Abstract
Agarwood leaf is a potential raw material for aromatic oil, yet it has lacked systematic research on the supercritical carbon dioxide (SC-CO2) extraction technique. In this study, aromatic oil from agarwood leaf was extracted by SC-CO2 extraction, hydrodistillation (HD), and Soxhlet extraction (SE), followed by a series of analysis. The kinetics models of SC-CO2 extraction were established with promising fitting quality. In aggregate, the yields, smells, and appearances of SC-CO2 extraction (45 ℃, 100 bar, 150 min) were better than the other two traditional extraction methods (SE and HD). Thirty and 18 constituents were identified by GC–MS and UPLC–QTOF–MS, respectively, which might explain the special aroma of agarwood leaf aromatic oil. Agarwood leaf aromatic oils contained abundant phenolic compounds and exhibited moderate antioxidant and AChE inhibitory activity, as well as significant anti-inflammatory and anti-melanogenesis activities. This work demonstrated that agarwood leaf aromatic oils extracted by SC-CO2 showed a great potential for developing as food additive or dietary supplement.
1. Introduction
Agarwood, a resinous part of the non-timber Aquilaria tree, is also a highly valuable product for medicine and fragrance purposes. The dark aromatic resin of the heartwood of Aquilaria species is called agarwood which is a well-known and prized fragrant wood. The most common economic species of agarwood are A. crassna, A. malaccensis, and A. sinensis, which are widely planted in southern China, India, and Southeast Asian countries and have been used as traditional medicines for more than a thousand years. However, agarwood tree has a long growth period, taking nearly a decade for planting to get the treasured fragrant heartwood, which seriously decrease the enthusiasm of enterprises and farmers. Fortunately, agarwood tree also offers high-yield raw aromatic materials with potential economic benefits every year, such as agarwood leaves.
Agarwood leaves have been developed into tea products and widely consumed in southern China, with local drinking standard. There are increasing studies on the development and utilization of agarwood leaves extracts, which have attracted growing attention because of their conducive health characteristics, such as antioxidant, anti-inflammatory, hypoglycemic and lipid-lowering. Given that agarwood leaves have strong fragrance similar to agarwood heartwood, agarwood leaves may have the potential to be developed as raw materials for aromatic oil, thus exerting more economic value. Conventionally, hydrodistillation and organic solvent extraction (hexane, petrol ether, diethyl ether) are widely applied in the recovery of plant oils. However, these traditional methods have some imperfection, such as low productivity, low purity, thermal degradation of bioactive and/or volatile compounds, and environmentally unfriendly.
The supercritical carbon dioxide (SC-CO2) extraction is a green technology with high selectivity, which can be used to efficiently extract heat-sensitive and easily oxidized compounds. Less organic solvent is used during extraction, leaving no toxic residues in the product. With these advantages, SC-CO2 extraction has been widely applied in bioactive compound extraction from natural plant materials or by-products. With the increasing demand for eco-friendly natural products, the market for natural flavors, including those from plant extraction and biotechnological origin, is estimated to grow by 6.0 % annually from 2023 to 2032. In this context, SC-CO2 extraction can become more competitive, and has been applied to industrial production. For example, the Indian company Synthite Industries Ltd. has a SC-CO2 extraction line that mainly extracts aromas and spices used in food, cosmetic, and nutraceutical applications, such as black tea, cassia, rosemary, vanilla, and coffee.
However, to the best of our knowledge, research on aromatic oils from agarwood leaf by SC-CO2 extraction is rare. Mingshi et al. explored the effects of different temperature, pressure, and extraction time on the yield of aromatic oils from agarwood leaf by SC-CO2 extraction preliminarily, but they did not study the extraction kinetics model. Moreover, studies about either the chemical composition or bioactivity of aromatic oils from agarwood leaf by SC-CO2 extraction are scarce in the literature. While chemical profiling and bioactivity assay are indispensable for the standardizing quality supervision of extracts in the research of food additives and phytomedicines. Therefore, it is necessary to determine the characteristics of aromatic oils from agarwood leaf by SC-CO2 extraction systematically for in-depth development.
In this study, we aim to explore the influence of various parameters (temperature, pressure, extraction time) on the extraction process of agarwood leaf (Aquilaria sinensis) by SC-CO2, so as to optimize the parameters and help the process scale-up. Secondly, the chemical profile (analyzed by GC–MS and UPLC-QTOF-MS) and biological activity (antioxidant, AChE inhibitory activity, anti-inflammatory, and anti-melanogenesis activities) of SC-CO2 extracts under different extraction conditions and traditional extraction methods extracts (hydrodistillation and Soxhlet extraction) were compared, that is, in our knowledge, the first systematically investigation of characteristics of aromatic oils from agarwood leaf by SC-CO2 extraction. The results of this study will provide a research basis for the standardization of agarwood leaf aromatic oils and offer direction for the in-depth development of agarwood leaf.
2. Materials and methods
2.1. Plant material
A commercial brand of dried agarwood leaf (Aquilaria sinensis, moisture content of 7 ± 0.5 %) was purchased from Nanfeng Town, Danzhou City, Hainan Province, China in 2021. In laboratory, the vacuum-packed agarwood leaves were ground in a pulverizer (Izara, model 800 A, China) at speed of 3500 rpm for 1 min, sieved through a No. 24 mesh sieve (850 µm ± 29 µm aperture), then immediately sealed in a hermetic bag and stored in a dry and dark place until use.
2.2. Reagents and solvents
HPLC-grade n-hexane (Fisher Scientific, USA) was employed in Soxhlet extraction and the dilution of extracts. Anhydrous sodium sulphate (Xilong Scientific, Shantou, China) was used to dehydrate the extracts. CO2 (99.9 %, Beijing Dongfang Medical Gas Co., Ltd. China) was used to carry out SC-CO2 extractions. Analytical grade ethanol (Beijing Chemical Works, China) was used for extract dilution. Folin & Ciocalteu's phenol reagent (Macklin, Shanghai, China), and anhydrous sodium carbonate (Xilong Scientific, Shantou, China) were used for total phenol content measurement; positive control assays were carried out with gallic acid (Macklin, Shanghai, China). Potassium persulfate, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH.) (Macklin, Shanghai, China) were used for the antioxidant assays; and positive control assays were carried out with standard-grade 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich Corp, USA). Sodium dodecyl sulfate (SDS), acetylthiocholine iodide (ATCh), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (Macklin, Shanghai, China), anhydrous sodium bicarbonate (NaHCO3, Xilong Scientific, Shantou, China), acetylcholinesterase (AChE, 220 U/g) and phosphate buffered saline (PBS, pH 7.2–7.4, a phosphate buffer which contains NaCl and KCl) (Solarbio, Beijing, China) were used for acetylcholinesterase bioassay; positive control assays were carried out with standard-grade (-)-huperzine A. A homologous series of n-alkanes (C7 - C40, Anpel, Shanghai, China) were used for retention index calculation in gas chromatography coupled to mass spectrometry (GC–MS) analysis.
