Comparative Metabolite Fingerprinting of Four Different Cinnamon Species Analyzed via UPLC–MS and GC–MS and Chemometric Tools

The present study aimed to assess metabolites heterogeneity among four major Cinnamomum species, including true cinnamon (Cinnamomum verum) and less explored species (C. cassia, C. iners, and C. tamala). UPLC-MS led to the annotation of 74 secondary metabolites belonging to different classes, including phenolic acids, tannins, flavonoids, and lignans. A new proanthocyanidin was identified for the first time in C. tamala, along with several glycosylated flavonoid and dicarboxylic fatty acids reported for the first time in cinnamon. Multivariate data analyses revealed, for cinnamates, an abundance in C. verum versus procyandins, dihydro-coumaroylglycosides, and coumarin in C. cassia. A total of 51 primary metabolites were detected using GC-MS analysis encompassing different classes, viz. sugars, fatty acids, and sugar alcohols, with true cinnamon from Malaysia suggested as a good sugar source for diabetic patients. Glycerol in C. tamala, erythritol in C. iners, and glucose and fructose in C. verum from Malaysia were major metabolites contributing to the discrimination among species.


Introduction
Cinnamon is produced mainly from the dried inner bark of various evergreen trees from the genus Cinnamomum [1]. The genus Cinnamomum, a member of the Lauraceae family, includes ca. 250 species cultivated widely in sub-tropical and tropical Asia, Africa, and South America for their culinary and medicinal attributes [2]. As it is traded on a global scale, cinnamon also has economic importance, with Sri Lanka considered the world's largest supplier of cinnamon products. It was reported that Sri Lanka exported cinnamon in various forms in 2016, with an estimated value of USD 167 million [3]. There are two distinct species of cinnamon, namely, C. verum (syn. C. zeylanicum), known as true cinnamon, and C. cassia (syn. C. aromaticum.), recognized as Chinese cinnamon [4]. True cinnamon, also known as Ceylon cinnamon, is indigenous to Sri Lanka [5], while Chinese cinnamon is native to South-East China [6]. True cinnamon has been substituted by Chinese cinnamon, available at a much lower price, albeit the latter encompassed higher levels of coumarin (ca. 0.31 mg/g), posing health risks when consumed regularly owing to its hepatotoxicity [7]. Other Cinnamomum species included C. tamala from North India [8] and C. iners from Central Malaysia [9]. UPLC-ESI-MS analysis was carried out in both negative and positive ionization modes, allowing the annotation of 74 metabolites (Figure 1) in the examined four Cinnamomum species from which C. verum was obtained from two different sources, in addition to C. cassia, C. iners, and C. tamala. Metabolites belonged to different classes, including phenolic acids, tannins, phenyl propanoids (i.e., hydroxycinnamates, coumaroyl derivatives), flavonoids, lignans, amides, terpenes, and fatty acids (i.e., dicarboxylated and tricarboxylated fatty acids). A list of identified peaks along with their chromatographic and spectroscopic data is presented in Table 1. The structures and fragmentation patterns of some identified metabolites discussed in the manuscript are shown in the Supplementary Material ( Supplementary Figures S1-S7). Metabolites were eluted based on their polarity in descending order. Positive and negative ionization modes provide greater coverage of the metabolome. The negative ionization mode showed better sensitivity than the positive mode, with lower noise and higher signal-to-noise ratios except for a few compounds (ca. 21 peaks), including alkaloids and some hydroxycinnamates, which readily ionized in the positive ion mode.    Figure S2), indicative for the presence of the A-type bond between the second and third flavan-3-ol. This tetramer was found only in C. cassia species. Therefore, peak L10 could serve as a marker for that species among cinnamon food products has yet to be confirmed.
Peak It was noticed that no PA trimers were detected in both C. verum accessions from either origin (Pakistan or Malaysia). This may account for its less astringent taste than C. cassia based on its tannins content [22].

