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Novel illudalane sesquiterpenes from Encephalartos villosus Lehm. antimicrobial activity

Abstract Phytochemical investigation of Encephalartos villosus Lehm. leaves afford two new illudalane sesquiterpenes namely Encephaldiene 1 and Encephaldiene 2 together with four known flavone glycosides, Luteolin-7-rutinoside, Luteolin-7-glucoside, Luteolin-7-rhamnoside and Apigenin-7-glucoside. The structures of the isolated compounds were elucidated by means of spectroscopic methods including 1D and 2D NMR experiments along with HRESIMS spectrometry. Antimicrobial activity of CHCl3 and MeOH extracts was investigated. Both extracts showed antibacterial activity against Gram-positive bacteria Streptococcus pneumonia and Bacillus subtilis, and antifungal activity against Aspergillus fumigatus. While CHCl3 extract showed additional activity against Gram-negative bacteria Escherichia coli.

Cycads have economic, ornamental, medicinal values and also have been used as a staple food source, as well as an emergency supply during periods of food shortage (Nair & Van Staden 2012).
One of the cycads group is the genus Encephalartos (zamiaceae) which is endemic to Africa and comprises 66 species (Donaldson 2003). South Africa is considered the regional centre of this type of cycads, it contains a diversity of about 37 species (Donaldson 2008).
The bark and stems of Encephalartos species are used in Africa for traditional medicine and are traded in traditional medicinal markets (Cousins et al., 2011). Ravele and Makhado (2002) reported the use of Encephalartos transvenosus decoction for the cure of stomach pain, heart attack and stroke by the local people around Mphaphuli in Southern Africa.
Encephalartos villosus Lehm. is one of the most common ornamental dwarf cycads in Southern Africa, it is known in common as a poor man's cycad due to its availability and low cost (Jones 2002).
Although genus Encephalartos has traditional medicinal importance, there is no phytochemical or biological evaluations of E. villosus plant, and hence this study was designed to investigate the phytochemical and biological activities of this plant.
In this study, the isolation and identification of two new illudalane sesquiterepens Encephaldiene 1 and Encephaldiene 2 together with four known flavone glycosides is reported along with antimicrobial evaluation of CHCl 3 (chloroformic) and MeOH (methanolic) extracts.
The protonated carbons were assigned by HSQC spectrum. The connectivity of carbons and groups was deduced from the 1H-1H COSY and HMBC spectra. H 2 -1 and H 2 -10 give HMBC correlations to C-11, C-12 and C-1 while H 3 -12 correlates to C-1, C-10 and C-13 indicated connectivity of C-11 to C-1 and C-10, with C-12 and C-13 connected to quaternary carbon C-11.
From COSY and HMBC NMR there were correlations from the 5-H 2 to C-6, C-7 prove the connection of -CH 2 -CH 2 OH fragment to C-6. Thus, the structure of 1 was assigned as shown in Figure 1.
The relative configuration of 1 was indicated by the correlations observed in the NOESY spectrum. The presence of NOE correlations between H-1β/H 3 -12, H-10β/H 3 -12 and H 3 -14/ H-6β and the absence of the correlations between H 3 -12/H-1α and H 3 -12/H-10α indicated the β-orientations of H-6, 14-Me and 12-Me, while 13-Me and 15-Me were in α-orientations.
The previous spectroscopic features are in agreement with those reported for illudalane sesquiterpenes (Becker et al. 1994;Nord et al. 2014) and the name Encephaldiene 1 is suggested for compound 1.
The exact molecular weight of compound 2 corresponds with the composition of C 14 H 22 O, and consequently an unsaturation index of four is assigned. The presence of four signals for unsaturated carbon atoms in the 13 C NMR spectrum showed that compound 2 is bicyclic. The first ring was assigned by the HMBC correlations from the protons 12-H 3 and 13-H 3 of the geminal methyl groups to C-1, C-10 and C-11. HMBC correlations from 8-H 2 to C-9, C-7 and C-6 as well as from 14-H 3 to C-2, C-3 and C-6, assigned the second ring ( Figure S1). The attachment of -CH 2 -CH 2 OH fragment to C-6 was supported by 5-H 2 correlations to C-6 and C-7. Comparison of the NMR spectroscopic data of compound 2 with those of 1 (Table S1) revealed that both the compounds have structure similarity with a difference of one point , which is the presence of the two germinal methyls (Me-12) and (Me-13) at C-11 assigned from 1 H NMR as singlet at δ H 0.91and 0.92, respectively, and the absence of one methyl singlet (Me-15) instead of the presence of one methyl doublet at δ H 0.89 assigned to Me-14. Therefore, compound 2 is a nor-illudalane sesquiterpene named Encephaldiene 2 ( Figure 1).
Compounds 1 has structure similarity with the previously reported illudalane sesquiterpenes riparol B isolated from Ripartites metrodii and pholiotic acid from Pholiota destruens (Becker et al. 1994;Weber et al. 2006), the points of differences are the presence of methyl group (Me-14) at C-3 instead of C-7 and the absence of a double bond at C-6 together with the presence of -CH 2 OH instead of C-4 carboxylic group in pholiotic acid.
CHCl 3 extract of E. villosus leaves showed antibacterial activity against Gram-positive bacteria Streptococcus pneumonia and Bacillus subtilis, Gram-negative bacteria Escherichia coli and exhibited antifungal activity against Aspergillus fumigatus; the highest activity was against S. pneumonia. MeOH extract showed antimicrobial activity against tested Grampositive bacteria S. pneumonia and B. subtilis and antifungal activity against A. fumigatus (Table 1, Figure S12). Sesquiterepens are group of plants, marine and fungi secondary metabolites, they have diverse structure and many pharmacological activities (Fraga 2007;Kuete 2013). In this study, we reported the isolation of two new illudalane type sesquiterepens from E. villosus. Few studies reported the isolation of illudane-type sesquiterpenes from plants (Castillo et al. 2003;Cáceres-Peña et al. 2013), while the isolation of illudalane-type sesquiterpenes is reported only from fungi (Becker et al. 1994;Weber et al. 2006) and this is the first report of illudalane skeleton from plants.
Microbial resistance threatens the effective treatment of a wide range of infectious diseases. It is considered a serious threat to public health because infections caused by resistant microorganisms often fail to respond to the standard treatment, resulting in prolonged illness, higher health care costs and a greater risk of death (Sosa et al. 2010;Cantas et al. 2013).
Natural products are considered the most consistently successful source for ideas of new drugs and in the way of seeking for new natural antimicrobial agents. The antimicrobial activity of E. villosus leaves was tested and it gave promising results (Table 1).
de Almeida Alves et al. (2000) suggested that inhibition zones >9 mm correspond to active antimicrobial samples. Accordingly, in the present work tested extracts showed a wide range of antimicrobial activity.
Several studies reported the antimicrobial activity of luteolin and luteolin glycosides particularly luteolin-7-O-glucoside (Chung et al. 2001;Chiruvella et al. 2007;Liu et al. 2010). Consequently, the presence of luteolin glycosides in E. villosus may contribute to the antimicrobial activity of the plant.
Further research is necessary to establish the pharmacological mechanisms of isolated compounds and methods to optimise their activity.