2.3. Soxhlet extraction (SE)
A Soxhlet extractor was used in the extraction procedure according to López-Bascón & Castro [16]. Approximately 6 g of agarwood leaf powder was extracted with 60 mL of n-hexane in Soxhlet for 3 h. After that, the n-hexane was removed at 40 ± 2 ℃ using a rapid solvent evaporation system (RK1012, GeneVac, UK). The dried Soxhlet extracts were weighed, sealed and stored at – 20 ℃ until analysis.
2.4. Hydrodistillation (HD)
The experiment was conducted using a Clevenger-type apparatus according to Ngo et al. with modification. First, 80 g of agarwood leaf powder was placed into a 2000 mL flask at a sample/water ratio of 1:12. The mixture was boiled for 5 h until no more essential oil was collected. The obtained mixtures of water and essential oil were then dehydrated with anhydrous sodium sulphate, weighed, and stored in dark bottles at – 20 ℃ until analysis.
2.5. Supercritical carbon dioxide (SC-CO2) extraction
SC-CO2 extraction was performed using a commercial automatic supercritical fluid extraction system (MV-10 ASFE System, Waters. USA), comprising fluid delivery module, extraction oven module, automatic backpressure-regulator, fraction collection module, and computer workstation. A total of 6 g of agarwood leaf powder and 6 g of glass beads (average OD: 3 mm) were mixed and packed into a 25 mL extraction vessel, leaving at least one fifth of the volume of the vessel empty. The function of adding glass beads and leaving empty volume is to prevent the agarwood leaf powder from being compacted, thereby increasing the contact area between the agarwood leaf powder and the solvent, as well as improving the extraction efficiency. The experiments were performed at temperatures of 35 ℃, 45 ℃, and 55 ℃ and pressures of 100 bar, 200 bar, and 300 bar. The extraction process was performed for 3 h in two stages alternately: one was the equilibration duration (supercritical CO2 and samples could fully contact at a CO2 flow rate of 1.0 mL/min), the other one was the dynamic duration (CO2 with extracts was released at a CO2 flow rate of 5.0 mL/min); the equilibration and dynamic processes were kept for 10 min in turn. The oil- containing gas from the extractor passed through a heated metering valve where the SC-CO2 was depressurized. Next, the extracted oil flowed into a cooling collection module while CO2 was vented to the waste gas treatment unit . For the extraction kinetics modelling experiments, every 20 min, SC-CO2 extracts were sequentially collected in different bottles and weighed. For biological activity and chemical analysis experiments, SC-CO2 extract was collected in one brown glass bottle during 3 h, then sealed and stored at – 20 ℃ until analysis.
The Broken and intact cells model proposed by Sovová was selected to describe the SC-CO2 extraction curves, which has been applied in the SC-CO2 extraction of feijoa leaf and Acacia dealbata flowers successfully. The model considers that some extractable substances are easy to contact with solvent, because they are found in broken cells, while the rest of the solutes remain trapped inside intact cells, where the solvent has to penetrate by diffusion to dissolve the soluble compounds. Therefore, the extraction process can be divided into three different periods: the constant extraction rate (CER) period, where easily extractable solutes are extracted mainly by convection at a constant rate; the falling extraction rate (FER) period, where mass transfer starts to be controlled by diffusion; the diffusion-controlled (DC) period, where easily solutes have been removed and mass transfer is governed by diffusion. Three straight lines corresponding with the three different periods formed the piecewise functions, and the actual data were fitted by the software IBM SPSS Statistics 26.0 (USA).
For t ≤ tCER (CER period):
For tCER ≤ t ≤ tFER (FER period):
For t ≥ tFER (DC period):where: y = accumulated yield in the respective period (%), yield is equal to SC-CO2 extracts mass (g) divided by raw agarwood leaf mass (g) and multiplied by 100 %; t = extraction time (min); b1 = y-intercept of the first line (%); k1 = slope of CER lines, k2 = slope of FER lines, and k3 = slope of DC lines, indicating the extraction rates (%.min−1); tCER = extraction time at the end of the CER period (min); tFER = extraction time at the end of the FER period.
2.6. GC–MS analysis
The volatile compounds of the hydrodistillation and SC-CO2 extracts were evaluated by gas chromatography coupled to mass spectrometry (GC–MS, Thermo Trace ISQ system, USA), under the following conditions: silica capillary column DB-5MS (30 m × 0.25 mm id, 0.25 µm film thickness, Angilent, USA); program temperature of 50–280 ℃ at a rate of 5 ℃/min and maintaining for 15 min; carrier gas: helium (99.999 %, 1 mL/min); injection port temperature of 200 ℃, splitless injection of 1.0 µL of the sample extracts diluted by 240 times in n-hexane and filtered through a 0.25-μm membrane filter; electronic impact ionization at 70 eV and 230 ℃; transmission line temperature of 250 ℃; mass spectrometer was programmed to scan from 50 to 500 m/z. Xcalibur software (Version 2.2, Thermo Scientific, USA) was used to analyze the raw data and preliminarily identify chromatographic peaks. Then, the results were further screened according to standard alkanes, mass spectrometry database, and literature.