Prodelphinidins
Prodelphinidin detection is particularly interesting as the pyrogallol group in gallo(epi)catechins (EG) is related to the biological activity of grape and tea polyphenols, as previously reported [32]. Thus, the identification of these substructures may explain some of the properties of cinnamon extracts. Among the studied extracts, it was detected only in C. tamala; thus, it may give this species more attention regarding its biological activity.
The main fragments detected in peak L51 [(M − H) − 467.097 (C 24 H 19 O 10 ) − ] were at m/z 313 (due to RDA fragmentation) and m/z 161 (due to loss of terminal (E)G unit) annotated as epigallocatechin-O-caffeate. It was previously detected in tea extract, albeit it is the first time that it has been detected in cinnamon. The pyrogallol moieties of (epi)gallocatechins (e.g., in tea) are more reactive than the catechol moieties of (epi)catechins regarding their antioxidant activity [62]. The fragmentation pattern suggested its annotation as a new procyanidin containing two (epi)catechin units attached to chrysin and a hexose moiety. Further investigation is required to confirm this identification using other spectroscopic tools.

Hydroxycinnamates (HCAs)
Hydroxycinnamates (HCAs) represent one of the characteristic constituents in cinnamon, which possess various biological activities such as antitumor, anti-inflammatory, antioxidant, and neuroprotective activities [63]. Eight cinnamyl derivatives (peaks L20, L24, from Malaysia and Pakistan. Cinnamic acid exhibited potential antibacterial activity [64], in addition to its anti-inflammatory and analgesic effect [65], posing these species to be further investigated for such indications. Peaks L20, L24, L27, and L62 were cinnamoyl glycosides showing characteristic sugar losses. Peak (L20) [ Whether peaks L27 and L63 could serve as markers for that species has yet to be confirmed. Peak L39 [(M + H) + m/z 133.0652 (C 9 H 9 O) + ] was identified as (E)-cinnamaldehyde that was reported to exhibit antibacterial, antifungal [66], antioxidant, and anti-inflammatory activities [67], in addition to its flavor imparting properties in cinnamon spice found most abundant in C. verum and C. iners compared to other species and to likely account for their pungent taste. Peak L42 [(M + H) + m/z 163.076 (C 10 H 11 O 2 ) + ] was identified as methoxy-cinnamaldehyde, found most prominent in C. verum species. C. verum was reported to exhibit antitumor activity due to its richness in methoxy-cinnamaldehyde [68] and rationalizing for its superiority among cinnamon drugs. Standardization of methoxy-cinnamaldehyde and cinnamaldehyde should provide a better indication of cinnamon's health benefits. Peak L43 [(M + H) + m/z 135.081 (C 9 H 11 O) + ] was identified as cinnamyl alcohol that was previously detected in bark and twigs of C. cassia [69].
Compounds L16, L17, L18, L19, and L33 were coumaroyl derivatives, a precursor to cinnamates. Peaks L18 and L19 showed the same molecular ions at m/z 327, whereas  [70], with the relative highest levels in C. iners species, posing its extract to be tested for these effects in the future. Compared to peak L14 showing UV maxima at 220, 280, and 310 nm, peak L21 showed absorbance at 270 and 310 nm, suggested of a substituted aporphine. Its mass spectrum (Supplementary Figure S7) showed (M + H) + at m/z 342.1678 (C 20 H 24 NO 4 ) + ] and a fragment ion peak at m/z 297 due to the opening of ring B and loss of methylene imine group as typical of aporphines and identified as corydine [39].

Lignans and Terpenes
Lignans from different plant sources were reported to show neuroprotective activities being useful in the treatment and prevention of neurodegenerative diseases [71]. In the present study, the negative ionization mode allowed the detection of two lignans, namely dihydroxy-tetramethoxy-epoxylignanol − )]), which were previously detected in the twigs of C. cassia [51]. The three compounds were detected in C. iners and C. verum from Pakistan. Different diterpenoids were previously detected in C. cassia with immunosuppressive activities that may play roles in the treatment or prevention of autoimmune diseases and chronic inflammatory disorders [59]. Herein two diterpenes were identified in all Cinnamomum

