Discovering COX-Inhibiting Constituents of Morus Root Bark: Activity-Guided versus Computer-Aided Methods

Abstract

The aim of this study was to compare the efficiency of two well known approaches for the discovery of the bioactive principle/s in medicinal plants, namely the activity-guided isolation versus the computer-aided drug discovery by means of virtual screening (VS) techniques. Morus root bark of Morus sp. L. (Moraceae) was selected as application example for the discovery of compounds with anti-inflammatory activity. The two cyclooxygenase isoenzymes COX-1 and COX-2 were chosen as targets and the corresponding pharmacophore models were generated by our research. The activity-guided fractionation of the methanol extract of the root bark resulted in the isolation of nine compounds. Their structures were elucidated by mass spectrometry, 1- and 2-dimensional NMR experiments and identified as moracins B, M, the regioisomers O/P as a mixture, and sanggenons B, C, D, E and O. The COX-1 and COX-2 inhibiting activities of these compounds were established in an enzyme assay and compared with the predicted hits obtained from the VS. Sanggenons C, E, and O, that were tested the first time for an inhibitory effect on COX-1 and -2, showed IC₅₀ values of 10 - 14 μM, and 40 - 50 μM, respectively. The results show that the COX activities obtained for the sanggenons are correctly predicted by the in silico filtering experiment. In the case of the isolated moracins, however, it failed because the COX inhibiting activities of moracins M and P/O were not retrieved by the VS. Structure-activity relationships of the isolated compounds are discussed as well as potential pitfalls and advantages of the applied strategies.

Abbreviations

COX:cyclooxygenase

EIA:enzyme immunoassay

HBA:hydrogen bond acceptor

PGE:prostaglandin-E

PGHS:prostaglandin-H synthase

VS:virtual screening

Activity-guided fractionation - Sang-Bai-Pi - virtual screening - pharmacophore model - sanggenons - moracins - cyclooxygenase - Morus sp. - Moraceae

