Essential oils are complex mixtures of volatile secondary metabolites of plants. They can be extracted from various organs: leaves, fruits, roots, etc. Essential oils often define plant taste, as well as its therapeutic potential. Numerous studies demonstrated their antimicrobial, antioxidant, spasmolytic, anti-inflammatory, and relaxing effects    .
Individual compositions of volatile compounds may be used to identify species or geographic origin of biomaterial. Moreover, rapid and reliable analytical methods may be developed for high-scale quality control for phytopharmacy. Many volatile compounds also act as signal substances (semiochemicals). Plant signals are often cross-specific: they are used to attract insect pollinators and to scare off pests   .
The common sea buckthorn (Hippophae rhamnoídes, Hippophae, Elaeagnaceae) is a dioecious shrub cultivated for its fruits that are used to produce juice, grist, and oil. Leaves can also be used for medical purposes    .
Hirvi and Honkanen  demonstrated that essential oils of H. rhamnoides fruit consist mainly of esters of carboxylic acids. All previous works are constructed on the research of essential oils of berries of a sea-buckthorn. In this study, we investigated essential oils of berries and green biomass of the sea buckthorn at various growth stages.
2. Material and Methods
2.1. Plant Material
Plant material of H. rhamnoídes ssp. mongolica was collected in 2016 in the experimental fields of the Institute of Cytology and Genetics SB RAS. Green spring shoots collected in April contained 37.5 mg of volatile compounds per kg of air-dry material; first-year seedlings without leaves, in June. Berries were harvested in the autumn and stored at −30˚C until used.
2.2. Extraction of Volatile Compounds
Dried seedlings or shoots (35.0 g) were suspended in 300 ml of deionized water, boiled in a flask with a reflux condenser for 30 min, and distilled with water vapor to a total volume of 200 ml. A total of 60.0 g of sodium chloride was added to the distillate, and the solution was extracted with 25.0 ml of ethyl acetate. The extract was dried with anhydrous sodium sulfate, filtered and analyzed by GC-MS. The same procedure was performed for 200 g of berries. For quantitation of volatile compounds, 1,2,3,4-tetrafluoronaphthalene was added to water distillates as a reference. All experiments are made in three replicas.
2.3. Chromatographic Analysis
Separation of compounds was performed with an Agilent Technologies 6890 chromatograph with a 5973 mass spectrometry detector. A 30 m capillary chromatographic column with the inner diameter of 0.25 mm was used. The speed of the carrier gas (helium) was 1.0 ml/min; the temperature of the sample heater was set at 250˚C; the thermostat was heated from 50˚C to 250˚C by 25˚C per min; sample input rate was 1.0 ml/min. The sample (1 µl) was applied to the column without flow separation.
Compounds were identified by comparing their mass-spectra with those obtained with NIST14 from the US National Institute of Technology and Standards (NIST) mass spectra libraries, the NIST Chemistry WebBook (https://webbook.nist.gov/), and the literary references from the latter database.
Linear RIs was determined in relation to a homologues series of n-alkanes (C6-C40) run under the same operating conditions. Relative percentage of the compounds was calculated based on the peak areas from the FID data without correction for response factors.
3. Results and Discussion
Shoots, in April, contained 37.5 mg of volatile compounds per kg of air-dry material; seedlings collected in June, 196.7 mg/kg. Berries contained 265 mg of volatile compounds per 1 kg of fresh biomass.
Figure 1 shows chromate grams of mixtries of volatile compounds isolated from different parts of H. rhamnoides.
All detected compounds are listed in Table 1.
The results are expressed as the mean ± standard deviation (n = 3). Values identified by an asterix indicate differences (p < 0.05).
Berries contained mostly carboxylic acids and their esters (Table 2), predominantly 3-methylbutyl ester of benzoic acid (11.63%), ethyl ester of hexanoic acid (9.07%), ethyl ester of (9Z)-hexadecenoic acid (8.85%), and 3-methylbutyl ester of 3-methylbutanoic acid (7.77%). The ratio of esters of aromatic to aliphatic acids was about 2:9.
Aromatic acids were represented by benzoic and phenylacetic acids. The alcoholic part of the esters was mostly derived from ethanol and 3-methylbutanol. Alkanes were scarce (0.29%), represented only by n-tricosane; the same was observed for alkenes (0.65%): (8Z, 11Z, 14Z)-heptadeca-1,8,11,14-tetraene and isomeric tricosenes. Phenylpropanoids were abundant in seedlings and shoots, but they accounted only for 1.5% of essential oils (β-asarone and (E)-isoelemicin) in berries. No monoterpene compounds were detected, but the following sesquiterpene hydrocarbons were identified: β-maaliene, γ-selinene, cadina-1(10),6,8-triene, and germacrene B. The content of oxidized terpene compounds was 5.12%, which included isocalamendiol, isolongifolanones, shyobunon and its isomers.
Our results were generally similar to those obtained by Hirvi and Honkanen  and Tiitinen et al. , but certain differences were detected. The results of  were similar to those of our study: caroboxylic acids and their esters accounted for 94.2% of the sample and were mainly represented by 3-methylbutyl ester of benzoic acid, ethyl ester of hexanoic acid, and 3-methylbutyl ester of 3-methylbutanoic acid (7.77%), as well as by 3-methylbutanoic acid. According to Tiitinen et al. , headspace volatiles form frozen berries of sea buckthorn contain 91.4% - 97.4% of caroboxylic acids and their esters, and the highly volatile ethanol and 3-methylbutanol accounted for the rest. They found less than 2.9% of esters of aromatic acids, while in  and our study they made up 17% of the sample. Both studies failed to detect alkanes, phenylpropanoids, and sesquiterpenes; however, in contrast to our data, they revealed approximately 1% of
Figure 1. Volatile compounds of different parts of H. rhamnoides, GC-MS analysis.
