Skip to main content
NCBI home page
Molecules logoLink to Molecules
. 2024 Aug 8;29(16):3760. doi: 10.3390/molecules29163760

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

Hema Lohani 1, Arvind Kumar 1,*, Vinod Bidarakundi 1, Lalit Agrawal 1, Syed Zafar Haider 1, Nirpendra Kumar Chauhan 1,*
Editor: Gavino Sanna1
PMCID: PMC11356901  PMID: 39202838

Abstract

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.

Keywords: Cinnamomum tamala, chemometrics, fatty acids, fatty acid amides, SC-CO2 extraction, UPLC-Q-TOF-MSE

1. 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-CO2) is a green extraction technique that has gained attraction as an alternative to traditional methods for extracting fatty acids [22,23]. SC-CO2 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 MSE 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-CO2 technique. A UPLC-Q-TOF-MSE 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-CO2 extracts of CTL.

2. Results and Discussion

2.1. Extraction Yield

Exhaustive drying experiments (110 °C, continued until no weight decrease was registered) showed that the average moisture content was 6.3 ± 0.28% of the shade-dried C. tamala leaves (CTL) powder. For efficient and appropriate SC-CO2 extraction, the optimized parameters, i.e., temperatures (55 °C), desired pressure (100, 150, 250, 300, and 500 bar), particle diameter (<1.0 mm), and tested extraction time (3 h), were applied in triplicate for each set of experiments. The extraction yields (%) of CTL extracts were 0.48 ± 0.04% at 100 bar/55 °C, 3.41 ± 0.56% at 150 bar/55 °C, 3.93 ± 0.01% at 250 bar/55 °C, 4.87 ± 0.54% at 300 bar/55 °C, and 7.94 ± 0.02% at 500 bar/55 °C, respectively.

2.2. UPLC-Q-TOF-MSE Analysis and Metabiltes Identification

Optimized chromatographic and mass spectral analysis were performed to characterize the bioactive compounds in the SC-CO2 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-CO2 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-MSE method in combination with databases to screen 166 compounds from CTL extracts.

Figure 1.

Figure 1

Open in a new tab

Base peak chromatograms (BPCs) of C. tamala leaf SC-CO2 extracts: (a) CTL1, (b) CTL2, (c) CTL3, (d) CTL4, and (e) CTL5 in positive ESI; (f) CTL1, (g) CTL2, (h) CTL3, (i) CTL4, and (j) CTL5 in negative ESI modes.

Table 1.

Tentative identification of chemical constituents in supercritical-CO2 extracts of C. tamala leaf using UPLC-Q-TOF-MSE in both positive and negative polarity.

