Interference of Linoleic Acid Fraction in Some Receptor Binding Assays K. Ingkaninan,* ,† J. K. von Frijtag Drabbe Ku ¨ nzel, ‡ A. P. IJzerman, ‡ and R. Verpoorte † Division of Pharmacognosy and Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands Received December 2, 1998 An extract of a suspension culture of Tabernaemontana pandacaqui Poir. was fractionated by centrifugal partition chromatography. Aliquots were tested in an adenosine A 1 receptor binding assay. This led to the isolation and identification of linoleic acid, which proved to be a noncompetitive inhibitor. This “false positive”effect also extended to some other binding assays. Currently, pharmaceutical industries are becoming in- creasingly interested in screening natural products for new biologically active compounds because of the availability of high-throughput screening methods using receptor bind- ing assays or enzyme assays. However, such screening can be hampered by the occurrence of ubiquitous compounds that have known bioactivities, or of compounds that cause nonspecific inhibition in the assay. Therefore, a prefrac- tionation prior to screening might be useful. Centrifugal partition chromatography (CPC) has been used as a tool for prefractionation of crude plant extracts in our laboratories. 1 CPC has particular advantages in natural product research. It combines higher sample- loading capacity with lower solvent consumption and a shorter run time compared to the conventional preparative LC techniques. Moreover, irreversible retention of a sample is eliminated since no solid support is used. CPC was first described by Murayama, 2 and the theoretical and the application aspects were extensively discussed by Fou- cault. 3 In the present study, we combined CPC with radioligand binding studies as a rapid screening assay. We were particularly interested in the adenosine A 1 receptor since many natural products such as purines and flavonoids show affinity for this receptor subtype. The affinities of the extracts from some plants and cell suspension cultures on the adenosine A 1 receptor were determined (Table 1). The ethanol extract from a Taber- naemontana pandacaqui Poir. cell suspension culture and the ethanol extract from a Catharanthus roseus (L.) G. Don cell suspension culture were two of the most active extracts, and were selected for further investigation. The previously selected CPC system, heptane/ethyl acetate/methanol/water 6:1:6:1 (v/v/v/v) 1 was used as the prefractionation step of an ethanol extract of a T. panda- caqui cell culture. Adenosine A 1 receptor binding assay guided fractionation led to the isolation of the noncompeti- tive inhibitor, linoleic acid. This compound was identified by means of 1 H NMR, MS, and by comparison of the 1 H NMR spectrum and the TLC chromatogram with a refer- ence compound. Its affinity for the adenosine A 1 receptor expressed as a half-maximal inhibiting concentration (IC 50 ) was 65 ( 14 μM (SD) (Figure 1). The maximum bindings observed from three separate saturation experiments car- ried out in the absence and in the presence of 56 μM linoleic acid were 603.4 ( 67.8 fmol/mg protein (SD) and 142.7 ( 56.1 fmol/mgprotein (SD) (Figure 2). This difference showed that linoleic acid acted as a noncompetitive inhibitor in the adenosine A 1 receptor binding assay. The activities of palmitic acid, stearic acid, oleic acid and arachidonic acid on the same assay were also tested. The unsaturated fatty acids showed a greater inhibitory effect than the two saturated fatty acids on this assay (Figure 3). Moreover, linoleic acid showed an effect in the opiate receptor binding assay and nucleoside transport protein binding assay (Table 2). In the opiate receptor binding assay, the amount of radioligand remaining in the assay increased in the presence of the high concentration of linoleic acid (10 -3 M), but decreased in the presence of 10 -4 M linoleic acid. On the other hand, in the nucleoside transport protein binding assay, only the high concentration of linoleic acid (10 -3 M) affected the assay by decreasing the amount of radioligand remaining in the assay. It has been reported before that fatty acids cause noncompetitive or mixed-noncompetitive * To whom correspondence should be addressed. Tel.: +31-71-5274503. # Fax: +31-71-5274511. E-mail: ingkanin@chem.leidenuniv.nl. † Division of Pharmacognosy. ‡ Division of Medicinal Chemistry. Table 1. Percentage Specific Binding of [ 3 H]DPCPX Remaining on Adenosine A1 Receptor after Incubation with 100 μL Aliquots of Some Plant and Plant Cell Culture Extracts (2.5 mg/mL) % specific binding a of [ 3 H] DPCPX water extract. ethanol extract. toluene extract. T. pandacaqui cell suspension culture 75 ( 26 6 ( 1 14 ( 4 C. roseus cell suspension culture nd b 2 ( 1 0 R. tinctorum cell suspension culture 72 ( 8 0 nd b T. pachysiphon dried leaves nd b 80 ( 8 96 ( 6 A. xhenryi roots nd b 19 ( 7 nd b a The results are the mean ( SD of 3 separate determinations in which duplicate samples were tested. b nd ) not determined. Figure 1. Affinity of linoleic acid on adenosine A1 receptor binding assay. The specific binding of [ 3 H]DPCPX remaining was determined as described in the Experimental Section. Values are means of one typical experiment performed in duplicate. 912 J. Nat. Prod. 1999, 62, 912-914 10.1021/np9805490 CCC: $18.00 © 1999 American Chemical Society and American Society of Pharmacognosy Published on Web 05/22/1999