Abstract
Introduction
Walnut, Juglans regia L., Juglandaceae, is a tree native to Peloponnesus, which has spread all over the Balkans and eastwards through the Caucasus up to the Himalayas.1,2 Walnut is also known under the names: common, Carpathian or Persian walnut. In Greek mythology, Juglans regia is dedicated to Carya or The Lady of Nut-Tree. 3 Walnut tree distribution in the Balkans is the result of favorable natural, soil and climatic conditions, which are suitable for its growth. Its biological and pomological characteristics in the Balkans have been investigated,4–9 and the biological remedies and herbal medicine information have been reported.10–13 The oils have an aromatic odor, with a somewhat bitter and harsh taste. 14 The leaves epicuticular waxes contain primary alcohols (41.6%), aldehydes (5.5%), hydrocarbons (3.0%), esters (3.5%), and fatty acids (8.4%). 15 Ascorbic acid is present in walnut leaves in less than a percent. 14 Other compounds present in the leaves can be divided into several groups: tannins, flavonoids, phenolic acids, and naphthalene derivatives. Leaves contain about one-tenth of tannins in the form of ellagitannins.12,14 Flavonoid compounds are present around 3%.14,16–18 Diverse phenolic acids were identified in leaves.16–19 The leaves also contain the naphthalene derivative hydrojuglone β-D-glucopyranoside.14,20,21 After hydrolysis, the aglycone obtained is hypothetically hydrojuglone, which after oxidation forms juglone, a more stable quinone. 22 Juglone, further polymerizes and in senescent and dry leaves only traces remain.17,23 The tree produces leaves in mid-spring, but they fall already in early autumn. Volatile compounds present in leaves mostly consist of monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes.24–30 The monoterpene hydrocarbons identified in all analyzed samples of walnut leaves are: α-pinene and β-pinene.24–30 The sesquiterpene hydrocarbon identified in all extracts was caryophyllene.24–30 Leaf senescence is a process involving remobilization of chemical compounds present in leaves to sink tissues. During leaf senescence, terpenoids are broken down into compounds which gradually fade, vanish, or are transported through the vascular plant system to sink tissues.
The aim of the study was to evaluate the differences in essential oil chemical composition of walnut leaves collected in 2 different seasons and obtain more insight into the mechanism of terpenoid fade.
Results and Discussion
The obtained walnut essential oil from green leaves was a green liquid, whereas that from yellow leaves was bright green. Their chemical compositions are depicted in Table 1.
Essential Oil Chemical Composition of J. regia Green and Yellow Leaves.
Compounds were identified by comparison of RI determined in relation to n-alkanes (C8-C30) and mass spectra.
Abbreviations: RI, retention index, experimentally determined; RIL, retention index, literature data. 31
Monoterpene hydrocarbons were not identified in yellow walnut leaves’ essential oil, while in green leaves they formed 22.5%. The explanation can be that at the beginning of leaf senescence an increase in the enzyme peroxidase is induced, and reactive oxygen species (ROS) are possible cause of monoterpene hydrocarbons oxidation into oxygenated monoterpenes. It was reported that in tobacco leaves, the activity of peroxidase was the indicator of senescence. 32 Peroxidase activity, during leaf senescence, was also observed in bean, sunflower, and rice leaves.33–35
Sesquiterpene hydrocarbons formed 1.1% of the essential oil of yellow walnut leaves, which is far less than that in green leaves (38.5%). The explanation can also be sought in the fact that the sesquiterpene hydrocarbons are oxidized into oxygenated sesquiterpenes. The most abundant compounds in fall leaves were oxygenated sesquiterpenes (75.2%). The acyclic diterpene alcohol, phytol, can originate from chlorophyll, after phytol hydrolysis from the core of the chlorophyll molecule. The analysis determined only 0.1% of phytol in the essential oil of yellow leaves. Germacrene D, a sesquiterpene hydrocarbon, was not identified in yellow walnut leaves while it was the most abundant sesquiterpene hydrocarbon in green walnut leaves’ essential oil (Supplemental Figure S1). The most abundant compound in yellow walnut leaves’ essential oil was β-eudesmol (Supplemental Figure S2).
