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Lipoxygenase Inhibiting Two New Sterols from Capparis spinosa.

Byline: Abdul Razaq, Ejaz Ahmed, Ahsan Sharif, Muhammad Azam Rasool, Muhammad Arshad, Muhammad Imran Anjum, Sajid Iqbal, Muhammad Sher and Zahid Nawaz

Summary: Spinosol A (1) and spinosol B (2) were isolated as new steroids from the chloroform soluble fraction of Capparis spinosa. The structures of both 1 and 2 were elucidated with the help of chemical tests and sophisticated spectroscopic techniques (1D and 2D NMR, EIMS, HREIMS). The compounds 1 and 2 showed significant inhibitory potential against the Lipoxygenase enzyme.

Keywords: Spinosol, Capparis spinosa, 1D, 2D-NMR and MS techniques, Lipoxygenase enzyme.

Introduction

Capparis spinosa belongs to the family capparaceae, which comprises 33 genera and 700 species, most of which are herbs, small shrubs and trees, found frequently in the tropical and subtropical regions with an aired climate of hemisphere. In Pakistan only two species Capparis spinose and Capparis decidua were found. C. spinosa is a perennial shrub with flashy leaves and white to pinkish-white large flowers. The plant is used by local practitioner for the treatment of rheumatism, osteoporosis, liver toxicity, urinary tract infections and treatment of anemia, arthritis and gout. Previously, alkaloids, nitrile glycosides and thio-glycosides have been isolated from this specie. The chemotaxonomic and ethnobotanic properties of the C. spinosa prompted us to isolate the new phytochemical constituents from this species [1-5].

In our present study, spinosol A (1) and spinosol B (2) are the two new steroids which have been found from the chloroform soluble fraction of C. spinosa and evaluated them as 2[alpha],3[beta]-dihydroxy-24-ethylcholest-5,22-diene and (24R)-ethyl-cholesta-5-ene-2[alpha],3[beta]-(22R)-triol respectively. Biological studies on spinosol A (1) and spinosol B (2) indicated their significant lipoxygenase inhibitory potential.

Experimental

Methods

Melting points were obtained on Buchi melting point apparatus and reported as uncorrected. Optical rotations were taken on a JASCO DIP 360 polarimeter. The IR spectra were recorded on a FTIR-8900 Shimadzu spectrophotometer. The 1D and 2D-NMR spectra were recorded in CDCl3 at 500 MHz on a Bruker Av 500 spectrometer. The chemical shift (I') values are reported in ppm units and coupling constant (J) are shown in Hz. EIMS, HREIMS and HRFABMS were recorded on a JMS-HX-110 with a data system on JMS-DA 500 mass spectrometer. Aluminum sheets pre-coated with silica gel 60F254 (20 x 20 cm, 0.2 mm thick, E-Merck) were used for TLC and silica gel (230-400 mesh) was used for column chromatography. Pre-coated RP-18 gel (E-Merck) glass plates were used for preparative TLC. GC-18A equipped with FID was used for GLC. For lipoxygenase (1.13.11.12) type 1-B (Soybean) inhibitory assay all chemicals were purchased from Sigma Aldrich Company (USA).

Plant Material

The whole plant Capparis spinosa was collected in November 2012 from Cholistan desert near Bahawalpur (Pakistan) and identified from Cholistan Institute of Desert Studies, The Islamia University Bahawalpur, Bahawalpur, Pakistan, where a voucher specimen has been deposited.

Isolation

The shade dried plant material (5 Kg) was chopped and soaked in methanol (3 x 20 L). The combined methanolic extract was evaporated under reduced pressure and a greenish sticky material (0.7 Kg) was obtained. The crude methanolic extract was fractionated between n-hexane, chloroform, ethyl acetate, n-butanol and water-soluble fractions. The chloroform soluble fraction (78 g) was subjected to column chromatography over silica gel in the increasing order of polarity to obtained 12 sub-fractions. The sub-fraction 4 obtained from n-hexane:chloroform (3:2), showed one major and several other minor spots on TLC. The sub-fraction 4 was again chromatographed over PTLC (Preparative Thin Layer Chromatography) using the solvent system n-hexane:acetone (3:1) to obtain spinosol A (1) as a major compound (21 mg), Rf = 0.45. The sub-fraction 5 was again chromatographed over PTLC using the solvent system n-hexane:acetone (7:3) to obtain spinosol B (2) (16 mg), Rf = 0.39.

