NPC Natural Product Communications Composition and Chemical Variability of Root Bark oil from Ivoirian Cleistopholis patens 2018 Vol. 13 No. 6 767 - 770 Zana A. Ouattara*ab, Thierry A. Yapic, Yves-Alain Békrob, Akhanovna J. Mamyrbékova-Békrob, Mathieu Paolia, Pierre Tomia, Joseph Casanovaa, Ange Bighellia and Félix Tomia a Université de Corse-CNRS, UMR 6134 SPE, Equipe Chimie et Biomasse, Route des Sanguinaires, 20000 Ajaccio, France b Laboratoire de Chimie Bio-Organique et de Substances Naturelles, UFR-SFA, Université Nangui Abrogoua, 02 BP 801 Abidjan 01 c Laboratoire de Chimie Organique Biologique, UFR-SSMT, Université Félix Houphouët-Boigny, BPV 34 Abidjan, Côte d’Ivoire zana150419@gmail.com Received: July 20th, 2017; Accepted: April 10th, 2018 The chemical composition of root bark oil of Cleistopholis patens (Benth.) Engl. & Diels from Côte d’Ivoire was determined by GC (in combination with retention indices), GC-MS and 13C NMR. The contents of the major compounds varied drastically from sample to sample: patchoulenone (0-70.5%), β-pinene (0-51.9%), bornyl acetate (0.5-31.2%), α-pinene (0.2-25.7%), juvenile hormone III (0.3-22.2%) and β-elemol (0-18.8%). Three chemical compositions may be differentiated, dominated by i) patchoulenone, ii) β-pinene and α-pinene, iii) juvenile hormone III, accompanied by bornyl acetate or β-elemol. 13C NMR data of patchoulenol (3-patchoulen-5-exo-ol) are reported. Keywords: Patchoulenone, Patchoulenol, Juvenile hormone III, β-Pinene, Chemical variability, 13C NMR. Cleistopholis is a small genus belonging to Annonaceae family and comprising three species: C. staudii, C. glauca and C. patens, present in tropical forest of Western and Central Africa. C. patens (synonyms: C. klaineana, C. pynaertii, C. lucens, C. verschuereni, C. brevipetala and Oxymitra patens) [1], is widely distributed from Senegal to Uganda and Democratic Republic of Congo and Cabinda (Angola). It is frequent in swampy forests, as well as in secondary forests. It prefers flat, disturbed and humid areas [2]. In Côte d’Ivoire, C. patens is present from the Southern forests to Northern forest galleries. In traditional medicine, decoction of the bark is used against stomach aches, diarrhea, tuberculosis, bronchitis and hepatitis. Bark pulp is employed to swellings, edemas and panaris, and bark juice is prescribed in nasal instillation to treat headaches, and in friction against rickets in children. Works carried out on solvent extracts of C. patens led to isolation and structure elucidation of several secondary metabolites: terpenes [3,4], alkaloids [3,5-7] and oligorhamnosides [8]. Few studies reported on the chemical compositions of C. patens essential oils: - The chemical compositions of essential oils isolated from various organs from Nigerian C. patens were characterized by the following main components: root oil, bornyl acetate and α-cadinol (content not mentioned) [9]; trunk bark oil, p-cymene (13.4%), germacrene D (12.5%) and myrcene (12.0%); fruit oil, linalool (23.1%) transand cis-linalool oxides (tetrahydrofuran) (17.7 and 17.0%, respectively); leaf oil, (E)-β-ocimene (31.0%), (E)-β-caryophyllene (12.8%) and linalool (8.5%) [10]. - C. patens from Cameroon produced sesquiterpene-rich essential oils: trunk bark oil, δ-cadinene (28.7%), α-copaene (16.9%) and germacrene B (7.4%); leaf oil, (E)-β-caryophyllene (17.5%), germacrene D (16.1%) and germacrene B (16.0%) [11]. - More recently, we got interested in the composition