No. RT Compound Chemical
Class		 Molecular Ion Observed Mass Error MS/MS Fragments SC-CO2 Extracts
CTL1 CTL2 CTL3 CTL4 CTL5
1 1.62 Protocatechuic acid CAD [M − H]− 153.0204 −0.7 109.0297 + − − − −
2 1.67 3-(4-Hydroxyphenyl)- lactic acid CAD [M − H]− 181.0496 2.4 119.0502 + + + + +
3 1.86 Oxodecanedioic acid DFA [M − H]− 215.0928 −1.4 197.0786
171.1076
155.0751		 − + + − +
4 1.88 Heptanedioic acid
(Pimelic acid I)		 DFA [M − H]− 159.0667 −2.5 141.0542
115.0772
97.0673		 + + + + −
5 1.90 Salicylic acid CAD [M − H]− 137.0244 0.0 93.0348 + + + + +
6 2.16 Heptanedioic acid
(Pimelic acid II)		 DFA [M − H]− 159.0665 −1.3 141.0542
115.0772
97.0673		 + + + + −
7 2.18 Octanedioic acid
(Suberic acid)		 DFA [M − H]− 173.082 −0.8 155.0687
129.0986
111.0816		 + + + + +
8 2.21 2-Hydroxyhydro-cinnamic acid CAD [M − H]− 165.0542 −1.3 119.0502
79.9562		 + + + + +
9 2.22 Hydroxysebacic acid DFA [M − H]− 217.1095 −6.5 199.0984
171.1049
155.1108		 + + + + +
10 2.32 3-Hydroxy-4-methoxy-cinnamic acid CAD [M − H]− 193.0517 −5.7 193.0517 + + − + +
11 2.33 Hydroxyundecanedioic acid DFA [M − H]− 231.1241 −1.3 213.1229
169.1233		 + + + + +
12 2.40 Syringaldehyde CAD [M + H]+ 183.0653 −0.1 155.0731
123.0470		 + + + + +
13 2.41 Oxododecanedioic
acid I		 DFA [M − H]− 243.1215 5.4 225.1170
207.1074
181.1243		 + + + + +
14 2.45 Decenedioic acid I DFA [M − H]− 199.0983 −3.5 181.0865
155.1055
137.0939		 + + + + +
15 2.47 Nonanedioic acid
(Azelaic acid)		 DFA [M − H] 187.0982 −3.2 169.0861
143.1065
125.0966		 + + + + +
16 2.50 Oxododecanedioic
acid II		 DFA [M − H]− 243.1215 5.4 225.1170
207.1074
181.1243		 + + + + +
17 2.60 Oxododecanedioic
acid III		 DFA [M − H]− 243.1214 5.8 225.1170
207.1074
181.1243		 + + + + +
18 2.65 Dodecenedioic acid I DFA [M − H]− 227.1301 −5.3 209.1197
183.1368
165.1287		 + + + + +
19 2.65 Decenedioic acid II DFA [M − H]− 199.0983 −3.5 181.0865
155.1055
137.0939		 + + + + +
20 2.65 Hydroxydodecanedioic acid DFA [M − H]− 245.1406 −4.9 227.1334
201.1317		 + + + + +
21 2.75 Sebacic acid DFA [M − H]− 201.113 1.2 183.1021
157.1214
139.1119		 + + + + +
22 2.77 4-Hydroxycinnamic acid CAD [M − H]− 163.0409 −5.0 119.0495 − − + − −
23 2.78 4-Methoxycinnamic acid CAD [M − H]− 177.0556 0.6 133.0653
103.0577
92.0285		 + + + + +
24 2.79 Nonendioic acid DFA [M − H]− 185.0815 2.2 167.0762,
141.0953,
123.0865		 − − − + −
25 2.79 Salicylic acid CAD [M − H]− 137.0243 0.7 119.0515
93.0348		 + + + + +
26 2.82 Abscisic acid CAD [M − H]− 263.1296 −2.7 219.1398
203.1083
153.0899		 + + + + +
27 2.82 p-Hydroxybenzoic acid CAD [M − H]− 137.0249 −3.1 93.0348 + + + + +
28 2.86 4-Hydroxy
cinnamaldehyde 		CAD [M − H]− 147.0457 −3.9 119.0481
117.0331		 + + + + +
29 2.92 Undecanedioic acid DFA [M − H]− 213.1128 1.9 195.1116
169.1233
151.1254		 + + + + +
30 2.93 Decenoic acid MFA [M − H]− 169.1233 0.6 169.1234
151.1153
125.1298		 + + + + +
31 2.94 Coumarin CAD [M + H]+ 147.0446 0.9 103.0540
91.0597 		+ + + + +
32 2.95 Oxodecenoic acid MFA [M − H]− 183.1028 −1.5 183.1027
147.0874
139.1129		 + + + + +
33 3.04 Decenedioic acid DFA [M − H]− 215.1292 −1.4 197.1188
171.1410
153.1279		 + + + + +
34 3.06 Cinnamic acid CAD [M − H]− 147.0457 0.5 103.0542 + + + + +
35 3.07 Dodecanedioic acid II DFA [M − H]− 227.1301 −5.3 209.1197
183.1368
165.1287		 + + + + +
36 3.15 9,10,13-Trihydroxy-11-octadecenoic acid MFA [M − H]− 329.2325 2.4 311.2269
293.2155
171.1046		 + + + + +
37 3.20 2-Methoxycinnamic
acid		 CAD [M − H]− 177.056 −1.5 133.0653
103.0577
92.0285		 + + + + +
38 3.21 Cinnamyl alcohol CAD [M + H]+ 135.0851 0.5 117.0695
91.0559 		− − + + −
39 3.33 Dihydroxyhexadecanoic acid MFA [M − H]− 287.2232 −1.4 269.2183
241.2277		 + + + + +
40 3.39 Dodecanedioic acid DFA [M − H]− 229.1439 2.9 211.1342
167.1434		 + + + + +
41 3.48 Cinnamaldehyde I CAD [M + H]+ 133.0648 0.9 103.0603
79.0593		 + + + + +
42 3.60 9,10,11-Trihydroxy-12-octadecenoic acid MFA [M − H]− 329.2325 2.4 311.2269
293.2155
171.1046		 + + + + +
43 3.72 Octadecanedioic acid I DFA [M − H]− 313.2375 3.2 295.2280
269.2425
251.2289		 + + + + +
44 3.87 Tridecanedioic acid DFA [M − H]− 243.1601 0.4 225.1506
199.1763
181.1609		 + + + + +
45 4.00 Nonanamide FAA [M + H]+ 158.1559 1.3 116.1119
69.0753		 + + + + +
46 4.00 Methylcinnamic acid CAD [M + H]+ 163.0757 −1.1 105.0356
103.0569
91.0519		 + + + + +
47 4.23 Cinnamyl acetate CAD [M + H]+ 177.0913 −1.2 105.0356
103.0569
91.0519		 + + + + +
48 4.41 Decanamide FAA [M + H]+ 172.1706 −5.8 128.0678
105.0731
69.0751		 + + + + +
49 4.49 Tetradecanedioic acid I DFA [M − H]− 257.1758 0.1 239.1580
213.1841
195.1700		 + + + + +
50 4.75 Cinnamyl alcohol II CAD [M + H]+ 135.0851 0.5 117.0695
91.0559 		+ + + + +
51 4.84 Hexadecanedioic acid DFA [M − H]− 283.1912 1.1 265.1766
221.1924		 + + + + +
52 4.94 Octadecanedioic acid II DFA [M − H]− 313.2375 3.2 295.2280
269.2425
251.2289		 + + + + +
53 5.08 Cinnamaldehyde II CAD [M + H]+ 133.0649 0.7 103.0582
77.0431		 + + + + +
54 5.26 Pentadecanedioic acid DFA [M − H]− 271.1915 0.0 253.1779
227.2038
209.1932 		+ + + + +
55 5.40 Octadecanedioic acid I DFA [M − H]− 311.2224 1.3 293.2123
267.2316
249.2220		 + + + + +
