Discrimination of four Cinnamomum species by proximate, antioxidant, and chemical profiling: towards quality assessment and authenticity

Abstract

Cinnamon (genus Cinnamomum) is a worldwide used spice. The highly valued, non-hepatotoxic C. verum (CV) is frequently adulterated with the cheaper hepatotoxic substitutes (C. burmannii (CB), C. cassia (CC), and C. loureiroi (CL)). Therefore, this study evaluated four major Cinnamomum species by proximate composition, antioxidant properties, and chemical analysis. The results showed that CB contained more ash and crude protein content. CC exhibited more moisture, crude fat, and nutritive value, while CV had more crude fiber and total carbohydrate content. The 80% methanol extracts of four Cinnamomum species exhibited the highest total phenolic contents (42.16 to 182.85 mg GAE/g), total flavonoid contents (0.80 to 1.07 mg QE/g), DPPH radical scavenging activities (EC₅₀, 0.94 to 3.98 mg/mL), and ABTS radical scavenging activities (EC₅₀, 0.09 to 0.33 mg/mL). The GC–MS based chemical profiling of CV was markedly different to those of CB, CC, and CL. Compared to the other three species, CV presented the highest eugenol content (5.77%) and the lowest coumarin content (1.90%). Principal component analysis (PCA) accounted for 94.91% of the variability, completely separating CV in quadrant I. Overall, nutritional and chemical profiles in combination with PCA could be effectively applied for monitoring Cinnamomum species, thereby ensuring food safety.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-023-05788-y.

Introduction

Cinnamon is an aromatic plant obtained from the inner bark of the tropical evergreen trees of the genus Cinnamomum. For many centuries, it has been widely preferred for its aroma and taste in food as well as in traditional medicines (Vasconcelos et al. 2018). Due to its numerous health benefits, cinnamon is considered a remedy for blood pressure, cancer, diabetes, Parkinson’s and Alzheimer’s diseases, gastrointestinal disorders, and urinary infections (Nabavi et al. 2015; Ribeiro-Santos et al. 2017). Almost every part of the cinnamon tree possesses some medicinal or culinary use, including the bark, flowers, fruits, leaves, and roots. Nowadays, cinnamon consumption in the form of herbal teas is increasing and is included in the diets of people all over the world (Ribeiro-Santos et al. 2017). According to the reports, the natural secondary metabolites present in cinnamon are approved as “Generally Recognized as Safe” (GRAS) materials for various food and nutraceutical applications with no side effects (Chávez-Delgado and Jacobo-Velázquez 2023; Suriyagoda et al. 2021). This awareness among consumers is boosting the global cinnamon market, which is expected to reach US$ 1.9 billion by 2025. With the rising demand, cases of adulteration and substitution are often encountered, creating health hazards, and affecting the quality and safety of the substance (Rana et al. 2021).

The bark of Cinnamomum species is used as a popular spice in South Asia, China, and Australia (Farag et al. 2018). The four commercially available Cinnamomum species include C. burmannii (Indonesian cinnamon; CB), C. cassia (syn. C. aromaticum or Chinese cinnamon; CC), C. loureiroi (Vietnamese cinnamon; CL), and C. verum (syn. C. zeylanicum or Ceylon cinnamon; CV). CV is regarded as highly valued and is non-hepatotoxic because of its trace amount of coumarin (about 0.004%). However, due to similar morphology, the other three cheaper and hepatotoxic substitutes, containing high coumarin content (up to 10.6%), are confused with CV (Lončar et al. 2020; Rana et al. 2021). A handful of scientific evidence points to the frequent usage of CC as an ingredient in various food and nutraceutical products (Vasconcelos et al. 2018). Eventually, consumption of such substitutes or adulterant species would surpass the maximum coumarin amount daily required, having detrimental effects and causing safety issues in the food and pharma sectors (Parihar et al. 2021). The European Food Safety Authority (EFSA) had limited the daily intake of coumarin to a maximum of 0.01 mg/kg body weight per day (De Silva et al. 2021). To disseminate the correct information on Cinnamomum species for various food and nutraceutical applications, it is important to investigate the morphological features, antioxidant properties, and chemical composition of Cinnamomum species (Jeremić et al. 2019; Weerasekera et al. 2021).

