Stress-induced biosynthesis of dicaffeoylquinic acids in globe artichoke

Leaf extracts from globe artichoke ( Cynara cardunculus L. var. scolymus) have been widely used in medicine as hepatoprotectant and choleretic agents. Globe artichoke leaves represent a natural source of phenolic acids with dicaffeoylquinic acids, such as cynarin (1,3-dicaffeoylquinic acid), along with its biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the most abundant molecules. This paper reports the development of an experimental system to induce caffeoylquinic acids. This system may serve to study the regulation of the biosynthesis of (poly)phenolic compounds in globe artichoke and the genetic basis of this metabolic regulation. By means of HPLC-PDA and accurate mass LC-QTOF MS and MS/MS analyses, the major phenolic compounds in globe artichoke leaves were identified: four isomers of dicaffeoylquinic acid, three isomers of caffeoylquinic acid, and the flavone luteolin 7-glucoside. Next, plant material was identified in which the concentration of phenolic compounds was comparable in the absence of particular treatments, with the aim to use this material to test the effect of stress application on the regulation of biosynthesis of caffeoylquinic acids. Using this material, the effect of UV-C, methyl jasmonate, and salicylic acid treatments on (poly)phenolic compounds was tested in different globe artichoke genotypes. UV-C exposure consistently increased the levels of dicaffeoylquinic acids in all genotypes, whereas the effect on compounds from the same biosynthetic pathway, for example, chlorogenic acid and luteolin-7-glucoside, was much less pronounced and was not statistically significant. No effect of methyl jasmonate or salicylic acid was found. Time-response experiments indicated that the level of dicaffeoylquinic acids reached a maximum at 24 h after UV radiation. On the basis of these results a role of dicaffeoylquinic acids in UV protection in globe artichoke is hypothesized.     

Identification and quantification of caffeoylquinic acids and flavonoids from artichoke (Cynara scolymus L.) heads, juice, and pomace by HPLC-DAD-ESI/MS(n)

A method for the identification and quantification of phenolic compounds from artichoke (Cynara scolymus L.) heads, juice, and pomace by HPLC with diode array and mass spectrometric detection was developed. Among the 22 major compounds, 11 caffeoylquinic acids and 8 flavonoids were detected. Quantification of individual compounds was carried out by external calibration. Apigenin 7-O-glucuronide was found to be the major flavonoid in all samples investigated. 1,5-Di-O-caffeoylquinic acid represented the major hydroxycinnamic acid, with 3890 mg/kg in artichoke heads and 3269 mg/kg in the pomace, whereas in the juice 1,3-di-O-caffeoylquinic acid (cynarin) was predominant, due to the isomerization during processing. Total phenolic contents of approximately 12 g/kg on a dry matter basis revealed that artichoke pomace is a promising source of phenolic compounds that might be recovered and used as natural antioxidants or functional food ingredients.     

Quantitative determination of phenolic compounds in artichoke-based dietary supplements and pharmaceuticals by high-performance liquid chromatography

Dietary supplements are among the most rapidly growing products in the food and personal care market with an estimated worldwide volume exceeding $60 billion. The main problem associated with dietary supplements is their legal classification. Being neither food nor medicine, they often inhabit a gray area between the two, which makes legal regulatory extremely difficult. Thus, a coexistence of products processed from the same botanical source on the same market as dietary supplement or pharmaceutical is possible. In the present study, various artichoke-based dietary supplements were investigated for their phenolic profile and compared to artichoke phytopharmaceuticals. Quantification of individual hydroxycinnamic acids and flavonoids was carried out by external calibration. For the first time, determination of several apigenin derivatives was included. Chlorogenic acid represented the major constituent in all samples investigated with the exception of juice derived from fresh flower heads, which exhibited a higher cynarin content. Furthermore, a distinction between products made from artichoke leaves or flower heads was possible. The results obtained revealed great diversity of pharmaceuticals and dietary supplements, highlighting the need of standardized quality requirements.     