of essential oils isolated from various parts of Ivoirian C. patens. The contents of the major components of leaf and trunk bark oils varied drastically from sample to sample. Therefore, statistical analysis, hierarchical clustering and principal components analysis, carried out on the compositions evidenced a fair chemical variability of the leaf oil (48 samples) and stem bark oil (36 samples) of this species. The 48 leaf oil samples were partitioned into three groups. The oil composition of the main group (group I, 33 samples) was dominated by (E)--caryophyllene and linalool. The oils of group II (eight samples) contained mainly -pinene and -pinene, while those of group III (seven samples) were dominated by sabinene, limonene and -phellandrene [12]. In parallel, three clusters have been distinguished within the compositions of 36 oil samples isolated from trunk bark. The composition of group I (ten samples) was dominated by β-pinene and α-pinene, group II (nine samples) was represented by α-phellandrene and p-cymene and group III (16 samples) by β-elemol [13]. In addition we reported on the identification and quantification of germacrene A, B and C in leaf and trunk bark oil of C. patens using a combination of GC(FID) and 13 C NMR. Indeed, we observed that these three heat-sensitive compounds partially or totally rearranged to -, - and -elemene, respectively, during GC analysis [14]. In the continuation of our work on the characterization of C. patens [12-14], as well as other aromatic plants of Côte d’Ivoire [15,16], the aim of this study was to determine the chemical composition of C. patens root bark oil and to evidence homogeneity or a chemical variability. Nine oil samples obtained by hydrodistillation (yield = 0.18-0.23%) of root bark harvested from individual trees of C. patens have been analyzed by GC (in combination with retention indices on two columns of different polarity) and by 13C NMR. Samples A, D, F and G have also been submitted to GC-MS analysis. In total, 55 compounds have been identified accounting for 78.8% to 95.8% of the total composition. 768 Natural Product Communications Vol. 13 (6) 2018 Ouattara et al. Table 1: Chemical composition of nine C. patens root bark oil samples. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Compounds a) α-Pinene Camphene Sabinene β-Pinene Myrcene α-Phellandrene -3-Carene p-Cymene Limonene* 1,8-Cineole* β-Phellandrene* γ-Terpinene Terpinolene Linalool Camphor Citronellal Terpinen-4-ol α-Terpineol Citronellol Geraniol Thymol Bornyl acetate Methyl geraniate δ-Elemene α-Copaene β-Elemene Cyperene (E)-β-Caryophyllene -Copaene trans--Bergamotene (E)--Farnesene Rotundene γ-Muurolene Germacrene D β-Selinene -Bisabolene γ-Cadinene* (Z)--Bisabolene* δ-Cadinene β-Elemol 5-endo-Methoxy-3-patchoulene Caryophyllene oxide 3-Patchoulen-5-endo-ol Patchoulenone Alismol τ-Cadinol -Muurolol β-Eudesmol α-Cadinol α-Eudesmol -Bisabolol -Bisabolol Methyl (2E,6E)-farnesoate Juvenile hormone III Methyl 10-oxo-(2E,6E)-farnesoate Total RIa b) 927 940 961 968 976 997 1001 1011 1017 1017 1017 1044 1075 1079 1117 1127 1157 1168 1204 1230 1263 1265 1296 1331 1372 1384 1396 1413 1425 1431 1446 1453 1465 1471 1476 1499 1506 1506 1513 1530 1541 1565 1590 1595 1606 1620 1627 1630 1633 1635 1654 1665 1756 1857 1884 RIp c) 1023 1067 1121 1113 1159 1169 1148 1270 1200 1208 1211 1244 1283 1541 1512 1476 1596 1690 1759 1839 2177 1576 1692 1468 1489 1587 1525 1594 1582 1578 1661 1636 1685 1704 1710 