56 5.46 Octadecanedioic acid III DFA [M − H]− 313.2375 3.2 295.2280
269.2425
251.2289		 + + + + +
57 5.50 Octadecanedioic acid II DFA [M − H]− 311.2224 1.3 293.2123
267.2316
249.2220		 + + + + +
58 5.53 Heptadecanedioic acid DFA [M − H]− 297.2067 1.4 279.1973
253.2210
235.2145		 + + + + +
59 5.70 Octadecanedioic acid III DFA [M − H]− 311.2224 1.3 293.2123
267.2316
249.2220		 + + + + +
60 5.98 Dihydroxystearic acid MFA [M − H]− 315.2544 −1.0 315.2544
297.2490		 + + + + +
61 6.03 Hydroxystearidonic
acid I		 MFA [M − H]− 291.1964 0.7 273.1883
255.2316
245.1916		 + + + + +
62 6.18 Hexadecanedioic acid DFA [M − H]− 285.2072 −0.35 267.1978
241.2069 		+ + + + +
63 6.32 Decanoic acid
(Capric acid) 		MFA [M − H]− 171.1392 −1.1 171.1396 + + + + +
64 6.40 Stearidonic acid I MFA [M − H]− 275.2027 −3.6 257.1952
231.2127
229.1872		 + + + + +
65 6.40 Lauramide FAA [M + H]+ 200.2015 −3.0 116.1121
102.0851
74.0631		 + + + + +
66 6.42 9-Hydroxy-12,14,16-octadecatrienoic acid MFA [M − H]− 293.2125 −1.0 275.2022
183.1399
171.1017		 + + + + +
67 6.57 Hydroxyoctadecatrienoic acid I MFA [M − H]− 293.2125 −1.0 275.2076
185.1206
171.1047		 + + + + +
68 6.57 Stearidonic acid II MFA [M − H]− 275.2027 −3.6 257.1952
229.1872		 + + + + +
69 6.80 Hydroxystearidonic
acid II		 MFA [M − H]− 291.1964 0.7 273.1883
255.2316
245.1916		 + + + + +
70 6.98 Hydroxystearidonic
acid III		 MFA [M − H]− 291.1964 0.7 273.1883
255.2316
245.1916		 + + + + +
71 7.16 Hydroxystearidonic
acid IV		 MFA [M − H]− 291.1964 0.7 273.1883
255.2316
245.1916		 + + + + +
72 7.17 Tridecanamide FAA [M + H]+ 214.2194 0.5 105.0761
91.0597
69.0781		 + + + + +
73 7.22 Heptadecanedioic acid I DFA [M − H]− 299.2242 −4.7 281.2143
255.2352
237.2166		 + + + + −
74 7.49 13-Hydroxy-9,11-octadecadienoic acid MFA [M − H]− 295.2278 0.3 277.2161
195.1418
113.0973		 + + + + +
75 7.85 Ricinoleic acid I MFA [M − H]− 297.2438 −1.0 279.2322
183.1396
93.0349		 + + + + +
76 8.30 Hydroxy-
octadecatrienoic acid II		 MFA [M − H]− 293.2125 −1.0 257.1911
171.1047		 + + + + +
77 8.33 Octadecanedioic acid IV DFA [M − H]− 313.2375 3.2 295.2280
269.2425
251.2289		 + + + + +
78 8.50 Hydroxy-
octadecatrienoic acid III		 MFA [M − H]− 293.2125 −1.0 275.2076
171.1047		 + + + + +
79 8.52 Ricinoleic acid II MFA [M − H]− 297.2438 −1.0 279.2322
183.1396
93.0349		 + + + + +
80 8.62 Ricinoleic acid III MFA [M − H]− 297.2438 −1.0 279.2322
183.1396
93.0349		 + + + + +
81 8.84 Dodecanoic acid
(Lauric acid) 		MFA [M − H]− 199.1704 −0.3 199.1704
181.1572		 + + + + +
82 9.01 Hydroxyhexadecenoic
acid I		 MFA [M − H]− 269.213 −3.0 251.2080
223.2160		 + + + + +
83 9.11 Palmitoleamide I FAA [M + H]+ 254.2483 −1.8 105.0752
91.0577
69.0753 		+ + + + +
84 9.14 Linoleamide FAA [M + H]+ 278.2471 2.7 189.1640
175.1480
91.0578 		+ + + + +
85 9.17 Tetradecanedioic acid II DFA [M − H]− 257.1758 0.1 239.1580
213.1841
195.1700		 − − + − +
86 9.26 9-Hydroxy-10,12-octadecadienoic acid MFA [M − H]− 295.2278 0.3 277.2229
183.0112
119.0509 		+ + + + +
87 9.29 Myristamide FAA [M + H]+ 228.2345 −1.3 116.1097
102.0963
88.0815		 + + + + +
88 9.36 9-Hydroxy-10,12-octadecadienoic acid MFA [M − H]− 295.2278 0.3 277.2229
183.0112
119.0509		 + + + + +
89 9.51 Nonadecanedioic acid DFA [M − H]− 327.2549 −2.4 309.2492
283.2639
265.2502		 + + + + +
90 9.81 Hydroxyhexadecenoic acid II MFA [M − H]− 269.213 −3.0 251.2080
223.2160		 + + + + +
91 9.96 Heptadecanedioic
acid II		 DFA [M − H]− 299.2242 −4.7 281.2143
255.2352
237.2166		 + + + + +
92 10.14 Dihydroxy-
octadecenoic acid 		MFA [M − H]− 313.2378 1.9 183.1315
129.0899		 + + + + +
93 10.16 Octadecanedioic acid V DFA [M − H]− 313.2375 3.2 295.2280
129.0899		 + + + + +
94 10.22 Tridecanoic acid MFA [M − H]− 213.1856 1.9 213.1856
195.1645		 + + + + +
95 10.27 Hydroxyhexadecenoic acid III MFA [M − H]− 269.213 −3.0 251.2080
225.2243
223.2160		 + + + + +
96 10.29 Hydroxyhexadecanoic
acid I		 MFA [M − H]− 271.2293 −5.2 271.2293
225.2244		 + + + + +
97 10.35 Pentadecanamide FAA [M + H]+ 242.2466 116.0578
102. 0954
91.0591		 + + + + +
98 10.50 Dihydroxy-
octadecadienoic acid I		 MFA [M − H]− 311.2222 1.9 183.1315
129.0899		 + + + + +
99 10.60 Palmitadienoic acid MFA [M − H]− 251.2016 0.4 251.2016 + + + + +
100 10.66 Linoleamide I FAA [M + H]+ 280.2631 1.4 88.0805
75.0431
57.0752		 + + + + +
101 10.70 Dihydroxy-
octadecadienoic acid II		 MFA [M − H]− 311.2222 1.9 293.2160
275.1958
257.2183		 + + + + +
102 10.74 Eicosanedioic acid DFA [M − H]− 341.2695 0.6 323.2603
297.2877
279.2632		 + + + + +
103 10.77 Nonadecanedioic acid DFA [M − H]− 325.2368 4.9 307.2291
281.2480
263.2364 		+ + + − +
104 11.01 Dihydroxy-
octadecadienoic acid III		 MFA [M − H]− 311.2222 1.9 293.2160
275.1958
257.2183		 + + + + +
105 11.10 Ceriporic acid I DFA [M − H]− 351.2534 1.9 333.2467
307.2613
289.2500		 + + + + +
106 11.14 Oleic acid I MFA [M − H]− 281.248 2.1 281.2481
263.2364
237.2231		 − + + + +
107 11.17 Pentacosanedioic
acid I		 DFA [M − H]− 411.3474 1.5 393.3307
367.3678
349.3567		 + + + + +
108 11.24 Stearic acid I MFA [M − H]− 283.2642 0.2 283.2642
265.2568		 + + + + +
109 11.35 Eicosenedioic acid DFA [M − H]− 339.2542 −0.3 321.2497
295.2707
277.2547		 + + + + +
