Identification of Fatty Acids, Amides and Cinnamic Acid Derivatives in Supercritical-CO2 Extracts of Cinnamomum tamala Leaves Using UPLC-Q-TOF-MSE Combined with Chemometrics

Cinnamomum tamala leaf (CTL), also known as Indian bay leaf, is used all over the world for seasoning, flavoring, and medicinal purposes. These characteristics could be explained by the presence of several essential bioactive substances and lipid derivatives. In this work, rapid screening and identification of the chemical compounds in supercritical (SC)-CO2 extracts of CTL by use of UPLC-Q-TOF-MSE with a multivariate statistical analysis approach was established in both negative and positive mode. A total of 166 metabolites, including 66 monocarboxylic fatty acids, 52 dicarboxylic fatty acids, 27 fatty acid amides, and 21 cinnamic acid derivatives, were tentatively identified based on accurate mass and the mass spectrometric fragmentation pattern, out of which 142 compounds were common in all SC-CO2 extracts of CTL. Further, PCA and cluster hierarchical analysis clearly discriminated the chemical profile of analyzed extracts and allowed the selection of SC-CO2 extract rich in fatty acids, fatty acid amides, and other bioactive constituents. The result showed that the higher number of compounds was detected in CTL4 (300 bar/55 °C) extract than the other CTL extracts. The mono- and di-carboxylic fatty acids, fatty acid amides, and cinnamic acid derivatives were identified in CTL for the first time. UPLC-Q-TOF-MSE combined with chemometric analysis is a powerful method to rapidly screen the metabolite profiling to justify the quality of CTL as a flavoring agent and in functional foods.


Introduction
Cinnamomum (Lauraceae) is a genus comprising over 250 species of evergreen trees found in sub-tropical and tropical Asia, Africa, and South America, valued for their culinary and medicinal uses [1].Among them, Cinnamomum tamala (Buch.-Ham.)T. Nees & Eberm., commonly known as Tejpat, Indian cassia, or Indian bay leaf, is one of the most commercially important species of the genus [2].This species is naturally distributed in the North-East Himalayas, North-Western Himalayas, and southern parts of the country from tropical to sub-tropical regions at altitudes of 900-2500 m [3,4].The leaves and bark of Cinnamomum trees are widely utilized as spices in cooking and for producing essential oils and have many applications in perfumery, flavoring, and pharmaceuticals industries [5].Cinnamomum tamala leaves (CTLs) are most popular as a food additive in numerous culinary preparations worldwide.In India, CTLs are used not only as spices and flavoring agents but also for their medicinal properties, addressing conditions such as diabetes, hyperlipidemia, inflammation, hepatotoxicity, and diarrhea [6].Since ancient times, CTLs have been traditionally utilized in Ayurvedic and Unani medicine to treat conditions related to scabies, the anus, rectum, liver, and spleen [7].
Moreover, research into the pharmacological activities of C. tamala has highlighted its various benefits, including antimicrobial, antioxidant, anti-inflammatory, analgesic, antiulcerogenic, antihypertensive, antidiabetic, antidiarrheal, antipyretic, anti-obesity, cardiovascular protective, and neuroprotective effects [3,6,8,9].Phytochemical studies of C. tamala extracts have identified several bioactive compounds such as terpenes, alkaloids, flavonoids, tannins, polyphenols, saponins, and fatty acids [10][11][12][13][14].Among these, fatty acids (FAs) are particularly notable due to their significant biological functions and health benefits, including roles in lipid metabolism, antioxidation, anti-inflammation, cholesterol lowering, and augmenting the liver detoxification process [15][16][17][18][19].For instance, linoleic and linolenic acids have been reported to offer protective effects against cardiovascular diseases, inflammatory conditions, and neurodegenerative disorders like Alzheimer's disease.When fatty acids combine with amines, they form fatty acid amides (FAAs), which have varying carbon lengths and unsaturation.These bioactive intracellular signaling molecules are regulated by fatty acid amide hydrolases, which convert FAAs back into their parent fatty acids [20,21].Despite their importance, there is limited research on the fatty acids in C. tamala.The study by Farag et al. in 2022 is the only one focusing on identifying fatty acids in C. tamala bark [12].Owing to the high medicinal value and effects of these components, it is crucial to characterize the fatty acids and FAAs in CTLs and develop an efficient green extraction method to minimize postprocessing requirements.
For this purpose, supercritical carbon dioxide (SC-CO 2 ) is a green extraction technique that has gained attraction as an alternative to traditional methods for extracting fatty acids [22,23].SC-CO 2 has advantages such as nontoxicity, selectivity, absence of solvent residues, and operation at low temperatures, making it suitable for extracting hydrophobic compounds without degrading active metabolites.Mass spectrometry (MS) has been extensively employed for the analysis of fatty acids, fatty acid amides, and fatty acid derivatives in targeted samples.Gas chromatography coupled with EI-MS is generally applied to analyze the fatty acids by derivatization to their respective fatty acid methyl esters (FAMEs) [24].In addition, liquid chromatography (LC)-MS is an effective tool for fatty acid analysis due to its high sensitivity, selectivity, and rapid analysis capabilities [25], and it also screens the chemical constituents in herbal extracts even at the sub ppm level [26].Q-TOF coupled with UPLC provides not only conventional MS and MS/MS data but also gives MS E for comprehensive accurate mass precursor and fragment ion information [27].This method can be used to consecutively scan by "low collision energy" and "high collision energy" in two channels, which provide the highly accurate information of parent ions and fragment ions within a single analysis.
This study aims to optimize the extraction conditions by investigating the metabolite profile of CTL extracts prepared by SC-CO 2 technique.A UPLC-Q-TOF-MS E technique combined with a chemometric approach will be used for the first time to rapidly screen and identify the fatty acids, fatty acid amides, and cinnamic acid derivatives in various different SC-CO 2 extracts of CTL.

