Gas chromatography for β-caryophyllene determination in St. John’s wort infused oil

INTRODUCTION

Hypericum Perforatum  L., commonly known as St. John’s wort, is one of the best-known medicinal herbs. It is famous for its neuroprotective, antineuralgic and antiviral properties, is beneficial for premenstrual syndrome, polycystic ovary syndrome, perimenopause and menopause, eczema and psoriasis. For a long time, St. John’s wort has been used in traditional medicines for healing skin wounds, burns, diseases of the alimentary tract, and psychological disorders, especially depression. St.  John’s wort has recently been shown to have antioxidant, anticonvulsant, analgesic, anti-inflammatory, cytotoxic and antidiabetic activities [1–4]. Due to its healing properties St. John’s wort is used as a nutraceutical, phytopharmaceutic, and an ingredient in cosmetics [5]. Its leaves and flowers since the ancient times are used to make herbal tea and to prepare infused oils. It is known that St. John’s wort infused oils help to overcome skin problems – relieve irritation, reduce itching, soften, disinfect, regenerate cells, act against herpes, hepatitis, flu viruses, relieve muscle, joint and bone pain. St. John’s wort infused oils are mostly used externally, but can be taken orally for stomach ulcers, are used as a food supplement [6].

Since many of the active components of St. John’s wort are poorly soluble in water but have a high hydrofobicity, it can be expected that the concentrations of these substances in St. John’s wort infused oils will be higher, and the medical properties of infused oils will be stronger than in herbal tee. Moreover, Hypericum extracts show a  significant antioxidant activity, inhibiting free radical generation and lipid peroxidation [7]. So St. John’s wort infused oils, in addition to their therapeutic efficacy, should also be more resistant to ageing.

There have been numerous investigations of St. John’s wort essential oils and they have shown a wide variability in composition. One of the main constituents of St. John’s wort is β-caryophyllene. Its quantity depends on the  region and varies in a broad range. For example, β-caryophyllene content in the  essential oil of St.  John’s wort from northern Turkey is 4.1–5.9% [8], from southeastern France 0.2–28.4% [9], from Greece 6.6–10.3% [10], from Uzbekistan 11.7%  [11] and from Lithuania 1.6–14.2% [12].

β-Caryophyllene as well as St.  John’s wort demonstrantes analgesic, antioxidant, anti-inflammatory, antidepressant and cancer cell growth inhibitory effects [13]. The similarity of the pharmacological properties of β-caryophyllene and of St.  John’s wort suggests that β-caryophyllene is one of the  principle substances responsible for the unique therapeutic effect of St. John’s wort.

β-Caryophyllene is usually determined by gas chromatography (GC) or gas chromatographymass spectrometry (GC-MS)  [14–16]. However, the preparation of multicomponent plant samples for gas chromatographic analysis is often complex and time-consuming. A perfect choice for the determination of volatile compounds is GC combined with headspace sampling [17, 18]. One of the most convenient mathods that combines GC and headspace sampling is static headspace gas chromatography (SHS-GC). It includes an isolation of a volatile analyte in a gas phase and subsequent automatic delivery of an aliquot of vapour to the GC system.

In the case of low concentrations of volatile analytes, before GC analysis they should be preconcentrated. An effective preconcentration technique is solid phase microextraction (SPME). With SPME, the analytes are absorbed on to an absorbent coated fused silica fibre, which is part of a syringe needle. After the partitioning of the analytes from the sample matrices to the extraction phases, the fibre is inserted directly into a GC injection port for thermal desorption [19].

The aim of this work was to prepare, compare and apply SHS-GC and SPME-GC-MS techniques for β-caryophyllene determination in St.  John’s wort infused oil.

EXPERIMENTAL

Reagents and samples

β-Caryophyllene (≥80%) was purchased from Sigma-Aldrich (Germany). Unrefined sunflower oil ‘Senoji aliejinė’ (Lithuania), refined sunflower oil (Ukraine) and coconut oil ‘Naturalisimo’ (the Netherlands) were purchased in a local supermarket.

β-Caryophyllene stock solution in coconut oil (10  mg/g) was prepared by weighting. Working β-caryophyllene solutions in coconut oil were prepared by dilution of the β-caryophyllene stock solution with coconut oil to a required concentration.