There has been a need for more stringent controls on the origin, safety, and quality of cinnamon recently. Studies have pointed out that the variations in the chemical composition of cinnamon plants or extracts contribute to a wide range of biological activities and their potential applications (Ribeiro-Santos et al. 2017). The chemical composition of different Cinnamomum species could play a crucial role in the identification and quality assessment of Cinnamomum (Parihar et al. 2021). Metabolomics, metabolite profiling, and fingerprinting are modern approaches that are increasingly used for the quality control analysis of herbal plants (Farag et al. 2018). Liquid chromatography-mass spectrometry (LC–MS), gas chromatography-mass spectrometry (GC–MS), and nuclear magnetic resonance (NMR) techniques have been widely used in metabolomic profiling studies. Among these, GC–MS has emerged as the most applicable, versatile, and robust method for comprehensive profiling of complex volatile as well as non-volatile metabolites in plant species through chemical derivatization (Singh and Sharma 2021). Studies have shown the potential of GC–MS to prove the quality of commercial cinnamon and identify volatile compounds from different parts of the cinnamon plant (Farag et al. 2018; Ribeiro-Santos et al. 2017).

To date, no study has been found on the quality assessment of four Cinnamomum species based on morphological characteristics, proximate analysis, or biochemical composition. The present study aimed to provide insight into cinnamon’s distinction of the four major plant resources, C. verum from C. burmannii, C. cassia, and C. loureiroi, using proximate, antioxidant properties, and GC–MS analysis. This study will provide a comprehensive profile of the major chemical compounds found in C. verum and its adulterants, and further discuss the relative quantification of these chemical compounds.

Materials and methods

Chemicals and reagents

Folin-Ciocalteu phenol reagent was obtained from Merck Chemicals (Darmstadt, Germany). Potassium persulfate was obtained from Riedel-de Haën^® (Seeize, Germany). Sodium carbonate and potassium acetate were purchased from Nihon Shiyaku Reagent (Tokyo, Japan). Aluminum chloride and BHT (butylated hydroxytoluene) were purchased from Showa Chemical Industry Co., Ltd. (Tokyo, Japan). Quercetin was obtained from Alfa Aesar (Heysham, England), and gallic acid was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)), and BHA (butylated hydroxyanisole) were purchased from Sigma-Aldrich GmbH (Steinheim, Germany). Methanol was purchased from Echo Chemical (Miaoli, Taiwan), and ethanol was purchased from Tobacco & Liquor Corporation (Pingtung Distillery, Taiwan). All the chemicals, solvents, and standards used in this study were of analytical/food grade.

Plant material collection and preparation

The dried tubular barks of four commercial Cinnamomum species were purchased from four different Asian countries in year 2018. CB was purchased from PT. Lion Super Indo, LLC, South Jakarta (Indonesia), while CC was obtained from Jofont Biotechnology Co., Ltd., Tainan (Taiwan). CL was obtained from Kim Nga, Ho Chi Minh City (Vietnam) and CV was obtained from The Grand Spices and Handicrafts, Kerala (India). All the samples were identified by Professor Yu-Chung Chiang at the Department of Biological Sciences, National Sun Yat-sen University, Taiwan. The samples were ground into powder using an electrical grinder (Yu Sheng Guang Food Machine, Taichung, Taiwan), sealed, and stored at − 80 °C until analysis.

Morphological description

The macroscopic evaluation was conducted on an intact dried bark sample by observing the characteristics (outer and inner bark appearance, shape, texture, thickness, and fracture) as described previously (Binitha et al. 2018; Nanda et al. 2015). The microscopic examination of the bark piece was performed according to Nanda et al. (2015). The stained and unstained sections were observed for cork and cortex, sclereids, oil cells, medullary rays, and starch grains under a compound microscope (Eclipse E100, Nikon Instruments Inc., Melville, NY, USA).