Vasomodulating potential of mediterranean wild plant extracts

The incidence of cardiovascular disease and endothelial dysfunction is low in the Mediterranean area, where the major proportion of daily calories comes from plant food, high in antioxidant polyphenols. It has been shown that a reduced production or enhanced inactivation of endothelium-derived nitric oxide (NO) is involved in the onset of endothelial dysfunction. We investigated the effects of Mediterranean wild plant, that is, wild artichoke and thyme, phenolic-rich extracts on NO release by porcine aortic endothelial cells (PAECs; by using indirect methods) and by cerebral cell membrane homogenates (by using direct NO detection). NO release by PAECs was significantly potentiated by 234% and 135% by wild artichoke and thyme extracts (10(-6) mol/L), respectively. Direct detection of NO release by brain membranes also showed significantly increased NO production after wild artichoke addition (+35.4%). Further, the release of another vasorelaxant factor by PAECs, that is, prostacyclin, was significantly increased by wild artichoke and thyme (10(-6) mol/L) (+269% and +190%, respectively). Investigation of the mechanism(s) of action of wild artichoke and thyme suggests maintenance of an intracellular reduced environment, as previously shown for ascorbate. Even though these data require in vivo confirmation, they suggest that regular intake of bioactive compounds from Mediterranean wild plants contributes to maintenance of proper vasomotion and to the low incidence of atherosclerosis and endothelial dysfunction recorded in the Mediterranean area.     

Time-resolved spectral studies of blue-green fluorescence of artichoke (Cynara cardunculus L. Var. Scolymus) leaves: identification of chlorogenic acid as one of the major fluorophores and age-mediated changes

Synchrotron radiation and the time-correlated single-photon counting technique were used to investigate the spectral and time-resolved characteristics of blue-green fluorescence (BGF) of artichoke leaves. Leaves emitted BGF under ultraviolet (UV) excitation; the abaxial side was much more fluorescent than the adaxial side, and in both cases, the youngest leaves were much more fluorescent than the oldest ones. The BGF of artichoke leaves was dominated by the presence of hydroxycinnamic acids. A decrease in the percentage of BGF attributable to the very short kinetic component (from 42 to 20%), in the shape of the BGF excitation spectra, and chlorogenic acid concentrations indicate that there is a loss of hydroxycinnamic acid with leaf age. Studies on excitation, emission, and synchronized fluorescence spectra of leaves and trichomes and chlorogenic acid contents indicate that chlorogenic acid is one of the main blue-green fluorophores in artichoke leaves. Results of the present study indicate that 20-42% (i.e., the very short kinetic component) of the overall BGF is emitted by chlorogenic acid. Time-resolved BGF measurements could be a means to extract information on chlorogenic acid fluorescence from the overall leaf BGF.     

菊芋氮磷钾吸收积累与分配特征研究

以“青芋1号”菊芋为试材,通过田间试验对菊芋全生育期的各器官内氮、磷、钾养分的吸收积累与分配特征进行了研究。结果表明,随生育期的延长菊芋各器官营养元素浓度呈下降趋势。营养元素吸收速率呈双峰曲线变化;营养元素积累总量表现为:氮钾磷。块茎形成前,营养元素主要存于茎、叶;块茎形成到块茎膨大始期,叶内营养元素分配量持续减少,块茎分配量持续增多,而茎内磷、钾则呈单峰曲线变化;块茎开始膨大后,营养元素在茎、叶的分配量均迅速减小,块茎分配量迅速增加。     

Plant Nutrition Aspects under Treated Wastewater Reuse Management

A greenhouse pot experiment was conducted in Agrinion, Greece, in 2009, using a Randomized Block Design. Treated Municipal Wastewater (TMWW) in five levels was applied to Brassica oleracea var. Capitata (cabbage) in four replications. The experiment aimed at investigating the effect of TMWW on: (a) the interrelationships of cabbage plant parts (roots, stems, leaves, and heads) heavy metal content, with the respective dry matter yield. (b) The relationship between each individual soil bioavailable diethylenetriaminepentaacetic acid (DTPA extractable) heavy metal, with the dry matter (dm) yield of the abovementioned cabbage plant parts. The heavy metals of cabbage plant parts dry matter were significantly related negatively and statistically with the respective dry matter yield. Similarly, the soil available DTPA-extractable heavy metals were generally negatively affecting the dry matter yield of roots, stems, leaves, and heads. Conversely, Co, Cr, and Cd were found to be positively associated with the dry matter yield increase of the aforementioned plant parts. This effect was indirect, probably being due to contribution in essential elements by the synergistic interactions of these heavy metals with plant nutrients, such as Ca, Mg, Fe, and Mn.     