Three samples of aereal parts of St. John’s wort plants were examined. One plant sample was purchased in a local pharmacy in 2021 (Ph2021) and two plant samples were collected in the flowering period from the field in the Varėna District (Lithuania) in 2020 (V2020) and in 2021 (V2021).

For St. John’s wort infused oil preparation, 100 g of sunflower oil (carrier oil) was added to 2  g of dried aereal parts of St. John’s wort.

Instrumentation and conditions

Headspace extraction and sample introduction was performed on a  PerkinElmer Headspace Sampler Turbomatrix 16 (PerkinElmer, USA) equipped with a  balanced pressure system. Twenty millilitre headspace vials were used in all experiments. A headspace vial was positioned in the HS autosampler and equilibrated at the  selected temperature. The  needle temperature and the  transition line temperature was 10°C higher than the headspace vial equilibration temperature. The  settings of the headspace sampler were 1 min for pressurization and 0.09  min for injection. Helium was employed as carrier gas with 16.7 psi column head pressure.

Headspace gas chromatographic analysis was performed on a PerkinElmer Clarus 580 series gas chromatograph (PerkinElmer, USA) equipped with a flame ionisation detector (temperature 250°C, hydrogen flow 40 ml/min, air flow 400 ml/min and auxiliary gas (helium) flow 30  ml/min). The  GC system was equipped with the  Rxi®-5Sil MS capillary column (30 m × 0.25 mm id, 0.25 µm film thickness). The  injector temperature was held at 110°C. The oven temperature was programmed as follows: 40°C for 1 min, from 40 to 60°C at 2°C/ min, from 60 to 200°C at 40°C/min, from 200 to 250°C at 10°C/min and held for 1 min.

Solid phase microextraction was performed with a Supelco CAR/PDMS (85 µm) fibre housed in a manual holder (Supelco Bellofonte, PA, USA). SPME was carried out in a 20 ml vial closed with a silicone rubber septum placed in a cap. The vial was placed in a water-jacketed vessel on a magnetic stirrer and kept at 80°C temperature for 20 min. Desorption was carried out in a GC-MS injection port at 250°C for 30 s.

Gas chromatographic-mass spectrometric analysis was performed on a PerkinElmer Clarus 580 series gas chromatograph equipped with a  programmable temperature vaporizer injector and coupled to a  PerkinElmer Clarus 560  S mass spectrometer (PerkinElmer, Shelton, USA). The system was equipped with the Elite-5MS capillary column (30 m × 0.25 mm id, 0.25 µm film thickness). Helium was employed as carrier gas with a  constant flow of 1  ml/min. Injection was performed in the split mode (10:1). The oven temperature was programmed as follows: from 45 to 100°C at 2°C/min, from 100 to 250°C at 5°C/ min and held for 5 min. The transfer line temperature was 280°C. The  electron ionisation ion source conditions were as follows: electron energy 70 eV and temperature 180°C. When GC-MS in the full scan mode was used, data acquisition was performed in a scan mode in a range of m/z 45–500. The  qualitative identification of different compounds was performed by comparing their mass spectra with those stored in the  NIST (National Institute of Standards and Technology) library. For β-caryophyllene quantification, the  selected ion monitoring (SIM) mode was used. The quantification ions (m/z values) were 93 and 133.

The samples were heated in a microwave reactor Monowave 450 (Anton Paar).

RESULTS AND DISCUSSION

Preliminary analysis of St. John’s wort

According to the literature, the quantitative composition of St. John’s wort depends on the region and the climatic conditions and varies in a broad range. In this work, three different samples of St. John’s wort have been investigated, two of them were collected in the Varėna District in 2020 and 2021, the third was bought at a pharmacy. The preliminary analysis was carried out using SPME and GC-MS.

For analysis, 0.2 g of dried arial part of St. John’s wort was placed in a 20 ml vial and subjected to SPME for 30  min at 80°C temperature. Desorption of the SPME fibre was carried out in the GCMS injection port at 250°C for 30 s.

The results of the analysis showed (Fig. 1, Table 1) that the  main volatile components of  St.  John’s wort from pharmacy were β-caryophyllene, γ  mu ro lene, caryophyllene oxide, γ-cadenen and δ-cadenen. The  main volatile components of St. John’s wort collected in 2021 in the Varėna District were γ-murolene, (Z)-β-farnesene, β-caryophyllene and δ-cadenene and the amounts of almost all components (except caryophyllene oxide) were higher (for nonane, 3-methylnonane, (Z)-β-farnesene and γ-murolene even more than 10 times higher) than in St. John’s wort purchased at the pharmacy. This can probably be explained by the fact that the samples were collected at different locations and dried under different conditions. In addition, St.  John’s wort collected in the Varėna District is dominated by flowers and leaves, while St. John’s wort purchased at the pharmacy is dominated by stems.