Proximate composition

The proximate compositions, including moisture, ash, crude protein, crude fat, and crude fiber were determined using Chinese National Standard (CNS) methods 2770-3, 2770-9, 2770-5, 2770-4, and 2770-8, respectively (CNS 1986). Total carbohydrate content was calculated by the difference method and nutritive value was determined by summing the multiplying percentages of different components according to Eqs. (1) and (2) and expressed as % and Kcal/100 g, respectively.

$$\text{Total carbohydrate content}\left(\text{\%}\right)=\left[100-\left(\text{moisture}+\text{ash}+\text{protein}+\text{fat}\right)\right]$$ 1

$$\text{Nutritive value}\left(\text{Kcal}/100\text{g}\right)=\left[4\left(\text{protein}\right)+4\left(\text{carbohydrate}\right)+9\left(\text{fat}\right)\right]$$ 2

Antioxidant properties

DPPH and ABTS radical scavenging activities

The free radical scavenging activities of the sample extracts were determined by the DPPH assay (Jayaprakasha et al. 2006). The sample extracts (0.1 mL) of varying concentrations (0.5, 1, 2, 3, and 4 mg/mL) were mixed with 0.1 mM DPPH solution (5 mL) and incubated in the dark for 20 min at room temperature. BHA was used as a standard. The absorbance was recorded at 517 nm against absolute methanol as a blank.

The ABTS scavenging activities of sample extracts were conducted according to Shan et al. (2005). The prepared working ABTS solution (1 mL) was added to the different concentrations (0.05, 0.10, 0.15, 0.20, and 0.30 mg/mL) of samples (0.1 mL). The reactive mixture was mixed thoroughly and incubated in the dark for 6 min at room temperature. BHT was used as a standard. The absorbance was measured at 734 nm. The percentage inhibitions of DPPH and ABTS radical scavenging activities were calculated according to the following Eq. (3).

$$\text{\% Inhibition}=\left[\frac{{\text{A}}_{\text{control}}-{\text{A}}_{\text{sample}/\text{standard}}}{{\text{A}}_{\text{control}}}\right]\times 100$$ 3

A_control is the absorbance of the control and A_{sample/standard} is the absorbance of the sample extract or the standard.

Extraction

The extraction was performed according to Dvorackova et al. (2015) with some modifications. Sample (2 g) was mixed with 20 mL of extracting solvents (80% ethanol, 80% methanol, or RO water) and sonicated (1510, Branson Ultrasonics Corp., Danbury, CT, USA) for 1 h at room temperature (25 ± 1 °C). Then, the homogenate was centrifuged (Himac CR 21F, Hitachi Koki Co., Ltd., Tokyo, Japan) at 14,380 × g and the supernatant was collected. The procedure was repeated, and the supernatants were combined and filtered. The extracts were stored at 4 °C until further analysis.

Total phenolic content (TPC) and total flavonoid content (TFC)

The TPC was determined according to Pourmorad et al. (2006). Briefly, diluted sample extract (0.5 mL) was mixed with 0.2 N Folin-Ciocalteu reagent (5 mL) and 1 M aqueous Na₂CO₃ (4 mL). The mixture was mixed well and kept in the dark for 15 min at room temperature. The absorbance was measured at 765 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan) and reported in mg of gallic acid equivalents (GAE) per gram (dry weight of the sample). The TFC was quantified using the method described by Assefa et al. (2018) with minor modifications. Diluted sample extract (0.5 mL) was mixed with absolute ethanol (1.5 mL), 10% aluminum chloride (0.1 mL), 1 M potassium acetate (0.1 mL), and RO water (2.8 mL). The reaction mixture was mixed thoroughly and incubated at room temperature for 30 min in the dark. The absorbance was taken at 415 nm. The results were expressed as mg of quercetin equivalents (QE) per gram (dry weight of the sample).

GC–MS analysis

The identification of chemical compounds in sample extracts was carried out by GC–MS (JMS-T200GC AccuTOF™ GCx, JEOL Ltd., Tokyo, Japan). The column used was a Rtx-WAX capillary column (30 m × 0.25 mm ID × 0.25 µm film thickness) and the mass detector was operated in electron impact (EI) mode with full scan (29 − 650 amu). The carrier gas was helium at a flow rate of 1 mL/min, the split ratio was 10:1, and the injector temperature was 250 °C. The column temperature was maintained at 40 °C for 1 min and then increased to 250 °C at a rate of 10 °C/min. The compounds were identified by comparing their mass spectra with the database from the National Institute Standard and Technology (NIST) library (Yu et al. 2020). The relative content of individual compounds was calculated based on the cumulative area of all peaks in the total ion chromatogram (TIC) as being 100% for a given sample.

Principal component analysis (PCA)

PCA was performed using IBM^® SPSS^® Statistics version 22.0 (IBM 2013) to classify Cinnamomum species and establish the relationship between the variables. A varimax rotation strategy was employed for the extraction of principal components (PCs).