Fertilization strategies on cultivars of globe artichoke: Effects on yield and quality performance

This paper refers on the yields and productive aspects of mineral and foliar nutrition of globe artichoke plants (cv. ‘Opal’ and ‘Madrigal’). Marketable yields were investigated in response to different nitrogen rates and foliar applications of biostimulating products. Field experiments were conducted over two seasons on an alluvial salty-clay soil in sub-humid climate conditions. Analyzed traits differed between years because of the age of plants while the lowest nitrogen rate and foliar spraying of biostimulants influenced significantly head weight, dry matter, and Soil Plant Analysis Division (SPAD) index. Biostimulants induced longer cultural cycle and heads lighter in weight. Cultivar ‘Madrigal’ was most productive than ‘Opal’, both in number (26.28% higher) and in weight (28.51%) of total heads. The two-year investigation showed that the synergic action was effective in improving some of the investigated productive traits of globe artichoke.     

Content of antioxidative caffeoylquinic acid derivatives in field-grown Ligularia fischeri (Ledeb.) Turcz and responses to sunlight

Ligularia fischeri (Ledeb.) Turcz, a commercial leafy vegetable, contains caffeoylquinic acid derivatives (CQAs) as major phenolic constituents. The HPLC chromatograms of leaf extracts collected from different areas in Korea showed a significant variation in CQA amount, and two tri-O-caffeoylquinic acids (triCQAs) were purified and structurally identified by NMR and MS from this plant. Radical scavenging activities among CQAs were found to be increased in proportion to the number of caffeoyl groups. Since this plant prefers damp and shady growth conditions, the effects of sunlight were investigated by growing plantlets in sunlight and shade for four weeks. Greater leaf thickness and higher phenolic contents were found for leaves grown in sunlight than in shade. Four major CQAs-5-mono-O-caffeoylquinic acid (5-monoCQA), and 3,4-, 3,5-, and 4,5-di-O-caffeoylquinic acid (diCQA)-were induced by solar irradiation, whereas the content of these compounds decreased steadily in shade leaves. The leaves of L. fischeri clearly showed adaptation responses to sunlight, and these characteristics can be exploited for cultivation of this plant for potential use as a nutraceutical and functional food.     

Solid-phase microextraction of volatile compounds from the chopped leaves of three species of Eucalyptus

Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography and ion-trap mass spectrometry has been used to identify biogenic volatile organic compounds present in the headspace of chopped leaves of Eucalyptus (E.) dunnii, E. citriodora, and E. saligna. A simple HS-SPME method entailing 30 min of extraction at 30 degrees C was developed for this purpose. Thirty compounds were identified in the headspace of 60 juvenile chopped Eucalyptus leaves, and another 30 were tentatively identified. The presence of compounds such as (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMNT), (E,E)-alpha-farnesene, (E,E,E)-3,7,11,15-tetramethyl-1,3,6,10,14-hexadecapentaene (TMHP), beta-caryophyllene, alpha-humulene, germacrene D, and beta-cubebene in the headspace of the leaves but not in the essential oils from the same Eucalyptus trees and information about the infochemical roles of some of these compounds in other living plant systems suggest they might play a bioactive role in Eucalyptus leaves.     

Effects of different cooking methods on antioxidant profile, antioxidant capacity, and physical characteristics of artichoke

In this study, the effects of three common cooking practices (i.e., boiling, steaming, and frying) on the artichoke phenolic compounds pattern were evaluated by LC-MS/MS analysis. The variation of carotenoids, antioxidant capacity, and artichoke physical properties after cooking was also investigated. The major phenolic compounds present in the raw sample were 5- O-caffeoylquinic and 1,5-di- O-caffeoylquinic acids; after cooking treatments, an increase of the overall caffeoylquinic acids concentration due to the formation of different dicaffeoylquinic acid isomers was observed. Steamed and fried samples showed similar patterns of dicaffeoylquinic concentrations, which were higher with respect to the boiled samples. On the other hand, all cooking practices, particularly frying, decreased flavonoid concentration. The antioxidant capacity of cooked artichokes, measured by three different assays, enormously increased after cooking, particularly after steaming (up to 15-fold) and boiling (up to 8-fold). The observed cooking effect on the artichoke antioxidant profile is probably due to matrix softening and increased extractability of compounds, but the increase of antioxidant capacity is much higher than the increase of antioxidant concentration. These results suggest that some common cooking treatments can be used to enhance the nutritional value of vegetables, increasing bioaccessibility of health-promoting constituents.     