2.7. UPLC-QTOF-MS analysis
The non-volatile compounds of the SC-CO2 extracts were evaluated by utra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS, I-class, Xevo 2-XS, Waters, USA), under the following conditions: HSS T3 C18 column (2.1 mm × 100 mm, 1.7 µm) (Waters Acquity, USA); mobile phase A was water with 0.1 % formic acid, mobile phase B was acetonitrile; column temperature was 35 ℃; the elution program was as follows: 0–15 min: 20–95 % B; 15–17 min: 95 % B; 17–18 min: 95–20 % B; and 18–23 min: 20 % B; flow rate of 0.3 mL/min; the sample extracts were diluted by 240 times in methanol and filtered through a 0.25-μm membrane filter, injection volume was 1.0 µL; ESI (electrospray ionization source) was set at negative mode; the analyses used leucine-enkephalin as lock mass ([M-H]– = 554.2615); the mass spectra were scanned from 50 to 1200 m/z; data acquisition mode was MSE. The raw data of UPLC-QTOF-MS was handled by Progenesis QI 2.3 software (Waters, USA), including import data, alignment review, experimental design setup, peak selection, deconvolution, and compound identification. The compound identification step of Progenesis QI 2.3 software was carried out by an online search of an in-house library of agarwood leaf (containing 338 compounds reported in literature) and a Progenesis MetaScope internal repository, the maximum allowable error was set to ± 5 ppm.
2.8. Total phenolic content (TPC)
Total phenolic content of extracts was quantified by the Folin-Ciocalteau method according to Ainsworth & Gillespie with modifications. The results were expressed as gallic acid equivalent, namely mg GAE/g extract, as well as mg GAE/100 g leaf when the global yield was taken into account. All experiments were performed in triplicate.
2.9. DPPH and ABTS antioxidant activity assay
DPPH assay was conducted following the procedure described by Mensor et al. with modifications. ABTS assay was carried out according to Re et al. , with modifications. The results were expressed as Trolox equivalent antioxidant capacity, namely mg TE/g extract, as well as mg TE/100 g leaf when the global yield was taken into account. All tests were carried out in triplicate.
2.10. Acetylcholinesterase (AChE) inhibitory activity assay
AChE inhibitory activity was assayed according to the Ellman method with modifications. The AChE inhibitory activity results were expressed as (-)-huperzine A equivalent antioxidant capacity, namely mg AE/g extract, as well as mg AE/kg leaf when the global yield was taken into account. All experiments were performed in triplicate.
2.11. Anti-inflammatory and anti-melanogenesis activities assay
Anti-inflammatory and anti-melanogenesis activities were determined according to Xianyao et al.. The effect of the samples on cyclooxygenase activity was assayed using the COX-2 Inhibitor Screening Kit (Beyotime Biotechnology, Shanghai, China) according to the instructions of manufacturer. Prostaglandin 2 (PGE-2, 20 μg/mL) was used to induce B16-F10 cell model with high melanin expression, and then the effect of the samples on the content of melanin and tyrosinase and the activity of tyrosinase was determined by bicinchoninic acid assay kit and mice tyrosinase (TyR) ELISA kit (Beyotime Biotechnology, Shanghai, China). Specific experimental materials and procedures were described by Xianyao et al. . All experiments were performed in triplicate.
2.12. Statistical analysis
Statistical analyses were conducted using the software GraphPad Prism 9 (California, USA). To confirm significant differences, ANOVA and Tukey tests, with α = 0.05 (significance level) were applied.
3. Results and discussion
3.1. SC-CO2 extraction kinetics modelling
In order to study the extraction kinetics models of SC-CO2 for extract from agarwood leaf, nine extraction experiments under different pressure and temperature conditions were carried out. The accumulated yields at successive time points were recorded, as shown in Fig. 1. The numerical modelling of SC-CO2 extraction process for agarwood leaf was conducive to predict the best extraction parameters and extended the process from laboratory to a larger scale. According to the high coefficient of determination (R2) shown in Fig. 1g, Piecewise function provided promising fitting quality between predictive models and actual data. Therefore, it was proved that Piecewise function was successfully applied to the SC-CO2 extraction process for agarwood leaf, and it could be recommended to be used in the kinetic model of the scale-up experiment.
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When the temperature remained constant, an increase in pressure facilitated an enhancement in the yield of SC-CO2 extracts, particularly at 55 ℃ (Fig. 1a–c). This trend in yield was probably due to the increased SC-CO2 density at higher pressure (Fig. 1g), which contributed to extraction process. Besides, it was observed that higher pressure both shortened constant extraction rate (tCER) period and falling extraction rate (tFER) period, accompanied by higher extraction rates (k1 and k2). This implies that a higher yield could be achieved more quickly in industrial production, thereby saving energy and reducing costs. The SC-CO2 extraction kinetics models constructed in this experiment provided a reference for large-scale procedures. For example, under 45 ℃/200 bar, the yield at 88 min (tFER) accounts for 80 % of the final yield at 180 min. Therefore, it is recommended to use 88 min as the extraction time.
It is more complicated that temperature affects the yield of the extraction process. With the increase of temperature, vapor pressure of the isolated compounds also increases, leading to higher solubility. However, SC-CO2 density decreases with the increase of temperature, resulting in lower solubility. As depicted in Fig. 1e,g, when pressure was 100 bar, the density of SC-CO2 was the highest at 35 ℃, but the yield was not the highest. At 55 ℃, the vapor pressure of the isolated compounds should be the highest, but the yield was actually the lowest. Conversely, at 45 ℃, the yield was the highest. It might be the result of comprehensive adjustment of temperature on both the vapor pressure of the isolated compounds and the density of SC-CO2. At a pressure of 200 bar, changes in temperature had little effect on yield (Fig. 1e). At pressure of 300 bar, increasing the temperature was beneficial for increasing yield (Fig. 1f), indicating that the influence of vapor pressure of the isolated compound was dominant at a higher pressure.
Based on the principles of the highest yield and the shortest extraction time, the best conditions found to be SFE-6 (yield = 1.75 %) and SFE-3 (tFER = 52 min). However, from the perspective of in-depth development, the overall characteristics, chemical composition and biological activity of agarwood leaf aromatic oils are also very important. Therefore, we comprehensively evaluated the extracts under different conditions, so as to choose the optimal extraction condition in the following experiments.