110 11.37 Hydroxyhexadecanoic acid II MFA [M − H]− 271.2293 1.5 271.2293
225.2244		 + + + + +
111 11.38 Pentadecenoic acid MFA [M − H]− 239.2015 0.8 239.2115
221.1918		 + + + + +
112 11.48 Linolenic acid MFA [M − H]− 277.2173 0.0 259.2143
233.2348
211.1382		 + + + + +
113 11.61 Myristic acid MFA [M − H]− 227.2015 0.7 227.2015
209.1939		 + + + + +
114 11.80 Oxotetra-
cosanedioic acid		 DFA [M − H]− 411.3118 −0.5 393.3081
375.2944
349.3106		 + + + + +
115 11.80 Palmitamide FAA [M + H]+ 256.2636 −0.4 116.1119
102.0963
88.0805 		+ + + + +
116 11.83 Heptadecadienoic acid MFA [M − H]− 265.2167 2.3 265.2167
247.2089		 + + + + +
117 11.88 Ceriporic acid II DFA [M − H]− 351.2534 1.9 333.2467
307.2613
289.2500		 − + + + −
118 11.88 Eicosadienoic acid I MFA [M − H]− 307.2649 −2.0 289.2500
263.2529
261.2602		 − − − + −
119 11.97 Heneicosanedioic acid DFA [M − H]− 355.285 1.1 337.2845
311.2908
293.2897		 + + + + +
120 12.10 Ceriporic acid III DFA [M − H]− 351.2534 1.9 333.2467
307.2613
289.2500		 − − − + −
121 12.25 Palmitoleic acid I MFA [M − H]− 253.2177 −1.6 253.2177
235.2183		 + + + + +
122 12.49 Ricinoleic acid IV MFA [M − H]− 297.2438 −1.0 279.2322
183.1396
93.0349		 + + + + +
123 12.51 Oleamide I FAA [M + H]+ 282.2787 1.4 135.1205
83.0896
69.0753 		+ + + + +
124 12.52 Arachidamide FAA [M + H]+ 312.3257 1.3 116.0678
102.0963
88.0597 		+ + + + +
125 12.70 Pentadecanoic acid MFA [M − H]− 241.2173 0.0 241.2173
223.2073		 + + + + +
126 12.70 Palmitic acid I MFA [M − H]− 255.2328 0.6 255.2351
237.2227		 + + + + +
127 12.70 Eicosenoic acid MFA [M − H]− 309.2783 5.2 309.2799
291.2735		 + − + + +
128 12.85 Heptadecanamide I FAA [M + H]+ 270.2778 4.8 116.0579
88.0597
57.0753 		+ + + + +
129 13.04 Linoleic acid MFA [M − H]− 279.2329 0.4 279.2329
261.2203
243.2081		 + + + + +
130 13.19 Docosanedioic acid DFA [M − H]− 369.301 0.0 335.3020
325.3030
307.2972		 + + + + +
131 13.22 Heptadecanamide II FAA [M + H]+ 270.2778 4.8 116.0579
88.0597
57.0753		 + + + + +
132 13.33 Palmitoleic acid II MFA [M − H]− 253.2177 −1.6 253.2177
235.2183		 + + + + +
133 13.40 Arachidinic acid I MFA [M − H]− 311.295 1.9 311.2950
293.2899
267.2970		 + + + + −
134 13.41 Heptadecenamide FAA [M + H]+ 268.2641 −2.3 116.0579
88.0597
57.0753		 + + + + +
135 13.41 Behenamide I FAA [M + H]+ 340.3575 −0.3 102.0963
88.0431
57.0752		 + + + + +
136 13.48 Palmitoleamide II FAA [M + H]+ 254.2481 −1.0 105.0752
91.0577
69.0753		 + + + + +
137 13.51 Erucamide I FAA [M + H]+ 338.3438 −6.1 321.2128
97.1100
83.0933 		+ + + + +
138 13.57 Heptadecenoic acid I MFA [M − H]− 267.2331 −0.4 267.2331
249.2276 		+ + + + +
139 13.66 Palmitoleic acid III MFA [M − H]− 253.2177 −1.6 253.2177
235.2183		 + + + + +
140 13.70 Palmitic acid II MFA [M − H]− 255.2328 0.6 255.2351
237.2227		 + + + + +
141 13.77 Heneicosanoic acid MFA [M − H]− 325.3113 −0.3 325.3113
307.3052
281.3201		 + + + + +
142 13.77 Heptadecanamide III FAA [M + H]+ 270.2778 4.8 115.0579
91.0597
69.0753 		+ + + + +
143 13.79 Oleamide II FAA [M + H]+ 282.2789 0.7 69.0753
55.0591		 + + + + +
144 13.82 Heptadecenoic acid II MFA [M − H]− 267.2331 −0.4 267.2331
249.2276 		+ − + + +
145 14.12 Arachidinic acid II MFA [M − H]− 311.295 1.9 311.2950
293.2899
267.2970		 + + + + +
146 14.30 Palmitic acid III MFA [M − H]− 255.2328 0.6 255.2351
237.2227		 + + + + +
147 14.37 Heptadecanoic acid I MFA [M − H]− 269.2482 1.5 269.2482
251.2439
225.2305		 + + + + +
148 14.39 Tricosanedioic acid DFA [M − H]− 383.3176 −2.4 365.3100
339.3257
321.3157		 + + + + +
149 14.41 Octadecanedioic acid VI DFA [M − H]− 313.2375 3.19 295.2280
269.2425
251.2289		 + + + + +
150 14.65 Stearamide FAA [M + H]+ 284.2957 −3.2 116.1121
102.0851
88.0821		 + + + + +
151 14.67 Erucamide II FAA [M + H]+ 338.3401 4.9 321.2128
97.1100
83.0933		 + + + + +
152 14.87 Stearic acid II MFA [M − H]− 283.2642 0.2 283.2642
265.2568		 + + + + +
153 14.87 Ocatdecanoic acid II MFA [M − H]− 281.2478 2.8 281.2478
263.2364		 + + + + +
154 14.95 Tetracosanoic acid MFA [M − H]− 367.3573 2.4 367.3573 + + + − −
155 14.95 Behenamide II FAA [M + H]+ 340.3575 −3.0 102.0963
88.0431
57.0752		 + + + + +
156 15.04 Nonadecanoic acid MFA [M − H]− 297.2798 0.3 297.2798
279.2667		 + + + + −
157 15.16 Eicosenamide FAA [M + H]+ 310.3092 3.8 256.2669
97.1100
69.0753 		+ + + + +
158 15.50 Tricosanoic acid MFA [M − H]− 353.3405 5.7 353.3405 + + − + −
159 15.56 Eicosadienoic acid II MFA [M − H]− 307.2649 −2.0 289.2500
263.2529
261.2602		 + + + + +
160 15.70 Docosanoic acid
(Behenic acid)		 MFA [M − H]− 339.3272 −0.9 295.3106
139.0407
119.0496		 + + + − +
161 15.70 Nonadecanamide II FAA [M + H]+ 298.3085 6.4 91.0597
69.0745		 + + + + +
162 15.70 Linoleamide II FAA [M + H]+ 280.2628 2.4 81.0513
69.0745
57.0752		 − + − − −
163 15.81 Tetracosanedioic acid DFA [M − H]− 397.3307 4.03 379.3195
353.3482
335.3321		 + + + + +
164 15.87 Heptadecanoic acid II
(Margaric acid)		 MFA [M − H]− 269.2482 1.5 269.2482
251.2439
225.2305		 + + + + +
165 16.07 Henicosanamide FAA [M + H]+ 326.3426 −2.6 91.0597
69.0753 		− + − + +
166 16.23 Pentacosanedioic acid II DFA [M − H]− 411.3474 1.16 393.3307
367.3678
349.3567		 + − + + +
Open in a new tab

