A Potential Low-Coumarin Cinnamon Substitute: Cinnamomum

Article pubs.acs.org/JAFC

A Potential Low-Coumarin Cinnamon Substitute: Cinnamomum osmophloeum Leaves Ting-Feng Yeh, Chun-Ya Lin, and Shang-Tzen Chang* School of Forestry and Resource Conservation, National Taiwan University, Taipei 10617, Taiwan ABSTRACT: The essential oils from leaves of Taiwan’s indigenous cinnamon (Cinnamomum osmophloeum ct. cinnamaldehyde) have similar constituents as compared to that from commercial bark cinnamons. This indigenous cinnamon has been proven to have excellent bioactivities. To understand whether this indigenous cinnamon contains a high level of the hepatotoxic compound, coumarin, as often seen in Cassia cinnamons, current research focused on determining the coumarin content in this indigenous cinnamon and screening the low-coumarin clones. The results demonstrated that the coumarin contents in all tested indigenous cinnamon clones were much lower than that found in Cassia cinnamons. In addition, this indigenous cinnamon contains about 80% (w/w) of cinnamaldehyde and 0.4−2.7% (w/w) of eugenol in its leaf essential oils. This combination could provide this indigenous cinnamon a better shelf life compared to that of regular commercial cinnamons. These results suggested that leaves of this indigenous cinnamon could be a potential resource for a safer cinnamon substitute. KEYWORDS: cinnamaldehyde, Cinnamomum osmophloeum, cinnamon, coumarin, eugenol, GC−MS, leaf

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tones.8 Coumarin was used as a flavoring ingredient in foods, alcoholic beverages, tobaccos, toothpastes, and detergents.9 However, the usage of coumarin as a food flavoring agent was banned in the 1950s due to the discovery of its hepatotoxic effects in laboratory animals consuming coumarin in the diet.10 Later on, coumarin-induced tumor formation was observed in high-dosage treated animals, and this compound was proved to have carcinogenic effects.9 The Council of the European Communities had set a maximum limit of 2 mg kg−1 for coumarin present in foodstuffs and beverages either naturally or following the addition of flavorings prepared from natural raw materials.11 Later, the European Food Safety Authority (EFSA), based on noobserved-adverse-effect level (NOAEL) for liver toxicity in the most sensitive animal species, established a Tolerable Daily Intake (TDI) of 0.1 mg of coumarin/kg body weight.12 In a revision of this issue in 2008, the EFSA suggested that exposure to coumarin resulting in an intake 3 times higher than the TDI for one to two weeks is not of safety concern; however the TDI stays unchanged.13 The coumarin contents in cinnamons or its related food products found in market places are generally more than the maximum allowed limit.5,14 It is also difficult for consumers to distinguish between Ceylon and Cassia cinnamon in their diets,6 even if they know that Ceylon cinnamon contains less coumarin than Cassia cinnamon. Moreover, a significant proportion of Asian populations seem to have deficiencies in proper coumarin metabolism, and they are susceptible to hepatotoxicity.15 Therefore, further efforts were suggested to be needed to harvest cinnamons with low coumarin level in the future.6

INTRODUCTION Cinnamomum osmophloeum Kaneth (C.o.) is an endemic hardwood species in Taiwan. It belongs to Cinnamomum of Lauraceae. It can grow to 12 m in height and about 40 cm in diameter and normally inhabits natural hardwood forests at elevations between 400 and 1500 m.1 Hu et al. once collected C.o. leaf samples randomly from 21 provenances of Taiwan and analyzed the major compositions of the leaf essential oils (EOs).2 They found that C.o. leaves from certain provenances contain cinnamaldehyde as the major constituent, and that those from some provenances contain linalool as the major compound. Based on the abundances of the chemical compounds in the leaf EOs, they classified C.o. into nine types: cassia type, cinnamaldehyde type, coumarin type, linalool type, eugenol type, camphor type, 4-terpineol type, linalool/ terpineol type, and mixed type.2 Among them, the leaves of the cinnamaldehyde type C.o. have been used by the local people as a substitute to commercial cinnamons due to the similar flavors. Commercial cinnamons are the dried inner barks of many specific Cinnamomum species, such as Ceylon cinnamon and Cassia cinnamon. Ceylon cinnamon usually refers to the dried bark of Cinnamomum verum J. S. Presl (syn Cinnamomum zeylanicum), and it is indigenous to Sri Lanka and southern India.3 Cassia cinnamons are from different species, such as Chinese cassia (Cinnamomum cassia J. S. Presl), Saigon cassia (Cinnamomum loureiroi Nees), and Indonesian cassia (Cinnamomum burmannii). Currently, more Cassia cinnamons are sold in the markets of many countries.4 Studies have showed that the coumarin contents in Cassia cinnamons are generally much higher than that in Ceylon cinnamon.4−6 The coumarin contents in Ceylon cinnamon have been reported in the range of 0−486 mg/kg of sample, whereas the coumarin contents in Cassia cinnamon were between 40 and 12180 mg/kg of sample.4−7 Coumarin is a natural flavoring found in many plants, and its aroma was described as a sweet, creamy vanilla odor with heavy nutlike © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1706