Statistical analysis

Data were expressed as mean ± standard deviation (SD) of triplicate analyses. The significant difference (p < 0.05) was determined by one-way analysis of variance (ANOVA) and Duncan’s multiple range tests using IBM^® SPSS^® Statistics version 22.0 (IBM 2013). Effective concentration (EC₅₀) values were calculated with a linear regression analysis using Microsoft Excel^® 2016 (Microsoft Corporation, Washington, USA).

Results and discussion

In the entire bark form, macroscopic and microscopic examination readily distinguish between the four Cinnamomum species. However, such differentiation by macroscopic or microscopic examination cannot be applied when dealing with cinnamon extracts. Therefore, a reliable chemistry-based method that generates reference chemical composition profiles is useful for cinnamon extract authentication. To achieve this objective, CB, CC, CL, and CV bark extracts were subjected to evaluation for antioxidant properties and identification of chemical compounds by GC–MS analysis. Finally, PCA was employed to provide good analytical accuracy and classify the cinnamon samples based on their similarity and diversity.

Morphological description

The barks of Cinnamomum plants from different species revealed visual variation based on bark morphology (Table 1 and Supplementary Fig. S1). The appearance of the outer bark and inner bark varied in four Cinnamomum species (Table 1). The shape was comprised of double quills (curls from both sides towards the center) in CB and CL, single quills (curls inwards from both sides with a rolling edge like a half-tube) in CC, and compound quill (curls from one side only and rolls up like a cigar) in CV (Farag et al. 2018; Jeremić et al. 2019; Menggala and Damme 2018). The texture of CV was smoother and brittler than that of CB, CC, and CL. The bark was comparatively thick in CB, CC, and CL as compared to CV. The fracture was short and tough in CB, CC, and CL, while it was fibrous and splintery in CV. These macroscopic descriptions agreed with the previous studies (Binitha et al. 2018; De Silva et al. 2021; Jeremić et al. 2019; Nanda et al. 2015), which proposed distinguishing macroscopic characters for Cinnamomum species.

Table 1.

Morphological description of four Cinnamomum species

Parameters	Cinnamomum species
	CB	CC	CL	CV
Macroscopic characters
Outer bark	Reddish brown with whitish patches of lichens and pale wavy longitudinal striations	Greyish brown with random greyish white patches of corks and irregular wrinkles	Dark brown with greyish patches of lichens and fine wrinkles	Dull yellowish brown with faint scars and wavy pale longitudinal striation
Inner bark	Dark blackish brown	Reddish brown	Reddish brown	Dark brown
Shape	Double quills	Single quills	Double quills	Compound quills
Texture	Smooth and very hard to break	Rough and hard to break	Rough and hard to break	Smooth and brittle
Thickness	1.22–2.89 mm	2.04–2.46 mm	3.68–4.30 mm	0.41–0.72 mm
Fracture	Short and tough	Short and tough	Short and tough	Short, fibrous, and splintery
Microscopic characters
Cork and cortex	Present	Present	Present	Cork absent Partial cortex present
Sclereids	Long and big	Long and big	Long and big	Small
Oil cells	Few	Few	Few	Many
Medullary rays	2–3 layered	2–3 layered	2–3 layered	Biseriate
Starch grains	Many	Many	Many	Few

CB C. burmannii; CC C. cassia; CL C. loureiroi; CV C. verum

CB, CC, and CL showed similar microscopic characteristics in the presence of cork andcortex, long and big-sized sclereids, few oil cells, 2−3 layered medullary rays, and many starch grains (Table 1 and Supplementary Fig. S2). In contrast, CV didn’t have cork but had partial cortex, small-sized sclereids, more oil cells, biseriate medullary rays, and fewer starch grains, which agreed with previous studies (Binitha et al. 2018; De Silva et al. 2021; Nanda et al. 2015). The presence of certain microscopic characters in different cinnamon species depends on the region of the plant stem (Jeremić et al. 2019). Based on our findings, it was possible to distinguish between Cinnamomum species by macroscopic and microscopic examination.