Multivariate Data Analysis of UPLC-ESI-MS Data
Although the visual inspection of the UPLC-MS chromatograms (Figure 1) of the examined species revealed different metabolite patterns. The data were further analyzed in a more holistic way using principal component analysis (PCA) to assess the variance within specimens in an untargeted manner. PCA is an unsupervised multivariate data analysis technique requiring no knowledge of the data set and was used to explain metabolite differences and possible discrimination between the studied species [72]. The PCA model for the studied species in negative mode (Figure 2a The UPLC-MS dataset from the positive ionization mode was also subjected to PCA analysis (Figure 2d-f), showing relatively different clustering for the studied cultivars with PC1 and PC2 to account for 44 and 32%, respectively. The score plot (Figure 2d) showed likewise that CI was the most segregated species among specimens, whereas CV failed to cluster with CVM as they represent the same species and opposite to negative ion mode results (Figure 2a). The rest of the cinnamon specimens were clustered together. The loading plot (Figure 2e) showed that CI was more enriched in (epi)catechins represented by methylenedioxy-dimethylepicatechin and epicatechintrimethylether, as well as alkaloids represented by norboldine and norisocorydine, and warrant for the profiling of plant extracts in different ionization modes. In contrast, methyl cinnamate and coumarin The UPLC-MS dataset from the positive ionization mode was also subjected to PCA analysis (Figure 2d-f), showing relatively different clustering for the studied cultivars with PC1 and PC2 to account for 44 and 32%, respectively. The score plot (Figure 2d) showed likewise that CI was the most segregated species among specimens, whereas CV failed to cluster with CVM as they represent the same species and opposite to negative ion mode results (Figure 2a). The rest of the cinnamon specimens were clustered together. The loading plot (Figure 2e) showed that CI was more enriched in (epi)catechins represented by methylenedioxy-dimethylepicatechin and epicatechintrimethylether, as well as alkaloids represented by norboldine and norisocorydine, and warrant for the profiling of plant extracts in different ionization modes. In contrast, methyl cinnamate and coumarin were most abundant in CV and CT, respectively. To identify whether variant metabolites revealed from PCA could serve as potential markers for examined species, supervised partial least squares-discriminant analysis (OPLS-DA) was employed.
The data from negative ion mode were first subjected to OPLS-DA analysis (Supplementary Figure S9a,b) using C. tamala (CT) as first-class against all other species to identify the markers that are most distant among examined cinnamon specimens. OPLS-DA, as a supervised multivariate data analysis technique, has greater potential in the identification of markers by providing the most relevant variables for the differentiation between two class groups. OPLS results confirmed PCA regarding the richness of C. tamala in protocatechuic acid at a p-value less than 0.05. In the positive ionization mode, another OPLS-DA model (Supplementary Figure S9c,d) using CI against other samples confirmed its richness in (epi)catechins and alkaloids concurrent with lower levels of coumarin. These findings rank CI as the closest species to CV, suggesting the former as a potential substitute for true cinnamon with minimal coumarin health risk. In an attempt to distinguish between true (CV and CVM) and Chinese cinnamon (CC), especially since CC is the common adulterant of true cinnamon, the OPLS-DA model was conducted in the negative (Supplementary Figure S10a Figure S10 revealed that (epi)catechin trimer A type, dihydrocoumaroylhexoside, dihydrocoumaroyl-O-pentosylhexoside, and coumarin were characteristic markers for Chinese cinnamon. Novel markers for true cinnamon revealed in this study included methyl cinnamate and cinnamoyl alcohol and are suggestive of the abundance of cinnamates in true cinnamon.
Lastly, to distinguish between true cinnamon of different origins that are CV and CVM, both specimens were modeled against each other using OPLS-DA ( Figure S11a) in negative ion mode with R2 and Q2 values of 0.97 and 0.96, respectively. The S-loading plot (Supplementary Figure S11b) showed the exact markers for true cinnamon from Malaysia belonged mostly to phenolic acids, i.e., Protocatechualdehyde, cinnamic acid and protocatechuic acid. The most discriminatory metabolites, as revealed from UPLC-ESI-MS and multivariate analysis, were then subjected to ANOVA analysis to confirm their statistical significance in differentiating between the samples under study (Supplementary  Table S2). C. iners showed a significantly higher level (p < 0.05) of (E)-Cinnamaldehyde and norboldine with the lowest level of coumarin. A significantly higher level (p < 0.05) of protocatechuic acid was observed in C. tamala, while true cinnamon showed its richness in cinnamate, including cinnamic acid, (E)-cinnamaldehyde, and methylcinnamic confirmed the results obtained from MVA.