November 25, 2013 January 20, 2014 January 29, 2014 January 29, 2014 dx.doi.org/10.1021/jf405312q | J. Agric. Food Chem. 2014, 62, 1706−1712

Journal of Agricultural and Food Chemistry

Article

The leaf EOs of the cinnamaldehyde-type C.o. have been proved to have excellent bioactivities, such as antifungal,16 antibacterial,17 antipathogenic,18 anti-inflammatory,19 antihyperuricemia,20 antidyslipidemic,21 and mosquito larvicidal activities.22 Although there are so many bioactivities in the cinnamaldehyde-type C.o., the information related to the coumarin contents of this species is very limited. To seek an alternative source for safe cinnamons without harvesting trees, the leaves of the cinnamaldehyde-type C.o. might have a great potential for this purpose. The objective of this study was to identify the coumarin level of this indigenous cinnamon. Samples from different geographical regions in Taiwan were collected, and the major constituents of essential oils from these samples were compared. Cassia cinnamon samples from local markets were also included in the comparison.

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MATERIALS AND METHODS

Plant Materials. Leaves of Cinnamomum osmophloeum ct. cinnamaldehyde (C.o.) from sixteen individual clones were collected from six different geographic sites in Taiwan in 2006 and 2010, and the trees were about 20 years old when harvested. All samples were identified by Sheng-Yu Lu and Yen-Ray Hsui, Taiwan Forestry Research Institute. Voucher specimens were deposited with the Laboratory of Wood Chemistry (School of Forestry and Resource Conservation, National Taiwan University). Table 1 lists the detailed

Table 1. Detailed Information of Cinnamomum osmophloeum ct. cinnamaldehyde (C.o.) and Cinnamomum cassia (C.c.) Collected species C.o.

C.c.

geographic sitea or source

sample type

no. of samples

A B C

leaf leaf leaf

5 5 2

D E F

leaf leaf leaf

1 1 2

G commercial source commercial source commercial source

leaf bark (powder) bark (twig) bark (stem)

sample ID

Figure 1. Geographic sites of Cinnamomum osmophloeum ct. cinnamaldehyde (C.o.) and Cinnamomum cassia (C.c.) samplings. A: Fushan. B: Lileng. C: Hualian. D: Changhua. E: Lugu. F: Puli. G: Sinsian.

