Launeugenol, a New Eugenol Derived Dimer from Launaea intybacea.
Summary: A eugenol derived dimer (1) has been isolated from the methanolic extract of the whole plant of Launaea intybacea along with the three known compounds. The structures of all the isolates (1-4) were established using 1D (1H, 13C) and 2D NMR (HMQC, HMBC, COSY) spectroscopy, mass spectrometry and comparison with the reported data in the literature. Compound 1 was tested for its DPPH radical scavenging and enzyme inhibitory activities against enzyme acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and lipoxygenase (LOX) and found to be moderately active against enzyme lipoxygenase with IC50 value of 56.3 uM.
Keywords: Launaea intybacea; Compositae; Secondary metabolite; Enzyme inhibition.
The genus Launaea of the family Compositae consists of 40 species growing in dry, saline and sandy habitats . In Pakistan this genus is represented by 20 species which possess galactagogue, soporific, diuretic, wound healing, antipyretic and aperients properties [2-4]. Most of the species of this genus possess antioxidant, antitumor, insecticidal and cytotoxic activities and are used in folk medicine against stomachic and skin diseases [5,6]. Launaea intybacea is a biennial herb commonly found in the coastal areas  of North and Central America, West Indies, tropical parts of Asia and Subcontinent . The plant is used by the local community as a folk remedy to treat jaundice, as blood purifier, galactogogue, hepatomegaly, dyspepsia, skin diseases, dry cough, galactoriya  and for the treatment of liver disorders [10, 11]. Previously we have isolated bioactive phenolics from this species .
Herein we report the isolation and characterization of a new eugenol derived dimer (1), together with lupeol (2) , b-sitosterol (3)  and oleanolic acid (4)  from the chloroform soluble fraction of the methanolic extract of the whole plant of Launaea intybacea (Fig. 1).
Results and discussion
Compound 1 was isolated as colorless oil. The IR spectrum showed absorption bands due to hydroxyl group (3550 cm-1), aromatic system (1610- 1500 cm-1) and allyl group (1645 cm-1). The UV spectrum showed the absorptions at 206 and 272 nm.
The HREIMS displayed molecular ion peak at m/z 342.1459 corresponding to the molecular formula C20H22O5 with 10 double bond equivalence. The aromatic region of the 1H NMR spectrum of 1 (Table- 1) displayed the signals at d 6.80 (d, J = 2.0 Hz, H-2,2') and 6.60 (d, J = 2.0 Hz, H-6,6'). The coupling constant (J = 2.0 Hz) and COSY correlations with each other suggested their meta position on a benzene ring. The olefinic region of the spectrum exhibited a methine at d 5.94 (tdd, J = 17.0, 10.0, 6.5 Hz, H- carbons . This data indicated an allyl moiety attached with the phenyl unit. The 13C NMR spectrum of 1 (Table-1) disclosed total 10 signals for one methyl, two methylene, three methine and four quaternary carbons.
The signal at d 150.2, 141.5, 138.6, 132.0, 116.5, 110.7 indicated the presence of 1,3,4,5-tetrasubstituted benzene ring. The presence of allyl group on the benzene ring was further confirmed by HMBC correlations of methylene protons (d 3.40) with aromatic carbons at d 116.5 and 110.7 and that of aromatic protons (d 6.80, 6.60) correlated with methylene carbons at d 40.7 (C-7). The methoxy protons (d 3.80) showed HMBC correlations with the carbon d 150.2 indicating its attachment with the benzene ring. The above discussed data revealed that 1 could be a 5-oxy derivative of eugenol. The 10 carbon signals in 13C NMR spectrum and 20 carbons in the molecular formula and mass fragmentation pattern (Fig. 2) confirmed that compound 1 (Fig. 1) is a dimer of 5- oxyeugenol and is named as launeugenol.
Eugenol is a major volatile component of clove oil and mainly used in the agricultural, cosmetic, fragrance, flavour and pharmaceutical industries. Pharmacologically, it has antimicrobial, anti-inflammatory, analgesic, anti-oxidant and anticancer activities. In addition, it is widely used in agricultural to protect foods from micro-organisms during storage . Keeping in view the uses and biological activities possessed by eugenol and its derivatives, we evaluated launeugenol (1) for its DPPH free radical scavenging potential and enzyme inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and lipoxygenase (LOX). At a concentration of 0.5mM, it showed weak antioxidant and enzyme inhibitory activity. The test compound was found moderate inhibitor of only the enzyme lipoxygenase with an IC50 value of 56.3 uM (Table-2).
Column chromatography was carried out using silica gel F254 (230-400 mesh). Aluminium sheets pre-coated with silica gel 60 F254 (20x20 cm,0.2 mm thick; E. Merck) were used for thin layer chromatography (TLC). TLC plates were visualized under UV lamp at 254 and 366 nm and by spraying with ceric sulphate solution followed by heating. UV spectra were scanned on a Hitachi UV-3200 spectrophotometer (lmax in nm).