Primary Metabolites Profiling Using GC-MS
Primary metabolites of cinnamon were assessed post-silylation using GC-MS analysis (Supplementary Figure S12) in order to account for the nutritive value of cinnamon. The results ( Table 2) revealed 51 primary metabolites categorized into 10 various chemical classes, i.e., sugars, esters, amino acids, phenolics, organic and fatty acids. All Cinnamomum species were enriched in sugars and esters, while amino acids were present at much lower levels. Table 2. Relative percentage of non-volatile metabolites detected in cinnamon barks using HS-SPME-GC-MS (peak numbers are preceded by G in text) measurements (n = 3) represented as average ± standard errors. Different letters indicate significant differences between cinnamon accessions according to the least significant difference analysis (p < 0.05; Tukey's test). CC: Cinnamomum cassia from Malaysia, CI: C. iners from Malaysia, CT: C. tamala from Pakistan, CV: C. verum from Pakistan, CVM: C. verum from Malaysia. (a)-(e) significantly different form the corresponding group. * Compounds confirmed by standards comparison.

Sugars
Sugars were the most abundant class in all Cinnamomum species except for CV (C. verum from Pakistan). The highest relative levels of total sugars were detected in CVM (C. verum from Malaysia) at ca. 64%, followed by CT at 53%. Monosaccharides were present at much higher levels compared to disaccharides represented by psicose (peaks G29, G30), glucose (G32, G33), and fructose (G31). Glucose (G32, G33) was the most dominant monosaccharide amounting to 23% in CVM versus the lowest levels in CI (C. iners) at ca. 2%. Psicose (G29, G30), a low-calorie monosaccharide sugar that is 70% as sweet as sucrose with anti-obesity and antidiabetic effects [73], was detected at the highest levels in true cinnamon from Malaysia, posing it as a good sugar source for diabetic patients. The only identified disaccharide sucrose (G34) was detected at 2% in CC (Chinese cinnamon) ca. three-folds higher than other cinnamon samples.
CI and CT contained the highest levels of sugar alcohols at 29 and 19%, respectively, versus the lowest levels (5%) in CVM (C. verum from Malaysia). Generally recognized as safe food additives, sugar alcohols are low digestible carbohydrates [74] and pose CI as a good source of sugar alcohols. Glycerol (G41) was the most abundant sugar alcohol in all Cinnamomum species except for CI (C. iners), where meso-erythritol (G43) and arabitol (G44, G45) were the major sugar alcohols detected at 16 and 6%, respectively. Among all sugar alcohols, meso-erythritol (43) and arabitol (G44, G45) provide the lowest calories (0.2 kcal/g) [75], posing CI as a potential low-calorie sweetener. Sensory analysis to compare taste preferences for CI compared to true cinnamon should now follow. As they possess antimicrobial activity [76], sugar acids were most enriched in C. verum from Malaysia (12%), while the lowest levels were detected in the same species from Pakistan (2%), suggesting geographical origin impact. However, such a hypothesis should be confirmed by analyzing true cinnamon samples from other origins. Major sugar acids detected in CVM included galactonic acid γ-lactone (G37, G38) at 12% of total metabolites composition.

Fatty Acids/Esters
Fatty acyl esters constituted the second major class in all Cinnamomum species (16-34%), reaching the highest content in CV. Major fatty acid esters included glycerol monostearate (G4) and followed by 1-monopalmitin (G3) in all Cinnamomum species. Glycerol monostearate (G4) is broadly used in bakery products to enhance the taste and appearance of flour foods owing to its anti-staling properties that rationalize the incorporation of cinnamon in pastry aside from its role as a natural flavor [77]. Monoglycerides generally act as emulsifiers resulting in a more stable air dispersed baked cake with a relatively soft crumb [78]. The abundance of esters in Cinnamomum species was affected by the levels of their corresponding fatty acids.
Fatty acids were present in all Cinnamomum species at considerable levels reaching 12% in CV and accounting for its fatty taste [79]. Stearic (G13) and palmitic (G9) acids were the main fatty acids at ca. 4%. Subsequently, these saturated fatty acids act as precursors for their counterpart major esters in cinnamon. CVM (C. verum from Malaysia) contained the least free fatty acids level [80].

