Isolation, structural elucidation and cytotoxicity evaluation of a new pentahydroxy-pimarane diterpenoid along with other chemical constituents from Aerva lanata

Aervalanata possesses various useful medicinal and pharmaceutical activities. Phytochemical investigation of the plant has now led to the isolation of a new 2α,3α,15,16,19-pentahydroxy pimar-8(14)-ene diterpenoid (1) together with 12 other known compounds identified as β-sitosterol (2), β-sitosterol-3-O-β-D-glucoside (3), canthin-6-one (4), 10-hydroxycanthin-6-one (aervine, 5), 10-methoxycanthin-6-one (methylaervine, 6), β-carboline-1-propionic acid (7), 1-O-β-D-glucopyranosyl-(2S,3R,8E)-2-[(2′R)-2-hydroxylpalmitoylamino]-8-octadecene-1,3-diol (8), 1-O-(β-D-glucopyranosyl)-(2S,3S,4R,8Z)-2-[(2′R)-2′-hydroxytetracosanoylamino]-8(Z)-octadene-1,3,4-triol (9), (2S,3S,4R,10E)-2-[(2′R)-2′-hydroxytetracosanoylamino]-10-octadecene-1,3,4-triol (10), 6′-O-(4″-hydroxy-trans-cinnamoyl)-kaempferol-3-O-β-D-glucopyranoside (tribuloside, 11), 3-cinnamoyltribuloside (12) and sulfonoquinovosyldiacylglyceride (13). Among these, six compounds (8–13) are reported for the first time from this plant. Cytotoxicity evaluation of the compounds against five cancer cell lines (CHO, HepG2, HeLa, A-431 and MCF-7) shows promising IC50 values for compounds 4, 6 and 12.


Introduction
India is a well known source for medicinal plants and their extracts which are used in Ayurvedic, Siddha, and Unani systems of medicine for treating different types of diseases. One of them is Aerva lanata (L.) A. L. Juss. ex Schultes from the family Amaranthaceae locally known as 'bui' or 'polpala'. It is an erect or prostrate under shrub with a long tap-root and many wollytomentose branches, found in the wild, with several names throughout India like Chaya (Bengali), Gorakhbuti (Hindi), Sirupulai (Tamil) and Bhadra (Sanskrit) (Khare 2007). It is also described in English as a stone breaking plant. A. lanata possessses medicinal and pharmaceutical importance (Ragavendran et al. 2012). A variety of pharmacological activities of this ethnomedicinally important plant has been reported as follows: Demulcent, antiinflammatory, expectorant, hepatoprotective and nephroprotective (Shirwaikar et al. 2004;Manokaran et al. 2008). Alcoholic extract of shoots of A. lanata has shown significant antidiabetic and antihyperglycaemic activities in rats (Deshmuk et al. 2008;Krishnan et al. 2009). Antimicrobial and cytotoxic (Chowdhury et al. 2002), diuretic and urolithiatic (Vetrichelvan et al. 2000;Soundararajan et al. 2006), antihyperlipidaemic and antiparasitic (Soundararajan et al. 2007;Anantha et al. 2010), antifertility and anti-diarrheal activities (Joanofarc & Vamsadhara 2003;Savadi & Alagawadi 2009) have also been reported.
As a part of our investigation of phytochemicals from Indian Medicinal Plants, A. lanata was selected for the study as a potential source of medicinal and pharmaceutical ingredients. This investigation deals with the isolation and structure elucidation of a new 2a,3a,15,16,19pentahydroxy-pimarane diterpenoid (1) and 12 other known compounds among which six are isolated for the first time from this plant.
Compound 1 was obtained as white crystals. The positive ion HR-ESI-MS (m/z 377.2315, calcd for C 20 H 34 O 5 Na, 377.2304) showed the molecular formula to be C 20 H 34 O 5 , representing an unsaturation value of four. The IR spectrum of 1 showed absorptions at 3419 and 1658 cm 21 corresponding to OH and CvC functional groups. In the 1 H NMR spectrum signals for three methyl groups located on quaternary carbons were observed as 3H singlets at d 0.97, 1.23 and 1.65. Two pairs of oxygenated methylene protons [d 4.21 (1H, bd, J ¼ 13.8 Hz) and 3.85 (1H, m), 4.19 (1H, bd, J ¼ 11.4 Hz) and 3.95 (1H, t, J ¼ 9.6 Hz)] and three oxygenated methine protons [d 3.87 (1H, m), 4.53 (1H, d, J ¼ 11.4 Hz) and 4.68 (1H, s)] signals were observed. A trisubstituted olefin proton peak was observed at d 5.71 (1H, s). The 13 C NMR and DEPT spectra displaying 20 carbon signals indicated a diterpene skeleton consisting of three methyls (at d 17.3, 23.5 and 24.1), seven methylene (two oxygenated, at d 63.8 and 65.4), six methine (three oxygenated, at d 66.9, 74.4, 80.1 and one olefinic, at d 129.6), and four quaternary carbon atoms (one olefinic, at d 137.7). Three methyl carbon signals were assigned to C-20, C-17 and C-18, respectively. Two oxygenated methylene carbon signals could be assigned to C-16 and C-19, respectively, whereas three oxygenated and one olefinic methine carbon signals were assigned to C-2, C-3, C-15 and C-14, respectively. The olefinic quaternary carbon signal at d 137.7 represents the C-8 carbon.  Figure 1. Chemical structure of compounds 1 -13 isolated from A. lanata.
Compared to the related pimarane diterpenoid darutigenol/kirenol/2b,3b,15,16-tetraydroxyent-pimar-8(14)-en (Herz et al. 1978;Jakupovic et al. 1987;Liu & Röder 1991) both C-2 and C-3 methylene peaks were replaced by downfield methine carbon peaks at d 66.9, 74.4 in 1, suggesting that two OH groups were present at C-2 and C-3. This conclusion was supported by the downfield shift of the C-1 carbon (d 41.7) as well as the C-4 carbon (d 45.5) signals. HMBC cross-peaks of H-3 with C-2, C-4, C-18, and C-1 peaks, of H-1 with C-2 and C-3 peaks as well as the COSY relationship of H-3 signal with H-2 along with the C-3 -OH proton (d 5.72) supported the above assignments. The coupling constant of H-2 (J ¼ 11.4 Hz) suggested it to be axial while H-3 must be equatorial to explain the absence of splitting. Furthermore, the absence of NOESY relationship between signals of H-5 and H-3 proved the equatorial orientation of H-3. Also the strong NOESY relationship in between H-2 and H-20 methyl proton suggested C-20 methyl to be axially-oriented. The NOESY correlation of H-11 (d 1.58) with both H-20 methyl proton (d 0.97) and H-17 methyl proton (d 1.22) confirms the axial-orientation of C-17 methyl group ( Figure S2).
The structure was ultimately confirmed by single crystal X-ray diffraction study of 1 where the b-orientation of H-2 and H-3 along with the C-19 oxygenated methylene and C-20, C-17 methyl were clearly visible ( Figure 2).

