Two Triterpenyl Fatty Acid Esters from Fagonia cretica as Lipoxygenase Inhibitors.
Summary: Two triterpenyl fatty acid esters were isolated from the chloroform soluble fraction of Fagonia cretica and their structures elucidated as 3[beta]-12-ursen-3yl-docosanoate (1) and 3[beta]-12-ursen-3yl-nonacosanoate (2) by 1D-NMR, 2D-NMR, mass spectrometric and chemical analyses. Both new compounds were also evaluated for their lipoxygenase inhibitory potential using Baicalein as a standard.
Keywords: Fagonia cretica, triterpenyl fatty acid esters, Baicalein, Lipoxygenase.
Fagonia cretica (locally known as Dhamma) is an important plant of zygophyllaceae family, which comprises 22 genera and 285 species, out of which 34 species are restricted to the humid and warm areas of all the continents. Only six species were present Pakistan . F. cretica usually exists in the form of small green spiny herb having woody branches as local plant used for the treatment of different diseases such as small pox, tumors and other swellings neck. [2-3]. Local practitioners use the leaves of F. cretica to cure the snakebite, whereas, aqueous decoction of aerial parts of the plant is a popular remedy for cancer in the early stage. This plant is also used for the treatment of some digestive tract diseases and blood vascular system. Crude extract of the leaves are used for treatment of snake bites . Literature survey revealed that triterpene glycosides and flavonoids have been reported from this plant [4-6].
In the present study, we have isolated two compounds from the chloroform soluble fraction of F. cretica. Their structures were elucidated as 3[beta]-12-ursen-3yl-docosanoate (1) and 3[beta]-12-ursen-3yl-nonacosanoate (2) by 1D-NMR (1H and 13C), 2D-NMR (HMQC, 1H-1H COSY, HMBC and NOESY), EIMS, HREIMS and chemical analyses. The medicinal significance of these new compounds (1 and 2) were evaluated by performing lipoxygenase inhibitory assay employing Baicalein as a standard, and both the compounds showed prominent inhibitory results.
Results and Discussion
Compound 1 was isolated as a colorless solid, which gave positive Liebermann-Burchard test for triterpene. The IR spectrum showed key absorption bands at 1741 and 1210 cm-1 for ester functionality, while at 1660, 810 cm-1 for individual olefinic moiety. The molecular formula C52H92O2 was established through the data of HREIMS showing [M+] peaks at m/z 748.7160 (calcd. for C52H92O2, 748.7179), also indicating seven degrees of unsaturation. The EIMS spectrum displayed distinctive peaks at m/z 218, 208, 207 and 203 characteristics for the retro Diels-Alder fragmentation in pentacyclic triterpene. The other important peaks in the EIMS spectrum were observed at m/z 530, 249, 426, 411, 408, 393, 340, 339, and 189 indicating the loss of docosanoyl moiety from the molecule.
The 1H-NMR spectrum exhibited a tri-substituted C=C bond at I' 5.13 (1H, t, J = 8.2 Hz). Six methyls were observed as singlet I' 1. 10, 1.04, 0.96, 0.90, 0.88 and 0.83 while two methyls were observed as doublet at I' 0.94 and 0.81 with coupling constants between 6.6-6.8 Hz. The 1H-NMR further showed a terminal methyl at I' 0.84 (t, J = 6.3 Hz) and a methylene at I' 2.31 as a triplet (2H, J = 7.5 Hz) indicating its attachment to the carbonyl of the ester group. An envelope of nineteen methylenes was observed at I' 1.25-1.28 (38 H) as a broad singlet. The 13C-NMR experiments (BB and DEPT) indicated the presence of six tertiary, two secondary and one primary methyls. The carbonyl carbon of the ester resonated at I' 171.8 while the olefinic carbon signals appeared at I'140.6 and 124.8. The signal at I' 80.5 was assigned to oxymethine carbon, whereas, the signals at I' 30.8-27.4 were indicative of several methylenes.
The 1H-NMR and 13C-NMR data clearly indicated the [alpha]-amyrin type skeleton along with a fatty acid ester moiety.
Hydrolysis of compound 1 with KOH in MeOH provided a free alcohol and methyl docosanoate. The alcohol showed molecular ion peak in HREIMS at m/z 426.3910 (calcd. for 426.3902) giving molecular formula C30H50O2. This compound was crystallized in chloroform (m.p. 186.5-187.5 AdegC) and optically active [[alpha]]D25 + 83.2. (c=1.0, CHCl3).
Physical and spectroscopic data resembles to those reported for 12-ursen-3[beta]-ol . The methyl docosanoate moiety was confirmed by the comparison of melting point (54-55.5 AdegC) and spectroscopic data with the reported molecules in the literature . The1H-1H COSY and HMBC correlations (Fig. 1) proved that the docosanoyl moiety was attached at 3-O-atom of the terpenoid nucleus. The equatorial 3[beta] configuration was assigned for H-3 based on coupling constants (1H, J = 10.5, 4.4 Hz). Hence, the structure of compound 1 was assigned as 3[beta]-12-ursen-3yl-docosanoate (Fig. 1) based on the combined spectral data and comparison with the literature data . To the best of our knowledge compound 1 was first time characterized by 1D-NMR (1H-NMR and 13C-NMR) and 2D-NMR (HMBC, HMQC, 1H-1H COSY 45Adeg and NOESY) data.