1716 1756 1722 1753 2070 1779 1973 2171 2091 2243 2160 2189 2217 2219 2208 2145 2207 2201 2491 2573 A d) 25.7 1.2 0.7 51.9 2.8 0.3 0.2 0.6 0.8 0.6 0.4 0.2 0.1 0.4 0.9 1.0 0.5 0.7 0.4 1.8 1.1 1.8 0.1 0.1 0.3 0.6 0.1 0.2 0.3 - B 23.7 4.9 0.7 46.8 1.8 1.3 0.6 0.9 1.1 0.1 0.5 0.2 0.4 0.1 tr 0.2 0.5 - 95.8 1.6 0.1 0.4 2.6 1.0 0.3 0.4 0.1 0.2 2.5 0.5 0.1 0.1 0.7 0.1 C 13.5 0.8 0.4 31.9 0.8 0.1 0.1 0.2 0.5 0.1 0.2 0.1 0.1 0.3 3.6 0.4 0.7 3.1 8.9 1.0 0.6 0.3 0.1 0.7 0.9 1.0 2.7 0.2 0.7 0.2 0.5 9.1 0.8 0.3 0.4 0.7 0.3 0.6 8.1 - D 12.6 7.5 0.4 28.9 2.2 3.2 1.1 1.0 1.6 0.5 0.3 0.9 0.1 0.3 0.8 1.3 6.3 0.3 0.6 0.9 7.4 1.9 1.0 0.5 0.7 0.5 0.4 0.1 0.2 3.8 - E 0.2 0.3 0.1 0.2 0.1 0.1 0.5 0.8 0.6 0.1 0.1 0.1 - F 0.6 4.9 0.4 0.1 0.2 0.2 0.4 0.7 0.6 0.2 - 94.5 95.0 6.9 0.2 1.8 0.8 1.3 4.3 1.7 0.8 4.1 0.4 0.3 18.8 3.6 0.5 3.2 0.7 1.2 4.8 0.7 0.8 0.5 0.2 22.2 1.1 G 0.2 1.0 0.2 0.1 0.6 0.1 0.3 0.2 0.1 tr 0.1 tr 4.3 1.7 3.3 0.3 1.3 5.9 1.0 1.0 2.7 4.1 0.3 7.4 0.8 0.9 0.1 2.0 2.4 23.6 0.2 1.0 2.2 1.3 4.0 1.2 0.3 1.0 1.6 H 0.2 0.1 0.2 0.1 0.3 0.6 0.4 0.2 2.6 1.2 0.2 0.9 3.3 0.6 0.3 0.7 0.3 0.3 0.4 0.5 0.8 0.2 0.7 65.7 2.6 0.2 0.9 0.3 0.3 1.0 - 31.2 1.3 0.4 1.1 6.9 2.7 1.2 0.7 1.0 0.5 2.9 4.4 1.0 2.2 2.0 9.5 0.1 0.4 1.6 15.8 0.9 88.9 91.0 I 0.4 3.2 0.1 0.1 0.3 0.2 2.1 0.1 1.0 - 0.2 0.1 0.7 0.6 0.2 1.8 0.1 1.0 0.1 0.6 2.9 0.5 0.3 0.2 0.4 0.3 0.6 0.5 0.2 0.5 70.5 1.3 0.3 0.6 0.1 0.6 0.4 89.2 78.8 96.1 91.5 Identification RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS RI, MS RI, MS, 13C NMR RI, MS RI, MS, 13C NMR RI, MS, 13C NMR RI, MS RI, MS, 13C NMR 13 RI, C NMR 13 RI, C NMR 13 RI, MS, C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR 13 RI, C NMR RI, MS, 13C NMR 13 RI, C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR RI, MS, 13C NMR a ) Order of elution and percentages of individual components are given on apolar column (BP-1), except those with an asterisk (percentage on polar column (BP-20) b) RIa retention indices determined on the apolar column (BP-1). c) RIp retention indices determined on the polar column (BP-20). d) Location of harvest: Adiopodoumé, samples A, B and D; Abengourou, samples C and F; Abidjan, sample E; Petit Yapo, samples G, H and I. As previously observed for leaf and trunk bark oil samples [12,13], the content of major components varied drastically from sample to sample, patchoulenone 44 (0-70.5%), β-pinene 4 (0-51.9%), bornyl acetate 22 (0.5-31.2%), α-pinene 1 (0.2-25.7%), juvenile hormone III 54 (0.3-22.2%) and β-elemol 40 (0-18.8%). Other components were present at appreciable contents: τ-cadinol 46 (up to 9.5%), geraniol 20 (up to 8.9%), cyperene 27 (up to 7.4%), camphene 2 (up to 7.5%), 5-endo-methoxy-3-patchoulene 41 (up to 4.4%), germacrene D 34 (up to 7.4%), citronellal 16 (up to 3.6%), 3patchoulen-5-endo-ol (iso-patchoulenol) 43 (up to 3.2%) and citronellol 19 (up to 3.1%). Juvenile hormone III 54 and methyl (2E,6E)-farnesoate 53 are insect hormones scarcely found in essential oil and already reported in C. patens trunk bark oil [13]. Two components with patchoulane skeleton 41 and 43 retained our attention (Figure 1). Indeed, 5-endo-methoxy-3-patchoulene 41 has recently been isolated from leaf oil of Xylopia aethiopica (Dunal) A. Rich. and its structure elucidated [17]. It accounted for 0.2-4.4% in five oil samples, out of nine. In parallel, compound 43 with RIs = 1590/2171, 0.5-3.2% in oil samples E, F and I, remained unidentified after GC-MS and 13C NMR analysis, being absent in MS (commercial and in-house) and 13C NMR (in-house) libraries at our disposal. 