Essential Oil Composition of Bupleurum praealtum and Bupleurum affine: New Natural Constituents

This study explores the chemical composition of essential oils from two Serbian Bupleurum species (Apiaceae), Bupleurum praealtum L. and Bupleurum affine L., traditionally recognized in Chinese medicine for their therapeutic potential but less studied for their essential oils. Through GC-MS analysis, we identified 230 constituents, revealing distinct profiles between the species. Perillyl 2-methylbutanoate was identified in B. affine oil for the first time, confirmed using synthetic approaches and characterized by advanced spectroscopic techniques, including two-dimensional NMR and spin-simulation of 1H NMR spectra. Additionally, new natural compounds, including tentatively identified 4-decyl acetate and 4-undecyl acetate, were discovered. The study also reports five stereoisomeric esters of tetradeca-5,7,9,11-tetraen-1-ol. These findings significantly contribute to the understanding of the phytochemical diversity within the genus Bupleurum and underscore potential differences in ecological adaptations or biosynthetic pathways among species.


Introduction
The genus Bupleurum L. (Apiaceae) encompasses a diverse array of plant species known for their aromatic and medicinal properties, and it is almost exclusively native to Europe and eastern Asia [1].Species of this genus are well-known for their over 2000-year long usage in traditional Chinese medicine as "liver tonics", for the treatment of feverproducing infections, common cold, inflammatory disorders, hepatitis, etc. [2,3] Radix Bupleuri is the most frequently mentioned ingredient of these preparations, and is derived from the dried roots of Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd., although many other Bupleurum species are also used under the same name (Bupleurum falcatum L. and Bupleurum yinchowense R.H.Shan and Yin Li).It has been found to possess anti-inflammatory [3], antiviral [4], antidepressant [5], antitumor [6], hepatoprotective [7], and immunoregulatory activities [8].
The chemical composition of plant species belonging to this genus has been extensively studied.Triterpene saikosaponins are the primary active constituents of these plants, responsible for a broad spectrum of pharmacological activities in preparations containing Radix Bupleuri [1].Polyacetylenes constitute a significant group of compounds found in plants of the Apiaceae family, exhibiting anti-inflammatory, antibacterial, anticancer, and antifungal properties, although some have been identified as toxic [9].Bupleurum longiradiatum, widely distributed in northeastern China and available in certain herb markets, is a toxic plant primarily containing the toxic polyacetylenes: bupleurotoxin and acetylbupleurotoxin, compounds absent in other Bupleurum species [9].Therefore, the polyacetylene profile can serve as a distinguishing feature among species within this genus.
Essential oils have gained attention for their diverse chemical compositions and therapeutic potential.The analysis of essential oils not only provides insights into the Plants 2024, 13, 2076 2 of 17 phytochemical profile of plants but also unlocks novel avenues for pharmacological exploration.Essential oils derived from the Bupleurum species have received comparatively less attention in scientific research.To date, essential oils from forty plant species within the genus Bupleurum have been chemically analyzed, with ten classified as annual and thirty as perennial species [10].Li and colleagues conducted a study focusing on ten Bupleurum species originating from China, revealing that the predominant constituents were aliphatic aldehydes and acids such as hexanol, heptanol, heptanoic acid, octanoic acid, and hexadecanoic acid [11].In addition to these, typical for Chinese species, the dominant compounds in the essential oil of B. marginatum were β-caryophyllene, β-caryophyllene oxide, and spathulenol [12].Conversely, essential oils from European Bupleurum species are characterized by elevated levels of α-and β-pinene, limonene, and 1,8-cineole, which might be attributed to environmental factors or genetic variations [1,10].
In this study, our objective is to enhance our understanding of the phytochemical diversity within the genus Bupleurum, which holds significant potential for both botanical classification and future pharmacological research.Utilizing comprehensive GC-MS analysis, we will investigate the chemical composition of essential oils extracted from B. praealtum schizocarps and, for the first time, B. affine aerial parts.Our primary focus will be on identifying and characterizing novel compounds, including conducting full NMR assignments.To verify the identity of selected constituents, we plan to perform appropriate synthesis and utilize the resulting standards for validating tentative identifications through co-injection experiments.