3.2. Yields, smells, and appearances of SE, HD, and SC-CO2 extracts
Soxhlet extraction (SE) has been the a standard technology for over a century, and is also the primary reference for measuring the performance of new extraction methods. Hydrodistillation (HD) is a traditional method to prepare essential oil which mainly uses water, so it is relatively environmental-friendly. Therefore, the aromatic oils of agarwood leaf obtained by SC-CO2 extraction under different conditions were compared with those obtained by the two traditional extraction methods (Soxhlet extraction and hydrodistillation) in this work.
Yields, smells, and appearances of extracts from agarwood leaf obtained by different extraction methods were described in Table S1. The order of yield was Soxhlet extraction (5.02 %) > SC-CO2 extraction (0.80–1.76 %) > hydrodistillation (0.1 %). The yield of SC-CO2 extraction in this experiment was similar to that of agarwood leaves and resinous heartwood reported in previous literature. Liu et al. reported that the yield of SC-CO2 extracts of agarwood leaf (Aquilaria sinensis) was 1.296 % (extraction condition: 30 ℃, 180 bar, 2 h). Other research investigated the SC-CO2 extracts from resinous heartwood of Aquilaria malaccensis, with their yields reported as 1.97 % (45–50 ℃, 30–38 bar, 4 h), 0.62 % (40 ℃, 180 bar, 2 h), and 1.89 % (46 ℃, 280 bar, 2 h), respectively.
As shown in Table S1, the hydrodistillation extract was a reddish-brown essential oil, with strong fragrance, but it had a scorched flavor and the lowest yield. The yield of the Soxhlet extract was the highest, but it was a blackish-green thick liquid with no fragrance. The SC-CO2 extracts were yellowish-green to blackish-green cream with a light fragrance or none. When the temperature increased to 55 ℃, the fragrance of SC-CO2 extracts was reduced or even absent. That's probably because the aroma substances were easily degraded by high temperature. With the increase of pressure, the green and black color of the SC-CO2 extracts gradually deepened. Previous studies have shown that the increase in pressure (>120 bar) may cause the coextraction of other compounds, leading to a decrease in the selectivity of SC-CO2 extraction of aroma compounds. The color and smells of SFE-1 (35 ℃/100 bar) and SFE-4 (45 ℃/100 bar) extracts were the best and most similar to “pure” essential oil. However, the yield of SFE-4 (1.55 %) was higher than that of SFE-1 (1.05 %). In conclusion, SFE-4 was the optimal condition for SC-CO2 extraction from agarwood leaf in terms of yield, color, and smell.
3.3. Volatile compounds identified by GC–MS
Table 1 compiles the volatile compounds of SC-CO2 and hydrodistillation extracts analyzed by GC–MS. Thirty constituents were identified by GC–MS, 16 of which had been reported existing in agarwood leaf previously. Lots of compounds found in hydrodistillation extracts had aroma, such as 2, 4-heptadienal, linalool, 1-furfurylpyrrole, safranal, alpha-farnesene, nerolidol, geranyl linalool, and phytol. These compounds were possibly one of the sources of the special aroma of hydrodistillation extracts, such as nutty, floral, roast, and woody. In particularly, linalool, alpha-farnesene, geranyl linalool, and phytol were detected in SC-CO2 extracts as well, which probably contributed to the floral and woody fragrance of SC-CO2 extracts. The specific values of constituent relative content (%) identified by GC–MS are shown in Table S2, which were calculated based on all peak areas detected. The greatest part of constituents identified in the SC-CO2 extracts were squalene (59.48–71.87 %), hentriacontane (5.59–10.2 %), friedelin (1.15–6.06 %), octacosane (1.30–4.71 %), lupenone (1.95–2.97 %), alpha-tocopherol (1.10–2.10 %), and beta-amyrin (1.19–1.44 %). The greatest part of constituents identified in the hydrodistillation extracts were phytol (35.72 %), squalene (23.70 %), and geranyl linalool (5.24 %). The relative content of squalene was the highest in SC-CO2 extracts and the second highest in hydrodistillation extracts, indicating that it was a very important constituent in agarwood leaf aromatic oil. Squalene has been detected in agarwood leaf in previous literature. For example, Khalil et al. identified squalene in methanolic extracts of agarwood leaf (Aquilaria malacensis) by GC–MS analysis. Jiashen et al. determined squalene in SC-CO2 extracts of agarwood leaf (Aquilaria sinensis) by GC–MS analysis. Hsiao et al. used methanol to extract agarwood leaf (Aquilaria sinensis) and partitioned with ethyl acetate and water, then authenticated squalene in the ethyl acetate layer by nuclear magnetic resonance and mass spectrometry analysis.
Table 1. Volatile compounds of aromatic oil from agarwood leaf identified by GC–MS.