Compound 2 was isolated as amorphous solid having triterpenoidal structure as indicated by positive Liebermann-Burchard test. The molecular formula was established by HREIMS which gave [M+] peak at m/z 846.8222 for C59H106O2 (calcd. for C59H106O2, 846.8274) presenting seven degrees of unsaturation. The other prominent peaks appeared at m/z 218 and 208 due to the retro Diels-Alder fragmentation in the molecule. The IR, ID (1H-NMR and 13C-NMR) and 2D-NMR (HMQC, HMBC, 1H-1H COSY and NOESY) spectral features were similar to 1 except for the side chain attached to the ester moiety in 2. Basic hydrolysis (KOH/MeOH) of 2 yielded a free alcohol and methyl nonacosanoate. The alcohol showed complete resemblance with 12-ursen-3[beta]-ol as in compound 1, while the methyl nonacosanoate moiety was identified by comparative TLC with an authentic sample, melting point (68-69.5 AdegC) and by exact agreement of its IR and NMR spectral data with those reported in the literature [9-10].
All these observations helped to assign the structure of 2 as 3[beta]-12-ursen-3yl-nonacosanoate (Fig. 1).
The enzyme inhibitory activity of 1 and 2 against lipoxygenase was determined using the reported Tappal method . The IC50 values were found to be 65.5 uM for 1 and 68.5 uM for 2 compared to the IC50 value 22.0 uM observed for Baicalein as a positive control (Table-1).
A digital polarimeter JASCO DIP-360 with 10 cm tube was used to measure the optical rotation.
The melting points were determined on a Gallenkemp melting point apparatus and are uncorrected. Infrared (IR) spectra were obtained on Hitachi U-3200 and JASCOA-320 spectrophotometer. 1H NMR spectra were recorded in CDCl3 using TMS as internal standard at 500 MHz on Bruker AM-500 nuclear magnetic resonance spectrometer. 13C-NMR spectra taken in CDCl3 at 125 MHz on Bruker AM-500 nuclear magnetic resonance spectrometer. The 2D-NMR (HMQC, HMBC, COSY and NOESY) spectra were recorded in CDCl3 on a Bruker AMX 500 NMR spectrometer. Low-resolution Electron Impact Mass Spectroscopy (EIMS) was performed on a Finnigan MAT 311 with MASSPEC data system. HREIMS measurements were made on Jeol JMS HX 110 mass spectrometer. Silica gel Si60 (230-400 mesh, E. Merck) was used as adsorbent in column chromatography for the purification of the compounds. All chemicals for comparative TLC and chemical tests were purchased from local supplier, deals with Sigma Aldrich Company (USA).
The whole plant (Fagonia cretica Linn) was collected from the Cholistan Desert near Bahawalpur (Pakistan) in November 2013, and identified from CIDS office, Cholistan Institute for Desert Studies (CIDS), The Islamia University of Bahawalpur (IUB), Bahawalpur, Pakistan where the voucher specimen has been deposited.
The air dried whole plant (8 Kg) was comprehensively extracted with 20-liter methanol and the process was repeated thrice. The combined extract was evaporated on rotavapor under reduced pressure to yield the residue (0.6 Kg), which was further partitioned between n-hexane (80 g), CHCl3 (130 g), ethyl acetate (55 g) n-butanol (85 g) and water fraction (60 g). The chloroform soluble fraction was chromatographed over silica gel using VLC (vacuum liquid chromatography) to obtain twelve fractions employing different ratios of n-hexane-CHCl3 solvent system in increasing order of polarity. The fraction 3 (n-hexane:CHCl3; 7:3) was further chromatographed over silica gel column chromatography using solvent system n-hexane-acetone in increasing order of polarity to obtain five sub fractions (3A-3E). The fraction 3C (n-hexane: acetone; 8.5:1.5) gave vital spots showing close Rf values on TLC.
The fraction 3C was again purified by preparative HP-TLC (RP-18; MeOH/H2O; 8.5:1.5) to afford 1 (18 mg) and 2 (11 mg) with Rf value 0.45 and 0.39, respectively.