Spectroscopic data

Spinosol A (1), colorless solid, m.p. 202-204 AdegC, [[alpha]]D25 -45.5 (c = 1.0, CHCl3). IR max cm-1 3410-3380 (OH), 2950 (C-H Str), 1655 (C = C Str), 870 (C-H bending) 1322 (C-H stretching, CH3 bending), 750 (C-C bending). EIMS m/z: [M+] 428 (11), 410 [M+ -18] (25), 396 (36), 392 (32), 297 (28), 257 (94), 243 (20), 180 (58), 139 (58), 105 (6) 57 (100). HREIMS M+ showing at m/z 428.6931, calcd. 428.6962. 1H and 13C-NMR (Table-1).

Spinosol B (2), Colorless amorphous solid, [[alpha]]D25 -62 (c = 1.0, CHCl3). IR max cm-1 3410-3382 (OH), 2950 (C-H Str), 1655 (C = C Str), 872 (C-H bending) 1322 (CH3 bending), 750 (C-C bending). EIMS m/z: [M+] 446 (8), 428 (15), 410 (16), 391 (28), 289 (58), 210 (74), 57 (100). HREIMS M+ showing at m/z 446.3728, calcd. 446.6962. 1H and 13C-NMR (Table-1).

Table-1: 1H, 13C-NMR) data of compound 1 and 2 in CDCl3.

###13C-NMR###1H-NMR Multiplicityand J (Hz)

Position###1H-NMR Multiplicity and J (Hz)###13C-NMR

###1###2

###2.29 dd (13.6, 10.4)###2.28 dd (13.6, 10.4)

###1###45.86###45.8

###1.84 dd (13.6, 4.1)###1.83 dd (13.6, 4.1)

###2###4.18 ddd (10.4, 8.8, 4.1)###71.81###4.18 ddd (10.4, 8.8, 4.1)###71.87

###3###3.80 ddd, (11.1, 8.8, 5.7)###76.90###3.81 ddd, (11.1, 8.8, 5.7)###76.9

###2.20 dd (13.3, 11.1)###2.21 dd (13.3, 11.1)

###4###40.46###40.49

###2.59 dd (11.1, 5.7)###2.59 dd (11.1, 5.7)

###5###-###140.27###-###140.25

###6###5.33 br d (5.3)###121.70###5.33 br d (5.3)###121.77

###1.53 m###1.52 m

###7###31.87###31.91

###1.49 m###1.50 m

###8###1.60 m###31.6###1.60 m###31.66

###9###1.05 m###50.15###1.04 m###50.19

###10###-###37.22###-###37.25

###1.45 m###1.46 m

###11###21.08###21.03

###1.41 m###1.41 m

###1.19 m###1.18 m

###12###39.60###39.66

###2.20 m###2.22 m

###13###-###43.51###-###43.58

###14###1.10 m###56.78###1.11 m###56.76

###1.63 m###1.63 m

###15###26.12###26.13

###1.06 m###1.07 m

###16###1.67, 1.30###28.70###1.68, 1.30###28.75

###17###1.36 m###56.0###1.35 m###56.04

###18###0.67 m###12.22###0.68 m###12.26

###19###1.12 m###19.3###1.12 m###19.37

###20###2.00 m###40.01###2.04 m###40.06

###21###1.01 d (6.7)###20.12###1.08 d (6.4)###12.40

###22###5.00 dd (15.1, 7.7)###138.29###3.55 ddd (10.2, 4.0, 1.9)###72.14

###23###5.17 dd (15.1, 7.5)###129.50###1.05 (dd, 13.5 10.2)###29.5

###24###1.41 m###51.23###1.75 m###41.8

###25###1.79 m###31.64###1.79 m###28.8

###26###0.87 d (6.1)###21.28###0.99 d (6.3)###17.65

###27###0.78 d (6.1)###21.28###0.88 d (6.2)###20.63

###28###1.26 m###25.34###1.28 m###23.70

###29###0.81 d (7.4)###12.36###0.84 d (7.4)###11.82

In Vitro Lipoxygenase inhibition assay

LOX inhibiting activity was measured by modifying the spectrophotometric method developed by Tappel (1962) [6] and already described in our previous work [7]. "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 C. 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".