| Tentative identification | RT (min) | Cas No. | Formula | Classes | Reported in agarwood leaf | Efficacy and application | Odor descriptions |
|---|---|---|---|---|---|---|---|
| 2, 4-heptadienal | 7.67 | 4313-03-5 | C7H10O | aldehydes | *NR | edible essence | nutty, fatty |
| cis-linalyl oxide | 9.34 | 5989-33-3 | C10H18O2 | monoterpenoids | NR | – | – |
| linalool | 10.00 | 78-70-6 | C11H24 | monoterpenoids | NR | antimicrobial and flavoring agent | citrus, floral, coriander, lemon |
| 1-furfurylpyrrole | 12.35 | 1438-94-4 | C9H9NO | pyrroles | NR | edible essence | cocoa, green, roast |
| safranal | 12.84 | 116-26-7 | C10H14O | monoterpenoids | [35] | edible essence | herb |
| beta-cyclocitral | 13.42 | 432-25-7 | C10H16O | monoterpenoids | NR | insect repellent | – |
| 1,3-di-tert-butylbenzene | 14.16 | 1014-60-4 | C14H22 | phenylpropanes | NR | – | – |
| tetradecane | 18.14 | 629-59-4 | C14H30 | alkanes | [36] | – | – |
| alpha-farnesene | 20.54 | 502-61-4 | C15H24 | sesquiterpenoids | NR | flavoring agent | boiled vegetable, floral, woody |
| nerolidol | 22.14 | 7212-44-4 | C15H26O | sesquiterpenoids | NR | neuroprotective; flavoring agent | fir, linoleum, pine |
| megastigmatrienone | 22.52 | 38818-55-2 | C13H18O | ketones | NR | – | – |
| phytone | 28.09 | 502-69-2 | C18H36O | ketones | [35] | – | – |
| phytol natural | 28.44 | 102608-53-7 | C20H40O | diterpenoids | [5] | synthesis raw material of vitamin E | – |
| phytan | 28.98 | 638-36-8 | C20H42 | alkanes | NR | – | – |
| farnesylacetone | 29.62 | 1117-52-8 | C18H30O | ketones | [35] | pharmaceutical intermediate | – |
| methyl palmitate | 29.77 | 112-39-0 | C17H34O2 | fatty acyls | [35] | – | – |
| isophytol | 30.44 | 505-32-8 | C20H40O | diterpenoids | NR | – | – |
| geranyl linalool | 32.22 | 1113-21-9 | C20H34O | diterpenoids | NR | flavoring agent | floral, woody |
| oleic acid | 32.94 | 112-80-1 | C18H34O2 | fatty acyls | NR | soften blood vessels; nutritional additives | – |
| phytol | 33.43 | 150-86-7 | C20H40O | diterpenoids | [4] | synthesis raw material of vitamin E | floral |
| trans-geranylgeraniol | 34.34 | 24034-73-9 | C20H34O | diterpenoids | NR | pharmaceutical intermediate | – |
| squalene | 44.61 | 111-02-4 | C30H50 | triterpenes | [5] | protect skin; nutritional additives | – |
| octacosane | 45.67 | 630-02-4 | C28H58 | alkanes | [6] | – | – |
| lupeol | 47.84 | 545-47-1 | C30H50O | pentacyclic triterpenoids | [37] | anti-inflammatory | – |
| hentriacontane | 48.94 | 630-04-6 | C34H70 | alkanes | [36] | – | – |
| alpha-tocopherol | 49.46 | 59-02-9 | C29H50O2 | prenol lipids | [35] | anti-ageing; nutritional additive | – |
| beta-amyrin | 53.36 | 559-70-6 | C30H50O | pentacyclic triterpenoids | [37] | pharmaceutical intermediate | – |
| lupenone | 54.61 | 1617-70-5 | C30H48O | pentacyclic triterpenoids | [35] | antidiabetic | – |
| stigmasterol | 56.71 | 83-48-7 | C29H48O | steroids | [38] | synthesis raw material of steroid hormone | – |
| friedelin | 59.72 | 559-74-0 | C30H50O | pentacyclic triterpenoids | [36] | pharmaceutical intermediate | – |
RT: retention time; *NR means that this compound has not been reported existing in agarwood leaf before; Efficacy and application information of each compound was obtained from Chemical Book (https://www.chemicalbook.com/ProductIndex.aspx) and PubChem (https://pubchem.ncbi.nlm.nih.gov/). Odor descriptions were obtained from LRI& odour database (http://www.odour.org.uk/lriindex.htmL) and Flavor Ingredient Library (https://www.femaflavor.org/flavor-library).
Squalene is a strong natural antioxidant, as well as a powerful intermediary and precursor for all steroid hormones and cholesterol production in both plants and animals. Squalene is widely used in food and cosmetics industries, however, the main commercial source of squalene is deep-sea fish liver, which may be harmful to marine ecology if further developed. To overcome this unsustainable practice, researchers began to look for sustainable resources of squalene in plants and microorganisms, such as olive, amaranth grain, and the bacterium Escherichia coli. The results of GC–MS analysis showed that squalene was the main volatile component in aromatic oil of agarwood leaf, suggesting that it could potentially serve as an alternative source of squalene. Agarwood leaf is a by-product in agarwood industry with very low cost. However, the yield of aromatic oil in agarwood leaf was still very poor (0.80–1.76 %). Future efforts should focus on optimizing cultivars optimizing cultivar, cultivation methods, and extraction techniques to make the yield of squalene in agarwood leaf can compensate for the expenses of their disposal. Other constituents identified by GC–MS also offer numerous health benefits, such as friedelin (anti-inflammation and antioxidation), lupenone (anti-virus and anti-diabetes), and alpha-tocopherol (nephroprotective property). In addition, many identified compounds are used in industry as edible essence, flavoring agent and pharmaceutical intermediates (Table 1).
As shown in Fig. 2, components identified by GC–MS in agarwood leaf extracted by SC-CO2 and hydrodistillation were divided into 13 classes, namely triterpenes (1), alkanes (4), pentacyclic triterpenoids (4), diterpenoids (5), sesquiterpenoids (2), ketones (3), fatty acyls (2), monoterpenoids (4), steroids (1), prenol lipids (1), pyrroles (1), phenylpropanes (1), and aldehydes (1). Representative GC–MS total ion chromatograms of SC-CO2 and hydrodistillation extracts are shown in Fig. 3a–b.
Fig. 2
Fig. 3The volatile compound profiles of SC-CO2 and hydrodistillation extracts were quite different (Fig. 2 and Table S2). SC-CO2 extracts were dominated by large-molecule and high boiling point compounds, such as triterpenes, long-chain alkanes, pentacyclic triterpenoids, and prenol lipids. Furthermore, most high boiling point compounds were only found in SC-CO2 extracts, including pentacyclic triterpenoids, long-chain alkanes (octacosane and hentriacontane), steroids, and prenol lipids. It suggested that high boiling point compounds were easy to extract under SC-CO2 extraction, because high pressure made it possible to extract compounds which were more firmly fixed on plant tissues . Both SC-CO2 extracts and hydrodistillation extracts detected triterpenes, diterpenoids, and fatty acyls compounds. Besides, hydrodistillation extracts still contained various small molecules such as ketones, sesquiterpenoids, monoterpenoids, pyrroles, and aldehydes, which were rare in SC-CO2 extracts. The possible reason was that CO2 stream might carry away some low boiling point compounds in the cooling collection module of the supercritical extraction system. Hierarchical cluster analysis showed that the volatile chemical profile of SFE-1 (35 ℃, 100 bar) was similar to hydrodistillation (Fig. 3d) the most. This was probably because low temperature and low pressure made SC-CO2 selectively extract more small-molecule and low boiling point compounds, but fewer large-molecule and high boiling point compounds.