IR spectra were measured on Shimadzu 460 spectrometer. 1H and 13C-NMR spectra were recorded on Bruker instrument operating at 500 and 125 MHz respectively. Mass spectra were measured on JMS Hx110 mass spectrometer with a data system and JMSA 500 mass spectrometers.
The whole plant L. intybacea Jacq. was collected from Cholistan Desert (District Bahawalpur, Punjab), Pakistan in April 2008 and was identified by Dr. Muhammad Arshad (Late), Ex- Taxonomist, Cholistan Institute for Desert Studies (CIDS), The Islamia University of Bahawalpur, Pakistan where a voucher specimen (0023- LI/CIDS/08) is deposited.
Extraction and Isolation
The methanolic extract of the L. intybacea was subjected to silica gel column chromatography eluting with n-hexane, n-hexane:dichloromethane (DCM), DCM, DCM:methanol and methanol in increasing order of polarity to get six fractions (1-6). The fraction 1 obtained from in n-hexane-DCM (3:7) showed two pots on TLC which on further silica gel column chromatography using same solvent system yielded lupeol (2, 65 mg) from the head fractions and b-sitosterol (3, 100 mg) from the tail fractions. The fraction 2 collected at n-hexane-DCM (1:9) subjected to column chromatography, eluted with pure DCM, yielded oleanolic acid (4, 95 mg). The fraction 3 obtained from pure DCM was subjected to silica gel column chromatography using DCM-MeOH (9.9:0.1) yielded launeugenol (1, 55 mg)
Launeugenol (1): Colorless oil (55 mg); UV (MeOH) lmax (log e) nm: 206 (4.58), 272 (4.46); IR (KBr) nmax cm-1: 3550, 1610-1500, 1645.
The 1H and 13C-NMR data, see Table-1; HREIMS m/z: 342.1459 (calcd. for C20H22O5, 342.1467).
DPPH Radical Scavenging Activity
The DPPH radical scavenging activities of pure compounds isolated from L. intybacea were examined by comparison with that of known antioxidant, butylated hydroxytoluene (BHT) using the method of Lee and Shibamoto . Briefly, various amounts of the compounds (500 ug/mL, 250 ug/mL, 125 ug/mL, 60 ug/mL, 30 ug/mL, 15 ug/mL) were mixed with 3 ml of methanolic solution of DPPH (0.1mM). The mixture was shaken vigorously and allowed to stand at room temperature for one an hour. Then absorbance was measured at 517 nm against methanol as a blank in the spectrophotometer. Lower absorbance of spectrophotometer indicated higher free radical scavenging activity.
The percent of DPPH decoloration of the samples was calculated according to the formula:
Antiradical activity = Acontrol - Asample/ Acontrol x100 Each sample was assayed in triplicate and mean values were calculated.
Acetylcholinesterase and Butyrylcholinesterase Assay
The Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) inhibition activity was performed according to the method used by Ellman  with slight modifications. Total volume of the reaction mixture was 100 uL. It contained 60 uL Na2HPO4 buffer with concentration of 50 mM and pH 7.7. Ten uL test compound (0.5 mM well-1) was added, followed by the addition of 10 uL (0.005 unit well-1) enzyme. The contents were mixed and pre- read at 405 nm. Then contents were pre-incubated for 10 min at 37oC. The reaction was initiated by the addition of 10 uL of 0.5 mM well-1 substrate (acetylthiocholine iodide), followed by the addition of 10 uL DTNB (0.5 mM well-1). After 30 min of incubation at 37oC, absorbance was measured at 405 nm. Synergy HT (BioTek, USA) 96-well plate reader was used in all experiments. All experiments were carried out with their respective controls in triplicate. Eserine (0.5 mM well-1) was used as a positive control.
The percent inhibition was calculated by the help of following equation.
Inhibition (%) = Control - Test x 100
Lipoxygenase (LOX) activity was assayed according to the Tappel method  with slight modifications. A total volume of 200 uL assay mixture contained 140 uL sodium phosphate buffer (100 mM, pH 8.0), 20 uL test compound and 15uL(600U) purified lipoxygenase enzyme (Sigma, USA). The contents were mixed and pre-read at 234 nm and pre incubated for 10 minutes at 25degC. The reaction was initiated by addition of 25 uL substrate solution. The change in absorbance was observed after 6 min at 234 nm. Synergy HT (BioTek, USA) 96-well plate reader was used in all experiments. All reactions were performed in triplicates. The positive and negative controls were included in the assay.
Baicalein (0.5 mM well ) was used as a positive control. The percentage inhibition was calculated by formula given below.
Inhibition (%) = Control - Test x 100
The authors are thankful to Higher Education Commission (HEC) Pakistan and Alaxander von Humboldt (AvH) Foundation, Germany for financial support. We are also obliged to Third World Academy of Science (TWAS), Italy for providing some of the basic lab facilities in Chemistry Department of IUB.
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|Author:||Saleem, Muhammad; Parveen, Shehla; Riaz, Naheed; Ejaz, Muhammad Ashraf Syeda Abida; Waris, Hafiz Muh|
|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Jun 30, 2013|
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