The seasonal variation study revealed that the J. regia essential oil monoterpene hydrocarbon content decreases from April to June, and in July the monoterpene hydrocarbon content is almost the same as in May. 28 From July to August monoterpene hydrocarbons decrease and in October their content is the lowest. 28 Oxygenated monoterpene content was the highest in September. 28 In this study, the essential oil obtained from yellow leaves showed an infinitesimal increase in oxygenated monoterpenes compared to the essential oil obtained from green leaves. The sesquiterpene hydrocarbons content showed alternating increase and decrease from April till July, from July to August an increase, and in the further months alternating decrease and increase. 28 The results obtained in this study showed significant decrease in sesquiterpene hydrocarbons, from green to yellow leaves. Oxygenated sesquiterpenes showed variation from April till October with significant increase in June and September. 28 The oxygenated sesquiterpenes present in the essential oil of yellow leaves, obtained in this study, were significantly higher than in the green leaves’ essential oil. The most abundant compounds in the essential oil obtained from leaves collected in May were eugenol, germacrene D, and methyl salicylate. 27 Germacrene D was present only in the essential oil of green leaves. The essential oil obtained from leaves collected in spring contained far more monoterpene hydrocarbons compared to oxygenated monoterpenes, and sesquiterpene hydrocarbons were more abundant than oxygenated sesquiterpenes. 26 In yellow leaves’ essential oil, monoterpene hydrocarbons were not present, while sesquiterpene hydrocarbons formed 1.1%, indicating their fade. The leaves collected in October, including the essential oil analysis of 4 varieties and 2 local cultivars, revealed that none of them contained oxygenated monoterpenes. 29 All varieties and 2 cultivars contained alcohols and esters, and only 1 local cultivar contained more oxygenated sesquiterpenes than sesquiterpene hydrocarbons. 29 In yellow leaves’ essential oil oxygenated sesquiterpenes dominated. Most of the investigations made indicate the variability in essential oil composition due to seasonal and vegetational development.
Conclusion
The conclusion is that the essential oil of yellow leaves contains far more oxygenated sesquiterpenes than green leaves’ essential oil. The results obtained indicate that oxidation is taking place in autumnal leaves, indicating the activity of the enzyme peroxidase and ROS. For the production of ROS and the hormonal signaling in leaves there is a lack of adequate models for studying leaf senescence. J. regia leaves can be a model species for the remaining questions in leaf senescence.
Material and Methods
Plant Material
Green and yellow walnut leaves were collected in June and September 2022, respectively. The leaves were collected from a tree located at 45°14′58″N, 19°49′34″E. Leaves were air dried and pulverized. The water content in the pulverized plant material was determined according to the AOAC Official Method 925.40 and was 6.8 ± 0.1%.
Hydrodistillation
The pulverized plant material (100 g) was hydrodistilled for 3 h using a Clevenger-type apparatus. The essential oil obtained was dried over anhydrous sodium sulfate.
GC-MS
The analysis was performed on a Hewlett Packard 5890 series II gas chromatograph coupled to a HP 5970 mass selective detector using a fused silica capillary column HP-5 (30 m × 0.25 mm, film thickness 0.25 μm). The column temperature was programmed from 40 to 260 °C at 4.0 °C·min−1 using helium as the carrier gas at a flow rate of 1.0 mL min−1. The ion source temperature was 300 °C and the electron energy 70 eV, in a m/z range of 45 to 450. The injected sample volume was 1 μL. Identification of individual oil components was accomplished by comparison of their retention indices with those of analytical standard substances of available terpenoids, using computer search and literature. 31
Supplemental Material
sj-docx-1-npx-10.1177_1934578X231191936 - Supplemental material for Essential Oils of Juglans regia Green and Yellow Leaves
Supplemental material, sj-docx-1-npx-10.1177_1934578X231191936 for Essential Oils of Juglans regia Green and Yellow Leaves by Nina Djapic in Natural Product Communications
Footnotes
Declaration of Conflicting Interests
The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical Approval
Not applicable. This article does not contain any studies with human or animal subjects.
Funding
The author received no financial support for the research, authorship, and/or publication of this article.
Statement of Human and Animal Rights
Not applicable. This article does not contain any guidelines followed for performing experimental procedures.
Statement of Informed Consent
Not applicable. This article does not contain any studies with human or animal subjects.
Supplemental Material
Supplemental material for this article is available online.
References
Supplementary Material
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