UPLC-Q-TOF-MS E Analysis and Metabiltes Identification
Optimized chromatographic and mass spectral analysis were performed to characterize the bioactive compounds in the SC-CO 2 extracts of CTL.Each extract (1.0 mg/mL, ca.1000 ppm) solution was prepared using HPLC analytical-grade solvent MeOH, filtered with a membrane disc filter, and then subjected to UPLC-Q-TOF-MS analysis.Isocratic and gradient UPLC methods were tested to optimize the conditions for maximum resolution of peaks.Different mobile phases (water/acetonitrile, 0.1% formic acid in water/acetonitrile, water/methanol, and 0.1% formic acid in water/methanol) at variable flow rates (0.25, 0.3, 0.4, and 0.5 mL/min) were examined and compared for better chromatographic separation and appropriate ionization.A mobile phase consisting of 0.1% aqueous formic acid and acetonitrile at a flow rate of 0.3 mL/min resulted in satisfactory separation in a short analysis time.CTL extracts were analyzed in the negative ionization modes using a Xevo G2-XS mass spectrometer, and the base peak chromatograms (BPCs) are shown in Figure 1.Due to the complexity of chemical composition in herbal extracts, we established a post-targeted screening strategy for the identification of lipids in different SC-CO 2 extracts of CTL.The accurate masses of targeted [M + H] + and/or [M − H] − ions of all possible fatty acids and fatty acid amides were extracted at the Waters Connect UNIFI workstation using a mass tolerance window of ±7 ppm, and the respective peak retention times (RT) are reported in Table 1.The mass spectra derived from these extracted ion chromatograms (EICs) show intense [M + H] + and/or [M − H] − ions with a mass error ≤ 6.5 ppm.The expected compound showed distinguishable MS/MS characteristic fragment ions with high mass accuracy.Compounds were tentatively identified by determining the elemental compositions of the precursor and product ions.The molecular formula and rational fragmentation patterns and pathways of these compounds were then identified based on a comparison of these data with chemical compound databases.In this way, we used the UPLC-Q-TOF-MS E method in combination with databases to screen 166 compounds from CTL extracts.