time collectedb or purchased

2 1

CoL1−5 CoL6−10 CoL11− 12 CoL13 CoL14 CoL15− 16 CcL1−2 CcBP

2010 2006 2010

2010 2011

1

CcTB

2011

1

CcSB

2011

apparatus. The essential oils were stored in airtight brown bottles at 4 °C before further GC or GC−MS analyses. Instrumentation. A Thermo Trace GC Ultra system (Thermo Scientific, Waltham, MA, USA) was used to quantify the major flavoring constituents of different samples. GC was performed on a 30 m DB-5 ms column (J&W Scientific, Folsom, CA, USA) with 0.25 μm film thickness and 0.25 mm inner diameter. The injection port was set to 250 °C, and the flame ionization detector (FID) was set to 300 °C. The helium flow was 1 mL/min, and the splitting ratio was 10:1. After a 1 min isothermal heating at 60 °C, the oven temperature was programmed at 4 °C/min to 220 °C, held for 2 min, increased at 20 °C/min to 250 °C, and held isocratic for 2 min. The peak areas of the target compounds were used to quantify the absolute contents (mg/kg of EO) compared to that of calibration samples with known concentrations. The same Thermo Trace GC Ultra system with a PolarisQ MSD mass spectrometer (Thermo Scientific, Waltham, MA, USA) was used for the compound authentication. The column and method used for GC−MS analysis were the same as those in the GC analysis, except the ion source was adjusted to 230 °C. Mass spectra were recorded from m/z 50 to 400 with an electron ionization of 70 eV. The compound authentication was achieved by the search results from NIST Mass Spectral Search Program V. 2.0 (National Institute of Standards and Technology, USA) and Wiley 7.0 mass database. Coinjection of the authentic compounds and calculation of the Kovats indices (KI)23 were also used for the compound authentication. Statistics. Different groups were compared all together by multiple means comparisons (Tukey HSD test, α = 0.05) to test for significant differences among groups (SAS 9.3, SAS Institute Inc., USA). All data are expressed as mean ± SE (n = 3).

2010 2010 2010

a

Please refer to Figure 1 for the geographic sites. bSamples were collected between October and November.

information of the C.o. leaf samples. The sites where these C.o. leaves harvested are also indicated in Figure 1. To compare the differences of flavoring constituents between Cassia cinnamon and C.o., three additional bark samples of Cinnamomum cassia (C.c.), commonly sold in the local markets, were used for comparison. Two C.c. leaf samples were also collected for comparison (Table 1). All samples were airdried at ambient temperature (23 °C) and stored at 4 °C for further experiments. Chemicals. The authentic compounds used to verify the flavoring constituents in current study, including trans-cinnamaldehyde, transcinnamyl acetate, trans-cinnamic acid, coumarin, and eugenol, are from TCI (Japan), Acros (Belgium), or Sigma-Aldrich (USA). The purities of these compounds are more than 95%. Distillation of Essential Oils. About 200 g of air-dried samples with 1000 mL of water were hydrodistilled for 6 h in a Clevenger-type

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RESULTS AND DISCUSSION Comparison of Main Compounds from the Essential Oils of C.o. Leaves and C.c. Samples. To know whether the 1707

dx.doi.org/10.1021/jf405312q | J. Agric. Food Chem. 2014, 62, 1706−1712


Article

Figure 2. GC−MS chromatograms of essential oils from C.o. leaves, C.c. bark, and C.c. leaves. 1: Cinnamaldehyde. 2: Eugenol. 3: Coumarin. 4: Cinnamyl acetate.

Figure 3. Variations of the main compounds from the essential oils of C.o. leaves (CoL) and C.c. samples (CcBP, bark powder; CcTB, twig bark; CcSB, stem bark; CcL, leaves). (A) Cinnamaldehyde. (B) Coumarin. (C) Eugenol. (D) Cinnamyl acetate. ND: not detected. All data are mean ± SE (n = 3). Please refer to Table 1 for the sample ID.

essential oils of C.o. leaves contain high levels of coumarin, C.o. leaves from five individual trees in one location (site A, Figure 1) were collected and the major compounds of their essential

oils were compared with that of the essential oils from C.c. samples. The typical chromatograms of the essential oils from C.o. leaves, C.c. bark, and C.c. leaves are shown in Figure 2. The 1708



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Figure 4. Variations of the main compounds from the essential oils of C.o. leaves in the same site. Solid bar: site A. Open bar: site B. (A) Cinnamaldehyde. (B) Coumarin. (C) Eugenol. (D) Cinnamyl acetate. All data are mean ± SE (n = 3). Please refer to Table 1 for the sample ID and site.