13C NMR spectra of samples E and F displayed a Natural Product Communications Vol. 13 (6) 2018 769 Ivoirian Cleistopholis patens root bark oil root bark oil displayed a fair chemical variability as previously observed for leaf oil and trunk bark oil [12,13]. From the analytical point of view, combined analysis of essential oils by GC(RI), GCMS and 13C NMR appeared useful. 1 3 5 HO HO Figure 1: Structure and stereochemistry of patchoulenol (3-patchoulen-5-exo-ol, left) and iso-patchoulenol (3-patchoulen-5-endo-ol, right) 43. series of 15 unassigned signals, with intensities in agreement with components that accounted for 2-3% of the whole composition. The 15 chemical shift values appeared very close to those of 5-endomethoxy-3-patchoulene 41 and owing to the lack of methoxy signal (57.69 ppm), the unidentified component could be the corresponding alcohol, 5-endo-hydroxy-3-patchoulene (synonyms: 3-patchoulen-5-endo-ol, iso-patchoulenol). In order to confirm this hypothesis, part of the oil sample I that contained 70% of patchoulenone, has been submitted to LiAlH4 reduction. Column chromatography of the bulk sample allowed isolation of a “pure” compound (98.5% on GC). In contrast, the 13C NMR spectrum showed two series of signals in 1:5 ratio. Search in the computerized literature demonstrated that the chemical shift values of the major compound belonged to 3-patchoulen-5-endo-ol, isolated for the first time from rhizomes of Cyperus rotundus [18] and named patchoulan-4-en-6-ol by the authors. The minor compound is the epimer of 43, namely 3-patchoulen-5-exo-ol, previously isolated from the essential oil of Cyperus scariosus and named patchoulenol by the authors who reported its 1H NMR data [19]. 13C NMR chemical shifts are given in the experimental part. Both epimers displayed close 13C NMR chemical shift values, except for those of carbon C11, up field shifted in the endo isomer ( steric effect of hydroxyl group). Although only nine samples have been isolated and analyzed, the composition of root oil from C. patens exhibited a fair chemical variability. Indeed, the compositions of two oil samples H and I were largely dominated by patchoulenone 44, 65.7% and 70.5%, respectively (23.6% in sample G), while this ketone was present at low content or absent in the remaining samples. Four other samples, A-D, contained mainly β-pinene 4 (28.9-51.9%) and α-pinene 1 (12.6-25.7%). However, the samples differed by the contents of various components, for instance: camphene (0.8-7.5%), citronellal (0-3.6%), citronellol (0-3.1%) and geraniol (0-8.9%). The two last oil samples, E and F, contained juvenile hormone III at appreciable contents (15.8% and 22.2%, respectively) accompanied either by bornyl acetate (sample E, 32.1%, main component, 6.9% in sample F) or by -elemol (sample F, 18.8%). Both samples contained fair amounts of τ-cadinol (9.5% and 4.8%). It could be pointed out that β-pinene was also the major component of some leaf oil samples and trunk bark oil samples from Ivoirian C. patens [12,13]. Juvenile hormone III and β-elemol were also reported as main components of some samples from C. patens trunk bark oil [13]. In parallel, bornyl acetate was among the major components of root bark oil from Nigerian C. patens [9]. In contrast, the composition dominated by patchoulenone is reported here for the first time in C. patens essential oils. This ketone has been previously