Determination of free inositols and other low molecular weight carbohydrates in vegetables

Different low molecular weight carbohydrates including saccharides, polyalcohols, sugar acids, and glycosides have been identified and quantified in different edible vegetables from Asteraceae, Amarantaceae, Amarylidaceae, Brassicaceae, Dioscoreaceae, and Solanaceae families by gas chromatography-mass spectrometry. Apart from glucose, fructose, and sucrose, other saccharides such as sedoheptulose in chicory, spinach, cabbage, purple yam, eggplant, radish, and oak leaf lettuce, rutinose in eggplant skin, and a glycosyl-inositol in spinach have been identified. chiro-Inositol was found in all vegetables of the Asteraceae family (3.1-32.6 mg 100 g(-1)), whereas scyllo-inositol was detected in those of purple yam, eggplant, artichoke, chicory, escarole, and endive (traces-23.2 mg 100 g(-1)). α-Galactosides, kestose, glucaric acid, and glycosyl-glycerols were also identified and quantified in some of the analyzed vegetables. Considering the bioactivity of most of these compounds, mainly chicory leaves, artichokes, lettuces, and purple yam could constitute beneficial sources for human health.     

烟粉虱取食对不同抗虫性辣椒品种营养物质和抗性物质的影响

为了探讨烟粉虱取食对辣椒体内营养物质和抗性物质的影响,选择抗虫辣椒品种(‘新一代三鹰椒’、‘新苏椒五号’)和感虫品种(‘苏椒13号’、‘苏椒15号’)各2个,每株辣椒分别接烟粉虱成虫0头(对照)、30头(低密度)、60头(中密度)和120头(高密度),取食24 h后测定辣椒叶片中叶绿素、可溶性糖、可溶性蛋白以及酚类、黄酮类化合物含量。结果发现,低密度的烟粉虱取食后,辣椒叶片叶绿素含量显著上升,随着虫口密度的提高,叶绿素含量有所下降,但仍高于对照;感虫辣椒品种的叶绿素含量增加幅度高于抗虫品种。低密度和高密度的烟粉虱取食后,辣椒叶片中可溶性糖含量出现明显下降趋势,抗虫品种的下降幅度大于感虫品种。低密度和中密度的烟粉虱取食后,辣椒叶片中可溶性蛋白含量显著下降,但高密度烟粉虱取食后辣椒叶片中可溶性蛋白含量明显上升;抗虫品种的变化幅度明显高于感虫品种。烟粉虱取食后,叶片中酚类化合物含量明显上升,感虫品种的上升幅度较抗虫品种上升幅度大;随着虫量的增加,辣椒叶片中酚类化合物含量呈明显上升趋势,但高密度烟粉虱取食后酚类化合物含量出现明显下降趋势。烟粉虱取食后,感虫辣椒品种‘苏椒15号’叶片中黄酮类化合物含量明显上升;其他3个品种在中等以下密度的烟粉虱取食时,黄酮类化合物与对照差异不显著,但在高密度烟粉虱取食时,黄酮类化合物明显高于对照。研究表明,烟粉虱取食可引起辣椒营养物质和抗性物质含量向着有利于提高抗虫性的方向改变,但不同虫口密度下不同辣椒品种的改变幅度不同。研究结果为进一步揭示寄主植物与烟粉虱之间的防御和反防御机制提供了基础。     

Nitrogen fertilization,irrigation, and harvest times affect biomass and energy value of Helianthus tuberosus L.

This study aimed to determine the effects of delayed harvest, irrigation and nitrogen fertilization on yield and fuel quality of the Jerusalem artichoke (JA) (Helianthus tuberosus L.). The biomass, calorific value, ash content and total calories per hectare of the Jerusalem artichoke were assessed in Inner Mongolia, China, at various harvest times, after irrigation and nitrogen application. The results showed that fresh and dry weights of tubers and underground biomass were higher when harvested after freezing; the dry yields of leaves and stems decreased with harvest time. In addition, irrigation significantly enhanced the yields of underground biomass, aboveground biomass and tubers, compared with non-irrigation conditions (p < 0.05). Interestingly, the highest yield was obtained with irrigation and treatment with nitrogenous fertilizers (20 to 50 kg ha^?1). The calorific values of tubers and roots were significantly higher for samples harvested after freezing (p < 0.05); the calorific values of leaves and stems significantly decreased with harvest time and without irrigation (p < 0.05). The calorific values of stems and leaves were higher than those of tubers and roots, when JA was harvested before freezing, and the opposite trend was obtained for harvest done after freezing. The highest calories per hectare was obtained in WN2 (585247.42 MJ ha^?1) on 30 September 2010 harvest. No correlation was found between the effects of water or nitrogenous fertilizers and ash content. However, the ash contents of stems, leafs, tubers and roots were significantly decreased (p < 0.05) with harvest time. Finally, in all treatment conditions, leaves produced the highest ash amounts among all plant parts, including stems, tubers, leaves and roots.     