3.4. Non-volatile compounds identified by UPLC-QTOF-MS
The non-volatile compound profile of SC-CO2 extracts from agarwood leaf was analyzed by UPLC-QTOF-MS, and the raw data was processed by Progenesis QI software. Each peak was assigned to several candidate compounds by Progenesis QI online identification. These candidate compounds were then further screen by comparing with literatures and various databases manually. For example, the peak at 7.40 min was further identified as genkwanin, because its adduct ion m/z 283.0608 (M-H) and typical fragment ions m/z 268.0386 (M-CH3) and m/z 240.0402 (M-CH3-CO) were consistent with the standard mass spectrum of genkwanin from Bruker Sumner MetaboBASE Plant Library. The peak at 7.67 min was tentatively identified as velutin, because its adduct ion m/z 313.0719 (M-H) and typical fragment ions m/z 298.0474 (M-CH3) and m/z 283.0231 (M-CH3-CH3) were consistent with the standard mass spectrum of velutin from MassBank High Quality Mass Spectral Database. All the other peaks were identified using the same method, only compounds that met the requirements were reported as shown in Table 2.
Table 2. Non-volatile compounds of SC-CO2 extracts of agarwood leaf identified by UPLC-QTOF-MS.
| RT (min) | Tentative identification | CAS NO. or PubChem SID | Formula | [M-H]- measured | Mass error (ppm) | Typical fragment ion | Identification basis | Classes | Reported in agarwood leaf | Efficacy and application | Odor descriptions |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 5.66 | genkwanin 5-glucoside | 11293760 | C22H22O10 | 491.1187 | –2.14 | 445.2131, 377.1780, 285.1244 | [47] | flavonoid glucosides | [47] | anti-inflammatory; antidermatosis | – |
| 6.46 | 2-(4-Methoxyphenethyl)− 6-methoxy-7-hydroxychromone | 102344869 | C19H18O5 | 325.1079 | –0.88 | 310.0842 | [4] | chromones | [4] | – | – |
| 7.26 | sakuranetin | 2957-21-3 | C16H14O5 | 285.0759 | –3.19 | 119.0501, 165.0200 | MetaboBASE | O-methylated flavonoids | *NR | anti-inflammatory; antidermatosis | – |
| 7.40 | genkwanin | 437-64-9 | C16H12O5 | 283.0608 | –1.36 | 240.0402, 268.0386 | MetaboBASE | O-methylated flavonoids | [4] | anti-inflammatory; antidermatosis | – |
| 7.57 | 7-methoxytricin | 107316-94-9 | C18H16O7 | 343.0818 | –1.40 | 328.0592, 283.0610 | [48] | O-methylated flavonoids | [48] | – | – |
| 7.67 | velutin | 25739-41-7 | C17H14O6 | 313.0719 | 0.44 | 298.0474, 283.0231 | MassBank | O-methylated flavonoids | [4] | antioxidant; inhibit tyrosinase and melanogenesis; anti-inflammatory; | – |
| 9.14 | (9E,15E)–12,13-dihydroxyoctadeca-9,15-dienoic acid | 139292049 | C18H32O4 | 311.2213 | –4.66 | 293.2041, 162.8332 | MassBank | fatty acyls | NR | – | – |
| 9.79 | 9-Hydroxy-10,12,15-octadecatrienoic acid | 89886-42-0 | C18H30O3 | 293.2113 | –3.28 | 275.1980, 235.1680, 183.1378 | MassBank | fatty acyls | NR | – | – |
| 10.11 | MGMG 18:3 | 139291884 | C27H46O9 | 559.3115 | 1.32 | 513.3101, 277.2121, 253.0903 | MassBank | glycosylglycerols | NR | – | – |
| 10.45 | FA 18:3 + 2 O | – | C18H30O4 | 291.1957 | –2.76 | 309.2160, 291.1954, 185.1268 | MassBank | fatty acyls | NR | – | – |
| 10.73 | FA 18:2 + 1 O | – | C18H32O3 | 295.2271 | –2.47 | 277.2155, 171.0999 | MassBank | fatty acyls | NR | – | – |
| 13.50 | farnesol | 4602-84-0 | C15H26O | 267.1958 | –3.30 | 220.8481, 205.8396, 162.8366， | MassBank | prenol lipids | NR | antimicrobial; edible essence | oil |
| 13.96 | myristic acid | 544-63-8 | C14H28O2 | 227.2012 | –1.97 | 186.103 | GNPS | fatty acyls | [35] | edible essence | wax, dairy |
| 14.83 | linoleic acid | 60-33-3 | C18H32O2 | 279.2330 | 0.001 | 347.2194, 379.1566, | MetaboBASE | fatty acyls | [35] | nutritional supplements | – |
| 15.69 | palmitic acid | 57-10-3 | C16H32O2 | 255.2327 | 3.85 | 323.2529, 355.1607 | MetaboBASE | fatty acyls | [5] | edible essence | – |
| 15.97 | oleic acid | 112-80-1 | C18H34O2 | 281.2485 | –0.42 | 381.1724, 349.2327 | MetaboBASE | fatty acyls | NR | soften blood vessels; nutritional additives | – |
| 17.33 | stearic acid | 57-11-4 | C18H36O2 | 283.2636 | –2.39 | 265.1473 | MassBank | fatty acyls | [49] | edible essence | dairy |
| 17.41 | hederagenin | 465-99-6 | C30H48O4 | 471.3493 | 2.69 | – | MetaboBASE | triterpenoids | [50] | – | – |
RT: retention time. Standard mass spectrum databases (https://mona.fiehnlab.ucdavis.edu/spectra/search): MetaboBASE, Bruker Sumner MetaboBASE Plant Library; MassBank, MassBank High Quality Mass Spectral Database; GNPS, Global Natural Product Social Molecular Networking Library. *NR: means that this compound has not been reported existing in agarwood leaf before. Efficacy and application information of each compound was obtained from Chemical Book (https://www.chemicalbook.com/ProductIndex.aspx) and PubChem (https://pubchem.ncbi.nlm.nih.gov/). Odor descriptions were obtained from LRI& odour database (http://www.odour.org.uk/lriindex.htmL) and Flavor Ingredient Library (https://www.femaflavor.org/flavor-library)
Finally, 18 compounds were putatively identified by UPLC-QTOF-MS, 10 of which had been reported existing in agarwood leaf previously . The aroma-active compounds identified by UPLC-QTOF-MS including farnesol, myristic acid, and stearic acid, which could possibly contribute to the wax and dairy aroma of agarwood leaf oils. The specific values of constituent relative content (%) identified by UPLC-QTOF-MS are shown in Table S3, which were calculated only based on the identified peak areas. The greatest part of constituents identified in the SC-CO2 extracts were velutin (25.06–51.88 %), palmitic acid (6.19–18.50 %), linoleic acid (8.56–18.33 %), oleic acid (3.59–12.24 %), and genkwanin (0.55–11.83 %).