Results and Discussion
GC-MS analysis of the essential oils of B. affine (BA) and B. praealtum (BP) led to the identification of 230 constituents (Table 1), amounting to 97.1% and 91.1% of the total detected GC-peak areas, respectively.The oil isolated from BA aerial parts exhibits only slightly lower overall percentages of sesquiterpene hydrocarbons (41.2%) compared to the schizocarps oil of BP (45.3%), indicating a similarity in the predominance of sesquiterpene hydrocarbons in both oils.Additionally, BP oil contains a higher percentage of structurally and biochemically distinct constituents ("others", 30.0%) compared to BA (12.7%), originating from a more diverse array of minor constituents.However, BP oil demonstrates a notably lower proportion of alkanes (5%) compared to BA (23.4%), implying potential differences in volatility and scent characteristics.The BP schizocarps essential oil predominantly consisted of germacrene D (24.0%), (E)-phytol (14.2%), and bicyclogermacrene (11.4%).Conversely, the primary constituents of the BA oil included undecane (21.0%), absent in the BP oil, along with germacrene D (18.6%) and (E)-phytol (5.0%).     2 Literature values of retention indices taken from Adams [18] or NIST [19] collection, if not stated otherwise. 3Compound identified based on mass spectra and retention indices matching with literature data, if not stated otherwise. 4Values are means of three individual analyses. 5A, alkanes; MH, monoterpene hydrocarbons; MO, oxygenated monoterpenes; SH, sesquiterpene hydrocarbons; SO, oxygenated sesquiterpenes; O, others. 6tr, trace amount (<0.05%). 7-, not detected. 8Constituent identity confirmed by co-injection of an authentic sample. 9Tentative identification based solely on MS comparison. 10see Section 3.3. 11Correct stereochemistry is unknown.
Additionally, the GC-MS analysis of the BA essential oil revealed the presence of one minor constituent (RI 1664), with an MS fragmentation pattern indicating a perillyl ester, and a molecular ion at m/z 236 (Supplementary Materials Figure S1), assumed to be the ester of perilla alcohol and a five-carbon atom acid.Previously, these esters were identified only once in the essential oil of another Apiaceae species, Kitagawia baicalensis (Redowsky ex Willd.)Pimenov [20].However, the paper did not specify the method used to confirm the identities of perillyl 2-methylbutanoate and perillyl 3-methylbutanoate.Solely comparing the retention indices provided (RI 1658 for perillyl 2-methylbutanoate and 1665 for perillyl 3-methylbutanoate) with the retention index of the unidentified component in the BA oil (RI 1664) does not definitively determine which of these two esters is present.Therefore, we opted to synthesize them for clarification.A reduction of the commercially available perilla aldehyde, followed by esterification with an appropriate acid gave the desired target esters (Figure 1).A co-injection experiment confirmed the occurrence of perillyl 2-methylbutanoate in the BA oil.The retention indices obtained from our synthesized standards do not align with those reported in the literature [20].This discrepancy suggests a Plants 2024, 13, 2076 10 of 17 potential confusion in the identity of these esters by Letchamo et al. [20], as our data indicate that Letchamo's 3-methylbutanoate closely matches our synthesized 2-methylbutanoate index.Consequently, we propose a reconsideration of the esters' identities.Our study represents the first definitive confirmation of the natural occurrence of 2-methylbutanoate in this context.The absence of perilla alcohol and perilla aldehyde in the essential oil is intriguing, as it is closely biosynthetically related to perillyl esters.Most likely, perillyl derivatives are derived from an enzymatic allylic oxidation of limonene present in the BA oil (1.0%).available perilla aldehyde, followed by esterification with an appropriate acid gave t desired target esters (Figure 1).A co-injection experiment confirmed the occurrence perillyl 2-methylbutanoate in the BA oil.The retention indices obtained from our synth sized standards do not align with those reported in the literature [20].This discrepan suggests a potential confusion in the identity of these esters by Letchamo et al. [20], as o data indicate that Letchamo's 3-methylbutanoate closely matches our synthesized methylbutanoate index.Consequently, we propose a reconsideration of the esters' iden ties.Our study represents the first definitive confirmation of the natural occurrence of methylbutanoate in this context.The