low EO yields of the C.c. leaf samples. Cassia cinnamon bark is known to contain a certain amount of coumarin,4−7,24 and the amounts vary by research ranging from 40 to 12180 mg/kg of sample. These amounts of coumarin far exceed 2 mg/kg of sample, the maximum limits of coumarin for food in general set by the Council of European Communities in 1988.11 Our results also confirmed that the C.c. bark samples contain high level of coumarin, whereas the C.o. leaf samples contain rather low levels of coumarin. Furthermore, the coumarin contents of the two C.o. leaf samples, CoL1 and CoL2, are remarkably lower than 2 mg/kg of sample, and they are 0.29 and 0.72 mg/ kg of sample, respectively. Identifying C.o. clones with lowcoumarin and high-cinnamaldehyde contents in their leaves as a potential source for bark cinnamon substitution would be very beneficial for reducing the coumarin-induced hepatotoxicity to certain groups of the populations. Eugenol (Figure 3C) constitutes about 4−27 g per kg of the C.o. leaf EOs (i.e., 118−455 mg/kg leaf sample), but was not detected for all C.c. samples tested. Cassia cinnamon was often reported to be without eugenol.4,6 Eugenol, the principal ingredient of cloves and their products, is widely used in agricultural industries as a food additive to protect foods from microbes during storage, and it is also routinely used in dental clinics as an antiseptic and analgesic.25 Although eugenol appears to induce cytotoxicity at levels far greater than those occurring in foods,26 when it is used as a food additive, it is listed as “generally recognized as safe (GRAS)” by the US Food and Drug Administration.27 Yeh et al. reported that C.o. leaf EOs containing eugenol have a longer shelf life and thermostability compared to those without eugenol. 28 Bevilacqua et al. also pointed out that the combination of eugenol and cinnamaldehyde has excellent antimicrobial activity and is good for food product preservation.29 This indicates that the shelf life of C.o. leaf samples could be longer than that of C.c. bark samples due to the existence of both eugenol and cinnamaldehyde within C.o. leaf EOs.

major compounds of essential oils from C.o. leaves (CoL1− CoL5 in Figure 3) detected by GC−MS are cinnamaldehyde, coumarin, eugenol, and cinnamyl acetate. About 80% (w/w) of the C.o. leaf EOs are composed of cinnamaldehyde (Figure 3A), and these values ranged from 769 to 809 g/kg of EOs, which correspond to about 8.9−26.1 g/kg of sample. The cinnamaldehyde contents from C.o. leaf EOs are rather comparable to that from C.c. bark EOs, ranging from 564 g/ kg of EO for the C.c. stem bark (CcSB in Figure 3A) to 855 g/ kg of EO for the C.c. bark powder (CcBP in Figure 3A). These values would be 23.6−33.8 g/kg based on sample weights. Woehrlin et al. reported that the cinnamaldehyde contents of C.c. powders were 12.0−42.6 g/kg of sample and that those of C.c. sticks were 8.93−54.3 g/kg of sample. 6 Similar cinnamaldehyde contents (15.4−22.3 g/kg) were also reported by Wang et al. when they analyzed C.c. bark samples.4 Both results are consistent with our measurements. The cinnamaldehyde contents of the C.c. leaf EOs (CcL1 and CcL2 in Figure 3A) are 325 g/kg of EO and 176 g/kg of EO, respectively. Both are much lower than the cinnamaldehyde contents of the C.o. leaf EOs and the C.c. bark EOs, indicating that C.c. leaves are not a good source for cinnamaldehyde flavoring materials. Researchers have pointed out that a good quality cinnamon would contain 60−90% of cinnamaldehyde as the major constituent in its volatile oil.3 C.o. leaf EOs contain about 80% of cinnamaldehyde as the major constituent, and hence C.o. leaves can be considered a good quality and also a great potential cinnamon substitute source to replace commercial bark cinnamons. The coumarin contents of the C.o. leaf EOs are about 0.02− 0.74 g/kg of EOs (Figure 3B), which correspond to 0.29−13.99 mg/kg leaf samples. The coumarin contents of the C.c. bark EOs are 6.69−9.46 g/kg of EOs (Figure 3B) and correspond to 94.1−374.6 mg/kg bark samples. The coumarin contents of the C.c. leaf EOs are 16.75−34.78 g/kg of EOs, however these values correspond to 26.8−97.4 mg/kg leaf samples due to very 1709



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Figure 5. Variations of the main compounds from the essential oils of C.o. leaves from different geographic sites. (A) Cinnamaldehyde. (B) Coumarin. (C) Eugenol. (D) Cinnamyl acetate. All data are mean ± SE (n = 3). Please refer to Table 1 for the sample ID and site.