Results and Discussion

Spinosol A (1) was isolated as colorless solid showing m.p. 202-204 AdegC and [[alpha]]D25 -45.5 (C = 1.0, CHCl3). The molecular formula was established as C29H48O2 through HREIMS showing M+ peak at m/z 428.6931, (calcd. 428.6962) indicating six degrees of unsaturation. The EIMS spectrum showed the intense peak at m/z 139 and 156 due to the loss of side chain as well as D ring fission, revealing the two oxygen atoms and an olefinic functionality in the steroidal nucleus and one C=C bond in the side chain [8]. The IR spectrum gave absorption bands of hydroxyl, olefinic and geminal dimethyl moieties at 3410, 1655 and 1322 cm-1, respectively, in the molecule. 1H-NMR spectrum showed the characteristic signal for olefinic proton of trisubstituted double bond at I' 5.33 (1H, br.d J = 5.3 Hz) and trans disubstituted double bond in which olefinic protons resonated at I' 5.17 (1H, dd, J = 15.1 and 7.7 Hz) and 5.00 (1H, dd, J = 15.4 and 7.5 Hz).

1H-NMR spectrum further showed the signals for two hydroxy methine protons at I' 4.18 (1H, ddd, J = 10.4, 8.8 and 4.1 Hz) and 3.80 (1H, ddd J = 11.1, 8.8 and 5.7 Hz). 1H-NMR also showed signals for six methyl groups, out of which two were tertiary (I' 1.12 and 0.67), three were secondary [I' 1.01 (d, J = 6.7 Hz), I' 0.87 (d, J = 6.1 Hz) and I' 0.78 (d, J = 6.1 Hz)] and one was a primary methyl at I' 0.81 (t, J = 7.4 Hz) indicating ethyl moiety. The molecular formula (C29H48O2) was supported by BB and DEPT 13C-NMR spectra showed 29 signals comprising 6 methyl, 12 methine, 8 methylene and 3 quaternary carbons. The four unsaturated carbons (2 olefinic moieties) were observed at I' 140.27, 138.29, 129.50 and 121.70, respectively while the oxymethine carbons observed at I' 71.81 and 76.90. In compound 1 the three fused six membered ring system was developed by the analysis of strong HMBC correlations of C-18 and C-19 angular methyls (Fig 1).

The positions of the hydroxyl moieties and the double bonds in the molecule were assigned by coupling constants, 1H-1H COSY and HMBC correlations. The oxymethine proton at I' 4.18 showed 1H-1H correlation with adjacent oxymethine proton at I' 3.80, which in turn showed the 1H-1H correlations with two other protons at I' 2.20 (1H, d, J = 13.3 and 11.1 Hz) and 2.59 (1H, d, J = 11.1 and 5.7 Hz) allowing us to assign the both hydroxyl groups to C-2 and C-3 respectively, which was further confirmed by HMBC correlations. The coupling constants of two oxymethine protons at I' 4.18 (1H, ddd, J = 10.4, 8.8 and 3.8 Hz) and 3.80 (1H ddd, J = 11.1, 8.8 and 5.7 Hz) suggested 1,2-glycolic functions with a trans configuration and 2[alpha],3[beta]-dihydroxy cholestane type skeleton [9-10]. On the basis of these evidences spinosol A (1) was assigned the structure as 2[alpha],3[beta]-dihydroxy-24-ethylcholest-5,22-diene (Fig. 1).

Spinosol B (2) was isolated as amorphous solid, [[alpha]]D25 -62 (c = 1.0, CHCl3) having molecular formula C29H50O3, based on HREIMS m/z 446.3728 (M+ calcd. 446.3760). It gave positive Salkowski and Liebermann Burchard tests indicating its steroidal nature. The IR, MS and NMR spectra were similar to compound 1 except for the one additional hydroxyl group and the lack of double bond in the side chain. EIMS showed prominent peaks at m/z 289 and 248 due to the loss of side chain and ring D fission, indicating the presence of two hydroxyl and one olefinic moiety in the steroidal nucleus, while other hydroxyl moiety in the side chain. The 1H and 13C data of the steroidal nucleus is similar to the compound 1. In 1H-NMR spectrum, one additional hydroxy methine proton was observed at I' 3.55 (ddd, J = 10.2, 4.0 and 1.9 Hz). 13C-NMR spectrum displayed 29 carbon signals comprising 6 methyl, 11 methine, 9 methylene and 3 quaternary carbons. The additional hydroxy methine carbon was observed at I' 72.14.