absence of perilla alcohol and perilla aldehyde in t essential oil is intriguing, as it is closely biosynthetically related to perillyl esters.M likely, perillyl derivatives are derived from an enzymatic allylic oxidation of limone present in the BA oil (1.0%).As there are two chiral centers in perillyl 2-methylbutanoate two diastereomers a possible.The synthetic sample was comprised of their unresolvable mixture on the D 5MS column, while the NMR signals of these two diastereomers were also practically distinct as will be described below.The spectra of the mixture of the synthesized est were assigned with the aid of 1 H NMR manual full spin spectral simulation (Figure Table 2).The full spin analysis was performed by manually adjusting δH and J values to the experimentally available values and further optimized using MestReNova 11.0.3 so ware (tools/spin simulation).Although the recorded spectra represent the superimpos spectra of diastereomers (Supplementary Materials Figures S3 and S4), while the sim lated spectra come from one diastereomer, the simulation outcome was in excellent agr ment with the experimental data of the synthetic compound.This can be explained by t fact that the chiral centers are distant from one another within the molecule, resulting no significant differences in the position and appearance of signals.These differen (mostly barely observable broadening) are visible only in certain signals, in the proxim of chiral centers (e.g., methyl group near the chiral center of the acidic part of the ester As there are two chiral centers in perillyl 2-methylbutanoate two diastereomers are possible.The synthetic sample was comprised of their unresolvable mixture on the DB-5MS column, while the NMR signals of these two diastereomers were also practically indistinct as will be described below.The spectra of the mixture of the synthesized esters were assigned with the aid of 1 H NMR manual full spin spectral simulation (Figure 2, Table 2).The full spin analysis was performed by manually adjusting δ H and J values to fit the experimentally available values and further optimized using MestReNova 11.0.3 software (tools/spin simulation).Although the recorded spectra represent the superimposed spectra of diastereomers (Supplementary Materials Figures S3 and S4), while the simulated spectra come from one diastereomer, the simulation outcome was in excellent agreement with the experimental data of the synthetic compound.This can be explained by the fact that the chiral centers are distant from one another within the molecule, resulting in no significant differences in the position and appearance of signals.These differences (mostly barely observable broadening) are visible only in certain signals, in the proximity of chiral centers (e.g., methyl group near the chiral center of the acidic part of the ester).1.8500 (ddddd, 2 J 6ax,6eq = −13.3, 3 J 6eq,7ax = 6.0, 3 J 5,6eq = 4.8, 4 J 4eq,6eq = 2.0, 3 J 6eq,7ax = 0.6, 1 H) 7ax 2.0770 (ddddtdd, 2 J 7ax,7eq = −17.5, 3 J 6ax,7ax = 10.0, 3 J 6eq,7ax = 6.0, 5 J 4ax,7ax = 4.0, 4 J 1a,7ax = 5 J 4eq,7ax = 1.8, 4 J 3,7ax = −1.6, 4 J 1b,7ax = 0. 1 Coupling constant values were initially inferred from 1 H homoselective decoupling NMR experiments and afterward refined through a manual iterative full spin analysis.For details, cf.Experimental part. 2 grHMBC correlations observed between the hydrogen in this row and the carbon in the listed position. 3Cross-peaks observed in the NOESY spectrum. Spectral simulation (Figure 2) allowed us to clearly discern the major couplings present among protons standardly buried within signals of higher order.The most significant coupling constants are shown in the structure in Figure 3. Three large constants, greater than 10 Hz, confirmed the approximately antiperiplanar position of hydrogens on the six-membered ring, placing the isopropylene group in a pseudo-equatorial position, as expected.Additionally, we noticed a large homoallylic constant of 4 Hz between the axial hydrogens in positions 4 and 7, besides two other homoallylic constants, of around 2 Hz.The reason for such a strong interaction between relatively distant hydrogen atoms can only be sought from their relative positions to the double bond, the parallel orientation of σ C-H and π C=C , which further confirms the depicted 3D structure (Figure 3).The large value of one more long-range constant, the W-coupling constant of around 2 Hz, between equatorial hydrogens in positions 4 and 6, also confirmed the reliability of the depicted 3D structure of perillyl ester. 