Many constituents of the SC-CO2 extracts of agarwood leaf identified by UPLC-QTOF-MS have various health benefits, such as velutin (antimicrobial activities and inhibitory effect on tyrosinase and melanogenesis), genkwanin, sakuranetin and their sugar derivatives (anti-inflammatory and antidermatosis activities), free fatty acids (essential for the formation of the epidermal barrier). Besides, some of the identified compounds were used in industry as edible essence, such as farnesol and stearic acid (Table 2).
As shown in Fig. 4, the components identified by UPLC-QTOF-MS in agarwood leaf extracted by SC-CO2 were divided into 7 classes, namely O-methylated flavonoids (4), fatty acyls (9), triterpenoids (1), flavonoid glucosides (1), chromones (1), prenol lipids (1), glycosylglycerols (1). Representative UPLC-QTOF-MS extract ion chromatogram of SC-CO2 extracts (SFE-4) is shown in Fig. 3c. The chemical profiles of SC-CO2 extracts at different conditions were largely consistent, mainly consisting of O-methylated flavonoids and fatty acyls. It was suggested that the abundant total phenolic content of SC-CO2 extracts mainly consisted of O-methylated flavonoids, as well as flavonoid glucosides and chromones. For SFE-1 (35 ℃, 100 bar) and SFE-4 (45 ℃, 100 bar), fatty acyls (relatively small polarity) accounted for more than O-methylated flavonoids (relatively large polarity), while other SFEs showed the opposite trend. Notably, hierarchical cluster analysis also suggested that the chemical profile of SFE-1 and SFE-4 were different to other SFEs (Fig. 3e), which was consistent with the fact that their fragrances were better than other extracts. It indicated that lower temperature and pressure of SC-CO2 were conducive to extract compounds with small polarity, which possibly contributed to the special fragrance of agarwood leaf oil.
Fig. 4It is worth noting that UHPLC-QTOF -MS was supposed to identify more compounds than GC–MS in metabolomics analysis, because they work on different principles. However, it seems that the result of SC-CO2 extracts chemical profiling was the opposite. One of the possible explanations was that SC-CO2 is a weak polar solvent, which will extract more weak polar compounds, and GC–MS is suitable for the analysis of weak polar compounds. Another reason was that the database (Section 2.7) used in compound identification step of Progenesis QI 2.3 software could not cover all the compounds in SC-CO2 extracts of agarwood leaf. Therefore, many peaks detected by UHPLC-QTOF -MS were remained unidentified in this work. It also proved that the chemical profiling of SC-CO2 extracts of agarwood leaf still has the possibility of further exploration in the future.
3.5. Total phenolic content (TPC)
Phenolic compounds have good antioxidant activity, as well as protect against many chronic diseases. The TPC of hydrodistillation extracts in this work was too low to be detected. TPC of SC-CO2 and Soxhlet extracts ranged from 2.71 to 7.49 mg GAE/g extract, with SEF-7 (55 ℃/100 bar) being the highest (Fig. S1a). When global yields were taken into account (Fig. S1b), the TPC of SC-CO2 extracts ranged from 4.20 to 7.05 mg GAE/100 g leaf, which increased with the increase in temperature and pressure. The TPC of Soxhlet extracts was particularly high (23.60 mg GAE/100 g leaf). It demonstrated that Soxhlet extraction method was more capable of extracting TPC than hydrodistillation and SC-CO2 extraction, which was consistent with the study of Latiff et al. So far by now, we have not found the report about the TPC of agarwood leaf by SC-CO2 extraction or Soxhlet extraction. Benito-Román et al. reported that the TPC of quinoa oil extracted by SC-CO2 (50 ℃ and 400 bar) was 3.3 mg/g oil. Bombardelli et al. studied the extraction of Araç á (Psidium cattleianum) fruits by SC-CO2 and determined the TPC of extracts as 0.327–0.363 mg GAE/g extract. Compared to these works of other plants, the TPC of agarwood leaf aromatic oil extracted by SC-CO2 was considerable in this work.
3.6. Antioxidant activity
The antioxidant activities of agarwood aromatic oil extracted by different methods were evaluated by DPPH and ABTS assays. The results are shown in Fig. 5a–d and Table S4. The hydrodistillation extracts exhibited the weakest scavenging capacity of DPPH and ABTS radicals, whether expressed as extract (Fig. 5a) or leaf (Fig. 5b). It was probably because the TPC of hydrodistillation extracts were the lowest (Fig. 3a,b), whereas phenolic compounds providing good antioxidant activity. In previous studies, the antioxidant activity of hydrodistillation extracts of Psidium cattleianum fruits and Aquilaria sinensis heartwood were weaker than SC-CO2 extracts as well.
Fig. 5The antioxidant activity of SC-CO2 extracts was higher than Soxhlet extracts generally when it expressed as extract (Fig. 5a,c), but the case was opposite when it expressed as leaf (Fig. 5b,d). A similar trend was observed for TPC (Fig. 3a,b). It indicated that the antioxidant activity of SC-CO2 and Soxhlet extracts corresponded to TPC. It can be deduced that the antioxidant compounds of SC-CO2 extracts were purer than those of Soxhlet extracts, but the antioxidant compounds extraction rates of Soxhlet extraction were higher than SC-CO2 extraction. Compared with DPPH radical, all extracts showed higher activity towards ABTS. This phenomenon was consistent with the research of Pavlić et al. , because the ABTS assay is regarded as a hydrophilic and lipophilic antioxidant system, while the DPPH assay is just a hydrophobic system.
