Composition and Bioactivity of Lipophilic Metabolites from Needles and Twigs of Korean and Siberian Pines (Pinus koraiensis Siebold & Zucc. and Pinus sibirica Du Tour)

Lipophilic extractive metabolites in different parts of the shoot system (needles and defoliated twigs) of Korean pine, Pinus koraiensis, and Siberian pine, Pinus sibirica, were studied by GC/MS. Korean pine needles comprised mainly bornyl p‐coumarate, heterocyclic 15‐O‐functionalized labdane type acids (lambertianic acid), 10‐nonacosanol, sterols and their esters. While Siberian pine needles contained less bornyl p‐coumarate, lambertianic acid, sterols and their esters, but were richer in other 15‐O‐functionalized labdane type acids. The major components of the twig extract of P. koraiensis were lambertianic acid, abietane and isopimarane type acids, cembrane type alcohols, 8‐O‐functionalized labdanoids, sterols, sterol esters, and acylglycerols. The same extract of P. sibirica differed in larger amounts of other 15‐O‐functionalized labdane type acids and pinolenic acid glycerides, but in less quantities of cembranoids and 8‐O‐functionalized labdanoids. The labdane type pinusolic acid was detected for the first time in Korean pine. P. koraiensis was found to be unique in the genus for an ability to synthesize phyllocladane diterpenoids. The content of bound Δ⁵‐unsaturated polymethylene‐interrupted fatty acids in the twig extracts of the both pines was similar or superior to that in their seed oil. Among the pines’ metabolites tested isocembrol was strongest in inhibition of both α‐glucosidase (IC₅₀ 2.9 μg/ml) and NO production in activated macrophages (IC₅₀ 3.6 μg/ml).     

Lipophilic Extracts from Needles and Defoliated Twigs of Pinus pumila from Two Different Populations

Hexane extracts of needles and defoliated twigs of Pinus pumila (Pall. ) Regel from two distant populations, located in the southwest and the east (i.e., Lake Baikal region and Sakhalin Island) of the species distribution range were studied by GC/MS analysis. Composition and retention indices of major components were determined. A drastic composition divergence for the extracts of P. pumila needles and defoliated twigs, depending on growth location, was established. Needle extracts from the eastern population sample contained mainly labdane‐type acids (anticopalic acid derivatives), whereas the predominant components of needle extracts from the other population sample were abietane‐type acids (abietic, neoabietic acids) and isopimarane‐type diterpenoids (sandaracopimaric acid, sandaracopimaradien‐3β‐ol). The main components of defoliated twig extracts from Sakhalin Island population sample were abietane‐type acids and cembrane‐type diterpenoids, while content of these compounds in the extracts of the southwestern marginal population sample was remarkably lower.     

Lipids and sterols of Pinus sylvestris L. sapwood and heartwood

Summary The lipid and sterol content and composition of three lipid fractions (free fatty acids/ sterols, triacylglycerols and sterol/triterpenoid esters) extracted from three stem discs of Pinus sylvestris were assessed to investigate metabolic changes related to heartwood formation. The wood was separated into (1) cambial zone, (2) outer sapwood, (3) inner sapwood, (4) transition zone, (5) outer heartwood and 6) inner heart-wood. The fractions were separated by thin-layer chromatography (TLC) and analysed by gas-liquid chromatography (GLC). The amount of fatty acids of sapwood triacylglycerols was about 1.5% (dry wt.) but a large reduction occurred in the transition zone. In contrast, noticeable amounts of free fatty acids were present only in the heart-wood. The most important fatty acids in the sapwood fractions were 16:0, 18:0, 18:1, 18:2 (the dominant fatty acid in all fractions), 18:3 and 20:3. Together 18:1 and 18:2 formed about 70% of the total triacylglycerol fatty acids. Of the sterol/ triterpenoid esters, 18:2 and 18:3 were predominant. The fatty acid composition of all fractions changed in the transition zone. The sterols found were sitosterol, stigmastanol, campesterol and campestanol. The amount of sterol esters increased towards the heartwood, and the amount of free sterols was lowest in the inner sapwood. Sitosterol was the dominant sterol in both free sterols and sterol esters.     