Comparing St. John’s wort collected in the Varėna District in 2020 and 2021, it turned out that there are more volatile components in St.  John’s wort collected in 2021 (β caryophyllene 6.8 times, α-caryophyllene 13 times and γ-murolene even 19 times more). This could be explained by different meteorological conditions. However, it is more likely that St. John’s wort loses some of its volatiles during long-term storage due to their evaporation or decomposition. One of the degradation products could be 2-methyloctane. Its amount in St. John’s wort collected in 2020 is 3 times higher than in 2021. In addition, twice as much caryophyllene oxide was found in St.  John’s wort collected in 2020. It is likely to occur by oxidising β-caryophyllene [20, 21].

Investigation of St. John’s wort infused oil

Since St. John’s wort has many healing properties, it is widely used to prepare tea. However, many active components of St.  John’s wort have low solubility in water. For example, the water solubility of β-caryophyllene is 0.05011  mg/l, of transβ-farnesene is less than 1 mg/l [22]. On the other hand, many St. John’s wort substances are highly soluble in oil (β-caryophyllene logP = 4.4; transβ-farnesene logP = 6.2), so the concentrations of these substances in St.  John’s wort infused oils should be higher, thus the  healing properties of the  infused oils should be stronger. Additionally, β-caryophyllene is known to have antioxidant properties, so it is likely that St. John’s wort-enriched oil oxidises more slowly.

St. John’s wort infused sunflower oil was chosen for the study.

Determination of β-caryophyllene in infused oil by SHS-GC

SHS-GC method is cheap, fast, fully automated and thus is promissing for the  determination of β-caryophyllene in complex matrices such as oils. When performing SHS-GC, it is important that the amount of analyte in the gas phase is as high as possible. This can be achieved by heating the sample. On the other hand, at high temperature, the  analyte can decompose. A  solution of β-caryophyllene in coconut oil was used to determine the  highest temperature at which β-caryophyllene is still stable. Coconut oil was chosen because it does not oxidise or form volatile by-products when heated to 180°C.

A  solution of β-caryophyllene in coconut oil (10 mg/g) was heated for 5 min in a microwave reactor at a  temperature of 80–180°C. Further, 1 g of the heated solution was taken for SHS-GC analysis. The headspace vial was heated for 10 min at 80°C temperature. The obtained results showed (Fig. 2) that when the temperature exceeded 120°C β-caryophyllene started to decompose. Moreover, it is interesting to note that when β-caryophyllene was heated at 180°C, its content was significantly reduced (Fig. 3). Therefore, in order to maintain the  valuable properties of β-caryophyllene, St.  John’s wort infused oil should not be heated above 120°C temperature.

Based on these results, a headspace vial heating temperature of 120°C was selected and the influence of the heating time on the amount of β-caryophyllene in the headspace was investigated. For that, a headspace vial was heated at 120°C temperature for 3–30  min. It was found that 10  min is enough to achieve equilibrium of the analyte between the solution and the  gas phase. Previous studies have shown [23] that the peak area of β-caryophyllene changes little when the sample content varies from 1 up to 5 g, so 1 g of solution was taken for analysis.

Quality parameters were determined under the  optimised conditions. The  calibration curve was drawn with 6 calibration points with three replicate injections and was linear in the concentration range of 0.1–10 mg/g of β-caryophyllene with the correlation coefficient 0.995. The limit of detection was calculated as three times the baseline noise and was 30 µg/g. The relative standard deviation was determined by five replication analysis of the sample with the β-caryophyllene concentration 1 mg/g and was 7.4%.

The prepared HSH-GC technique was attempted for the  determination of β-caryophyllene in St. John’s wort infused sunflower oil. Unfortunately, the β-caryophyllene peak was not seen in the chromatogram because of the high detection limit.

Determination of β-caryophyllene in infused oil by SPME-GC-MS

In order to reduce a  detection limit of β-caryophyllene, a  preconcentration by SPME was introduced. Moreover, gas chromatographic analysis was accomplished using mass spectrometric detection in the SIR mode.