General experimental procedures
HRESIMS were acquired in positive ion mode on a Q-TOF premier spectrometer equipped with a nanoelectrospray ion source (Waters-Milford, MA, uSA). NMR experiments were performed on Bruker AM-400, DRX-500. Chemical shifts (d) were expressed in ppm with reference to the solvent signals. Standard pulse sequences and phase cycling were used for DQF-COSY, HSQC, HMBC and ROESY experiments. TLC was performed on precoated Kieselgel

Plant material
Leaves of E. villosus were collected in June 2012 from El-zoharia Botanical Garden in Cairo, Egypt. It is mentioned in the garden reference book and was identified by Dr Mamdouh Shokry, the botanist of the garden. Voucher sample number (2012-2E) under the name E. villosus was reserved in the Department of Pharmacognosy, Faculty of Pharmacy (Girls), Al-Azhar university, Cairo, Egypt.
CHCl 3 extract was subjected to vacuum liquid chromatography using silica gel (200 mesh) as stationary phase and CH 2 Cl 2 followed by CH 2 Cl 2 -MeOH (9:1) as mobile phase. Fractions of 30 mL were collected and similar fraction by TLC were grouped to give four major fractions.

Antimicrobial activity
The antimicrobial activity was tested against two Gram-positive and two Gram-negative bacteria and two fungi, respectively: S. pneumonia (RCB 010,010), B. subtilis (RCMB 010,067), E. coli (RCMB 010,052), Pseudomonas aeruginosa (RCMB 010,043), A. fumigatus (RCMB 02,568) and Candida albicans (RCMB 05,036). Agar diffusion method was adopted according to Bauer et al. (1966) using trypticase Soy agar (Difco) medium inoculated with the suspension of the test organisms. Discs (6 mm) were impregnated with the tested extracts (5 mg/mL), dimethyl sulphoxide (100 μL) as a negative control and with ampicillin, gentamicin and amphotericin B as reference standards for the antibacterial and antifungal activities, respectively. The impregnated discs were placed on the culture medium surface and were incubated at 25 °C for 48 h in case of fungi and at 35-37 °C for 24-48 h in case of bacteria. After incubation, the diameters of inhibition zones were recorded in mm. The results were expressed by mean zone of inhibition in mm ± SD in Table 1.