Pliocene white pine (Pinus subgenus Strobus) needles from western Yunnan,southwestern China

Abstract

The fossil records of Pinus L. are abundant since the Late Cretaceous, especially for the subgenus Pinus L. (the hard pines). However, those of the subgenus Strobus (D. Don) Lemmon (the white or soft pines) are not well documented. In this study, we describe a new species of white pines, Pinus plioarmandii sp. nov., from the Pliocene of western Yunnan, southwestern China. This species mostly resembles extant P. armandii Franch. in gross morphology and cuticular structure of needles. Molecular dating showed that the Asian white pines split into two lineages around the Late Miocene, and the Pinus armandii clade diverged at the Early Pliocene. The present fossil occurrence indicates that Pinus armandii might originate in southwest China and probably in the western Yunnan. The glacial events during the Pleistocene might be the major factors for the retreat of the white pines from western Yunnan, and the rapid uplift of the Yunnan Plateau and deep incision of the river valleys since the Pliocene had posed a significant geographic barrier for their reconnection in the subsequent warm climate condition.     

Sterols in Germinating Seeds and Developing seedlings of Longleaf Pine, Pinus palustris

Sterols in germinating embryos and young seedlings of longleaf pine (Pinus palustris Mill.) were identified and quantities determined for different periods after germination. Sterol analyses were performed by gas-liquid chromatography (GLC) and verified by combination of GLC-mass spectrometry. Campesterol and β-sitosterol were two major sterols which accounted for most of the sterol composition while stigmasterol was present in very small amounts. No cholesterol was revealed by GLC-mass spectrometry although there was a minor peak appearing on the sterol gas-liquid chromatograms with a retention time close to that of authentic cholesterol. By fractionation, three different forms of sterols were obtained: steryl esters, steryl glycosides, and free sterols. The sterols were mainly found in the esterified fraction, while steryl glycosides and free sterols only made up a small portion of the total sterol value. The total sterol content in general increased during seedling development, and this increase reflected mainly a change in steryl esters. The low levels of both free and glycosidic sterols remained nearly unchanged throughout the experimental germination period.     

Free and bound sterols in seedlings of Cucumis sativus

Free sterols, sterol esters, sterol monoglucosides and sterol acylmonoglucosides have been obtained from 10 days old seedlings of Cucumis sativus. Free sterols and sterol esters consist mainly of Δ⁷ di- and triunsaturated sterols, whereas Δ⁷ mono-unsaturated and Δ⁵ mono- and diunsaturated sterols predominate in the glucosides and acylglucosides. Both acetates and derivatives of higher fatty acids, mainly linoleic and linolenic acids, have been found in the sterol esters. Sterol acylglucosides contain mostly saturated fatty acids, palmitic and stearic acids being the main components.     

Diterpene resin acids from the needle oleoresin of pinus strobus

The resin acid components of P. strobus needles were isolated, and the major constituents identified as labdenoic diterpenes. In addition to anticopalic and strobic acids previously reported in the needles, 3-oxoanticopalic, 3β-acetoxyanticopalic, 3β-hydroxyanticopalic acids and the 8α-hydroxy derivative of anticopalic acid were found along with two new compounds. The structures of the two new compounds, a cycloanticopalic acid and an abcoanticopalic acid, were determined by NMR. The composition of the diterpene resin acids was obtained for several samples of P. strobus needles from provenance tests.     

Characterization of two chemotypes of <Emphasis Type="Italic">Pinus pinaster</Emphasis> by their terpene and acid patterns in needles

The existence of two chemotypes of Pinus pinaster, on the basis of the chemical composition of the resin acids in their needles, is known. An investigation was performed on 54 samples of needles of Spanish Pinus pinaster to study the differences between these chemotypes on the basis of monoterpene, sesquiterpene, neutral diterpene, fatty acid, and resin acid composition. One-hundred and twelve compounds were identified by GC–FID and GC–MS. Statistical analysis of the results established the existence of two groups or chemotypes, in the ratio of 5:1. In one chemotype, total acid compounds were more abundant than neutral compounds, whereas in the other the concentrations of both neutral and acid compounds were similar. Distinction of the chemotypes was based on the presence/absence of a sesquiterpene (germacrene d-4-ol acetate), neutral diterpenes (8(14),13(15)-abietadiene, anticopalol, an isomer of anticopalol, and pimarol), fatty acids (10-octadecenoic, 14-hydroxy-10-octadecenoic, and 13-hydroxy-9-octadenoic acids and an unidentified fatty acid), and resin acids (levopimaric + palustric, eperuic, and anticopalic acids, and three isomers of anticopalic acid); and on the different relative percentages of other compounds of these types. This study gives a wide view of the composition of the needles of Pinus pinaster, improving the differentiation of chemotypes on the basis of terpene and acid composition.     