(CTL1) 100 bar/55 °C; (CTL2) 15 0 bar/55 °C; (CTL3) 250 bar/55 °C; (CTL4) 300 bar/55 °C; (CTL5) 500 bar/55 °C; monocarboxylic fatty acid (MFA); dicarboxylic fatty acid (DFA); fatty acid amide (FAA); cinnamic acid derivative (CAD); I–VI indicates presence of isomers; (a) identification based on mass spectrometry data and comparison with the online database with the reference standards; (+)/(–) indicates presence/absence of compound in corresponding extract.

2.2.1. 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 MSE experiment up to 85 eV. They showed characterization ions corresponding to [M − H − 18]−, [M − H − 46]−, and [M − H − 44]− ions, resulting from a loss of one water molecule, loss of -HCOOH, and decarboxylation from quasimolecular ions, respectively (Figure 2). Eleven peaks at 66, 67, 74, 76, 106, 108, 112, 129, 146, 153, and 158 have been detected as the most abundant monocarboxylic FAs in five different SC-CO2 CTL extracts and tentatively identified as 9-hydroxy-12,14,16-octadecatrienoic acid (tR = 6.42 min), hydroxyoctadecatrienoic acid (tR = 6.57 min), 13-hydroxy-9,11-octadecadienoic acid (tR = 7.49 min), hydroxyoctadecatrienoic acid II (tR = 8.30 min), oleic acid (tR = 11.14 min), stearic acid I (tR = 11.24 min), linolenic acid (tR = 11.48 min), linoleic acid (tR = 13.04 min), palmitic acid III (tR = 14.30 min), ocatdecanoic acid II (tR = 14.87 min), tetracosanoic acid (tR = 15.50 min), and docosanoic acid or behenic acid (tR = 15.70 min), respectively, based on exact mass and MS/MS data [30]. Monocarboxylic FAs have been detected as the most abundant in CTL4 (300 bar/55 °C) SC-CO2 extract compared to other extracts.