Organic Acids
Another primary metabolite class posing quantitative differences among examined cinnamon specimens was organic acids to impart a slightly bitter taste, especially in CT (C. tamala), which has the highest levels (8%). Oxalic (G18), (E)-cinnamic (G23), and quinic (G25) acids were the major constituents of this class. Oxalic acid (G18) is considered an anti-nutrient, whereas quinic acid (G25) exhibits anti-inflammatory and immune-enhancing activities [81]. As it was detected at a seven-fold higher level in CVM than CV, (E)-cinnamic acid (G23) has a honey-like odor with anti-obesity effects [82]. The elevated levels of (E)-cinnamic acid (G23) in CVM (C. verum from Malaysia) compared to CV (C. verum from Pakistan) were in agreement with UPLC-MS results in negative ion mode (Supplementary Figure S11). Moreover, a direct correlation was observed between (E)-cinnamaldehyde and its precursor (E)-cinnamic acid, which is more enriched in CVM than CV.

Phenolics
Phenolics were more abundant in CT (5%) and CV (2%) than in other cinnamon samples, and they are considered natural antioxidants [83]. Major phenolics detected using GC-MS included catechin (G27) and protocatechuic acid (G26) in all Cinnamomum species. Catechin (G27), a predominant component in tea, exhibited a bitter taste [84], though with many health benefits, including antioxidant and antidiabetic activities [85] contributing to the overall biological effects of cinnamon. Protocatechuic acid (G26) was present at much higher levels in CT at 5% as indicated by OPLS-DA-UPLC-MS results (Figure 2b) and in accordance with GC-MS results posing this accession as a potential antioxidant.

Multivariate Data Analysis Using GC-MS Data
The GC-MS data were likewise analyzed using PCA (Figure 3) to assess the variance within cinnamon specimens in an untargeted manner and to compare the classification potential of GC-MS compared to the UPLC-MS platform. The PCA model for the studied species (Figure 3a) explained 47% of the total variance in PC1, whereas the second principal component, PC2, explained 30% of the variance. HCA (Figure 3b) showed that CT was the most distant among other samples in agreement with UPLC-MS results (Figure 2a). However, this model failed to cluster CV and CVM together, which are of the same genotype and appear together from the UPLC-MS-based PCA model (Figure 2a). Examination of the loadings plot (Figure 3c) pointed out that glycerol (G41) and protocatechuic acid (G26) were more abundant in CT. Moreover, Cl (C. iners) was more enriched in sugar alcohols represented by meso-erythritol (G43), while CV (C. verum from Pakistan) encompassed more fatty acyl esters, i.e., glycerol monostearate (G4) and 1-monopalmitin (G3). On the right side of the loading plot along PC1, CVM (C. verum from Malaysia) was characterized by higher levels of sugar acids viz. galactonic acid γ-lactone (G37) and its isomer (G38) in addition to sugars viz., fructopyranose (G31), psicofuranose (G29/G30), and glucopyranose (32/33). Sugars are of low taxonomic value and are thus not clear markers for distinguishing CV and CVM accessions, which are dependent on agricultural practices or growing conditions [86].
Considering CT distant segregation in the PCA model, it was modeled as one class using OPLS-DA analysis (Supplementary Figure S13) versus all other species in order to identify its significant markers at a p-value less than 0.001. The OPLS-DA score plot (Supplementary Figure S13a On the right side of the loading plot along PC1, CVM (C. verum from Malaysia) was characterized by higher levels of sugar acids viz. galactonic acid γ-lactone (G37) and its isomer (G38) in addition to sugars viz., fructopyranose (G31), psicofuranose (G29/G30), and glucopyranose (32/33). Sugars are of low taxonomic value and are thus not clear markers for distinguishing CV and CVM accessions, which are dependent on agricultural practices or growing conditions [86].

Plant Material
Bark specimens of four different Cinnamomum species viz., C. cassia, C. iners, C. tamala, and C. verum were obtained from different sources with sample information presented in Supplementary Table S1. The bark from each specimen was separately homogenized with a mortar and pestle under liquid nitrogen and then stored in tight glass containers at −20 • C until further analysis. Vouchers of cinnamon specimens are deposited at the College of Pharmacy Herbarium, Cairo University, Egypt.

Chemicals
Formic acid and acetonitrile (HPLC grade) were provided by Baker (The Netherlands). All other solvents, standards, and chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA).