The clonal variations of eugenol and cinnamyl acetate contents from either site A or site B show differences within the same site (Tukey HSD test, α = 0.05). The eugenol content variations of the C.o. clones in site A range between 4 and 27 g/ kg of EOs, and those of clones in site B range between 8 and 24 g/kg of EOs (Figure 4C). The C.o. leaves from site A contain 12−38 g of cinnamyl acetate per kg of EOs, however, those from site B contain 0.4−17 g of cinnamyl acetate per kg of EOs (Figure 4C). Franz pointed out that composition variation within a species seems to be the principle in essential oil crops,30 and hence the variation seen within site A or site B is likely under the individual genetic variability of the C.o. plant itself. Variations of Main Compounds from the EOs of C.o. Leaves from Different Geographic Sites. Additional samples from 4 different geographic sites were collected and compared with the low-coumarin clones (CoL1 and CoL2) from site A. The results showed that the cinnamaldehyde contents in the C.o. leaves from different sites were no different under Tukey HSD test at α = 0.05 level (Figure 5A), indicating that cinnamaldehyde contents of the C.o. leaves were quite consistent and stable between different geographic sites. The cinnamaldehyde contents of the C.o. leaves from different sites were all above 60% EO, which meets the criterion as a good quality cinnamon source indicated by Poole and Poole.3 The C.o. leaves from sites C, D, and E show relatively higher level of coumarin contents compared to those from sites A (CoL1 and CoL2) and F (CoL15 and CoL16) (Figure 5B). The coumarin contents of the C.o. leaves do vary by different geographic sites (Tukey HSD test, α = 0.05). The coumarin contents of the C.o. leaves from sites A (CoL1 and CoL2) and F (CoL15 and CoL16) were all around 0.01−0.02 g/kg of EOs. If we calculate them on the basis of leaf sample weight, they are all significantly lower than 2 mg/kg of sample, and are 0.29, 0.72, 0.13, and 0.15 mg/kg of leaf samples, respectively. The coumarin contents of many cinnamon species including C. cassia, C. verum, C. burmannii, C. loureiroi, Cinnamomum

Cinnamyl acetate (Figure 3D) constitutes about 12−38 g/kg of EOs of the C.o. leaves (i.e., 136−694 mg/kg of C.o. leaf samples). The C.c. stem bark sample contains cinnamyl acetate in the level of about 7 g/kg of bark EO (i.e., 290 mg/kg of C.c. stem bark sample), and this level is higher in the C.c. leaf samples, about 122−288 g/kg of leaf EOs (i.e., 195−807 mg/kg of C.c. leaf samples). We did not detect any cinnamic acid (data not show) in the C.o. leaves’ EOs, however, we did detect cinnamic acid of 88 and 132 g/kg of EOs (i.e., 960 and 5506 mg/kg of sample) from the C.c. twig bark and stem bark samples. Clonal Variations of Essential Oils in C.o. Leaves from the Same Site. To know the clonal variations of the essential oils in C.o. leaves from the same geographic region, 5 additional C.o. clone samples from the second site (site B) were compared and the compositions of their leaf EOs were analyzed (Figure 4). The results showed that the cinnamaldehyde contents of the C.o. leaves within the same site, either site A or site B, are quite similar (Tukey HSD test, α = 0.05). They are all about 80% (w/w) of EOs in Figure 4A, except for that of CoL9 sample in site B. Although the cinnamaldehyde content of CoL9 sample is in the margin of 62.3% EO, it is still more than 60% of EO, which can also be considered as a good quality cinnamon source. The clonal variations of the coumarin contents in C.o. leaves showed significant difference within site A (Tukey HSD test, α = 0.05), except for CoL1 and CoL2 samples (Figure 4B), whereas this big variation was less significant between different samples from site B (0.03−0.10 g/kg of EOs), indicating that the coumarin contents of the C.o. clones in site B are generally low-coumarin clones compared to that from site A. If C.o. clones from site A will be selected for future massive propagation for commercial purposes, a further coumarin screening for individual clones will be needed to ensure lowcoumarin C.o. clones are selected for propagation to reduce the potential risk of coumarin-induced toxicity. 1710