Figure 2.

Figure 2

Open in a new tab

MS/MS spectra of hydroxyl derivatives of monocarboxylic fatty acids: (a) 9,10,13-trihydroxy-11-octadenoic acid, (b) 9,10,11-trihydroxy-12-octadenoic acid, (c) 13-hydroxy-9,11-octadecadienoic acid, and (d) 9-hydroxy-10,12-octadecadienoic acid.

Similarly, total 52 dicarboxylic fatty acids have been tentatively identified in CTL extracts. Monitoring the high-energy CID channel, fragment spectra revealed no fragmentation for many fatty acids, while the formation of [M − H − 18]−, [M − H − 44]−, and [M − H − 18 − 44]− ions was observed in low intensity, resulting from a loss of water molecules, decarboxylation, and simultaneous loss of water and CO2 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 (tR = 11.10 min), 117 (tR = 11.88 min), and 120 (tR = 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 163 have been identified as the most abundant peaks corresponding to azelaic acid (m/z 187.0982), octadecenedioic acid I (m/z 311.2224), octadecanedioic acid (m/z 313.2375), octadecenedioic acid II (m/z 311.2224), octadecenedioic acid III (m/z 311.2224), pentacosanedioic acid (m/z 411.3474), heneicosanedioic acid (m/z 355.2850), docosanedioic acid (m/z 369.3010), tricosanedioic acid (m/z 383.3176), octadecanedioic acid VI (m/z 313.2375), and tetracosanedioic acid (m/z 397.3307), respectively. Trihydroxyoctadecanoic acid showed main MS/MS fragments (Figure 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-CO2 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.

2.2.2. 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 C9 to C22. 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 [C6H14NO]+, m/z 102.0897 [C5H12NO]+, m/z 88.0739 [C4H10NO]+, and m/z 74.0631 [C3H8NO]+ corresponding to the cleavage of acyl chain (Figure 3); accordingly, these peaks were identified as lauramide (tR = 6.40 min), palmitamide (tR = 11.80 min), myristamide (tR = 9.29 min), and stearamide (tR = 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, tR = 9.11 min) (m/z 254.2483), heptadecenamide (C17:1, tR = 13.41 min) (m/z 268.2641), oleamide (C18:1, tR = 12.51 min) (m/z 282.2787), eicosenamide (C20:1, tR = 15.16 min) (m/z 310.3092), and erucamide (C22:1, tR = 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 (tR = 10.66 min) at m/z 280.2631 and 162 (tR = 15.70 min) at m/z 280.2628 were observed as [M + H]+ ion with empirical formula [C18H34NO]+ 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 (tR = 9.14 min) at m/z 278.2471, empirical formula [C18H32NO]+, 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].

Figure 3.

Figure 3

Open in a new tab

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 (tR = 3.21 min) with [(M + H)+ m/z 135.081 (C9H11O)+] and fragment, was observed at m/z 117.0695 [M + H − H2O]+ and identified as cinnamyl alcohol with the reference compound [12]. Peak 34 (tR = 3.06 min) at m/z 147.0457 was observed as [M − H]− ion with empirical formula [C9H8O2]−, confirmed as cinnamic acid, which was supported by its characteristic fragment ions of m/z 103.0553 [M − H − CO2]− (Figure 4). Peak 31 (tR = 2.94 min), 41 (tR = 3.48 min), and 53 (tR = 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 [C9H7O2]+, 133.0648 [C9H9O]+, and 133.0649 [C9H9O]+, 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 (C9H7O2) 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 − CO2]+ 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 extracts. Peak 26 (tR = 2.82 min) was detected as [M − H]− ion at m/z 263.1296 [C15H19O4]− 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-CO2 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 4.