For SPME of β-caryophyllene, 1  g of β-caryophyllene solution in coconut oil was placed into a 20 ml volume vial and sealed with a silicone rubber septa. The septa was pearsed with a SPME needle, the SPME fibre was exposed to the headspace of the solution and the extraction was carried out at 80°C temperature using 200 rpm stirring rate. As it was demonstrated above, β-caryophyllene starts to decompose when the  temperature exceeds 120°C. However, for simplicity the heating of the vial was carried out in a water termostate that did not allow to reach 120°C heating temperature. The  SPME extraction time was examined and it was found that after 20 min the peak area of β-caryophyllene became stable. Thus 20 min were stabilised as an optimum SPME time. The  desorption was carried out in a GC injection port at 250°C for 30 s.

The calibration curve was drawn with 6 calibration points with three replicate injections. In order to improve sensitivity and reduce interferences, the  peak areas of β-caryophyllene were recorded using the SIR mode (m/z 93 and 133). The calibration curve was linear in a concentration range of 0.017–100 mg/kg of β-caryophyllene with the correlation coefficient 0.999. The limit of detection was calculated as three times the baseline noise and was 5 µg/kg. The relative standard deviation was determined by five replication analysis of the  sample with the  β-caryophyllene concentration 10 mg/kg and was 6.1%.

The method was applied for the determination of β-caryophyllene in St. John’s wort infused oil. It was determined that after 30 days infusion sunflower oil contained 35 µg/kg of β-caryophyllene and after 130 days infusion 109 µg/kg of β-caryophyllene.

Effect of heating on the composition of volatile components of St. John’s wort infused oil

It was studied how heating affects St. John’s wort infused sunflower oil and sunflower oil that does not contain St. John’s wort. The oils were heated in a microwave reactor for 10 min at 180°C temperature and analysed by SPME-GC-MS in the full scan mode. Peak areas of volatile components of the oils are presented in Table 2, the chromatograms of unheated and heated St. John’s wort infused sunflower oil are presented in Fig. 4.

As can be seen from the results presented in Table  2, after heating, large amounts of 2-propenal, 2-hexenal, 1-hexene, 4,5-dimethyl-, 1-octen-3-ol and 2,4-decadienal were formed. These substances were not detected in the unheated oil. Also, in the heated oil, quantities of hexanal, heptanal, 2-heptenal, 2-octenal and nonanal significantly increased. Interestingly, heating increases the  amount of hexanal in pure sunflower oil by 5.8 times, and in St.  John’s wort-infused oil only by 3.6 times. Hexanal is a marker of lipid oxidation degree as it is the main secondary oxidation product of linoleic acid which is one of the principle fatty acids of many edible oils [24]. Therefore, it can be concluded that St. John’s wort slowed down the oxidation of sunflower oil. The  amount of β-caryophyllene decreased in the  heated St.  John’s wort oil, with the  peak area of β-caryophyllene before heating being 504979 and after heating being 166240. It is likely that β-caryophyllene was converted to caryophyllene oxide by temperature exposure. Before heating the caryophyllene oxide peak area was 46699, after heating it was 57412.

CONCLUSIONS

β-Caryophyllene is one of its main active components of St. John’s wort, which is one of the bestknown medicinal herbs with various healing properties. Many of the active components of St. John’s wort are poorly soluble in water but exhibit a high hydrofobicity. Thus, the  concentrations of these substances in St.  John’s wort infused oils should be higher, and the medical properties of infused oils should be stronger than in herbal tee.

In this work, two gas chromatographic techniques, SHS-GC and SPME-GC-MS, were proposed for the determination of β-caryophyllene in St. John’s wort infused oils. A comparison of these two techniques revealed that SPME-GC-MS can be successfully applied to the analysis of edible oil. The other method, SHS-GC, is simple and fast, but because of its high detection limit, it can only be applied to oil samples with β-caryophyllene concentration higher than 0.1 mg/g.

St. John’s wort has been shown to partially inhibit the oxidation of sunflower oil. In addition, heating at 180°C has been shown to reduce the amount of many active components in St. John’s wort infused sunflower oil.

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Vida Vičkačkaitė, Sandra Petrokaitė, Vilius Poškus

DUJŲ CHROMATOGRAFIJOS METODAI β-KARIOFILENUI NUSTATYTI JONAŽOLIŲ EKSTRAKTUOSE ALIEJUJE