Variations in morphological and agronomic traits among Jerusalem artichoke (Helianthus tuberosus L.) accessions

Jerusalem artichoke is a diversely-utilized crop. Selection for high yield, inulin content and other economically important traits are useful for improving this crop. The objectives of the present study were to evaluate genetic variability for qualitative and quantitative traits among Jerusalem artichoke accessions and to identify different groups of accessions using morphological and agronomic traits. Seventy-nine accessions were evaluated in a randomized complete block design with two replications in the late rainy season 2008, the early rainy season 2009 and the late rainy season 2009 at Khon Kaen University agronomy farm, Thailand. Morphological and agronomic characteristics were evaluated for genetic variations. High variations were found among Jerusalem artichoke accessions for qualitative and quantitative characters, and selection for these characters is possible. High variations were observed for tuber width, number of tubers/plant, biomass, fresh tuber yield and tuber size. Correlation coefficient between fresh tuber yield and tuber size was positive and significant (0.58, P ≤ 0.01). Improvement of tuber size is a means to improve yield and tuber quality. Based on morphological and agronomic characteristics, Jerusalem artichoke accessions were clustered into four distinct groups (R² = 0.88). These groups may be used as parental material to generate progenies for further improvement of this crop. This information will enable breeders to make informed decisions about possible heterotic groups for their breeding programs and germplasm conservation.     

Composition of essential oil of costmary [Balsamita major (L.) Desf. ] at different growth phases

The essential oils from leaves and flowers of costmary, Balsamita major (L.) Desf. (syn. Chrysanthemum balsamita L.), were analyzed at various phases of plant growth. The highest contents of oil both in leaves and in flowers were determined before full blooming, 1.15 and 1.34% (w/w), respectively. Seventy-eight volatile compounds have been identified in the oils of Balsamita major, of which 58 (19 tentatively, 39 positively) have not been reported in this plant previously. Carvone and alpha-thujone were found to be dominating compounds constituting from 51.8 to 68.0% and from 9.0 to 16.1% in the total oil, respectively. Seasonal variations in the oil compositions were not considerable except for the starting phase (May 25, 1995), when the content of carvone was lower and the content of alpha-thujone and sesquiterpenes higher. The content of sesquiterpenes was approximately 2 times higher in flowers than in the leaves. Absolute amount of most components was highest at the bud formation period.     

Jerusalem artichoke growth,development, and field storage. I. Numerical assessment of plant part development and dry matter acquisition and allocation

Abstract

Expanding commercial interest in the use of inulin as a bulking agent for artificial sweeteners, dietary fiber health supplement, fat replacement for processed foods, feed stock for fructose syrups, and a wide range of potential industrial products, has stimulated research on inulin‐containing crops such as the Jerusalem artichoke. To better understand the developmental physiology of the crop and to identify potential breeding objectives, the temporal pattern of development of individual plant parts (shoots, branches, leaves, flowers, stolons, tubers, and roots) and the allocation of dry matter into the same plant parts were monitored in the cultivar ‘Sunchoke’ over the entire growing season and during in situ field storage during the early winter, 32 weeks after planting. While number of shoots (～9) peaked in week 10, the number of branches (42.8), stolons (49.4), and tubers (85.5) reached a maximum 24–28 weeks after planting. Number of leaves (～525) peaked between weeks 20–24 after planting, as did number of flowers (～55). The Jerusalem artichoke allocated the major portion of its dry matter (dm) into aboveground plant parts during the first half of the growing season. Approximately 16 weeks after planting, the pattern of allocation shifted dramatically with: a) near cessation in the acquisition of dry matter; and b) the reallocation of existing dry matter from the aboveground organs into the tubers. By the 16th week after planting, 85% of the total dm was in the aboveground plant parts, but declined to 28% by the 30th week. Of the total dm, 92% was accrued during the first 16 weeks and only 8% thereafter. The shift in dry matter resources coincided with a dramatic decrease in leaf number and in leaf and branch dry weight. By the end of the season, the harvest index reached 0.70 and the tuber yield 14.61 dm ha^?1. Yield improvement could potentially be facilitated through lengthening the logarithmic period of carbon fixation and by earlier tuber induction and development.     