Identification of Fatty Acids
FAs are a group of chemical compounds that contain a carboxylic acid functional group (-COOH) at one end of their hydrocarbon chain.In this study, two types of FAs were detected, with one being a monocarboxylic FA containing one -COOH group, while the second one was a dicarboxylic FA, containing two -COOH groups [28].A total of 66 peaks have been extracted from TICs and tentatively identified as monocarboxylic FAs.A total of 19 peaks out of 66 have been observed to be saturated monocarboxylic FA, as they contain no double bonds in their carbon chain, based on their HRMS, empirical formula, and double bond equivalents (DBE).Saturated FAs showed a positive relationship between retention time and the length of FA, which indicates that the elusion time increases as the carbon length of fatty acid increases.Also, they showed strong [M − H] − ion in both channels, i.e., low-energy CID and high-energy CID.On the other hand, the lack of detection of fragment ions of the linear hydrocarbon backbone is in accordance with the previous reports [29].In the high-energy CID channel, the [M − H] − ion did not lead to a decrease when using the highest energy in MS E   and simultaneous loss of water and CO 2 molecules, respectively (Table 1 and Figure 2).Around 28 peaks out of 52 have been tentatively identified as saturated dicarboxylic fatty acids having a carbon chain length of 7 to 25.The [M − H] − ion of 13 peaks was tentatively identified as unsaturated dicarboxylic FA having one unsaturation, while three peaks at 105 (t R = 11.10 min), 117 (t R = 11.88 min), and 120 (t R = 12.10 min) has two unsaturations.Moreover, eight peaks have been identified as oxygenated dicarboxylic FA based on their exact mass, empirical formula, DBE, characteristic fragment ions, and literature support.Peaks 15,55,56,57,59,107,119,130,148,149,and 2) at m/z 311 and 293 due to subsequent loss of two water molecules and a main fragment at m/z 211 due to C15\C16 bond cleavage [31].Dicarboxylic FAs have also been detected at maximum intensity in CTL4 (300 bar/55 • C) SC-CO 2 extract.Recently, Farag et al., in their 2022 study, have reported several fatty acids (mono-and di-carboxylated) from the bark of different cinnamon species, including C. tamala [12].To the best of our knowledge, there is no report on the identification of fatty acids in CTL.

Identification of Fatty Acid Amides
Generally, FAAs are bioactive lipid signaling molecules that play key roles in biological activities such as analgesic, antianxiety, anti-convulsion, anti-epilepsy, neuroprotection, and weight loss functions.In our study, 27 peaks were observed as the [M + H] + ion in positive ion mode (ESI+) and their empirical formula assigned to C, H, O, and single N atoms that are present in the structure.Out of these, 16 peaks were tentatively identified as saturated FAAs based on their exact mass, empirical formula, and one double bond equivalent (DBE) and they were similar regardless of the acyl chain length ranging from C 9 to C 22 .Also, they were discovered to have similar fragment ion peaks containing carbon, hydrogen, oxygen, and nitrogen, which were fragments having the amide head group with variation in the acyl fragmentation site.The MS/MS spectra of the [M + H] + ion of these peaks showed the fragment ions at the m/z 116.1123 [C 6 H 14 NO] + , m/z 102.0897 [C 5 H 12 NO] + , m/z 88.0739 [C 4 H 10 NO] + , and m/z 74.0631 [C 3 H 8 NO] + corresponding to the cleavage of acyl chain (Figure 3); accordingly, these peaks were identified as lauramide (t R = 6.40 min), palmitamide (t R = 11.80 min), myristamide (t R = 9.29 min), and stearamide (t R = 14.65 min), respectively [25,32].The empirical formula of the [M + H] + ion of eight peaks (83, 123, 134, 136, 137, 143, 151, and 157) were found to be two double bond equivalents (DBE), one corresponding to an amide group and one corresponding to unsaturation in the acyl chain.The MS/MS spectra of these compounds showed fragments corresponding to the cleavage of the acyl fragmentation site.Palmitoleamide (C16:1, t R = 9.11 min) (m/z 254.2483), heptadecenamide (C17:1, t R = 13.41 min) (m/z 268.2641), oleamide (C18:1, t R = 12.51 min) (m/z 282.2787), eicosenamide (C20:1, t R = 15.16 min) (m/z 310.3092), and erucamide (C22:1, t R = 13.51 min) (m/z 338.3438) were tentatively identified as monosaturated FAAs in CTL extracts based on their exact mass and literature support [30].In addition to saturated and monosaturated FAAs, di-and trisaturated FAAs were also identified in CTL extracts based on their exact mass, empirical formula, and DBE.Peaks 100 (t R = 10.66 min) at m/z 280.2631 and 162 (t R = 15.70 min) at m/z 280.2628 were observed as [M + H] + ion with empirical formula [C 18 H 34 NO] + and three DBE.The MS/MS spectra of these peaks showed similar fragment ions, showing the presence of isomeric peaks.These peaks were tentatively assigned as linoleamide (C18:2) based on their fragment ion reported earlier [33].Peak 87 (t R = 9.14 min) at m/z 278.2471, empirical formula [C 18 H 32 NO] + , showed four DBE (i.e., three double bonds in the acyl chain) and was tentatively assigned as linolenamide (C18:3) based on fragment ions, which were observed due to cleavages of the acyl chain.
Observed molecules such as oleamide, palmitamide, and linoleamide have been reported for their hypnotic effects, analgesic effect, and potential to inhibit the migration of cancer cells, prevent Alzheimer's disease, cardiovascular disease, inflammation, etc. [25,34,35].Cinnamaldehyde has been reported to exhibit antibacterial, antifungal [36], antioxidant, and anti-inflammatory activities [37], including its flavor-imparting properties due to its pungent taste.Peak 31 (C 9 H 7 O 2 ) obtained a quasi-ionic peak at m/z 147.0446 in ESI (+) mode, and the matching fragments were mainly m/z 103.0541 [M + H − CO 2 ] + and m/z 91.0540 [M + H − 2CO] + (Table 1), which was consistent with the cleavage fragment of coumarin in the literature [38] and standard, so the peak was confirmed to be coumarin.These compounds, however, were detected as the major component in CTL and tentatively identified as plant hormone abscisic acid with the assistance of the library and database [12].They were found to be most intense in CTL2 (150 bar/55 • C) SC-CO 2 extract.Previously, various cinnamic acid derivatives, such as cinnamyl alcohol, cinnamic acid, cinnamaldehyde, and cinnamyl acetate, have been identified in C. tamala, which is in appropriate agreement with our finding [12,14].