Figure 4

Open in a new tab

MS/MS spectra of (a) 3-(4-hydroxyphenyl)lactic acid, (b) 2-hydroxyhydrocinnamic acid, (c) 4-hydroxycinnamic acid, and (d) cinnamic acid.

2.3. 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 SCCO2 extracts at different pressures are graphically represented in Figure 5 and Figure 6. From Figure 5a and Figure 6a, it can be observed that the SC-CO2 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.

Figure 5.

Figure 5

Open in a new tab

Data representing the (a) PCA biplot, (b) heatmap representing the cluster hierarchical analysis, and (c) correlation among different SC-CO2 extracts of C. tamala leaf in (−)-ESI mode.

Figure 6.

Figure 6

Open in a new tab

Data representing the (a) PCA biplot, (b) heatmap representing the cluster hierarchical analysis, and (c) correlation among different SC-CO2 extracts of C. tamala leaf in (+)-ESI mode.

In negative ionization mode, among all extracts (CTL1–CTL5), CTL4 extract acts differently and contributes to the maximum variation from the other SC-CO2 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 (Figure 5b and Figure 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 (Figure 5c and Figure 6c), on the other hand, exhibited a correlation between the qualitative analysis of different extracts. From Figure 6c, a good correlation (R2 > 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 (R2 < 0.7) with other SC-CO2 extracts (Figure 5c). A Venn diagram was constructed to summarize the number of metabolites that differentially accumulated in different SC-CO2 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-CO2 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-CO2 extracts of CTL in both ionization modes. Moreover, it could also be concluded that the SC-CO2 extraction parameters used in CTL4 are the optimum to achieve maximum fatty acids, fatty amides, and cinnamic acid derivatives in the present study.

Figure 7.

Figure 7

Open in a new tab

Venn diagram representing untargeted metabolites distribution in different SC-CO2 extracts of CTL leaves.

3. Experimental

3.1. Chemicals and Materials

Cinnamaldehyde (93%), cinnamyl alcohol (98%), cinnamyl acetate (99%), and coumarin (99%) were purchased from Sigma Aldrich (St Louis, MO, USA). Acetonitrile and methanol (LC-MS grade) were obtained from J.T. Baker (Deventer, The Netherlands). Formic acid (LC-MS grade) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Type 1 grade water, produced by Adrona Crystal, was used for all experimental procedures. High-purity gases (99.995%) for extraction were obtained from Linde (Dehradun, Uttarakhand, India).

3.2. 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.

3.3. Supercritical Fluid (CO2) 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 CO2 (food grade) at 0.9–1.0 kg/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 CO2 (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-CO2 CTL extracts was prepared in methanol and filtered through a 0.22 µm nylon syringe filter (AXIVA Sichem Biotech, Delhi, India) prior to analysis.

3.4. UPLC-Q-TOF-MSE Analysis

The UPLC analysis was performed on a Waters Acquity UPLCTM system (Waters, Milford, MA, USA) interfaced with a Waters Xevo G2-XS Quadrupole time-of-flight mass spectrometer (Waters Corporation, Milford, MI, USA) equipped with an electrospray ion source. The Waters Acquity UPLCTM system was equipped with a binary solvent manager, sample manager, column oven, and photodiode array detector. A Waters ACQUITY UPLC HSS T3 analytical column (100 mm × 2.1 mm, 1.8 μm) was used for chromatographic separation of compounds in SC-CO2 extract of CTL. The chromatographic parameters were set as follows: column temperature, 40 °C; flow rate, 0.3 mL/min; temperature of the autosampler, 4 °C; mobile phase, solvent A (0.1% formic acid in water) and solvent B (acetonitrile). A linear gradient was applied for elution as follows: 0–1 min, 10–30% B; 1–2 min, 30–50% B; 2–8 min, 50–70% B; 8–13 min, 70–85% B; 13–15 min, 85% B; 15–19 min, 85–10% B; 19–20 min, 10% B. The injection volume of the blank (methanol) and sample was 2 μL. The PDA spectra were obtained by scanning the samples in the range of 190–400 nm.

The mass spectrometric (MS) data were acquired in MSE 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 MSE 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.

3.5. Chemometric Analysis

For the analysis of qualitative data, the PCA, correlation plots, and hierarchical cluster analysis heatmap diagrams were made with the open-source R software version 3.5.1 by using ggplot2 (https://ggplot2.tidyverse.org/), factoextra (https://cran.r-project.org/web/packages/factoextra/index.html), and ggcorrplot (https://cran.r-project.org/web/packages/ggcorrplot/readme/README.html) packages from the Comprehensive R Archive Network (CRAN) database accessed on 10 June 2023. Venn diagrams were generated using a web tool.

4. Conclusions

The present study combined the chromatographic (UPLC-Q-TOF-MSE) separation technique with chemometric analysis to establish optimized SC-CO2 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-CO2 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-CO2 extracts analyzed by UPLC-Q-TOF-MS and justifying the quality of CTL as a flavoring agent and in functional foods.

Acknowledgments

We are sincerely thankful to technical field staff Bhupendra Singh and Sonal Bisht for the collection of plant material and assistance.