1 Coupling constant values were initially inferred from 1 H homoselective decoupling NMR experiments and afterward refined through a manual iterative full spin analysis.For details, cf.Experimental part. 2 grHMBC correlations observed between the hydrogen in this row and the carbon in the listed position. 3Cross-peaks observed in the NOESY spectrum.Spectral simulation (Figure 2) allowed us to clearly discern the major couplings present among protons standardly buried within signals of higher order.The most significant coupling constants are shown in the structure in Figure 3. Three large constants, greater than 10 Hz, confirmed the approximately antiperiplanar position of hydrogens on the sixmembered ring, placing the isopropylene group in a pseudo-equatorial position, as expected.Additionally, we noticed a large homoallylic constant of 4 Hz between the axial hydrogens in positions 4 and 7, besides two other homoallylic constants, of around 2 Hz.The reason for such a strong interaction between relatively distant hydrogen atoms can only be sought from their relative positions to the double bond, the parallel orientation of σC-H and πC=C, which further confirms the depicted 3D structure (Figure 3).The large value of one more long-range constant, the W-coupling constant of around 2 Hz, between equatorial hydrogens in positions 4 and 6, also confirmed the reliability of the depicted 3D structure of perillyl ester.The four possible stereoisomers of perillyl 2-methylbutanoate could be expected to have different scents as well as potentially different biological activities.The synthesized mixture of these isomers (all four) had a floral-menthol scent.Synthesizing these esters using chirally pure alcohols and acids would allow us to determine the scent of each individual stereoisomer.
In the BA essential oil, the presence of numerous components with identical or similar mass spectra to esters of tetradec-4,6,8,10-tetraen-1-ol and acids with five carbon atoms, previously detected in the BP diethyl ether extract (praealtaesters A, B, C, and D), was noted.It is presumed that along the known esters, the remaining detected esters represent related constituents differing in the configuration of double bonds in the alcohol part of the molecule.It is interesting to note that such compounds were not detected in the BP essential oil.This discrepancy could be attributed to environmental factors or the fact that the essential oil was derived from the fruits of this plant species, while these polyunsaturated compounds were identified in the diethyl ether extract of the whole aerial parts.All the detected isomers would represent new natural products.
Furthermore, similar MS fragmentation patterns of two minor constituents of the BA oil (RI 1304 and 1394, and a base ion at m/z 43, which is indicative of acetates), and secondin-intensity ion at m/z 115 suggested that these constituents represent homologous acetates of long-chain saturated 4-alkanols.The alternative α-fragmentation of the 4-alkyl acetates observed at m/z 157, i.e., m/z 171, in the two spectra, led to the possible number of carbon atoms in the chains to be 10 and 11, respectively.The presence of 4-decyl acetate and 4-undecyl acetate, new natural compounds, was confirmed using the correlation of The four possible stereoisomers of perillyl 2-methylbutanoate could be expected to have different scents as well as potentially different biological activities.The synthesized mixture of these isomers (all four) had a floral-menthol scent.Synthesizing these esters using chirally pure alcohols and acids would allow us to determine the scent of each individual stereoisomer.
In the BA essential oil, the presence of numerous components with identical or similar mass spectra to esters of tetradec-4,6,8,10-tetraen-1-ol and acids with five carbon atoms, previously detected in the BP diethyl ether extract (praealtaesters A, B, C, and D), was noted.It is presumed that along the known esters, the remaining detected esters represent related constituents differing in the configuration of double bonds in the alcohol part of the molecule.It is interesting to note that such compounds were not detected in the BP essential oil.This discrepancy could be attributed to environmental factors or the fact that the essential oil was derived from the fruits of this plant species, while these polyunsaturated compounds were identified in the diethyl ether extract of the whole aerial parts.All the detected isomers would represent new natural products.