The position of the hydroxyl moiety was proved at C-22 by the inspection of 1H-1H COSY spectrum. The signal at I' 3.55 indicated the connectivity with C-20 protons resonating at I' 1.74. In HMBC spectral observation it was also confirmed at C-22 (Fig. 2). The [alpha] configuration of C-22 hydroxyl moiety was supported by strong NOEs correlations between C-17, C-20 and C-22. The relative stereochemistry 'R' of C-22 was assigned on the comparison of the 13C-NMR data of the similar compounds reported in literature [11]. On the basis of these evidences, compound 2 was assigned as (24R)-ethyl-cholesta-5-ene-2[alpha],3[beta]-(22R)-triol (Fig. 1). The inhibitory activity of 1 and 2 against lipoxygenase enzyme was determined using the method developed by Tappal [6]. The IC50 values were found to be 55.5% (1) and 62.5% (2) uM as against IC50 value of 22.0 uM observed for Baicalein as a positive control.

Table-2: Invitro quantitative inhibition of lipoxygenase by compound 1 and 2.

###Compound###IC50 +- SMEa) [uM]

###1###55.5 +- 0.10

###2###62.5 +- 0.10

###Baicaleinb###22.0 +- 0.04

Conclusion

From the present study it was concluded that Capparis spinosa contain potent lipoxygenase inhibitory constituents which can be taken up by the local herbal pharmaceutical industry for further drug designing.

Acknowledgments

We are grateful to International Center of Chemical and Biological Sciences, Karachi University, Karachi for NMR and MS data acquisition. We also want to thank Professor Dr. Muhammad Ashraf (The Islamia University Bahawalpur, Pakistan) for his guidance and suggestions during the biological evaluation of the compounds. The authors declare no conflict of interest.

References

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3. H. M. Said, Hamdard Pharmacopeia of Eastern Medicine, Time Press, Karachi, P. 44 (1969).

4. V. U. Ahmed, S. Arif, A. R. Amber, K. Usman Ghani and G. A. Miana, A new spremidine alkaloid from Capparis deciduas. Heterocycles, 23, 3015 (1985).

5. T. Gull, B. Sultana, I. A. Bhatti and A. Jamil, Antibacterial potential of Capparis spinosa and Capparis decidua extracts. Int. J. Agri. Bio., 17, 727 (2015).

6. A. l. Tappel, Method in Enzymology, Academic press, New York, 5, P-539 (1962).

7. M. H. Kazim, Ejaz Ahmed, S. Hameed, A. Malik and M. Ashraf, Cashmirols A and B, new lipoxygenase inhibiting triterpenes from Sorbus cashmariana, Chemistry and Biodiversity., 9, 1471 (2009).

8. A. Sharif, E. Ahmed and A. Malik, Recurvosides A and B, antifungal novel steroidal glucosides from Haloxylon recurvum. Z. Natureforsch. 61b, 1148 (2006).

9. C. D. Jerrassi., D. B. Thomas, A. L. Ton Livings, C. R. Thompson, Terpenoids. XXXI. The structure and stereochemistry of medicagenic acid. J. Ame. Chem. Soc., 79, 5292, (1957).

10. H. T. Cheung, D. G. William Son, NMR Signals of Methyl Groups of Triterpenes with oxygen functions at positions 2, 3 and 23. Tetrahedron, 25, 119, (1969).

11. Y. Li, M. Ishibashi, M. Satake, M. Chen, X. Oshima, Y. Ohizumi. Sterol and triterpenoid constituents of Verbena littoralis with NGF-potentiating activity, J. Nat. Prod., 66, 698 (2003).
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Author:Razaq, Abdul; Ahmed, Ejaz; Sharif, Ahsan; Rasool, Muhammad Azam; Arshad, Muhammad; Anjum, Muhammad I
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Dec 31, 2017
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