Proximate composition

As shown in Table 2, the proximate composition of Cinnamomum species differed significantly (p < 0.05). The moisture content of different cinnamon samples varied from 11.18 to 15.83%, the highest for CC and the lowest for CV, while ash content varied from 3.47 to 6.11%, the highest for CB and the lowest for CC (Table 2). Jeong et al. (2014) reported a comparable moisture content ranged from 9.09 to 16.23% in various cinnamon powders. The high ash content in a cinnamon sample indicates the abundance of mineral elements (Emelike et al. 2020). The crude protein content was significantly (p < 0.05) high in CB (4.45%), followed by CV (3.82%), CL (3.45%), and CC (3.07%). The crude fat varied significantly from 1.90 to 5.67%, where CC and CV had the highest and lowest fat content, respectively. The increase in fat content could be due to good sources of essential oils (Emelike et al. 2020). The crude fiber varied between 15.34% for CB and 33.15% for CV. Total carbohydrate content ranged between 71.96% (CC) and 78.52% (CV), while nutritive value ranged between 342.56 kcal/100 g (CL) and 351.15 kcal/100 g (CC). The high nutritive value of cinnamon could be attributed to a high amount of protein and carbohydrates (Al-Numair et al. 2007).

Table 2.

Proximate composition of four Cinnamomum species

Parameters	Cinnamomum species
	CB	CC	CL	CV
Moisture content (%)	11.69 ± 0.02c	15.83 ± 0.35a	13.32 ± 0.20b	11.18 ± 0.17c
Ash content (%)	6.11 ± 0.06a	3.47 ± 0.09d	5.11 ± 0.16b	4.59 ± 0.17c
Crude protein (%)	4.45 ± 0.06a	3.07 ± 0.01d	3.45 ± 0.06c	3.82 ± 0.06b
Crude fat (%)	3.56 ± 0.04b	5.67 ± 0.13a	3.25 ± 0.07c	1.90 ± 0.05d
Crude fiber (%)	15.34 ± 0.08c	15.37 ± 0.13c	17.38 ± 0.57b	33.15 ± 0.08a
Total carbohydrate content (%)	74.18 ± 0.01b	71.96 ± 0.56c	74.87 ± 0.37b	78.52 ± 0.12a
Nutritive value (Kcal/100 g)	346.57 ± 0.54b	351.15 ± 1.13a	342.56 ± 1.10c	346.42 ± 0.20b

Results are expressed as mean ± SD (n = 3). Mean values with different superscripts within the same row are significantly different (p < 0.05) based on Duncan’s multiple range test

CB C. burmannii; CC C. cassia; CL C. loureiroi; CV C. verum

TPC and TFC

The effects of different solvents (80% ethanol, 80% methanol, and water) on TPC and TFC of Cinnamomum species are presented in Table 3. The highest values of TPC were recorded in 80% methanol extracts (42.16 to 182.85 mg GAE/g), followed by 80% ethanol extracts (39.61 to 182.66 mg GAE/g), while the lowest was in water extracts (7.73 to 64.93 mg GAE/g). CC and CB showed the lowest and highest TPC contents in all three solvent extraction systems, respectively. Chan et al. (2016) indicated TPC values of 80% methanol extract from CC ranging from 32 to 56.80 mg GAE/g. Variations in TPC values in various solvent extracts from cinnamon species were also reported (Georgieva and Mihaylova 2014; Manju and Gupta 2015; Shan et al. 2005). These discrepancies in the results could be attributed to differences in extraction times, growing conditions or processing methods, and the age and developmental stage of the plants (Chan et al. 2016; Madiha et al. 2017; Ribeiro-Santos et al. 2017).

Table 3.

Total phenolic and flavonoid contents of four Cinnamomum species extracted by different solvents

Solvent	TPC (mg GAE/g extract)				TFC (mg QE/g extract)
	CB	CC	CL	CV	CB	CC	CL	CV
80% Ethanol	182.66 ± 3.61aA	39.61 ± 0.94aD	54.94 ± 0.40bC	65.11 ± 0.72bB	0.85 ± 0.18aA	0.84 ± 0.23aA	0.70 ± 0.10bA	0.83 ± 0.01bA
80% Methanol	182.85 ± 3.53aA	42.16 ± 2.26aD	65.72 ± 2.05aC	75.37 ± 0.33aB	0.96 ± 0.02aA	0.80 ± 0.08aB	1.07 ± 0.06aA	1.03 ± 0.09aA
RO water	64.93 ± 1.31bA	7.73 ± 0.53bD	20.62 ± 0.78cC	40.56 ± 1.96cB	0.46 ± 0.10bB	0.15 ± 0.06bC	0.32 ± 0.12cBC	0.66 ± 0.05cA