There are few literatures evaluating antioxidant activity of agarwood leaf SC-CO2 extracts. Some articles reported the antioxidant activity of organic solvent extracts of agarwood, but they did not use Trolox equivalent to express the results, making it hard to compare this work with them. Nevertheless, we can still estimate the antioxidant activity level of agarwood leaf SC-CO2 extracts through comparing with other spice plants. Pavlić et al. reported that the antioxidant activity of peppermint extracted by SC-CO2 was 20.55–33.27 mg TE/g extract (by DPPH) and 24.82–47.50 mg TE/g extract (by ABTS). Oliveira et al. reported that the antioxidant activity of Piper divaricatum leaf extracted by SC-CO2 was 116.17–296.86 mg TE/g extract (by DPPH). In our work, the antioxidant activity of agarwood leaf extracted by SC-CO2 was 22.27–54.89 mg TE/g extract (by DPPH) and 30.15 – 76.66 mg TE/g extract (by ABTS). Thus, the antioxidant activity of agarwood leaf aromatic oil seems to be at a moderate level among spice plants.
3.7. Acetylcholinesterase (AChE) inhibitory activity
The development of neurodegenerative diseases such as Alzheimer's disease is usually mediated by acetylcholine metabolic defect. Inhibition of AChE is a common method to treat and prevent these diseases, because acetylcholinesterase (AChE) is in charge of the hydrolysis of acetylcholine. Natural plant oils contain abundant lipophilic substances, such as terpenoids, which can easily cross the blood-brain barrier. Besides, many plant oils have been reported to have AChE inhibitory activity, such as peppermint oil and Pinus species essential oils. Therefore, the AChE inhibitory activity of agarwood leaf was assayed to explore its potential as a healthy food for preventing Alzheimer's disease.
As shown in Fig. 5e,f, the order of AChE inhibitory activity was SC-CO2 extracts (0.69–1.79 mg AE/100 g extract)>Soxhlet extracts (0.49 mg AE/100 g extract) > hydrodistillation (0.12 mg AE/100 g extract). Pavlić et al. reported that the AChE inhibitory activity of SC-CO2 extracts of peppermint were 5.07 − 5.73 mg galatamine equivalent/g extract. Therefore, agarwood leaf aromatic oil only exhibited relatively weak AChE inhibitory activity.
3.8. Anti-inflammatory and anti-melanogenesis activities
Skin inflammation is strongly associated to COX-2 secretion, so the anti-inflammatory effect of aromatic oil can be evaluated according to the inhibition rate of COX-2 activity. SFE-1 inhibited COX-2 activity in a concentration-dependent manner, indicating that it contained bioactive compounds which inhibit COX-2 activity (Fig. 5g). PGE-2 is another major proinflammatory cytokine in skin inflammation which could motivate melanocyte synthesis, enhance melanin secretion, and initiate pigmentation. In human body, tyrosinase in melanocytes is the rate-limiting enzyme in the biosynthesis of melanin biosynthesis, so a secure and efficient tyrosinase inhibitor may be a fine candidate for the treatment of hyperpigmentation. The cell survival rates of B16-F10 cells treated with SFE-1 at 25, 50 and 100 μg/mL were 93.51 %, 92.53 %, and 92.38 %, respectively. Thus, a concentration of 100 μg/mL was used in subsequent experiments. SFE-1 significantly inhibited melanin and tyrosinase production, as well as tyrosinase activity in PGE-2-stimulated B16-F10 cells (Fig. 5h). Agarwood leaf aromatic oil exhibited significant anti-inflammatory and anti-melanogenesis activities, which indicated that it was a potential hyperpigmentation inhibitor.
4. Conclusion
In this study, the extraction kinetics models of SC-CO2 for extract from agarwood leaf under different temperature and pressure were obtained with good fitting quality, which would provide a reference for large-scale procedures. In aggregate, the yields, smells, and appearances of SC-CO2 extracts (45 ℃, 100 bar, 150 min) were better than those of the other two traditional extraction (Soxhlet extraction and hydrodistillation). The chemical profiling showed that the TPC of agarwood leaf aromatic oil was abundant. In addition, 30 and 18 constituents from agarwood leaf aromatic oil were rapidly identified by GC–MS and UPLC-QTOF-MS, respectively, which might explain the special aroma of agarwood leaf aromatic oil. The biological activity assay results showed that agarwood leaf aromatic oils exhibited moderate antioxidant and AChE inhibitory activity, as well as significant anti-inflammatory and anti-melanogenesis activities.
In summary, our work demonstrated that agarwood leaf aromatic oils extracted by SC-CO2 showed a great potential for developing as food additives or dietary supplements in food industry from the aspects of extraction process, chemical composition and biological activity. The results of this report provided more laboratory basis for large-scale SC-CO2 extraction. In the future, we will carry out chemical profiling and bioactivity assay of agarwood leaf aromatic oil from different cultivars and producing areas. This will provide data for the quality control and standardization of agarwood leaf aromatic oils by SC-CO2 extraction, and ultimately contribute to enhancing the circular economy of the agarwood grove.
CRediT authorship contribution statement
Peiling Wu: Data curation; Formal analysis; Writing – original draft; Conceptualization; Methodology. Le Sun: Writing review; Supervision, Formal analysis. Shunyao Qi: Validation; Visualization. Tiexin Zeng: Software; Visualization. Ziyu Hou: Data curation; Validation. Yun Yang: Data curation; Resources. Lijia Xu: Supervision, Conceptualization; Methodology; Writing review; Project administration; Resources. Jianhe Wei: Funding acquisition; Conceptualization. Peigen Xiao: Project administration; Conceptualization; Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work in ''Extraction process, chemical profile, and biological activity of aromatic oil from agarwood leaf (Aquilaria sinensis) by supercritical carbon dioxide extraction''.
Acknowledgements
This work was supported by Hainan Academician Innovation Platform Scientific Research Project, CAMS Innovation Fund for Medical Sciences (CIFMS: 2022-I2M-2-002), and Guangxi Science and Technology base and talent project (NO. AD22080012). The authors would like to sincerely appreciate Qingli Wu for contribution of the language polish and revision. The authors would like to sincerely appreciate Narda Aquilaria Research Institute (Hainan) Co., Ltd. for offering the agarwood leaf samples.
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