Long Chain Alkyl and Alkenyl Phenols from the Roots of Ozoroa insignis

Ozoroa insignis Del. (Heeria insignis Del.) (Anacardiaceae) is a shrub or tree to 6.5 m high, of the soudanian savanna from Senegal to Niger and Nigeria, and across Africa to Ethiopia, Zaire and E. Africa. This species has a wide range of healing properties, namely, in the treatment of diarrhea and venereal diseases, tapeworm and hookworm, schistosomiasis, and kidney trouble. The anthelmintic effect of root bark and leaves of O. insignis was evidenced in vitro against the worms Schistosoma mansoni and Hymenolepis diminuta, whereas a bark extract displayed cytotoxic activity against Hep-G2, MDA-MB231, and 5637 human cancer cell lines. Stembark and stemwood were also investigated for in vitro topoisomerase inhibition. In Guinea-Bissau, the infusion of roots of O. insignis is taken by women after childbirth to increase lactation. Although other traditional uses of the roots of O. insignis have been reported, so far, only the leaves, flowers, twigs, and bark have been submitted to a limited phytochemical investigation that led to the isolation of essential oils, anacardic acid, and ginkgolic acid. In a previous antitumor screening of medicinal plants from Guinea-Bissau, we have observed that an ethanolic root extract of O. insignis showed moderate activity in KB, A 549 and MDA-MB cell lines, with IC 50 values of 30.5, 22.0 and 15.5 μg mL, respectively. In the present paper, we describe the isolation and structural determination of a series of alkyl and alkenylphenols from nonpolar fractions of the ethanolic extract, whose activity was monitored by the brine shrimp assay.


Introduction
Ozoroa insignis Del. (Heeria insignis Del.) (Anacardiaceae) is a shrub or tree to 6.5 m high, of the soudanian savanna from Senegal to Niger and Nigeria, and across Africa to Ethiopia, Zaire and E. Africa. 1 This species has a wide range of healing properties, namely, in the treatment of diarrhea and venereal diseases, 2 tapeworm and hookworm, 3 schistosomiasis, 4,5 and kidney trouble. 6The anthelmintic effect of root bark and leaves of O. insignis was evidenced in vitro against the worms Schistosoma mansoni and Hymenolepis diminuta, 5 whereas a bark extract displayed cytotoxic activity against Hep-G2, MDA-MB-231, and 5637 human cancer cell lines. 2Stembark and stemwood were also investigated for in vitro topoisomerase inhibition. 7In Guinea-Bissau, the infusion of roots of O. insignis is taken by women after childbirth to increase lactation. 8Although other traditional uses of the roots of O. insignis have been reported, 1 so far, only the leaves, flowers, twigs, and bark have been submitted to a limited phytochemical investigation that led to the isolation of essential oils, anacardic acid, and ginkgolic acid. 2,6,9n a previous antitumor screening of medicinal plants from Guinea-Bissau, 8 we have observed that an ethanolic root extract of O. insignis showed moderate activity in KB, A 549 and MDA-MB cell lines, with IC 50 values of 30.5, 22.0 and 15.5 μg mL -1 , respectively.In the present paper, we describe the isolation and structural determination of a series of alkyl and alkenylphenols from nonpolar fractions of the ethanolic extract, whose activity was monitored by the brine shrimp assay.