Results are represented as mean ± SD (n = 3). Mean values with different lower-case superscripts in the same column indicate significant differences (p < 0.05) within the extracting solvents and different upper-case superscripts in each row indicate significant differences (p < 0.05) within the species

TPC total phenolic content; TFC total flavonoid content; GAE gallic acid equivalent; QE quercetin equivalent; CB C. burmannii; CC C. cassia; CL C. loureiroi; CV C. verum

Similarly, the TFC in 80% methanol extracts (0.80 to 1.07 mg QE/g) was higher than that of 80% ethanol extracts (0.70 to 0.85 mg QE/g) (Table 3). The lowest TFC values obtained in the water extracts were in the range of 0.15–0.66 mg QE/g. Assefa et al. (2018) reported a TFC value of 1.05 mg QE/g in 80% methanol extract of cinnamon, which agreed with our findings. Our results concluded that the effect of solvents on the TFC extraction of cinnamon species was similar to TPC.

DPPH and ABTS radical scavenging activities

The DPPH and ABTS radical scavenging activities of different solvent extracts of Cinnamomum species were increased in a dose-dependent manner (Supplementary Fig. S3). Among the solvent extracts shown in Table 4, the highest DPPH radical scavenging activity was exhibited by 80% methanol extracts (EC₅₀ of 0.94 to 3.98 mg/mL), followed by 80% ethanol extracts (EC₅₀ of 1.01 to 4.33 mg/mL) and water extracts (EC₅₀ of 3.65 to 20.87 mg/mL). CC and CB showed the lowest and highest DPPH radical scavenging activities, respectively. The highest antioxidant activities exhibited by 80% methanol extracts could be due to the differences in the viscosity and polarity of the different solvents (Annegowda et al. 2012). This would mean that the antioxidant compounds were more soluble in polar solvents like 80% methanol, since 80% methanol has more polar organic properties (Chigayo et al. 2016).

Table 4.

Effective concentration (EC₅₀) values in antioxidant activities of four Cinnamomum species extracted by different solvents

Solvent	DPPH EC50 (mg/mL)				ABTS EC50 (mg/mL)
	CB	CC	CL	CV	CB	CC	CL	CV
80% Ethanol	1.01 ± 0.11bD	4.33 ± 0.10bA	3.11 ± 0.12bB	2.69 ± 0.08bC	0.11 ± 0.00bC	0.39 ± 0.03bA	0.25 ± 0.02bB	0.23 ± 0.01bB
80% Methanol	0.94 ± 0.10bD	3.98 ± 0.14bA	2.68 ± 0.15bB	2.39 ± 0.12cC	0.09 ± 0.01bC	0.33 ± 0.02bA	0.24 ± 0.01bB	0.21 ± 0.01bB
RO water	3.65 ± 0.56aC	20.87 ± 1.51aA	6.29 ± 0.43aB	4.29 ± 0.17aC	0.27 ± 0.03aB	1.05 ± 0.53aA	0.40 ± 0.05aB	0.29 ± 0.02aB
BHA	0.21 ± 0.01				NA
BHT	NA				0.04 ± 0.00

Results are expressed as mean ± SD (n = 3). Mean values with different lower-case superscripts in the same column are significantly different (p < 0.05) within the extracting solvents and different upper-case superscripts in each row indicate significant differences (p < 0.05) within the species

DPPH 2,2-diphenyl-1-picrylhydrazyl radical assay; ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical assay; BHA butylated hydroxyanisole; BHT butylated hydroxytoluene; NA not applicable; CB C. burmannii; CC C. cassia; CL C. loureiroi; CV C. verum

The ABTS radical scavenging followed a similar trend to that with DPPH (Table 4). The EC₅₀ of 80% methanol extracts (0.09 to 0.33 mg/mL) showed higher antioxidant activity than 80% ethanol extracts (0.11 to 0.39 mg/mL), followed by water extracts (0.27 to 1.05 mg/mL). Overall, DPPH and ABTS radical scavenging activities of different cinnamon species in 80% methanol decreased in the order of CB, CV, CL, and CC. According to Butsat and Siriamornpun (2016), a significant correlation exists between the antioxidant activity of plant extracts and their TPC and TFC. This fact supports our findings that 80% methanol extracts showed the highest TPC and TFC and exhibited the highest antioxidant activities (DPPH and ABTS) compared to 80% ethanol and water extracts. Thus, 80% methanol was the most suitable solvent for phenolic and flavonoid compound extraction in the present study.

