New ent-kaurane diterpenoid dimer from Pulicaria inuloides

Abstract A new naturally occurring ent-kaurane diterpenoid dimer, 15β, 15′β-oxybis (ent-kaur-16-en-19-oic acid) (1) along with six known compounds, 15β-hydroxy-ent-kaur-16-en-19-oic acid (2), 15β-hydroxy-ent-kaur-16-en-19-oate-β-d-glucopyranoside (3), 6-hydroxykaempferol-3, 7-dimethyl ether (4), quercetagetin 3, 7, 3′-trimethyl ether (5), β-sitosterol (6) and β-sitosterol glucoside (daucosterol) (7) were isolated from the aerial parts of Pulicaria inuloides DC. Compounds 2–5 were isolated for the first time from genus Pulicaria. The structures of compounds 1–7 were established on the basis of extensive 1D and 2D NMR spectroscopic techniques in combination with ESI-MS. The antimicrobial activity of the isolated compounds was evaluated against Staphylococcus aureus, Escherichia coli and Candida albicans. Sulphorhodamine B cytotoxic assay against HepG2 (liver cancer) cell line and ABTS antioxidant assay were carried out.


Introduction
The genus Pulicaria belongs to subtribe inulinae, tribe inuleae, family Asteraceae. It is represented by 100 species widespread all around the world, more than 10 of which grow in Yemen (Liu et al. 2010;. Previous researches indicated that the genus Pulicaria is chemically not homogenous. Several species afforded monoterpenoids, sesquiterpenoids, diterpenoids, triterpenoids, flavonoids, steroids and essential oils (Singh et al. 1985;Al-Hazimi & Al-Khathlan 1992;Marco et al. 1992;Liu et al. 2010;Ragab & Raafat 2016;Casiglia et al. 2016). Traditionally, plants of this genus are used as carminative, insect repellant, anthelmintic and diuretic. It is also used for the treatment of inflammation, intestinal disorders, menstrual cramps, dysentery and diarrhoea (Liu et al. 2010;Mothana et al. 2011;Al-Hajj, Rashid et al. 2014). Recent investigations disclosed that some plants of this genus exhibit anticancer, antioxidant, antimicrobial, antifungal and antimalarial activities (Mothana et al. 2007(Mothana et al. , 2011Al-Hajj, Rashid et al. 2014). Pulicaria inuloides DC. is a perennial herb growing wildly in Taiz mountains, Yemen. The flowers and leaves of the plant are used as spice and in herbal tea (Al-Hajj, Rashid et al. 2014). To the best of our knowledge, few reports about the chemical constituents of P. inuloides DC. were characterised. As part of our ongoing research programme for the discovery of bioactive compounds, seven compounds were isolated from the aerial parts of P. inuloides DC. This paper reports the isolation, structure characterisation and the evaluation of the antimicrobial, antioxidant and cytotoxic activities of the isolated compounds.
The 1 H NMR and 13 C NMR spectra of compound 1 were almost identical to that of compound 2, but the completely different R f values of them indicated that they are different compounds. The R f values of compounds 1 and 2 were (0.27 & 0.09) and (0.38 & 0.16) using systems III and IV as TLC solvent systems, respectively. The ESI-MS − of compound 1 showed a molecular ion peak at m/z 617.2 [M − H] − which was consistent with a molecular formula C 40 H 58 O 5 indicating that compound 1 is a homodimer consisting of two monomers of compound 2. The ESI-MS signal at 617.2 [2M (for 2) − H − H 2 O] − suggested the presence of ether or ester linkage between the two units of the symmetric dimer. As compound 1 showed the presence of only one free carboxylic carbon signal at δ C 181.0 assigned for C-19 & C-19′ and the absence of the characteristic esterified carboxylic group signal at about δ C 170-175. Therefore, the attachment between the two units forming the dimer was suggested to be through ether linkage between the two hydroxyl groups at C-15. This was further confirmed by the upfield shift of C-15 and C-15′ from 88.1 in compound 2 to 86.5 in compound 1. Compound 1, therefore, was determined as 15β, 15′β-oxybis (ent-kaur-16-en-19-oic acid).

General details
uV spectra were measured in methanol using Shimadzu 1601 PC (TCC240, Japan) spectrophotometer. Chromatographic separations were performed using silica gel (E-Merck, Germany). TLC was performed on silica gel G60F 254 (E-Merck, Germany). The 1 H and 13 C NMR spectra were recorded in DMSO-d 6 and CD 3 OD using TMS as an internal standard, on a JEOL Eclipse-400 NMR spectrometer, operating at 400 MHz for 1 H and 100 MHz for 13 C NMR and on Bruker AV-400 spectrometer at 400 MHz for 1 H and 100 MHz for 13 C NMR. ESI-MS was recorded on JOEL, JMS-AX500 mass spectrometer. For TLC analysis, the following solvent systems were used: hexane-ethyl acetate (9:1, system I), hexane-ethyl acetate (8:2, system II), methylene chloride-methanol (8:2, system III) and ethyl acetate-methanol-water (100:16.5:13.5, system IV)

Plant material
The aerial parts of P. inuloides DC. were collected from Taiz area, Yemen in March, 2010. The plant was authenticated by Dr Ramzy Mothanna, Professor of pharmacognosy, Department of pharmacognosy, Faculty of pharmacy, Sanaa university, Yemen. A voucher specimen (Astpul-Ap-1000-43) is kept in pharmacognosy Department, Faculty of Pharmacy, Mansoura university, Egypt.

Extraction and isolation
The air-dried aerial parts of P. inuloides DC. (540 g) were powdered and extracted by maceration in methanol at room temperature (4 × 2 L). The collected methanolic extract was evaporated under reduced pressure to give 15 g of green viscous residue (A). The marc was re-extracted with methanol-water (1:1), the obtained hydroalcoholic extract was evaporated under reduced pressure then coarsely fractionated on a diaion HP-20 packed column using water followed by methanol then the methanolic effluent was evaporated under reduced pressure to give (3 g) dark brown solid residue (B). The methanolic extract (A) was chromatographed on a silica gel column eluting with petroleum ether-ethyl acetate (100:0 to 0:100) and ethyl acetate-methanol (100:0 to 0:100) sequentially to give four subfractions I-IV, monitored by TLC using solvent systems (I-III).
The hydroalcoholic fraction (B) was chromatographed on silica gel column eluting with ethyl acetate-methanol in increasing polarity, monitored by TLC and similar subfractions were collected together. Subfractions 7-11 eluted with ethyl acetate-methanol (90:10) were further purified on Sephadex LH-20 using methanol for elution to yield compound 1 (10 mg).

Antioxidant activity screening assay
The antioxidant activity was evaluated using a colorimetric method described by Lissi et al. (1999) and Yen and Chen (1995).

Sulphorhodamine (SRB) cytotoxic activity screening assay
This method was carried out according to Skehan and Storeng (1990).

Antimicrobial activity
The antimicrobial assay was carried out according to Stylianakis et al. (2003).