Chemotaxonomic Implications of the n‐Alkane Composition and the Nonacosan‐10‐ol Content in Picea omorika,Pinus heldreichii,and Pinus peuce

The n‐alkane composition and the nonacosan‐10‐ol content in the needle cuticular waxes of Serbian spruce (Picea omorika), Bosnian pine (Pinus heldreichii), and Macedonian pine (Pinus peuce) were compared. The amount of nonacosan‐10‐ol in the needle waxes of P. omorika was higher than those in P. heldreichii and P. peuce. The range of n‐alkanes was also wider in P. omorika (C₁₈–C₃₅) than in P. heldreichii and P. peuce (C₁₈–C₃₃). The dominant n‐alkanes were C₂₉ in the needle waxes of P. omorika, C₂₃, C₂₇, and C₂₅ in those of P. heldreichii, and C₂₉, C₂₅, C₂₇, and C₂₃ in those of P. peuce. The waxes of P. omorika contained higher amounts of n‐alkanes C₂₉, C₃₁, and C₃₃, while those of P. heldreichii and P. peuce had higher contents of n‐alkanes C₂₁, C₂₂, C₂₃, C₂₄, and C₂₆. The principal component analysis of the contents of nine n‐alkanes showed a clear separation of the Serbian spruce populations from those of the two investigated pine species, which partially overlapped. The separation of the species was due to high contents of the n‐alkanes C₂₉ and C₃₁ (P. omorika), C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, and C₂₄ (P. heldreichii), and C₂₈ (P. peuce). Cluster analysis also showed a clear separation between the P. omorika populations on one side and the P. heldreichii and P. peuce populations on the other side. The n‐alkane and terpene compositions are discussed in the light of their usefulness in chemotaxonomy as well as with regard to the biogeography and phylogeny of these rare and endemic conifers.     

Sterols of the heterotrophic dinoflagellate,pfiesteria piscicida (dinophyceae): is there a lipid biomarker?1

Within U.S. waters, blooms of the dinoflagellate, Pfiesteria piscicida, have been recorded on an almost regular basis in the Chesapeake Bay and surrounding mid‐Atlantic regions for the last two decades. Despite the apparent significance of such blooms to the environment and human health and the attendant economic consequences, little work has addressed the physiology and biochemistry, particularly that of sterol composition, of P. piscicida. GC‐MS characterization of trimethylsilyl ether derivatives of sterols from free sterol and sterol ester fractions was performed in an effort to determine whether P. piscicida produces unique sterols that may serve as potential biomarkers. This characterization revealed that like most dinoflagellates, the majority of sterols was present as free sterols. Furthermore, the profile of free sterols was found to resemble those of photosynthetic dinoflagellates, with the dominant compound being the previously reported dinoflagellate sterol, dinosterol. A number of other 4α‐methyl‐substituted sterols and steroidal ketones common to other dinoflagellates were also identified. No strong candidate(s) for a unique sterol biomarker was present.     

Secondary Metabolites from Pinus mugo Turra subsp. mugo Growing in the Majella National Park (Central Apennines,Italy)

In this study, we examined the composition regarding secondary metabolites of P. mugo Turra ssp. mugo growing in the protected area of Majella National Park, which is the southernmost station of the habitat of this species. Both the nonpolar and polar fractions were considered. In particular, the essential‐oil composition showed a high variety of compounds, and 109 compounds were detected, and 101 were identified, among which abietane‐type compounds have a taxonomic relevance. Abietanes were also isolated from the polar fraction, together with an acylated flavonol and a remarkably high amount of shikimic acid.     