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(6) Woehrlin, F.; Fry, H.; Abraham, K.; Preiss-Weigert, A. Quantification of flavoring constituents in cinnamon: high variation of coumarin in cassia bark from the German retail market and in authentic samples from Indonesia. J. Agric. Food Chem. 2010, 58, 10568−10575. (7) He, Z. D.; Qiao, C. F.; Han, Q. B.; Cheng, C. L.; Xu, H. X.; Jiang, R. W.; But, P. P. H.; Shaw, P. C. Authentication and quantitative analysis on the chemical profile of cassia bark (Cortex cinnamomi) by high-pressure liquid chromatography. J. Agric. Food Chem. 2005, 53, 2424−2428. (8) Clark, G. S. Coumarin, an aroma chemical profile. Perfum. Flavor. 1995, 20, 23−34. (9) Lake, B. G. Coumarin metabolism, toxicity and carcinogenicity: Relevance for human risk assessment. Food Chem. Toxicol. 1999, 37, 423−453. (10) Hazleton, L. W.; Tusing, T. W.; Zeitlin, B. R.; Thiessen, R.; Murer, H. K. Toxicity of coumarin. J. Pharmacol. Exp. Ther. 1956, 118, 348−358. (11) Council of the European Communities. On the approximation of the laws of the Member States relating to flavourings for use in foodstuffs and to source materials for their production. Council Directive 88/388/EEC 1988, L184, 61−66. (12) European Food Safety Authority. Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contacts with food (AFC) on a request from the commission related to coumarin. EFSA J. 2004, 104, 1−36. (13) European Food Safety Authority. Coumarin in flavourings and other food ingredients with flavouring properties. EFSA J. 2008, 793, 1−15. (14) Fotland, T. O.; Paulsen, J. E.; Sanner, T.; Alexander, J.; Husoy, T. Risk assessment of coumarin using the bench mark dose (BMD) approach: Children in Norway which regularly eat oatmeal porridge with cinnamon may exceed the TDI for coumarin with several folds. Food Chem. Toxicol. 2012, 50, 903−912. (15) Abraham, K.; Wohrlin, F.; Lindtner, O.; Heinemeyer, G.; Lampen, A. Toxicology and risk assessment of coumarin: Focus on human data. Mol. Nutr. Food Res. 2010, 54, 228−239. (16) Lee, H. C.; Cheng, S. S.; Chang, S. T. Antifungal property of the essential oils and their constituents from Cinnamomum osmophloeum leaf against tree pathogenic fungi. J. Sci. Food Agric. 2005, 85, 2047− 2053. (17) Chang, S. T.; Chen, P. F.; Chang, S. C. Antibacterial activity of leaf essential oils and their constituents from Cinnamomum osmophloeum. J. Ethnopharmacol. 2001, 77, 123−127. (18) Cheng, S. S.; Chung, M. J.; Chen, Y. J.; Chang, S. T. Antipathogenic activities and chemical composition of Cinnamomum osmophloeum and Cinnamomum zeylanicum leaf essential oils. J. Wood Chem. Technol. 2011, 31, 73−87. (19) Chao, L. K.; Hua, K. F.; Hsu, H. Y.; Cheng, S. S.; Lin, I. F.; Chen, C. J.; Chen, S. T.; Chang, S. T. Cinnamaldehyde inhibits proinflammatory cytokines secretion from monocytes/macrophages through suppression of intracellular signaling. Food Chem. Toxicol. 2008, 46, 220−231. (20) Wang, S. Y.; Yang, C. W.; Liao, J. W.; Zhen, W. W.; Chu, F. H.; Chang, S. T. Essential oil from leaves of Cinnamomum osmophloeum acts as a xanthine oxidase inhibitor and reduces the serum uric acid levels in oxonate-induced mice. Phytomedicine 2008, 15, 940−945. (21) Lin, T. Y.; Liao, J. W.; Chang, S. T.; Wang, S. Y. Antidyslipidemic activity of hot-water extracts from leaves of Cinnamomum osmophloeum Kaneh. Phytother. Res. 2011, 25, 1317− 1322. (22) Cheng, S. S.; Liu, J. Y.; Tsai, K. H.; Chen, W. J.; Chang, S. T. Chemical composition and mosquito larvicidal activity of essential oils from leaves of different Cinnamomum osmophloeum provenances. J. Agric. Food Chem. 2004, 52, 4395−4400. (23) Adams, R. P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry; Allured Pub Corp: Carol Stream, IL, 2007.