UPLC-ESI-QTOF-MS Analysis and Metabolites Identification
Dried finely pulverized cinnamon specimens (10 mg) were extracted by adding 2 mL 70% MeOH, containing 10 µg mL −1 umbelliferone as an internal standard sonicated for 20 min with frequent shaking, then centrifuged at 12,000× g for 10 min to remove debris. The filtered extract through a 0.22 µm filter was subjected to solid-phase extraction using a C 18 cartridge (Sep-Pack, Waters, Milford, MA, USA) as previously described [87]. Cinnamon bark methanol extracts (2 µL) were injected on an HSS T3 column (100 × 1.0 mm, particle size 1.8 µm; Waters, Milford, MA, USA) installed on an ACQUITY UPLC system (Waters, Milford, MA, USA) equipped with a 6540 Agilent Ultra-High-Definition (UHD) Accurate-Mass Q-TOF-LC-MS (Palo Alto, CA, USA) coupled to an ESI interface, operated in positive or negative ion mode under the exact conditions of our previous work [56]. Characterization of metabolites was performed using their UV-VIS spectra (220-600 nm), exact masses, in addition to MS 2 in both ionization modes, RT data, and reference literature and searching the phytochemical dictionary of natural products [88].

GC-MS Analysis of Silylated Primary Metabolites and Identification
Dried finely pulverized cinnamon specimens (100 mg) were extracted by adding 5 mL 100% MeOH, sonicated for 30 min with frequent shaking, then centrifuged at 12,000× g for 10 min to remove debris. Next, 100 µL of the methanol extract was transferred into screw-cap vials and evaporated under nitrogen gas until complete dryness. Then, 150 µL of MSTFA (N-methyl-N-(trimethylsilyl)-trifluoroacetamide), previously diluted 1:1 (v/v) with anhydrous pyridine, was added and incubated for 45 min at 60 • C for derivatization. Silylated products were separated by an Rtx-5MS column (30 m length, 0.25 mm i.d., and 0.25 µm film) [89]. For evaluation of biological replicates, under the same conditions, three separate samples were analyzed for each cinnamon specimen. Non-volatile silylated components were identified by comparing their Kovats indices (KI) relative to the C6-C20 n-alkane series, as well as matching the mass spectra obtained with the NIST and WILEY libraries and with standards when available. Before mass spectral matching, peaks were first deconvoluted through AMDIS software (www.amdis.net, accessed on 16 October 2020), and their abundance data were extracted using the MET-IDEA tool [90].

Multivariate Data (MVA) and Statistical Analyses
Each cinnamon group's data were represented as the mean ± standard deviation (SD) of three replicates. One-way analysis of variance (ANOVA) was employed through IBM SPSS Statistics, Version 28.0. (Armonk, NY, USA: IBM Corp) with a p-value less than 0.05 to indicate significance between groups. The data table of MS abundances generated from either UPLC-MS or GC-MS was subjected to modeling, i.e., PCA (principal component analysis), HCA (hierarchical clustering analysis), and OPLS-DA (partial least-squares discriminant analysis) using SIMCA-P version 13.0 software package (Umetrics, Umeå, Sweden). Subsequently, markers were determined by analyzing the S-plot, which revealed covariance (p) and correlation (pcor). All variables were Pareto scaled and mean-centered. Validation of models was evaluated by computing the diagnostic indices, i.e., Q2 and R2 values, and permutation testing of iterations.