Sterols of the Toxic Marine Dinoflagellate,Pyrodinium bahamense

Pyrodinium bahamense is a dinoflagellate of concern in subtropical and tropical coastal environments. To date, there is only a single published study on its fatty acids, but no published data on its sterol composition. Sterols, which are membrane‐reinforcing lipids in eukaryotes, display a great diversity of structures in dinoflagellates, with some serving as chemotaxonomic markers. We have examined the sterol compositions of two isolates of P. bahamense from Indian River Lagoon and Tampa Bay, Florida, and have found both to produce three sterols: cholesterol, dinosterol, and 4α‐methylgorgostanol. All three sterols are found in closely related, armored taxa.     

Concentration gradients of free sterols,steryl esters and lipid phosphorus in the trunkwood of Scot's pine (Pinus sylvestris L.)

The amounts of free sterols, steryl esters and lipid phosphorus were determined in the sapwood and heartwood of mature, and in the outer and inner sapwood of young Pinus sylvestris trees. In the mature trees (up to 70 years old) the heartwood contains significantly higher amounts of free sterols than the sapwood. No radial gradient can be demonstrated in the amounts of steryl esters. Lipids extracted from the sapwood contain higher amounts of phosphorus than those from the heartwood. Stems of young Pinus sylvestris trees (up to 13 years old) show in the inner sapwood higher amounts of both free sterols and steryl esters than the peripheral younger wood zone. The inner sapwood of the young stems shows slightly higher amounts of lipid phosphorus than the outer sapwood. The results indicate that Pinus sylvestris accumulates both free sterols and steryl esters in the stems at a very early stage of the life cycle. Sterol accumulation in the innermost parts of the stems seems not to depend on heartwood formation.     

Morphological and Chemical Differentiation between Tunisian Populations of Pinus halepensis,Pinus brutia,and Pinus pinaster

The lipid fraction of seeds from different pine species and populations was studied regarding total lipid content, fatty acid profile and vitamin E composition. The investigated seeds contained a high percentage of lipid (13.6 to 31.5 %). Lipid fractions were found to be rich in vitamin E, which varied significantly among species and populations. P. halepensis (Ph−Hn) showed the highest content of vitamin E (256.3 mg/kg of seeds) and the uppermost content of α-tocopherol (44 mg/kg). However, P. halepensis (Ph−Kas) was the richest in γ-tocopherol (204.9 mg/kg). Lipid fractions had a low content of δ-tocopherol (1.2 to 3.6 mg/kg. The highest content of γ-tocotrienol (∼18 %) was determined for P. halepensis (Ph−Dc and Ph−Hn). Thirteen fatty acids were identified by GC-FID with significant variation between the investigated species. The linoleic acid was the major fatty acid followed by oleic acid and palmitic acid. The chemical differentiation among species for the composition of fatty acids and vitamin E was confirmed by PCA. Significant correlations were observed between the content of vitamin E and fatty acids and ecological parameters of P. halepensis populations.     

基于SRAP标记的紫溪山华山松种子园无性系遗传多样性分析

为明确云南楚雄市紫溪山华山松种子园内不同种源无性系间的遗传背景,该研究收集了园内6个种源的60个华山松无性系单株针叶,采用改良CTAB法提取其总DNA,并利用SRAP分子标记对其进行遗传多样性分析。结果表明:(1)从100对引物组合中共筛选出15对具有多态性的SRAP引物,经SRAP-PCR扩增后,共获得出194个位点,多态位点百分率(PPB)为85.05%,Nei’s基因多样性指数(H)为0.233 7,Shannon’s信息指数(I)为0.341 9,种源间的遗传分化系数(GST)为0.355 5。(2)华山松6个种源遗传多样性较高,且遗传变异主要存在于华山松种源内,种源地会泽(HZ)与巍山(WS)种源的遗传距离最近(D=0.050 1),会泽(HZ)与宜良(YL)种源的遗传距离最远(D=0.361 8)。(3)聚类分析显示将6个华山松种源一共聚为3类:会泽(HZ)和巍山(WS)种源聚为一类;楚雄(CX)、南华(NH)和宜良(YL)种源聚为一类;腾冲(TC)种源单独为一类。综合上述结果显示,紫溪山华山松种子园内无性系间的遗传分化处于较高水平,为华山松杂交育种时亲本的选配及种质资源的...     

