Summary:
The present thesis is an in-depth study of the chemical profile of C. brevifolia (Hook.f.) Elwes & A. Henry (Pinaceae) and seeks on the detailed phytochemical analysis of the secondary metabolites that appear to have been overlooked in the only prior study on this species (Creţu et al., 2013). Formerly, C. brevifolia was considered as a subspecies of C. libani A. Rich. The present study contributes to the chemotaxonomic study of C. brevifolia in comparaison to the other Cedrus species and more specifically to C. libani.
In the present investigation were carried out the following:
Phytochemical analysis, i.e. isolation and structure elucidation of the secondary metabolites from the:
- dichloromethane, methanol, methanol: H2O (5:1) needle extracts,
- cyclohexane, dichloromethane, methanol bark extracts
- cyclohexane, ethanol, ethanol: H2O: (1:1) resin extracts.
In vitro biological assays for the evaluation of the antioxidant and anti-inflammatory activities of the extracts and the isolated secondary metabolites, as cedar products have been traditionally used in several inflammation-related diseases, since antiquity.
Analysis of the volatile constituents of the resin, as well as of the heartwood and sapwood of the trunk.
Dendrochronology of Cedrus brevifolia.
In total, 40 secondary metabolites were isolated:
I. Dichloromethane extract of needles:
Eight diterpenes and one phytosterol were isolated and identified:
manoyl oxide (3), phytyl acetate (4), dehydroabietic acid (5), dehydroabietinal (6), methyl dehydroabietate (7), 9,13α-epidioxy-8(14)-abieten-18-oic acid (8), 13-hydroxy-8,11,13-podocarpatrien-18-oic acid (9), 15-hydroxy-dehydroabietic acid (10), (3β,24R)-campesterol (11)
II. Methanol extract of needles:
Eleven phenolic derivatives, one apocarotenoid and three flavonoids and one tannin were isolated and identified:
trans-p-coumaric acid (13), p-coumaroyl-4-Ο-β-D-glucoside (17), p-anisic acid (18), trans-vaginoside (19), benzoate glucoside (20), benzyl-β-D-glucoside (21), benzyl-β-D-rutinoside (22), 2-methoxy-phenyl-β-D-glucoside (23), 3,4-dimethoxyphenyl-β-D-glucoside (24), β-D- glucoside of raspberry ketone (25), 4-hydroxybenzoic acid 4-O-β-D-glucoside (26), abscisic alcohol- β-D-glucoside (12), astragalin (29), isorhamnetin 3-O-β-D-glucoside (30), taxifolin (36), (-) (2S,3R)-catechin (40)
III. Methanol: Η2Ο (5:1) extract of needles:
four flavonoids were isolated and identified:
nicotiflorin (31), kaempferide-3-O- β-rutinoside (32), tiliroside (33), syringetin-3-Ο-β-D-glucoside (34)
IV. Dichloromethane extract of bark:
One phenolic acid was isolated and identified: (E)-ferulic acid (15), which was the main secondary metabolite of the extract
V. Methanol extract of bark:
Two phenolic acids, two flavonoids and one hydrolyzed chalcone were isoalated and identified: (E)-ferulic acid (15), syringic acid (16), quercetin (28), eriodictyol (35), cilicione-b (37)
VI. Cyclohexane extract of resin:
Three diterpenes were isolated and identified:
manool (1), 3β-hydroxy-manool (2), dehydroabietic acid (5)
VII. Ethanol extract of resin:
Two phenolic compounds and two lignans were isolated and identified:
(E)-caffeic acid (14), dibutyl phthalate (27), (+) lariciresinol (38), (+) 9-acetyl-lariciresinol (39)
VIII. Ethanol: Η2Ο (1:1) of resin:
One phenolic derivative was isolated and identified: (+) lariciresinol (38)
In a previous study, Crețu et al. (2013) reported the presence only of substances 36 and 40 in C. brevifolia bark, detected by HPLC-DAD-ESI-MS.
The extracts and the isolated compounds were tested in vitro for their antioxidant and anti-inflammatory activity. The methanol, methanol: H2O (5: 1) extracts of the needles, the bark methanol extract, the ethanol and ethanol: H2O (1: 1) resin extracts showed positive antioxidant effects in all biological tests. In particular, the bark methanol extract, the resin ethanol and the ethanol: H2O (1:1) extracts interacted with the free radical DPPH and exhibited better reducing ability compared to the reference compound (NDGA) 93 %, 89% and 85%, respectively. Moreover, all of them presented strong inhibition of the linoleic acid induced sodium peroxidation (AAPH) at 97% 100% and 84% compared to the reference compound (trolox). As for the anti-inflammatory activity based to the inhibition of soybean lipoxygenase (LOX), the bark methanol extract was the most active (80%). The isolated compounds submitted to the same biological assays exhibited different levels of bioactivity. Among them, 14, 17, 40, 28, 35, 36, 37, 38, 39 showed remarkable interaction with DPPH, while 1, 14, 17, 35, 39 were the most potent against LOX inhibition. Substances 17, 38, 39 exhibited inhibition of AAPH-induced sodium hydroxide peroxidation. The results corroborate to the ethnopharmacological uses of the plant.
Qualitative and quantitative analysis of the volatile constituents by GC-MS of the trunk segments, as well as of the resin, revealed the presence of sixteen compounds in the heartwood, fourteen compounds in sapwood covering 93.2% and 99.5%, respectively, while seven compounds were found in the resin in percentage of 99.9% of its total volatile constituents, all of them belonging to the terpenoids.
During tree dating, a thick disc of C. brevifolia wood was examined. Being only 52 years old and in comparison, to the age that Cedrus species can reach, was rather young when cut.
The last two processes, the dendrochronology and the qualitative and quantitative analysis of the volatile constituents of the trunk and resin, were performed in order to correlate its age with the biosynthesis of volatile secondary metabolites.
Keywords:
Cedrus brevifolia, phytochemical investigation, needles, bark, resin, terpenoids, sterols, apocarotenoids, phenolic acid, flavonoids, lignans, tannins, NMR, anti-oxidant activity, anti-inflammatory activity, essential oils, GC-MS, dendrochronology