Furthermore, similar MS fragmentation patterns of two minor constituents of the BA oil (RI 1304 and 1394, and a base ion at m/z 43, which is indicative of acetates), and second-in-intensity ion at m/z 115 suggested that these constituents represent homologous acetates of long-chain saturated 4-alkanols.The alternative α-fragmentation of the 4-alkyl acetates observed at m/z 157, i.e., m/z 171, in the two spectra, led to the possible number of carbon atoms in the chains to be 10 and 11, respectively.The presence of 4-decyl acetate and 4-undecyl acetate, new natural compounds, was confirmed using the correlation of experimental RI data with available data from the literature in the case of 4-nonyl acetate [21].In addition, isomeric undecanols (differing in the position of the alcohol group) were detected in the BA essential oil, likely formed through the hydroxylation of undecane present in the oil.
Interestingly, the essential oils of Hypericum spp.(Hypericaceae) and Scandix pectenveneris L. (Apiaceae) also showcase a significant presence of C 9 -C 15 alkanes, mirroring the composition of BA oil.For example, the essential oils extracted from Hypericum species from Bulgaria predominantly featured 2-methyloctane, ranging from 9.13% to 40.9%, alongside nonane and undecane [22].Similarly, the essential oils from different Hypericum species from Serbia unveiled substantial alkane content, with H. hirsutum exhibiting heightened levels of nonane and undecane [23].The alkane fraction in the essential oil of S. pecten-veneris was particularly prominent in samples obtained from aerial parts and roots, constituting 47.8% to 78.1% of the oils [24].Although these compounds were also present in the fruits, their relative abundance was significantly lower (11.1%).Notably, there was a remarkably high concentration of tridecane and pentadecane in the oils of this plant species.This composition aligns with the findings observed in BA oil, where undecane is identified as one of the principal components (21%), whereas BP lacks undecane and similar chain-length alkanes.It is notable that the previously analyzed essential oil from B. praealtum aerial parts contained significant compounds such as (+)-spathulenol, (-)-(E)-caryophyllene oxide, and octyl 2-methylbutanoate, which were either present in significantly lower quantities or absent in the schizocarp essential oil investigated in this study.The study by Kapetanos et al. [15] did not specify which parts of the plant constituted the aerial parts they utilized, but based on the collection date (June 2003) from natural populations, it can be inferred that during this period, the plants were not in the fruit-bearing phase and thus did not contain schizocarps.This difference in plant phase could also explain the observed disparity in chemical composition between the schizocarp oil analyzed in this study and the previously analyzed aerial parts oil.
All the essential oils isolated from the Bupleurum species within the Juncea subsection, including B. cappadocicum, B. gerardii, and B. pauciradiatum, were characterized by a high content of undecane [10].However, also significant differences were noted among these oils.For instance, in B. cappadocicum, the flower oil additionally contained high levels of heptanal, whereas the fruit oil was rich in spathulenol, and the root oil featured hexadecanoic acid [25].In contrast, B. gerardii oils showed varying levels of hexanal across different plant parts, with undecane consistently present in high amounts [25,26].Similarly, in B. pauciradiatum, germacrene D dominated in flower oils, β-pinene in fruit oils, and spathulenol in root oils, highlighting distinct chemical profiles influenced by plant organ specificity within the same subsection [27].These findings underscore the variability in chemical profiles among Bupleurum species within the Juncea subsection, influenced by both genetic factors and environmental conditions.The two species analyzed in this study exhibit chemical traits similar to those observed in previously investigated oils from taxa within this subsection.It seems that there may be speciation within these species concerning the accumulation or biosynthesis of volatile alkanes or sesquiterpenes, which are major constituents of the oils.This warrants further investigation and could potentially yield chemotaxonomically significant traits.The plant material was identified by the late Professor Vladimir Ran delović.