Population Variability of Nonacosan‐10‐ol and n‐Alkanes in Needle Cuticular Waxes of Macedonian Pine (Pinus peuce Griseb.)

This is the first report on population variability of nonacosan‐10‐ol and n‐alkanes in needle epicuticular waxes of Macedonian pine (Pinus peuce Griseb .) Hexane extracts of needle samples, originating from two natural populations in Montenegro (Zeletin and Sjekirica) and from one population in Serbia (Mokra Gora) were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The amount of nonacosan‐10‐ol varied individually from 41.3 to 72.31% (average 55.9%), with the Sjekirica population being statistically divergent (64.4% on average). The results showed n‐alkanes in epicuticular waxes ranging from C₁₈ to C₃₃. The most abundant alkanes were C₂₉, C₂₅, C₂₇, and C₂₃ (15.5, 11.1, 10.6, and 10.5% on average, resp.). The carbon preference index of Pinus peuce ranged from 1.0 to 4.3 (1.9 on average). Average chain length ranged from 18.4 to 27.7 (average 25.7). A high level of inidividual quantitative variation in all of these hydrocarbon parameters was also detected. These results were compared with published data on other species from the Pinus genus.     

Effects of previous defoliation on pine looper larval performance

1 During outbreaks of the pine looper, Bupalus piniarius, its host, Pinus sylvestris, is severely defoliated. The larvae of this geometrid normally feed on mature needles. However, because trees are totally defoliated during outbreaks, the next generation is forced to feed on current-year needles. 2 Bupalus piniarius larvae feeding on previously defoliated trees may show lower performance either because of induced resistance or because larvae have to feed on needles not normally fed upon (current instead of mature). 3 These hypotheses were tested in an experiment where larvae were reared on (i) shoots naturally defoliated the previous year, and thus, bearing only current-year needles, (ii) non-defoliated shoots where larvae had access only to current-year needles, and (iii) control shoots with access to both current and mature needles. 4 There was no support for the induction hypothesis. Survival was lower on naturally defoliated shoots than on control shoots (81.3 vs. 90.9%), but survival was lower also on non-defoliated shoots where larvae had access only to current-year needles (78.8%). Data on larval feeding distribution showed a strong preference for mature needles. 5 Needle nitrogen concentration of current-year needles was 38% higher on defoliated trees than on non-defoliated trees. 6 It is concluded that defoliation affected larval performance primarily through the removal of the preferred type of needles and not because of an induced resistance. Effects of increased concentrations of allelochemicals in defoliated shoots, if present, were probably cancelled out by increased nitrogen concentrations.     

Reduced realised fecundity in the pine looper Bupalus piniarius caused by host plant defoliation

1. Whether the potential fecundity of herbivorous insects is realised or not may depend on female behaviour, which in turn may be influenced by host plant acceptability. Female Bupalus piniarius were observed to discriminate against needles growing out the year following defoliation (current‐year needles) of its host plant Pinus sylvestris. 2. It was hypothesised that the discriminatory behaviour was due to current‐year needles being less secure as a substrate. Field and laboratory experiments were designed to test this hypothesis and to estimate the discrepancy between potential and realised fecundity when females were offered defoliated branches. 3. In a laboratory oviposition experiment, B. piniarius females were exposed to branches bearing either current‐year needles only or both mature and current‐year needles. Daily oviposition rate, egg batch size, longevity, and mature eggs and fat retained at death were recorded for each female. In field experiments, the rate at which eggs dropped from expanding needles and the capacity of neonate larvae hatching from the dropped eggs to colonise a tree were assessed. 4. Significantly fewer eggs were laid when females were exposed to defoliated branches. 5. Twenty‐six and 16% of the eggs laid on current‐year needles dropped from the needles in 1998 and 1999 respectively, whereas no eggs dropped from mature needles in 1998 and only one egg (< 1%) dropped in 1999. 6. A very small proportion of larvae hatching on the forest floor (simulated egg drop) was able to recolonise host trees. 7. These results emphasise the importance of oviposition behaviour on realised fecundity when analysing insect population dynamics. In the case of B. piniarius, egg placement, although a minor detail during the normal course of events, became of key importance when defoliation deprived females of their preferred egg attachment site.     

