure food poisoning and the leaves are used as an anti-inflammatory (Ruangrungsi & Mangkhla 2004). This species contained coumarins, terpenoids and steroids (Wohlrabe & Hänsel 1977). In this study, the chemical constituents of the fruits of this plant have been investigated. All isolated compounds were evaluated for antifungal activity against Pythium insidiosum. P. insidiosum is the etiological agent of pythiosis, a granulomatous disease characterised by cutaneous and subcutaneous lesion, corneal ulcer, keratitis and vascular disease. Conventional drugs are usually ineffective because of their cytoplasmic membrane lacking of ergosterol (Mendoza 2005). Radical excision of infected tissues or organs is the main option for the treatment of pythiosis. The significance of searching treatment from a novel microorganism or medicinal plants found in environmental area can be found from studying a natural process. The report of meroterpenes and furanocoumarin, which isolated from the leaves of culen (Psoralea glandulosa), has been reported for their antiphytopathogenicity against Botrytis cinerea and Phytophthora cinnamomi (Madrid Villegas et al. 2015). It has been reported the cytotoxicity of crude extract of an endophytic fungus against Artemia salina (Dame et al. 2016). The report of antifungal activity of phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH P) from Pseudomonas chlororaphis subsp. aureofaciens strain M71 against plant pathogenic fungi and oomycetes has been reported (Puopolo et al. 2013). Due to the need of effective treatments for pythiosis without using antifungal drugs by using the effective compounds, we aim to find the bioactive compounds against this fungus-like organism and our attention has been focused on the targeted medical plants such as S. taccada.
According to the high mortality and fatality rates of this disease caused by P. insidiosum, several experiments both in vivo and in vitro have been studied for the new alternative therapies especially from the medicinal plants products. The antifungal activity of essential oils from the Origanum vulgare, Origanum majorana, Mentha piperita and Rosmarinus officinalis has been reported for their minimum inhibitory concentration (MIC) to P. insidiosum ranging from 0.05 to 1.75 mg/mL (Fonseca et al. 2015). All isolated compounds in this study were also evaluated and compounds 5 and 7 demonstrated strong activity by showing MIC values of 5 and 10 μg/mL, which give the inhibition of 100% of mycelium growth at the lower concentration, while the standard drugs, terbinafine, showed MIC values of eight (Table 2). This indicated that our compounds have a higher potential against the growth of P. insidiosum. In vivo investigations, the use of topical formulation of essential oils of O. vulgare and M. piperita both singly, associated and in combination with immunotherapy had little or no action on the evolution of the disease. However, our study did not perform in vivo study, the formulation of compounds 5 and 7 might be test in vivo in the near future.

General experimental procedures
A SANYO Gallenkamp (uK) melting point apparatus was used to determine melting points. A JASCO DIP-1000 digital polarimeter was used to measure the optical rotation. An Agilent 8453 uV-Visible spectrophotometer (Germany) was used to record the uV spectra. IR spectra were recorded as thin films using a Perkin elmer Spectrum One FT-IR spectrophotometer (uK). The NMR spectra were recorded on a Varian Mercury plus spectrometer (uK) operating at 400 MHz ( 1 H) and at 100 MHz ( 13 C). The solvent residual peak was used for chemical shift referencing (δ H 3.31, δ C 49.0 for methanol-d 4 and δ H 7.26, δ C 77.2 for CDCl 3 ). Mass spectra were obtained on a Micromass Q-TOF 2 hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer with a Z-spray eS source (Micromass, uK). CC was carried out using silica gel 60 (100-200 mesh, Merck). TLC was performed on silica gel 60 F 254 (Merck) precoated aluminium sheets. The compounds were visualised under uV light and by spraying with acidic anisaldehyde solution followed by heating. Gel filtration was carried out over Sephadex LH-20 (Pharmacia) suspended in MeOH. Distilled solvents were used throughout the separation process.

Fungal isolates
P. insidiosum strain SIMI6666, a clinical isolated from a cornea pus of patient with ocular pythiosis who live in Kampang Phet province, Thailand . The identities of this isolated was confirmed by a polymerase chain reaction based assay and its physiological characteristic of the reproductive structure by zoosporogenesis technique. The fungal strain was subculture on Sabouraud dextrose agar (SDA) (Oxoid, uK) slant and stored at 25 °C.

Susceptibility testing
The susceptibility of the P. insidiosum strains to the tested compounds was tested by disc diffusion assay and applied following the CLSI M-51-P. The entire surface of each 100 mm-diameter non-supplemented Sabouraud Dextrose Agar (Oxoid, uK) plate was inoculated with the hyphal block of 1 × 1 cm of P.insidiosum. The tested compounds were dissolved at a concentration of 10 μg/mL and performed 2-fold serial dilution range from 0.0780 to 10 μg/ mL. Then, 20 μL of the tested compounds were impregnated on sterilised discs (6.0 mm) (Whatman, england) and placed on a SDA plate (Oxoid, uK). Terbinafine (20 mg/100 μL; 20 μL/disc) (Sigma-Aldrich, uSA) and a disc with etOAc or methanol only were used as control discs. Plates were kept at room temperature for 2 h in a laminar flow cabinet and incubated at 25 °C for 3, 6 and 9 days. The MIC of each compound was determined by visual observation and represents the inhibition of 100% of mycelium growth of P. insidiosum.

Conclusion
Chemical investigation of the etOAc and MeOH extracts from the fruits of S. taccada led to the isolation of 11 compounds. A new furanocoumarin, scataccanol (1) and 10 known compounds were discovered. Antifungal activity against P. insidiosum was testing by disc diffusion assay. Compounds 5 and 7 displayed antifungal activity with MIC values of 5 and 10 μg/mL, respectively, while the other compounds showed inactive against P. insidiosum.