Isolation of Essential Oils
Dried above-ground parts of B. affine (120 g) and schizocarps of B. praealtum (100 g) were subjected to hydrodistillation for 2.5 h using the original Clevenger-type apparatus, and yielded 0.06% (w/w) and 0.01% (w/w) of essential oil, respectively.The distillation procedure was conducted in triplicate.The oils were taken in 2 mL of GC-grade pentane, dried with anhydrous Na 2 SO 4 , and immediately analyzed.

General Experimental Procedures
All used chemicals and solvents were obtained from commercial sources (Sigma-Aldrich, St. Louis, MO, USA; Merck, Darmstadt, Germany; Fisher Scientific, Waltham, MA, USA) and used as received, except for the solvents, which were predistilled and dried before use.Silica gel 60, particle size distribution 40-63 mm (Acros Organics, Geel, Belgium), was used for dry-flash chromatography, whereas precoated Al silica gel plates (Merck, Darmstadt, Germany), Kieselgel 60 F 254 , 0.2 mm) were used for analytical TLC analyses.The spots on TLC were visualized by spraying with 50% (v/v) aq.H 2 SO 4 followed by heating.Elemental analysis (microanalysis of carbon and hydrogen) was carried out with a Carlo Erba Elemental Analyzer model 1106 (Carlo Erba Strumentazione, Milan, Italy). 1 H and 13 C NMR spectra were recorded on a Bruker Avance III 400 MHz NMR spectrometer (Fällanden, Switzerland; 1 H at 400 MHz, 13 C at 100.6 MHz), equipped with a 5 mm dual 13 C/ 1 H probe head at 20 • C. All the NMR spectra were recorded in chloroform-d (Sigma-Aldrich, St. Louis, MO, USA) with tetramethylsilane as the internal standard.Chemical shifts (δ) are reported in ppm and referenced to tetramethylsilane (δ H = 0.00 ppm), or the (residual) solvent signal (CHCl 3 ), and 13 CDCl 3 , in 1 H NMR and 13 C NMR and heteronuclear 2D spectra, respectively.Scalar couplings are reported in Hertz (Hz).The acquired NMR experiments, both 1D and 2D, were recorded using standard Bruker built-in pulse sequences. 1H NMR full spin analysis of perillyl 2-methylbutanoate was performed by manually adjusting δ H and J values to fit the experimentally available values and further optimized using MestReNova 11.0.3 software (tools/spin simulation).This procedure led to a systematic refinement of all calculated NMR parameters until the simulation outcome was in excellent agreement (NRMSD < 0.05%) with the experimental data of the isolated compounds.

Gas Chromatography-Mass Spectrometry (GC-MS) Analyses
GC-MS analyses (3 repetitions) were carried out using a Hewlett-Packard 6890N gas chromatograph equipped with a fused silica capillary column DB-5MS (5% diphenylsiloxane and 95% dimethylsiloxane, 30 m × 0.25 mm, film thickness 0.25 µm, Agilent Technologies, Palo Alto, CA, USA) and coupled with a 5975B mass selective detector from the same company.The injector and interface were operated at 250 and 300 • C, respectively.Oven temperature was raised from 70 to 290 • C at a heating rate of 5 • C/min and the program ended with an isothermal period of 10 min.As a carrier gas helium at 1.0 mL/min was used.The samples, 1.0 µL of essential oil solutions in diethyl ether (1.0 mg of an essential oil sample per 1.0 mL of solvent), were injected in a pulsed split mode (the flow was 1.5 mL/min for the first 0.5 min and then set to 1.0 mL/min throughout the remainder of the analysis; split ratio 40:1).MS conditions were as follows: ionization voltage 70 eV, acquisition mass range m/z 35-650, scan time 0.32 s.Constituents were identified by comparison of their linear retention indices (relative to C 8 -C 40 n-alkanes on a DB-5MS column) with literature values and their mass spectra with those of authentic standards, as well as those from Wiley 6, NIST11, MassFinder 2.3, and a homemade MS library, except in the cases of 4-nonyl acetate [21] and dodecyl benzoate [28], with the spectra corresponding to pure substances and components of known oils, and wherever possible, by co-injection with an authentic sample.