Identification of chemical compounds by GC–MS

A total of 19 chemical compounds were identified by GC–MS (Table 5). The chromatograms are shown in Supplementary Fig. S4. The compounds, belonging to the classes of monoterpene alcohols, sesquiterpenes, phenolic monoterpenoids, ketones, aldehydes, esters, and carboxylic acids, varied in Cinnamomum species. There were 11, 11, 13, and 7 compounds identified in CB, CC, CL, and CV, respectively. The aldehydes, predominantly from (E)-cinnamaldehyde, represented one of the most important classes of chemical compounds in four Cinnamomum species, accounting for 77.12%, 84.94%, 73.39%, and 73.59% in CB, CC, CL, and CV, respectively. Ketones were the second dominant class of compounds in CB (6.47%), CC (5.64%), and CL (10.29%), while phenolic monoterpenoids were in CV (8.07%). Other compounds, including esters, monoterpene alcohols, sesquiterpenes, and carboxylic acids, were either present in small quantities in some species or absent. The major compound found in all Cinnamomum species was (E)-cinnamaldehyde, as reported in the earlier studies (Liang et al. 2019; Yu et al. 2020). The compound composition variations in different Cinnamomum species may be due to differences in extraction methods and geographical origin (Liang et al. 2019).

Table 5.

Relative quantities of chemical compounds in four Cinnamomum species using GC–MS analysis

Compounds	Relative content (%)
	CB	CC	CL	CV
Eucalyptol	0.46 ± 0.08	–	–	–
Acetic acid	–	–	0.47 ± 0.12	–
Copaene	1.03 ± 0.07	0.41 ± 0.04	0.20 ± 0.17	–
Benzaldehyde	0.09 ± 0.16	0.06 ± 0.11	0.08 ± 0.13	–
Linalool	–	–	–	1.08 ± 0.11
Bornyl acetate	0.55 ± 0.07	–	–	–
(E)-Caryophyllene	0.74 ± 0.11	–	0.07 ± 0.13	0.55 ± 0.48
α-Terpineol	0.21 ± 0.19	–	–	–
γ-Muurolene	–	–	0.14 ± 0.12	–
α-Muurolene	–	0.37 ± 0.02	0.26 ± 0.03	–
δ-Cadinene	0.46 ± 0.07	0.28 ± 0.07	0.16 ± 0.15	–
(E)-Calamenene	–	0.19 ± 0.01	–	–
(Z)-Cinnamaldehyde	0.29 ± 0.06	0.20 ± 0.02	0.20 ± 0.01	–
(E)-Cinnamaldehyde	76.74 ± 3.31	84.68 ± 2.57	73.11 ± 1.24	70.58 ± 2.88
(E)-Cinnamyl acetate	2.31 ± 0.16	0.32 ± 0.02	1.54 ± 0.09	–
Eugenol	–	0.41 ± 0.01	0.43 ± 0.10	5.77 ± 0.42
o-Methoxycinnamaldehyde	–	–	–	3.01 ± 0.13
Coumarin	6.47 ± 0.39	5.64 ± 0.11	10.29 ± 0.16	1.90 ± 0.99
Methoxy eugenol	–	0.94 ± 0.03	0.92 ± 0.20	2.30 ± 0.27
Monoterpene alcohols	0.67	–	–	1.08
Sesquiterpenes	2.23	1.25	0.83	0.55
Phenolic monoterpenoids	–	1.35	1.35	8.07
Ketones	6.47	5.64	10.29	1.90
Aldehydes	77.12	84.94	73.39	73.59
Esters	2.86	0.32	1.54	–
Carboxylic acids	–	–	0.47	–
Total	89.35	93.50	87.87	85.19

Monoterpene alcohols	0.67	–	–	1.08
Sesquiterpenes	2.23	1.25	0.83	0.55
Phenolic monoterpenoids	–	1.35	1.35	8.07
Ketones	6.47	5.64	10.29	1.90
Aldehydes	77.12	84.94	73.39	73.59
Esters	2.86	0.32	1.54	–
Carboxylic acids	–	–	0.47	–
Total	89.35	93.50	87.87	85.19

Results are expressed as mean ± SD (n = 3)

CB C. burmannii; CC C. cassia; CL C. loureiroi; CV C. verum

Compared to the other three species, CV had the lowest content of coumarin (1.90%) and the highest content of eugenol (5.77%). Chairunnisa et al. (2017) demonstrated that (E)-cinnamaldehyde was the most abundant component present in CB bark, while eugenol was absent. However, eugenol is among the main substances found in CV oil and has strong antibacterial properties (Yu et al. 2020).