Results and Discussion
The ethanol extract of O. insignis was fractionated according to Scheme 1, and the chromatographic fractions monitored by TLC and the brine shrimp assay. 10,11 1H and 13 C NMR, and GLC analysis indicated the presence of five mixtures (I-V) of long chain alkyl and alkenylphenols (Figure 1), which were further characterized by GCMS of their α,β-bis-methylthiolated derivatives.][14][15][16][17] GCMS of mixture I showed peaks of [M] + at m/z 232, 276, 304, and 332, three peaks with the same [M] + at m/z 302, two peaks of [M] + at m/z 330, and one peak of [M] + at m/z 442.These data indicated the presence of cardanols 1, 3, and 5, with saturated side chains (Table 1), and seven cardanols (6, 16-18, 21, 22, and 29) with monounsaturated side chains differing in the position of the double bond.In all cases, the mass spectra displayed a base peak at m/z 108 due to benzylic cleavage of alkyl 3-hydroxybenzene. 15Location of the double bond in the alkenyl side chain was deduced from mass spectral analysis of the corresponding α,β-bis-methylthiolated derivatives, which provide readily recognizable fragments on electron bombardment (Table 1; Figure 2a). 18,19The Z stereochemistry of the double bond was assigned from the appearance of a low resolution multiplet in the 1 H NMR spectrum at δ 5.40 (2H, m), and carbons at allylic positions at δ 27.2. 17he NMR profiles of mixtures II and III were identical to those of I.In mixture II, thirteen cardanols with monounsaturated side chains (7, 8, 10, 13-15, 19, 20, 23-26, and 31) could be characterized from their GCMS and mass spectra fragmentation of the methylthiolated derivatives (Table 1; Figure 2a), 18,19 whereas in mixture III, in addition to five cardanols with saturated side chains (1-5), and six with monounsaturated side chains (9, 11, 12, 27, 28, 31) (Table 1), one compound with a diunsaturated side-chain (30) was identified.In this case, the derivatization reaction leads to a cyclic methylthiolated derivative, whose mass spectrum showed two fragments at m/z 279 (base peak) and m/z 201, indicative of the presence of two double bonds separated by two methylene groups (Figure 2b, o = 2). 19he NMR data of mixture IV, when compared to those of I-III, showed a different substitution pattern of the aromatic ring.Three coupled aromatic protons were observed at δ 6.72 (d, J 7.6 Hz), 6.85 (d, J 8.0 Hz) and δ 7.27 (t, J 8.0 Hz), and, in addition to the presence of the alkyl side chain and a phenolic hydroxyl group, a carboxyl methyl ester was assigned from a HMBC correlation of a carbonyl resonance at δ C 172.0 with a methyl group at δ 3.96.Analysis of COSY, DEPT, HMQC, HMBC and NOESY spectra, and comparison with literature data allowed to confirm a basic structure of anacardic acid methyl esters for the constituents of mixture IV. 6,[13][14][15][16][17] Its GCMS indicated the presence of three compounds (32, 35, and 36) with saturated side chains and three others (39-41) with one Z double bond. 17Mixture V was comprised of cardanol 29, and anacardic acid methyl esters 32-38, which were identified as described above.
1][22] These compounds are known to possess a wide range of biological properties, such as antimicrobial, antitumoral, molluscicide, antifungal, insectide, regulator of gene expression, antioxidant, lipoxygenase and uncoupling activity of oxidative phosphorylation.They inhibit the generation of superoxide radicals by xanthine oxidase, and lower the levels of serum cholesterol in rats. 2,14,21-276-Oxa isosteres of anacardic acids proved to be among the most potent inhibitors of the bacterial two-component regulatory systems KinA/SpoOF and NRII/NRI, reported to date. 28They also constitute some of the active principles of Ginkgo biloba, 29 with a recognized effect as inhibitors of prolyl endopeptidase and glycerol-3phosphate dehydrogenase, 30,31 antifeedant and cytotoxic activities. 17,32,33In this plant they occur together with cardanols (or ginkgols), whose antitumor activity has also been demonstrated. 17he pool of anacardic acid methyl esters and cardanols identified in the present work possess some unusual structural features.Their alkyl side chains varies from 13 to 25 carbons

Plant material
Ozoroa insignis Delile (Anacardiaceae), was collected in January 1994 at Contuboel, Guinea-Bissau, and identified at the Herbarium of Botany Centre (LISC), where the voucher specimen nº 854 is deposited.

Extraction and fractionation
The air dried roots of O. insignis (1.65 kg) were powdered, and extracted in a Soxhlet apparatus with EtOH (5 L) at room temperature.Evaporation of the solvent under reduced pressure afforded an oily residue (83.3 g), that was submitted to fractionation according to Scheme 1.

Brine shrimp assay
The brine shrimp (Artemia salina) lethality assay was carried out according to a described method. 10,11xture I (  1.

Methylthiolation of mixtures I-V
A 200-500 ng portion (0.4-1.0 nmol) of mixtures I-V was solubilized in 50 μL of DMDS, and 50 μL of carbon disulfide, and 300 μg (1.18 μmol) of iodine were added.The reaction mixture was kept at 60 °C for 40 h in smallvolume sealed vials.The reaction was quenched with aqueous Na 2 S 2 O 3 (3 × 10 -4 mol L -1 ).The organic phase was extracted and evaporated to dryness under a nitrogen stream.The residue was dissolved in hexane and analyzed by GCMS.

Table 1 .
Mass Spectra data of cardanols 1-31 and anacardic acid methyl esters 32-41 Δ 10,14 cardanol (30) was also identified.In alkylphenols isolated from other Anacardiaceae the length do not exceeds 17 carbons, and when only one double bond is present, the position is most frequently Δ 8 .