wilsonii, and Cinnamomum japonicum have been reported by many researchers,3,4,6,7 and only C. verum samples (Ceylon cinnamon) might contain coumarin lower than 2 mg/kg of sample. All other samples were reported to exceed the 2 mg of coumarin per kg of sample regulation.3,4,6,7 Our samples show that specific C.o. clones from some areas did show very low coumarin contents (lower than 2 mg/kg of sample). The eugenol contents of the C.o. leaves from different geographic regions were from 4 to 27 g/kg of EOs (Figure 5C), and showed no significant difference between different sites except that from site C (Tukey HSD test, α = 0.05). However the eugenol contents from all C.o. leaf samples were higher than those from the C.c. samples (Figure 3C), indicating that the shelf life of the C.o. leaf samples could be longer compared to that of the C.c. samples. The cinnamyl acetate contents of the C.o. leaves from different geographic sites showed no significant difference between different sites (Tukey HSD test, α = 0.05) (Figure 5D). Our results clearly suggested that leaves of Cinnamomum osmophloeum ct. cinnamaldehyde could be a potential renewable source for a better cinnamon substitute due to its low coumarin content. It will be worth selecting the highcinnamaldehyde, low-coumarin, and eugenol-containing clones from the specific sites, and conducting further field tests using clonal propagations in different sites to test if these highcinnamaldehyde, low-coumarin, and eugenol-containing clones are stable over different geographic regions and seasons. This will promote these special clones for further commercial purposes.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +886-2-33664626. Fax: +886-2-23654520. E-mail: [email protected]. Funding

This study was sponsored part by a grant (101AS-13.2.2-FB-e1) from the Forestry Bureau, Council of Agriculture, Taiwan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge Sheng-Yu Lu and Yen-Ray Hsui of Taiwan Forestry Research Institute for their supports in sample identification and Li-Yuan Liu of National Taiwan University for her technical assistance.

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REFERENCES

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(24) Lv, G. P.; Huang, W. H.; Yang, F. Q.; Li, J.; Li, S. P. Pressurized liquid extraction and GC-MS analysis for simultaneous determination of seven components in Cinnamomum cassia and the effect of sample preparation. J. Sep. Sci. 2010, 33, 2341−2348. (25) Kamatou, G. P.; Vermaak, I.; Viljoen, A. M. Eugenol-from the remote Maluku Islands to the international market place: A review of a remarkable and versatile molecule. Molecules 2012, 17, 6953−6981. (26) Maralhas, A.; Monteiro, A.; Martins, C.; Kranendonk, M.; Laires, A.; Rueff, J.; Rodrigues, A. S. Genotoxicity and endoreduplication inducing activity of the food flavouring eugenol. Mutagenesis 2006, 21, 199−204. (27) Code for Federal Regulations. Part 182 substances generally recognized as safe. In Title 21 Food and Drugs; U.S. Government Printing Office: Washington, DC, 2013; Vol. 3. (28) Yeh, H. F.; Luo, C. Y.; Lin, C. Y.; Cheng, S. S.; Hsu, Y. R.; Chang, S. T. Methods for thermal stability enhancement of leaf essential oils and their main consituents from indigenous cinnamon (Cinnamomum osmophloeum). J. Agric. Food Chem. 2013, 61, 6293− 6298. (29) Bevilacqua, A.; Corbo, M. R.; Sinigaglia, M. Combining eugenol and cinnamaldehyde to control the growth of Alicyclobacillus acidoterrestris. Food Control 2010, 21, 172−177. (30) Franz, C. Genetics. In Volatile Oil Crops: Their Biology, Biochemistry and Production; Hay, R. K. M., Waterman, P. G., Eds.; Longman: Harlow, U.K., 1993; pp 63−96.

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A Potential Low-Coumarin Cinnamon Substitute: Cinnamomum

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