水煤浆气化渣对毛乌素沙地土壤改良与菊芋生长的促进效应研究

我国水煤浆气化渣生产量巨大,农业利用是提高其综合利用水平的重要途径之一。本文通过大田试验研究了水煤浆气化渣用于改良砂土、栽培菊芋的效果;大田试验设计施用水煤浆气化渣（10500 kg hm?2）和不施用施用水煤浆气化渣（对照）2个处理。结果表明:播种前,与对照处理相比,施用水煤浆气化渣处理的0 ~ 20 cm土层土壤pH值提高2.09%、全盐含量降低9.5%,速效钾、碱解氮、全氮、有效磷的含量分别提高了31.91%、254.2%、108.00%、52.74%,土壤含水量增加显著;种植菊芋结束后,施用水煤浆气化渣处理的0 ~ 20 cm土层土壤速效钾、全氮、有效磷含量分别比对照处理提高了26.19%、440%、29.8%和416%,pH值和全盐含量降低6.38%和27.8%;同时施用水煤浆气化渣,土壤pH值升高了8.65%,全盐含量下降13.04%,碱解氮降低了83.90%,土壤全氮、有效磷分别提高了211.86%和64.00%;施用水煤浆气化渣处理的菊芋株高、根深、茎粗、地上生物量和产量比对照提高18.56%、26.77%、3.54%、12.57%和6.21%。说明施用水煤浆气化渣和种植菊芋具有改良沙化土壤的作用,且施用水煤浆气化渣能够明显促进菊芋生长。     

Phenolic compounds from the leaf extract of artichoke (Cynara scolymus L.) and their antimicrobial activities

A preliminary antimicrobial disk assay of chloroform, ethyl acetate, and n-butanol extracts of artichoke (Cynara scolymus L.) leaf extracts showed that the n-butanol fraction exhibited the most significant antimicrobial activities against seven bacteria species, four yeasts, and four molds. Eight phenolic compounds were isolated from the n-butanol soluble fraction of artichoke leaf extracts. On the basis of high-performance liquid chromatography/electrospray ionization mass spectrometry, tandem mass spectrometry, and nuclear magnetic resonance techniques, the structures of the isolated compounds were determined as the four caffeoylquinic acid derivatives, chlorogenic acid (1), cynarin (2), 3,5-di-O-caffeoylquinic acid (3), and 4,5-di-O-caffeoylquinic acid (4), and the four flavonoids, luteolin-7-rutinoside (5), cynaroside (6), apigenin-7-rutinoside (7), and apigenin-7-O-beta-D-glucopyranoside (8), respectively. The isolated compounds were examined for their antimicrobial activities on the above microorganisms, indicating that all eight phenolic compounds showed activity against most of the tested organisms. Among them, chlorogenic acid, cynarin, luteolin-7-rutinoside, and cynaroside exhibited a relatively higher activity than other compounds; in addition, they were more effective against fungi than bacteria. The minimum inhibitory concentrations of these compounds were between 50 and 200 microg/mL.     

Variation in essential oil composition within individual leaves of sweet basil (Ocimum basilicum L.) is more affected by leaf position than by leaf age

The aroma in sweet basil is a factor affecting the commercial value of the crop. In previous studies leaf age was considered to be a factor that influences the composition of essential oil (EO). In this study it was hypothesized that a single observation of the EO content in leaves from different positions on the main stem (young vs old) could predict the developmental changes in the plant during its life cycle. Plants harvested at week 16 demonstrated an exponential increase (R(2) = 0.92) in EO concentration in leaves on the main stem and lateral shoots, indicating higher EO concentrations in younger than in older leaves. Eugenol and methyleugenol predominated (28-77%) in the extract. Eugenol levels were higher in younger leaves (～53%), and methyl-eugenol levels predominated in older leaves (～68%). Linalool was lower in mature leaves than in younger leaves. This suggested that eugenol converted into methyleugenol and linalool decreased as leaf mature. However, in weekly monitored plants, the levels of these compounds in the EO had limited variation in the maturing leaf regardless of its position on the stem. This proposed that the EO composition in an individual leaf is mostly affected by the leaf position on the stem and not by its maturation process. Because leaf position is related to plant development, it is probable that the plant's physiological age at the time of leaf formation from the primordial tissue is the factor affecting the EO composition. It was concluded that interpretation of scientific observations should be carried out with caution and that hypotheses should be tested utilizing multifaceted approaches.