Chemometric Analysis
Data representing the chemometric distribution of fatty acid and fatty acid amides obtained in positive and negative ionization mode in UPLC-Q-TOF-MS from the SCCO 2 extracts at different pressures are graphically represented in Figures 5 and 6.From Figures 5a and 6a, it can be observed that the SC-CO 2 extracts act differently in both modes.Two principal components (PC1 and PC2) contribute to 91.9% and 86.6% variation for both positive and negative ionization mode, respectively.
In negative ionization mode, among all extracts (CTL1-CTL5), CTL4 extract acts differently and contributes to the maximum variation from the other SC-CO 2 extracts, whereas, in positive ionization mode (Figure 6a), the least variation was observed between CTL2 and CTL4, as they were clustered together and the other three extracts were clustered together.These results are supported by multivariate heatmap (Figures 5b and 6b) clusters drawn based on a ward clustering method, where the rows and column are distanced apart based on the Euclidean distance.From the heatmap, it can be observed that CTL4 extract is grouped in a single separate cluster, whereas the other three extracts perform similarly and are grouped in a separate cluster.Correlation plots (Figures 5c and 6c), on the other hand, exhibited a correlation between the qualitative analysis of different extracts.From Figure 6c, a good correlation (R 2 > 0.7) can be observed between CTL3, CTL4, and CTL5, whereas a low correlation of these extracts with CTL2 and CTL4 extracts can be observed as they are separating them from each other.Conversely, for negative ESI mode, CTL4 extract behaves differently from other extracts and exhibits a low correlation (R 2 < 0.7) with other SC-CO 2 extracts (Figure 5c).A Venn diagram was constructed to summarize the number of metabolites that differentially accumulated in different SC-CO 2 extracts of CTL leaves, which relatively overlap between each set of metabolites (Figure 7).A total of 166 metabolites were identified in leaves extracts; out of these, 142 metabolites were common to all five CTL extracts, projected in the center of the diagram.Notably, a highly bioactive compound known as protocatechuic acid was found exclusively in CTL1 extract (100 bar/55 • C).Protocatechuic acid has been reported to have various biological activities, for example, anti-inflammatory, neuroprotective, antiviral, anticancer, and antiaging activities [39].It is also reported to have a protective effect against metabolic syndrome and preservation of liver, kidneys, and reproductive functions [39].On the other hand, CTL2 (150 bar/55 • C) has linoleamide II as a fatty acid amide, which has been reported to exert sedative and hypnotic effects and inhibits the migration of cancer cells in humans [25,40].An exclusive compound 4-hydroxycinnamic acid (HCA) was found in CTL3 (250 bar/55 • C), which is well known for its health-beneficial effects and use as cosmeceutical ingredients.HCA is mainly recognized as a potent antioxidant and is involved in the prevention of several diseases connected to oxidative stress, i.e., cardiovascular and neurodegenerative diseases and cancer [41].Nonanedioic acid is an alpha, omega-dicarboxylic acid having a role as an antibacterial agent, an antineoplastic agent, a dermatologic drug, and a plant metabolite.Nonendioic acid, eicosadienoic acid I, and ceriporic acid III were identified in CTL4 (300 bar/55 • C).Surprisingly, CTL5 (500 bar/55 • C) extract did not have any exclusive compounds; further, it has least 151 compounds as compared to other extracts.The lower number of compounds may be due to the SC-CO 2 extraction parameters (pressure/temperature), because high selectivity of lipophilic bioactive compounds can be easily achieved by lowering the pressure and/or temperature in the separator [42].Based on the chemometric data, it can be observed that CTL4 extract has performed differently from the other SC-CO 2 extracts of CTL in both ionization modes.Moreover, it could also be concluded that the SC-CO 2 extraction parameters used in CTL4 are the optimum to achieve maximum fatty acids, fatty amides, and cinnamic acid derivatives in the present study.