PCA

PCA was conducted to establish the correlation among the variables of Cinnamomum species. The only first five principal components extracted (PC1–5 with eigenvalues greater than one) explained 94.91% of the total variance. The PC1, PC2, PC3, PC4, and PC5 contributed to 28.35%, 27.33%, 21.62%, 13.40%, and 4.21% of the total variance, respectively. The variables that correlated the most with PC1 were TPC (0.977), ABTS (− 0.946), DPPH (− 0.938), eucalyptol (0.926), and bornyl acetate (0.926) (Supplementary Table S1). The o-methoxycinnamaldehyde (− 0.985), linalool (− 0.985), eugenol (− 0.983), and (Z)-cinnamaldehyde (0.936) dominated PC2. The nutritive value (0.999), total carbohydrate content (0.999), crude protein (0.976), moisture content (0.967), and ash content (0.948) loaded highly in PC3. The TFC (0.852) and acetic acid (0.809) were high in PC4, while benzaldehyde (0.946) contributed to build PC5. Moreover, the PCA score plot revealed the separation of Cinnamomum species into four groups, as shown in Fig. 1a. CV (group 1) was present in quadrant I and CB (group 2) in quadrant III of the score plot. CL (group 3) and CC (group 4) were positioned in quadrant IV of the score plot. The corresponding loading plot (Fig. 1b) described the variables related to the separation of Cinnamomum species. The variables TPC, ABTS, DPPH, eucalyptol, bornyl acetate, o-methoxycinnamaldehyde, linalool, eugenol, (Z)-cinnamaldehyde, nutritive value, total carbohydrate content, crude protein, moisture content, ash content, TFC, acetic acid, and benzaldehyde can be regarded as the characteristic variables in Cinnamomum species. Therefore, these variables may be identified as the referents of Cinnamomum species.

Fig. 1.

Principal component analysis (PCA) result for four Cinnamomum species. a PCA score plot and b loading plot. CB C. burmannii; CC C. cassia; CL C. loureiroi; CV C. verum

Conclusion

This is the first study for the quality evaluation of four major cinnamon resources by proximate composition, antioxidant properties, and chemical analysis, with the inclusion of an exploratory examination of the macro-microscopic characteristics of bark. The macro-microscopic results elucidated the distinguished features of CV from its adulterants (CB, CC, and CL). Moreover, the nutritional and chemical profile in combination with PCA recognized 17 referent variables (TPC, ABTS, DPPH, eucalyptol, bornyl acetate, o-methoxycinnamaldehyde, linalool, eugenol, (Z)-cinnamaldehyde, nutritive value, total carbohydrate content, crude protein, moisture content, ash content, TFC, acetic acid, and benzaldehyde) that could be regarded as reference markers for the discrimination of four Cinnamomum species. In the future, this method will benefit the food and nutraceutical industries by ensuring food quality and safety for a variety of cinnamon-containing foodstuffs and medicinal products.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CB

C. burmannii

CC

C. cassia

CL

C. loureiroi

CV

C. verum

TPC

Total phenolic content

TFC

Total flavonoid content

DPPH

2,2-diphenyl-1-picrylhydrazyl

ABTS

2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)

GC–MS

Gas chromatography–mass spectrometry

ANOVA

Analysis of variance

PCA

Principal component analysis

PC

Principal component

Author contributions

PR carried out the work, responsible for conceptualization, methodology, software, investigation, and writing of original draft. S-CS responsible for validation, supervised, reviewed, edited draft, and provided resources and funding.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

All data generated or analysed during this study are included in this published article (and its supplementary information files).

Code availability

Not applicable.

Declarations

Conflict of interest

Authors declare that they have no conflict of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

All authors are willing for publication of this manuscript.