Conclusions
This study provides the most holistic map of cinnamon spice primary and secondary metabolites composition using a multiplex approach of UPLC-MS and GC-MS techniques analyzed using chemometric tools. Such metabolite profiling justifies the premium value of C. verum as a flavoring agent and in functional foods. UPLC-MS analysis allowed the identification of 74 metabolites, of which a new proanthocyanidin suggested to encompass catechin, chrysin, catechin, and hexose was detected for the first time, trihydroxylated fatty acid (trihydroxyoctadecaenoic acid) and three dicarboxylic fatty acids (hexadecanedioic acid, octadecenedioic acid, and hexadecanedioic acid methyl ester) were detected for the first time in cinnamon, albeit, though other spectroscopic analysis, i.e., NMR still required for complete elucidation of these metabolites. In addition, a number of newly identified flavonoid glycosides included naringenin di-O-hexoside, isorhamnetin-O-pentosyldeoxyhexoside, and luteolin-O-hexosyl-C-hexoside. It revealed the richness of Chinese cinnamon in coumarin, while C. verum and C. tamala were rich sources of cinna-mates. Norboldine, an aporphine alkaloid of potential inhibitory activity against type I HIV, was detected at high levels in C. iners species, warranting further assays of its extract against different viruses. Despite the great proximity between C. verum of both origins, UPLC-MS allowed the detection of a number of compounds that accounted for differences between both origins, including dihydrocoumaroyl-O-hexoside and lignans. The palatability and agreeable taste of cinnamon spice pose it as an ingredient in nutraceuticals. According to the UPLC-MS profile, C. iners was the closest species to official C. verum concurrent with a low level of coumarin with a relatively high level of cinnamaldehyde, suggesting the former as a potential substitute for true cinnamon regarding minimal health hazards.
Primary metabolites analysis by GC-MS revealed true cinnamon richness in fatty acids and acyl esters, though with qualitative variation among different origins. Our findings also revealed that sugars were the most discriminatory metabolites among Cinnamomum species, with true cinnamon encompassing the highest levels compared to other specimens. Whereas C. iners showed the healthiest low-calorie sugar profile with lower sugars and high sugar alcohol levels at 29%, viz., meso-erythritol (16%) and arabitol (6%) and thus posing it as a sugar source for diabetics.
MVA of GC-MS and UPLC-MS detected in negative ion mode data revealed that C. tamala was the most chemically distinctive species attributed to the elevated dihydrocinnacasside pentoside, protocatechuic acid, and glycerol. In contrast, positive ion UPLC-MS mode revealed that C. iners was the most distant species, as it is rich in catechins and alkaloids, i.e., norboldine and norisocorydine. Among GC-MS and UPLC-MS employed analytical platforms, UPLC-MS in negative ion mode provided the most rational classification, with close segregation of CV and CVM specimens, and not observed in other PCA models. Novel markers revealed from this study to identify adulteration of true cinnamon (CV) with Chinese cinnamon (CC) included dihydrocoumaroyl-O-hexoside and dihydrocoumaroyl-O-pentosylhexoside in addition to the well-recognized coumarin. On the other hand, cinnamates represented by methyl cinnamate, (E)-cinnamaldehyde, and cinnamoyl alcohol were enriched in true cinnamon. Such chemical marker should aid in the detection of adulteration in true cinnamon, especially when present in extract lacking the typical morphological features to distinguish it from its allied drugs, i.e., Chinese type.
Although the selected Cinnamomum species do not represent all accessions of cinnamon worldwide, our approach is certainly feasible for analyzing other Cinnamomum species from such further sources to exploit factors that might impact the metabolic makeup, i.e., storage, seasonal variation and growth stage. Combining our variable metabolite profile data with gene expression can further assist in exploring involved genes, evaluating biosynthetic pathways, and ultimately enhancing breeding. The isolation and complete identification of the discriminative chemo-markers along with the newly highlighted metabolites should follow on as future work.  [1] against the correlation p(cor) [1] of the variables of the discriminating component; Figure S11: UPLC-MS OPLS-DA (a) score plot and (b) loading S-plots derived from modelling CV (C. verum from Pakistan) versus CVM (C. verum from Malaysia) on negative ion mode revealing the covariance p [1] against the correlation p(cor) [1] of the variables of the discriminating component; Figure S12: Representative SPME-GC-MS chromatograms of cinnamon primary metabolites, acquired from (a) CI (C. iners from Malaysia), (b) CT (C. tamala from Pakistan) and (c) CV (C. verum from Pakistan); Figure  S13: GC-MS OPLS-DA (a) score plot and (b) loading S-plots derived from modelling CT (C. tamala from Pakistan) versus all other samples revealing the covariance p [1] against the correlation p(cor) [1] of the variables of the discriminating component; Figure S14: GC-MS OPLS-DA (a) score plot and (b) loading S-plots derived from modelling CC (Cinnamomum cassia from Malaysia) versus CV (C. verum from Pakistan) revealing the covariance p [1] against the correlation p(cor) [1] of the variables of the discriminating component; Table S1: Origin of the different species of cinnamon barks used in the analysis; Table S2: Relative quantification of the most discriminatory metabolites in the studied Cinnamomum species identified by UPLC-ESI-MS and multivariate analysis. Values are represented as average (n = 3) of normalized peak areas × 10 3 to umbelliferon (internal standard) ± standard error. Different letters indicate significant differences between cinnamon accessions according to the least significant difference analysis (p < 0.05; Tukey's test).