Gas Chromatography-Flame Ionization Detector (GC-FID) Analyses
The GC-FID analyses (three repetitions of each sample) were carried out using an Agilent 7890A GC system equipped with a single injector, one flame ionization detector (FID), and a fused silica capillary column HP-5MS (5% diphenylsiloxane and 95% dimethylsiloxane, 30 m × 0.32 mm, film thickness 0.25 µm, Agilent Technologies, Palo Alto, CA, USA).The oven temperature was programmed from 70 • C to 300 • C at 15 • C/min and then held isothermally at 300 • C for 5 min; carrier gas was nitrogen at 3.0 mL/min; the injector temperature was held at 250 • C. The samples, 1.0 µL of the corresponding solutions, were injected in a splitless mode.The parameters of the FID detector were as follows: heater temperature-300 • C, H 2 flow-30 mL/min, air flow-400 mL/min, makeup flow-23.5 mL/min, data collection-Agilent GC Chemstation with a digitization rate of 20 Hz.

Synthesis of Perilla Alcohol
A mixture of perilla aldehyde (450 mg, 3 mmol) and NaBH 4 (456 mg, 12 mmol) in anhydrous methanol (25 mL) was stirred at 0 • C for one hour, then the ice bath was removed, and the stirring was continued for one hour at room temperature.The reaction mixture was quenched by slowly adding 1 M HCl until the excess borohydride was destroyed.The mixture was extracted with Et 2 O (3 × 50 mL).The organic layers were combined, washed with brine, dried with anhydrous MgSO 4 and the solvent was removed in vacuo, giving 387 mg of perilla alcohol (yield 85%).Mass spectrum and RI of the synthesized alcohol ((4-(prop-1-en-2-yl)cyclohex-1-en-1-yl)methanol) matched with the data available in the literature [18].
A solution of perilla alcohol (15.2 mg, 0.1 mmol), 3-methylbutanoic acid (10.2 mg, 0.1 mmol), 4-(dimethylamino)pyridine (DMAP, 2.4 mg, 0.02 mmol), and N,N ′ -dicyclohexylcarbodiimide (DCC, 20.6 mg, 0.1 mmol) in 1 mL of dry CH 2 Cl 2 was stirred in a round bottom flask overnight at room temperature in a GC vial.Afterward, the reaction mixture was filtered through a thin layer of Celite ® , and the resulting residue was analyzed by GC-MS, without isolation, to obtain the MS and RI data.The resulting reaction mixture was purified by silica gel column chromatography giving 19.5 mg (83% yield) of perillyl 3-methylbutanoate and was used to obtain NMR data.

Conclusions
In conclusion, our study presents a detailed characterization of the essential oils extracted from B. praealtum (BP) and B. affine (BA), revealing their distinct chemical compositions through comprehensive GC-MS analysis.We identified a total of 230 constituents across both oils.In BP schizocarps oil, major components included germacrene D (24.0%), (E)-phytol (14.2%), and bicyclogermacrene (11.4%).In contrast, BA oil was characterized

Figure 3 .
Figure 3. Three-dimensional structure of perillyl 2-methylbutanoate and the analysis of coupling constants disclosed using spin simulation.

Figure 3 .
Figure 3. Three-dimensional structure of perillyl 2-methylbutanoate and the analysis of coupling constants disclosed using spin simulation.
The above-ground plant parts of B. affine in the intermediate flowering-fruit-bearing phase were collected in September 2016 on the slopes of Suva Planina Mt. (near Niš, southeastern Serbia, 43 • 11 ′ 53.1 ′′ N 22 • 08 ′ 33.6 ′′ E), and the schizocarps of B. praealtum were collected in September 2023 in the village Sićevo (southeastern Serbia), both from single populations.Voucher specimens have been deposited in the Herbarium of the Faculty of Sciences and Mathematics, University of Niš (voucher nos.HMN 12112 and HMN 18286).

Table 1 .
Chemical composition of B. affine and B. praealtum essential oils.
1Retention indices determined experimentally on a DB-5MS column relative to a series of C 7 -C 40 n-alkanes.