Plant Materials
C. tamala leaves were collected from the experimental field of Centre for Aromatic Plants (CAP) under Doon Valley climatic conditions of Uttarakhand (30 • 36 ′ 22.13 ′′ N, 77 • 84 ′ 95.38 ′′ E) in the month of October 2021.The plant was authenticated by plant taxonomist Dr. Sunil Sah (Senior Scientist) and a voucher specimen deposited in the CAP Herbarium.Leaves were washed thoroughly with normal tap water followed by deionized water and dried at room temperature (25-30 • C).All dried leaves were crushed into coarsely ground powder (particle size < 1.0 mm, 18 mesh) using a pulverizer (Decibel, Lab Willey Grinder, Model No. DB 5581-4, New Delhi, India) and stored in an airtight container at room temperature until analysis.The moisture content of the powder was estimated to be 6.3 ± 2.8% on a dry weight basis.

Supercritical Fluid (CO 2 ) Extraction and Sample Preparation
The coarsely ground leaves powder (2.5 kg) was charged into a 12 L extraction vessel (SS316) with a maintained constant flow rate of CO 2 (food grade) at 0.9-1.0kg/min (Thar SFE 2000-2-FMC50, Thar Instruments, Pittsburgh, PA, USA) for the first 15 min and the system was on a static period.After completion of the static period, the system was run at a continuous flow of CO 2 (1.0 kg/min, 120 min), which connected to a collection chambers (separators 1 and 2), where pressure was reduced to 8.0 MPa (80 bar).The optimized extraction parameters, temperatures (55 • C), and desired pressure (100, 150, 250, 300, and 500 bar) were applied in triplicate for each set of experiments.The pressure in both the extraction and separation vessels was controlled by a pressure regulator valve.The extract in the form of oleoresin was collected from the separator and the average amount (%) of extracts was calculated.All extracts were stored in amber-colored screw-capped glass vials at 4 • C until further analysis.In total, 1.0 mg/mL solution of the dried SC-CO 2 CTL extracts was prepared in methanol and filtered through a 0.22 µm nylon syringe filter (AXIVA Sichem Biotech, Delhi, India) prior to analysis.
The mass spectrometric (MS) data were acquired in MS E experiment under sensitivity mode in both positive and negative electrospray ionization (ESI+/−).The acquisition parameters for MS were set as follows: capillary voltage, 2.5 kV; sample cone voltage, 30.0 V; source temperature, 120 • C; desolvation temperature, 450 • C; cone gas flow rate, 50 L/h; desolvation gas flow rate, 900 L/h; source offset, 80 V; acquisition time, 20 min for both polarities.The low-energy collision-induced dissociation (CID) of the MS E experiment was 6 eV, the high-energy CID was 30-85 eV, and the scanning range was m/z 50-1200.Nitrogen was used as the drying, nebulizing, and collision gas.Leucine enkephalin (200 pg/mL, 5 µL/min) was used as the reference compound in order to obtain exact mass accuracy, with [(M + H) + m/z 556.2766] as the positive ion and [(M − H) − m/z 554.2620] as the negative ion.The lock-spray scan time was set at 0.25 s, with an interval of 30 s.The data were acquired and processed by Waters Connect UNIFI version 3.0.0.15.