Table 1. Volatile compounds composition of different parts of H. rhamnoides.
Table 2. Major components of essential oils of different parts of H. rhamnoides.
C10H16 monoterpene hydrocarbons.
Socaci et al.  studied essential oils of berries and juice of both wild and cultivated H. rhamnoides carpatica. The main components were again 3-methylbutyl ester of 3-methylbutanoic acid and ethyl ester of hexanoic acid, as well as ethyl esters of 2-methyl and 3-methylbutyric acids. The share of terpene compounds, limonene, and cis-ozymene was below 1%.
The differences of our results from those obtained by Cakir  were more pronounced. The last found that volatile compounds except octyl acetate consist mostly of ethyl esters of various aliphatic acids, while phenylpropanoids, terpenes and esters of isomeric valeric acids, were absent. The content of aliphatic hydrocarbons was 13.5%, and of aliphatic alcohols, 15.7%.
In the study of Yue et al. , palmitic acid accounted for the major part of the acid-ester fraction. However, we consider these results highly questionable based on the reported retention times do not match the corresponding Kovacs indices.
Vítová et al.  found the majority of the volatile compounds of the sea buckthorn berries to be represented by aliphatic alcohols with low boiling point (C2-C6), oct-2-en-3-ol, octanal, esters (butyl acetate, ethyl acetate and ethyl hexanoate), and ketones (pentane-2-one, and 3-hydroxybutan-2-one). Alkanes, phenylpropanoids, and terpene compounds were not detected.
It is hard to explain the discrepancies among these studies. Hirvi & Honkanen  analyzed essential oils from berries of two varieties of the sea buckthorn from Finland, of H. rhamnoides ssp. mongolica from Russia and the local H. rhamnoides ssp. rhamnoides in two consecutive seasons. Both varieties are cultivated in southern and southwestern Finland. The authors used the Principal Component Analysis (PCA) and found that the differences were mainly observed among seasons, not among the varieties. However, a similar study of Vítova et al.  yielded opposite results. PCA analysis of Socaci et al.  indicated that essential oils of seeds and juice from various sea buckthorn varieties consist of the same substances but in highly varying ratios. On the whole, these studies yielded strongly differing composition of essential oils, which might be attributed to regional (climatic) differences. All aforementioned studies used different methods: solid-phase microextraction (SPME) in , vapor SPME in , vapor distillation in , and hydroextraction distillation in  . However, we failed to detect any interconnections caused by differences in extraction procedures.
We used hydrodistillation to extract essential oils from seedlings and shoots of the sea buckthorn. Similar to alkanes, phenylpropanoids prevailed, although their total content was lower in shoots than in seedlings (25.9% vs. 72.9%). Among phenylpropanoids, β-аsarone was the most abundant (64.4%), followed by its isomers (α- and γ-asarons), elemicin and cis- and trans-isoelemicins (a total of 8.08%), as well as low amounts of O-methyleugenol. The observed high content of phenylpropanoids is remarkable, as these substances are increasingly used due to their antiplatelet and anticoagulant activity  .
Compared to berries, essential oils from seedlings had more long chain alkanes (С22-С29), small amounts of oxidized monoterpenes of linalool, β-cyclocitral and camphor (a total of 0.53%), and significantly more oxidized sesquiterpenes (7.5%), almost similar to those found in berries. A small portion of sesquiterpene hydrocarbons was represented by copaene, β-elemen, and α-calacorene. Carboxylic acids and their esters were absent.
In the essential oils isolated from shoots of the sea buckthorn, phenylpropanoids were represented only by β- and γ-asarons. Isomers of elemicin were absent, while small amounts of methyl-cis-isoeugenol were detected. Content of n-alkanes (С22-С30) was found to change during vegetative development, from 15.84% in seedlings to 29.86% in shoots. It is well known that cuticular wax that protects shoots from the elements, pathogenic microorganism and phytophagous insects contains long-chain alkanes  . Piasentier et al.  found that n-alkane profiles change during vegetative development, and these changes vary in different tree species. The maximum alkane length in the green mass of the sea buckthorn was n = 29  (Kukina et al., 2016).
We also found the following alkenes in essential oils from the shoots of the sea buckthorn: 1-dodecene, 1-tetradecene, 1-octadecene, and 1-docosene (total content of 4.4%), which were probably formed from the corresponding terminal alkanols; retene (0.47%), the marker substance of forest fires; squalene (4.39%), which is often found in plants; and (8Z, 11Z, 14Z)-Heptadeca-1,8,11,14-tetraene (1.23%), which is characteristic for all essential oils. We failed to found carboxylic acids and their esters that are abundant in berries; however, we detected methylabietate (1.18%) and dibutyl phthalate (0.45%). We believe the latter to be a human-introduced admixture. We also detected sesquiterpenes: β-elemen (0.38%) and caryophyllene (0.20%). We identified several compounds also found in berries, but in higher relative amounts (a total of 17.07%): isocalamendiol, isolongifolenes, shyobunon, and its isomers.
It is well known that shyobunon and its analogues may act as repellents and insecticides  . Moreover, Yue et al. demonstrated that essential oils from various parts of the sea buckthorn are effective against pathogenic bacteria: Staphylococcus aureus was equally suppressed by oils from all studied material, oil from berries had the highest impact on Bacillus subtilis and B. coagulans, and oils from seedlings, on E. coli  .
Therefore, in this study we extracted essential oils from different parts of the sea buckthorn Hippophae rhamnoides, L. by hydrodistillation, identified them using GC-MS, and performed comparative analysis.
This research was ﬁnancially supported by the budget project ICG SB RAS 0324-2019-0040.
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