Chemodiversity of Nonacosan‐10‐ol and n‐Alkanes in the Needle Wax of Pinus heldreichii

This is the first report of individual variability and population diversity of the contents of nonacosan‐10‐ol and n‐alkanes in the needle cuticular waxes of Bosnian pines originated from Montenegro, regarded as Pinus heldreichii var. leucodermis, and from Serbia, regarded as P. heldreichii var. pan?i?i. The amount of nonacosan‐10‐ol varied individually from 27.4 to 73.2% (55.5% in average), but differences between the four investigated populations were not statistically confirmed. The size of the n‐alkanes ranged from C₁₈ to C₃₃. The most abundant n‐alkanes were C₂₃, C₂₇, and C₂₅ (12.2, 11.2, and 10.8% in average, resp.). The carbon preference index (CPI) of the n‐alkanes ranged from 0.8 to 3.1 (1.6 in average), while the average chain length (ACL) ranged from 20.9 to 26.5 (24.4 in average). Long‐chain and mid‐chain n‐alkanes prevailed (49.6 and 37.9% in average, resp.). It was also found that the populations of P. heldreichii var. leucodermis had predominantly a narrower range of n‐alkanes (C₁₈? C₃₁) than the trees of the variety pan?i?i (C₁₈? C₃₃). Differences between the varieties were also significant for most of the other characteristics of the n‐alkane pattern (e.g., most abundant n‐alkanes, CPI, ACL, and relative proportion of short‐, mid‐, and long‐chain n‐alkanes). The principle component and cluster analyses of eleven n‐alkanes confirmed the significant diversity of these two varieties.     

Lipids in roots of Pinus sylvestris seedlings and in mycelia of Pisolithus tinctorius during ectomycorrhiza formation: changes in fatty acid and sterol composition

The composition of fatty acids and sterols in soil lipid fractions is often used as a global indicator for the status and changes of soil microbial communities. In order to validate such analyses in the context of ectomycorrhizal communities, an experiment was performed in which seedlings of Pinus sylvestris and the fungus Pisolithus tinctorius were grown separately, or combined to form ectomycorrhiza under axenic conditions. Fatty acids of the neutral lipid fraction (NLFAs) and the phospholipid fraction (PLFAs) as well as sterols were identified and quantified by gas chromatography–mass spectrometry. When grown separately, the two organisms differed strongly with respect to the sterol composition. Sterols had a much higher relative abundance in the fungus in comparison with the plant, and the two main fungal sterols, ergosterol and 24‐ethyllanosta‐8,24(24′)‐diene‐3beta,22zeta‐diol (Et lano 8,24), as well as six minor fungal sterols were not found in the plant. On the other hand, the three sterols found in plant roots were absent from the fungus. With regard to fatty acids, the lipids of both organisms contained the same three major PLFAs, namely n16:0, 18:2–9,12c, and 18:1–9c. However, plant lipids contained, in addition, eight PLFAs and five NLFAs that were not present in the fungus. On the other hand, the fungus contained two PLFAs and two NLFAs that were not present in the plant. When the fungus and the plant were brought together, there was a drastic change in the lipid composition of the root: within a day, all the saturated fatty acids in the NLFA fraction increased very strongly and then slowly decreased but remained at an elevated level throughout the experiment. All these saturated fatty acids also started to appear in the extraradical fungal mycelium; they increased steadily and reached their highest levels at the end of the experiment. These results indicate that in symbiosis, the fungus transports plant lipids from the symbiotic interface to the extraradical mycelium. Concerning sterols, the extraradical mycelium acquired only a small amount of plant‐specific sterols. However, its ergosterol content steadily decreased whereas the content of Et lano 8,24 remained high, causing the ratio of these two sterols to decrease from 1 : 7 to 1 : 20, whereas in the ectomycorrhizal root, the opposite phenomenon occurred, so that the ratio increased to a value of almost 1 : 1. The marked changes in the composition of the extraradical mycelium were well reflected in a principal component analysis of all lipid components. The present results show that a given ectomycorrhizal fungus may display markedly different lipid compositions in its intraradical and extraradical parts. In addition, they highlight a potential role of plant lipid transfer from the root to the fungus in the functioning of the ectomycorrhizal symbiosis.