Conclusions
The present study combined the chromatographic (UPLC-Q-TOF-MS E ) separation technique with chemometric analysis to establish optimized SC-CO 2 extraction conditions to achieve maximum fatty acids, fatty amides, and cinnamic acid derivatives from Uttarakhand C. tamala leaves.A total of 166 metabolites, of which 118 were fatty acids, 27 fatty amides, and 21 cinnamic acid derivatives, were identified in both positive and negative ion mode, out of which 142 compounds were common and found in all five extracts.This rapid and high-quality chemical analysis revealed that the SC-CO 2 extraction parameters used in CTL4 were the most optimized in the present study.Moreover, these metabolites possess a lot of interest because of their diverse spectrum of biological functions, especially in the fields of nutraceuticals.To the best of our knowledge, this is the first study to detect the different metabolites in SC-CO 2 extracts analyzed by UPLC-Q-TOF-MS and justifying the quality of CTL as a flavoring agent and in functional foods.

Figure 3 .
Figure 3. MS/MS spectra of fatty acid amides: (a) lauramide, (b) palmitamide, (c) myristamide, and (d) stearamide.2.2.3.Identification of Cinnamic Acid Derivatives Apart from FAs and FAAs, a total of 21 cinnamic acid derivatives have also been tentatively identified in CTL extracts.Out of these, 12 compounds have been tentatively identified based on their HR-MS, MS/MS, and literature support.Nine peaks out of twelve were detected as [M − H] − ion in (-)-ESI, while two peaks were detected as [M + H] + ion.The major identification, Peak 38 (t R = 3.21 min) with [(M + H) + m/z 135.081 (C 9 H 11 O) + ] and fragment, was observed at m/z 117.0695 [M + H − H 2 O] + and identified as cinnamyl alcohol with the reference compound [12].Peak 34 (t R = 3.06 min) at m/z 147.0457 was observed as [M − H] − ion with empirical formula [C 9 H 8 O 2 ] − , confirmed as cinnamic acid, which was supported by its characteristic fragment ions of m/z 103.0553 [M − H − CO 2 ] − (Figure 4).Peak 31 (t R = 2.94 min), 41 (t R = 3.48 min), and 53 (t R = 5.08 min) were confirmed as coumarin, trans-cinnamaldehyde, and cis-cinnamaldehyde with the reference compounds as [M + H] + ion at m/z 147.0446 [C 9 H 7 O 2 ] + , 133.0648 [C 9 H 9 O] + , and 133.0649 [C 9 H 9 O] + , respectively.Cinnamaldehyde has been reported to exhibit antibacterial, antifungal [36], antioxidant, and anti-inflammatory activities [37], including its flavor-imparting properties due to its pungent taste.Peak 31 (C 9 H 7 O 2 ) obtained a quasi-ionic peak at m/z 147.0446 in ESI (+) mode, and the matching fragments were mainly m/z 103.0541 [M + H − CO 2 ] + and m/z 91.0540 [M + H − 2CO] + (Table1), which was consistent with the cleavage fragment of coumarin in the literature[38] and standard, so the peak was confirmed to be coumarin.These compounds, however, were detected as the major component in CTL extracts.Peak 26 (t R = 2.82 min) was detected as [M − H] − ion at m/z 263.1296 [C 15 H 19 O 4 ] −and tentatively identified as plant hormone abscisic acid with the assistance of the library and database[12].They were found to be most intense in CTL2 (150 bar/55 • C) SC-CO 2 extract.Previously, various cinnamic acid derivatives, such as cinnamyl alcohol, cinnamic acid, cinnamaldehyde, and cinnamyl acetate, have been identified in C. tamala, which is in appropriate agreement with our finding[12,14].

Figure 7 .
Figure 7. Venn diagram representing untargeted metabolites distribution in different SC-CO 2 extracts of CTL leaves.

Table 1 .
Tentative identification of chemical constituents in supercritical-CO 2 extracts of C. tamala leaf using UPLC-Q-TOF-MS E in both positive and negative polarity.