References

• 1 Heinrich M, Gibbons S. Ethnopharmacology in drug discovery: an analysis of its role and potential contribution.  J Pharm Pharmacol. 2001;  53 425-32
• 2 Rollinger J M, Haupt S, Stuppner H; Langer T. Combining ethnopharmacology and virtual screening for lead structure discovery: COX-inhibitors as application example.  J Chem Inf Comp Sci. 2004;  44 480-8
• 3 Berendes J. Des Pedanios Dioskurides aus Anazarbos Arneimittellehre in fünf Büchern. Stuttgart; 1902; reprint: Vaduz: Sändig Reprints Verlag 1997: pp 144-5
• 4 Yamatake Y, Shibata M, Nagai M. Pharmacological studies on root bark of mulberry tree (Morus alba L.)  Japan J Pharmacol. 1976;  26 461-9
• 5 Nomura T. The chemistry and biosynthesis of isoprenylated flavonoids from moraceous plants.  Pure Appl Chem. 1999;  71 1115-8
• 6 Reininger E, Bauer R. New PGHS-1 and -2 microtiter assays for the screening of herbal drugs and in vitro inhibition of PGHS-isoenzymes by polyunsaturated fatty acids from Platycodi radix . Phytomedicine 2005: in press
• 7 Fiebich B L, Grozdeva M, Hess S, Hüll M, Danesch U, Bodensieck A, Bauer R. Petasites hybridus extracts in vitro inhibit COX-2 and PGE2 release by direct interaction with the enzyme and by preventing p42/44 MAP kinase activation in rat primary microglial cells.  Planta Med. 2005;  71 12-9
• 8 Nomura T, Fukai T, Hano Y, Uzawa J. Structure of sanggenon C, a natural hypotensive Diels-Alder adduct from Chinese crude drug ”Sang Bai-Pi” (Morus root barks).  Heterocycles. 1981;  16 2141-8
• 9 Hano Y, Nomura T. Structure of sanggenon O, a natural Diels-Alder type adduct from the Chinese crude drug ”Sang-Bai-Pi” (Morus root bark).  Heterocycles. 1985;  23 2499-503
• 10 Shi Y Q, Fukai T, Nomura T. Structure of sanggenon O, a Diels-Alder type adduct derived from a chalcone and a dehydroprenylated sanggenon-type flavanone from Morus cathayana .  Heterocycles. 2001;  54 639-46
• 11 Shi Y Q, Fukai T, Ochiai M, Nomura T. Absolute structures of 3-hydroxy-2-prenylflavanones with an ether linkage between the 2′- and 3-positions from moraceous plants.  Heterocycles. 2001;  55 13-20
• 12 Shen R, Lin M. Diels-Alder type adducts from Morus cathayana .  Phytochemistry. 2001;  57 1231-5
• 13 Nomura T, Fukai T, Hano Y, Uzawa J. Structure of sanggenon D, a natural hypotensive Diels-Alder adduct from Chinese crude drug ”Sang-Bai-Pi” (Morus root barks).  Heterocycles. 1982;  17 381-9
• 14 Hano Y, Kohno H, Suzuki S, Nomura T. Constituents of the cultivated mulberry tree. XXXVII. Constituents of the Chinese crude drug Sang-Bai-Pi (Morus root bark). VIII. Structures of sanggenons E and P, two new Diels-Alder-type adducts from the Chinese crude drug Sang-Bai-Pi (Morus root bark).  Heterocycles. 1986;  24 2285-91
• 15 Hano Y, Itoh M, Fukai T, Nomura T, Urano S. Revised structure of sanggenon B.  Heterocycles. 1985;  23 1691-6
• 16 Hano Y, Kanzaki R, Fukai , Nomura T. Revised structure of sanggenon A.  Heterocycles. 1997;  45 867-74
• 17 Takasugi M, Nagao S, Masamune T, Shirata A, Takahashi K. Structure of moracin A and B, new phytoalexins from diseased mulberry. Tetrahedron Lett 1978: 797-8
• 18 Wanjala C CW, Juma B F, Bojase G, Gashe B A, Majinda R RT. Erythrinaline alkaloids and antimicrobial flavonoids from Erythrina latissima .  Planta Med. 2002;  68 640-2
• 19 Deshpande V H, Srinivasan R, Rao A VR. Wood phenolics of Morus species. IV. Phenolics of the heartwood of five Morus species.  Ind J Chem. 1975;  13 453-7
• 20 Zhou C X, Tanaka J, Cheng C HK, Higa T, Tan R X. Steroidal alkaloids and stilbenoids from Veratrum taliense .  Planta Med. 1999;  65 480-2
• 21 Hirakura K, Fujimoto Y, Fukai T, Nomura T. Constituents of the cultivated mulberry tree. 30. Two phenolic glycosides from the root bark of the cultivated mulberry tree (Morus lhou).  J Nat Prod. 1986;  49 218-24
• 22 Chi Y S, Jong H G, Son K H, Chang H W, Kang S S, Kim H P. Effects of naturally occurring prenylated flavonoids on enzymes metabolizing arachidonic acid: Cyclooxygenases and lipoxygenases.  Biochem Pharmacol. 2001;  62 1185-91
• 23 Su B -N, Cuendet M, Hawthorne M E, Kardono L BS, Riswan S, Fong H HS, Mehta R G, Pezzuto J M, Kinghorn A D. Constituents of the bark and twigs of Artocarpus dadah with cyclooxygenase inhibitory activity.  J Nat Prod. 2002;  65 163-9
• 24 Basnet P, Kadota S, Terashima S. Shimizu M, Namba T. Two new 2-arylbenzofuran derivatives from hypoglycemic activity-bearing fractions of Morus insignis .  Chem Pharm Bull. 1993;  41 1238-43
• 25 Kimura Y, Okuda H, Nomura T, Fukai T, Arichi S. Effects of flavonoids and related compounds from mulberry tree on arachidonate metabolism in rat platelet homogenates.  Chem Pharm Bull (Tokyo). 1986;  34 1223-27
• 26 Kubinyi H. Drug research: myths, hype and reality.  Nature Rev Drug Discov. 2003;  2 665-8