Author Contributions

H.L. and N.K.C., conceptualization and methodology. A.K. and V.B. designed the experiments and acquired and analyzed data. A.K. and L.A. wrote the paper and interpreted the data. S.Z.H. helped in statistical experiments and editing the manuscript. H.L. and N.K.C. provided guidance for the experiments. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

References

  • 1.Jayaprakasha G., Rao L.J.M. Chemistry, biogenesis, and biological activities of Cinnamomum zeylanicum. Crit. Rev. Food Sci. Nutr. 2011;51:547–562. doi: 10.1080/10408391003699550. [DOI] [PubMed] [Google Scholar]
  • 2.Bisht V.K. Cinnamomum tamala (Buch.-Ham.) T. Nees & Eberm.: An alternative source of natural linalool. Natl. Acad. Sci. Lett. 2021;44:59–61. [Google Scholar]
  • 3.Sharma V., Lingamallu J.M.R. An overview on chemical composition, bioactivity and processing of leaves of Cinnamomum tamala. Crit. Rev. Food Sci. Nutr. 2014;54:433–448. doi: 10.1080/10408398.2011.587615. [DOI] [PubMed] [Google Scholar]
  • 4.Ahmed A., Choudhary M.I., Farooq A., Demirci B., Demirci F., Başer K.H.C. Essential oil constituents of the spice Cinnamomum tamala (Ham.) Nees & Eberm. Flavour Fragr. J. 2000;15:388–390. [Google Scholar]
  • 5.Haider S.Z., Lohani H., Bhandari U., Naik G., Chauhan N. Nutritional value and volatile composition of leaf and bark of Cinnamomum tamala from Uttarakhand (India) J. Essent. Oil-Bear. Plants. 2018;21:732–740. doi: 10.1080/0972060X.2018.1497546. [DOI] [Google Scholar]
  • 6.Kumar S., Sharma S., Vasudeva N. Chemical compositions of Cinnamomum tamala oil from two different regions of India. Asian Pac. J. Trop. Dis. 2012;2:S761–S764. doi: 10.1016/S2222-1808(12)60260-6. [DOI] [Google Scholar]
  • 7.Jain A., Dubey M., Gupta A., Mahajan S. Antimicrobial activity of Cinnamomum tamala (Tejpat) against some bacterial and fungal pathogens. J. Pharm. Res. 2011;4:3975–3977. [Google Scholar]
  • 8.Sudan R., Bhagat M., Gupta S., Chitrarakha D.T. Comparative analysis of cytotoxic and antioxidant potential of edible Cinnamomum verum (bark) and Cinnamomum tamala (Indian bay leaf) Free. Radic. Antioxid. 2013;3:S71–S734. doi: 10.1016/j.fra.2013.05.005. [DOI] [Google Scholar]
  • 9.Tiwari S., Talreja S. Importance of Cinnamomum tamala in the treatment of various diseases. Pharmacogn. J. 2020;12:1792–1796. doi: 10.5530/pj.2020.12.241. [DOI] [Google Scholar]
  • 10.Hassan W., Kazmi S.N.Z., Noreen H., Riaz A., Zaman B. Antimicrobial activity of Cinnamomum tamala leaves. J. Nutr. Disord. Ther. 2016;6:190–195. doi: 10.4172/2161-0509.1000190. [DOI] [Google Scholar]
  • 11.Narath S., Koroth S.K., Shankar S.S., George B., Mutta V., Wacławek S., Cerník M., Padil V.V.T., Varma R.S. Cinnamomum tamala leaf extract stabilized zinc oxide nanoparticles: A promising photocatalyst for methylene blue degradation. Nanomaterials. 2021;11:1558–1575. doi: 10.3390/nano11061558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Farag M.A., Kabbash E.M., Mediani A., Doll S., Esatbeyoglu T., Afifi S.M. Comparative metabolite fingerprinting of four different Cinnamon species analyzed via UPLC-MS and GC-MS and chemometric tools. Molecules. 2022;27:2935. doi: 10.3390/molecules27092935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Khan A.D., Singh M.K., Lavhale P.M., Kaushik R. Phytochemical screening and HPTLC analysis of bio-active markers of ethanol extract of Indian Bay leaves. J. Herbs Spices Med. Plants. 2023;29:156–167. doi: 10.1080/10496475.2022.2117253. [DOI] [Google Scholar]
  • 14.Champati B.B., Das P.K., Sahoo C., Ray A., Jena S., Sahoo A., Nayak S., Lata S., Panda P.C. Chemical fingerprinting and multicomponent quantitative analysis for quality control of Cinnamomum tamala collected from Western Himalaya by HPLC-DAD. Heliyon. 2024;10:e30361. doi: 10.1016/j.heliyon.2024.e30361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Boutros C., Somasundar P., Razzak A., Helton S., Espat N.J. Omega-3 fatty acids: Investigations from cytokine regulation to pancreatic cancer gene suppression. Arch. Surg. 2010;145:515–520. doi: 10.1001/archsurg.2010.91. [DOI] [PubMed] [Google Scholar]
  • 16.Guil-Guerrero J.L., Delgado A.D., Gonzalez M.C.M., Isasa M.E.T. Fatty acids and carotenes in some ber (Ziziphus jujuba Mill) varieties. Plant Foods Hum. Nutr. 2004;59:23–27. doi: 10.1007/s11130-004-0017-2. [DOI] [PubMed] [Google Scholar]
  • 17.Sears B., Perry M. The role of fatty acids in insulin resistance. Lipids Health Dis. 2015;14:121. doi: 10.1186/s12944-015-0123-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Mizunoya W., Haramizu S., Shibakusa T., Okabe Y., Fushiki T. Dietary conjugated linoleic acid increases endurance capacity and fat oxidation in mice during exercise. Lipids. 2005;40:265–271. doi: 10.1007/s11745-005-1381-5. [DOI] [PubMed] [Google Scholar]
  • 19.Savych A., Basaraba R., Muzyka N., Ilashchuk P. Analysis of fatty acid composition content in the plant components of antidiabetic herbal mixture by GC-MS. Pharmacia. 2021;68:433–439. doi: 10.3897/pharmacia.68.e66693. [DOI] [Google Scholar]
  • 20.Murkar A., Koninck J.D., Merali Z. Cannabinoids: Revealing their complexity and role in central networks of fear and anxi-ety. Neurosci. Biobehav. Rev. 2021;131:30–46. doi: 10.1016/j.neubiorev.2021.09.002. [DOI] [PubMed] [Google Scholar]
  • 21.Hermes D.J., Xu C., Poklis J.L., Niphakis M.J., Cravatt B.F., Mackie K., Lichtman A.H., Ignatowska-Jankowska B.M., Fit-ting S. Neuroprotective effects of fatty acid amide hydrolase catabolic enzyme inhibition in a HIV-1 Tat model of neuroAIDS. Neuropharmacology. 2018;141:55–65. doi: 10.1016/j.neuropharm.2018.08.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Cvitkovic D., Skarica I., Dragovic-Uzelac V., Balbino S. Supercritical CO2 extraction of fatty acids, phytosterols, and volatiles from Myrtle (Myrtus communis L.) fruit. Molecules. 2024;29:1755. doi: 10.3390/molecules29081755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Idris S.A., Rosli N.R., Aris R.M.A.R. Supercritical carbon dioxide extraction of fatty acids compounds from tamarind seeds. Mater. Today Proc. 2022;63:S462–S466. doi: 10.1016/j.matpr.2022.04.129. [DOI] [Google Scholar]
  • 24.Mok H.J., Lee J.W., Bandu R., Kang H.S., Kimb K.-H., Kim K.P. A rapid and sensitive profiling of free fatty acids using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) after chemical derivatization. RSC Adv. 2016;6:32130–32139. doi: 10.1039/C6RA01344A. [DOI] [Google Scholar]
  • 25.Li Z., Dong F., Sun Y., Sun Z., Song X., Dong Y., Huang X., Zhong J., Zhang R., Wang M., et al. Qualitative and quantitative analysis of six fatty acid amides in 11 edible vegetable oils using Liquid Chromatography-Mass Spectrometry. Front. Nutr. 2022;9:857858. doi: 10.3389/fnut.2022.857858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kumar S., Singh A., Kumar B. Identification and characterization of phenolics and terpenoids from ethanolic extracts of Phyllanthus species by HPLC-ESI-QTOF-MS/MS. J. Pharm. Anal. 2017;7:214–222. doi: 10.1016/j.jpha.2017.01.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ma F., Cui Q., Bai G. Combining UPLC/Q-TOF-MS/MS with biological evaluation for NF-κB inhibitors in Uyghur medicine Althaea rosea flowers. Front. Plant Sci. 2019;9:19751984. doi: 10.3389/fpls.2018.01975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Mantzourani C., Kokotou M.G. Liquid Chromatography-Mass Spectrometry (LC-MS) derivatization-based methods for the determination of fatty acids in biological samples. Molecules. 2022;27:5717. doi: 10.3390/molecules27175717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Hu T., An Z., Shi C., Lie P., Liu L. A sensitive and efficient method for simultaneous profiling of bile acids and fatty acids by UPLC-MS/MS. J. Pharmaceut. Biomed. 2020;178:112815. doi: 10.1016/j.jpba.2019.112815. [DOI] [PubMed] [Google Scholar]
  • 30.Koch E., Wiebel M., Hopmann C., Kampschulte N., Schebb N.H. Rapid quantification of fatty acids in plant oils and biological samples by LC-MS. Anal. Bioanal. Chem. 2021;413:5439–5451. doi: 10.1007/s00216-021-03525-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Serag A., Baky M.H., Dolland S., Farag M.A. UHPLC-MS metabolome based classification of umbelliferous fruit taxa: A prospect for phytoequivalency of its different accessions and in response to roasting. RSC Adv. 2020;10:76–85. doi: 10.1039/C9RA07841J. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Divito E.B., Davic A.P., Johnson M.E., Cascio M. Electrospray ionization and collision induced dissociation mass spectrometry of primary fatty acid amides. Anal. Chem. 2012;84:2388–2394. doi: 10.1021/ac203158u. [DOI] [PubMed] [Google Scholar]
  • 33.Nichols K.K., Ham B.M., Nichols J.J., Ziegler C., Green-Church K.B. Identification of fatty acids and fatty acid amides in human meibomian gland secretions. Investig. Ophthalmol. Vis. Sci. 2007;48:34–39. doi: 10.1167/iovs.06-0753. [DOI] [PubMed] [Google Scholar]
  • 34.Yerlikaya S., Baloglu M.C., Diuzheva A., Jekő J., Cziáky Z., Zengin G. Investigation of chemical profile, biological properties of Lotus corniculatus L. extracts and their apoptotic-autophagic effects on breast cancer cells. J. Pharm. Biomed. Anal. 2019;174:286–299. doi: 10.1016/j.jpba.2019.05.068. [DOI] [PubMed] [Google Scholar]
  • 35.Hernandez-Diaz C., Juarez-Oropeza M.A., Mascher D., Pavón N., Regla I., Paredes-Carbajal M.C. Effects of oleamide on the vasomotor responses in the rat. Cannabis Cannabinoid Res. 2020;5:42–50. doi: 10.1089/can.2019.0014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Wang Y., Zhang Y., Shi Y.-Q., Pan X.-H., Lu Y.-H., Cao P. Antibacterial effects of cinnamon (Cinnamomum zeylanicum) bark essential oil on Porphyromonas gingivalis. Microb. Pathog. 2018;116:26–32. doi: 10.1016/j.micpath.2018.01.009. [DOI] [PubMed] [Google Scholar]
  • 37.Kim M.S., Kim J.Y. Cinnamon subcritical water extract attenuates intestinal inflammation and enhances intestinal tight junc-tion in a Caco-2 and RAW264. 7 co-culture model. Food Funct. 2019;10:4350–4360. doi: 10.1039/C9FO00302A. [DOI] [PubMed] [Google Scholar]
  • 38.Wang P., Chi J., Guo H., Wang S.-X., Wang J., Xu E.-P., Dai L.-P., Wang Z.-M. Identification of differential compositions of aqueous extracts of Cinnamomi ramulus and Cinnamomi cortex. Molecules. 2023;28:2015–2036. doi: 10.3390/molecules28052015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Song J., He Y., Luo C., Feng B., Ran F., Xu H., Ci Z., Xu R., Han L., Zhang D. New progress in the pharmacology of pro-tocatechuic acid: A compound ingested in daily foods and herbs frequently and heavily. Pharmacol. Res. 2020;161:105109. doi: 10.1016/j.phrs.2020.105109. [DOI] [PubMed] [Google Scholar]
  • 40.Huang J.K., Jan C.R. Linoleamide, a brain lipid that induces sleep, increases cytosolic Ca2+ levels in MDCK renal tubular cells. Life Sci. 2001;68:997–1004. doi: 10.1016/S0024-3205(00)01002-X. [DOI] [PubMed] [Google Scholar]
  • 41.Sova M., Saso L. Natural sources, pharmacokinetics, biological activities and health benefits of hydroxycinnamic acids and their metabolites. Nutrients. 2020;12:2190–2219. doi: 10.3390/nu12082190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Sojic B., Putnik P., Danilovic B., Teslic N., Kovacevic D.B., Pavlic B. Lipid extracts obtained by supercritical fluid extraction and their application in meat products. Antioxidants. 2022;11:716–734. doi: 10.3390/antiox11040716. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data are contained within the article.

Articles from Molecules are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

RESOURCES