Colorless amorphous solid, [[alpha]]D +31.5 (c = 1.0, CHCl3); M.P. 155-156 AdegC; HREIMS, [M+] at m/z 748.7179 (calcd. for C52H92O2 748.7199); IR (CHCl3) max cm-1 1741 (ester carbonyl), 1660 (olefin), 1210 (C-O); 1H-NMR (CDCl3, 500 MHz) I': 0.81 (3H, d, J = 6.8, Me-29), 0.82 (3H, s, Me-30), 0.84 (3H, t, J = 6.3, Me-22') 0.88 (3H, s, Me-25), 0.90 (3H, s, Me-23), 0.94 (3H, d, J = 6.6, Me-28), 0.96 (3H, s, Me-26), 1.04 (3H, s, Me-24), 1.10 (3H, s, Me-25), 1.25-1.28 (br.s, H-3'-21'), 2.31 (2H, t, J = 7.5, H = 2'), 4.41 (1H, dd, J = 10.5, 4.4, H-3), 5.13 (1H, t, J = 8.2, H-12);
13C-NMR (CDCl3, 125 MHz) I': 39.1 (C-1), 25.0 (C-2), 80.5 (C-3), 38.2 (C-4), 54.6 (C-5), 18.3 (C-6), 32.5 (C-7), 40.7 (C-8), 47.3 (C-9), 37.1 (C-10), 23.5 (C-11), 124.8 (C-12), 140.6 (C-13), 42.1 (C-14), 26.4 (C-15), 26.9 (C-16), 33.9 (C-17), 58.1 (C-18), 46.7 (C-19), 39.2 (C-20), 32.7 (C-21), 41.5 (C-22), 18.6 (C-23), 28.6 (C-24), 15.9 (C-25), 15.8 (C-26), 25.5 (C-27), 18.1 (C-28), 21.1 (C-29), 25.9 (C-30), 171.8 (C-1'), 32.9 (C-2'), 27.4-30.8 (C-3'-21'), 14.2 (C-22'); EIMS, [M+] m/z 748 (11), 530 (26), 426 (66), 411 (59), 409 (77), 408 (46), 393 (37), 340 (45), 339 (51), 249 (38), 218 (100), 203 (91) 208 (85), 207 (72), 189 (86).
Colorless amorphous solid, [[alpha]]D + 29.8 (c = 1.0, CHCl3); M.P. 171-172 AdegC; HREIMS, [M+] at m/z 846.8222 (calcd. for C59H106O2 846.8274); IR (CHCl3) max cm-1 1748 (ester carbonyl), 1655 (olefin), 1212 (C-O); 1H-NMR (CDCl3, 500 MHz) I': 0.82 (3H, d, J = 6.6, Me-29), 0.85 (3H, s, Me-30), 0.84 (3H, t, J = 6.5, Me-29') 0.89 (3H, s, Me-25), 0.92 (3H, s, Me-23), 0.95 (3H, d, J = 6.8, Me-28), 0.97 (3H, s, Me-26), 1.03 (3H, s, Me-24), 1.11 (3H, s, Me-25), 1.24-1.27 (m, H-3'-28'), 2.32 (2H, t, J = 7.2, H-2'), 4.40 (1H, dd, J = 10.5, 4.4, H-3), 5.13 (1H, t, J = 8.0, H-12);
13C-NMR CDCl3, 125 MHz) I': 39.1 (C-1), 25.2 (C-2), 80.6 (C-3), 38.3 (C-4), 54.4 (C-5), 18.1 (C-6), 32.5 (C-7), 40.6 (C-8), 47.3 (C-9), 37.0 (C-10), 23.3 (C-11), 124.6 (C-12), 140.4 (C-13), 42.1 (C-14), 26.3 (C-15), 26.8 (C-16), 33.7 (C-17), 58.3 (C-18), 46.6 (C-19), 39.2 (C-20), 32.7 (C-21), 41.4 (C-22), 18.5 (C-23), 28.5 (C-24), 16.1 (C-25), 16.0 (C-26), 25.4 (C-27), 18.0 (C-28), 21.3 (C-29), 26.0 (C-30), 171.6 (C-1'), 32.6 (C-2'), 27.2-29.9 (C-3'-28'), 14.1 (C-29'); EIMS, [M+] m/z 846 (10), 629 (30), 530 (37), 438 (69), 426 (70), 411 (44), 408 (57), 393 (21), 218 (100), 249 (37), 208 (80), 207 (77).
In Vitro Lipoxygenase inhibition assay
LOX inhibiting activity was measured by modifying the spectrophotometric method developed by Tappel (1962)  and already described in our previous work . "Reaction mixtures containing 160 uL (100 mM) sodium phosphate buffer (pH 8.0), 10 uL of test-compound solution and 20 uL of lipoxygenase solution was mixed and incubated for 10 min at 25 AdegC. The reaction was then initiated by the addition of 10 uL linoleic acid (substrate) solution, with the formation of (9Z,11E)-(13S)-13-hydroperoxy-octadeca-9,11-dienoate, and the change of absorbance at 234 nm was followed for 6 min. All the inhibition experiments were performed in triplicate in 96-well micro-plates in Spectra Max 340 (Molecular Devices, USA). The IC50 values were calculated using the EZ-Fit Enzyme kinetics program (Perrella Scientific Inc. Amherst, USA).
The percentage (%) inhibition was calculated as follows (E - S) / E x 100, where E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound".
Table-1: In vitro quantitative inhibition of lipoxygenase enzyme by compound 1 and 2.
Compound No.###IC50 +- SMEa) [uM]
###1###65.5 +- 0.10
###2###68.5 +- 0.10
Baicaleinb###22.0 +- 0.04
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|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Apr 30, 2018|
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