Biological activities
Cytotoxicity evaluation of the isolated thirteen compounds against five cancer lines (CHO, HepG2, HeLa, A-431 and MCF-7) was performed via MTT assay using doxorubicin as standard. Compounds 4, 6 and 12 are quite promising as indicated from their IC 50 values and further evaluation is in progress (Table 1).

General experimental procedure
Melting points were determined in capillaries and are uncorrected. IR spectra were recorded as KBr pellets using a JASCO 410 FTIR spectrometer (Tokyo, Japan). The NMR spectra were recorded using a Bruker 600 DPX spectrometer (Coventry, UK) operating at 600 MHz for 1 H and 150 MHz for 13 C in Py-d 5 with TMS as internal standard and the chemical shifts are reported in d units. Mass spectra (positive mode) were obtained on a LC-ESI-Q-TOF micro mass spectrometer in the electro spray ionization mode. All other solvents and chromatographic absorbents were procured from E. Merck (Darmstadt, Germany) and SRL (Mumbai, India) Ltd. unless otherwise indicated. Thin layer chromatography was performed on pre-coated silica gel 60 F 254 aluminum sheets (E. Merck, Darmstadt, Germany) using various solvent systems (1%, 5% and 10% MeOH in CHCl 3 ) and spots were developed using UV irradiation, iodine and Liebermann-Burchard reagent.

Plant material
Mature whole aerial part of A. lanata was collected from the Muzaffarpur district, Bihar, India during November 2012 and identified by Botanical Survey of India, Howrah, West Bengal. A voucher specimen (No.YB-2) was deposited in the Chemistry department, Indian Institute of Chemical Biology, Kolkata.

Conclusion
In conclusion, a new 2a, 3a,15,16,19-pentahydroxy pimar-8(14)-ene diterpenoid (1) in addition to 12 other known compounds (2 -13) were isolated and characterized using through spectrometric analysis (IR, 1D and 2D NMR, Mass) and comparison of the literature data for the known compounds. Among these 6 compounds (8-13) are reported for the first time from this plant. Cytotoxicity evaluation of the isolated thirteen compounds against five cancer cell lines were performed which shows promising IC 50 values for compound 4, 6 and 12.
Supplementary data 1 H, 13 C NMR, IR and HRMS data along with spectrometric copy of compound 1 associated with this article can be found in the online version. Crystallographic data in CIF format are available free of charge via the Internet at CCDC 1014285. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: þ 44 1223 336033; or deposit@ccdc. cam.ac.uk).