identified at a lesser extent (33.2%) in C. glauca root bark oil [20]. In conclusion, the composition of six Ivoirian C. patens root bark oil samples is reported here for the first time. Various chemical compositions have been observed, dominated either by patchoulenone or by β-pinene and -pinene; or by juvenile hormone III accompanied by bornyl acetate or β-elemol. Therefore, C. patens Experimental Plant material and essential oil isolation: Root bark was harvested on nine individual trees of C. patens (Benth.) Engl. & Diels, growing wild in four localities; Abengourou (C and F), Abidjan (E), Adiopodoumé (A, B and D) and Petit Yapo (G, H and I) (figure 2). The identification of the plant material was confirmed by regretted Prof. L. Aké Assi (Centre National de Floristique, Université Felix Houphouët Boigny, Abidjan). A voucher specimen has been deposited in the Herbarium of the CNF, (reference LAA 10612). Abengourou● ● Petit Yapo Abidjan Adiopodoumé ●  Figure 2: Locations of harvest of C. patens root bark. Fresh material (1.0-1.5 kg) was submitted to hydrodistillation during 3h using a Clevenger-type apparatus. The essential oil samples were dried over anhydrous Na2SO4, and then conserved in refrigerator (4-5°C). Patchoulenol and iso-patchoulenol 43: Sample I (54 mg) in dry Et2O (20mL) was added dropwise to a suspension of LiAlH4 (19 mg) in dry Et2O. The mixture was stirred at room temperature for 1 h and then a solution of 10% NaOH was added. The organic layer was separated, washed with water and dried over anhydrous MgSO4. Evaporation of the solvent under reduced pressure followed by CC (silica gel, 35–70 μm, 20 g) yielded patchoulenol epimers (40 mg, 98.5%, GC). Analyses: GC, GC-MS and 13C NMR analyses were performed as previously reported [20, 21]. Identification of individual components: component identification was based on: (a) comparison of their GC retention indices (RI) on polar and apolar columns determined relative to the retention times of a series of n-alkanes (C7-C28) with linear interpolation (Target Compounds software from Perkin Elmer) with those of authentic compounds or literature data [22], (b) on computer matching with a laboratory-made and commercial mass spectral libraries and comparison of spectra with literature data [23], (c) on comparison of the signals in the 13C NMR spectra of essential oils with those of reference spectra compiled in the laboratory spectral library with the help of laboratory-developed software [24]. In the investigated samples, individual components were identified by 13C-NMR at contents as low as 0.4 - 0.5% 13 C NMR data of 3-patchoulen-5-endo-ol 43: δC, CDCl3: 143.50 (C), 135.87 (C), 68.74 (CH), 65.00 (C), 53.61 (CH), 42.26 (CH2), 770 Natural Product Communications Vol. 13 (6) 2018 Ouattara et al. 41.37 (C), 35.39 (CH), 29.20 (CH2), 27.19 (CH2), 26.11 (CH3), 21.82 (CH2), 20.44 (CH3), 18.17 (CH3), 14.26 (CH3). (C), 34.88 (CH), 28.66 (CH2), 27.38 (CH3), 26.40 (CH2), 26.05 (CH2), 20.17 (CH3), 17.79 (CH3), 14.64 (CH3). 13 Acknowledgments - The authors wish to thank the Ministère de l’Enseignement Supérieur de Côte d’Ivoire for providing research grants to Z. A. O. and T. A. Y. C NMR data of 3-patchoulen-5-exo-ol: δC, CDCl3: 145.70 (C), 132.50 (C), 72.97 (CH), 67.47 (C), 58.31 (CH), 42.74 (CH2), 40.49 References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] Le Thomas A. (1969) Flore du Gabon, Volume 16: Annonaceae. 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